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Sample records for high throughput quantitative

  1. Fluorescent foci quantitation for high-throughput analysis

    Directory of Open Access Journals (Sweden)

    Elena Ledesma-Fernández

    2015-06-01

    Full Text Available 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.

  2. Compound Cytotoxicity Profiling Using Quantitative High-Throughput Screening

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

    2008-01-01

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

  3. Quantitative High-Throughput Screening Using a Coincidence Reporter Biocircuit.

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    Schuck, Brittany W; MacArthur, Ryan; Inglese, James

    2017-04-10

    Reporter-biased artifacts-i.e., compounds that interact directly with the reporter enzyme used in a high-throughput screening (HTS) assay and not the biological process or pharmacology being interrogated-are now widely recognized to reduce the efficiency and quality of HTS used for chemical probe and therapeutic development. Furthermore, narrow or single-concentration HTS perpetuates false negatives during primary screening campaigns. Titration-based HTS, or quantitative HTS (qHTS), and coincidence reporter technology can be employed to reduce false negatives and false positives, respectively, thereby increasing the quality and efficiency of primary screening efforts, where the number of compounds investigated can range from tens of thousands to millions. The three protocols described here allow for generation of a coincidence reporter (CR) biocircuit to interrogate a biological or pharmacological question of interest, generation of a stable cell line expressing the CR biocircuit, and qHTS using the CR biocircuit to efficiently identify high-quality biologically active small molecules. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  4. Multidimensional NMR approaches towards highly resolved, sensitive and high-throughput quantitative metabolomics.

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    Marchand, Jérémy; Martineau, Estelle; Guitton, Yann; Dervilly-Pinel, Gaud; Giraudeau, Patrick

    2017-02-01

    Multi-dimensional NMR is an appealing approach for dealing with the challenging complexity of biological samples in metabolomics. This article describes how spectroscopists have recently challenged their imagination in order to make 2D NMR a powerful tool for quantitative metabolomics, based on innovative pulse sequences combined with meticulous analytical chemistry approaches. Clever time-saving strategies have also been explored to make 2D NMR a high-throughput tool for metabolomics, relying on alternative data acquisition schemes such as ultrafast NMR. Currently, much work is aimed at drastically boosting the NMR sensitivity thanks to hyperpolarisation techniques, which have been used in combination with fast acquisition methods and could greatly expand the application potential of NMR metabolomics. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Streptococcus mutans Protein Synthesis during Mixed-Species Biofilm Development by High-Throughput Quantitative Proteomics

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    Klein, Marlise I.; Xiao, Jin; Lu, Bingwen; Delahunty, Claire M.; Yates, John R.; Koo, Hyun

    2012-01-01

    Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins...

  6. 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.

  7. Development of High-Throughput Quantitative Assays for Glucose Uptake in Cancer Cell Lines

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    Hassanein, Mohamed; Weidow, Brandy; Koehler, Elizabeth; Bakane, Naimish; Garbett, Shawn; Shyr, Yu; Quaranta, Vito

    2013-01-01

    Purpose Metabolism, and especially glucose uptake, is a key quantitative cell trait that is closely linked to cancer initiation and progression. Therefore, developing high-throughput assays for measuring glucose uptake in cancer cells would be enviable for simultaneous comparisons of multiple cell lines and microenvironmental conditions. This study was designed with two specific aims in mind: the first was to develop and validate a high-throughput screening method for quantitative assessment of glucose uptake in “normal” and tumor cells using the fluorescent 2-deoxyglucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), and the second was to develop an image-based, quantitative, single-cell assay for measuring glucose uptake using the same probe to dissect the full spectrum of metabolic variability within populations of tumor cells in vitro in higher resolution. Procedure The kinetics of population-based glucose uptake was evaluated for MCF10A mammary epithelial and CA1d breast cancer cell lines, using 2-NBDG and a fluorometric microplate reader. Glucose uptake for the same cell lines was also examined at the single-cell level using high-content automated microscopy coupled with semi-automated cell-cytometric image analysis approaches. Statistical treatments were also implemented to analyze intra-population variability. Results Our results demonstrate that the high-throughput fluorometric assay using 2-NBDG is a reliable method to assess population-level kinetics of glucose uptake in cell lines in vitro. Similarly, single-cell image-based assays and analyses of 2-NBDG fluorescence proved an effective and accurate means for assessing glucose uptake, which revealed that breast tumor cell lines display intra-population variability that is modulated by growth conditions. Conclusions These studies indicate that 2-NBDG can be used to aid in the high-throughput analysis of the influence of chemotherapeutics on glucose uptake in cancer

  8. Emerging flow injection mass spectrometry methods for high-throughput quantitative analysis.

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    Nanita, Sergio C; Kaldon, Laura G

    2016-01-01

    Where does flow injection analysis mass spectrometry (FIA-MS) stand relative to ambient mass spectrometry (MS) and chromatography-MS? Improvements in FIA-MS methods have resulted in fast-expanding uses of this technique. Key advantages of FIA-MS over chromatography-MS are fast analysis (typical run time method simplicity, and FIA-MS offers high-throughput without compromising sensitivity, precision and accuracy as much as ambient MS techniques. Consequently, FIA-MS is increasingly becoming recognized as a suitable technique for applications where quantitative screening of chemicals needs to be performed rapidly and reliably. The FIA-MS methods discussed herein have demonstrated quantitation of diverse analytes, including pharmaceuticals, pesticides, environmental contaminants, and endogenous compounds, at levels ranging from parts-per-billion (ppb) to parts-per-million (ppm) in very complex matrices (such as blood, urine, and a variety of foods of plant and animal origin), allowing successful applications of the technique in clinical diagnostics, metabolomics, environmental sciences, toxicology, and detection of adulterated/counterfeited goods. The recent boom in applications of FIA-MS for high-throughput quantitative analysis has been driven in part by (1) the continuous improvements in sensitivity and selectivity of MS instrumentation, (2) the introduction of novel sample preparation procedures compatible with standalone mass spectrometric analysis such as salting out assisted liquid-liquid extraction (SALLE) with volatile solutes and NH4(+) QuEChERS, and (3) the need to improve efficiency of laboratories to satisfy increasing analytical demand while lowering operational cost. The advantages and drawbacks of quantitative analysis by FIA-MS are discussed in comparison to chromatography-MS and ambient MS (e.g., DESI, LAESI, DART). Generally, FIA-MS sits 'in the middle' between ambient MS and chromatography-MS, offering a balance between analytical capability and

  9. Quantitative Live-Cell Confocal Imaging of 3D Spheroids in a High-Throughput Format.

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    Leary, Elizabeth; Rhee, Claire; Wilks, Benjamin T; Morgan, Jeffrey R

    2018-02-01

    Accurately predicting the human response to new compounds is critical to a wide variety of industries. Standard screening pipelines (including both in vitro and in vivo models) often lack predictive power. Three-dimensional (3D) culture systems of human cells, a more physiologically relevant platform, could provide a high-throughput, automated means to test the efficacy and/or toxicity of novel substances. However, the challenge of obtaining high-magnification, confocal z stacks of 3D spheroids and understanding their respective quantitative limitations must be overcome first. To address this challenge, we developed a method to form spheroids of reproducible size at precise spatial locations across a 96-well plate. Spheroids of variable radii were labeled with four different fluorescent dyes and imaged with a high-throughput confocal microscope. 3D renderings of the spheroid had a complex bowl-like appearance. We systematically analyzed these confocal z stacks to determine the depth of imaging and the effect of spheroid size and dyes on quantitation. Furthermore, we have shown that this loss of fluorescence can be addressed through the use of ratio imaging. Overall, understanding both the limitations of confocal imaging and the tools to correct for these limits is critical for developing accurate quantitative assays using 3D spheroids.

  10. Quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death.

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    Zhu, Ping Jun; Hobson, John P; Southall, Noel; Qiu, Cunping; Thomas, Craig J; Lu, Jiamo; Inglese, James; Zheng, Wei; Leppla, Stephen H; Bugge, Thomas H; Austin, Christopher P; Liu, Shihui

    2009-07-15

    Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a beta-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin. Further efforts demonstrated that these compounds impaired the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor, but not the related TEM8 receptor, indicating that these compounds likely interfere with toxin internalization.

  11. Curating and Preparing High-Throughput Screening Data for Quantitative Structure-Activity Relationship Modeling.

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    Kim, Marlene T; Wang, Wenyi; Sedykh, Alexander; Zhu, Hao

    2016-01-01

    Publicly available bioassay data often contains errors. Curating massive bioassay data, especially high-throughput screening (HTS) data, for Quantitative Structure-Activity Relationship (QSAR) modeling requires the assistance of automated data curation tools. Using automated data curation tools are beneficial to users, especially ones without prior computer skills, because many platforms have been developed and optimized based on standardized requirements. As a result, the users do not need to extensively configure the curation tool prior to the application procedure. In this chapter, a freely available automatic tool to curate and prepare HTS data for QSAR modeling purposes will be described.

