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Sample records for high throughput vol-oxidizer

  1. High throughput screening informatics.

    PubMed

    Ling, Xuefeng Bruce

    2008-03-01

    High throughput screening (HTS), an industrial effort to leverage developments in the areas of modern robotics, data analysis and control software, liquid handling devices, and sensitive detectors, has played a pivotal role in the drug discovery process, allowing researchers to efficiently screen millions of compounds to identify tractable small molecule modulators of a given biological process or disease state and advance them into high quality leads. As HTS throughput has significantly increased the volume, complexity, and information content of datasets, lead discovery research demands a clear corporate strategy for scientific computing and subsequent establishment of robust enterprise-wide (usually global) informatics platforms, which enable complicated HTS work flows, facilitate HTS data mining, and drive effective decision-making. The purpose of this review is, from the data analysis and handling perspective, to examine key elements in HTS operations and some essential data-related activities supporting or interfacing the screening process, and outline properties that various enabling software should have. Additionally, some general advice for corporate managers with system procurement responsibilities is offered.

  2. High-throughput proteomics

    NASA Astrophysics Data System (ADS)

    Lesley, Scott A.; Nasoff, Marc; Kreusch, Andreas; Spraggon, Glen

    2001-04-01

    Proteomics has become a major focus as researchers attempt to understand the vast amount of genomic information. Protein complexity makes identifying and understanding gene function inherently difficult. The challenge of studying proteins in a global way is driving the development of new technologies for systematic and comprehensive analysis of protein structure and function. We are addressing this challenge through instrumentation and approaches to rapidly express, purify, crystallize, and mutate large numbers of human gene products. Our approach applies the principles of HTS technologies commonly used in pharmaceutical development. Genes are cloned, expressed, and purified in parallel to achieve a throughput potential of hundreds per day. Our instrumentation allows us to produce tens of milligrams of protein from 96 separate clones simultaneously. Purified protein is used for several applications including a high-throughput crystallographic screening approach for structure determination using automated image analysis. To further understand protein function, we are integrating a mutagenesis and screening approach. By combining these key technologies, we hope to provide a fundamental basis for understanding gene function at the protein level.

  3. High throughput continuous cryopump

    SciTech Connect

    Foster, C.A.

    1986-01-01

    A cryocondensation pump with a unique regeneration mechanism that allows continuous operation has been constructed and tested. The pump features a device referred to as the ''Snail'' which removes the cryofrost layer as it is moved over the pumping surfaces. A forepump pumps the sublimed gas generated inside the Snail. The compression ratio of the pump is the ratio of the cryopump speed to the leakage conductance of the Snail. Deuterium had been pumped continuously at 30 torr.L/s at a speed of 2000 L/s and a compression ratio of 100. The pump, being all metal sealed and free of lubricating fluids, has many potential applications where untraclean high throughput pumping is desirable. Since the pump regenerates on a time scale of 60 seconds, the inventory in the pump is minimized - an important consideration when pumping radioactive materials such as tritium. Test data and a videotape of the Snail removing the cryofrost will be shown.

  4. High-Throughput Proteomics

    NASA Astrophysics Data System (ADS)

    Zhang, Zhaorui; Wu, Si; Stenoien, David L.; Paša-Tolić, Ljiljana

    2014-06-01

    Mass spectrometry (MS)-based high-throughput proteomics is the core technique for large-scale protein characterization. Due to the extreme complexity of proteomes, sophisticated separation techniques and advanced MS instrumentation have been developed to extend coverage and enhance dynamic range and sensitivity. In this review, we discuss the separation and prefractionation techniques applied for large-scale analysis in both bottom-up (i.e., peptide-level) and top-down (i.e., protein-level) proteomics. Different approaches for quantifying peptides or intact proteins, including label-free and stable-isotope-labeling strategies, are also discussed. In addition, we present a brief overview of different types of mass analyzers and fragmentation techniques as well as selected emerging techniques.

  5. High throughput optical scanner

    DOEpatents

    Basiji, David A.; van den Engh, Gerrit J.

    2001-01-01

    A scanning apparatus is provided to obtain automated, rapid and sensitive scanning of substrate fluorescence, optical density or phosphorescence. The scanner uses a constant path length optical train, which enables the combination of a moving beam for high speed scanning with phase-sensitive detection for noise reduction, comprising a light source, a scanning mirror to receive light from the light source and sweep it across a steering mirror, a steering mirror to receive light from the scanning mirror and reflect it to the substrate, whereby it is swept across the substrate along a scan arc, and a photodetector to receive emitted or scattered light from the substrate, wherein the optical path length from the light source to the photodetector is substantially constant throughout the sweep across the substrate. The optical train can further include a waveguide or mirror to collect emitted or scattered light from the substrate and direct it to the photodetector. For phase-sensitive detection the light source is intensity modulated and the detector is connected to phase-sensitive detection electronics. A scanner using a substrate translator is also provided. For two dimensional imaging the substrate is translated in one dimension while the scanning mirror scans the beam in a second dimension. For a high throughput scanner, stacks of substrates are loaded onto a conveyor belt from a tray feeder.

  6. High-throughput protein crystallization.

    PubMed

    Stevens, R C

    2000-10-01

    The combinatorial chemistry industry has made major advances in the handling and mixing of small volumes, and in the development of robust liquid-handling systems. In addition, developments have been made in the area of material handling for the high-throughput drug screening and combinatorial chemistry fields. Lastly, improvements in beamline optics at synchrotron sources have enabled the use of flash-frozen micron-sized (10-50 microm) crystals. The combination of these and other recent advances will make high-throughput protein crystallography possible. Further advances in high-throughput methods of protein crystallography will require application of the above developments and the accumulation of success/failure data in a more systematic manner. Major changes in crystallography technology will emerge based on the data collected by first-generation high-throughput systems.

  7. High-throughput continuous cryopump

    SciTech Connect

    Foster, C.A.

    1986-01-01

    A cryopump with a unique method of regeneration which allows continuous operation at high throughput has been constructed and tested. Deuterium was pumped continuously at a throughput of 30 Torr.L/s at a speed of 2000 L/s and a compression ratio of 200. Argon was pumped at a throughput of 60 Torr.L/s at a speed of 1275 L/s. To produce continuous operation of the pump, a method of regeneration that does not thermally cycle the pump is employed. A small chamber (the ''snail'') passes over the pumping surface and removes the frost from it either by mechanical action with a scraper or by local heating. The material removed is topologically in a secondary vacuum system with low conductance into the primary vacuum; thus, the exhaust can be pumped at pressures up to an effective compression ratio determined by the ratio of the pumping speed to the leakage conductance of the snail. The pump, which is all-metal-sealed and dry and which regenerates every 60 s, would be an ideal system for pumping tritium. Potential fusion applications are for mpmp limiters, for repeating pneumatic pellet injection lines, and for the centrifuge pellet injector spin tank, all of which will require pumping tritium at high throughput. Industrial applications requiring ultraclean pumping of corrosive gases at high throughput, such as the reactive ion etch semiconductor process, may also be feasible.

  8. High throughput protein production screening

    DOEpatents

    Beernink, Peter T.; Coleman, Matthew A.; Segelke, Brent W.

    2009-09-08

    Methods, compositions, and kits for the cell-free production and analysis of proteins are provided. The invention allows for the production of proteins from prokaryotic sequences or eukaryotic sequences, including human cDNAs using PCR and IVT methods and detecting the proteins through fluorescence or immunoblot techniques. This invention can be used to identify optimized PCR and WT conditions, codon usages and mutations. The methods are readily automated and can be used for high throughput analysis of protein expression levels, interactions, and functional states.

  9. High-Throughput Sequencing Technologies

    PubMed Central

    Reuter, Jason A.; Spacek, Damek; Snyder, Michael P.

    2015-01-01

    Summary The human genome sequence has profoundly altered our understanding of biology, human diversity and disease. The path from the first draft sequence to our nascent era of personal genomes and genomic medicine has been made possible only because of the extraordinary advancements in DNA sequencing technologies over the past ten years. Here, we discuss commonly used high-throughput sequencing platforms, the growing array of sequencing assays developed around them as well as the challenges facing current sequencing platforms and their clinical application. PMID:26000844

  10. High throughput vacuum chemical epitaxy

    NASA Astrophysics Data System (ADS)

    Fraas, L. M.; Malocsay, E.; Sundaram, V.; Baird, R. W.; Mao, B. Y.; Lee, G. Y.

    1990-10-01

    We have developed a vacuum chemical epitaxy (VCE) reactor which avoids the use of arsine and allows multiple wafers to be coated at one time. Our vacuum chemical epitaxy reactor closely resembles a molecular beam epitaxy system in that wafers are loaded into a stainless steel vacuum chamber through a load chamber. Also as in MBE, arsenic vapors are supplied as reactant by heating solid arsenic sources thereby avoiding the use of arsine. However, in our VCE reactor, a large number of wafers are coated at one time in a vacuum system by the substitution of Group III alkyl sources for the elemental metal sources traditionally used in MBE. Higher wafer throughput results because in VCE, the metal-alkyl sources for Ga, Al, and dopants can be mixed at room temperature and distributed uniformly though a large area injector to multiple substrates as a homogeneous array of mixed element molecular beams. The VCE reactor that we have built and that we shall describe here uniformly deposits films on 7 inch diameter substrate platters. Each platter contains seven two inch or three 3 inch diameter wafers. The load chamber contains up to nine platters. The vacuum chamber is equipped with two VCE growth zones and two arsenic ovens, one per growth zone. Finally, each oven has a 1 kg arsenic capacity. As of this writing, mirror smooth GaAs films have been grown at up to 4 μm/h growth rate on multiple wafers with good thickness uniformity. The background doping is p-type with a typical hole concentration and mobility of 1 × 10 16/cm 3 and 350 cm 2/V·s. This background doping level is low enough for the fabrication of MESFETs, solar cells, and photocathodes as well as other types of devices. We have fabricated MESFET devices using VCE-grown epi wafers with peak extrinsic transconductance as high as 210 mS/mm for a threshold voltage of - 3 V and a 0.6 μm gate length. We have also recently grown AlGaAs epi layers with up to 80% aluminum using TEAl as the aluminum alkyl source. The Al

  11. Nanoliter high throughput quantitative PCR

    PubMed Central

    Morrison, Tom; Hurley, James; Garcia, Javier; Yoder, Karl; Katz, Arrin; Roberts, Douglas; Cho, Jamie; Kanigan, Tanya; Ilyin, Sergey E.; Horowitz, Daniel; Dixon, James M.; Brenan, Colin J.H.

    2006-01-01

    Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols. PMID:17000636

  12. High-Throughput Nonlinear Optical Microscopy

    PubMed Central

    So, Peter T.C.; Yew, Elijah Y.S.; Rowlands, Christopher

    2013-01-01

    High-resolution microscopy methods based on different nonlinear optical (NLO) contrast mechanisms are finding numerous applications in biology and medicine. While the basic implementations of these microscopy methods are relatively mature, an important direction of continuing technological innovation lies in improving the throughput of these systems. Throughput improvement is expected to be important for studying fast kinetic processes, for enabling clinical diagnosis and treatment, and for extending the field of image informatics. This review will provide an overview of the fundamental limitations on NLO microscopy throughput. We will further cover several important classes of high-throughput NLO microscope designs with discussions on their strengths and weaknesses and their key biomedical applications. Finally, this review will close with a perspective of potential future technological improvements in this field. PMID:24359736

  13. High throughput network for multiprocessor interconnections

    NASA Astrophysics Data System (ADS)

    Raatikainen, Pertti; Zidbeck, Juha

    1993-05-01

    Multiprocessor architectures are needed to support modern broadband applications, since traditional bus structures are not capable of providing high throughput. New bus structures are needed, especially in the area of network components and terminals. A study to find an efficient and cost effective interconnection topology for the future high speed products is presented. The most common bus topologies are introduced, and their characteristics are estimated to decide which one of them offers best performance and lowest implementation cost. The ring topology is chosen to be studied in more detail. Four competing bus access schemes for the high throughput ring are introduced as well as simulation models for each of them. Using transfer delay and throughput results, as well as keeping the implementation point of view in mind, the best candidate is selected to be studied and experimented in the succeeding research project.

  14. High-throughput phenotyping of plant shoots.

    PubMed

    Berger, Bettina; de Regt, Bas; Tester, Mark

    2012-01-01

    Advances in automated plant handling and image acquisition now make it possible to use digital imaging for the high-throughput phenotyping of plants. Various traits can be extracted from individual images. However, the potential of this technology lies in the acquisition of time series. Since whole shoot imaging is nondestructive, plants can now be monitored throughout their lifecycle, and dynamic traits such as plant growth and development can be captured and quantified. The technique is applicable to a wide range of plants and research areas and makes high-throughput screens possible, reducing the time and labor needed for the phenotypic characterization of plants.

  15. Economic consequences of high throughput maskless lithography

    NASA Astrophysics Data System (ADS)

    Hartley, John G.; Govindaraju, Lakshmi

    2005-11-01

    Many people in the semiconductor industry bemoan the high costs of masks and view mask cost as one of the significant barriers to bringing new chip designs to market. All that is needed is a viable maskless technology and the problem will go away. Numerous sites around the world are working on maskless lithography but inevitably, the question asked is "Wouldn't a one wafer per hour maskless tool make a really good mask writer?" Of course, the answer is yes, the hesitation you hear in the answer isn't based on technology concerns, it's financial. The industry needs maskless lithography because mask costs are too high. Mask costs are too high because mask pattern generators (PG's) are slow and expensive. If mask PG's become much faster, mask costs go down, the maskless market goes away and the PG supplier is faced with an even smaller tool demand from the mask shops. Technical success becomes financial suicide - or does it? In this paper we will present the results of a model that examines some of the consequences of introducing high throughput maskless pattern generation. Specific features in the model include tool throughput for masks and wafers, market segmentation by node for masks and wafers and mask cost as an entry barrier to new chip designs. How does the availability of low cost masks and maskless tools affect the industries tool makeup and what is the ultimate potential market for high throughput maskless pattern generators?

  16. High-throughput neuro-imaging informatics

    PubMed Central

    Miller, Michael I.; Faria, Andreia V.; Oishi, Kenichi; Mori, Susumu

    2013-01-01

    This paper describes neuroinformatics technologies at 1 mm anatomical scale based on high-throughput 3D functional and structural imaging technologies of the human brain. The core is an abstract pipeline for converting functional and structural imagery into their high-dimensional neuroinformatic representation index containing O(1000–10,000) discriminating dimensions. The pipeline is based on advanced image analysis coupled to digital knowledge representations in the form of dense atlases of the human brain at gross anatomical scale. We demonstrate the integration of these high-dimensional representations with machine learning methods, which have become the mainstay of other fields of science including genomics as well as social networks. Such high-throughput facilities have the potential to alter the way medical images are stored and utilized in radiological workflows. The neuroinformatics pipeline is used to examine cross-sectional and personalized analyses of neuropsychiatric illnesses in clinical applications as well as longitudinal studies. We demonstrate the use of high-throughput machine learning methods for supporting (i) cross-sectional image analysis to evaluate the health status of individual subjects with respect to the population data, (ii) integration of image and personal medical record non-image information for diagnosis and prognosis. PMID:24381556

  17. Network medicine and high throughput screening.

    PubMed

    Smith, Robert E; Tran, Kevin; Vocque, Ralph H

    2013-09-01

    A new paradigm is emerging in modern drug discovery. It is a fusion of traditional and modern medicine, phenotypic and targeted drug discovery, or systems and reductionist thinking. This is exemplified by using a combination of network medicine and high throughput screening. It blends the use of physiologically relevant biological systems with the high throughput and statistical robustness of modern assay technologies. The basic principles of network theory and tools of network medicine are described. Scale-free networks and their organizing principles are discussed. They are emergent properties of living, autopoietic systems. This includes networks of people who do high throughput screening (HTS), and microscopic networks of ions, metabolites, DNA, RNA, proteins, lipids, carbohydrates, viruses, bacteria, fungi, human cells and tissues. Databases have been constructed based on the metabolome, genome, transcriptome, proteome, lipidome, glycocode, virome, bacteriome and many others. Modern HTS can be used to examine the interactions of many parts of the complex human network. High content screening (HCS) can look at perturbations that occur when test compounds are added to single cells. Allo-network drugs can have effects far beyond a single protein and can be transmitted to other cells. Interactions and hidden connections can be revealed, with the goal of developing new drugs that have few, if any harmful side effects and are effective against multi-drug resistant cancer cells or bacteria.

  18. Viral detection by high-throughput sequencing.

    PubMed

    Motooka, Daisuke; Nakamura, Shota; Hagiwara, Katsuro; Nakaya, Takaaki

    2015-01-01

    We applied a high-throughput sequencing platform, Ion PGM, for viral detection in fecal samples from adult cows collected in Hokkaido, Japan. Random RT-PCR was performed to amplify RNA extracted from 0.25 ml of fecal specimens (N = 8), and more than 5 μg of cDNA was synthesized. Unbiased high-throughput sequencing using the 318 v2 semiconductor chip of these eight samples yielded 57-580 K (average: 270 K, after data analysis) reads in a single run. As a result, viral genome sequences were detected in each specimen. In addition to bacteriophage, mammal- and insect-derived viruses, partial genome sequences of plant, algal, and protozoal viruses were detected. Thus, this metagenomic analysis of fecal specimens could be useful to comprehensively understand viral populations of the intestine and food sources in animals. PMID:25287501

  19. Microfabricated high-throughput electronic particle detector.

    PubMed

    Wood, D K; Requa, M V; Cleland, A N

    2007-10-01

    We describe the design, fabrication, and use of a radio frequency reflectometer integrated with a microfluidic system, applied to the very high-throughput measurement of micron-scale particles, passing in a microfluidic channel through the sensor region. The device operates as a microfabricated Coulter counter [U.S. Patent No. 2656508 (1953)], similar to a design we have described previously, but here with significantly improved electrode geometry as well as including electronic tuning of the reflectometer; the two improvements yielding an improvement by more than a factor of 10 in the signal to noise and in the diametric discrimination of single particles. We demonstrate the high-throughput discrimination of polystyrene beads with diameters in the 4-10 microm range, achieving diametric resolutions comparable to the intrinsic spread of diameters in the bead distribution, at rates in excess of 15 x 10(6) beads/h.

  20. Viral detection by high-throughput sequencing.

    PubMed

    Motooka, Daisuke; Nakamura, Shota; Hagiwara, Katsuro; Nakaya, Takaaki

    2015-01-01

    We applied a high-throughput sequencing platform, Ion PGM, for viral detection in fecal samples from adult cows collected in Hokkaido, Japan. Random RT-PCR was performed to amplify RNA extracted from 0.25 ml of fecal specimens (N = 8), and more than 5 μg of cDNA was synthesized. Unbiased high-throughput sequencing using the 318 v2 semiconductor chip of these eight samples yielded 57-580 K (average: 270 K, after data analysis) reads in a single run. As a result, viral genome sequences were detected in each specimen. In addition to bacteriophage, mammal- and insect-derived viruses, partial genome sequences of plant, algal, and protozoal viruses were detected. Thus, this metagenomic analysis of fecal specimens could be useful to comprehensively understand viral populations of the intestine and food sources in animals.

  1. High Throughput Screening For Hazard and Risk of Environmental Contaminants

    EPA Science Inventory

    High throughput toxicity testing provides detailed mechanistic information on the concentration response of environmental contaminants in numerous potential toxicity pathways. High throughput screening (HTS) has several key advantages: (1) expense orders of magnitude less than an...

  2. Automated High Throughput Drug Target Crystallography

    SciTech Connect

    Rupp, B

    2005-02-18

    The molecular structures of drug target proteins and receptors form the basis for 'rational' or structure guided drug design. The majority of target structures are experimentally determined by protein X-ray crystallography, which as evolved into a highly automated, high throughput drug discovery and screening tool. Process automation has accelerated tasks from parallel protein expression, fully automated crystallization, and rapid data collection to highly efficient structure determination methods. A thoroughly designed automation technology platform supported by a powerful informatics infrastructure forms the basis for optimal workflow implementation and the data mining and analysis tools to generate new leads from experimental protein drug target structures.

  3. High throughput chemical munitions treatment system

    DOEpatents

    Haroldsen, Brent L.; Stofleth, Jerome H.; Didlake, Jr., John E.; Wu, Benjamin C-P

    2011-11-01

    A new High-Throughput Explosive Destruction System is disclosed. The new system is comprised of two side-by-side detonation containment vessels each comprising first and second halves that feed into a single agent treatment vessel. Both detonation containment vessels further comprise a surrounding ventilation facility. Moreover, the detonation containment vessels are designed to separate into two half-shells, wherein one shell can be moved axially away from the fixed, second half for ease of access and loading. The vessels are closed by means of a surrounding, clam-shell type locking seal mechanisms.

  4. High throughput screening technologies for ion channels

    PubMed Central

    Yu, Hai-bo; Li, Min; Wang, Wei-ping; Wang, Xiao-liang

    2016-01-01

    Ion channels are involved in a variety of fundamental physiological processes, and their malfunction causes numerous human diseases. Therefore, ion channels represent a class of attractive drug targets and a class of important off-targets for in vitro pharmacological profiling. In the past decades, the rapid progress in developing functional assays and instrumentation has enabled high throughput screening (HTS) campaigns on an expanding list of channel types. Chronologically, HTS methods for ion channels include the ligand binding assay, flux-based assay, fluorescence-based assay, and automated electrophysiological assay. In this review we summarize the current HTS technologies for different ion channel classes and their applications. PMID:26657056

  5. Clustering of High Throughput Gene Expression Data

    PubMed Central

    Pirim, Harun; Ekşioğlu, Burak; Perkins, Andy; Yüceer, Çetin

    2012-01-01

    High throughput biological data need to be processed, analyzed, and interpreted to address problems in life sciences. Bioinformatics, computational biology, and systems biology deal with biological problems using computational methods. Clustering is one of the methods used to gain insight into biological processes, particularly at the genomics level. Clearly, clustering can be used in many areas of biological data analysis. However, this paper presents a review of the current clustering algorithms designed especially for analyzing gene expression data. It is also intended to introduce one of the main problems in bioinformatics - clustering gene expression data - to the operations research community. PMID:23144527

  6. High-Throughput Methods for Electron Crystallography

    PubMed Central

    Stokes, David L.; Ubarretxena-Belandia, Iban; Gonen, Tamir; Engel, Andreas

    2013-01-01

    Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing the natural environment of a lipid membrane. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, images and diffraction can be recorded by electron microscopy. The corresponding data can be combined to produce a three-dimensional reconstruction which, under favorable conditions, can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative and potentially complementary methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on detergent complexation by cyclodextrin; a specialized pipetting robot has been designed not only to titrate cyclodextrin, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described. PMID:23132066

  7. High-throughput methods for electron crystallography.

    PubMed

    Stokes, David L; Ubarretxena-Belandia, Iban; Gonen, Tamir; Engel, Andreas

    2013-01-01

    Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing a native lipid environment for these proteins. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, electron microscopy can be used to collect images and diffraction and the corresponding data can be combined to produce a three-dimensional reconstruction, which under favorable conditions can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on titration of cyclodextrin as a chelating agent for detergent; a specialized pipetting robot has been designed not only to add cyclodextrin in a systematic way, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described.

  8. High Throughput Optimization of Stem Cell Microenvironments

    PubMed Central

    Yang, Fan; Mei, Ying; Langer, Robert; Anderson, Daniel G.

    2009-01-01

    Stem cells have great potential as cell sources for regenerative medicine due to both their self-renewal and multi-lineage differentiation capacity. Despite advances in the field of stem cell biology, major challenges remain before stem cells can be widely used for therapeutic purposes. One challenge is to develop reproducible methods to control stem cell growth and differentiation. The niche in which stem cells reside is a complex, multi-factorial environment. In contrast to using cells alone, biomaterials can provide initial structural support, and allow cells to adhere, proliferate and differentiate in a three-dimensional environment. Researchers have incorporated signals into the biomaterials that can promote desired cell functions in a spatially and temporally controlled manner. Despite progress in biomaterial design and methods to modulate cellular behavior, many of the complex signal networks that regulate cell-material interactions remain unclear. Due to the vast numbers of material properties to be explored and the complexity of cell-surface interactions, it is often difficult to optimize stem cell microenvironments using conventional, iterative approaches. To address these challenges, high throughput screening of combinatorial libraries has emerged as a novel approach to achieve rapid screening with reduced materials and costs. In this review, we discuss recent research in the area of high throughput approaches for characterization and optimization of cellular interactions with their microenvironments. In contrast to conventional approaches, screening combinatorial libraries can result in the discovery of unexpected material solutions to these complex problems. PMID:19601753

  9. Preliminary High-Throughput Metagenome Assembly

    SciTech Connect

    Dusheyko, Serge; Furman, Craig; Pangilinan, Jasmyn; Shapiro, Harris; Tu, Hank

    2007-03-26

    Metagenome data sets present a qualitatively different assembly problem than traditional single-organism whole-genome shotgun (WGS) assembly. The unique aspects of such projects include the presence of a potentially large number of distinct organisms and their representation in the data set at widely different fractions. In addition, multiple closely related strains could be present, which would be difficult to assemble separately. Failure to take these issues into account can result in poor assemblies that either jumble together different strains or which fail to yield useful results. The DOE Joint Genome Institute has sequenced a number of metagenomic projects and plans to considerably increase this number in the coming year. As a result, the JGI has a need for high-throughput tools and techniques for handling metagenome projects. We present the techniques developed to handle metagenome assemblies in a high-throughput environment. This includes a streamlined assembly wrapper, based on the JGI?s in-house WGS assembler, Jazz. It also includes the selection of sensible defaults targeted for metagenome data sets, as well as quality control automation for cleaning up the raw results. While analysis is ongoing, we will discuss preliminary assessments of the quality of the assembly results (http://fames.jgi-psf.org).

  10. High throughput sample processing and automated scoring.

    PubMed

    Brunborg, Gunnar; Jackson, Petra; Shaposhnikov, Sergey; Dahl, Hildegunn; Azqueta, Amaya; Collins, Andrew R; Gutzkow, Kristine B

    2014-01-01

    The comet assay is a sensitive and versatile method for assessing DNA damage in cells. In the traditional version of the assay, there are many manual steps involved and few samples can be treated in one experiment. High throughput (HT) modifications have been developed during recent years, and they are reviewed and discussed. These modifications include accelerated scoring of comets; other important elements that have been studied and adapted to HT are cultivation and manipulation of cells or tissues before and after exposure, and freezing of treated samples until comet analysis and scoring. HT methods save time and money but they are useful also for other reasons: large-scale experiments may be performed which are otherwise not practicable (e.g., analysis of many organs from exposed animals, and human biomonitoring studies), and automation gives more uniform sample treatment and less dependence on operator performance. The HT modifications now available vary largely in their versatility, capacity, complexity, and costs. The bottleneck for further increase of throughput appears to be the scoring. PMID:25389434

  11. High-throughput rod-induced electrospinning

    NASA Astrophysics Data System (ADS)

    Wu, Dezhi; Xiao, Zhiming; Teh, Kwok Siong; Han, Zhibin; Luo, Guoxi; Shi, Chuan; Sun, Daoheng; Zhao, Jinbao; Lin, Liwei

    2016-09-01

    A high throughput electrospinning process, directly from flat polymer solution surfaces induced by a moving insulating rod, has been proposed and demonstrated. Different rods made of either phenolic resin or paper with a diameter of 1–3 cm and a resistance of about 100–500 MΩ, has been successfully utilized in the process. The rod is placed approximately 10 mm above the flat polymer solution surface with a moving speed of 0.005–0.4 m s‑1 this causes the solution to generate multiple liquid jets under an applied voltage of 15–60 kV for the tip-less electrospinning process. The local electric field induced by the rod can boost electrohydrodynamic instability in order to generate Taylor cones and liquid jets. Experimentally, it is found that a large rod diameter and a small solution-to-rod distance can enhance the local electrical field to reduce the magnitude of the applied voltage. In the prototype setup with poly (ethylene oxide) polymer solution, an area of 5 cm  ×  10 cm and under an applied voltage of 60 kV, the maximum throughput of nanofibers is recorded to be approximately144 g m‑2 h‑1.

  12. High-throughput rod-induced electrospinning

    NASA Astrophysics Data System (ADS)

    Wu, Dezhi; Xiao, Zhiming; Teh, Kwok Siong; Han, Zhibin; Luo, Guoxi; Shi, Chuan; Sun, Daoheng; Zhao, Jinbao; Lin, Liwei

    2016-09-01

    A high throughput electrospinning process, directly from flat polymer solution surfaces induced by a moving insulating rod, has been proposed and demonstrated. Different rods made of either phenolic resin or paper with a diameter of 1-3 cm and a resistance of about 100-500 MΩ, has been successfully utilized in the process. The rod is placed approximately 10 mm above the flat polymer solution surface with a moving speed of 0.005-0.4 m s-1 this causes the solution to generate multiple liquid jets under an applied voltage of 15-60 kV for the tip-less electrospinning process. The local electric field induced by the rod can boost electrohydrodynamic instability in order to generate Taylor cones and liquid jets. Experimentally, it is found that a large rod diameter and a small solution-to-rod distance can enhance the local electrical field to reduce the magnitude of the applied voltage. In the prototype setup with poly (ethylene oxide) polymer solution, an area of 5 cm  ×  10 cm and under an applied voltage of 60 kV, the maximum throughput of nanofibers is recorded to be approximately144 g m-2 h-1.

  13. High Throughput Screening Tools for Thermoelectric Materials

    NASA Astrophysics Data System (ADS)

    Wong-Ng, W.; Yan, Y.; Otani, M.; Martin, J.; Talley, K. R.; Barron, S.; Carroll, D. L.; Hewitt, C.; Joress, H.; Thomas, E. L.; Green, M. L.; Tang, X. F.

    2015-06-01

    A suite of complementary high-throughput screening systems for combinatorial films was developed at National Institute of Standards and Technology to facilitate the search for efficient thermoelectric materials. These custom-designed capabilities include a facility for combinatorial thin film synthesis and a suite of tools for screening the Seebeck coefficient, electrical resistance (electrical resistivity), and thermal effusivity (thermal conductivity) of these films. The Seebeck coefficient and resistance are measured via custom-built automated apparatus at both ambient and high temperatures. Thermal effusivity is measured using a frequency domain thermoreflectance technique. This paper will discuss applications using these tools on representative thermoelectric materials, including combinatorial composition-spread films, conventional films, single crystals, and ribbons.

  14. High-Throughput Analysis of Enzyme Activities

    SciTech Connect

    Lu, Guoxin

    2007-01-01

    High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different

  15. A high-throughput neutron spectrometer

    NASA Astrophysics Data System (ADS)

    Stampfl, Anton; Noakes, Terry; Bartsch, Friedl; Bertinshaw, Joel; Veliscek-Carolan, Jessica; Nateghi, Ebrahim; Raeside, Tyler; Yethiraj, Mohana; Danilkin, Sergey; Kearley, Gordon

    2010-03-01

    A cross-disciplinary high-throughput neutron spectrometer is currently under construction at OPAL, ANSTO's open pool light-water research reactor. The spectrometer is based on the design of a Be-filter spectrometer (FANS) that is operating at the National Institute of Standards research reactor in the USA. The ANSTO filter-spectrometer will be switched in and out with another neutron spectrometer, the triple-axis spectrometer, Taipan. Thus two distinct types of neutron spectrometers will be accessible: one specialised to perform phonon dispersion analysis and the other, the filter-spectrometer, designed specifically to measure vibrational density of states. A summary of the design will be given along with a detailed ray-tracing analysis. Some preliminary results will be presented from the spectrometer.

  16. High-throughput cellular RNA device engineering.

    PubMed

    Townshend, Brent; Kennedy, Andrew B; Xiang, Joy S; Smolke, Christina D

    2015-10-01

    Methods for rapidly assessing sequence-structure-function landscapes and developing conditional gene-regulatory devices are critical to our ability to manipulate and interface with biology. We describe a framework for engineering RNA devices from preexisting aptamers that exhibit ligand-responsive ribozyme tertiary interactions. Our methodology utilizes cell sorting, high-throughput sequencing and statistical data analyses to enable parallel measurements of the activities of hundreds of thousands of sequences from RNA device libraries in the absence and presence of ligands. Our tertiary-interaction RNA devices performed better in terms of gene silencing, activation ratio and ligand sensitivity than optimized RNA devices that rely on secondary-structure changes. We applied our method to build biosensors for diverse ligands and determine consensus sequences that enable ligand-responsive tertiary interactions. These methods advance our ability to develop broadly applicable genetic tools and to elucidate the underlying sequence-structure-function relationships that empower rational design of complex biomolecules. PMID:26258292

  17. High throughput assays for analyzing transcription factors.

    PubMed

    Li, Xianqiang; Jiang, Xin; Yaoi, Takuro

    2006-06-01

    Transcription factors are a group of proteins that modulate the expression of genes involved in many biological processes, such as cell growth and differentiation. Alterations in transcription factor function are associated with many human diseases, and therefore these proteins are attractive potential drug targets. A key issue in the development of such therapeutics is the generation of effective tools that can be used for high throughput discovery of the critical transcription factors involved in human diseases, and the measurement of their activities in a variety of disease or compound-treated samples. Here, a number of innovative arrays and 96-well format assays for profiling and measuring the activities of transcription factors will be discussed. PMID:16834538

  18. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Forsythe, Elizabeth; Achari, Aniruddha

    2006-01-01

    We have shown that by covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and the presence of the probe at low concentrations does not affect the X-ray data quality or the crystallization behavior. The presence of the trace fluorescent label gives a number of advantages when used with high throughput crystallizations. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a dark background. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Brightly fluorescent crystals are readily found against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. We are now testing the use of high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that kinetics leading to non-structured phases may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Preliminary experiments with test proteins have resulted in the extraction of a number of crystallization conditions from screening outcomes based solely on the presence of bright fluorescent regions. Subsequent experiments will test this approach using a wider

  19. Data Analysis for High-Throughput RNAi Screening.

    PubMed

    Azorsa, David O; Turnidge, Megan A; Arora, Shilpi

    2016-01-01

    High-throughput RNA interference (HT-RNAi) screening is an effective technology to help identify important genes and pathways involved in a biological process. Analysis of high-throughput RNAi screening data is a critical part of this technology, and many analysis methods have been described. Here, we summarize the workflow and types of analyses commonly used in high-throughput RNAi screening. PMID:27581298

  20. High Throughput Screening and Selection Methods for Directed Enzyme Evolution

    PubMed Central

    2015-01-01

    Successful evolutionary enzyme engineering requires a high throughput screening or selection method, which considerably increases the chance of obtaining desired properties and reduces the time and cost. In this review, a series of high throughput screening and selection methods are illustrated with significant and recent examples. These high throughput strategies are also discussed with an emphasis on compatibility with phenotypic analysis during directed enzyme evolution. Lastly, certain limitations of current methods, as well as future developments, are briefly summarized. PMID:26074668

  1. High-Throughput Screening in Primary Neurons

    PubMed Central

    Sharma, Punita; Ando, D. Michael; Daub, Aaron; Kaye, Julia A.; Finkbeiner, Steven

    2013-01-01

    Despite years of incremental progress in our understanding of diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), there are still no disease-modifying therapeutics. The discrepancy between the number of lead compounds and approved drugs may partially be a result of the methods used to generate the leads and highlights the need for new technology to obtain more detailed and physiologically relevant information on cellular processes in normal and diseased states. Our high-throughput screening (HTS) system in a primary neuron model can help address this unmet need. HTS allows scientists to assay thousands of conditions in a short period of time which can reveal completely new aspects of biology and identify potential therapeutics in the span of a few months when conventional methods could take years or fail all together. HTS in primary neurons combines the advantages of HTS with the biological relevance of intact, fully differentiated neurons which can capture the critical cellular events or homeostatic states that make neurons uniquely susceptible to disease-associated proteins. We detail methodologies of our primary neuron HTS assay workflow from sample preparation to data reporting. We also discuss our adaptation of our HTS system into high-content screening (HCS), a type of HTS that uses multichannel fluorescence images to capture biological events in situ, and is uniquely suited to study dynamical processes in living cells. PMID:22341232

  2. High-Throughput Baculovirus Expression System for Membrane Protein Production.

    PubMed

    Kalathur, Ravi C; Panganiban, Marinela; Bruni, Renato

    2016-01-01

    The ease of use, robustness, cost-effectiveness, and posttranslational machinery make baculovirus expression system a popular choice for production of eukaryotic membrane proteins. This system can be readily adapted for high-throughput operations. This chapter outlines the techniques and procedures for cloning, transfection, small-scale production, and purification of membrane protein samples in a high-throughput manner. PMID:27485337

  3. High-Throughput Enzyme Kinetics Using Microarrays

    SciTech Connect

    Guoxin Lu; Edward S. Yeung

    2007-11-01

    We report a microanalytical method to study enzyme kinetics. The technique involves immobilizing horseradish peroxidase on a poly-L-lysine (PLL)- coated glass slide in a microarray format, followed by applying substrate solution onto the enzyme microarray. Enzyme molecules are immobilized on the PLL-coated glass slide through electrostatic interactions, and no further modification of the enzyme or glass slide is needed. In situ detection of the products generated on the enzyme spots is made possible by monitoring the light intensity of each spot using a scientific-grade charged-coupled device (CCD). Reactions of substrate solutions of various types and concentrations can be carried out sequentially on one enzyme microarray. To account for the loss of enzyme from washing in between runs, a standard substrate solution is used for calibration. Substantially reduced amounts of substrate solution are consumed for each reaction on each enzyme spot. The Michaelis constant K{sub m} obtained by using this method is comparable to the result for homogeneous solutions. Absorbance detection allows universal monitoring, and no chemical modification of the substrate is needed. High-throughput studies of native enzyme kinetics for multiple enzymes are therefore possible in a simple, rapid, and low-cost manner.

  4. Orthogonal NGS for High Throughput Clinical Diagnostics.

    PubMed

    Chennagiri, Niru; White, Eric J; Frieden, Alexander; Lopez, Edgardo; Lieber, Daniel S; Nikiforov, Anastasia; Ross, Tristen; Batorsky, Rebecca; Hansen, Sherry; Lip, Va; Luquette, Lovelace J; Mauceli, Evan; Margulies, David; Milos, Patrice M; Napolitano, Nichole; Nizzari, Marcia M; Yu, Timothy; Thompson, John F

    2016-04-19

    Next generation sequencing is a transformative technology for discovering and diagnosing genetic disorders. However, high-throughput sequencing remains error-prone, necessitating variant confirmation in order to meet the exacting demands of clinical diagnostic sequencing. To address this, we devised an orthogonal, dual platform approach employing complementary target capture and sequencing chemistries to improve speed and accuracy of variant calls at a genomic scale. We combined DNA selection by bait-based hybridization followed by Illumina NextSeq reversible terminator sequencing with DNA selection by amplification followed by Ion Proton semiconductor sequencing. This approach yields genomic scale orthogonal confirmation of ~95% of exome variants. Overall variant sensitivity improves as each method covers thousands of coding exons missed by the other. We conclude that orthogonal NGS offers improvements in variant calling sensitivity when two platforms are used, better specificity for variants identified on both platforms, and greatly reduces the time and expense of Sanger follow-up, thus enabling physicians to act on genomic results more quickly.

  5. Orthogonal NGS for High Throughput Clinical Diagnostics

    PubMed Central

    Chennagiri, Niru; White, Eric J.; Frieden, Alexander; Lopez, Edgardo; Lieber, Daniel S.; Nikiforov, Anastasia; Ross, Tristen; Batorsky, Rebecca; Hansen, Sherry; Lip, Va; Luquette, Lovelace J.; Mauceli, Evan; Margulies, David; Milos, Patrice M.; Napolitano, Nichole; Nizzari, Marcia M.; Yu, Timothy; Thompson, John F.

    2016-01-01

    Next generation sequencing is a transformative technology for discovering and diagnosing genetic disorders. However, high-throughput sequencing remains error-prone, necessitating variant confirmation in order to meet the exacting demands of clinical diagnostic sequencing. To address this, we devised an orthogonal, dual platform approach employing complementary target capture and sequencing chemistries to improve speed and accuracy of variant calls at a genomic scale. We combined DNA selection by bait-based hybridization followed by Illumina NextSeq reversible terminator sequencing with DNA selection by amplification followed by Ion Proton semiconductor sequencing. This approach yields genomic scale orthogonal confirmation of ~95% of exome variants. Overall variant sensitivity improves as each method covers thousands of coding exons missed by the other. We conclude that orthogonal NGS offers improvements in variant calling sensitivity when two platforms are used, better specificity for variants identified on both platforms, and greatly reduces the time and expense of Sanger follow-up, thus enabling physicians to act on genomic results more quickly. PMID:27090146

  6. High Throughput Bent-Pipe Processor Demonstrator

    NASA Astrophysics Data System (ADS)

    Tabacco, P.; Vernucci, A.; Russo, L.; Cangini, P.; Botticchio, T.; Angeletti, P.

    2008-08-01

    The work associated to this article is a study initiative sponsored by ESA/ESTEC that responds to the crucial need of developing new Satellite payload aimed at making rapid progresses in handling large amounts of data at a competitive price with respect to terrestrial one in the telecommunication field. Considering the quite limited band allowed to space communications at Ka band, reusing the same band in a large number of beams is mandatory: therefore beam-forming is the right technological answer. Technological progresses - mainly in the digital domain - also help greatly in increasing the satellite capacity. Next Satellite payload target are set in throughput range of 50Gbps. Despite the fact that the implementation of a wideband transparent processor for a high capacity communication payload is a very challenging task, Space Engineering team in the frame of this ESA study proposed an intermediate step of development for a scalable unit able to demonstrate both the capacity and flexibility objectives for different type of Wideband Beamforming antennas designs. To this aim the article describes the features of Wideband HW (analog and digital) platform purposely developed by Space Engineering in the frame of this ESA/ESTEC contract ("WDBFN" contract) with some preliminary system test results. The same platform and part of the associated SW will be used in the development and demonstration of the real payload digital front end Mux and Demux algorithms as well as the Beam Forming and on Board channel switching in frequency domain. At the time of this article writing, despite new FPGA and new ADC and DAC converters have become available as choices for wideband system implementation, the two HW platforms developed by Space Engineering, namely WDBFN ADC and DAC Boards, represent still the most performing units in terms of analog bandwidth, processing capability (in terms of FPGA module density), SERDES (SERiliazer DESerializers) external links density, integration form

  7. Technological advances in high-throughput screening.

    PubMed

    Liu, Bailing; Li, Songjun; Hu, Jie

    2004-01-01

    High-throughput screening (HTS) is the process of testing a large number of diverse chemical structures against disease targets to identify 'hits'. Compared to traditional drug screening methods, HTS is characterized by its simplicity, rapidness, low cost, and high efficiency, taking the ligand-target interactions as the principle, as well as leading to a higher information harvest. As a multidisciplinary field, HTS involves an automated operation-platform, highly sensitive testing system, specific screening model (in vitro), an abundant components library, and a data acquisition and processing system. Various technologies, especially the novel technologies such as fluorescence, nuclear-magnetic resonance, affinity chromatography, surface plasmon resonance, and DNA microarray, are now available, and the screening of more than 100,000 samples per day is already possible. Fluorescence-based assays include the scintillation proximity assay, time-resolved energy transfer, fluorescence anisotropy, fluorescence correlation spectroscopy, and fluorescence fluctuation spectroscopy. Fluorescence-based techniques are likely to be among the most important detection approaches used for HTS due to their high sensitivity and amenability to automation, giving the industry-wide drive to simplify, miniaturize, and speed up assays. The application of NMR technology to HTS is another recent trend in drug research. One advantage afforded by NMR technology is that it can provide direct information on the affinity of the screening compounds and the binding location of protein. The structure-activity relationship acquired from NMR analysis can sharpen the library design, which will be very important in furnishing HTS with well-defined drug candidates. Affinity chromatography used for library screening will provide the information on the fundamental processes of drug action, such as absorption, distribution, excretion, and receptor activation; also the eluting curve can give directly the

  8. Creating an Infrastructure for High-Throughput High-Resolution Cryogenic Electron Microscopy

    PubMed Central

    Shrum, Donald C.; Woodruff, Brent W.

    2012-01-01

    New instrumentation for three-dimensional electron microscopy is facilitating an increase in the throughput of data collection and reconstruction. The increase in throughput creates bottlenecks in the workflow for storing and processing the image data. Here we describe the creation and quantify the throughput of a high-throughput infrastructure supporting collection of three-dimensional data collection. PMID:22842049

  9. Creating an infrastructure for high-throughput high-resolution cryogenic electron microscopy.

    PubMed

    Shrum, Donald C; Woodruff, Brent W; Stagg, Scott M

    2012-10-01

    New instrumentation for three-dimensional electron microscopy is facilitating an increase in the throughput of data collection and reconstruction. The increase in throughput creates bottlenecks in the workflow for storing and processing the image data. Here we describe the creation and quantify the throughput of a high-throughput infrastructure supporting collection of three-dimensional data collection.

  10. High-throughput acoustic separation of platelets from whole blood.

    PubMed

    Chen, Yuchao; Wu, Mengxi; Ren, Liqiang; Liu, Jiayang; Whitley, Pamela H; Wang, Lin; Huang, Tony Jun

    2016-09-21

    Platelets contain growth factors which are important in biomedical and clinical applications. In this work, we present an acoustic separation device for high-throughput, non-invasive platelet isolation. In particular, we separated platelets from whole blood at a 10 mL min(-1) throughput, which is three orders of magnitude greater than that of existing acoustic-based platelet separation techniques. Without sample dilution, we observed more than 80% RBC/WBC removal and platelet recovery. High throughput, high separation efficiency, and biocompatibility make this device useful for many clinical applications. PMID:27477388

  11. High-throughput acoustic separation of platelets from whole blood.

    PubMed

    Chen, Yuchao; Wu, Mengxi; Ren, Liqiang; Liu, Jiayang; Whitley, Pamela H; Wang, Lin; Huang, Tony Jun

    2016-09-21

    Platelets contain growth factors which are important in biomedical and clinical applications. In this work, we present an acoustic separation device for high-throughput, non-invasive platelet isolation. In particular, we separated platelets from whole blood at a 10 mL min(-1) throughput, which is three orders of magnitude greater than that of existing acoustic-based platelet separation techniques. Without sample dilution, we observed more than 80% RBC/WBC removal and platelet recovery. High throughput, high separation efficiency, and biocompatibility make this device useful for many clinical applications.

  12. Development of A High Throughput Method Incorporating Traditional Analytical Devices

    PubMed Central

    White, C. C.; Embree, E.; Byrd, W. E; Patel, A. R.

    2004-01-01

    A high-throughput (high throughput is the ability to process large numbers of samples) and companion informatics system has been developed and implemented. High throughput is defined as the ability to autonomously evaluate large numbers of samples, while an informatics system provides the software control of the physical devices, in addition to the organization and storage of the generated electronic data. This high throughput system includes both an ultra-violet and visible light spectrometer (UV-Vis) and a Fourier transform infrared spectrometer (FTIR) integrated with a multi sample positioning table. This method is designed to quantify changes in polymeric materials occurring from controlled temperature, humidity and high flux UV exposures. The integration of the software control of these analytical instruments within a single computer system is presented. Challenges in enhancing the system to include additional analytical devices are discussed. PMID:27366626

  13. C. elegans in high-throughput drug discovery

    PubMed Central

    O’Reilly, Linda P.; Luke, Cliff J.; Perlmutter, David H.; Silverman, Gary A.; Pak, Stephen C.

    2014-01-01

    C. elegans has proven to be a useful model organism for investigating molecular and cellular aspects of numerous human diseases. More recently, investigators have explored the use of this organism as a tool for drug discovery. Although earlier drug screens were labor-intensive and low in throughput, recent advances in high-throughput liquid workflows, imaging platforms and data analysis software have made C. elegans a viable option for automated high-throughput drug screens. This review will outline the evolution of C. elegans-based drug screening, discuss the inherent challenges of using C. elegans, and highlight recent technological advances that have paved the way for future drug screens. PMID:24333896

  14. Extended length microchannels for high density high throughput electrophoresis systems

    DOEpatents

    Davidson, James C.; Balch, Joseph W.

    2000-01-01

    High throughput electrophoresis systems which provide extended well-to-read distances on smaller substrates, thus compacting the overall systems. The electrophoresis systems utilize a high density array of microchannels for electrophoresis analysis with extended read lengths. The microchannel geometry can be used individually or in conjunction to increase the effective length of a separation channel while minimally impacting the packing density of channels. One embodiment uses sinusoidal microchannels, while another embodiment uses plural microchannels interconnected by a via. The extended channel systems can be applied to virtually any type of channel confined chromatography.

  15. High-Throughput Sequencing in Mitochondrial DNA Research

    PubMed Central

    Ye, Fei; Samuels, David C.; Clark, Travis; Guo, Yan

    2014-01-01

    Next-generation sequencing, also known as high-throughput sequencing, has greatly enhanced researchers’ ability to conduct biomedical research on all levels. Mitochondrial research has also benefitted greatly from high-throughput sequencing; sequencing technology now allows for screening of all 16569 base pairs of the mitochondrial genome simultaneously for SNPs and low level heteroplasmy and, in some cases, the estimation of mitochondrial DNA copy number. It is important to realize the full potential of high-throughput sequencing for the advancement of mitochondrial research. To this end, we review how high-throughput sequencing has impacted mitochondrial research in the categories of SNPs, low level heteroplasmy, copy number, and structural variants. We also discuss the different types of mitochondrial DNA sequencing and their pros and cons. Based on previous studies conducted by various groups, we provide strategies for processing mitochondrial DNA sequencing data, including assembly, variant calling, and quality control. PMID:24859348

  16. High-Throughput Pharmacokinetics for Environmental Chemicals (SOT)

    EPA Science Inventory

    High throughput screening (HTS) promises to allow prioritization of thousands of environmental chemicals with little or no in vivo information. For bioactivity identified by HTS, toxicokinetic (TK) models are essential to predict exposure thresholds below which no significant bio...

  17. High-throughput sequencing in mitochondrial DNA research.

    PubMed

    Ye, Fei; Samuels, David C; Clark, Travis; Guo, Yan

    2014-07-01

    Next-generation sequencing, also known as high-throughput sequencing, has greatly enhanced researchers' ability to conduct biomedical research on all levels. Mitochondrial research has also benefitted greatly from high-throughput sequencing; sequencing technology now allows for screening of all 16,569 base pairs of the mitochondrial genome simultaneously for SNPs and low level heteroplasmy and, in some cases, the estimation of mitochondrial DNA copy number. It is important to realize the full potential of high-throughput sequencing for the advancement of mitochondrial research. To this end, we review how high-throughput sequencing has impacted mitochondrial research in the categories of SNPs, low level heteroplasmy, copy number, and structural variants. We also discuss the different types of mitochondrial DNA sequencing and their pros and cons. Based on previous studies conducted by various groups, we provide strategies for processing mitochondrial DNA sequencing data, including assembly, variant calling, and quality control.

  18. MIPHENO: Data normalization for high throughput metabolic analysis.

    EPA Science Inventory

    High throughput methodologies such as microarrays, mass spectrometry and plate-based small molecule screens are increasingly used to facilitate discoveries from gene function to drug candidate identification. These large-scale experiments are typically carried out over the course...

  19. AOPs & Biomarkers: Bridging High Throughput Screening and Regulatory Decision Making.

    EPA Science Inventory

    As high throughput screening (HTS) approaches play a larger role in toxicity testing, computational toxicology has emerged as a critical component in interpreting the large volume of data produced. Computational models for this purpose are becoming increasingly more sophisticated...

  20. HIGH THROUGHPUT ASSESSMENTS OF CONVENTIONAL AND ALTERNATIVE COMPOUNDS

    EPA Science Inventory

    High throughput approaches for quantifying chemical hazard, exposure, and sustainability have the potential to dramatically impact the pace and nature of risk assessments. Integrated evaluation strategies developed at the US EPA incorporate inherency,bioactivity,bioavailability, ...

  1. High throughput growth and characterization of thin film materials

    NASA Astrophysics Data System (ADS)

    Mao, Samuel S.

    2013-09-01

    It usually takes more than 10 years for a new material from initial research to its first commercial application. Therefore, accelerating the pace of discovery of new materials is critical to tackling challenges in areas ranging from clean energy to national security. As discovery of new materials has not kept pace with the product design cycles in many sectors of industry, there is a pressing need to develop and utilize high throughput screening and discovery technologies for the growth and characterization of new materials. This article presents two distinctive types of high throughput thin film material growth approaches, along with a number of high throughput characterization techniques, established in the author's group. These approaches include a second-generation "discrete" combinatorial semiconductor discovery technology that enables the creation of arrays of individually separated thin film semiconductor materials of different compositions, and a "continuous" high throughput thin film material screening technology that enables the realization of ternary alloy libraries with continuously varying elemental ratios.

  2. Evaluating Rapid Models for High-Throughput Exposure Forecasting (SOT)

    EPA Science Inventory

    High throughput exposure screening models can provide quantitative predictions for thousands of chemicals; however these predictions must be systematically evaluated for predictive ability. Without the capability to make quantitative, albeit uncertain, forecasts of exposure, the ...

  3. High resolution hyperspectral imaging with a high throughput virtual slit

    NASA Astrophysics Data System (ADS)

    Gooding, Edward A.; Gunn, Thomas; Cenko, Andrew T.; Hajian, Arsen R.

    2016-05-01

    Hyperspectral imaging (HSI) device users often require both high spectral resolution, on the order of 1 nm, and high light-gathering power. A wide entrance slit assures reasonable étendue but degrades spectral resolution. Spectrometers built using High Throughput Virtual Slit™ (HTVS) technology optimize both parameters simultaneously. Two remote sensing use cases that require high spectral resolution are discussed. First, detection of atmospheric gases with intrinsically narrow absorption lines, such as hydrocarbon vapors or combustion exhaust gases such as NOx and CO2. Detecting exhaust gas species with high precision has become increasingly important in the light of recent events in the automobile industry. Second, distinguishing reflected daylight from emission spectra in the visible and NIR (VNIR) regions is most easily accomplished using the Fraunhofer absorption lines in solar spectra. While ground reflectance spectral features in the VNIR are generally quite broad, the Fraunhofer lines are narrow and provide a signature of intrinsic vs. extrinsic illumination. The High Throughput Virtual Slit enables higher spectral resolution than is achievable with conventional spectrometers by manipulating the beam profile in pupil space. By reshaping the instrument pupil with reflective optics, HTVS-equipped instruments create a tall, narrow image profile at the exit focal plane, typically delivering 5X or better the spectral resolution achievable with a conventional design.

  4. Informatics solutions for high-throughput proteomics.

    PubMed

    Topaloglou, Thodoros

    2006-06-01

    The success of mass-spectrometry-based proteomics as a method for analyzing proteins in biological samples is accompanied by challenges owning to demands for increased throughput. These challenges arise from the vast volume of data generated by proteomics experiments combined with the heterogeneity in data formats, processing methods, software tools and databases that are involved in the translation of spectral data into relevant and actionable information for scientists. Informatics aims to provide answers to these challenges by transferring existing solutions from information management to proteomics and/or by generating novel computational methods for automation of proteomics data processing.

  5. Towards A Fully Automated High-Throughput Phototransfection System

    PubMed Central

    Cappelleri, David J.; Halasz, Adam; Sul, Jai-Yoon; Kim, Tae Kyung; Eberwine, James; Kumar, Vijay

    2010-01-01

    We have designed and implemented a framework for creating a fully automated high-throughput phototransfection system. Integrated image processing, laser target position calculation, and stage movements show a throughput increase of > 23X over the current manual phototransfection method while the potential for even greater throughput improvements (> 110X) is described. A software tool for automated off-line single cell morphological measurements, as well as real-time image segmentation analysis, has also been constructed and shown to be able quantify changes in the cell before and after the process, successfully characterizing them, using metrics such as cell perimeter, area, major and minor axis length, and eccentricity values. PMID:20706617

  6. Multiple-injection high-throughput gas chromatography analysis.

    PubMed

    Schafer, Wes; Wang, Heather; Welch, Christopher J

    2016-08-01

    Multiple-injection techniques have been shown to be a simple way to perform high-throughput analysis where the entire experiment resides in a single chromatogram, simplifying the data analysis and interpretation. In this study, multiple-injection techniques are applied to gas chromatography with flame ionization detection and mass detection to significantly increase sample throughput. The unique issues of implementing a traditional "Fast" injection mode of multiple-injection techniques with gas chromatography and mass spectrometry are discussed. Stacked injections are also discussed as means to increase the throughput of longer methods where mass detection is unable to distinguish between analytes of the same mass and longer retentions are required to resolve components of interest. Multiple-injection techniques are shown to increase instrument throughput by up to 70% and to simplify data analysis, allowing hits in multiple parallel experiments to be identified easily. PMID:27292909

  7. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Forsythe, Elizabeth; Achari, Amiruddha

    2005-01-01

    cost optics, further increasing throughput at synchrotrons. Preliminary experiments show that the presence of the fluorescent probe does not affect the nucleation process or the quality of the X-ray data obtained.

  8. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Forsythe, Elizabeth

    2004-01-01

    cost optics, further increasing throughput at synchrotrons. This presentation will focus on the methodology for fluorescent labeling, the crystallization results, and the effects of the trace labeling on the crystal quality.

  9. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Forsythe, Elizabeth

    2005-01-01

    , further increasing throughput at synchrotrons. This presentation will focus on the methodology for fluorescent labeling, the crystallization results, and the effects of the trace labeling on the crystal quality.

  10. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

    Minamitani, Elizabeth Forsythe; Pusey, Marc L.

    2004-01-01

    using relatively low cost optics, further increasing throughput at synchrotrons. This presentation will focus on the methodology for fluorescent labeling, the crystallization results, and the effects of the trace labeling on the crystal quality.

  11. The high throughput biomedicine unit at the institute for molecular medicine Finland: high throughput screening meets precision medicine.

    PubMed

    Pietiainen, Vilja; Saarela, Jani; von Schantz, Carina; Turunen, Laura; Ostling, Paivi; Wennerberg, Krister

    2014-05-01

    The High Throughput Biomedicine (HTB) unit at the Institute for Molecular Medicine Finland FIMM was established in 2010 to serve as a national and international academic screening unit providing access to state of the art instrumentation for chemical and RNAi-based high throughput screening. The initial focus of the unit was multiwell plate based chemical screening and high content microarray-based siRNA screening. However, over the first four years of operation, the unit has moved to a more flexible service platform where both chemical and siRNA screening is performed at different scales primarily in multiwell plate-based assays with a wide range of readout possibilities with a focus on ultraminiaturization to allow for affordable screening for the academic users. In addition to high throughput screening, the equipment of the unit is also used to support miniaturized, multiplexed and high throughput applications for other types of research such as genomics, sequencing and biobanking operations. Importantly, with the translational research goals at FIMM, an increasing part of the operations at the HTB unit is being focused on high throughput systems biological platforms for functional profiling of patient cells in personalized and precision medicine projects.

