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Sample records for automated high throughput

  1. Automated High Throughput Drug Target Crystallography

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    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.

  2. A fully automated high-throughput training system for rodents.

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    Rajesh Poddar

    Full Text Available Addressing the neural mechanisms underlying complex learned behaviors requires training animals in well-controlled tasks, an often time-consuming and labor-intensive process that can severely limit the feasibility of such studies. To overcome this constraint, we developed a fully computer-controlled general purpose system for high-throughput training of rodents. By standardizing and automating the implementation of predefined training protocols within the animal's home-cage our system dramatically reduces the efforts involved in animal training while also removing human errors and biases from the process. We deployed this system to train rats in a variety of sensorimotor tasks, achieving learning rates comparable to existing, but more laborious, methods. By incrementally and systematically increasing the difficulty of the task over weeks of training, rats were able to master motor tasks that, in complexity and structure, resemble ones used in primate studies of motor sequence learning. By enabling fully automated training of rodents in a home-cage setting this low-cost and modular system increases the utility of rodents for studying the neural underpinnings of a variety of complex behaviors.

  3. Implementation of an Automated High-Throughput Plasmid DNA Production Pipeline.

    Science.gov (United States)

    Billeci, Karen; Suh, Christopher; Di Ioia, Tina; Singh, Lovejit; Abraham, Ryan; Baldwin, Anne; Monteclaro, Stephen

    2016-12-01

    Biologics sample management facilities are often responsible for a diversity of large-molecule reagent types, such as DNA, RNAi, and protein libraries. Historically, the management of large molecules was dispersed into multiple laboratories. As methodologies to support pathway discovery, antibody discovery, and protein production have become high throughput, the implementation of automation and centralized inventory management tools has become important. To this end, to improve sample tracking, throughput, and accuracy, we have implemented a module-based automation system integrated into inventory management software using multiple platforms (Hamilton, Hudson, Dynamic Devices, and Brooks). Here we describe the implementation of these systems with a focus on high-throughput plasmid DNA production management.

  4. An industrial engineering approach to laboratory automation for high throughput screening

    OpenAIRE

    Menke, Karl 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.

  5. Upscaling and automation of electrophysiology: toward high throughput screening in ion channel drug discovery.

    Science.gov (United States)

    Asmild, Margit; Oswald, Nicholas; Krzywkowski, Karen M; Friis, Søren; Jacobsen, Rasmus B; Reuter, Dirk; Taboryski, Rafael; Kutchinsky, Jonathan; Vestergaard, Ras K; Schrøder, Rikke L; Sørensen, Claus B; Bech, Morten; Korsgaard, Mads P G; Willumsen, Niels J

    2003-01-01

    Effective screening of large compound libraries in ion channel drug discovery requires the development of new electrophysiological techniques with substantially increased throughputs compared to the conventional patch clamp technique. Sophion Bioscience is aiming to meet this challenge by developing two lines of automated patch clamp products, a traditional pipette-based system called Apatchi-1, and a silicon chip-based system QPatch. The degree of automation spans from semi-automation (Apatchi-1) where a trained technician interacts with the system in a limited way, to a complete automation (QPatch 96) where the system works continuously and unattended until screening of a full compound library is completed. The performance of the systems range from medium to high throughputs.

  6. High Throughput Facility

    Data.gov (United States)

    Federal Laboratory Consortium — Argonne?s high throughput facility provides highly automated and parallel approaches to material and materials chemistry development. The facility allows scientists...

  7. Automated High Throughput Protein Crystallization Screening at Nanoliter Scale and Protein Structural Study on Lactate Dehydrogenase

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    Li, Fenglei [Iowa State Univ., Ames, IA (United States)

    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

  8. Development of a semi-automated high throughput transient transfection system.

    Science.gov (United States)

    Bos, Aaron B; Duque, Joseph N; Bhakta, Sunil; Farahi, Farzam; Chirdon, Lindsay A; Junutula, Jagath R; Harms, Peter D; Wong, Athena W

    2014-06-20

    Transient transfection of mammalian cells provides a rapid method of producing protein for research purposes. Combining the transient transfection protein expression system with new automation technologies developed for the biotechnology industry would enable a high throughput protein production platform that could be utilized to generate a variety of different proteins in a short amount of time. These proteins could be used for an assortment of studies including proof of concept, antibody development, and biological structure and function. Here we describe such a platform: a semi-automated process for PEI-mediated transient protein production in tubespins at a throughput of 96 transfections at a time using a Biomek FX(P) liquid handling system. In one batch, 96 different proteins can be produced in milligram amounts by PEI transfection of HEK293 cells cultured in 50 mL tubespins. Methods were developed for the liquid handling system to automate the different processes associated with transient transfections such as initial cell seeding, DNA:PEI complex activation and DNA:PEI complex addition to the cells. Increasing DNA:PEI complex incubation time resulted in lower protein expression. To minimize protein production variability, the methods were further optimized to achieve consistent cell seeding, control the DNA:PEI incubation time and prevent cross-contamination among different tubespins. This semi-automated transfection process was applied to express 520 variants of a human IgG1 (hu IgG1) antibody.

  9. Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

    Science.gov (United States)

    Cheng, Yu-Heng; Chen, Yu-Chih; Brien, Riley; Yoon, Euisik

    2016-10-07

    Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

  10. Automated analysis of NF-κB nuclear translocation kinetics in high-throughput screening.

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    Zi Di

    Full Text Available Nuclear entry and exit of the NF-κB family of dimeric transcription factors plays an essential role in regulating cellular responses to inflammatory stress. The dynamics of this nuclear translocation can vary significantly within a cell population and may dramatically change e.g. upon drug exposure. Furthermore, there is significant heterogeneity in individual cell response upon stress signaling. In order to systematically determine factors that define NF-κB translocation dynamics, high-throughput screens that enable the analysis of dynamic NF-κB responses in individual cells in real time are essential. Thus far, only NF-κB downstream signaling responses of whole cell populations at the transcriptional level are in high-throughput mode. In this study, we developed a fully automated image analysis method to determine the time-course of NF-κB translocation in individual cells, suitable for high-throughput screenings in the context of compound screening and functional genomics. Two novel segmentation methods were used for defining the individual nuclear and cytoplasmic regions: watershed masked clustering (WMC and best-fit ellipse of Voronoi cell (BEVC. The dynamic NFκB oscillatory response at the single cell and population level was coupled to automated extraction of 26 analogue translocation parameters including number of peaks, time to reach each peak, and amplitude of each peak. Our automated image analysis method was validated through a series of statistical tests demonstrating computational efficient and accurate NF-κB translocation dynamics quantification of our algorithm. Both pharmacological inhibition of NF-κB and short interfering RNAs targeting the inhibitor of NFκB, IκBα, demonstrated the ability of our method to identify compounds and genetic players that interfere with the nuclear transition of NF-κB.

  11. A High-Throughput Automated Microfluidic Platform for Calcium Imaging of Taste Sensing

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    Yi-Hsing Hsiao

    2016-07-01

    Full Text Available The human enteroendocrine L cell line NCI-H716, expressing taste receptors and taste signaling elements, constitutes a unique model for the studies of cellular responses to glucose, appetite regulation, gastrointestinal motility, and insulin secretion. Targeting these gut taste receptors may provide novel treatments for diabetes and obesity. However, NCI-H716 cells are cultured in suspension and tend to form multicellular aggregates, preventing high-throughput calcium imaging due to interferences caused by laborious immobilization and stimulus delivery procedures. Here, we have developed an automated microfluidic platform that is capable of trapping more than 500 single cells into microwells with a loading efficiency of 77% within two minutes, delivering multiple chemical stimuli and performing calcium imaging with enhanced spatial and temporal resolutions when compared to bath perfusion systems. Results revealed the presence of heterogeneity in cellular responses to the type, concentration, and order of applied sweet and bitter stimuli. Sucralose and denatonium benzoate elicited robust increases in the intracellular Ca2+ concentration. However, glucose evoked a rapid elevation of intracellular Ca2+ followed by reduced responses to subsequent glucose stimulation. Using Gymnema sylvestre as a blocking agent for the sweet taste receptor confirmed that different taste receptors were utilized for sweet and bitter tastes. This automated microfluidic platform is cost-effective, easy to fabricate and operate, and may be generally applicable for high-throughput and high-content single-cell analysis and drug screening.

  12. OptoDyCE: Automated system for high-throughput all-optical dynamic cardiac electrophysiology

    Science.gov (United States)

    Klimas, Aleksandra; Yu, Jinzhu; Ambrosi, Christina M.; Williams, John C.; Bien, Harold; Entcheva, Emilia

    2016-02-01

    In the last two decades, drugs withdrawals from the market were due to cardiac toxicity, where unintended interactions with ion channels disrupt the heart's normal electrical function. Consequently, all new drugs must undergo preclinical testing for cardiac liability, adding to an already expensive and lengthy process. Recognition that proarrhythmic effects often result from drug action on multiple ion channels demonstrates a need for integrative and comprehensive measurements. Additionally, patient-specific therapies relying on emerging technologies employing stem-cell derived cardiomyocytes (e.g. induced pluripotent stem-cell-derived cardiomyocytes, iPSC-CMs) require better screening methods to become practical. However, a high-throughput, cost-effective approach for cellular cardiac electrophysiology has not been feasible. Optical techniques for manipulation and recording provide a contactless means of dynamic, high-throughput testing of cells and tissues. Here, we consider the requirements for all-optical electrophysiology for drug testing, and we implement and validate OptoDyCE, a fully automated system for all-optical cardiac electrophysiology. We demonstrate the high-throughput capabilities using multicellular samples in 96-well format by combining optogenetic actuation with simultaneous fast high-resolution optical sensing of voltage or intracellular calcium. The system can also be implemented using iPSC-CMs and other cell-types by delivery of optogenetic drivers, or through the modular use of dedicated light-sensitive somatic cells in conjunction with non-modified cells. OptoDyCE provides a truly modular and dynamic screening system, capable of fully-automated acquisition of high-content information integral for improved discovery and development of new drugs and biologics, as well as providing a means of better understanding of electrical disturbances in the heart.

  13. An Automated High Throughput Proteolysis and Desalting Platform for Quantitative Proteomic Analysis

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    Albert-Baskar Arul

    2013-06-01

    Full Text Available Proteomics for biomarker validation needs high throughput instrumentation to analyze huge set of clinical samples for quantitative and reproducible analysis at a minimum time without manual experimental errors. Sample preparation, a vital step in proteomics plays a major role in identification and quantification of proteins from biological samples. Tryptic digestion a major check point in sample preparation for mass spectrometry based proteomics needs to be more accurate with rapid processing time. The present study focuses on establishing a high throughput automated online system for proteolytic digestion and desalting of proteins from biological samples quantitatively and qualitatively in a reproducible manner. The present study compares online protein digestion and desalting of BSA with conventional off-line (in-solution method and validated for real time sample for reproducibility. Proteins were identified using SEQUEST data base search engine and the data were quantified using IDEALQ software. The present study shows that the online system capable of handling high throughput samples in 96 well formats carries out protein digestion and peptide desalting efficiently in a reproducible and quantitative manner. Label free quantification showed clear increase of peptide quantities with increase in concentration with much linearity compared to off line method. Hence we would like to suggest that inclusion of this online system in proteomic pipeline will be effective in quantification of proteins in comparative proteomics were the quantification is really very crucial.

  14. Semi-Automated Library Preparation for High-Throughput DNA Sequencing Platforms

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    Eveline Farias-Hesson

    2010-01-01

    Full Text Available Next-generation sequencing platforms are powerful technologies, providing gigabases of genetic information in a single run. An important prerequisite for high-throughput DNA sequencing is the development of robust and cost-effective preprocessing protocols for DNA sample library construction. Here we report the development of a semi-automated sample preparation protocol to produce adaptor-ligated fragment libraries. Using a liquid-handling robot in conjunction with Carboxy Terminated Magnetic Beads, we labeled each library sample using a unique 6 bp DNA barcode, which allowed multiplex sample processing and sequencing of 32 libraries in a single run using Applied Biosystems' SOLiD sequencer. We applied our semi-automated pipeline to targeted medical resequencing of nuclear candidate genes in individuals affected by mitochondrial disorders. This novel method is capable of preparing as much as 32 DNA libraries in 2.01 days (8-hour workday for emulsion PCR/high throughput DNA sequencing, increasing sample preparation production by 8-fold.

  15. Improvement of an automated protein crystal exchange system PAM for high-throughput data collection

    Energy Technology Data Exchange (ETDEWEB)

    Hiraki, Masahiko, E-mail: masahiko.hiraki@kek.jp; Yamada, Yusuke; Chavas, Leonard M. G. [High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801 (Japan); Wakatsuki, Soichi [High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801 (Japan); SLAC National Accelerator Laboratory, 2575 Sand Hill Road, MS 69, Menlo Park, CA 94025-7015 (United States); Stanford University, Beckman Center B105, Stanford, CA 94305-5126 (United States); Matsugaki, Naohiro [High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801 (Japan)

    2013-11-01

    A special liquid-nitrogen Dewar with double capacity for the sample-exchange robot has been created at AR-NE3A at the Photon Factory, allowing continuous fully automated data collection. In this work, this new system is described and the stability of its calibration is discussed. Photon Factory Automated Mounting system (PAM) protein crystal exchange systems are available at the following Photon Factory macromolecular beamlines: BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. The beamline AR-NE3A has been constructed for high-throughput macromolecular crystallography and is dedicated to structure-based drug design. The PAM liquid-nitrogen Dewar can store a maximum of three SSRL cassettes. Therefore, users have to interrupt their experiments and replace the cassettes when using four or more of them during their beam time. As a result of investigation, four or more cassettes were used in AR-NE3A alone. For continuous automated data collection, the size of the liquid-nitrogen Dewar for the AR-NE3A PAM was increased, doubling the capacity. In order to check the calibration with the new Dewar and the cassette stand, calibration experiments were repeatedly performed. Compared with the current system, the parameters of the novel system are shown to be stable.

  16. Development of automated high throughput single molecular microfluidic detection platform for signal transduction analysis

    Science.gov (United States)

    Huang, Po-Jung; Baghbani Kordmahale, Sina; Chou, Chao-Kai; Yamaguchi, Hirohito; Hung, Mien-Chie; Kameoka, Jun

    2016-03-01

    Signal transductions including multiple protein post-translational modifications (PTM), protein-protein interactions (PPI), and protein-nucleic acid interaction (PNI) play critical roles for cell proliferation and differentiation that are directly related to the cancer biology. Traditional methods, like mass spectrometry, immunoprecipitation, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy require a large amount of sample and long processing time. "microchannel for multiple-parameter analysis of proteins in single-complex (mMAPS)"we proposed can reduce the process time and sample volume because this system is composed by microfluidic channels, fluorescence microscopy, and computerized data analysis. In this paper, we will present an automated mMAPS including integrated microfluidic device, automated stage and electrical relay for high-throughput clinical screening. Based on this result, we estimated that this automated detection system will be able to screen approximately 150 patient samples in a 24-hour period, providing a practical application to analyze tissue samples in a clinical setting.

  17. Robo-Lector – a novel platform for automated high-throughput cultivations in microtiter plates with high information content

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    Kensy Frank

    2009-08-01

    Full Text Available Abstract Background In industry and academic research, there is an increasing demand for flexible automated microfermentation platforms with advanced sensing technology. However, up to now, conventional platforms cannot generate continuous data in high-throughput cultivations, in particular for monitoring biomass and fluorescent proteins. Furthermore, microfermentation platforms are needed that can easily combine cost-effective, disposable microbioreactors with downstream processing and analytical assays. Results To meet this demand, a novel automated microfermentation platform consisting of a BioLector and a liquid-handling robot (Robo-Lector was sucessfully built and tested. The BioLector provides a cultivation system that is able to permanently monitor microbial growth and the fluorescence of reporter proteins under defined conditions in microtiter plates. Three examplary methods were programed on the Robo-Lector platform to study in detail high-throughput cultivation processes and especially recombinant protein expression. The host/vector system E. coli BL21(DE3 pRhotHi-2-EcFbFP, expressing the fluorescence protein EcFbFP, was hereby investigated. With the method 'induction profiling' it was possible to conduct 96 different induction experiments (varying inducer concentrations from 0 to 1.5 mM IPTG at 8 different induction times simultaneously in an automated way. The method 'biomass-specific induction' allowed to automatically induce cultures with different growth kinetics in a microtiter plate at the same biomass concentration, which resulted in a relative standard deviation of the EcFbFP production of only ± 7%. The third method 'biomass-specific replication' enabled to generate equal initial biomass concentrations in main cultures from precultures with different growth kinetics. This was realized by automatically transferring an appropiate inoculum volume from the different preculture microtiter wells to respective wells of the main

  18. Automated degenerate PCR primer design for high-throughput sequencing improves efficiency of viral sequencing

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    Li Kelvin

    2012-11-01

    Full Text Available Abstract Background In a high-throughput environment, to PCR amplify and sequence a large set of viral isolates from populations that are potentially heterogeneous and continuously evolving, the use of degenerate PCR primers is an important strategy. Degenerate primers allow for the PCR amplification of a wider range of viral isolates with only one set of pre-mixed primers, thus increasing amplification success rates and minimizing the necessity for genome finishing activities. To successfully select a large set of degenerate PCR primers necessary to tile across an entire viral genome and maximize their success, this process is best performed computationally. Results We have developed a fully automated degenerate PCR primer design system that plays a key role in the J. Craig Venter Institute’s (JCVI high-throughput viral sequencing pipeline. A consensus viral genome, or a set of consensus segment sequences in the case of a segmented virus, is specified using IUPAC ambiguity codes in the consensus template sequence to represent the allelic diversity of the target population. PCR primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the full length of the specified target region. As part of the tiling process, primer pairs are computationally screened to meet the criteria for successful PCR with one of two described amplification protocols. The actual sequencing success rates for designed primers for measles virus, mumps virus, human parainfluenza virus 1 and 3, human respiratory syncytial virus A and B and human metapneumovirus are described, where >90% of designed primer pairs were able to consistently successfully amplify >75% of the isolates. Conclusions Augmenting our previously developed and published JCVI Primer Design Pipeline, we achieved similarly high sequencing success rates with only minor software modifications. The recommended methodology for the construction of the consensus

  19. Automated high-throughput quantification of mitotic spindle positioning from DIC movies of Caenorhabditis embryos.

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    David Cluet

    Full Text Available The mitotic spindle is a microtubule-based structure that elongates to accurately segregate chromosomes during anaphase. Its position within the cell also dictates the future cell cleavage plan, thereby determining daughter cell orientation within a tissue or cell fate adoption for polarized cells. Therefore, the mitotic spindle ensures at the same time proper cell division and developmental precision. Consequently, spindle dynamics is the matter of intensive research. Among the different cellular models that have been explored, the one-cell stage C. elegans embryo has been an essential and powerful system to dissect the molecular and biophysical basis of spindle elongation and positioning. Indeed, in this large and transparent cell, spindle poles (or centrosomes can be easily detected from simple DIC microscopy by human eyes. To perform quantitative and high-throughput analysis of spindle motion, we developed a computer program ACT for Automated-Centrosome-Tracking from DIC movies of C. elegans embryos. We therefore offer an alternative to the image acquisition and processing of transgenic lines expressing fluorescent spindle markers. Consequently, experiments on large sets of cells can be performed with a simple setup using inexpensive microscopes. Moreover, analysis of any mutant or wild-type backgrounds is accessible because laborious rounds of crosses with transgenic lines become unnecessary. Last, our program allows spindle detection in other nematode species, offering the same quality of DIC images but for which techniques of transgenesis are not accessible. Thus, our program also opens the way towards a quantitative evolutionary approach of spindle dynamics. Overall, our computer program is a unique macro for the image- and movie-processing platform ImageJ. It is user-friendly and freely available under an open-source licence. ACT allows batch-wise analysis of large sets of mitosis events. Within 2 minutes, a single movie is processed

  20. Automated high-throughput infusion ESI-MS with direct coupling to a microtiter plate.

    Science.gov (United States)

    Felten, C; Foret, F; Minarik, M; Goetzinger, W; Karger, B L

    2001-04-01

    This paper describes the design and application of instrumentation for automated high-throughput infusion ESI-mass spectrometry. The approach, based on a subatmospheric ESI interface, allows sample introduction from a commercially available microtiter plate without the need for a separate fluid delivery system. The microtiter plate was placed vertically on a three-dimensional translation stage in front of the sampling ESI interface. A single, 7-cm, 20-microm-i.d. fused-silica capillary (total volume, 70 nL), with a tapered tip, served as a combination of sample delivery and spraying capillary. The tapered tip of the capillary was enclosed in a subatmospheric chamber attached in front of the orifice of the mass spectrometer. The sample aspiration rate (flow rate) was regulated by computer-controlled pneumatic valves, which allowed fast switching of the pressure in the subatmospheric ESI chamber. A flow-through wash device was positioned between the microtiter plate and the ESI interface. This design allowed alternate filling of the capillary with (a) sample from the wells and (b) wash solution from the wash device. Sample turnaround times of 10 s/sample, with a 120-nL sample consumption/analysis, and a duty cycle (percentage of total analysis time spent acquiring data) of 40% were achieved. The infusion system was demonstrated in the analysis of preparative HPLC fractions from a small molecule combinatorial library.

  1. Upscaling and automation of electrophysiology: toward high throughput screening in ion channel drug discovery

    DEFF Research Database (Denmark)

    Asmild, Margit; Oswald, Nicholas; Krzywkowski, Karen M

    2003-01-01

    Effective screening of large compound libraries in ion channel drug discovery requires the development of new electrophysiological techniques with substantially increased throughputs compared to the conventional patch clamp technique. Sophion Bioscience is aiming to meet this challenge...... (QPatch 96) where the system works continuously and unattended until screening of a full compound library is completed. The performance of the systems range from medium to high throughputs....

  2. Automated cantilever exchange and optical alignment for High-throughput, parallel atomic force microscopy

    CERN Document Server

    Bijnagte, Tom; Kramer, Lukas; Dekker, Bert; Herfst, Rodolf; Sadeghian, Hamed

    2016-01-01

    In atomic force microscopy (AFM), the exchange and alignment of the AFM cantilever with respect to the optical beam and position-sensitive detector (PSD) are often performed manually. This process is tedious and time-consuming and sometimes damages the cantilever or tip. To increase the throughput of AFM in industrial applications, the ability to automatically exchange and align the cantilever in a very short time with sufficient accuracy is required. In this paper, we present the development of an automated cantilever exchange and optical alignment instrument. We present an experimental proof of principle by exchanging various types of AFM cantilevers in 6 seconds with an accuracy better than 2 um. The exchange and alignment unit is miniaturized to allow for integration in a parallel AFM. The reliability of the demonstrator has also been evaluated. Ten thousand continuous exchange and alignment cycles were performed without failure. The automated exchange and alignment of the AFM cantilever overcome a large ...

  3. Automated high-throughput protein purification using an ÄKTApurifier and a CETAC autosampler.

    Science.gov (United States)

    Yoo, Daniel; Provchy, Justin; Park, Cynthia; Schulz, Craig; Walker, Kenneth

    2014-05-30

    As the pace of drug discovery accelerates there is an increased focus on screening larger numbers of protein therapeutic candidates to identify those that are functionally superior and to assess manufacturability earlier in the process. Although there have been advances toward high throughput (HT) cloning and expression, protein purification is still an area where improvements can be made to conventional techniques. Current methodologies for purification often involve a tradeoff between HT automation or capacity and quality. We present an ÄKTA combined with an autosampler, the ÄKTA-AS, which has the capability of purifying up to 240 samples in two chromatographic dimensions without the need for user intervention. The ÄKTA-AS has been shown to be reliable with sample volumes between 0.5 mL and 100 mL, and the innovative use of a uniquely configured loading valve ensures reliability by efficiently removing air from the system as well as preventing sample cross contamination. Incorporation of a sample pump flush minimizes sample loss and enables recoveries ranging from the low tens of micrograms to milligram quantities of protein. In addition, when used in an affinity capture-buffer exchange format the final samples are formulated in a buffer compatible with most assays without requirement of additional downstream processing. The system is designed to capture samples in 96-well microplate format allowing for seamless integration of downstream HT analytic processes such as microfluidic or HPLC analysis. Most notably, there is minimal operator intervention to operate this system, thereby increasing efficiency, sample consistency and reducing the risk of human error.

  4. Fully Automated Electro Membrane Extraction Autosampler for LC-MS Systems Allowing Soft Extractions for High-Throughput Applications.

    Science.gov (United States)

    Fuchs, David; Pedersen-Bjergaard, Stig; Jensen, Henrik; Rand, Kasper D; Honoré Hansen, Steen; Petersen, Nickolaj Jacob

    2016-07-05

    The current work describes the implementation of electro membrane extraction (EME) into an autosampler for high-throughput analysis of samples by EME-LC-MS. The extraction probe was built into a luer lock adapter connected to a HTC PAL autosampler syringe. As the autosampler drew sample solution, analytes were extracted into the lumen of the extraction probe and transferred to a LC-MS system for further analysis. Various parameters affecting extraction efficacy were investigated including syringe fill strokes, syringe pull up volume, pull up delay and volume in the sample vial. The system was optimized for soft extraction of analytes and high sample throughput. Further, it was demonstrated that by flushing the EME-syringe with acidic wash buffer and reverting the applied electric potential, carry-over between samples can be reduced to below 1%. Performance of the system was characterized (RSD, high extraction speed of EME, a complete analytical workflow of purification, separation, and analysis of sample could be achieved within only 5.5 min. With the developed system large sequences of samples could be analyzed in a completely automated manner. This high degree of automation makes the developed EME-autosampler a powerful tool for a wide range of applications where high-throughput extractions are required before sample analysis.

  5. An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and Automated Data Analysis Software

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    Shah, Pranav; Kerns, Edward; Nguyen, Dac-Trung; Obach, R. Scott; Wang, Amy Q.; Zakharov, Alexey; McKew, John; Simeonov, Anton; Hop, Cornelis E. C. A.

    2016-01-01

    Advancement of in silico tools would be enabled by the availability of data for metabolic reaction rates and intrinsic clearance (CLint) of a diverse compound structure data set by specific metabolic enzymes. Our goal is to measure CLint for a large set of compounds with each major human cytochrome P450 (P450) isozyme. To achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle a large volume of compound sets. The substrate depletion method [in vitro half-life (t1/2) method] was chosen to determine CLint. The assay (384-well format) consisted of three parts: 1) a robotic system for incubation and sample cleanup; 2) two different integrated, ultraperformance liquid chromatography/mass spectrometry (UPLC/MS) platforms to determine the percent remaining of parent compound, and 3) an automated data analysis system. The CYP3A4 assay was evaluated using two long t1/2 compounds, carbamazepine and antipyrine (t1/2 > 30 minutes); one moderate t1/2 compound, ketoconazole (10 < t1/2 < 30 minutes); and two short t1/2 compounds, loperamide and buspirone (t½ < 10 minutes). Interday and intraday precision and accuracy of the assay were within acceptable range (∼12%) for the linear range observed. Using this assay, CYP3A4 CLint and t1/2 values for more than 3000 compounds were measured. This high-throughput, automated, and robust assay allows for rapid metabolic stability screening of large compound sets and enables advanced computational modeling for individual human P450 isozymes. PMID:27417180

  6. High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection.

    Directory of Open Access Journals (Sweden)

    Priya Choudhry

    Full Text Available Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays.

  7. High-throughput isolation of giant viruses in liquid medium using automated flow cytometry and fluorescence staining.

    Directory of Open Access Journals (Sweden)

    Jacques Yaacoub Bou Khalil

    2016-01-01

    Full Text Available The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than ten strains of previously known species of giant viruses and 7 new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed.

  8. High-Throughput Isolation of Giant Viruses in Liquid Medium Using Automated Flow Cytometry and Fluorescence Staining

    Science.gov (United States)

    Khalil, Jacques Y. B.; Robert, Stephane; Reteno, Dorine G.; Andreani, Julien; Raoult, Didier; La Scola, Bernard

    2016-01-01

    The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than 10 strains of previously known species of giant viruses and seven new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed. PMID:26858703

  9. Automated SNP Genotype Clustering Algorithm to Improve Data Completeness in High-Throughput SNP Genotyping Datasets from Custom Arrays

    Institute of Scientific and Technical Information of China (English)

    Edward; M.; Smith; Jack; Littrell; Michael; Olivier

    2007-01-01

    High-throughput SNP genotyping platforms use automated genotype calling algo- rithms to assign genotypes. While these algorithms work efficiently for individual platforms, they are not compatible with other platforms, and have individual biases that result in missed genotype calls. Here we present data on the use of a second complementary SNP genotype clustering algorithm. The algorithm was originally designed for individual fluorescent SNP genotyping assays, and has been opti- mized to permit the clustering of large datasets generated from custom-designed Affymetrix SNP panels. In an analysis of data from a 3K array genotyped on 1,560 samples, the additional analysis increased the overall number of genotypes by over 45,000, significantly improving the completeness of the experimental data. This analysis suggests that the use of multiple genotype calling algorithms may be ad- visable in high-throughput SNP genotyping experiments. The software is written in Perl and is available from the corresponding author.

  10. Automated, high-throughput, motility analysis in Caenorhabditis elegans and parasitic nematodes: Applications in the search for new anthelmintics

    Directory of Open Access Journals (Sweden)

    Steven D. Buckingham

    2014-12-01

    Full Text Available The scale of the damage worldwide to human health, animal health and agricultural crops resulting from parasitic nematodes, together with the paucity of treatments and the threat of developing resistance to the limited set of widely-deployed chemical tools, underlines the urgent need to develop novel drugs and chemicals to control nematode parasites. Robust chemical screens which can be automated are a key part of that discovery process. Hitherto, the successful automation of nematode behaviours has been a bottleneck in the chemical discovery process. As the measurement of nematode motility can provide a direct scalar readout of the activity of the neuromuscular system and an indirect measure of the health of the animal, this omission is acute. Motility offers a useful assay for high-throughput, phenotypic drug/chemical screening and several recent developments have helped realise, at least in part, the potential of nematode-based drug screening. Here we review the challenges encountered in automating nematode motility and some important developments in the application of machine vision, statistical imaging and tracking approaches which enable the automated characterisation of nematode movement. Such developments facilitate automated screening for new drugs and chemicals aimed at controlling human and animal nematode parasites (anthelmintics and plant nematode parasites (nematicides.

  11. State-of-the-art automated patch clamp devices: Heat activation, action potentials and high throughput in ion channel screening

    Directory of Open Access Journals (Sweden)

    Sonja eStoelzle

    2011-11-01

    Full Text Available Ion channels are essential in a wide range of cellular functions and their malfunction underlies many disease states making them important targets in drug discovery. Diverse automated patch clamp systems with high-throughput capabilities are available for drug screening, but there are limitations in the application range. For example, solution exchange time, temperature control and the availability of the current clamp mode can be limiting factors. However, further development of existing devices and introduction of new systems widen the range of possible experiments and increase throughput. Here we introduce new features and platforms that meet the needs of drug discovery researchers and basic researchers alike.The Patchliner is an automated patch clamp system capable of recording up to 8 cells simultaneously with high success rates. Novel features such as temperature control and recordings in the current clamp mode are described here. Standard cell lines and excitable cells such as stem cell-derived cardiomyocytes have been used in the voltage clamp and current clamp modes with the view to finding new drug candidates and safety testing methods in a more physiologically relevant environment. The SyncroPatch 96, is a screening platform capable of recording from 96 cells in parallel and offers a throughput of 5000 data points per day. Full dose response curves can be acquired from individual cells reducing the cost per data point. The system is an ideal tool for secondary screening efforts and for safety testing on ligand- and voltage-gated ion channels.The Patchliner and SyncroPatch 96 are ideal platforms for drug discovery, ion channel research and safety testing, combining long awaited features such as parallel action potential recordings and temperature control with extensively increased throughput.

  12. Automated cleaning and pre-processing of immunoglobulin gene sequences from high-throughput sequencing

    Directory of Open Access Journals (Sweden)

    Miri eMichaeli

    2012-12-01

    Full Text Available High throughput sequencing (HTS yields tens of thousands to millions of sequences that require a large amount of pre-processing work to clean various artifacts. Such cleaning cannot be performed manually. Existing programs are not suitable for immunoglobulin (Ig genes, which are variable and often highly mutated. This paper describes Ig-HTS-Cleaner (Ig High Throughput Sequencing Cleaner, a program containing a simple cleaning procedure that successfully deals with pre-processing of Ig sequences derived from HTS, and Ig-Indel-Identifier (Ig Insertion – Deletion Identifier, a program for identifying legitimate and artifact insertions and/or deletions (indels. Our programs were designed for analyzing Ig gene sequences obtained by 454 sequencing, but they are applicable to all types of sequences and sequencing platforms. Ig-HTS-Cleaner and Ig-Indel-Identifier have been implemented in Java and saved as executable JAR files, supported on Linux and MS Windows. No special requirements are needed in order to run the programs, except for correctly constructing the input files as explained in the text. The programs' performance has been tested and validated on real and simulated data sets.

  13. Towards high-throughput automated targeted femtosecond laser-based transfection of adherent cells

    Science.gov (United States)

    Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Gunn-Moore, Frank; Dholakia, Kishan

    2011-03-01

    Femtosecond laser induced cell membrane poration has proven to be an attractive alternative to the classical methods of drug and gene delivery. It is a selective, sterile, non-contact technique that offers a highly localized operation, low toxicity and consistent performance. However, its broader application still requires the development of robust, high-throughput and user-friendly systems. We present a system capable of unassisted enhanced targeted optoinjection and phototransfection of adherent mammalian cells with a femtosecond laser. We demonstrate the advantages of a dynamic diffractive optical element, namely a spatial light modulator (SLM) for precise three dimensional positioning of the beam. It enables the implementation of a "point-and-shoot" system in which using the software interface a user simply points at the cell and a predefined sequence of precisely positioned doses can be applied. We show that irradiation in three axial positions alleviates the problem of exact beam positioning on the cell membrane and doubles the number of viably optoinjected cells when compared with a single dose. The presented system enables untargeted raster scan irradiation which provides transfection of adherent cells at the throughput of 1 cell per second.

  14. High throughput detection of Coxiella burnetii by real-time PCR with internal control system and automated DNA preparation

    Directory of Open Access Journals (Sweden)

    Kramme Stefanie

    2008-05-01

    Full Text Available Abstract Background Coxiella burnetii is the causative agent of Q-fever, a widespread zoonosis. Due to its high environmental stability and infectivity it is regarded as a category B biological weapon agent. In domestic animals infection remains either asymptomatic or presents as infertility or abortion. Clinical presentation in humans can range from mild flu-like illness to acute pneumonia and hepatitis. Endocarditis represents the most common form of chronic Q-fever. In humans serology is the gold standard for diagnosis but is inadequate for early case detection. In order to serve as a diagnostic tool in an eventual biological weapon attack or in local epidemics we developed a real-time 5'nuclease based PCR assay with an internal control system. To facilitate high-throughput an automated extraction procedure was evaluated. Results To determine the minimum number of copies that are detectable at 95% chance probit analysis was used. Limit of detection in blood was 2,881 copies/ml [95%CI, 2,188–4,745 copies/ml] with a manual extraction procedure and 4,235 copies/ml [95%CI, 3,143–7,428 copies/ml] with a fully automated extraction procedure, respectively. To demonstrate clinical application a total of 72 specimens of animal origin were compared with respect to manual and automated extraction. A strong correlation between both methods was observed rendering both methods suitable. Testing of 247 follow up specimens of animal origin from a local Q-fever epidemic rendered real-time PCR more sensitive than conventional PCR. Conclusion A sensitive and thoroughly evaluated real-time PCR was established. Its high-throughput mode may show a useful approach to rapidly screen samples in local outbreaks for other organisms relevant for humans or animals. Compared to a conventional PCR assay sensitivity of real-time PCR was higher after testing samples from a local Q-fever outbreak.

  15. Automated Sample Preparation for Radiogenic and Non-Traditional Metal Isotopes: Removing an Analytical Barrier for High Sample Throughput

    Science.gov (United States)

    Field, M. Paul; Romaniello, Stephen; Gordon, Gwyneth W.; Anbar, Ariel D.; Herrmann, Achim; Martinez-Boti, Miguel A.; Anagnostou, Eleni; Foster, Gavin L.

    2014-05-01

    MC-ICP-MS has dramatically improved the analytical throughput for high-precision radiogenic and non-traditional isotope ratio measurements, compared to TIMS. The generation of large data sets, however, remains hampered by tedious manual drip chromatography required for sample purification. A new, automated chromatography system reduces the laboratory bottle neck and expands the utility of high-precision isotope analyses in applications where large data sets are required: geochemistry, forensic anthropology, nuclear forensics, medical research and food authentication. We have developed protocols to automate ion exchange purification for several isotopic systems (B, Ca, Fe, Cu, Zn, Sr, Cd, Pb and U) using the new prepFAST-MC™ (ESI, Nebraska, Omaha). The system is not only inert (all-flouropolymer flow paths), but is also very flexible and can easily facilitate different resins, samples, and reagent types. When programmed, precise and accurate user defined volumes and flow rates are implemented to automatically load samples, wash the column, condition the column and elute fractions. Unattended, the automated, low-pressure ion exchange chromatography system can process up to 60 samples overnight. Excellent reproducibility, reliability, recovery, with low blank and carry over for samples in a variety of different matrices, have been demonstrated to give accurate and precise isotopic ratios within analytical error for several isotopic systems (B, Ca, Fe, Cu, Zn, Sr, Cd, Pb and U). This illustrates the potential of the new prepFAST-MC™ (ESI, Nebraska, Omaha) as a powerful tool in radiogenic and non-traditional isotope research.

  16. Towards automated high-throughput screening of C. elegans on agar

    CERN Document Server

    Kabra, Mayank; O'Rourke, Eyleen J; Xie, Xin; Ljosa, Vebjorn; Jones, Thouis R; Ausubel, Frederick M; Ruvkun, Gary; Carpenter, Anne E; Freund, Yoav

    2010-01-01

    High-throughput screening (HTS) using model organisms is a promising method to identify a small number of genes or drugs potentially relevant to human biology or disease. In HTS experiments, robots and computers do a significant portion of the experimental work. However, one remaining major bottleneck is the manual analysis of experimental results, which is commonly in the form of microscopy images. This manual inspection is labor intensive, slow and subjective. Here we report our progress towards applying computer vision and machine learning methods to analyze HTS experiments that use Caenorhabditis elegans (C. elegans) worms grown on agar. Our main contribution is a robust segmentation algorithm for separating the worms from the background using brightfield images. We also show that by combining the output of this segmentation algorithm with an algorithm to detect the fluorescent dye, Nile Red, we can reliably distinguish different fluorescence-based phenotypes even though the visual differences are subtle....

  17. PLAN: a web platform for automating high-throughput BLAST searches and for managing and mining results

    Directory of Open Access Journals (Sweden)

    Zhao Xuechun

    2007-02-01

    Full Text Available Abstract Background BLAST searches are widely used for sequence alignment. The search results are commonly adopted for various functional and comparative genomics tasks such as annotating unknown sequences, investigating gene models and comparing two sequence sets. Advances in sequencing technologies pose challenges for high-throughput analysis of large-scale sequence data. A number of programs and hardware solutions exist for efficient BLAST searching, but there is a lack of generic software solutions for mining and personalized management of the results. Systematically reviewing the results and identifying information of interest remains tedious and time-consuming. Results Personal BLAST Navigator (PLAN is a versatile web platform that helps users to carry out various personalized pre- and post-BLAST tasks, including: (1 query and target sequence database management, (2 automated high-throughput BLAST searching, (3 indexing and searching of results, (4 filtering results online, (5 managing results of personal interest in favorite categories, (6 automated sequence annotation (such as NCBI NR and ontology-based annotation. PLAN integrates, by default, the Decypher hardware-based BLAST solution provided by Active Motif Inc. with a greatly improved efficiency over conventional BLAST software. BLAST results are visualized by spreadsheets and graphs and are full-text searchable. BLAST results and sequence annotations can be exported, in part or in full, in various formats including Microsoft Excel and FASTA. Sequences and BLAST results are organized in projects, the data publication levels of which are controlled by the registered project owners. In addition, all analytical functions are provided to public users without registration. Conclusion PLAN has proved a valuable addition to the community for automated high-throughput BLAST searches, and, more importantly, for knowledge discovery, management and sharing based on sequence alignment results

  18. Automated reporter quantification in vivo: high-throughput screening method for reporter-based assays in zebrafish.

    Directory of Open Access Journals (Sweden)

    Steven L Walker

    Full Text Available Reporter-based assays underlie many high-throughput screening (HTS platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv. ARQiv differs from current "high-content" (e.g., confocal imaging-based whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1 Rapid; achieving true HTS capacities (i.e., >50,000 units per day, 2 Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5, and 3 Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1 Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors, 2 Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3 Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current "high-content" whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform.

  19. A High Throughput, 384-Well, Semi-Automated, Hepatocyte Intrinsic Clearance Assay for Screening New Molecular Entities in Drug Discovery.

    Science.gov (United States)

    Heinle, Lance; Peterkin, Vincent; de Morais, Sonia M; Jenkins, Gary J; Badagnani, Ilaria

    2015-01-01

    A high throughput, semi-automated clearance screening assay in hepatocytes was developed allowing a scientist to generate data for 96 compounds in one week. The 384-well format assay utilizes a Thermo Multidrop Combi and an optimized LC-MS/MS method. The previously reported LCMS/ MS method reduced the analytical run time by 3-fold, down to 1.2 min injection-to-injection. The Multidrop was able to deliver hepatocytes to 384-well plates with minimal viability loss. Comparison of results from the new 384-well and historical 24-well assays yielded a correlation of 0.95. In addition, results obtained for 25 marketed drugs with various metabolism pathways had a correlation of 0.75 when compared with literature values. Precision was maintained in the new format as 8 compounds tested in ≥39 independent experiments had coefficients of variation ≤21%. The ability to predict in vivo clearances using the new stability assay format was also investigated using 22 marketed drugs and 26 AbbVie compounds. Correction of intrinsic clearance values with binding to hepatocytes (in vitro data) and plasma (in vivo data) resulted in a higher in vitro to in vivo correlation when comparing 22 marketed compounds in human (0.80 vs 0.35) and 26 AbbVie Discovery compounds in rat (0.56 vs 0.17), demonstrating the importance of correcting for binding in clearance studies. This newly developed high throughput, semi-automated clearance assay allows for rapid screening of Discovery compounds to enable Structure Activity Relationship (SAR) analysis based on high quality hepatocyte stability data in sufficient quantity and quality to drive the next round of compound synthesis.

  20. Robofurnace: A semi-automated laboratory chemical vapor deposition system for high-throughput nanomaterial synthesis and process discovery

    Science.gov (United States)

    Oliver, C. Ryan; Westrick, William; Koehler, Jeremy; Brieland-Shoultz, Anna; Anagnostopoulos-Politis, Ilias; Cruz-Gonzalez, Tizoc; Hart, A. John

    2013-11-01

    Laboratory research and development on new materials, such as nanostructured thin films, often utilizes manual equipment such as tube furnaces due to its relatively low cost and ease of setup. However, these systems can be prone to inconsistent outcomes due to variations in standard operating procedures and limitations in performance such as heating and cooling rates restrict the parameter space that can be explored. Perhaps more importantly, maximization of research throughput and the successful and efficient translation of materials processing knowledge to production-scale systems, relies on the attainment of consistent outcomes. In response to this need, we present a semi-automated lab-scale chemical vapor deposition (CVD) furnace system, called "Robofurnace." Robofurnace is an automated CVD system built around a standard tube furnace, which automates sample insertion and removal and uses motion of the furnace to achieve rapid heating and cooling. The system has a 10-sample magazine and motorized transfer arm, which isolates the samples from the lab atmosphere and enables highly repeatable placement of the sample within the tube. The system is designed to enable continuous operation of the CVD reactor, with asynchronous loading/unloading of samples. To demonstrate its performance, Robofurnace is used to develop a rapid CVD recipe for carbon nanotube (CNT) forest growth, achieving a 10-fold improvement in CNT forest mass density compared to a benchmark recipe using a manual tube furnace. In the long run, multiple systems like Robofurnace may be linked to share data among laboratories by methods such as Twitter. Our hope is Robofurnace and like automation will enable machine learning to optimize and discover relationships in complex material synthesis processes.

  1. Automated neural network classifiers for identifying micrometastases in peripheral blood via high-throughput microscopy

    Science.gov (United States)

    Agustin, Ramses M.; Azimi, Behrad; Price, Jeffrey H.

    2007-02-01

    Tissue-specific biomarkers have been studied to identify micrometastases in bone marrow and/or peripheral blood. Many studies, however, have shown conflicting results for sensitivity and specificity of detection, forestalling translation of these findings into routine clinical use for prognosis or diagnosis. Genetic instability and heterogeneity of cancers may make using an absolute set of differential expression markers difficult, if not impossible, for accurate detection of rare cancer cells via a simple blood test. The literature is rich with examples of pathologists using morphology to identify cancer in tissue sections. We hypothesize that morphological features based on fluorescent staining of common subcellular compartments, in particular, the nucleus, may be useful for detection and classification. Highthroughput/ high-content image cytometry and computer-automated classification can aid pathologists to find suspicious cells, independent of biomarkers. Feature data are collected from an in vitro spiked model of breast cancer in the circulation; prestaining with CellTracker Orange creates a gold standard for assessing cancer origin. A neural network classifier is designed using seven nuclear morphology features thought a priori to be important for classification. With adequate training data, sensitive and specific detection may be achieved. Neural networks may be robustly trained to assist pathologists in detecting a wide variety of cancers.

  2. Automated high-throughput assessment of prostate biopsy tissue using infrared spectroscopic chemical imaging

    Science.gov (United States)

    Bassan, Paul; Sachdeva, Ashwin; Shanks, Jonathan H.; Brown, Mick D.; Clarke, Noel W.; Gardner, Peter

    2014-03-01

    Fourier transform infrared (FT-IR) chemical imaging has been demonstrated as a promising technique to complement histopathological assessment of biomedical tissue samples. Current histopathology practice involves preparing thin tissue sections and staining them using hematoxylin and eosin (H&E) after which a histopathologist manually assess the tissue architecture under a visible microscope. Studies have shown that there is disagreement between operators viewing the same tissue suggesting that a complementary technique for verification could improve the robustness of the evaluation, and improve patient care. FT-IR chemical imaging allows the spatial distribution of chemistry to be rapidly imaged at a high (diffraction-limited) spatial resolution where each pixel represents an area of 5.5 × 5.5 μm2 and contains a full infrared spectrum providing a chemical fingerprint which studies have shown contains the diagnostic potential to discriminate between different cell-types, and even the benign or malignant state of prostatic epithelial cells. We report a label-free (i.e. no chemical de-waxing, or staining) method of imaging large pieces of prostate tissue (typically 1 cm × 2 cm) in tens of minutes (at a rate of 0.704 × 0.704 mm2 every 14.5 s) yielding images containing millions of spectra. Due to refractive index matching between sample and surrounding paraffin, minimal signal processing is required to recover spectra with their natural profile as opposed to harsh baseline correction methods, paving the way for future quantitative analysis of biochemical signatures. The quality of the spectral information is demonstrated by building and testing an automated cell-type classifier based upon spectral features.

  3. High-Throughput, Automated Protein A Purification Platform with Multiattribute LC-MS Analysis for Advanced Cell Culture Process Monitoring.

    Science.gov (United States)

    Dong, Jia; Migliore, Nicole; Mehrman, Steven J; Cunningham, John; Lewis, Michael J; Hu, Ping

    2016-09-06

    The levels of many product related variants observed during the production of monoclonal antibodies are dependent on control of the manufacturing process, especially the cell culture process. However, it is difficult to characterize samples pulled from the bioreactor due to the low levels of product during the early stages of the process and the high levels of interfering reagents. Furthermore, analytical results are often not available for several days, which slows the process development cycle and prevents "real time" adjustments to the manufacturing process. To reduce the delay and enhance our ability to achieve quality targets, we have developed a low-volume, high-throughput, and high-content analytical platform for at-line product quality analysis. This workflow includes an automated, 96-well plate protein A purification step to isolate antibody product from the cell culture fermentation broth, followed by rapid, multiattribute LC-MS analysis. We have demonstrated quantitative correlations between particular process parameters with the levels of glycosylated and glycated species in a series of small scale experiments, but the platform could be used to monitor other attributes and applied across the biopharmaceutical industry.

  4. High-throughput automated system for statistical biosensing employing microcantilevers arrays

    DEFF Research Database (Denmark)

    Bosco, Filippo; Chen, Ching H.; Hwu, En T.

    2011-01-01

    In this paper we present a completely new and fully automated system for parallel microcantilever-based biosensing. Our platform is able to monitor simultaneously the change of resonance frequency (dynamic mode), of deflection (static mode), and of surface roughness of hundreds of cantilevers...... in a very short time over multiple biochemical reactions. We have proven that our system is capable to measure 900 independent microsensors in less than a second. Here, we report statistical biosensing results performed over a haptens-antibody assay, where complete characterization of the biochemical...

  5. High throughput DNA sequence variant detection by conformation sensitive capillary electrophoresis and automated peak comparison.

    Science.gov (United States)

    Davies, Helen; Dicks, Ed; Stephens, Philip; Cox, Charles; Teague, Jon; Greenman, Chris; Bignell, Graham; O'meara, Sarah; Edkins, Sarah; Parker, Adrian; Stevens, Claire; Menzies, Andrew; Blow, Matt; Bottomley, Bill; Dronsfield, Mark; Futreal, P Andrew; Stratton, Michael R; Wooster, Richard

    2006-03-01

    We report the development of a heteroduplex-based mutation detection method using multicapillary automated sequencers, known as conformation-sensitive capillary electrophoresis (CSCE). Our optimized CSCE protocol detected 93 of 95 known base substitution sequence variants. Since the optimization of the method, we have analyzed 215 Mb of DNA and identified 3397 unique variants. An analysis of this data set indicates that the sensitivity of CSCE is above 95% in the central 56% of the average PCR product. To fully exploit the mutation detection capacity of this method, we have developed software, canplot, which automatically compares normal and test results to prioritize samples that are most likely to contain variants. Using multiple fluorescent dyes, CSCE has the capacity to screen over 2.2 Mb on one ABI3730 each day. Therefore this technique is suitable for projects where a rapid and sensitive DNA mutation detection system is required.

  6. Factors to be considered for robust high-throughput automated DNA sequencing using a multiple-capillary array instrument

    Science.gov (United States)

    Carrilho, Emanuel; Miller, Arthur W.; Ruiz-Martinez, Marie C.; Kotler, Lev; Kesilman, Jeffrey; Karger, Barry L.