  12. Quantitative dot blot analysis (QDB), a versatile high throughput immunoblot method.

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    Tian, Geng; Tang, Fangrong; Yang, Chunhua; Zhang, Wenfeng; Bergquist, Jonas; Wang, Bin; Mi, Jia; Zhang, Jiandi

    2017-08-29

    Lacking access to an affordable method of high throughput immunoblot analysis for daily use remains a big challenge for scientists worldwide. We proposed here Quantitative Dot Blot analysis (QDB) to meet this demand. With the defined linear range, QDB analysis fundamentally transforms traditional immunoblot method into a true quantitative assay. Its convenience in analyzing large number of samples also enables bench scientists to examine protein expression levels from multiple parameters. In addition, the small amount of sample lysates needed for analysis means significant saving in research sources and efforts. This method was evaluated at both cellular and tissue levels with unexpected observations otherwise would be hard to achieve using conventional immunoblot methods like Western blot analysis. Using QDB technique, we were able to observed an age-dependent significant alteration of CAPG protein expression level in TRAMP mice. We believe that the adoption of QDB analysis would have immediate impact on biological and biomedical research to provide much needed high-throughput information at protein level in this "Big Data" era.

  13. Transcriptional biomarkers--high throughput screening, quantitative verification, and bioinformatical validation methods.

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    Riedmaier, Irmgard; Pfaffl, Michael W

    2013-01-01

    Molecular biomarkers found their way into many research fields, especially in molecular medicine, medical diagnostics, disease prognosis, risk assessment but also in other areas like food safety. Different definitions for the term biomarker exist, but on the whole biomarkers are measureable biological molecules that are characteristic for a specific physiological status including drug intervention, normal or pathological processes. There are various examples for molecular biomarkers that are already successfully used in clinical diagnostics, especially as prognostic or diagnostic tool for diseases. Molecular biomarkers can be identified on different molecular levels, namely the genome, the epigenome, the transcriptome, the proteome, the metabolome and the lipidome. With special "omic" technologies, nowadays often high throughput technologies, these molecular biomarkers can be identified and quantitatively measured. This article describes the different molecular levels on which biomarker research is possible including some biomarker candidates that have already been identified. Hereby the transcriptomic approach will be described in detail including available high throughput methods, molecular levels, quantitative verification, and biostatistical requirements for transcriptional biomarker identification and validation. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Mass spectrometry for high throughput quantitative proteomics in plant research: lessons from thylakoid membranes.

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    Whitelegge, Julian P

    2004-12-01

    Proteomics seeks to monitor the flux of protein through cells under variable developmental and environmental influences as programmed by the genome. Consequently, it is necessary to measure changes in protein abundance and turnover rate as faithfully as possible. In the absence of non-invasive technologies, the majority of proteomics approaches involve destructive sampling at various time points to obtain 'snapshots' that periodically report the genomes's product. The work has fallen to separations technologies coupled to mass spectrometry, for high throughput protein identification. Quantitation has become the major challenge facing proteomics as the field matures. Because of the variability of day-to-day measurements of protein quantities by mass spectrometry, a common feature of quantitative proteomics is the use of stable isotope coding to distinguish control and experimental samples in a mixture that can be profiled in a single experiment. To address limitations with separation technologies such as 2D-gel electrophoresis, alternative systems are being introduced including multi-dimensional chromatography. Strategies that accelerate throughput for mass spectrometry are also emerging and the benefits of these 'shotgun' protocols will be considered in the context of the thylakoid membrane and photosynthesis. High resolution Fourier-transform mass spectrometry is bringing increasingly accurate mass measurements to peptides and a variety of gas-phase dissociation mechanisms are permitting 'top-down' sequencing of intact proteins. Finally, a versatile workflow for sub-cellular compartments including membranes is presented that allows for intact protein mass measurements, localization of post-translational modifications and relative quantitation or turnover measurement.

  15. Bayesian analysis of high-throughput quantitative measurement of protein-DNA interactions.

    Directory of Open Access Journals (Sweden)

    David D Pollock

    Full Text Available Transcriptional regulation depends upon the binding of transcription factor (TF proteins to DNA in a sequence-dependent manner. Although many experimental methods address the interaction between DNA and proteins, they generally do not comprehensively and accurately assess the full binding repertoire (the complete set of sequences that might be bound with at least moderate strength. Here, we develop and evaluate through simulation an experimental approach that allows simultaneous high-throughput quantitative analysis of TF binding affinity to thousands of potential DNA ligands. Tens of thousands of putative binding targets can be mixed with a TF, and both the pre-bound and bound target pools sequenced. A hierarchical Bayesian Markov chain Monte Carlo approach determines posterior estimates for the dissociation constants, sequence-specific binding energies, and free TF concentrations. A unique feature of our approach is that dissociation constants are jointly estimated from their inferred degree of binding and from a model of binding energetics, depending on how many sequence reads are available and the explanatory power of the energy model. Careful experimental design is necessary to obtain accurate results over a wide range of dissociation constants. This approach, which we call Simultaneous Ultra high-throughput Ligand Dissociation EXperiment (SULDEX, is theoretically capable of rapid and accurate elucidation of an entire TF-binding repertoire.

  16. Integrating high-throughput pyrosequencing and quantitative real-time PCR to analyze complex microbial communities.

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    Zhang, Husen; Parameswaran, Prathap; Badalamenti, Jonathan; Rittmann, Bruce E; Krajmalnik-Brown, Rosa

    2011-01-01

    New high-throughput technologies continue to emerge for studying complex microbial communities. In particular, massively parallel pyrosequencing enables very high numbers of sequences, providing a more complete view of community structures and a more accurate inference of the functions than has been possible just a few years ago. In parallel, quantitative real-time PCR (QPCR) allows quantitative monitoring of specific community members over time, space, or different environmental conditions. In this review, we discuss the principles of these two methods and their complementary applications in studying microbial ecology in bioenvironmental systems. We explain parallel sequencing of amplicon libraries and using bar codes to differentiate multiple samples in a pyrosequencing run. We also describe best procedures and chemistries for QPCR amplifications and address advantages of applying automation to increase accuracy. We provide three examples in which we used pyrosequencing and QPCR together to define and quantify members of microbial communities: in the human large intestine, in a methanogenic digester whose sludge was made more bioavailable by a high-voltage pretreatment, and on the biofilm anode of a microbial electrolytic cell. We highlight our key findings in these systems and how both methods were used in concert to achieve those findings. Finally, we supply detailed methods for generating PCR amplicon libraries for pyrosequencing, pyrosequencing data analysis, QPCR methodology, instrumentation, and automation.

  17. Recent advances in quantitative high throughput and high content data analysis.

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    Moutsatsos, Ioannis K; Parker, Christian N

    2016-01-01

    High throughput screening has become a basic technique with which to explore biological systems. Advances in technology, including increased screening capacity, as well as methods that generate multiparametric readouts, are driving the need for improvements in the analysis of data sets derived from such screens. This article covers the recent advances in the analysis of high throughput screening data sets from arrayed samples, as well as the recent advances in the analysis of cell-by-cell data sets derived from image or flow cytometry application. Screening multiple genomic reagents targeting any given gene creates additional challenges and so methods that prioritize individual gene targets have been developed. The article reviews many of the open source data analysis methods that are now available and which are helping to define a consensus on the best practices to use when analyzing screening data. As data sets become larger, and more complex, the need for easily accessible data analysis tools will continue to grow. The presentation of such complex data sets, to facilitate quality control monitoring and interpretation of the results will require the development of novel visualizations. In addition, advanced statistical and machine learning algorithms that can help identify patterns, correlations and the best features in massive data sets will be required. The ease of use for these tools will be important, as they will need to be used iteratively by laboratory scientists to improve the outcomes of complex analyses.

  18. High throughput quantitative phenotyping of plant resistance using chlorophyll fluorescence image analysis.

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    Rousseau, Céline; Belin, Etienne; Bove, Edouard; Rousseau, David; Fabre, Frédéric; Berruyer, Romain; Guillaumès, Jacky; Manceau, Charles; Jacques, Marie-Agnès; Boureau, Tristan

    2013-06-13

    In order to select for quantitative plant resistance to pathogens, high throughput approaches that can precisely quantify disease severity are needed. Automation and use of calibrated image analysis should provide more accurate, objective and faster analyses than visual assessments. In contrast to conventional visible imaging, chlorophyll fluorescence imaging is not sensitive to environmental light variations and provides single-channel images prone to a segmentation analysis by simple thresholding approaches. Among the various parameters used in chlorophyll fluorescence imaging, the maximum quantum yield of photosystem II photochemistry (Fv/Fm) is well adapted to phenotyping disease severity. Fv/Fm is an indicator of plant stress that displays a robust contrast between infected and healthy tissues. In the present paper, we aimed at the segmentation of Fv/Fm images to quantify disease severity. Based on the Fv/Fm values of each pixel of the image, a thresholding approach was developed to delimit diseased areas. A first step consisted in setting up thresholds to reproduce visual observations by trained raters of symptoms caused by Xanthomonas fuscans subsp. fuscans (Xff) CFBP4834-R on Phaseolus vulgaris cv. Flavert. In order to develop a thresholding approach valuable on any cultivars or species, a second step was based on modeling pixel-wise Fv/Fm-distributions as mixtures of Gaussian distributions. Such a modeling may discriminate various stages of the symptom development but over-weights artifacts that can occur on mock-inoculated samples. Therefore, we developed a thresholding approach based on the probability of misclassification of a healthy pixel. Then, a clustering step is performed on the diseased areas to discriminate between various stages of alteration of plant tissues. Notably, the use of chlorophyll fluorescence imaging could detect pre-symptomatic area. The interest of this image analysis procedure for assessing the levels of quantitative resistance

  19. Analysis of JC virus DNA replication using a quantitative and high-throughput assay.

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    Shin, Jong; Phelan, Paul J; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A

    2014-11-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Quantitative X-ray microradiography for high-throughput phenotyping of osteoarthritis in mice.