  12. Inkjet printing for high-throughput cell patterning.

    PubMed

    Roth, E A; Xu, T; Das, M; Gregory, C; Hickman, J J; Boland, T

    2004-08-01

    The adaptation of inkjet printing technology to the complex fields of tissue engineering and biomaterial development presents the potential to increase progress in these emerging technologies through the implementation of this high-throughput capability via automated processes to enable precise control and repeatability. In this paper, a method of applying high-throughput inkjet printing to control cellular attachment and proliferation by precise, automated deposition of collagen is presented. The results indicate that commercial inkjet printing technology can be used to create viable cellular patterns with a resolution of 350 microm through the deposition of biologically active proteins. This method demonstrates a combination of off-the-shelf inkjet printing and biomaterials and has potential to be adapted to tissue engineering and colony patterning applications. Adapting this method into the three-dimensional construction of cellular structures for eventual high-throughput tissue engineering using a bottom-up approach is possible.

  13. Combinatorial and high-throughput screening approaches for strain engineering.

    PubMed

    Liu, Wenshan; Jiang, Rongrong

    2015-03-01

    Microbes have long been used in the industry to produce valuable biochemicals. Combinatorial engineering approaches, new strain engineering tools derived from inverse metabolic engineering, have started to attract attention in recent years, including genome shuffling, error-prone DNA polymerase, global transcription machinery engineering (gTME), random knockout/overexpression libraries, ribosome engineering, multiplex automated genome engineering (MAGE), customized optimization of metabolic pathways by combinatorial transcriptional engineering (COMPACTER), and library construction of "tunable intergenic regions" (TIGR). Since combinatorial approaches and high-throughput screening methods are fundamentally interconnected, color/fluorescence-based, growth-based, and biosensor-based high-throughput screening methods have been reviewed. We believe that with the help of metabolic engineering tools and new combinatorial approaches, plus effective high-throughput screening methods, researchers will be able to achieve better results on improving microorganism performance under stress or enhancing biochemical yield.

  14. Insights to transcriptional networks by using high throughput RNAi strategies

    PubMed Central

    Mattila, Jaakko

    2010-01-01

    RNA interference (RNAi) is a powerful method to unravel the role of a given gene in eukaryotic cells. The development of high throughput assay platforms such as fluorescence plate readers and high throughput microscopy has allowed the design of genome wide RNAi screens to systemically discern members of regulatory networks around various cellular processes. Here we summarize the different strategies employed in RNAi screens to reveal regulators of transcriptional networks. We focus our discussion in experimental approaches designed to uncover regulatory interactions modulating transcription factor activity. PMID:19952073

  15. Advances in high throughput DNA sequence data compression.

    PubMed

    Sardaraz, Muhammad; Tahir, Muhammad; Ikram, Ataul Aziz

    2016-06-01

    Advances in high throughput sequencing technologies and reduction in cost of sequencing have led to exponential growth in high throughput DNA sequence data. This growth has posed challenges such as storage, retrieval, and transmission of sequencing data. Data compression is used to cope with these challenges. Various methods have been developed to compress genomic and sequencing data. In this article, we present a comprehensive review of compression methods for genome and reads compression. Algorithms are categorized as referential or reference free. Experimental results and comparative analysis of various methods for data compression are presented. Finally, key challenges and research directions in DNA sequence data compression are highlighted. PMID:26846812

  16. CHALLENGES IN SECONDARY ANALYSIS OF HIGH THROUGHPUT SCREENING DATA

    PubMed Central

    BLUCHER, AURORA S.; MCWEENEY, SHANNON K.

    2014-01-01

    Repurposing an existing drug for an alternative use is not only a cost effective method of development, but also a faster process due to the drug's previous clinical testing and established pharmokinetic profiles. A potentially rich resource for computational drug repositioning approaches is publically available high throughput screening data, available in databases such as PubChem Bioassay and ChemBank. We examine statistical and computational considerations for secondary analysis of publicly available high throughput screening (HTS) data with respect to metadata, data quality, and completeness. We discuss developing methods and best practices that can help to ameliorate these issues. PMID:24297539

  17. High-throughput screening to identify inhibitors of lysine demethylases.

    PubMed

    Gale, Molly; Yan, Qin

    2015-01-01

    Lysine demethylases (KDMs) are epigenetic regulators whose dysfunction is implicated in the pathology of many human diseases including various types of cancer, inflammation and X-linked intellectual disability. Particular demethylases have been identified as promising therapeutic targets, and tremendous efforts are being devoted toward developing suitable small-molecule inhibitors for clinical and research use. Several High-throughput screening strategies have been developed to screen for small-molecule inhibitors of KDMs, each with advantages and disadvantages in terms of time, cost, effort, reliability and sensitivity. In this Special Report, we review and evaluate the High-throughput screening methods utilized for discovery of novel small-molecule KDM inhibitors.

  18. High-throughput screening for modulators of cellular contractile force†

    PubMed Central

    Park, Chan Young; Zhou, Enhua H.; Tambe, Dhananjay; Chen, Bohao; Lavoie, Tera; Dowell, Maria; Simeonov, Anton; Maloney, David J.; Marinkovic, Aleksandar; Tschumperlin, Daniel J.; Burger, Stephanie; Frykenberg, Matthew; Butler, James P.; Stamer, W. Daniel; Johnson, Mark; Solway, Julian; Fredberg, Jeffrey J.

    2015-01-01

    When cellular contractile forces are central to pathophysiology, these forces comprise a logical target of therapy. Nevertheless, existing high-throughput screens are limited to upstream signalling intermediates with poorly defined relationships to such a physiological endpoint. Using cellular force as the target, here we report a new screening technology and demonstrate its applications using human airway smooth muscle cells in the context of asthma and Schlemm's canal endothelial cells in the context of glaucoma. This approach identified several drug candidates for both asthma and glaucoma. We attained rates of 1000 compounds per screening day, thus establishing a force-based cellular platform for high-throughput drug discovery. PMID:25953078

  19. Screening and synthesis: high throughput technologies applied to parasitology.

    PubMed

    Morgan, R E; Westwood, N J

    2004-01-01

    High throughput technologies continue to develop in response to the challenges set by the genome projects. This article discusses how the techniques of both high throughput screening (HTS) and synthesis can influence research in parasitology. Examples of the use of targeted and phenotype-based HTS using unbiased compound collections are provided. The important issue of identifying the protein target(s) of bioactive compounds is discussed from the synthetic chemist's perspective. This article concludes by reviewing recent examples of successful target identification studies in parasitology.

  20. The synergy between combinatorial chemistry and high-throughput screening.

    PubMed

    Diller, David J

    2008-05-01

    Despite the initial promise of combinatorial chemistry, particularly large library combinatorial chemistry, to greatly accelerate drug discovery, this approach has not been fully utilized as a means to build the compound collections of pharmaceutical and biotechnology companies. This review highlights some of the strengths of large library combinatorial chemistry as a means of generating molecules for lead discovery, such as providing rich and robust structure-activity relationships around each hit series. The challenges and concepts emerging from traditional high-throughput screening and fragment-based drug design, how these methods influence the design of large combinatorial libraries and the interpretation of the ensuing high-throughput screening data are also highlighted.

  1. Perspective: Data infrastructure for high throughput materials discovery

    NASA Astrophysics Data System (ADS)

    Pfeif, E. A.; Kroenlein, K.

    2016-05-01

    Computational capability has enabled materials design to evolve from trial-and-error towards more informed methodologies that require large amounts of data. Expert-designed tools and their underlying databases facilitate modern-day high throughput computational methods. Standard data formats and communication standards increase the impact of traditional data, and applying these technologies to a high throughput experimental design provides dense, targeted materials data that are valuable for material discovery. Integrated computational materials engineering requires both experimentally and computationally derived data. Harvesting these comprehensively requires different methods of varying degrees of automation to accommodate variety and volume. Issues of data quality persist independent of type.

  2. Substrate independent ATPase activity may complicate high throughput screening.

    PubMed

    Tuntland, Micheal L; Fung, L W-M

    2016-10-01

    Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting. Based on the published mechanism, we designed a protocol for our screening for inhibitors of SAICAR synthetase (PurC), and we found a gradual increase in [Pi] in positive control samples over the course of the day. Further investigation indicated that hydrolysis of ATP catalyzed by PurC, rather than substrate-related phosphate release, was responsible for a partial contribution to the signals in the control samples. Thus substrate-independent ATPase activity may complicate high throughput screening. PMID:27430931

  3. A high-throughput label-free nanoparticle analyser

    NASA Astrophysics Data System (ADS)

    Fraikin, Jean-Luc; Teesalu, Tambet; McKenney, Christopher M.; Ruoslahti, Erkki; Cleland, Andrew N.

    2011-05-01

    Synthetic nanoparticles and genetically modified viruses are used in a range of applications, but high-throughput analytical tools for the physical characterization of these objects are needed. Here we present a microfluidic analyser that detects individual nanoparticles and characterizes complex, unlabelled nanoparticle suspensions. We demonstrate the detection, concentration analysis and sizing of individual synthetic nanoparticles in a multicomponent mixture with sufficient throughput to analyse 500,000 particles per second. We also report the rapid size and titre analysis of unlabelled bacteriophage T7 in both salt solution and mouse blood plasma, using just ~1 × 10-6 l of analyte. Unexpectedly, in the native blood plasma we discover a large background of naturally occurring nanoparticles with a power-law size distribution. The high-throughput detection capability, scalable fabrication and simple electronics of this instrument make it well suited for diverse applications.

  4. High-throughput screening, predictive modeling and computational embryology - Abstract

    EPA Science Inventory

    High-throughput screening (HTS) studies are providing a rich source of data that can be applied to chemical profiling to address sensitivity and specificity of molecular targets, biological pathways, cellular and developmental processes. EPA’s ToxCast project is testing 960 uniq...

  5. Accounting For Uncertainty in The Application Of High Throughput Datasets

    EPA Science Inventory

    The use of high throughput screening (HTS) datasets will need to adequately account for uncertainties in the data generation process and propagate these uncertainties through to ultimate use. Uncertainty arises at multiple levels in the construction of predictors using in vitro ...

  6. High Throughput Assays and Exposure Science (ISES annual meeting)

    EPA Science Inventory

    High throughput screening (HTS) data characterizing chemical-induced biological activity has been generated for thousands of environmentally-relevant chemicals by the US inter-agency Tox21 and the US EPA ToxCast programs. For a limited set of chemicals, bioactive concentrations r...

  7. High Throughput Exposure Estimation Using NHANES Data (SOT)

    EPA Science Inventory

    In the ExpoCast project, high throughput (HT) exposure models enable rapid screening of large numbers of chemicals for exposure potential. Evaluation of these models requires empirical exposure data and due to the paucity of human metabolism/exposure data such evaluations includ...

  8. High-throughput glycoanalytical technology for systems glycobiology.

    PubMed

    Liu, Li; Telford, Jayne E; Knezevic, Ana; Rudd, Pauline M

    2010-10-01

    The development of glycoanalytical HPLC-based high-throughput technology has greatly enhanced the study of glycobiology, facilitating the discovery of disease-related solutions and providing an informative view of glycosylation and its relationship with other biological disciplines in a systems biology approach.

  9. New High Throughput Methods to Estimate Chemical Exposure

    EPA Science Inventory

    EPA has made many recent advances in high throughput bioactivity testing. However, concurrent advances in rapid, quantitative prediction of human and ecological exposures have been lacking, despite the clear importance of both measures for a risk-based approach to prioritizing an...

  10. Environmental Impact on Vascular Development Predicted by High Throughput Screening

    EPA Science Inventory

    Understanding health risks to embryonic development from exposure to environmental chemicals is a significant challenge given the diverse chemical landscape and paucity of data for most of these compounds. High throughput screening (HTS) in EPA’s ToxCastTM project provides vast d...

  11. High-throughput production of two disulphide-bridge toxins.

    PubMed

    Upert, Grégory; Mourier, Gilles; Pastor, Alexandra; Verdenaud, Marion; Alili, Doria; Servent, Denis; Gilles, Nicolas

    2014-08-01

    A quick and efficient production method compatible with high-throughput screening was developed using 36 toxins belonging to four different families of two disulphide-bridge toxins. Final toxins were characterized using HPLC co-elution, CD and pharmacological studies.

  12. High-Throughput Screening for Streptomyces Antibiotic Biosynthesis Activators

    PubMed Central

    Chen, Li; Wang, Yemin; Guo, Hang; Xu, Min; Deng, Zixin

    2012-01-01

    A genomic cosmid library of Streptomyces clavuligerus was constructed and transferred efficiently by conjugation to Streptomyces lividans, and 12 distinct groups of overlapping cosmid clones that activated the silent actinorhodin biosynthesis gene cluster were identified. This generally applicable high-throughput screening procedure greatly facilitates the identification of antibiotic biosynthesis activators. PMID:22504805

  13. High-Throughput Sequencing and Rare Genetic Diseases

    PubMed Central

    Makrythanasis, P.; Antonarakis, S.E.

    2012-01-01

    High-throughput sequencing has drastically changed the research of genes responsible for genetic disorders and is now gradually introduced as an additional genetic diagnostic testing in clinical practice. The current debates on the emerging technical, medical and ethical issues as well as the potential optimum use of the available technology are discussed. PMID:23293577

  14. High-throughput screening, predictive modeling and computational embryology

    EPA Science Inventory

    High-throughput screening (HTS) studies are providing a rich source of data that can be applied to profile thousands of chemical compounds for biological activity and potential toxicity. EPA’s ToxCast™ project, and the broader Tox21 consortium, in addition to projects worldwide,...

  15. Fully Bayesian Analysis of High-throughput Targeted Metabolomics Assays

    EPA Science Inventory

    High-throughput metabolomic assays that allow simultaneous targeted screening of hundreds of metabolites have recently become available in kit form. Such assays provide a window into understanding changes to biochemical pathways due to chemical exposure or disease, and are usefu...

  16. A high throughput droplet based electroporation system

    NASA Astrophysics Data System (ADS)

    Yoo, Byeongsun; Ahn, Myungmo; Im, Dojin; Kang, Inseok

    2014-11-01

    Delivery of exogenous genetic materials across the cell membrane is a powerful and popular research tool for bioengineering. Among conventional non-viral DNA delivery methods, electroporation (EP) is one of the most widely used technologies and is a standard lab procedure in molecular biology. We developed a novel digital microfluidic electroporation system which has higher efficiency of transgene expression and better cell viability than that of conventional EP techniques. We present the successful performance of digital EP system for transformation of various cell lines by investigating effects of the EP conditions such as electric pulse voltage, number, and duration on the cell viability and transfection efficiency in comparison with a conventional bulk EP system. Through the numerical analysis, we have also calculated the electric field distribution around the cells precisely to verify the effect of the electric field on the high efficiency of the digital EP system. Furthermore, the parallelization of the EP processes has been developed to increase the transformation productivity. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (Grant Number: 2013R1A1A2011956).

  17. High-throughput titration of luciferase-expressing recombinant viruses.

    PubMed

    Garcia, Vanessa; Krishnan, Ramya; Davis, Colin; Batenchuk, Cory; Le Boeuf, Fabrice; Abdelbary, Hesham; Diallo, Jean-Simon

    2014-01-01

    Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

  18. Higher throughput high resolution multi-worm tracker

    NASA Astrophysics Data System (ADS)

    Javer, Avelino; Li, Kezhi; Gyenes, Bertalan; Brown, Andre; Behavioural Genomics Team

    2015-03-01

    We have developed a high throughput imaging system for tracking multiple nematode worms at high resolution. The tracker consists of 6 cameras mounted on a motorized gantry so that up to 48 plates (each with approximately 30 worms) can be imaged without user intervention. To deal with the high data rate of the cameras we use real time processing to find worms and only save the immediately surrounding pixels. The system is also equipped with automatic oxygen and carbon dioxide control for observing stimulus response behaviour. We will describe the design and performance of the new system, some of the challenges of truly high throughput behaviour recording, and report preliminary results on inter-individual variation in behaviour as well as a quantitative analysis of C. elegans response to hypoxia, oxygen reperfusion, and carbon dioxide. Funding provided by the Medical Research Council.

  19. High-throughput theoretical design of lithium battery materials

    NASA Astrophysics Data System (ADS)

    Shi-Gang, Ling; Jian, Gao; Rui-Juan, Xiao; Li-Quan, Chen

    2016-01-01

    The rapid evolution of high-throughput theoretical design schemes to discover new lithium battery materials is reviewed, including high-capacity cathodes, low-strain cathodes, anodes, solid state electrolytes, and electrolyte additives. With the development of efficient theoretical methods and inexpensive computers, high-throughput theoretical calculations have played an increasingly important role in the discovery of new materials. With the help of automatic simulation flow, many types of materials can be screened, optimized and designed from a structural database according to specific search criteria. In advanced cell technology, new materials for next generation lithium batteries are of great significance to achieve performance, and some representative criteria are: higher energy density, better safety, and faster charge/discharge speed. Project supported by the National Natural Science Foundation of China (Grant Nos. 11234013 and 51172274) and the National High Technology Research and Development Program of China (Grant No. 2015AA034201).

  20. A Multidisciplinary Approach to High Throughput Nuclear Magnetic Resonance Spectroscopy

    PubMed Central

    Pourmodheji, Hossein; Ghafar-Zadeh, Ebrahim; Magierowski, Sebastian

    2016-01-01

    Nuclear Magnetic Resonance (NMR) is a non-contact, powerful structure-elucidation technique for biochemical analysis. NMR spectroscopy is used extensively in a variety of life science applications including drug discovery. However, existing NMR technology is limited in that it cannot run a large number of experiments simultaneously in one unit. Recent advances in micro-fabrication technologies have attracted the attention of researchers to overcome these limitations and significantly accelerate the drug discovery process by developing the next generation of high-throughput NMR spectrometers using Complementary Metal Oxide Semiconductor (CMOS). In this paper, we examine this paradigm shift and explore new design strategies for the development of the next generation of high-throughput NMR spectrometers using CMOS technology. A CMOS NMR system consists of an array of high sensitivity micro-coils integrated with interfacing radio-frequency circuits on the same chip. Herein, we first discuss the key challenges and recent advances in the field of CMOS NMR technology, and then a new design strategy is put forward for the design and implementation of highly sensitive and high-throughput CMOS NMR spectrometers. We thereafter discuss the functionality and applicability of the proposed techniques by demonstrating the results. For microelectronic researchers starting to work in the field of CMOS NMR technology, this paper serves as a tutorial with comprehensive review of state-of-the-art technologies and their performance levels. Based on these levels, the CMOS NMR approach offers unique advantages for high resolution, time-sensitive and high-throughput bimolecular analysis required in a variety of life science applications including drug discovery. PMID:27294925

  1. A Multidisciplinary Approach to High Throughput Nuclear Magnetic Resonance Spectroscopy.

    PubMed

    Pourmodheji, Hossein; Ghafar-Zadeh, Ebrahim; Magierowski, Sebastian

    2016-06-09

    Nuclear Magnetic Resonance (NMR) is a non-contact, powerful structure-elucidation technique for biochemical analysis. NMR spectroscopy is used extensively in a variety of life science applications including drug discovery. However, existing NMR technology is limited in that it cannot run a large number of experiments simultaneously in one unit. Recent advances in micro-fabrication technologies have attracted the attention of researchers to overcome these limitations and significantly accelerate the drug discovery process by developing the next generation of high-throughput NMR spectrometers using Complementary Metal Oxide Semiconductor (CMOS). In this paper, we examine this paradigm shift and explore new design strategies for the development of the next generation of high-throughput NMR spectrometers using CMOS technology. A CMOS NMR system consists of an array of high sensitivity micro-coils integrated with interfacing radio-frequency circuits on the same chip. Herein, we first discuss the key challenges and recent advances in the field of CMOS NMR technology, and then a new design strategy is put forward for the design and implementation of highly sensitive and high-throughput CMOS NMR spectrometers. We thereafter discuss the functionality and applicability of the proposed techniques by demonstrating the results. For microelectronic researchers starting to work in the field of CMOS NMR technology, this paper serves as a tutorial with comprehensive review of state-of-the-art technologies and their performance levels. Based on these levels, the CMOS NMR approach offers unique advantages for high resolution, time-sensitive and high-throughput bimolecular analysis required in a variety of life science applications including drug discovery.

  2. Multiple column high-throughput e-beam inspection (EBI)

    NASA Astrophysics Data System (ADS)

    Lam, David K.; Monahan, Kevin M.; Liu, Enden D.; Tran, Cong; Prescop, Ted

    2012-03-01

    Single-column e-beam systems are used in production for the detection of electrical defects, but are too slow to be used for the detection of small physical defects, and can't meet future inspection requirements. This paper presents a multiplecolumn e-beam technology for high throughput wafer inspection. Multibeam has developed all-electrostatic columns for high-resolution imaging. The elimination of magnetic coils enables the columns to be small; e-beam deflection is faster in the absence of magnetic hysteresis. Multiple miniaturecolumns are assembled in an array. An array of 100 columns covers the entire surface of a 300mm wafer, affording simultaneous cross-wafer sampling. Column performance simulations and system architecture are presented. Also provided are examples of high throughput, more efficient, multiple-column wafer inspection.

  3. A high-throughput microRNA expression profiling system.

    PubMed

    Guo, Yanwen; Mastriano, Stephen; Lu, Jun

    2014-01-01

    As small noncoding RNAs, microRNAs (miRNAs) regulate diverse biological functions, including physiological and pathological processes. The expression and deregulation of miRNA levels contain rich information with diagnostic and prognostic relevance and can reflect pharmacological responses. The increasing interest in miRNA-related research demands global miRNA expression profiling on large numbers of samples. We describe here a robust protocol that supports high-throughput sample labeling and detection on hundreds of samples simultaneously. This method employs 96-well-based miRNA capturing from total RNA samples and on-site biochemical reactions, coupled with bead-based detection in 96-well format for hundreds of miRNAs per sample. With low-cost, high-throughput, high detection specificity, and flexibility to profile both small and large numbers of samples, this protocol can be adapted in a wide range of laboratory settings. PMID:25030917

  4. Direct assembling methodologies for high-throughput bioscreening

    PubMed Central

    Rodríguez-Dévora, Jorge I.; Shi, Zhi-dong; Xu, Tao

    2012-01-01

    Over the last few decades, high-throughput (HT) bioscreening, a technique that allows rapid screening of biochemical compound libraries against biological targets, has been widely used in drug discovery, stem cell research, development of new biomaterials, and genomics research. To achieve these ambitions, scaffold-free (or direct) assembly of biological entities of interest has become critical. Appropriate assembling methodologies are required to build an efficient HT bioscreening platform. The development of contact and non-contact assembling systems as a practical solution has been driven by a variety of essential attributes of the bioscreening system, such as miniaturization, high throughput, and high precision. The present article reviews recent progress on these assembling technologies utilized for the construction of HT bioscreening platforms. PMID:22021162

  5. A high-throughput multiplex method adapted for GMO detection.

    PubMed

    Chaouachi, Maher; Chupeau, Gaëlle; Berard, Aurélie; McKhann, Heather; Romaniuk, Marcel; Giancola, Sandra; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

    2008-12-24

    A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

  6. High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella pneumophila

    PubMed Central

    Chiaraviglio, Lucius

    2015-01-01

    Legionella pneumophila is a Gram-negative opportunistic human pathogen that causes a severe pneumonia known as Legionnaires' disease. Notably, in the human host, the organism is believed to replicate solely within an intracellular compartment, predominantly within pulmonary macrophages. Consequently, successful therapy is predicated on antimicrobials penetrating into this intracellular growth niche. However, standard antimicrobial susceptibility testing methods test solely for extracellular growth inhibition. Here, we make use of a high-throughput assay to characterize intracellular growth inhibition activity of known antimicrobials. For select antimicrobials, high-resolution dose-response analysis was then performed to characterize and compare activity levels in both macrophage infection and axenic growth assays. Results support the superiority of several classes of nonpolar antimicrobials in abrogating intracellular growth. Importantly, our assay results show excellent correlations with prior clinical observations of antimicrobial efficacy. Furthermore, we also show the applicability of high-throughput automation to two- and three-dimensional synergy testing. High-resolution isocontour isobolograms provide in vitro support for specific combination antimicrobial therapy. Taken together, findings suggest that high-throughput screening technology may be successfully applied to identify and characterize antimicrobials that target bacterial pathogens that make use of an intracellular growth niche. PMID:26392509

  7. Human transcriptome array for high-throughput clinical studies.

    PubMed

    Xu, Weihong; Seok, Junhee; Mindrinos, Michael N; Schweitzer, Anthony C; Jiang, Hui; Wilhelmy, Julie; Clark, Tyson A; Kapur, Karen; Xing, Yi; Faham, Malek; Storey, John D; Moldawer, Lyle L; Maier, Ronald V; Tompkins, Ronald G; Wong, Wing Hung; Davis, Ronald W; Xiao, Wenzhong

    2011-03-01

    A 6.9 million-feature oligonucleotide array of the human transcriptome [Glue Grant human transcriptome (GG-H array)] has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing as well as detection of coding SNPs and noncoding transcripts. The performance of the array was examined and compared with mRNA sequencing (RNA-Seq) results over multiple independent replicates of liver and muscle samples. Compared with RNA-Seq of 46 million uniquely mappable reads per replicate, the GG-H array is highly reproducible in estimating gene and exon abundance. Although both platforms detect similar expression changes at the gene level, the GG-H array is more sensitive at the exon level. Deeper sequencing is required to adequately cover low-abundance transcripts. The array has been implemented in a multicenter clinical program and has generated high-quality, reproducible data. Considering the clinical trial requirements of cost, sample availability, and throughput, the GG-H array has a wide range of applications. An emerging approach for large-scale clinical genomic studies is to first use RNA-Seq to the sufficient depth for the discovery of transcriptome elements relevant to the disease process followed by high-throughput and reliable screening of these elements on thousands of patient samples using custom-designed arrays.

  8. Human transcriptome array for high-throughput clinical studies

    PubMed Central

    Xu, Weihong; Seok, Junhee; Mindrinos, Michael N.; Schweitzer, Anthony C.; Jiang, Hui; Wilhelmy, Julie; Clark, Tyson A.; Kapur, Karen; Xing, Yi; Faham, Malek; Storey, John D.; Moldawer, Lyle L.; Maier, Ronald V.; Tompkins, Ronald G.; Wong, Wing Hung; Davis, Ronald W.; Xiao, Wenzhong; Toner, Mehmet; Warren, H. Shaw; Schoenfeld, David A.; Rahme, Laurence; McDonald-Smith, Grace P.; Hayden, Douglas; Mason, Philip; Fagan, Shawn; Yu, Yong-Ming; Cobb, J. Perren; Remick, Daniel G.; Mannick, John A.; Lederer, James A.; Gamelli, Richard L.; Silver, Geoffrey M.; West, Michael A.; Shapiro, Michael B.; Smith, Richard; Camp, David G.; Qian, Weijun; Tibshirani, Rob; Lowry, Stephen; Calvano, Steven; Chaudry, Irshad; Cohen, Mitchell; Moore, Ernest E.; Johnson, Jeffrey; Baker, Henry V.; Efron, Philip A.; Balis, Ulysses G. J.; Billiar, Timothy R.; Ochoa, Juan B.; Sperry, Jason L.; Miller-Graziano, Carol L.; De, Asit K.; Bankey, Paul E.; Herndon, David N.; Finnerty, Celeste C.; Jeschke, Marc G.; Minei, Joseph P.; Arnoldo, Brett D.; Hunt, John L.; Horton, Jureta; Cobb, J. Perren; Brownstein, Bernard; Freeman, Bradley; Nathens, Avery B.; Cuschieri, Joseph; Gibran, Nicole; Klein, Matthew; O'Keefe, Grant

    2011-01-01

    A 6.9 million-feature oligonucleotide array of the human transcriptome [Glue Grant human transcriptome (GG-H array)] has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing as well as detection of coding SNPs and noncoding transcripts. The performance of the array was examined and compared with mRNA sequencing (RNA-Seq) results over multiple independent replicates of liver and muscle samples. Compared with RNA-Seq of 46 million uniquely mappable reads per replicate, the GG-H array is highly reproducible in estimating gene and exon abundance. Although both platforms detect similar expression changes at the gene level, the GG-H array is more sensitive at the exon level. Deeper sequencing is required to adequately cover low-abundance transcripts. The array has been implemented in a multicenter clinical program and has generated high-quality, reproducible data. Considering the clinical trial requirements of cost, sample availability, and throughput, the GG-H array has a wide range of applications. An emerging approach for large-scale clinical genomic studies is to first use RNA-Seq to the sufficient depth for the discovery of transcriptome elements relevant to the disease process followed by high-throughput and reliable screening of these elements on thousands of patient samples using custom-designed arrays. PMID:21317363

  9. High-Throughput Optical Sensing Immunoassays on Smartphone.

    PubMed

    Wang, Li-Ju; Sun, Rongrong; Vasile, Tina; Chang, Yu-Chung; Li, Lei

    2016-08-16

    We present an optical sensing platform on a smartphone for high-throughput screening immunoassays. For the first time, a designed microprism array is utilized to achieve a one-time screening of 64 samples. To demonstrate the capability and the reliability of this optical sensing platform on smartphone, human interleukin 6 (IL-6) protein and six types of plant viruses are immunoassayed. The ability of quantification is shown by a sigmoidal dose-response curve fitting to analyze IL-6 protein. The accuracy in measuring the concentrations of IL-6 protein achieves 99.1%. On the other hand, to validate on-field immunoassays by our device, a total of 1030 samples are assayed using three immunoassay methods to detect six types of plant viruses. The accuracy is up to 96.2-99.9%; in addition, there is a high degree of agreement with lab instruments. The total cost for this high-throughput optical screening platform is ∼$50 USD. The reading time is only 2 s for 64 samples. The size is just as big as a portable hard drive. Our optical sensing platform on the smartphone offers a route toward in situ high-throughput screening immunoassays for viruses, pathogens, biomarkers, and toxins by decentralizing laboratory tests. With this mobile point-of-care optical platform, the spread of disease can be timely stopped within a very short turnaround time. PMID:27434250

  10. Computational analysis of high-throughput flow cytometry data

    PubMed Central

    Robinson, J Paul; Rajwa, Bartek; Patsekin, Valery; Davisson, Vincent Jo

    2015-01-01

    Introduction Flow cytometry has been around for over 40 years, but only recently has the opportunity arisen to move into the high-throughput domain. The technology is now available and is highly competitive with imaging tools under the right conditions. Flow cytometry has, however, been a technology that has focused on its unique ability to study single cells and appropriate analytical tools are readily available to handle this traditional role of the technology. Areas covered Expansion of flow cytometry to a high-throughput (HT) and high-content technology requires both advances in hardware and analytical tools. The historical perspective of flow cytometry operation as well as how the field has changed and what the key changes have been discussed. The authors provide a background and compelling arguments for moving toward HT flow, where there are many innovative opportunities. With alternative approaches now available for flow cytometry, there will be a considerable number of new applications. These opportunities show strong capability for drug screening and functional studies with cells in suspension. Expert opinion There is no doubt that HT flow is a rich technology awaiting acceptance by the pharmaceutical community. It can provide a powerful phenotypic analytical toolset that has the capacity to change many current approaches to HT screening. The previous restrictions on the technology, based on its reduced capacity for sample throughput, are no longer a major issue. Overcoming this barrier has transformed a mature technology into one that can focus on systems biology questions not previously considered possible. PMID:22708834

  11. High throughput screening of starch structures using carbohydrate microarrays.

    PubMed

    Tanackovic, Vanja; Rydahl, Maja Gro; Pedersen, Henriette Lodberg; Motawia, Mohammed Saddik; Shaik, Shahnoor Sultana; Mikkelsen, Maria Dalgaard; Krunic, Susanne Langgaard; Fangel, Jonatan Ulrik; Willats, William George Tycho; Blennow, Andreas

    2016-01-01

    In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated maltooligosaccharides, pure starch samples including a variety of different structures with variations in the amylopectin branching pattern, amylose content and phosphate content, enzymatically modified starches and glycogen were included. Using this technique, different important structures, including amylose content and branching degrees could be differentiated in a high throughput fashion. The screening method was validated using transgenic barley grain analysed during development and subjected to germination. Typically, extreme branching or linearity were detected less than normal starch structures. The method offers the potential for rapidly analysing resistant and slowly digested dietary starches. PMID:27468930

  12. High throughput screening of starch structures using carbohydrate microarrays

    PubMed Central

    Tanackovic, Vanja; Rydahl, Maja Gro; Pedersen, Henriette Lodberg; Motawia, Mohammed Saddik; Shaik, Shahnoor Sultana; Mikkelsen, Maria Dalgaard; Krunic, Susanne Langgaard; Fangel, Jonatan Ulrik; Willats, William George Tycho; Blennow, Andreas

    2016-01-01

    In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated maltooligosaccharides, pure starch samples including a variety of different structures with variations in the amylopectin branching pattern, amylose content and phosphate content, enzymatically modified starches and glycogen were included. Using this technique, different important structures, including amylose content and branching degrees could be differentiated in a high throughput fashion. The screening method was validated using transgenic barley grain analysed during development and subjected to germination. Typically, extreme branching or linearity were detected less than normal starch structures. The method offers the potential for rapidly analysing resistant and slowly digested dietary starches. PMID:27468930

  13. Portable thermo-powered high-throughput visual electrochemiluminescence sensor.

    PubMed

    Hao, Nan; Xiong, Meng; Zhang, Jia-dong; Xu, Jing-Juan; Chen, Hong-Yuan

    2013-12-17

    This paper describes a portable thermo-powered high-throughput visual electrochemiluminescence (ECL) sensor for the first time. This sensor is composed of a tiny power supply device based on thermal-electrical conversion and a facile prepared array electrode. The ECL detection could be conducted with thermo-power, which is easily accessible. For example, hot water, a bonfire, or a lighted candle enables the detection to be conducted. And the assay can be directly monitored by the naked eye semiquantitatively or smart phones quantitatively. Combined with transparent electrode and array microreactors, a portable high-throughput sensor was achieved. The portable device, avoiding the use of an electrochemical workstation to generate potential and a photomultiplier tube to receive the signal, is not only a valuable addition for traditional methods but also a suitable device for field operation or point-of-care testing. PMID:24215560

  14. High-throughput screening to identify inhibitors of lysine demethylases

    PubMed Central

    Gale, Molly; Yan, Qin

    2015-01-01

    Lysine demethylases (KDMs) are epigenetic regulators whose dysfunction is implicated in the pathology of many human diseases including various types of cancer, inflammation and X-linked intellectual disability. Particular demethylases have been identified as promising therapeutic targets, and tremendous efforts are being devoted toward developing suitable small-molecule inhibitors for clinical and research use. Several high-throughput screening strategies have been developed to screen for small-molecule inhibitors of KDMs, each with advantages and disadvantages in terms of time, cost, effort, reliability and sensitivity. In this Special Report, we review and evaluate the high-throughput screening methods utilized for discovery of novel small-molecule KDM inhibitors. PMID:25687466

  15. High-throughput patterning of photonic structures with tunable periodicity

    PubMed Central

    Kempa, Thomas J.; Bediako, D. Kwabena; Kim, Sun-Kyung; Park, Hong-Gyu; Nocera, Daniel G.

    2015-01-01

    A patterning method termed “RIPPLE” (reactive interface patterning promoted by lithographic electrochemistry) is applied to the fabrication of arrays of dielectric and metallic optical elements. This method uses cyclic voltammetry to impart patterns onto the working electrode of a standard three-electrode electrochemical setup. Using this technique and a template stripping process, periodic arrays of Ag circular Bragg gratings are patterned in a high-throughput fashion over large substrate areas. By varying the scan rate of the cyclically applied voltage ramps, the periodicity of the gratings can be tuned in situ over micrometer and submicrometer length scales. Characterization of the periodic arrays of periodic gratings identified point-like and annular scattering modes at different planes above the structured surface. Facile, reliable, and rapid patterning techniques like RIPPLE may enable the high-throughput and low-cost fabrication of photonic elements and metasurfaces for energy conversion and sensing applications. PMID:25870280

  16. High Throughput siRNA Screening Using Reverse Transfection.

    PubMed

    von Schantz, Carina; Saarela, Jani

    2016-01-01

    RNA interference (RNAi) is a commonly used technique to knockdown gene function. Here, we describe a high throughput screening method for siRNA mediated gene silencing of the breast cancer cell line MDA-MB-231 using reverse transfection. Furthermore, we describe the setup for two separate methods for detecting viable and dead cells using either homogenous assays or image-based analysis. PMID:27581282

  17. A fully automated robotic system for high throughput fermentation.

    PubMed

    Zimmermann, Hartmut F; Rieth, Jochen

    2007-03-01

    High throughput robotic systems have been used since the 1990s to carry out biochemical assays in microtiter plates. However, before the application of such systems in industrial fermentation process development, some important specific demands should be taken into account. These are sufficient oxygen supply, optimal growth temperature, minimized sample evaporation, avoidance of contaminations, and simple but reliable process monitoring. A fully automated solution where all these aspects have been taken into account is presented.

  18. High-throughput evaluation of synthetic metabolic pathways

    PubMed Central

    Klesmith, Justin R.; Whitehead, Timothy A.

    2016-01-01

    A central challenge in the field of metabolic engineering is the efficient identification of a metabolic pathway genotype that maximizes specific productivity over a robust range of process conditions. Here we review current methods for optimizing specific productivity of metabolic pathways in living cells. New tools for library generation, computational analysis of pathway sequence-flux space, and high-throughput screening and selection techniques are discussed. PMID:27453919

  19. THINK Back: KNowledge-based Interpretation of High Throughput data.

    PubMed

    Farfán, Fernando; Ma, Jun; Sartor, Maureen A; Michailidis, George; Jagadish, Hosagrahar V

    2012-01-01

    Results of high throughput experiments can be challenging to interpret. Current approaches have relied on bulk processing the set of expression levels, in conjunction with easily obtained external evidence, such as co-occurrence. While such techniques can be used to reason probabilistically, they are not designed to shed light on what any individual gene, or a network of genes acting together, may be doing. Our belief is that today we have the information extraction ability and the computational power to perform more sophisticated analyses that consider the individual situation of each gene. The use of such techniques should lead to qualitatively superior results. The specific aim of this project is to develop computational techniques to generate a small number of biologically meaningful hypotheses based on observed results from high throughput microarray experiments, gene sequences, and next-generation sequences. Through the use of relevant known biomedical knowledge, as represented in published literature and public databases, we can generate meaningful hypotheses that will aide biologists to interpret their experimental data. We are currently developing novel approaches that exploit the rich information encapsulated in biological pathway graphs. Our methods perform a thorough and rigorous analysis of biological pathways, using complex factors such as the topology of the pathway graph and the frequency in which genes appear on different pathways, to provide more meaningful hypotheses to describe the biological phenomena captured by high throughput experiments, when compared to other existing methods that only consider partial information captured by biological pathways. PMID:22536867

  20. THINK Back: KNowledge-based Interpretation of High Throughput data

    PubMed Central

    2012-01-01

    Results of high throughput experiments can be challenging to interpret. Current approaches have relied on bulk processing the set of expression levels, in conjunction with easily obtained external evidence, such as co-occurrence. While such techniques can be used to reason probabilistically, they are not designed to shed light on what any individual gene, or a network of genes acting together, may be doing. Our belief is that today we have the information extraction ability and the computational power to perform more sophisticated analyses that consider the individual situation of each gene. The use of such techniques should lead to qualitatively superior results. The specific aim of this project is to develop computational techniques to generate a small number of biologically meaningful hypotheses based on observed results from high throughput microarray experiments, gene sequences, and next-generation sequences. Through the use of relevant known biomedical knowledge, as represented in published literature and public databases, we can generate meaningful hypotheses that will aide biologists to interpret their experimental data. We are currently developing novel approaches that exploit the rich information encapsulated in biological pathway graphs. Our methods perform a thorough and rigorous analysis of biological pathways, using complex factors such as the topology of the pathway graph and the frequency in which genes appear on different pathways, to provide more meaningful hypotheses to describe the biological phenomena captured by high throughput experiments, when compared to other existing methods that only consider partial information captured by biological pathways. PMID:22536867

  1. Promises and Pitfalls of High-Throughput Biological Assays.

    PubMed

    Finak, Greg; Gottardo, Raphael

    2016-01-01

    This chapter discusses some of the pitfalls encountered when performing biomedical research involving high-throughput "omics" data and presents some strategies and guidelines that researchers should follow when undertaking such studies. We discuss common errors in experimental design and data analysis that lead to irreproducible and non-replicable research and provide some guidelines to avoid these common mistakes so that researchers may have confidence in study outcomes, even if the results are negative. We discuss the importance of ranking and prespecifying hypotheses, performing power analysis, careful experimental design, and preplanning of statistical analyses in order to avoid the "fishing expedition" data analysis strategy, which is doomed to fail. The impact of multiple testing on false-positive rates is discussed, particularly in the context of the analysis of high-throughput data, and methods to correct for it are presented, as well as approaches to detect and correct for experimental biases and batch effects, which often plague high-throughput assays. We highlight the importance of sharing data and analysis code to facilitate reproducibility and present tools and software that are appropriate for this purpose. PMID:27115636

  2. THINK Back: KNowledge-based Interpretation of High Throughput data.

    PubMed

    Farfán, Fernando; Ma, Jun; Sartor, Maureen A; Michailidis, George; Jagadish, Hosagrahar V

    2012-03-13

    Results of high throughput experiments can be challenging to interpret. Current approaches have relied on bulk processing the set of expression levels, in conjunction with easily obtained external evidence, such as co-occurrence. While such techniques can be used to reason probabilistically, they are not designed to shed light on what any individual gene, or a network of genes acting together, may be doing. Our belief is that today we have the information extraction ability and the computational power to perform more sophisticated analyses that consider the individual situation of each gene. The use of such techniques should lead to qualitatively superior results. The specific aim of this project is to develop computational techniques to generate a small number of biologically meaningful hypotheses based on observed results from high throughput microarray experiments, gene sequences, and next-generation sequences. Through the use of relevant known biomedical knowledge, as represented in published literature and public databases, we can generate meaningful hypotheses that will aide biologists to interpret their experimental data. We are currently developing novel approaches that exploit the rich information encapsulated in biological pathway graphs. Our methods perform a thorough and rigorous analysis of biological pathways, using complex factors such as the topology of the pathway graph and the frequency in which genes appear on different pathways, to provide more meaningful hypotheses to describe the biological phenomena captured by high throughput experiments, when compared to other existing methods that only consider partial information captured by biological pathways.

  3. NCBI GEO: archive for high-throughput functional genomic data.

    PubMed

    Barrett, Tanya; Troup, Dennis B; Wilhite, Stephen E; Ledoux, Pierre; Rudnev, Dmitry; Evangelista, Carlos; Kim, Irene F; Soboleva, Alexandra; Tomashevsky, Maxim; Marshall, Kimberly A; Phillippy, Katherine H; Sherman, Patti M; Muertter, Rolf N; Edgar, Ron

    2009-01-01

    The Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) is the largest public repository for high-throughput gene expression data. Additionally, GEO hosts other categories of high-throughput functional genomic data, including those that examine genome copy number variations, chromatin structure, methylation status and transcription factor binding. These data are generated by the research community using high-throughput technologies like microarrays and, more recently, next-generation sequencing. The database has a flexible infrastructure that can capture fully annotated raw and processed data, enabling compliance with major community-derived scientific reporting standards such as 'Minimum Information About a Microarray Experiment' (MIAME). In addition to serving as a centralized data storage hub, GEO offers many tools and features that allow users to effectively explore, analyze and download expression data from both gene-centric and experiment-centric perspectives. This article summarizes the GEO repository structure, content and operating procedures, as well as recently introduced data mining features. GEO is freely accessible at http://www.ncbi.nlm.nih.gov/geo/.

  4. High throughput and miniaturised systems for biodegradability assessments.

    PubMed

    Cregut, Mickael; Jouanneau, Sulivan; Brillet, François; Durand, Marie-José; Sweetlove, Cyril; Chenèble, Jean-Charles; L'Haridon, Jacques; Thouand, Gérald

    2014-01-01

    The society demands safer products with a better ecological profile. Regulatory criteria have been developed to prevent risks for human health and the environment, for example, within the framework of the European regulation REACH (Regulation (EC) No 1907, 2006). This has driven industry to consider the development of high throughput screening methodologies for assessing chemical biodegradability. These new screening methodologies must be scalable for miniaturisation, reproducible and as reliable as existing procedures for enhanced biodegradability assessment. Here, we evaluate two alternative systems that can be scaled for high throughput screening and conveniently miniaturised to limit costs in comparison with traditional testing. These systems are based on two dyes as follows: an invasive fluorescent dyes that serves as a cellular activity marker (a resazurin-like dye reagent) and a noninvasive fluorescent oxygen optosensor dye (an optical sensor). The advantages and limitations of these platforms for biodegradability assessment are presented. Our results confirm the feasibility of these systems for evaluating and screening chemicals for ready biodegradability. The optosensor is a miniaturised version of a component already used in traditional ready biodegradability testing, whereas the resazurin dye offers an interesting new screening mechanism for chemical concentrations greater than 10 mg/l that are not amenable to traditional closed bottle tests. The use of these approaches allows generalisation of high throughput screening methodologies to meet the need of developing new compounds with a favourable ecological profile and also assessment for regulatory purpose.

  5. Condor-COPASI: high-throughput computing for biochemical networks

    PubMed Central

    2012-01-01

    Background Mathematical modelling has become a standard technique to improve our understanding of complex biological systems. As models become larger and more complex, simulations and analyses require increasing amounts of computational power. Clusters of computers in a high-throughput computing environment can help to provide the resources required for computationally expensive model analysis. However, exploiting such a system can be difficult for users without the necessary expertise. Results We present Condor-COPASI, a server-based software tool that integrates COPASI, a biological pathway simulation tool, with Condor, a high-throughput computing environment. Condor-COPASI provides a web-based interface, which makes it extremely easy for a user to run a number of model simulation and analysis tasks in parallel. Tasks are transparently split into smaller parts, and submitted for execution on a Condor pool. Result output is presented to the user in a number of formats, including tables and interactive graphical displays. Conclusions Condor-COPASI can effectively use a Condor high-throughput computing environment to provide significant gains in performance for a number of model simulation and analysis tasks. Condor-COPASI is free, open source software, released under the Artistic License 2.0, and is suitable for use by any institution with access to a Condor pool. Source code is freely available for download at http://code.google.com/p/condor-copasi/, along with full instructions on deployment and usage. PMID:22834945

  6. Computational approaches to phenotyping: high-throughput phenomics.

    PubMed

    Lussier, Yves A; Liu, Yang

    2007-01-01

    The recent completion of the Human Genome Project has made possible a high-throughput "systems approach" for accelerating the elucidation of molecular underpinnings of human diseases, and subsequent derivation of molecular-based strategies to more effectively prevent, diagnose, and treat these diseases. Although altered phenotypes are among the most reliable manifestations of altered gene functions, research using systematic analysis of phenotype relationships to study human biology is still in its infancy. This article focuses on the emerging field of high-throughput phenotyping (HTP) phenomics research, which aims to capitalize on novel high-throughput computation and informatics technology developments to derive genomewide molecular networks of genotype-phenotype associations, or "phenomic associations." The HTP phenomics research field faces the challenge of technological research and development to generate novel tools in computation and informatics that will allow researchers to amass, access, integrate, organize, and manage phenotypic databases across species and enable genomewide analysis to associate phenotypic information with genomic data at different scales of biology. Key state-of-the-art technological advancements critical for HTP phenomics research are covered in this review. In particular, we highlight the power of computational approaches to conduct large-scale phenomics studies.

  7. Design of a High-Throughput Plasma-Processing System

    SciTech Connect

    Darkazalli, Ghazi; Matthei, Keith; Ruby, Douglas S.

    1999-07-20

    Sandia National Laboratories has demonstrated significant performance gains in crystalline silicon solar cell technology through the use of plasma-processing for the deposition of silicon nitride by Plasma Enhanced Chemical Vapor Deposition (PECVD), plasma-hydrogenation of the nitride layer, and reactive-ion etching of the silicon surface prior to the deposition to decrease the reflectivity of the surface. One of the major problems of implementing plasma processing into a cell production line is the batch configuration and/or low throughput of the systems currently available. This report describes the concept of a new in-line plasma processing system that could meet the industrial requirements for a high-throughput and cost effective solution for mass production of solar cells.

  8. A high throughput mechanical screening device for cartilage tissue engineering.

    PubMed

    Mohanraj, Bhavana; Hou, Chieh; Meloni, Gregory R; Cosgrove, Brian D; Dodge, George R; Mauck, Robert L

    2014-06-27

    Articular cartilage enables efficient and near-frictionless load transmission, but suffers from poor inherent healing capacity. As such, cartilage tissue engineering strategies have focused on mimicking both compositional and mechanical properties of native tissue in order to provide effective repair materials for the treatment of damaged or degenerated joint surfaces. However, given the large number design parameters available (e.g. cell sources, scaffold designs, and growth factors), it is difficult to conduct combinatorial experiments of engineered cartilage. This is particularly exacerbated when mechanical properties are a primary outcome, given the long time required for testing of individual samples. High throughput screening is utilized widely in the pharmaceutical industry to rapidly and cost-effectively assess the effects of thousands of compounds for therapeutic discovery. Here we adapted this approach to develop a high throughput mechanical screening (HTMS) system capable of measuring the mechanical properties of up to 48 materials simultaneously. The HTMS device was validated by testing various biomaterials and engineered cartilage constructs and by comparing the HTMS results to those derived from conventional single sample compression tests. Further evaluation showed that the HTMS system was capable of distinguishing and identifying 'hits', or factors that influence the degree of tissue maturation. Future iterations of this device will focus on reducing data variability, increasing force sensitivity and range, as well as scaling-up to even larger (96-well) formats. This HTMS device provides a novel tool for cartilage tissue engineering, freeing experimental design from the limitations of mechanical testing throughput.

  9. High-throughput insect cell protein expression applications.

    PubMed

    Buchs, Mirjam; Kim, Ernie; Pouliquen, Yann; Sachs, Michael; Geisse, Sabine; Mahnke, Marion; Hunt, Ian

    2009-01-01

    The Baculovirus Expression Vector System (BEVS) is one of the most efficient systems for production of recombinant proteins and consequently its application is wide-spread in industry as well as in academia. Since the early 1970s, when the first stable insect cell lines were established and the infectivity of bacu-lovirus in an in vitro culture system was demonstrated (1, 2), virtually thousands of reports have been published on the successful expression of proteins using this system as well as on method improvement. However, despite its popularity the system is labor intensive and time consuming. Moreover, adaptation of the system to multi-parallel (high-throughput) expression is much more difficult to achieve than with E. coli due to its far more complex nature. However, recent years have seen the development of strategies that have greatly enhanced the stream-lining and speed of baculovirus protein expression for increased throughput via use of automation and miniaturization. This chapter therefore tries to collate these developments in a series of protocols (which are modifications to standard procedure plus several new approaches) that will allow the user to expedite the speed and throughput of baculovirus-mediated protein expression and facilitate true multi-parallel, high-throughput protein expression profiling in insect cells. In addition we also provide a series of optimized protocols for small and large-scale transient insect cell expression that allow for both the rapid analysis of multiple constructs and the concomitant scale-up of those selected for on-going analysis. Since this approach is independent of viral propagation, the timelines for this approach are markedly shorter and offer a significant advantage over standard bacu-lovirus expression approach strategies in the context of HT applications.

  10. High throughput computing: a solution for scientific analysis

    USGS Publications Warehouse

    O'Donnell, M.

    2011-01-01

    handle job failures due to hardware, software, or network interruptions (obviating the need to manually resubmit the job after each stoppage); be affordable; and most importantly, allow us to complete very large, complex analyses that otherwise would not even be possible. In short, we envisioned a job-management system that would take advantage of unused FORT CPUs within a local area network (LAN) to effectively distribute and run highly complex analytical processes. What we found was a solution that uses High Throughput Computing (HTC) and High Performance Computing (HPC) systems to do exactly that (Figure 1).

  11. Achieving High Throughput for Data Transfer over ATM Networks

    NASA Technical Reports Server (NTRS)

    Johnson, Marjory J.; Townsend, Jeffrey N.

    1996-01-01

    File-transfer rates for ftp are often reported to be relatively slow, compared to the raw bandwidth available in emerging gigabit networks. While a major bottleneck is disk I/O, protocol issues impact performance as well. Ftp was developed and optimized for use over the TCP/IP protocol stack of the Internet. However, TCP has been shown to run inefficiently over ATM. In an effort to maximize network throughput, data-transfer protocols can be developed to run over UDP or directly over IP, rather than over TCP. If error-free transmission is required, techniques for achieving reliable transmission can be included as part of the transfer protocol. However, selected image-processing applications can tolerate a low level of errors in images that are transmitted over a network. In this paper we report on experimental work to develop a high-throughput protocol for unreliable data transfer over ATM networks. We attempt to maximize throughput by keeping the communications pipe full, but still keep packet loss under five percent. We use the Bay Area Gigabit Network Testbed as our experimental platform.

  12. Controlling high-throughput manufacturing at the nano-scale

    NASA Astrophysics Data System (ADS)

    Cooper, Khershed P.

    2013-09-01

    Interest in nano-scale manufacturing research and development is growing. The reason is to accelerate the translation of discoveries and inventions of nanoscience and nanotechnology into products that would benefit industry, economy and society. Ongoing research in nanomanufacturing is focused primarily on developing novel nanofabrication techniques for a variety of applications—materials, energy, electronics, photonics, biomedical, etc. Our goal is to foster the development of high-throughput methods of fabricating nano-enabled products. Large-area parallel processing and highspeed continuous processing are high-throughput means for mass production. An example of large-area processing is step-and-repeat nanoimprinting, by which nanostructures are reproduced again and again over a large area, such as a 12 in wafer. Roll-to-roll processing is an example of continuous processing, by which it is possible to print and imprint multi-level nanostructures and nanodevices on a moving flexible substrate. The big pay-off is high-volume production and low unit cost. However, the anticipated cost benefits can only be realized if the increased production rate is accompanied by high yields of high quality products. To ensure product quality, we need to design and construct manufacturing systems such that the processes can be closely monitored and controlled. One approach is to bring cyber-physical systems (CPS) concepts to nanomanufacturing. CPS involves the control of a physical system such as manufacturing through modeling, computation, communication and control. Such a closely coupled system will involve in-situ metrology and closed-loop control of the physical processes guided by physics-based models and driven by appropriate instrumentation, sensing and actuation. This paper will discuss these ideas in the context of controlling high-throughput manufacturing at the nano-scale.

  13. SSFinder: high throughput CRISPR-Cas target sites prediction tool.

    PubMed

    Upadhyay, Santosh Kumar; Sharma, Shailesh

    2014-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system facilitates targeted genome editing in organisms. Despite high demand of this system, finding a reliable tool for the determination of specific target sites in large genomic data remained challenging. Here, we report SSFinder, a python script to perform high throughput detection of specific target sites in large nucleotide datasets. The SSFinder is a user-friendly tool, compatible with Windows, Mac OS, and Linux operating systems, and freely available online. PMID:25089276

  14. Developing High-Throughput HIV Incidence Assay with Pyrosequencing Platform

    PubMed Central

    Park, Sung Yong; Goeken, Nolan; Lee, Hyo Jin; Bolan, Robert; Dubé, Michael P.

    2014-01-01

    ABSTRACT Human immunodeficiency virus (HIV) incidence is an important measure for monitoring the epidemic and evaluating the efficacy of intervention and prevention trials. This study developed a high-throughput, single-measure incidence assay by implementing a pyrosequencing platform. We devised a signal-masking bioinformatics pipeline, which yielded a process error rate of 5.8 × 10−4 per base. The pipeline was then applied to analyze 18,434 envelope gene segments (HXB2 7212 to 7601) obtained from 12 incident and 24 chronic patients who had documented HIV-negative and/or -positive tests. The pyrosequencing data were cross-checked by using the single-genome-amplification (SGA) method to independently obtain 302 sequences from 13 patients. Using two genomic biomarkers that probe for the presence of similar sequences, the pyrosequencing platform correctly classified all 12 incident subjects (100% sensitivity) and 23 of 24 chronic subjects (96% specificity). One misclassified subject's chronic infection was correctly classified by conducting the same analysis with SGA data. The biomarkers were statistically associated across the two platforms, suggesting the assay's reproducibility and robustness. Sampling simulations showed that the biomarkers were tolerant of sequencing errors and template resampling, two factors most likely to affect the accuracy of pyrosequencing results. We observed comparable biomarker scores between AIDS and non-AIDS chronic patients (multivariate analysis of variance [MANOVA], P = 0.12), indicating that the stage of HIV disease itself does not affect the classification scheme. The high-throughput genomic HIV incidence marks a significant step toward determining incidence from a single measure in cross-sectional surveys. IMPORTANCE Annual HIV incidence, the number of newly infected individuals within a year, is the key measure of monitoring the epidemic's rise and decline. Developing reliable assays differentiating recent from chronic

  15. IRAS: High-Throughput Identification of Novel Alternative Splicing Regulators.

    PubMed

    Zheng, S

    2016-01-01

    Alternative splicing is a fundamental regulatory process of gene expression. Defects in alternative splicing can lead to various diseases, and modification of disease-causing splicing events presents great therapeutic promise. Splicing outcome is commonly affected by extracellular stimuli and signaling cascades that converge on RNA-binding splicing regulators. These trans-acting factors recognize cis-elements in pre-mRNA transcripts to affect spliceosome assembly and splice site choices. Identification of these splicing regulators and/or upstream modulators has been difficult and traditionally done by piecemeal. High-throughput screening strategies to find multiple regulators of exon splicing have great potential to accelerate the discovery process, but typically confront low sensitivity and low specificity of screening assays. Here we describe a unique screening strategy, IRAS (identifying regulators of alternative splicing), using a pair of dual-output minigene reporters to allow for sensitive detection of exon splicing changes. Each dual-output reporter produces green fluorescent protein (GFP) and red fluorescent protein (RFP) fluorescent signals to assay the two spliced isoforms exclusively. The two complementary minigene reporters alter GFP/RFP output ratios in the opposite direction in response to splicing change. Applying IRAS in cell-based high-throughput screens allows sensitive and specific identification of splicing regulators and modulators for any alternative exons of interest. In comparison to previous high-throughput screening methods, IRAS substantially enhances the specificity of the screening assay. This strategy significantly eliminates false positives without sacrificing sensitive identification of true regulators of splicing. PMID:27241759

  16. High-throughput sequencing: a roadmap toward community ecology.

    PubMed

    Poisot, Timothée; Péquin, Bérangère; Gravel, Dominique

    2013-04-01

    High-throughput sequencing is becoming increasingly important in microbial ecology, yet it is surprisingly under-used to generate or test biogeographic hypotheses. In this contribution, we highlight how adding these methods to the ecologist toolbox will allow the detection of new patterns, and will help our understanding of the structure and dynamics of diversity. Starting with a review of ecological questions that can be addressed, we move on to the technical and analytical issues that will benefit from an increased collaboration between different disciplines. PMID:23610649

  17. Adaptive Sampling for High Throughput Data Using Similarity Measures

    SciTech Connect

    Bulaevskaya, V.; Sales, A. P.