    1997-05-01

    The overall goal of our program is to develop a robust, high throughput, fully automated DNA sequencing instrument based on replaceable polymer solutions using a multicapillary array. Significant effort has already been devoted to column and polymer chemistry in order to obtain long read lengths per run in fast analysis time. In this paper we report on progress in instrument considerations and data processing software. A simple instrument design, based on no moving parts for continuous illumination of the capillaries and detection of the fluorescent light was used for this study. Our polymer solution replacement system with the permanent connection between the buffer/chamber manifold and capillary columns on the detector side is designed to prevent the trapping of air bubbles during matrix solution replacement. A special construction of a column-electrode couple on the injection side precludes air trapping during sample injection from small sample volumes. Our in-house software now features the significant reduction of the crosstalk signal from neighbor columns, which may be a potential problem in densely packed large capillary array sequencers.

  7. High-throughput automated microfluidic sample preparation for accurate microbial genomics

    Science.gov (United States)

    Kim, Soohong; De Jonghe, Joachim; Kulesa, Anthony B.; Feldman, David; Vatanen, Tommi; Bhattacharyya, Roby P.; Berdy, Brittany; Gomez, James; Nolan, Jill; Epstein, Slava; Blainey, Paul C.

    2017-01-01

    Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (∼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence ∼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications. PMID:28128213

  8. Integrated Automation of High-Throughput Screening and Reverse Phase Protein Array Sample Preparation

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    multiplexing readouts, but this has a natural limitation. High-content screening via image acquisition and analysis allows multiplexing of few parameters, but is connected to substantial time consumption and complex logistics. We report on integration of Reverse Phase Protein Arrays (RPPA)-based readouts...

  9. Automated discovery of novel drug formulations using predictive iterated high throughput experimentation.

    Directory of Open Access Journals (Sweden)

    Filippo Caschera

    Full Text Available BACKGROUND: We consider the problem of optimizing a liposomal drug formulation: a complex chemical system with many components (e.g., elements of a lipid library that interact nonlinearly and synergistically in ways that cannot be predicted from first principles. METHODOLOGY/PRINCIPAL FINDINGS: The optimization criterion in our experiments was the percent encapsulation of a target drug, Amphotericin B, detected experimentally via spectrophotometric assay. Optimization of such a complex system requires strategies that efficiently discover solutions in extremely large volumes of potential experimental space. We have designed and implemented a new strategy of evolutionary design of experiments (Evo-DoE, that efficiently explores high-dimensional spaces by coupling the power of computer and statistical modeling with experimentally measured responses in an iterative loop. CONCLUSIONS: We demonstrate how iterative looping of modeling and experimentation can quickly produce new discoveries with significantly better experimental response, and how such looping can discover the chemical landscape underlying complex chemical systems.

  10. Fully Automated Electro Membrane Extraction Autosampler for LC-MS Systems Allowing Soft Extractions for High-Throughput Applications

    DEFF Research Database (Denmark)

    Fuchs, David; Pedersen-Bjergaard, Stig; Jensen, Henrik

    2016-01-01

    The current work describes the implementation of electro membrane extraction (EME) into an autosampler for high-throughput analysis of samples by EME-LC-MS. The extraction probe was built into a luer lock adapter connected to a HTC PAL autosampler syringe. As the autosampler drew sample solution,...

  11. Automation of High-Throughput Mass Spectrometry-Based Plasma N-Glycome Analysis with Linkage-Specific Sialic Acid Esterification.

    Science.gov (United States)

    Bladergroen, Marco R; Reiding, Karli R; Hipgrave Ederveen, Agnes L; Vreeker, Gerda C M; Clerc, Florent; Holst, Stephanie; Bondt, Albert; Wuhrer, Manfred; van der Burgt, Yuri E M

    2015-09-04

    Glycosylation is a post-translational modification of key importance with heterogeneous structural characteristics. Previously, we have developed a robust, high-throughput MALDI-TOF-MS method for the comprehensive profiling of human plasma N-glycans. In this approach, sialic acid residues are derivatized with linkage-specificity, namely the ethylation of α2,6-linked sialic acid residues with parallel lactone formation of α2,3-linked sialic acids. In the current study, this procedure was used as a starting point for the automation of all steps on a liquid-handling robot system. This resulted in a time-efficient and fully standardized procedure with throughput times of 2.5 h for a first set of 96 samples and approximately 1 h extra for each additional sample plate. The mass analysis of the thus-obtained glycans was highly reproducible in terms of relative quantification, with improved interday repeatability as compared to that of manual processing.

  12. Selective Detection and Automated Counting of Fluorescently-Labeled Chrysotile Asbestos Using a Dual-Mode High-Throughput Microscopy (DM-HTM Method

    Directory of Open Access Journals (Sweden)

    Jung Kyung Kim

    2013-05-01

    Full Text Available Phase contrast microscopy (PCM is a widely used analytical method for airborne asbestos, but it is unable to distinguish asbestos from non-asbestos fibers and requires time-consuming and laborious manual counting of fibers. Previously, we developed a high-throughput microscopy (HTM method that could greatly reduce human intervention and analysis time through automated image acquisition and counting of fibers. In this study, we designed a dual-mode HTM (DM-HTM device for the combined reflection and fluorescence imaging of asbestos, and automated a series of built-in image processing commands of ImageJ software to test its capabilities. We used DksA, a chrysotile-adhesive protein, for selective detection of chrysotile fibers in the mixed dust-free suspension of crysotile and amosite prepared in the laboratory. We demonstrate that fluorescently-stained chrysotile and total fibers can be identified and enumerated automatically in a high-throughput manner by the DM-HTM system. Combined with more advanced software that can correctly identify overlapping and branching fibers and distinguish between fibers and elongated dust particles, the DM-HTM method should enable fully automated counting of airborne asbestos.

  13. High throughput drug profiling

    OpenAIRE

    Entzeroth, Michael; Chapelain, Béatrice; Guilbert, Jacques; Hamon, Valérie

    2000-01-01

    High throughput screening has significantly contributed to advances in drug discovery. The great increase in the number of samples screened has been accompanied by increases in costs and in the data required for the investigated compounds. High throughput profiling addresses the issues of compound selectivity and specificity. It combines conventional screening with data mining technologies to give a full set of data, enabling development candidates to be more fully compared.

  14. Adaption of a fragment analysis technique to an automated high-throughput multicapillary electrophoresis device for the precise qualitative and quantitative characterization of microbial communities.

    Science.gov (United States)

    Trotha, René; Reichl, Udo; Thies, Frank L; Sperling, Danuta; König, Wolfgang; König, Brigitte

    2002-04-01

    The analysis of microbial communities is of increasing importance in life sciences and bioengineering. Traditional techniques of investigations like culture or cloning methods suffer from many disadvantages. They are unable to give a complete qualitative and quantitative view of the total amount of microorganisms themselves, their interactions among each other and with their environment. Obviously, the determination of static or dynamic balances among microorganisms is of fast growing interest. The generation of species specific and fluorescently labeled 16S ribosomal DNA (rDNA) fragments by the terminal restriction fragment length polymorphism (T-RFLP) technique is a suitable tool to overcome the problems other methods have. For the separation of these fragments polyacrylamide gel sequencers are preferred as compared to capillary sequencers using linear polymers until now because of their higher electrophoretic resolution and therefore sizing accuracy. But modern capillary sequencers, especially multicapillary sequencers, offer an advanced grade of automation and an increased throughput necessary for the investigation of complex communities in long-time studies. Therefore, we adapted a T-RFLP technique to an automated high-throughput multicapillary electrophoresis device (ABI 3100 Genetic Analysis) with regard to a precise qualitative and quantitative characterization of microbial communities.

  15. An automated, high-throughput, 384 well Cytochrome P450 cocktail IC50 assay using a rapid resolution LC-MS/MS end-point.

    Science.gov (United States)

    Youdim, Kuresh A; Lyons, Richard; Payne, Leon; Jones, Barry C; Saunders, Kenneth

    2008-09-10

    The current study focused on the development of an automated IC50 cocktail assay in a miniaturized 384 well assay format. This was developed in combination with a significantly shorter high pressure liquid chromatography (HPLC) separation and liquid chromatography-mass spectrometry (LC-MS/MS) run-time; than those currently reported in the literature. The 384-well assay used human liver microsomes in conjunction with a cocktail of probe substrates metabolized by the five major CYPs (tacrine for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4). To validate the usefulness of the automated and analytical methodologies, IC50 determinations were performed for a series of test compounds known to exhibit inhibition across these five major P450s. Eight compounds (sertraline, disulfuram, ticlopidine fluconazole, fluvoxamine, ketoconazole, miconazole, paroxetine, flunitrazepam) were studied as part of a cocktail assay, and against each CYPs individually. The data showed that the IC50s generated with cocktail incubations did not differ to a great extent from those obtained in the single probe experiments and hence unlikely to significantly influence the predicted clinical DDI risk. In addition the present method offered a significant advantage over some of the existing cocktail analytical methodology in that separation can be achieved with run times as short as 1 min without compromising data integrity. Although numerous studies have been reported to measure CYP inhibition in a cocktail format the need to support growing discovery libraries not only relies on higher throughput assays but quicker analytical run times. The current study reports a miniaturized high-throughput cocktail IC50 assay, in conjunction with a robust, rapid resolution LC-MS/MS end-point offered increased sample throughput without compromising analytical sensitivity or analyte resolution.

  16. Selection and development of representative simple sequence repeat primers and multiplex SSR sets for high throughput automated genotyping in maize

    Institute of Scientific and Technical Information of China (English)

    WANG FengGe; ZHAO JiuRan; DAI JingRui; YI HongMei; KUANG Meng; SUN YanMei; YU XinYan; GUO JingLun; WANG Lu

    2007-01-01

    In the current study, 1900 maize simple sequence repeat (SSR) primers published in MaizeGDB were screened utilizing reference literature, 15 representative Chinese maize inbred lines and 15 Chinese maize hybrids from national regional testing. In total, 500 highly polymorphic primers were identified and used to construct a genetic map. 100 evenly distributed primers, 10 primers per chromosome, were further selected as a set of universal SSR core primers, recommended as preferred primers for general studies. These core primers were then redesigned and used to construct a high throughput multiplex PCR system based on a five-color fluorescence capillary detection system. We report here that two sets of ten-plex PCR combinations have been constructed, each consisting of 10 primers, with one primer per chromosome.

  17. Validation of a novel, fully automated high throughput high-performance liquid chromatographic/tandem mass Spectrometric method for quantification of pantoprazole in human plasma.

    Science.gov (United States)

    Dotsikas, Yannis; Apostolou, Constantinos; Soumelas, Stefanos; Kolocouri, Filomila; Ziaka, Afroditi; Kousoulos, Constantinos; Loukas, Yannis L

    2010-01-01

    An automated high-throughput HPLC/MS/MS method was developed for the quantitative determination of pantoprazole in human plasma. Only 100 microL plasma was placed in 2.2 mL 96 deep-well plates, and both pantoprazole and omeprazole (IS) were extracted from human plasma by liquid-liquid extraction, using diethyl ether-dichloromethane (70:30, v/v) as the organic solvent. Robotic liquid-handling workstations were used for all liquid transfer and solution preparation steps and resulted in a short sample preparation time. After vortexing, centrifugation, and freezing, the supernatant organic solvent was evaporated and reconstituted in a small volume of reconstitution solution. Sample analysis was performed by utilizing the combination of RP-HPLC/MS/MS, with positive-ion electrospray ionization and multiple reaction monitoring detection. The chromatographic run time was set at 1.8 min with a flow rate of 0.6 mL/min on a Nucleosil octylsilyl (C8) analytical column. The method was proven to be sensitive, specific, accurate, and precise for the determination of pantoprazole in human plasma. The method was applied to a bioequivalence study after per os administration of a 40 mg pantoprazole gastric retentive tablet.

  18. Automated vector selection of SIVQ and parallel computing integration MATLAB TM : Innovations supporting large-scale and high-throughput image analysis studies

    Directory of Open Access Journals (Sweden)

    Jerome Cheng

    2011-01-01

    Full Text Available Introduction: Spatially invariant vector quantization (SIVQ is a texture and color-based image matching algorithm that queries the image space through the use of ring vectors. In prior studies, the selection of one or more optimal vectors for a particular feature of interest required a manual process, with the user initially stochastically selecting candidate vectors and subsequently testing them upon other regions of the image to verify the vector′s sensitivity and specificity properties (typically by reviewing a resultant heat map. In carrying out the prior efforts, the SIVQ algorithm was noted to exhibit highly scalable computational properties, where each region of analysis can take place independently of others, making a compelling case for the exploration of its deployment on high-throughput computing platforms, with the hypothesis that such an exercise will result in performance gains that scale linearly with increasing processor count. Methods: An automated process was developed for the selection of optimal ring vectors to serve as the predicate matching operator in defining histopathological features of interest. Briefly, candidate vectors were generated from every possible coordinate origin within a user-defined vector selection area (VSA and subsequently compared against user-identified positive and negative "ground truth" regions on the same image. Each vector from the VSA was assessed for its goodness-of-fit to both the positive and negative areas via the use of the receiver operating characteristic (ROC transfer function, with each assessment resulting in an associated area-under-the-curve (AUC figure of merit. Results: Use of the above-mentioned automated vector selection process was demonstrated in two cases of use: First, to identify malignant colonic epithelium, and second, to identify soft tissue sarcoma. For both examples, a very satisfactory optimized vector was identified, as defined by the AUC metric. Finally, as an

  19. Qgui: A high-throughput interface for automated setup and analysis of free energy calculations and empirical valence bond simulations in biological systems.

    Science.gov (United States)

    Isaksen, Geir Villy; Andberg, Tor Arne Heim; Åqvist, Johan; Brandsdal, Bjørn Olav

    2015-07-01

    Structural information and activity data has increased rapidly for many protein targets during the last decades. In this paper, we present a high-throughput interface (Qgui) for automated free energy and empirical valence bond (EVB) calculations that use molecular dynamics (MD) simulations for conformational sampling. Applications to ligand binding using both the linear interaction energy (LIE) method and the free energy perturbation (FEP) technique are given using the estrogen receptor (ERα) as a model system. Examples of free energy profiles obtained using the EVB method for the rate-limiting step of the enzymatic reaction catalyzed by trypsin are also shown. In addition, we present calculation of high-precision Arrhenius plots to obtain the thermodynamic activation enthalpy and entropy with Qgui from running a large number of EVB simulations.

  20. An Improved Method for Measuring Quantitative Resistance to the Wheat Pathogen Zymoseptoria tritici Using High-Throughput Automated Image Analysis.

    Science.gov (United States)

    Stewart, Ethan L; Hagerty, Christina H; Mikaberidze, Alexey; Mundt, Christopher C; Zhong, Ziming; McDonald, Bruce A

    2016-07-01

    Zymoseptoria tritici causes Septoria tritici blotch (STB) on wheat. An improved method of quantifying STB symptoms was developed based on automated analysis of diseased leaf images made using a flatbed scanner. Naturally infected leaves (n = 949) sampled from fungicide-treated field plots comprising 39 wheat cultivars grown in Switzerland and 9 recombinant inbred lines (RIL) grown in Oregon were included in these analyses. Measures of quantitative resistance were percent leaf area covered by lesions, pycnidia size and gray value, and pycnidia density per leaf and lesion. These measures were obtained automatically with a batch-processing macro utilizing the image-processing software ImageJ. All phenotypes in both locations showed a continuous distribution, as expected for a quantitative trait. The trait distributions at both sites were largely overlapping even though the field and host environments were quite different. Cultivars and RILs could be assigned to two or more statistically different groups for each measured phenotype. Traditional visual assessments of field resistance were highly correlated with quantitative resistance measures based on image analysis for the Oregon RILs. These results show that automated image analysis provides a promising tool for assessing quantitative resistance to Z. tritici under field conditions.

  1. 2dx_automator: implementation of a semiautomatic high-throughput high-resolution cryo-electron crystallography pipeline.

    Science.gov (United States)

    Scherer, Sebastian; Kowal, Julia; Chami, Mohamed; Dandey, Venkata; Arheit, Marcel; Ringler, Philippe; Stahlberg, Henning

    2014-05-01

    The introduction of direct electron detectors (DED) to cryo-electron microscopy has tremendously increased the signal-to-noise ratio (SNR) and quality of the recorded images. We discuss the optimal use of DEDs for cryo-electron crystallography, introduce a new automatic image processing pipeline, and demonstrate the vast improvement in the resolution achieved by the use of both together, especially for highly tilted samples. The new processing pipeline (now included in the software package 2dx) exploits the high SNR and frame readout frequency of DEDs to automatically correct for beam-induced sample movement, and reliably processes individual crystal images without human interaction as data are being acquired. A new graphical user interface (GUI) condenses all information required for quality assessment in one window, allowing the imaging conditions to be verified and adjusted during the data collection session. With this new pipeline an automatically generated unit cell projection map of each recorded 2D crystal is available less than 5 min after the image was recorded. The entire processing procedure yielded a three-dimensional reconstruction of the 2D-crystallized ion-channel membrane protein MloK1 with a much-improved resolution of 5Å in-plane and 7Å in the z-direction, within 2 days of data acquisition and simultaneous processing. The results obtained are superior to those delivered by conventional photographic film-based methodology of the same sample, and demonstrate the importance of drift-correction.

  2. metaBIT, an integrative and automated metagenomic pipeline for analysing microbial profiles from high-throughput sequencing shotgun data.

    Science.gov (United States)

    Louvel, Guillaume; Der Sarkissian, Clio; Hanghøj, Kristian; Orlando, Ludovic

    2016-11-01

    Micro-organisms account for most of the Earth's biodiversity and yet remain largely unknown. The complexity and diversity of microbial communities present in clinical and environmental samples can now be robustly investigated in record times and prices thanks to recent advances in high-throughput DNA sequencing (HTS). Here, we develop metaBIT, an open-source computational pipeline automatizing routine microbial profiling of shotgun HTS data. Customizable by the user at different stringency levels, it performs robust taxonomy-based assignment and relative abundance calculation of microbial taxa, as well as cross-sample statistical analyses of microbial diversity distributions. We demonstrate the versatility of metaBIT within a range of published HTS data sets sampled from the environment (soil and seawater) and the human body (skin and gut), but also from archaeological specimens. We present the diversity of outputs provided by the pipeline for the visualization of microbial profiles (barplots, heatmaps) and for their characterization and comparison (diversity indices, hierarchical clustering and principal coordinates analyses). We show that metaBIT allows an automatic, fast and user-friendly profiling of the microbial DNA present in HTS shotgun data sets. The applications of metaBIT are vast, from monitoring of laboratory errors and contaminations, to the reconstruction of past and present microbiota, and the detection of candidate species, including pathogens.

  3. An algorithmic approach to automated high-throughput identification of disulfide connectivity in proteins using tandem mass spectrometry.

    Science.gov (United States)

    Lee, Timothy; Singh, Rahul; Yen, Ten-Yang; Macher, Bruce

    2007-01-01

    Knowledge of the pattern of disulfide linkages in a protein leads to a better understanding of its tertiary structure and biological function. At the state-of-the-art, liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) can produce spectra of the peptides in a protein that are putatively joined by a disulfide bond. In this setting, efficient algorithms are required for matching the theoretical mass spaces of all possible bonded peptide fragments to the experimentally derived spectra to determine the number and location of the disulfide bonds. The algorithmic solution must also account for issues associated with interpreting experimental data from mass spectrometry, such as noise, isotopic variation, neutral loss, and charge state uncertainty. In this paper, we propose a algorithmic approach to high-throughput disulfide bond identification using data from mass spectrometry, that addresses all the aforementioned issues in a unified framework. The complexity of the proposed solution is of the order of the input spectra. The efficacy and efficiency of the method was validated using experimental data derived from proteins with with diverse disulfide linkage patterns.

  4. Automation and integration of polymerase chain reaction with capillary electrophoresis for high throughput genotyping and disease diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, N.

    1999-02-12

    Genotyping is to detect specific loci in the human genome. These loci provide important information for forensic testing, construction of genetic linkage maps, gene related disease diagnosis and pharmacogenetic research. Genotyping is becoming more and more popular after these loci can be easily amplified by polymerase chain reaction (PCR). Capillary electrophoresis has its unique advantages for DNA analysis due to its fast heat dissipation and ease of automation. Four projects are described in which genotyping is performed by capillary electrophoresis emphasizing different aspects. First, the author demonstrates a principle to determine the genotype based on capillary electrophoresis system. VNTR polymorphism in the human D1S80 locus was studied. Second, the separation of four short tandem repeat (STR) loci vWF, THO1, TPOX and CSF1PO (CTTv) by using poly(ethylene oxide) (PEO) was studied in achieving high resolution and preventing rehybridization of the DNA fragments. Separation under denaturing, non-denaturing conditions and at elevated temperature was discussed. Third, a 250 {micro}m i.d., 365 {micro}m o.d. fused silica capillary was used as the microreactor for PCR. Fourth, direct PCR from blood was studied to simplify the sample preparation for genotyping to minimum.

  5. Automated mini-column solid-phase extraction cleanup for high-throughput analysis of chemical contaminants in foods by low-pressure gas chromatography – tandem mass spectrometry

    Science.gov (United States)

    This study demonstrated the application of an automated high-throughput mini-cartridge solid-phase extraction (mini-SPE) cleanup for the rapid low-pressure gas chromatography – tandem mass spectrometry (LPGC-MS/MS) analysis of pesticides and environmental contaminants in QuEChERS extracts of foods. ...

  6. High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

    Directory of Open Access Journals (Sweden)

    Qureshi Nasib

    2006-05-01

    Full Text Available Abstract Background The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins. Results We used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved Cel

  7. Robofurnace: A semi-automated laboratory chemical vapor deposition system for high-throughput nanomaterial synthesis and process discovery

    OpenAIRE

    Oliver, C. Ryan; Westrick, William; Koehler, Jeremy; Brieland-Shoultz, Anna; Anagnostopoulos-Politis, Ilias; Cruz-Gonzalez, Tizoc; Hart, A. John

    2013-01-01

    Laboratory research and development on new materials, such as nanostructured thin films, often utilizes manual equipment such as tube furnaces due to its relatively low cost and ease of setup. However, these systems can be prone to inconsistent outcomes due to variations in standard operating procedures and limitations in performance such as heating and cooling rates restrict the parameter space that can be explored. Perhaps more importantly, maximization of research throughput and the succes...

  8. High-Throughput Screening of Na(V)1.7 Modulators Using a Giga-Seal Automated Patch Clamp Instrument.

    Science.gov (United States)

    Chambers, Chris; Witton, Ian; Adams, Cathryn; Marrington, Luke; Kammonen, Juha

    2016-03-01

    Voltage-gated sodium (Na(V)) channels have an essential role in the initiation and propagation of action potentials in excitable cells, such as neurons. Of these channels, Na(V)1.7 has been indicated as a key channel for pain sensation. While extensive efforts have gone into discovering novel Na(V)1.7 modulating compounds for the treatment of pain, none has reached the market yet. In the last two years, new compound screening technologies have been introduced, which may speed up the discovery of such compounds. The Sophion Qube(®) is a next-generation 384-well giga-seal automated patch clamp (APC) screening instrument, capable of testing thousands of compounds per day. By combining high-throughput screening and follow-up compound testing on the same APC platform, it should be possible to accelerate the hit-to-lead stage of ion channel drug discovery and help identify the most interesting compounds faster. Following a period of instrument beta-testing, a Na(V)1.7 high-throughput screen was run with two Pfizer plate-based compound subsets. In total, data were generated for 158,000 compounds at a median success rate of 83%, which can be considered high in APC screening. In parallel, IC50 assay validation and protocol optimization was completed with a set of reference compounds to understand how the IC50 potencies generated on the Qube correlate with data generated on the more established Sophion QPatch(®) APC platform. In summary, the results presented here demonstrate that the Qube provides a comparable but much faster approach to study Na(V)1.7 in a robust and reliable APC assay for compound screening.

  9. Maximizing throughput in an automated test system

    Institute of Scientific and Technical Information of China (English)

    朱君

    2007-01-01

    @@ Overview This guide is collection of whitepapers designed to help you develop test systems that lower your cost, increase your test throughput, and can scale with future requirements. This whitepaper provides strategies for maximizing system throughput. To download the complete developers guide (120 pages), visit ni. com/automatedtest.

  10. A high throughput MATLAB program for automated force-curve processing using the AdG polymer model.

    Science.gov (United States)

    O'Connor, Samantha; Gaddis, Rebecca; Anderson, Evan; Camesano, Terri A; Burnham, Nancy A

    2015-02-01

    Research in understanding biofilm formation is dependent on accurate and representative measurements of the steric forces related to brush on bacterial surfaces. A MATLAB program to analyze force curves from an AFM efficiently, accurately, and with minimal user bias has been developed. The analysis is based on a modified version of the Alexander and de Gennes (AdG) polymer model, which is a function of equilibrium polymer brush length, probe radius, temperature, separation distance, and a density variable. Automating the analysis reduces the amount of time required to process 100 force curves from several days to less than 2min. The use of this program to crop and fit force curves to the AdG model will allow researchers to ensure proper processing of large amounts of experimental data and reduce the time required for analysis and comparison of data, thereby enabling higher quality results in a shorter period of time.

  11. Validation of a fully automated high throughput liquid chromatographic/tandem mass spectrometric method for roxithromycin quantification in human plasma. Application to a bioequivalence study.

    Science.gov (United States)

    Kousoulos, Constantinos; Tsatsou, Georgia; Dotsikas, Yannis; Apostolou, Constantinos; Loukas, Yannis L

    2008-05-01

    A fully automated high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the determination of roxithromycin in human plasma. The plasma samples were treated by liquid-liquid extraction (LLE) in 2.2 mL 96-deep-well plates. Roxithromycin and the internal standard clarithromycin were extracted from 100 microL of human plasma by LLE, using methyl t-butyl ether as the organic solvent. All liquid transfer steps were performed automatically using robotic liquid handling workstations. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated and reconstituted. Sample analysis was performed by reversed-phase LC-MS/MS, with positive ion electrospray ionization, using multiple-reaction monitoring. The method had a very short chromatographic run time of 1.6 min. The calibration curve was linear for the range of concentrations 50.0-20.0x10(3) ng mL(-1). The proposed method was fully validated and it was proven to be selective, accurate, precise, reproducible and suitable for the determination of roxithromycin in human plasma. Therefore, it was applied to the rapid and reliable determination of roxithromycin in a bioequivalence study after per os administration of 300 mg tablet formulations of roxithromycin.

  12. Semi-automated high-throughput fluorescent intercalator displacement-based discovery of cytotoxic DNA binding agents from a large compound library.

    Science.gov (United States)

    Glass, Lateca S; Bapat, Aditi; Kelley, Mark R; Georgiadis, Millie M; Long, Eric C

    2010-03-01

    High-throughput fluorescent intercalator displacement (HT-FID) was adapted to the semi-automated screening of a commercial compound library containing 60,000 molecules resulting in the discovery of cytotoxic DNA-targeted agents. Although commercial libraries are routinely screened in drug discovery efforts, the DNA binding potential of the compounds they contain has largely been overlooked. HT-FID led to the rapid identification of a number of compounds for which DNA binding properties were validated through demonstration of concentration-dependent DNA binding and increased thermal melting of A/T- or G/C-rich DNA sequences. Selected compounds were assayed further for cell proliferation inhibition in glioblastoma cells. Seven distinct compounds emerged from this screening procedure that represent structures unknown previously to be capable of targeting DNA leading to cell death. These agents may represent structures worthy of further modification to optimally explore their potential as cytotoxic anti-cancer agents. In addition, the general screening strategy described may find broader impact toward the rapid discovery of DNA targeted agents with biological activity.

  13. Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening

    Science.gov (United States)

    Ebert, Maximilian C. C. J. C.; Mugford, Paul F.; Pelletier, Joelle N.

    2017-01-01

    The Golden Gate strategy entails the use of type IIS restriction enzymes, which cut outside of their recognition sequence. It enables unrestricted design of unique DNA fragments that can be readily and seamlessly recombined. Successfully employed in other synthetic biology applications, we demonstrate its advantageous use to engineer a biocatalyst. Hot-spots for mutations were individuated in three distinct regions of Candida antarctica lipase A (Cal-A), the biocatalyst chosen as a target to demonstrate the versatility of this recombination method. The three corresponding gene segments were subjected to the most appropriate method of mutagenesis (targeted or random). Their straightforward reassembly allowed combining products of different mutagenesis methods in a single round for rapid production of a series of diverse libraries, thus facilitating directed evolution. Screening to improve discrimination of short-chain versus long-chain fatty acid substrates was aided by development of a general, automated method for visual discrimination of the hydrolysis of varied substrates by whole cells. PMID:28178357

  14. C. elegans in high-throughput drug discovery

    OpenAIRE

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

    2013-01-01

    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 ...

  15. metaBIT, an integrative and automated metagenomic pipeline for analysing microbial profiles from high-throughput sequencing shotgun data

    DEFF Research Database (Denmark)

    Louvel, Guillaume; Der Sarkissian, Clio; Hanghøj, Kristian Ebbesen

    2016-01-01

    Micro-organisms account for most of the Earth's biodiversity and yet remain largely unknown. The complexity and diversity of microbial communities present in clinical and environmental samples can now be robustly investigated in record times and prices thanks to recent advances in high......, as well as cross-sample statistical analyses of microbial diversity distributions. We demonstrate the versatility of metaBIT within a range of published HTS data sets sampled from the environment (soil and seawater) and the human body (skin and gut), but also from archaeological specimens. We present...

  16. Integrated automation for continuous high-throughput synthetic chromosome assembly and transformation to identify improved yeast strains for industrial production of peptide sweetener brazzein

    Science.gov (United States)

    Production and recycling of recombinant sweetener peptides in industrial biorefineries involves the evaluation of large numbers of genes and proteins. High-throughput integrated robotic molecular biology platforms that have the capacity to rapidly synthesize, clone, and express heterologous gene ope...

  17. High throughput production of mouse monoclonal antibodies using antigen microarrays

    DEFF Research Database (Denmark)

    De Masi, Federico; Chiarella, P.; Wilhelm, H.;

    2005-01-01

    Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification....... Monoclonal antibodies were raised to different targets in single batch runs of 6-10 wk using multiplexed immunisations, automated fusion and cell-culture, and a novel antigen-coated microarray-screening assay. In a large-scale experiment, where eight mice were immunized with ten antigens each, we generated...

  18. Efficient Management of High-Throughput Screening Libraries with SAVANAH

    DEFF Research Database (Denmark)

    List, Markus; Elnegaard, Marlene Pedersen; Schmidt, Steffen;

    2016-01-01

    High-throughput screening (HTS) has become an indispensable tool for the pharmaceutical industry and for biomedical research. A high degree of automation allows for experiments in the range of a few hundred up to several hundred thousand to be performed in close succession. The basis for such scr...

  19. Fluorescent Approaches to High Throughput Crystallography

    Science.gov (United States)

    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

  20. High-Throughput, Large-Scale SNP Genotyping: Bioinformatics Considerations

    OpenAIRE

    Margetic, Nino

    2004-01-01

    In order to provide a high-throughput, large-scale genotyping facility at the national level we have developed a set of inter-dependent information systems. A combination of commercial, publicly-available and in-house developed tools links a series of data repositories based both on flat files and relational databases providing an almost complete semi-automated pipeline.

  1. Preliminary High-Throughput Metagenome Assembly

    Energy Technology Data Exchange (ETDEWEB)

    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).

  2. Reverse Phase Protein Arrays for High-throughput Toxicity Screening

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    High-throughput screening is extensively applied for identification of drug targets and drug discovery and recently it found entry into toxicity testing. Reverse phase protein arrays (RPPAs) are used widespread for quantification of protein markers. We reasoned that RPPAs also can be utilized...... beneficially in automated high-throughput toxicity testing. An advantage of using RPPAs is that, in addition to the baseline toxicity readout, they allow testing of multiple markers of toxicity, such as inflammatory responses, which do not necessarily cumulate in cell death. We used transfection of si...... a robotic screening platform. Furthermore, we automated sample tracking and data analysis by developing a bundled bioinformatics tool named “MIRACLE”. Automation and RPPA-based viability/toxicity readouts enable rapid testing of large sample numbers, while granting the possibility for flexible consecutive...

  3. Perspective: Data infrastructure for high throughput materials discovery

    Science.gov (United States)

    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.

  4. Efficient Management of High-Throughput Screening Libraries with SAVANAH

    DEFF Research Database (Denmark)

    List, Markus; Elnegaard, Marlene Pedersen; Schmidt, Steffen;

    2017-01-01

    High-throughput screening (HTS) has become an indispensable tool for the pharmaceutical industry and for biomedical research. A high degree of automation allows for experiments in the range of a few hundred up to several hundred thousand to be performed in close succession. The basis for such scr......High-throughput screening (HTS) has become an indispensable tool for the pharmaceutical industry and for biomedical research. A high degree of automation allows for experiments in the range of a few hundred up to several hundred thousand to be performed in close succession. The basis...... for such screens are molecular libraries, that is, microtiter plates with solubilized reagents such as siRNAs, shRNAs, miRNA inhibitors or mimics, and sgRNAs, or small compounds, that is, drugs. These reagents are typically condensed to provide enough material for covering several screens. Library plates thus need...

  5. High-throughput and automated diagnosis of antimicrobial resistance using a cost-effective cellphone-based micro-plate reader

    Science.gov (United States)

    Feng, Steve; Tseng, Derek; di Carlo, Dino; Garner, Omai B.; Ozcan, Aydogan

    2016-12-01

    Routine antimicrobial susceptibility testing (AST) can prevent deaths due to bacteria and reduce the spread of multi-drug-resistance, but cannot be regularly performed in resource-limited-settings due to technological challenges, high-costs, and lack of trained professionals. We demonstrate an automated and cost-effective cellphone-based 96-well microtiter-plate (MTP) reader, capable of performing AST without the need for trained diagnosticians. Our system includes a 3D-printed smartphone attachment that holds and illuminates the MTP using a light-emitting-diode array. An inexpensive optical fiber-array enables the capture of the transmitted light of each well through the smartphone camera. A custom-designed application sends the captured image to a server to automatically determine well-turbidity, with results returned to the smartphone in ~1 minute. We tested this mobile-reader using MTPs prepared with 17 antibiotics targeting Gram-negative bacteria on clinical isolates of Klebsiella pneumoniae, containing highly-resistant antimicrobial profiles. Using 78 patient isolate test-plates, we demonstrated that our mobile-reader meets the FDA-defined AST criteria, with a well-turbidity detection accuracy of 98.21%, minimum-inhibitory-concentration accuracy of 95.12%, and a drug-susceptibility interpretation accuracy of 99.23%, with no very major errors. This mobile-reader could eliminate the need for trained diagnosticians to perform AST, reduce the cost-barrier for routine testing, and assist in spatio-temporal tracking of bacterial resistance.

  6. High Throughput Plasma Water Treatment

    Science.gov (United States)

    Mujovic, Selman; Foster, John

    2016-10-01

    The troublesome emergence of new classes of micro-pollutants, such as pharmaceuticals and endocrine disruptors, poses challenges for conventional water treatment systems. In an effort to address these contaminants and to support water reuse in drought stricken regions, new technologies must be introduced. The interaction of water with plasma rapidly mineralizes organics by inducing advanced oxidation in addition to other chemical, physical and radiative processes. The primary barrier to the implementation of plasma-based water treatment is process volume scale up. In this work, we investigate a potentially scalable, high throughput plasma water reactor that utilizes a packed bed dielectric barrier-like geometry to maximize the plasma-water interface. Here, the water serves as the dielectric medium. High-speed imaging and emission spectroscopy are used to characterize the reactor discharges. Changes in methylene blue concentration and basic water parameters are mapped as a function of plasma treatment time. Experimental results are compared to electrostatic and plasma chemistry computations, which will provide insight into the reactor's operation so that efficiency can be assessed. Supported by NSF (CBET 1336375).

  7. Galaxy High Throughput Genotyping Pipeline for GeneTitan.

    Science.gov (United States)

    Karpenko, Oleksiy; Bahroos, Neil; Chukhman, Morris; Dong, Xiao; Kanabar, Pinal; Arbieva, Zarema; Jackson, Tommie; Hendrickson, William

    2013-01-01

    Latest genotyping solutions allow for rapid testing of more than two million markers in one experiment. Fully automated instruments such as Affymetrix GeneTitan enable processing of large numbers of samples in a truly high-throughput manner. In concert with solutions like Axiom, fully customizable array plates can now utilize automated workflows that can leverage multi-channel instrumentation like the GeneTitan. With the growing size of raw data output, the serial computational architecture of the software, typically distributed by the vendors on turnkey desktop solutions for quality control and genotype calling, becomes legacy rather than an advantage. Advanced software techniques provide power, flexibility, and can be deployed in an HPC environment, but become technically inconvenient for biologists to use. Here we present a pipeline that uses Galaxy as a mechanism to lower the barrier for complex analysis, and increase efficiency by leveraging high-throughput computing.

  8. High-Throughput Analysis of Enzyme Activities

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Guoxin [Iowa State Univ., Ames, IA (United States)

    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

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

    Science.gov (United States)

    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.

  10. BioXTAS RAW, a software program for high-throughput automated small-angle X-ray scattering data reduction and preliminary analysis

    DEFF Research Database (Denmark)

    Nielsen, S.S.; Toft, K.N.; Snakenborg, Detlef

    2009-01-01

    -dimensional data in terms of the indirect Fourier transform using the objective Bayesian approach to obtain the pair-distance distribution function, PDDF, and is thereby a free and open-source alternative to existing PDDF estimation software. Apart from the TIFF input format, the program also accepts ASCII......A fully open source software program for automated two-dimensional and one-dimensional data reduction and preliminary analysis of isotropic small-angle X-ray scattering (SAXS) data is presented. The program is freely distributed, following the open-source philosophy, and does not rely on any...... commercial software packages. BioXTAS RAW is a fully automated program that, via an online feature, reads raw two-dimensional SAXS detector output files and processes and plots data as the data files are created during measurement sessions. The software handles all steps in the data reduction. This includes...

  11. Automated microfluidic sample-preparation platform for high-throughput structural investigation of proteins by small-angle X-ray scattering

    DEFF Research Database (Denmark)

    Lafleur, Josiane P.; Snakenborg, Detlef; Nielsen, Søren Skou

    2011-01-01

    A new microfluidic sample-preparation system is presented for the structural investigation of proteins using small-angle X-ray scattering (SAXS) at synchrotrons. The system includes hardware and software features for precise fluidic control, sample mixing by diffusion, automated X-ray exposure...... control, UV absorbance measurements and automated data analysis. As little as 15 l of sample is required to perform a complete analysis cycle, including sample mixing, SAXS measurement, continuous UV absorbance measurements, and cleaning of the channels and X-ray cell with buffer. The complete analysis...... cycle can be performed in less than 3 min. Bovine serum albumin was used as a model protein to characterize the mixing efficiency and sample consumption of the system. The N2 fragment of an adaptor protein (p120-RasGAP) was used to demonstrate how the device can be used to survey the structural space...

  12. High-throughput mass spectrometric cytochrome P450 inhibition screening.

    Science.gov (United States)

    Lim, Kheng B; Ozbal, Can C; Kassel, Daniel B

    2013-01-01

    We describe here a high-throughput assay to support rapid evaluation of drug discovery compounds for possible drug-drug interaction (DDI). Each compound is evaluated for its DDI potential by incubating over a range of eight concentrations and against a panel of six cytochrome P450 (CYP) enzymes: 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4. The method utilizes automated liquid handling for sample preparation, and online solid-phase extraction/tandem mass spectrometry (SPE/MS/MS) for sample analyses. The system is capable of generating two 96-well assay plates in 30 min, and completes the data acquisition and analysis of both plates in about 30 min. Many laboratories that perform the CYP inhibition screening automate only part of the processes leaving a throughput bottleneck within the workflow. The protocols described in this chapter are aimed to streamline the entire process from assay to data acquisition and processing by incorporating automation and utilizing high-precision instrument to maximize throughput and minimize bottleneck.

  13. High-throughput screening in the C. elegans nervous system.

    Science.gov (United States)

    Kinser, Holly E; Pincus, Zachary

    2016-06-03

    The nematode Caenorhabditis elegans is widely used as a model organism in the field of neurobiology. The wiring of the C. elegans nervous system has been entirely mapped, and the animal's optical transparency allows for in vivo observation of neuronal activity. The nematode is also small in size, self-fertilizing, and inexpensive to cultivate and maintain, greatly lending to its utility as a whole-animal model for high-throughput screening (HTS) in the nervous system. However, the use of this organism in large-scale screens presents unique technical challenges, including reversible immobilization of the animal, parallel single-animal culture and containment, automation of laser surgery, and high-throughput image acquisition and phenotyping. These obstacles require significant modification of existing techniques and the creation of new C. elegans-based HTS platforms. In this review, we outline these challenges in detail and survey the novel technologies and methods that have been developed to address them.

  14. Spotsizer: High-throughput quantitative analysis of microbial growth

    Science.gov (United States)

    Jeffares, Daniel C.; Arzhaeva, Yulia; Bähler, Jürg

    2017-01-01

    Microbial colony growth can serve as a useful readout in assays for studying complex genetic interactions or the effects of chemical compounds. Although computational tools for acquiring quantitative measurements of microbial colonies have been developed, their utility can be compromised by inflexible input image requirements, non-trivial installation procedures, or complicated operation. Here, we present the Spotsizer software tool for automated colony size measurements in images of robotically arrayed microbial colonies. Spotsizer features a convenient graphical user interface (GUI), has both single-image and batch-processing capabilities, and works with multiple input image formats and different colony grid types. We demonstrate how Spotsizer can be used for high-throughput quantitative analysis of fission yeast growth. The user-friendly Spotsizer tool provides rapid, accurate, and robust quantitative analyses of microbial growth in a high-throughput format. Spotsizer is freely available at https://data.csiro.au/dap/landingpage?pid=csiro:15330 under a proprietary CSIRO license. PMID:27712582

  15. Data Management for High-Throughput Genomics

    CERN Document Server

    Roehm, Uwe

    2009-01-01

    Today's sequencing technology allows sequencing an individual genome within a few weeks for a fraction of the costs of the original Human Genome project. Genomics labs are faced with dozens of TB of data per week that have to be automatically processed and made available to scientists for further analysis. This paper explores the potential and the limitations of using relational database systems as the data processing platform for high-throughput genomics. In particular, we are interested in the storage management for high-throughput sequence data and in leveraging SQL and user-defined functions for data analysis inside a database system. We give an overview of a database design for high-throughput genomics, how we used a SQL Server database in some unconventional ways to prototype this scenario, and we will discuss some initial findings about the scalability and performance of such a more database-centric approach.

  16. High-throughput computing in the sciences.

    Science.gov (United States)

    Morgan, Mark; Grimshaw, Andrew

    2009-01-01

    While it is true that the modern computer is many orders of magnitude faster than that of yesteryear; this tremendous growth in CPU clock rates is now over. Unfortunately, however, the growth in demand for computational power has not abated; whereas researchers a decade ago could simply wait for computers to get faster, today the only solution to the growing need for more powerful computational resource lies in the exploitation of parallelism. Software parallelization falls generally into two broad categories--"true parallel" and high-throughput computing. This chapter focuses on the latter of these two types of parallelism. With high-throughput computing, users can run many copies of their software at the same time across many different computers. This technique for achieving parallelism is powerful in its ability to provide high degrees of parallelism, yet simple in its conceptual implementation. This chapter covers various patterns of high-throughput computing usage and the skills and techniques necessary to take full advantage of them. By utilizing numerous examples and sample codes and scripts, we hope to provide the reader not only with a deeper understanding of the principles behind high-throughput computing, but also with a set of tools and references that will prove invaluable as she explores software parallelism with her own software applications and research.

  17. INTRODUCTION OF THE HIGH THROUGHPUT SCREENING SYSTEM

    Institute of Scientific and Technical Information of China (English)

    李元

    2001-01-01

    In this article, we introduce the system of high throughput screening (HTS). Its role in new drug study and current development is described. The relationship between research achievements of genome study and new type screening model of new drugs is emphasized. The personal opinions of current problems about HTS study in China are raised.

  18. INTRODUCTION OF THE HIGH THROUGHPUT SCREENING SYSTEM

    Institute of Scientific and Technical Information of China (English)

    李元

    2001-01-01

    In this article, we introduce the system of high throughput screening (HTS). Its role in new drug study and current development is described. The relationship between research achievements of genome study and new type screening model of new drugs is emphasized. The personal opinions of current problems about HTS study in China are raised.``

  19. High Throughput Analysis of Photocatalytic Water Purification

    NARCIS (Netherlands)

    Romao, Joana; Barata, David; Habibovic, Pamela; Mul, Guido; Baltrusaitis, Jonas

    2014-01-01

    We present a novel high throughput photocatalyst efficiency assessment method based on 96-well microplates and UV-Vis spectroscopy. We demonstrate the reproducibility of the method using methyl orange (MO) decomposition, and compare kinetic data obtained with those provided in the literature for lar

  20. High-throughput diffusion multiples

    Directory of Open Access Journals (Sweden)

    J.-C. Zhao

    2005-10-01

    Full Text Available A diffusion multiple is an assembly of three or more different metal blocks, in intimate interfacial contact, subjected to high temperature to allow thermal interdiffusion to create solid-solution compositions and intermetallic compounds. Using microscale probes, composition-structure-phase-property relationships can be established with an efficiency orders of magnitude higher than conventional one-composition-at-a-time practice. For structural materials, such relationships include phase diagrams, diffusion coefficients, precipitation kinetics, solution strengthening effects, and precipitation strengthening effects. Many microscale probes can also be used to study several materials phenomena. For instance, microscale thermal conductivity measurements can be used to study order-disordering transformation, site preference in intermetallic compounds, solid-solution effect on conductivity, and compositional point defect propensity. This article will use a few examples to illustrate the capabilities and developmental needs of this approach.

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

    Science.gov (United States)

    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.

  2. COMPUTER APPROACHES TO WHEAT HIGH-THROUGHPUT PHENOTYPING

    Directory of Open Access Journals (Sweden)

    Afonnikov D.

    2012-08-01

    Full Text Available The growing need for rapid and accurate approaches for large-scale assessment of phenotypic characters in plants becomes more and more obvious in the studies looking into relationships between genotype and phenotype. This need is due to the advent of high throughput methods for analysis of genomes. Nowadays, any genetic experiment involves data on thousands and dozens of thousands of plants. Traditional ways of assessing most phenotypic characteristics (those with reliance on the eye, the touch, the ruler are little effective on samples of such sizes. Modern approaches seek to take advantage of automated phenotyping, which warrants a much more rapid data acquisition, higher accuracy of the assessment of phenotypic features, measurement of new parameters of these features and exclusion of human subjectivity from the process. Additionally, automation allows measurement data to be rapidly loaded into computer databases, which reduces data processing time.In this work, we present the WheatPGE information system designed to solve the problem of integration of genotypic and phenotypic data and parameters of the environment, as well as to analyze the relationships between the genotype and phenotype in wheat. The system is used to consolidate miscellaneous data on a plant for storing and processing various morphological traits and genotypes of wheat plants as well as data on various environmental factors. The system is available at www.wheatdb.org. Its potential in genetic experiments has been demonstrated in high-throughput phenotyping of wheat leaf pubescence.

  3. A high-throughput cidality screen for Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Parvinder Kaur

    Full Text Available Exposure to Mycobacterium tuberculosis (Mtb aerosols is a major threat to tuberculosis (TB researchers, even in bio-safety level-3 (BSL-3 facilities. Automation and high-throughput screens (HTS in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well 'spot-assay' for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets. The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808. An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process.

  4. High throughput-per-footprint inertial focusing.

    Science.gov (United States)

    Ciftlik, Ata Tuna; Ettori, Maxime; Gijs, Martin A M

    2013-08-26

    Matching the scale of microfluidic flow systems with that of microelectronic chips for realizing monolithically integrated systems still needs to be accomplished. However, this is appealing only if such re-scaling does not compromise the fluidic throughput. This is related to the fact that the cost of microelectronic circuits primarily depends on the layout footprint, while the performance of many microfluidic systems, like flow cytometers, is measured by the throughput. The simple operation of inertial particle focusing makes it a promising technique for use in such integrated flow cytometer applications, however, microfluidic footprints demonstrated so far preclude monolithic integration. Here, the scaling limits of throughput-per-footprint (TPFP) in using inertial focusing are explored by studying the interplay between theory, the effect of channel Reynolds numbers up to 1500 on focusing, the entry length for the laminar flow to develop, and pressure resistance of the microchannels. Inertial particle focusing is demonstrated with a TPFP up to 0.3 L/(min cm²) in high aspect-ratio rectangular microfluidic channels that are readily fabricated with a post-CMOS integratable process, suggesting at least a 100-fold improvement compared to previously demonstrated techniques. Not only can this be an enabling technology for realizing cost-effective monolithically integrated flow cytometry devices, but the methodology represented here can also open perspectives for miniaturization of many biomedical microfluidic applications requiring monolithic integration with microelectronics without compromising the throughput.

  5. High Throughput Neuro-Imaging Informatics

    Directory of Open Access Journals (Sweden)

    Michael I Miller

    2013-12-01

    Full Text Available 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 representations index containing O(E3-E4 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 non-image information for diagnosis and prognosis.

  6. HIGH THROUGHPUT DRILLING OF TITANIUM ALLOYS

    Institute of Scientific and Technical Information of China (English)

    LI Rui; SHIH Albert Jau-Min

    2007-01-01

    The experiments of high throughput drilling of Ti-6Al-4V at 183 m/min cutting speed and 156 mm3/s material removal rate using a 4 mm diameter WC-Co spiral point drill are conducted. At this material removal rate, it took only 0.57 s to drill a hole in a 6.35 mm thick Ti plate. Supplying the cutting fluid via through-the-drill holes and the balance of cutting speed and feed have proven to be critical for drill life. An inverse heat transfer model is developed to predict the heat flux and the drill temperature distribution in drilling. A three-dimensional finite element modeling of drilling is conducted to predict the thrust force and torque. Experimental result demonstrates that, using proper machining process parameters, tool geometry, and fine-grained WC-Co tool material, the high throughput machining of Ti alloy is technically feasible.