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    Waung, J A; Maynard, S A; Gopal, S; Gogakos, A; Logan, J G; Williams, G R; Bassett, J H D

    2014-10-01

    To investigate and validate digital X-ray microradiography as a novel, high-throughput and cost-effective screening approach to identify abnormal joint phenotypes in mice. Digital X-ray microradiography was used to quantify the subchondral bone mineral content (BMC) in the medial tibial plateau. Accuracy and reproducibility of the method were determined in 22 samples from C57BL/6(B6Brd;B6Dnk;B6N-Tyr(c-Brd)) wild-type mice. The method was then validated in wild-type mice that had undergone surgical destabilisation of medial meniscus (DMM) and in a genetically modified mouse strain with an established increase in trabecular bone mass. The measurement of subchondral BMC by digital X-ray microradiography had a coefficient of variation of 3.6%. Digital X-ray microradiography was able to demonstrate significantly increased subchondral BMC in the medial tibial plateau of male mice 4 and 8 weeks after DMM surgery and in female mice 8 weeks after surgery. Furthermore, digital X-ray microradiography also detected the increase in subchondral BMC in a genetically modified mouse strain with high trabecular bone mass. Quantitation of subchondral BMC by digital X-ray microradiography is a rapid, sensitive and cost-effective method to identify abnormal joint phenotypes in mice of both genders at several ages. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    Science.gov (United States)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

    2015-01-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. PMID:25155200

  2. Using Weighted Entropy to Rank Chemicals in Quantitative High Throughput Screening Experiments

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    Shockley, Keith R.

    2014-01-01

    Quantitative high throughput screening (qHTS) experiments can simultaneously produce concentration-response profiles for thousands of chemicals. In a typical qHTS study, a large chemical library is subjected to a primary screen in order to identify candidate hits for secondary screening, validation studies or prediction modeling. Different algorithms, usually based on the Hill equation logistic model, have been used to classify compounds as active or inactive (or inconclusive). However, observed concentration-response activity relationships may not adequately fit a sigmoidal curve. Furthermore, it is unclear how to prioritize chemicals for follow-up studies given the large uncertainties that often accompany parameter estimates from nonlinear models. Weighted Shannon entropy can address these concerns by ranking compounds according to profile-specific statistics derived from estimates of the probability mass distribution of response at the tested concentration levels. This strategy can be used to rank all tested chemicals in the absence of a pre-specified model structure or the approach can complement existing activity call algorithms by ranking the returned candidate hits. The weighted entropy approach was evaluated here using data simulated from the Hill equation model. The procedure was then applied to a chemical genomics profiling data set interrogating compounds for androgen receptor agonist activity. PMID:24056003

  3. Quantitative High-Throughput Identification of Drugs as Modulators of Human Constitutive Androstane Receptor

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    Lynch, Caitlin; Zhao, Jinghua; Huang, Ruili; Xiao, Jingwei; Li, Linhao; Heyward, Scott; Xia, Menghang; Wang, Hongbing

    2015-01-01

    The constitutive androstane receptor (CAR, NR1I3) plays a key role in governing the transcription of numerous hepatic genes that involve xenobiotic metabolism/clearance, energy homeostasis, and cell proliferation. Thus, identification of novel human CAR (hCAR) modulators may not only enhance early prediction of drug-drug interactions but also offer potentially novel therapeutics for diseases such as metabolic disorders and cancer. In this study, we have generated a double stable cell line expressing both hCAR and a CYP2B6-driven luciferase reporter for quantitative high-throughput screening (qHTS) of hCAR modulators. Approximately 2800 compounds from the NIH Chemical Genomics Center Pharmaceutical Collection were screened employing both the activation and deactivation modes of the qHTS. Activators (115) and deactivators (152) of hCAR were identified from the primary qHTS, among which 10 agonists and 10 antagonists were further validated in the physiologically relevant human primary hepatocytes for compound-mediated hCAR nuclear translocation and target gene expression. Collectively, our results reveal that hCAR modulators can be efficiently identified through this newly established qHTS assay. Profiling drug collections for hCAR activity would facilitate the prediction of metabolism-based drug-drug interactions, and may lead to the identification of potential novel therapeutics. PMID:25993555

  4. Sensitive, quantitative, and high-throughput detection of angiogenic markers using shape-coded hydrogel microparticles.

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    Al-Ameen, Mohammad Ali; Li, Ji; Beer, David G; Ghosh, Gargi

    2015-07-07

    Elevated serum concentrations of angiogenic markers including vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) have been correlated with various clinical disorders including cancer, cardiovascular diseases, diabetes mellitus, and liver fibrosis. In addition, the correlation between the serum concentrations of these factors, clinical diagnosis, prognosis, and response to therapeutic agents is significant. Thereby suggesting high-throughput detection of serum levels of angiogenic markers has important implications in early detection of different clinical disorders as well as for subsequent therapy monitoring. Here, we demonstrate the feasibility of utilization of shape-coded hydrogel microparticle based suspension arrays for quantitative and reproducible measurement of VEGF, FGF, and PDGF in single and multiplexed assays. Bio-inert PEG hydrogel attenuated the background signal thereby improving the sensitivity of the detection method as well as eliminating the need for blocking the proteins. In the singleplexed assay, the detection limits of 1.7 pg ml(-1), 1.4 pg ml(-1), and 1.5 pg ml(-1) for VEGF, FGF, and PDGF respectively indicated that the sensitivity of the developed method exceeds that of the conventional technologies. We also demonstrated that in the multiplexed assays, recovery of the proteins was within 20% of the expected values. The practical applicability of the hydrogel microparticle based detection system was established by demonstrating the ability of the system to quantify the production of VEGF, FGF, and PDGF by breast cancer cells (MDA-MB-231).

  5. Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification.

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    Fu, Wei; Zhu, Pengyu; Wei, Shuang; Zhixin, Du; Wang, Chenguang; Wu, Xiyang; Li, Feiwu; Zhu, Shuifang

    2017-04-01

    Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for

  6. Quantitative description on structure-property relationships of Li-ion battery materials for high-throughput computations.

    Science.gov (United States)

    Wang, Youwei; Zhang, Wenqing; Chen, Lidong; Shi, Siqi; Liu, Jianjun

    2017-01-01

    Li-ion batteries are a key technology for addressing the global challenge of clean renewable energy and environment pollution. Their contemporary applications, for portable electronic devices, electric vehicles, and large-scale power grids, stimulate the development of high-performance battery materials with high energy density, high power, good safety, and long lifetime. High-throughput calculations provide a practical strategy to discover new battery materials and optimize currently known material performances. Most cathode materials screened by the previous high-throughput calculations cannot meet the requirement of practical applications because only capacity, voltage and volume change of bulk were considered. It is important to include more structure-property relationships, such as point defects, surface and interface, doping and metal-mixture and nanosize effects, in high-throughput calculations. In this review, we established quantitative description of structure-property relationships in Li-ion battery materials by the intrinsic bulk parameters, which can be applied in future high-throughput calculations to screen Li-ion battery materials. Based on these parameterized structure-property relationships, a possible high-throughput computational screening flow path is proposed to obtain high-performance battery materials.

  7. Quantitative description on structure–property relationships of Li-ion battery materials for high-throughput computations

    Science.gov (United States)

    Wang, Youwei; Zhang, Wenqing; Chen, Lidong; Shi, Siqi; Liu, Jianjun

    2017-01-01

    Abstract Li-ion batteries are a key technology for addressing the global challenge of clean renewable energy and environment pollution. Their contemporary applications, for portable electronic devices, electric vehicles, and large-scale power grids, stimulate the development of high-performance battery materials with high energy density, high power, good safety, and long lifetime. High-throughput calculations provide a practical strategy to discover new battery materials and optimize currently known material performances. Most cathode materials screened by the previous high-throughput calculations cannot meet the requirement of practical applications because only capacity, voltage and volume change of bulk were considered. It is important to include more structure–property relationships, such as point defects, surface and interface, doping and metal-mixture and nanosize effects, in high-throughput calculations. In this review, we established quantitative description of structure–property relationships in Li-ion battery materials by the intrinsic bulk parameters, which can be applied in future high-throughput calculations to screen Li-ion battery materials. Based on these parameterized structure–property relationships, a possible high-throughput computational screening flow path is proposed to obtain high-performance battery materials. PMID:28458737

  8. Quantitative monitoring of Arabidopsis thaliana growth and development using high-throughput plant phenotyping.

    Science.gov (United States)

    Arend, Daniel; Lange, Matthias; Pape, Jean-Michel; Weigelt-Fischer, Kathleen; Arana-Ceballos, Fernando; Mücke, Ingo; Klukas, Christian; Altmann, Thomas; Scholz, Uwe; Junker, Astrid

    2016-08-16

    With the implementation of novel automated, high throughput methods and facilities in the last years, plant phenomics has developed into a highly interdisciplinary research domain integrating biology, engineering and bioinformatics. Here we present a dataset of a non-invasive high throughput plant phenotyping experiment, which uses image- and image analysis- based approaches to monitor the growth and development of 484 Arabidopsis thaliana plants (thale cress). The result is a comprehensive dataset of images and extracted phenotypical features. Such datasets require detailed documentation, standardized description of experimental metadata as well as sustainable data storage and publication in order to ensure the reproducibility of experiments, data reuse and comparability among the scientific community. Therefore the here presented dataset has been annotated using the standardized ISA-Tab format and considering the recently published recommendations for the semantical description of plant phenotyping experiments.

  9. Towards cracking the epigenetic code using a combination of high-throughput epigenomics and quantitative mass spectrometry-based proteomics.