    2015-05-06

    The need for adaptive sampling arises in the context of high throughput data because the rates of data arrival are many orders of magnitude larger than the rates at which they can be analyzed. A very fast decision must therefore be made regarding the value of each incoming observation and its inclusion in the analysis. In this report we discuss one approach to adaptive sampling, based on the new data point’s similarity to the other data points being considered for inclusion. We present preliminary results for one real and one synthetic data set.

  18. Live Cell Optical Sensing for High Throughput Applications

    NASA Astrophysics Data System (ADS)

    Fang, Ye

    Live cell optical sensing employs label-free optical biosensors to non-invasively measure stimulus-induced dynamic mass redistribution (DMR) in live cells within the sensing volume of the biosensor. The resultant DMR signal is an integrated cellular response, and reflects cell signaling mediated through the cellular target(s) with which the stimulus intervenes. This article describes the uses of live cell optical sensing for probing cell biology and ligand pharmacology, with an emphasis of resonant waveguide grating biosensor cellular assays for high throughput applications.

  19. Orchestrating high-throughput genomic analysis with Bioconductor.

    PubMed

    Huber, Wolfgang; Carey, Vincent J; Gentleman, Robert; Anders, Simon; Carlson, Marc; Carvalho, Benilton S; Bravo, Hector Corrada; Davis, Sean; Gatto, Laurent; Girke, Thomas; Gottardo, Raphael; Hahne, Florian; Hansen, Kasper D; Irizarry, Rafael A; Lawrence, Michael; Love, Michael I; MacDonald, James; Obenchain, Valerie; Oleś, Andrzej K; Pagès, Hervé; Reyes, Alejandro; Shannon, Paul; Smyth, Gordon K; Tenenbaum, Dan; Waldron, Levi; Morgan, Martin

    2015-02-01

    Bioconductor is an open-source, open-development software project for the analysis and comprehension of high-throughput data in genomics and molecular biology. The project aims to enable interdisciplinary research, collaboration and rapid development of scientific software. Based on the statistical programming language R, Bioconductor comprises 934 interoperable packages contributed by a large, diverse community of scientists. Packages cover a range of bioinformatic and statistical applications. They undergo formal initial review and continuous automated testing. We present an overview for prospective users and contributors. PMID:25633503

  20. Analysis of High-Throughput ELISA Microarray Data

    SciTech Connect

    White, Amanda M.; Daly, Don S.; Zangar, Richard C.

    2011-02-23

    Our research group develops analytical methods and software for the high-throughput analysis of quantitative enzyme-linked immunosorbent assay (ELISA) microarrays. ELISA microarrays differ from DNA microarrays in several fundamental aspects and most algorithms for analysis of DNA microarray data are not applicable to ELISA microarrays. In this review, we provide an overview of the steps involved in ELISA microarray data analysis and how the statistically sound algorithms we have developed provide an integrated software suite to address the needs of each data-processing step. The algorithms discussed are available in a set of open-source software tools (http://www.pnl.gov/statistics/ProMAT).

  1. Computational Proteomics: High-throughput Analysis for Systems Biology

    SciTech Connect

    Cannon, William R.; Webb-Robertson, Bobbie-Jo M.

    2007-01-03

    High-throughput (HTP) proteomics is a rapidly developing field that offers the global profiling of proteins from a biological system. The HTP technological advances are fueling a revolution in biology, enabling analyses at the scales of entire systems (e.g., whole cells, tumors, or environmental communities). However, simply identifying the proteins in a cell is insufficient for understanding the underlying complexity and operating mechanisms of the overall system. Systems level investigations are relying more and more on computational analyses, especially in the field of proteomics generating large-scale global data.

  2. High-throughput crystallography for lead discovery in drug design.

    PubMed

    Blundell, Tom L; Jhoti, Harren; Abell, Chris

    2002-01-01

    Knowledge of the three-dimensional structures of protein targets now emerging from genomic data has the potential to accelerate drug discovery greatly. X-ray crystallography is the most widely used technique for protein structure determination, but technical challenges and time constraints have traditionally limited its use primarily to lead optimization. Here, we describe how significant advances in process automation and informatics have aided the development of high-throughput X-ray crystallography, and discuss the use of this technique for structure-based lead discovery.

  3. High-throughput DNA sequencing: a genomic data manufacturing process.

    PubMed

    Huang, G M

    1999-01-01

    The progress trends in automated DNA sequencing operation are reviewed. Technological development in sequencing instruments, enzymatic chemistry and robotic stations has resulted in ever-increasing capacity of sequence data production. This progress leads to a higher demand on laboratory information management and data quality assessment. High-throughput laboratories face the challenge of organizational management, as well as technology management. Engineering principles of process control should be adopted in this biological data manufacturing procedure. While various systems attempt to provide solutions to automate different parts of, or even the entire process, new technical advances will continue to change the paradigm and provide new challenges.

  4. Orchestrating high-throughput genomic analysis with Bioconductor

    PubMed Central

    Huber, Wolfgang; Carey, Vincent J.; Gentleman, Robert; Anders, Simon; Carlson, Marc; Carvalho, Benilton S.; Bravo, Hector Corrada; Davis, Sean; Gatto, Laurent; Girke, Thomas; Gottardo, Raphael; Hahne, Florian; Hansen, Kasper D.; Irizarry, Rafael A.; Lawrence, Michael; Love, Michael I.; MacDonald, James; Obenchain, Valerie; Oleś, Andrzej K.; Pagès, Hervé; Reyes, Alejandro; Shannon, Paul; Smyth, Gordon K.; Tenenbaum, Dan; Waldron, Levi; Morgan, Martin

    2015-01-01

    Bioconductor is an open-source, open-development software project for the analysis and comprehension of high-throughput data in genomics and molecular biology. The project aims to enable interdisciplinary research, collaboration and rapid development of scientific software. Based on the statistical programming language R, Bioconductor comprises 934 interoperable packages contributed by a large, diverse community of scientists. Packages cover a range of bioinformatic and statistical applications. They undergo formal initial review and continuous automated testing. We present an overview for prospective users and contributors. PMID:25633503

  5. Benchmarking Procedures for High-Throughput Context Specific Reconstruction Algorithms

    PubMed Central

    Pacheco, Maria P.; Pfau, Thomas; Sauter, Thomas

    2016-01-01

    Recent progress in high-throughput data acquisition has shifted the focus from data generation to processing and understanding of how to integrate collected information. Context specific reconstruction based on generic genome scale models like ReconX or HMR has the potential to become a diagnostic and treatment tool tailored to the analysis of specific individuals. The respective computational algorithms require a high level of predictive power, robustness and sensitivity. Although multiple context specific reconstruction algorithms were published in the last 10 years, only a fraction of them is suitable for model building based on human high-throughput data. Beside other reasons, this might be due to problems arising from the limitation to only one metabolic target function or arbitrary thresholding. This review describes and analyses common validation methods used for testing model building algorithms. Two major methods can be distinguished: consistency testing and comparison based testing. The first is concerned with robustness against noise, e.g., missing data due to the impossibility to distinguish between the signal and the background of non-specific binding of probes in a microarray experiment, and whether distinct sets of input expressed genes corresponding to i.e., different tissues yield distinct models. The latter covers methods comparing sets of functionalities, comparison with existing networks or additional databases. We test those methods on several available algorithms and deduce properties of these algorithms that can be compared with future developments. The set of tests performed, can therefore serve as a benchmarking procedure for future algorithms. PMID:26834640

  6. High-throughput GPU-based LDPC decoding

    NASA Astrophysics Data System (ADS)

    Chang, Yang-Lang; Chang, Cheng-Chun; Huang, Min-Yu; Huang, Bormin

    2010-08-01

    Low-density parity-check (LDPC) code is a linear block code known to approach the Shannon limit via the iterative sum-product algorithm. LDPC codes have been adopted in most current communication systems such as DVB-S2, WiMAX, WI-FI and 10GBASE-T. LDPC for the needs of reliable and flexible communication links for a wide variety of communication standards and configurations have inspired the demand for high-performance and flexibility computing. Accordingly, finding a fast and reconfigurable developing platform for designing the high-throughput LDPC decoder has become important especially for rapidly changing communication standards and configurations. In this paper, a new graphic-processing-unit (GPU) LDPC decoding platform with the asynchronous data transfer is proposed to realize this practical implementation. Experimental results showed that the proposed GPU-based decoder achieved 271x speedup compared to its CPU-based counterpart. It can serve as a high-throughput LDPC decoder.

  7. Benchmarking Procedures for High-Throughput Context Specific Reconstruction Algorithms.

    PubMed

    Pacheco, Maria P; Pfau, Thomas; Sauter, Thomas

    2015-01-01

    Recent progress in high-throughput data acquisition has shifted the focus from data generation to processing and understanding of how to integrate collected information. Context specific reconstruction based on generic genome scale models like ReconX or HMR has the potential to become a diagnostic and treatment tool tailored to the analysis of specific individuals. The respective computational algorithms require a high level of predictive power, robustness and sensitivity. Although multiple context specific reconstruction algorithms were published in the last 10 years, only a fraction of them is suitable for model building based on human high-throughput data. Beside other reasons, this might be due to problems arising from the limitation to only one metabolic target function or arbitrary thresholding. This review describes and analyses common validation methods used for testing model building algorithms. Two major methods can be distinguished: consistency testing and comparison based testing. The first is concerned with robustness against noise, e.g., missing data due to the impossibility to distinguish between the signal and the background of non-specific binding of probes in a microarray experiment, and whether distinct sets of input expressed genes corresponding to i.e., different tissues yield distinct models. The latter covers methods comparing sets of functionalities, comparison with existing networks or additional databases. We test those methods on several available algorithms and deduce properties of these algorithms that can be compared with future developments. The set of tests performed, can therefore serve as a benchmarking procedure for future algorithms.

  8. Probabilistic Assessment of High-Throughput Wireless Sensor Networks.

    PubMed

    Kim, Robin E; Mechitov, Kirill; Sim, Sung-Han; Spencer, Billie F; Song, Junho

    2016-01-01

    Structural health monitoring (SHM) using wireless smart sensors (WSS) has the potential to provide rich information on the state of a structure. However, because of their distributed nature, maintaining highly robust and reliable networks can be challenging. Assessing WSS network communication quality before and after finalizing a deployment is critical to achieve a successful WSS network for SHM purposes. Early studies on WSS network reliability mostly used temporal signal indicators, composed of a smaller number of packets, to assess the network reliability. However, because the WSS networks for SHM purpose often require high data throughput, i.e., a larger number of packets are delivered within the communication, such an approach is not sufficient. Instead, in this study, a model that can assess, probabilistically, the long-term performance of the network is proposed. The proposed model is based on readily-available measured data sets that represent communication quality during high-throughput data transfer. Then, an empirical limit-state function is determined, which is further used to estimate the probability of network communication failure. Monte Carlo simulation is adopted in this paper and applied to a small and a full-bridge wireless networks. By performing the proposed analysis in complex sensor networks, an optimized sensor topology can be achieved. PMID:27258270

  9. Probabilistic Assessment of High-Throughput Wireless Sensor Networks

    PubMed Central

    Kim, Robin E.; Mechitov, Kirill; Sim, Sung-Han; Spencer, Billie F.; Song, Junho

    2016-01-01

    Structural health monitoring (SHM) using wireless smart sensors (WSS) has the potential to provide rich information on the state of a structure. However, because of their distributed nature, maintaining highly robust and reliable networks can be challenging. Assessing WSS network communication quality before and after finalizing a deployment is critical to achieve a successful WSS network for SHM purposes. Early studies on WSS network reliability mostly used temporal signal indicators, composed of a smaller number of packets, to assess the network reliability. However, because the WSS networks for SHM purpose often require high data throughput, i.e., a larger number of packets are delivered within the communication, such an approach is not sufficient. Instead, in this study, a model that can assess, probabilistically, the long-term performance of the network is proposed. The proposed model is based on readily-available measured data sets that represent communication quality during high-throughput data transfer. Then, an empirical limit-state function is determined, which is further used to estimate the probability of network communication failure. Monte Carlo simulation is adopted in this paper and applied to a small and a full-bridge wireless networks. By performing the proposed analysis in complex sensor networks, an optimized sensor topology can be achieved. PMID:27258270

  10. Probabilistic Assessment of High-Throughput Wireless Sensor Networks.

    PubMed

    Kim, Robin E; Mechitov, Kirill; Sim, Sung-Han; Spencer, Billie F; Song, Junho

    2016-01-01

    Structural health monitoring (SHM) using wireless smart sensors (WSS) has the potential to provide rich information on the state of a structure. However, because of their distributed nature, maintaining highly robust and reliable networks can be challenging. Assessing WSS network communication quality before and after finalizing a deployment is critical to achieve a successful WSS network for SHM purposes. Early studies on WSS network reliability mostly used temporal signal indicators, composed of a smaller number of packets, to assess the network reliability. However, because the WSS networks for SHM purpose often require high data throughput, i.e., a larger number of packets are delivered within the communication, such an approach is not sufficient. Instead, in this study, a model that can assess, probabilistically, the long-term performance of the network is proposed. The proposed model is based on readily-available measured data sets that represent communication quality during high-throughput data transfer. Then, an empirical limit-state function is determined, which is further used to estimate the probability of network communication failure. Monte Carlo simulation is adopted in this paper and applied to a small and a full-bridge wireless networks. By performing the proposed analysis in complex sensor networks, an optimized sensor topology can be achieved.

  11. High-throughput screening to enhance oncolytic virus immunotherapy.

    PubMed

    Allan, K J; Stojdl, David F; Swift, S L

    2016-01-01

    High-throughput screens can rapidly scan and capture large amounts of information across multiple biological parameters. Although many screens have been designed to uncover potential new therapeutic targets capable of crippling viruses that cause disease, there have been relatively few directed at improving the efficacy of viruses that are used to treat disease. Oncolytic viruses (OVs) are biotherapeutic agents with an inherent specificity for treating malignant disease. Certain OV platforms - including those based on herpes simplex virus, reovirus, and vaccinia virus - have shown success against solid tumors in advanced clinical trials. Yet, many of these OVs have only undergone minimal engineering to solidify tumor specificity, with few extra modifications to manipulate additional factors. Several aspects of the interaction between an OV and a tumor-bearing host have clear value as targets to improve therapeutic outcomes. At the virus level, these include delivery to the tumor, infectivity, productivity, oncolysis, bystander killing, spread, and persistence. At the host level, these include engaging the immune system and manipulating the tumor microenvironment. Here, we review the chemical- and genome-based high-throughput screens that have been performed to manipulate such parameters during OV infection and analyze their impact on therapeutic efficacy. We further explore emerging themes that represent key areas of focus for future research. PMID:27579293

  12. Fluorescent foci quantitation for high-throughput analysis

    PubMed Central

    Ledesma-Fernández, Elena; Thorpe, Peter H.

    2015-01-01

    A number of cellular proteins localize to discrete foci within cells, for example DNA repair proteins, microtubule organizing centers, P bodies or kinetochores. It is often possible to measure the fluorescence emission from tagged proteins within these foci as a surrogate for the concentration of that specific protein. We wished to develop tools that would allow quantitation of fluorescence foci intensities in high-throughput studies. As proof of principle we have examined the kinetochore, a large multi-subunit complex that is critical for the accurate segregation of chromosomes during cell division. Kinetochore perturbations lead to aneuploidy, which is a hallmark of cancer cells. Hence, understanding kinetochore homeostasis and regulation are important for a global understanding of cell division and genome integrity. The 16 budding yeast kinetochores colocalize within the nucleus to form a single focus. Here we have created a set of freely-available tools to allow high-throughput quantitation of kinetochore foci fluorescence. We use this ‘FociQuant’ tool to compare methods of kinetochore quantitation and we show proof of principle that FociQuant can be used to identify changes in kinetochore protein levels in a mutant that affects kinetochore function. This analysis can be applied to any protein that forms discrete foci in cells. PMID:26290880

  13. High-Throughput Characterization of Vapor-Deposited Organic Glasses

    NASA Astrophysics Data System (ADS)

    Dalal, Shakeel S.

    Glasses are non-equilibrium materials which on short timescales behave like solids, and on long timescales betray their liquid-like structure. The most common way of preparing a glass is to cool the liquid faster than it can structurally rearrange. Until recently, most preparation schemes for a glass were considered to result in materials with undifferentiable structure and properties. This thesis utilizes a particular preparation method, physical vapor deposition, in order to prepare glasses of organic molecules with properties otherwise considered to be unobtainable. The glasses are characterized using spectroscopic ellipsometry, both as a dilatometric technique and as a reporter of molecular packing. The results reported here develop ellipsometry as a dilatometric technique on a pair of model glass formers, alpha,alpha,beta-trisnaphthylbenzene and indomethacin. It is found that the molecular orientation, as measured by birefringence, can be tuned by changing the substrate temperature during the deposition. In order to efficiently characterize the properties of vapor-deposited indomethacin as a function of substrate temperature, a high-throughput method is developed to capture the entire interesting range of substrate temperatures in just a few experiments. This high-throughput method is then leveraged to describe molecular mobility in vapor-deposited indomethacin. It is also used to demonstrate that the behavior of organic semiconducting molecules agrees with indomethacin quantitatively, and this agreement has implications for emerging technologies such as light-emitting diodes, photovoltaics and thin-film transistors made from organic molecules.

  14. Prospective, high-throughput molecular profiling of human gliomas

    PubMed Central

    Batchelor, Tracy T.; Dias-Santagata, Dora; Borger, Darrell; Stiles, Charles D.; Wang, Daphne L.; Curry, William T.; Wen, Patrick Y.; Ligon, Keith L.; Ellisen, Leif; Louis, David N.; Iafrate, A. John

    2013-01-01

    Gliomas consist of multiple histologic and molecular subtypes with different clinical phenotypes and responsiveness to treatment. However, enrollment criteria for clinical trials still largely do not take into account these underlying molecular differences. We have incorporated a high-throughput tumor genotyping program based on the ABI SNaPshot platform as well as other molecular diagnostic tests into the standard evaluation of glioma patients in order to assess whether prospective molecular profiling would allow rational patient selection onto clinical trials. From 218 gliomas we prospectively collected SNaPshot genotyping data on 68 mutated loci from 15 key cancer genes along with data from clinical assays for gene amplification (EGFR, PDGFRA, MET), 1p/19q co-deletion and MGMT promoter methylation. SNaPshot mutations and focal gene amplifications were detected in 38.5 and 47.1 % of glioblastomas, respectively. Genetic alterations in EGFR, IDH1 and PIK3CA closely matched frequencies reported in recent studies. In addition, we identified events that are rare in gliomas although are known driver mutations in other cancer types, such as mutations of AKT1, BRAF and KRAS. Patients with genetic alterations that activate signaling pathways were enrolled onto genetically selective clinical trials for malignant glioma as well as for other solid cancers. High-throughput molecular profiling incorporated into the routine clinical evaluation of glioma patients may enable the rational selection of patients for targeted therapy clinical trials and thereby improve the likelihood that such trials succeed. PMID:22821383

  15. High throughput instruments, methods, and informatics for systems biology.

    SciTech Connect

    Sinclair, Michael B.; Cowie, Jim R.; Van Benthem, Mark Hilary; Wylie, Brian Neil; Davidson, George S.; Haaland, David Michael; Timlin, Jerilyn Ann; Aragon, Anthony D.; Keenan, Michael Robert; Boyack, Kevin W.; Thomas, Edward Victor; Werner-Washburne, Margaret C.; Mosquera-Caro, Monica P.; Martinez, M. Juanita; Martin, Shawn Bryan; Willman, Cheryl L.

    2003-12-01

    High throughput instruments and analysis techniques are required in order to make good use of the genomic sequences that have recently become available for many species, including humans. These instruments and methods must work with tens of thousands of genes simultaneously, and must be able to identify the small subsets of those genes that are implicated in the observed phenotypes, or, for instance, in responses to therapies. Microarrays represent one such high throughput method, which continue to find increasingly broad application. This project has improved microarray technology in several important areas. First, we developed the hyperspectral scanner, which has discovered and diagnosed numerous flaws in techniques broadly employed by microarray researchers. Second, we used a series of statistically designed experiments to identify and correct errors in our microarray data to dramatically improve the accuracy, precision, and repeatability of the microarray gene expression data. Third, our research developed new informatics techniques to identify genes with significantly different expression levels. Finally, natural language processing techniques were applied to improve our ability to make use of online literature annotating the important genes. In combination, this research has improved the reliability and precision of laboratory methods and instruments, while also enabling substantially faster analysis and discovery.

  16. Advances, practice, and clinical perspectives in high-throughput sequencing.

    PubMed

    Park, S-J; Saito-Adachi, M; Komiyama, Y; Nakai, K

    2016-07-01

    Remarkable advances in high-throughput sequencing technologies have fundamentally changed our understanding of the genetic and epigenetic molecular bases underlying human health and diseases. As these technologies continue to revolutionize molecular biology leading to fresh perspectives, it is imperative to thoroughly consider the enormous excitement surrounding the technologies by highlighting the characteristics of platforms and their global trends as well as potential benefits and limitations. To date, with a variety of platforms, the technologies provide an impressive range of applications, including sequencing of whole genomes and transcriptomes, identifying of genome modifications, and profiling of protein interactions. Because these applications produce a flood of data, simultaneous development of bioinformatics tools is required to efficiently deal with the big data and to comprehensively analyze them. This review covers the major achievements and performances of the high-throughput sequencing and further summarizes the characteristics of their applications along with introducing applicable bioinformatics tools. Moreover, a step-by-step procedure for a practical transcriptome analysis is described employing an analytical pipeline. Clinical perspectives with special consideration to human oral health and diseases are also covered. PMID:26602181

  17. A quantitative high throughput assay for identifying gametocytocidal compounds.

    PubMed

    Tanaka, Takeshi Q; Dehdashti, Seameen J; Nguyen, Dac-Trung; McKew, John C; Zheng, Wei; Williamson, Kim C

    2013-03-01

    Current antimalarial drug treatment does not effectively kill mature Plasmodium falciparum gametocytes, the parasite stage responsible for malaria transmission from human to human via a mosquito. Consequently, following standard therapy malaria can still be transmitted for over a week after the clearance of asexual parasites. A new generation of malaria drugs with gametocytocidal properties, or a gametocytocidal drug that could be used in combinational therapy with currently available antimalarials, is needed to control the spread of the disease and facilitate eradication efforts. We have developed a 1536-well gametocyte viability assay for the high throughput screening of large compound collections to identify novel compounds with gametocytocidal activity. The signal-to-basal ratio and Z'-factor for this assay were 3.2-fold and 0.68, respectively. The IC(50) value of epoxomicin, the positive control compound, was 1.42±0.09 nM that is comparable to previously reported values. This miniaturized assay significantly reduces the number of gametocytes required for the AlamarBlue viability assay, and enables high throughput screening for lead discovery efforts. Additionally, the screen does not require a specialized parasite line, gametocytes from any strain, including field isolates, can be tested. A pilot screen utilizing the commercially available LOPAC library, consisting of 1280 known compounds, revealed two selective gametocytocidal compounds having 54- and 7.8-fold gametocytocidal selectivity in comparison to their cell cytotoxicity effect against the mammalian SH-SY5Y cell line.

  18. A Quantitative High Throughput Assay for Identifying Gametocytocidal Compounds

    PubMed Central

    Tanaka, Takeshi Q.; Dehdashti, Seameen J.; Nguyen, Dac-Trung; McKew, John C.; Zheng, Wei; Williamson, Kim C.

    2013-01-01

    Current antimalarial drug treatment does not effectively kill mature Plasmodium falciparum gametocytes, the parasite stage responsible for malaria transmission from human to human via a mosquito. Consequently, following standard therapy malaria can still be transmitted for over a week after the clearance of asexual parasites. A new generation of malaria drugs with gametocytocidal properties, or a gametocytocidal drug that could be used in combinational therapy with currently available antimalarials, is needed to control the spread of the disease and facilitate eradication efforts. We have developed a 1,536-well gametocyte viability assay for the high throughput screening of large compound collections to identify novel compounds with gametocytocidal activity. The signal-to-basal ratio and Z′-factor for this assay were 3.2-fold and 0.68, respectively. The IC50 value of epoxomicin, the positive control compound, was 1.42 ± 0.09 nM that is comparable to previously reported values. This miniaturized assay significantly reduces the number of gametocytes required for the alamarBlue viability assay, and enables high throughput screening for lead discovery efforts. Additionally, the screen does not require a specialized parasite line, gametocytes from any strain, including field isolates, can be tested. A pilot screen utilizing the commercially available LOPAC library, consisting of 1,280 known compounds, revealed two selective gametocytocidal compounds having 54 and 7.8-fold gametocytocidal selectivity in comparison to their cell cytotoxicity effect against the mammalian SH-SY5Y cell line. PMID:23454872

  19. High-throughput screening to enhance oncolytic virus immunotherapy

    PubMed Central

    Allan, KJ; Stojdl, David F; Swift, SL

    2016-01-01

    High-throughput screens can rapidly scan and capture large amounts of information across multiple biological parameters. Although many screens have been designed to uncover potential new therapeutic targets capable of crippling viruses that cause disease, there have been relatively few directed at improving the efficacy of viruses that are used to treat disease. Oncolytic viruses (OVs) are biotherapeutic agents with an inherent specificity for treating malignant disease. Certain OV platforms – including those based on herpes simplex virus, reovirus, and vaccinia virus – have shown success against solid tumors in advanced clinical trials. Yet, many of these OVs have only undergone minimal engineering to solidify tumor specificity, with few extra modifications to manipulate additional factors. Several aspects of the interaction between an OV and a tumor-bearing host have clear value as targets to improve therapeutic outcomes. At the virus level, these include delivery to the tumor, infectivity, productivity, oncolysis, bystander killing, spread, and persistence. At the host level, these include engaging the immune system and manipulating the tumor microenvironment. Here, we review the chemical- and genome-based high-throughput screens that have been performed to manipulate such parameters during OV infection and analyze their impact on therapeutic efficacy. We further explore emerging themes that represent key areas of focus for future research. PMID:27579293

  20. High-throughput characterization for solar fuels materials discovery

    NASA Astrophysics Data System (ADS)

    Mitrovic, Slobodan; Becerra, Natalie; Cornell, Earl; Guevarra, Dan; Haber, Joel; Jin, Jian; Jones, Ryan; Kan, Kevin; Marcin, Martin; Newhouse, Paul; Soedarmadji, Edwin; Suram, Santosh; Xiang, Chengxiang; Gregoire, John; High-Throughput Experimentation Team

    2014-03-01

    In this talk I will present the status of the High-Throughput Experimentation (HTE) project of the Joint Center for Artificial Photosynthesis (JCAP). JCAP is an Energy Innovation Hub of the U.S. Department of Energy with a mandate to deliver a solar fuel generator based on an integrated photoelectrochemical cell (PEC). However, efficient and commercially viable catalysts or light absorbers for the PEC do not exist. The mission of HTE is to provide the accelerated discovery through combinatorial synthesis and rapid screening of material properties. The HTE pipeline also features high-throughput material characterization using x-ray diffraction and x-ray photoemission spectroscopy (XPS). In this talk I present the currently operating pipeline and focus on our combinatorial XPS efforts to build the largest free database of spectra from mixed-metal oxides, nitrides, sulfides and alloys. This work was performed at Joint Center for Artificial Photosynthesis, a DOE Energy Innovation Hub, supported through the Office of Science of the U.S. Department of Energy under Award No. DE-SC0004993.

  1. High-throughput siRNA-based functional target validation.

    PubMed

    Xin, Hong; Bernal, Alejandro; Amato, Frank A; Pinhasov, Albert; Kauffman, Jack; Brenneman, Douglas E; Derian, Claudia K; Andrade-Gordon, Patricia; Plata-Salamán, Carlos R; Ilyin, Sergey E

    2004-06-01

    The drug discovery process pursued by major pharmaceutical companies for many years starts with target identification followed by high-throughput screening (HTS) with the goal of identifying lead compounds. To accomplish this goal, significant resources are invested into automation of the screening process or HTS. Robotic systems capable of handling thousands of data points per day are implemented across the pharmaceutical sector. Many of these systems are amenable to handling cell-based screening protocols as well. On the other hand, as companies strive to develop innovative products based on novel mechanisms of action(s), one of the current bottlenecks of the industry is the target validation process. Traditionally, bioinformatics and HTS groups operate separately at different stages of the drug discovery process. The authors describe the convergence and integration of HTS and bioinformatics to perform high-throughput target functional identification and validation. As an example of this approach, they initiated a project with a functional cell-based screen for a biological process of interest using libraries of small interfering RNA (siRNA) molecules. In this protocol, siRNAs function as potent gene-specific inhibitors. siRNA-mediated knockdown of the target genes is confirmed by TaqMan analysis, and genes with impacts on biological functions of interest are selected for further analysis. Once the genes are confirmed and further validated, they may be used for HTS to yield lead compounds.

  2. Polymer Microarrays for High Throughput Discovery of Biomaterials

    PubMed Central

    Hook, Andrew L.; Chang, Chien-Yi; Yang, Jing; Scurr, David J.; Langer, Robert; Anderson, Daniel G.; Atkinson, Steve; Williams, Paul; Davies, Martyn C.; Alexander, Morgan R.

    2012-01-01

    The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3. PMID:22314927

  3. High-throughput screening with micro-x-ray fluorescence

    SciTech Connect

    Havrilla, George J.; Miller, Thomasin C.

    2005-06-15

    Micro-x-ray fluorescence (MXRF) is a useful characterization tool for high-throughput screening of combinatorial libraries. Due to the increasing threat of use of chemical warfare (CW) agents both in military actions and against civilians by terrorist extremists, there is a strong push to improve existing methods and develop means for the detection of a broad spectrum of CW agents in a minimal amount of time to increase national security. This paper describes a combinatorial high-throughput screening technique for CW receptor discovery to aid in sensor development. MXRF can screen materials for elemental composition at the mesoscale level (tens to hundreds of micrometers). The key aspect of this work is the use of commercial MXRF instrumentation coupled with the inherent heteroatom elements within the target molecules of the combinatorial reaction to provide rapid and specific identification of lead species. The method is demonstrated by screening an 11-mer oligopeptide library for selective binding of the degradation products of the nerve agent VX. The identified oligopeptides can be used as selective molecular receptors for sensor development. The MXRF screening method is nondestructive, requires minimal sample preparation or special tags for analysis, and the screening time depends on the desired sensitivity.

  4. Management of High-Throughput DNA Sequencing Projects: Alpheus.

    PubMed

    Miller, Neil A; Kingsmore, Stephen F; Farmer, Andrew; Langley, Raymond J; Mudge, Joann; Crow, John A; Gonzalez, Alvaro J; Schilkey, Faye D; Kim, Ryan J; van Velkinburgh, Jennifer; May, Gregory D; Black, C Forrest; Myers, M Kathy; Utsey, John P; Frost, Nicholas S; Sugarbaker, David J; Bueno, Raphael; Gullans, Stephen R; Baxter, Susan M; Day, Steve W; Retzel, Ernest F

    2008-12-26

    High-throughput DNA sequencing has enabled systems biology to begin to address areas in health, agricultural and basic biological research. Concomitant with the opportunities is an absolute necessity to manage significant volumes of high-dimensional and inter-related data and analysis. Alpheus is an analysis pipeline, database and visualization software for use with massively parallel DNA sequencing technologies that feature multi-gigabase throughput characterized by relatively short reads, such as Illumina-Solexa (sequencing-by-synthesis), Roche-454 (pyrosequencing) and Applied Biosystem's SOLiD (sequencing-by-ligation). Alpheus enables alignment to reference sequence(s), detection of variants and enumeration of sequence abundance, including expression levels in transcriptome sequence. Alpheus is able to detect several types of variants, including non-synonymous and synonymous single nucleotide polymorphisms (SNPs), insertions/deletions (indels), premature stop codons, and splice isoforms. Variant detection is aided by the ability to filter variant calls based on consistency, expected allele frequency, sequence quality, coverage, and variant type in order to minimize false positives while maximizing the identification of true positives. Alpheus also enables comparisons of genes with variants between cases and controls or bulk segregant pools. Sequence-based differential expression comparisons can be developed, with data export to SAS JMP Genomics for statistical analysis. PMID:20151039

  5. Structuring intuition with theory: The high-throughput way

    NASA Astrophysics Data System (ADS)

    Fornari, Marco

    2015-03-01

    First principles methodologies have grown in accuracy and applicability to the point where large databases can be built, shared, and analyzed with the goal of predicting novel compositions, optimizing functional properties, and discovering unexpected relationships between the data. In order to be useful to a large community of users, data should be standardized, validated, and distributed. In addition, tools to easily manage large datasets should be made available to effectively lead to materials development. Within the AFLOW consortium we have developed a simple frame to expand, validate, and mine data repositories: the MTFrame. Our minimalistic approach complement AFLOW and other existing high-throughput infrastructures and aims to integrate data generation with data analysis. We present few examples from our work on materials for energy conversion. Our intent s to pinpoint the usefulness of high-throughput methodologies to guide the discovery process by quantitatively structuring the scientific intuition. This work was supported by ONR-MURI under Contract N00014-13-1-0635 and the Duke University Center for Materials Genomics.

  6. Evaluation of a High Throughput Starch Analysis Optimised for Wood

    PubMed Central

    Bellasio, Chandra; Fini, Alessio; Ferrini, Francesco

    2014-01-01

    Starch is the most important long-term reserve in trees, and the analysis of starch is therefore useful source of physiological information. Currently published protocols for wood starch analysis impose several limitations, such as long procedures and a neutralization step. The high-throughput standard protocols for starch analysis in food and feed represent a valuable alternative. However, they have not been optimised or tested with woody samples. These have particular chemical and structural characteristics, including the presence of interfering secondary metabolites, low reactivity of starch, and low starch content. In this study, a standard method for starch analysis used for food and feed (AOAC standard method 996.11) was optimised to improve precision and accuracy for the analysis of starch in wood. Key modifications were introduced in the digestion conditions and in the glucose assay. The optimised protocol was then evaluated through 430 starch analyses of standards at known starch content, matrix polysaccharides, and wood collected from three organs (roots, twigs, mature wood) of four species (coniferous and flowering plants). The optimised protocol proved to be remarkably precise and accurate (3%), suitable for a high throughput routine analysis (35 samples a day) of specimens with a starch content between 40 mg and 21 µg. Samples may include lignified organs of coniferous and flowering plants and non-lignified organs, such as leaves, fruits and rhizomes. PMID:24523863

  7. High-Throughput Screening Uncovers Novel Botulinum Neurotoxin Inhibitor Chemotypes.

    PubMed

    Bompiani, Kristin M; Caglič, Dejan; Krutein, Michelle C; Benoni, Galit; Hrones, Morgan; Lairson, Luke L; Bian, Haiyan; Smith, Garry R; Dickerson, Tobin J

    2016-08-01

    Botulism is caused by potent and specific bacterial neurotoxins that infect host neurons and block neurotransmitter release. Treatment for botulism is limited to administration of an antitoxin within a short time window, before the toxin enters neurons. Alternatively, current botulism drug development targets the toxin light chain, which is a zinc-dependent metalloprotease that is delivered into neurons and mediates long-term pathology. Several groups have identified inhibitory small molecules, peptides, or aptamers, although no molecule has advanced to the clinic due to a lack of efficacy in advanced models. Here we used a homogeneous high-throughput enzyme assay to screen three libraries of drug-like small molecules for new chemotypes that modulate recombinant botulinum neurotoxin light chain activity. High-throughput screening of 97088 compounds identified numerous small molecules that activate or inhibit metalloprotease activity. We describe four major classes of inhibitory compounds identified, detail their structure-activity relationships, and assess their relative inhibitory potency. A previously unreported chemotype in any context of enzyme inhibition is described with potent submicromolar inhibition (Ki = 200-300 nM). Additional detailed kinetic analyses and cellular cytotoxicity assays indicate the best compound from this series is a competitive inhibitor with cytotoxicity values around 4-5 μM. Given the potency and drug-like character of these lead compounds, further studies, including cellular activity assays and DMPK analysis, are justified. PMID:27314875

  8. Printing Proteins as Microarrays for High-Throughput Function Determination

    NASA Astrophysics Data System (ADS)

    MacBeath, Gavin; Schreiber, Stuart L.

    2000-09-01

    Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

  9. Adaptation to high throughput batch chromatography enhances multivariate screening.

    PubMed

    Barker, Gregory A; Calzada, Joseph; Herzer, Sibylle; Rieble, Siegfried

    2015-09-01

    High throughput process development offers unique approaches to explore complex process design spaces with relatively low material consumption. Batch chromatography is one technique that can be used to screen chromatographic conditions in a 96-well plate. Typical batch chromatography workflows examine variations in buffer conditions or comparison of multiple resins in a given process, as opposed to the assessment of protein loading conditions in combination with other factors. A modification to the batch chromatography paradigm is described here where experimental planning, programming, and a staggered loading approach increase the multivariate space that can be explored with a liquid handling system. The iterative batch chromatography (IBC) approach is described, which treats every well in a 96-well plate as an individual experiment, wherein protein loading conditions can be varied alongside other factors such as wash and elution buffer conditions. As all of these factors are explored in the same experiment, the interactions between them are characterized and the number of follow-up confirmatory experiments is reduced. This in turn improves statistical power and throughput. Two examples of the IBC method are shown and the impact of the load conditions are assessed in combination with the other factors explored.

  10. A High-Throughput Screen for Antibiotic Drug Discovery

    PubMed Central

    Scanlon, Thomas C.; Dostal, Sarah M.; Griswold, Karl E.

    2014-01-01

    We describe an ultra-high-throughput screening platform enabling discovery and/or engineering of natural product antibiotics. The methodology involves creation of hydrogel-in-oil emulsions in which recombinant microorganisms are co-emulsified with bacterial pathogens; antibiotic activity is assayed by use of a fluorescent viability dye. We have successfully utilized both bulk emulsification and microfluidic technology for the generation of hydrogel microdroplets that are size-compatible with conventional flow cytometry. Hydrogel droplets are ~25 pL in volume, and can be synthesized and sorted at rates exceeding 3,000 drops/s. Using this technique, we have achieved screening throughputs exceeding 5 million clones/day. Proof-of-concept experiments demonstrate efficient selection of antibiotic-secreting yeast from a vast excess of negative controls. In addition, we have successfully used this technique to screen a metagenomic library for secreted antibiotics that kill the human pathogen Staphylococcus aureus. Our results establish the practical utility of the screening platform, and we anticipate that the accessible nature of our methods will enable others seeking to identify and engineer the next generation of antibacterial biomolecules. PMID:23955804

  11. Measuring intracellular calcium fluxes in high throughput mode.

    PubMed

    Chambers, Chris; Smith, Fiona; Williams, Christine; Marcos, Sandra; Liu, Zhen Han; Hayter, Paul; Ciaramella, Giuseppe; Keighley, Wilma; Gribbon, Phil; Sewing, Andreas

    2003-06-01

    The measurement of intracellular calcium fluxes in real time is widely applied within the pharmaceutical industry to measure the activation of G-protein coupled receptors (GPCRhyp;s), either for pharmacological characterisation or to screen for new surrogate ligands. Initially restricted to G(q) coupled GPCRs, the introduction of promiscuous and chimeric G-proteins has further widened the application of these assays. The development of new calcium sensitive dyes and assays has provided sensitive, homogeneous assays which can be readily applied to high throughput screening (HTS). In this paper we describe the full automation of this assay type using a fluorometric imaging plate reader (FLIPR ) integrated into a Beckman/Sagian system to establish a simple robotic system that is well suited for the current medium throughput screening in this area of lead discovery. Using a recently completed HTS we discuss important determinants for FLIPR based screening, highlight some limitations of the current approach, and look at the requirements for future automated systems capable of keeping up with expanding compound files.

  12. A High-Throughput Yeast Halo Assay for Bioactive Compounds.

    PubMed

    Bray, Walter; Lokey, R Scott

    2016-01-01

    When a disk of filter paper is impregnated with a cytotoxic or cytostatic drug and added to solid medium seeded with yeast, a visible clear zone forms around the disk whose size depends on the concentration and potency of the drug. This is the traditional "halo" assay and provides a convenient, if low-throughput, read-out of biological activity that has been the mainstay of antifungal and antibiotic testing for decades. Here, we describe a protocol for a high-throughput version of the halo assay, which uses an array of 384 pins to deliver ∼200 nL of stock solutions from compound plates onto single-well plates seeded with yeast. Using a plate reader in the absorbance mode, the resulting halos can be quantified and the data archived in the form of flat files that can be connected to compound databases with standard software. This assay has the convenience associated with the visual readout of the traditional halo assay but uses far less material and can be automated to screen thousands of compounds per day. PMID:27587777

  13. A robust robotic high-throughput antibody purification platform.

    PubMed

    Schmidt, Peter M; Abdo, Michael; Butcher, Rebecca E; Yap, Min-Yin; Scotney, Pierre D; Ramunno, Melanie L; Martin-Roussety, Genevieve; Owczarek, Catherine; Hardy, Matthew P; Chen, Chao-Guang; Fabri, Louis J

    2016-07-15

    Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant.

  14. High-throughput microcavitation bubble induced cellular mechanotransduction

    NASA Astrophysics Data System (ADS)

    Compton, Jonathan Lee

    inhibitor to IP 3 induced Ca2+ release. This capability opens the development of a high-throughput screening platform for molecules that modulate cellular mechanotransduction. We have applied this approach to screen the effects of a small set of small molecules, in a 96-well plate in less than an hour. These detailed studies offer a basis for the design, development, and implementation of a novel high-throughput mechanotransduction assay to rapidly screen the effect of small molecules on cellular mechanotransduction at high throughput.

  15. Machine Learning for High-Throughput Stress Phenotyping in Plants.

    PubMed

    Singh, Arti; Ganapathysubramanian, Baskar; Singh, Asheesh Kumar; Sarkar, Soumik

    2016-02-01

    Advances in automated and high-throughput imaging technologies have resulted in a deluge of high-resolution images and sensor data of plants. However, extracting patterns and features from this large corpus of data requires the use of machine learning (ML) tools to enable data assimilation and feature identification for stress phenotyping. Four stages of the decision cycle in plant stress phenotyping and plant breeding activities where different ML approaches can be deployed are (i) identification, (ii) classification, (iii) quantification, and (iv) prediction (ICQP). We provide here a comprehensive overview and user-friendly taxonomy of ML tools to enable the plant community to correctly and easily apply the appropriate ML tools and best-practice guidelines for various biotic and abiotic stress traits.

  16. Learning robust cell signalling models from high throughput proteomic data

    PubMed Central

    Koch, Mitchell; Broom, Bradley M.; Subramanian, Devika

    2015-01-01

    We propose a framework for learning robust Bayesian network models of cell signalling from high-throughput proteomic data. We show that model averaging using Bayesian bootstrap resampling generates more robust structures than procedures that learn structures using all of the data. We also develop an algorithm for ranking the importance of network features using bootstrap resample data. We apply our algorithms to derive the T-cell signalling network from the flow cytometry data of Sachs et al. (2005). Our learning algorithm has identified, with high confidence, several new crosstalk mechanisms in the T-cell signalling network. Many of them have already been confirmed experimentally in the recent literature and six new crosstalk mechanisms await experimental validation. PMID:19525198

  17. Numerical techniques for high-throughput reflectance interference biosensing

    NASA Astrophysics Data System (ADS)

    Sevenler, Derin; Ünlü, M. Selim

    2016-06-01

    We have developed a robust and rapid computational method for processing the raw spectral data collected from thin film optical interference biosensors. We have applied this method to Interference Reflectance Imaging Sensor (IRIS) measurements and observed a 10,000 fold improvement in processing time, unlocking a variety of clinical and scientific applications. Interference biosensors have advantages over similar technologies in certain applications, for example highly multiplexed measurements of molecular kinetics. However, processing raw IRIS data into useful measurements has been prohibitively time consuming for high-throughput studies. Here we describe the implementation of a lookup table (LUT) technique that provides accurate results in far less time than naive methods. We also discuss an additional benefit that the LUT method can be used with a wider range of interference layer thickness and experimental configurations that are incompatible with methods that require fitting the spectral response.

  18. Quantitative High-Throughput Luciferase Screening in Identifying CAR Modulators.

    PubMed

    Lynch, Caitlin; Zhao, Jinghua; Wang, Hongbing; Xia, Menghang

    2016-01-01

    The constitutive androstane receptor (CAR, NR1I3) is responsible for the transcription of multiple drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both of these mechanisms require translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active in immortalized cell lines due to the basal nuclear location of this receptor. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the high basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify hCAR1 modulators through the employment of a double stable cell line. Using this line, we are able to identify activators, as well as deactivators, of the challenging nuclear receptor, CAR. PMID:27518621

  19. Automated, high-throughput IgG-antibody glycoprofiling platform.

    PubMed

    Stöckmann, Henning; Adamczyk, Barbara; Hayes, Jerrard; Rudd, Pauline M

    2013-09-17

    One of today's key challenges is the ability to decode the functions of complex carbohydrates in various biological contexts. To generate high-quality glycomics data in a high-throughput fashion, we developed a robotized and low-cost N-glycan analysis platform for glycoprofiling of immunoglobulin G antibodies (IgG), which are central players of the immune system and of vital importance in the biopharmaceutical industry. The key features include (a) rapid IgG affinity purification and sample concentration, (b) protein denaturation and glycan release on a multiwell filtration device, (c) glycan purification on solid-supported hydrazide, and (d) glycan quantification by ultra performance liquid chromatography. The sample preparation workflow was automated using a robotic liquid-handling workstation, allowing the preparation of 96 samples (or multiples thereof) in 22 h with excellent reproducibility and, thus, should greatly facilitate biomarker discovery and glycosylation monitoring of therapeutic IgGs.

  20. High-throughput human metabolism and toxicity analysis.

    PubMed

    Lee, Moo-Yeal; Dordick, Jonathan S

    2006-12-01

    Poor drug candidate safety profiles are often identified late in the drug development process, manifesting themselves in the preclinical and clinical phases and significantly contributing to the high cost and low yield of drug discovery. As a result, new tools are needed to accelerate the assessment of drug candidate toxicity and human metabolism earlier in the drug development process, from primary drug candidate screening to lead optimization. Although high-throughput screens exist for much of the discovery phase of drug development, translating such screening techniques into platforms that can accurately mimic the human in vivo response and predict the impact of drug candidates on human toxicology has proven difficult. Nevertheless, some success has been achieved in recent years, which may ultimately yield widespread acceptance in the pharmaceutical industry.

  1. Multifunctional encoded particles for high-throughput biomolecule analysis.

    PubMed

    Pregibon, Daniel C; Toner, Mehmet; Doyle, Patrick S

    2007-03-01

    High-throughput screening for genetic analysis, combinatorial chemistry, and clinical diagnostics benefits from multiplexing, which allows for the simultaneous assay of several analytes but necessitates an encoding scheme for molecular identification. Current approaches for multiplexed analysis involve complicated or expensive processes for encoding, functionalizing, or decoding active substrates (particles or surfaces) and often yield a very limited number of analyte-specific codes. We present a method based on continuous-flow lithography that combines particle synthesis and encoding and probe incorporation into a single process to generate multifunctional particles bearing over a million unique codes. By using such particles, we demonstrate a multiplexed, single-fluorescence detection of DNA oligomers with encoded particle libraries that can be scanned rapidly in a flow-through microfluidic channel. Furthermore, we demonstrate with high specificity the same multiplexed detection using individual multiprobe particles.

  2. Predicting Novel Bulk Metallic Glasses via High- Throughput Calculations

    NASA Astrophysics Data System (ADS)

    Perim, E.; Lee, D.; Liu, Y.; Toher, C.; Gong, P.; Li, Y.; Simmons, W. N.; Levy, O.; Vlassak, J.; Schroers, J.; Curtarolo, S.

    Bulk metallic glasses (BMGs) are materials which may combine key properties from crystalline metals, such as high hardness, with others typically presented by plastics, such as easy processability. However, the cost of the known BMGs poses a significant obstacle for the development of applications, which has lead to a long search for novel, economically viable, BMGs. The emergence of high-throughput DFT calculations, such as the library provided by the AFLOWLIB consortium, has provided new tools for materials discovery. We have used this data to develop a new glass forming descriptor combining structural factors with thermodynamics in order to quickly screen through a large number of alloy systems in the AFLOWLIB database, identifying the most promising systems and the optimal compositions for glass formation. National Science Foundation (DMR-1436151, DMR-1435820, DMR-1436268).

  3. High-throughput flow cytometry for drug discovery.

    PubMed

    Edwards, Bruce S; Young, Susan M; Saunders, Matthew J; Bologa, Cristian; Oprea, Tudor I; Ye, Richard D; Prossnitz, Eric R; Graves, Steven W; Sklar, Larry A

    2007-05-01

    High-throughput flow cytometry exploits a novel many-samples/one-file approach to dramatically speed data acquisition, limit aspirated sample volume to as little as 2 μl/well and produce multisample data sets that facilitate automated analysis of samples in groups as well as individually. It has been successfully applied to both cell- and microsphere-based bioassays in 96- and 384-well formats, to screen tens-of-thousands of compounds and identify novel bioactive structures. High-content multiparametric analysis capabilities have been exploited for assay multiplexing, allowing the assessment of biologic selectivity and specificity to be an integral component of primary screens. These and other advances in the last decade have contributed to the application of flow cytometry as a uniquely powerful tool for probing biologic and chemical diversity and complex systems biology.

  4. Statistically invalid classification of high throughput gene expression data.

    PubMed

    Barbash, Shahar; Soreq, Hermona

    2013-01-01

    Classification analysis based on high throughput data is a common feature in neuroscience and other fields of science, with a rapidly increasing impact on both basic biology and disease-related studies. The outcome of such classifications often serves to delineate novel biochemical mechanisms in health and disease states, identify new targets for therapeutic interference, and develop innovative diagnostic approaches. Given the importance of this type of studies, we screened 111 recently-published high-impact manuscripts involving classification analysis of gene expression, and found that 58 of them (53%) based their conclusions on a statistically invalid method which can lead to bias in a statistical sense (lower true classification accuracy then the reported classification accuracy). In this report we characterize the potential methodological error and its scope, investigate how it is influenced by different experimental parameters, and describe statistically valid methods for avoiding such classification mistakes.

  5. A primer on high-throughput computing for genomic selection.

    PubMed

    Wu, Xiao-Lin; Beissinger, Timothy M; Bauck, Stewart; Woodward, Brent; Rosa, Guilherme J M; Weigel, Kent A; Gatti, Natalia de Leon; Gianola, Daniel

    2011-01-01

    High-throughput computing (HTC) uses computer clusters to solve advanced computational problems, with the goal of accomplishing high-throughput over relatively long periods of time. In genomic selection, for example, a set of markers covering the entire genome is used to train a model based on known data, and the resulting model is used to predict the genetic merit of selection candidates. Sophisticated models are very computationally demanding and, with several traits to be evaluated sequentially, computing time is long, and output is low. In this paper, we present scenarios and basic principles of how HTC can be used in genomic selection, implemented using various techniques from simple batch processing to pipelining in distributed computer clusters. Various scripting languages, such as shell scripting, Perl, and R, are also very useful to devise pipelines. By pipelining, we can reduce total computing time and consequently increase throughput. In comparison to the traditional data processing pipeline residing on the central processors, performing general-purpose computation on a graphics processing unit provide a new-generation approach to massive parallel computing in genomic selection. While the concept of HTC may still be new to many researchers in animal breeding, plant breeding, and genetics, HTC infrastructures have already been built in many institutions, such as the University of Wisconsin-Madison, which can be leveraged for genomic selection, in terms of central processing unit capacity, network connectivity, storage availability, and middleware connectivity. Exploring existing HTC infrastructures as well as general-purpose computing environments will further expand our capability to meet increasing computing demands posed by unprecedented genomic data that we have today. We anticipate that HTC will impact genomic selection via better statistical models, faster solutions, and more competitive products (e.g., from design of marker panels to realized

  6. A Primer on High-Throughput Computing for Genomic Selection

    PubMed Central

    Wu, Xiao-Lin; Beissinger, Timothy M.; Bauck, Stewart; Woodward, Brent; Rosa, Guilherme J. M.; Weigel, Kent A.; Gatti, Natalia de Leon; Gianola, Daniel

    2011-01-01

    High-throughput computing (HTC) uses computer clusters to solve advanced computational problems, with the goal of accomplishing high-throughput over relatively long periods of time. In genomic selection, for example, a set of markers covering the entire genome is used to train a model based on known data, and the resulting model is used to predict the genetic merit of selection candidates. Sophisticated models are very computationally demanding and, with several traits to be evaluated sequentially, computing time is long, and output is low. In this paper, we present scenarios and basic principles of how HTC can be used in genomic selection, implemented using various techniques from simple batch processing to pipelining in distributed computer clusters. Various scripting languages, such as shell scripting, Perl, and R, are also very useful to devise pipelines. By pipelining, we can reduce total computing time and consequently increase throughput. In comparison to the traditional data processing pipeline residing on the central processors, performing general-purpose computation on a graphics processing unit provide a new-generation approach to massive parallel computing in genomic selection. While the concept of HTC may still be new to many researchers in animal breeding, plant breeding, and genetics, HTC infrastructures have already been built in many institutions, such as the University of Wisconsin–Madison, which can be leveraged for genomic selection, in terms of central processing unit capacity, network connectivity, storage availability, and middleware connectivity. Exploring existing HTC infrastructures as well as general-purpose computing environments will further expand our capability to meet increasing computing demands posed by unprecedented genomic data that we have today. We anticipate that HTC will impact genomic selection via better statistical models, faster solutions, and more competitive products (e.g., from design of marker panels to realized

  7. A high-throughput 3-D composite dielectrophoretic separator.

    PubMed

    Fatoyinbo, Henry O; Kamchis, David; Whattingham, Reginald; Ogin, Stephen L; Hughes, Michael P

    2005-07-01

    Dielectrophoresis has great potential to offer a range of diverse fields from bioprocessing to clinical medicine, but is hampered by low throughput rates to the micrometer scale of the electrodes required to generate highly nonuniform fields. Here we describe a novel approach to electrode construction, using a drilled laminated structure to form channels bearing electrodes 30 microm across and 150 microm apart. Since these electrodes appear along all sides of the drilled bore, the trapping efficiency is improved over conventional devices. We have developed and demonstrated a separator capable of sorting a 50:50 mixture of viable and nonviable yeast cells into an 86:14 mixture at 25 mLhr(-1).

  8. High Throughput Screening Method to Explore Protein Interactions with Nanoparticles

    PubMed Central

    Nasir, Irem; Fatih, Warda; Svensson, Anja; Radu, Dennis; Linse, Sara; Cabaleiro Lago, Celia; Lundqvist, Martin

    2015-01-01

    The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions. PMID:26313757

  9. A high-throughput screening for phosphatases using specific substrates.

    PubMed

    Senn, Alejandro M; Wolosiuk, Ricardo A

    2005-04-01

    A high-throughput screening was developed for the detection of phosphatase activity in bacterial colonies. Unlike other methods, the current procedure can be applied to any phosphatase because it uses physiological substrates and detects the compelled product of all phosphatase reactions, that is, orthophosphate. In this method, substrates diffuse from a filter paper across a nitrocellulose membrane to bacterial colonies situated on the opposite face, and then reaction products flow back to the paper. Finally, a colorimetric reagent discloses the presence of orthophosphate in the filter paper. We validated the performance of this assay with several substrates and experimental conditions and with different phosphatases, including a library of randomly mutagenized rapeseed chloroplast fructose-1,6-bisphosphatase. This procedure could be extended to other enzymatic activities provided that an appropriate detection of reaction products is available.

  10. Proteomics equipped with multiplexing toward ultra high throughput.

    PubMed

    Kim, Min-Sik

    2015-01-01

    MS-based quantitative proteomics is a powerful technology to study virtually almost all biological and clinical samples. Although it has been known to be a high-throughput method, an MS analysis of a higher number of samples remains to be challenging practically and economically. In this issue, the use of multiplexing strategy for quantitative analysis of proteomes and phosphoproteomes has been demonstrated by Paulo et al. (Proteomics 2015, 15, 462-473) to better understand in vivo effects of two small molecule inhibitors on a mouse model. Within the short period of drug treatment, it has been found that the protein alteration is minimal in three tissues tested, whereas the phosphorylation level was widely altered. PMID:25522341

  11. High-throughput rheology in a microfluidic device

    NASA Astrophysics Data System (ADS)

    Furst, Eric; Schultz, Kelly; Han, Hyejin; Kim, Chongyoup

    2011-11-01

    High-throughput rheological measurements in a microfluidic device are demonstrated. A series of microrheology samples is generated as droplets in an immiscible spacer fluid using a microfluidic T-junction. The compositions of the sample droplets are continuously varied over a wide range. Rheology measurements are made in each droplet using multiple particle tracking microrheology. We review critical design and operating parameters, including the droplet size, flow rates and rapid fabrication methods. Validation experiments are performed by measuring the solution viscosity of glycerine and the biopolymer heparin as a function of concentration. Finally, an analysis of droplet mixing is performed in order to optimize the device performance. Overall, the combination of microrheology with microfluidics maximizes the number of rheological measurements while simultaneously minimizing the sample preparation time and amount of material, and should be particularly suited to the characterization of scarce or expensive materials. We acknowledge financial support from the NSF (CBET-0730292).

  12. Interactive Visual Analysis of High Throughput Text Streams

    SciTech Connect

    Steed, Chad A; Potok, Thomas E; Patton, Robert M; Goodall, John R; Maness, Christopher S; Senter, James K; Potok, Thomas E

    2012-01-01

    The scale, velocity, and dynamic nature of large scale social media systems like Twitter demand a new set of visual analytics techniques that support near real-time situational awareness. Social media systems are credited with escalating social protest during recent large scale riots. Virtual communities form rapidly in these online systems, and they occasionally foster violence and unrest which is conveyed in the users language. Techniques for analyzing broad trends over these networks or reconstructing conversations within small groups have been demonstrated in recent years, but state-of- the-art tools are inadequate at supporting near real-time analysis of these high throughput streams of unstructured information. In this paper, we present an adaptive system to discover and interactively explore these virtual networks, as well as detect sentiment, highlight change, and discover spatio- temporal patterns.