  7. High-throughput sample preparation for the quantitation of acephate, methamidophos, omethoate, dimethoate, ethylenethiourea, and propylenethiourea in human urine using 96-well-plate automated extraction and high-performance liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Jayatilaka, Nayana K; Angela Montesano, M; Whitehead, Ralph D; Schloth, Sara J; Needham, Larry L; Barr, Dana Boyd

    2011-07-01

    Acephate, methamidophos, o-methoate, and dimethoate are organophosphorus pesticides, and ethylenethiouria and propylenethiourea are two metabolites from the bisdithiocarbamate fungicide family. They are some of the most widely used pesticides and fungicides in agriculture both domestically and abroad. The existing high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) method for the measurement of these compounds in human urine was improved by using a 96-well plate format sample preparation; the use of HPLC-MS/MS was comparable with a concentration range of 0.125 to 50 ng/ml. Deuterium-labeled acephate, ethylenethiouria, and methamidophos were used as internal standards. The sample preparation procedure, in the 96-well format with a 0.8-ml urine sample size, uses lyophilization of samples, followed by extraction with dichloromethane. The analytes were chromatographed on a Zorbax SB-C3 (4.6 × 150 mm, 5.0-μm) column with gradient elution by using 0.1% formic acid in aqueous solution (solvent A) and 0.1% formic acid in methanol (solvent B) mobile phase at a flow rate of 1 ml/min. Quantitative analysis was performed by atmospheric pressure chemical ionization source in positive ion mode using multiple-reaction monitoring of the precursor-to-product ion pairs for the analytes on a TSQ Quantum Ultra HPLC-MS/MS. Repeated analyses of urine samples spiked with high (15 ng/ml), medium (5 ng/ml), and low (1 ng/ml) concentrations of the analytes gave relative SDs of <13%. The limits of detection were in the range of 0.004-0.01 ng/ml. The method also has high accuracy, high precision, and excellent extraction recovery. Furthermore, the improved sample preparation method decreased the cost and labor required while effectively doubling the analytic throughput with minimal matrix effect.

  8. Field high-throughput phenotyping: the new crop breeding frontier.

    Science.gov (United States)

    Araus, José Luis; Cairns, Jill E

    2014-01-01

    Constraints in field phenotyping capability limit our ability to dissect the genetics of quantitative traits, particularly those related to yield and stress tolerance (e.g., yield potential as well as increased drought, heat tolerance, and nutrient efficiency, etc.). The development of effective field-based high-throughput phenotyping platforms (HTPPs) remains a bottleneck for future breeding advances. However, progress in sensors, aeronautics, and high-performance computing are paving the way. Here, we review recent advances in field HTPPs, which should combine at an affordable cost, high capacity for data recording, scoring and processing, and non-invasive remote sensing methods, together with automated environmental data collection. Laboratory analyses of key plant parts may complement direct phenotyping under field conditions. Improvements in user-friendly data management together with a more powerful interpretation of results should increase the use of field HTPPs, therefore increasing the efficiency of crop genetic improvement to meet the needs of future generations.

  9. Warehouse automation

    OpenAIRE

    Pogačnik, Jure

    2017-01-01

    An automated high bay warehouse is commonly used for storing large number of material with a high throughput. In an automated warehouse pallet movements are mainly performed by a number of automated devices like conveyors systems, trolleys, and stacker cranes. From the introduction of the material to the automated warehouse system to its dispatch the system requires no operator input or intervention since all material movements are done automatically. This allows the automated warehouse to op...

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

    Science.gov (United States)

    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.

  11. High-throughput protein analysis integrating bioinformatics and experimental assays.

    Science.gov (United States)

    del Val, Coral; Mehrle, Alexander; Falkenhahn, Mechthild; Seiler, Markus; Glatting, Karl-Heinz; Poustka, Annemarie; Suhai, Sandor; Wiemann, Stefan

    2004-01-01

    The wealth of transcript information that has been made publicly available in recent years requires the development of high-throughput functional genomics and proteomics approaches for its analysis. Such approaches need suitable data integration procedures and a high level of automation in order to gain maximum benefit from the results generated. We have designed an automatic pipeline to analyse annotated open reading frames (ORFs) stemming from full-length cDNAs produced mainly by the German cDNA Consortium. The ORFs are cloned into expression vectors for use in large-scale assays such as the determination of subcellular protein localization or kinase reaction specificity. Additionally, all identified ORFs undergo exhaustive bioinformatic analysis such as similarity searches, protein domain architecture determination and prediction of physicochemical characteristics and secondary structure, using a wide variety of bioinformatic methods in combination with the most up-to-date public databases (e.g. PRINTS, BLOCKS, INTERPRO, PROSITE SWISSPROT). Data from experimental results and from the bioinformatic analysis are integrated and stored in a relational database (MS SQL-Server), which makes it possible for researchers to find answers to biological questions easily, thereby speeding up the selection of targets for further analysis. The designed pipeline constitutes a new automatic approach to obtaining and administrating relevant biological data from high-throughput investigations of cDNAs in order to systematically identify and characterize novel genes, as well as to comprehensively describe the function of the encoded proteins.

  12. High-throughput methods for electron crystallography.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Wu, Henry; Mayeshiba, Tam; Morgan, Dane

    2016-07-01

    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.

  14. New technologies for ultra-high throughput genotyping in plants.

    Science.gov (United States)

    Appleby, Nikki; Edwards, David; Batley, Jacqueline

    2009-01-01

    Molecular genetic markers represent one of the most powerful tools for the analysis of plant genomes and the association of heritable traits with underlying genetic variation. Molecular marker technology has developed rapidly over the last decade, with the development of high-throughput genotyping methods. Two forms of sequence-based marker, simple sequence repeats (SSRs), also known as microsatellites and single nucleotide polymorphisms (SNPs) now predominate applications in modern plant genetic analysis, along the anonymous marker systems such as amplified fragment length polymorphisms (AFLPs) and diversity array technology (DArT). The reducing cost of DNA sequencing and increasing availability of large sequence data sets permits the mining of this data for large numbers of SSRs and SNPs. These may then be used in applications such as genetic linkage analysis and trait mapping, diversity analysis, association studies and marker-assisted selection. Here, we describe automated methods for the discovery of molecular markers and new technologies for high-throughput, low-cost molecular marker genotyping. Genotyping examples include multiplexing of SSRs using Multiplex-Ready marker technology (MRT); DArT genotyping; SNP genotyping using the Invader assay, the single base extension (SBE), oligonucleotide ligation assay (OLA) SNPlex system, and Illumina GoldenGate and Infinium methods.

  15. Organic chemistry. Nanomole-scale high-throughput chemistry for the synthesis of complex molecules.

    Science.gov (United States)

    Buitrago Santanilla, Alexander; Regalado, Erik L; Pereira, Tony; Shevlin, Michael; Bateman, Kevin; Campeau, Louis-Charles; Schneeweis, Jonathan; Berritt, Simon; Shi, Zhi-Cai; Nantermet, Philippe; Liu, Yong; Helmy, Roy; Welch, Christopher J; Vachal, Petr; Davies, Ian W; Cernak, Tim; Dreher, Spencer D

    2015-01-02

    At the forefront of new synthetic endeavors, such as drug discovery or natural product synthesis, large quantities of material are rarely available and timelines are tight. A miniaturized automation platform enabling high-throughput experimentation for synthetic route scouting to identify conditions for preparative reaction scale-up would be a transformative advance. Because automated, miniaturized chemistry is difficult to carry out in the presence of solids or volatile organic solvents, most of the synthetic "toolkit" cannot be readily miniaturized. Using palladium-catalyzed cross-coupling reactions as a test case, we developed automation-friendly reactions to run in dimethyl sulfoxide at room temperature. This advance enabled us to couple the robotics used in biotechnology with emerging mass spectrometry-based high-throughput analysis techniques. More than 1500 chemistry experiments were carried out in less than a day, using as little as 0.02 milligrams of material per reaction.

  16. High throughput screening of physicochemical properties and in vitro ADME profiling in drug discovery.

    Science.gov (United States)

    Wan, Hong; Holmén, Anders G

    2009-03-01

    Current advances of new technologies with robotic automated assays combined with highly selective and sensitive LC-MS enable high-speed screening of lead series libraries in many in vitro assays. In this review, we summarize state of the art high throughput assays for screening of key physicochemical properties such as solubility, lipophilicity, pKa, drug-plasma protein binding and brain tissue binding as well as in vitro ADME profiling. We discuss two primary approaches for high throughput screening of solubility, i.e. an automated 96-well plate assay integrated with LC-MS and a rapid multi-wavelength UV plate reader. We address the advantages of newly developed miniaturized techniques for high throughput pKa screening by capillary electrophoresis combined with mass spectrometry (CE-MS) with automated data analysis flow. Several new lipophilicity approaches other than octanol-water partitioning are critically reviewed, including rapid liquid chromatographic retention based approach, immobilized artificial membrane (IAM) partitioning and liposome, and potential microemulsion electrokinetic chromatography (MEEKC) for accurate screening of LogP. We highlight the sample pooling (namely cassette dosing, all-in-one, cocktail) as an efficient approach for high throughput screening of physicochemical properties and in vitro ADME profiling with emphasis on the benefit of on-line quality control. This cassette dosing approach has been widely adapted in drug discovery for rapid screening of in vivo pharmacokinetic parameters with significantly increased capacity and dramatically reduced animal usage.

  17. High-throughput rod-induced electrospinning

    Science.gov (United States)

    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.

  18. Achieving High Data Throughput in Research Networks

    Institute of Scientific and Technical Information of China (English)

    WarrenMatthews; LesCottrell

    2001-01-01

    After less than a year of operation ,the BaBar experiment at SLAC has collected almost 100 million particle collision events in a database approaching 165TB.Around 20 TB of data has been exported via the Internet to the BaBar regional center at IN2P3 in Lyon,France,and around 40TB of simulated data has been imported from the Lawrence Livermore National Laboratory(LLNL),BaBar Collaborators plan to double data collection each year and export a third of the data to IN2P3.So within a few years the SLAC OC3 (155Mbps) connection will be fully utilized by file transfer to France alone.Upgrades to infrastructure is essential and detailed understanding of performance issues and the requirements for reliable high throughput transfers is critical.In this talk results from active and passive monitoring and direct measurements of throughput will be reviewed.Methods for achieving the ambitious requirements will be discussed.

  19. High Throughput Screening for Neurodegeneration and Complex Disease Phenotypes

    OpenAIRE

    Varma, Hemant; Lo, Donald C.; Stockwell, Brent R.

    2008-01-01

    High throughput screening (HTS) for complex diseases is challenging. This stems from the fact that complex phenotypes are difficult to adapt to rapid, high throughput assays. We describe the recent development of high throughput and high-content screens (HCS) for neurodegenerative diseases, with a focus on inherited neurodegenerative disorders, such as Huntington's disease. We describe, among others, HTS assays based on protein aggregation, neuronal death, caspase activation and mutant protei...

  20. A high-throughput neutron spectrometer

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  2. Automated metrology and NDE measurements for increased throughput in aerospace component manufacture

    Science.gov (United States)

    MacLeod, Charles N.; Pierce, S. Gareth; Morozov, Maxim; Summan, Rahul; Dobie, Gordon; McCubbin, Paul; McCubbin, Coreen; Dearie, Scott; Munro, Gavin

    2015-03-01

    Composite materials, particularly Carbon-Fibre-Reinforced Polymer (CFRP), find extensive use in construction of modern airframe structures. Quality and conformance checks can be a serious limitation on production throughput in aerospace manufacturing. Traditionally Non-Destructive Evaluation (NDE) and metrology measurements are undertaken at different stages of a product manufacture cycle using specific dedicated equipment and personnel. However, since both processes involve direct interaction with the component's surface, an opportunity exists to combine these to potentially reduce overall cycle time. In addition when considering moves towards automation of both inspection processes, it is clear that measured metrology data is an essential input parameter to the automated NDE workflow. The authors present the findings of a proof of concept combined sub-scale NDE and Metrology demonstrator cell for aerospace components. Permitting a maximum part area size of 3 × 1 m2, KUKA KR5 6 degree of freedom robotic manipulators were utilised to deploy two inspection payloads. Firstly automated non-contact photogrammetric metrology measurement was employed to inspect the structure for conformance of dimension in relation to reference designs (available from CAD). Secondly automated phased array technology was deployed to inspect and produce ultrasonic thickness mapping of components of nominal 20mm thickness. Parameters such as overall cycle time, part dimensional accuracy, robotic path accuracy and data registration are assessed in the paper to highlight both the current state of the art performance available and the future direction of required research focus.

  3. metAlignID: a high-throughput software tool set for automated detection of trace level contaminants in comprehensive LECO two-dimensional gas chromatography time-of-flight mass spectrometry data.

    Science.gov (United States)

    Lommen, Arjen; van der Kamp, Henk J; Kools, Harrie J; van der Lee, Martijn K; van der Weg, Guido; Mol, Hans G J

    2012-11-09

    A new alternative data processing tool set, metAlignID, is developed for automated pre-processing and library-based identification and concentration estimation of target compounds after analysis by comprehensive two-dimensional gas chromatography with mass spectrometric detection. The tool set has been developed for and tested on LECO data. The software is developed to run multi-threaded (one thread per processor core) on a standard PC (personal computer) under different operating systems and is as such capable of processing multiple data sets simultaneously. Raw data files are converted into netCDF (network Common Data Form) format using a fast conversion tool. They are then preprocessed using previously developed algorithms originating from metAlign software. Next, the resulting reduced data files are searched against a user-composed library (derived from user or commercial NIST-compatible libraries) (NIST=National Institute of Standards and Technology) and the identified compounds, including an indicative concentration, are reported in Excel format. Data can be processed batch wise. The overall time needed for conversion together with processing and searching of 30 raw data sets for 560 compounds is routinely within an hour. The screening performance is evaluated for detection of pesticides and contaminants in raw data obtained after analysis of soil and plant samples. Results are compared to the existing data-handling routine based on proprietary software (LECO, ChromaTOF). The developed software tool set, which is freely downloadable at www.metalign.nl, greatly accelerates data-analysis and offers more options for fine-tuning automated identification toward specific application needs. The quality of the results obtained is slightly better than the standard processing and also adds a quantitative estimate. The software tool set in combination with two-dimensional gas chromatography coupled to time-of-flight mass spectrometry shows great potential as a highly-automated

  4. Continuous-flow high pressure hydrogenation reactor for optimization and high-throughput synthesis.

    Science.gov (United States)

    Jones, Richard V; Godorhazy, Lajos; Varga, Norbert; Szalay, Daniel; Urge, Laszlo; Darvas, Ferenc

    2006-01-01

    This paper reports on a novel continuous-flow hydrogenation reactor and its integration with a liquid handler to generate a fully automated high-throughput hydrogenation system for library synthesis. The reactor, named the H-Cube, combines endogenous hydrogen generation from the electrolysis of water with a continuous flow-through system. The system makes significant advances over current batch hydrogenation reactors in terms of safety, reaction validation efficiency, and rates of reaction. The hydrogenation process is described along with a detailed description of the device's main parts. The reduction of a series of functional groups, varying in difficulty up to 70 degrees C and 70 bar are also described. The paper concludes with the integration of the device into an automated liquid handler followed by the reduction of a nitro compound in a high throughput manner. The system is fully automated and can conduct 5 reactions in the time it takes to perform and workup one reaction manually on a standard batch reactor.

  5. Antileishmanial High-Throughput Drug Screening Reveals Drug Candidates with New Scaffolds

    OpenAIRE

    Siqueira-Neto, Jair L; Ok-Ryul Song; Hyunrim Oh; Jeong-Hun Sohn; Gyongseon Yang; Jiyoun Nam; Jiyeon Jang; Jonathan Cechetto; Chang Bok Lee; Seunghyun Moon; Auguste Genovesio; Eric Chatelain; Thierry Christophe; Freitas-Junior, Lucio H.

    2010-01-01

    International audience; Drugs currently available for leishmaniasis treatment often show parasite resistance, highly toxic side effects and prohibitive costs commonly incompatible with patients from the tropical endemic countries. In this sense, there is an urgent need for new drugs as a treatment solution for this neglected disease. Here we show the development and implementation of an automated high-throughput viability screening assay for the discovery of new drugs against Leishmania. Assa...

  6. High Throughput Direct Detection Doppler Lidar Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Lite Cycles, Inc. (LCI) proposes to develop a direct-detection Doppler lidar (D3L) technology called ELITE that improves the system optical throughput by more than...

  7. A high-throughput chemically induced inflammation assay in zebrafish

    Directory of Open Access Journals (Sweden)

    Liebel Urban

    2010-12-01

    Full Text Available Abstract Background Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening. Results Here we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction. Conclusions This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay. See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.

  8. High Volume Throughput Computing: Identifying and Characterizing Throughput Oriented Workloads in Data Centers

    CERN Document Server

    Zhan, Jianfeng; Sun, Ninghui; Wang, Lei; Jia, Zhen; Luo, Chunjie

    2012-01-01

    For the first time, this paper systematically identifies three categories of throughput oriented workloads in data centers: services, data processing applications, and interactive real-time applications, whose targets are to increase the volume of throughput in terms of processed requests or data, or supported maximum number of simultaneous subscribers, respectively, and we coins a new term high volume throughput computing (in short HVC) to describe those workloads and data center systems designed for them. We characterize and compare HVC with other computing paradigms, e.g., high throughput computing, warehouse-scale computing, and cloud computing, in terms of levels, workloads, metrics, coupling degree, data scales, and number of jobs or service instances. We also preliminarily report our ongoing work on the metrics and benchmarks for HVC systems, which is the foundation of designing innovative data center systems for HVC workloads.

  9. Ultraspecific probes for high throughput HLA typing

    Directory of Open Access Journals (Sweden)

    Eggers Rick

    2009-02-01

    Full Text Available Abstract Background The variations within an individual's HLA (Human Leukocyte Antigen genes have been linked to many immunological events, e.g. susceptibility to disease, response to vaccines, and the success of blood, tissue, and organ transplants. Although the microarray format has the potential to achieve high-resolution typing, this has yet to be attained due to inefficiencies of current probe design strategies. Results We present a novel three-step approach for the design of high-throughput microarray assays for HLA typing. This approach first selects sequences containing the SNPs present in all alleles of the locus of interest and next calculates the number of base changes necessary to convert a candidate probe sequences to the closest subsequence within the set of sequences that are likely to be present in the sample including the remainder of the human genome in order to identify those candidate probes which are "ultraspecific" for the allele of interest. Due to the high specificity of these sequences, it is possible that preliminary steps such as PCR amplification are no longer necessary. Lastly, the minimum number of these ultraspecific probes is selected such that the highest resolution typing can be achieved for the minimal cost of production. As an example, an array was designed and in silico results were obtained for typing of the HLA-B locus. Conclusion The assay presented here provides a higher resolution than has previously been developed and includes more alleles than previously considered. Based upon the in silico and preliminary experimental results, we believe that the proposed approach can be readily applied to any highly polymorphic gene system.

  10. Orthogonal NGS for High Throughput Clinical Diagnostics.

    Science.gov (United States)

    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.

  11. High-throughput data pipelines for metabolic flux analysis in plants.

    Science.gov (United States)

    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.

  12. Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)

    Energy Technology Data Exchange (ETDEWEB)

    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 BSL-4 High-Throughput Screen Identifies Sulfonamide Inhibitors of Nipah Virus

    OpenAIRE

    2014-01-01

    Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-...

  14. Computational chemistry, data mining, high-throughput synthesis and screening - informatics and integration in drug discovery

    OpenAIRE

    Manly, Charles J.

    2001-01-01

    Drug discovery today includes considerable focus of laboratory automation and other resources on both combinatorial chemistry and high-throughput screening, and computational chemistry has been a part of pharmaceutical research for many years. The real benefit of these technologies is beyond the exploitation of each individually. Only recently have significant efforts focused on effectively integrating these and other discovery disciplines to realize their larger potential. This technical not...

  15. High Throughput Screen for Novel Antimicrobials using a Whole Animal Infection Model

    OpenAIRE

    Moy, Terence I.; Annie L Conery; Larkins-Ford, Jonah; Wu, Gang; Mazitschek, Ralph; Casadei, Gabriele; Lewis, Kim; Carpenter, Anne E.; Ausubel, Frederick M.

    2009-01-01

    The nematode Caenorhabditis elegans is a unique whole animal model system for identifying small molecules with in vivo anti-infective properties. C. elegans can be infected with a broad range of human pathogens, including Enterococcus faecalis, an important human nosocomial pathogen with a mortality rate of up to 37% that is increasingly acquiring resistance to antibiotics. Here, we describe an automated, high throughput screen of 37,200 compounds and natural product extracts for those that e...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. High throughput screening for anti-Trypanosoma cruzi drug discovery.

    Science.gov (United States)

    Alonso-Padilla, Julio; Rodríguez, Ana

    2014-12-01

    The discovery of new therapeutic options against Trypanosoma cruzi, the causative agent of Chagas disease, stands as a fundamental need. Currently, there are only two drugs available to treat this neglected disease, which represents a major public health problem in Latin America. Both available therapies, benznidazole and nifurtimox, have significant toxic side effects and their efficacy against the life-threatening symptomatic chronic stage of the disease is variable. Thus, there is an urgent need for new, improved anti-T. cruzi drugs. With the objective to reliably accelerate the drug discovery process against Chagas disease, several advances have been made in the last few years. Availability of engineered reporter gene expressing parasites triggered the development of phenotypic in vitro assays suitable for high throughput screening (HTS) as well as the establishment of new in vivo protocols that allow faster experimental outcomes. Recently, automated high content microscopy approaches have also been used to identify new parasitic inhibitors. These in vitro and in vivo early drug discovery approaches, which hopefully will contribute to bring better anti-T. cruzi drug entities in the near future, are reviewed here.

  18. High throughput screening for anti-Trypanosoma cruzi drug discovery.

    Directory of Open Access Journals (Sweden)

    Julio Alonso-Padilla

    2014-12-01

    Full Text Available The discovery of new therapeutic options against Trypanosoma cruzi, the causative agent of Chagas disease, stands as a fundamental need. Currently, there are only two drugs available to treat this neglected disease, which represents a major public health problem in Latin America. Both available therapies, benznidazole and nifurtimox, have significant toxic side effects and their efficacy against the life-threatening symptomatic chronic stage of the disease is variable. Thus, there is an urgent need for new, improved anti-T. cruzi drugs. With the objective to reliably accelerate the drug discovery process against Chagas disease, several advances have been made in the last few years. Availability of engineered reporter gene expressing parasites triggered the development of phenotypic in vitro assays suitable for high throughput screening (HTS as well as the establishment of new in vivo protocols that allow faster experimental outcomes. Recently, automated high content microscopy approaches have also been used to identify new parasitic inhibitors. These in vitro and in vivo early drug discovery approaches, which hopefully will contribute to bring better anti-T. cruzi drug entities in the near future, are reviewed here.

  19. High Throughput Bent-Pipe Processor Demonstrator

    Science.gov (United States)

    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

  20. High-throughput automated confocal microscopy imaging screen of a kinase-focused library to identify p38 mitogen-activated protein kinase inhibitors using the GE InCell 3000 analyzer.

    Science.gov (United States)

    Trask, O Joseph; Nickischer, Debra; Burton, Audrey; Williams, Rhonda Gates; Kandasamy, Ramani A; Johnston, Patricia A; Johnston, Paul A

    2009-01-01

    The integration of fluorescent microscopy imaging technologies and image analysis into high-content screening (HCS) has been applied throughout the drug discovery pipeline to identify, evaluate, and advance compounds from early lead generation through preclinical candidate selection. In this chapter we describe the development, validation, and implementation of an HCS assay to screen compounds from a kinase-focused small-molecule library to identify inhibitors of the p38 pathway using the GE InCell 3000 automated imaging platform. The assay utilized a genetically modified HeLa cell line stably expressing mitogen-activated, protein-activating protein kinase-2 fused to enhanced green fluorescent protein (MK2-EGFP) and measured the subcellular distribution of the MK2-EGFP as a direct readout of p38 activation. The MK2-EGFP translocation assay performed in 384-well glass bottom microtiter plates exhibited a robust Z-factor of 0.46 and reproducible EC50 and IC50 determinations for activators and inhibitors, respectively. A total of 32,891 compounds were screened in singlicate at 50 microM and 156 were confirmed as inhibitors of p38-mediated MK2-EGFP translocation in follow-up IC50 concentration response curves. Thirty-one compounds exhibited IC50s less than 1 microM, and at least one novel structural class of p38 inhibitor was identified using this HCA/HCS chemical biology screening approach.

  1. Applications of ambient mass spectrometry in high-throughput screening.

    Science.gov (United States)

    Li, Li-Ping; Feng, Bao-Sheng; Yang, Jian-Wang; Chang, Cui-Lan; Bai, Yu; Liu, Hu-Wei

    2013-06-07

    The development of rapid screening and identification techniques is of great importance for drug discovery, doping control, forensic identification, food safety and quality control. Ambient mass spectrometry (AMS) allows rapid and direct analysis of various samples in open air with little sample preparation. Recently, its applications in high-throughput screening have been in rapid progress. During the past decade, various ambient ionization techniques have been developed and applied in high-throughput screening. This review discusses typical applications of AMS, including DESI (desorption electrospray ionization), DART (direct analysis in real time), EESI (extractive electrospray ionization), etc., in high-throughput screening (HTS).

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

    Directory of Open Access Journals (Sweden)

    Panagiotis Tsakanikas

    Full Text Available 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.

  3. High Throughput T Epitope Mapping and Vaccine Development

    Directory of Open Access Journals (Sweden)

    Giuseppina Li Pira

    2010-01-01

    Full Text Available Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th and by cytolytic T lymphocytes (CTL is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost.

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

    Science.gov (United States)

    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.

  5. Resolution- and throughput-enhanced spectroscopy using high-throughput computational slit

    CERN Document Server

    Kazemzadeh, Farnoud

    2016-01-01

    There exists a fundamental tradeoff between spectral resolution and the efficiency or throughput for all optical spectrometers. The primary factors affecting the spectral resolution and throughput of an optical spectrometer are the size of the entrance aperture and the optical power of the focusing element. Thus far collective optimization of the above mentioned has proven difficult. Here, we introduce the concept of high-throughput computational slits (HTCS) for improving both the effective spectral resolution and efficiency of a spectrometer. The proposed HTCS approach was experimentally validated using an optical spectrometer configured with a 200 um entrance aperture, test, and a 50 um entrance aperture, control, demonstrating improvements in spectral resolution of the spectrum by ~20% over the control spectral resolution and improvements in efficiency of > 2 times the efficiency of the largest entrance aperture used in the study while producing highly accurate spectra.

  6. HIGH THROUGHPUT OF MAP PROCESSOR USING PIPELINE WINDOW DECODING

    Directory of Open Access Journals (Sweden)

    P. Nithya

    2012-11-01

    Full Text Available Turbo codes are one of the most efficient error correcting code which approaches the Shannon limit.The high throughput in turbo decoder can be achieved by parallelizing several soft Input Soft Output(SISOunits together.In this way,multiple SISO decoders work on the same data frame at the same values and delievers soft outputs can be split into three terms like the soft channel and a priori input and the extrinsic value.The extrinsic value is used for the next iteration.The high throughput of Max-Log-MAP processor tha supports both single Binary(SBand Double-binary(DB convolutional turbo codes.Decoding of these codes however an iterative processing is requires high computation rate and latency.Thus in order to achieve high throughput and to reduce latency by using serial processing techniques.The pipeline window(PWdecoding is introduced to support arbitrary frame sizes with high throughput.

  7. High-throughput Saccharification assay for lignocellulosic materials.

    Science.gov (United States)

    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

  8. Integrated Analysis Platform: An Open-Source Information System for High-Throughput Plant Phenotyping.

    Science.gov (United States)

    Klukas, Christian; Chen, Dijun; Pape, Jean-Michel

    2014-06-01

    High-throughput phenotyping is emerging as an important technology to dissect phenotypic components in plants. Efficient image processing and feature extraction are prerequisites to quantify plant growth and performance based on phenotypic traits. Issues include data management, image analysis, and result visualization of large-scale phenotypic data sets. Here, we present Integrated Analysis Platform (IAP), an open-source framework for high-throughput plant phenotyping. IAP provides user-friendly interfaces, and its core functions are highly adaptable. Our system supports image data transfer from different acquisition environments and large-scale image analysis for different plant species based on real-time imaging data obtained from different spectra. Due to the huge amount of data to manage, we utilized a common data structure for efficient storage and organization of data for both input data and result data. We implemented a block-based method for automated image processing to extract a representative list of plant phenotypic traits. We also provide tools for build-in data plotting and result export. For validation of IAP, we performed an example experiment that contains 33 maize (Zea mays 'Fernandez') plants, which were grown for 9 weeks in an automated greenhouse with nondestructive imaging. Subsequently, the image data were subjected to automated analysis with the maize pipeline implemented in our system. We found that the computed digital volume and number of leaves correlate with our manually measured data in high accuracy up to 0.98 and 0.95, respectively. In summary, IAP provides a multiple set of functionalities for import/export, management, and automated analysis of high-throughput plant phenotyping data, and its analysis results are highly reliable.

  9. High Throughput Hall Thruster for Small Spacecraft Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Busek is developing a high throughput nominal 100-W Hall Effect Thruster. This device is well sized for spacecraft ranging in size from several tens of kilograms to...

  10. High Throughput Hall Thruster for Small Spacecraft Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Busek Co. Inc. proposes to develop a high throughput, nominal 100 W Hall Effect Thruster (HET). This HET will be sized for small spacecraft (< 180 kg), including...

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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...

  13. Inferential literacy for experimental high-throughput biology.

    Science.gov (United States)

    Miron, Mathieu; Nadon, Robert

    2006-02-01

    Many biologists believe that data analysis expertise lags behind the capacity for producing high-throughput data. One view within the bioinformatics community is that biological scientists need to develop algorithmic skills to meet the demands of the new technologies. In this article, we argue that the broader concept of inferential literacy, which includes understanding of data characteristics, experimental design and statistical analysis, in addition to computation, more adequately encompasses what is needed for efficient progress in high-throughput biology.

  14. FLASH Assembly of TALENs Enables High-Throughput Genome Editing

    OpenAIRE

    Reyon, Deepak; Tsai, Shengdar Q.; Khayter, Cyd; Foden, Jennifer A.; Sander, Jeffry D.; Joung, J. Keith

    2012-01-01

    Engineered transcription activator-like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) platform, a rapid and cost-effective method we developed to enable ...

  15. Virtual high throughput screening (vHTS) - A perspective

    OpenAIRE

    Subramaniam, Sangeetha; Mehrotra, Monica; Gupta, Dinesh,

    2008-01-01

    With the exponential rise in the number of viable novel drug targets, computational methods are being increasingly applied to accelerate the drug discovery process. Virtual High Throughput Screening (vHTS) is one such established methodology to identify drug candidates from large collection of compound libraries. Although it complements the expensive and time consuming High Throughput Screening (HTS) of compound libraries, vHTS possess inherent challenges. The successful vHTS requires the car...

  16. Protocol: A high-throughput DNA extraction system suitable for conifers

    Directory of Open Access Journals (Sweden)

    Rajora Om P

    2008-08-01

    Full Text Available Abstract Background High throughput DNA isolation from plants is a major bottleneck for most studies requiring large sample sizes. A variety of protocols have been developed for DNA isolation from plants. However, many species, including conifers, have high contents of secondary metabolites that interfere with the extraction process or the subsequent analysis steps. Here, we describe a procedure for high-throughput DNA isolation from conifers. Results We have developed a high-throughput DNA extraction protocol for conifers using an automated liquid handler and modifying the Qiagen MagAttract Plant Kit protocol. The modifications involve change to the buffer system and improving the protocol so that it almost doubles the number of samples processed per kit, which significantly reduces the overall costs. We describe two versions of the protocol: one for medium-throughput (MTP and another for high-throughput (HTP DNA isolation. The HTP version works from start to end in the industry-standard 96-well format, while the MTP version provides higher DNA yields per sample processed. We have successfully used the protocol for DNA extraction and genotyping of thousands of individuals of several spruce and a pine species. Conclusion A high-throughput system for DNA extraction from conifer needles and seeds has been developed and validated. The quality of the isolated DNA was comparable with that obtained from two commonly used methods: the silica-spin column and the classic CTAB protocol. Our protocol provides a fully automatable and cost effective solution for processing large numbers of conifer samples.

  17. High-throughput culturing of fungi from plant litter by a dilution-to-extinction technique.

    Science.gov (United States)

    Collado, Javier; Platas, Gonzalo; Paulus, Barbara; Bills, Gerald F

    2007-06-01

    High-throughput bacterial cultivation has improved the recovery of slow-growing and previously uncultured bacteria. The most robust high-throughput methods are based on techniques of 'dilution to extinction' or 'extinction culturing'. The low-density partitioning of CFUs in tubes or microwells exploits the fact that the number of culturable species typically increases as inoculum density decreases. Bacterial high-throughput culturing methods were adapted to fungi to generate large numbers of fungal extinction cultures. The efficiency of extinction culturing was assessed by comparing it with particle filtration and automated plate-streaking. Equal volumes of particle suspension from five litter collections of the New Zealand forest tree Elaeocarpus dentatus were compared. Dilute particle suspensions of litter were pipetted into 48-well tissue culture plates containing 1 mL of agar medium per well. Particle volumes from the same samples were applied to continuous agar surfaces in Omnitray plates by automated streaking, and fungal diversity and richness were measured. The spectrum of isolates was assessed by microscopy and sequencing of the ITS or 28S region of the rRNA gene. Estimates of species diversity between the two methods were comparable, but extinction culturing increased species richness. Compared with plating methods using continuous surfaces, extinction culturing distributes fungal propagules over partitioned surfaces. Intercolony interactions are reduced, permitting longer incubation times, and colony initiation and recovery improved. Effort to evaluate and recover colonies from fungal isolation plates was substantially reduced.

  18. High throughput electrophysiology: new perspectives for ion channel drug discovery

    DEFF Research Database (Denmark)

    Willumsen, Niels J; Bech, Morten; Olesen, Søren-Peter

    2003-01-01

    . A cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct...... channel targets accessible for drug screening. Specifically, genuine HTS parallel processing techniques based on arrays of planar silicon chips are being developed, but also lower throughput sequential techniques may be of value in compound screening, lead optimization, and safety screening...... characterization of ion channel properties. However, patch clamp is a slow, labor-intensive, and thus expensive, technique. New techniques combining the reliability and high information content of patch clamping with the virtues of high throughput philosophy are emerging and predicted to make a number of ion...

  19. Miniature high-throughput chemosensing of yield, ee, and absolute configuration from crude reaction mixtures

    Science.gov (United States)

    Bentley, Keith W.; Zhang, Peng; Wolf, Christian

    2016-01-01

    High-throughput experimentation (HTE) has emerged as a widely used technology that accelerates discovery and optimization processes with parallel small-scale reaction setups. A high-throughput screening (HTS) method capable of comprehensive analysis of crude asymmetric reaction mixtures (eliminating product derivatization or isolation) would provide transformative impact by matching the pace of HTE. We report how spontaneous in situ construction of stereodynamic metal probes from readily available, inexpensive starting materials can be applied to chiroptical chemosensing of the total amount, enantiomeric excess (ee), and absolute configuration of a wide variety of amines, diamines, amino alcohols, amino acids, carboxylic acids, α-hydroxy acids, and diols. This advance and HTS potential are highlighted with the analysis of 1 mg of crude reaction mixtures of a catalytic asymmetric reaction. This operationally simple assay uses a robust mix-and-measure protocol, is amenable to microscale platforms and automation, and provides critical time efficiency and sustainability advantages over traditional serial methods. PMID:26933684

  20. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EF

  1. Towards a high throughput droplet-based agglutination assay

    KAUST Repository

    Kodzius, Rimantas

    2013-10-22

    This work demonstrates the detection method for a high throughput droplet based agglutination assay system. Using simple hydrodynamic forces to mix and aggregate functionalized microbeads we avoid the need to use magnetic assistance or mixing structures. The concentration of our target molecules was estimated by agglutination strength, obtained through optical image analysis. Agglutination in droplets was performed with flow rates of 150 µl/min and occurred in under a minute, with potential to perform high-throughput measurements. The lowest target concentration detected in droplet microfluidics was 0.17 nM, which is three orders of magnitude more sensitive than a conventional card based agglutination assay.

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

    Science.gov (United States)

    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.

  3. High-throughput Binary Vectors for Plant Gene Function Analysis

    Institute of Scientific and Technical Information of China (English)

    Zhi-Yong Lei; Ping Zhao; Min-Jie Cao; Rong Cui; Xi Chen; Li-Zhong Xiong; Qi-Fa Zhang; David J. Oliver; Cheng-Bin Xiang

    2007-01-01

    A series of high-throughput binary cloning vectors were constructed to facilitate gene function analysis in higher plants. This vector series consists of plasmids designed for plant expression, promoter analysis, gene silencing,and green fluorescent protein fusions for protein localization. These vectors provide for high-throughput and efficient cloning utilizing sites for λ phage integrase/excisionase. In addition, unique restriction sites are incorporated in a multiple cloning site and enable promoter replacement. The entire vector series are available with complete sequence information and detailed annotations and are freely distributed to the scientific community for non-commercial uses.

  4. Droplet microfluidics for high-throughput biological assays.

    Science.gov (United States)

    Guo, Mira T; Rotem, Assaf; Heyman, John A; Weitz, David A

    2012-06-21

    Droplet microfluidics offers significant advantages for performing high-throughput screens and sensitive assays. Droplets allow sample volumes to be significantly reduced, leading to concomitant reductions in cost. Manipulation and measurement at kilohertz speeds enable up to 10(8) samples to be screened in one day. Compartmentalization in droplets increases assay sensitivity by increasing the effective concentration of rare species and decreasing the time required to reach detection thresholds. Droplet microfluidics combines these powerful features to enable currently inaccessible high-throughput screening applications, including single-cell and single-molecule assays.

  5. High throughput recombinant protein production of fungal secreted proteins

    DEFF Research Database (Denmark)

    Vala, Andrea Lages Lino; Roth, Doris; Grell, Morten Nedergaard

    2011-01-01

    a high-throughput protein production system with a special focus on fungal secreted proteins. We use a ligation independent cloning to clone target genes into expression vectors for E. coli and P. pastoris and a small scale test expression to identify constructs producing soluble protein. Expressed...... interaction), between fungi of the order Entomophthorales and aphids (pathogenic interaction), and in the mycoparasitic interaction between the oomycetes Pythium oligandrum and P. ultimum. In general, the high-throughput protein production system can lead to a better understanding of fungal/host interactions...

  6. Development and Operation of High-throughput Accurate-wavelength Lens-based Spectrometer

    Energy Technology Data Exchange (ETDEWEB)

    Bell, Ronald E

    2014-07-01

    A high-throughput spectrometer for the 400-820 nm wavelength range has been developed for charge exchange recombination spectroscopy or general spectroscopy. A large 2160 mm-1 grating is matched with fast f /1.8 200 mm lenses, which provide stigmatic imaging. A precision optical encoder measures the grating angle with an accuracy < 0.075 arc seconds. A high quantum efficiency low-etaloning CCD detector allows operation at longer wavelengths. A patch panel allows input fibers to interface with interchangeable fiber holders that attach to a kinematic mount behind the entrance slit. Computer-controlled hardware allows automated control of wavelength, timing, f-number, automated data collection, and wavelength calibration.

  7. A high-throughput label-free nanoparticle analyser

    Science.gov (United States)

    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.

  8. High-throughput screening for industrial enzyme production hosts by droplet microfluidics

    DEFF Research Database (Denmark)

    Sjostrom, Staffan L.; Bai, Yunpeng; Huang, Mingtao

    2014-01-01

    A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its α...... in contrast to previous droplet-based directed evolution that has focused on improving enzyme protein structure. In the workflow presented, enzyme producing single cells are encapsulated in 20 pL droplets with a fluorogenic reporter substrate. The coupling of a desired phenotype (secreted enzyme concentration......) with the genotype (contained in the cell) inside a droplet enables selection of single cells with improved enzyme production capacity by droplet sorting. The platform has a throughput over 300 times higher than that of the current industry standard, an automated microtiter plate screening system. At the same time...

  9. Simple Protein Complex Purification and Identification Method Suitable for High- throughput Mapping of Protein Interaction Networks

    Energy Technology Data Exchange (ETDEWEB)

    Markillie, Lye Meng; Lin, Chiann Tso; Adkins, Joshua N.; Auberry, Deanna L.; Hill, Eric A.; Hooker, Brian S.; Moore, Priscilla A.; Moore, Ronald J.; Shi, Liang; Wiley, H. S.; Kery, Vladimir

    2005-04-11

    Most of the current methods for purification and identification of protein complexes use endogenous expression of affinity tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, gel separation, in-gel digestion and mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pulldown assay with denaturing elution, trypsin digestion in organic solvent and LC ESI MS/MS protein identification using SEQUEST analysis. Our method is simple, easy to scale up and automate thus suitable for high throughput mapping of protein interaction networks and functional proteomics.

  10. Development of scalable high throughput fermentation approaches for physiological characterisation of yeast and filamentous fungi

    DEFF Research Database (Denmark)

    Knudsen, Peter Boldsen

    asan indication of comparable growth physiology in both microtiter plates and bioreactors, which was substantiated by metabolic flux analysis resulting in identical flux distributions over micro-scale and lab scale cultivations. The thesis further presents a novel automated high throughput method......, demonstrated superior yields and productivities for an A. nidulans strain where two copies of the 6-MSA gene had been chromosomally integrated. Implementation of a chemostat cultivation strategy for A. nidulans proved successful for conditional comparison, while metabolic oscillations and low productivities...

  11. High-throughput bioinformatics with the Cyrille2 pipeline system.

    NARCIS (Netherlands)

    Fiers, M.W.E.J.; Burgt, van der A.; Datema, E.; Groot, de J.C.W.; Ham, van R.C.H.J.

    2008-01-01

    Background - Modern omics research involves the application of high-throughput technologies that generate vast volumes of data. These data need to be pre-processed, analyzed and integrated with existing knowledge through the use of diverse sets of software tools, models and databases. The analyses a

  12. An improved high throughput sequencing method for studying oomycete communities

    DEFF Research Database (Denmark)

    Sapkota, Rumakanta; Nicolaisen, Mogens

    2015-01-01

    Culture-independent studies using next generation sequencing have revolutionizedmicrobial ecology, however, oomycete ecology in soils is severely lagging behind. The aimof this study was to improve and validate standard techniques for using high throughput sequencing as a tool for studying oomyce...

  13. High-throughput cloning and expression in recalcitrant bacteria

    NARCIS (Netherlands)

    Geertsma, Eric R.; Poolman, Bert

    2007-01-01

    We developed a generic method for high-throughput cloning in bacteria that are less amenable to conventional DNA manipulations. The method involves ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector-backbone exchange (VBEx) into an organism

  14. High-throughput STR analysis for DNA database using direct PCR.

    Science.gov (United States)

    Sim, Jeong Eun; Park, Su Jeong; Lee, Han Chul; Kim, Se-Yong; Kim, Jong Yeol; Lee, Seung Hwan

    2013-07-01

    Since the Korean criminal DNA database was launched in 2010, we have focused on establishing an automated DNA database profiling system that analyzes short tandem repeat loci in a high-throughput and cost-effective manner. We established a DNA database profiling system without DNA purification using a direct PCR buffer system. The quality of direct PCR procedures was compared with that of conventional PCR system under their respective optimized conditions. The results revealed not only perfect concordance but also an excellent PCR success rate, good electropherogram quality, and an optimal intra/inter-loci peak height ratio. In particular, the proportion of DNA extraction required due to direct PCR failure could be minimized to direct PCR system can be adopted for automated DNA database profiling systems to replace or supplement conventional PCR system in a time- and cost-saving manner.

  15. MIPHENO: data normalization for high throughput metabolite analysis

    Directory of Open Access Journals (Sweden)

    Bell Shannon M

    2012-01-01

    Full Text Available Abstract Background 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 of months and years, often without the controls needed to compare directly across the dataset. Few methods are available to facilitate comparisons of high throughput metabolic data generated in batches where explicit in-group controls for normalization are lacking. Results Here we describe MIPHENO (Mutant Identification by Probabilistic High throughput-Enabled Normalization, an approach for post-hoc normalization of quantitative first-pass screening data in the absence of explicit in-group controls. This approach includes a quality control step and facilitates cross-experiment comparisons that decrease the false non-discovery rates, while maintaining the high accuracy needed to limit false positives in first-pass screening. Results from simulation show an improvement in both accuracy and false non-discovery rate over a range of population parameters (p -16 and a modest but significant (p -16 improvement in area under the receiver operator characteristic curve of 0.955 for MIPHENO vs 0.923 for a group-based statistic (z-score. Analysis of the high throughput phenotypic data from the Arabidopsis Chloroplast 2010 Project (http://www.plastid.msu.edu/ showed ~ 4-fold increase in the ability to detect previously described or expected phenotypes over the group based statistic. Conclusions Results demonstrate MIPHENO offers substantial benefit in improving the ability to detect putative mutant phenotypes from post-hoc analysis of large data sets. Additionally, it facilitates data interpretation and permits cross-dataset comparison where group-based controls are missing. MIPHENO is applicable to a wide range of high throughput screenings and the code is

  16. High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography.

    Science.gov (United States)

    Desmarais, Samantha M; Tropini, Carolina; Miguel, Amanda; Cava, Felipe; Monds, Russell D; de Pedro, Miguel A; Huang, Kerwyn Casey

    2015-12-25

    The bacterial cell wall is a network of glycan strands cross-linked by short peptides (peptidoglycan); it is responsible for the mechanical integrity of the cell and shape determination. Liquid chromatography can be used to measure the abundance of the muropeptide subunits composing the cell wall. Characteristics such as the degree of cross-linking and average glycan strand length are known to vary across species. However, a systematic comparison among strains of a given species has yet to be undertaken, making it difficult to assess the origins of variability in peptidoglycan composition. We present a protocol for muropeptide analysis using ultra performance liquid chromatography (UPLC) and demonstrate that UPLC achieves resolution comparable with that of HPLC while requiring orders of magnitude less injection volume and a fraction of the elution time. We also developed a software platform to automate the identification and quantification of chromatographic peaks, which we demonstrate has improved accuracy relative to other software. This combined experimental and computational methodology revealed that peptidoglycan composition was approximately maintained across strains from three Gram-negative species despite taxonomical and morphological differences. Peptidoglycan composition and density were maintained after we systematically altered cell size in Escherichia coli using the antibiotic A22, indicating that cell shape is largely decoupled from the biochemistry of peptidoglycan synthesis. High-throughput, sensitive UPLC combined with our automated software for chromatographic analysis will accelerate the discovery of peptidoglycan composition and the molecular mechanisms of cell wall structure determination.

  17. High throughput modular chambers for rapid evaluation of anesthetic sensitivity

    Directory of Open Access Journals (Sweden)

    Eckmann David M

    2006-11-01

    Full Text Available Abstract Background Anesthetic sensitivity is determined by the interaction of multiple genes. Hence, a dissection of genetic contributors would be aided by precise and high throughput behavioral screens. Traditionally, anesthetic phenotyping has addressed only induction of anesthesia, evaluated with dose-response curves, while ignoring potentially important data on emergence from anesthesia. Methods We designed and built a controlled environment apparatus to permit rapid phenotyping of twenty-four mice simultaneously. We used the loss of righting reflex to indicate anesthetic-induced unconsciousness. After fitting the data to a sigmoidal dose-response curve with variable slope, we calculated the MACLORR (EC50, the Hill coefficient, and the 95% confidence intervals bracketing these values. Upon termination of the anesthetic, Emergence timeRR was determined and expressed as the mean ± standard error for each inhaled anesthetic. Results In agreement with several previously published reports we find that the MACLORR of halothane, isoflurane, and sevoflurane in 8–12 week old C57BL/6J mice is 0.79% (95% confidence interval = 0.78 – 0.79%, 0.91% (95% confidence interval = 0.90 – 0.93%, and 1.96% (95% confidence interval = 1.94 – 1.97%, respectively. Hill coefficients for halothane, isoflurane, and sevoflurane are 24.7 (95% confidence interval = 19.8 – 29.7%, 19.2 (95% confidence interval = 14.0 – 24.3%, and 33.1 (95% confidence interval = 27.3 – 38.8%, respectively. After roughly 2.5 MACLORR • hr exposures, mice take 16.00 ± 1.07, 6.19 ± 0.32, and 2.15 ± 0.12 minutes to emerge from halothane, isoflurane, and sevoflurane, respectively. Conclusion This system enabled assessment of inhaled anesthetic responsiveness with a higher precision than that previously reported. It is broadly adaptable for delivering an inhaled therapeutic (or toxin to a population while monitoring its vital signs, motor reflexes, and providing precise control

  18. Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus.

    Science.gov (United States)

    Urbański, Dorian Fabian; Małolepszy, Anna; Stougaard, Jens; Andersen, Stig Uggerhøj

    2012-02-01

    Use of insertion mutants facilitates functional analysis of genes, but it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics in most plant species. The main challenge is developing efficient high-throughput procedures for both mutagenesis and identification of insertion sites. To date, only floral-dip T-DNA transformation of Arabidopsis has produced independent germinal insertions, thereby allowing generation of mutant populations from seeds of single plants. In addition, advances in insertion detection have been hampered by a lack of protocols, including software for automated data analysis, that take full advantage of high-throughput next-generation sequencing. We have addressed these challenges by developing the FSTpoolit protocol and software package, and here we demonstrate its efficacy by detecting 8935 LORE1 insertions in 3744 Lotus japonicus plants. The identified insertions show that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to homogenous gene targeting and exonic insertion preference. As LORE1 transposition occurs in the germline, harvesting seeds from a single founder line and cultivating progeny generates a complete mutant population. This ease of LORE1 mutagenesis, combined with the efficient FSTpoolit protocol, which exploits 2D pooling, Illumina sequencing and automated data analysis, allows highly cost-efficient development of a comprehensive reverse genetic resource.