    Science.gov (United States)

    Stunnenberg, Hendrik G; Vermeulen, Michiel

    2011-07-01

    High-throughput genomic sequencing and quantitative mass spectrometry (MS)-based proteomics technology have recently emerged as powerful tools, increasing our understanding of chromatin structure and function. Both of these approaches require substantial investments and expertise in terms of instrumentation, experimental methodology, bioinformatics, and data interpretation and are, therefore, usually applied independently from each other by dedicated research groups. However, when applied reiteratively in the context of epigenetics research these approaches are strongly synergistic in nature. Copyright © 2011 WILEY Periodicals, Inc.

  10. Quantitative high-throughput screen identifies inhibitors of the Schistosoma mansoni redox cascade.

    Directory of Open Access Journals (Sweden)

    Anton Simeonov

    2008-01-01

    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

  11. Streptococcus mutans protein synthesis during mixed-species biofilm development by high-throughput quantitative proteomics.

    Directory of Open Access Journals (Sweden)

    Marlise I Klein

    Full Text Available Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA and glucan-binding (gbpB during this transition (P<0.05. Furthermore, S. mutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism, and molecular chaperones (GroEL. Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms demonstrating fundamental differences in the matrix assembly, survival and biofilm

  12. Streptococcus mutans Protein Synthesis during Mixed-Species Biofilm Development by High-Throughput Quantitative Proteomics

    Science.gov (United States)

    Klein, Marlise I.; Xiao, Jin; Lu, Bingwen; Delahunty, Claire M.; Yates, John R.; Koo, Hyun

    2012-01-01

    Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v) was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT) approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA) and glucan-binding (gbpB) during this transition (Pmutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism), and molecular chaperones (GroEL). Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms) demonstrating fundamental differences in the matrix assembly, survival and biofilm maintenance in the presence of other

  13. A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis

    Science.gov (United States)

    Taggart, David J.; Camerlengo, Terry L.; Harrison, Jason K.; Sherrer, Shanen M.; Kshetry, Ajay K.; Taylor, John-Stephen; Huang, Kun; Suo, Zucai

    2013-01-01

    Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis–syn thymidine–thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases. PMID:23470999

  14. 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...

  15. A method for high-throughput quantitative analysis of yeast chronological life span.

    Science.gov (United States)

    Murakami, Christopher J; Burtner, Christopher R; Kennedy, Brian K; Kaeberlein, Matt

    2008-02-01

    Chronological aging in yeast has been studied by maintaining cells in a quiescent-like stationary phase culture and monitoring cell survival over time. The composition of the growth medium can have a profound influence on chronological aging. For example, dietary restriction accomplished by lowering the glucose concentration of the medium significantly increases life span. Here we report a novel high-throughput method for measuring yeast chronological life span by monitoring outgrowth of aging cells using a Bioscreen C MBR machine. We show that this method provides survival data comparable to traditional methods, but with decreased variability. In addition to reducing the glucose concentration, we find that elevated amino acid levels or increased osmolarity of the growth medium is sufficient to increase chronological life span. We also report that life-span extension from dietary restriction does not require any of the five yeast sirtuins (Sir2, Hst1, Hst2, Hst3, or Hst4) either alone or in combination.

  16. A novel high-throughput assay for the quantitative assessment of receptor trafficking.

    Science.gov (United States)

    Grimsey, Natasha L; Narayan, Pritika J; Dragunow, Mike; Glass, Michelle

    2008-11-01

    1. Receptor transport between intracellular compartments has important consequences for receptor function and is an exciting area of current study. Existing methods for studying receptor trafficking often require labour-intensive techniques or are difficult to quantify reliably. We report a novel high-throughput method that uses automated imaging and analysis tools to accurately quantify cannabinoid CB1 receptor trafficking. 2. Haemagglutinin (HA)-tagged CB1 was stably expressed in HEK-293 cells and cell surface or total receptors were detected immunocytochemically. Images of receptor and nuclear staining were acquired with an automated fluorescent microscope (Discovery-1; Molecular Devices, Sunnyvale, CA, USA) and quantified at high throughput with MetaMorph (Molecular Devices) software. The 'Granularity' assay measured internalization by counting receptor clusters that appear during receptor endocytosis, a well-established approach. Our assay, referred to as 'Total Grey Value per Cell' (TGVC), measures the total fluorescence above background, normalized to cell count. 3. Incubation with the cannabinoid agonist HU-210 (100 nmol/L) resulted in rapid CB1 internalization, reaching a maximum within 20 min. Whether quantified by Granularity or TGVC, the time-course of endocytosis could be modelled with exponentially derived curves and with similar half-lives. We demonstrate the sensitivity of our TGVC method by measuring the concentration dependence of CB1 internalization and its versatility by measuring downregulation following chronic agonist exposure, whereby total CB1 was reduced to approximately 55% of basal after 3 h. 4. The TGVC quantification method described is efficient, accurate and versatile and is likely to provide a valuable tool in receptor trafficking studies.

  17. High Throughput Protein Quantitation using MRM Viewer Software and Dynamic MRM on a Triple Quadruple Mass Spectrometer

    Science.gov (United States)

    Miller, C.; Waddell, K.; Tang, N.

    2010-01-01

    RP-122 Peptide quantitation using Multiple Reaction Monitoring (MRM) has been established as an important methodology for biomarker verification andvalidation.This requires high throughput combined with high sensitivity to analyze potentially thousands of target peptides in each sample.Dynamic MRM allows the system to only acquire the required MRMs of the peptide during a retention window corresponding to when each peptide is eluting. This reduces the number of concurrent MRM and therefore improves quantitation and sensitivity. MRM Selector allows the user to generate an MRM transition list with retention time information from discovery data obtained on a QTOF MS system.This list can be directly imported into the triple quadrupole acquisition software.However, situations can exist where a) the list of MRMs contain an excess of MRM transitions allowable under the ideal acquisition conditions chosen ( allowing for cycle time and chromatography conditions), or b) too many transitions in a certain retention time region which would result in an unacceptably low dwell time and cycle time.A new tool - MRM viewer has been developed to help users automatically generate multiple dynamic MRM methods from a single MRM list.In this study, a list of 3293 MRM transitions from a human plasma sample was compiled.A single dynamic MRM method with 3293 transitions results in a minimum dwell time of 2.18ms.Using MRM viewer we can generate three dynamic MRM methods with a minimum dwell time of 20ms which can give a better quality MRM quantitation.This tool facilitates both high throughput and high sensitivity for MRM quantitation.

  18. High-throughput label-free screening of euglena gracilis with optofluidic time-stretch quantitative phase microscopy

    Science.gov (United States)

    Guo, Baoshan; Lei, Cheng; Ito, Takuro; Yaxiaer, Yalikun; Kobayashi, Hirofumi; Jiang, Yiyue; Tanaka, Yo; Ozeki, Yasuyuki; Goda, Keisuke

    2017-02-01

    The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, microalgal biofuel is expected to play a key role in reducing the detrimental effects of global warming since microalgae absorb atmospheric CO2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid contents and fail to characterize a diverse population of microalgal cells with single-cell resolution in a noninvasive and interference-free manner. Here we demonstrate high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy. In particular, we use Euglena gracilis - an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement) within lipid droplets. Our optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch phase-contrast microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase contents of every single cell at a high throughput of 10,000 cells/s. We characterize heterogeneous populations of E. gracilis cells under two different culture conditions to evaluate their lipid production efficiency. Our method holds promise as an effective analytical tool for microalgaebased biofuel production.

  19. A Fluorescence-Based High-Throughput Coupled Enzymatic Assay for Quantitation of Isoaspartate in Proteins and Peptides.

    Science.gov (United States)

    Puri, Aastha; Quan, Yong; Narang, Ajit S; Adams, Monica; Gandhi, Rajesh; Nashine, Vishal C

    2017-04-01

    Formation of isoaspartate (IsoAsp) from spontaneous asparagine (Asn) deamidation or aspartate (Asp) isomerization is one of the most common non-enzymatic pathways of chemical degradation of protein and peptide pharmaceuticals. Rapid quantitation of IsoAsp formation can enable rank-ordering of potential drug candidates, mutants, and formulations as well as support shelf life prediction and stability requirements. A coupled enzymatic fluorescence-based IsoAsp assay (CEFIA) was developed as a high-throughput method for quantitation of IsoAsp in peptides and proteins. In this note, application of this method to two therapeutic candidate proteins with distinct structural scaffolds is described. In addition, the results obtained with this method are compared to those from conventional assays.

  20. A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae.

    Science.gov (United States)

    Chen, Wei; Zhang, Chengwu; Song, Lirong; Sommerfeld, Milton; Hu, Qiang

    2009-04-01

    Isolation of high neutral lipid-containing microalgae is key to the commercial success of microalgae-based biofuel production. The Nile red fluorescence method has been successfully applied to the determination of lipids in certain microalgae, but has been unsuccessful in many others, particularly those with thick, rigid cell walls that prevent the penetration of the fluorescence dye. The conventional "one sample at a time" method was also time-consuming. In this study, the solvent dimethyl sulfoxide (DMSO) was introduced to microalgal samples as the stain carrier at an elevated temperature. The cellular neutral lipids were determined and quantified using a 96-well plate on a fluorescence spectrophotometer with an excitation wavelength of 530 nm and an emission wavelength of 575 nm. An optimized procedure yielded a high correlation coefficient (R(2)=0.998) with the lipid standard triolein and repeated measurements of replicates. Application of the improved method to several green algal strains gave very reproducible results with relative standard errors of 8.5%, 3.9% and 8.6%, 4.5% for repeatability and reproducibility at two concentration levels (2.0 microg/mL and 20 microg/mL), respectively. Moreover, the detection and quantification limits of the improved Nile red staining method were 0.8 microg/mL and 2.0 microg/mL for the neutral lipid standard triolein, respectively. The modified method and a conventional gravimetric determination method provided similar results on replicate samples. The 96-well plate-based Nile red method can be used as a high throughput technique for rapid screening of a broader spectrum of naturally-occurring and genetically-modified algal strains and mutants for high neutral lipid/oil production.