  13. UAV-based high-throughput phenotyping in legume crops

    NASA Astrophysics Data System (ADS)

    Sankaran, Sindhuja; Khot, Lav R.; Quirós, Juan; Vandemark, George J.; McGee, Rebecca J.

    2016-05-01

    In plant breeding, one of the biggest obstacles in genetic improvement is the lack of proven rapid methods for measuring plant responses in field conditions. Therefore, the major objective of this research was to evaluate the feasibility of utilizing high-throughput remote sensing technology for rapid measurement of phenotyping traits in legume crops. The plant responses of several chickpea and peas varieties to the environment were assessed with an unmanned aerial vehicle (UAV) integrated with multispectral imaging sensors. Our preliminary assessment showed that the vegetation indices are strongly correlated (p<0.05) with seed yield of legume crops. Results endorse the potential of UAS-based sensing technology to rapidly measure those phenotyping traits.

  14. High Throughput Screening Method to Explore Protein Interactions with Nanoparticles.

    PubMed

    Nasir, Irem; Fatih, Warda; Svensson, Anja; Radu, Dennis; Linse, Sara; Cabaleiro Lago, Celia; Lundqvist, Martin

    2015-01-01

    The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions. PMID:26313757

  15. High-throughput process development for biopharmaceutical drug substances.

    PubMed

    Bhambure, Rahul; Kumar, Kaushal; Rathore, Anurag S

    2011-03-01

    Quality by Design (QbD) is gaining industry acceptance as an approach towards development and commercialization of biotechnology therapeutic products that are expressed via microbial or mammalian cell lines. In QbD, the process is designed and controlled to deliver specified quality attributes consistently. To acquire the enhanced understanding that is necessary to achieve the above, however, requires more extensive experimentation to establish the design space for the process and the product. With biotechnology companies operating under ever-increasing pressure towards lowering the cost of manufacturing, the use of high-throughput tools has emerged as a necessary enabler of QbD in a time- and resource-constrained environment. We review this topic for those in academia and industry that are engaged in drug substance process development.

  16. Estimating Protistan Diversity Using High-Throughput Sequencing.

    PubMed

    Hu, Sarah K; Liu, Zhenfeng; Lie, Alle A Y; Countway, Peter D; Kim, Diane Y; Jones, Adriane C; Gast, Rebecca J; Cary, S Craig; Sherr, Evelyn B; Sherr, Barry F; Caron, David A

    2015-01-01

    Sequencing hypervariable regions from the 18S rRNA gene is commonly employed to characterize protistan biodiversity, yet there are concerns that short reads do not provide the same taxonomic resolution as full-length sequences. A total of 7,432 full-length sequences were used to perform an in silico analysis of how sequences of various lengths and target regions impact downstream ecological interpretations. Sequences that were longer than 400 nucleotides and included the V4 hypervariable region generated results similar to those derived from full-length 18S rRNA gene sequences. Present high-throughput sequencing capabilities are approaching protistan diversity estimation comparable to whole gene sequences.

  17. High-throughput determination of RNA structure by proximity ligation.

    PubMed

    Ramani, Vijay; Qiu, Ruolan; Shendure, Jay

    2015-09-01

    We present an unbiased method to globally resolve RNA structures through pairwise contact measurements between interacting regions. RNA proximity ligation (RPL) uses proximity ligation of native RNA followed by deep sequencing to yield chimeric reads with ligation junctions in the vicinity of structurally proximate bases. We apply RPL in both baker's yeast (Saccharomyces cerevisiae) and human cells and generate contact probability maps for ribosomal and other abundant RNAs, including yeast snoRNAs, the RNA subunit of the signal recognition particle and the yeast U2 spliceosomal RNA homolog. RPL measurements correlate with established secondary structures for these RNA molecules, including stem-loop structures and long-range pseudoknots. We anticipate that RPL will complement the current repertoire of computational and experimental approaches in enabling the high-throughput determination of secondary and tertiary RNA structures. PMID:26237516

  18. High-Throughput Sequencing of Complete Mitochondrial Genomes.

    PubMed

    Briscoe, Andrew George; Hopkins, Kevin Peter; Waeschenbach, Andrea

    2016-01-01

    Next-generation sequencing has revolutionized mitogenomics, turning a cottage industry into a high throughput process. This chapter outlines methodologies used to sequence, assemble, and annotate mitogenomes of non-model organisms using Illumina sequencing technology, utilizing either long-range PCR amplicons or gDNA as starting template. Instructions are given on how to extract DNA, conduct long-range PCR amplifications, generate short Sanger barcode tag sequences, prepare equimolar sample pools, construct and assess quality library preparations, assemble Illumina reads using either seeded reference mapping or de novo assembly, and annotate mitogenomes in the absence of an automated pipeline. Notes and recommendations, derived from our own experience, are given throughout this chapter. PMID:27460369

  19. A Colloidal Stability Assay Suitable for High-Throughput Screening.

    PubMed

    Abarca, Carla; Ali, M Monsur; Yang, Songtao; Dong, Xiaofei; Pelton, Robert H

    2016-03-01

    A library of 32 polystyrene copolymer latexes, with diameters ranging between 53 and 387 nm, was used to develop and demonstrate a high-throughput assay using a 96-well microplate platform to measure critical coagulation concentrations, a measure of colloidal stability. The most robust assay involved an automated centrifugation-decantation step to remove latex aggregates before absorbance measurements, eliminating aggregate interference with optical measurements made through the base of the multiwell plates. For smaller nanoparticles (diameter <150 nm), the centrifugation-decantation step was not required as the interference was less than with larger particles. Parallel measurements with a ChemiDoc MP plate scanner gave indications of aggregation; however, the results were less sensitive than the absorbance measurements. PMID:26857643

  20. High-throughput ab-initio dilute solute diffusion database

    PubMed Central

    Wu, Henry; Mayeshiba, Tam; Morgan, Dane

    2016-01-01

    We demonstrate automated generation of diffusion databases from high-throughput density functional theory (DFT) calculations. A total of more than 230 dilute solute diffusion systems in Mg, Al, Cu, Ni, Pd, and Pt host lattices have been determined using multi-frequency diffusion models. We apply a correction method for solute diffusion in alloys using experimental and simulated values of host self-diffusivity. We find good agreement with experimental solute diffusion data, obtaining a weighted activation barrier RMS error of 0.176 eV when excluding magnetic solutes in non-magnetic alloys. The compiled database is the largest collection of consistently calculated ab-initio solute diffusion data in the world. PMID:27434308

  1. High-throughput virtual screening for drug discovery in parallel.

    PubMed

    Toledo-Sherman, Leticia M; Chen, Deqi

    2002-05-01

    With the influx of targets generated by genomics and proteomics initiatives, a new drug discovery paradigm is emerging. Many companies are setting up target family platforms that tackle multiple targets and therapeutic areas simultaneously. Virtual screening (VS) techniques are a fundamental component of such platforms for in silico filtering of compound collections and prioritization of chemistry and screening efforts. At the heart of these, structure-based docking and scoring methods are especially effective in identifying bioactive molecules if the structure of a target is available. As structural genomics maps the structural space of the proteome, these techniques are expected to become commonplace. In light of this, an overview of the latest developments in VS methodology is given here. In particular, emphasis is placed on those techniques adaptable to high-throughput VS in parallel drug discovery platforms. The first examples of docking across multiple targets have already appeared in the literature and will be reviewed here.

  2. Microfluidic cell chips for high-throughput drug screening.

    PubMed

    Chi, Chun-Wei; Ahmed, Ah Rezwanuddin; Dereli-Korkut, Zeynep; Wang, Sihong

    2016-05-01

    The current state of screening methods for drug discovery is still riddled with several inefficiencies. Although some widely used high-throughput screening platforms may enhance the drug screening process, their cost and oversimplification of cell-drug interactions pose a translational difficulty. Microfluidic cell-chips resolve many issues found in conventional HTS technology, providing benefits such as reduced sample quantity and integration of 3D cell culture physically more representative of the physiological/pathological microenvironment. In this review, we introduce the advantages of microfluidic devices in drug screening, and outline the critical factors which influence device design, highlighting recent innovations and advances in the field including a summary of commercialization efforts on microfluidic cell chips. Future perspectives of microfluidic cell devices are also provided based on considerations of present technological limitations and translational barriers. PMID:27071838

  3. High-throughput DNA extraction of forensic adhesive tapes.

    PubMed

    Forsberg, Christina; Jansson, Linda; Ansell, Ricky; Hedman, Johannes

    2016-09-01

    Tape-lifting has since its introduction in the early 2000's become a well-established sampling method in forensic DNA analysis. Sampling is quick and straightforward while the following DNA extraction is more challenging due to the "stickiness", rigidity and size of the tape. We have developed, validated and implemented a simple and efficient direct lysis DNA extraction protocol for adhesive tapes that requires limited manual labour. The method uses Chelex beads and is applied with SceneSafe FAST tape. This direct lysis protocol provided higher mean DNA yields than PrepFiler Express BTA on Automate Express, although the differences were not significant when using clothes worn in a controlled fashion as reference material (p=0.13 and p=0.34 for T-shirts and button-down shirts, respectively). Through in-house validation we show that the method is fit-for-purpose for application in casework, as it provides high DNA yields and amplifiability, as well as good reproducibility and DNA extract stability. After implementation in casework, the proportion of extracts with DNA concentrations above 0.01ng/μL increased from 71% to 76%. Apart from providing higher DNA yields compared with the previous method, the introduction of the developed direct lysis protocol also reduced the amount of manual labour by half and doubled the potential throughput for tapes at the laboratory. Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions when the aim is to enable high-throughput DNA extraction of complex crime scene samples. PMID:27448236

  4. High-throughput DNA extraction of forensic adhesive tapes.

    PubMed

    Forsberg, Christina; Jansson, Linda; Ansell, Ricky; Hedman, Johannes

    2016-09-01

    Tape-lifting has since its introduction in the early 2000's become a well-established sampling method in forensic DNA analysis. Sampling is quick and straightforward while the following DNA extraction is more challenging due to the "stickiness", rigidity and size of the tape. We have developed, validated and implemented a simple and efficient direct lysis DNA extraction protocol for adhesive tapes that requires limited manual labour. The method uses Chelex beads and is applied with SceneSafe FAST tape. This direct lysis protocol provided higher mean DNA yields than PrepFiler Express BTA on Automate Express, although the differences were not significant when using clothes worn in a controlled fashion as reference material (p=0.13 and p=0.34 for T-shirts and button-down shirts, respectively). Through in-house validation we show that the method is fit-for-purpose for application in casework, as it provides high DNA yields and amplifiability, as well as good reproducibility and DNA extract stability. After implementation in casework, the proportion of extracts with DNA concentrations above 0.01ng/μL increased from 71% to 76%. Apart from providing higher DNA yields compared with the previous method, the introduction of the developed direct lysis protocol also reduced the amount of manual labour by half and doubled the potential throughput for tapes at the laboratory. Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions when the aim is to enable high-throughput DNA extraction of complex crime scene samples.

  5. Compound Cytotoxicity Profiling Using Quantitative High-Throughput Screening

    PubMed Central

    Xia, Menghang; Huang, Ruili; Witt, Kristine L.; Southall, Noel; Fostel, Jennifer; Cho, Ming-Hsuang; Jadhav, Ajit; Smith, Cynthia S.; Inglese, James; Portier, Christopher J.; Tice, Raymond R.; Austin, Christopher P.

    2008-01-01

    Background The propensity of compounds to produce adverse health effects in humans is generally evaluated using animal-based test methods. Such methods can be relatively expensive, low-throughput, and associated with pain suffered by the treated animals. In addition, differences in species biology may confound extrapolation to human health effects. Objective The National Toxicology Program and the National Institutes of Health Chemical Genomics Center are collaborating to identify a battery of cell-based screens to prioritize compounds for further toxicologic evaluation. Methods A collection of 1,408 compounds previously tested in one or more traditional toxicologic assays were profiled for cytotoxicity using quantitative high-throughput screening (qHTS) in 13 human and rodent cell types derived from six common targets of xenobiotic toxicity (liver, blood, kidney, nerve, lung, skin). Selected cytotoxicants were further tested to define response kinetics. Results qHTS of these compounds produced robust and reproducible results, which allowed cross-compound, cross-cell type, and cross-species comparisons. Some compounds were cytotoxic to all cell types at similar concentrations, whereas others exhibited species- or cell type–specific cytotoxicity. Closely related cell types and analogous cell types in human and rodent frequently showed different patterns of cytotoxicity. Some compounds inducing similar levels of cytotoxicity showed distinct time dependence in kinetic studies, consistent with known mechanisms of toxicity. Conclusions The generation of high-quality cytotoxicity data on this large library of known compounds using qHTS demonstrates the potential of this methodology to profile a much broader array of assays and compounds, which, in aggregate, may be valuable for prioritizing compounds for further toxicologic evaluation, identifying compounds with particular mechanisms of action, and potentially predicting in vivo biological response. PMID:18335092

  6. A rapid, inexpensive high throughput screen method for neurite outgrowth.

    PubMed

    Yeyeodu, Susan T; Witherspoon, Sam M; Gilyazova, Nailya; Ibeanu, Gordon C

    2010-01-01

    Neurite outgrowth assays are the most common phenotypic screen to assess chemical effects on neuronal cells. Current automated assays involve expensive equipment, lengthy sample preparation and handling, costly reagents and slow rates of data acquisition and analysis. We have developed a high throughput screen (HTS) for neurite outgrowth using a robust neuronal cell model coupled to fast and inexpensive visualization methods, reduced data volume and rapid data analysis. Neuroscreen-1 (NS-1) cell, a subclone of PC12, possessing rapid growth and enhanced sensitivity to NGF was used as a model neuron. This method reduces preparation time by using cells expressing GFP or native cells stained with HCS CellMask(™) Red in a multiplexed 30 min fixation and staining step. A 2x2 camera binning process reduced both image data files and analysis times by 75% and 60% respectively, compared to current protocols. In addition, eliminating autofocus steps during montage generation reduced data collection time. Pharmacological profiles for stimulation and inhibition of neurite outgrowth by NGF and SU6656 were comparable to current standard method utilizing immunofluorescence detection of tubulin. Potentiation of NGF-induced neurite outgrowth by members of a 1,120-member Prestwick compound library as assayed using this method identified six molecules, including etoposide, isoflupredone acetate, fludrocortisone acetate, thioguanosine, oxyphenbutazone and gibberellic acid, that more than doubled the neurite mass primed by 2 ng/ml NGF. This simple procedure represents an important routine approach in high throughput screening of large chemical libraries using the neurite outgrowth phenotype as a measure of the effects of chemical molecules on neuronal cells. PMID:21347208

  7. Commercially available high-throughput Dip Pen Nanolithography

    NASA Astrophysics Data System (ADS)

    Haaheim, J. R.; Tevaarwerk, E. R.; Fragala, J.; Shile, R.

    2008-04-01

    Dip Pen Nanolithography ® (DPN ®) is an inherently additive SPM-based technique which operates under ambient conditions, making it suitable to deposit a wide range of biological and inorganic materials. Massively parallel two-dimensional nanopatterning with DPN is now commercially available via NanoInk's 2D nano PrintArray TM, making DPN a high-throughput, flexible and versatile method for precision nanoscale pattern formation. By fabricating 55,000 tip-cantilevers across a 1 cm2 chip, we leverage the inherent versatility of DPN and demonstrate large area surface coverage, routinely achieving throughputs of 3x10 7 μm2 per hour. Further, we have engineered the device to be easy to use, wire-free, and fully integrated with the NSCRIPTOR's scanner, stage, and sophisticated lithography routines. In this talk we discuss the methods of operating this commercially available device, subsequent results showing sub-100 nm feature sizes and excellent uniformity (standard deviation < 16%), and our continuing development work. Simultaneous multiplexed deposition of a variety of molecules is a fundamental goal of massively parallel 2D nanopatterning, and we will discuss our progress on this front, including ink delivery methods, tip coating, and patterning techniques to generate combinatorial libraries of nanoscale patterns. Another fundamental challenge includes planar leveling of the 2D nano PrintArray, and herein we describe our successful implementation of device viewports and integrated software leveling routines that monitor cantilever deflection to achieve planarity and uniform surface contact. Finally, we will discuss the results of 2D nanopatterning applications such as: 1) rapidly and flexibly generating nanostructures; 2) chemically directed assembly and 3) directly writing biological materials.

  8. High throughput optoelectronic smart pixel systems using diffractive optics

    NASA Astrophysics Data System (ADS)

    Chen, Chih-Hao

    1999-12-01

    Recent developments in digital video, multimedia technology and data networks have greatly increased the demand for high bandwidth communication channels and high throughput data processing. Electronics is particularly suited for switching, amplification and logic functions, while optics is more suitable for interconnections and communications with lower energy and crosstalk. In this research, we present the design, testing, integration and demonstration of several optoelectronic smart pixel devices and system architectures. These systems integrate electronic switching/processing capability with parallel optical interconnections to provide high throughput network communication and pipeline data processing. The Smart Pixel Array Cellular Logic processor (SPARCL) is designed in 0.8 m m CMOS and hybrid integrated with Multiple-Quantum-Well (MQW) devices for pipeline image processing. The Smart Pixel Network Interface (SAPIENT) is designed in 0.6 m m GaAs and monolithically integrated with LEDs to implement a highly parallel optical interconnection network. The Translucent Smart Pixel Array (TRANSPAR) design is implemented in two different versions. The first version, TRANSPAR-MQW, is designed in 0.5 m m CMOS and flip-chip integrated with MQW devices to provide 2-D pipeline processing and translucent networking using the Carrier- Sense-MultipleAccess/Collision-Detection (CSMA/CD) protocol. The other version, TRANSPAR-VM, is designed in 1.2 m m CMOS and discretely integrated with VCSEL-MSM (Vertical-Cavity-Surface- Emitting-Laser and Metal-Semiconductor-Metal detectors) chips and driver/receiver chips on a printed circuit board. The TRANSPAR-VM provides an option of using the token ring network protocol in addition to the embedded functions of TRANSPAR-MQW. These optoelectronic smart pixel systems also require micro-optics devices to provide high resolution, high quality optical interconnections and external source arrays. In this research, we describe an innovative

  9. High-Throughput In Vitro Glycoside Hydrolase (HIGH) Screening for Enzyme Discovery

    SciTech Connect

    Kim, Tae-Wan; Chokhawala, Harshal A.; Hess, Matthias; Dana, Craig M.; Baer, Zachary; Sczyrba, Alexander; Rubin, Edward M.; Blanch, Harvey W.; Clark, Douglas S.

    2011-09-16

    A high-throughput protein-expression and screening method (HIGH method, see picture) provides a rapid approach to the discovery of active glycoside hydrolases in environmental samples. Finally, HIGH screening combines cloning, protein expression, and enzyme hydrolysis in one pot; thus, the entire process from gene expression to activity detection requires only three hours.

  10. Surface free energy activated high-throughput cell sorting.

    PubMed

    Zhang, Xinru; Zhang, Qian; Yan, Tao; Jiang, Zeyi; Zhang, Xinxin; Zuo, Yi Y

    2014-09-16

    Cell sorting is an important screening process in microbiology, biotechnology, and clinical research. Existing methods are mainly based on single-cell analysis as in flow cytometric and microfluidic cell sorters. Here we report a label-free bulk method for sorting cells by differentiating their characteristic surface free energies (SFEs). We demonstrated the feasibility of this method by sorting model binary cell mixtures of various bacterial species, including Pseudomonas putida KT2440, Enterococcus faecalis ATCC 29212, Salmonella Typhimurium ATCC 14028, and Escherichia coli DH5α. This method can effectively separate 10(10) bacterial cells within 30 min. Individual bacterial species can be sorted with up to 96% efficiency, and the cell viability ratio can be as high as 99%. In addition to its capacity of sorting evenly mixed bacterial cells, we demonstrated the feasibility of this method in selecting and enriching cells of minor populations in the mixture (presenting at only 1% in quantity) to a purity as high as 99%. This SFE-activated method may be used as a stand-alone method for quickly sorting a large quantity of bacterial cells or as a prescreening tool for microbial discrimination. Given its advantages of label-free, high-throughput, low cost, and simplicity, this SFE-activated cell sorting method has potential in various applications of sorting cells and abiotic particles. PMID:25184988

  11. High-throughput process development: I. Process chromatography.

    PubMed

    Rathore, Anurag S; Bhambure, Rahul

    2014-01-01

    Chromatographic separation serves as "a workhorse" for downstream process development and plays a key role in removal of product-related, host cell-related, and process-related impurities. Complex and poorly characterized raw materials and feed material, low feed concentration, product instability, and poor mechanistic understanding of the processes are some of the critical challenges that are faced during development of a chromatographic step. Traditional process development is performed as trial-and-error-based evaluation and often leads to a suboptimal process. High-throughput process development (HTPD) platform involves an integration of miniaturization, automation, and parallelization and provides a systematic approach for time- and resource-efficient chromatography process development. Creation of such platforms requires integration of mechanistic knowledge of the process with various statistical tools for data analysis. The relevance of such a platform is high in view of the constraints with respect to time and resources that the biopharma industry faces today. This protocol describes the steps involved in performing HTPD of process chromatography step. It described operation of a commercially available device (PreDictor™ plates from GE Healthcare). This device is available in 96-well format with 2 or 6 μL well size. We also discuss the challenges that one faces when performing such experiments as well as possible solutions to alleviate them. Besides describing the operation of the device, the protocol also presents an approach for statistical analysis of the data that is gathered from such a platform. A case study involving use of the protocol for examining ion-exchange chromatography of granulocyte colony-stimulating factor (GCSF), a therapeutic product, is briefly discussed. This is intended to demonstrate the usefulness of this protocol in generating data that is representative of the data obtained at the traditional lab scale. The agreement in the

  12. High-throughput, high-resolution Echelle deep-UV Raman spectrometer.

    PubMed

    Bykov, Sergei V; Sharma, Bhavya; Asher, Sanford A

    2013-08-01

    We constructed an ultrahigh-throughput, high-resolution ultraviolet (UV) Raman spectrograph that utilizes a high-efficiency filter-stage monochromator and a high-dispersion Echelle spectrograph. The spectrograph utilizes a total of six mirrors and two gratings, with an overall efficiency at 229 nm of ~18%. The limiting resolution of our spectrometer is 0.6 cm⁻¹ full width half-maximum (FWHM), as measured for 229 nm Rayleigh scattering. Use of a 1 mm-wide entrance slit gives rise to an approximately 10 cm⁻¹ FWHM resolution at 229 nm. The ultrahigh spectrograph throughput enables ultrahigh signal-to-noise ratio, deep UV Raman spectra that allow us to monitor <1% changes in peptide bond composition. The throughput is measured to be 35-fold greater than conventional deep UV Raman spectrometers.

  13. High-Throughput Screening Based Identification of Paramyxovirus Inhibitors

    PubMed Central

    Yoon, Jeong-Jeong; Chawla, Dhruv; Paal, Tanja; Ndungu, Maina; Du, Yuhong; Kurtkaya, Serdar; Sun, Aiming; Snyder, James P; Plemper, Richard K

    2008-01-01

    Paramyxoviruses are negative strand non-segmented RNA viruses. Several members of this family constitute major human pathogens that, collectively, are responsible for major morbidity and mortality worldwide. In an effort to ultimately develop novel therapeutics against measles virus (MV), a prominent member of the paramyxovirus family, we report a high-throughput screening protocol that allows hit identification using non-recombinant primary MV strains as targets. Implementation of the assay has yielded 60 hit candidates from a 137,500-entry library. Counterscreening and generation of dose-response curves narrows this pool to 35 compounds with active concentrations ≤15.3 μM against the MV-Alaska strain and specificity indices ranging from 36 to >500. Library mining for structural analogs of several confirmed hits combined with re-testing of identified candidates reveals a low false-negative rate and, thus, a high accuracy of primary hit identification. Eleven of the confirmed hits were found to interfere with the viral entry machinery, while the remaining 24 compounds target post-entry steps of the viral life cycle. Activity testing against selected members of the paramyxovirus family reveals three patterns of activity: 1) exclusively MV-specific blockers; 2) inhibitors of MV and related viruses of the same genus; 3) broader-range inhibitors with activity against a different paramyxovirinae genus. Representatives of the last class may open avenues for the development of broad-range paramyxovirus inhibitors through hit-to-lead chemistry. PMID:18626114

  14. High-Throughput, Multi-Image Cryohistology of Mineralized Tissues.

    PubMed

    Dyment, Nathaniel A; Jiang, Xi; Chen, Li; Hong, Seung-Hyun; Adams, Douglas J; Ackert-Bicknell, Cheryl; Shin, Dong-Guk; Rowe, David W

    2016-01-01

    There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward. PMID:27684089

  15. BOOGIE: Predicting Blood Groups from High Throughput Sequencing Data

    PubMed Central

    Giollo, Manuel; Minervini, Giovanni; Scalzotto, Marta; Leonardi, Emanuela; Ferrari, Carlo; Tosatto, Silvio C. E.

    2015-01-01

    Over the last decade, we have witnessed an incredible growth in the amount of available genotype data due to high throughput sequencing (HTS) techniques. This information may be used to predict phenotypes of medical relevance, and pave the way towards personalized medicine. Blood phenotypes (e.g. ABO and Rh) are a purely genetic trait that has been extensively studied for decades, with currently over thirty known blood groups. Given the public availability of blood group data, it is of interest to predict these phenotypes from HTS data which may translate into more accurate blood typing in clinical practice. Here we propose BOOGIE, a fast predictor for the inference of blood groups from single nucleotide variant (SNV) databases. We focus on the prediction of thirty blood groups ranging from the well known ABO and Rh, to the less studied Junior or Diego. BOOGIE correctly predicted the blood group with 94% accuracy for the Personal Genome Project whole genome profiles where good quality SNV annotation was available. Additionally, our tool produces a high quality haplotype phase, which is of interest in the context of ethnicity-specific polymorphisms or traits. The versatility and simplicity of the analysis make it easily interpretable and allow easy extension of the protocol towards other phenotypes. BOOGIE can be downloaded from URL http://protein.bio.unipd.it/download/. PMID:25893845

  16. High-throughput multiparameter analysis of individual mitochondria.

    PubMed

    Zhang, Shuyue; Zhu, Shaobin; Yang, Lingling; Zheng, Yan; Gao, Min; Wang, Shuo; Zeng, Jin-zhang; Yan, Xiaomei

    2012-08-01

    Mitochondria are one of the most important organelles responsible for cellular energy metabolism and apoptosis regulation. However, single-mitochondrion analysis is challenging, because of their small sizes and the low content of organelle constituents. Here, we report the development of a sensitive and versatile platform for high-throughput multiparameter analysis of individual mitochondria. Employing specific fluorescent staining with a laboratory-built high-sensitivity flow cytometer (HSFCM), we demonstrate the simultaneous detection of side scatter, cardiolipin, and mitochondria DNA (mtDNA) of a single mitochondrion. Simultaneous measurements of side scatter, porin, and cytochrome c of individual mitochondria are reported for the first time. Correlation analysis among multiple attributes on an organelle-by-organelle basis could provide a more definitive assessment of the purity, structure integrity, and apoptosis-related proteins of isolated mitochondria than bulk measurement. This work represents a significant advancement in single-mitochondrion analysis. We believe that the HSFCM holds great potential for studying apoptotic signal transduction pathways at the single-mitochondrion level.

  17. High Throughput, Continuous, Mass Production of Photovoltaic Modules

    SciTech Connect

    Kurt Barth

    2008-02-06

    AVA Solar has developed a very low cost solar photovoltaic (PV) manufacturing process and has demonstrated the significant economic and commercial potential of this technology. This I & I Category 3 project provided significant assistance toward accomplishing these milestones. The original goals of this project were to design, construct and test a production prototype system, fabricate PV modules and test the module performance. The original module manufacturing costs in the proposal were estimated at $2/Watt. The objectives of this project have been exceeded. An advanced processing line was designed, fabricated and installed. Using this automated, high throughput system, high efficiency devices and fully encapsulated modules were manufactured. AVA Solar has obtained 2 rounds of private equity funding, expand to 50 people and initiated the development of a large scale factory for 100+ megawatts of annual production. Modules will be manufactured at an industry leading cost which will enable AVA Solar's modules to produce power that is cost-competitive with traditional energy resources. With low manufacturing costs and the ability to scale manufacturing, AVA Solar has been contacted by some of the largest customers in the PV industry to negotiate long-term supply contracts. The current market for PV has continued to grow at 40%+ per year for nearly a decade and is projected to reach $40-$60 Billion by 2012. Currently, a crystalline silicon raw material supply shortage is limiting growth and raising costs. Our process does not use silicon, eliminating these limitations.

  18. High-throughput charge exchange recombination spectroscopy system on MAST

    SciTech Connect

    Conway, N. J.; Carolan, P. G.; McCone, J.; Walsh, M. J.; Wisse, M.

    2006-10-15

    A major upgrade to the charge exchange recombination spectroscopy system on MAST has recently been implemented. The new system consists of a high-throughput spectrometer coupled to a total of 224 spatial channels, including toroidal and poloidal views of both neutral heating beams on MAST. Radial resolution is {approx}1 cm, comparable to the ion Larmor radius. The toroidal views are configured with 64 channels per beam, while the poloidal views have 32 channels per beam. Background channels for both poloidal and toroidal views are also provided. A large transmission grating is at the heart of the new spectrometer, with high quality single lens reflex lenses providing excellent imaging performance and permitting the full exploitation of the available etendue of the camera sensor. The charge-coupled device camera chosen has four-tap readout at a maximum aggregate speed of 8.8 MHz, and it is capable of reading out the full set of 224 channels in less than 4 ms. The system normally operates at 529 nm, viewing the C{sup 5+} emission line, but can operate at any wavelength in the range of 400-700 nm. Results from operating the system on MAST are shown, including impurity ion temperature and velocity profiles. The system's excellent spatial resolution is ideal for the study of transport barrier phenomena on MAST, an activity which has already been advanced significantly by data from the new diagnostic.

  19. High-Throughput FRET Assay Yields Allosteric SERCA Activators

    PubMed Central

    Cornea, Razvan L.; Lockamy, Elizabeth L.; Gruber, Simon J.; Muretta, Joseph M.; Jin, Dongzhu; Chen, Jiqiu; Dahl, Russell; Bartfai, Tamas; Zsebo, Krisztina M.; Gillispie, Gregory D.; Thomas, David D.

    2013-01-01

    Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarco-/endoplasmic reticulum Ca-ATPase (SERCA) by its endogenous regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca2+ regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20,000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 primary hits (0.2%), 31 (72%) were found to be false positives upon more thorough testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and pre-clinical testing. We were concerned about the high rate of false positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HT. PMID:22923787

  20. High-throughput protein crystallography and drug discovery.

    PubMed

    Tickle, Ian; Sharff, Andrew; Vinkovic, Mladen; Yon, Jeff; Jhoti, Harren

    2004-10-20

    Single crystal X-ray diffraction is the technique of choice for studying the interactions of small organic molecules with proteins by determining their three-dimensional structures; however the requirement for highly purified protein and lack of process automation have traditionally limited its use in this field. Despite these shortcomings, the use of crystal structures of therapeutically relevant drug targets in pharmaceutical research has increased significantly over the last decade. The application of structure-based drug design has resulted in several marketed drugs and is now an established discipline in most pharmaceutical companies. Furthermore, the recently published full genome sequences of Homo sapiens and a number of micro-organisms have provided a plethora of new potential drug targets that could be utilised in structure-based drug design programs. In order to take maximum advantage of this explosion of information, techniques have been developed to automate and speed up the various procedures required to obtain protein crystals of suitable quality, to collect and process the raw X-ray diffraction data into usable structural information, and to use three-dimensional protein structure as a basis for drug discovery and lead optimisation. This tutorial review covers the various technologies involved in the process pipeline for high-throughput protein crystallography as it is currently being applied to drug discovery. It is aimed at synthetic and computational chemists, as well as structural biologists, in both academia and industry, who are interested in structure-based drug design.

  1. Towards Prebiotic Catalytic Amyloids Using High Throughput Screening

    PubMed Central

    Friedmann, Michael P.; Torbeev, Vladimir; Zelenay, Viviane; Sobol, Alexander; Greenwald, Jason; Riek, Roland

    2015-01-01

    Enzymes are capable of directing complex stereospecific transformations and of accelerating reaction rates many orders of magnitude. As even the simplest known enzymes comprise thousands of atoms, the question arises as to how such exquisite catalysts evolved. A logical predecessor would be shorter peptides, but they lack the defined structure and size that are apparently necessary for enzyme functions. However, some very short peptides are able to assemble into amyloids, thereby forming a well-defined tertiary structure called the cross-β-sheet, which bestows unique properties upon the peptides. We have hypothesized that amyloids could have been the catalytically active precursor to modern enzymes. To test this hypothesis, we designed an amyloid peptide library that could be screened for catalytic activity. Our approach, amenable to high-throughput methodologies, allowed us to find several peptides and peptide mixtures that form amyloids with esterase activity. These results indicate that amyloids, with their stability in a wide range of conditions and their potential as catalysts with low sequence specificity, would indeed be fitting precursors to modern enzymes. Furthermore, our approach can be efficiently expanded upon in library size, screening conditions, and target activity to yield novel amyloid catalysts with potential applications in aqueous-organic mixtures, at high temperature and in other extreme conditions that could be advantageous for industrial applications. PMID:26650386

  2. High throughput illumination systems for solar simulators and photoresist exposure

    NASA Astrophysics Data System (ADS)

    Feldman, Arkady

    2010-08-01

    High throughput illumination systems are critical component in photolithography, solar simulators, UV curing, microscopy, and spectral analysis. A good refractive condenser system has F/# .60, or N.A .80, but it captures only 10 to 15% of energy emitted by an incandescent or gas-discharge lamp, as these sources emit light in all directions. Systems with ellipsoidal or parabolic reflectors are much more efficient, they capture up to 80% of total energy emitted by lamps. However, these reflectors have large aberrations when working with real sources of finite dimensions, resulting in poor light concentrating capability. These aberrations also increase beam divergence, collimation, and affect edge definition in flood exposure systems. The problem is aggravated by the geometry of high power Arc lamps where, for thermal considerations, the anode has a larger diameter than the cathode and absorbs and obscures part of the energy. This results in an asymmetrical energy distribution emitted by the lamp and makes efficiency of Lamp - reflector configuration dependent on orientation of lamp in the reflector. This paper presents the analysis of different configurations of Lamp - Reflector systems of different power levels and their energy distribution in the image plane. Configuration, which results in significant improvement of brightness, is derived.

  3. Validation of high throughput sequencing and microbial forensics applications

    PubMed Central

    2014-01-01

    High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security. PMID:25101166

  4. Analysis of DNA Sequence Variants Detected by High Throughput Sequencing

    PubMed Central

    Adams, David R; Sincan, Murat; Fajardo, Karin Fuentes; Mullikin, James C; Pierson, Tyler M; Toro, Camilo; Boerkoel, Cornelius F; Tifft, Cynthia J; Gahl, William A; Markello, Tom C

    2014-01-01

    The Undiagnosed Diseases Program at the National Institutes of Health uses High Throughput Sequencing (HTS) to diagnose rare and novel diseases. HTS techniques generate large numbers of DNA sequence variants, which must be analyzed and filtered to find candidates for disease causation. Despite the publication of an increasing number of successful exome-based projects, there has been little formal discussion of the analytic steps applied to HTS variant lists. We present the results of our experience with over 30 families for whom HTS sequencing was used in an attempt to find clinical diagnoses. For each family, exome sequence was augmented with high-density SNP-array data. We present a discussion of the theory and practical application of each analytic step and provide example data to illustrate our approach. The paper is designed to provide an analytic roadmap for variant analysis, thereby enabling a wide range of researchers and clinical genetics practitioners to perform direct analysis of HTS data for their patients and projects. PMID:22290882

  5. High-Throughput, Multi-Image Cryohistology of Mineralized Tissues.

    PubMed

    Dyment, Nathaniel A; Jiang, Xi; Chen, Li; Hong, Seung-Hyun; Adams, Douglas J; Ackert-Bicknell, Cheryl; Shin, Dong-Guk; Rowe, David W

    2016-01-01

    There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

  6. High-throughput FRET assay yields allosteric SERCA activators.

    PubMed

    Cornea, Razvan L; Gruber, Simon J; Lockamy, Elizabeth L; Muretta, Joseph M; Jin, Dongzhu; Chen, Jiqiu; Dahl, Russell; Bartfai, Tamas; Zsebo, Krisztina M; Gillispie, Gregory D; Thomas, David D

    2013-01-01

    Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarcoplasmic reticulum Ca-ATPase (SERCA) by its cardiac regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca(2+) regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20 000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 hits (0.2%), 31 (72%) were found to be false-positives upon more thorough FRET testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and preclinical testing. We were concerned about the high rate of false-positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HTS.

  7. High-throughput Protein Purification and Quality Assessment for Crystallization

    PubMed Central

    Kim, Youngchang; Babnigg, Gyorgy; Jedrzejczak, Robert; Eschenfeldt, William H.; Li, Hui; Maltseva, Natalia; Hatzos-Skintges, Catherine; Gu, Minyi; Makowska-Grzyska, Magdalena; Wu, Ruiying; An, Hao; Chhor, Gekleng; Joachimiak, Andrzej

    2012-01-01

    The ultimate goal of structural biology is to understand the structural basis of proteins in cellular processes. In structural biology, the most critical issue is the availability of high-quality samples. “Structural biology-grade” proteins must be generated in the quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. With structural genomics emphasizing a genome-based approach in understanding protein structure and function, a number of unique structures covering most of the protein folding space have been determined and new technologies with high efficiency have been developed. At the Midwest Center for Structural Genomics (MCSG), we have developed semi-automated protocols for high-throughput parallel protein expression and purification. A protein, expressed as a fusion with a cleavable affinity tag, is purified in two consecutive immobilized metal affinity chromatography (IMAC) steps: (i) the first step is an IMAC coupled with buffer-exchange, or size exclusion chromatography (IMAC-I), followed by the cleavage of the affinity tag using the highly specific Tobacco Etch Virus (TEV) protease; [1] the second step is IMAC and buffer exchange (IMAC-II) to remove the cleaved tag and tagged TEV protease. These protocols have been implemented on multidimensional chromatography workstations and, as we have shown, many proteins can be successfully produced in large-scale. All methods and protocols used for purification, some developed by MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Diseases (CSGID) purification pipeline, are

  8. High-Throughput/High-Content Screening Assays with Engineered Nanomaterials in ToxCast

    EPA Science Inventory

    High-throughput and high-content screens are attractive approaches for prioritizing nanomaterial hazards and informing targeted testing due to the impracticality of using traditional toxicological testing on the large numbers and varieties of nanomaterials. The ToxCast program a...

  9. High-Resolution and High-Throughput Plasmonic Photopatterning of Complex Molecular Orientations in Liquid Crystals.

    PubMed

    Guo, Yubing; Jiang, Miao; Peng, Chenhui; Sun, Kai; Yaroshchuk, Oleg; Lavrentovich, Oleg; Wei, Qi-Huo

    2016-03-23

    A plasmonic photopatterning technique is proposed and demonstrated for aligning the molecular orientation in liquid crystals (LCs) in patterns with designer complexity. Using plasmonic metamasks in which target molecular directors are encoded, LC alignments of arbitrary planar patterns can be achieved in a repeatable and scalable fashion withunprecedentedly high spatial resolution and high throughput.

  10. High Resolution Genotyping of Campylobacter Using PCR and High-Throughput Mass Spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this work we report a high throughput mass spectrometry-based technique for rapid high resolution strain identification of Campylobacter jejuni. This method readily distinguishes C. jejuni from C. coli, has comparable resolving power to multi-locus sequence typing (MLST), is applicable to mixtur...

  11. Optimization of high-throughput nanomaterial developmental toxicity testing in zebrafish embryos

    EPA Science Inventory

    Nanomaterial (NM) developmental toxicities are largely unknown. With an extensive variety of NMs available, high-throughput screening methods may be of value for initial characterization of potential hazard. We optimized a zebrafish embryo test as an in vivo high-throughput assay...

  12. High-throughput microfluidic line scan imaging for cytological characterization

    NASA Astrophysics Data System (ADS)

    Hutcheson, Joshua A.; Powless, Amy J.; Majid, Aneeka A.; Claycomb, Adair; Fritsch, Ingrid; Balachandran, Kartik; Muldoon, Timothy J.

    2015-03-01

    Imaging cells in a microfluidic chamber with an area scan camera is difficult due to motion blur and data loss during frame readout causing discontinuity of data acquisition as cells move at relatively high speeds through the chamber. We have developed a method to continuously acquire high-resolution images of cells in motion through a microfluidics chamber using a high-speed line scan camera. The sensor acquires images in a line-by-line fashion in order to continuously image moving objects without motion blur. The optical setup comprises an epi-illuminated microscope with a 40X oil immersion, 1.4 NA objective and a 150 mm tube lens focused on a microfluidic channel. Samples containing suspended cells fluorescently stained with 0.01% (w/v) proflavine in saline are introduced into the microfluidics chamber via a syringe pump; illumination is provided by a blue LED (455 nm). Images were taken of samples at the focal plane using an ELiiXA+ 8k/4k monochrome line-scan camera at a line rate of up to 40 kHz. The system's line rate and fluid velocity are tightly controlled to reduce image distortion and are validated using fluorescent microspheres. Image acquisition was controlled via MATLAB's Image Acquisition toolbox. Data sets comprise discrete images of every detectable cell which may be subsequently mined for morphological statistics and definable features by a custom texture analysis algorithm. This high-throughput screening method, comparable to cell counting by flow cytometry, provided efficient examination including counting, classification, and differentiation of saliva, blood, and cultured human cancer cells.

  13. HAPIscreen, a method for high-throughput aptamer identification

    PubMed Central

    2011-01-01

    Background Aptamers are oligonucleotides displaying specific binding properties for a predetermined target. They are selected from libraries of randomly synthesized candidates through an in vitro selection process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment) alternating selection and amplification steps. SELEX is followed by cloning and sequencing of the enriched pool of oligonucleotides to enable comparison of the selected sequences. The most represented candidates are then synthesized and their binding properties are individually evaluated thus leading to the identification of aptamers. These post-selection steps are time consuming and introduce a bias to the expense of poorly amplified binders that might be of high affinity and are consequently underrepresented. A method that would circumvent these limitations would be highly valuable. Results We describe a novel homogeneous solution-based method for screening large populations of oligonucleotide candidates generated from SELEX. This approach, based on the AlphaScreen® technology, is carried out on the exclusive basis of the binding properties of the selected candidates without the needs of performing a priori sequencing. It therefore enables the functional identification of high affinity aptamers. We validated the HAPIscreen (High throughput APtamer Identification screen) methodology using aptamers targeted to RNA hairpins, previously identified in our laboratory. We then screened pools of candidates issued from SELEX rounds in a 384 well microplate format and identify new RNA aptamers to pre-microRNAs. Conclusions HAPIscreen, an Alphascreen®-based methodology for the identification of aptamers is faster and less biased than current procedures based on sequence comparison of selected oligonucleotides and sampling binding properties of few individuals. Moreover this methodology allows for screening larger number of candidates. Used here for selecting anti-premiR aptamers, HAPIscreen

  14. Virtual high throughput screening in new lead identification.

    PubMed

    Badrinarayan, Preethi; Sastry, G Narahari

    2011-12-01

    Drug discovery continues to be one of the greatest contemporary challenges and rational application of modelling approaches is the first important step to obtain lead compounds, which can be optimised further. Virtual high throughput screening (VHTS) is one of the efficient approaches to obtain lead structures for a given target. Strategic application of different screening filters like pharmacophore mapping, shape-based, ligand-based, molecular similarity etc., in combination with other drug design protocols provide invaluable insights in lead identification and optimization. Screening of large databases using these computational methods provides potential lead compounds, thus triggering a meaningful interplay between computations and experiments. In this review, we present a critical account on the relevance of molecular modelling approaches in general, lead optimization and virtual screening methods in particular for new lead identification. The importance of developing reliable scoring functions for non-bonded interactions has been highlighted, as it is an extremely important measure for the reliability of scoring function. The lead optimization and new lead design has also been illustrated with examples. The importance of employing a combination of general and target specific screening protocols has also been highlighted. PMID:21843146

  15. High Throughput Interrogation of Behavioral Transitions in C. elegans

    NASA Astrophysics Data System (ADS)

    Liu, Mochi; Shaevitz, Joshua; Leifer, Andrew

    We present a high-throughput method to probe transformations from neural activity to behavior in Caenorhabditis elegans to better understand how organisms change behavioral states. We optogenetically deliver white-noise stimuli to target sensory or inter neurons while simultaneously recording the movement of a population of worms. Using all the postural movement data collected, we computationally classify stereotyped behaviors in C. elegans by clustering based on the spectral properties of the instantaneous posture. (Berman et al., 2014) Transitions between these behavioral clusters indicate discrete behavioral changes. To study the neural correlates dictating these transitions, we perform model-driven experiments and employ Linear-Nonlinear-Poisson cascades that take the white-noise stimulus as the input. The parameters of these models are fitted by reverse-correlation from our measurements. The parameterized models of behavioral transitions predict the worm's response to novel stimuli and reveal the internal computations the animal makes before carrying out behavioral decisions. Preliminary results are shown that describe the neural-behavioral transformation between neural activity in mechanosensory neurons and reversal behavior.

  16. Translational informatics: enabling high-throughput research paradigms

    PubMed Central

    Embi, Peter J.; Sen, Chandan K.

    2009-01-01

    A common thread throughout the clinical and translational research domains is the need to collect, manage, integrate, analyze, and disseminate large-scale, heterogeneous biomedical data sets. However, well-established and broadly adopted theoretical and practical frameworks and models intended to address such needs are conspicuously absent in the published literature or other reputable knowledge sources. Instead, the development and execution of multidisciplinary, clinical, or translational studies are significantly limited by the propagation of “silos” of both data and expertise. Motivated by this fundamental challenge, we report upon the current state and evolution of biomedical informatics as it pertains to the conduct of high-throughput clinical and translational research and will present both a conceptual and practical framework for the design and execution of informatics-enabled studies. The objective of presenting such findings and constructs is to provide the clinical and translational research community with a common frame of reference for discussing and expanding upon such models and methodologies. PMID:19737991

  17. High-Throughput, Data-Rich Cellular RNA Device Engineering

    PubMed Central

    Townshend, Brent; Kennedy, Andrew B.; Xiang, Joy S.; Smolke, Christina D.

    2015-01-01

    Methods for rapidly assessing sequence-structure-function landscapes and developing conditional gene-regulatory devices are critical to our ability to manipulate and interface with biology. We describe a framework for engineering RNA devices from preexisting aptamers that exhibit ligand-responsive ribozyme tertiary interactions. Our methodology utilizes cell sorting, high-throughput sequencing, and statistical data analyses to enable parallel measurements of the activities of hundreds of thousands of sequences from RNA device libraries in the absence and presence of ligands. Our tertiary interaction RNA devices exhibit improved performance in terms of gene silencing, activation ratio, and ligand sensitivity as compared to optimized RNA devices that rely on secondary structure changes. We apply our method to building biosensors for diverse ligands and determine consensus sequences that enable ligand-responsive tertiary interactions. These methods advance our ability to develop broadly applicable genetic tools and to elucidate understanding of the underlying sequence-structure-function relationships that empower rational design of complex biomolecules. PMID:26258292

  18. Multiplexing a high-throughput liability assay to leverage efficiencies.

    PubMed

    Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

    2009-06-01

    In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

  19. High Throughput Profiling of Molecular Shapes in Crystals

    NASA Astrophysics Data System (ADS)

    Spackman, Peter R.; Thomas, Sajesh P.; Jayatilaka, Dylan

    2016-02-01

    Molecular shape is important in both crystallisation and supramolecular assembly, yet its role is not completely understood. We present a computationally efficient scheme to describe and classify the molecular shapes in crystals. The method involves rotation invariant description of Hirshfeld surfaces in terms of of spherical harmonic functions. Hirshfeld surfaces represent the boundaries of a molecule in the crystalline environment, and are widely used to visualise and interpret crystalline interactions. The spherical harmonic description of molecular shapes are compared and classified by means of principal component analysis and cluster analysis. When applied to a series of metals, the method results in a clear classification based on their lattice type. When applied to around 300 crystal structures comprising of series of substituted benzenes, naphthalenes and phenylbenzamide it shows the capacity to classify structures based on chemical scaffolds, chemical isosterism, and conformational similarity. The computational efficiency of the method is demonstrated with an application to over 14 thousand crystal structures. High throughput screening of molecular shapes and interaction surfaces in the Cambridge Structural Database (CSD) using this method has direct applications in drug discovery, supramolecular chemistry and materials design.

  20. Advances in High Throughput Screening of Biomass Recalcitrance (Poster)

    SciTech Connect

    Turner, G. B.; Decker, S. R.; Tucker, M. P.; Law, C.; Doeppke, C.; Sykes, R. W.; Davis, M. F.; Ziebell, A.

    2012-06-01

    This was a poster displayed at the Symposium. Advances on previous high throughput screening of biomass recalcitrance methods have resulted in improved conversion and replicate precision. Changes in plate reactor metallurgy, improved preparation of control biomass, species-specific pretreatment conditions, and enzymatic hydrolysis parameters have reduced overall coefficients of variation to an average of 6% for sample replicates. These method changes have improved plate-to-plate variation of control biomass recalcitrance and improved confidence in sugar release differences between samples. With smaller errors plant researchers can have a higher degree of assurance more low recalcitrance candidates can be identified. Significant changes in plate reactor, control biomass preparation, pretreatment conditions and enzyme have significantly reduced sample and control replicate variability. Reactor plate metallurgy significantly impacts sugar release aluminum leaching into reaction during pretreatment degrades sugars and inhibits enzyme activity. Removal of starch and extractives significantly decreases control biomass variability. New enzyme formulations give more consistent and higher conversion levels, however required re-optimization for switchgrass. Pretreatment time and temperature (severity) should be adjusted to specific biomass types i.e. woody vs. herbaceous. Desalting of enzyme preps to remove low molecular weight stabilizers and improved conversion levels likely due to water activity impacts on enzyme structure and substrate interactions not attempted here due to need to continually desalt and validate precise enzyme concentration and activity.

  1. High-Throughput Preparation of New Photoactive Nanocomposites.

    PubMed

    Conterosito, Eleonora; Benesperi, Iacopo; Toson, Valentina; Saccone, Davide; Barbero, Nadia; Palin, Luca; Barolo, Claudia; Gianotti, Valentina; Milanesio, Marco

    2016-06-01

    New low-cost photoactive hybrid materials based on organic luminescent molecules inserted into hydrotalcite (layered double hydroxides; LDH) were produced, which exploit the high-throughput liquid-assisted grinding (LAG) method. These materials are conceived for applications in dye-sensitized solar cells (DSSCs) as a co-absorbers and in silicon photovoltaic (PV) panels to improve their efficiency as they are able to emit where PV modules show the maximum efficiency. A molecule that shows a large Stokes' shift was designed, synthesized, and intercalated into LDH. Two dyes already used in DSSCs were also intercalated to produce two new nanocomposites. LDH intercalation allows the stability of organic dyes to be improved and their direct use in polymer melt blending. The prepared nanocomposites absorb sunlight from UV to visible and emit from blue to near-IR and thus can be exploited for light-energy management. Finally one nanocomposite was dispersed by melt blending into a poly(methyl methacrylate)-block-poly(n-butyl acrylate) copolymer to obtain a photoactive film.

  2. High-throughput measurements of hydrogel tissue construct mechanics.

    PubMed

    Marquez, Juan Pablo; Legant, Wesley; Lam, Vy; Cayemberg, Amy; Elson, Elliot; Wakatsuki, Tetsuro

    2009-06-01

    Engineered tissues represent a natural environment for studying cell physiology, mechanics, and function. Cellular interactions with the extracellular matrix proteins are important determinants of cell physiology and tissue mechanics. Dysregulation of these parameters can result in diseases such as cardiac fibrosis and atherosclerosis. In this report we present a novel system to produce hydrogel tissue constructs (HTCs) and to characterize their mechanical properties. HTCs are grown in custom chambers and a robotic system is used to indent them and measure the resulting forces. Force measurements are then used to estimate HTC pretension (cellular contractility). Pretension was reduced in a dose-dependent manner by cytochalasin D (CD) treatment; the highest concentration (2microM) resulted in 10-fold decrease. On the other hand, treatment with fetal bovine serum (20%) resulted in approximately threefold increase in pretension. Excellent repeatability and precision were observed in measurements from replicate HTCs. The coefficient of statistical variance of quantified pretension ranged from 7% to 15% (n=4). Due to the small size (4x4x0.8mm) of the HTCs, this system of profiling HTC mechanics can readily be used in high-throughput applications. In particular, it can be used for screening chemical libraries in search of drugs that can alter tissue mechanics.

  3. A microfluidic, high throughput protein crystal growth method for microgravity.

    PubMed

    Carruthers, Carl W; Gerdts, Cory; Johnson, Michael D; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions' microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 10(3) cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories. PMID:24278480

  4. High throughput virus plaque quantitation using a flatbed scanner.

    PubMed

    Sullivan, Kate; Kloess, Johannes; Qian, Chen; Bell, Donald; Hay, Alan; Lin, Yi Pu; Gu, Yan

    2012-01-01

    The plaque assay is a standard technique for measuring influenza virus infectivity and inhibition of virus replication. Counting plaque numbers and quantifying virus infection of cells in multiwell plates quickly, accurately and automatically remain a challenge. Visual inspection relies upon experience, is subjective, often time consuming, and has less reproducibility than automated methods. In this paper, a simple, high throughput imaging-based alternative is proposed which uses a flatbed scanner and image processing software to quantify the infected cell population and plaque formation. Quantitation results were evaluated with reference to visual counting and achieved better than 80% agreement. The method was shown to be particularly advantageous in titration of the number of plaques and infected cells when influenza viruses produce a heterogeneous population of small plaques. It was also shown to be insensitive to the densities of plaques in determination of neutralization titres and IC(50)s of drug susceptibility. In comparison to other available techniques, this approach is cost-effective, relatively accurate, and readily available.

  5. A Microfluidic, High Throughput Protein Crystal Growth Method for Microgravity

    PubMed Central

    Carruthers Jr, Carl W.; Gerdts, Cory; Johnson, Michael D.; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions’ microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 103 cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories. PMID:24278480

  6. High Throughput Multispectral Image Processing with Applications in Food Science.

    PubMed

    Tsakanikas, Panagiotis; Pavlidis, Dimitris; Nychas, George-John

    2015-01-01

    Recently, machine vision is gaining attention in food science as well as in food industry concerning food quality assessment and monitoring. Into the framework of implementation of Process Analytical Technology (PAT) in the food industry, image processing can be used not only in estimation and even prediction of food quality but also in detection of adulteration. Towards these applications on food science, we present here a novel methodology for automated image analysis of several kinds of food products e.g. meat, vanilla crème and table olives, so as to increase objectivity, data reproducibility, low cost information extraction and faster quality assessment, without human intervention. Image processing's outcome will be propagated to the downstream analysis. The developed multispectral image processing method is based on unsupervised machine learning approach (Gaussian Mixture Models) and a novel unsupervised scheme of spectral band selection for segmentation process optimization. Through the evaluation we prove its efficiency and robustness against the currently available semi-manual software, showing that the developed method is a high throughput approach appropriate for massive data extraction from food samples. PMID:26466349

  7. High Throughput Multispectral Image Processing with Applications in Food Science.

    PubMed

    Tsakanikas, Panagiotis; Pavlidis, Dimitris; Nychas, George-John

    2015-01-01

    Recently, machine vision is gaining attention in food science as well as in food industry concerning food quality assessment and monitoring. Into the framework of implementation of Process Analytical Technology (PAT) in the food industry, image processing can be used not only in estimation and even prediction of food quality but also in detection of adulteration. Towards these applications on food science, we present here a novel methodology for automated image analysis of several kinds of food products e.g. meat, vanilla crème and table olives, so as to increase objectivity, data reproducibility, low cost information extraction and faster quality assessment, without human intervention. Image processing's outcome will be propagated to the downstream analysis. The developed multispectral image processing method is based on unsupervised machine learning approach (Gaussian Mixture Models) and a novel unsupervised scheme of spectral band selection for segmentation process optimization. Through the evaluation we prove its efficiency and robustness against the currently available semi-manual software, showing that the developed method is a high throughput approach appropriate for massive data extraction from food samples.

  8. High-throughput optical screening of cellular mechanotransduction

    NASA Astrophysics Data System (ADS)

    Compton, Jonathan L.; Luo, Justin C.; Ma, Huan; Botvinick, Elliot; Venugopalan, Vasan

    2014-09-01

    We introduce an optical platform for rapid, high-throughput screening of exogenous molecules that affect cellular mechanotransduction. Our method initiates mechanotransduction in adherent cells using single laser-microbeam generated microcavitation bubbles without requiring flow chambers or microfluidics. These microcavitation bubbles expose adherent cells to a microtsunami, a transient microscale burst of hydrodynamic shear stress, which stimulates cells over areas approaching 1 mm2. We demonstrate microtsunami-initiated mechanosignalling in primary human endothelial cells. This observed signalling is consistent with G-protein-coupled receptor stimulation, resulting in Ca2+ release by the endoplasmic reticulum. Moreover, we demonstrate the dose-dependent modulation of microtsunami-induced Ca2+ signalling by introducing a known inhibitor to this pathway. The imaging of Ca2+ signalling and its modulation by exogenous molecules demonstrates the capacity to initiate and assess cellular mechanosignalling in real time. We utilize this capability to screen the effects of a set of small molecules on cellular mechanotransduction in 96-well plates using standard imaging cytometry.

  9. High-throughput screening in the diagnostics industry.

    PubMed

    Wilson, S; Howell, S

    2002-08-01

    The diagnostics industry is constantly under pressure to bring innovation quicker to market and so the impetus to speed up product-development cycle times becomes greater. There are a number of steps in the product-development cycle where the application of high-throughput screening can help. In the case of lateral-flow immuno-diagnostics the selection of antibody reagents is paramount. In particular, rapid identification of antibody pairs that are able to 'sandwich' around the target antigen is required. One screen that has been applied successfully is the use of surface plasmon resonance biosensors like Biacore. Using such a system one can evaluate over 400 antibody pairings in under 5 days. Conventional approaches to screen this number of antibody pairs would take many months. Other automated screening systems like DELFIA can be used in processing the vast amount of tests required for clinical trials. In addition, the use of robotics to automate routine product testing can be used to shorten the product-development cycle.