  19. Semiautomated Alignment of High-Throughput Metabolite Profiles with Chemometric Tools

    Science.gov (United States)

    Xiao, Zi-dan; Liang, Yi-zeng; Chau, Foo-tim; Chan, Hoi-yan

    2017-01-01

    The rapid increase in the use of metabolite profiling/fingerprinting techniques to resolve complicated issues in metabolomics has stimulated demand for data processing techniques, such as alignment, to extract detailed information. In this study, a new and automated method was developed to correct the retention time shift of high-dimensional and high-throughput data sets. Information from the target chromatographic profiles was used to determine the standard profile as a reference for alignment. A novel, piecewise data partition strategy was applied for the determination of the target components in the standard profile as markers for alignment. An automated target search (ATS) method was proposed to find the exact retention times of the selected targets in other profiles for alignment. The linear interpolation technique (LIT) was employed to align the profiles prior to pattern recognition, comprehensive comparison analysis, and other data processing steps. In total, 94 metabolite profiles of ginseng were studied, including the most volatile secondary metabolites. The method used in this article could be an essential step in the extraction of information from high-throughput data acquired in the study of systems biology, metabolomics, and biomarker discovery. PMID:28168083

  20. Fast high-throughput screening of temoporfin-loaded liposomal formulations prepared by ethanol injection method.

    Science.gov (United States)

    Yang, Kewei; Delaney, Joseph T; Schubert, Ulrich S; Fahr, Alfred

    2012-03-01

    A new strategy for fast, convenient high-throughput screening of liposomal formulations was developed, utilizing the automation of the so-called ethanol-injection method. This strategy was illustrated by the preparation and screening of the liposomal formulation library of a potent second-generation photosensitizer, temoporfin. Numerous liposomal formulations were efficiently prepared using a pipetting robot, followed by automated size characterization, using a dynamic light scattering plate reader. Incorporation efficiency of temoporfin and zeta potential were also detected in selected cases. To optimize the formulation, different parameters were investigated, including lipid types, lipid concentration in injected ethanol, ratio of ethanol to aqueous solution, ratio of drug to lipid, and the addition of functional phospholipid. Step-by-step small liposomes were prepared with high incorporation efficiency. At last, an optimized formulation was obtained for each lipid in the following condition: 36.4 mg·mL(-1) lipid, 13.1 mg·mL(-1) mPEG(2000)-DSPE, and 1:4 ethanol:buffer ratio. These liposomes were unilamellar spheres, with a diameter of approximately 50 nm, and were very stable for over 20 weeks. The results illustrate this approach to be promising for fast high-throughput screening of liposomal formulations.

  1. CellSegm - a MATLAB toolbox for high-throughput 3D cell segmentation.

    Science.gov (United States)

    Hodneland, Erlend; Kögel, Tanja; Frei, Dominik Michael; Gerdes, Hans-Hermann; Lundervold, Arvid

    2013-08-09

    : The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening.

  2. An improved high-throughput lipid extraction method for the analysis of human brain lipids.

    Science.gov (United States)

    Abbott, Sarah K; Jenner, Andrew M; Mitchell, Todd W; Brown, Simon H J; Halliday, Glenda M; Garner, Brett

    2013-03-01

    We have developed a protocol suitable for high-throughput lipidomic analysis of human brain samples. The traditional Folch extraction (using chloroform and glass-glass homogenization) was compared to a high-throughput method combining methyl-tert-butyl ether (MTBE) extraction with mechanical homogenization utilizing ceramic beads. This high-throughput method significantly reduced sample handling time and increased efficiency compared to glass-glass homogenizing. Furthermore, replacing chloroform with MTBE is safer (less carcinogenic/toxic), with lipids dissolving in the upper phase, allowing for easier pipetting and the potential for automation (i.e., robotics). Both methods were applied to the analysis of human occipital cortex. Lipid species (including ceramides, sphingomyelins, choline glycerophospholipids, ethanolamine glycerophospholipids and phosphatidylserines) were analyzed via electrospray ionization mass spectrometry and sterol species were analyzed using gas chromatography mass spectrometry. No differences in lipid species composition were evident when the lipid extraction protocols were compared, indicating that MTBE extraction with mechanical bead homogenization provides an improved method for the lipidomic profiling of human brain tissue.

  3. High throughput workflow for coacervate formation and characterization in shampoo systems.

    Science.gov (United States)

    Kalantar, T H; Tucker, C J; Zalusky, A S; Boomgaard, T A; Wilson, B E; Ladika, M; Jordan, S L; Li, W K; Zhang, X; Goh, C G

    2007-01-01

    Cationic cellulosic polymers find wide utility as benefit agents in shampoo. Deposition of these polymers onto hair has been shown to mend split-ends, improve appearance and wet combing, as well as provide controlled delivery of insoluble actives. The deposition is thought to be enhanced by the formation of a polymer/surfactant complex that phase-separates from the bulk solution upon dilution. A standard characterization method has been developed to characterize the coacervate formation upon dilution, but the test is time and material prohibitive. We have developed a semi-automated high throughput workflow to characterize the coacervate-forming behavior of different shampoo formulations. A procedure that allows testing of real use shampoo dilutions without first formulating a complete shampoo was identified. This procedure was adapted to a Tecan liquid handler by optimizing the parameters for liquid dispensing as well as for mixing. The high throughput workflow enabled preparation and testing of hundreds of formulations with different types and levels of cationic cellulosic polymers and surfactants, and for each formulation a haze diagram was constructed. Optimal formulations and their dilutions that give substantial coacervate formation (determined by haze measurements) were identified. Results from this high throughput workflow were shown to reproduce standard haze and bench-top turbidity measurements, and this workflow has the advantages of using less material and allowing more variables to be tested with significant time savings.

  4. High-throughput theoretical design of lithium battery materials

    Science.gov (United States)

    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).

  5. Reproducible, high-throughput synthesis of colloidal nanocrystals for optimization in multidimensional parameter space.

    Science.gov (United States)

    Chan, Emory M; Xu, Chenxu; Mao, Alvin W; Han, Gang; Owen, Jonathan S; Cohen, Bruce E; Milliron, Delia J

    2010-05-12

    While colloidal nanocrystals hold tremendous potential for both enhancing fundamental understanding of materials scaling and enabling advanced technologies, progress in both realms can be inhibited by the limited reproducibility of traditional synthetic methods and by the difficulty of optimizing syntheses over a large number of synthetic parameters. Here, we describe an automated platform for the reproducible synthesis of colloidal nanocrystals and for the high-throughput optimization of physical properties relevant to emerging applications of nanomaterials. This robotic platform enables precise control over reaction conditions while performing workflows analogous to those of traditional flask syntheses. We demonstrate control over the size, size distribution, kinetics, and concentration of reactions by synthesizing CdSe nanocrystals with 0.2% coefficient of variation in the mean diameters across an array of batch reactors and over multiple runs. Leveraging this precise control along with high-throughput optical and diffraction characterization, we effectively map multidimensional parameter space to tune the size and polydispersity of CdSe nanocrystals, to maximize the photoluminescence efficiency of CdTe nanocrystals, and to control the crystal phase and maximize the upconverted luminescence of lanthanide-doped NaYF(4) nanocrystals. On the basis of these demonstrative examples, we conclude that this automated synthesis approach will be of great utility for the development of diverse colloidal nanomaterials for electronic assemblies, luminescent biological labels, electroluminescent devices, and other emerging applications.

  6. A new ultrasonic high-throughput instrument for rapid DNA release from microorganisms.

    Science.gov (United States)

    Hohnadel, Marisa; Felden, Luc; Fijuljanin, Demir; Jouette, Sébastien; Chollet, Renaud

    2014-04-01

    Rapid detection methods for microbiological contamination are requested by many industries that respond to public health concerns. The control laboratories are replacing traditional culturing methods with faster assays based on nucleic acid amplification technologies, such as real-time PCR. However, an optimal nucleic acid sample preparation method is critical for the sensitivity and specificity of such tools. A high-throughput automated external ultrasonic device was developed for rapid lysing of microorganisms. Based on Ct values obtained from real-time PCR, there was efficient DNA release from the 16 microorganisms tested, including Gram positive and negative bacteria, bacterial spores, yeasts and spores of molds. Linearity of the lysis method was also demonstrated for Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica, Candida albicans and the spores of Aspergillus brasiliensis, with correlation coefficients (r(2)) between 0.90 and 0.98. After sonication, PCR analysis of the lysates revealed earlier Ct values (0.83 to 7.49) for S. aureus, P. aeruginosa and S. enterica compared to the bead-beating method of lysis. These results demonstrated more efficient DNA release from bacteria with the ultrasonication system. Nevertheless, for fungi, the Ct values were 0.94 to 1.61 later for sonication than for bead beating. This study demonstrates that 4min of sonication with this new automated high-throughput instrument allows for the efficient lysis of a large range of microorganisms.

  7. Investigation of ternary subsystems of superalloys by thin-film combinatorial synthesis and high-throughput analysis

    Directory of Open Access Journals (Sweden)

    König Dennis

    2014-01-01

    Full Text Available A Co-Ti-W thin film materials library was fabricated by magnetron sputtering. By using automated high-throughput measurement techniques (resistance mapping, automated XRD measurements and cluster analysis a yet unknown phase region was revealed. The existence region of the new ternary phase is close to the composition Co60Ti15W25. In order to transfer the results from thin film to bulk material, a bulk sample was prepared by arc melting and subsequent heat treatment. Scanning electron microscopy and chemical micro-analysis data support that a yet unknown ternary phase exists in the system Co-Ti-W.

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

    Directory of Open Access Journals (Sweden)

    Hossein Pourmodheji

    2016-06-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  12. A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma

    DEFF Research Database (Denmark)

    Cham, Gerald K K; Kurtis, Jonathan; Lusingu, John;

    2008-01-01

    BACKGROUND: The level of antibodies against PfEMP1 is routinely quantified by the conventional microtitre enzyme-linked immunosorbent assay (ELISA). However, ELISA only measures one analyte at a time and requires a relatively large plasma volume if the complete antibody profile of the sample...... of twenty nine PfEMP1 domains were PCR amplified from 3D7 genomic DNA, expressed in the Baculovirus system and purified by metal-affinity chromatography. The antibody reactivity level to the recombinant PfEMP1 proteins in human hyper-immune plasma was measured by ELISA. In parallel, these recombinant PfEMP1......-based assay was sensitive, accurate and reproducible. Four recombinant PfEMP1 proteins C17, D5, D9 and D12, selected on the basis that they showed a spread of median fluorescent intensity (MFI) values from low to high when analysed by the bead-based assay were analysed by ELISA and the results from both...

  13. High-throughput sequence alignment using Graphics Processing Units

    Directory of Open Access Journals (Sweden)

    Trapnell Cole

    2007-12-01

    Full Text Available Abstract Background The recent availability of new, less expensive high-throughput DNA sequencing technologies has yielded a dramatic increase in the volume of sequence data that must be analyzed. These data are being generated for several purposes, including genotyping, genome resequencing, metagenomics, and de novo genome assembly projects. Sequence alignment programs such as MUMmer have proven essential for analysis of these data, but researchers will need ever faster, high-throughput alignment tools running on inexpensive hardware to keep up with new sequence technologies. Results This paper describes MUMmerGPU, an open-source high-throughput parallel pairwise local sequence alignment program that runs on commodity Graphics Processing Units (GPUs in common workstations. MUMmerGPU uses the new Compute Unified Device Architecture (CUDA from nVidia to align multiple query sequences against a single reference sequence stored as a suffix tree. By processing the queries in parallel on the highly parallel graphics card, MUMmerGPU achieves more than a 10-fold speedup over a serial CPU version of the sequence alignment kernel, and outperforms the exact alignment component of MUMmer on a high end CPU by 3.5-fold in total application time when aligning reads from recent sequencing projects using Solexa/Illumina, 454, and Sanger sequencing technologies. Conclusion MUMmerGPU is a low cost, ultra-fast sequence alignment program designed to handle the increasing volume of data produced by new, high-throughput sequencing technologies. MUMmerGPU demonstrates that even memory-intensive applications can run significantly faster on the relatively low-cost GPU than on the CPU.

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

    Science.gov (United States)

    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.

  15. Computational analysis of high-throughput flow cytometry data

    Science.gov (United States)

    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

  16. High throughput electrophysiology: new perspectives for ion channel drug discovery.

    Science.gov (United States)

    Willumsen, Niels J; Bech, Morten; Olesen, Søren-Peter; Jensen, Bo Skaaning; Korsgaard, Mads P G; Christophersen, Palle

    2003-01-01

    Proper function of ion channels is crucial for all living cells. Ion channel dysfunction may lead to a number of diseases, so-called channelopathies, and a number of common diseases, including epilepsy, arrhythmia, and type II diabetes, are primarily treated by drugs that modulate ion channels. A cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct characterization of ion channel properties. However, patch clamp is a slow, labor-intensive, and thus expensive, technique. New techniques combining the reliability and high information content of patch clamping with the virtues of high throughput philosophy are emerging and predicted to make a number of ion channel targets accessible for drug screening. Specifically, genuine HTS parallel processing techniques based on arrays of planar silicon chips are being developed, but also lower throughput sequential techniques may be of value in compound screening, lead optimization, and safety screening. The introduction of new powerful HTS electrophysiological techniques is predicted to cause a revolution in ion channel drug discovery.

  17. Fluorescent biosensors for high throughput screening of protein kinase inhibitors.

    Science.gov (United States)

    Prével, Camille; Pellerano, Morgan; Van, Thi Nhu Ngoc; Morris, May C

    2014-02-01

    High throughput screening assays aim to identify small molecules that interfere with protein function, activity, or conformation, which can serve as effective tools for chemical biology studies of targets involved in physiological processes or pathways of interest or disease models, as well as templates for development of therapeutics in medicinal chemistry. Fluorescent biosensors constitute attractive and powerful tools for drug discovery programs, from high throughput screening assays, to postscreen characterization of hits, optimization of lead compounds, and preclinical evaluation of candidate drugs. They provide a means of screening for inhibitors that selectively target enzymatic activity, conformation, and/or function in vitro. Moreover, fluorescent biosensors constitute useful tools for cell- and image-based, multiplex and multiparametric, high-content screening. Application of fluorescence-based sensors to screen large and complex libraries of compounds in vitro, in cell-based formats or whole organisms requires several levels of optimization to establish robust and reproducible assays. In this review, we describe the different fluorescent biosensor technologies which have been applied to high throughput screens, and discuss the prerequisite criteria underlying their successful application. Special emphasis is placed on protein kinase biosensors, since these enzymes constitute one of the most important classes of therapeutic targets in drug discovery.

  18. A CRISPR CASe for High-Throughput Silencing

    Directory of Open Access Journals (Sweden)

    Jacob eHeintze

    2013-10-01

    Full Text Available Manipulation of gene expression on a genome-wide level is one of the most important systematic tools in the post-genome era. Such manipulations have largely been enabled by expression cloning approaches using sequence-verified cDNA libraries, large-scale RNA interference libraries (shRNA or siRNA and zinc finger nuclease technologies. More recently, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated (Cas9-mediated gene editing technology has been described that holds great promise for future use of this technology in genomic manipulation. It was suggested that the CRISPR system has the potential to be used in high-throughput, large-scale loss of function screening. Here we discuss some of the challenges in engineering of CRISPR/Cas genomic libraries and some of the aspects that need to be addressed in order to use this technology on a high-throughput scale.

  19. High throughput screening of starch structures using carbohydrate microarrays

    DEFF Research Database (Denmark)

    Tanackovic, Vanja; Rydahl, Maja Gro; Pedersen, Henriette Lodberg

    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...

  20. Sensitivity study of reliable, high-throughput resolution metricsfor photoresists

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Christopher N.; Naulleau, Patrick P.

    2007-07-30

    The resolution of chemically amplified resists is becoming an increasing concern, especially for lithography in the extreme ultraviolet (EUV) regime. Large-scale screening and performance-based down-selection is currently underway to identify resist platforms that can support shrinking feature sizes. Resist screening efforts, however, are hampered by the absence of reliable resolution metrics that can objectively quantify resist resolution in a high-throughput fashion. Here we examine two high-throughput metrics for resist resolution determination. After summarizing their details and justifying their utility, we characterize the sensitivity of both metrics to two of the main experimental uncertainties associated with lithographic exposure tools, namely: limited focus control and limited knowledge of optical aberrations. For an implementation at EUV wavelengths, we report aberration and focus limited error bars in extracted resolution of {approx} 1.25 nm RMS for both metrics making them attractive candidates for future screening and down-selection efforts.

  1. High-throughput screening for modulators of cellular contractile force

    CERN Document Server

    Park, Chan Young; 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; Krishnan, Ramaswamy

    2014-01-01

    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 signaling intermediates with poorly defined relationship to such a physiological endpoint. Using cellular force as the target, here we screened libraries to identify novel drug candidates in the case of human airway smooth muscle cells in the context of asthma, and also in the case of 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.

  2. High‐throughput automated scoring of Ki67 in breast cancer tissue microarrays from the Breast Cancer Association Consortium

    Science.gov (United States)

    Howat, William J; Daley, Frances; Zabaglo, Lila; McDuffus, Leigh‐Anne; Blows, Fiona; Coulson, Penny; Raza Ali, H; Benitez, Javier; Milne, Roger; Brenner, Herman; Stegmaier, Christa; Mannermaa, Arto; Chang‐Claude, Jenny; Rudolph, Anja; Sinn, Peter; Couch, Fergus J; Tollenaar, Rob A.E.M.; Devilee, Peter; Figueroa, Jonine; Sherman, Mark E; Lissowska, Jolanta; Hewitt, Stephen; Eccles, Diana; Hooning, Maartje J; Hollestelle, Antoinette; WM Martens, John; HM van Deurzen, Carolien; Investigators, kConFab; Bolla, Manjeet K; Wang, Qin; Jones, Michael; Schoemaker, Minouk; Broeks, Annegien; van Leeuwen, Flora E; Van't Veer, Laura; Swerdlow, Anthony J; Orr, Nick; Dowsett, Mitch; Easton, Douglas; Schmidt, Marjanka K; Pharoah, Paul D; Garcia‐Closas, Montserrat

    2016-01-01

    Abstract Automated methods are needed to facilitate high‐throughput and reproducible scoring of Ki67 and other markers in breast cancer tissue microarrays (TMAs) in large‐scale studies. To address this need, we developed an automated protocol for Ki67 scoring and evaluated its performance in studies from the Breast Cancer Association Consortium. We utilized 166 TMAs containing 16,953 tumour cores representing 9,059 breast cancer cases, from 13 studies, with information on other clinical and pathological characteristics. TMAs were stained for Ki67 using standard immunohistochemical procedures, and scanned and digitized using the Ariol system. An automated algorithm was developed for the scoring of Ki67, and scores were compared to computer assisted visual (CAV) scores in a subset of 15 TMAs in a training set. We also assessed the correlation between automated Ki67 scores and other clinical and pathological characteristics. Overall, we observed good discriminatory accuracy (AUC = 85%) and good agreement (kappa = 0.64) between the automated and CAV scoring methods in the training set. The performance of the automated method varied by TMA (kappa range= 0.37–0.87) and study (kappa range = 0.39–0.69). The automated method performed better in satisfactory cores (kappa = 0.68) than suboptimal (kappa = 0.51) cores (p‐value for comparison = 0.005); and among cores with higher total nuclei counted by the machine (4,000–4,500 cells: kappa = 0.78) than those with lower counts (50–500 cells: kappa = 0.41; p‐value = 0.010). Among the 9,059 cases in this study, the correlations between automated Ki67 and clinical and pathological characteristics were found to be in the expected directions. Our findings indicate that automated scoring of Ki67 can be an efficient method to obtain good quality data across large numbers of TMAs from multicentre studies. However, robust algorithm development and rigorous pre‐ and post

  3. WormScan: a technique for high-throughput phenotypic analysis of Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Mark D Mathew

    Full Text Available BACKGROUND: There are four main phenotypes that are assessed in whole organism studies of Caenorhabditis elegans; mortality, movement, fecundity and size. Procedures have been developed that focus on the digital analysis of some, but not all of these phenotypes and may be limited by expense and limited throughput. We have developed WormScan, an automated image acquisition system that allows quantitative analysis of each of these four phenotypes on standard NGM plates seeded with E. coli. This system is very easy to implement and has the capacity to be used in high-throughput analysis. METHODOLOGY/PRINCIPAL FINDINGS: Our system employs a readily available consumer grade flatbed scanner. The method uses light stimulus from the scanner rather than physical stimulus to induce movement. With two sequential scans it is possible to quantify the induced phototactic response. To demonstrate the utility of the method, we measured the phenotypic response of C. elegans to phosphine gas exposure. We found that stimulation of movement by the light of the scanner was equivalent to physical stimulation for the determination of mortality. WormScan also provided a quantitative assessment of health for the survivors. Habituation from light stimulation of continuous scans was similar to habituation caused by physical stimulus. CONCLUSIONS/SIGNIFICANCE: There are existing systems for the automated phenotypic data collection of C. elegans. The specific advantages of our method over existing systems are high-throughput assessment of a greater range of phenotypic endpoints including determination of mortality and quantification of the mobility of survivors. Our system is also inexpensive and very easy to implement. Even though we have focused on demonstrating the usefulness of WormScan in toxicology, it can be used in a wide range of additional C. elegans studies including lifespan determination, development, pathology and behavior. Moreover, we have even adapted the

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

    OpenAIRE

    Kent Edward; Hoops Stefan; Mendes Pedro

    2012-01-01

    Abstract 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 experti...

  5. Intel: High Throughput Computing Collaboration: A CERN openlab / Intel collaboration

    CERN Document Server

    CERN. Geneva

    2015-01-01

    The Intel/CERN High Throughput Computing Collaboration studies the application of upcoming Intel technologies to the very challenging environment of the LHC trigger and data-acquisition systems. These systems will need to transport and process many terabits of data every second, in some cases with tight latency constraints. Parallelisation and tight integration of accelerators and classical CPU via Intel's OmniPath fabric are the key elements in this project.

  6. Generating barcoded libraries for multiplex high-throughput sequencing.

    Science.gov (United States)

    Knapp, Michael; Stiller, Mathias; Meyer, Matthias

    2012-01-01

    Molecular barcoding is an essential tool to use the high throughput of next generation sequencing platforms optimally in studies involving more than one sample. Various barcoding strategies allow for the incorporation of short recognition sequences (barcodes) into sequencing libraries, either by ligation or polymerase chain reaction (PCR). Here, we present two approaches optimized for generating barcoded sequencing libraries from low copy number extracts and amplification products typical of ancient DNA studies.

  7. Systematic error detection in experimental high-throughput screening

    OpenAIRE

    2011-01-01

    Abstract Background High-throughput screening (HTS) is a key part of the drug discovery process during which thousands of chemical compounds are screened and their activity levels measured in order to identify potential drug candidates (i.e., hits). Many technical, procedural or environmental factors can cause systematic measurement error or inequalities in the conditions in which the measurements are taken. Such systematic error has the potential to critically affect the hit selection proces...

  8. Mass spectrometry for high-throughput metabolomics analysis of urine

    OpenAIRE

    Abdelrazig, Salah M.A.

    2015-01-01

    Direct electrospray ionisation-mass spectrometry (direct ESI-MS), by omitting the chromatographic step, has great potential for application as a high-throughput approach for untargeted urine metabolomics analysis compared to liquid chromatography-mass spectrometry (LC-MS). The rapid development and technical innovations revealed in the field of ambient ionisation MS such as nanoelectrospray ionisation (nanoESI) chip-based infusion and liquid extraction surface analysis mass spectrometry (LESA...

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

    Science.gov (United States)

    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/.

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

    Directory of Open Access Journals (Sweden)

    Kent Edward

    2012-07-01

    Full Text Available Abstract 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.

  11. High-throughput bioinformatics with the Cyrille2 pipeline system

    Directory of Open Access Journals (Sweden)

    de Groot Joost CW

    2008-02-01

    Full Text Available Abstract Background Modern omics research involves the application of high-throughput technologies that generate vast volumes of data. These data need to be pre-processed, analyzed and integrated with existing knowledge through the use of diverse sets of software tools, models and databases. The analyses are often interdependent and chained together to form complex workflows or pipelines. Given the volume of the data used and the multitude of computational resources available, specialized pipeline software is required to make high-throughput analysis of large-scale omics datasets feasible. Results We have developed a generic pipeline system called Cyrille2. The system is modular in design and consists of three functionally distinct parts: 1 a web based, graphical user interface (GUI that enables a pipeline operator to manage the system; 2 the Scheduler, which forms the functional core of the system and which tracks what data enters the system and determines what jobs must be scheduled for execution, and; 3 the Executor, which searches for scheduled jobs and executes these on a compute cluster. Conclusion The Cyrille2 system is an extensible, modular system, implementing the stated requirements. Cyrille2 enables easy creation and execution of high throughput, flexible bioinformatics pipelines.

  12. A microdroplet dilutor for high-throughput screening

    Science.gov (United States)

    Niu, Xize; Gielen, Fabrice; Edel, Joshua B.; Demello, Andrew J.

    2011-06-01

    Pipetting and dilution are universal processes used in chemical and biological laboratories to assay and experiment. In microfluidics such operations are equally in demand, but difficult to implement. Recently, droplet-based microfluidics has emerged as an exciting new platform for high-throughput experimentation. However, it is challenging to vary the concentration of droplets rapidly and controllably. To this end, we developed a dilution module for high-throughput screening using droplet-based microfluidics. Briefly, a nanolitre-sized sample droplet of defined concentration is trapped within a microfluidic chamber. Through a process of droplet merging, mixing and re-splitting, this droplet is combined with a series of smaller buffer droplets to generate a sequence of output droplets that define a digital concentration gradient. Importantly, the formed droplets can be merged with other reagent droplets to enable rapid chemical and biological screens. As a proof of concept, we used the dilutor to perform a high-throughput homogeneous DNA-binding assay using only nanolitres of sample.

  13. FLASH assembly of TALENs for high-throughput genome editing.

    Science.gov (United States)

    Reyon, Deepak; Tsai, Shengdar Q; Khayter, Cyd; Foden, Jennifer A; Sander, Jeffry D; Joung, J Keith

    2012-05-01

    Engineered transcription activator–like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published, and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the fast ligation-based automatable solid-phase high-throughput (FLASH) system, a rapid and cost-effective method for large-scale assembly of TALENs. We tested 48 FLASH-assembled TALEN pairs in a human cell–based EGFP reporter system and found that all 48 possessed efficient gene-modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with other genome modification technologies.

  14. High Throughput and High Speed Organic Synthesis in Drug Discovery:An Integration of Chemistry and Technology

    Institute of Scientific and Technical Information of China (English)

    Li Chen

    2004-01-01

    Drug discovery is a complicated process that involves multiple synthetic chemistry tasks.Among them, lead generation and optimization is the core business in the discovery research.During the stage of lead generation, a large library of many thousands individual compounds will be screened against a biological target to identify a set of hits that showed desirable activity. Once a hit has been identified, analog synthesis and development of SAR around this hit and establishment of relationship between targeted protein and its cellular function become the key activity in the drug discovery project. During this process, focused libraries of a few hundred compounds (X00) will be synthesized and tested. For synthetic chemistry tasks, the efficiency can be enhanced through "parallel processing or high throughput chemisay" using automation technology with which a large number of compounds can be synthesized, purified and plated at same time for biological and pharmaceutical screenings. To accelerate the lead optimization high speed synthesis is conducted when necessary and to improve "single process" efficiency using automation technology, when the rate limiting step become developing synthetic method to produce large amount of compounds (in gram quantity) that can be used in vivo pharmacology study. With these in mind, we designed integrated HTC technology platform which supports the key chemical synthesis activities in discovery research. In this presentation, several examples will be presented to illustrate the applications of high throughput and high speed organic synthesis in drug discovery.

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

    Science.gov (United States)

    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.

  16. Discovery of novel targets with high throughput RNA interference screening.

    Science.gov (United States)

    Kassner, Paul D

    2008-03-01

    High throughput technologies have the potential to affect all aspects of drug discovery. Considerable attention is paid to high throughput screening (HTS) for small molecule lead compounds. The identification of the targets that enter those HTS campaigns had been driven by basic research until the advent of genomics level data acquisition such as sequencing and gene expression microarrays. Large-scale profiling approaches (e.g., microarrays, protein analysis by mass spectrometry, and metabolite profiling) can yield vast quantities of data and important information. However, these approaches usually require painstaking in silico analysis and low-throughput basic wet-lab research to identify the function of a gene and validate the gene product as a potential therapeutic drug target. Functional genomic screening offers the promise of direct identification of genes involved in phenotypes of interest. In this review, RNA interference (RNAi) mediated loss-of-function screens will be discussed and as well as their utility in target identification. Some of the genes identified in these screens should produce similar phenotypes if their gene products are antagonized with drugs. With a carefully chosen phenotype, an understanding of the biology of RNAi and appreciation of the limitations of RNAi screening, there is great potential for the discovery of new drug targets.

  17. High-throughput Accurate-wavelength Lens-based Visible Spectrometera

    Energy Technology Data Exchange (ETDEWEB)

    Ronald E. Belll and Filippo Scotti

    2010-06-04

    A scanning visible spectrometer has been prototyped to complement fixed-wavelength transmission grating spectrometers for charge exchange recombination spectroscopy. Fast f/1.8 200 mm commercial lenses are used with a large 2160 mm-1 grating for high throughput. A stepping-motor controlled sine drive positions the grating, which is mounted on a precision rotary table. A high-resolution optical encoder on the grating stage allows the grating angle to be measured with an absolute accuracy of 0.075 arcsec, corresponding to a wavelength error ≤ 0.005 Å. At this precision, changes in grating groove density due to thermal expansion and variations in the refractive index of air are important. An automated calibration procedure determines all relevant spectrometer parameters to high accuracy. Changes in bulk grating temperature, atmospheric temperature and pressure are monitored between the time of calibration and the time of measurement to insure a persistent wavelength calibration

  18. Drug Discovery Goes Three-Dimensional: Goodbye to Flat High-Throughput Screening?

    Science.gov (United States)

    Eglen, Richard M; Randle, David H

    2015-06-01

    Immortalized cells, generated from two-dimensional cell culture techniques, are widely used in compound screening, lead optimization, and drug candidate selection. However, such cells lack many characteristics of cells in vivo. This could account for the high failure rates of lead candidates in clinical evaluation. New approaches from cell biology, materials science, and bioengineering are increasing the utility of three-dimensional (3D) culture. These approaches have become more compatible with automation and, thus, provide more physiologically relevant cells for high-throughput/high-content screening, notably in oncology drug discovery. Techniques range from simple 3D spheroids, comprising one or more cell types, to complex multitissue organoids cultured in extracellular matrix gels or microfabricated chips. Furthermore, each approach can be applied to stem cells, such as induced pluripotent stem cells, thereby providing additional phenotypic relevance and the exciting potential to enable screening in disease-specific cell types.

  19. A production throughput forecasting system in an automated hard disk drive test operation using GRNN

    Directory of Open Access Journals (Sweden)

    Nara Samattapapong

    2016-04-01

    Originality/value: The production throughput volume is a key performance index of hard disk drive manufacturing systems that need to be forecast. Because of the production throughput forecasting result is useful information for management team to respond to any changing in production processes and resources allocation. However, a practically forecasting system for production throughput has not been described in detail yet. The experiments were conducted on a real data set from the final testing operation of hard disk drive manufacturing factory by using Visual Basics Application on Microsoft Excel© to develop preliminary forecasting system on testing and verification process. The experimental result shows that the proposed model is superior to the performance of the current forecasting system.

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

    Science.gov (United States)

    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.

  1. SSFinder: High Throughput CRISPR-Cas Target Sites Prediction Tool

    Directory of Open Access Journals (Sweden)

    Santosh Kumar Upadhyay

    2014-01-01

    Full Text Available 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.

  2. HTRF(®): pioneering technology for high-throughput screening.

    Science.gov (United States)

    Degorce, François

    2006-12-01

    Cisbio international pioneered the field of homogeneous fluorescence methodologies and time-resolved fluorescence resonance in particular, through its proprietary technology, HTRF(®). The development was based on Prof. Jean-Marie Lehn's research on rare earth fluorescence properties (awarded the Nobel Prize in Chemistry in 1987) and on Cisbio's expertise in homogenous time-resolved fluorescence (HTRF). The technology is used in assay development and drug screening, most notably in high-throughput screening applications. This highly powerful technology is particularly applied to the areas of G-protein-coupled receptor and kinase screening, as well as a series of targets related to inflammation, metabolic diseases and CNS disorders.

  3. Receptor-based high-throughput screening and identification of estrogens in dietary supplements using bioaffinity liquid-chromatography ion mobility mass spectrometry

    NARCIS (Netherlands)

    Aqai, P.; Gómez Blesa, N.; Major, H.; Pedotti, P.; Varani, L.; Ferrero, V.E.V.; Haasnoot, W.; Nielen, M.W.F.

    2013-01-01

    A high-throughput bioaffinity liquid chromatography-mass spectrometry (BioMS) approach was developed and applied for the screening and identification of recombinant human estrogen receptor a (ERa) ligands in dietary supplements. For screening, a semi-automated mass spectrometric ligand binding assay

  4. An Automatic Quality Control Pipeline for High-Throughput Screening Hit Identification.

    Science.gov (United States)

    Zhai, Yufeng; Chen, Kaisheng; Zhong, Yang; Zhou, Bin; Ainscow, Edward; Wu, Ying-Ta; Zhou, Yingyao

    2016-09-01

    The correction or removal of signal errors in high-throughput screening (HTS) data is critical to the identification of high-quality lead candidates. Although a number of strategies have been previously developed to correct systematic errors and to remove screening artifacts, they are not universally effective and still require fair amount of human intervention. We introduce a fully automated quality control (QC) pipeline that can correct generic interplate systematic errors and remove intraplate random artifacts. The new pipeline was first applied to ~100 large-scale historical HTS assays; in silico analysis showed auto-QC led to a noticeably stronger structure-activity relationship. The method was further tested in several independent HTS runs, where QC results were sampled for experimental validation. Significantly increased hit confirmation rates were obtained after the QC steps, confirming that the proposed method was effective in enriching true-positive hits. An implementation of the algorithm is available to the screening community.

  5. High-throughput retrotransposon-based fluorescent markers: improved information content and allele discrimination

    Directory of Open Access Journals (Sweden)

    Baker David

    2009-07-01

    Full Text Available Abstract Background Dense genetic maps, together with the efficiency and accuracy of their construction, are integral to genetic studies and marker assisted selection for plant breeding. High-throughput multiplex markers that are robust and reproducible can contribute to both efficiency and accuracy. Multiplex markers are often dominant and so have low information content, this coupled with the pressure to find alternatives to radio-labelling, has led us to adapt the SSAP (sequence specific amplified polymorphism marker method from a 33P labelling procedure to fluorescently tagged markers analysed from an automated ABI 3730 xl platform. This method is illustrated for multiplexed SSAP markers based on retrotransposon insertions of pea and is applicable for the rapid and efficient generation of markers from genomes where repetitive element sequence information is available for primer design. We cross-reference SSAP markers previously generated using the 33P manual PAGE system to fluorescent peaks, and use these high-throughput fluorescent SSAP markers for further genetic studies in Pisum. Results The optimal conditions for the fluorescent-labelling method used a triplex set of primers in the PCR. These included a fluorescently labelled specific primer together with its unlabelled counterpart, plus an adapter-based primer with two bases of selection on the 3' end. The introduction of the unlabelled specific primer helped to optimise the fluorescent signal across the range of fragment sizes expected, and eliminated the need for extensive dilutions of PCR amplicons. The software (GeneMarker Version 1.6 used for the high-throughput data analysis provided an assessment of amplicon size in nucleotides, peak areas and fluorescence intensity in a table format, so providing additional information content for each marker. The method has been tested in a small-scale study with 12 pea accessions resulting in 467 polymorphic fluorescent SSAP markers of which

  6. An integrated bioanalytical platform for supporting high-throughput serum protein binding screening.

    Science.gov (United States)

    Zhang, Jun; Shou, Wilson Z; Vath, Marianne; Kieltyka, Kasia; Maloney, Jennifer; Elvebak, Larry; Stewart, Jeremy; Herbst, John; Weller, Harold N

    2010-12-30

    Quantification of small molecules using liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a triple quadrupole mass spectrometer has become a common practice in bioanalytical support of in vitro adsorption, distribution, metabolism and excretion (ADME) screening. The bioanalysis process involves primarily three indispensable steps: MS/MS optimization for a large number of new chemical compounds undergoing various screening assays in early drug discovery, high-throughput sample analysis with LC/MS/MS for those chemically diverse compounds using the optimized MS/MS conditions, and post-acquisition data review and reporting. To improve overall efficiency of ADME bioanalysis, an integrated system was proposed featuring an automated and unattended MS/MS optimization, a staggered parallel LC/MS/MS for high-throughput sample analysis, and a sophisticated software tool for LC/MS/MS raw data review as well as biological data calculation and reporting. The integrated platform has been used in bioanalytical support of a serum protein binding screening assay with high speed, high capacity, and good robustness. In this new platform, a unique sample dilution scheme was also introduced. With this dilution design, the total number of analytical samples was reduced; therefore, the total operation time was reduced and the overall throughput was further improved. The performance of the protein binding screening assay was monitored with two controls representing high and low binding properties and an acceptable inter-assay consistency was achieved. This platform has been successfully used for the determination of serum protein binding in multiple species for more than 4000 compounds.

  7. High-throughput screening to enhance oncolytic virus immunotherapy

    Directory of Open Access Journals (Sweden)

    Allan KJ

    2016-04-01

    Full Text Available KJ Allan,1,2 David F Stojdl,1–3 SL Swift1 1Children’s Hospital of Eastern Ontario (CHEO Research Institute, 2Department of Biology, Microbiology and Immunology, 3Department of Pediatrics, University of Ottawa, Ottawa, ON, Canada Abstract: 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. Keywords: oncolytic, virus, screen, high-throughput, cancer, chemical, genomic, immunotherapy

  8. Spectrophotometric Enzyme Assays for High-Throughput Screening

    Directory of Open Access Journals (Sweden)

    Jean-Louis Reymond

    2004-01-01

    Full Text Available This paper reviews high-throughput screening enzyme assays developed in our laboratory over the last ten years. These enzyme assays were initially developed for the purpose of discovering catalytic antibodies by screening cell culture supernatants, but have proved generally useful for testing enzyme activities. Examples include TLC-based screening using acridone-labeled substrates, fluorogenic assays based on the β-elimination of umbelliferone or nitrophenol, and indirect assays such as the back-titration method with adrenaline and the copper-calcein fluorescence assay for aminoacids.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. A High-Throughput SU-8Microfluidic Magnetic Bead Separator

    DEFF Research Database (Denmark)

    Bu, Minqiang; Christensen, T. B.; Smistrup, Kristian

    2007-01-01

    We present a novel microfluidic magnetic bead separator based on SU-8 fabrication technique for high through-put applications. The experimental results show that magnetic beads can be captured at an efficiency of 91 % and 54 % at flow rates of 1 mL/min and 4 mL/min, respectively. Integration...... of soft magnetic elements in the chip leads to a slightly higher capturing efficiency and a more uniform distribution of captured beads over the separation chamber than the system without soft magnetic elements....

  11. High-throughput synthesis and analysis of acylated cyanohydrins.

    Science.gov (United States)

    Hamberg, Anders; Lundgren, Stina; Wingstrand, Erica; Moberg, Christina; Hult, Karl

    2007-01-01

    The yields and optical purities of products obtained from chiral Lewis acid/Lewis base-catalysed additions of alpha-ketonitriles to prochiral aldehydes could be accurately determined by an enzymatic method. The amount of remaining aldehyde was determined after its reduction to an alcohol, whilst the two product enantiomers were analysed after subsequent hydrolysis first by the (S)-selective Candida antarctica lipase B and then by the unselective pig liver esterase. The method could be used for analysis of products obtained from a number of aromatic aldehydes and aliphatic ketonitriles. Microreactor technology was successfully combined with high-throughput analysis for efficient catalyst optimization.

  12. Adaptive Sampling for High Throughput Data Using Similarity Measures

    Energy Technology Data Exchange (ETDEWEB)

    Bulaevskaya, V. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Sales, A. P. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    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.

  13. High-throughput epitope identification for snakebite antivenom

    DEFF Research Database (Denmark)

    Engmark, Mikael; De Masi, Federico; Laustsen, Andreas Hougaard

    Insight into the epitopic recognition pattern for polyclonal antivenoms is a strong tool for accurate prediction of antivenom cross-reactivity and provides a basis for design of novel antivenoms. In this work, a high-throughput approach was applied to characterize linear epitopes in 966 individua...... toxins from pit vipers (Crotalidae) using the ICP Crotalidae antivenom. Due to an abundance of snake venom metalloproteinases and phospholipase A2s in the venoms used for production of the investigated antivenom, this study focuses on these toxin families....

  14. High-Throughput Continuous Flow Synthesis of Nickel Nanoparticles for the Catalytic Hydrodeoxygenation of Guaiacol

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, Emily J.; Habas, Susan E.; Wang, Lu; Ruddy, Daniel A.; White, Erick A.; Baddour, Frederick G.; Griffin, Michael B.; Schaidle, Joshua A.; Malmstadt, Noah; Brutchey, Richard L.

    2016-11-07

    The translation of batch chemistries to high-throughput continuous flow methods dresses scaling, automation, and reproducibility concerns associated with the implementation of colloidally prepared nanoparticle (NP) catalysts for industrial catalytic processes. Nickel NPs were synthesized by the high-temperature amine reduction of a Ni2+ precursor using a continuous millifluidic (mF) flow method, achieving yields greater than 60%. The resulting Ni NP catalysts were compared against catalysts prepared in a batch reaction under conditions analogous to the continuous flow conditions with respect to total reaction volume, time, and temperature and by traditional incipient wetness (IW) impregnation for the hydrodeoxygenation (HDO) of guaiacol under ex situ catalytic fast pyrolysis conditions. Compared to the IW method, the colloidally prepared NPs displayed increased morphological control and narrowed size distributions, and the NPs prepared by both methods showed similar size, shape, and crystallinity. The Ni NP catalyst synthesized by the continuous flow method exhibited similar H-adsorption site densities, site-time yields, and selectivities towards deoxygenated products as compared to the analogous batch reaction, and outperformed the IW catalyst with respect to higher selectivity to lower oxygen content products and a 6.9-fold slower deactivation rate. These results demonstrate the utility of synthesizing colloidal Ni NP catalysts using continuous flow methods while maintaining the catalytic properties displayed by the batch equivalent. This methodology can be extended to other catalytically relevant base metals for the high-throughput synthesis of metal NPs for the catalytic production of biofuels.

  15. A BSL-4 high-throughput screen identifies sulfonamide inhibitors of Nipah virus.

    Science.gov (United States)

    Tigabu, Bersabeh; Rasmussen, Lynn; White, E Lucile; Tower, Nichole; Saeed, Mohammad; Bukreyev, Alexander; Rockx, Barry; LeDuc, James W; Noah, James W

    2014-04-01

    Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 μM and selectivities >10. Three sulfonamide compounds with EC50 values BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses.

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

    Directory of Open Access Journals (Sweden)

    Xiao-Lin eWu

    2011-02-01

    Full Text Available 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 (GPU 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 (CPU 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

  17. New high-throughput methods of investigating polymer electrolytes

    Science.gov (United States)

    Alcock, Hannah J.; White, Oliver C.; Jegelevicius, Grazvydas; Roberts, Matthew R.; Owen, John R.

    2011-03-01

    Polymer electrolyte films have been prepared by solution casting techniques from precursor solutions of a poly(vinylidene fluoride-co-hexafluoropropylene) (PVdF-HFP), lithium-bis(trifluoromethane) sulfonimide (LiTFSI), and propylene carbonate (PC). Arrays of graded composition were characterised by electrochemical impedance spectroscopy (EIS), differential scanning calorimetry (DSC) and X-ray diffraction (XRD) using high throughput techniques. Impedance analysis showed the resistance of the films as a function of LiTFSI, PC and polymer content. The ternary plot of conductivity shows an area that combines a solid-like mechanical stability with high conductivity, 1 × 10-5 S cm-1 at the composition 0.55/0.15/0.30 wt% PVdF-HFP/LiTFSI/PC, increasing with PC content. In regions with less than a 50 wt% fraction of PVdF-HFP the films were too soft to give meaningful results by this method. The DSC measurements on solvent free, salt-doped polymers show a reduced crystallinity, and high throughput XRD patterns show that non-polar crystalline phases are suppressed by the presence of LiTFSI and PC.

  18. High throughput discovery of new fouling-resistant surfaces†

    Science.gov (United States)

    Zhou, Mingyan; Liu, Hongwei; Venkiteshwaran, Adith; Kilduff, James; Anderson, Daniel G.; Langer, Robert; Belfort, Georges

    2017-01-01

    A novel high throughput method for synthesis and screening of customized protein-resistant surfaces was developed. This method is an inexpensive, fast, reproducible and scalable approach to synthesize and screen protein-resistant surfaces appropriate for a specific feed. The method is illustrated here by combining a high throughput platform (HTP) approach together with our patented photo-induced graft polymerization (PGP) method developed for facile modification of commercial poly(aryl sulfone) membranes. We demonstrate that the HTP–PGP approach to synthesize and screen fouling-resistant surfaces is general, and thus provides the capability to develop surfaces optimized for specific feeds. Surfaces were prepared via graft polymerization onto poly(ether sulfone) (PES) membranes and were evaluated using a protein adsorption assay followed by pressure-driven filtration. We have employed the HTP–PGP approach to confirm previously reported successful monomers and to develop new antifouling surfaces from a library of 66 monomers for four different challenges of interest to the biotechnology community: hen egg-white lysozyme, supernatant from Chinese Hamster Ovary (CHO) cells in phosphate buffered saline (PBS) solution as a model cell suspension, and immunoglobulin G (IgG) precipitated in the absence and presence of bovine serum albumin (BSA) in high salt solution as a model precipitation process.

  19. High-throughput fragment screening by affinity LC-MS.

    Science.gov (United States)

    Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

    2013-02-01

    Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in 3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K (D)) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.

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

    Science.gov (United States)

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

    2016-05-31

    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.

  1. Benchmarking procedures for high-throughput context specific reconstruction algorithms

    Directory of Open Access Journals (Sweden)

    Maria ePires Pacheco

    2016-01-01

    Full Text Available 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 (Duarte et al., 2007; Thiele et al., 2013 or HMR (Agren et al., 2013 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 ten 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 former includes methods like cross validation or testing with artificial networks. 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

  2. Advances in Automation and Throughput of the Mars Organic Analyzer Microchip Capillary Electrophoresis System

    Science.gov (United States)

    Haldeman, B. J.; Skelley, A. M.; Scherer, J. R.; Jayarajah, C.; Mathies, R. A.

    2005-12-01

    We have previously demonstrated the design, construction and testing of a portable microchip capillary electrophoresis (CE) instrument called the Mars Organic Analyzer (MOA) for analysis of amino acids and amine containing organic molecules (1). This instrument is designed to accept organic compounds isolated from samples by sublimation or by subcritical water extraction, to label the amine groups with fluorescamine, and to perform high resolution electrophoretic analysis. The CE instrument has shown remarkable robustness during successful field tests last year in the Panoche Valley, CA (1) and more recently in the Atacama Desert, Chile (2). For successful operation on Mars, however, it is necessary to operate autonomously and to analyze large numbers of samples, blanks, and standards. Toward this end we present here two advances in the MOA system that test key aspects of an eventual flight prototype. First, we have developed an automated microfluidic system and method for the autonomous loading, running and cleaning of the CE chip on the single channel MOA instrument. The integration of microfabricated PDMS valves and pumps with all-glass separation channels in a multilayer design enabled creation of structures for complex fluidic routing. Twenty sequential analyses of an amino acid standard were performed with an automated cleaning procedure between runs. In addition, dilutions were performed on-chip, and blanks were run to demonstrate the elimination of carry-over from run to run. These results demonstrate an important advance of the technology readiness level of the MOA. Second, we have designed, constructed and successfully tested a lab version of the multichannel instrument we initially proposed for the MSL opportunity. The portable Multi-Channel Mars Organic Analyzer (McMOA, 25 by 30 by 15 cm), was designed to sequentially interrogate eight radially oriented CE separation channels on a single wafer. Since each channel can be used to analyze 20 or more

  3. Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus

    DEFF Research Database (Denmark)

    Urbanski, Dorian Fabian; Malolepszy, Anna; Stougaard, Jens;

    2012-01-01

    and insertion site identification. So far, only floral dip T-DNA transformation of Arabidopsis has produced independent germinal insertions, thereby allowing generation of mutant populations from seeds of single plants. In addition, advances in insertion detection have been hampered by a lack of protocols...... including software for automated data analysis, which take full advantage of high next-generation sequencing throughput. Here we address these challenges by developing the FSTpoolit protocol and software package and we demonstrate its efficacy by detecting 8,935 LORE1 insertions in 3,744 Lotus japonicus...... plants. The identified insertions showed that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to its homogenous gene targeting and exonic insertion preference. Since LORE1 transposition occurs in the germline, harvesting seeds from a single founder line and cultivating...

  4. High-throughput optofluidic system for the laser microsurgery of oocytes

    Science.gov (United States)

    Chandsawangbhuwana, Charlie; Shi, Linda Z.; Zhu, Qingyuan; Alliegro, Mark C.; Berns, Michael W.

    2012-01-01

    This study combines microfluidics with optical microablation in a microscopy system that allows for high-throughput manipulation of oocytes, automated media exchange, and long-term oocyte observation. The microfluidic component of the system transports oocytes from an inlet port into multiple flow channels. Within each channel, oocytes are confined against a microfluidic barrier using a steady fluid flow provided by an external computer-controlled syringe pump. This allows for easy media replacement without disturbing the oocyte location. The microfluidic and optical-laser microbeam ablation capabilities of the system were validated using surf clam (Spisula solidissima) oocytes that were immobilized in order to permit ablation of the 5 μm diameter nucleolinus within the oocyte nucleolus. Oocytes were the followed and assayed for polar body ejection.

  5. High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana

    DEFF Research Database (Denmark)

    Bach, Søren Spanner; Bassard, Jean-Étienne André; Andersen-Ranberg, Johan;

    2014-01-01

    To respond to the rapidly growing number of genes putatively involved in terpenoid metabolism, a robust high-throughput platform for functional testing is needed. An in planta expression system offers several advantages such as the capacity to produce correctly folded and active enzymes localized...... describe an expression platform for rapid testing of candidate terpenoid biosynthetic genes based on Agrobacterium mediated gene expression in N. benthamiana leaves. Simultaneous expression of multiple genes is facilitated by co-infiltration of leaves with several engineered Agrobacterium strains, possibly...... making this the fastest and most convenient system for the assembly of plant terpenoid biosynthetic routes. Tools for cloning of expression plasmids, N. benthamiana culturing, Agrobacterium preparation, leaf infiltration, metabolite extraction, and automated GC-MS data mining are provided. With all steps...

  6. Robotic high-throughput purification of affinity-tagged recombinant proteins.

    Science.gov (United States)

    Wiesler, Simone C; Weinzierl, Robert O J

    2015-01-01

    Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories.

  7. Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

    DEFF Research Database (Denmark)

    Lundqvist, Magnus; Edfors, Fredrik; Sivertsson, Åsa;

    2015-01-01

    We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We...... present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments...... is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts...