  1. Optimizing experimental procedures for quantitative evaluation of crop plant performance in high throughput phenotyping systems

    Directory of Open Access Journals (Sweden)

    Astrid eJunker

    2015-01-01

    Full Text Available Detailed and standardized protocols for plant cultivation in environmentally controlled conditions are an essential prerequisite to conduct reproducible experiments with precisely defined treatments. Setting up appropriate and well defined experimental procedures is thus crucial for the generation of solid evidence and is thus indispensable for successful plant research. Non-invasive and high throughput (HT phenotyping technologies offer the opportunity to monitor and quantify performance dynamics of several hundreds of plants at a time. Compared to small scale plant cultivations, HT systems have much higher demands, from a conceptual and a logistic point of view, on experimental design, as well as the actual plant cultivation conditions, and the image analysis and statistical methods for data evaluation. Furthermore, cultivation conditions need to be designed that elicit plant performance characteristics corresponding to those under natural conditions. This manuscript describes critical steps in the optimization of procedures for HT plant phenotyping systems. Starting with the model plant Arabidopsis, HT-compatible methods were tested, and optimized with regard to growth substrate, soil coverage, watering regime, experimental design (considering environmental inhomogeneities in automated plant cultivation and imaging systems. As revealed by metabolite profiling, plant movement did not affect the plants’ physiological status. Based on these results, procedures for maize HT cultivation and monitoring were established. Variation of maize vegetative growth in the HT phenotyping system did match well with that observed in the field. The presented results outline important issues to be considered in the design of HT phenotyping experiments for model and crop plants. It thereby provides guidelines for the setup of HT experimental procedures, which are required for the generation of reliable and reproducible data of phenotypic variation for a broad

  2. Optimizing experimental procedures for quantitative evaluation of crop plant performance in high throughput phenotyping systems.

    Science.gov (United States)

    Junker, Astrid; Muraya, Moses M; Weigelt-Fischer, Kathleen; Arana-Ceballos, Fernando; Klukas, Christian; Melchinger, Albrecht E; Meyer, Rhonda C; Riewe, David; Altmann, Thomas

    2014-01-01

    Detailed and standardized protocols for plant cultivation in environmentally controlled conditions are an essential prerequisite to conduct reproducible experiments with precisely defined treatments. Setting up appropriate and well defined experimental procedures is thus crucial for the generation of solid evidence and indispensable for successful plant research. Non-invasive and high throughput (HT) phenotyping technologies offer the opportunity to monitor and quantify performance dynamics of several hundreds of plants at a time. Compared to small scale plant cultivations, HT systems have much higher demands, from a conceptual and a logistic point of view, on experimental design, as well as the actual plant cultivation conditions, and the image analysis and statistical methods for data evaluation. Furthermore, cultivation conditions need to be designed that elicit plant performance characteristics corresponding to those under natural conditions. This manuscript describes critical steps in the optimization of procedures for HT plant phenotyping systems. Starting with the model plant Arabidopsis, HT-compatible methods were tested, and optimized with regard to growth substrate, soil coverage, watering regime, experimental design (considering environmental inhomogeneities) in automated plant cultivation and imaging systems. As revealed by metabolite profiling, plant movement did not affect the plants' physiological status. Based on these results, procedures for maize HT cultivation and monitoring were established. Variation of maize vegetative growth in the HT phenotyping system did match well with that observed in the field. The presented results outline important issues to be considered in the design of HT phenotyping experiments for model and crop plants. It thereby provides guidelines for the setup of HT experimental procedures, which are required for the generation of reliable and reproducible data of phenotypic variation for a broad range of applications.

  3. Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis

    Directory of Open Access Journals (Sweden)

    Ludovico eSilvestri

    2015-05-01

    Full Text Available Characterizing the cytoarchitecture of mammalian central nervous system on a brain-wide scale is becoming a compelling need in neuroscience. For example, realistic modeling of brain activity requires the definition of quantitative features of large neuronal populations in the whole brain. Quantitative anatomical maps will also be crucial to classify the cytoarchtitectonic abnormalities associated with neuronal pathologies in a high reproducible and reliable manner. In this paper, we apply recent advances in optical microscopy and image analysis to characterize the spatial distribution of Purkinje cells across the whole cerebellum. Light sheet microscopy was used to image with micron-scale resolution a fixed and cleared cerebellum of an L7-GFP transgenic mouse, in which all Purkinje cells are fluorescently labeled. A fast and scalable algorithm for fully automated cell identification was applied on the image to extract the position of all the fluorescent Purkinje cells. This vectorized representation of the cell population allows a thorough characterization of the complex three-dimensional distribution of the neurons, highlighting the presence of gaps inside the lamellar organization of Purkinje cells, whose density is believed to play a significant role in autism spectrum disorders. Furthermore, clustering analysis of the localized somata permits dividing the whole cerebellum in groups of Purkinje cells with high spatial correlation, suggesting new possibilities of anatomical partition. The quantitative approach presented here can be extended to study the distribution of different types of cell in many brain regions and across the whole encephalon, providing a robust base for building realistic computational models of the brain, and for unbiased morphological tissue screening in presence of pathologies and/or drug treatments.

  4. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. High-Throughput Quantitation of Proline Betaine in Foods and Suitability as a Valid Biomarker for Citrus Consumption.

    Science.gov (United States)

    Lang, Roman; Lang, Tatjana; Bader, Matthias; Beusch, Anja; Schlagbauer, Verena; Hofmann, Thomas

    2017-03-01

    Proline betaine has been proposed as a candidate dietary biomarker for citrus intake. To validate its suitability as a dietary biomarker and to gain insight into the range of this per-methylated amino acid in foods and beverages, a quick and accurate stable isotope dilution assay was developed for quantitative high-throughput HILIC-MS/MS screening of proline betaine in foods and urine after solvent-mediated matrix precipitation. Quantitative analysis of a variety of foods confirmed substantial amounts of proline betaine in citrus juices (140-1100 mg/L) and revealed high abundance in tubers of the vegetable Stachys affinis, also known as Chinese artichocke (∼700 mg/kg). Seafood including clams, shrimp, and lobster contained limited amounts (1-95 mg/kg), whereas only traces were detected in fish, cuttlefish, fresh meat, dairy products, fresh vegetable (citrus products or the exclusion of citrus products in the wash-out phase of an intervention study. Moreover, proline betaine measurement can contribute to the establishment of a toolbox of valid dietary biomarkers reflecting wider aspects of diet to assess metabolic profiles as measures of dietary exposure and indicators of dietary patterns, dietary changes, or effectiveness of dietary interventions.

  6. Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell–derived medium spiny neurons

    Directory of Open Access Journals (Sweden)

    Marco Straccia

    2015-01-01

    Full Text Available A systematic characterization of the spatio-temporal gene expression during human neurodevelopment is essential to understand brain function in both physiological and pathological conditions. In recent years, stem cell technology has provided an in vitro tool to recapitulate human development, permitting also the generation of human models for many diseases. The correct differentiation of human pluripotent stem cell (hPSC into specific cell types should be evaluated by comparison with specific cells/tissue profiles from the equivalent adult in vivo organ. Here, we define by a quantitative high-throughput gene expression analysis the subset of specific genes of the whole ganglionic eminence (WGE and adult human striatum. Our results demonstrate that not only the number of specific genes is crucial but also their relative expression levels between brain areas. We next used these gene profiles to characterize the differentiation of hPSCs. Our findings demonstrate a temporal progression of gene expression during striatal differentiation of hPSCs from a WGE toward an adult striatum identity. Present results establish a gene expression profile to qualitatively and quantitatively evaluate the telencephalic hPSC-derived progenitors eventually used for transplantation and mature striatal neurons for disease modeling and drug-screening.

  7. Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods.

    Science.gov (United States)

    Piñol, J; Mir, G; Gomez-Polo, P; Agustí, N

    2015-07-01

    The quantification of the biological diversity in environmental samples using high-throughput DNA sequencing is hindered by the PCR bias caused by variable primer-template mismatches of the individual species. In some dietary studies, there is the added problem that samples are enriched with predator DNA, so often a predator-specific blocking oligonucleotide is used to alleviate the problem. However, specific blocking oligonucleotides could coblock nontarget species to some degree. Here, we accurately estimate the extent of the PCR biases induced by universal and blocking primers on a mock community prepared with DNA of twelve species of terrestrial arthropods. We also compare universal and blocking primer biases with those induced by variable annealing temperature and number of PCR cycles. The results show that reads of all species were recovered after PCR enrichment at our control conditions (no blocking oligonucleotide, 45 °C annealing temperature and 40 cycles) and high-throughput sequencing. They also show that the four factors considered biased the final proportions of the species to some degree. Among these factors, the number of primer-template mismatches of each species had a disproportionate effect (up to five orders of magnitude) on the amplification efficiency. In particular, the number of primer-template mismatches explained most of the variation (~3/4) in the amplification efficiency of the species. The effect of blocking oligonucleotide concentration on nontarget species relative abundance was also significant, but less important (below one order of magnitude). Considering the results reported here, the quantitative potential of the technique is limited, and only qualitative results (the species list) are reliable, at least when targeting the barcoding COI region. © 2014 John Wiley & Sons Ltd.