  10. High-throughput optical screening of cellular mechanotransduction

    PubMed Central

    Compton, Jonathan L.; Luo, Justin C.; Ma, Huan; Botvinick, Elliot; Venugopalan, Vasan

    2014-01-01

    We introduce an optical platform for rapid, high-throughput screening of exogenous molecules that affect cellular mechanotransduction. Our method initiates mechanotransduction in adherent cells using single laser-microbeam generated micro-cavitation bubbles (μCBs) without requiring flow chambers or microfluidics. These μCBs expose adherent cells to a microTsunami, a transient microscale burst of hydrodynamic shear stress, which stimulates cells over areas approaching 1mm2. We demonstrate microTsunami-initiated mechanosignalling in primary human endothelial cells. This observed signalling is consistent with G-protein-coupled receptor stimulation resulting in Ca2+ release by the endoplasmic reticulum. Moreover, we demonstrate the dose-dependent modulation of microTsunami-induced Ca2+ signalling by introducing a known inhibitor to this pathway. The imaging of Ca2+ signalling, and its modulation by exogenous molecules, demonstrates the capacity to initiate and assess cellular mechanosignalling in real-time. We utilize this capability to screen the effects of a set of small molecules on cellular mechanotransduction in 96-well plates using standard imaging cytometry. PMID:25309621

  11. Comprehensive analysis of high-throughput screening data

    NASA Astrophysics Data System (ADS)

    Heyse, Stephan

    2002-06-01

    High-Throughput Screening (HTS) data in its entirety is a valuable raw material for the drug-discovery process. It provides the most compete information about the biological activity of a company's compounds. However, its quantity, complexity and heterogeneity require novel, sophisticated approaches in data analysis. At GeneData, we are developing methods for large-scale, synoptical mining of screening data in a five-step analysis: (1) Quality Assurance: Checking data for experimental artifacts and eliminating low quality data. (2) Biological Profiling: Clustering and ranking of compounds based on their biological activity, taking into account specific characteristics of HTS data. (3) Rule-based Classification: Applying user-defined rules to biological and chemical properties, and providing hypotheses on the biological mode-of-action of compounds. (4) Joint Biological-Chemical Analysis: Associating chemical compound data to HTS data, providing hypotheses for structure- activity relationships. (5) integration with Genomic and Gene Expression Data: Linking into other components of GeneData's bioinformatics platform, and assessing the compounds' modes-of-action, toxicity, and metabolic properties. These analyses address issues that are crucial for a correct interpretation and full exploitation of screening data. They lead to a sound rating of assays and compounds at an early state of the lead-finding process.

  12. High-Throughput Preparation of New Photoactive Nanocomposites.

    PubMed

    Conterosito, Eleonora; Benesperi, Iacopo; Toson, Valentina; Saccone, Davide; Barbero, Nadia; Palin, Luca; Barolo, Claudia; Gianotti, Valentina; Milanesio, Marco

    2016-06-01

    New low-cost photoactive hybrid materials based on organic luminescent molecules inserted into hydrotalcite (layered double hydroxides; LDH) were produced, which exploit the high-throughput liquid-assisted grinding (LAG) method. These materials are conceived for applications in dye-sensitized solar cells (DSSCs) as a co-absorbers and in silicon photovoltaic (PV) panels to improve their efficiency as they are able to emit where PV modules show the maximum efficiency. A molecule that shows a large Stokes' shift was designed, synthesized, and intercalated into LDH. Two dyes already used in DSSCs were also intercalated to produce two new nanocomposites. LDH intercalation allows the stability of organic dyes to be improved and their direct use in polymer melt blending. The prepared nanocomposites absorb sunlight from UV to visible and emit from blue to near-IR and thus can be exploited for light-energy management. Finally one nanocomposite was dispersed by melt blending into a poly(methyl methacrylate)-block-poly(n-butyl acrylate) copolymer to obtain a photoactive film. PMID:27137753

  13. Salmonella serotype determination utilizing high-throughput genome sequencing data.

    PubMed

    Zhang, Shaokang; Yin, Yanlong; Jones, Marcus B; Zhang, Zhenzhen; Deatherage Kaiser, Brooke L; Dinsmore, Blake A; Fitzgerald, Collette; Fields, Patricia I; Deng, Xiangyu

    2015-05-01

    Serotyping forms the basis of national and international surveillance networks for Salmonella, one of the most prevalent foodborne pathogens worldwide (1-3). Public health microbiology is currently being transformed by whole-genome sequencing (WGS), which opens the door to serotype determination using WGS data. SeqSero (www.denglab.info/SeqSero) is a novel Web-based tool for determining Salmonella serotypes using high-throughput genome sequencing data. SeqSero is based on curated databases of Salmonella serotype determinants (rfb gene cluster, fliC and fljB alleles) and is predicted to determine serotype rapidly and accurately for nearly the full spectrum of Salmonella serotypes (more than 2,300 serotypes), from both raw sequencing reads and genome assemblies. The performance of SeqSero was evaluated by testing (i) raw reads from genomes of 308 Salmonella isolates of known serotype; (ii) raw reads from genomes of 3,306 Salmonella isolates sequenced and made publicly available by GenomeTrakr, a U.S. national monitoring network operated by the Food and Drug Administration; and (iii) 354 other publicly available draft or complete Salmonella genomes. We also demonstrated Salmonella serotype determination from raw sequencing reads of fecal metagenomes from mice orally infected with this pathogen. SeqSero can help to maintain the well-established utility of Salmonella serotyping when integrated into a platform of WGS-based pathogen subtyping and characterization.

  14. A High-Throughput Screen for Alpha Particle Radiation Protectants

    PubMed Central

    Seideman, Jonathan H.; Shum, David; Djaballah, Hakim

    2010-01-01

    Abstract Alpha-particle-emitting elements are of increasing importance as environmental and occupational carcinogens, toxic components of radiation dispersal devices and accidents, and potent therapeutics in oncology. Alpha particle radiation differs from radiations of lower linear energy transfer in that it predominantly damages DNA via direct action. Because of this, radical scavengers effective for other radiations have had only limited effect in mitigating alpha particle toxicity. We describe here a simple assay and a pilot screen of 3,119 compounds in a high-throughput screen (HTS), using the alpha-particle-emitting isotope, 225Ac, for the discovery of compounds that might protect mammalian cells from alpha particles through novel mechanisms. The assay, which monitored the viability of a myeloid leukemic cell line upon alpha particle exposure, was robust and reproducible, yielding a Z' factor of 0.66 and a signal-to-noise ratio of nearly 10 to 1. Surprisingly, 1 compound emerged from this screen, epoxy-4,5-α-dihydroxysantonin (EDHS), that showed considerable protective activity. While the value of EDHS remains to be determined, its discovery is a proof of concept and validation of the utility of this HTS methodology. Further application of the described assay could yield compounds useful in minimizing the toxicity and carcinogenesis associated with alpha particle exposure. PMID:20658946

  15. High-throughput screening in academia: the Harvard experience.

    PubMed

    Stein, Ross L

    2003-12-01

    To identify small-molecule modulators of biologic systems, academic scientists are beginning to use high-throughput screening (HTS) approaches that have traditionally been used only in industry. The HTS laboratories that are being established in universities, while differing in details of staffing, equipment, and size, have all been created to attain 1 or more of 3 principal goals: drug discovery, chemical genetics, or training. This article will examine the role that these activities play in 4 HTS laboratories that have been created within the academic community of Harvard Medical School and its affiliated institutions. First, the 3 activities will be defined with special attention paid to describing the impact they are having on how academic biologic science is conducted today. Next, the histories and operations of the 4 Harvard laboratories are reviewed. In the course of these summaries, emphasis is placed on understanding the motivational role that the 3 activities initially played in the creation of the 4 Harvard facilities and the roles that the activities continue to play in their day-to-day operations. Finally, several concerns are identified that must be attended to for the successful establishment and operation of an academic biologic science that has yet to be fully determined. HTS has the ability to provide the tools to test previously untestable hypotheses and can thereby allow the discovery of the unanticipated and the truly novel.

  16. High Throughput Multispectral Image Processing with Applications in Food Science

    PubMed Central

    Tsakanikas, Panagiotis; Pavlidis, Dimitris; Nychas, George-John

    2015-01-01

    Recently, machine vision is gaining attention in food science as well as in food industry concerning food quality assessment and monitoring. Into the framework of implementation of Process Analytical Technology (PAT) in the food industry, image processing can be used not only in estimation and even prediction of food quality but also in detection of adulteration. Towards these applications on food science, we present here a novel methodology for automated image analysis of several kinds of food products e.g. meat, vanilla crème and table olives, so as to increase objectivity, data reproducibility, low cost information extraction and faster quality assessment, without human intervention. Image processing’s outcome will be propagated to the downstream analysis. The developed multispectral image processing method is based on unsupervised machine learning approach (Gaussian Mixture Models) and a novel unsupervised scheme of spectral band selection for segmentation process optimization. Through the evaluation we prove its efficiency and robustness against the currently available semi-manual software, showing that the developed method is a high throughput approach appropriate for massive data extraction from food samples. PMID:26466349

  17. High Throughput Profiling of Molecular Shapes in Crystals

    PubMed Central

    Spackman, Peter R.; Thomas, Sajesh P.; Jayatilaka, Dylan

    2016-01-01

    Molecular shape is important in both crystallisation and supramolecular assembly, yet its role is not completely understood. We present a computationally efficient scheme to describe and classify the molecular shapes in crystals. The method involves rotation invariant description of Hirshfeld surfaces in terms of of spherical harmonic functions. Hirshfeld surfaces represent the boundaries of a molecule in the crystalline environment, and are widely used to visualise and interpret crystalline interactions. The spherical harmonic description of molecular shapes are compared and classified by means of principal component analysis and cluster analysis. When applied to a series of metals, the method results in a clear classification based on their lattice type. When applied to around 300 crystal structures comprising of series of substituted benzenes, naphthalenes and phenylbenzamide it shows the capacity to classify structures based on chemical scaffolds, chemical isosterism, and conformational similarity. The computational efficiency of the method is demonstrated with an application to over 14 thousand crystal structures. High throughput screening of molecular shapes and interaction surfaces in the Cambridge Structural Database (CSD) using this method has direct applications in drug discovery, supramolecular chemistry and materials design. PMID:26908351

  18. Detecting Alu insertions from high-throughput sequencing data

    PubMed Central

    David, Matei; Mustafa, Harun; Brudno, Michael

    2013-01-01

    High-throughput sequencing technologies have allowed for the cataloguing of variation in personal human genomes. In this manuscript, we present alu-detect, a tool that combines read-pair and split-read information to detect novel Alus and their precise breakpoints directly from either whole-genome or whole-exome sequencing data while also identifying insertions directly in the vicinity of existing Alus. To set the parameters of our method, we use simulation of a faux reference, which allows us to compute the precision and recall of various parameter settings using real sequencing data. Applying our method to 100 bp paired Illumina data from seven individuals, including two trios, we detected on average 1519 novel Alus per sample. Based on the faux-reference simulation, we estimate that our method has 97% precision and 85% recall. We identify 808 novel Alus not previously described in other studies. We also demonstrate the use of alu-detect to study the local sequence and global location preferences for novel Alu insertions. PMID:23921633

  19. High throughput LSPR and SERS analysis of aminoglycoside antibiotics.

    PubMed

    McKeating, Kristy S; Couture, Maxime; Dinel, Marie-Pier; Garneau-Tsodikova, Sylvie; Masson, Jean-Francois

    2016-08-15

    Aminoglycoside antibiotics are used in the treatment of infections caused by Gram-negative bacteria, and are often dispensed only in severe cases due to their adverse side effects. Patients undergoing treatment with these antibiotics are therefore commonly subjected to therapeutic drug monitoring (TDM) to ensure a safe and effective personalised dosage. The ability to detect these antibiotics in a rapid and sensitive manner in human fluids is therefore of the utmost importance in order to provide effective monitoring of these drugs, which could potentially allow for a more widespread use of this class of antibiotics. Herein, we report on the detection of various aminoglycosides, by exploiting their ability to aggregate gold nanoparticles. The number and position of the amino groups of aminoglycoside antibiotics controlled the aggregation process. We investigated the complementary techniques of surface enhanced Raman spectroscopy (SERS) and localized surface plasmon resonance (LSPR) for dual detection of these aminoglycoside antibiotics and performed an in-depth study of the feasibility of carrying out TDM of tobramycin using a platform amenable to high throughput analysis. Herein, we also demonstrate dual detection of tobramycin using both LSPR and SERS in a single platform and within the clinically relevant concentration range needed for TDM of this particular aminoglycoside. Additionally we provide evidence that tobramycin can be detected in spiked human serum using only functionalised nanoparticles and SERS analysis. PMID:27412506

  20. High-throughput screening technologies for drug glucuronidation profiling.

    PubMed

    Trubetskoy, Olga; Finel, Moshe; Trubetskoy, Vladimir

    2008-08-01

    A significant number of endogenous and exogenous compounds, including many therapeutic agents, are metabolized in humans via glucuronidation, catalysed by uridine diphosphoglucuronosyltransferases (UGTs). The study of the UGTs is a growing field of research, with constantly accumulated and updated information regarding UGT structure, purification, substrate specificity and inhibition, including clinically relevant drug interactions. Development of reliable UGT assays for the assessment of individual isoform substrate specificity and for the discovery of novel isoform-specific substrates and inhibitors is crucial for understanding the function and regulation of the UGT enzyme family and its clinical and pharmacological relevance. High-throughput screening (HTS) is a powerful technology used to search for novel substrates and inhibitors for a wide variety of targets. However, application of HTS in the context of UGTs is complicated because of the poor stability, low levels of expression, low affinity and broad substrate specificity of the enzymes, combined with difficulties in obtaining individual UGT isoforms in purified format, and insufficient information regarding isoform-specific substrates and inhibitors. This review examines the current status of HTS assays used in the search for novel UGT substrates and inhibitors, emphasizing advancements and challenges in HTS technologies for drug glucuronidation profiling, and discusses possible avenues for future advancement of the field.

  1. High Throughput Profiling of Molecular Shapes in Crystals.

    PubMed

    Spackman, Peter R; Thomas, Sajesh P; Jayatilaka, Dylan

    2016-01-01

    Molecular shape is important in both crystallisation and supramolecular assembly, yet its role is not completely understood. We present a computationally efficient scheme to describe and classify the molecular shapes in crystals. The method involves rotation invariant description of Hirshfeld surfaces in terms of of spherical harmonic functions. Hirshfeld surfaces represent the boundaries of a molecule in the crystalline environment, and are widely used to visualise and interpret crystalline interactions. The spherical harmonic description of molecular shapes are compared and classified by means of principal component analysis and cluster analysis. When applied to a series of metals, the method results in a clear classification based on their lattice type. When applied to around 300 crystal structures comprising of series of substituted benzenes, naphthalenes and phenylbenzamide it shows the capacity to classify structures based on chemical scaffolds, chemical isosterism, and conformational similarity. The computational efficiency of the method is demonstrated with an application to over 14 thousand crystal structures. High throughput screening of molecular shapes and interaction surfaces in the Cambridge Structural Database (CSD) using this method has direct applications in drug discovery, supramolecular chemistry and materials design. PMID:26908351

  2. The JCSG high-throughput structural biology pipeline.

    PubMed

    Elsliger, Marc André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wooley, John; Wüthrich, Kurt; Wilson, Ian A

    2010-10-01

    The Joint Center for Structural Genomics high-throughput structural biology pipeline has delivered more than 1000 structures to the community over the past ten years. The JCSG has made a significant contribution to the overall goal of the NIH Protein Structure Initiative (PSI) of expanding structural coverage of the protein universe, as well as making substantial inroads into structural coverage of an entire organism. Targets are processed through an extensive combination of bioinformatics and biophysical analyses to efficiently characterize and optimize each target prior to selection for structure determination. The pipeline uses parallel processing methods at almost every step in the process and can adapt to a wide range of protein targets from bacterial to human. The construction, expansion and optimization of the JCSG gene-to-structure pipeline over the years have resulted in many technological and methodological advances and developments. The vast number of targets and the enormous amounts of associated data processed through the multiple stages of the experimental pipeline required the development of variety of valuable resources that, wherever feasible, have been converted to free-access web-based tools and applications.

  3. New Lung Cancer Panel for High-Throughput Targeted Resequencing

    PubMed Central

    Kim, Eun-Hye; Lee, Sunghoon; Park, Jongsun; Lee, Kyusang; Bhak, Jong

    2014-01-01

    We present a new next-generation sequencing-based method to identify somatic mutations of lung cancer. It is a comprehensive mutation profiling protocol to detect somatic mutations in 30 genes found frequently in lung adenocarcinoma. The total length of the target regions is 107 kb, and a capture assay was designed to cover 99% of it. This method exhibited about 97% mean coverage at 30× sequencing depth and 42% average specificity when sequencing of more than 3.25 Gb was carried out for the normal sample. We discovered 513 variations from targeted exome sequencing of lung cancer cells, which is 3.9-fold higher than in the normal sample. The variations in cancer cells included previously reported somatic mutations in the COSMIC database, such as variations in TP53, KRAS, and STK11 of sample H-23 and in EGFR of sample H-1650, especially with more than 1,000× coverage. Among the somatic mutations, up to 91% of single nucleotide polymorphisms from the two cancer samples were validated by DNA microarray-based genotyping. Our results demonstrated the feasibility of high-throughput mutation profiling with lung adenocarcinoma samples, and the profiling method can be used as a robust and effective protocol for somatic variant screening. PMID:25031567

  4. A microfluidic, high throughput protein crystal growth method for microgravity.

    PubMed

    Carruthers, Carl W; Gerdts, Cory; Johnson, Michael D; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions' microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 10(3) cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories.

  5. Microfluidic hydrodynamic focusing for high-throughput applications

    NASA Astrophysics Data System (ADS)

    Zhao, Jingjing; You, Zheng

    2015-12-01

    Microfluidic hydrodynamic focusing is critical for chip-based bioanalytical systems to increase throughput and sensitivity, especially for microflow cytometers, enabling a sample flow to be confined to the center of a microchannel with a narrow cross-section. Current microfluidic hydrodynamic focusing designs are usually unable to maintain stable focusing in high flow velocity conditions, resulting in a large cross-section or even failed focusing. To overcome this challenge, this paper aims to develop a design that can achieve effective microfluidic hydrodynamic focusing at high velocity with favorable performance. For this purpose, specially designed structures and arc-shaped channels are used. Two focusing regions are modeled and optimized mathematically, and flow behavior is investigated using numerical simulations. The functional relationship between flow rates and the cross-sectional dimensions of the focused sample flow is explored, and a measurement method for testing the dimensions is developed. The design is implemented in glass chips and characterized experimentally. In a rectangular channel with a cross-section of 300 μm  ×  150 μm the sample flow can be focused down to 5-11 μm horizontally and 10-21 μm vertically at a roughly constant velocity of 4.4 m s-1 when the sample flow rate varies between 10 and 60 μl min-1. Effective focusing is accessible within a wide velocity range from 0.7 to 10 m s-1. The experimental results validate that the focusing design performs better than existing microfluidic designs at high velocities, while its performance is close to that of the designs used in conventional flow cytometers with much less volume and a simpler structure. The focusing design can serve as the basis for microflow cytometers or it can be integrated into various microfluidic systems where complete focusing is needed.

  6. Solion ion source for high-efficiency, high-throughput solar cell manufacturing

    SciTech Connect

    Koo, John Binns, Brant; Miller, Timothy; Krause, Stephen; Skinner, Wesley; Mullin, James

    2014-02-15

    In this paper, we introduce the Solion ion source for high-throughput solar cell doping. As the source power is increased to enable higher throughput, negative effects degrade the lifetime of the plasma chamber and the extraction electrodes. In order to improve efficiency, we have explored a wide range of electron energies and determined the conditions which best suit production. To extend the lifetime of the source we have developed an in situ cleaning method using only existing hardware. With these combinations, source life-times of >200 h for phosphorous and >100 h for boron ion beams have been achieved while maintaining 1100 cell-per-hour production.

  7. Towards high throughput screening of electrochemical stability of battery electrolytes.

    PubMed

    Borodin, Oleg; Olguin, Marco; Spear, Carrie E; Leiter, Kenneth W; Knap, Jaroslaw

    2015-09-01

    High throughput screening of solvents and additives with potential applications in lithium batteries is reported. The initial test set is limited to carbonate and phosphate-based compounds and focused on their electrochemical properties. Solvent stability towards first and second reduction and oxidation is reported from density functional theory (DFT) calculations performed on isolated solvents surrounded by implicit solvent. The reorganization energy is estimated from the difference between vertical and adiabatic redox energies and found to be especially important for the accurate prediction of reduction stability. A majority of tested compounds had the second reduction potential higher than the first reduction potential indicating that the second reduction reaction might play an important role in the passivation layer formation. Similarly, the second oxidation potential was smaller for a significant subset of tested molecules than the first oxidation potential. A number of potential sources of errors introduced during screening of the electrolyte electrochemical properties were examined. The formation of lithium fluoride during reduction of semifluorinated solvents such as fluoroethylene carbonate and the H-transfer during oxidation of solvents were found to shift the electrochemical potential by 1.5-2 V and could shrink the electrochemical stability window by as much as 3.5 V when such reactions are included in the screening procedure. The initial oxidation reaction of ethylene carbonate and dimethyl carbonate at the surface of the completely de-lithiated LiNi0.5Mn1.5O4 high voltage spinel cathode was examined using DFT. Depending on the molecular orientation at the cathode surface, a carbonate molecule either exhibited deprotonation or was found bound to the transition metal via its carbonyl oxygen. PMID:26266636

  8. Towards high throughput screening of electrochemical stability of battery electrolytes

    NASA Astrophysics Data System (ADS)

    Borodin, Oleg; Olguin, Marco; Spear, Carrie E.; Leiter, Kenneth W.; Knap, Jaroslaw

    2015-09-01

    High throughput screening of solvents and additives with potential applications in lithium batteries is reported. The initial test set is limited to carbonate and phosphate-based compounds and focused on their electrochemical properties. Solvent stability towards first and second reduction and oxidation is reported from density functional theory (DFT) calculations performed on isolated solvents surrounded by implicit solvent. The reorganization energy is estimated from the difference between vertical and adiabatic redox energies and found to be especially important for the accurate prediction of reduction stability. A majority of tested compounds had the second reduction potential higher than the first reduction potential indicating that the second reduction reaction might play an important role in the passivation layer formation. Similarly, the second oxidation potential was smaller for a significant subset of tested molecules than the first oxidation potential. A number of potential sources of errors introduced during screening of the electrolyte electrochemical properties were examined. The formation of lithium fluoride during reduction of semifluorinated solvents such as fluoroethylene carbonate and the H-transfer during oxidation of solvents were found to shift the electrochemical potential by 1.5-2 V and could shrink the electrochemical stability window by as much as 3.5 V when such reactions are included in the screening procedure. The initial oxidation reaction of ethylene carbonate and dimethyl carbonate at the surface of the completely de-lithiated LiNi0.5Mn1.5O4 high voltage spinel cathode was examined using DFT. Depending on the molecular orientation at the cathode surface, a carbonate molecule either exhibited deprotonation or was found bound to the transition metal via its carbonyl oxygen.

  9. Towards high throughput screening of electrochemical stability of battery electrolytes.

    PubMed

    Borodin, Oleg; Olguin, Marco; Spear, Carrie E; Leiter, Kenneth W; Knap, Jaroslaw

    2015-09-01

    High throughput screening of solvents and additives with potential applications in lithium batteries is reported. The initial test set is limited to carbonate and phosphate-based compounds and focused on their electrochemical properties. Solvent stability towards first and second reduction and oxidation is reported from density functional theory (DFT) calculations performed on isolated solvents surrounded by implicit solvent. The reorganization energy is estimated from the difference between vertical and adiabatic redox energies and found to be especially important for the accurate prediction of reduction stability. A majority of tested compounds had the second reduction potential higher than the first reduction potential indicating that the second reduction reaction might play an important role in the passivation layer formation. Similarly, the second oxidation potential was smaller for a significant subset of tested molecules than the first oxidation potential. A number of potential sources of errors introduced during screening of the electrolyte electrochemical properties were examined. The formation of lithium fluoride during reduction of semifluorinated solvents such as fluoroethylene carbonate and the H-transfer during oxidation of solvents were found to shift the electrochemical potential by 1.5-2 V and could shrink the electrochemical stability window by as much as 3.5 V when such reactions are included in the screening procedure. The initial oxidation reaction of ethylene carbonate and dimethyl carbonate at the surface of the completely de-lithiated LiNi0.5Mn1.5O4 high voltage spinel cathode was examined using DFT. Depending on the molecular orientation at the cathode surface, a carbonate molecule either exhibited deprotonation or was found bound to the transition metal via its carbonyl oxygen.

  10. Mining Chemical Activity Status from High-Throughput Screening Assays

    PubMed Central

    Soufan, Othman; Ba-alawi, Wail; Afeef, Moataz; Essack, Magbubah; Rodionov, Valentin; Kalnis, Panos; Bajic, Vladimir B.

    2015-01-01

    High-throughput screening (HTS) experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive) in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR) in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare. PMID:26658480

  11. High-throughput screening method for lipases/esterases.

    PubMed

    Mateos-Díaz, Eduardo; Rodríguez, Jorge Alberto; de Los Ángeles Camacho-Ruiz, María; Mateos-Díaz, Juan Carlos

    2012-01-01

    High-throughput screening (HTS) methods for lipases and esterases are generally performed by using synthetic chromogenic substrates (e.g., p-nitrophenyl, resorufin, and umbelliferyl esters) which may be misleading since they are not their natural substrates (e.g., partially or insoluble triglycerides). In previous works, we have shown that soluble nonchromogenic substrates and p-nitrophenol (as a pH indicator) can be used to quantify the hydrolysis and estimate the substrate selectivity of lipases and esterases from several sources. However, in order to implement a spectrophotometric HTS method using partially or insoluble triglycerides, it is necessary to find particular conditions which allow a quantitative detection of the enzymatic activity. In this work, we used Triton X-100, CHAPS, and N-lauroyl sarcosine as emulsifiers, β-cyclodextrin as a fatty acid captor, and two substrate concentrations, 1 mM of tributyrin (TC4) and 5 mM of trioctanoin (TC8), to improve the test conditions. To demonstrate the utility of this method, we screened 12 enzymes (commercial preparations and culture broth extracts) for the hydrolysis of TC4 and TC8, which are both classical substrates for lipases and esterases (for esterases, only TC4 may be hydrolyzed). Subsequent pH-stat experiments were performed to confirm the preference of substrate hydrolysis with the hydrolases tested. We have shown that this method is very useful for screening a high number of lipases (hydrolysis of TC4 and TC8) or esterases (only hydrolysis of TC4) from wild isolates or variants generated by directed evolution using nonchromogenic triglycerides directly in the test.

  12. Anchored hybrid enrichment for massively high-throughput phylogenomics.

    PubMed

    Lemmon, Alan R; Emme, Sandra A; Lemmon, Emily Moriarty

    2012-10-01

    The field of phylogenetics is on the cusp of a major revolution, enabled by new methods of data collection that leverage both genomic resources and recent advances in DNA sequencing. Previous phylogenetic work has required labor-intensive marker development coupled with single-locus polymerase chain reaction and DNA sequencing on clade-by-clade and locus-by-locus basis. Here, we present a new, cost-efficient, and rapid approach to obtaining data from hundreds of loci for potentially hundreds of individuals for deep and shallow phylogenetic studies. Specifically, we designed probes for target enrichment of >500 loci in highly conserved anchor regions of vertebrate genomes (flanked by less conserved regions) from five model species and tested enrichment efficiency in nonmodel species up to 508 million years divergent from the nearest model. We found that hybrid enrichment using conserved probes (anchored enrichment) can recover a large number of unlinked loci that are useful at a diversity of phylogenetic timescales. This new approach has the potential not only to expedite resolution of deep-scale portions of the Tree of Life but also to greatly accelerate resolution of the large number of shallow clades that remain unresolved. The combination of low cost (~1% of the cost of traditional Sanger sequencing and ~3.5% of the cost of high-throughput amplicon sequencing for projects on the scale of 500 loci × 100 individuals) and rapid data collection (~2 weeks of laboratory time) are expected to make this approach tractable even for researchers working on systems with limited or nonexistent genomic resources. PMID:22605266

  13. High-throughput neuroimaging-genetics computational infrastructure

    PubMed Central

    Dinov, Ivo D.; Petrosyan, Petros; Liu, Zhizhong; Eggert, Paul; Hobel, Sam; Vespa, Paul; Woo Moon, Seok; Van Horn, John D.; Franco, Joseph; Toga, Arthur W.

    2014-01-01

    Many contemporary neuroscientific investigations face significant challenges in terms of data management, computational processing, data mining, and results interpretation. These four pillars define the core infrastructure necessary to plan, organize, orchestrate, validate, and disseminate novel scientific methods, computational resources, and translational healthcare findings. Data management includes protocols for data acquisition, archival, query, transfer, retrieval, and aggregation. Computational processing involves the necessary software, hardware, and networking infrastructure required to handle large amounts of heterogeneous neuroimaging, genetics, clinical, and phenotypic data and meta-data. Data mining refers to the process of automatically extracting data features, characteristics and associations, which are not readily visible by human exploration of the raw dataset. Result interpretation includes scientific visualization, community validation of findings and reproducible findings. In this manuscript we describe the novel high-throughput neuroimaging-genetics computational infrastructure available at the Institute for Neuroimaging and Informatics (INI) and the Laboratory of Neuro Imaging (LONI) at University of Southern California (USC). INI and LONI include ultra-high-field and standard-field MRI brain scanners along with an imaging-genetics database for storing the complete provenance of the raw and derived data and meta-data. In addition, the institute provides a large number of software tools for image and shape analysis, mathematical modeling, genomic sequence processing, and scientific visualization. A unique feature of this architecture is the Pipeline environment, which integrates the data management, processing, transfer, and visualization. Through its client-server architecture, the Pipeline environment provides a graphical user interface for designing, executing, monitoring validating, and disseminating of complex protocols that utilize

  14. Applications of Biophysics in High-Throughput Screening Hit Validation.

    PubMed

    Genick, Christine Clougherty; Barlier, Danielle; Monna, Dominique; Brunner, Reto; Bé, Céline; Scheufler, Clemens; Ottl, Johannes

    2014-06-01

    For approximately a decade, biophysical methods have been used to validate positive hits selected from high-throughput screening (HTS) campaigns with the goal to verify binding interactions using label-free assays. By applying label-free readouts, screen artifacts created by compound interference and fluorescence are discovered, enabling further characterization of the hits for their target specificity and selectivity. The use of several biophysical methods to extract this type of high-content information is required to prevent the promotion of false positives to the next level of hit validation and to select the best candidates for further chemical optimization. The typical technologies applied in this arena include dynamic light scattering, turbidometry, resonance waveguide, surface plasmon resonance, differential scanning fluorimetry, mass spectrometry, and others. Each technology can provide different types of information to enable the characterization of the binding interaction. Thus, these technologies can be incorporated in a hit-validation strategy not only according to the profile of chemical matter that is desired by the medicinal chemists, but also in a manner that is in agreement with the target protein's amenability to the screening format. Here, we present the results of screening strategies using biophysics with the objective to evaluate the approaches, discuss the advantages and challenges, and summarize the benefits in reference to lead discovery. In summary, the biophysics screens presented here demonstrated various hit rates from a list of ~2000 preselected, IC50-validated hits from HTS (an IC50 is the inhibitor concentration at which 50% inhibition of activity is observed). There are several lessons learned from these biophysical screens, which will be discussed in this article.

  15. High-throughput screening method for lipases/esterases.

    PubMed

    Mateos-Díaz, Eduardo; Rodríguez, Jorge Alberto; de Los Ángeles Camacho-Ruiz, María; Mateos-Díaz, Juan Carlos

    2012-01-01

    High-throughput screening (HTS) methods for lipases and esterases are generally performed by using synthetic chromogenic substrates (e.g., p-nitrophenyl, resorufin, and umbelliferyl esters) which may be misleading since they are not their natural substrates (e.g., partially or insoluble triglycerides). In previous works, we have shown that soluble nonchromogenic substrates and p-nitrophenol (as a pH indicator) can be used to quantify the hydrolysis and estimate the substrate selectivity of lipases and esterases from several sources. However, in order to implement a spectrophotometric HTS method using partially or insoluble triglycerides, it is necessary to find particular conditions which allow a quantitative detection of the enzymatic activity. In this work, we used Triton X-100, CHAPS, and N-lauroyl sarcosine as emulsifiers, β-cyclodextrin as a fatty acid captor, and two substrate concentrations, 1 mM of tributyrin (TC4) and 5 mM of trioctanoin (TC8), to improve the test conditions. To demonstrate the utility of this method, we screened 12 enzymes (commercial preparations and culture broth extracts) for the hydrolysis of TC4 and TC8, which are both classical substrates for lipases and esterases (for esterases, only TC4 may be hydrolyzed). Subsequent pH-stat experiments were performed to confirm the preference of substrate hydrolysis with the hydrolases tested. We have shown that this method is very useful for screening a high number of lipases (hydrolysis of TC4 and TC8) or esterases (only hydrolysis of TC4) from wild isolates or variants generated by directed evolution using nonchromogenic triglycerides directly in the test. PMID:22426713

  16. High Throughput Screening for Drugs that Modulate Intermediate Filament Proteins

    PubMed Central

    Sun, Jingyuan; Groppi, Vincent E.; Gui, Honglian; Chen, Lu; Xie, Qing; Liu, Li

    2016-01-01

    Intermediate filament (IF) proteins have unique and complex cell and tissue distribution. Importantly, IF gene mutations cause or predispose to more than 80 human tissue-specific diseases (IF-pathies), with the most severe disease phenotypes being due to mutations at conserved residues that result in a disrupted IF network. A critical need for the entire IF-pathy field is the identification of drugs that can ameliorate or cure these diseases, particularly since all current therapies target the IF-pathy complication, such as diabetes or cardiovascular disease, rather than the mutant IF protein or gene. We describe a high throughput approach to identify drugs that can normalize disrupted IF proteins. This approach utilizes transduction of lentivirus that expresses green-fluorescent-protein-tagged keratin 18 (K18) R90C in A549 cells. The readout is drug ‘hits’ that convert the dot-like keratin filament distribution, due to the R90C mutation, to a wildtype-like filamentous array. A similar strategy can be used to screen thousands of compounds and can be utilized for practically any IF protein with a filament-disrupting mutation, and could therefore potentially target many IF-pathies. ‘Hits’ of interest require validation in cell culture then using in vivo experimental models. Approaches to study the mechanism of mutant-IF normalization by potential drugs of interest are also described. The ultimate goal of this drug screening approach is to identify effective and safe compounds that can potentially be tested for clinical efficacy in patients. PMID:26795471

  17. Virtual High-Throughput Screening To Identify Novel Activin Antagonists

    PubMed Central

    Zhu, Jie; Mishra, Rama K.; Schiltz, Gary E.; Makanji, Yogeshwar; Scheidt, Karl A.; Mazar, Andrew P.; Woodruff, Teresa K.

    2015-01-01

    Activin belongs to the TGFβ superfamily, which is associated with several disease conditions, including cancer-related cachexia, preterm labor with delivery, and osteoporosis. Targeting activin and its related signaling pathways holds promise as a therapeutic approach to these diseases. A small-molecule ligand-binding groove was identified in the interface between the two activin βA subunits and was used for a virtual high-throughput in silico screening of the ZINC database to identify hits. Thirty-nine compounds without significant toxicity were tested in two well-established activin assays: FSHβ transcription and HepG2 cell apoptosis. This screening workflow resulted in two lead compounds: NUCC-474 and NUCC-555. These potential activin antagonists were then shown to inhibit activin A-mediated cell proliferation in ex vivo ovary cultures. In vivo testing showed that our most potent compound (NUCC-555) caused a dose-dependent decrease in FSH levels in ovariectomized mice. The Blitz competition binding assay confirmed target binding of NUCC-555 to the activin A:ActRII that disrupts the activin A:ActRII complex’s binding with ALK4-ECD-Fc in a dose-dependent manner. The NUCC-555 also specifically binds to activin A compared with other TGFβ superfamily member myostatin (GDF8). These data demonstrate a new in silico-based strategy for identifying small-molecule activin antagonists. Our approach is the first to identify a first-in-class small-molecule antagonist of activin binding to ALK4, which opens a completely new approach to inhibiting the activity of TGFβ receptor superfamily members. in addition, the lead compound can serve as a starting point for lead optimization toward the goal of a compound that may be effective in activin-mediated diseases. PMID:26098096

  18. Towards Chip Scale Liquid Chromatography and High Throughput Immunosensing

    SciTech Connect

    Ni, J.

    2000-09-21

    This work describes several research projects aimed towards developing new instruments and novel methods for high throughput chemical and biological analysis. Approaches are taken in two directions. The first direction takes advantage of well-established semiconductor fabrication techniques and applies them to miniaturize instruments that are workhorses in analytical laboratories. Specifically, the first part of this work focused on the development of micropumps and microvalves for controlled fluid delivery. The mechanism of these micropumps and microvalves relies on the electrochemically-induced surface tension change at a mercury/electrolyte interface. A miniaturized flow injection analysis device was integrated and flow injection analyses were demonstrated. In the second part of this work, microfluidic chips were also designed, fabricated, and tested. Separations of two fluorescent dyes were demonstrated in microfabricated channels, based on an open-tubular liquid chromatography (OT LC) or an electrochemically-modulated liquid chromatography (EMLC) format. A reduction in instrument size can potentially increase analysis speed, and allow exceedingly small amounts of sample to be analyzed under diverse separation conditions. The second direction explores the surface enhanced Raman spectroscopy (SERS) as a signal transduction method for immunoassay analysis. It takes advantage of the improved detection sensitivity as a result of surface enhancement on colloidal gold, the narrow width of Raman band, and the stability of Raman scattering signals to distinguish several different species simultaneously without exploiting spatially-separated addresses on a biochip. By labeling gold nanoparticles with different Raman reporters in conjunction with different detection antibodies, a simultaneous detection of a dual-analyte immunoassay was demonstrated. Using this scheme for quantitative analysis was also studied and preliminary dose-response curves from an immunoassay of a

  19. Potentiometric sensor for the high throughput determination of tetramisole hydrochloride.

    PubMed

    Gupta, Vinod Kumar; Singh, Ashok Kumar; Gupta, Barkha

    2007-08-01

    The electrochemical response characteristics of poly(vinyl)chloride (PVC) based membrane sensors for determination of tetramisole hydrochloride (TmCl) is described. The membranes of these electrodes consist of tetramisole-tetraphenyl borate (Tm-TPB), chlorophenyl borate (Tm-ClPB), and phosphotungstate (Tm(3)-PT) ion associations dispersed in a PVC matrix with dibutylpthalate as a plasticizer. The electrodes were fully characterized in terms of composition, life span, usable pH range, and working concentration range and ionic strength. The electrodes showed Nernstian response over the concentration ranges of 7.4 x 10(-7) to 1.0 x 10(-2) M, 1.7 x 10(-6) to 1.0 x 10(-2) M, and 5.6 x 10(-6) to 1.0 x 10(-2) M TmCl, respectively, and were applied to the potentiometric determination of tetramisole ion in pure solutions and pharmaceutical preparations. The potentiometric determination was also used in the determination of tetramisole in pharmaceutical preparations in four batches of different expiration dates. The electrodes exhibited good selectivity for TmCl with respect to a large number of excipients such as inorganic cations, organic cations, amino acids, and sugars. The solubility product of the ion-pair and the formation constant of the precipitation reaction leading to the ion-pair formation were determined conductometrically. The new potentiometric method offers the advantages of high-throughput determination, simplicity, accuracy, automation feasibility, and applicability to turbid and colored sample solutions. PMID:17979641

  20. Low inlet gas velocity high throughput biomass gasifier

    DOEpatents

    Feldmann, Herman F.; Paisley, Mark A.

    1989-01-01

    The present invention discloses a novel method of operating a gasifier for production of fuel gas from carbonaceous fuels. The process disclosed enables operating in an entrained mode using inlet gas velocities of less than 7 feet per second, feedstock throughputs exceeding 4000 lbs/ft.sup.2 -hr, and pressures below 100 psia.

  1. HT-Paxos: High Throughput State-Machine Replication Protocol for Large Clustered Data Centers

    PubMed Central

    Agarwal, Ajay

    2015-01-01

    Paxos is a prominent theory of state-machine replication. Recent data intensive systems that implement state-machine replication generally require high throughput. Earlier versions of Paxos as few of them are classical Paxos, fast Paxos, and generalized Paxos have a major focus on fault tolerance and latency but lacking in terms of throughput and scalability. A major reason for this is the heavyweight leader. Through offloading the leader, we can further increase throughput of the system. Ring Paxos, Multiring Paxos, and S-Paxos are few prominent attempts in this direction for clustered data centers. In this paper, we are proposing HT-Paxos, a variant of Paxos that is the best suitable for any large clustered data center. HT-Paxos further offloads the leader very significantly and hence increases the throughput and scalability of the system, while at the same time, among high throughput state-machine replication protocols, it provides reasonably low latency and response time. PMID:25821856

  2. High-sensitivity high-throughput chip based biosensor array for multiplexed detection of heavy metals

    NASA Astrophysics Data System (ADS)

    Yan, Hai; Tang, Naimei; Jairo, Grace A.; Chakravarty, Swapnajit; Blake, Diane A.; Chen, Ray T.

    2016-03-01

    Heavy metal ions released into the environment from industrial processes lead to various health hazards. We propose an on-chip label-free detection approach that allows high-sensitivity and high-throughput detection of heavy metals. The sensing device consists of 2-dimensional photonic crystal microcavities that are combined by multimode interferometer to form a sensor array. We experimentally demonstrate the detection of cadmium-chelate conjugate with concentration as low as 5 parts-per-billion (ppb).

  3. High-throughput Saccharification assay for lignocellulosic materials.

    PubMed

    Gomez, Leonardo D; Whitehead, Caragh; Roberts, Philip; McQueen-Mason, Simon J

    2011-07-03

    Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the phenolic polymer lignin, that is also difficult to digest (1). In order to improve the digestibility of plant materials we need to discover the main bottlenecks for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification (2). These tasks require a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system. This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2

  4. High-throughput detection method for influenza virus.

    PubMed

    Kumar, Pawan; Bartoszek, Allison E; Moran, Thomas M; Gorski, Jack; Bhattacharyya, Sanjib; Navidad, Jose F; Thakar, Monica S; Malarkannan, Subramaniam

    2012-01-01

    Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture (1,2). Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus (3) to test the efficacy of this technique using MDCK cells (4). MDCK cells (10(4) or 5 x 10(3) per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 (5) and hemagglutinin (1) are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 10(2)-10(5) PFU could be consistently observed. Minimal but detectable positivity consistently seen with 10(2)-10(3) PFU PR8 viral titers demonstrated the high

  5. Integration of Dosimetry, Exposure and High-Throughput Screening Data in Chemical Toxicity Assessment

    EPA Science Inventory

    High-throughput in vitro toxicity screening can provide an efficient way to identify potential biological targets for chemicals. However, relying on nominal assay concentrations may misrepresent potential in vivo effects of these chemicals due to differences in bioavailability, c...

  6. High-Throughput Industrial Coatings Research at The Dow Chemical Company.

    PubMed

    Kuo, Tzu-Chi; Malvadkar, Niranjan A; Drumright, Ray; Cesaretti, Richard; Bishop, Matthew T

    2016-09-12

    At The Dow Chemical Company, high-throughput research is an active area for developing new industrial coatings products. Using the principles of automation (i.e., using robotic instruments), parallel processing (i.e., prepare, process, and evaluate samples in parallel), and miniaturization (i.e., reduce sample size), high-throughput tools for synthesizing, formulating, and applying coating compositions have been developed at Dow. In addition, high-throughput workflows for measuring various coating properties, such as cure speed, hardness development, scratch resistance, impact toughness, resin compatibility, pot-life, surface defects, among others have also been developed in-house. These workflows correlate well with the traditional coatings tests, but they do not necessarily mimic those tests. The use of such high-throughput workflows in combination with smart experimental designs allows accelerated discovery and commercialization.

  7. High-Throughput Industrial Coatings Research at The Dow Chemical Company.

    PubMed

    Kuo, Tzu-Chi; Malvadkar, Niranjan A; Drumright, Ray; Cesaretti, Richard; Bishop, Matthew T

    2016-09-12

    At The Dow Chemical Company, high-throughput research is an active area for developing new industrial coatings products. Using the principles of automation (i.e., using robotic instruments), parallel processing (i.e., prepare, process, and evaluate samples in parallel), and miniaturization (i.e., reduce sample size), high-throughput tools for synthesizing, formulating, and applying coating compositions have been developed at Dow. In addition, high-throughput workflows for measuring various coating properties, such as cure speed, hardness development, scratch resistance, impact toughness, resin compatibility, pot-life, surface defects, among others have also been developed in-house. These workflows correlate well with the traditional coatings tests, but they do not necessarily mimic those tests. The use of such high-throughput workflows in combination with smart experimental designs allows accelerated discovery and commercialization. PMID:27440008

  8. Establishment of local searching methods for orbitrap-based high throughput metabolomics analysis.

    PubMed

    Tang, Haiping; Wang, Xueying; Xu, Lina; Ran, Xiaorong; Li, Xiangjun; Chen, Ligong; Zhao, Xinbin; Deng, Haiteng; Liu, Xiaohui

    2016-08-15

    Our method aims to establish local endogenous metabolite databases economically without purchasing chemical standards, giving strong bases for following orbitrap based high throughput untargeted metabolomics analysis. A new approach here is introduced to construct metabolite databases on the base of biological sample analysis and mathematic extrapolation. Building local metabolite databases traditionally requires expensive chemical standards, which is barely affordable for most research labs. As a result, most labs working on metabolomics analysis have to refer public libraries, which is time consuming and limited for high throughput analysis. Using this strategy, a high throughput orbitrap based metabolomics platform can be established at almost no cost within a couple of months. It enables to facilitate the application of high throughput metabolomics analysis to identify disease-related biomarkers or investigate biological functions using orbitrap. PMID:27260449

  9. Microscopy with microlens arrays: high throughput, high resolution and light-field imaging.

    PubMed

    Orth, Antony; Crozier, Kenneth

    2012-06-01

    We demonstrate highly parallelized fluorescence scanning microscopy using a refractive microlens array. Fluorescent beads and rat femur tissue are imaged over a 5.5 mm x 5.5 mm field of view at a pixel throughput of up to 4 megapixels/s and a resolution of 706 nm. We also demonstrate the ability to extract different perspective views of a pile of microspheres.

  10. Miniaturized FRET assays and microfluidics: key components for ultra-high-throughput screening.

    PubMed

    Mere; Bennett; Coassin; England; Hamman; Rink; Zimmerman; Negulescu

    1999-08-01

    Assay miniaturization applicable across a wide range of target classes, along with automation and process integration, are well-recognized goals for ultra-high-throughput screening on an industrial scale. This report summarizes the implementation of fluorescence resonance energy transfer (FRET)-based biochemical and cell-based assays in 3456-well NanoWelltrade mark assay plates using key components of Aurora's ultra-high-throughput screening system.

  11. High-throughput screening of antagonists for the orphan G-protein coupled receptor GPR139

    PubMed Central

    Wang, Jia; Zhu, Lin-yun; Liu, Qing; Hentzer, Morten; Smith, Garrick Paul; Wang, Ming-wei

    2015-01-01

    Aim: To discover antagonists of the orphan G-protein coupled receptor GPR139 through high-throughput screening of a collection of diverse small molecules. Methods: Calcium mobilization assays were used to identify initial hits and for subsequent confirmation studies. Results: Five small molecule antagonists, representing 4 different scaffolds, were identified following high-throughput screening of 16 000 synthetic compounds. Conclusion: The findings provide important tools for further study of this orphan G-protein coupled receptor. PMID:26027661

  12. High throughput electrospinning of high-quality nanofibers via an aluminum disk spinneret

    NASA Astrophysics Data System (ADS)

    Zheng, Guokuo

    In this work, a simple and efficient needleless high throughput electrospinning process using an aluminum disk spinneret with 24 holes is described. Electrospun mats produced by this setup consisted of fine fibers (nano-sized) of the highest quality while the productivity (yield) was many times that obtained from conventional single-needle electrospinning. The goal was to produce scaled-up amounts of the same or better quality nanofibers under variable concentration, voltage, and the working distance than those produced with the single needle lab setting. The fiber mats produced were either polymer or ceramic (such as molybdenum trioxide nanofibers). Through experimentation the optimum process conditions were defined to be: 24 kilovolt, a distance to collector of 15cm. More diluted solutions resulted in smaller diameter fibers. Comparing the morphologies of the nanofibers of MoO3 produced by both the traditional and the high throughput set up it was found that they were very similar. Moreover, the nanofibers production rate is nearly 10 times than that of traditional needle electrospinning. Thus, the high throughput process has the potential to become an industrial nanomanufacturing process and the materials processed by it may be used as filtration devices, in tissue engineering, and as sensors.

  13. High throughput on-chip analysis of high-energy charged particle tracks using lensfree imaging

    SciTech Connect

    Luo, Wei; Shabbir, Faizan; Gong, Chao; Gulec, Cagatay; Pigeon, Jeremy; Shaw, Jessica; Greenbaum, Alon; Tochitsky, Sergei; Joshi, Chandrashekhar; Ozcan, Aydogan

    2015-04-13

    We demonstrate a high-throughput charged particle analysis platform, which is based on lensfree on-chip microscopy for rapid ion track analysis using allyl diglycol carbonate, i.e., CR-39 plastic polymer as the sensing medium. By adopting a wide-area opto-electronic image sensor together with a source-shifting based pixel super-resolution technique, a large CR-39 sample volume (i.e., 4 cm × 4 cm × 0.1 cm) can be imaged in less than 1 min using a compact lensfree on-chip microscope, which detects partially coherent in-line holograms of the ion tracks recorded within the CR-39 detector. After the image capture, using highly parallelized reconstruction and ion track analysis algorithms running on graphics processing units, we reconstruct and analyze the entire volume of a CR-39 detector within ∼1.5 min. This significant reduction in the entire imaging and ion track analysis time not only increases our throughput but also allows us to perform time-resolved analysis of the etching process to monitor and optimize the growth of ion tracks during etching. This computational lensfree imaging platform can provide a much higher throughput and more cost-effective alternative to traditional lens-based scanning optical microscopes for ion track analysis using CR-39 and other passive high energy particle detectors.

  14. Review article: high-throughput affinity-based technologies for small-molecule drug discovery.

    PubMed

    Zhu, Zhengrong; Cuozzo, John

    2009-12-01

    High-throughput affinity-based technologies are rapidly growing in use as primary screening methods in drug discovery. In this review, their principles and applications are described and their impact on small-molecule drug discovery is evaluated. In general, these technologies can be divided into 2 groups: those that detect binding interactions by measuring changes to the protein target and those that detect bound compounds. Technologies detecting binding interactions by focusing on the protein have limited throughput but can reveal mechanistic information about the binding interaction; technologies detecting bound compounds have very high throughput, some even significantly higher than current high-throughput screening technologies, but offer limited information about the binding interaction. In addition, the appropriate use of affinity-based technologies is discussed. Finally, nanotechnology is predicted to generate a significant impact on the future of affinity-based technologies. PMID:19822881

  15. A GUINIER CAMERA FOR SR POWDER DIFFRACTION: HIGH RESOLUTION AND HIGH THROUGHPUT.

    SciTech Connect

    SIDDONS,D.P.; HULBERT, S.L.; STEPHENS, P.W.

    2006-05-28

    The paper describe a new powder diffraction instrument for synchrotron radiation sources which combines the high throughput of a position-sensitive detector system with the high resolution normally only provided by a crystal analyzer. It uses the Guinier geometry which is traditionally used with an x-ray tube source. This geometry adapts well to the synchrotron source, provided proper beam conditioning is applied. The high brightness of the SR source allows a high resolution to be achieved. When combined with a photon-counting silicon microstrip detector array, the system becomes a powerful instrument for radiation-sensitive samples or time-dependent phase transition studies.

  16. Lessons from high-throughput protein crystallization screening: 10 years of practical experience

    PubMed Central

    JR, Luft; EH, Snell; GT, DeTitta

    2011-01-01

    Introduction X-ray crystallography provides the majority of our structural biological knowledge at a molecular level and in terms of pharmaceutical design is a valuable tool to accelerate discovery. It is the premier technique in the field, but its usefulness is significantly limited by the need to grow well-diffracting crystals. It is for this reason that high-throughput crystallization has become a key technology that has matured over the past 10 years through the field of structural genomics. Areas covered The authors describe their experiences in high-throughput crystallization screening in the context of structural genomics and the general biomedical community. They focus on the lessons learnt from the operation of a high-throughput crystallization screening laboratory, which to date has screened over 12,500 biological macromolecules. They also describe the approaches taken to maximize the success while minimizing the effort. Through this, the authors hope that the reader will gain an insight into the efficient design of a laboratory and protocols to accomplish high-throughput crystallization on a single-, multiuser-laboratory or industrial scale. Expert Opinion High-throughput crystallization screening is readily available but, despite the power of the crystallographic technique, getting crystals is still not a solved problem. High-throughput approaches can help when used skillfully; however, they still require human input in the detailed analysis and interpretation of results to be more successful. PMID:22646073

  17. Outlook for development of high-throughput cryopreservation for small-bodied biomedical model fishes.

    PubMed

    Tiersch, Terrence R; Yang, Huiping; Hu, E

    2011-08-01

    With the development of genomic research technologies, comparative genome studies among vertebrate species are becoming commonplace for human biomedical research. Fish offer unlimited versatility for biomedical research. Extensive studies are done using these fish models, yielding tens of thousands of specific strains and lines, and the number is increasing every day. Thus, high-throughput sperm cryopreservation is urgently needed to preserve these genetic resources. Although high-throughput processing has been widely applied for sperm cryopreservation in livestock for decades, application in biomedical model fishes is still in the concept-development stage because of the limited sample volumes and the biological characteristics of fish sperm. High-throughput processing in livestock was developed based on advances made in the laboratory and was scaled up for increased processing speed, capability for mass production, and uniformity and quality assurance. Cryopreserved germplasm combined with high-throughput processing constitutes an independent industry encompassing animal breeding, preservation of genetic diversity, and medical research. Currently, there is no specifically engineered system available for high-throughput of cryopreserved germplasm for aquatic species. This review is to discuss the concepts and needs for high-throughput technology for model fishes, propose approaches for technical development, and overview future directions of this approach.

  18. Construction and Analysis of High-Density Linkage Map Using High-Throughput Sequencing Data

    PubMed Central

    Liu, Min; Liu, Hui; Zeng, Huaping; Deng, Dejing; Xin, Huaigen; Song, Jun; Xu, Chunhua; Sun, Xiaowen; Hou, Xilin; Wang, Xiaowu; Zheng, Hongkun

    2014-01-01

    Linkage maps enable the study of important biological questions. The construction of high-density linkage maps appears more feasible since the advent of next-generation sequencing (NGS), which eases SNP discovery and high-throughput genotyping of large population. However, the marker number explosion and genotyping errors from NGS data challenge the computational efficiency and linkage map quality of linkage study methods. Here we report the HighMap method for constructing high-density linkage maps from NGS data. HighMap employs an iterative ordering and error correction strategy based on a k-nearest neighbor algorithm and a Monte Carlo multipoint maximum likelihood algorithm. Simulation study shows HighMap can create a linkage map with three times as many markers as ordering-only methods while offering more accurate marker orders and stable genetic distances. Using HighMap, we constructed a common carp linkage map with 10,004 markers. The singleton rate was less than one-ninth of that generated by JoinMap4.1. Its total map distance was 5,908 cM, consistent with reports on low-density maps. HighMap is an efficient method for constructing high-density, high-quality linkage maps from high-throughput population NGS data. It will facilitate genome assembling, comparative genomic analysis, and QTL studies. HighMap is available at http://highmap.biomarker.com.cn/. PMID:24905985

  19. Advancing a distributed multi-scale computing framework for large-scale high-throughput discovery in materials science.

    PubMed

    Knap, J; Spear, C E; Borodin, O; Leiter, K W

    2015-10-30

    We describe the development of a large-scale high-throughput application for discovery in materials science. Our point of departure is a computational framework for distributed multi-scale computation. We augment the original framework with a specialized module whose role is to route evaluation requests needed by the high-throughput application to a collection of available computational resources. We evaluate the feasibility and performance of the resulting high-throughput computational framework by carrying out a high-throughput study of battery solvents. Our results indicate that distributed multi-scale computing, by virtue of its adaptive nature, is particularly well-suited for building high-throughput applications.

  20. Compressed data organization for high throughput parallel entropy coding

    NASA Astrophysics Data System (ADS)

    Said, Amir; Mahfoodh, Abo-Talib; Yea, Sehoon

    2015-09-01

    The difficulty of parallelizing entropy coding is increasingly limiting the data throughputs achievable in media compression. In this work we analyze what are the fundamental limitations, using finite-state-machine models for identifying the best manner of separating tasks that can be processed independently, while minimizing compression losses. This analysis confirms previous works showing that effective parallelization is feasible only if the compressed data is organized in a proper way, which is quite different from conventional formats. The proposed new formats exploit the fact that optimal compression is not affected by the arrangement of coded bits, but it goes further in exploiting the decreasing cost of data processing and memory. Additional advantages include the ability to use, within this framework, increasingly more complex data modeling techniques, and the freedom to mix different types of coding. We confirm the parallelization effectiveness using coding simulations that run on multi-core processors, and show how throughput scales with the number of cores, and analyze the additional bit-rate overhead.

  1. High-Throughput Assessment of Cellular Mechanical Properties.

    PubMed

    Darling, Eric M; Di Carlo, Dino

    2015-01-01

    Traditionally, cell analysis has focused on using molecular biomarkers for basic research, cell preparation, and clinical diagnostics; however, new microtechnologies are enabling evaluation of the mechanical properties of cells at throughputs that make them amenable to widespread use. We review the current understanding of how the mechanical characteristics of cells relate to underlying molecular and architectural changes, describe how these changes evolve with cell-state and disease processes, and propose promising biomedical applications that will be facilitated by the increased throughput of mechanical testing: from diagnosing cancer and monitoring immune states to preparing cells for regenerative medicine. We provide background about techniques that laid the groundwork for the quantitative understanding of cell mechanics and discuss current efforts to develop robust techniques for rapid analysis that aim to implement mechanophenotyping as a routine tool in biomedicine. Looking forward, we describe additional milestones that will facilitate broad adoption, as well as new directions not only in mechanically assessing cells but also in perturbing them to passively engineer cell state.