  8. A High-Throughput Method for the Analysis of Larval Developmental Phenotypes in Caenorhabditis elegans.

    Science.gov (United States)

    Olmedo, María; Geibel, Mirjam; Artal-Sanz, Marta; Merrow, Martha

    2015-10-01

    Caenorhabditis elegans postembryonic development consists of four discrete larval stages separated by molts. Typically, the speed of progression through these larval stages is investigated by visual inspection of the molting process. Here, we describe an automated method to monitor the timing of these discrete phases of C. elegans maturation, from the first larval stage through adulthood, using bioluminescence. The method was validated with a lin-42 mutant strain that shows delayed development relative to wild-type animals and with a daf-2 mutant that shows an extended second larval stage. This new method is inherently high-throughput and will finally allow dissecting the molecular machinery governing the speed of the developmental clock, which has so far been hampered by the lack of a method suitable for genetic screens.

  9. High-throughput screening to enhance oncolytic virus immunotherapy

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  11. Evaluation of a high throughput starch analysis optimised for wood.

    Directory of Open Access Journals (Sweden)

    Chandra Bellasio

    Full Text Available 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.

  12. Evaluation of a high throughput starch analysis optimised for wood.

    Science.gov (United States)

    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.

  13. A scalable approach for high throughput branch flow filtration.

    Science.gov (United States)

    Inglis, David W; Herman, Nick

    2013-05-07

    Microfluidic continuous flow filtration methods have the potential for very high size resolution using minimum feature sizes that are larger than the separation size, thereby circumventing the problem of clogging. Branch flow filtration is particularly promising because it has an unlimited dynamic range (ratio of largest passable particle to the smallest separated particle) but suffers from very poor volume throughput because when many branches are used, they cannot be identical if each is to have the same size cut-off. We describe a new iterative approach to the design of branch filtration devices able to overcome this limitation without large dead volumes. This is demonstrated by numerical modelling, fabrication and testing of devices with 20 branches, with dynamic ranges up to 6.9, and high filtration ratios (14-29%) on beads and fungal spores. The filters have a sharp size cutoff (10× depletion for 12% size difference), with large particle rejection equivalent to a 20th order Butterworth low pass filter. The devices are fully scalable, enabling higher throughput and smaller cutoff sizes and they are compatible with ultra low cost fabrication.

  14. Fluorescent foci quantitation for high-throughput analysis

    Science.gov (United States)

    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

  15. MassCode liquid arrays as a tool for multiplexed high-throughput genetic profiling.

    Directory of Open Access Journals (Sweden)

    Gregory S Richmond

    Full Text Available Multiplexed detection assays that analyze a modest number of nucleic acid targets over large sample sets are emerging as the preferred testing approach in such applications as routine pathogen typing, outbreak monitoring, and diagnostics. However, very few DNA testing platforms have proven to offer a solution for mid-plexed analysis that is high-throughput, sensitive, and with a low cost per test. In this work, an enhanced genotyping method based on MassCode technology was devised and integrated as part of a high-throughput mid-plexing analytical system that facilitates robust qualitative differential detection of DNA targets. Samples are first analyzed using MassCode PCR (MC-PCR performed with an array of primer sets encoded with unique mass tags. Lambda exonuclease and an array of MassCode probes are then contacted with MC-PCR products for further interrogation and target sequences are specifically identified. Primer and probe hybridizations occur in homogeneous solution, a clear advantage over micro- or nanoparticle suspension arrays. The two cognate tags coupled to resultant MassCode hybrids are detected in an automated process using a benchtop single quadrupole mass spectrometer. The prospective value of using MassCode probe arrays for multiplexed bioanalysis was demonstrated after developing a 14plex proof of concept assay designed to subtype a select panel of Salmonella enterica serogroups and serovars. This MassCode system is very flexible and test panels can be customized to include more, less, or different markers.

  16. A grid algorithm for high throughput fitting of dose-response curve data.

    Science.gov (United States)

    Wang, Yuhong; Jadhav, Ajit; Southal, Noel; Huang, Ruili; Nguyen, Dac-Trung

    2010-10-21

    We describe a novel algorithm, Grid algorithm, and the corresponding computer program for high throughput fitting of dose-response curves that are described by the four-parameter symmetric logistic dose-response model. The Grid algorithm searches through all points in a grid of four dimensions (parameters) and finds the optimum one that corresponds to the best fit. Using simulated dose-response curves, we examined the Grid program's performance in reproducing the actual values that were used to generate the simulated data and compared it with the DRC package for the language and environment R and the XLfit add-in for Microsoft Excel. The Grid program was robust and consistently recovered the actual values for both complete and partial curves with or without noise. Both DRC and XLfit performed well on data without noise, but they were sensitive to and their performance degraded rapidly with increasing noise. The Grid program is automated and scalable to millions of dose-response curves, and it is able to process 100,000 dose-response curves from high throughput screening experiment per CPU hour. The Grid program has the potential of greatly increasing the productivity of large-scale dose-response data analysis and early drug discovery processes, and it is also applicable to many other curve fitting problems in chemical, biological, and medical sciences.

  17. High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification

    Directory of Open Access Journals (Sweden)

    Sun Zhenyu

    2001-08-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation. Results SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe. Conclusions Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.

  18. SNP high-throughput screening in grapevine using the SNPlex™ genotyping system

    Directory of Open Access Journals (Sweden)

    Velasco Riccardo

    2008-01-01

    Full Text Available Abstract Background Until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism (SNP discovery and genotyping in grapevine (Vitis vinifera L.. However, following completion of the sequence of the highly heterozygous genome of Pinot Noir, it has been possible to identify millions of electronic SNPs (eSNPs thus providing a valuable source for high-throughput genotyping methods. Results Herein we report the first application of the SNPlex™ genotyping system in grapevine aiming at the anchoring of an eukaryotic genome. This approach combines robust SNP detection with automated assay readout and data analysis. 813 candidate eSNPs were developed from non-repetitive contigs of the assembled genome of Pinot Noir and tested in 90 progeny of Syrah × Pinot Noir cross. 563 new SNP-based markers were obtained and mapped. The efficiency rate of 69% was enhanced to 80% when multiple displacement amplification (MDA methods were used for preparation of genomic DNA for the SNPlex assay. Conclusion Unlike other SNP genotyping methods used to investigate thousands of SNPs in a few genotypes, or a few SNPs in around a thousand genotypes, the SNPlex genotyping system represents a good compromise to investigate several hundred SNPs in a hundred or more samples simultaneously. Therefore, the use of the SNPlex assay, coupled with whole genome amplification (WGA, is a good solution for future applications in well-equipped laboratories.

  19. High-Throughput Peptide Epitope Mapping Using Carbon Nanotube Field-Effect Transistors

    Directory of Open Access Journals (Sweden)

    Steingrimur Stefansson

    2013-01-01

    Full Text Available Label-free and real-time detection technologies can dramatically reduce the time and cost of pharmaceutical testing and development. However, to reach their full promise, these technologies need to be adaptable to high-throughput automation. To demonstrate the potential of single-walled carbon nanotube field-effect transistors (SWCNT-FETs for high-throughput peptide-based assays, we have designed circuits arranged in an 8 × 12 (96-well format that are accessible to standard multichannel pipettors. We performed epitope mapping of two HIV-1 gp160 antibodies using an overlapping gp160 15-mer peptide library coated onto nonfunctionalized SWCNTs. The 15-mer peptides did not require a linker to adhere to the non-functionalized SWCNTs, and binding data was obtained in real time for all 96 circuits. Despite some sequence differences in the HIV strains used to generate these antibodies and the overlapping peptide library, respectively, our results using these antibodies are in good agreement with known data, indicating that peptides immobilized onto SWCNT are accessible and that linear epitope mapping can be performed in minutes using SWCNT-FET.

  20. Solid-phase cloning for high-throughput assembly of single and multiple DNA parts.

    Science.gov (United States)

    Lundqvist, Magnus; Edfors, Fredrik; Sivertsson, Åsa; Hallström, Björn M; Hudson, Elton P; Tegel, Hanna; Holmberg, Anders; Uhlén, Mathias; Rockberg, Johan

    2015-04-20

    We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.

  1. High-throughput process development of chromatography steps: advantages and limitations of different formats used.

    Science.gov (United States)

    Łącki, Karol M

    2012-10-01

    In the past, development of a chromatographic separation method has been accomplished by performing a series of experiments using either manual or automated chromatography systems. The screening of a vast experimental space became very expensive because all experiments had to be performed in a serial manner, and the chromatography systems used were designed for relatively large columns and, therefore, the experiments required large sample volumes. To address these issues, high-throughput miniaturized methods employing different operating principles and/or formats have been introduced. Herein, a technical review of the most common high-throughput formats used for the development of chromatographic purification steps is presented. The formats considered include minicolumns, prefilled pipette tips, and microtiter filter plates prefilled with chromatography resins. Advantages and limitations of each format are discussed through the prism of chromatographic theory, engineering principles, and known mass-transfer mechanisms. A roadmap for applicability of the different formats for process development purposes and implementation of a Quality by Design initiative for designing/optimization of chromatography steps is also discussed.

  2. Automatic classification and pattern discovery in high-throughput protein crystallization trials.

    Science.gov (United States)

    Cumbaa, Christian; Jurisica, Igor

    2005-01-01

    Conceptually, protein crystallization can be divided into two phases search and optimization. Robotic protein crystallization screening can speed up the search phase, and has a potential to increase process quality. Automated image classification helps to increase throughput and consistently generate objective results. Although the classification accuracy can always be improved, our image analysis system can classify images from 1,536-well plates with high classification accuracy (85%) and ROC score (0.87), as evaluated on 127 human-classified protein screens containing 5,600 crystal images and 189,472 non-crystal images. Data mining can integrate results from high-throughput screens with information about crystallizing conditions, intrinsic protein properties, and results from crystallization optimization. We apply association mining, a data mining approach that identifies frequently occurring patterns among variables and their values. This approach segregates proteins into groups based on how they react in a broad range of conditions, and clusters cocktails to reflect their potential to achieve crystallization. These results may lead to crystallization screen optimization, and reveal associations between protein properties and crystallization conditions. We also postulate that past experience may lead us to the identification of initial conditions favorable to crystallization for novel proteins.

  3. High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization.

    Science.gov (United States)

    Holden, Seamus J; Pengo, Thomas; Meibom, Karin L; Fernandez Fernandez, Carmen; Collier, Justine; Manley, Suliana

    2014-03-25

    We created a high-throughput modality of photoactivated localization microscopy (PALM) that enables automated 3D PALM imaging of hundreds of synchronized bacteria during all stages of the cell cycle. We used high-throughput PALM to investigate the nanoscale organization of the bacterial cell division protein FtsZ in live Caulobacter crescentus. We observed that FtsZ predominantly localizes as a patchy midcell band, and only rarely as a continuous ring, supporting a model of "Z-ring" organization whereby FtsZ protofilaments are randomly distributed within the band and interact only weakly. We found evidence for a previously unidentified period of rapid ring contraction in the final stages of the cell cycle. We also found that DNA damage resulted in production of high-density continuous Z-rings, which may obstruct cytokinesis. Our results provide a detailed quantitative picture of in vivo Z-ring organization.

  4. A high volume, high throughput volumetric sorption analyzer

    Science.gov (United States)

    Soo, Y. C.; Beckner, M.; Romanos, J.; Wexler, C.; Pfeifer, P.; Buckley, P.; Clement, J.

    2011-03-01

    In this talk we will present an overview of our new Hydrogen Test Fixture (HTF) constructed by the Midwest Research Institute for The Alliance for Collaborative Research in Alternative Fuel Technology to test activated carbon monoliths for hydrogen gas storage. The HTF is an automated, computer-controlled volumetric instrument for rapid screening and manipulation of monoliths under an inert atmosphere (to exclude degradation of carbon from exposure to oxygen). The HTF allows us to measure large quantity (up to 500 g) of sample in a 0.5 l test tank, making our results less sensitive to sample inhomogeneity. The HTF can measure isotherms at pressures ranging from 1 to 300 bar at room temperature. For comparison, other volumetric instruments such as Hiden Isochema's HTP-1 Volumetric Analyser can only measure carbon samples up to 150 mg at pressures up to 200 bar. Work supported by the US DOD Contract # N00164-08-C-GS37.

  5. A high throughput screen for biomining cellulase activity from metagenomic libraries.

    Science.gov (United States)

    Mewis, Keith; Taupp, Marcus; Hallam, Steven J

    2011-02-01

    Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production (1). Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils (2), buffalo rumen (3) and the termite hind-gut (4) using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood (5)). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio (6). Other methods have been reported (7,8) but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate (9). Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction (10). The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

  6. Towards high-throughput microfluidic Raman-activated cell sorting.

    Science.gov (United States)

    Zhang, Qiang; Zhang, Peiran; Gou, Honglei; Mou, Chunbo; Huang, Wei E; Yang, Menglong; Xu, Jian; Ma, Bo

    2015-09-21

    Raman-activated cell sorting (RACS) is a promising single-cell analysis technology that is able to identify and isolate individual cells of targeted type, state or environment from an isogenic population or complex consortium of cells, in a label-free and non-invasive manner. However, compared with those widely used yet labeling-required or staining-dependent cell sorting technologies such as FACS and MACS, the weak Raman signal greatly limits the further development of the existing RACS systems to achieve higher throughput. Strategies that can tackle this bottleneck include, first, improvement of Raman-acquisition efficiency and quality based on advanced Raman spectrometers and enhanced Raman techniques; second, development of novel microfluidic devices for cell sorting followed by integration into a complete RACS system. Exploiting these strategies, prototypes for a new generation of RACS have been demonstrated, such as flow-based OT-RACS, DEP-RACS, and SERS/CARS flow cytometry. Such high-throughput microfluidic RACS can provide biologists with a powerful single-cell analysis tool to explore the scientific questions or applications that have been beyond the reach of FACS and MACS.

  7. High-throughput comet assay using 96 minigels.

    Science.gov (United States)

    Gutzkow, Kristine B; Langleite, Torgrim M; Meier, Silja; Graupner, Anne; Collins, Andrew R; Brunborg, Gunnar

    2013-05-01

    The single-cell gel electrophoresis--the comet assay--has proved to be a sensitive and relatively simple method that is much used in research for the analysis of specific types of DNA damage, and its use in genotoxicity testing is increasing. The efficiency of the comet assay, in terms of number of samples processed per experiment, has been rather poor, and both research and toxicological testing should profit from an increased throughput. We have designed and validated a format involving 96 agarose minigels supported by a hydrophilic polyester film. Using simple technology, hundreds of samples may be processed in one experiment by one person, with less time needed for processing, less use of chemicals and requiring fewer cells per sample. Controlled electrophoresis, including circulation of the electrophoresis solution, improves the homogeneity between replicate samples in the 96-minigel format. The high-throughput method described in this paper should greatly increase the overall capacity, versatility and robustness of the comet assay.

  8. High-throughput microcavitation bubble induced cellular mechanotransduction

    Science.gov (United States)

    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.

  9. FBI Fingerprint Image Capture System High-Speed-Front-End throughput modeling

    Energy Technology Data Exchange (ETDEWEB)

    Rathke, P.M.

    1993-09-01

    The Federal Bureau of Investigation (FBI) has undertaken a major modernization effort called the Integrated Automated Fingerprint Identification System (IAFISS). This system will provide centralized identification services using automated fingerprint, subject descriptor, mugshot, and document processing. A high-speed Fingerprint Image Capture System (FICS) is under development as part of the IAFIS program. The FICS will capture digital and microfilm images of FBI fingerprint cards for input into a central database. One FICS design supports two front-end scanning subsystems, known as the High-Speed-Front-End (HSFE) and Low-Speed-Front-End, to supply image data to a common data processing subsystem. The production rate of the HSFE is critical to meeting the FBI`s fingerprint card processing schedule. A model of the HSFE has been developed to help identify the issues driving the production rate, assist in the development of component specifications, and guide the evolution of an operations plan. A description of the model development is given, the assumptions are presented, and some HSFE throughput analysis is performed.

  10. An Efficient High Throughput Metabotyping Platform for Screening of Biomass Willows

    Directory of Open Access Journals (Sweden)

    Delia I. Corol

    2014-10-01

    Full Text Available Future improvement of woody biomass crops such as willow and poplar relies on our ability to select for metabolic traits that sequester more atmospheric carbon into biomass, or into useful products to replace petrochemical streams. We describe the development of metabotyping screens for willow, using combined 1D 1H-NMR-MS. A protocol was developed to overcome 1D 1H-NMR spectral alignment problems caused by variable pH and peak broadening arising from high organic acid levels and metal cations. The outcome was a robust method to allow direct statistical comparison of profiles arising from source (leaf and sink (stem tissues allowing data to be normalised to a constant weight of the soluble metabolome. We also describe the analysis of two willow biomass varieties, demonstrating how fingerprints from 1D 1H-NMR-MS vary from the top to the bottom of the plant. Automated extraction of quantitative data of 56 primary and secondary metabolites from 1D 1H-NMR spectra was realised by the construction and application of a Salix metabolite spectral library using the Chenomx software suite. The optimised metabotyping screen in conjunction with automated quantitation will enable high-throughput screening of genetic collections. It also provides genotype and tissue specific data for future modelling of carbon flow in metabolic networks.

  11. Numerical techniques for high-throughput reflectance interference biosensing

    Science.gov (United States)

    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.

  12. The Principals and Practice of Distributed High Throughput Computing

    CERN Document Server

    CERN. Geneva

    2016-01-01

    The potential of Distributed Processing Systems to deliver computing capabilities with qualities ranging from high availability and reliability to easy expansion in functionality and capacity were recognized and formalized in the 1970’s. For more three decade these principals Distributed Computing guided the development of the HTCondor resource and job management system. The widely adopted suite of software tools offered by HTCondor are based on novel distributed computing technologies and are driven by the evolving needs of High Throughput scientific applications. We will review the principals that underpin our work, the distributed computing frameworks and technologies we developed and the lessons we learned from delivering effective and dependable software tools in an ever changing landscape computing technologies and needs that range today from a desktop computer to tens of thousands of cores offered by commercial clouds. About the speaker Miron Livny received a B.Sc. degree in Physics and Mat...

  13. Single-platelet nanomechanics measured by high-throughput cytometry

    Science.gov (United States)

    Myers, David R.; Qiu, Yongzhi; Fay, Meredith E.; Tennenbaum, Michael; Chester, Daniel; Cuadrado, Jonas; Sakurai, Yumiko; Baek, Jong; Tran, Reginald; Ciciliano, Jordan C.; Ahn, Byungwook; Mannino, Robert G.; Bunting, Silvia T.; Bennett, Carolyn; Briones, Michael; Fernandez-Nieves, Alberto; Smith, Michael L.; Brown, Ashley C.; Sulchek, Todd; Lam, Wilbur A.

    2016-10-01

    Haemostasis occurs at sites of vascular injury, where flowing blood forms a clot, a dynamic and heterogeneous fibrin-based biomaterial. Paramount in the clot's capability to stem haemorrhage are its changing mechanical properties, the major drivers of which are the contractile forces exerted by platelets against the fibrin scaffold. However, how platelets transduce microenvironmental cues to mediate contraction and alter clot mechanics is unknown. This is clinically relevant, as overly softened and stiffened clots are associated with bleeding and thrombotic disorders. Here, we report a high-throughput hydrogel-based platelet-contraction cytometer that quantifies single-platelet contraction forces in different clot microenvironments. We also show that platelets, via the Rho/ROCK pathway, synergistically couple mechanical and biochemical inputs to mediate contraction. Moreover, highly contractile platelet subpopulations present in healthy controls are conspicuously absent in a subset of patients with undiagnosed bleeding disorders, and therefore may function as a clinical diagnostic biophysical biomarker.

  14. A high throughput DNA extraction method with high yield and quality

    Directory of Open Access Journals (Sweden)

    Xin Zhanguo

    2012-07-01

    Full Text Available Abstract Background Preparation of large quantity and high quality genomic DNA from a large number of plant samples is a major bottleneck for most genetic and genomic analyses, such as, genetic mapping, TILLING (Targeting Induced Local Lesion IN Genome, and next-generation sequencing directly from sheared genomic DNA. A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield, or costly. Here, we describe a method for high throughput genomic DNA isolation from sorghum [Sorghum bicolor (L. Moench] leaves and dry seeds with high yield, high quality, and affordable cost. Results We developed a high throughput DNA isolation method by combining a high yield CTAB extraction method with an improved cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum leaves and dry seeds. The DNA yield was improved by nearly 30 fold with 4 times less consumption of MagAttract beads. The method can also be used in other plant species, including cotton leaves and pine needles. Conclusion A high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. The main advantages of the method are low cost, high yield, high quality, and high throughput. One person can process two 96-well plates in a working day at a cost of $0.10 per sample of magnetic beads plus other consumables that other methods will also need.

  15. A high-throughput strategy to screen 2D crystallization trials of membrane proteins.

    Science.gov (United States)

    Vink, Martin; Derr, Kd; Love, James; Stokes, David L; Ubarretxena-Belandia, Iban

    2007-12-01

    Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1 week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24 h, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography.

  16. Magnetic Alignment of Microelements Containing Cultured Neuronal Networks for High-Throughput Screening.

    Science.gov (United States)

    Gordon, Kent R; Wang, Yuli; Allbritton, Nancy L; Taylor, Anne Marion

    2015-10-01

    High-throughput screening (HTS) on neurons presents unique difficulties because they are postmitotic, limited in supply, and challenging to harvest from animals or generate from stem cells. These limitations have hindered neurological drug discovery, leaving an unmet need to develop cost-effective technology for HTS using neurons. Traditional screening methods use up to 20,000 neurons per well in 384-well plates. To increase throughput, we use "microraft" arrays, consisting of 1600 square, releasable, paramagnetic, polystyrene microelements (microrafts), each providing a culture surface for 500-700 neurons. These microrafts can be detached from the array and transferred to 384-well plates for HTS; however, they must be centered within wells for automated imaging. Here, we developed a magnet array plate, compatible with HTS fluid-handling systems, to center microrafts within wells. We used finite element analysis to select an effective size of the magnets and confirmed that adjacent magnetic fields do not interfere. We then experimentally tested the plate's centering ability and found a centering efficiency of 100%, compared with 4.35% using a flat magnet. We concluded that microrafts could be centered after settling randomly within the well, overcoming friction, and confirmed these results by centering microrafts containing hippocampal neurons cultured for 8 days.

  17. High-throughput DNA droplet assays using picoliter reactor volumes.

    Science.gov (United States)

    Srisa-Art, Monpichar; deMello, Andrew J; Edel, Joshua B

    2007-09-01

    The online characterization and detection of individual droplets at high speeds, low analyte concentrations, and perfect detection efficiencies is a significant challenge underpinning the application of microfluidic droplet reactors to high-throughput chemistry and biology. Herein, we describe the integration of confocal fluorescence spectroscopy as a high-efficiency detection method for droplet-based microfluidics. Issues such as surface contamination, rapid mixing, and rapid detection, as well as low detections limits have been addressed with the approach described when compared to conventional laminar flow-based fluidics. Using such a system, droplet size, droplet shape, droplet formation frequencies, and droplet compositions can be measured accurately and precisely at kilohertz frequencies. Taking advantage of this approach, we demonstrate a high-throughput biological assay based on fluorescence resonance energy transfer (FRET). By attaching a FRET donor (Alexa Fluor 488) to streptavidin and labeling a FRET acceptor (Alexa Fluor 647) on one DNA strand and biotin on the complementary strand, donor and acceptor molecules are brought in proximity due to streptavidin-biotin binding, resulting in FRET. Fluorescence bursts of the donor and acceptor from each droplet can be monitored simultaneously using separate avalanche photodiode detectors operating in single photon counting mode. Binding assays were investigated and compared between fixed streptavidin and DNA concentrations. Binding curves fit perfectly to Hill-Waud models, and the binding ratio between streptavidin and biotin was evaluated and found to be in agreement with the biotin binding sites on streptavidin. FRET efficiency for this FRET pair was also investigated from the binding results. Efficiency results show that this detection system can precisely measure FRET even at low FRET efficiencies.

  18. Utility of lab-on-a-chip technology for high-throughput nucleic acid and protein analysis

    DEFF Research Database (Denmark)

    Hawtin, Paul; Hardern, Ian; Wittig, Rainer

    2005-01-01

    On-chip electrophoresis can provide size separations of nucleic acids and proteins similar to more traditional slab gel electrophoresis. Lab-on-a-chip (LoaC) systems utilize on-chip electrophoresis in conjunction with sizing calibration, sensitive detection schemes, and sophisticated data analysis...... to achieve rapid analysis times (C systems to enable and augment systems biology investigations. RNA quality, as assessed by an RNA integrity number score, is compared to existing quality control (QC) measurements. High-throughput DNA analysis of multiplex PCR...... samples is used to stratify gene sets for disease discovery. Finally, the applicability of a high-throughput LoaC system for assessing protein purification is demonstrated. The improvements in workflow processes, speed of analysis, data accuracy and reproducibility, and automated data analysis...

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

    Science.gov (United States)

    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

  20. Combinatorial Synthesis of and high-throughput protein release from polymer film and nanoparticle libraries.

    Science.gov (United States)

    Petersen, Latrisha K; Chavez-Santoscoy, Ana V; Narasimhan, Balaji

    2012-09-06

    Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides(1). This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously(1). This combinatorial platform has been validated with conventional methods(2) and the polyanhydride film and nanoparticle libraries have been characterized with (1)H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and

  1. High throughput sequencing reveals a novel fabavirus infecting sweet cherry.

    Science.gov (United States)

    Villamor, D E V; Pillai, S S; Eastwell, K C

    2017-03-01

    The genus Fabavirus currently consists of five species represented by viruses that infect a wide range of hosts but none reported from temperate climate fruit trees. A virus with genomic features resembling fabaviruses (tentatively named Prunus virus F, PrVF) was revealed by high throughput sequencing of extracts from a sweet cherry tree (Prunus avium). PrVF was subsequently shown to be graft transmissible and further identified in three other non-symptomatic Prunus spp. from different geographical locations. Two genetic variants of RNA1 and RNA2 coexisted in the same samples. RNA1 consisted of 6,165 and 6,163 nucleotides, and RNA2 consisted of 3,622 and 3,468 nucleotides.

  2. High-throughput profiling in the hematopoietic system.

    Science.gov (United States)

    Fabbri, Muller; Spizzo, Riccardo; Calin, George A

    2010-01-01

    The expression profile of microRNAs significantly varies in physiological and pathological conditions. Increasing evidence from the literature shows that abnormalities of the miRNome (defined as the full spectrum of miRNAs expressed in a genome) occur in almost all human diseases and have important pathogenetic, prognostic, and therapeutic implications. The study of the aberrancies of the miRNome has become possible by developing high-throughput profiling techniques that allow the simultaneous detection of differences in miRNA expression between normal and pathologic tissues or simply tissues at different stages of differentiation. These techniques provide the basis for further investigations focused on the miRNAs, which are most frequently and widely differentially expressed under the different investigated conditions.

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

    Science.gov (United States)

    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.

  4. High-throughput sequencing in veterinary infection biology and diagnostics.

    Science.gov (United States)

    Belák, S; Karlsson, O E; Leijon, M; Granberg, F

    2013-12-01

    Sequencing methods have improved rapidly since the first versions of the Sanger techniques, facilitating the development of very powerful tools for detecting and identifying various pathogens, such as viruses, bacteria and other microbes. The ongoing development of high-throughput sequencing (HTS; also known as next-generation sequencing) technologies has resulted in a dramatic reduction in DNA sequencing costs, making the technology more accessible to the average laboratory. In this White Paper of the World Organisation for Animal Health (OIE) Collaborating Centre for the Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine (Uppsala, Sweden), several approaches and examples of HTS are summarised, and their diagnostic applicability is briefly discussed. Selected future aspects of HTS are outlined, including the need for bioinformatic resources, with a focus on improving the diagnosis and control of infectious diseases in veterinary medicine.

  5. High-throughput Identification of Phage-derived Imaging Agents

    Directory of Open Access Journals (Sweden)

    Kimberly A. Kelly

    2006-01-01

    Full Text Available The use of phage-displayed peptide libraries is a powerful method for selecting peptides with desired binding properties. However, the validation and prioritization of “hits” obtained from this screening approach remains challenging. Here, we describe the development and testing of a new analysis method to identify and display hits from phage-display experiments and high-throughput enzyme-linked immunosorbent assay screens. We test the method using a phage screen against activated macrophages to develop imaging agents with higher specificity for active disease processes. The new methodology should be useful in identifying phage hits and is extendable to other library screening methods such as small-molecule and nanoparticle libraries.

  6. High-Throughput Screening Using Fourier-Transform Infrared Imaging

    Directory of Open Access Journals (Sweden)

    Erdem Sasmaz

    2015-06-01

    Full Text Available Efficient parallel screening of combinatorial libraries is one of the most challenging aspects of the high-throughput (HT heterogeneous catalysis workflow. Today, a number of methods have been used in HT catalyst studies, including various optical, mass-spectrometry, and gas-chromatography techniques. Of these, rapid-scanning Fourier-transform infrared (FTIR imaging is one of the fastest and most versatile screening techniques. Here, the new design of the 16-channel HT reactor is presented and test results for its accuracy and reproducibility are shown. The performance of the system was evaluated through the oxidation of CO over commercial Pd/Al2O3 and cobalt oxide nanoparticles synthesized with different reducer-reductant molar ratios, surfactant types, metal and surfactant concentrations, synthesis temperatures, and ramp rates.

  7. Interactive Visual Analysis of High Throughput Text Streams

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

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

    Science.gov (United States)

    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.

  9. EDITORIAL: Combinatorial and High-Throughput Materials Research

    Science.gov (United States)

    Potyrailo, Radislav A.; Takeuchi, Ichiro

    2005-01-01

    The success of combinatorial and high-throughput methodologies relies greatly on the availability of various characterization tools with new and improved capabilities [1]. Indeed, how useful can a combinatorial library of 250, 400, 25 000 or 2 000 000 compounds be [2-5] if one is unable to characterize its properties of interest fairly quickly? How useful can a set of thousands of spectra or chromatograms be if one is unable to analyse them in a timely manner? For these reasons, the development of new approaches for materials characterization is one of the most active areas in combinatorial materials science. The importance of this aspect of research in the field has been discussed in numerous conferences including the Pittsburgh Conferences, the American Chemical Society Meetings, the American Physical Society Meetings, the Materials Research Society Symposia and various Gordon Research Conferences. Naturally, the development of new measurement instrumentation attracts the attention not only of practitioners of combinatorial materials science but also of those who design new software for data manipulation and mining. Experimental designs of combinatorial libraries are pursued with available and realistic synthetic and characterization capabilities in mind. It is becoming increasingly critical to link the design of new equipment for high-throughput parallel materials synthesis with integrated measurement tools in order to enhance the efficacy of the overall experimental strategy. We have received an overwhelming response to our proposal and call for papers for this Special Issue on Combinatorial Materials Science. The papers in this issue of Measurement Science and Technology are a very timely collection that captures the state of modern combinatorial materials science. They demonstrate the significant advances that are taking place in the field. In some cases, characterization tools are now being operated in the factory mode. At the same time, major challenges

  10. High throughput phenotyping for aphid resistance in large plant collections

    Directory of Open Access Journals (Sweden)

    Chen Xi

    2012-08-01

    Full Text Available Abstract Background Phloem-feeding insects are among the most devastating pests worldwide. They not only cause damage by feeding from the phloem, thereby depleting the plant from photo-assimilates, but also by vectoring viruses. Until now, the main way to prevent such problems is the frequent use of insecticides. Applying resistant varieties would be a more environmental friendly and sustainable solution. For this, resistant sources need to be identified first. Up to now there were no methods suitable for high throughput phenotyping of plant germplasm to identify sources of resistance towards phloem-feeding insects. Results In this paper we present a high throughput screening system to identify plants with an increased resistance against aphids. Its versatility is demonstrated using an Arabidopsis thaliana activation tag mutant line collection. This system consists of the green peach aphid Myzus persicae (Sulzer and the circulative virus Turnip yellows virus (TuYV. In an initial screening, with one plant representing one mutant line, 13 virus-free mutant lines were identified by ELISA. Using seeds produced from these lines, the putative candidates were re-evaluated and characterized, resulting in nine lines with increased resistance towards the aphid. Conclusions This M. persicae-TuYV screening system is an efficient, reliable and quick procedure to identify among thousands of mutated lines those resistant to aphids. In our study, nine mutant lines with increased resistance against the aphid were selected among 5160 mutant lines in just 5 months by one person. The system can be extended to other phloem-feeding insects and circulative viruses to identify insect resistant sources from several collections, including for example genebanks and artificially prepared mutant collections.

  11. High-throughput miniaturized bioreactors for cell culture process development: reproducibility, scalability, and control.

    Science.gov (United States)

    Rameez, Shahid; Mostafa, Sigma S; Miller, Christopher; Shukla, Abhinav A

    2014-01-01

    Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr™) is an automated micro-bioreactor system with miniature single-use bioreactors with a 10-15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in-line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr™ resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr™ was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr™ system as a high throughput system for cell culture process development.

  12. Computer controlled cryo-electron microscopy--TOM² a software package for high-throughput applications.

    Science.gov (United States)

    Korinek, Andreas; Beck, Florian; Baumeister, Wolfgang; Nickell, Stephan; Plitzko, Jürgen M

    2011-09-01

    Automated data acquisition expedites structural studies by electron microscopy and it allows to collect data sets of unprecedented size and consistent quality. In electron tomography it greatly facilitates the systematic exploration of large cellular landscapes and in single particle analysis it allows to generate data sets for an exhaustive classification of coexisting molecular states. Here we describe a novel software philosophy and architecture that can be used for a great variety of automated data acquisition scenarios. Based on our original software package TOM, the new TOM(2) package has been designed in an object-oriented way. The whole program can be seen as a collection of self-sufficient modules with defined relationships acting in a concerted manner. It subdivides data acquisition into a set of hierarchical tasks, bonding data structure and the operations to be performed tightly together. To demonstrate its capacity for high-throughput data acquisition it has been used in conjunction with instrumentation combining the latest technological achievements in electron optics, cryogenics and robotics. Its performance is demonstrated with a single particle analysis case study and with a batch tomography application.

  13. Laboratory Information Management Software for genotyping workflows: applications in high throughput crop genotyping

    Directory of Open Access Journals (Sweden)

    Prasanth VP

    2006-08-01

    Full Text Available Abstract Background With the advances in DNA sequencer-based technologies, it has become possible to automate several steps of the genotyping process leading to increased throughput. To efficiently handle the large amounts of genotypic data generated and help with quality control, there is a strong need for a software system that can help with the tracking of samples and capture and management of data at different steps of the process. Such systems, while serving to manage the workflow precisely, also encourage good laboratory practice by standardizing protocols, recording and annotating data from every step of the workflow. Results A laboratory information management system (LIMS has been designed and implemented at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT that meets the requirements of a moderately high throughput molecular genotyping facility. The application is designed as modules and is simple to learn and use. The application leads the user through each step of the process from starting an experiment to the storing of output data from the genotype detection step with auto-binning of alleles; thus ensuring that every DNA sample is handled in an identical manner and all the necessary data are captured. The application keeps track of DNA samples and generated data. Data entry into the system is through the use of forms for file uploads. The LIMS provides functions to trace back to the electrophoresis gel files or sample source for any genotypic data and for repeating experiments. The LIMS is being presently used for the capture of high throughput SSR (simple-sequence repeat genotyping data from the legume (chickpea, groundnut and pigeonpea and cereal (sorghum and millets crops of importance in the semi-arid tropics. Conclusion A laboratory information management system is available that has been found useful in the management of microsatellite genotype data in a moderately high throughput genotyping

  14. A pre-validation trial - testing genotoxicity of several chemicals using standard, medium- and high-throughput comet formats.

    Directory of Open Access Journals (Sweden)

    Kristine Bjerve Gutzkow

    2015-06-01

    Results obtained with the three systems (standard, medium- and high-throughput were essentially the same. The 96-minigel format was analysed with the fully automated scoring system IMSTAR and comparable results were achieved with the semi-automated scoring system from Perceptives. The known genotoxic chemicals MNU, B(aP, 4-NQO and cyclophosphamide showed little consistent sign of genotoxicity at concentrations causing limited cytotoxicity. D-mannitol and Triton X-100 were, as expected, non-genotoxic (though Triton X-100, at high concentrations, caused DNA breaks as an apparent secondary effect of cytotoxicity. Etoposide and bleomycin gave significant increase in DNA strand break at borderline cytotoxic concentrations. The limitation of the assay to detect damaged bases by known genotoxins may be overcome by incorporating a DNA repair enzyme, such as formamidopyrimidine-DNA-glycosylase (FPG, to convert damaged bases into breaks as shown by Azqueta A et al., Mutagenesis vol. 28 no. 3 pp. 271–277, 2013 .

  15. A High-Temperature, High-Throughput Method for Monitoring Residual Formaldehyde in Vaccine Formulations.

    Science.gov (United States)

    Stallings, Kendra D; Kitchener, Rebecca L; Hentz, Nathaniel G

    2014-06-01

    Formaldehyde has long been used in the chemical inactivation of viral material during vaccine production. Viral inactivation is required so that the vaccine does not infect the patient. Formaldehyde is diluted during the vaccine manufacturing process, but residual quantities of formaldehyde are still present in some current vaccines. Although formaldehyde is considered safe for use in vaccines by the Food and Drug Administration, excessive exposure to this chemical may lead to cancer or other health-related issues. An assay was developed that is capable of detecting levels of residual formaldehyde in influenza vaccine samples. The assay employs incubation of dosage formulation suspensions with hydralazine hydrochloride under mildly acidic conditions and elevated temperatures, where formaldehyde is derivatized to yield fluorescent s-triazolo-[3,4-a]-phthalazine. The assay has been traditionally run by high-performance liquid chromatography, where runtimes of 15 minutes per sample can be expected. Our laboratory has developed a plate-based version that drastically improved the throughput to a runtime of 96 samples per minute. The assay was characterized and validated with respect to reaction temperature, evaporation, stability, and selectivity to monitor residual formaldehyde in various influenza vaccine samples, including in-process samples. Heat transfer and evaporation will be especially considered in this work. Since the assay is plate based, it is automation friendly. The new assay format has attained detection limits of 0.01 µg/mL residual formaldehyde, which is easily able to detect and quantify formaldehyde at levels used in many current vaccine formulations (<5 µg/0.5-mL dose).

  16. Development and qualification of a high sensitivity, high throughput Q-PCR assay for quantitation of residual host cell DNA in purification process intermediate and drug substance samples.

    Science.gov (United States)

    Zhang, Wei; Wu, Meng; Menesale, Emily; Lu, Tongjun; Magliola, Aeona; Bergelson, Svetlana

    2014-11-01

    Methods of high sensitivity, accuracy and throughput are needed for quantitation of low level residual host cell DNA in purification process intermediates and drug substances of therapeutic proteins. In this study, we designed primer/probe sets targeting repetitive Alu repeats or Alu-equivalent sequences in the human, Chinese hamster and murine genomes. When used in quantitative polymerase chain reactions (Q-PCRs), these primer/probe sets showed high species specificity and gave significantly higher sensitivity compared to those targeting the low copy number GAPDH gene. This allowed for detection of residual host cell DNA of much lower concentrations and, for some samples, eliminated the need for DNA extraction. By combining the high sensitivity Alu Q-PCR with high throughput automated DNA extraction using an automated MagMAX magnetic particle processor, we successfully developed and qualified a highly accurate, specific, sensitive and efficient method for the quantitation of residual host cell DNA in process intermediates and drug substances of multiple therapeutic proteins purified from cells of multiple species. Compared to the previous method using manual DNA extraction and primer/probe sets targeting the GAPDH gene, this new method increased our DNA extraction throughput by over sevenfold, and lowered the lower limit of quantitation by up to eightfold.

  17. Disubstituted 1-aryl-4-aminopiperidine library synthesis using computational drug design and high-throughput batch and flow technologies.

    Science.gov (United States)

    Bryan, Marian C; Hein, Christopher D; Gao, Hua; Xia, Xiaoyang; Eastwood, Heather; Bruenner, Bernd A; Louie, Steven W; Doherty, Elizabeth M

    2013-09-09

    A platform that incorporates computational library design, parallel solution-phase synthesis, continuous flow hydrogenation, and automated high throughput purification and reformatting technologies was applied to the production of a 120-member library of 1-aryl-4-aminopiperidine analogues for drug discovery screening. The application described herein demonstrates the advantages of computational library design coupled with a flexible, modular approach to library synthesis. The enabling technologies described can be readily adopted by the traditional medicinal chemist without extensive training and lengthy process development times.

  18. High throughput optoelectronic smart pixel systems using diffractive optics

    Science.gov (United States)

    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

  19. A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production

    Directory of Open Access Journals (Sweden)

    Bottomley Stephen P

    2006-03-01

    Full Text Available Abstract Background In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol. Results The set of E. coli expression vectors, encode for either a hexa-histidine tag or the three most commonly used solubility tags (GST, MBP, NusA and all with an N-terminal hexa-histidine sequence. The result is two-fold: the His-tag facilitates purification by immobilised metal affinity chromatography, whilst the fusion domains act primarily as solubility aids during expression, in addition to providing an optional purification step. We have also incorporated a TEV recognition sequence following the solubility tag domain, which allows for highly specific cleavage (using TEV protease of the fusion protein to yield native protein. These vectors are also designed for ligation-independent cloning and they possess a high-level expressing T7 promoter, which is suitable for auto-induction. To validate our vector system, we have cloned four different genes and also one gene into all four vectors and used small-scale expression and purification techniques. We demonstrate that the vectors are capable of high levels of expression and that efficient screening of new proteins can be readily achieved at the laboratory level. Conclusion The result is a set of four rationally designed vectors, which can be used for streamlined cloning, expression and purification of target proteins in the laboratory and have the potential for being adaptable to a high-throughput

  20. Automated tests of ANA immunofluorescence as throughput autoantibody detection technology: strengths and limitations.

    Science.gov (United States)

    Meroni, Pier Luigi; Bizzaro, Nicola; Cavazzana, Ilaria; Borghi, Maria Orietta; Tincani, Angela

    2014-03-03

    Anti-nuclear antibody (ANA) assay is a screening test used for almost all autoimmune rheumatic diseases, and in a number of these cases, it is a diagnostic/classification parameter. In addition, ANA is also a useful test for additional autoimmune disorders. The indirect immunofluorescence technique on monolayers of cultured epithelial cells is the current recommended method because it has higher sensitivity than solid phase assays. However, the technique is time-consuming and requires skilled operators. Automated ANA reading systems have recently been developed, which offer the advantage of faster and much easier performance as well as better harmonization in the interpretation of the results. Preliminary validation studies of these systems have given promising results in terms of analytical specificity and reproducibility. However, these techniques require further validation in clinical studies and need improvement in their recognition of mixed or less common staining patterns.

  1. Leveraging the power of high performance computing for next generation sequencing data analysis: tricks and twists from a high throughput exome workflow.

    Science.gov (United States)

    Kawalia, Amit; Motameny, Susanne; Wonczak, Stephan; Thiele, Holger; Nieroda, Lech; Jabbari, Kamel; Borowski, Stefan; Sinha, Vishal; Gunia, Wilfried; Lang, Ulrich; Achter, Viktor; Nürnberg, Peter

    2015-01-01

    Next generation sequencing (NGS) has been a great success and is now a standard method of research in the life sciences. With this technology, dozens of whole genomes or hundreds of exomes can be sequenced in rather short time, producing huge amounts of data. Complex bioinformatics analyses are required to turn these data into scientific findings. In order to run these analyses fast, automated workflows implemented on high performance computers are state of the art. While providing sufficient compute power and storage to meet the NGS data challenge, high performance computing (HPC) systems require special care when utilized for high throughput processing. This is especially true if the HPC system is shared by different users. Here, stability, robustness and maintainability are as important for automated workflows as speed and throughput. To achieve all of these aims, dedicated solutions have to be developed. In this paper, we present the tricks and twists that we utilized in the implementation of our exome data processing workflow. It may serve as a guideline for other high throughput data analysis projects using a similar infrastructure. The code implementing our solutions is provided in the supporting information files.

  2. High throughput jet singlet oxygen generator for multi kilowatt SCOIL

    Science.gov (United States)

    Rajesh, R.; Singhal, Gaurav; Mainuddin; Tyagi, R. K.; Dawar, A. L.

    2010-06-01

    A jet flow singlet oxygen generator (JSOG) capable of handling chlorine flows of nearly 1.5 mol s -1 has been designed, developed, and tested. The generator is designed in a modular configuration taking into consideration the practical aspects of handling high throughput flows without catastrophic BHP carry over. While for such high flow rates a cross-flow configuration has been reported, the generator utilized in the present study is a counter flow configuration. A near vertical extraction of singlet oxygen is effected at the generator exit, followed by a 90° rotation of the flow forming a novel verti-horizontal COIL scheme. This allows the COIL to be operated with a vertical extraction SOG followed by the horizontal arrangement of subsequent COIL systems such as supersonic nozzle, cavity, supersonic diffuser, etc. This enables a more uniform weight distribution from point of view of mobile and other platform mounted systems, which is highly relevant for large scale systems. The present study discusses the design aspects of the jet singlet oxygen generator along with its test results for various operating ranges. Typically, for the intended design flow rates, the chlorine utilization and singlet oxygen yield have been observed to be ˜94% and ˜64%, respectively.

  3. High-Throughput Network Communication with NetIO

    CERN Document Server

    Schumacher, J\\"orn; The ATLAS collaboration; Vandelli, Wainer

    2016-01-01

    HPC network technologies like Infiniband, TrueScale or OmniPath provide low-latency and high-throughput communication between hosts, which makes them attractive options for data-acquisition systems in large-scale high-energy physics experiments. Like HPC networks, DAQ networks are local and include a well specified number of systems. Unfortunately traditional network communication APIs for HPC clusters like MPI or PGAS target exclusively the HPC community and are not suited well for DAQ applications. It is possible to build distributed DAQ applications using low-level system APIs like Infiniband Verbs (and this has been done), but it requires a non negligible effort and expert knowledge. On the other hand, message services like 0MQ have gained popularity in the HEP community. Such APIs allow to build distributed applications with a high-level approach and provide good performance. Unfortunately their usage usually limits developers to TCP/IP-based networks. While it is possible to operate a TCP/IP stack on to...

  4. High-Throughput Screening Using Mass Spectrometry within Drug Discovery.

    Science.gov (United States)

    Rohman, Mattias; Wingfield, Jonathan

    2016-01-01

    In order to detect a biochemical analyte with a mass spectrometer (MS) it is necessary to ionize the analyte of interest. The analyte can be ionized by a number of different mechanisms, however, one common method is electrospray ionization (ESI). Droplets of analyte are sprayed through a highly charged field, the droplets pick up charge, and this is transferred to the analyte. High levels of salt in the assay buffer will potentially steal charge from the analyte and suppress the MS signal. In order to avoid this suppression of signal, salt is often removed from the sample prior to injection into the MS. Traditional ESI MS relies on liquid chromatography (LC) to remove the salt and reduce matrix effects, however, this is a lengthy process. Here we describe the use of RapidFire™ coupled to a triple-quadrupole MS for high-throughput screening. This system uses solid-phase extraction to de-salt samples prior to injection, reducing processing time such that a sample is injected into the MS ~every 10 s.

  5. High-throughput Transcriptome analysis, CAGE and beyond

    KAUST Repository

    Kodzius, Rimantas

    2008-11-25

    1. Current research - PhD work on discovery of new allergens - Postdoctoral work on Transcriptional Start Sites a) Tag based technologies allow higher throughput b) CAGE technology to define promoters c) CAGE data analysis to understand Transcription - Wo

  6. Hydrodynamic Cell Trapping for High Throughput Single-Cell Applications

    Directory of Open Access Journals (Sweden)

    Amin Abbaszadeh Banaeiyan

    2013-12-01

    Full Text Available The possibility to conduct complete cell assays under a precisely controlled environment while consuming minor amounts of chemicals and precious drugs have made microfluidics an interesting candidate for quantitative single-cell studies. Here, we present an application-specific microfluidic device, cellcomb, capable of conducting high-throughput single-cell experiments. The system employs pure hydrodynamic forces for easy cell trapping and is readily fabricated in polydimethylsiloxane (PDMS using soft lithography techniques. The cell-trapping array consists of V-shaped pockets designed to accommodate up to six Saccharomyces cerevisiae (yeast cells with the average diameter of 4 μm. We used this platform to monitor the impact of flow rate modulation on the arsenite (As(III uptake in yeast. Redistribution of a green fluorescent protein (GFP-tagged version of the heat shock protein Hsp104 was followed over time as read out. Results showed a clear reverse correlation between the arsenite uptake and three different adjusted low = 25 nL min−1, moderate = 50 nL min−1, and high = 100 nL min−1 flow rates. We consider the presented device as the first building block of a future integrated application-specific cell-trapping array that can be used to conduct complete single cell experiments on different cell types.

  7. Development of automatic image analysis methods for high-throughput and high-content screening

    NARCIS (Netherlands)

    Di, Zi

    2013-01-01

    This thesis focuses on the development of image analysis methods for ultra-high content analysis of high-throughput screens where cellular phenotype responses to various genetic or chemical perturbations that are under investigation. Our primary goal is to deliver efficient and robust image analysis

  8. Recent advances in high-throughput molecular marker identification for superficial and invasive bladder cancers

    DEFF Research Database (Denmark)

    Andersen, Lars Dyrskjøt; Zieger, Karsten; Ørntoft, Torben Falck

    2007-01-01

    individually contributed to the management of the disease. However, the development of high-throughput techniques for simultaneous assessment of a large number of markers has allowed classification of tumors into clinically relevant molecular subgroups beyond those possible by pathological classification. Here......, we review the recent advances in high-throughput molecular marker identification for superficial and invasive bladder cancers....

  9. High throughput label-free platform for statistical bio-molecular sensing

    DEFF Research Database (Denmark)

    Bosco, Filippo; Hwu, En-Te; Chen, Ching-Hsiu

    2011-01-01

    Sensors are crucial in many daily operations including security, environmental control, human diagnostics and patient monitoring. Screening and online monitoring require reliable and high-throughput sensing. We report on the demonstration of a high-throughput label-free sensor platform utilizing...

  10. Application of an active attachment model as a high-throughput demineralization biofilm model

    NARCIS (Netherlands)

    Silva, T.C.; Pereira, A.F.F.; Exterkate, R.A.M.; Bagnato, V.S.; Buzalaf, M.A.R.; de A.M. Machado, M.A.; ten Cate, J.M.; Crielaard, W.; Deng, D.M.