  8. Development and validation of a quantitative, high-throughput, fluorescent-based bioassay to detect schistosoma viability.

    Directory of Open Access Journals (Sweden)

    Emily Peak

    2010-07-01

    Full Text Available Schistosomiasis, caused by infection with the blood fluke Schistosoma, is responsible for greater than 200,000 human deaths per annum. Objective high-throughput screens for detecting novel anti-schistosomal targets will drive 'genome to drug' lead translational science at an unprecedented rate. Current methods for detecting schistosome viability rely on qualitative microscopic criteria, which require an understanding of parasite morphology, and most importantly, must be subjectively interpreted. These limitations, in the current state of the art, have significantly impeded progress into whole schistosome screening for next generation chemotherapies.We present here a microtiter plate-based method for reproducibly detecting schistosomula viability that takes advantage of the differential uptake of fluorophores (propidium iodide and fluorescein diacetate by living organisms. We validate this high-throughput system in detecting schistosomula viability using auranofin (a known inhibitor of thioredoxin glutathione reductase, praziquantel and a range of small compounds with previously-described (gambogic acid, sodium salinomycin, ethinyl estradiol, fluoxetidine hydrochloride, miconazole nitrate, chlorpromazine hydrochloride, amphotericin b, niclosamide or suggested (bepridil, ciclopirox, rescinnamine, flucytosine, vinblastine and carbidopa anti-schistosomal activities. This developed method is sensitive (200 schistosomula/well can be assayed, relevant to industrial (384-well microtiter plate compatibility and academic (96-well microtiter plate compatibility settings, translatable to functional genomics screens and drug assays, does not require a priori knowledge of schistosome biology and is quantitative.The wide-scale application of this fluorescence-based bioassay will greatly accelerate the objective identification of novel therapeutic lead targets/compounds to combat schistosomiasis. Adapting this bioassay for use with other parasitic worm species

  9. Quantitative insertion-site sequencing (QIseq) for high throughput phenotyping of transposon mutants.

    Science.gov (United States)

    Bronner, Iraad F; Otto, Thomas D; Zhang, Min; Udenze, Kenneth; Wang, Chengqi; Quail, Michael A; Jiang, Rays H Y; Adams, John H; Rayner, Julian C

    2016-07-01

    Genetic screening using random transposon insertions has been a powerful tool for uncovering biology in prokaryotes, where whole-genome saturating screens have been performed in multiple organisms. In eukaryotes, such screens have proven more problematic, in part because of the lack of a sensitive and robust system for identifying transposon insertion sites. We here describe quantitative insertion-site sequencing, or QIseq, which uses custom library preparation and Illumina sequencing technology and is able to identify insertion sites from both the 5' and 3' ends of the transposon, providing an inbuilt level of validation. The approach was developed using piggyBac mutants in the human malaria parasite Plasmodium falciparum but should be applicable to many other eukaryotic genomes. QIseq proved accurate, confirming known sites in >100 mutants, and sensitive, identifying and monitoring sites over a >10,000-fold dynamic range of sequence counts. Applying QIseq to uncloned parasites shortly after transfections revealed multiple insertions in mixed populations and suggests that >4000 independent mutants could be generated from relatively modest scales of transfection, providing a clear pathway to genome-scale screens in P. falciparum QIseq was also used to monitor the growth of pools of previously cloned mutants and reproducibly differentiated between deleterious and neutral mutations in competitive growth. Among the mutants with fitness defects was a mutant with a piggyBac insertion immediately upstream of the kelch protein K13 gene associated with artemisinin resistance, implying mutants in this gene may have competitive fitness costs. QIseq has the potential to enable the scale-up of piggyBac-mediated genetics across multiple eukaryotic systems. © 2016 Bronner et al.; Published by Cold Spring Harbor Laboratory Press.

  10. High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping

    Directory of Open Access Journals (Sweden)

    Esteras Cristina

    2012-02-01

    Full Text Available Abstract Background Cucurbita pepo is a member of the Cucurbitaceae family, the second- most important horticultural family in terms of economic importance after Solanaceae. The "summer squash" types, including Zucchini and Scallop, rank among the highest-valued vegetables worldwide. There are few genomic tools available for this species. The first Cucurbita transcriptome, along with a large collection of Single Nucleotide Polymorphisms (SNP, was recently generated using massive sequencing. A set of 384 SNP was selected to generate an Illumina GoldenGate assay in order to construct the first SNP-based genetic map of Cucurbita and map quantitative trait loci (QTL. Results We herein present the construction of the first SNP-based genetic map of Cucurbita pepo using a population derived from the cross of two varieties with contrasting phenotypes, representing the main cultivar groups of the species' two subspecies: Zucchini (subsp. pepo × Scallop (subsp. ovifera. The mapping population was genotyped with 384 SNP, a set of selected EST-SNP identified in silico after massive sequencing of the transcriptomes of both parents, using the Illumina GoldenGate platform. The global success rate of the assay was higher than 85%. In total, 304 SNP were mapped, along with 11 SSR from a previous map, giving a map density of 5.56 cM/marker. This map was used to infer syntenic relationships between C. pepo and cucumber and to successfully map QTL that control plant, flowering and fruit traits that are of benefit to squash breeding. The QTL effects were validated in backcross populations. Conclusion Our results show that massive sequencing in different genotypes is an excellent tool for SNP discovery, and that the Illumina GoldenGate platform can be successfully applied to constructing genetic maps and performing QTL analysis in Cucurbita. This is the first SNP-based genetic map in the Cucurbita genus and is an invaluable new tool for biological research

  11. High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping.

    Science.gov (United States)

    Esteras, Cristina; Gómez, Pedro; Monforte, Antonio J; Blanca, José; Vicente-Dólera, Nelly; Roig, Cristina; Nuez, Fernando; Picó, Belén

    2012-02-22

    Cucurbita pepo is a member of the Cucurbitaceae family, the second- most important horticultural family in terms of economic importance after Solanaceae. The "summer squash" types, including Zucchini and Scallop, rank among the highest-valued vegetables worldwide. There are few genomic tools available for this species.The first Cucurbita transcriptome, along with a large collection of Single Nucleotide Polymorphisms (SNP), was recently generated using massive sequencing. A set of 384 SNP was selected to generate an Illumina GoldenGate assay in order to construct the first SNP-based genetic map of Cucurbita and map quantitative trait loci (QTL). We herein present the construction of the first SNP-based genetic map of Cucurbita pepo using a population derived from the cross of two varieties with contrasting phenotypes, representing the main cultivar groups of the species' two subspecies: Zucchini (subsp. pepo) × Scallop (subsp. ovifera). The mapping population was genotyped with 384 SNP, a set of selected EST-SNP identified in silico after massive sequencing of the transcriptomes of both parents, using the Illumina GoldenGate platform. The global success rate of the assay was higher than 85%. In total, 304 SNP were mapped, along with 11 SSR from a previous map, giving a map density of 5.56 cM/marker. This map was used to infer syntenic relationships between C. pepo and cucumber and to successfully map QTL that control plant, flowering and fruit traits that are of benefit to squash breeding. The QTL effects were validated in backcross populations. Our results show that massive sequencing in different genotypes is an excellent tool for SNP discovery, and that the Illumina GoldenGate platform can be successfully applied to constructing genetic maps and performing QTL analysis in Cucurbita. This is the first SNP-based genetic map in the Cucurbita genus and is an invaluable new tool for biological research, especially considering that most of these markers are located in

  12. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries.

    Science.gov (United States)

    Inglese, James; Auld, Douglas S; Jadhav, Ajit; Johnson, Ronald L; Simeonov, Anton; Yasgar, Adam; Zheng, Wei; Austin, Christopher P

    2006-08-01

    High-throughput screening (HTS) of chemical compounds to identify modulators of molecular targets is a mainstay of pharmaceutical development. Increasingly, HTS is being used to identify chemical probes of gene, pathway, and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal will require methodologies that efficiently generate pharmacological data from the primary screen and reliably profile the range of biological activities associated with large chemical libraries. Traditional HTS, which tests compounds at a single concentration, is not suited to this task, because HTS is burdened by frequent false positives and false negatives and requires extensive follow-up testing. We have developed a paradigm, quantitative HTS (qHTS), tested with the enzyme pyruvate kinase, to generate concentration-response curves for >60,000 compounds in a single experiment. We show that this method is precise, refractory to variations in sample preparation, and identifies compounds with a wide range of activities. Concentration-response curves were classified to rapidly identify pyruvate kinase activators and inhibitors with a variety of potencies and efficacies and elucidate structure-activity relationships directly from the primary screen. Comparison of qHTS with traditional single-concentration HTS revealed a high prevalence of false negatives in the single-point screen. This study demonstrates the feasibility of qHTS for accurately profiling every compound in large chemical libraries (>10(5) compounds). qHTS produces rich data sets that can be immediately mined for reliable biological activities, thereby providing a platform for chemical genomics and accelerating the identification of leads for drug discovery.