  2. High-throughput screening for lead optimization: a rational approach.

    PubMed

    Bajpai, M; Adkison, K K

    2000-01-01

    Genetics, combinatorial chemistry and automation have greatly increased the number of therapeutic programs and compounds in the pharmaceutical industry pipeline. The increase in the number of new molecular entities (NMEs) has led to changes in the process by which compounds are evaluated during drug discovery and selected for clinical development. There is a need for the earlier determination of the absorption, distribution and elimination characteristics of NMEs, and drug metabolism scientists are working to develop higher-throughput in vitro screens for absorption, distribution and metabolism of compounds. These screens rely on advancements in analytical technology and molecular biology, and frequently use human or 'humanized' tissues. Throughput to determine in vivo pharmacokinetics has also progressed with the use of mixture dosing and sample pooling methods. The continued refinement of in vitro and in vivo ADME methods will allow the industry to evaluate the absorption and disposition characteristics of larger numbers of molecules and will ultimately allow the prediction of human pharmacokinetics at early stages of the development process.

  3. High-throughput miniaturized microfluidic microscopy with radially parallelized channel geometry.

    PubMed

    Jagannadh, Veerendra Kalyan; Bhat, Bindu Prabhath; Nirupa Julius, Lourdes Albina; Gorthi, Sai Siva

    2016-03-01

    In this article, we present a novel approach to throughput enhancement in miniaturized microfluidic microscopy systems. Using the presented approach, we demonstrate an inexpensive yet high-throughput analytical instrument. Using the high-throughput analytical instrument, we have been able to achieve about 125,880 cells per minute (more than one hundred and twenty five thousand cells per minute), even while employing cost-effective low frame rate cameras (120 fps). The throughput achieved here is a notable progression in the field of diagnostics as it enables rapid quantitative testing and analysis. We demonstrate the applicability of the instrument to point-of-care diagnostics, by performing blood cell counting. We report a comparative analysis between the counts (in cells per μl) obtained from our instrument, with that of a commercially available hematology analyzer. PMID:26781098

  4. High-throughput approaches for evaluating absorption, distribution, metabolism and excretion properties of lead compounds.

    PubMed

    Tarbit, M H; Berman, J

    1998-06-01

    Combinatorial chemistry methods and high-throughput screening for leads in industrial drug discovery have generated a potential bottleneck in the optimisation processes that seek to align potency with good pharmacokinetics in order to produce good medicines. This has resulted in the need for higher throughput methods of screening for absorption, distribution, metabolism and excretion properties. Significant progress has been made in throughput of in vivo pharmacokinetic studies, with the introduction of cassette, or multiple-in-one, protocols. In this technique, typically up to ten compounds are administered in one dose and analysed concomitantly on the mass spectrometer. High-throughput methods in in vitro absorption, distribution, metabolism and excretion are less well-developed as yet, and current approaches comprise automation of well-established methods for absorption using cell lines and metabolism using liver microsomes.

  5. A high-throughput in vitro ring assay for vasoactivity using magnetic 3D bioprinting.

    PubMed

    Tseng, Hubert; Gage, Jacob A; Haisler, William L; Neeley, Shane K; Shen, Tsaiwei; Hebel, Chris; Barthlow, Herbert G; Wagoner, Matthew; Souza, Glauco R

    2016-01-01

    Vasoactive liabilities are typically assayed using wire myography, which is limited by its high cost and low throughput. To meet the demand for higher throughput in vitro alternatives, this study introduces a magnetic 3D bioprinting-based vasoactivity assay. The principle behind this assay is the magnetic printing of vascular smooth muscle cells into 3D rings that functionally represent blood vessel segments, whose contraction can be altered by vasodilators and vasoconstrictors. A cost-effective imaging modality employing a mobile device is used to capture contraction with high throughput. The goal of this study was to validate ring contraction as a measure of vasoactivity, using a small panel of known vasoactive drugs. In vitro responses of the rings matched outcomes predicted by in vivo pharmacology, and were supported by immunohistochemistry. Altogether, this ring assay robustly models vasoactivity, which could meet the need for higher throughput in vitro alternatives. PMID:27477945

  6. A high-throughput in vitro ring assay for vasoactivity using magnetic 3D bioprinting

    PubMed Central

    Tseng, Hubert; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Shen, Tsaiwei; Hebel, Chris; Barthlow, Herbert G.; Wagoner, Matthew; Souza, Glauco R.

    2016-01-01

    Vasoactive liabilities are typically assayed using wire myography, which is limited by its high cost and low throughput. To meet the demand for higher throughput in vitro alternatives, this study introduces a magnetic 3D bioprinting-based vasoactivity assay. The principle behind this assay is the magnetic printing of vascular smooth muscle cells into 3D rings that functionally represent blood vessel segments, whose contraction can be altered by vasodilators and vasoconstrictors. A cost-effective imaging modality employing a mobile device is used to capture contraction with high throughput. The goal of this study was to validate ring contraction as a measure of vasoactivity, using a small panel of known vasoactive drugs. In vitro responses of the rings matched outcomes predicted by in vivo pharmacology, and were supported by immunohistochemistry. Altogether, this ring assay robustly models vasoactivity, which could meet the need for higher throughput in vitro alternatives. PMID:27477945

  7. Space Link Extension Protocol Emulation for High-Throughput, High-Latency Network Connections

    NASA Technical Reports Server (NTRS)

    Tchorowski, Nicole; Murawski, Robert

    2014-01-01

    New space missions require higher data rates and new protocols to meet these requirements. These high data rate space communication links push the limitations of not only the space communication links, but of the ground communication networks and protocols which forward user data to remote ground stations (GS) for transmission. The Consultative Committee for Space Data Systems, (CCSDS) Space Link Extension (SLE) standard protocol is one protocol that has been proposed for use by the NASA Space Network (SN) Ground Segment Sustainment (SGSS) program. New protocol implementations must be carefully tested to ensure that they provide the required functionality, especially because of the remote nature of spacecraft. The SLE protocol standard has been tested in the NASA Glenn Research Center's SCENIC Emulation Lab in order to observe its operation under realistic network delay conditions. More specifically, the delay between then NASA Integrated Services Network (NISN) and spacecraft has been emulated. The round trip time (RTT) delay for the continental NISN network has been shown to be up to 120ms; as such the SLE protocol was tested with network delays ranging from 0ms to 200ms. Both a base network condition and an SLE connection were tested with these RTT delays, and the reaction of both network tests to the delay conditions were recorded. Throughput for both of these links was set at 1.2Gbps. The results will show that, in the presence of realistic network delay, the SLE link throughput is significantly reduced while the base network throughput however remained at the 1.2Gbps specification. The decrease in SLE throughput has been attributed to the implementation's use of blocking calls. The decrease in throughput is not acceptable for high data rate links, as the link requires constant data a flow in order for spacecraft and ground radios to stay synchronized, unless significant data is queued a the ground station. In cases where queuing the data is not an option

  8. High-throughput allogeneic antibody detection using protein microarrays.

    PubMed

    Paul, Jed; Sahaf, Bita; Perloff, Spenser; Schoenrock, Kelsi; Wu, Fang; Nakasone, Hideki; Coller, John; Miklos, David

    2016-05-01

    Enzyme-linked immunosorbent assays (ELISAs) have traditionally been used to detect alloantibodies in patient plasma samples post hematopoietic cell transplantation (HCT); however, protein microarrays have the potential to be multiplexed, more sensitive, and higher throughput than ELISAs. Here, we describe the development of a novel and sensitive microarray method for detection of allogeneic antibodies against minor histocompatibility antigens encoded on the Y chromosome, called HY antigens. Six microarray surfaces were tested for their ability to bind recombinant protein and peptide HY antigens. Significant allogeneic immune responses were determined in male patients with female donors by considering normal male donor responses as baseline. HY microarray results were also compared with our previous ELISA results. Our overall goal was to maximize antibody detection for both recombinant protein and peptide epitopes. For detection of HY antigens, the Epoxy (Schott) protein microarray surface was both most sensitive and reliable and has become the standard surface in our microarray platform. PMID:26902899

  9. A high-throughput, high-quality plant genomic DNA extraction protocol.

    PubMed

    Li, H; Li, J; Cong, X H; Duan, Y B; Li, L; Wei, P C; Lu, X Z; Yang, J B

    2013-01-01

    The isolation of high-quality genomic DNA (gDNA) is a crucial technique in plant molecular biology. The quality of gDNA determines the reliability of real-time polymerase chain reaction (PCR) analysis. In this paper, we reported a high-quality gDNA extraction protocol optimized for real-time PCR in a variety of plant species. Performed in a 96-well block, our protocol provides high throughput. Without the need for phenol-chloroform and liquid nitrogen or dry ice, our protocol is safer and more cost-efficient than traditional DNA extraction methods. The method takes 10 mg leaf tissue to yield 5-10 µg high-quality gDNA. Spectral measurement and electrophoresis were used to demonstrate gDNA purity. The extracted DNA was qualified in a restriction enzyme digestion assay and conventional PCR. The real-time PCR amplification was sufficiently sensitive to detect gDNA at very low concentrations (3 pg/µL). The standard curve of gDNA dilutions from our phenol-chloroform-free protocol showed better linearity (R(2) = 0.9967) than the phenol-chloroform protocol (R(2) = 0.9876). The results indicate that the gDNA was of high quality and fit for real-time PCR. This safe, high-throughput plant gDNA extraction protocol could be used to isolate high-quality gDNA for real-time PCR and other downstream molecular applications. PMID:24222228

  10. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control

    NASA Astrophysics Data System (ADS)

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L. James; Lu, Wu

    2015-12-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk

  11. Forecasting Ecological Genomics: High-Tech Animal Instrumentation Meets High-Throughput Sequencing

    PubMed Central

    Shafer, Aaron B. A.; Northrup, Joseph M.; Wikelski, Martin; Wittemyer, George; Wolf, Jochen B. W.

    2016-01-01

    Recent advancements in animal tracking technology and high-throughput sequencing are rapidly changing the questions and scope of research in the biological sciences. The integration of genomic data with high-tech animal instrumentation comes as a natural progression of traditional work in ecological genetics, and we provide a framework for linking the separate data streams from these technologies. Such a merger will elucidate the genetic basis of adaptive behaviors like migration and hibernation and advance our understanding of fundamental ecological and evolutionary processes such as pathogen transmission, population responses to environmental change, and communication in natural populations. PMID:26745372

  12. Forecasting Ecological Genomics: High-Tech Animal Instrumentation Meets High-Throughput Sequencing.

    PubMed

    Shafer, Aaron B A; Northrup, Joseph M; Wikelski, Martin; Wittemyer, George; Wolf, Jochen B W

    2016-01-01

    Recent advancements in animal tracking technology and high-throughput sequencing are rapidly changing the questions and scope of research in the biological sciences. The integration of genomic data with high-tech animal instrumentation comes as a natural progression of traditional work in ecological genetics, and we provide a framework for linking the separate data streams from these technologies. Such a merger will elucidate the genetic basis of adaptive behaviors like migration and hibernation and advance our understanding of fundamental ecological and evolutionary processes such as pathogen transmission, population responses to environmental change, and communication in natural populations.

  13. High-throughput analysis of yeast replicative aging using a microfluidic system

    PubMed Central

    Jo, Myeong Chan; Liu, Wei; Gu, Liang; Dang, Weiwei; Qin, Lidong

    2015-01-01

    Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction. PMID:26170317

  14. High-throughput analysis of yeast replicative aging using a microfluidic system.

    PubMed

    Jo, Myeong Chan; Liu, Wei; Gu, Liang; Dang, Weiwei; Qin, Lidong

    2015-07-28

    Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction. PMID:26170317

  15. High throughput screening of ligand binding to macromolecules using high resolution powder diffraction

    DOEpatents

    Von Dreele, Robert B.; D'Amico, Kevin

    2006-10-31

    A process is provided for the high throughput screening of binding of ligands to macromolecules using high resolution powder diffraction data including producing a first sample slurry of a selected polycrystalline macromolecule material and a solvent, producing a second sample slurry of a selected polycrystalline macromolecule material, one or more ligands and the solvent, obtaining a high resolution powder diffraction pattern on each of said first sample slurry and the second sample slurry, and, comparing the high resolution powder diffraction pattern of the first sample slurry and the high resolution powder diffraction pattern of the second sample slurry whereby a difference in the high resolution powder diffraction patterns of the first sample slurry and the second sample slurry provides a positive indication for the formation of a complex between the selected polycrystalline macromolecule material and at least one of the one or more ligands.

  16. Chemically modified solid state nanopores for high throughput nanoparticle separation

    NASA Astrophysics Data System (ADS)

    Prabhu, Anmiv S.; Jubery, Talukder Zaki N.; Freedman, Kevin J.; Mulero, Rafael; Dutta, Prashanta; Kim, Min Jun

    2010-11-01

    The separation of biomolecules and other nanoparticles is a vital step in several analytical and diagnostic techniques. Towards this end we present a solid state nanopore-based set-up as an efficient separation platform. The translocation of charged particles through a nanopore was first modeled mathematically using the multi-ion model and the surface charge density of the nanopore membrane was identified as a critical parameter that determines the selectivity of the membrane and the throughput of the separation process. Drawing from these simulations a single 150 nm pore was fabricated in a 50 nm thick free-standing silicon nitride membrane by focused-ion-beam milling and was chemically modified with (3-aminopropyl)triethoxysilane to change its surface charge density. This chemically modified membrane was then used to separate 22 and 58 nm polystyrene nanoparticles in solution. Once optimized, this approach can readily be scaled up to nanopore arrays which would function as a key component of next-generation nanosieving systems.

  17. High-throughput, high-fidelity HLA genotyping with deep sequencing.

    PubMed

    Wang, Chunlin; Krishnakumar, Sujatha; Wilhelmy, Julie; Babrzadeh, Farbod; Stepanyan, Lilit; Su, Laura F; Levinson, Douglas; Fernandez-Viña, Marcelo A; Davis, Ronald W; Davis, Mark M; Mindrinos, Michael

    2012-05-29

    Human leukocyte antigen (HLA) genes are the most polymorphic in the human genome. They play a pivotal role in the immune response and have been implicated in numerous human pathologies, especially autoimmunity and infectious diseases. Despite their importance, however, they are rarely characterized comprehensively because of the prohibitive cost of standard technologies and the technical challenges of accurately discriminating between these highly related genes and their many allelles. Here we demonstrate a high-resolution, and cost-effective methodology to type HLA genes by sequencing, which combines the advantage of long-range amplification, the power of high-throughput sequencing platforms, and a unique genotyping algorithm. We calibrated our method for HLA-A, -B, -C, and -DRB1 genes with both reference cell lines and clinical samples and identified several previously undescribed alleles with mismatches, insertions, and deletions. We have further demonstrated the utility of this method in a clinical setting by typing five clinical samples in an Illumina MiSeq instrument with a 5-d turnaround. Overall, this technology has the capacity to deliver low-cost, high-throughput, and accurate HLA typing by multiplexing thousands of samples in a single sequencing run, which will enable comprehensive disease-association studies with large cohorts. Furthermore, this approach can also be extended to include other polymorphic genes.

  18. High-throughput, high-fidelity HLA genotyping with deep sequencing

    PubMed Central

    Wang, Chunlin; Krishnakumar, Sujatha; Wilhelmy, Julie; Babrzadeh, Farbod; Stepanyan, Lilit; Su, Laura F.; Levinson, Douglas; Fernandez-Viña, Marcelo A.; Davis, Ronald W.; Davis, Mark M.; Mindrinos, Michael

    2012-01-01

    Human leukocyte antigen (HLA) genes are the most polymorphic in the human genome. They play a pivotal role in the immune response and have been implicated in numerous human pathologies, especially autoimmunity and infectious diseases. Despite their importance, however, they are rarely characterized comprehensively because of the prohibitive cost of standard technologies and the technical challenges of accurately discriminating between these highly related genes and their many allelles. Here we demonstrate a high-resolution, and cost-effective methodology to type HLA genes by sequencing, which combines the advantage of long-range amplification, the power of high-throughput sequencing platforms, and a unique genotyping algorithm. We calibrated our method for HLA-A, -B, -C, and -DRB1 genes with both reference cell lines and clinical samples and identified several previously undescribed alleles with mismatches, insertions, and deletions. We have further demonstrated the utility of this method in a clinical setting by typing five clinical samples in an Illumina MiSeq instrument with a 5-d turnaround. Overall, this technology has the capacity to deliver low-cost, high-throughput, and accurate HLA typing by multiplexing thousands of samples in a single sequencing run, which will enable comprehensive disease-association studies with large cohorts. Furthermore, this approach can also be extended to include other polymorphic genes. PMID:22589303

  19. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control.

    PubMed

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L James; Lu, Wu

    2016-01-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a "3D nano-channel electroporation (NEP) chip" on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40,000 cells per cm(2) on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.

  20. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control.

    PubMed

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L James; Lu, Wu

    2016-01-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a "3D nano-channel electroporation (NEP) chip" on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40,000 cells per cm(2) on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells. PMID:26309218

  1. PALM and STORM: Into large fields and high-throughput microscopy with sCMOS detectors.

    PubMed

    Almada, Pedro; Culley, Siân; Henriques, Ricardo

    2015-10-15

    Single Molecule Localization Microscopy (SMLM) techniques such as Photo-Activation Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) enable fluorescence microscopy super-resolution: the overcoming of the resolution barrier imposed by the diffraction of light. These techniques are based on acquiring hundreds or thousands of images of single molecules, locating them and reconstructing a higher-resolution image from the high-precision localizations. These methods generally imply a considerable trade-off between imaging speed and resolution, limiting their applicability to high-throughput workflows. Recent advancements in scientific Complementary Metal-Oxide Semiconductor (sCMOS) camera sensors and localization algorithms reduce the temporal requirements for SMLM, pushing it toward high-throughput microscopy. Here we outline the decisions researchers face when considering how to adapt hardware on a new system for sCMOS sensors with high-throughput in mind. PMID:26079924

  2. Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing

    PubMed Central

    Chandran, Anandhakumar; Syed, Junetha; Taylor, Rhys D.; Kashiwazaki, Gengo; Sato, Shinsuke; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

    2016-01-01

    Chemically engineered small molecules targeting specific genomic sequences play an important role in drug development research. Pyrrole-imidazole polyamides (PIPs) are a group of molecules that can bind to the DNA minor-groove and can be engineered to target specific sequences. Their biological effects rely primarily on their selective DNA binding. However, the binding mechanism of PIPs at the chromatinized genome level is poorly understood. Herein, we report a method using high-throughput sequencing to identify the DNA-alkylating sites of PIP-indole-seco-CBI conjugates. High-throughput sequencing analysis of conjugate 2 showed highly similar DNA-alkylating sites on synthetic oligos (histone-free DNA) and on human genomes (chromatinized DNA context). To our knowledge, this is the first report identifying alkylation sites across genomic DNA by alkylating PIP conjugates using high-throughput sequencing. PMID:27098039

  3. High-throughput measurements of the optical redox ratio using a commercial microplate reader

    NASA Astrophysics Data System (ADS)

    Cannon, Taylor M.; Shah, Amy T.; Walsh, Alex J.; Skala, Melissa C.

    2015-01-01

    There is a need for accurate, high-throughput, functional measures to gauge the efficacy of potential drugs in living cells. As an early marker of drug response in cells, cellular metabolism provides an attractive platform for high-throughput drug testing. Optical techniques can noninvasively monitor NADH and FAD, two autofluorescent metabolic coenzymes. The autofluorescent redox ratio, defined as the autofluorescence intensity of NADH divided by that of FAD, quantifies relative rates of cellular glycolysis and oxidative phosphorylation. However, current microscopy methods for redox ratio quantification are time-intensive and low-throughput, limiting their practicality in drug screening. Alternatively, high-throughput commercial microplate readers quickly measure fluorescence intensities for hundreds of wells. This study found that a commercial microplate reader can differentiate the receptor status of breast cancer cell lines (p<0.05) based on redox ratio measurements without extrinsic contrast agents. Furthermore, microplate reader redox ratio measurements resolve response (p<0.05) and lack of response (p>0.05) in cell lines that are responsive and nonresponsive, respectively, to the breast cancer drug trastuzumab. These studies indicate that the microplate readers can be used to measure the redox ratio in a high-throughput manner and are sensitive enough to detect differences in cellular metabolism that are consistent with microscopy results.

  4. A High-Throughput Biological Calorimetry Core: Steps to Startup, Run, and Maintain a Multiuser Facility.

    PubMed

    Yennawar, Neela H; Fecko, Julia A; Showalter, Scott A; Bevilacqua, Philip C

    2016-01-01

    Many labs have conventional calorimeters where denaturation and binding experiments are setup and run one at a time. While these systems are highly informative to biopolymer folding and ligand interaction, they require considerable manual intervention for cleaning and setup. As such, the throughput for such setups is limited typically to a few runs a day. With a large number of experimental parameters to explore including different buffers, macromolecule concentrations, temperatures, ligands, mutants, controls, replicates, and instrument tests, the need for high-throughput automated calorimeters is on the rise. Lower sample volume requirements and reduced user intervention time compared to the manual instruments have improved turnover of calorimetry experiments in a high-throughput format where 25 or more runs can be conducted per day. The cost and efforts to maintain high-throughput equipment typically demands that these instruments be housed in a multiuser core facility. We describe here the steps taken to successfully start and run an automated biological calorimetry facility at Pennsylvania State University. Scientists from various departments at Penn State including Chemistry, Biochemistry and Molecular Biology, Bioengineering, Biology, Food Science, and Chemical Engineering are benefiting from this core facility. Samples studied include proteins, nucleic acids, sugars, lipids, synthetic polymers, small molecules, natural products, and virus capsids. This facility has led to higher throughput of data, which has been leveraged into grant support, attracting new faculty hire and has led to some exciting publications.

  5. High Throughput Proteomics Using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

    SciTech Connect

    Qian, Weijun; Camp, David G.; Smith, Richard D.

    2004-06-01

    The advent of high throughput proteomics technology for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of cellular machinery. Here, we review recent advances in high-resolution capillary liquid chromatography coupled to Fourier transform ion cyclotron resonance (FTICR) mass spectrometry along with its potential application to high throughput proteomics. These technological advances combined with quantitative stable isotope labeling methodologies provide powerful tools for expanding our understanding of biology at the system-level.

  6. High-throughput proteomics using Fourier transform ion cyclotron resonance mass spectrometry.

    PubMed

    Qian, Wei-Jun; Camp, David G; Smith, Richard D

    2004-06-01

    The advent of high-throughput proteomic technologies for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of the cellular machinery. Here, recent advances in high-resolution capillary liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry are reviewed along with its potential application to high-throughput proteomics. These technological advances combined with quantitative stable isotope labeling methodologies provide powerful tools for expanding our understanding of biology at the system level.

  7. High-throughput screening approaches for investigating drug metabolism and pharmacokinetics.

    PubMed

    Roberts, S A

    2001-01-01

    1. High-throughput screening approaches have been adopted throughout the pharmaceutical industry to aid in the rapid discovery of new chemical entities. Because it is now well recognized that the selection of a robust candidate requires a balance of potency, safety and pharmacokinetics, the role of drug metabolism departments has widened from their traditional one of supporting drug development to include the screening of compounds during the discovery process. To put drug metabolism and pharmacokinetic (DMPK) studies in context, the evolving role of DMPK screening in the drug discovery strategy of pharmaceutical companies will be discussed and a generalized approach will be presented. 2. With the increasing numbers of compounds requiring screening, DMPK optimization methods have had to be adapted for high throughput. There have been many developments in this field over the past decade and this review will focus on the high-throughput DMPK screening methodologies used today and in the recent past. 3. In vitro and in silico (computer-based) methods have proven most amenable to high-throughput approaches and these will firm the bulk of the review, but some advances with in vivo methods will also be discussed. As there has been a vast increase in published material on the topic of high-throughput DMPK methodologies in the past 10 years, it would be impossible to cover every method in detail, so this review will concentrate on the key areas and refer the reader to other, more detailed reviews wherever possible. 4. Most high-throughput methods would not be possible without the enabling technologies of computing, automation, new sample preparation technologies, and highly sensitive and selective detection systems, and these will also be reviewed. 5. The advantages and disadvantages of the screening methods will be presented, in particular the issue of handling the false-positives and -negatives that arise. 6. In concluding the review, future developments in this field

  8. Parallelized ultra-high throughput microfluidic emulsifier for multiplex kinetic assays

    PubMed Central

    Lim, Jiseok; Caen, Ouriel; Vrignon, Jérémy; Konrad, Manfred; Baret, Jean-Christophe

    2015-01-01

    Droplet-based microfluidic technologies are powerful tools for applications requiring high-throughput, for example, in biochemistry or material sciences. Several systems have been proposed for the high-throughput production of monodisperse emulsions by parallelizing multiple droplet makers. However, these systems have two main limitations: (1) they allow the use of only a single disperse phase; (2) they are based on multiple layer microfabrication techniques. We present here a pipette-and-play solution offering the possibility of manipulating simultaneously 10 different disperse phases on a single layer device. This system allows high-throughput emulsion production using aqueous flow rates of up to 26 ml/h (>110 000 drops/s) leading to emulsions with user-defined complex chemical composition. We demonstrate the multiplex capabilities of our system by measuring the kinetics of β-galactosidase in droplets using nine different concentrations of a fluorogenic substrate. PMID:26015838

  9. High-throughput parallel SPM for metrology, defect, and mask inspection

    NASA Astrophysics Data System (ADS)

    Sadeghian, H.; Herfst, R. W.; van den Dool, T. C.; Crowcombe, W. E.; Winters, J.; Kramer, G. F. I. J.

    2014-10-01

    Scanning probe microscopy (SPM) is a promising candidate for accurate assessment of metrology and defects on wafers and masks, however it has traditionally been too slow for high-throughput applications, although recent developments have significantly pushed the speed of SPM [1,2]. In this paper we present new results obtained with our previously presented high-throughput parallel SPM system [3,4] that showcase two key advances that are required for a successful deployment of SPM in high-throughput metrology, defect and mask inspection. The first is a very fast (up to 40 lines/s) image acquisition and a comparison of the image quality as function of speed. Secondly, a fast approach method: measurements of the scan-head approaching the sample from 0.2 and 1.0 mm distance in under 1.4 and 6 seconds respectively.

  10. Graphene oxide-based micropatterns via high-throughput multiphoton-induced reduction and ablation.

    PubMed

    Li, Yi-Cheng; Yeh, Te-Fu; Huang, Hsin-Chieh; Chang, Hsin-Yu; Lin, Chun-Yu; Cheng, Li-Chung; Chang, Chia-Yuan; Teng, Hsisheng; Chen, Shean-Jen

    2014-08-11

    In this study, a developed temporal focusing-based femtosecond laser system provides high-throughput multiphoton-induced reduction and ablation of graphene oxide (GO) films. Integrated with a digital micromirror device to locally control the laser pulse numbers, GO-based micropatterns can be quickly achieved instantly. Furthermore, the degree of reduction and ablation can be precisely adjusted via controlling the laser wavelength, power, and pulse number. Compared to point-by-point scanning laser direct writing, this approach offers a high-throughput and multiple-function approach to accomplish a large area of micro-scale patterns on GO films. The high-throughput micropatterning of GO via the temporal focusing-based femtosecond laser system fulfills the requirement of mass production for GO-based applications in microelectronic devices. PMID:25321055

  11. High throughput screening to investigate the interaction of stem cells with their extracellular microenvironment

    PubMed Central

    Ankam, Soneela; Teo, Benjamin KK; Kukumberg, Marek; Yim, Evelyn KF

    2013-01-01

    Stem cells in vivo are housed within a functional microenvironment termed the “stem cell niche.” As the niche components can modulate stem cell behaviors like proliferation, migration and differentiation, evaluating these components would be important to determine the most optimal platform for their maintenance or differentiation. In this review, we have discussed methods and technologies that have aided in the development of high throughput screening assays for stem cell research, including enabling technologies such as the well-established multiwell/microwell plates and robotic spotting, and emerging technologies like microfluidics, micro-contact printing and lithography. We also discuss the studies that utilized high throughput screening platform to investigate stem cell response to extracellular matrix, topography, biomaterials and stiffness gradients in the stem cell niche. The combination of the aforementioned techniques could lay the foundation for new perspectives in further development of high throughput technology and stem cell research. PMID:23899508

  12. Ultrathin glass filter fabricated by femtosecond laser processing for high-throughput microparticle filtering

    NASA Astrophysics Data System (ADS)

    Yalikun, Yaxiaer; Tanaka, Nobuyuki; Hosokawa, Yoichiroh; Iino, Takanori; Tanaka, Yo

    2016-06-01

    In this study, we developed a method of fabricating totally glass-based filters having micrometer-scale through holes for high-throughput filtration using a femtosecond laser. Filtration using a membrane-type filter is an indispensable technique for biological, chemical, and physical analysis fields. A larger flow rate or stronger driving pressure will result in a faster filtration. However, conventional high-throughput filtering methods often use a relatively slow flow rate or low pressure owing to the fracture toughness of the filter material. In this study, we introduce a customizable 4-µm-thick glass filter that could be used for high-throughput microparticle filtering at a flow velocity of 4 m/s.

  13. High Throughput Fluorescent Screening of Membrane Potential under Variable Gravity Conditions

    NASA Astrophysics Data System (ADS)

    Kohn, F. P. M.

    2013-02-01

    In bilayer and patch-clamp experiments it was shown that the electrophysiological parameters of neuronal cells, as there are ion channel activity, intracellular ion concentrations and membrane potential, respond to gravity changes. Due to technical limitations (e.g. time-consuming manual operations) of electrophysiological techniques the amount of acquired data is limited. Optical techniques as fluorescence and fluorometric measurements can also be used to investigate electrophysiological properties of cells as sensitive fluorescent probes for these properties have been developed. On ground various high-throughput fluorometric systems are commercially available. Such a high throughput system would significantly increase the possible data yield and facilitate a lot of future experiments in micro- and hypergravity research. Therefore a FlexStation® 1 from Molecular Devices, a high-throughput multiwell reader, was adapted to parabolic flight conditions. In a first series of experiments the membrane potential of neuronal cells was investigated to verify the system.

  14. Microfluidics for cell-based high throughput screening platforms - A review.

    PubMed

    Du, Guansheng; Fang, Qun; den Toonder, Jaap M J

    2016-01-15

    In the last decades, the basic techniques of microfluidics for the study of cells such as cell culture, cell separation, and cell lysis, have been well developed. Based on cell handling techniques, microfluidics has been widely applied in the field of PCR (Polymerase Chain Reaction), immunoassays, organ-on-chip, stem cell research, and analysis and identification of circulating tumor cells. As a major step in drug discovery, high-throughput screening allows rapid analysis of thousands of chemical, biochemical, genetic or pharmacological tests in parallel. In this review, we summarize the application of microfluidics in cell-based high throughput screening. The screening methods mentioned in this paper include approaches using the perfusion flow mode, the droplet mode, and the microarray mode. We also discuss the future development of microfluidic based high throughput screening platform for drug discovery.

  15. Image-based high-throughput screening for inhibitors of angiogenesis.

    PubMed

    Evensen, Lasse; Link, Wolfgang; Lorens, James B

    2013-01-01

    Automated multicolor fluorescence microscopy facilitates high-throughput quantitation of cellular parameters of complex, organotypic systems. In vitro co-cultured vascular cells form capillary-like networks that model facets of angiogenesis, making it an attractive alternative for anti-angiogenic drug discovery. We have adapted this angiogenesis assay system to a high-throughput format to enable automated image-based high-throughput screening of live primary human vascular cell co-cultures with chemical libraries for anti-angiogenic drug discovery. Protocols are described for setup of a fluorescence-based co-culture assay, live cell image acquisition, image analysis of morphological parameters, and screening data handling. PMID:23027002

  16. Three-Dimensional Spheroid Cell Culture Model for Target Identification Utilizing High-Throughput RNAi Screens.

    PubMed

    Iles, LaKesla R; Bartholomeusz, Geoffrey A

    2016-01-01

    The intrinsic limitations of 2D monolayer cell culture models have prompted the development of 3D cell culture model systems for in vitro studies. Multicellular tumor spheroid (MCTS) models closely simulate the pathophysiological milieu of solid tumors and are providing new insights into tumor biology as well as differentiation, tissue organization, and homeostasis. They are straightforward to apply in high-throughput screens and there is a great need for the development of reliable and robust 3D spheroid-based assays for high-throughput RNAi screening for target identification and cell signaling studies highlighting their potential in cancer research and treatment. In this chapter we describe a stringent standard operating procedure for the use of MCTS for high-throughput RNAi screens. PMID:27581289

  17. Applications of High-Throughput Clonogenic Survival Assays in High-LET Particle Microbeams

    PubMed Central

    Georgantzoglou, Antonios; Merchant, Michael J.; Jeynes, Jonathan C. G.; Mayhead, Natalie; Punia, Natasha; Butler, Rachel E.; Jena, Rajesh

    2016-01-01

    Charged particle therapy is increasingly becoming a valuable tool in cancer treatment, mainly due to the favorable interaction of particle radiation with matter. Its application is still limited due, in part, to lack of data regarding the radiosensitivity of certain cell lines to this radiation type, especially to high-linear energy transfer (LET) particles. From the earliest days of radiation biology, the clonogenic survival assay has been used to provide radiation response data. This method produces reliable data but it is not optimized for high-throughput microbeam studies with high-LET radiation where high levels of cell killing lead to a very low probability of maintaining cells’ clonogenic potential. A new method, therefore, is proposed in this paper, which could potentially allow these experiments to be conducted in a high-throughput fashion. Cells are seeded in special polypropylene dishes and bright-field illumination provides cell visualization. Digital images are obtained and cell detection is applied based on corner detection, generating individual cell targets as x–y points. These points in the dish are then irradiated individually by a micron field size high-LET microbeam. Post-irradiation, time-lapse imaging follows cells’ response. All irradiated cells are tracked by linking trajectories in all time-frames, based on finding their nearest position. Cell divisions are detected based on cell appearance and individual cell temporary corner density. The number of divisions anticipated is low due to the high probability of cell killing from high-LET irradiation. Survival curves are produced based on cell’s capacity to divide at least four to five times. The process is repeated for a range of doses of radiation. Validation shows the efficiency of the proposed cell detection and tracking method in finding cell divisions. PMID:26835414

  18. Applications of High-Throughput Clonogenic Survival Assays in High-LET Particle Microbeams.

    PubMed

    Georgantzoglou, Antonios; Merchant, Michael J; Jeynes, Jonathan C G; Mayhead, Natalie; Punia, Natasha; Butler, Rachel E; Jena, Rajesh

    2015-01-01

    Charged particle therapy is increasingly becoming a valuable tool in cancer treatment, mainly due to the favorable interaction of particle radiation with matter. Its application is still limited due, in part, to lack of data regarding the radiosensitivity of certain cell lines to this radiation type, especially to high-linear energy transfer (LET) particles. From the earliest days of radiation biology, the clonogenic survival assay has been used to provide radiation response data. This method produces reliable data but it is not optimized for high-throughput microbeam studies with high-LET radiation where high levels of cell killing lead to a very low probability of maintaining cells' clonogenic potential. A new method, therefore, is proposed in this paper, which could potentially allow these experiments to be conducted in a high-throughput fashion. Cells are seeded in special polypropylene dishes and bright-field illumination provides cell visualization. Digital images are obtained and cell detection is applied based on corner detection, generating individual cell targets as x-y points. These points in the dish are then irradiated individually by a micron field size high-LET microbeam. Post-irradiation, time-lapse imaging follows cells' response. All irradiated cells are tracked by linking trajectories in all time-frames, based on finding their nearest position. Cell divisions are detected based on cell appearance and individual cell temporary corner density. The number of divisions anticipated is low due to the high probability of cell killing from high-LET irradiation. Survival curves are produced based on cell's capacity to divide at least four to five times. The process is repeated for a range of doses of radiation. Validation shows the efficiency of the proposed cell detection and tracking method in finding cell divisions. PMID:26835414

  19. High-Throughput High-Resolution Class I HLA Genotyping in East Africa

    PubMed Central

    Koehler, Rebecca N.; Walsh, Anne M.; Sanders-Buell, Eric E.; Eller, Leigh Anne; Eller, Michael; Currier, Jeffrey R.; Bautista, Christian T.; Wabwire-Mangen, Fred; Hoelscher, Michael; Maboko, Leonard; Kim, Jerome; Michael, Nelson L.; Robb, Merlin L.; McCutchan, Francine E.; Kijak, Gustavo H.

    2010-01-01

    HLA, the most genetically diverse loci in the human genome, play a crucial role in host-pathogen interaction by mediating innate and adaptive cellular immune responses. A vast number of infectious diseases affect East Africa, including HIV/AIDS, malaria, and tuberculosis, but the HLA genetic diversity in this region remains incompletely described. This is a major obstacle for the design and evaluation of preventive vaccines. Available HLA typing techniques, that provide the 4-digit level resolution needed to interpret immune responses, lack sufficient throughput for large immunoepidemiological studies. Here we present a novel HLA typing assay bridging the gap between high resolution and high throughput. The assay is based on real-time PCR using sequence-specific primers (SSP) and can genotype carriers of the 49 most common East African class I HLA-A, -B, and -C alleles, at the 4-digit level. Using a validation panel of 175 samples from Kampala, Uganda, previously defined by sequence-based typing, the new assay performed with 100% sensitivity and specificity. The assay was also implemented to define the HLA genetic complexity of a previously uncharacterized Tanzanian population, demonstrating its inclusion in the major East African genetic cluster. The availability of genotyping tools with this capacity will be extremely useful in the identification of correlates of immune protection and the evaluation of candidate vaccine efficacy. PMID:20505773

  20. High-throughput high-resolution microscopic slide digitization for pathology

    NASA Astrophysics Data System (ADS)

    Beckstead, Jeffrey A.; Dawson, Robert; Feineigle, Patricia A.; Gilbertson, John, Jr.; Hauser, Christopher; McVaugh, Timothy; Palmieri, Francesco; Sholehvar, David; Wetzel, Arthur

    2003-07-01

    Pathologist study tissue samples to determine the presence and nature of diseases. Morphology is a critical component to identifying cellular and tissue structures and the functional changes produced by disease. Technical advances in the field of pathology have primarily been in the areas of tissue preparation and the staining process that enhances the pathologist's identification of these structures. Pathologist's primary tool for diagnosis has remained the same for over a century--the optical microscope. Radiology has made tremendous advances with digitization and the ease of exchange and image analysis that comes with digital data and today's computer technology. Pathology is primed to enter the digital era as well. The major hurdles to wide spread acceptance of conversion to digital pathological imaging have been image resolution, scanner throughput, image file size and image display rates. InterScope Technologies, Inc. has developed a high-throughput, high-resolution microscopic slide digitization system that is well suited for pathological examination and diagnosis. This system is fully automated, captures at 0.3 μm per pixel, and can capture a slide in under 3 minutes, and has the potential to capture much faster. This paper will present the technical challenges associated with digital pathological imaging and how InterScope has addressed these challenges in the development of their digital scanner.

  1. ToxCast Workflow: High-throughput screening assay data processing, analysis and management (SOT)

    EPA Science Inventory

    US EPA’s ToxCast program is generating data in high-throughput screening (HTS) and high-content screening (HCS) assays for thousands of environmental chemicals, for use in developing predictive toxicity models. Currently the ToxCast screening program includes over 1800 unique c...

  2. Accelerating the design of solar thermal fuel materials through high throughput simulations.

    PubMed

    Liu, Yun; Grossman, Jeffrey C

    2014-12-10

    Solar thermal fuels (STF) store the energy of sunlight, which can then be released later in the form of heat, offering an emission-free and renewable solution for both solar energy conversion and storage. However, this approach is currently limited by the lack of low-cost materials with high energy density and high stability. In this Letter, we present an ab initio high-throughput computational approach to accelerate the design process and allow for searches over a broad class of materials. The high-throughput screening platform we have developed can run through large numbers of molecules composed of earth-abundant elements and identifies possible metastable structures of a given material. Corresponding isomerization enthalpies associated with the metastable structures are then computed. Using this high-throughput simulation approach, we have discovered molecular structures with high isomerization enthalpies that have the potential to be new candidates for high-energy density STF. We have also discovered physical principles to guide further STF materials design through structural analysis. More broadly, our results illustrate the potential of using high-throughput ab initio simulations to design materials that undergo targeted structural transitions.

  3. High Throughput Genotoxicity Profiling of the US EPA ToxCast Chemical Library

    EPA Science Inventory

    A key aim of the ToxCast project is to investigate modern molecular and genetic high content and high throughput screening (HTS) assays, along with various computational tools to supplement and perhaps replace traditional assays for evaluating chemical toxicity. Genotoxicity is a...

  4. Accelerating the Design of Solar Thermal Fuel Materials through High Throughput Simulations

    SciTech Connect

    Liu, Y; Grossman, JC

    2014-12-01

    Solar thermal fuels (STF) store the energy of sunlight, which can then be released later in the form of heat, offering an emission-free and renewable solution for both solar energy conversion and storage. However, this approach is currently limited by the lack of low-cost materials with high energy density and high stability. In this Letter, we present an ab initio high-throughput computational approach to accelerate the design process and allow for searches over a broad class of materials. The high-throughput screening platform we have developed can run through large numbers of molecules composed of earth-abundant elements and identifies possible metastable structures of a given material. Corresponding isomerization enthalpies associated with the metastable structures are then computed. Using this high-throughput simulation approach, we have discovered molecular structures with high isomerization enthalpies that have the potential to be new candidates for high-energy density STF. We have also discovered physical principles to guide further STF materials design through structural analysis. More broadly, our results illustrate the potential of using high-throughput ab initio simulations to design materials that undergo targeted structural transitions.

  5. Accelerating the design of solar thermal fuel materials through high throughput simulations.

    PubMed

    Liu, Yun; Grossman, Jeffrey C

    2014-12-10

    Solar thermal fuels (STF) store the energy of sunlight, which can then be released later in the form of heat, offering an emission-free and renewable solution for both solar energy conversion and storage. However, this approach is currently limited by the lack of low-cost materials with high energy density and high stability. In this Letter, we present an ab initio high-throughput computational approach to accelerate the design process and allow for searches over a broad class of materials. The high-throughput screening platform we have developed can run through large numbers of molecules composed of earth-abundant elements and identifies possible metastable structures of a given material. Corresponding isomerization enthalpies associated with the metastable structures are then computed. Using this high-throughput simulation approach, we have discovered molecular structures with high isomerization enthalpies that have the potential to be new candidates for high-energy density STF. We have also discovered physical principles to guide further STF materials design through structural analysis. More broadly, our results illustrate the potential of using high-throughput ab initio simulations to design materials that undergo targeted structural transitions. PMID:25372463

  6. Perspective: Composition-structure-property mapping in high-throughput experiments: Turning data into knowledge

    NASA Astrophysics Data System (ADS)

    Hattrick-Simpers, Jason R.; Gregoire, John M.; Kusne, A. Gilad

    2016-05-01

    With their ability to rapidly elucidate composition-structure-property relationships, high-throughput experimental studies have revolutionized how materials are discovered, optimized, and commercialized. It is now possible to synthesize and characterize high-throughput libraries that systematically address thousands of individual cuts of fabrication parameter space. An unresolved issue remains transforming structural characterization data into phase mappings. This difficulty is related to the complex information present in diffraction and spectroscopic data and its variation with composition and processing. We review the field of automated phase diagram attribution and discuss the impact that emerging computational approaches will have in the generation of phase diagrams and beyond.

  7. Microsphere-enhanced label-free high-throughput molecular detection

    NASA Astrophysics Data System (ADS)

    Deng, Cheng; Huang, Guoliang; Xu, Shukuan; Zhu, Jing; Ma, Rui; Han, Chao; Chen, Shengyi; Ma, Chen

    2008-12-01

    In this paper, we developed a microsphere enhanced and label free high throughput molecular detection based on SPRI and microarray chips, and a self-built surface plasmon resonance imaging instrument was set. One label-free protein chip forming a 7× probe array was designed and fabricated using a commercial microarray robot spotter on chemical modified gold-coated glass slide. Antibody molecules were successfully detected in label-free and high-throughput method by using this chip. The detection signals on the chip was successfully enhanced by using microspheres with a diameter of 1 μm.

  8. Dynamic evaluation and control of blood clotting using a microfluidic platform for high-throughput diagnostics

    NASA Astrophysics Data System (ADS)

    Combariza, Miguel E.; Yu, Xinghuo; Nesbitt, Warwick; Tovar-Lopez, Francisco; Rabus, Dominik G.; Mitchell, Arnan

    2015-12-01

    Microfluidic technology has the potential to revolutionise blood-clotting diagnostics by incorporating key physiological blood flow conditions like shear rate. In this paper we present a customised dynamic microfluidic system, which evaluates the blood clotting response to multiple conditions of shear rate on a single microchannel. The system can achieve high-throughput testing through use of an advanced fluid control system, which provides with rapid and precise regulation of the blood flow conditions in the platform. We present experimental results that demonstrate the potential of this platform to develop into a high-throughput, low-cost, blood-clotting diagnostics device.

  9. Medium to high throughput screening: microfabrication and chip-based technology.

    PubMed

    Wen, Yuan; Zhang, Xudong; Yang, Shang-Tian

    2012-01-01

    Medium to high throughput screening for toxicity testing can provide a wealth of information with significant time and cost savings. New technologies, such as microfabrication, microfluidics and chip-based technology, combined with advanced cell culture and detection techniques, open up new opportunities in toxicity testing. In this chapter, fundamentals of microfabrication and microfluidics are discussed with a focus on the broad and novel applications on toxicity studies enabled by these technologies. Emphasis is placed on microscale cell and tissue culture models for medium and high throughput systemic toxicity studies in vitro.

  10. High-throughput detection of DNA double-strand breaks using image cytometry

    PubMed Central

    Fowler, Tyler L.; Bailey, Alison M.; Bednarz, Bryan P.; Kimple, Randall J.

    2015-01-01

    Assessment of γH2AX expression for studying DNA double-strand break formation is often performed by manual counting of foci using immunofluorescence microscopy, an approach that is laborious and subject to significant foci selection bias. Here we present a novel high-throughput method for detecting DNA double-strand breaks using automated image cytometry assessment of cell average γH2AX immunofluorescence. Our technique provides an expedient, high-throughput, objective, and cost-effective method for γH2AX analysis. PMID:25605579

  11. Statistical and Computational Methods for High-Throughput Sequencing Data Analysis of Alternative Splicing

    PubMed Central

    2013-01-01

    The burgeoning field of high-throughput sequencing significantly improves our ability to understand the complexity of transcriptomes. Alternative splicing, as one of the most important driving forces for transcriptome diversity, can now be studied at an unprecedent resolution. Efficient and powerful computational and statistical methods are in urgent need to facilitate the characterization and quantification of alternative splicing events. Here we discuss methods in splice junction read mapping, and methods in exon-centric or isoform-centric quantification of alternative splicing. In addition, we discuss HITS-CLIP and splicing QTL analyses which are novel high-throughput sequencing based approaches in the dissection of splicing regulation. PMID:24058384

  12. High-throughput analysis of total nitrogen content that replaces the classic Kjeldahl method.

    PubMed

    Yasuhara, T; Nokihara, K

    2001-10-01

    A high-throughput method for determination of total nitrogen content has been developed. The method involves decomposition of samples, followed by trapping and quantitative colorimetric determination of the resulting ammonia. The present method is rapid, facile, and economical. Thus, it can replace the classic Kjeldahl method through its higher efficiency for determining multiple samples. Compared to the classic method, the present method is economical and environmentally friendly. Based on the present method, a novel reactor was constructed to realize routine high-throughput analyses of multiple samples such as those found for pharmaceutical materials, foods, and/or excrements.

  13. Accelerating Virtual High-Throughput Ligand Docking: current technology and case study on a petascale supercomputer

    PubMed Central

    Ellingson, Sally R.; Dakshanamurthy, Sivanesan; Brown, Milton; Smith, Jeremy C.

    2013-01-01

    In this paper we give the current state of high-throughput virtual screening. We describe a case study of using a task-parallel MPI (Message Passing Interface) version of Autodock4 [1], [2] to run a virtual high-throughput screen of one-million compounds on the Jaguar Cray XK6 Supercomputer at Oak Ridge National Laboratory. We include a description of scripts developed to increase the efficiency of the predocking file preparation and postdocking analysis. A detailed tutorial, scripts, and source code for this MPI version of Autodock4 are available online at http://www.bio.utk.edu/baudrylab/autodockmpi.htm. PMID:24729746

  14. Rapid and high-throughput detection of highly pathogenic bacteria by Ibis PLEX-ID technology.

    PubMed

    Jacob, Daniela; Sauer, Uschi; Housley, Roberta; Washington, Cicely; Sannes-Lowery, Kristin; Ecker, David J; Sampath, Rangarajan; Grunow, Roland

    2012-01-01

    In this manuscript, we describe the identification of highly pathogenic bacteria using an assay coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS) run on an Ibis PLEX-ID high-throughput platform. The biothreat cluster assay identifies most of the potential bioterrorism-relevant microorganisms including Bacillus anthracis, Francisella tularensis, Yersinia pestis, Burkholderia mallei and pseudomallei, Brucella species, and Coxiella burnetii. DNA from 45 different reference materials with different formulations and different concentrations were chosen and sent to a service screening laboratory that uses the PCR/ESI-MS platform to provide a microbial identification service. The standard reference materials were produced out of a repository built up in the framework of the EU funded project "Establishment of Quality Assurances for Detection of Highly Pathogenic Bacteria of Potential Bioterrorism Risk" (EQADeBa). All samples were correctly identified at least to the genus level.

  15. Achieving high mass-throughput of therapeutic proteins through parvovirus retentive filters.

    PubMed

    Bolton, Glen R; Basha, Jonida; Lacasse, Daniel P

    2010-01-01

    Parvovirus retentive filters that assure removal of viruses and virus-like particles during the production of therapeutic proteins significantly contribute to total manufacturing costs. Operational approaches that can increase throughput and reduce filtration area would result in a significant cost savings. A combination of methods was used to achieve high throughputs of an antibody or therapeutic protein solution through three parvovirus retentive filters. These methods included evaluation of diatomaceous earth or size-based prefilters, the addition of additives, and the optimization of protein concentration, temperature, buffer composition, and solution pH. An optimum temperature of 35°C was found for maximizing throughput through the Virosart CPV and Viresolve Pro filters. Mass-throughput values of 7.3, 26.4, and 76.2 kg/m(2) were achieved through the Asahi Planova 20N, Virosart CPV, and Viresolve Pro filters, respectively, in 4 h of processing. Mass-throughput values of 73, 137, and 192 kg/m(2) were achieved through a Millipore Viresolve Pro filter in 4.0, 8.8, and 22.1 h of processing, respectively, during a single experiment. However, large-scale parvovirus filtration operations are typically controlled to limit volumetric throughput to below the level achieved during small-scale virus spiking experiments. The virus spike may cause significant filter plugging, limiting throughput. Therefore newer parvovirus filter spiking strategies should be adopted that may lead to more representative viral clearance data and higher utilization of large-scale filter capacity.

  16. Microfluidic-Enabled Print-to-Screen Platform for High-Throughput Screening of Combinatorial Chemotherapy.

    PubMed

    Ding, Yuzhe; Li, Jiannan; Xiao, Wenwu; Xiao, Kai; Lee, Joyce; Bhardwaj, Urvashi; Zhu, Zijie; Digiglio, Philip; Yang, Gaomai; Lam, Kit S; Pan, Tingrui

    2015-10-20

    Since the 1960s, combination chemotherapy has been widely utilized as a standard method to treat cancer. However, because of the potentially enormous number of drug candidates and combinations, conventional identification methods of the effective drug combinations are usually associated with significantly high operational costs, low throughput screening, laborious and time-consuming procedures, and ethical concerns. In this paper, we present a low-cost, high-efficiency microfluidic print-to-screen (P2S) platform, which integrates combinatorial screening with biomolecular printing for high-throughput screening of anticancer drug combinations. This P2S platform provides several distinct advantages and features, including automatic combinatorial printing, high-throughput parallel drug screening, modular disposable cartridge, and biocompatibility, which can potentially speed up the entire discovery cycle of potent drug combinations. Microfluidic impact printing utilizing plug-and-play microfluidic cartridges is experimentally characterized with controllable droplet volume and accurate positioning. Furthermore, the combinatorial print-to-screen assay is demonstrated in a proof-of-concept biological experiment which can identify the positive hits among the entire drug combination library in a parallel and rapid manner. Overall, this microfluidic print-to-screen platform offers a simple, low-cost, high-efficiency solution for high-throughput large-scale combinatorial screening and can be applicable for various emerging applications in drug cocktail discovery. PMID:26334956

  17. High Throughput Atomic Layer Deposition Processes: High Pressure Operations, New Reactor Designs, and Novel Metal Processing

    NASA Astrophysics Data System (ADS)

    Mousa, MoatazBellah Mahmoud

    Atomic Layer Deposition (ALD) is a vapor phase nano-coating process that deposits very uniform and conformal thin film materials with sub-angstrom level thickness control on various substrates. These unique properties made ALD a platform technology for numerous products and applications. However, most of these applications are limited to the lab scale due to the low process throughput relative to the other deposition techniques, which hinders its industrial adoption. In addition to the low throughput, the process development for certain applications usually faces other obstacles, such as: a required new processing mode (e.g., batch vs continuous) or process conditions (e.g., low temperature), absence of an appropriate reactor design for a specific substrate and sometimes the lack of a suitable chemistry. This dissertation studies different aspects of ALD process development for prospect applications in the semiconductor, textiles, and battery industries, as well as novel organic-inorganic hybrid materials. The investigation of a high pressure, low temperature ALD process for metal oxides deposition using multiple process chemistry revealed the vital importance of the gas velocity over the substrate to achieve fast depositions at these challenging processing conditions. Also in this work, two unique high throughput ALD reactor designs are reported. The first is a continuous roll-to-roll ALD reactor for ultra-fast coatings on porous, flexible substrates with very high surface area. While the second reactor is an ALD delivery head that allows for in loco ALD coatings that can be executed under ambient conditions (even outdoors) on large surfaces while still maintaining very high deposition rates. As a proof of concept, part of a parked automobile window was coated using the ALD delivery head. Another process development shown herein is the improvement achieved in the selective synthesis of organic-inorganic materials using an ALD based process called sequential vapor

  18. Spatial tuning of acoustofluidic pressure nodes by altering net sonic velocity enables high-throughput, efficient cell sorting

    SciTech Connect

    Jung, Seung-Yong; Notton, Timothy; Fong, Erika; Shusteff, Maxim; Weinberger, Leor S.

    2015-01-07

    Particle sorting using acoustofluidics has enormous potential but widespread adoption has been limited by complex device designs and low throughput. Here, we report high-throughput separation of particles and T lymphocytes (600 μL min-1) by altering the net sonic velocity to reposition acoustic pressure nodes in a simple two-channel device. Finally, the approach is generalizable to other microfluidic platforms for rapid, high-throughput analysis.

  19. Spatial tuning of acoustofluidic pressure nodes by altering net sonic velocity enables high-throughput, efficient cell sorting

    DOE PAGES

    Jung, Seung-Yong; Notton, Timothy; Fong, Erika; Shusteff, Maxim; Weinberger, Leor S.

    2015-01-07

    Particle sorting using acoustofluidics has enormous potential but widespread adoption has been limited by complex device designs and low throughput. Here, we report high-throughput separation of particles and T lymphocytes (600 μL min-1) by altering the net sonic velocity to reposition acoustic pressure nodes in a simple two-channel device. Finally, the approach is generalizable to other microfluidic platforms for rapid, high-throughput analysis.

  20. Environmental surveillance and monitoring--The next frontiers for high-throughput toxicology.

    PubMed

    Schroeder, Anthony L; Ankley, Gerald T; Houck, Keith A; Villeneuve, Daniel L

    2016-03-01

    High-throughput toxicity testing technologies along with the World Wide Web are revolutionizing both generation of and access to data regarding the biological activities that chemicals can elicit when they interact with specific proteins, genes, or other targets in the body of an organism. To date, however, most of the focus has been on the application of such data to assessment of individual chemicals. The authors suggest that environmental surveillance and monitoring represent the next frontiers for high-throughput toxicity testing. Resources already exist in curated databases of chemical-biological interactions, including highly standardized quantitative dose-response data generated from nascent high-throughput toxicity testing programs such as ToxCast and Tox21, to link chemicals detected through environmental analytical chemistry to known biological activities. The emergence of the adverse outcome pathway framework and the associated knowledge base for linking molecular-level or pathway-level perturbations of biological systems to adverse outcomes traditionally considered in risk assessment and regulatory decision-making through a series of measurable biological changes provides a critical link between activity and hazard. Furthermore, environmental samples can be directly analyzed via high-throughput toxicity testing platforms to provide an unprecedented breadth of biological activity characterization that integrates the effects of all compounds present in a mixture, whether known or not. Novel application of these chemical-biological interaction data provides an opportunity to transform scientific characterization of potential hazards associated with exposure to complex mixtures of environmental contaminants. PMID:26923854

  1. High-throughput data pipelines for metabolic flux analysis in plants.

    PubMed

    Poskar, C Hart; Huege, Jan; Krach, Christian; Shachar-Hill, Yair; Junker, Björn H

    2014-01-01

    In this chapter we illustrate the methodology for high-throughput metabolic flux analysis. Central to this is developing an end to end data pipeline, crucial for integrating the wet lab experiments and analytics, combining hardware and software automation, and standardizing data representation providing importers and exporters to support third party tools. The use of existing software at the start, data extraction from the chromatogram, and the end, MFA analysis, allows for the most flexibility in this workflow. Developing iMS2Flux provided a standard, extensible, platform independent tool to act as the "glue" between these end points. Most importantly this tool can be easily adapted to support different data formats, data verification and data correction steps allowing it to be central to managing the data necessary for high-throughput MFA. An additional tool was needed to automate the MFA software and in particular to take advantage of the course grained parallel nature of high-throughput analysis and available high performance computing facilities.In combination these methods show the development of high-throughput pipelines that allow metabolic flux analysis to join as a full member of the omics family.