    2012-01-01

    Objectives To investigate the potential of an active attachment biofilm model as a high-throughput demineralization biofilm model for the evaluation of caries-preventive agents. Methods Streptococcus mutans UA159 biofilms were grown on bovine dentine discs in a high-throughput active attachment mode

  11. High throughput comet assay to study genotoxicity of nanomaterials

    Directory of Open Access Journals (Sweden)

    Naouale El Yamani

    2015-06-01

    Full Text Available The unique physicochemical properties of engineered nanomaterials (NMs have accelerated their use in diverse industrial and domestic products. Although their presence in consumer products represents a major concern for public health safety, their potential impact on human health is poorly understood. There is therefore an urgent need to clarify the toxic effects of NMs and to elucidate the mechanisms involved. In view of the large number of NMs currently being used, high throughput (HTP screening technologies are clearly needed for efficient assessment of toxicity. The comet assay is the most used method in nanogenotoxicity studies and has great potential for increasing throughput as it is fast, versatile and robust; simple technical modifications of the assay make it possible to test many compounds (NMs in a single experiment. The standard gel of 70-100 μL contains thousands of cells, of which only a tiny fraction are actually scored. Reducing the gel to a volume of 5 μL, with just a few hundred cells, allows twelve gels to be set on a standard slide, or 96 as a standard 8x12 array. For the 12 gel format, standard slides precoated with agarose are placed on a metal template and gels are set on the positions marked on the template. The HTP comet assay, incorporating digestion of DNA with formamidopyrimidine DNA glycosylase (FPG to detect oxidised purines, has recently been applied to study the potential induction of genotoxicity by NMs via reactive oxygen. In the NanoTEST project we investigated the genotoxic potential of several well-characterized metal and polymeric nanoparticles with the comet assay. All in vitro studies were harmonized; i.e. NMs were from the same batch, and identical dispersion protocols, exposure time, concentration range, culture conditions, and time-courses were used. As a kidney model, Cos-1 fibroblast-like kidney cells were treated with different concentrations of iron oxide NMs, and cells embedded in minigels (12

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

    Science.gov (United States)

    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. Model-based high-throughput design of ion exchange protein chromatography.

    Science.gov (United States)

    Khalaf, Rushd; Heymann, Julia; LeSaout, Xavier; Monard, Florence; Costioli, Matteo; Morbidelli, Massimo

    2016-08-12

    This work describes the development of a model-based high-throughput design (MHD) tool for the operating space determination of a chromatographic cation-exchange protein purification process. Based on a previously developed thermodynamic mechanistic model, the MHD tool generates a large amount of system knowledge and thereby permits minimizing the required experimental workload. In particular, each new experiment is designed to generate information needed to help refine and improve the model. Unnecessary experiments that do not increase system knowledge are avoided. Instead of aspiring to a perfectly parameterized model, the goal of this design tool is to use early model parameter estimates to find interesting experimental spaces, and to refine the model parameter estimates with each new experiment until a satisfactory set of process parameters is found. The MHD tool is split into four sections: (1) prediction, high throughput experimentation using experiments in (2) diluted conditions and (3) robotic automated liquid handling workstations (robotic workstation), and (4) operating space determination and validation. (1) Protein and resin information, in conjunction with the thermodynamic model, is used to predict protein resin capacity. (2) The predicted model parameters are refined based on gradient experiments in diluted conditions. (3) Experiments on the robotic workstation are used to further refine the model parameters. (4) The refined model is used to determine operating parameter space that allows for satisfactory purification of the protein of interest on the HPLC scale. Each section of the MHD tool is used to define the adequate experimental procedures for the next section, thus avoiding any unnecessary experimental work. We used the MHD tool to design a polishing step for two proteins, a monoclonal antibody and a fusion protein, on two chromatographic resins, in order to demonstrate it has the ability to strongly accelerate the early phases of process

  14. PTMScout, a Web resource for analysis of high throughput post-translational proteomics studies.

    Science.gov (United States)

    Naegle, Kristen M; Gymrek, Melissa; Joughin, Brian A; Wagner, Joel P; Welsch, Roy E; Yaffe, Michael B; Lauffenburger, Douglas A; White, Forest M

    2010-11-01

    The rate of discovery of post-translational modification (PTM) sites is increasing rapidly and is significantly outpacing our biological understanding of the function and regulation of those modifications. To help meet this challenge, we have created PTMScout, a web-based interface for viewing, manipulating, and analyzing high throughput experimental measurements of PTMs in an effort to facilitate biological understanding of protein modifications in signaling networks. PTMScout is constructed around a custom database of PTM experiments and contains information from external protein and post-translational resources, including gene ontology annotations, Pfam domains, and Scansite predictions of kinase and phosphopeptide binding domain interactions. PTMScout functionality comprises data set comparison tools, data set summary views, and tools for protein assignments of peptides identified by mass spectrometry. Analysis tools in PTMScout focus on informed subset selection via common criteria and on automated hypothesis generation through subset labeling derived from identification of statistically significant enrichment of other annotations in the experiment. Subset selection can be applied through the PTMScout flexible query interface available for quantitative data measurements and data annotations as well as an interface for importing data set groupings by external means, such as unsupervised learning. We exemplify the various functions of PTMScout in application to data sets that contain relative quantitative measurements as well as data sets lacking quantitative measurements, producing a set of interesting biological hypotheses. PTMScout is designed to be a widely accessible tool, enabling generation of multiple types of biological hypotheses from high throughput PTM experiments and advancing functional assignment of novel PTM sites. PTMScout is available at http://ptmscout.mit.edu.

  15. Development of a facile method for high throughput screening with reporter gene assays.

    Science.gov (United States)

    Goetz, A S; Andrews, J L; Littleton, T R; Ignar, D M

    2000-10-01

    This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.

  16. Airborne TDMA for High Throughput and Fast Weather Conditions Notification

    Directory of Open Access Journals (Sweden)

    Hyungjun Jang

    2011-05-01

    Full Text Available As air traffic grows significantly, aircraft accidents increase. Many aviation accidents could be prevented if the precise aircraft positions and weather conditions on the aircraft’s route were known. Existing studies propose determining the precise aircraft positions via a VHF channel with an air-to-air radio relay system that is based on mobile ad-hoc networks. However, due to the long propagation delay, the existing TDMA MAC schemes underutilize the networks. The existing TDMA MAC sends data and receives ACK in one time slot, which requires two guard times in one time slot. Since aeronautical communications spans a significant distance, the guard time occupies a significantly large portion of the slot. To solve this problem, we propose a piggybacking mechanism ACK. Our proposed MAC has one guard time in one time slot, which enables the transmission of more data. Using this additional data, we can send weather conditions that pertain to the aircraft’s current position. Our analysis shows that this proposed MAC performs better than the existing MAC, since it offers better throughput and network utilization. In addition, our weather condition notification model achieves a much lower transmission delay than a HF (high frequency voice communication.

  17. High-throughput translational medicine: challenges and solutions.

    Science.gov (United States)

    Sulakhe, Dinanath; Balasubramanian, Sandhya; Xie, Bingqing; Berrocal, Eduardo; Feng, Bo; Taylor, Andrew; Chitturi, Bhadrachalam; Dave, Utpal; Agam, Gady; Xu, Jinbo; Börnigen, Daniela; Dubchak, Inna; Gilliam, T Conrad; Maltsev, Natalia

    2014-01-01

    Recent technological advances in genomics now allow producing biological data at unprecedented tera- and petabyte scales. Yet, the extraction of useful knowledge from this voluminous data presents a significant challenge to a scientific community. Efficient mining of vast and complex data sets for the needs of biomedical research critically depends on seamless integration of clinical, genomic, and experimental information with prior knowledge about genotype-phenotype relationships accumulated in a plethora of publicly available databases. Furthermore, such experimental data should be accessible to a variety of algorithms and analytical pipelines that drive computational analysis and data mining. Translational projects require sophisticated approaches that coordinate and perform various analytical steps involved in the extraction of useful knowledge from accumulated clinical and experimental data in an orderly semiautomated manner. It presents a number of challenges such as (1) high-throughput data management involving data transfer, data storage, and access control; (2) scalable computational infrastructure; and (3) analysis of large-scale multidimensional data for the extraction of actionable knowledge.We present a scalable computational platform based on crosscutting requirements from multiple scientific groups for data integration, management, and analysis. The goal of this integrated platform is to address the challenges and to support the end-to-end analytical needs of various translational projects.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    Carl W Carruthers

    Full Text Available 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.

  20. An apparatus for high throughput nanomechanical muscle cell experimentation.

    Science.gov (United States)

    Garcia-Webb, M; Hunter, I; Taberner, A

    2004-01-01

    An array of independent muscle cell testing modules is being developed to explore the mechanics of cardiac myocytes. The instrument will be able to perform established physiological tests and utilize novel system identification techniques to measure the dynamic stiffness and stress frequency response of single cells with possible applications in the pharmaceutical industry for high throughput screening. Currently, each module consists of two independently controlled Lorentz force actuators in the form of stainless steel cantilevers with dimensions 0.025 mm x 0.8 mm x 3 mm, 0.1 m/N compliance and 1.5 kHz resonant frequency. Confocal position sensors focused on each cantilever provide position and force resolution 0.1 mm and forces > 0.1 mN. A custom Visual Basic.Net software interface to a National Instruments data acquisition card implements real time digital control over 4 input channels and 2 output channels at 20 kHz. In addition, algorithms for both swept sine and stochastic system identification have been written to probe mechanical systems. The device has been used to find the dynamic stiffness of a 5 microm diameter polymer fiber between 0 and 500 Hz.

  1. Assessing the utility and limitations of high throughput virtual screening

    Directory of Open Access Journals (Sweden)

    Paul Daniel Phillips

    2016-05-01

    Full Text Available Due to low cost, speed, and unmatched ability to explore large numbers of compounds, high throughput virtual screening and molecular docking engines have become widely utilized by computational scientists. It is generally accepted that docking engines, such as AutoDock, produce reliable qualitative results for ligand-macromolecular receptor binding, and molecular docking results are commonly reported in literature in the absence of complementary wet lab experimental data. In this investigation, three variants of the sixteen amino acid peptide, α-conotoxin MII, were docked to a homology model of the a3β2-nicotinic acetylcholine receptor. DockoMatic version 2.0 was used to perform a virtual screen of each peptide ligand to the receptor for ten docking trials consisting of 100 AutoDock cycles per trial. The results were analyzed for both variation in the calculated binding energy obtained from AutoDock, and the orientation of bound peptide within the receptor. The results show that, while no clear correlation exists between consistent ligand binding pose and the calculated binding energy, AutoDock is able to determine a consistent positioning of bound peptide in the majority of trials when at least ten trials were evaluated.

  2. Comprehensive analysis of high-throughput screening data

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  4. High throughput miniature drug-screening platform using bioprinting technology.

    Science.gov (United States)

    Rodríguez-Dévora, Jorge I; Zhang, Bimeng; Reyna, Daniel; Shi, Zhi-dong; Xu, Tao

    2012-09-01

    In the pharmaceutical industry, new drugs are tested to find appropriate compounds for therapeutic purposes for contemporary diseases. Unfortunately, novel compounds emerge at expensive prices and current target evaluation processes have limited throughput, thus leading to an increase of cost and time for drug development. This work shows the development of the novel inkjet-based deposition method for assembling a miniature drug-screening platform, which can realistically and inexpensively evaluate biochemical reactions in a picoliter-scale volume at a high speed rate. As proof of concept, applying a modified Hewlett Packard model 5360 compact disc printer, green fluorescent protein expressing Escherichia coli cells along with alginate gel solution have been arrayed on a coverslip chip under a repeatable volume of 180% ± 26% picoliters per droplet; subsequently, different antibiotic droplets were patterned on the spots of cells to evaluate the inhibition of bacteria for antibiotic screening. The proposed platform was compared to the current screening process, validating its effectiveness. The viability and basic function of the printed cells were evaluated, resulting in cell viability above 98% and insignificant or no DNA damage to human kidney cells transfected. Based on the reduction of investment and compound volume used by this platform, this technique has the potential to improve the actual drug discovery process at its target evaluation stage.

  5. High-Throughput Preparation of New Photoactive Nanocomposites.

    Science.gov (United States)

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

    2016-06-08

    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.

  6. The JCSG high-throughput structural biology pipeline.

    Science.gov (United States)

    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.

  7. Hypothesis testing in high-throughput screening for drug discovery.

    Science.gov (United States)

    Prummer, Michael

    2012-04-01

    Following the success of small-molecule high-throughput screening (HTS) in drug discovery, other large-scale screening techniques are currently revolutionizing the biological sciences. Powerful new statistical tools have been developed to analyze the vast amounts of data in DNA chip studies, but have not yet found their way into compound screening. In HTS, characterization of single-point hit lists is often done only in retrospect after the results of confirmation experiments are available. However, for prioritization, for optimal use of resources, for quality control, and for comparison of screens it would be extremely valuable to predict the rates of false positives and false negatives directly from the primary screening results. Making full use of the available information about compounds and controls contained in HTS results and replicated pilot runs, the Z score and from it the p value can be estimated for each measurement. Based on this consideration, we have applied the concept of p-value distribution analysis (PVDA), which was originally developed for gene expression studies, to HTS data. PVDA allowed prediction of all relevant error rates as well as the rate of true inactives, and excellent agreement with confirmation experiments was found.

  8. High Throughput Profiling of Molecular Shapes in Crystals

    Science.gov (United States)

    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.

  9. High throughput screening for drug discovery of autophagy modulators.

    Science.gov (United States)

    Shu, Chih-Wen; Liu, Pei-Feng; Huang, Chun-Ming

    2012-11-01

    Autophagy is an evolutionally conserved process in cells for cleaning abnormal proteins and organelles in a lysosome dependent manner. Growing studies have shown that defects or induced autophagy contributes to many diseases including aging, neurodegeneration, pathogen infection, and cancer. However, the precise involvement of autophagy in health and disease remains controversial because the theories are built on limited assays and chemical modulators, indicating that the role of autophagy in diseases may require further verification. Many food and drug administration (FDA) approved drugs modulate autophagy signaling, suggesting that modulation of autophagy with pharmacological agonists or antagonists provides a potential therapy for autophagy-related diseases. This suggestion raises an attractive issue on drug discovery for exploring chemical modulators of autophagy. High throughput screening (HTS) is becoming a powerful tool for drug discovery that may accelerate screening specific autophagy modulators to clarify the role of autophagy in diseases. Herein, this review lays out current autophagy assays to specifically measure autophagy components such as LC3 (mammalian homologue of yeast Atg8) and Atg4. These assays are feasible or successful for HTS with certain chemical libraries, which might be informative for this intensively growing field as research tools and hopefully developing new drugs for autophagy-related diseases.

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

    Science.gov (United States)

    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.

  11. Development and implementation of a high-throughput compound screening assay for targeting disrupted ER calcium homeostasis in Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Kamran Honarnejad

    Full Text Available Disrupted intracellular calcium homeostasis is believed to occur early in the cascade of events leading to Alzheimer's disease (AD pathology. Particularly familial AD mutations linked to Presenilins result in exaggerated agonist-evoked calcium release from endoplasmic reticulum (ER. Here we report the development of a fully automated high-throughput calcium imaging assay utilizing a genetically-encoded FRET-based calcium indicator at single cell resolution for compound screening. The established high-throughput screening assay offers several advantages over conventional high-throughput calcium imaging technologies. We employed this assay for drug discovery in AD by screening compound libraries consisting of over 20,000 small molecules followed by structure-activity-relationship analysis. This led to the identification of Bepridil, a calcium channel antagonist drug in addition to four further lead structures capable of normalizing the potentiated FAD-PS1-induced calcium release from ER. Interestingly, it has recently been reported that Bepridil can reduce Aβ production by lowering BACE1 activity. Indeed, we also detected lowered Aβ, increased sAPPα and decreased sAPPβ fragment levels upon Bepridil treatment. The latter findings suggest that Bepridil may provide a multifactorial therapeutic modality for AD by simultaneously addressing multiple aspects of the disease.

  12. Microfluidic hydrodynamic focusing for high-throughput applications

    Science.gov (United States)

    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.

  13. Characterization of metalloproteins by high-throughput X-ray absorption spectroscopy.

    Science.gov (United States)

    Shi, Wuxian; Punta, Marco; Bohon, Jen; Sauder, J Michael; D'Mello, Rhijuta; Sullivan, Mike; Toomey, John; Abel, Don; Lippi, Marco; Passerini, Andrea; Frasconi, Paolo; Burley, Stephen K; Rost, Burkhard; Chance, Mark R

    2011-06-01

    High-throughput X-ray absorption spectroscopy was used to measure transition metal content based on quantitative detection of X-ray fluorescence signals for 3879 purified proteins from several hundred different protein families generated by the New York SGX Research Center for Structural Genomics. Approximately 9% of the proteins analyzed showed the presence of transition metal atoms (Zn, Cu, Ni, Co, Fe, or Mn) in stoichiometric amounts. The method is highly automated and highly reliable based on comparison of the results to crystal structure data derived from the same protein set. To leverage the experimental metalloprotein annotations, we used a sequence-based de novo prediction method, MetalDetector, to identify Cys and His residues that bind to transition metals for the redundancy reduced subset of 2411 sequences sharing metalloproteins to validate predicted metalloprotein binding site structures. This combination of experimental and bioinformatics approaches provides comprehensive active site analysis on the genome scale for metalloproteins as a class, revealing new insights into metalloprotein structure and function.

  14. Highly Automated Arrival Management and Control System Suitable for Early NextGen

    Science.gov (United States)

    Swenson, Harry N.; Jung, Jaewoo

    2013-01-01

    This is a presentation of previously published work conducted in the development of the Terminal Area Precision Scheduling and Spacing (TAPSS) system. Included are concept and technical descriptions of the TAPSS system and results from human in the loop simulations conducted at Ames Research Center. The Terminal Area Precision Scheduling and Spacing system has demonstrated through research and extensive high-fidelity simulation studies to have benefits in airport arrival throughput, supporting efficient arrival descents, and enabling mixed aircraft navigation capability operations during periods of high congestion. NASA is currently porting the TAPSS system into the FAA TBFM and STARS system prototypes to ensure its ability to operate in the FAA automation Infrastructure. NASA ATM Demonstration Project is using the the TAPSS technologies to provide the ground-based automation tools to enable airborne Interval Management (IM) capabilities. NASA and the FAA have initiated a Research Transition Team to enable potential TAPSS and IM Technology Transfer.

  15. High-throughput neuroimaging-genetics computational infrastructure.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    KAUST Repository

    Soufan, Othman

    2015-12-14

    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.

  18. High-Throughput Neuroimaging-Genetics Computational Infrastructure

    Directory of Open Access Journals (Sweden)

    Ivo D Dinov

    2014-04-01

    Full Text Available 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. 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. 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 diverse suites of software tools and web-services. These pipeline workflows are represented as portable XML objects which transfer the execution instructions and user specifications from the client user machine to remote pipeline servers for distributed computing. Using Alzheimer’s and Parkinson’s data, we provide several examples of translational applications using this infrastructure.

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

    Science.gov (United States)

    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.

  20. A perspective on high throughput analysis of pesticide residues in foods

    Institute of Scientific and Technical Information of China (English)

    Kai ZHANG; Jon W WONG; Perry G WANG

    2011-01-01

    The screening of pesticide residues plays a vital role in food safety. Applications of high throughput analytical procedures are desirable for screening a large number of pesticides and food samples in a time-effi- cient and cost-effective manner. This review discusses how sample throughput of pesticide analysis could be improved with an emphasis on sample preparation, instrumentation and data analysis.

  1. Lab-on-a-chip-based high-throughput screening of the genotoxicity of engineered nanomaterials.

    Science.gov (United States)

    Vecchio, Giuseppe; Fenech, Michael; Pompa, Pier Paolo; Voelcker, Nicolas H

    2014-07-09

    The continuous increasing of engineered nanomaterials (ENMs) in our environment, their combinatorial diversity, and the associated genotoxic risks, highlight the urgent need to better define the possible toxicological effects of ENMs. In this context, we present a new high-throughput screening (HTS) platform based on the cytokinesis-block micronucleus (CBMN) assay, lab-on-chip cell sorting, and automated image analysis. This HTS platform has been successfully applied to the evaluation of the cytotoxic and genotoxic effects of silver nanoparticles (AgNPs) and silica nanoparticles (SiO2NPs). In particular, our results demonstrate the high cyto- and genotoxicity induced by AgNPs and the biocompatibility of SiO2NPs, in primary human lymphocytes. Moreover, our data reveal that the toxic effects are also dependent on size, surface coating, and surface charge. Most importantly, our HTS platform shows that AgNP-induced genotoxicity is lymphocyte sub-type dependent and is particularly pronounced in CD2+ and CD4+ cells.

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

    Science.gov (United States)

    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

  3. Hypoxia-sensitive reporter system for high-throughput screening.

    Science.gov (United States)

    Tsujita, Tadayuki; Kawaguchi, Shin-ichi; Dan, Takashi; Baird, Liam; Miyata, Toshio; Yamamoto, Masayuki

    2015-01-01

    The induction of anti-hypoxic stress enzymes and proteins has the potential to be a potent therapeutic strategy to prevent the progression of ischemic heart, kidney or brain diseases. To realize this idea, small chemical compounds, which mimic hypoxic conditions by activating the PHD-HIF-α system, have been developed. However, to date, none of these compounds were identified by monitoring the transcriptional activation of hypoxia-inducible factors (HIFs). Thus, to facilitate the discovery of potent inducers of HIF-α, we have developed an effective high-throughput screening (HTS) system to directly monitor the output of HIF-α transcription. We generated a HIF-α-dependent reporter system that responds to hypoxic stimuli in a concentration- and time-dependent manner. This system was developed through multiple optimization steps, resulting in the generation of a construct that consists of the secretion-type luciferase gene (Metridia luciferase, MLuc) under the transcriptional regulation of an enhancer containing 7 copies of 40-bp hypoxia responsive element (HRE) upstream of a mini-TATA promoter. This construct was stably integrated into the human neuroblastoma cell line, SK-N-BE(2)c, to generate a reporter system, named SKN:HRE-MLuc. To improve this system and to increase its suitability for the HTS platform, we incorporated the next generation luciferase, Nano luciferase (NLuc), whose longer half-life provides us with flexibility for the use of this reporter. We thus generated a stably transformed clone with NLuc, named SKN:HRE-NLuc, and found that it showed significantly improved reporter activity compared to SKN:HRE-MLuc. In this study, we have successfully developed the SKN:HRE-NLuc screening system as an efficient platform for future HTS.

  4. Scanning fluorescence detector for high-throughput DNA genotyping

    Science.gov (United States)

    Rusch, Terry L.; Petsinger, Jeremy; Christensen, Carl; Vaske, David A.; Brumley, Robert L., Jr.; Luckey, John A.; Weber, James L.

    1996-04-01

    A new scanning fluorescence detector (SCAFUD) was developed for high-throughput genotyping of short tandem repeat polymorphisms (STRPs). Fluorescent dyes are incorporated into relatively short DNA fragments via polymerase chain reaction (PCR) and are separated by electrophoresis in short, wide polyacrylamide gels (144 lanes with well to read distances of 14 cm). Excitation light from an argon laser with primary lines at 488 and 514 nm is introduced into the gel through a fiber optic cable, dichroic mirror, and 40X microscope objective. Emitted fluorescent light is collected confocally through a second fiber. The confocal head is translated across the bottom of the gel at 0.5 Hz. The detection unit utilizes dichroic mirrors and band pass filters to direct light with 10 - 20 nm bandwidths to four photomultiplier tubes (PMTs). PMT signals are independently amplified with variable gain and then sampled at a rate of 2500 points per scan using a computer based A/D board. LabView software (National Instruments) is used for instrument operation. Currently, three fluorescent dyes (Fam, Hex and Rox) are simultaneously detected with peak detection wavelengths of 543, 567, and 613 nm, respectively. The detection limit for fluorescein-labeled primers is about 100 attomoles. Planned SCAFUD upgrades include rearrangement of laser head geometry, use of additional excitation lasers for simultaneous detection of more dyes, and the use of detector arrays instead of individual PMTs. Extensive software has been written for automatic analysis of SCAFUD images. The software enables background subtraction, band identification, multiple- dye signal resolution, lane finding, band sizing and allele calling. Whole genome screens are currently underway to search for loci influencing such complex diseases as diabetes, asthma, and hypertension. Seven production SCAFUDs are currently in operation. Genotyping output for the coming year is projected to be about one million total genotypes (DNA

  5. Towards Chip Scale Liquid Chromatography and High Throughput Immunosensing

    Energy Technology Data Exchange (ETDEWEB)

    Ni, Jing [Iowa State Univ., Ames, IA (United States)

    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

  6. Engineering customized TALE nucleases (TALENs) and TALE transcription factors by fast ligation-based automatable solid-phase high-throughput (FLASH) assembly.

    Science.gov (United States)

    Reyon, Deepak; Maeder, Morgan L; Khayter, Cyd; Tsai, Shengdar Q; Foley, Jonathan E; Sander, Jeffry D; Joung, J Keith

    2013-07-01

    Customized DNA-binding domains made using transcription activator-like effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in various organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or downregulate expression of endogenous genes in human cells and plants. This unit describes a detailed protocol for the recently described fast ligation-based automatable solid-phase high-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multichannel pipet. Using the automated approach, a single researcher can construct up to 96 DNA fragments encoding TALE repeat arrays of various lengths in a single day, and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in a week or less. Plasmids required for FLASH are available by request from the Joung lab (http://eGenome.org). This unit also describes improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) web server (http://ZiFiT.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high throughput.

  7. Complete blood cell count in psittaciformes by using high-throughput image cytometry: a pilot study.

    Science.gov (United States)

    Beaufrère, Hugues; Ammersbach, Mélanie; Tully, Thomas N

    2013-09-01

    The avian hemogram is usually performed in veterinary diagnostic laboratories by using manual cell counting techniques and differential counts determined by light microscopy. There is no standard automated technique for avian blood cell count and differentiation to date. These shortcomings in birds are primarily because erythrocytes and thrombocytes are nucleated, which precludes the use of automated analyzers programmed to perform mammal complete blood cell counts. In addition, there is no standard avian antibody panel, which would allow cell differentiation by immunophenotyping across all commonly seen bird species. We report an alternative hematologic approach for quantification and differentiation of avian blood cells by using high-throughput image cytometry on blood smears in psittacine bird species. A pilot study was designed with 70 blood smears of different psittacine bird species stained with a Wright-Giemsa stain. The slides were scanned at 0.23 microm/pixel. The open-source softwares CellProfiler and CellProfiler Analyst were used for analyzing and sorting each cell by image cytometry. A "pipeline" was constructed in the CellProfiler by using different modules to identify and export hundreds of measures per cell for shape, intensity, and texture. Rules for classifying the different blood cell phenotypes were then determined based on these measurements by iterative feedback and machine learning by using CellProfiler Analyst. Although this approach shows promises, avian Leukopet results could not be duplicated when using this technique as is. Further studies and more standardized prospective investigations may be needed to refine the "pipeline" strategy and the machine learning algorithm.

  8. Automation or De-automation

    Science.gov (United States)

    Gorlach, Igor; Wessel, Oliver

    2008-09-01

    In the global automotive industry, for decades, vehicle manufacturers have continually increased the level of automation of production systems in order to be competitive. However, there is a new trend to decrease the level of automation, especially in final car assembly, for reasons of economy and flexibility. In this research, the final car assembly lines at three production sites of Volkswagen are analysed in order to determine the best level of automation for each, in terms of manufacturing costs, productivity, quality and flexibility. The case study is based on the methodology proposed by the Fraunhofer Institute. The results of the analysis indicate that fully automated assembly systems are not necessarily the best option in terms of cost, productivity and quality combined, which is attributed to high complexity of final car assembly systems; some de-automation is therefore recommended. On the other hand, the analysis shows that low automation can result in poor product quality due to reasons related to plant location, such as inadequate workers' skills, motivation, etc. Hence, the automation strategy should be formulated on the basis of analysis of all relevant aspects of the manufacturing process, such as costs, quality, productivity and flexibility in relation to the local context. A more balanced combination of automated and manual assembly operations provides better utilisation of equipment, reduces production costs and improves throughput.

  9. A High-Throughput Pipeline for the Design of Real-Time PCR Signatures

    Science.gov (United States)

    2010-06-23

    available soon. A high-throughput pipeline for the design of real - time PCR signatures BMC Bioinformatics 2010, 11:340 doi:10.1186/1471-2105-11-340 Ravi...AND SUBTITLE A high-throughput pipeline for the design of real - time PCR signatures 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER...Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 1 A high-throughput pipeline for the design of real - time PCR signatures Ravi Vijaya Satya

  10. PhenStat: A Tool Kit for Standardized Analysis of High Throughput Phenotypic Data.

    Directory of Open Access Journals (Sweden)

    Natalja Kurbatova

    Full Text Available The lack of reproducibility with animal phenotyping experiments is a growing concern among the biomedical community. One contributing factor is the inadequate description of statistical analysis methods that prevents researchers from replicating results even when the original data are provided. Here we present PhenStat--a freely available R package that provides a variety of statistical methods for the identification of phenotypic associations. The methods have been developed for high throughput phenotyping pipelines implemented across various experimental designs with an emphasis on managing temporal variation. PhenStat is targeted to two user groups: small-scale users who wish to interact and test data from large resources and large-scale users who require an automated statistical analysis pipeline. The software provides guidance to the user for selecting appropriate analysis methods based on the dataset and is designed to allow for additions and modifications as needed. The package was tested on mouse and rat data and is used by the International Mouse Phenotyping Consortium (IMPC. By providing raw data and the version of PhenStat used, resources like the IMPC give users the ability to replicate and explore results within their own computing environment.

  11. Commentary: Roles for Pathologists in a High-throughput Image Analysis Team.

    Science.gov (United States)

    Aeffner, Famke; Wilson, Kristin; Bolon, Brad; Kanaly, Suzanne; Mahrt, Charles R; Rudmann, Dan; Charles, Elaine; Young, G David

    2016-08-01

    Historically, pathologists perform manual evaluation of H&E- or immunohistochemically-stained slides, which can be subjective, inconsistent, and, at best, semiquantitative. As the complexity of staining and demand for increased precision of manual evaluation increase, the pathologist's assessment will include automated analyses (i.e., "digital pathology") to increase the accuracy, efficiency, and speed of diagnosis and hypothesis testing and as an important biomedical research and diagnostic tool. This commentary introduces the many roles for pathologists in designing and conducting high-throughput digital image analysis. Pathology review is central to the entire course of a digital pathology study, including experimental design, sample quality verification, specimen annotation, analytical algorithm development, and report preparation. The pathologist performs these roles by reviewing work undertaken by technicians and scientists with training and expertise in image analysis instruments and software. These roles require regular, face-to-face interactions between team members and the lead pathologist. Traditional pathology training is suitable preparation for entry-level participation on image analysis teams. The future of pathology is very exciting, with the expanding utilization of digital image analysis set to expand pathology roles in research and drug development with increasing and new career opportunities for pathologists.

  12. FASTDEF: fast defocus and astigmatism estimation for high-throughput transmission electron microscopy.

    Science.gov (United States)

    Vargas, J; Otón, J; Marabini, R; Jonic, S; de la Rosa-Trevín, J M; Carazo, J M; Sorzano, C O S

    2013-02-01

    In this work we present a fast and automated algorithm for estimating the contrast transfer function (CTF) of a transmission electron microscope. The approach is very suitable for High Throughput work because: (a) it does not require any initial defocus estimation, (b) it is almost an order of magnitude faster than existing approaches, (c) it opens the way to well-defined extensions to the estimation of higher order aberrations, at the same time that provides defocus and astigmatism estimations comparable in accuracy to well established methods, such as Xmipp and CTFFIND3 approaches. The new algorithm is based on obtaining the wrapped modulating phase of the power spectra density pattern by the use of a quadrature filter. This phase is further unwrapped in order to obtain the continuous and smooth absolute phase map; then a Zernike polynomial fitting is performed and the defocus and astigmatism parameters are determined. While the method does not require an initial estimation of the defocus parameters or any non-linear optimization procedure, these approaches can be used if further refinement is desired. Results of the CTF estimation method are presented for standard negative stained images, cryo-electron microscopy images in the absence of carbon support, as well as micrographs with only ice. Additionally, we have also tested the proposed method with micrographs acquired from tilted and untilted samples, obtaining good results. The algorithm is freely available as a part of the Xmipp package [http://xmipp.cnb.csic.es].

  13. High Throughput Method to Quantify Anterior-Posterior Polarity of T-Cells and Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Susan J. Marriott

    2011-11-01

    Full Text Available The virologic synapse (VS, which is formed between a virus-infected and uninfected cell, plays a central role in the transmission of certain viruses, such as HIV and HTLV-1. During VS formation, HTLV-1-infected T-cells polarize cellular and viral proteins toward the uninfected T-cell. This polarization resembles anterior-posterior cell polarity induced by immunological synapse (IS formation, which is more extensively characterized than VS formation and occurs when a T-cell interacts with an antigen-presenting cell. One measure of cell polarity induced by both IS or VS formation is the repositioning of the microtubule organizing center (MTOC relative to the contact point with the interacting cell. Here we describe an automated, high throughput system to score repositioning of the MTOC and thereby cell polarity establishment. The method rapidly and accurately calculates the angle between the MTOC and the IS for thousands of cells. We also show that the system can be adapted to score anterior-posterior polarity establishment of epithelial cells. This general approach represents a significant advancement over manual cell polarity scoring, which is subject to experimenter bias and requires more time and effort to evaluate large numbers of cells.

  14. PhenStat: A Tool Kit for Standardized Analysis of High Throughput Phenotypic Data.

    Science.gov (United States)

    Kurbatova, Natalja; Mason, Jeremy C; Morgan, Hugh; Meehan, Terrence F; Karp, Natasha A

    2015-01-01

    The lack of reproducibility with animal phenotyping experiments is a growing concern among the biomedical community. One contributing factor is the inadequate description of statistical analysis methods that prevents researchers from replicating results even when the original data are provided. Here we present PhenStat--a freely available R package that provides a variety of statistical methods for the identification of phenotypic associations. The methods have been developed for high throughput phenotyping pipelines implemented across various experimental designs with an emphasis on managing temporal variation. PhenStat is targeted to two user groups: small-scale users who wish to interact and test data from large resources and large-scale users who require an automated statistical analysis pipeline. The software provides guidance to the user for selecting appropriate analysis methods based on the dataset and is designed to allow for additions and modifications as needed. The package was tested on mouse and rat data and is used by the International Mouse Phenotyping Consortium (IMPC). By providing raw data and the version of PhenStat used, resources like the IMPC give users the ability to replicate and explore results within their own computing environment.

  15. Corifungin, a new drug lead against Naegleria, identified from a high-throughput screen.

    Science.gov (United States)

    Debnath, Anjan; Tunac, Josefino B; Galindo-Gómez, Silvia; Silva-Olivares, Angélica; Shibayama, Mineko; McKerrow, James H

    2012-11-01

    Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. The drug of choice in treating PAM is the antifungal antibiotic amphotericin B, but its use is associated with severe adverse effects. Moreover, few patients treated with amphotericin B have survived PAM. Therefore, fast-acting and efficient drugs are urgently needed for the treatment of PAM. To facilitate drug screening for this pathogen, an automated, high-throughput screening methodology was developed and validated for the closely related species Naegleria gruberi. Five kinase inhibitors and an NF-kappaB inhibitor were hits identified in primary screens of three compound libraries. Most importantly for a preclinical drug discovery pipeline, we identified corifungin, a water-soluble polyene macrolide with a higher activity against Naegleria than that of amphotericin B. Transmission electron microscopy of N. fowleri trophozoites incubated with different concentrations of corifungin showed disruption of cytoplasmic and plasma membranes and alterations in mitochondria, followed by complete lysis of amebae. In vivo efficacy of corifungin in a mouse model of PAM was confirmed by an absence of detectable amebae in the brain and 100% survival of mice for 17 days postinfection for a single daily intraperitoneal dose of 9 mg/kg of body weight given for 10 days. The same dose of amphotericin B did not reduce ameba growth, and mouse survival was compromised. Based on these results, the U.S. FDA has approved orphan drug status for corifungin for the treatment of PAM.

  16. High-throughput single-molecule force spectroscopy for membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bosshart, Patrick D; Casagrande, Fabio; Frederix, Patrick L T M; Engel, Andreas; Fotiadis, Dimitrios [M E Mueller Institute for Structural Biology, Biozentrum of the University of Basel, CH-4056 Basel (Switzerland); Ratera, Merce; Palacin, Manuel [Institute for Research in Biomedicine, Barcelona Science Park, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona and Centro de Investigacion Biomedica en Red de Enfermedades Raras, E-08028 Barcelona (Spain); Bippes, Christian A; Mueller, Daniel J [BioTechnology Center, Technical University, Tatzberg 47, D-01307 Dresden (Germany)], E-mail: andreas.engel@unibas.ch, E-mail: dimitrios.fotiadis@mci.unibe.ch

    2008-09-24

    Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether {approx}400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with {approx}200 (AdiC) and {approx}400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

  17. High-Throughput Screening for Bioactive Molecules Using Primary Cell Culture of Transgenic Zebrafish Embryos

    Directory of Open Access Journals (Sweden)

    Haigen Huang

    2012-09-01

    Full Text Available Transgenic zebrafish embryos expressing tissue-specific green fluorescent protein (GFP can provide an unlimited supply of primary embryonic cells. Agents that promote the differentiation of these cells may be beneficial for therapeutics. We report a high-throughput approach for screening small molecules that regulate cell differentiation using lineage-specific GFP transgenic zebrafish embryonic cells. After validating several known regulators of the differentiation of endothelial and other cell types, we performed a screen for proangiogenic molecules using undifferentiated primary cells from flk1-GFP transgenic zebrafish embryos. Cells were grown in 384-well plates with 12,128 individual small molecules, and GFP expression was analyzed by means of an automated imaging system, which allowed us to screen thousands of compounds weekly. As a result, 23 molecules were confirmed to enhance angiogenesis, and 11 of them were validated to promote the proliferation of mammalian human umbilical vascular endothelial cells and induce Flk1+ cells from murine embryonic stem cells. We demonstrated the general applicability of this strategy by analyzing additional cell lineages using zebrafish expressing GFP in pancreatic, cardiac, and dopaminergic cells.

  18. Adaptation of high-throughput screening in drug discovery-toxicological screening tests.

    Science.gov (United States)

    Szymański, Paweł; Markowicz, Magdalena; Mikiciuk-Olasik, Elżbieta

    2012-01-01

    High-throughput screening (HTS) is one of the newest techniques used in drug design and may be applied in biological and chemical sciences. This method, due to utilization of robots, detectors and software that regulate the whole process, enables a series of analyses of chemical compounds to be conducted in a short time and the affinity of biological structures which is often related to toxicity to be defined. Since 2008 we have implemented the automation of this technique and as a consequence, the possibility to examine 100,000 compounds per day. The HTS method is more frequently utilized in conjunction with analytical techniques such as NMR or coupled methods e.g., LC-MS/MS. Series of studies enable the establishment of the rate of affinity for targets or the level of toxicity. Moreover, researches are conducted concerning conjugation of nanoparticles with drugs and the determination of the toxicity of such structures. For these purposes there are frequently used cell lines. Due to the miniaturization of all systems, it is possible to examine the compound's toxicity having only 1-3 mg of this compound. Determination of cytotoxicity in this way leads to a significant decrease in the expenditure and to a reduction in the length of the study.

  19. Adaptation of High-Throughput Screening in Drug Discovery—Toxicological Screening Tests

    Directory of Open Access Journals (Sweden)

    Paweł Szymański

    2011-12-01

    Full Text Available High-throughput screening (HTS is one of the newest techniques used in drug design and may be applied in biological and chemical sciences. This method, due to utilization of robots, detectors and software that regulate the whole process, enables a series of analyses of chemical compounds to be conducted in a short time and the affinity of biological structures which is often related to toxicity to be defined. Since 2008 we have implemented the automation of this technique and as a consequence, the possibility to examine 100,000 compounds per day. The HTS method is more frequently utilized in conjunction with analytical techniques such as NMR or coupled methods e.g., LC-MS/MS. Series of studies enable the establishment of the rate of affinity for targets or the level of toxicity. Moreover, researches are conducted concerning conjugation of nanoparticles with drugs and the determination of the toxicity of such structures. For these purposes there are frequently used cell lines. Due to the miniaturization of all systems, it is possible to examine the compound’s toxicity having only 1–3 mg of this compound. Determination of cytotoxicity in this way leads to a significant decrease in the expenditure and to a reduction in the length of the study.

  20. Reconciling Phylogeny and Function During Plant Litter Decomposition by High-Throughput Functional Metagenomics

    Science.gov (United States)

    Nyyssonen, M.; Weihe, C.; Goulden, M.; Treseder, K. K.; Martiny, J.; Martiny, A.; Allison, S. D.; Brodie, E. L.

    2012-12-01

    Integrating information on microbial diversity and functionality with ecosystem processes may be critical to predicting how ecosystems respond to environmental change. While theoretical models can be used to link microbial processes to environmental responses and rates, accurate predictions of ecosystem functioning would benefit from detailed information on microbial community composition and function. In this study, our aim was to identify functional traits involved in plant litter decomposition, a model process for carbon cycling, from decomposing plant litter. The overall goal is then to link these traits with individual microbial taxa and use this information to build predictive trait-based models of ecosystem responses to global change. In order to identify activities involved in plant litter decomposition we used automated high-throughput assays for functional screening of metagenomic fosmid libraries prepared from decomposing plant litter. Litter was collected over 15 month period from a global change field experiment undergoing rainfall and nitrogen manipulations. We identified over 600 cellulose, hemicellulose, chitin and starch hydrolyzing clones following screening of over 300,000 clones. The frequency of positive clones was ten times lower during dry season but no significant differences in hit rates were observed between different treatments. The positive clones were shotgun sequenced on the Illumina sequencing platform and the identified hydrolytic genes were shown to represent variety bacterial taxonomic groups including Proteobacteria and Bacteroidetes.

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

    Science.gov (United States)

    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).

  2. ARQiv-HTS, a versatile whole-organism screening platform enabling in vivo drug discovery at high-throughput rates.

    Science.gov (United States)

    White, David T; Eroglu, Arife Unal; Wang, Guohua; Zhang, Liyun; Sengupta, Sumitra; Ding, Ding; Rajpurohit, Surendra K; Walker, Steven L; Ji, Hongkai; Qian, Jiang; Mumm, Jeff S

    2016-12-01

    The zebrafish has emerged as an important model for whole-organism small-molecule screening. However, most zebrafish-based chemical screens have achieved only mid-throughput rates. Here we describe a versatile whole-organism drug discovery platform that can achieve true high-throughput screening (HTS) capacities. This system combines our automated reporter quantification in vivo (ARQiv) system with customized robotics, and is termed 'ARQiv-HTS'. We detail the process of establishing and implementing ARQiv-HTS: (i) assay design and optimization, (ii) calculation of sample size and hit criteria, (iii) large-scale egg production, (iv) automated compound titration, (v) dispensing of embryos into microtiter plates, and (vi) reporter quantification. We also outline what we see as best practice strategies for leveraging the power of ARQiv-HTS for zebrafish-based drug discovery, and address technical challenges of applying zebrafish to large-scale chemical screens. Finally, we provide a detailed protocol for a recently completed inaugural ARQiv-HTS effort, which involved the identification of compounds that elevate insulin reporter activity. Compounds that increased the number of insulin-producing pancreatic beta cells represent potential new therapeutics for diabetic patients. For this effort, individual screening sessions took 1 week to conclude, and sessions were performed iteratively approximately every other day to increase throughput. At the conclusion of the screen, more than a half million drug-treated larvae had been evaluated. Beyond this initial example, however, the ARQiv-HTS platform is adaptable to almost any reporter-based assay designed to evaluate the effects of chemical compounds in living small-animal models. ARQiv-HTS thus enables large-scale whole-organism drug discovery for a variety of model species and from numerous disease-oriented perspectives.

  3. Development of a high-throughput Candida albicans biofilm chip.

    Directory of Open Access Journals (Sweden)

    Anand Srinivasan

    Full Text Available 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.

  4. Development of a high-throughput Candida albicans biofilm chip.

    Science.gov (United States)

    Srinivasan, Anand; Uppuluri, Priya; Lopez-Ribot, Jose; Ramasubramanian, Anand K

    2011-04-22

    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.

  5. High throughput RNAi assay optimization using adherent cell cytometry

    Directory of Open Access Journals (Sweden)

    Pradhan Leena

    2011-04-01

    Full Text Available Abstract Background siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC. Methods AoSMC were seeded at a density of 3000-8000 cells/well of a 96well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM, or non-targeting labeled siRNA, siGLO Red (5 or 50 nM using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo® was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell. Results After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19. Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs. Conclusion This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate in vitro cell assays and thus save costs.

  6. Emerging high throughput analyses of cyanobacterial toxins and toxic cyanobacteria.

    Science.gov (United States)

    Sivonen, Kaarina

    2008-01-01

    The common occurrence of toxic cyanobacteria causes problems for health of animals and human beings. More research and good monitoring systems are needed to protect water users. It is important to have rapid, reliable and accurate analysis i.e. high throughput methods to identify the toxins as well as toxin producers in the environment. Excellent methods, such as ELISA already exist to analyse cyanobacterial hepatotoxins and saxitoxins, and PPIA for microcystins and nodularins. The LC/MS method can be fast in identifying the toxicants in the samples. Further development of this area should resolve the problems with sampling and sample preparation, which still are the bottlenecks of rapid analyses. In addition, the availability of reliable reference materials and standards should be resolved. Molecular detection methods are now routine in clinical and criminal laboratories and may also become important in environmental diagnostics. One prerequisite for the development of molecular analysis is that pure cultures of the producer organisms are available for identification of the biosynthetic genes responsible for toxin production and for proper testing of the diagnostic methods. Good methods are already available for the microcystin and nodularin-producing cyanobacteria such as conventional PCR, quantitative real-time PCR and microarrays/DNA chips. The DNA-chip technology offers an attractive monitoring system for toxic and non-toxic cyanobacteria. Only with these new technologies (PCR + DNA-chips) will we be able to study toxic cyanobacteria populations in situ and the effects of environmental factors on the occurrence and proliferation of especially toxic cyanobacteria. This is likely to yield important information for mitigation purposes. Further development of these methods should include all cyanobacterial biodiversity, including all toxin producers and primers/probes to detect producers of neurotoxins, cylindrospermopsins etc. (genes are unknown). The on

  7. Automation of Library Preparation for High-resolution ChIP-seq Profiling

    Science.gov (United States)

    Henry, Isabelle M.; Cotterman, Rebecca; Knoepfler, Paul; Comai, Luca; Kim, Ryan W.; O'Geen, Henriette

    2013-01-01

    The dynamic modification of DNA and histones plays a key role in transcriptional regulationthrough altering the packaging of DNA and modifying the nucleosome surface. These chromatin states, also referred to as the epigenome, are distinctive for different tissues, developmental stages, and disease states and can also be altered by environmental influences. New technologies allow the genome-wide visualization of the information encoded in theepigenome. For example, the chromatin immunoprecipitation (ChIP) assay allows investigators to characterize DNA–protein interactions in vivo. ChIP followed by high-throughput sequencing (ChIP-seq) is a powerful tool to identify genome-wide profiles of transcription factors, histone modifications, DNA methylation, and nucleosome positioning. The low yield of ChIP assays presents a challenge for reproducible and high quality library preparation for high throughput sequencing. Using the automated library preparation system from IntegenX, we prepared ChIP-seq libraries from as little as 1 ng ChIP DNA material. Sequencing of biological replicates on the Illumina platform confirmed a 95%-98% overlap of identified binding sites. Progress of ChIP experiments using limited tissue amounts from rice seedlings as well as other applications of the library automation system will be discussed.

  8. Large-scale microfluidics providing high-resolution and high-throughput screening of Caenorhabditis elegans poly-glutamine aggregation model

    Science.gov (United States)

    Mondal, Sudip; Hegarty, Evan; Martin, Chris; Gökçe, Sertan Kutal; Ghorashian, Navid; Ben-Yakar, Adela

    2016-10-01

    Next generation drug screening could benefit greatly from in vivo studies, using small animal models such as Caenorhabditis elegans for hit identification and lead optimization. Current in vivo assays can operate either at low throughput with high resolution or with low resolution at high throughput. To enable both high-throughput and high-resolution imaging of C. elegans, we developed an automated microfluidic platform. This platform can image 15 z-stacks of ~4,000 C. elegans from 96 different populations using a large-scale chip with a micron resolution in 16 min. Using this platform, we screened ~100,000 animals of the poly-glutamine aggregation model on 25 chips. We tested the efficacy of ~1,000 FDA-approved drugs in improving the aggregation phenotype of the model and identified four confirmed hits. This robust platform now enables high-content screening of various C. elegans disease models at the speed and cost of in vitro cell-based assays.

  9. Validation of high-throughput real time polymerase chain reaction assays for simultaneous detection of invasive citrus pathogens.

    Science.gov (United States)

    Saponari, Maria; Loconsole, Giuliana; Liao, Hui-Hong; Jiang, Bo; Savino, Vito; Yokomi, Raymond K

    2013-11-01

    A number of important citrus pathogens are spread by graft propagation, arthropod vector transmission and inadvertent import and dissemination of infected plants. For these reasons, citrus disease management and clean stock programs require pathogen detection systems which are economical and sensitive to maintain a healthy industry. To this end, multiplex quantitative real-time PCR (qPCR) assays were developed allowing high-throughput and simultaneous detection of some major invasive citrus pathogens. Automated high-throughput extraction comparing several bead-based commercial extraction kits were tested and compared with tissue print and manual extraction to obtain nucleic acids from healthy and pathogen-infected citrus trees from greenhouse in planta collections and field. Total nucleic acids were used as templates for pathogen detection. Multiplex reverse transcription-qPCR (RT-qPCR) assays were developed for simultaneous detection of six targets including a virus, two viroids, a bacterium associated with huanglongbing and a citrus RNA internal control. Specifically, two one-step TaqMan-based multiplex RT-qPCR assays were developed and tested with target templates to determine sensitivity and detection efficiency. The first assay included primers and probes for 'Candidatus Liberibacter asiaticus' (CLas) and Citrus tristeza virus (CTV) broad spectrum detection and genotype differentiation (VT- and T3-like genotypes). The second assay contained primers and probes for Hop stunt viroid (HSVd), Citrus exocortis viroid (CEVd) and the mitochondrial NADH dehydrogenase (nad5) mRNA as an internal citrus host control. Primers and TaqMan probes for the viroids were designed in this work; whereas those for the other pathogens were from reports of others. Based on quantitation cycle values, automated high-throughput extraction of samples proved to be as suitable as manual extraction. The multiplex RT-qPCR assays detected both RNA and DNA pathogens in the same dilution series

  10. Wide Throttling, High Throughput Hall Thruster for Science and Exploration Missions Project

    Data.gov (United States)

    National Aeronautics and Space Administration — In response to Topic S3.04 "Propulsion Systems," Busek Co. Inc. will develop a high throughput Hall effect thruster with a nominal peak power of 1-kW and wide...