  13. High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli.

    Science.gov (United States)

    Saez, Natalie J; Nozach, Hervé; Blemont, Marilyne; Vincentelli, Renaud

    2014-07-30

    Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment. Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive

  14. A community resource for high-throughput quantitative RT-PCR analysis of transcription factor gene expression in Medicago truncatula

    Directory of Open Access Journals (Sweden)

    Redman Julia C

    2008-07-01

    Full Text Available Abstract Background Medicago truncatula is a model legume species that is currently the focus of an international genome sequencing effort. Although several different oligonucleotide and cDNA arrays have been produced for genome-wide transcript analysis of this species, intrinsic limitations in the sensitivity of hybridization-based technologies mean that transcripts of genes expressed at low-levels cannot be measured accurately with these tools. Amongst such genes are many encoding transcription factors (TFs, which are arguably the most important class of regulatory proteins. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR is the most sensitive method currently available for transcript quantification, and one that can be scaled up to analyze transcripts of thousands of genes in parallel. Thus, qRT-PCR is an ideal method to tackle the problem of TF transcript quantification in Medicago and other plants. Results We established a bioinformatics pipeline to identify putative TF genes in Medicago truncatula and to design gene-specific oligonucleotide primers for qRT-PCR analysis of TF transcripts. We validated the efficacy and gene-specificity of over 1000 TF primer pairs and utilized these to identify sets of organ-enhanced TF genes that may play important roles in organ development or differentiation in this species. This community resource will be developed further as more genome sequence becomes available, with the ultimate goal of producing validated, gene-specific primers for all Medicago TF genes. Conclusion High-throughput qRT-PCR using a 384-well plate format enables rapid, flexible, and sensitive quantification of all predicted Medicago transcription factor mRNAs. This resource has been utilized recently by several groups in Europe, Australia, and the USA, and we expect that it will become the 'gold-standard' for TF transcript profiling in Medicago truncatula.

  15. High-throughput quantitative analysis of domoic acid directly from mussel tissue using Laser Ablation Electrospray Ionization - tandem mass spectrometry.

    Science.gov (United States)

    Beach, Daniel G; Walsh, Callee M; McCarron, Pearse

    2014-12-15

    Eliminating sample extraction or liquid chromatography steps from methods for analysis of the neurotoxin Domoic Acid (DA) in shellfish could greatly increase throughput in food safety testing laboratories worldwide. To this end, we have investigated the use of Laser Ablation Electrospray Ionization (LAESI) with tandem mass spectrometry (MS/MS) detection for DA analysis directly from mussel tissue homogenates without sample extraction, cleanup or separation. DA could be selectively detected directly from mussel tissue homogenates using MS/MS in selected reaction monitoring scan mode. The quantitative capabilities of LAESI-MS/MS for DA analysis from mussel tissue were evaluated by analysis of four mussel tissue reference materials using matrix-matched calibration. Linear response was observed from 1 mg/kg to 40 mg/kg and the method limit of detection was 1 mg/kg. Results for DA analysis in tissue within the linear range were in good agreement with two established methods, LC-UV and LC-MS/MS (recoveries from 103 to 125%). Beyond the linear range, extraction and clean-up were required to achieve good quantitation. Most notable is the extremely rapid analysis time of about 10 s per sample by LAESI-MS/MS, which corresponds to a significant increase in sample throughput compared with existing methodology for routine DA analysis. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

  16. Quantitative digital image analysis of chromogenic assays for high throughput screening of alpha-amylase mutant libraries.

    Science.gov (United States)

    Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

    2009-08-01

    An image analysis-based method for high throughput screening of an alpha-amylase mutant library using chromogenic assays was developed. Assays were performed in microplates and high resolution images of the assay plates were read using the Virtual Microplate Reader (VMR) script to quantify the concentration of the chromogen. This method is fast and sensitive in quantifying 0.025-0.3 mg starch/ml as well as 0.05-0.75 mg glucose/ml. It was also an effective screening method for improved alpha-amylase activity with a coefficient of variance of 18%.

  17. High-throughput quantitative biochemical characterization of algal biomass by NIR spectroscopy; multiple linear regression and multivariate linear regression analysis.

    Science.gov (United States)

    Laurens, L M L; Wolfrum, E J

    2013-12-18

    One of the challenges associated with microalgal biomass characterization and the comparison of microalgal strains and conversion processes is the rapid determination of the composition of algae. We have developed and applied a high-throughput screening technology based on near-infrared (NIR) spectroscopy for the rapid and accurate determination of algal biomass composition. We show that NIR spectroscopy can accurately predict the full composition using multivariate linear regression analysis of varying lipid, protein, and carbohydrate content of algal biomass samples from three strains. We also demonstrate a high quality of predictions of an independent validation set. A high-throughput 96-well configuration for spectroscopy gives equally good prediction relative to a ring-cup configuration, and thus, spectra can be obtained from as little as 10-20 mg of material. We found that lipids exhibit a dominant, distinct, and unique fingerprint in the NIR spectrum that allows for the use of single and multiple linear regression of respective wavelengths for the prediction of the biomass lipid content. This is not the case for carbohydrate and protein content, and thus, the use of multivariate statistical modeling approaches remains necessary.

  18. Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum

    Science.gov (United States)

    Krastins, Bryan; Prakash, Amol; Sarracino, David A.; Nedelkov, Dobrin; Niederkofler, Eric E.; Kiernan, Urban A.; Nelson, Randall; Vogelsang, Maryann S.; Vadali, Gouri; Garces, Alejandra; Sutton, Jennifer N.; Peterman, Scott; Byram, Gregory; Darbouret, Bruno; Pérusse, Joëlle R.; Seidah, Nabil G.; Coulombe, Benoit; Gobom, Johan; Portelius, Erik; Pannee, Josef; Blennow, Kaj; Kulasingam, Vathany; Couchman, Lewis; Moniz, Caje; Lopez, Mary F.

    2013-01-01

    Objectives The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. Design and methods The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. Results In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67–0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. Conclusions We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications. PMID:23313081

  19. Quantitative Mass Spectrometry for Bacterial Protein Toxins — A Sensitive, Specific, High-Throughput Tool for Detection and Diagnosis

    Directory of Open Access Journals (Sweden)

    Suzanne Kalb

    2011-03-01

    Full Text Available Matrix-assisted laser-desorption time-of-flight (MALDI-TOF mass spectrometry (MS is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins. The two binary toxins produced by B. anthracis consist of protective antigen (PA which combines with lethal factor (LF and edema factor (EF, forming lethal toxin and edema toxin respectively. LF is a zinc-dependent endoprotease which hydrolyzes specific proteins involved in inflammation and immunity. EF is an adenylyl cyclase which converts ATP to cyclic-AMP. Toxin-specific enzyme activity for a strategically designed substrate, amplifies reaction products which are detected by MALDI-TOF-MS and LC-ESI-MS/MS. Pre-concentration/purification with toxin specific monoclonal antibodies provides additional specificity. These combined technologies have achieved high specificity, ultrasensitive detection and quantification of the anthrax toxins. We also describe potential applications to diseases of high public health impact, including Clostridium difficile glucosylating toxins and the Bordetella pertussis adenylyl cyclase.

  20. Analysis of nuclear organization with TANGO, software for high-throughput quantitative analysis of 3D fluorescence microscopy images.

    Science.gov (United States)

    Ollion, Jean; Cochennec, Julien; Loll, François; Escudé, Christophe; Boudier, Thomas

    2015-01-01

    The cell nucleus is a highly organized cellular organelle that contains the genome. An important step to understand the relationships between genome positioning and genome functions is to extract quantitative data from three-dimensional (3D) fluorescence imaging. However, such approaches are limited by the requirement for processing and analyzing large sets of images. Here we present a practical approach using TANGO (Tools for Analysis of Nuclear Genome Organization), an image analysis tool dedicated to the study of nuclear architecture. TANGO is a generic tool able to process large sets of images, allowing quantitative study of nuclear organization. In this chapter a practical description of the software is drawn in order to give an overview of its different concepts and functionalities. This description is illustrated with a precise example that can be performed step-by-step on experimental data provided on the website http://biophysique.mnhn.fr/tango/HomePage.

  1. High-throughput, label-free, single-cell, microalgal lipid screening by machine-learning-equipped optofluidic time-stretch quantitative phase microscopy.

    Science.gov (United States)

    Guo, Baoshan; Lei, Cheng; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Jiang, Yiyue; Tanaka, Yo; Ozeki, Yasuyuki; Goda, Keisuke

    2017-05-01

    The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO 2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single-cell resolution in a non-invasive and interference-free manner. Here high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch quantitative phase microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase maps of every single cell at a high throughput of 10,000 cells/s, enabling accurate cell classification without the need for fluorescent staining. Specifically, the dataset was used to characterize heterogeneous populations of E. gracilis cells under two different culture conditions (nitrogen-sufficient and nitrogen-deficient) and achieve the cell classification with an error rate of only 2.15%. The method holds promise as an effective analytical tool for microalgae-based biofuel production. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  2. A high-throughput qPCR system for simultaneous quantitative detection of dairy Lactococcus lactis and Leuconostoc bacteriophages

    DEFF Research Database (Denmark)

    Muhammed, Musemma Kedir; Krych, Lukasz; Nielsen, Dennis Sandris

    2017-01-01

    Simultaneous quantitative detection of Lactococcus (Lc.) lactis and Leuconostoc species bacteriophages (phages) has not been reported in dairies using undefined mixed-strain DL-starters, probably due to the lack of applicable methods. We optimized a high-throughput qPCR system that allows simulta...... vat system. High levels of 936 and P335 phages were detected in the mother culture and the bulk starter, but also in the whey samples. Low levels of phages were detected in the cheese milk samples....... 107 to 9 x 103 phage particles mL-1 without any additional up-concentrating steps. The amplification efficiencies of the corresponding assays were 100.1±2.6, 98.7±2.3, 101.0±2.3 and 96.2±6.2. The qPCR system was tested on samples obtained from a dairy plant that employed traditional mother-bulk-cheese...