  2. A high-throughput standing surface acoustic wave (SSAW)-based cell sorter

    PubMed Central

    Li, Peng; Mao, Zhangming; Huang, Po-Hsun; Rufo, Joseph; Guo, Feng; Wang, Lin; McCoy, J. Philip; Levine, Stewart J.; Huang, Tony Jun

    2015-01-01

    Acoustic-based fluorescence activated cell sorters (FACS) have drawn increased attention in recent years due to their versatility, high biocompatibility, high controllability, and simple design. However, the sorting throughput for existing acoustic cell sorters is far from optimum for practical applications. Here we report a high-throughput cell sorting method based on standing surface acoustic waves (SSAWs). We utilized a pair of focused interdigital transducers (FIDTs) to generate SSAW with high resolution and high energy efficiency. As a result, the sorting throughput is improved significantly from conventional acoustic-based cell sorting methods. We demonstrated the successful sorting of 10 μm polystyrene particles with a minimum actuation time of 72 μs, which translates to a potential sorting rate of more than 13,800 events/s. Without using a cell-detection unit, we were able to demonstrate an actual sorting throughput of 3,300 events/s. Our sorting method can be conveniently integrated with upstream detection units, and it represents an important development towards a functional acoustic-based FACS system. PMID:26289231

  3. High-throughput time-stretch microscopy with morphological and chemical specificity

    NASA Astrophysics Data System (ADS)

    Lei, Cheng; Ugawa, Masashi; Nozawa, Taisuke; Ideguchi, Takuro; Di Carlo, Dino; Ota, Sadao; Ozeki, Yasuyuki; Goda, Keisuke

    2016-03-01

    Particle analysis is an effective method in analytical chemistry for sizing and counting microparticles such as emulsions, colloids, and biological cells. However, conventional methods for particle analysis, which fall into two extreme categories, have severe limitations. Sieving and Coulter counting are capable of analyzing particles with high throughput, but due to their lack of detailed information such as morphological and chemical characteristics, they can only provide statistical results with low specificity. On the other hand, CCD or CMOS image sensors can be used to analyze individual microparticles with high content, but due to their slow charge download, the frame rate (hence, the throughput) is significantly limited. Here by integrating a time-stretch optical microscope with a three-color fluorescent analyzer on top of an inertial-focusing microfluidic device, we demonstrate an optofluidic particle analyzer with a sub-micrometer spatial resolution down to 780 nm and a high throughput of 10,000 particles/s. In addition to its morphological specificity, the particle analyzer provides chemical specificity to identify chemical expressions of particles via fluorescence detection. Our results indicate that we can identify different species of microparticles with high specificity without sacrificing throughput. Our method holds promise for high-precision statistical particle analysis in chemical industry and pharmaceutics.

  4. A targeted proteomics toolkit for high-throughput absolute quantification of Escherichia coli proteins.

    PubMed

    Batth, Tanveer S; Singh, Pragya; Ramakrishnan, Vikram R; Sousa, Mirta M L; Chan, Leanne Jade G; Tran, Huu M; Luning, Eric G; Pan, Eva H Y; Vuu, Khanh M; Keasling, Jay D; Adams, Paul D; Petzold, Christopher J

    2014-11-01

    Transformation of engineered Escherichia coli into a robust microbial factory is contingent on precise control of metabolism. Yet, the throughput of omics technologies used to characterize cell components has lagged far behind our ability to engineer novel strains. To expand the utility of quantitative proteomics for metabolic engineering, we validated and optimized targeted proteomics methods for over 400 proteins from more than 20 major pathways in E. coli metabolism. Complementing these methods, we constructed a series of synthetic genes to produce concatenated peptides (QconCAT) for absolute quantification of the proteins and made them available through the Addgene plasmid repository (www.addgene.org). To facilitate high sample throughput, we developed a fast, analytical-flow chromatography method using a 5.5-min gradient (10 min total run time). Overall this toolkit provides an invaluable resource for metabolic engineering by increasing sample throughput, minimizing development time and providing peptide standards for absolute quantification of E. coli proteins.

  5. Ultra high-throughput single molecule spectroscopy with a 1024 pixel SPAD

    PubMed Central

    Colyer, Ryan A.; Scalia, Giuseppe; Villa, Federica A.; Guerrieri, Fabrizio; Tisa, Simone; Zappa, Franco; Cova, Sergio; Weiss, Shimon; Michalet, Xavier

    2013-01-01

    Single-molecule spectroscopy is a powerful approach to measuring molecular properties such as size, brightness, conformation, and binding constants. Due to the low concentrations in the single-molecule regime, measurements with good statistical accuracy require long acquisition times. Previously we showed a factor of 8 improvement in acquisition speed using a custom-CMOS 8x1 SPAD array. Here we present preliminary results with a 64X improvement in throughput obtained using a liquid crystal on silicon spatial light modulator (LCOS-SLM) and a novel standard CMOS 1024 pixel SPAD array, opening the way to truly high-throughput single-molecule spectroscopy. PMID:24386535

  6. Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization

    PubMed Central

    Fan, Beiyuan; Li, Xiufeng; Chen, Deyong; Peng, Hongshang; Wang, Junbo; Chen, Jian

    2016-01-01

    This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on three key performance parameters (absolute quantification, sensitivity, and throughput). PMID:26891303

  7. Applications of high throughput (combinatorial) methodologies to electronic, magnetic, optical, and energy-related materials

    NASA Astrophysics Data System (ADS)

    Green, Martin L.; Takeuchi, Ichiro; Hattrick-Simpers, Jason R.

    2013-06-01

    High throughput (combinatorial) materials science methodology is a relatively new research paradigm that offers the promise of rapid and efficient materials screening, optimization, and discovery. The paradigm started in the pharmaceutical industry but was rapidly adopted to accelerate materials research in a wide variety of areas. High throughput experiments are characterized by synthesis of a "library" sample that contains the materials variation of interest (typically composition), and rapid and localized measurement schemes that result in massive data sets. Because the data are collected at the same time on the same "library" sample, they can be highly uniform with respect to fixed processing parameters. This article critically reviews the literature pertaining to applications of combinatorial materials science for electronic, magnetic, optical, and energy-related materials. It is expected that high throughput methodologies will facilitate commercialization of novel materials for these critically important applications. Despite the overwhelming evidence presented in this paper that high throughput studies can effectively inform commercial practice, in our perception, it remains an underutilized research and development tool. Part of this perception may be due to the inaccessibility of proprietary industrial research and development practices, but clearly the initial cost and availability of high throughput laboratory equipment plays a role. Combinatorial materials science has traditionally been focused on materials discovery, screening, and optimization to combat the extremely high cost and long development times for new materials and their introduction into commerce. Going forward, combinatorial materials science will also be driven by other needs such as materials substitution and experimental verification of materials properties predicted by modeling and simulation, which have recently received much attention with the advent of the Materials Genome

  8. Organic light-emitting diodes: High-throughput virtual screening

    NASA Astrophysics Data System (ADS)

    Hirata, Shuzo; Shizu, Katsuyuki

    2016-10-01

    Computer networks, trained with data from delayed-fluorescence materials that have been successfully used in organic light-emitting diodes, facilitate the high-speed prediction of good emitters for display and lighting applications.

  9. Holographic memory module with ultra-high capacity and throughput

    SciTech Connect

    Vladimir A. Markov, Ph.D.

    2000-06-04

    High capacity, high transfer rate, random access memory systems are needed to archive and distribute the tremendous volume of digital information being generated, for example, the human genome mapping and online libraries. The development of multi-gigabit per second networks underscores the need for next-generation archival memory systems. During Phase I we conducted the theoretical analysis and accomplished experimental tests that validated the key aspects of the ultra-high density holographic data storage module with high transfer rate. We also inspected the secure nature of the encoding method and estimated the performance of full-scale system. Two basic architectures were considered, allowing for reversible compact solid-state configuration with limited capacity, and very large capacity write once read many memory system.

  10. Automatic Dendritic Length Quantification for High Throughput Screening of Mature Neurons

    PubMed Central

    Smafield, Timothy; Pasupuleti, Venkat; Sharma, Kamal; Huganir, Richard L.; Ye, Bing

    2015-01-01

    High-throughput automated fluorescent imaging and screening are important for studying neuronal development, functions, and pathogenesis. An automatic approach of analyzing images acquired in automated fashion, and quantifying dendritic characteristics is critical for making such screens high-throughput. However, automatic and effective algorithms and tools, especially for the images of mature mammalian neurons with complex arbors, have been lacking. Here, we present algorithms and a tool for quantifying dendritic length that is fundamental for analyzing growth of neuronal network. We employ a divide-and-conquer framework that tackles the challenges of high-throughput images of neurons and enables the integration of multiple automatic algorithms. Within this framework, we developed algorithms that adapt to local properties to detect faint branches. We also developed a path search that can preserve the curvature change to accurately measure dendritic length with arbor branches and turns. In addition, we proposed an ensemble strategy of three estimation algorithms to further improve the overall efficacy. We tested our tool on images for cultured mouse hippocampal neurons immunostained with a dendritic marker for high-throughput screen. Results demonstrate the effectiveness of our proposed method when comparing the accuracy with previous methods. The software has been implemented as an ImageJ plugin and available for use. PMID:25854493

  11. Use of High-Throughput Testing and Approaches for Evaluating Chemical Risk-Relevance to Humans

    EPA Science Inventory

    ToxCast is profiling the bioactivity of thousands of chemicals based on high-throughput screening (HTS) and computational models that integrate knowledge of biological systems and in vivo toxicities. Many of these assays probe signaling pathways and cellular processes critical to...

  12. An Improved, high-throughput method for detection of bluetongue virus RNA in Culicodes midges

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new rapid (less than 6 h from insect-to-results) high-throughput assay is reported that is sensitive and specific for detecting BTV RNA in Culicoides biting midges. Homogenization and extraction of nucleic acids from individual Culicoides specimens were performed in a 96-well plate format using sp...

  13. Improved high-throughput bioassay for Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As we gain more information through functional genomic studies of Rhyzopertha dominica (F.), we need a high throughput bioassay system to screen potential biopesticides. R. dominica is an internal feeder during immature stages and presents unique challenges with traditional bioassay methods. Our pri...

  14. High-Throughput Models for Exposure-Based Chemical Prioritization in the ExpoCast Project

    EPA Science Inventory

    The United States Environmental Protection Agency (U.S. EPA) must characterize potential risks to human health and the environment associated with manufacture and use of thousands of chemicals. High-throughput screening (HTS) for biological activity allows the ToxCast research pr...

  15. Environmental surveillance and monitoring the next frontier for pathway-based high throughput screening

    EPA Science Inventory

    In response to a proposed vision and strategy for toxicity testing in the 21st century nascent high throughput toxicology (HTT) programs have tested thousands of chemicals in hundreds of pathway-based biological assays. Although, to date, use of HTT data for safety assessment of ...

  16. Thawing the Landscape of the Era of High Throughput: Signs of Spring?

    PubMed

    Brady, Donald W

    2015-09-01

    In his latest book, Dr. Kenneth Ludmerer examines the history of graduate medical education (GME) in the United States, including its "era of high throughput" during which residents admitted more patients for shorter periods of time as hospitals focused on decreasing length of stay secondary to prospective payment reform. The author of this Commentary considers the implications of the era of high throughput and how the U.S. health care system must change to address its lasting effects.The era of high throughput initially had incomplete penetrance across the health care system landscape and a variable effect on GME. Trainees were variably aware of the financial forces bearing down on the health care system. Over time, the pervasiveness of the financial pressures and managed care became more complete, and the ubiquity of information through the Internet and social media ensured that residents became more acutely aware of how the changes to the health care system were affecting their education. There is now an opportunity for GME to be the nidus for ushering in an era of cost consciousness focused on patient needs and higher-quality GME rather than on the financial pressures that characterized the era of high throughput. PMID:26164641

  17. Evaluation of food-relevant chemicals in the ToxCast high-throughput screening program

    EPA Science Inventory

    There are thousands of chemicals that are directly added to or come in contact with food, many of which have undergone little to no toxicological evaluation. The ToxCast high-throughput screening (HTS) program has evaluated over 1,800 chemicals in concentration-response across ~8...

  18. HIGH-THROUGHPUT IDENTIFICATION OF CATALYTIC REDOX-ACTIVE CYSTEINE RESIDUES

    EPA Science Inventory

    Cysteine (Cys) residues often play critical roles in proteins; however, identification of their specific functions has been limited to case-by-case experimental approaches. We developed a procedure for high-throughput identification of catalytic redox-active Cys in proteins by se...

  19. Incorporating Population Variability and Susceptible Subpopulations into Dosimetry for High-Throughput Toxicity Testing

    EPA Science Inventory

    Momentum is growing worldwide to use in vitro high-throughput screening (HTS) to evaluate human health effects of chemicals. However, the integration of dosimetry into HTS assays and incorporation of population variability will be essential before its application in a risk assess...

  20. AOPs and Biomarkers: Bridging High Throughput Screening and Regulatory Decision Making

    EPA Science Inventory

    As high throughput screening (HTS) plays a larger role in toxicity testing, camputational toxicology has emerged as a critical component in interpreting the large volume of data produced. Computational models designed to quantify potential adverse effects based on HTS data will b...

  1. High-Throughput Simulation of Environmental Chemical Fate for Exposure Prioritization

    EPA Science Inventory

    The U.S. EPA must consider lists of hundreds to thousands of chemicals when allocating resources to identify risk in human populations and the environment. High-throughput screening assays to characterize biological activity in vitro have allowed the ToxCastTM program to identify...

  2. High-Throughput Exposure Potential Prioritization for ToxCast Chemicals

    EPA Science Inventory

    The U.S. EPA must consider lists of hundreds to thousands of chemicals when prioritizing research resources in order to identify risk to human populations and the environment. High-throughput assays to identify biological activity in vitro have allowed the ToxCastTM program to i...

  3. High-Throughput Simulation of Environmental Chemical Fate for Exposure Prioritization (Annual Meeting of ISES)

    EPA Science Inventory

    The U.S. EPA must consider thousands of chemicals when allocating resources to assess risk in human populations and the environment. High-throughput screening assays to characterize biological activity in vitro are being implemented in the ToxCastTM program to rapidly characteri...

  4. High-Throughput Toxicokinetics (HTTK) R package (CompTox CoP presentation)

    EPA Science Inventory

    Toxicokinetics (TK) provides a bridge between HTS and HTE by predicting tissue concentrations due to exposure, but traditional TK methods are resource intensive. Relatively high throughput TK (HTTK) methods have been used by the pharmaceutical industry to determine range of effic...

  5. High Throughput Prioritization for Integrated Toxicity Testing Based on ToxCast Chemical Profiling

    EPA Science Inventory

    The rational prioritization of chemicals for integrated toxicity testing is a central goal of the U.S. EPA’s ToxCast™ program (http://epa.gov/ncct/toxcast/). ToxCast includes a wide-ranging battery of over 500 in vitro high-throughput screening assays which in Phase I was used to...

  6. A high-throughput, precipitating colorimetric sandwich ELISA microarray for shiga toxins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies)...

  7. High-throughput sequencing to decipher the genetic heterogeneity of deafness

    PubMed Central

    2012-01-01

    Identifying genes causing non-syndromic hearing loss has been challenging using traditional approaches. We describe the impact that high-throughput sequencing approaches are having in discovery of genes related to hearing loss and the implications for clinical diagnosis. PMID:22647651

  8. High-Throughput Dietary Exposure Predictions for Chemical Migrants from Food Packaging Materials

    EPA Science Inventory

    United States Environmental Protection Agency researchers have developed a Stochastic Human Exposure and Dose Simulation High -Throughput (SHEDS-HT) model for use in prioritization of chemicals under the ExpoCast program. In this research, new methods were implemented in SHEDS-HT...

  9. A high-throughput screening assay for distinguishing nitrile hydratases from nitrilases.

    PubMed

    Angelini, Leticia Mara Lima; da Silva, Amanda Ribeiro Martins; Rocco, Lucas de Freitas Coli; Milagre, Cintia Duarte de Freitas

    2015-03-01

    A modified colorimetric high-throughput screen based on pH changes combined with an amidase inhibitor capable of distinguishing between nitrilases and nitrile hydratases. This enzymatic screening is based on a binary response and is suitable for the first step of hierarchical screening projects.

  10. A high-throughput screening assay for distinguishing nitrile hydratases from nitrilases

    PubMed Central

    Angelini, Leticia Mara Lima; da Silva, Amanda Ribeiro Martins; Rocco, Lucas de Freitas Coli; Milagre, Cintia Duarte de Freitas

    2015-01-01

    A modified colorimetric high-throughput screen based on pH changes combined with an amidase inhibitor capable of distinguishing between nitrilases and nitrile hydratases. This enzymatic screening is based on a binary response and is suitable for the first step of hierarchical screening projects. PMID:26221095

  11. [New methods in pharmaceutical research: combinatorial chemistry and high throughput screening].

    PubMed

    Schirlin, Daniel; Galvan, Martin; Le Fur, Gérard

    2007-01-01

    New lead-identification methodologies such as high-throughput screening and combinatorial chemistry have been integrated into pharmaceutical research over the past 5-10 years. More rational use in the selection of potential preclinical candidates for some difficult targets has increased the chances of success.

  12. An industrial engineering approach to laboratory automation for high throughput screening.

    PubMed

    Menke, K C

    2000-01-01

    Across the pharmaceutical industry, there are a variety of approaches to laboratory automation for high throughput screening. At Sphinx Pharmaceuticals, the principles of industrial engineering have been applied to systematically identify and develop those automated solutions that provide the greatest value to the scientists engaged in lead generation.

  13. The Impact of Data Fragmentation on High-Throughput Clinical Phenotyping

    ERIC Educational Resources Information Center

    Wei, Weiqi

    2012-01-01

    Subject selection is essential and has become the rate-limiting step for harvesting knowledge to advance healthcare through clinical research. Present manual approaches inhibit researchers from conducting deep and broad studies and drawing confident conclusions. High-throughput clinical phenotyping (HTCP), a recently proposed approach, leverages…

  14. Environmental surveillance and monitoring. The next frontiers for high-throughput toxicology

    EPA Science Inventory

    High throughput toxicity testing (HTT) technologies along with the world-wide web are revolutionizing both generation and access to data regarding the bioactivities that chemicals can elicit when they interact with specific proteins, genes, or other targets in the body of an orga...

  15. High-throughput genotyping of hop (Humulus lupulus L.) utilising diversity arrays technology (DArT)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Implementation of molecular methods in hop breeding is dependent on the availability of sizeable numbers of polymorphic markers and a comprehensive understanding of genetic variation. Diversity Arrays Technology (DArT) is a high-throughput cost-effective method for the discovery of large numbers of...

  16. Quantitative high-throughput population dynamics in continuous-culture by automated microscopy.

    PubMed

    Merritt, Jason; Kuehn, Seppe

    2016-01-01

    We present a high-throughput method to measure abundance dynamics in microbial communities sustained in continuous-culture. Our method uses custom epi-fluorescence microscopes to automatically image single cells drawn from a continuously-cultured population while precisely controlling culture conditions. For clonal populations of Escherichia coli our instrument reveals history-dependent resilience and growth rate dependent aggregation. PMID:27616752

  17. Perspectives on Validation of High-Throughput Assays Supporting 21st Century Toxicity Testing

    EPA Science Inventory

    In vitro high-throughput screening (HTS) assays are seeing increasing use in toxicity testing. HTS assays can simultaneously test many chemicals but have seen limited use in the regulatory arena, in part because of the need to undergo rigorous, time-consuming formal validation. ...

  18. Optical microplates for photonic high throughput screening of algal photosynthesis and biofuel production.

    PubMed

    Mertiri, Taulant; Chen, Meng; Holland, Thomas; Basu, Amar S

    2011-01-01

    Biological systems respond not only to chemical stimuli (drugs, proteins) but also to physical stimuli (light, heat, stress). Though there are many high throughput tools for screening chemical stimuli, no such tool exists for screening of physical stimuli. This paper presents a novel instrument for photonic high throughput screening of photosynthesis, a light-driven bioprocess. The optical microplate has a footprint identical to a standard 96 well plate, and it provides temporal and intensity control of light in each individual well. Intensity control provides 128 dimming levels (7-bit resolution), with maximum intensity 120 mE/cm(2). Temporal modulation, used for studying dynamics and regulation of photosynthesis, can be as low as 10 μs. We used photonic screening for high throughput studies of algal growth rates and photosynthetic efficiency, using the model organism Dunaliella tertiolecta, a lipid producing algae of interest in biofuel production. Due to the ability to conduct 96 studies in parallel, experiments that would require 2 years using conventional tools can be completed in 1 week. This instrument opens up novel high throughput protocols for photobiology and the growing field of phenomics.

  19. A practical fluorogenic substrate for high-throughput screening of glutathione S-transferase inhibitors.

    PubMed

    Fujikawa, Yuuta; Morisaki, Fumika; Ogura, Asami; Morohashi, Kana; Enya, Sora; Niwa, Ryusuke; Goto, Shinji; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo; Inoue, Hideshi

    2015-07-21

    We report a new fluorogenic substrate for glutathione S-transferase (GST), 3,4-DNADCF, enabling the assay with a low level of nonenzymatic background reaction. Inhibitors against Noppera-bo/GSTe14 from Drosophila melanogaster were identified by high throughput screening using 3,4-DNADCF, demonstrating the utility of this substrate.

  20. A novel high-throughput irradiator for in vitro radiation sensitivity bioassays

    NASA Astrophysics Data System (ADS)

    Fowler, Tyler L.

    Given the emphasis on more personalized radiation therapy there is an ongoing and compelling need to develop high-throughput screening tools to further examine the biological effects of ionizing radiation on cells, tissues and organ systems in either the research or clinical setting. Conventional x-ray irradiators are designed to provide maximum versatility to radiobiology researchers, typically accommodating small animals, tissue or blood samples, and cellular applications. This added versatility often impedes the overall sensitivity and specificity of an experiment resulting in a trade-off between the number of absorbed doses (or dose rates) and biological endpoints that can be investigated in vitro in a reasonable amount of time. Therefore, modern irradiator designs are incompatible with current high-throughput bioassay technologies. Furthermore, important dosimetry and calibration characteristics (i.e. dose build-up region, beam attenuation, and beam scatter) of these irradiators are typically unknown to the end user, which can lead to significant deviation between delivered dose and intended dose to cells that adversely impact experimental results. Therefore, the overarching goal of this research is to design and develop a robust and fully automated high-throughput irradiator for in vitro radiation sensitivity investigations. Additionally, in vitro biological validation of this system was performed by assessing intracellular reactive oxygen species production, physical DNA double strand breaks, and activation of cellular DNA repair mechanisms. Finally, the high-throughput irradiator was used to investigate autophagic flux, a cellular adaptive response, as a potential biomarker of radiation sensitivity.

  1. High-throughput synthesis and characterization of nanocrystalline porphyrinic zirconium metal-organic frameworks.

    PubMed

    Kelty, M L; Morris, W; Gallagher, A T; Anderson, J S; Brown, K A; Mirkin, C A; Harris, T D

    2016-06-14

    We describe and employ a high-throughput screening method to accelerate the synthesis and identification of pure-phase, nanocrystalline metal-organic frameworks (MOFs). We demonstrate the efficacy of this method through its application to a series of porphyrinic zirconium MOFs, resulting in the isolation of MOF-525, MOF-545, and PCN-223 on the nanoscale. PMID:27247981

  2. Incorporating Human Dosimetry and Exposure into High-Throughput In Vitro Toxicity Screening

    EPA Science Inventory

    Many chemicals in commerce today have undergone limited or no safety testing. To reduce the number of untested chemicals and prioritize limited testing resources, several governmental programs are using high-throughput in vitro screens for assessing chemical effects across multip...

  3. Development of a thyroperoxidase inhibition assay for high-throughput screening

    EPA Science Inventory

    High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluores...

  4. High-throughput synthesis and characterization of nanocrystalline porphyrinic zirconium metal-organic frameworks.

    PubMed

    Kelty, M L; Morris, W; Gallagher, A T; Anderson, J S; Brown, K A; Mirkin, C A; Harris, T D

    2016-06-14

    We describe and employ a high-throughput screening method to accelerate the synthesis and identification of pure-phase, nanocrystalline metal-organic frameworks (MOFs). We demonstrate the efficacy of this method through its application to a series of porphyrinic zirconium MOFs, resulting in the isolation of MOF-525, MOF-545, and PCN-223 on the nanoscale.

  5. HTPheno: An image analysis pipeline for high-throughput plant phenotyping

    PubMed Central

    2011-01-01

    Background In the last few years high-throughput analysis methods have become state-of-the-art in the life sciences. One of the latest developments is automated greenhouse systems for high-throughput plant phenotyping. Such systems allow the non-destructive screening of plants over a period of time by means of image acquisition techniques. During such screening different images of each plant are recorded and must be analysed by applying sophisticated image analysis algorithms. Results This paper presents an image analysis pipeline (HTPheno) for high-throughput plant phenotyping. HTPheno is implemented as a plugin for ImageJ, an open source image processing software. It provides the possibility to analyse colour images of plants which are taken in two different views (top view and side view) during a screening. Within the analysis different phenotypical parameters for each plant such as height, width and projected shoot area of the plants are calculated for the duration of the screening. HTPheno is applied to analyse two barley cultivars. Conclusions HTPheno, an open source image analysis pipeline, supplies a flexible and adaptable ImageJ plugin which can be used for automated image analysis in high-throughput plant phenotyping and therefore to derive new biological insights, such as determination of fitness. PMID:21569390

  6. Predictive Model of Rat Reproductive Toxicity from ToxCast High Throughput Screening

    EPA Science Inventory

    The EPA ToxCast research program uses high throughput screening for bioactivity profiling and predicting the toxicity of large numbers of chemicals. ToxCast Phase‐I tested 309 well‐characterized chemicals in over 500 assays for a wide range of molecular targets and cellular respo...

  7. PLASMA PROTEIN PROFILING AS A HIGH THROUGHPUT TOOL FOR CHEMICAL SCREENING USING A SMALL FISH MODEL

    EPA Science Inventory

    Hudson, R. Tod, Michael J. Hemmer, Kimberly A. Salinas, Sherry S. Wilkinson, James Watts, James T. Winstead, Peggy S. Harris, Amy Kirkpatrick and Calvin C. Walker. In press. Plasma Protein Profiling as a High Throughput Tool for Chemical Screening Using a Small Fish Model (Abstra...

  8. Detecting adulterants in milk powder using high-throughput Raman chemical imaging

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study used a line-scan high-throughput Raman imaging system to authenticate milk powder. A 5 W 785 nm line laser (240 mm long and 1 mm wide) was used as a Raman excitation source. The system was used to acquire hyperspectral Raman images in a wavenumber range of 103–2881 cm-1 from the skim milk...

  9. High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death

    EPA Science Inventory

    Human ATAD5 is an excellent biomarker for identifying genotoxic compounds because ATADS protein levels increase post-transcriptionally following exposure to a variety of DNA damaging agents. Here we report a novel quantitative high-throughput ATAD5-Iuciferase assay that can moni...

  10. Monitoring of four DNA extraction methods upstream of high-throughput sequencing of Anisakidae nematodes.

    PubMed

    Seesao, Y; Audebert, C; Verrez-Bagnis, V; Merlin, S; Jérôme, M; Viscogliosi, E; Dei-Cas, E; Aliouat-Denis, C M; Gay, M

    2014-07-01

    Different methods were evaluated to extract DNA from pooled nematodes belonging to Anisakis, Contracaecum, Pseudoterranova and Hysterothylacium genera isolated from edible fish. Pooled DNA extraction is the first and compulsory step to allow the identification of a large number of samples through high-throughput DNA sequencing with drastic time and cost reductions. PMID:24845469

  11. Estimating Toxicity Pathway Activating Doses for High Throughput Chemical Risk Assessments

    EPA Science Inventory

    Estimating a Toxicity Pathway Activating Dose (TPAD) from in vitro assays as an analog to a reference dose (RfD) derived from in vivo toxicity tests would facilitate high throughput risk assessments of thousands of data-poor environmental chemicals. Estimating a TPAD requires def...

  12. 20160314 - Evaluation of sequencing approaches for high-throughput toxicogenomics (SOT)

    EPA Science Inventory

    Whole-genome in vitro transcriptomics has shown the capability to identify mechanisms of action and estimates of potency for chemical-mediated effects in a toxicological framework, but with limited throughput and high cost. We present the evaluation of three toxicogenomics platfo...

  13. Quantitative high-throughput population dynamics in continuous-culture by automated microscopy

    PubMed Central

    Merritt, Jason; Kuehn, Seppe

    2016-01-01

    We present a high-throughput method to measure abundance dynamics in microbial communities sustained in continuous-culture. Our method uses custom epi-fluorescence microscopes to automatically image single cells drawn from a continuously-cultured population while precisely controlling culture conditions. For clonal populations of Escherichia coli our instrument reveals history-dependent resilience and growth rate dependent aggregation. PMID:27616752

  14. A high-throughput-compatible assay to measure the degradation of endogenous Huntingtin proteins

    PubMed Central

    Wu, Peng; Lu, Ming-xing; Cui, Xiao-tian; Yang, He-qing; Yu, Shen-liang; Zhu, Jian-bin; Sun, Xiao-li; Lu, Boxun

    2016-01-01

    Aim: The accumulation of disease-causing proteins is a common hallmark of many neurodegenerative disorders. Measuring the degradation of such proteins using high-throughput-compatible assays is highly desired for the identification of genetic and chemical modulators of degradation. For example, Huntington's disease (HD) is an incurable hereditary neurodegenerative disorder caused by the cytotoxicity of mutant huntingtin protein (mHTT). The high-throughput measurement of mHTT degradation is important in HD drug discovery and research. Existing methods for such purposes have limitations due to their dependence on protein tags or pan protein synthesis inhibitors. Here, we report a high-throughput-compatible pulse-chase method (CH-chase) for the measurement of endogenous tag-free huntingtin protein (HTT) degradation based on Click chemistry and Homogeneous Time Resolved Fluorescence (HTRF) technologies. Methods: The pulsed-labeled proteins were conjugated with biotin using the click reaction strain-promoted alkyne-azide cycloaddition (SPAAC), and the chase signals were calculated by measuring the reduction percentage of the HTT HTRF signals after pull-down with streptavidin beads. Results: We validated that the signals were within the linear detection range and were HTT-specific. We successfully measured the degradation of endogenous HTT in a high-throughput-compatible format using 96-well plates. The predicted changes of HTT degradation by known modifiers were observed, which confirmed that the assay is suitable for the identification of HTT degradation modifiers. Conclusion: We have established the first high-throughput-compatible assay capable of measuring endogenous, tag-free HTT degradation, providing a valuable tool for HD research and drug discovery. The method could be applied to other proteins and can facilitate research on other neurodegenerative disorders and proteinopathies. PMID:27264314

  15. Advances in surface plasmon resonance-based high throughput biochips

    NASA Astrophysics Data System (ADS)

    Zhang, San-Jun; Berguiga, Lotfi; Elezgaray, Juan; Hugo, Nicolas; Li, Wen-Xue; Roland, Thibault; Zeng, He-Ping; Argoul, Francoise

    2009-12-01

    This article reviews our recent advances in surface plasmon resonance (SPR) based biochips. It includes four issues, which are the preparation and characterization of high quality gold film, the preparation and characterization of self-assembled monolayer (SAM), dynamics of DNA adsorption on SAMs, and SPR-based microscopies. Numerous topics related to SPR, such as, the modeling of SPR by transmission matrix, effective medium theory, applications of SPR in biology, and SPR-based novel microscopies, are discussed. A novel electrochemical technique, which is extremely useful for the preparation and characterization of high quality SAMs, is also discussed.

  16. A Highly Sensitive, High-Throughput Assay for the Detection of Turner Syndrome

    PubMed Central

    Hager, Karl; Hosono, Seiyu; Wise, Anastasia; Li, Peining; Rinder, Henry M.; Gruen, Jeffrey R.

    2011-01-01

    Objective: Turner syndrome (TS) occurs when an X-chromosome is completely or partially deleted or when X-chromosomal mosaicism is present. Girls with TS benefit from early diagnosis and treatment with GH; however, many girls with TS are not detected until after 10 yr of age, resulting in delayed evaluation and treatment. Methods: We developed a high-throughput test for TS, based on a quantitative method of genotyping to detect X-chromosome abnormalities. This test uses pyrosequencing to quantitate relative allele strength (RAS) from single-nucleotide polymorphisms using 18 informative single-nucleotide polymorphisms markers that span the X-chromosome and one marker for the detection of Y-chromosome material. Results: Cutoff ranges for heterozygous, homozygous, or out-of-range RAS values were established from a cohort of 496 males and females. Positive TS scoring criteria were defined as the presence of homozygosity for all 18 markers or the presence of at least one out-of-range RAS value. To determine the validity of this rapid test for TS detection, we undertook a large-scale study using DNA from 132 females without TS and 74 females with TS for whom karyotypes were available. TS was identified with 96.0% sensitivity and 97.0% specificity in this cohort. We also tested buccal swab DNA from a group of 19 females without TS and 69 females with TS. In this group, TS was identified with 97.1% sensitivity and 84.2% specificity. Conclusions: These results demonstrate the validity of a high-throughput, pyrosequencing based test for the accurate detection of TS, providing a potential alternative to karyotype testing. PMID:21177792

  17. HIGH THROUGHPUT SPECTRAL IMAGING SYSTEM FOR WHOLESOMENESS INSPECTION OF CHICKEN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An online line-scan imaging system containing an electron-multiplying charge-coupled device detector and line-scan spectrograph was used for identifying wholesome and unwholesome freshly slaughtered chicken carcasses on high-speed commercial chicken processing lines. Hyperspectral images were acqui...

  18. Embedded image enhancement for high-throughput cameras

    NASA Astrophysics Data System (ADS)

    Geerts, Stan J. C.; Cornelissen, Dion; de With, Peter H. N.

    2014-03-01

    This paper presents image enhancement for a novel Ultra-High-Definition (UHD) video camera offering 4K images and higher. Conventional image enhancement techniques need to be reconsidered for the high-resolution images and the low-light sensitivity of the new sensor. We study two image enhancement functions and evaluate and optimize the algorithms for embedded implementation in programmable logic (FPGA). The enhancement study involves high-quality Auto White Balancing (AWB) and Local Contrast Enhancement (LCE). We have compared multiple algorithms from literature, both with objective and subjective metrics. In order to objectively compare Local Contrast (LC), an existing LC metric is modified for LC measurement in UHD images. For AWB, we have found that color histogram stretching offers a subjective high image quality and it is among the algorithms with the lowest complexity, while giving only a small balancing error. We impose a color-to-color gain constraint, which improves robustness of low-light images. For local contrast enhancement, a combination of contrast preserving gamma and single-scale Retinex is selected. A modified bilateral filter is designed to prevent halo artifacts, while significantly reducing the complexity and simultaneously preserving quality. We show that by cascading contrast preserving gamma and single-scale Retinex, the visibility of details is improved towards the level appropriate for high-quality surveillance applications. The user is offered control over the amount of enhancement. Also, we discuss the mapping of those functions on a heterogeneous platform to come to an effective implementation while preserving quality and robustness.

  19. A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes.

    PubMed

    Na, Hong; Laver, John D; Jeon, Jouhyun; Singh, Fateh; Ancevicius, Kristin; Fan, Yujie; Cao, Wen Xi; Nie, Kun; Yang, Zhenglin; Luo, Hua; Wang, Miranda; Rissland, Olivia; Westwood, J Timothy; Kim, Philip M; Smibert, Craig A; Lipshitz, Howard D; Sidhu, Sachdev S

    2016-04-01

    Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins.

  20. Digital fragment analysis of short tandem repeats by high-throughput amplicon sequencing.

    PubMed

    Darby, Brian J; Erickson, Shay F; Hervey, Samuel D; Ellis-Felege, Susan N

    2016-07-01

    High-throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high-throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high-throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost-effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence-aware microsatellite data. PMID:27386092

  1. A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes.

    PubMed

    Na, Hong; Laver, John D; Jeon, Jouhyun; Singh, Fateh; Ancevicius, Kristin; Fan, Yujie; Cao, Wen Xi; Nie, Kun; Yang, Zhenglin; Luo, Hua; Wang, Miranda; Rissland, Olivia; Westwood, J Timothy; Kim, Philip M; Smibert, Craig A; Lipshitz, Howard D; Sidhu, Sachdev S

    2016-04-01

    Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins. PMID:26847261

  2. High Throughput Label Free Measurement of Cancer Cell Adhesion Kinetics Under Hemodynamic Flow

    PubMed Central

    Spencer, Adrianne; Baker, Aaron B.

    2016-01-01

    The kinetics of receptor-mediated cell adhesion to extracellular matrix and adherent cell monolayers plays a key role in many physiological and pathological processes including cancer metastasis. Within this process the presence of fluidic shear forces is a key regulator of binding equilibrium and kinetics of cell adhesion. Current techniques to examine the kinetics of cell adhesion are either performed in the absence of flow or are low throughput, limiting their application to pharmacological compound screening or the high throughput investigation of biological mechanisms. We developed a high throughput flow device that applies flow in a multi-well format and interfaced this system with electric cell-substrate impedance sensing (ECIS) system to allow label free detection of cell adhesion. We demonstrate that this combined system is capable of making real time measurements of cancer cell adhesion to extracellular matrix and immobilized platelets. In addition, we examined the dependence of the kinetics of binding of cancer cells on the level of shear stress and in the presence of small molecule inhibitors to adhesion-related pathways. This versatile system is broadly adaptable to the high throughput study of cell adhesion kinetics for many applications including drug screening and the investigation of the mechanisms of cancer metastasis. PMID:26816215

  3. A synchronous Gigabit Ethernet protocol stack for high-throughput UDP/IP applications

    NASA Astrophysics Data System (ADS)

    Födisch, P.; Lange, B.; Sandmann, J.; Büchner, A.; Enghardt, W.; Kaever, P.

    2016-01-01

    State of the art detector readout electronics require high-throughput data acquisition (DAQ) systems. In many applications, e. g. for medical imaging, the front-end electronics are set up as separate modules in a distributed DAQ. A standardized interface between the modules and a central data unit is essential. The requirements on such an interface are varied, but demand almost always a high throughput of data. Beyond this challenge, a Gigabit Ethernet interface is predestined for the broad requirements of Systems-on-a-Chip (SoC) up to large-scale DAQ systems. We have implemented an embedded protocol stack for a Field Programmable Gate Array (FPGA) capable of high-throughput data transmission and clock synchronization. A versatile stack architecture for the User Datagram Protocol (UDP) and Internet Control Message Protocol (ICMP) over Internet Protocol (IP) such as Address Resolution Protocol (ARP) as well as Precision Time Protocol (PTP) is presented. With a point-to-point connection to a host in a MicroTCA system we achieved the theoretical maximum data throughput limited by UDP both for 1000BASE-T and 1000BASE-KX links. Furthermore, we show that the random jitter of a synchronous clock over a 1000BASE-T link for a PTP application is below 60 ps.

  4. (abstract) A High Throughput 3-D Inner Product Processor

    NASA Technical Reports Server (NTRS)

    Daud, Tuan

    1996-01-01

    A particularily challenging image processing application is the real time scene acquisition and object discrimination. It requires spatio-temporal recognition of point and resolved objects at high speeds with parallel processing algorithms. Neural network paradigms provide fine grain parallism and, when implemented in hardware, offer orders of magnitude speed up. However, neural networks implemented on a VLSI chip are planer architectures capable of efficient processing of linear vector signals rather than 2-D images. Therefore, for processing of images, a 3-D stack of neural-net ICs receiving planar inputs and consuming minimal power are required. Details of the circuits with chip architectures will be described with need to develop ultralow-power electronics. Further, use of the architecture in a system for high-speed processing will be illustrated.

  5. High-throughput method for a kinetics analysis of the high-pressure inactivation of microorganisms using microplates.

    PubMed

    Hasegawa, Toshimi; Hayashi, Manabu; Nomura, Kazuki; Hayashi, Mayumi; Kido, Miyuki; Ohmori, Tsuneo; Fukuda, Masao; Iguchi, Akinori; Ueno, Shigeaki; Shigematsu, Toru; Hirayama, Masao; Fujii, Tomoyuki

    2012-06-01

    Using microplates as pressure and cultivation vessels, a high-throughput method was developed for analyzing the high-pressure inactivation kinetics of microorganisms. The loss of viability from a high-pressure treatment, measured based on the growth delay during microplate cultivation, showed reproducibility with the conventional agar plate method and was applicable for the kinetics analysis.

  6. Melter Throughput Enhancements for High-Iron HLW

    SciTech Connect

    Kruger, A. A.; Gan, Hoa; Joseph, Innocent; Pegg, Ian L.; Matlack, Keith S.; Chaudhuri, Malabika; Kot, Wing

    2012-12-26

    This report describes work performed to develop and test new glass and feed formulations in order to increase glass melting rates in high waste loading glass formulations for HLW with high concentrations of iron. Testing was designed to identify glass and melter feed formulations that optimize waste loading and waste processing rate while meeting all processing and product quality requirements. The work included preparation and characterization of crucible melts to assess melt rate using a vertical gradient furnace system and to develop new formulations with enhanced melt rate. Testing evaluated the effects of waste loading on glass properties and the maximum waste loading that can be achieved. The results from crucible-scale testing supported subsequent DuraMelter 100 (DM100) tests designed to examine the effects of enhanced glass and feed formulations on waste processing rate and product quality. The DM100 was selected as the platform for these tests due to its extensive previous use in processing rate determination for various HLW streams and glass compositions.

  7. High-throughput variation detection and genotyping using microarrays.

    PubMed

    Cutler, D J; Zwick, M E; Carrasquillo, M M; Yohn, C T; Tobin, K P; Kashuk, C; Mathews, D J; Shah, N A; Eichler, E E; Warrington, J A; Chakravarti, A

    2001-11-01

    The genetic dissection of complex traits may ultimately require a large number of SNPs to be genotyped in multiple individuals who exhibit phenotypic variation in a trait of interest. Microarray technology can enable rapid genotyping of variation specific to study samples. To facilitate their use, we have developed an automated statistical method (ABACUS) to analyze microarray hybridization data and applied this method to Affymetrix Variation Detection Arrays (VDAs). ABACUS provides a quality score to individual genotypes, allowing investigators to focus their attention on sites that give accurate information. We have applied ABACUS to an experiment encompassing 32 autosomal and eight X-linked genomic regions, each consisting of approximately 50 kb of unique sequence spanning a 100-kb region, in 40 humans. At sufficiently high-quality scores, we are able to read approximately 80% of all sites. To assess the accuracy of SNP detection, 108 of 108 SNPs have been experimentally confirmed; an additional 371 SNPs have been confirmed electronically. To access the accuracy of diploid genotypes at segregating autosomal sites, we confirmed 1515 of 1515 homozygous calls, and 420 of 423 (99.29%) heterozygotes. In replicate experiments, consisting of independent amplification of identical samples followed by hybridization to distinct microarrays of the same design, genotyping is highly repeatable. In an autosomal replicate experiment, 813,295 of 813,295 genotypes are called identically (including 351 heterozygotes); at an X-linked locus in males (haploid), 841,236 of 841,236 sites are called identically.

  8. A droplet-based, optofluidic device for high-throughput, quantitative bioanalysis.

    PubMed

    Guo, Feng; Lapsley, Michael Ian; Nawaz, Ahmad Ahsan; Zhao, Yanhui; Lin, Sz-Chin Steven; Chen, Yuchao; Yang, Shikuan; Zhao, Xing-Zhong; Huang, Tony Jun

    2012-12-18

    Analysis of chemical or biomolecular contents in a tiny amount of specimen presents a significant challenge in many biochemical studies and diagnostic applications. In this work, we present a single-layer, optofluidic device for real-time, high-throughput, quantitative analysis of droplet contents. Our device integrates an optical fiber-based, on-chip detection unit with a droplet-based microfluidic unit. It can quantitatively analyze the contents of individual droplets in real-time. It also achieves a detection throughput of 2000 droplets per second, a detection limit of 20 nM, and an excellent reproducibility in its detection results. In a proof-of-concept study, we demonstrate that our device can be used to perform detection of DNA and its mutations by monitoring the fluorescent signal changes of the target DNA/molecular beacon complex in single droplets. Our approach can be immediately extended to a real-time, high-throughput detection of other biomolecules (such as proteins and viruses) in droplets. With its advantages in throughput, functionality, cost, size, and reliability, the droplet-based optofluidic device presented here can be a valuable tool for many medical diagnostic applications.

  9. High-Throughput Mutational Analysis of a Twister Ribozyme.

    PubMed

    Kobori, Shungo; Yokobayashi, Yohei

    2016-08-22

    Recent discoveries of new classes of self-cleaving ribozymes in diverse organisms have triggered renewed interest in the chemistry and biology of ribozymes. Functional analysis and engineering of ribozymes often involve performing biochemical assays on multiple ribozyme mutants. However, because each ribozyme mutant must be individually prepared and assayed, the number and variety of mutants that can be studied are severely limited. All of the single and double mutants of a twister ribozyme (a total of 10 296 mutants) were generated and assayed for their self-cleaving activity by exploiting deep sequencing to count the numbers of cleaved and uncleaved sequences for every mutant. Interestingly, we found that the ribozyme is highly robust against mutations such that 71 % and 30 % of all single and double mutants, respectively, retain detectable activity under the assay conditions. It was also observed that the structural elements that comprise the ribozyme exhibit distinct sensitivity to mutations. PMID:27461281

  10. High-throughput cell analysis using multiplexed array technologies.

    PubMed

    Beske, Oren E; Goldbard, Simon

    2002-09-15

    The desire for more biologically relevant data from primary screening has resulted in a dramatic increase of cell-based assays in HTS labs. Consequently, new cell-array technologies are being developed to increase the quality and quantity of data emerging from such screens. These technologies take the form of both positional and non-positional formats, each with their own advantages. Notably, screens using these technologies generate databases of high-quality data that can be analyzed in ways currently not possible. The power of cell-based assays combined with new array and analytical technologies will enable the condensation of serial drug discovery processes, thereby decreasing the time and cost of taking a hit compound into clinical trials. Here, we compare array strategies being developed towards the goal of integrating multiplexed cell-based assays into HTS. PMID:12546879

  11. High throughput microfluidic rapid and low cost prototyping packaging methods.

    PubMed

    Miled, Amine; Sawan, Mohamad

    2013-12-23

    In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques. The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture. Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 μl/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

  12. Automated High Throughput Protein Crystallization Screening at Nanoliter Scale and Protein Structural Study on Lactate Dehydrogenase

    SciTech Connect

    Li, Fenglei

    2006-08-09

    The purposes of our research were: (1) To develop an economical, easy to use, automated, high throughput system for large scale protein crystallization screening. (2) To develop a new protein crystallization method with high screening efficiency, low protein consumption and complete compatibility with high throughput screening system. (3) To determine the structure of lactate dehydrogenase complexed with NADH by x-ray protein crystallography to study its inherent structural properties. Firstly, we demonstrated large scale protein crystallization screening can be performed in a high throughput manner with low cost, easy operation. The overall system integrates liquid dispensing, crystallization and detection and serves as a whole solution to protein crystallization screening. The system can dispense protein and multiple different precipitants in nanoliter scale and in parallel. A new detection scheme, native fluorescence, has been developed in this system to form a two-detector system with a visible light detector for detecting protein crystallization screening results. This detection scheme has capability of eliminating common false positives by distinguishing protein crystals from inorganic crystals in a high throughput and non-destructive manner. The entire system from liquid dispensing, crystallization to crystal detection is essentially parallel, high throughput and compatible with automation. The system was successfully demonstrated by lysozyme crystallization screening. Secondly, we developed a new crystallization method with high screening efficiency, low protein consumption and compatibility with automation and high throughput. In this crystallization method, a gas permeable membrane is employed to achieve the gentle evaporation required by protein crystallization. Protein consumption is significantly reduced to nanoliter scale for each condition and thus permits exploring more conditions in a phase diagram for given amount of protein. In addition

  13. Ultra-high-throughput Production of III-V/Si Wafer for Electronic and Photonic Applications

    NASA Astrophysics Data System (ADS)

    Geum, Dae-Myeong; Park, Min-Su; Lim, Ju Young; Yang, Hyun-Duk; Song, Jin Dong; Kim, Chang Zoo; Yoon, Euijoon; Kim, Sanghyeon; Choi, Won Jun

    2016-02-01

    Si-based integrated circuits have been intensively developed over the past several decades through ultimate device scaling. However, the Si technology has reached the physical limitations of the scaling. These limitations have fuelled the search for alternative active materials (for transistors) and the introduction of optical interconnects (called “Si photonics”). A series of attempts to circumvent the Si technology limits are based on the use of III-V compound semiconductor due to their superior benefits, such as high electron mobility and direct bandgap. To use their physical properties on a Si platform, the formation of high-quality III-V films on the Si (III-V/Si) is the basic technology ; however, implementing this technology using a high-throughput process is not easy. Here, we report new concepts for an ultra-high-throughput heterogeneous integration of high-quality III-V films on the Si using the wafer bonding and epitaxial lift off (ELO) technique. We describe the ultra-fast ELO and also the re-use of the III-V donor wafer after III-V/Si formation. These approaches provide an ultra-high-throughput fabrication of III-V/Si substrates with a high-quality film, which leads to a dramatic cost reduction. As proof-of-concept devices, this paper demonstrates GaAs-based high electron mobility transistors (HEMTs), solar cells, and hetero-junction phototransistors on Si substrates.

  14. Ultra-high-throughput Production of III-V/Si Wafer for Electronic and Photonic Applications

    PubMed Central

    Geum, Dae-Myeong; Park, Min-Su; Lim, Ju Young; Yang, Hyun-Duk; Song, Jin Dong; Kim, Chang Zoo; Yoon, Euijoon; Kim, SangHyeon; Choi, Won Jun

    2016-01-01

    Si-based integrated circuits have been intensively developed over the past several decades through ultimate device scaling. However, the Si technology has reached the physical limitations of the scaling. These limitations have fuelled the search for alternative active materials (for transistors) and the introduction of optical interconnects (called “Si photonics”). A series of attempts to circumvent the Si technology limits are based on the use of III-V compound semiconductor due to their superior benefits, such as high electron mobility and direct bandgap. To use their physical properties on a Si platform, the formation of high-quality III-V films on the Si (III-V/Si) is the basic technology ; however, implementing this technology using a high-throughput process is not easy. Here, we report new concepts for an ultra-high-throughput heterogeneous integration of high-quality III-V films on the Si using the wafer bonding and epitaxial lift off (ELO) technique. We describe the ultra-fast ELO and also the re-use of the III-V donor wafer after III-V/Si formation. These approaches provide an ultra-high-throughput fabrication of III-V/Si substrates with a high-quality film, which leads to a dramatic cost reduction. As proof-of-concept devices, this paper demonstrates GaAs-based high electron mobility transistors (HEMTs), solar cells, and hetero-junction phototransistors on Si substrates. PMID:26864968

  15. Ultra-high-throughput Production of III-V/Si Wafer for Electronic and Photonic Applications.

    PubMed

    Geum, Dae-Myeong; Park, Min-Su; Lim, Ju Young; Yang, Hyun-Duk; Song, Jin Dong; Kim, Chang Zoo; Yoon, Euijoon; Kim, SangHyeon; Choi, Won Jun

    2016-02-11

    Si-based integrated circuits have been intensively developed over the past several decades through ultimate device scaling. However, the Si technology has reached the physical limitations of the scaling. These limitations have fuelled the search for alternative active materials (for transistors) and the introduction of optical interconnects (called "Si photonics"). A series of attempts to circumvent the Si technology limits are based on the use of III-V compound semiconductor due to their superior benefits, such as high electron mobility and direct bandgap. To use their physical properties on a Si platform, the formation of high-quality III-V films on the Si (III-V/Si) is the basic technology ; however, implementing this technology using a high-throughput process is not easy. Here, we report new concepts for an ultra-high-throughput heterogeneous integration of high-quality III-V films on the Si using the wafer bonding and epitaxial lift off (ELO) technique. We describe the ultra-fast ELO and also the re-use of the III-V donor wafer after III-V/Si formation. These approaches provide an ultra-high-throughput fabrication of III-V/Si substrates with a high-quality film, which leads to a dramatic cost reduction. As proof-of-concept devices, this paper demonstrates GaAs-based high electron mobility transistors (HEMTs), solar cells, and hetero-junction phototransistors on Si substrates.

  16. High-Throughput Plasmid cDNA Library Screening

    SciTech Connect

    Wan, Kenneth H.; Yu, Charles; George, Reed A.; Carlson, JosephW.; Hoskins, Roger A.; Svirskas, Robert; Stapleton, Mark; Celniker, SusanE.

    2006-05-24

    Libraries of cDNA clones are valuable resources foranalysing the expression, structure, and regulation of genes, as well asfor studying protein functions and interactions. Full-length cDNA clonesprovide information about intron and exon structures, splice junctionsand 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs)derived from cDNA clones can be used to generate constructs allowingexpression of native proteins and N- or C-terminally tagged proteins.Thus, obtaining full-length cDNA clones and sequences for most or allgenes in an organism is critical for understanding genome functions.Expressed sequence tag (EST) sequencing samples cDNA libraries at random,which is most useful at the beginning of large-scale screening projects.However, as projects progress towards completion, the probability ofidentifying unique cDNAs via EST sequencing diminishes, resulting in poorrecovery of rare transcripts. We describe an adapted, high-throughputprotocol intended for recovery of specific, full-length clones fromplasmid cDNA libraries in five days.

  17. Label-free high-throughput imaging flow cytometry

    NASA Astrophysics Data System (ADS)

    Mahjoubfar, A.; Chen, C.; Niazi, K. R.; Rabizadeh, S.; Jalali, B.

    2014-03-01

    Flow cytometry is an optical method for studying cells based on their individual physical and chemical characteristics. It is widely used in clinical diagnosis, medical research, and biotechnology for analysis of blood cells and other cells in suspension. Conventional flow cytometers aim a laser beam at a stream of cells and measure the elastic scattering of light at forward and side angles. They also perform single-point measurements of fluorescent emissions from labeled cells. However, many reagents used in cell labeling reduce cellular viability or change the behavior of the target cells through the activation of undesired cellular processes or inhibition of normal cellular activity. Therefore, labeled cells are not completely representative of their unaltered form nor are they fully reliable for downstream studies. To remove the requirement of cell labeling in flow cytometry, while still meeting the classification sensitivity and specificity goals, measurement of additional biophysical parameters is essential. Here, we introduce an interferometric imaging flow cytometer based on the world's fastest continuous-time camera. Our system simultaneously measures cellular size, scattering, and protein concentration as supplementary biophysical parameters for label-free cell classification. It exploits the wide bandwidth of ultrafast laser pulses to perform blur-free quantitative phase and intensity imaging at flow speeds as high as 10 meters per second and achieves nanometer-scale optical path length resolution for precise measurements of cellular protein concentration.

  18. High-throughput search for new permanent magnet materials.

    PubMed

    Goll, D; Loeffler, R; Herbst, J; Karimi, R; Schneider, G

    2014-02-12

    The currently highest-performance Fe-Nd-B magnets show limited cost-effectiveness and lifetime due to their rare-earth (RE) content. The demand for novel hard magnetic phases with more widely available RE metals, reduced RE content or, even better, completely free of RE metals is therefore tremendous. The chances are that such materials still exist given the large number of as yet unexplored alloy systems. To discover such phases, an elaborate concept is necessary which can restrict and prioritize the search field while making use of efficient synthesis and analysis methods. It is shown that an efficient synthesis of new phases using heterogeneous non-equilibrium diffusion couples and reaction sintering is possible. Quantitative microstructure analysis of the domain pattern of the hard magnetic phases can be used to estimate the intrinsic magnetic parameters (saturation polarization from the domain contrast, anisotropy constant from the domain width, Curie temperature from the temperature dependence of the domain contrast). The probability of detecting TM-rich phases for a given system is high, therefore the approach enables one to scan through even higher component systems with one single sample. The visualization of newly occurring hard magnetic phases via their typical domain structure and the correlation existing between domain structure and intrinsic magnetic properties allows an evaluation of the industrial relevance of these novel phases.

  19. Informatics and High Throughput Screening of Thermophysical Properties

    NASA Technical Reports Server (NTRS)

    Hyers, Robert W.; Rogers, Jan R.

    2008-01-01

    The combination of computer-aided experiments with computational modeling enables a new class of powerful tools for materials research. A non-contact method for measuring density, thermal expansion, and creep of undercooled and high-temperature materials has been developed, using electrostatic levitation and optical diagnostics, including digital video. These experiments were designed to take advantage of the large volume of data (many gigabytes/experiment, terabytes/campaign) to gain additional information about the samples. For example, using sub-pixel interpolation to measure about 1000 vectors per image of the sample's surface allows the density of an axisymmetric sample to be determined to an accuracy of about 200 ppm (0.02%). A similar analysis applied to the surface shape of a rapidly rotating sample is combined with finite element modeling to determine the stress-dependence of creep in the sample in a single test. Details of the methods for both the computer-aided experiments and computational models will be discussed.

  20. High-Throughput Sequencing of a South American Amerindian

    PubMed Central

    Almeida, Renan; Alencar, Dayse O.; Barbosa, Maria Silvanira; Gusmão, Leonor; Silva, Wilson A.; de Souza, Sandro J.; Silva, Artur; Ribeiro-dos-Santos, Ândrea; Darnet, Sylvain; Santos, Sidney

    2013-01-01

    The emergence of next-generation sequencing technologies allowed access to the vast amounts of information that are contained in the human genome. This information has contributed to the understanding of individual and population-based variability and improved the understanding of the evolutionary history of different human groups. However, the genome of a representative of the Amerindian populations had not been previously sequenced. Thus, the genome of an individual from a South American tribe was completely sequenced to further the understanding of the genetic variability of Amerindians. A total of 36.8 giga base pairs (Gbp) were sequenced and aligned with the human genome. These Gbp corresponded to 95.92% of the human genome with an estimated miscall rate of 0.0035 per sequenced bp. The data obtained from the alignment were used for SNP (single-nucleotide) and INDEL (insertion-deletion) calling, which resulted in the identification of 502,017 polymorphisms, of which 32,275 were potentially new high-confidence SNPs and 33,795 new INDELs, specific of South Native American populations. The authenticity of the sample as a member of the South Native American populations was confirmed through the analysis of the uniparental (maternal and paternal) lineages. The autosomal comparison distinguished the investigated sample from others continental populations and revealed a close relation to the Eastern Asian populations and Aboriginal Australian. Although, the findings did not discard the classical model of America settlement; it brought new insides to the understanding of the human population history. The present study indicates a remarkable genetic variability in human populations that must still be identified and contributes to the understanding of the genetic variability of South Native American populations and of the human populations history. PMID:24386182

  1. Development of a High-Throughput Candida albicans Biofilm Chip

    PubMed Central

    Srinivasan, Anand; Uppuluri, Priya; Lopez-Ribot, Jose; Ramasubramanian, Anand K.