  11. Wide Throttling, High Throughput Hall Thruster for Science and Exploration Missions Project

    Data.gov (United States)

    National Aeronautics and Space Administration — In response to Topic S3-04 "Propulsion Systems," Busek proposes to develop a high throughput Hall effect thruster with a nominal peak power of 1-kW and wide...

  12. NMRbot: Python scripts enable high-throughput data collection on current Bruker BioSpin NMR spectrometers.

    Science.gov (United States)

    Clos, Lawrence J; Jofre, M Fransisca; Ellinger, James J; Westler, William M; Markley, John L

    2013-06-01

    To facilitate the high-throughput acquisition of nuclear magnetic resonance (NMR) experimental data on large sets of samples, we have developed a simple and straightforward automated methodology that capitalizes on recent advances in Bruker BioSpin NMR spectrometer hardware and software. Given the daunting challenge for non-NMR experts to collect quality spectra, our goal was to increase user accessibility, provide customized functionality, and improve the consistency and reliability of resultant data. This methodology, NMRbot, is encoded in a set of scripts written in the Python programming language accessible within the Bruker BioSpin TopSpin™ software. NMRbot improves automated data acquisition and offers novel tools for use in optimizing experimental parameters on the fly. This automated procedure has been successfully implemented for investigations in metabolomics, small-molecule library profiling, and protein-ligand titrations on four Bruker BioSpin NMR spectrometers at the National Magnetic Resonance Facility at Madison. The investigators reported benefits from ease of setup, improved spectral quality, convenient customizations, and overall time savings.

  13. A multi-endpoint, high-throughput study of nanomaterial toxicity in Caenorhabditis elegans

    OpenAIRE

    Jung, Sang-Kyu; Qu, Xiaolei; Aleman-Meza, Boanerges; Wang, Tianxiao; Riepe, Celeste; Liu, Zheng; Li, Qilin; Zhong, Weiwei

    2015-01-01

    The booming nanotech industry has raised public concerns about the environmental health and safety impact of engineered nanomaterials (ENMs). High-throughput assays are needed to obtain toxicity data for the rapidly increasing number of ENMs. Here we present a suite of high-throughput methods to study nanotoxicity in intact animals using Caenorhabditis elegans as a model. At the population level, our system measures food consumption of thousands of animals to evaluate population fitness. At t...

  14. Self-encoding Functional Resin Applying for Combinatorial Chemistry and High Throughput Screening

    Institute of Scientific and Technical Information of China (English)

    DU Lei; CHEN Tong-sheng

    2004-01-01

    A novel solid phase organic synthesis resin was synthesized for combinatorial high-throughput screening,which based on FTIR spectra self-encoding functional resin technology. A new deconvolution strategy termed position encoding deconvolution had illustrated and was compared with some popular combinatorial deconvolution strategies in efficiency and information content. The mimic high throughput screening of hexapeptide library successfully proved the applying of the self-encoding functional resin technology and the position encoding deconvolution strategy.

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

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Wei; Shabbir, Faizan; Gong, Chao; Gulec, Cagatay; Pigeon, Jeremy; Shaw, Jessica; Greenbaum, Alon; Tochitsky, Sergei; Joshi, Chandrashekhar [Electrical Engineering Department, University of California, Los Angeles, California 90095 (United States); Ozcan, Aydogan, E-mail: ozcan@ucla.edu [Electrical Engineering Department, University of California, Los Angeles, California 90095 (United States); Bioengineering Department, University of California, Los Angeles, California 90095 (United States); California NanoSystems Institute (CNSI), University of California, Los Angeles, California 90095 (United States)

    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.

  16. High-Throughput Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat Genotyping for Mycobacterium tuberculosis Epidemiological Studies

    Science.gov (United States)

    Bidault, Floriane; Mosnier, Amandine; Bablishvili, Nino; Tukvadze, Nestani; Somphavong, Silaphet; Paboriboune, Phimpha; Ocheretina, Oksana; Pape, Jean William; Paranhos-Baccala, Glaucia; Berland, Jean-Luc

    2014-01-01

    The emergence of drug-resistant forms of tuberculosis (TB) represents a major public health concern. Understanding the transmission routes of the disease is a key factor for its control and for the implementation of efficient interventions. Mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) marker typing is a well-described method for lineage identification and transmission tracking. However, the conventional manual genotyping technique is cumbersome and time-consuming and entails many risks for errors, thus hindering its implementation and dissemination. We describe here a new approach using the QIAxcel system, an automated high-throughput capillary electrophoresis system that also carries out allele calling. This automated method was assessed on 1,824 amplicons from 82 TB isolates and tested with sets of markers of 15 or 24 loci. Overall allele-calling concordance between the methods from 140 to 1,317 bp was 98.9%. DNA concentrations and repeatability and reproducibility performances showed no biases in allele calling. Furthermore, turnaround time using this automated system was reduced by 81% compared to the conventional manual agarose gel method. In sum, this new automated method facilitates MIRU-VNTR genotyping and provides reliable results. Therefore, it is well suited for field genotyping. The implementation of this method will help to achieve accurate and cost-effective epidemiological studies, especially in countries with a high prevalence of TB, where the high number of strains complicates the surveillance of circulating lineages and requires efficient interventions to be carried out in an urgent manner. PMID:25428144

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  19. High Throughput 600 Watt Hall Effect Thruster for Space Exploration

    Science.gov (United States)

    Szabo, James; Pote, Bruce; Tedrake, Rachel; Paintal, Surjeet; Byrne, Lawrence; Hruby, Vlad; Kamhawi, Hani; Smith, Tim

    2016-01-01

    A nominal 600-Watt Hall Effect Thruster was developed to propel unmanned space vehicles. Both xenon and iodine compatible versions were demonstrated. With xenon, peak measured thruster efficiency is 46-48% at 600-W, with specific impulse from 1400 s to 1700 s. Evolution of the thruster channel due to ion erosion was predicted through numerical models and calibrated with experimental measurements. Estimated xenon throughput is greater than 100 kg. The thruster is well sized for satellite station keeping and orbit maneuvering, either by itself or within a cluster.

  20. High-throughput studies of protein shapes and interactions by synchrotron small-angle X-ray scattering.

    Science.gov (United States)

    Jeffries, Cy M; Svergun, Dmitri I

    2015-01-01

    Solution-based small angle X-ray scattering (SAXS) affords the opportunity to extract accurate structural parameters and global shape information from diverse biological macromolecular systems. SAXS is an ideal complementary technique to other structural and biophysical methods but it can also be applied alone to access structural information that is otherwise unobtainable using high-resolution methods. Macromolecular structures ranging from kilodaltons to gigadaltons can be analyzed, which encompasses the size of most proteins and functional cellular complexes. The SAXS analysis is performed using only a few microliters of solution containing microgram quantities of purified material in sample environments that can be tailored to mimic physiological conditions or altered to suit a particular question. High-brilliance synchrotron X-ray sources and parallel advances in hardware and computing have reduced data acquisition times to the millisecond range and the application of automated methods have allowed data processing and low resolution shape modelling to be completed within minutes. These developments have paved the way for high-throughput studies that generate significant quantities of structural information over a short period of time. Here, we briefly consider the basics of SAXS and describe major methods and protocols employed in high-throughput SAXS studies.

  1. Automated large-scale culture and medium-throughput chemical screen for modulators of proliferation and viability of human induced pluripotent stem cell-derived neuroepithelial-like stem cells.

    Science.gov (United States)

    McLaren, Donna; Gorba, Thorsten; Marguerie de Rotrou, Anita; Pillai, Gopalan; Chappell, Clare; Stacey, Alison; Lingard, Sarah; Falk, Anna; Smith, Austin; Koch, Philipp; Brüstle, Oliver; Vickers, Richard; Tinsley, Jon; Flanders, David; Bello, Paul; Craig, Stewart

    2013-03-01

    The aim of this study was to demonstrate proof-of-concept feasibility for the use of human neural stem cells (NSCs) for high-throughput screening (HTS) applications. For this study, an adherent human induced pluripotent stem (iPS) cell-derived long-term, self-renewing, neuroepithelial-like stem (lt-NES) cell line was selected as a representative NSC. Here, we describe the automated large-scale serum-free culture ("scale-up") of human lt-NES cells on the CompacT SelecT cell culture robotic platform, followed by their subsequent automated "scale-out" into a microwell plate format. We also report a medium-throughput screen of 1000 compounds to identify modulators of neural stem cell proliferation and/or survival. The screen was performed on two independent occasions using a cell viability assay with end-point reading resulting in the identification of 24 potential hit compounds, 5 of which were found to increase the proliferation and/or survival of human lt-NES on both occasions. Follow-up studies confirmed a dose-dependent effect of one of the hit compounds, which was a Cdk-2 modulator. This approach could be further developed as part of a strategy to screen compounds to either improve the procedures for the in vitro expansion of neural stem cells or to potentially modulate endogenous neural stem cell behavior in the diseased nervous system.

  2. High-content live cell imaging with RNA probes: advancements in high-throughput antimalarial drug discovery

    Directory of Open Access Journals (Sweden)

    Cervantes Serena

    2009-06-01

    Full Text Available Abstract Background Malaria, a major public health issue in developing nations, is responsible for more than one million deaths a year. The most lethal species, Plasmodium falciparum, causes up to 90% of fatalities. Drug resistant strains to common therapies have emerged worldwide and recent artemisinin-based combination therapy failures hasten the need for new antimalarial drugs. Discovering novel compounds to be used as antimalarials is expedited by the use of a high-throughput screen (HTS to detect parasite growth and proliferation. Fluorescent dyes that bind to DNA have replaced expensive traditional radioisotope incorporation for HTS growth assays, but do not give additional information regarding the parasite stage affected by the drug and a better indication of the drug's mode of action. Live cell imaging with RNA dyes, which correlates with cell growth and proliferation, has been limited by the availability of successful commercial dyes. Results After screening a library of newly synthesized stryrl dyes, we discovered three RNA binding dyes that provide morphological details of live parasites. Utilizing an inverted confocal imaging platform, live cell imaging of parasites increases parasite detection, improves the spatial and temporal resolution of the parasite under drug treatments, and can resolve morphological changes in individual cells. Conclusion This simple one-step technique is suitable for automation in a microplate format for novel antimalarial compound HTS. We have developed a new P. falciparum RNA high-content imaging growth inhibition assay that is robust with time and energy efficiency.

  3. Analysis of high-throughput sequencing and annotation strategies for phage genomes.

    Directory of Open Access Journals (Sweden)

    Matthew R Henn

    Full Text Available BACKGROUND: Bacterial viruses (phages play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage. METHODOLOGY/PRINCIPAL FINDINGS: To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles, and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL or of a whole genome shotgun library (WGSL, or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling. CONCLUSIONS/SIGNIFICANCE: These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.

  4. Characterization of Metalloproteins by High-throughput X-ray Absorption Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    W Shi; M Punta; J Bohon; J Sauder; R DMello; M Sullivan; J Toomey; D Abel; M Lippi; et al.

    2011-12-31

    High-throughput X-ray absorption spectroscopy was used to measure transition metal content based on quantitative detection of X-ray fluorescence signals for 3879 purified proteins from several hundred different protein families generated by the New York SGX Research Center for Structural Genomics. Approximately 9% of the proteins analyzed showed the presence of transition metal atoms (Zn, Cu, Ni, Co, Fe, or Mn) in stoichiometric amounts. The method is highly automated and highly reliable based on comparison of the results to crystal structure data derived from the same protein set. To leverage the experimental metalloprotein annotations, we used a sequence-based de novo prediction method, MetalDetector, to identify Cys and His residues that bind to transition metals for the redundancy reduced subset of 2411 sequences sharing <70% sequence identity and having at least one His or Cys. As the HT-XAS identifies metal type and protein binding, while the bioinformatics analysis identifies metal-binding residues, the results were combined to identify putative metal-binding sites in the proteins and their associated families. We explored the combination of this data with homology models to generate detailed structure models of metal-binding sites for representative proteins. Finally, we used extended X-ray absorption fine structure data from two of the purified Zn metalloproteins to validate predicted metalloprotein binding site structures. This combination of experimental and bioinformatics approaches provides comprehensive active site analysis on the genome scale for metalloproteins as a class, revealing new insights into metalloprotein structure and function.

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

    Science.gov (United States)

    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 Discrete Time Markov Chain Model for High Throughput Bidirectional Fano Decoders

    CERN Document Server

    Xu, Ran; Morris, Kevin; Kocak, Taskin

    2010-01-01

    The bidirectional Fano algorithm (BFA) can achieve at least two times decoding throughput compared to the conventional unidirectional Fano algorithm (UFA). In this paper, bidirectional Fano decoding is examined from the queuing theory perspective. A Discrete Time Markov Chain (DTMC) is employed to model the BFA decoder with a finite input buffer. The relationship between the input data rate, the input buffer size and the clock speed of the BFA decoder is established. The DTMC based modelling can be used in designing a high throughput parallel BFA decoding system. It is shown that there is a tradeoff between the number of BFA decoders and the input buffer size, and an optimal input buffer size can be chosen to minimize the hardware complexity for a target decoding throughput in designing a high throughput parallel BFA decoding system.

  7. Throughput Analysis for a High-Performance FPGA-Accelerated Real-Time Search Application

    Directory of Open Access Journals (Sweden)

    Wim Vanderbauwhede

    2012-01-01

    Full Text Available We propose an FPGA design for the relevancy computation part of a high-throughput real-time search application. The application matches terms in a stream of documents against a static profile, held in off-chip memory. We present a mathematical analysis of the throughput of the application and apply it to the problem of scaling the Bloom filter used to discard nonmatches.

  8. Development and clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria

    OpenAIRE

    Perera, Rushini S.; Ding, Xavier C; Tully, Frank; Oliver, James; Bright, Nigel; Bell, David; Chiodini, Peter L; Gonzalez, Iveth J.; Spencer D Polley

    2017-01-01

    Background Accurate and efficient detection of sub-microscopic malaria infections is crucial for enabling rapid treatment and interruption of transmission. Commercially available malaria LAMP kits have excellent diagnostic performance, though throughput is limited by the need to prepare samples individually. Here, we evaluate the clinical performance of a newly developed high throughput (HTP) sample processing system for use in conjunction with the Eiken malaria LAMP kit. Methods The HTP syst...

  9. Estimation of immune cell densities in immune cell conglomerates: an approach for high-throughput quantification.

    Directory of Open Access Journals (Sweden)

    Niels Halama

    Full Text Available BACKGROUND: Determining the correct number of positive immune cells in immunohistological sections of colorectal cancer and other tumor entities is emerging as an important clinical predictor and therapy selector for an individual patient. This task is usually obstructed by cell conglomerates of various sizes. We here show that at least in colorectal cancer the inclusion of immune cell conglomerates is indispensable for estimating reliable patient cell counts. Integrating virtual microscopy and image processing principally allows the high-throughput evaluation of complete tissue slides. METHODOLOGY/PRINCIPAL FINDINGS: For such large-scale systems we demonstrate a robust quantitative image processing algorithm for the reproducible quantification of cell conglomerates on CD3 positive T cells in colorectal cancer. While isolated cells (28 to 80 microm(2 are counted directly, the number of cells contained in a conglomerate is estimated by dividing the area of the conglomerate in thin tissues sections (< or =6 microm by the median area covered by an isolated T cell which we determined as 58 microm(2. We applied our algorithm to large numbers of CD3 positive T cell conglomerates and compared the results to cell counts obtained manually by two independent observers. While especially for high cell counts, the manual counting showed a deviation of up to 400 cells/mm(2 (41% variation, algorithm-determined T cell numbers generally lay in between the manually observed cell numbers but with perfect reproducibility. CONCLUSION: In summary, we recommend our approach as an objective and robust strategy for quantifying immune cell densities in immunohistological sections which can be directly implemented into automated full slide image processing systems.

  10. A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders

    Science.gov (United States)

    Simeoni, Ilenia; Stephens, Jonathan C.; Hu, Fengyuan; Deevi, Sri V. V.; Megy, Karyn; Bariana, Tadbir K.; Lentaigne, Claire; Schulman, Sol; Sivapalaratnam, Suthesh; Vries, Minka J. A.; Westbury, Sarah K.; Greene, Daniel; Papadia, Sofia; Alessi, Marie-Christine; Attwood, Antony P.; Ballmaier, Matthias; Baynam, Gareth; Bermejo, Emilse; Bertoli, Marta; Bray, Paul F.; Bury, Loredana; Cattaneo, Marco; Collins, Peter; Daugherty, Louise C.; Favier, Rémi; French, Deborah L.; Furie, Bruce; Gattens, Michael; Germeshausen, Manuela; Ghevaert, Cedric; Goodeve, Anne C.; Guerrero, Jose A.; Hampshire, Daniel J.; Hart, Daniel P.; Heemskerk, Johan W. M.; Henskens, Yvonne M. C.; Hill, Marian; Hogg, Nancy; Jolley, Jennifer D.; Kahr, Walter H.; Kelly, Anne M.; Kerr, Ron; Kostadima, Myrto; Kunishima, Shinji; Lambert, Michele P.; Liesner, Ri; López, José A.; Mapeta, Rutendo P.; Mathias, Mary; Millar, Carolyn M.; Nathwani, Amit; Neerman-Arbez, Marguerite; Nurden, Alan T.; Nurden, Paquita; Othman, Maha; Peerlinck, Kathelijne; Perry, David J.; Poudel, Pawan; Reitsma, Pieter; Rondina, Matthew T.; Smethurst, Peter A.; Stevenson, William; Szkotak, Artur; Tuna, Salih; van Geet, Christel; Whitehorn, Deborah; Wilcox, David A.; Zhang, Bin; Revel-Vilk, Shoshana; Gresele, Paolo; Bellissimo, Daniel B.; Penkett, Christopher J.; Laffan, Michael A.; Mumford, Andrew D.; Rendon, Augusto; Freson, Kathleen; Ouwehand, Willem H.; Turro, Ernest

    2016-01-01

    Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD. PMID:27084890

  11. High-speed CMOS image sensor for high-throughput lensless microfluidic imaging system

    Science.gov (United States)

    Yan, Mei; Huang, Xiwei; Jia, Qixiang; Nadipalli, Revanth; Wang, Tongxi; Shang, Yang; Yu, Hao; Je, Minkyu; Yeo, Kiatseng

    2012-03-01

    The integration of CMOS image sensor and microfluidics becomes a promising technology for point-of-care (POC) diagnosis. However, commercial image sensors usually have limited speed and low-light sensitivity. One high-speed and high-sensitivity CMOS image sensor chip is introduced in this paper, targeted for high-throughput microfluidic imaging system. Firstly, high speed image sensor architecture is introduced with design of column-parallel single-slope analog-todigital converter (ADC) with digital correlated double sampling (CDS). The frame rate can be achieved to 2400 frames/second (fps) with resolution of 128×96 for high-throughput microfluidic imaging. Secondly, the designed system has superior low-light sensitivity, which is achieved by large pixel size (10μm×10μm, 56% fill factor). Pixel peak signalnoise- ratio (SNR) reaches to 50dB with 10dB improvement compared to the commercial pixel (2.2μm×2.2μm). The degradation of pixel resolution is compensated by super-resolution image processing algorithm. By reconstructing single image with multiple low-resolution frames, we can equivalently achieve 2μm resolution with physical 10μm pixel. Thirdly, the system-on-chip (SoC) integration results in a real-time controlled intelligent imaging system without expensive data storage and time-consuming computer analysis. This initial sensor prototype with timing-control makes it possible to develop high-throughput lensless microfluidic imaging system for POC diagnosis.

  12. High-Throughput Atomic Force Microscopes Operating in Parallel

    CERN Document Server

    Sadeghian, H; Dekker, B; Winters, J; Bijnagte, T; Rijnbeek, R

    2016-01-01

    Atomic force microscopy (AFM) is an essential nanoinstrument technique for several applications such as cell biology and nanoelectronics metrology and inspection. The need for statistically significant sample sizes means that data collection can be an extremely lengthy process in AFM. The use of a single AFM instrument is known for its very low speed and not being suitable for scanning large areas, resulting in very-low-throughput measurement. We address this challenge by parallelizing AFM instruments. The parallelization is achieved by miniaturizing the AFM instrument and operating many of them simultaneously. This nanoinstrument has the advantages that each miniaturized AFM can be operated independently and that the advances in the field of AFM, both in terms of speed and imaging modalities, can be implemented more easily. Moreover, a parallel AFM instrument also allows one to measure several physical parameters simultaneously; while one instrument measures nano-scale topography, another instrument can meas...

  13. Achieving Timeliness and High Throughput Metrics in Dissemination Systems

    Directory of Open Access Journals (Sweden)

    MANNE ANUSHA

    2012-09-01

    Full Text Available Existing systems for information dissemination is inadequate and typically results in information gaps. The lack of a clear concise system for information dissemination makes it difficult to determine the most efficient and effective way to pass information especially in the fields of ecommerce and security alerting systems to the appropriate parties. These systems usually require that the desired information be matched between numerous sources and sinks based on established subscriptions. Timeliness and Throughput are performance metrics used for evaluation. And these existing systems fail to achieve a balance between the two. So a much better system termed INFOD (INFOrmation Dissemination was proposed earlier that achieves a balance between performance metrics. We observed that an Integrated Control Loop used by admission control scheme of INFOD employs PL/SQL stored procedures that are huge computation overhead. We propose to replace them with Java stored procedures that can tremendously increase the performance.

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

    Science.gov (United States)

    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.

  15. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control

    Science.gov (United States)

    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

  16. Regulatory pathway analysis by high-throughput in situ hybridization.

    Directory of Open Access Journals (Sweden)

    Axel Visel

    2007-10-01

    Full Text Available Automated in situ hybridization enables the construction of comprehensive atlases of gene expression patterns in mammals. Such atlases can become Web-searchable digital expression maps of individual genes and thus offer an entryway to elucidate genetic interactions and signaling pathways. Towards this end, an atlas housing approximately 1,000 spatial gene expression patterns of the midgestation mouse embryo was generated. Patterns were textually annotated using a controlled vocabulary comprising >90 anatomical features. Hierarchical clustering of annotations was carried out using distance scores calculated from the similarity between pairs of patterns across all anatomical structures. This process ordered hundreds of complex expression patterns into a matrix that reflects the embryonic architecture and the relatedness of patterns of expression. Clustering yielded 12 distinct groups of expression patterns. Because of the similarity of expression patterns within a group, members of each group may be components of regulatory cascades. We focused on the group containing Pax6, an evolutionary conserved transcriptional master mediator of development. Seventeen of the 82 genes in this group showed a change of expression in the developing neocortex of Pax6-deficient embryos. Electromobility shift assays were used to test for the presence of Pax6-paired domain binding sites. This led to the identification of 12 genes not previously known as potential targets of Pax6 regulation. These findings suggest that cluster analysis of annotated gene expression patterns obtained by automated in situ hybridization is a novel approach for identifying components of signaling cascades.

  17. Development and Application of a High-Throughput Screening Method to Evaluate Antifungal Activity against Trichophyton tonsurans.

    Science.gov (United States)

    Preuett, Barry; Leeder, J Steven; Abdel-Rahman, Susan

    2015-10-01

    There exist relatively few drug classes on the market to treat dermatophyte infections. This investigation was designed to develop and validate high-throughput methodology for screening and confirmation of chemicals for activity against Trichophyton tonsurans. Growth characteristics were examined on two platforms (96- and 384-well) in three media at eight spore concentrations over a period of up to 120 h. Microspectrophotometry was used to automate plate reads. The 384-well platform was used to screen more than 7000 compounds from six chemical libraries. Z-scores for optical density relative to positive growth controls were used to flag compounds of interest and activity confirmed in separate assays. The final conditions selected for both screening and confirmation with minimum inhibitory concentration (MIC) determination were growth for 48 h at 32 °C in SabDex with 1 × 10(4) spores per reaction. Sensitivity and specificity averaged 99.2% (range, 95.2%-100%) and 99.8% (range, 99.1%-100%), respectively. MICs for known antifungals were similar to those reported by others using Clinical and Laboratory Standards Institute methods. Several novel compound classes were identified to have activity against T. tonsurans with potency comparable to known antifungals. A robust, reproducible assay is described that permits high-throughput screening in T. tonsurans.

  18. Accelerator mass spectrometry targets of submilligram carbonaceous samples using the high-throughput Zn reduction method.

    Science.gov (United States)

    Kim, Seung-Hyun; Kelly, Peter B; Clifford, Andrew J

    2009-07-15

    The high-throughput Zn reduction method was developed and optimized for various biological/biomedical accelerator mass spectrometry (AMS) applications of mg of C size samples. However, high levels of background carbon from the high-throughput Zn reduction method were not suitable for sub-mg of C size samples in environmental, geochronology, and biological/biomedical AMS applications. This study investigated the effect of background carbon mass (mc) and background 14C level (Fc) from the high-throughput Zn reduction method. Background mc was 0.011 mg of C and background Fc was 1.5445. Background subtraction, two-component mixing, and expanded formulas were used for background correction. All three formulas accurately corrected for backgrounds to 0.025 mg of C in the aerosol standard (NIST SRM 1648a). Only the background subtraction and the two-component mixing formulas accurately corrected for backgrounds to 0.1 mg of C in the IAEA-C6 and -C7 standards. After the background corrections, our high-throughput Zn reduction method was suitable for biological (diet)/biomedical (drug) and environmental (fine particulate matter) applications of sub-mg of C samples (> or = 0.1 mg of C) in keeping with a balance between throughput (270 samples/day/analyst) and sensitivity/accuracy/precision of AMS measurement. The development of a high-throughput method for examination of > or = 0.1 mg of C size samples opens up a range of applications for 14C AMS studies. While other methods do exist for > or = 0.1 mg of C size samples, the low throughput has made them cost prohibitive for many applications.

  19. A high-throughput screening assay to identify bacterial antagonists against Fusarium verticillioides.

    Science.gov (United States)

    Figueroa-López, Alejandro Miguel; Cordero-Ramírez, Jesús Damián; Quiroz-Figueroa, Francisco Roberto; Maldonado-Mendoza, Ignacio Eduardo

    2014-07-01

    A high-throughput antagonistic assay was developed to screen for bacterial isolates capable of controlling the maize fungal phytopathogen Fusarium verticillioides. This assay combines a straightforward methodology, in which the fungus is challenged with bacterial isolates in liquid medium, with a novel approach that uses the plant lectin wheat germ agglutinin (WGA) coupled to a fluorophore (Alexa-Fluor® 488) under the commercial name of WGA, Alexa Fluor® 488 conjugate. The assay is performed in a 96-well plate format, which reduces the required laboratory space and streamlines quantitation and automation of the process, making it fast and accurate. The basis of our assay is that fungal biomass can be assessed by WGA, Alexa Fluor® 488 conjugate staining, which recognizes the chitin in the fungal cell wall and thus permits the identification of potential antagonistic bacteria that inhibit fungal growth. This principle was validated by chitin-competition binding assays against WGA, Alexa Fluor® 488 conjugate; confocal laser microscopy confirmed that the fluorescent WGA, Alexa Fluor® 488 conjugate binds to the chitin of the fungal cell wall. The majority of bacterial isolates did not bind to the WGA, Alexa Fluor® 488 conjugate. Furthermore, including washing steps significantly reduced any bacterial staining to background levels, even in the rare cases where bacterial isolates were capable of binding to WGA. Confirmatory conventional agar plate antagonistic assays were also conducted to validate our technique. We are now successfully employing this large-scale antagonistic assay as a pre-screening step for potential fungal antagonists in extensive bacteria collections (on the order of thousands of isolates).

  20. "Enzyme Test Bench," a high-throughput enzyme characterization technique including the long-term stability.

    Science.gov (United States)

    Rachinskiy, Kirill; Schultze, Hergen; Boy, Matthias; Bornscheuer, Uwe; Büchs, Jochen

    2009-06-01

    A new high throughput technique for enzyme characterization with specific attention to the long term stability, called "Enzyme Test Bench," is presented. The concept of the Enzyme Test Bench consists of short term enzyme tests in 96-well microtiter plates under partly extreme conditions to predict the enzyme long term stability under moderate conditions. The technique is based on the mathematical modeling of temperature dependent enzyme activation and deactivation. Adapting the temperature profiles in sequential experiments by optimal non-linear experimental design, the long term deactivation effects can be purposefully accelerated and detected within hours. During the experiment the enzyme activity is measured online to estimate the model parameters from the obtained data. Thus, the enzyme activity and long term stability can be calculated as a function of temperature. The engineered instrumentation provides for simultaneous automated assaying by fluorescent measurements, mixing and homogenous temperature control in the range of 10-85 +/- 0.5 degrees C. A universal fluorescent assay for online acquisition of ester hydrolysis reactions by pH-shift is developed and established. The developed instrumentation and assay are applied to characterize two esterases. The results of the characterization, carried out in microtiter plates applying short term experiments of hours, are in good agreement with the results of long term experiments at different temperatures in 1 L stirred tank reactors of a week. Thus, the new technique allows for both: the enzyme screening with regard to the long term stability and the choice of the optimal process temperature regarding such process parameters as turn over number, space time yield or optimal process duration. The comparison of the temperature dependent behavior of both characterized enzymes clearly demonstrates that the frequently applied estimation of long term stability at moderate temperatures by simple activity measurements

  1. A low cost and high throughput magnetic bead-based immuno-agglutination assay in confined droplets.

    Science.gov (United States)

    Teste, Bruno; Ali-Cherif, Anaïs; Viovy, Jean Louis; Malaquin, Laurent

    2013-06-21

    Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.

  2. A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility.

    Directory of Open Access Journals (Sweden)

    Michael J Smout

    Full Text Available BACKGROUND: Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a novel application for a real-time cell monitoring device (xCELLigence that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC(50 values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and -resistant isolates of H. contortus. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing.

  3. High-throughput synthesis equipment applied to polymer research

    Science.gov (United States)

    Hoogenboom, Richard; Schubert, Ulrich S.

    2005-06-01

    To speed up synthetic polymer research, a workflow dedicated to automated polymer synthesis and characterization was developed. The workflow consists of several synthesis robots with online and offline analytical equipment. For screening of reaction parameters and for library synthesis, robots equipped with glass reactors and vortex agitation are applied that match very well the classical (small-scale) laboratory experiments. For the screening and library preparation also a microwave synthesizer can be used. For upscaling purposes and process development, a robot containing 100mL tank reactors with mechanical stirring is utilized. This robot also offers the possibility to continuously feed reagents to the reactor and to operate in batch, semibatch or continuous mode.

  4. A high-throughput, multi-channel photon-counting detector with picosecond timing

    CERN Document Server

    Lapington, J S; Miller, G M; Ashton, T J R; Jarron, P; Despeisse, M; Powolny, F; Howorth, J; Milnes, J

    2009-01-01

    High-throughput photon counting with high time resolution is a niche application area where vacuum tubes can still outperform solid-state devices. Applications in the life sciences utilizing time-resolved spectroscopies, particularly in the growing field of proteomics, will benefit greatly from performance enhancements in event timing and detector throughput. The HiContent project is a collaboration between the University of Leicester Space Research Centre, the Microelectronics Group at CERN, Photek Ltd., and end-users at the Gray Cancer Institute and the University of Manchester. The goal is to develop a detector system specifically designed for optical proteomics, capable of high content (multi-parametric) analysis at high throughput. The HiContent detector system is being developed to exploit this niche market. It combines multi-channel, high time resolution photon counting in a single miniaturized detector system with integrated electronics. The combination of enabling technologies; small pore microchanne...

  5. High-throughput Phenotyping and Genomic Selection: The Frontiers of Crop Breeding Converge

    Institute of Scientific and Technical Information of China (English)

    Llorenc Cabrera-Bosquet; José Crossa; Jarislav von Zitzewitz; Maria Dolors Serret; José Luis Araus

    2012-01-01

    Genomic selection (GS) and high-throughput phenotyping have recently been captivating the interest of the crop breeding community from both the public and private sectors world-wide.Both approaches promise to revolutionize the prediction of complex traits,including growth,yield and adaptation to stress.Whereas high-throughput phenotyping may help to improve understanding of crop physiology,most powerful techniques for high-throughput field phenotyping are empirical rather than analytical and comparable to genomic selection.Despite the fact that the two methodological approaches represent the extremes of what is understood as the breeding process (phenotype versus genome),they both consider the targeted traits (e.g.grain yield,growth,phenology,plant adaptation to stress) as a black box instead of dissecting them as a set of secondary traits (i.e.physiological) putatively related to the target trait.Both GS and high-throughput phenotyping have in common their empirical approach enabling breeders to use genome profile or phenotype without understanding the underlying biology.This short review discusses the main aspects of both approaches and focuses on the case of genomic selection of maize flowering traits and near-infrared spectroscopy (NIRS) and plant spectral reflectance as high-throughput field phenotyping methods for complex traits such as crop growth and yield.

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

    Science.gov (United States)

    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.

  7. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control.

    Science.gov (United States)

    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.

  8. High-throughput atomic force microscopes operating in parallel

    Science.gov (United States)

    Sadeghian, Hamed; Herfst, Rodolf; Dekker, Bert; Winters, Jasper; Bijnagte, Tom; Rijnbeek, Ramon

    2017-03-01

    Atomic force microscopy (AFM) is an essential nanoinstrument technique for several applications such as cell biology and nanoelectronics metrology and inspection. The need for statistically significant sample sizes means that data collection can be an extremely lengthy process in AFM. The use of a single AFM instrument is known for its very low speed and not being suitable for scanning large areas, resulting in a very-low-throughput measurement. We address this challenge by parallelizing AFM instruments. The parallelization is achieved by miniaturizing the AFM instrument and operating many of them simultaneously. This instrument has the advantages that each miniaturized AFM can be operated independently and that the advances in the field of AFM, both in terms of speed and imaging modalities, can be implemented more easily. Moreover, a parallel AFM instrument also allows one to measure several physical parameters simultaneously; while one instrument measures nano-scale topography, another instrument can measure mechanical, electrical, or thermal properties, making it a lab-on-an-instrument. In this paper, a proof of principle of such a parallel AFM instrument has been demonstrated by analyzing the topography of large samples such as semiconductor wafers. This nanoinstrument provides new research opportunities in the nanometrology of wafers and nanolithography masks by enabling real die-to-die and wafer-level measurements and in cell biology by measuring the nano-scale properties of a large number of cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    Prabhu, Anmiv S; Kim, Min Jun [School of Biomedical Engineering and Health Science, Drexel University, Philadelphia, PA 19104 (United States); Jubery, Talukder Zaki N; Dutta, Prashanta [School of Mechanical and Materials Engineering, Washington State University, Pullman, WA 99164 (United States); Freedman, Kevin J; Mulero, Rafael, E-mail: mkim@coe.drexel.ed [Department of Mechanical Engineering and Mechanics, Drexel University, Philadelphia, PA 19104 (United States)

    2010-11-17

    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.

  10. A robust, high-throughput method for computing maize ear, cob, and kernel attributes automatically from images.

    Science.gov (United States)

    Miller, Nathan D; Haase, Nicholas J; Lee, Jonghyun; Kaeppler, Shawn M; de Leon, Natalia; Spalding, Edgar P

    2017-01-01

    Grain yield of the maize plant depends on the sizes, shapes, and numbers of ears and the kernels they bear. An automated pipeline that can measure these components of yield from easily-obtained digital images is needed to advance our understanding of this globally important crop. Here we present three custom algorithms designed to compute such yield components automatically from digital images acquired by a low-cost platform. One algorithm determines the average space each kernel occupies along the cob axis using a sliding-window Fourier transform analysis of image intensity features. A second counts individual kernels removed from ears, including those in clusters. A third measures each kernel's major and minor axis after a Bayesian analysis of contour points identifies the kernel tip. Dimensionless ear and kernel shape traits that may interrelate yield components are measured by principal components analysis of contour point sets. Increased objectivity and speed compared to typical manual methods are achieved without loss of accuracy as evidenced by high correlations with ground truth measurements and simulated data. Millimeter-scale differences among ear, cob, and kernel traits that ranged more than 2.5-fold across a diverse group of inbred maize lines were resolved. This system for measuring maize ear, cob, and kernel attributes is being used by multiple research groups as an automated Web service running on community high-throughput computing and distributed data storage infrastructure. Users may create their own workflow using the source code that is staged for download on a public repository.

  11. A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes.

    Science.gov (United States)

    Schmid-Burgk, Jonathan L; Schmidt, Tobias; Kaiser, Vera; Höning, Klara; Hornung, Veit

    2013-01-01

    Transcription activator–like (TAL) effector proteins derived from Xanthomonas species have emerged as versatile scaffolds for engineering DNA-binding proteins of user-defined specificity and functionality. Here we describe a rapid, simple, ligation-independent cloning (LIC) technique for synthesis of TAL effector genes. Our approach is based on a library of DNA constructs encoding individual TAL effector repeat unit combinations that can be processed to contain long, unique single-stranded DNA overhangs suitable for LIC. Assembly of TAL effector arrays requires only the combinatorial mixing of fluids and has exceptional fidelity. TAL effector nucleases (TALENs) produced by this method had high genome-editing activity at endogenous loci in HEK 293T cells (64% were active). To maximize throughput, we generated a comprehensive 5-mer TAL effector repeat unit fragment library that allows automated assembly of >600 TALEN genes in a single day. Given its simplicity, throughput and fidelity, LIC assembly will permit the generation of TAL effector gene libraries for large-scale functional genomics studies.

  12. First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic β-cell mass.

    Science.gov (United States)

    Wang, Guangliang; Rajpurohit, Surendra K; Delaspre, Fabien; Walker, Steven L; White, David T; Ceasrine, Alexis; Kuruvilla, Rejji; Li, Ruo-Jing; Shim, Joong S; Liu, Jun O; Parsons, Michael J; Mumm, Jeff S

    2015-07-28

    Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in >500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing β cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of β-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating β-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling β-cell mass, potential therapeutic targets for treating diabetes.

  13. Recent Progress Using High-throughput Sequencing Technologies in Plant Molecular Breeding

    Institute of Scientific and Technical Information of China (English)

    Qiang Gao; Guidong Yue; Wenqi Li; Junyi Wang; Jiaohui Xu; Ye Yin

    2012-01-01

    High-throughput sequencing is a revolutionary technological innovation in DNA sequencing.This technology has an ultra-low cost per base of sequencing and an overwhelmingly high data output.High-throughput sequencing has brought novel research methods and solutions to the research fields of genomics and post-genomics.Furthermore,this technology is leading to a new molecular breeding revolution that has landmark significance for scientific research and enables us to launch multi-level,multifaceted,and multi-extent studies in the fields of crop genetics,genomics,and crop breeding.In this paper,we review progress in the application of high-throughput sequencing technologies to plant molecular breeding studies.

  14. High-throughput imaging: Focusing in on drug discovery in 3D.

    Science.gov (United States)

    Li, Linfeng; Zhou, Qiong; Voss, Ty C; Quick, Kevin L; LaBarbera, Daniel V

    2016-03-01

    3D organotypic culture models such as organoids and multicellular tumor spheroids (MCTS) are becoming more widely used for drug discovery and toxicology screening. As a result, 3D culture technologies adapted for high-throughput screening formats are prevalent. While a multitude of assays have been reported and validated for high-throughput imaging (HTI) and high-content screening (HCS) for novel drug discovery and toxicology, limited HTI/HCS with large compound libraries have been reported. Nonetheless, 3D HTI instrumentation technology is advancing and this technology is now on the verge of allowing for 3D HCS of thousands of samples. This review focuses on the state-of-the-art high-throughput imaging systems, including hardware and software, and recent literature examples of 3D organotypic culture models employing this technology for drug discovery and toxicology screening.

  15. Filtering high-throughput protein-protein interaction data using a combination of genomic features

    Directory of Open Access Journals (Sweden)

    Patil Ashwini

    2005-04-01

    Full Text Available Abstract Background Protein-protein interaction data used in the creation or prediction of molecular networks is usually obtained from large scale or high-throughput experiments. This experimental data is liable to contain a large number of spurious interactions. Hence, there is a need to validate the interactions and filter out the incorrect data before using them in prediction studies. Results In this study, we use a combination of 3 genomic features – structurally known interacting Pfam domains, Gene Ontology annotations and sequence homology – as a means to assign reliability to the protein-protein interactions in Saccharomyces cerevisiae determined by high-throughput experiments. Using Bayesian network approaches, we show that protein-protein interactions from high-throughput data supported by one or more genomic features have a higher likelihood ratio and hence are more likely to be real interactions. Our method has a high sensitivity (90% and good specificity (63%. We show that 56% of the interactions from high-throughput experiments in Saccharomyces cerevisiae have high reliability. We use the method to estimate the number of true interactions in the high-throughput protein-protein interaction data sets in Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens to be 27%, 18% and 68% respectively. Our results are available for searching and downloading at http://helix.protein.osaka-u.ac.jp/htp/. Conclusion A combination of genomic features that include sequence, structure and annotation information is a good predictor of true interactions in large and noisy high-throughput data sets. The method has a very high sensitivity and good specificity and can be used to assign a likelihood ratio, corresponding to the reliability, to each interaction.

  16. High-precision, high-throughput stability determinations facilitated by robotics and a semiautomated titrating fluorometer.

    Science.gov (United States)

    Edgell, Marshall Hall; Sims, Dorothy A; Pielak, Gary J; Yi, Fang

    2003-06-24

    The use of statistical modeling to test hypotheses concerning the determinants of protein structure requires stability data (e.g., the free energy of denaturation in H(2)O, DeltaG(HOH)) from hundreds of protein mutants. Fluorescence-monitored chemical denaturation provides a convenient method for high-precision, high-throughput DeltaG(HOH) determination. For eglin c we find that a throughput of about 20 min per protein can be attained in a two-channel semiautomated titrating fluorometer. We find also that the use of robotics for protein purification and preparation of the solutions for chemical denaturation gives highly precise DeltaG(HOH) values in which the standard deviation of values from multiple preparations (+/-0.051 kcal/mol) differs very little from multiple measurements from a single preparation (+/-0.040 kcal/mol). Since the variance introduced into model fitting by DeltaG(HOH) increases as the square of measurement error, there is a premium on precision. In fact, the fraction of stability behavior explicable by otherwise perfect models goes from 98% to only 50% over the error range commonly reported for chemical denaturation measurements (0.1-0.6 kcal/mol). We have found that the precision of chemical denaturation DeltaG(HOH) measurements depends most heavily on the precision of the instrument used, followed by protein purity and the capacity to precisely prepare the solutions used for titrations.

  17. A novel imaging-based high-throughput screening approach to anti-angiogenic drug discovery.

    Science.gov (United States)

    Evensen, Lasse; Micklem, David R; Link, Wolfgang; Lorens, James B

    2010-01-01

    The successful progression to the clinic of angiogenesis inhibitors for cancer treatment has spurred interest in developing new classes of anti-angiogenic compounds. The resulting surge in available candidate therapeutics highlights the need for robust, high-throughput angiogenesis screening systems that adequately capture the complexity of new vessel formation while providing quantitative evaluation of the potency of these agents. Available in vitro angiogenesis assays are either cumbersome, impeding adaptation to high-throughput screening formats, or inadequately model the complex multistep process of new vessel formation. We therefore developed an organotypic endothelial-mural cell co-culture assay system that reflects several facets of angiogenesis while remaining compatible with high-throughput/high-content image screening. Co-culture of primary human endothelial cells (EC) and vascular smooth muscle cells (vSMC) results in assembly of a network of tubular endothelial structures enveloped with vascular basement membrane proteins, thus, comprising the three main components of blood vessels. Initially, EC are dependent on vSMC-derived VEGF and sensitive to clinical anti-angiogenic therapeutics. A subsequent phenotypic VEGF-switch renders EC networks resistant to anti-VEGF therapeutics, demarcating a mature vascular phenotype. Conversely, mature EC networks remain sensitive to vascular disrupting agents. Therefore, candidate anti-angiogenic compounds can be interrogated for their relative potency on immature and mature networks and classified as either vascular normalizing or vascular disrupting agents. Here, we demonstrate that the EC-vSMC co-culture assay represents a robust high-content imaging high-throughput screening system for identification of novel anti-angiogenic agents. A pilot high-throughput screening campaign was used to define informative imaging parameters and develop a follow-up dose-response scheme for hit characterization. High-throughput

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

    Science.gov (United States)

    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.

  19. High throughput route selection in multi-rate wireless mesh networks

    Institute of Scientific and Technical Information of China (English)

    WEI Yi-fei; GUO Xiang-li; SONG Mei; SONG Jun-de

    2008-01-01

    Most existing Ad-hoc routing protocols use the shortest path algorithm with a hop count metric to select paths. It is appropriate in single-rate wireless networks, but has a tendency to select paths containing long-distance links that have low data rates and reduced reliability in multi-rate networks. This article introduces a high throughput routing algorithm utilizing the multi-rate capability and some mesh characteristics in wireless fidelity (WiFi) mesh networks. It uses the medium access control (MAC) transmission time as the routing metric, which is estimated by the information passed up from the physical layer. When the proposed algorithm is adopted, the Ad-hoc on-demand distance vector (AODV) routing can be improved as high throughput AODV (HT-AODV). Simulation results show that HT-AODV is capable of establishing a route that has high data-rate, short end-to-end delay and great network throughput.

  20. Current impact and future directions of high throughput sequencing in plant virus diagnostics.

    Science.gov (United States)

    Massart, Sebastien; Olmos, Antonio; Jijakli, Haissam; Candresse, Thierry

    2014-08-08

    The ability to provide a fast, inexpensive and reliable diagnostic for any given viral infection is a key parameter in efforts to fight and control these ubiquitous pathogens. The recent developments of high-throughput sequencing (also called Next Generation Sequencing - NGS) technologies and bioinformatics have drastically changed the research on viral pathogens. It is now raising a growing interest for virus diagnostics. This review provides a snapshot vision on the current use and impact of high throughput sequencing approaches in plant virus characterization. More specifically, this review highlights the potential of these new technologies and their interplay with current protocols in the future of molecular diagnostic of plant viruses. The current limitations that will need to be addressed for a wider adoption of high-throughput sequencing in plant virus diagnostics are thoroughly discussed.

  1. High-throughput screening of small molecule libraries using SAMDI mass spectrometry.

    Science.gov (United States)

    Gurard-Levin, Zachary A; Scholle, Michael D; Eisenberg, Adam H; Mrksich, Milan

    2011-07-11

    High-throughput screening is a common strategy used to identify compounds that modulate biochemical activities, but many approaches depend on cumbersome fluorescent reporters or antibodies and often produce false-positive hits. The development of "label-free" assays addresses many of these limitations, but current approaches still lack the throughput needed for applications in drug discovery. This paper describes a high-throughput, label-free assay that combines self-assembled monolayers with mass spectrometry, in a technique called SAMDI, as a tool for screening libraries of 100,000 compounds in one day. This method is fast, has high discrimination, and is amenable to a broad range of chemical and biological applications.

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

    Science.gov (United States)

    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.

  3. Applications of high-throughput clonogenic survival assays in high-LET particle microbeams

    Directory of Open Access Journals (Sweden)

    Antonios eGeorgantzoglou

    2016-01-01

    Full Text Available 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-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 4-5 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.

  4. A Warm Near-Infrared High-Resolution Spectrograph with Very High Throughput (WINERED)

    CERN Document Server

    Kondo, Sohei; Kobayashi, Naoto; Yasui, Chikako; Mito, Hiroyuki; Fukue, Kei; Nakanishi, Kenshi; Kawanishi, Takafumi; Nakaoka, Tetsuya; Otsubo, Shogo; Kinoshita, Masaomi; Kitano, Ayaka; Hamano, Satoshi; Mizumoto, Misaki; Yamamoto, Ryo; Izumi, Natsuko; Matsunaga, Noriyuki; Kawakita, Hideyo

    2015-01-01

    WINERED is a newly built high-efficiency (throughput$ > 25-30\\%$) and high-resolution spectrograph customized for short NIR bands at 0.9-1.35 ${\\rm \\mu}$m. WINERED is equipped with ambient temperature optics and a cryogenic camera using a 1.7 ${\\rm \\mu}$m cut-off HgCdTe HAWAII-2RG array detector. WINERED has two grating modes: one with a conventional reflective echelle grating (R$\\sim$28,300), which covers 0.9-1.35 $\\mu$m simultaneously, the other with ZnSe or ZnS immersion grating (R$\\sim$100,000). We have completed the development of WINERED except for the immersion grating, and started engineering and science observations at the Nasmyth platform of the 1.3 m Araki Telescope at Koyama Astronomical Observatory of Kyoto-Sangyo University in Japan. We confirmed that the spectral resolution ($R\\sim$ 28,300) and the throughput ($>$ 40\\% w/o telescope/atmosphere/array QE) meet our specifications. We measured ambient thermal backgrounds (e.g., 0.06 ${\\rm [e^{-}/sec/pixel]}$ at 287 K), which are roughly consistent ...

  5. A Validated High-Throughput Fluorometric Method for Determination of Omeprazole in Quality Control Laboratory via Charge Transfer Sensitized Fluorescence.

    Science.gov (United States)

    Mahmoud, Ashraf M; Ahmed, Sameh A

    2016-03-01

    A high-throughput 96-microwell plate fluorometric method was developed and validated to determine omeprazole (OMZ) in its dosage forms. The method was based on the charge-transfer (CT) sensitized fluorescence reaction of OMZ with 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ). This fluorescence reaction provided a new approach for simple, sensitive and selective determinations of OMZ in pharmaceutical preparations. In the present method, the fluorescence reaction was carried out in 96-microwell plates as reaction vessels in order to increase the automation of the methodology and the efficiency of its use in quality control laboratories. All factors affecting the fluorescence reaction were carefully studied and the conditions were optimized. The stoichiometry of the fluorescence reaction between OMZ and DDQ was determined and the reaction mechanism was suggested. Under the optimum conditions, the linear range was 100-6000 ng/ml with the lowest LOD of 33 ng/ml. Analytical performance of the proposed assay, in terms of accuracy and precision, was statistically validated and the results were satisfactory; RSD was <2.6 % and the accuracy was 98.6-101.6 %. The method was successfully applied to the analysis of OMZ in its dosage forms; the recovery values were 98.26-99.60 ± 0.95-2.22 %. The developed methodology may provide a safer, automated and economic tool for the analysis of OMZ in quality control laboratories.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  7. Accelerating Virtual High-Throughput Ligand Docking: current technology and case study on a petascale supercomputer.

    Science.gov (United States)

    Ellingson, Sally R; Dakshanamurthy, Sivanesan; Brown, Milton; Smith, Jeremy C; Baudry, Jerome

    2014-04-25

    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.

  8. High-throughput tri-colour flow cytometry technique to assess Plasmodium falciparum parasitaemia in bioassays

    DEFF Research Database (Denmark)

    Tiendrebeogo, Regis W; Adu, Bright; Singh, Susheel K

    2014-01-01

    BACKGROUND: Unbiased flow cytometry-based methods have become the technique of choice in many laboratories for high-throughput, accurate assessments of malaria parasites in bioassays. A method to quantify live parasites based on mitotracker red CMXRos was recently described but consistent...... distinction of early ring stages of Plasmodium falciparum from uninfected red blood cells (uRBC) remains a challenge. METHODS: Here, a high-throughput, three-parameter (tri-colour) flow cytometry technique based on mitotracker red dye, the nucleic acid dye coriphosphine O (CPO) and the leucocyte marker CD45...

  9. High-throughput analysis of total nitrogen content that replaces the classic Kjeldahl method.

    Science.gov (United States)

    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.

  10. High-throughput exposure modeling to support prioritization of chemicals in personal care products

    DEFF Research Database (Denmark)

    Csiszar, Susan A.; Ernstoff, Alexi; Fantke, Peter;

    2016-01-01

    We demonstrate the application of a high-throughput modeling framework to estimate exposure to chemicals used in personal care products (PCPs). As a basis for estimating exposure, we use the product intake fraction (PiF), defined as the mass of chemical taken by an individual or population per mass...... intakes were associated with body lotion. Bioactive doses derived from high-throughput in vitro toxicity data were combined with the estimated PiFs to demonstrate an approach to estimate bioactive equivalent chemical content and to screen chemicals for risk....

  11. Rapid and high-throughput detection of highly pathogenic bacteria by Ibis PLEX-ID technology.

    Directory of Open Access Journals (Sweden)

    Daniela Jacob

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Vinit Kumar

    2015-01-01

    Full Text Available 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.

  13. Achieving high mass-throughput of therapeutic proteins through parvovirus retentive filters.

    Science.gov (United States)

    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.

  14. Fully Automated One-Step Production of Functional 3D Tumor Spheroids for High-Content Screening.

    Science.gov (United States)

    Monjaret, François; Fernandes, Mathieu; Duchemin-Pelletier, Eve; Argento, Amelie; Degot, Sébastien; Young, Joanne

    2016-04-01

    Adoption of spheroids within high-content screening (HCS) has lagged behind high-throughput screening (HTS) due to issues with running complex assays on large three-dimensional (3D) structures.To enable multiplexed imaging and analysis of spheroids, different cancer cell lines were grown in 3D on micropatterned 96-well plates with automated production of nine uniform spheroids per well. Spheroids achieve diameters of up to 600 µm, and reproducibility was experimentally validated (interwell and interplate CV(diameter) integration of micropatterned spheroid models within fundamental research and drug discovery applications.

  15. Microfluidic-Enabled Print-to-Screen Platform for High-Throughput Screening of Combinatorial Chemotherapy.

    Science.gov (United States)

    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.

  16. High-throughput magnetic flow sorting of human cells selected on the basis of magnetophoretic mobility

    Science.gov (United States)

    Reece, Lisa M.; Sanders, Lehanna; Kennedy, David; Guernsey, Byron; Todd, Paul; Leary, James F.

    2010-02-01

    We have shown the potential of a new method for optimizing the separation of human stem cell subsets from peripheral blood based on a novel cell labeling technique that leverages the capabilities of a new commercially available high speed magnetic cell sorting system (IKOTECH LLC, New Albany, IN). This new system sorts cells in a continuously flowing manner using a Quadrupole Magnetic cell Sorter (QMS). The sorting mechanism is based upon the magnetophoretic mobility of the cells, a property related to the relative binding distributions of magnetic particles per cell, as determined by the utilization of a Magnetic Cell Tracking Velocimeter (MCTV). KG-1 cells were competitively labeled with anti-CD34 magnetic beads and anti-CD34 FITC to obtain an optimal level of magnetophoretic mobility as visualized by the MCTV for high throughput sort recovery in the QMS. In QMS sorting, the concept of split-flow thin channel (SPLITT) separation technology is applied by having a sample stream enter a vertical annular flow channel near the channel's interior wall followed by another sheath flow entering near the exterior wall. The two flows are initially separated by a flow splitter. They pass through the bore of a Halbach permanent quadrupole magnet assembly, which draws magnetized cells outward and deflects them into a positive outflow, while negative cells continue straight out via the inner flow lamina. QMS sorts cells based upon their magnetophoretic mobility, or the velocity of a cell per unit ponderomotive force, the counterpart of fluorescence intensity in flow cytometry. The magnetophoretic mobility distribution of a cell population, measured by automated MCTV, is used as input data for the algorithmic control of sample, sheath, and outlet flow velocities of the QMS. In this study, the relative binding distributions of magnetic particles per cell were determined by MCTV using novel sorting and sizing algorithms. The resulting mobility histograms were used to set the QMS

  17. Candida albicans biofilm chip (CaBChip) for high-throughput antifungal drug screening.

    Science.gov (United States)

    Srinivasan, Anand; Lopez-Ribot, Jose L; Ramasubramanian, Anand K

    2012-07-18

    Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 °C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance). Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation

  18. High Throughput Atomic Layer Deposition Processes: High Pressure Operations, New Reactor Designs, and Novel Metal Processing

    Science.gov (United States)

    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

  19. A 96-well screen filter plate for high-throughput biological sample preparation and LC-MS/MS analysis.

    Science.gov (United States)

    Peng, Sean X; Cousineau, Martin; Juzwin, Stephen J; Ritchie, David M

    2006-01-01

    A novel 96-well screen filter plate (patent pending) has been invented to eliminate a time-consuming and labor-intensive step in preparation of in vivo study samples--to remove blood or plasma clots. These clots plug the pipet tips during a manual or automated sample-transfer step causing inaccurate pipetting or total pipetting failure. Traditionally, these blood and plasma clots are removed by picking them out manually one by one from each sample tube before any sample transfer can be made. This has significantly slowed the sample preparation process and has become a bottleneck for automated high-throughput sample preparation using robotic liquid handlers. Our novel screen filter plate was developed to solve this problem. The 96-well screen filter plate consists of 96 stainless steel wire-mesh screen tubes connected to the 96 openings of a top plate so that the screen filter plate can be readily inserted into a 96-well sample storage plate. Upon insertion, the blood and plasma clots are excluded from entering the screen tube while clear sample solutions flow freely into it. In this way, sample transfer can be easily completed by either manual or automated pipetting methods. In this report, three structurally diverse compounds were selected to evaluate and validate the use of the screen filter plate. The plasma samples of these compounds were transferred and processed in the presence and absence of the screen filter plate and then analyzed by LC-MS/MS methods. Our results showed a good agreement between the samples prepared with and without the screen filter plate, demonstrating the utility and efficiency of this novel device for preparation of blood and plasma samples. The device is simple, easy to use, and reusable. It can be employed for sample preparation of other biological fluids that contain floating particulates or aggregates.

  20. A ground-up approach to High Throughput Cloud Computing in High-Energy Physics

    CERN Document Server

    AUTHOR|(INSPIRE)INSPIRE-00245123; Ganis, Gerardo; Bagnasco, Stefano

    The thesis explores various practical approaches in making existing High Throughput computing applications common in High Energy Physics work on cloud-provided resources, as well as opening the possibility for running new applications. The work is divided into two parts: firstly we describe the work done at the computing facility hosted by INFN Torino to entirely convert former Grid resources into cloud ones, eventually running Grid use cases on top along with many others in a more flexible way. Integration and conversion problems are duly described. The second part covers the development of solutions for automatizing the orchestration of cloud workers based on the load of a batch queue and the development of HEP applications based on ROOT's PROOF that can adapt at runtime to a changing number of workers.

  1. Validation of a High-Throughput Multiplex Genetic Detection System for Helicobacter pylori Identification, Quantification, Virulence, and Resistance Analysis

    OpenAIRE

    Zhang, Yanmei; Zhao, Fuju; Kong, Mimi; Wang, Shiwen; Nan, Li; Hu, Binjie; Olszewski, Michal A.; Miao, Yingxin; Ji, Danian; Jiang, Wenrong; Fang, Yi; Zhang, Jinghao; Chen, Fei; Xiang, Ping; Wu, Yong

    2016-01-01

    Helicobacter pylori (H. pylori) infection is closely related to various gastroduodenal diseases. Virulence factors and bacterial load of H. pylori are associated with clinical outcomes, and drug-resistance severely impacts the clinical efficacy of eradication treatment. Existing detection methods are low-throughput, time-consuming and labor intensive. Therefore, a rapid and high-throughput method is needed for clinical diagnosis, treatment, and monitoring for H. pylori. High-throughput Multip...

  2. A targeted proteomics toolkit for high-throughput absolute quantification of Escherichia coli proteins.

    Science.gov (United States)

    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.

  3. Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization

    Science.gov (United States)

    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

  4. Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization

    Directory of Open Access Journals (Sweden)

    Beiyuan Fan

    2016-02-01

    Full Text Available 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.

  5. Considerations for the design and reporting of enzyme assays in high-throughput screening applications

    Directory of Open Access Journals (Sweden)

    Michael G. Acker

    2014-05-01

    Full Text Available This review describes the key steps and methods which are used to develop enzyme assays suitable for high-throughput screening (HTS applications. The goals of HTS enzyme assays are defined relative to lower-throughput bench top assays and important aspects which go into constructing robust and sensitive enzyme assays are described. Methods that have been applied to common enzyme classes are reviewed and pitfalls related to assay artifacts are discussed. We also suggest a reporting format to describe the steps in HTS enzyme assays.

  6. Development of Droplet Microfluidics Enabling High-Throughput Single-Cell Analysis

    Directory of Open Access Journals (Sweden)

    Na Wen

    2016-07-01

    Full Text Available This article reviews recent developments in droplet microfluidics enabling high-throughput single-cell analysis. Five key aspects in this field are included in this review: (1 prototype demonstration of single-cell encapsulation in microfluidic droplets; (2 technical improvements of single-cell encapsulation in microfluidic droplets; (3 microfluidic droplets enabling single-cell proteomic analysis; (4 microfluidic droplets enabling single-cell genomic analysis; and (5 integrated microfluidic droplet systems enabling single-cell screening. We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on key performances of throughput, multifunctionality, and absolute quantification.

  7. Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA

    Science.gov (United States)

    TakYu, Zeta; Guan, Huijiao; Ki Cheung, Mei; McHugh, Walker M.; Cornell, Timothy T.; Shanley, Thomas P.; Kurabayashi, Katsuo; Fu, Jianping

    2015-06-01

    Immunoassays represent one of the most popular analytical methods for detection and quantification of biomolecules. However, conventional immunoassays such as ELISA and flow cytometry, even though providing high sensitivity and specificity and multiplexing capability, can be labor-intensive and prone to human error, making them unsuitable for standardized clinical diagnoses. Using a commercialized no-wash, homogeneous immunoassay technology (‘AlphaLISA’) in conjunction with integrated microfluidics, herein we developed a microfluidic immunoassay chip capable of rapid, automated, parallel immunoassays of microliter quantities of samples. Operation of the microfluidic immunoassay chip entailed rapid mixing and conjugation of AlphaLISA components with target analytes before quantitative imaging for analyte detections in up to eight samples simultaneously. Aspects such as fluid handling and operation, surface passivation, imaging uniformity, and detection sensitivity of the microfluidic immunoassay chip using AlphaLISA were investigated. The microfluidic immunoassay chip could detect one target analyte simultaneously for up to eight samples in 45 min with a limit of detection down to 10 pg mL-1. The microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo. Together, the microfluidic immunoassay chip provides a promising high-throughput, high-content platform for rapid, automated, parallel quantitative immunosensing applications.

  8. A high-throughput lab-on-a-chip interface for zebrafish embryo tests in drug discovery and ecotoxicology

    Science.gov (United States)

    Zhu, Feng; Akagi, Jin; Hall, Chris J.; Crosier, Kathryn E.; Crosier, Philip S.; Delaage, Pierre; Wlodkowic, Donald

    2013-12-01

    Drug discovery screenings performed on zebrafish embryos mirror with a high level of accuracy. The tests usually performed on mammalian animal models, and the fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, conventional methods utilising 96-well microtiter plates and manual dispensing of fish embryos are very time-consuming. They rely on laborious and iterative manual pipetting that is a main source of analytical errors and low throughput. In this work, we present development of a miniaturised and high-throughput Lab-on-a-Chip (LOC) platform for automation of FET assays. The 3D high-density LOC array was fabricated in poly-methyl methacrylate (PMMA) transparent thermoplastic using infrared laser micromachining while the off-chip interfaces were fabricated using additive manufacturing processes (FDM and SLA). The system's design facilitates rapid loading and immobilization of a large number of embryos in predefined clusters of traps during continuous microperfusion of drugs/toxins. It has been conceptually designed to seamlessly interface with both upright and inverted fluorescent imaging systems and also to directly interface with conventional microtiter plate readers that accept 96-well plates. We also present proof-of-concept interfacing with a high-speed imaging cytometer Plate RUNNER HD® capable of multispectral image acquisition with resolution of up to 8192 x 8192 pixels and depth of field of about 40 μm. Furthermore, we developed a miniaturized and self-contained analytical device interfaced with a miniaturized USB microscope. This system modification is capable of performing rapid imaging of multiple embryos at a low resolution for drug toxicity analysis.

  9. Quantitative high-throughput screen identifies inhibitors of the Schistosoma mansoni redox cascade.

    Directory of Open Access Journals (Sweden)

    Anton Simeonov

    Full Text Available Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR and peroxiredoxin (Prx and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 microL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC(50s ranging from micromolar to the assay response limit ( approximately 25 nM. This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump

  10. HIGH-THROUGHPUT IDENTIFICATION OF CATALYTIC REDOX-ACTIVE CYSTEINE RESIDUES

    Science.gov (United States)

    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...

  11. 3D high throughput screening and profiling of embryoid bodies in thermoformed microwell plates

    NARCIS (Netherlands)

    Vrij, E. J.; Espinoza, S.; Heilig, M.; Kolew, A.; Schneider, M.; Van Blitterswijk, C. A.; Truckenmüller, R. K.; Rivron, N. C.

    2016-01-01

    3D organoids using stem cells to study development and disease are now widespread. These models are powerful to mimic in vivo situations but are currently associated with high variability and low throughput. For biomedical research, platforms are thus necessary to increase reproducibility and allow

  12. New approach for high-throughput screening of drug activity on Plasmodium liver stages.

    NARCIS (Netherlands)

    Gego, A.; Silvie, O.; Franetich, J.F.; Farhati, K.; Hannoun, L.; Luty, A.J.F.; Sauerwein, R.W.; Boucheix, C.; Rubinstein, E.; Mazier, D.

    2006-01-01

    Plasmodium liver stages represent potential targets for antimalarial prophylactic drugs. Nevertheless, there is a lack of molecules active on these stages. We have now developed a new approach for the high-throughput screening of drug activity on Plasmodium liver stages in vitro, based on an infrare

  13. Reverse Phase Protein Arrays for High-Throughput Protein Measurements in Mammospheres

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    Protein Array (RPPA)-based readout format integrated into robotic siRNA screening. This technique would allow post-screening high-throughput quantification of protein changes. Recently, breast cancer stem cells (BCSCs) have attracted much attention, as a tumor- and metastasis-driving subpopulation...

  14. An integrated framework for discovery and genotyping of genomic variants from high-throughput sequencing experiments

    NARCIS (Netherlands)

    Duitama, Jorge; Quintero, Juan Camilo; Cruz, Daniel Felipe; Quintero, Constanza; Hubmann, Georg; Foulquié-Moreno, Maria R.; Verstrepen, Kevin J.; Thevelein, Johan M.; Tohme, Joe

    2014-01-01

    Recent advances in high-throughput sequencing (HTS) technologies and computing capacity have produced unprecedented amounts of genomic data that have unraveled the genetics of phenotypic variability in several species. However, operating and integrating current software tools for data analysis still

  15. A high-throughput, precipitating colorimetric sandwich ELISA microarray for shiga toxins

    Science.gov (United States)

    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)...

  16. High-throughput open source computational methods for genetics and genomics

    NARCIS (Netherlands)

    Prins, J.C.P.

    2015-01-01

    Biology is increasingly data driven by virtue of the development of high-throughput technologies, such as DNA and RNA sequencing. Computational biology and bioinformatics are scientific disciplines that cross-over between the disciplines of biology, informatics and statistics; which is clearly refle

  17. Cancer panomics: computational methods and infrastructure for integrative analysis of cancer high-throughput "omics" data

    DEFF Research Database (Denmark)

    Brunak, Søren; De La Vega, Francisco M.; Rätsch, Gunnar

    2014-01-01

    Targeted cancer treatment is becoming the goal of newly developed oncology medicines and has already shown promise in some spectacular cases such as the case of BRAF kinase inhibitors in BRAF-mutant (e.g. V600E) melanoma. These developments are driven by the advent of high-throughput sequencing...

  18. High-throughput screening of carbohydrate-degrading enzymes using novel insoluble chromogenic substrate assay kits

    DEFF Research Database (Denmark)

    Schückel, Julia; Kracun, Stjepan Kresimir; Willats, William George Tycho

    2016-01-01

    for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay...

  19. High-Throughput Dietary Exposure Predictions for Chemical Migrants from Food Packaging Materials

    Science.gov (United States)

    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...

  20. A practical fluorogenic substrate for high-throughput screening of glutathione S-transferase inhibitors.

    Science.gov (United States)

    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.

  1. A High-Throughput MALDI-TOF Mass Spectrometry-Based Assay of Chitinase Activity

    Science.gov (United States)

    A high-throughput MALDI-TOF mass spectrometric assay is described for assay of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates, and is readily achievable on a microliter scale (2 µL total volume, containing 2 µg of substrate and 1 ng of protein). The speed a...

  2. Comprehensive analysis of high-throughput screens with HiTSeekR

    DEFF Research Database (Denmark)

    List, Markus; Schmidt, Steffen; Christiansen, Helle;

    2016-01-01

    High-throughput screening (HTS) is an indispensable tool for drug (target) discovery that currently lacks user-friendly software tools for the robust identification of putative hits from HTS experiments and for the interpretation of these findings in the context of systems biology. We developed H...

  3. High-throughput analysis of the impact of antibiotics on the human intestinal microbiota composition

    NARCIS (Netherlands)

    Ladirat, S.E.; Schols, H.A.; Nauta, A.; Schoterman, M.H.C.; Keijser, B.J.F.; Montijn, R.C.; Gruppen, H.; Schuren, F.H.J.

    2013-01-01

    Antibiotic treatments can lead to a disruption of the human microbiota. In this in-vitro study, the impact of antibiotics on adult intestinal microbiota was monitored in a new high-throughput approach: a fermentation screening-platform was coupled with a phylogenetic microarray analysis (Intestinal-

  4. Computational and statistical methods for high-throughput analysis of post-translational modifications of proteins

    DEFF Research Database (Denmark)

    Schwämmle, Veit; Braga, Thiago Verano; Roepstorff, Peter

    2015-01-01

    for high-throughput experiments allow large-scale identification and quantification of several PTM types. This review addresses the concurrently emerging challenges for the computational analysis of the resulting data and presents PTM-centered approaches for spectra identification, statistical analysis...

  5. A high-throughput method for GMO multi-detection using a microfluidic dynamic array

    NARCIS (Netherlands)

    Brod, F.C.A.; Dijk, van J.P.; Voorhuijzen, M.M.; Dinon, A.Z.; Guimarães, L.H.S.; Scholtens, I.M.J.; Arisi, A.C.M.; Kok, E.J.

    2014-01-01

    The ever-increasing production of genetically modified crops generates a demand for high-throughput DNAbased methods for the enforcement of genetically modified organisms (GMO) labelling requirements. The application of standard real-time PCR will become increasingly costly with the growth of the nu

  6. High-throughput exposure modeling to support prioritization of chemicals in personal care products

    DEFF Research Database (Denmark)

    Csiszar, Susan A.; Ernstoff, Alexi; Fantke, Peter

    2016-01-01

    We demonstrate the application of a high-throughput modeling framework to estimate exposure to chemicals used in personal care products (PCPs). As a basis for estimating exposure, we use the product intake fraction (PiF), defined as the mass of chemical taken by an individual or population per mass...

  7. Roche genome sequencer FLX based high-throughput sequencing of ancient DNA

    DEFF Research Database (Denmark)

    Alquezar-Planas, David E; Fordyce, Sarah Louise

    2012-01-01

    Since the development of so-called "next generation" high-throughput sequencing in 2005, this technology has been applied to a variety of fields. Such applications include disease studies, evolutionary investigations, and ancient DNA. Each application requires a specialized protocol to ensure tha...

  8. High-throughput semiquantitative analysis of insertional mutations in heterogeneous tumors

    NARCIS (Netherlands)

    Koudijs, M.J.; Klijn, C.; van der Weyden, L.; Kool, J.; ten Hoeve, J.; Sie, D.; Prasetyanti, P.R.; Schut, E.; Kas, S.; Whipp, T.; Cuppen, E.; Wessels, L.; Adams, D.J.; Jonkers, J.

    2011-01-01

    Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on

  9. High-throughput synthesis and characterization of nanocrystalline porphyrinic zirconium metal-organic frameworks.

    Science.gov (United States)

    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.

  10. PLASMA PROTEIN PROFILING AS A HIGH THROUGHPUT TOOL FOR CHEMICAL SCREENING USING A SMALL FISH MODEL

    Science.gov (United States)

    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...

  11. Investigation of non-halogenated solvent mixtures for high throughput fabrication of polymerfullerene solar cells

    NARCIS (Netherlands)

    Schmidt-Hansberg, B.; Sanyal, M.; Grossiord, N.; Galagan, Y.O.; Baunach, M.; Klein, M.F.G.; Colsmann, A.; Scharfer, P.; Lemmer, U.; Dosch, H.; Michels, J.J; Barrena, E.; Schabel, W.

    2012-01-01

    The rapidly increasing power conversion efficiencies of organic solar cells are an important prerequisite towards low cost photovoltaic fabricated in high throughput. In this work we suggest indane as a non-halogenated replacement for the commonly used halogenated solvent o-dichlorobenzene. Indane w

  12. Organic light-emitting diodes: High-throughput virtual screening

    Science.gov (United States)

    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.

  13. Subtyping Animal Influenza Virus with General Multiplex RT-PCR and Liquichip High Throughput (GMPLex)

    Institute of Scientific and Technical Information of China (English)

    Zhi-feng Qin; Bing Cheng; Zhou-xi Ruan; Ying-zuo Bi; Joseph J Giambrone; Hong-zhuan Wu; Jie Sun; Ti-kang Lu; Shao-ling Zeng; Qun-yi Hua; Qing-yan Ling; Shu-kun Chen; Jian-qiang Lv; Cai-hong Zhang

    2012-01-01

    This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses.Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1,H3,H5,H7,H9 subtypes,and NA gene of the N1 and N2 subtypes.Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR).It included three stages of RT-PCR amplification,and then the RT-PCR products were further tested by LiquiChip probe,combined to give an influenza virus (Ⅳ) rapid high throughput subtyping test,designated as GMPLex.The IV GMPLex rapid high throughput subtyping test presents the following features:high throughput,able to determine the subtypes of 9 target genes in H1,H3,H5,H7,H9,N1,and N2 subtypes of the influenza A virus at one time; rapid,completing the influenza subtyping within 6 hours; high specificity,ensured the specificity of the different subtypes by using two nested degenerate primers and one probe,no cross reaction occurring between the subtypes,no non-specific reactions with other pathogens and high sensitivity.When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene,the GMPLex test showed a sensitivity of 105(=280ELD50) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12,respectively.When used to detect the product of GM RT-PCR for HSN1 strain at the same time,both showed a sensitivity of 10-4(=2800 ELD50).The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.

  14. High-throughput screening of high Monascus pigment-producing strain based on digital image processing.

    Science.gov (United States)

    Xia, Meng-lei; Wang, Lan; Yang, Zhi-xia; Chen, Hong-zhang

    2016-04-01

    This work proposed a new method which applied image processing and support vector machine (SVM) for screening of mold strains. Taking Monascus as example, morphological characteristics of Monascus colony were quantified by image processing. And the association between the characteristics and pigment production capability was determined by SVM. On this basis, a highly automated screening strategy was achieved. The accuracy of the proposed strategy is 80.6 %, which is compatible with the existing methods (81.1 % for microplate and 85.4 % for flask). Meanwhile, the screening of 500 colonies only takes 20-30 min, which is the highest rate among all published results. By applying this automated method, 13 strains with high-predicted production were obtained and the best one produced as 2.8-fold (226 U/mL) of pigment and 1.9-fold (51 mg/L) of lovastatin compared with the parent strain. The current study provides us with an effective and promising method for strain improvement.

  15. A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes.

    Science.gov (United States)

    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.

  16. Designs and concept reliance of a fully automated high-content screening platform.

    Science.gov (United States)

    Radu, Constantin; Adrar, Hosna Sana; Alamir, Ab; Hatherley, Ian; Trinh, Trung; Djaballah, Hakim

    2012-10-01

    High-content screening (HCS) is becoming an accepted platform in academic and industry screening labs and does require slightly different logistics for execution. To automate our stand-alone HCS microscopes, namely, an alpha IN Cell Analyzer 3000 (INCA3000), originally a Praelux unit hooked to a Hudson Plate Crane with a maximum capacity of 50 plates per run, and the IN Cell Analyzer 2000 (INCA2000), in which up to 320 plates could be fed per run using the Thermo Fisher Scientific Orbitor, we opted for a 4 m linear track system harboring both microscopes, plate washer, bulk dispensers, and a high-capacity incubator allowing us to perform both live and fixed cell-based assays while accessing both microscopes on deck. Considerations in design were given to the integration of the alpha INCA3000, a new gripper concept to access the onboard nest, and peripheral locations on deck to ensure a self-reliant system capable of achieving higher throughput. The resulting system, referred to as Hestia, has been fully operational since the new year, has an onboard capacity of 504 plates, and harbors the only fully automated alpha INCA3000 unit in the world.

  17. Embedded image enhancement for high-throughput cameras

    Science.gov (United States)

    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.

  18. Ensembler: Enabling High-Throughput Molecular Simulations at the Superfamily Scale.

    Directory of Open Access Journals (Sweden)

    Daniel L Parton

    2016-06-01

    Full Text Available The rapidly expanding body of available genomic and protein structural data provides a rich resource for understanding protein dynamics with biomolecular simulation. While computational infrastructure has grown rapidly, simulations on an omics scale are not yet widespread, primarily because software infrastructure to enable simulations at this scale has not kept pace. It should now be possible to study protein dynamics across entire (superfamilies, exploiting both available structural biology data and conformational similarities across homologous proteins. Here, we present a new tool for enabling high-throughput simulation in the genomics era. Ensembler takes any set of sequences-from a single sequence to an entire superfamily-and shepherds them through various stages of modeling and refinement to produce simulation-ready structures. This includes comparative modeling to all relevant PDB structures (which may span multiple conformational states of interest, reconstruction of missing loops, addition of missing atoms, culling of nearly identical structures, assignment of appropriate protonation states, solvation in explicit solvent, and refinement and filtering with molecular simulation to ensure stable simulation. The output of this pipeline is an ensemble of structures ready for subsequent molecular simulations using computer clusters, supercomputers, or distributed computing projects like Folding@home. Ensembler thus automates much of the time-consuming process of preparing protein models suitable for simulation, while allowing scalability up to entire superfamilies. A particular advantage of this approach can be found in the construction of kinetic models of conformational dynamics-such as Markov state models (MSMs-which benefit from a diverse array of initial configurations that span the accessible conformational states to aid sampling. We demonstrate the power of this approach by constructing models for all catalytic domains in the human

  19. Ensembler: Enabling High-Throughput Molecular Simulations at the Superfamily Scale.

    Science.gov (United States)

    Parton, Daniel L; Grinaway, Patrick B; Hanson, Sonya M; Beauchamp, Kyle A; Chodera, John D

    2016-06-01

    The rapidly expanding body of available genomic and protein structural data provides a rich resource for understanding protein dynamics with biomolecular simulation. While computational infrastructure has grown rapidly, simulations on an omics scale are not yet widespread, primarily because software infrastructure to enable simulations at this scale has not kept pace. It should now be possible to study protein dynamics across entire (super)families, exploiting both available structural biology data and conformational similarities across homologous proteins. Here, we present a new tool for enabling high-throughput simulation in the genomics era. Ensembler takes any set of sequences-from a single sequence to an entire superfamily-and shepherds them through various stages of modeling and refinement to produce simulation-ready structures. This includes comparative modeling to all relevant PDB structures (which may span multiple conformational states of interest), reconstruction of missing loops, addition of missing atoms, culling of nearly identical structures, assignment of appropriate protonation states, solvation in explicit solvent, and refinement and filtering with molecular simulation to ensure stable simulation. The output of this pipeline is an ensemble of structures ready for subsequent molecular simulations using computer clusters, supercomputers, or distributed computing projects like Folding@home. Ensembler thus automates much of the time-consuming process of preparing protein models suitable for simulation, while allowing scalability up to entire superfamilies. A particular advantage of this approach can be found in the construction of kinetic models of conformational dynamics-such as Markov state models (MSMs)-which benefit from a diverse array of initial configurations that span the accessible conformational states to aid sampling. We demonstrate the power of this approach by constructing models for all catalytic domains in the human tyrosine kinase

  20. A high-throughput, in-vitro assay for Bacillus thuringiensis insecticidal proteins.

    Science.gov (United States)

    Izumi Willcoxon, Michi; Dennis, Jaclyn R; Lau, Sabina I; Xie, Weiping; You, You; Leng, Song; Fong, Ryan C; Yamamoto, Takashi

    2016-01-10

    A high-throughput, in-vitro assay for Bacillus thuringiensis (Bt) insecticidal proteins designated as Cry was developed and evaluated for screening a large number of Cry protein variants produced by DNA shuffling. This automation-amenable assay exploits an insect cell line expressing a single receptor of Bt Cry proteins. The Cry toxin used to develop this assay is a variant of the Cry1Ab protein called IP1-88, which was produced previously by DNA shuffling. Cell mortality caused by the activated Bt Cry toxin was determined by chemical cell viability assay in 96/384-well microtiter plates utilizing CellTiter 96(®) obtained from Promega. A widely-accepted mode-of-action theory of certain Bt Cry proteins suggests that the activated toxin binds to one or more receptors and forms a pore through the insect gut epithelial cell apical membrane. A number of insect proteins such as cadherin-like protein (Cad), aminopeptidase-N (APN), alkaline phosphatase (ALP) and ABC transporter (ABCC) have been identified as the receptors of Bt Cry toxins. In this study, Bt Cry toxin receptors Ostrinia nubilalis (European corn borer) cadherin-like protein (On-Cad) and aminopeptidase-N 1 and 3 (On-APN1, On-APN3) and Spodoptera frugiperda (fall armyworm) cadherin-like protein (Sf-Cad) were cloned in an insect cell line, Sf21, and a mammalian cell line, Expi293F. It was observed by ligand blotting and immunofluorescence microscopy that trypsin-activated IP1-88 bound to On-Cad and On-APN1, but not Sf-Cad or On-APN3. In contrast, IP1-88 bound only to APN1 in BBMV (Brush Border Membrane Vesicles) prepared from the third and fourth-instar O. nubilalis larval midgut. The sensitivity of the recombinant cells to the toxin was then tested. IP1-88 showed no toxicity to non-recombinant Sf21 and Expi293F. Toxicity was observed only when the On-Cad gene was cloned and expressed. Sf-Cad and On-APN1 were not able to make those cells sensitive to the toxin. Since the expression of On-Cad alone was

  1. Ensembler: Enabling High-Throughput Molecular Simulations at the Superfamily Scale

    Science.gov (United States)

    Parton, Daniel L.; Grinaway, Patrick B.; Hanson, Sonya M.; Beauchamp, Kyle A.; Chodera, John D.

    2016-01-01

    The rapidly expanding body of available genomic and protein structural data provides a rich resource for understanding protein dynamics with biomolecular simulation. While computational infrastructure has grown rapidly, simulations on an omics scale are not yet widespread, primarily because software infrastructure to enable simulations at this scale has not kept pace. It should now be possible to study protein dynamics across entire (super)families, exploiting both available structural biology data and conformational similarities across homologous proteins. Here, we present a new tool for enabling high-throughput simulation in the genomics era. Ensembler takes any set of sequences—from a single sequence to an entire superfamily—and shepherds them through various stages of modeling and refinement to produce simulation-ready structures. This includes comparative modeling to all relevant PDB structures (which may span multiple conformational states of interest), reconstruction of missing loops, addition of missing atoms, culling of nearly identical structures, assignment of appropriate protonation states, solvation in explicit solvent, and refinement and filtering with molecular simulation to ensure stable simulation. The output of this pipeline is an ensemble of structures ready for subsequent molecular simulations using computer clusters, supercomputers, or distributed computing projects like Folding@home. Ensembler thus automates much of the time-consuming process of preparing protein models suitable for simulation, while allowing scalability up to entire superfamilies. A particular advantage of this approach can be found in the construction of kinetic models of conformational dynamics—such as Markov state models (MSMs)—which benefit from a diverse array of initial configurations that span the accessible conformational states to aid sampling. We demonstrate the power of this approach by constructing models for all catalytic domains in the human tyrosine

  2. A high-throughput bioinformatics distributed computing platform

    OpenAIRE

    Keane, Thomas M; Page, Andrew J.; McInerney, James O; Naughton, Thomas J.

    2005-01-01

    In the past number of years the demand for high performance computing has greatly increased in the area of bioinformatics. The huge increase in size of many genomic databases has meant that many common tasks in bioinformatics are not possible to complete in a reasonable amount of time on a single processor. Recently distributed computing has emerged as an inexpensive alternative to dedicated parallel computing. We have developed a general-purpose distributed computing platform ...

  3. High-throughput antibody development and retrospective epitope mapping

    DEFF Research Database (Denmark)

    Rydahl, Maja Gro

    Plant cell walls are composed of an interlinked network of polysaccharides, glycoproteins and phenolic polymers. When addressing the diverse polysaccharides in green plants, including land plants and the ancestral green algae, there are significant overlaps in the cell wall structures. Yet...... the binding profile - in more or less high resolution - of two small molecular probes, 11 carbohydrate binding modules and 24 monoclonal antibodies. This was made possible by combining the HTP multiplexing capacity of carbohydrate microarrays with diverse glycomic tools, to downstream characterize...

  4. Micropatterned comet assay enables high throughput and sensitive DNA damage quantification.

    Science.gov (United States)

    Ge, Jing; Chow, Danielle N; Fessler, Jessica L; Weingeist, David M; Wood, David K; Engelward, Bevin P

    2015-01-01

    The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H2O2-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies.

  5. A high throughput approach to quantify protein adsorption on combinatorial metal/metal oxide surfaces using electron microprobe and spectroscopic ellipsometry

    Science.gov (United States)

    Byrne, T.; Lohstreter, L.; Filiaggi, M. J.; Bai, Zhijun; Dahn, J. R.

    2008-09-01

    Although metallic biomaterials are widely used, systematic studies of protein adsorption onto such materials are generally lacking. Combinatorial binary films of Al 1-xTi x and Al 1-xNb x (0 ⩽ x ⩽ 1) and corresponding pure element films were produced on glass substrates using a unique magnetron sputtering technique. Fibrinogen and albumin adsorption amounts were measured by wavelength-dispersive spectroscopy (WDS) and spectroscopic ellipsometry (SE) equipment, both high throughput techniques with automated motion stage capabilities. X-ray diffraction revealed that the binary films have crystalline phases present near the ends of the compositional gradient with an amorphous region throughout the interior of the gradient. X-ray photoelectron spectroscopy provided the surface chemistry along the binary films and showed that Al 2O 3 preferentially formed at the surface. Protein adsorption onto these films was found to be closely correlated to the alumina surface fraction, with high alumina content at the surface leading to low amounts of adsorbed fibrinogen and albumin. Protein adsorption amounts obtained with WDS and SE were in excellent agreement for all films. This suggests that this combinatorial materials approach combined with these state-of-the-art, automated high throughput instruments provides a novel way to accurately monitor protein adsorption taking place at the surfaces of these metal/metal oxide materials.

  6. Melter Throughput Enhancements for High-Iron HLW

    Energy Technology Data Exchange (ETDEWEB)

    Kruger, A. A. [Department of Energy, Office of River Protection, Richland, Washington (United States); Gan, Hoa [The Catholic University of America, Washington, DC (United States); Joseph, Innocent [The Catholic University of America, Washington, DC (United States); Pegg, Ian L. [The Catholic University of America, Washington, DC (United States); Matlack, Keith S. [The Catholic University of America, Washington, DC (United States); Chaudhuri, Malabika [The Catholic University of America, Washington, DC (United States); Kot, Wing [The Catholic University of America, Washington, DC (United States)

    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. Multiplexed labeling system for high-throughput cell sorting.

    Science.gov (United States)

    Shin, Seung Won; Park, Kyung Soo; Song, In Hyun; Shin, Woo Jung; Kim, Byung Woo; Kim, Dong-Ik; Um, Soong Ho

    2016-09-01

    Flow cytometry and fluorescence activated cell sorting techniques were designed to realize configurable classification and separation of target cells. A number of cell phenotypes with different functionalities have recently been revealed. Before simultaneous selective capture of cells, it is desirable to label different samples with the corresponding dyes in a multiplexing manner to allow for a single analysis. However, few methods to obtain multiple fluorescent colors for various cell types have been developed. Even when restricted laser sources are employed, a small number of color codes can be expressed simultaneously. In this study, we demonstrate the ability to manifest DNA nanostructure-based multifluorescent colors formed by a complex of dyes. Highly precise self-assembly of fluorescent dye-conjugated oligonucleotides gives anisotropic DNA nanostructures, Y- and tree-shaped DNA (Y-DNA and T-DNA, respectively), which may be used as platforms for fluorescent codes. As a proof of concept, we have demonstrated seven different fluorescent codes with only two different fluorescent dyes using T-DNA. This method provides maximum efficiency for current flow cytometry. We are confident that this system will provide highly efficient multiplexed fluorescent detection for bioanalysis compared with one-to-one fluorescent correspondence for specific marker detection.

  8. Application of parallel liquid chromatography/mass spectrometry for high throughput microsomal stability screening of compound libraries.

    Science.gov (United States)

    Xu, Rongda; Nemes, Csaba; Jenkins, Kelly M; Rourick, Robyn A; Kassel, Daniel B; Liu, Charles Z C

    2002-02-01

    Solution-phase and solid-phase parallel synthesis and high throughput screening have enabled biologically active and selective compounds to be identified at an unprecedented rate. The challenge has been to convert these hits into viable development candidates. To accelerate the conversion of these hits into lead development candidates, early assessment of the physicochemical and pharmacological properties of these compounds is being made. In particular, in vitro absorption, distribution, metabolism, and elimination (ADME) assays are being conducted at earlier and earlier stages of discovery with the goal of reducing the attrition rate of these potential drug candidates as they progress through development. In this report, we present an eight-channel parallel liquid chromatography/mass spectrometry (LC/MS) system in combination with custom Visual Basic and Applescript automated data processing applications for high throughput early ADME. The parallel LC/MS system was configured with one set of gradient LC pumps and an eight-channel multiple probe autosampler. The flow was split equivalently into eight streams before the multiple probe autosampler and recombined after the eight columns and just prior to the mass spectrometer ion source. The system was tested for column-to-column variation and for reproducibility over a 17 h period (approximately 500 injections per column). The variations in retention time and peak area were determined to be less than 2 and 10%, respectively, in both tests. The parallel LC/MS system described permits time-course microsomal incubations (t(o), t5, t15, t30) to be measured in triplicate and enables estimations of t 1/2 microsomal stability. The parallel LC/MS system is capable of analyzing up to 240 samples per hour and permits the complete profiling up to two microtiter plates of compounds per day (i.e., 176 test substrate compounds + sixteen controls).

  9. A High Throughput Interferometeric Technique For Planet Detection

    Science.gov (United States)

    Mahadevan, Suvrath

    2007-12-01

    We have developed a novel instrument called the Exoplanet Tracker (ET) that can measure precise radial velocities. ET is installed at the Kitt Peak 2.1m telescope and uses a Michelson interferometer in series with a medium resolution spectrograph, enabling high light-collection effeciency. A survey for exoplanet systems using ET has lead to the discovery of a hot-Jupiter planet around the star HD102195. We have also built a stable monolithic Michelson interferometer for ET, enabling higher stability and precision. The development of the fixed-delay interferometer technology with the ET prototype has lead to the development of a large multi-object instrument capable of observing over 60 objects simultaneoulsy in the 3 degree field of view of the Sloan telescope. An upgraded version of this instrument will be used to conduct a large-scale survey for exoplanetary systems starting July 2008.

  10. High-throughput discovery of novel developmental phenotypes.

    Science.gov (United States)

    Dickinson, Mary E; Flenniken, Ann M; Ji, Xiao; Teboul, Lydia; Wong, Michael D; White, Jacqueline K; Meehan, Terrence F; Weninger, Wolfgang J; Westerberg, Henrik; Adissu, Hibret; Baker, Candice N; Bower, Lynette; Brown, James M; Caddle, L Brianna; Chiani, Francesco; Clary, Dave; Cleak, James; Daly, Mark J; Denegre, James M; Doe, Brendan; Dolan, Mary E; Edie, Sarah M; Fuchs, Helmut; Gailus-Durner, Valerie; Galli, Antonella; Gambadoro, Alessia; Gallegos, Juan; Guo, Shiying; Horner, Neil R; Hsu, Chih-Wei; Johnson, Sara J; Kalaga, Sowmya; Keith, Lance C; Lanoue, Louise; Lawson, Thomas N; Lek, Monkol; Mark, Manuel; Marschall, Susan; Mason, Jeremy; McElwee, Melissa L; Newbigging, Susan; Nutter, Lauryl M J; Peterson, Kevin A; Ramirez-Solis, Ramiro; Rowland, Douglas J; Ryder, Edward; Samocha, Kaitlin E; Seavitt, John R; Selloum, Mohammed; Szoke-Kovacs, Zsombor; Tamura, Masaru; Trainor, Amanda G; Tudose, Ilinca; Wakana, Shigeharu; Warren, Jonathan; Wendling, Olivia; West, David B; Wong, Leeyean; Yoshiki, Atsushi; MacArthur, Daniel G; Tocchini-Valentini, Glauco P; Gao, Xiang; Flicek, Paul; Bradley, Allan; Skarnes, William C; Justice, Monica J; Parkinson, Helen E; Moore, Mark; Wells, Sara; Braun, Robert E; Svenson, Karen L; de Angelis, Martin Hrabe; Herault, Yann; Mohun, Tim; Mallon, Ann-Marie; Henkelman, R Mark; Brown, Steve D M; Adams, David J; Lloyd, K C Kent; McKerlie, Colin; Beaudet, Arthur L; Bućan, Maja; Murray, Stephen A

    2016-09-22

    Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.

  11. Ultra-high-throughput Production of III-V/Si Wafer for Electronic and Photonic Applications.

    Science.gov (United States)

    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.

  12. A High Throughput On-Demand Routing Protocol for Multirate Ad Hoc Wireless Networks

    Science.gov (United States)

    Rahman, Md. Mustafizur; Hong, Choong Seon; Lee, Sungwon

    Routing in wireless ad hoc networks is a challenging issue because it dynamically controls the network topology and determines the network performance. Most of the available protocols are based on single-rate radio networks and they use hop-count as the routing metric. There have been some efforts for multirate radios as well that use transmission-time of a packet as the routing metric. However, neither the hop-count nor the transmission-time may be a sufficient criterion for discovering a high-throughput path in a multirate wireless ad hoc network. Hop-count based routing metrics usually select a low-rate bound path whereas the transmission-time based metrics may select a path with a comparatively large number of hops. The trade-off between transmission time and effective transmission range of a data rate can be another key criterion for finding a high-throughput path in such environments. In this paper, we introduce a novel routing metric based on the efficiency of a data rate that balances the required time and covering distance by a transmission and results in increased throughput. Using the new metric, we propose an on-demand routing protocol for multirate wireless environment, dubbed MR-AODV, to discover high-throughput paths in the network. A key feature of MR-AODV is that it controls the data rate in transmitting both the data and control packets. Rate control during the route discovery phase minimizes the route request (RREQ) avalanche. We use simulations to evaluate the performance of the proposed MR-AODV protocol and results reveal significant improvements in end-to-end throughput and minimization of routing overhead.

  13. New technologies for high throughput screening of effective traditional Chinese medicine components%中药活性成分的高通量筛选新技术

    Institute of Scientific and Technical Information of China (English)

    丁璇; 洪战英; 柴逸峰

    2015-01-01

    The research progress on new technologies for high throughput screening of effective traditional Chinese med-icine (TCM) components was summarized based on the recent documents at home and abroad ,among which bio-chromatogra-phy ,chip-technology and computer-aided virtual screen technology were widely used .Compared with traditional screening technology ,those new ones had shown advantages in efficiency ,automation and high-throughput ,providing new ways to screen effective components of TCM with high throughput .%以近年来国内外研究文献为基础,总结概括了近5年来中药活性成分的高通量筛选新技术的进展情况。其中生物色谱技术、芯片技术和计算机辅助虚拟筛选等技术得到了广泛应用。这些新技术在中药活性成分筛选方面,比起传统方法具有效率高、自动化、通量高的优点,可以为中药活性成分的高通量筛选提供新思路。

  14. A sensitive high throughput ELISA for human eosinophil peroxidase: a specific assay to quantify eosinophil degranulation from patient-derived sources.

    Science.gov (United States)

    Ochkur, Sergei I; Kim, John Dongil; Protheroe, Cheryl A; Colbert, Dana; Condjella, Rachel M; Bersoux, Sophie; Helmers, Richard A; Moqbel, Redwan; Lacy, Paige; Kelly, Elizabeth A; Jarjour, Nizar N; Kern, Robert; Peters, Anju; Schleimer, Robert P; Furuta, Glenn T; Nair, Parameswaran; Lee, James J; Lee, Nancy A

    2012-10-31

    Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patients in a clinical setting have been limited to ELISA assessments for the presence of the prominent granule ribonucleases, ECP and EDN. However, the demonstration that these ribonucleases are expressed by leukocytes other than eosinophils, as well as cells of non-hematopoietic origin, limits the usefulness of these assays. Two novel monoclonal antibodies recognizing eosinophil peroxidase (EPX) were used to develop an eosinophil-specific and sensitive sandwich ELISA. The sensitivity of this EPX-based ELISA was shown to be similar to that of the commercially available ELISA kits for ECP and EDN. More importantly, evidence is also presented confirming that among these granule protein detection options, EPX-based ELISA is the only eosinophil-specific assay. The utility of this high throughput assay to detect released EPX was shown in ex vivo degranulation studies with isolated human eosinophils. In addition, EPX-based ELISA was used to detect and quantify eosinophil degranulation in several in vivo patient settings, including bronchoalveolar lavage fluid obtained following segmental allergen challenge of subjects with allergic asthma, induced sputum derived from respiratory subjects following hypotonic saline inhalation, and nasal lavage of chronic rhinosinusitis patients. This unique EPX-based ELISA thus provides an eosinophil-specific assay that is sensitive, reproducible, and quantitative. In addition, this assay is adaptable to high throughput formats (e.g., automated assays utilizing microtiter plates) using the diverse patient fluid samples typically available in research and clinical settings.

  15. High-throughput machining using high average power ultrashort pulse lasers and ultrafast polygon scanner

    Science.gov (United States)

    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.

  16. High-throughput search for new permanent magnet materials.

    Science.gov (United States)

    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.

  17. High-Throughput Plasmid cDNA Library Screening

    Energy Technology Data Exchange (ETDEWEB)

    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.

  18. Scrutinizing virus genome termini by high-throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Shasha Li

    Full Text Available Analysis of genomic terminal sequences has been a major step in studies on viral DNA replication and packaging mechanisms. However, traditional methods to study genome termini are challenging due to the time-consuming protocols and their inefficiency where critical details are lost easily. Recent advances in next generation sequencing (NGS have enabled it to be a powerful tool to study genome termini. In this study, using NGS we sequenced one iridovirus genome and twenty phage genomes and confirmed for the first time that the high frequency sequences (HFSs found in the NGS reads are indeed the terminal sequences of viral genomes. Further, we established a criterion to distinguish the type of termini and the viral packaging mode. We also obtained additional terminal details such as terminal repeats, multi-termini, asymmetric termini. With this approach, we were able to simultaneously detect details of the genome termini as well as obtain the complete sequence of bacteriophage genomes. Theoretically, this application can be further extended to analyze larger and more complicated genomes of plant and animal viruses. This study proposed a novel and efficient method for research on viral replication, packaging, terminase activity, transcription regulation, and metabolism of the host cell.

  19. High throughput interferometric Doppler technique for planet detection

    Science.gov (United States)

    Mahadevan, Suvrath

    We have developed a novel instrument called the Exoplanet Tracker (ET) that can measure precise differential radial velocities, as well as barycentric radial velocities. ET is installed at the Kitt Peak 2.1 meter telescope and uses a Michelson interferometer in series with a medium resolution spectrograph. This instrument allows stellar radial velocities to be measured precisely without the use of a high resolution spectrograph. This allows the instrument to be very efficient in collecting light from the telescope. ET can achieve a radial velocity precision of 5-10 m s-1 over a 10 day observing run. A survey for extrasolar planets using the ET instrument has led to the detection of radial velocity variability for the star HD102195. Using photometry, CaII HK measurements, and precision radial velocities we demonstrate that these radial velocity variations are caused by a giant planet in a 4.11 day orbit around HD102195. A prototype monolithic interferometer has also been built for the ET instrument and is capable of delivering precise radial velocities. A large multi-object radial velocity instrument based on the ET instrument has been built and installed at the wide field Sloan 2.5 m telescope. This instrument, called the W. M. Keck Exoplanet Tracker, is capable of obtaining precise radial velocities for 59 stars simultaneously. Over the next few years this multi-object instrument will be used to conduct an All Sky ExoPlanet Survey capable of efficiently searching thousands of stars for planets.

  20. Label-free high-throughput imaging flow cytometry

    Science.gov (United States)

    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.