    2011-01-01

    We have developed a high-density microarray platform consisting of nano-biofilms of Candida albicans. A robotic microarrayer was used to print yeast cells of C. albicans encapsulated in a collagen matrix at a volume as low as 50 nL onto surface-modified microscope slides. Upon incubation, the cells grow into fully formed “nano-biofilms”. The morphological and architectural complexity of these biofilms were evaluated by scanning electron and confocal scanning laser microscopy. The extent of biofilm formation was determined using a microarray scanner from changes in fluorescence intensities due to FUN 1 metabolic processing. This staining technique was also adapted for antifungal susceptibility testing, which demonstrated that, similar to regular biofilms, cells within the on-chip biofilms displayed elevated levels of resistance against antifungal agents (fluconazole and amphotericin B). Thus, results from structural analyses and antifungal susceptibility testing indicated that despite miniaturization, these biofilms display the typical phenotypic properties associated with the biofilm mode of growth. In its final format, the C. albicans biofilm chip (CaBChip) is composed of 768 equivalent and spatially distinct nano-biofilms on a single slide; multiple chips can be printed and processed simultaneously. Compared to current methods for the formation of microbial biofilms, namely the 96-well microtiter plate model, this fungal biofilm chip has advantages in terms of miniaturization and automation, which combine to cut reagent use and analysis time, minimize labor intensive steps, and dramatically reduce assay costs. Such a chip should accelerate the antifungal drug discovery process by enabling rapid, convenient and inexpensive screening of hundreds-to-thousands of compounds simultaneously. PMID:21544190

  2. High-throughput machining using high average power ultrashort pulse lasers and ultrafast polygon scanner

    NASA Astrophysics Data System (ADS)

    Schille, Joerg; Schneider, Lutz; Streek, André; Kloetzer, Sascha; Loeschner, Udo

    2016-03-01

    In this paper, high-throughput ultrashort pulse laser machining is investigated on various industrial grade metals (Aluminium, Copper, Stainless steel) and Al2O3 ceramic at unprecedented processing speeds. This is achieved by using a high pulse repetition frequency picosecond laser with maximum average output power of 270 W in conjunction with a unique, in-house developed two-axis polygon scanner. Initially, different concepts of polygon scanners are engineered and tested to find out the optimal architecture for ultrafast and precision laser beam scanning. Remarkable 1,000 m/s scan speed is achieved on the substrate, and thanks to the resulting low pulse overlap, thermal accumulation and plasma absorption effects are avoided at up to 20 MHz pulse repetition frequencies. In order to identify optimum processing conditions for efficient high-average power laser machining, the depths of cavities produced under varied parameter settings are analyzed and, from the results obtained, the characteristic removal values are specified. The maximum removal rate is achieved as high as 27.8 mm3/min for Aluminium, 21.4 mm3/min for Copper, 15.3 mm3/min for Stainless steel and 129.1 mm3/min for Al2O3 when full available laser power is irradiated at optimum pulse repetition frequency.

  3. Three dimensional microfluidics with embedded microball lenses for parallel and high throughput multicolor fluorescence detection

    PubMed Central

    Fan, Y. J.; Wu, Y. C.; Chen, Y.; Kung, Y. C.; Wu, T. H.; Huang, K. W.; Sheen, H. J.; Chiou, P. Y.

    2013-01-01

    We report a 3D microfluidic device with 32 detection channels and 64 sheath flow channels and embedded microball lens array for high throughput multicolor fluorescence detection. A throughput of 358 400 cells/s has been accomplished. This device is realized by utilizing solid immersion micro ball lens arrays for high sensitivity and parallel fluorescence detection. High refractive index micro ball lenses (n = 2.1) are embedded underneath PDMS channels close to cell detection zones in channels. This design permits patterning high N.A. micro ball lenses in a compact fashion for parallel fluorescence detection on a small footprint device. This device also utilizes 3D microfluidic fabrication to address fluid routing issues in two-dimensional parallel sheath focusing and allows simultaneous pumping of 32 sample channels and 64 sheath flow channels with only two inlets. PMID:24404054

  4. Design of novel solar thermal fuels with high-throughput ab initio simulations

    NASA Astrophysics Data System (ADS)

    Liu, Yun; Grossman, Jeffrey

    2014-03-01

    Solar thermal fuels (STF) store the energy of sunlight, which can then be released later in the form of heat, offering an emission-free and renewable solution for both solar energy conversion and storage. However, this approach is currently limited by the lack of low-cost materials with high energy density and high stability. Previously we have predicted a new class of functional materials that have the potential to address these challenges. Recently, we have developed an ab initio high-throughput computational approach to accelerate the design process and allow for searches over a broad class of materials. The high-throughput screening algorithm we have developed can run through large numbers of molecules composed of earth-abundant elements, and identifies possible metastable structures of a given material. Corresponding isomerization enthalpies associated with the metastable structures are then computed. Using this high-throughput simulation approach, we have discovered molecular structures with high isomerization enthalpies that have the potential to be new candidates for high-energy density STF. We have also discovered physical design principles to guide further STF materials design through the correlation between isomerization enthalpy and structural properties.

  5. SPIM-fluid: open source light-sheet based platform for high-throughput imaging.

    PubMed

    Gualda, Emilio J; Pereira, Hugo; Vale, Tiago; Estrada, Marta Falcão; Brito, Catarina; Moreno, Nuno

    2015-11-01

    Light sheet fluorescence microscopy has recently emerged as the technique of choice for obtaining high quality 3D images of whole organisms/embryos with low photodamage and fast acquisition rates. Here we present an open source unified implementation based on Arduino and Micromanager, which is capable of operating Light Sheet Microscopes for automatized 3D high-throughput imaging on three-dimensional cell cultures and model organisms like zebrafish, oriented to massive drug screening. PMID:26601007

  6. 'Shotgun DNA synthesis' for the high-throughput construction of large DNA molecules.

    PubMed

    Kim, Hwangbeom; Han, Hyojun; Ahn, Jinwoo; Lee, Joongoo; Cho, Namjin; Jang, Hoon; Kim, Hyoki; Kwon, Sunghoon; Bang, Duhee

    2012-10-01

    We developed a highly scalable 'shotgun' DNA synthesis technology by utilizing microchip oligonucleotides, shotgun assembly and next-generation sequencing technology. A pool of microchip oligonucleotides targeting a penicillin biosynthetic gene cluster were assembled into numerous random fragments, and tagged with 20 bp degenerate barcode primer pairs. An optimal set of error-free fragments were identified by high-throughput DNA sequencing, selectively amplified using the barcode sequences, and successfully assembled into the target gene cluster.

  7. SPIM-fluid: open source light-sheet based platform for high-throughput imaging

    PubMed Central

    Gualda, Emilio J.; Pereira, Hugo; Vale, Tiago; Estrada, Marta Falcão; Brito, Catarina; Moreno, Nuno

    2015-01-01

    Light sheet fluorescence microscopy has recently emerged as the technique of choice for obtaining high quality 3D images of whole organisms/embryos with low photodamage and fast acquisition rates. Here we present an open source unified implementation based on Arduino and Micromanager, which is capable of operating Light Sheet Microscopes for automatized 3D high-throughput imaging on three-dimensional cell cultures and model organisms like zebrafish, oriented to massive drug screening. PMID:26601007

  8. Cosputtered composition-spread reproducibility established by high-throughput x-ray fluorescence

    SciTech Connect

    Gregoire, John M.; Dale, Darren; Kazimirov, Alexander; DiSalvo, Francis J.; Dover, R. Bruce van

    2010-09-15

    We describe the characterization of sputtered yttria-zirconia composition spread thin films by x-ray fluorescence (XRF). We also discuss our automated analysis of the XRF data, which was collected in a high throughput experiment at the Cornell High Energy Synchrotron Source. The results indicate that both the composition reproducibility of the library deposition and the composition measurements have a precision of better than 1 atomic percent.

  9. A High-Throughput, Adaptive FFT Architecture for FPGA-Based Space-Borne Data Processors

    NASA Technical Reports Server (NTRS)

    Nguyen, Kayla; Zheng, Jason; He, Yutao; Shah, Biren

    2010-01-01

    Historically, computationally-intensive data processing for space-borne instruments has heavily relied on ground-based computing resources. But with recent advances in functional densities of Field-Programmable Gate-Arrays (FPGAs), there has been an increasing desire to shift more processing on-board; therefore relaxing the downlink data bandwidth requirements. Fast Fourier Transforms (FFTs) are commonly used building blocks for data processing applications, with a growing need to increase the FFT block size. Many existing FFT architectures have mainly emphasized on low power consumption or resource usage; but as the block size of the FFT grows, the throughput is often compromised first. In addition to power and resource constraints, space-borne digital systems are also limited to a small set of space-qualified memory elements, which typically lag behind the commercially available counterparts in capacity and bandwidth. The bandwidth limitation of the external memory creates a bottleneck for a large, high-throughput FFT design with large block size. In this paper, we present the Multi-Pass Wide Kernel FFT (MPWK-FFT) architecture for a moderately large block size (32K) with considerations to power consumption and resource usage, as well as throughput. We will also show that the architecture can be easily adapted for different FFT block sizes with different throughput and power requirements. The result is completely contained within an FPGA without relying on external memories. Implementation results are summarized.

  10. Evaluation of Compatibility of ToxCast High-Throughput/High-Content Screening Assays with Engineered Nanomaterials

    EPA Science Inventory

    High-throughput and high-content screens are attractive approaches for prioritizing nanomaterial hazards and informing targeted testing due to the impracticality of using traditional toxicological testing on the large numbers and varieties of nanomaterials. The ToxCast program a...

  11. Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method.

    PubMed

    Lin, Jia-Ren; Fallahi-Sichani, Mohammad; Sorger, Peter K

    2015-01-01

    Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, particularly in the case of highly multiplexed methods such as mass cytometry (CyTOF) able to correlate the levels of multiple signalling, differentiation and cell fate markers. Immunofluorescence (IF) microscopy adds information on cell morphology and the microenvironment that are not obtained using flow-based techniques, but the multiplicity of conventional IF is limited. This has motivated development of imaging methods that require specialized instrumentation, exotic reagents or proprietary protocols that are difficult to reproduce in most laboratories. Here we report a public-domain method for achieving high multiplicity single-cell IF using cyclic immunofluorescence (CycIF), a simple and versatile procedure in which four-colour staining alternates with chemical inactivation of fluorophores to progressively build a multichannel image. Because CycIF uses standard reagents and instrumentation and is no more expensive than conventional IF, it is suitable for high-throughput assays and screening applications. PMID:26399630

  12. Design of a high throughput electronics module for high energy physics experiments

    NASA Astrophysics Data System (ADS)

    Wang, Chun-Jie; Liu, Zhen-An; Zhao, Jing-Zhou; Liu, Zhao

    2016-06-01

    High-energy physics experiments enable us to explore and understand particle properties and interactions. An increase in luminosity in the accelerator, which allows us to study particles in higher energy ranges, demands faster data transmission and processing. Aimed at this, a high throughput uTCA-compliant electronics module, based on the latest FPGAs, has been designed. It contains 48 10.0 Gb/s optical fiber input channels and 24 10.0 Gb/s optical fiber output channels, supporting up to 480 Gb/s input bandwidth and 240 Gb/s output bandwidth. It complies with the uTCA standards, providing high speed data exchange capability and functioning as a compact and key module in a trigger and DAQ system for a large experiment. A reliable 10.0 Gb/s data transmission among two boards has been verified and one functionality that merges 6 1.6 Gb/s data channels into one single 10.0 Gb/s channel has been achieved. The hardware, firmware and software together with a performance evaluation are given in this paper. Supported by National Natural Science Foundation of China (11435013, 11461141011)

  13. An Enzymatic Assay for High-Throughput Screening of Cytidine-Producing Microbial Strains

    PubMed Central

    Dong, Huina; Liu, Yongfei; Zu, Xin; Li, Ning; Li, Feiran; Zhang, Dawei

    2015-01-01

    Cytidine is an industrially useful precursor for the production of antiviral compounds and a variety of industrial compounds. Interest in the microbial production of cytidine has grown recently and high-throughput screening of cytidine over-producers is an important approach in large-scale industrial production using microorganisms. An enzymatic assay for cytidine was developed combining cytidine deaminase (CDA) and indophenol method. CDA catalyzes the cleavage of cytidine to uridine and NH3, the latter of which can be accurately determined using the indophenol method. The assay was performed in 96-well plates and had a linear detection range of cytidine of 0.058 - 10 mM. This assay was used to determine the amount of cytidine in fermentation flasks and the results were compared with that of High Perfomance Liquid Chromatography (HPLC) method. The detection range of the CDA method is not as wide as that of the HPLC, furthermore the correlation factor of CDA method is not as high as that of HPLC. However, it was suitable for the detection of large numbers of crude samples and was applied to high-throughput screening for high cytidine-producing strains using 96-well deep-hole culture plates. This assay was proved to be simple, accurate, specific and suitable for cytidine detection and high-throughput screening of cytidine-producing strains in large numbers of samples (96 well or more). PMID:25816248

  14. A Novel High-Throughput Approach to Measure Hydroxyl Radicals Induced by Airborne Particulate Matter

    PubMed Central

    Son, Yeongkwon; Mishin, Vladimir; Welsh, William; Lu, Shou-En; Laskin, Jeffrey D.; Kipen, Howard; Meng, Qingyu

    2015-01-01

    Oxidative stress is one of the key mechanisms linking ambient particulate matter (PM) exposure with various adverse health effects. The oxidative potential of PM has been used to characterize the ability of PM induced oxidative stress. Hydroxyl radical (•OH) is the most destructive radical produced by PM. However, there is currently no high-throughput approach which can rapidly measure PM-induced •OH for a large number of samples with an automated system. This study evaluated four existing molecular probes (disodium terephthalate, 3′-p-(aminophenyl)fluorescein, coumarin-3-carboxylic acid, and sodium benzoate) for their applicability to measure •OH induced by PM in a high-throughput cell-free system using fluorescence techniques, based on both our experiments and on an assessment of the physicochemical properties of the probes reported in the literature. Disodium terephthalate (TPT) was the most applicable molecular probe to measure •OH induced by PM, due to its high solubility, high stability of the corresponding fluorescent product (i.e., 2-hydroxyterephthalic acid), high yield compared with the other molecular probes, and stable fluorescence intensity in a wide range of pH environments. TPT was applied in a high-throughput format to measure PM (NIST 1648a)-induced •OH, in phosphate buffered saline. The formed fluorescent product was measured at designated time points up to 2 h. The fluorescent product of TPT had a detection limit of 17.59 nM. The soluble fraction of PM contributed approximately 76.9% of the •OH induced by total PM, and the soluble metal ions of PM contributed 57.4% of the overall •OH formation. This study provides a promising cost-effective high-throughput method to measure •OH induced by PM on a routine basis. PMID:26516887

  15. Quantitative monitoring of Arabidopsis thaliana growth and development using high-throughput plant phenotyping

    PubMed Central

    Arend, Daniel; Lange, Matthias; Pape, Jean-Michel; Weigelt-Fischer, Kathleen; Arana-Ceballos, Fernando; Mücke, Ingo; Klukas, Christian; Altmann, Thomas; Scholz, Uwe; Junker, Astrid

    2016-01-01

    With the implementation of novel automated, high throughput methods and facilities in the last years, plant phenomics has developed into a highly interdisciplinary research domain integrating biology, engineering and bioinformatics. Here we present a dataset of a non-invasive high throughput plant phenotyping experiment, which uses image- and image analysis- based approaches to monitor the growth and development of 484 Arabidopsis thaliana plants (thale cress). The result is a comprehensive dataset of images and extracted phenotypical features. Such datasets require detailed documentation, standardized description of experimental metadata as well as sustainable data storage and publication in order to ensure the reproducibility of experiments, data reuse and comparability among the scientific community. Therefore the here presented dataset has been annotated using the standardized ISA-Tab format and considering the recently published recommendations for the semantical description of plant phenotyping experiments. PMID:27529152

  16. High-Throughput Metagenomic Technologies for Complex Microbial Community Analysis: Open and Closed Formats

    PubMed Central

    He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G.; Alvarez-Cohen, Lisa

    2015-01-01

    ABSTRACT   Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied “open-format” and “closed-format” detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions. PMID:25626903

  17. High-throughput metagenomic technologies for complex microbial community analysis. Open and closed formats

    SciTech Connect

    Zhou, Jizhong; He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G.; Alvarez-Cohen, Lisa

    2015-01-27

    Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied “open-format” and “closed-format” detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions.

  18. High-Throughput Continuous Flow Production of Nanoscale Liposomes by Microfluidic Vertical Flow Focusing.

    PubMed

    Hood, Renee R; DeVoe, Don L

    2015-11-18

    Liposomes represent a leading class of nanoparticles for drug delivery. While a variety of techniques for liposome synthesis have been reported that take advantage of microfluidic flow elements to achieve precise control over the size and polydispersity of nanoscale liposomes, with important implications for nanomedicine applications, these methods suffer from extremely limited throughput, making them impractical for large-scale nanoparticle synthesis. High aspect ratio microfluidic vertical flow focusing is investigated here as a new approach to overcoming the throughput limits of established microfluidic nanoparticle synthesis techniques. Here the vertical flow focusing technique is utilized to generate populations of small, unilamellar, and nearly monodisperse liposomal nanoparticles with exceptionally high production rates and remarkable sample homogeneity. By leveraging this platform, liposomes with modal diameters ranging from 80 to 200 nm are prepared at production rates as high as 1.6 mg min(-1) in a simple flow-through process.

  19. Molecular classification of fatty liver by high-throughput profiling of protein post-translational modifications.

    PubMed

    Urasaki, Yasuyo; Fiscus, Ronald R; Le, Thuc T

    2016-04-01

    We describe an alternative approach to classifying fatty liver by profiling protein post-translational modifications (PTMs) with high-throughput capillary isoelectric focusing (cIEF) immunoassays. Four strains of mice were studied, with fatty livers induced by different causes, such as ageing, genetic mutation, acute drug usage, and high-fat diet. Nutrient-sensitive PTMs of a panel of 12 liver metabolic and signalling proteins were simultaneously evaluated with cIEF immunoassays, using nanograms of total cellular protein per assay. Changes to liver protein acetylation, phosphorylation, and O-N-acetylglucosamine glycosylation were quantified and compared between normal and diseased states. Fatty liver tissues could be distinguished from one another by distinctive protein PTM profiles. Fatty liver is currently classified by morphological assessment of lipid droplets, without identifying the underlying molecular causes. In contrast, high-throughput profiling of protein PTMs has the potential to provide molecular classification of fatty liver.

  20. Turning tumor-promoting copper into an anti-cancer weapon via high-throughput chemistry.

    PubMed

    Wang, F; Jiao, P; Qi, M; Frezza, M; Dou, Q P; Yan, B

    2010-01-01

    Copper is an essential element for multiple biological processes. Its concentration is elevated to a very high level in cancer tissues for promoting cancer development through processes such as angiogenesis. Organic chelators of copper can passively reduce cellular copper and serve the role as inhibitors of angiogenesis. However, they can also actively attack cellular targets such as proteasome, which plays a critical role in cancer development and survival. The discovery of such molecules initially relied on a step by step synthesis followed by biological assays. Today high-throughput chemistry and high-throughput screening have significantly expedited the copper-binding molecules discovery to turn "cancer-promoting" copper into anti-cancer agents.

  1. High-throughput multiphoton-induced three-dimensional ablation and imaging for biotissues

    PubMed Central

    Lin, Chun-Yu; Li, Pei-Kao; Cheng, Li-Chung; Li, Yi-Cheng; Chang, Chia-Yuan; Chiang, Ann-Shyn; Dong, Chen Yuan; Chen, Shean-Jen

    2015-01-01

    In this study, a temporal focusing-based high-throughput multiphoton-induced ablation system with axially-resolved widefield multiphoton excitation has been successfully applied to rapidly disrupt biotissues. Experimental results demonstrate that this technique features high efficiency for achieving large-area laser ablation without causing serious photothermal damage in non-ablated regions. Furthermore, the rate of tissue processing can reach around 1.6 × 106 μm3/s in chicken tendon. Moreover, the temporal focusing-based multiphoton system can be efficiently utilized in optical imaging through iterating high-throughput multiphoton-induced ablation machining followed by widefield optical sectioning; hence, it has the potential to obtain molecular images for a whole bio-specimen. PMID:25780739

  2. High-throughput metagenomic technologies for complex microbial community analysis: open and closed formats.

    PubMed

    Zhou, Jizhong; He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G; Alvarez-Cohen, Lisa

    2015-01-27

    Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied "open-format" and "closed-format" detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions.

  3. Life in the fast lane: high-throughput chemistry for lead generation and optimisation.

    PubMed

    Hunter, D

    2001-01-01

    The pharmaceutical industry has come under increasing pressure due to regulatory restrictions on the marketing and pricing of drugs, competition, and the escalating costs of developing new drugs. These forces can be addressed by the identification of novel targets, reductions in the development time of new drugs, and increased productivity. Emphasis has been placed on identifying and validating new targets and on lead generation: the response from industry has been very evident in genomics and high throughput screening, where new technologies have been applied, usually coupled with a high degree of automation. The combination of numerous new potential biological targets and the ability to screen large numbers of compounds against many of these targets has generated the need for large diverse compound collections. To address this requirement, high-throughput chemistry has become an integral part of the drug discovery process.

  4. Turning Tumor-Promoting Copper into an Anti-Cancer Weapon via High-Throughput Chemistry

    PubMed Central

    Wang, F.; Jiao, P.; Qi, M.; Frezza, M.; Dou, Q.P.; Yan, B.

    2013-01-01

    Copper is an essential element for multiple biological processes. Its concentration is elevated to a very high level in cancer tissues for promoting cancer development through processes such as angiogenesis. Organic chelators of copper can passively reduce cellular copper and serve the role as inhibitors of angiogenesis. However, they can also actively attack cellular targets such as proteasome, which plays a critical role in cancer development and survival. The discovery of such molecules initially relied on a step by step synthesis followed by biological assays. Today high-throughput chemistry and high-throughput screening have significantly expedited the copper-binding molecules discovery to turn “cancer-promoting” copper into anti-cancer agents. PMID:20586723

  5. Standard finishing categories for high-throughput sequencing of viral genomes.

    PubMed

    Ladner, J T; Kuhn, J H; Palacios, G

    2016-04-01

    Viral genome sequencing has become the cornerstone of almost all aspects of virology. In particular, high-throughput, next-generation viral genome sequencing has become an integral part of molecular epidemiological investigations into outbreaks of viral disease, such as the recent outbreaks of Middle Eastern respiratory syndrome, Ebola virus disease and Zika virus infection. Multiple institutes have acquired the expertise and necessary infrastructure to perform such investigations, as evidenced by the accumulation of thousands of novel viral sequences over progressively shorter time periods. The authors recently proposed a nomenclature comprised of five high-throughput sequencing standard categories to describe the quality of determined viral genome sequences. These five categories (standard draft, high quality, coding complete, complete and finished) cover all levels of viral genome finishing and can be applied to sequences determined by any technology platform or assembly technique.

  6. High-throughput sequencing for detection of subpopulations of bacteria not previously associated with artisanal cheeses.

    PubMed

    Quigley, Lisa; O'Sullivan, Orla; Beresford, Tom P; Ross, R Paul; Fitzgerald, Gerald F; Cotter, Paul D

    2012-08-01

    Here, high-throughput sequencing was employed to reveal the highly diverse bacterial populations present in 62 Irish artisanal cheeses and, in some cases, associated cheese rinds. Using this approach, we revealed the presence of several genera not previously associated with cheese, including Faecalibacterium, Prevotella, and Helcococcus and, for the first time, detected the presence of Arthrobacter and Brachybacterium in goats' milk cheese. Our analysis confirmed many previously observed patterns, such as the dominance of typical cheese bacteria, the fact that the microbiota of raw and pasteurized milk cheeses differ, and that the level of cheese maturation has a significant influence on Lactobacillus populations. It was also noted that cheeses containing adjunct ingredients had lower proportions of Lactococcus species. It is thus apparent that high-throughput sequencing-based investigations can provide valuable insights into the microbial populations of artisanal foods.

  7. Quantitative monitoring of Arabidopsis thaliana growth and development using high-throughput plant phenotyping.

    PubMed

    Arend, Daniel; Lange, Matthias; Pape, Jean-Michel; Weigelt-Fischer, Kathleen; Arana-Ceballos, Fernando; Mücke, Ingo; Klukas, Christian; Altmann, Thomas; Scholz, Uwe; Junker, Astrid

    2016-08-16

    With the implementation of novel automated, high throughput methods and facilities in the last years, plant phenomics has developed into a highly interdisciplinary research domain integrating biology, engineering and bioinformatics. Here we present a dataset of a non-invasive high throughput plant phenotyping experiment, which uses image- and image analysis- based approaches to monitor the growth and development of 484 Arabidopsis thaliana plants (thale cress). The result is a comprehensive dataset of images and extracted phenotypical features. Such datasets require detailed documentation, standardized description of experimental metadata as well as sustainable data storage and publication in order to ensure the reproducibility of experiments, data reuse and comparability among the scientific community. Therefore the here presented dataset has been annotated using the standardized ISA-Tab format and considering the recently published recommendations for the semantical description of plant phenotyping experiments.

  8. Standard finishing categories for high-throughput sequencing of viral genomes.

    PubMed

    Ladner, J T; Kuhn, J H; Palacios, G

    2016-04-01

    Viral genome sequencing has become the cornerstone of almost all aspects of virology. In particular, high-throughput, next-generation viral genome sequencing has become an integral part of molecular epidemiological investigations into outbreaks of viral disease, such as the recent outbreaks of Middle Eastern respiratory syndrome, Ebola virus disease and Zika virus infection. Multiple institutes have acquired the expertise and necessary infrastructure to perform such investigations, as evidenced by the accumulation of thousands of novel viral sequences over progressively shorter time periods. The authors recently proposed a nomenclature comprised of five high-throughput sequencing standard categories to describe the quality of determined viral genome sequences. These five categories (standard draft, high quality, coding complete, complete and finished) cover all levels of viral genome finishing and can be applied to sequences determined by any technology platform or assembly technique. PMID:27217167

  9. Quantitative monitoring of Arabidopsis thaliana growth and development using high-throughput plant phenotyping.

    PubMed

    Arend, Daniel; Lange, Matthias; Pape, Jean-Michel; Weigelt-Fischer, Kathleen; Arana-Ceballos, Fernando; Mücke, Ingo; Klukas, Christian; Altmann, Thomas; Scholz, Uwe; Junker, Astrid

    2016-01-01

    With the implementation of novel automated, high throughput methods and facilities in the last years, plant phenomics has developed into a highly interdisciplinary research domain integrating biology, engineering and bioinformatics. Here we present a dataset of a non-invasive high throughput plant phenotyping experiment, which uses image- and image analysis- based approaches to monitor the growth and development of 484 Arabidopsis thaliana plants (thale cress). The result is a comprehensive dataset of images and extracted phenotypical features. Such datasets require detailed documentation, standardized description of experimental metadata as well as sustainable data storage and publication in order to ensure the reproducibility of experiments, data reuse and comparability among the scientific community. Therefore the here presented dataset has been annotated using the standardized ISA-Tab format and considering the recently published recommendations for the semantical description of plant phenotyping experiments. PMID:27529152

  10. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing

    PubMed Central

    Woodruff, Kristina; Maerkl, Sebastian J.

    2016-01-01

    Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies. PMID:27030663

  11. Automated disposable small scale reactor for high throughput bioprocess development: a proof of concept study.

    PubMed

    Bareither, Rachel; Bargh, Neil; Oakeshott, Robert; Watts, Kathryn; Pollard, David

    2013-12-01

    The acceleration of bioprocess development for biologics and vaccines can be enabled by automated high throughput technologies. This will alleviate the significant resource burden from the multi-factorial statistical experimentation required for controlling product quality attributes of complex biologics. Recent technology advances have improved clone evaluation and screening, but have struggled to combine the scale down criteria required for both high cell density cell culture and microbial processes, with sufficient automation and disposable technologies to accelerate process development. This article describes the proof of concept evaluations of an automated disposable small scale reactor for high throughput upstream process development. Characterization studies established the small scale stirred tank disposable 250 mL reactor as similar to those of lab and pilot scale. The reactor generated equivalent process performance for industrial biologics processes for therapeutic protein and monoclonal antibody production using CHO cell culture, Pichia pastoris and E. coli. This included similar growth, cell viability, product titer, and product quality. The technology was shown to be robust across multiple runs and met the requirements for the ability to run high cell density processes (>400 g/L wet cell weight) with exponential feeds and sophisticated event triggered processes. Combining this reactor into an automated array of reactors will ultimately be part of a high throughput process development strategy. This will combine upstream, small scale purification with rapid analytics that will dramatically shorten timelines and costs of developing biological processes.

  12. A new high-resolution, high-throughput spectrometer: first experience as applied to Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Meade, Jeffrey T.; Behr, Bradford B.; Hajian, Arsen R.

    2012-06-01

    The classic trade-off between resolution and throughput in a dispersive spectrometer is overcome using virtual slit technology. An optimized spectrometer designed from the ground up to incorporate a virtual slit is experimentally demonstrated by Raman experiments.

  13. Low-Cost, High-Throughput Sequencing of DNA Assemblies Using a Highly Multiplexed Nextera Process.

    PubMed

    Shapland, Elaine B; Holmes, Victor; Reeves, Christopher D; Sorokin, Elena; Durot, Maxime; Platt, Darren; Allen, Christopher; Dean, Jed; Serber, Zach; Newman, Jack; Chandran, Sunil

    2015-07-17

    In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrial-scale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15× coverage), which is a 20-fold reduction over using Sanger sequencing (2× coverage). We reduced the volume of the Nextera tagmentation reaction by 100-fold and developed an automated workflow to prepare thousands of samples for sequencing. We also developed software to track the samples and associated sequence data and to rapidly identify correctly assembled constructs having the fewest defects. As DNA synthesis and assembly become a centralized commodity, this NGS quality control (QC) process will be essential to groups operating high-throughput pipelines for DNA construction.

  14. Protective Polymer Coatings for High-Throughput, High-Purity Cellular Isolation

    PubMed Central

    2016-01-01

    Cell-based therapies are emerging as the next frontier of medicine, offering a plausible path forward in the treatment of many devastating diseases. Critically, current methods for antigen positive cell sorting lack a high throughput method for delivering ultrahigh purity populations, prohibiting the application of some cell-based therapies to widespread diseases. Here we show the first use of targeted, protective polymer coatings on cells for the high speed enrichment of cells. Individual, antigen-positive cells are coated with a biocompatible hydrogel which protects the cells from a surfactant solution, while uncoated cells are immediately lysed. After lysis, the polymer coating is removed through orthogonal photochemistry, and the isolate has >50% yield of viable cells and these cells proliferate at rates comparable to control cells. Minority cell populations are enriched from erythrocyte-depleted blood to >99% purity, whereas the entire batch process requires 1 h and <$2000 in equipment. Batch scale-up is only contingent on irradiation area for the coating photopolymerization, as surfactant-based lysis can be easily achieved on any scale. PMID:26244409

  15. SwellGel: a sample preparation affinity chromatography technology for high throughput proteomic applications.

    PubMed

    Haney, Paul J; Draveling, Connie; Durski, Wendy; Romanowich, Kathryn; Qoronfleh, M Walid

    2003-04-01

    Development of high throughput systems for purification and analysis of proteins is essential for the success of today's proteomic research. We have developed an affinity chromatography technology that allows the customization of high capacity/high throughput chromatographic separation of proteins. This technology utilizes selected chromatography media that are dehydrated to form uniform SwellGel discs. Unlike wet resin slurries, these discs are easily adaptable to a variety of custom formats, eliminating problems associated with resin dispensing, equilibration, or leakage. Discs can be made in assorted sizes (resin volume 15 microl-3 ml) dispensed in various formats (384-, 96-, 48-, and 24-well microplates or columns) and different ligands can be attached to the matrix. SwellGel discs rapidly hydrate upon addition of either water or the protein sample, providing dramatically increased capacity compared to coated plates. At the same time, the discs offer greater stability, reproducibility, and ease of handling than standard wet chromatography resins. We previously reported the development of SwellGel for the purification of 6x His- and glutathione-S-transferase (GST)-tagged fusion proteins [Prot. Exp. Purif. 22 (2001) 359-366]. In this paper, we discuss an expanded list of SwellGel stabilized chromatographic methods that have been adapted to high throughput formats for processing protein samples ranging from 10 microl to 10 ml (1 microg to 50 mg protein). Data are presented applying SwellGel discs to high throughput proteomic applications such as affinity tag purification, protein desalting, the removal of abundant proteins from serum including albumin and immunoglobulin, and the isolation of phosphorylated peptides for mass spectrometry. PMID:12699691

  16. High-throughput micro-scale cultivations and chromatography modeling: Powerful tools for integrated process development.

    PubMed

    Baumann, Pascal; Hahn, Tobias; Hubbuch, Jürgen

    2015-10-01

    Upstream processes are rather complex to design and the productivity of cells under suitable cultivation conditions is hard to predict. The method of choice for examining the design space is to execute high-throughput cultivation screenings in micro-scale format. Various predictive in silico models have been developed for many downstream processes, leading to a reduction of time and material costs. This paper presents a combined optimization approach based on high-throughput micro-scale cultivation experiments and chromatography modeling. The overall optimized system must not necessarily be the one with highest product titers, but the one resulting in an overall superior process performance in up- and downstream. The methodology is presented in a case study for the Cherry-tagged enzyme Glutathione-S-Transferase from Escherichia coli SE1. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for direct product analytics by simple VIS absorption measurements. High-throughput cultivations were carried out in a 48-well format in a BioLector micro-scale cultivation system (m2p-Labs, Germany). The downstream process optimization for a set of randomly picked upstream conditions producing high yields was performed in silico using a chromatography modeling software developed in-house (ChromX). The suggested in silico-optimized operational modes for product capturing were validated subsequently. The overall best system was chosen based on a combination of excellent up- and downstream performance.

  17. High-throughput microplate enzymatic assays for fast sugar and acid quantification in apple and tomato.

    PubMed

    Vermeir, S; Nicolaï, B M; Jans, K; Maes, G; Lammertyn, J

    2007-05-01

    In this article, we report on the use of miniaturized and automated enzymatic assays as an alternative technology for fast sugar and acid quantification in apples and tomatoes. Enzymatic assays for d-glucose, d-fructose, sucrose, D-sorbitol/xylitol, L-malic acid, citric acid, succinic acid, and L-glutamic acid were miniaturized from the standard 3 mL assays in cuvettes into assays of 200 microL or lower in 96 or 384 well microplates. The miniaturization and the automation were achieved with a four channel automatic liquid handling system in order to reduce the dispensing errors and to obtain an increased sample throughput. Performance factors (limit of detection, linearity of calibration curve, and repeatability) of the assays with standard solutions were proven to be satisfactory. The automated and miniaturized assays were validated with high-pressure liquid chromatography (HPLC) analyses for the quantification of sugars and acids in tomato and apple extracts. The high correlation between the two techniques for the different components indicates that the high-throughput microplate enzymatic assays can serve as a fast, reliable, and inexpensive alternative for HPLC as the standard analysis technique in the taste characterization of fruit and vegetables. In addition to the analysis of extracts, the high-throughput microplate enzymatic assays were used for the direct analysis of centrifuged and filtered tomato juice with an additional advantage that the sample preparation time and analysis costs are reduced significantly.

  18. High-Throughput Quantitative Lipidomics Analysis of Nonesterified Fatty Acids in Human Plasma.

    PubMed

    Christinat, Nicolas; Morin-Rivron, Delphine; Masoodi, Mojgan

    2016-07-01

    We present a high-throughput, nontargeted lipidomics approach using liquid chromatography coupled to high-resolution mass spectrometry for quantitative analysis of nonesterified fatty acids. We applied this method to screen a wide range of fatty acids from medium-chain to very long-chain (8 to 24 carbon atoms) in human plasma samples. The method enables us to chromatographically separate branched-chain species from their straight-chain isomers as well as separate biologically important ω-3 and ω-6 polyunsaturated fatty acids. We used 51 fatty acid species to demonstrate the quantitative capability of this method with quantification limits in the nanomolar range; however, this method is not limited only to these fatty acid species. High-throughput sample preparation was developed and carried out on a robotic platform that allows extraction of 96 samples simultaneously within 3 h. This high-throughput platform was used to assess the influence of different types of human plasma collection and preparation on the nonesterified fatty acid profile of healthy donors. Use of the anticoagulants EDTA and heparin has been compared with simple clotting, and only limited changes have been detected in most nonesterified fatty acid concentrations. PMID:27185515

  19. High-throughput SHAPE and hydroxyl radical analysis of RNA structure and ribonucleoprotein assembly.

    PubMed

    McGinnis, Jennifer L; Duncan, Caia D S; Weeks, Kevin M

    2009-01-01

    RNA folds to form complex structures vital to many cellular functions. Proteins facilitate RNA folding at both the secondary and tertiary structure levels. An absolute prerequisite for understanding RNA folding and ribonucleoprotein (RNP) assembly reactions is a complete understanding of the RNA structure at each stage of the folding or assembly process. Here we provide a guide for comprehensive and high-throughput analysis of RNA secondary and tertiary structure using SHAPE and hydroxyl radical footprinting. As an example of the strong and sometimes surprising conclusions that can emerge from high-throughput analysis of RNA folding and RNP assembly, we summarize the structure of the bI3 group I intron RNA in four distinct states. Dramatic structural rearrangements occur in both secondary and tertiary structure as the RNA folds from the free state to the active, six-component, RNP complex. As high-throughput and high-resolution approaches are applied broadly to large protein-RNA complexes, other proteins previously viewed as making simple contributions to RNA folding are also likely to be found to exert multifaceted, long-range, cooperative, and nonadditive effects on RNA folding. These protein-induced contributions add another level of control, and potential regulatory function, in RNP complexes.

  20. [Advances in high-throughput transcriptome research of traditional Chinese medicines].

    PubMed

    Zhang, Zhao-Bao; Hou, Lin; Pan, Qing; Wang, Xu-Min; Cui, Qing-Hua; Tian, Jing-Zhen; Ma, Lu-Yu

    2014-05-01

    Traditional Chinese medicine is a treasure of Chinese culture, absorbing the wisdom of the Chinese people. Continuous application of new technologies makes traditional Chinese medicine research advance with the times. After several years of development, high-throughput transcriptome study has become a mature research tool in biology. This paper reviewed the advances in medicine transcriptome study, and compared two sequencing platforms, Roche's GS FLX platform and Illumina's HiSeq 2000 platform. Moreover, this paper introduced medicine transcriptome analysis process, with Panax quinquefolius and Lonicera japonica for examples, showing the characteristics of traditional Chinese medicine transcriptome studies. High-throughput transcriptome studies facilitate traditional Chinese medicine research with overall understand of functional genes, give clear elucidation of metabolic pathways, lay molecular foundation for the traditional Chinese medicine research and offer modern interpretation for traditional Chinese medicine theory. However, the current study faces several difficulties, including weak molecular basis, high sequencing cost and staff shortages in data anaysis. In the future, with the development in sequencing technology, the combination of transcriptome and other genomics, such as proteome and metabolome, will lay a solid foundation for the new high-throughput screening and developing model for the traditional Chinese medicine industry.

  1. Acoustic Droplet Ejection Applications for High-Throughput Screening of Infectious Agents.

    PubMed

    Rasmussen, Lynn; White, E Lucile; Bostwick, James R

    2016-02-01

    When acoustic droplet ejection technology was first introduced for high-throughput applications, it was used primarily for dispensing compounds dissolved in DMSO. The high precision and accuracy achieved for low-volume transfers in this application were noted by those working outside of the compound management area, and interest was generated in expanding the scope of the technology to include other liquid types. Later-generation instruments included calibrations for several aqueous buffers that were applicable to the life sciences. The High Throughput Screening Center at Southern Research has made use of this range of liquid calibrations for the Infectious Disease Program. The original calibration for DMSO has allowed the preparation of assay-ready plates that can be sent to remote locations. This process was used as part of the collaboration between Southern Research and Galveston National Laboratory, University of Texas Medical Branch, to develop high-throughput screening for biological safety level 4 containment and to provide compounds for two pilot screens that were run there with BSL-4-level pathogens. The aqueous calibrations have been instrumental in miniaturizing assays used for infectious disease, such as qPCR, tissue culture infectious dose 50, and bacterial motility, to make them compatible with HTS operations. PMID:26663786

  2. Subnuclear foci quantification using high-throughput 3D image cytometry

    NASA Astrophysics Data System (ADS)

    Wadduwage, Dushan N.; Parrish, Marcus; Choi, Heejin; Engelward, Bevin P.; Matsudaira, Paul; So, Peter T. C.

    2015-07-01

    Ionising radiation causes various types of DNA damages including double strand breaks (DSBs). DSBs are often recognized by DNA repair protein ATM which forms gamma-H2AX foci at the site of the DSBs that can be visualized using immunohistochemistry. However most of such experiments are of low throughput in terms of imaging and image analysis techniques. Most of the studies still use manual counting or classification. Hence they are limited to counting a low number of foci per cell (5 foci per nucleus) as the quantification process is extremely labour intensive. Therefore we have developed a high throughput instrumentation and computational pipeline specialized for gamma-H2AX foci quantification. A population of cells with highly clustered foci inside nuclei were imaged, in 3D with submicron resolution, using an in-house developed high throughput image cytometer. Imaging speeds as high as 800 cells/second in 3D were achieved by using HiLo wide-field depth resolved imaging and a remote z-scanning technique. Then the number of foci per cell nucleus were quantified using a 3D extended maxima transform based algorithm. Our results suggests that while most of the other 2D imaging and manual quantification studies can count only up to about 5 foci per nucleus our method is capable of counting more than 100. Moreover we show that 3D analysis is significantly superior compared to the 2D techniques.

  3. Selection of recombinant anti-SH3 domain antibodies by high-throughput phage display.

    PubMed

    Huang, Haiming; Economopoulos, Nicolas O; Liu, Bernard A; Uetrecht, Andrea; Gu, Jun; Jarvik, Nick; Nadeem, Vincent; Pawson, Tony; Moffat, Jason; Miersch, Shane; Sidhu, Sachdev S

    2015-11-01

    Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high-throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel. Binding selections using a phage-displayed synthetic antigen-binding fragment (Fab) library against 110 human SH3 domains yielded hundreds of Fabs targeting 58 antigens. Affinity assays demonstrated that representative Fabs bind tightly and specifically to their targets. Furthermore, we developed an efficient affinity maturation strategy adaptable to high-throughput, which increased affinity dramatically but did not compromise specificity. Finally, we tested Fabs in common cell biology applications and confirmed recognition of the full-length antigen in immunoprecipitation, immunoblotting and immunofluorescence assays. In summary, we have established a rapid and robust high-throughput methodology that can be applied to generate highly functional and renewable antibodies targeting protein domains on a proteome-wide scale.

  4. Droplet microfluidic technology for single-cell high-throughput screening.

    PubMed

    Brouzes, Eric; Medkova, Martina; Savenelli, Neal; Marran, Dave; Twardowski, Mariusz; Hutchison, J Brian; Rothberg, Jonathan M; Link, Darren R; Perrimon, Norbert; Samuels, Michael L

    2009-08-25

    We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses. PMID:19617544

  5. A versatile toolkit for high throughput functional genomics with Trichoderma reesei

    SciTech Connect

    Schuster, Andre; Bruno, Kenneth S.; Collett, James R.; Baker, Scott E.; Seiboth, Bernhard; Kubicek, Christian P.; Schmoll, Monika

    2012-01-02

    The ascomycete fungus, Trichoderma reesei (anamorph of Hypocrea jecorina), represents a biotechnological workhorse and is currently one of the most proficient cellulase producers. While strain improvement was traditionally accomplished by random mutagenesis, a detailed understanding of cellulase regulation can only be gained using recombinant technologies. RESULTS: Aiming at high efficiency and high throughput methods, we present here a construction kit for gene knock out in T. reesei. We provide a primer database for gene deletion using the pyr4, amdS and hph selection markers. For high throughput generation of gene knock outs, we constructed vectors using yeast mediated recombination and then transformed a T. reesei strain deficient in non-homologous end joining (NHEJ) by spore electroporation. This NHEJ-defect was subsequently removed by crossing of mutants with a sexually competent strain derived from the parental strain, QM9414.CONCLUSIONS:Using this strategy and the materials provided, high throughput gene deletion in T. reesei becomes feasible. Moreover, with the application of sexual development, the NHEJ-defect can be removed efficiently and without the need for additional selection markers. The same advantages apply for the construction of multiple mutants by crossing of strains with different gene deletions, which is now possible with considerably less hands-on time and minimal screening effort compared to a transformation approach. Consequently this toolkit can considerably boost research towards efficient exploitation of the resources of T. reesei for cellulase expression and hence second generation biofuel production.

  6. High-throughput olfactory conditioning and memory retention test show variation in Nasonia parasitic wasps

    PubMed Central

    Hoedjes, K M; Steidle, J L M; Werren, J H; Vet, L E M; Smid, H M

    2012-01-01

    Most of our knowledge on learning and memory formation results from extensive studies on a small number of animal species. Although features and cellular pathways of learning and memory are highly similar in this diverse group of species, there are also subtle differences. Closely related species of parasitic wasps display substantial variation in memory dynamics and can be instrumental to understanding both the adaptive benefit of and mechanisms underlying this variation. Parasitic wasps of the genus Nasonia offer excellent opportunities for multidisciplinary research on this topic. Genetic and genomic resources available for Nasonia are unrivaled among parasitic wasps, providing tools for genetic dissection of mechanisms that cause differences in learning. This study presents a robust, high-throughput method for olfactory conditioning of Nasonia using a host encounter as reward. A T-maze olfactometer facilitates high-throughput memory retention testing and employs standardized odors of equal detectability, as quantified by electroantennogram recordings. Using this setup, differences in memory retention between Nasonia species were shown. In both Nasonia vitripennis and Nasonia longicornis, memory was observed up to at least 5 days after a single conditioning trial, whereas Nasonia giraulti lost its memory after 2 days. This difference in learning may be an adaptation to species-specific differences in ecological factors, for example, host preference. The high-throughput methods for conditioning and memory retention testing are essential tools to study both ultimate and proximate factors that cause variation in learning and memory formation in Nasonia and other parasitic wasp species. PMID:22804968

  7. High-Throughput CRISPR Typing of Mycobacterium tuberculosis Complex and Salmonella enterica Serotype Typhimurium.

    PubMed

    Sola, Christophe; Abadia, Edgar; Le Hello, Simon; Weill, François-Xavier

    2015-01-01

    Spoligotyping was developed almost 18 years ago and still remains a popular first-lane genotyping technique to identify and subtype Mycobacterium tuberculosis complex (MTC) clinical isolates at a phylogeographic level. For other pathogens, such as Salmonella enterica, recent studies suggest that specifically designed spoligotyping techniques could be interesting for public health purposes. Spoligotyping was in its original format a reverse line-blot hybridization method using capture probes designed on "spacers" and attached to a membrane's surface and a PCR product obtained from clustered regularly interspaced short palindromic repeats (CRISPRs). Cowan et al. and Fabre et al. were the first to propose a high-throughput Spoligotyping method based on microbeads for MTC and S. enterica serotype Typhimurium, respectively. The main advantages of the high-throughput Spoligotyping techniques we describe here are their low cost, their robustness, and the existence (at least for MTC) of very large databases that allow comparisons between spoligotypes from anywhere.

  8. Bioinformatics of Cancer ncRNA in High Throughput Sequencing: Present State and Challenges

    PubMed Central

    Jorge, Natasha Andressa Nogueira; Ferreira, Carlos Gil; Passetti, Fabio

    2012-01-01

    The numerous genome sequencing projects produced unprecedented amount of data providing significant information to the discovery of novel non-coding RNA (ncRNA). Several ncRNAs have been described to control gene expression and display important role during cell differentiation and homeostasis. In the last decade, high throughput methods in conjunction with approaches in bioinformatics have been used to identify, classify, and evaluate the expression of hundreds of ncRNA in normal and pathological states, such as cancer. Patient outcomes have been already associated with differential expression of ncRNAs in normal and tumoral tissues, providing new insights in the development of innovative therapeutic strategies in oncology. In this review, we present and discuss bioinformatics advances in the development of computational approaches to analyze and discover ncRNA data in oncology using high throughput sequencing technologies. PMID:23251139

  9. High-Throughput CRISPR Typing of Mycobacterium tuberculosis Complex and Salmonella enterica Serotype Typhimurium.

    PubMed

    Sola, Christophe; Abadia, Edgar; Le Hello, Simon; Weill, François-Xavier

    2015-01-01

    Spoligotyping was developed almost 18 years ago and still remains a popular first-lane genotyping technique to identify and subtype Mycobacterium tuberculosis complex (MTC) clinical isolates at a phylogeographic level. For other pathogens, such as Salmonella enterica, recent studies suggest that specifically designed spoligotyping techniques could be interesting for public health purposes. Spoligotyping was in its original format a reverse line-blot hybridization method using capture probes designed on "spacers" and attached to a membrane's surface and a PCR product obtained from clustered regularly interspaced short palindromic repeats (CRISPRs). Cowan et al. and Fabre et al. were the first to propose a high-throughput Spoligotyping method based on microbeads for MTC and S. enterica serotype Typhimurium, respectively. The main advantages of the high-throughput Spoligotyping techniques we describe here are their low cost, their robustness, and the existence (at least for MTC) of very large databases that allow comparisons between spoligotypes from anywhere. PMID:25981468

  10. Current developments in high-throughput analysis for microalgae cellular contents.

    PubMed

    Lee, Tsung-Hua; Chang, Jo-Shu; Wang, Hsiang-Yu

    2013-11-01

    Microalgae have emerged as one of the most promising feedstocks for biofuels and bio-based chemical production. However, due to the lack of effective tools enabling rapid and high-throughput analysis of the content of microalgae biomass, the efficiency of screening and identification of microalgae with desired functional components from the natural environment is usually quite low. Moreover, the real-time monitoring of the production of target components from microalgae is also difficult. Recently, research efforts focusing on overcoming this limitation have started. In this review, the recent development of high-throughput methods for analyzing microalgae cellular contents is summarized. The future prospects and impacts of these detection methods in microalgae-related processing and industries are also addressed.

  11. Next-Generation High-Throughput Functional Annotation of Microbial Genomes

    PubMed Central

    Baric, Ralph S.; Damania, Blossom; Miller, Samuel I.; Rubin, Eric J.

    2016-01-01

    ABSTRACT Host infection by microbial pathogens cues global changes in microbial and host cell biology that facilitate microbial replication and disease. The complete maps of thousands of bacterial and viral genomes have recently been defined; however, the rate at which physiological or biochemical functions have been assigned to genes has greatly lagged. The National Institute of Allergy and Infectious Diseases (NIAID) addressed this gap by creating functional genomics centers dedicated to developing high-throughput approaches to assign gene function. These centers require broad-based and collaborative research programs to generate and integrate diverse data to achieve a comprehensive understanding of microbial pathogenesis. High-throughput functional genomics can lead to new therapeutics and better understanding of the next generation of emerging pathogens by rapidly defining new general mechanisms by which organisms cause disease and replicate in host tissues and by facilitating the rate at which functional data reach the scientific community. PMID:27703071

  12. Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)

    SciTech Connect

    Hura, Greg L.; Menon, Angeli L.; Hammel, Michal; Rambo, Robert P.; Poole II, Farris L.; Tsutakawa, Susan E.; Jenney Jr, Francis E.; Classen, Scott; Frankel, Kenneth A.; Hopkins, Robert C.; Yang, Sungjae; Scott, Joseph W.; Dillard, Bret D.; Adams, Michael W. W.; Tainer, John A.

    2009-07-20

    We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.

  13. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors

    PubMed Central

    Colot, Hildur V.; Park, Gyungsoon; Turner, Gloria E.; Ringelberg, Carol; Crew, Christopher M.; Litvinkova, Liubov; Weiss, Richard L.; Borkovich, Katherine A.; Dunlap, Jay C.

    2006-01-01

    The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination. PMID:16801547

  14. High-throughput automated image analysis of neuroinflammation and neurodegeneration enables quantitative assessment of virus neurovirulence

    PubMed Central

    Maximova, Olga A.; Murphy, Brian R.; Pletnev, Alexander G.

    2010-01-01

    Historically, the safety of live attenuated vaccine candidates against neurotropic viruses was assessed by semi-quantitative analysis of virus-induced histopathology in the central nervous system of monkeys. We have developed a high-throughput automated image analysis (AIA) for the quantitative assessment of virus-induced neuroinflammation and neurodegeneration. Evaluation of the results generated by AIA showed that quantitative estimates of lymphocytic infiltration, microglial activation, and neurodegeneration strongly and significantly correlated with results of traditional histopathological scoring. In addition, we show that AIA is a targeted, objective, accurate, and time-efficient approach that provides reliable differentiation of virus neurovirulence. As such, it may become a useful tool in establishing consistent analytical standards across research and development laboratories and regulatory agencies, and may improve the safety evaluation of live virus vaccines. The implementation of this high-throughput AIA will markedly advance many fields of research including virology, neuroinflammation, neuroscience, and vaccinology. PMID:20688036

  15. Development and Application of a High Throughput Protein Unfolding Kinetic Assay

    PubMed Central

    Wang, Qiang; Waterhouse, Nicklas; Feyijinmi, Olusegun; Dominguez, Matthew J.; Martinez, Lisa M.; Sharp, Zoey; Service, Rachel; Bothe, Jameson R.; Stollar, Elliott J.

    2016-01-01

    The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses. PMID:26745729

  16. Evaluation of high-throughput assays for in vitro drug susceptibility testing of Tritrichomonas foetus trophozoites.

    PubMed

    Bader, Chris; Jesudoss Chelladurai, Jeba; Thompson, Kylie; Hall, Cindy; Carlson, Steve A; Brewer, Matthew T

    2016-06-15

    Tritrichomonas foetus is a sexually transmitted protozoan parasite that causes abortions in cattle and results in severe economic losses. In the United States, there are no safe and effective treatments for this parasite and infected animals are typically culled. In order to expedite drug discovery efforts, we investigated in vitro trophozoite killing assays amenable to high-throughput screening in 96 well plate formats. We evaluated the reduction of resorufin, incorporation of propidium iodide, and a luminescence-based ATP detection assay. Of these methods, reduction of resorufin was found to be the most reliable predictor of trophozoite concentrations. We further validated this method by conducting dose-response experiments suitable for calculation of EC50 values for two established compounds with known activity against trophozoites in vitro, namely, metronidazole and ronidazole. Our results demonstrate that the resorufin method is suitable for high-throughput screening and could be used to enhance efforts targeting new treatments for bovine trichomoniasis. PMID:27198774

  17. Establishing an Infrastructure for High-Throughput Short-Interfering RNA Screening.

    PubMed

    Yin, Hongwei; Sereduk, Chris; Tang, Nanyun

    2016-01-01

    RNA interference (RNAi) is a readily available research tool that can be used to accelerate the identification and functional validation of a multitude of new candidate drug targets by experimentally perturbing gene expression and function. High-throughput RNAi technology using libraries of short-interfering RNA (siRNA) makes it possible to rapidly identify genes and biomarkers associated with biological processes such as diseases or a cellular response to therapy. Thus, RNAi-based screening is an extremely powerful technology that can provide tremendous insights into the mechanisms of action and contexts of vulnerability of a particular drug treatment. This chapter describes the infrastructure requirements needed to successfully perform HT-RNAi screening. Information on the methodology, instrumentation, experimental design, and workflow aspects is provided, as well as insights on how to successfully implement a high-throughput RNAi screen. PMID:27581280

  18. A pulse-front-tilt-compensated streaked optical spectrometer with high throughput and picosecond time resolution

    NASA Astrophysics Data System (ADS)

    Katz, J.; Boni, R.; Rivlis, R.; Muir, C.; Froula, D. H.

    2016-11-01

    A high-throughput, broadband optical spectrometer coupled to the Rochester optical streak system equipped with a Photonis P820 streak tube was designed to record time-resolved spectra with 1-ps time resolution. Spectral resolution of 0.8 nm is achieved over a wavelength coverage range of 480 to 580 nm, using a 300-groove/mm diffraction grating in conjunction with a pair of 225-mm-focal-length doublets operating at an f/2.9 aperture. Overall pulse-front tilt across the beam diameter generated by the diffraction grating is reduced by preferentially delaying discrete segments of the collimated input beam using a 34-element reflective echelon optic. The introduced delay temporally aligns the beam segments and the net pulse-front tilt is limited to the accumulation across an individual sub-element. The resulting spectrometer design balances resolving power and pulse-front tilt while maintaining high throughput.

  19. High Throughput Screening Identifies Novel Lead Compounds with Activity against Larval, Juvenile and Adult Schistosoma mansoni.

    PubMed

    Mansour, Nuha R; Paveley, Ross; Gardner, J Mark F; Bell, Andrew S; Parkinson, Tanya; Bickle, Quentin

    2016-04-01

    An estimated 600 million people are affected by the helminth disease schistosomiasis caused by parasites of the genus Schistosoma. There is currently only one drug recommended for treating schistosomiasis, praziquantel (PZQ), which is effective against adult worms but not against the juvenile stage. In an attempt to identify improved drugs for treating the disease, we have carried out high throughput screening of a number of small molecule libraries with the aim of identifying lead compounds with balanced activity against all life stages of Schistosoma. A total of almost 300,000 compounds were screened using a high throughput assay based on motility of worm larvae and image analysis of assay plates. Hits were screened against juvenile and adult worms to identify broadly active compounds and against a mammalian cell line to assess cytotoxicity. A number of compounds were identified as promising leads for further chemical optimization. PMID:27128493

  20. Miniaturization of High-Throughput Epigenetic Methyltransferase Assays with Acoustic Liquid Handling.

    PubMed

    Edwards, Bonnie; Lesnick, John; Wang, Jing; Tang, Nga; Peters, Carl

    2016-02-01

    Epigenetics continues to emerge as an important target class for drug discovery and cancer research. As programs scale to evaluate many new targets related to epigenetic expression, new tools and techniques are required to enable efficient and reproducible high-throughput epigenetic screening. Assay miniaturization increases screening throughput and reduces operating costs. Echo liquid handlers can transfer compounds, samples, reagents, and beads in submicroliter volumes to high-density assay formats using only acoustic energy-no contact or tips required. This eliminates tip costs and reduces the risk of reagent carryover. In this study, we demonstrate the miniaturization of a methyltransferase assay using Echo liquid handlers and two different assay technologies: AlphaLISA from PerkinElmer and EPIgeneous HTRF from Cisbio.