  3. A method for quantitative analysis of standard and high-throughput qPCR expression data based on input sample quantity.

    Directory of Open Access Journals (Sweden)

    Mateusz G Adamski

    Full Text Available Over the past decade rapid advances have occurred in the understanding of RNA expression and its regulation. Quantitative polymerase chain reactions (qPCR have become the gold standard for quantifying gene expression. Microfluidic next generation, high throughput qPCR now permits the detection of transcript copy number in thousands of reactions simultaneously, dramatically increasing the sensitivity over standard qPCR. Here we present a gene expression analysis method applicable to both standard polymerase chain reactions (qPCR and high throughput qPCR. This technique is adjusted to the input sample quantity (e.g., the number of cells and is independent of control gene expression. It is efficiency-corrected and with the use of a universal reference sample (commercial complementary DNA (cDNA permits the normalization of results between different batches and between different instruments--regardless of potential differences in transcript amplification efficiency. Modifications of the input quantity method include (1 the achievement of absolute quantification and (2 a non-efficiency corrected analysis. When compared to other commonly used algorithms the input quantity method proved to be valid. This method is of particular value for clinical studies of whole blood and circulating leukocytes where cell counts are readily available.

  4. Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications

    OpenAIRE

    Rutledge, R. G.

    2004-01-01

    Quantitative real-time PCR has revolutionized many aspects of genetic research, biomedical diagnostics and pathogen detection. Nevertheless, the full potential of this technology has yet to be realized, primarily due to the limitations of the threshold-based methodologies that are currently used for quantitative analysis. Prone to errors caused by variations in reaction preparation and amplification conditions, these approaches necessitate construction of standard curves for each target seque...

  5. High Throughput Transcriptomics @ USEPA (Toxicology ...

    Science.gov (United States)

    The ideal chemical testing approach will provide complete coverage of all relevant toxicological responses. It should be sensitive and specific It should identify the mechanism/mode-of-action (with dose-dependence). It should identify responses relevant to the species of interest. Responses should ideally be translated into tissue-, organ-, and organism-level effects. It must be economical and scalable. Using a High Throughput Transcriptomics platform within US EPA provides broader coverage of biological activity space and toxicological MOAs and helps fill the toxicological data gap. Slide presentation at the 2016 ToxForum on using High Throughput Transcriptomics at US EPA for broader coverage biological activity space and toxicological MOAs.

  6. Bayesian estimation and use of high-throughput remote sensing indices for quantitative genetic analyses of leaf growth.

    Science.gov (United States)

    Baker, Robert L; Leong, Wen Fung; An, Nan; Brock, Marcus T; Rubin, Matthew J; Welch, Stephen; Weinig, Cynthia

    2017-10-20

    We develop Bayesian function-valued trait models that mathematically isolate genetic mechanisms underlying leaf growth trajectories by factoring out genotype-specific differences in photosynthesis. Remote sensing data can be used instead of leaf-level physiological measurements. Characterizing the genetic basis of traits that vary during ontogeny and affect plant performance is a major goal in evolutionary biology and agronomy. Describing genetic programs that specifically regulate morphological traits can be complicated by genotypic differences in physiological traits. We describe the growth trajectories of leaves using novel Bayesian function-valued trait (FVT) modeling approaches in Brassica rapa recombinant inbred lines raised in heterogeneous field settings. While frequentist approaches estimate parameter values by treating each experimental replicate discretely, Bayesian models can utilize information in the global dataset, potentially leading to more robust trait estimation. We illustrate this principle by estimating growth asymptotes in the face of missing data and comparing heritabilities of growth trajectory parameters estimated by Bayesian and frequentist approaches. Using pseudo-Bayes factors, we compare the performance of an initial Bayesian logistic growth model and a model that incorporates carbon assimilation (A max) as a cofactor, thus statistically accounting for genotypic differences in carbon resources. We further evaluate two remotely sensed spectroradiometric indices, photochemical reflectance (pri2) and MERIS Terrestrial Chlorophyll Index (mtci) as covariates in lieu of A max, because these two indices were genetically correlated with A max across years and treatments yet allow much higher throughput compared to direct leaf-level gas-exchange measurements. For leaf lengths in uncrowded settings, including A max improves model fit over the initial model. The mtci and pri2 indices also outperform direct A max measurements. Of particular

  7. High-throughput screening and quantitation of guanidino and ureido compounds using liquid chromatography-drift tube ion mobility spectrometry-mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Ruo-Jing [National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032 (China); Zhang, Fang, E-mail: fzhang@sioc.ac.cn [National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032 (China); Chen, Xiu-Ping; Qi, Wan-Shu [National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032 (China); Guan, Qing [Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center, Shanghai 200032 (China); Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032 (China); Sun, Tuan-Qi, E-mail: tuanqisun@163.com [Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center, Shanghai 200032 (China); Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032 (China); Guo, Yin-Long, E-mail: ylguo@sioc.ac.cn [National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032 (China)

    2017-04-08

    The present work focused on the high-throughput screening and quantitation of guanidino compounds (GCs) and ureido compounds (UCs) in human thyroid tissues. The strategy employed benzylic rearrangement stable isotope labeling (BRSIL) for the sample preparation and then detection using liquid chromatography-drift tube ion mobility spectrometry-quadrupole time of flight mass spectrometry (LC-DTIMS-QTOF MS). A short reversed-phase LC realized an on-line desalting and a measurement cycle of 5.0 min. DTIMS separation enhanced the better specificity and selectivity for the benzil labeled GCs and UCs. The elevated mass resolution of QTOF MS enabled measure of the characteristic ions at accurate mass in MS and tandem MS spectra. Collision cross section (CCS) from DTIMS and accurate mass from QTOF MS were used as two qualifiers for the profiling and identification of GCs and UCs. In addition, an integral abundance arising from 3-D ion features (retention time, drift time, m/z) was applied to quantify the GCs and UCs in human thyroid tissues. The quantitative validation indicated good linearity (coefficient values ≥ 0.9981), good precision (1.0%–12.3% for intra-day and 0.9%–7.8% for inter-day) and good accuracy (91%–109%). The results demonstrated that the developed BRSIL coupled with LC-DTIMS-QTOF MS can be a powerful analysis platform to investigate GCs and UCs in human thyroid tissues. - Highlights: • The separation power of DTIMS-MS enhanced peak capacity, spectral clarity, and specificity of benzil labeled GCs and UCs. • Short-column LC for on-line desalting increased the throughput with a measurement cycle of 5.0 min. • CCS and accurate mass as a pair of qualifiers were used for the profiling and identification of GCs and UCs. • An integral abundance arising from 3-D ion features (RT, DT, m/z) was used as a novel quantifier for quantitation. • The developed method was applied to screen and quantify the GCs and UCs in human thyroid tissues.

  8. Quantitation and Identification of Intact Major Milk Proteins for High-Throughput LC-ESI-Q-TOF MS Analyses.

    Science.gov (United States)

    Vincent, Delphine; Elkins, Aaron; Condina, Mark R; Ezernieks, Vilnis; Rochfort, Simone

    2016-01-01

    Cow's milk is an important source of proteins in human nutrition. On average, cow's milk contains 3.5% protein. The most abundant proteins in bovine milk are caseins and some of the whey proteins, namely beta-lactoglobulin, alpha-lactalbumin, and serum albumin. A number of allelic variants and post-translationally modified forms of these proteins have been identified. Their occurrence varies with breed, individuality, stage of lactation, and health and nutritional status of the animal. It is therefore essential to have reliable methods of detection and quantitation of these proteins. Traditionally, major milk proteins are quantified using liquid chromatography (LC) and ultra violet detection method. However, as these protein variants co-elute to some degree, another dimension of separation is beneficial to accurately measure their amounts. Mass spectrometry (MS) offers such a tool. In this study, we tested several RP-HPLC and MS parameters to optimise the analysis of intact bovine proteins from milk. From our tests, we developed an optimum method that includes a 20-28-40% phase B gradient with 0.02% TFA in both mobile phases, at 0.2 mL/min flow rate, using 75°C for the C8 column temperature, scanning every 3 sec over a 600-3000 m/z window. The optimisations were performed using external standards commercially purchased for which ionisation efficiency, linearity of calibration, LOD, LOQ, sensitivity, selectivity, precision, reproducibility, and mass accuracy were demonstrated. From the MS analysis, we can use extracted ion chromatograms (EICs) of specific ion series of known proteins and integrate peaks at defined retention time (RT) window for quantitation purposes. This optimum quantitative method was successfully applied to two bulk milk samples from different breeds, Holstein-Friesian and Jersey, to assess differences in protein variant levels.

  9. Quantitation and Identification of Intact Major Milk Proteins for High-Throughput LC-ESI-Q-TOF MS Analyses.

    Directory of Open Access Journals (Sweden)

    Delphine Vincent

    Full Text Available Cow's milk is an important source of proteins in human nutrition. On average, cow's milk contains 3.5% protein. The most abundant proteins in bovine milk are caseins and some of the whey proteins, namely beta-lactoglobulin, alpha-lactalbumin, and serum albumin. A number of allelic variants and post-translationally modified forms of these proteins have been identified. Their occurrence varies with breed, individuality, stage of lactation, and health and nutritional status of the animal. It is therefore essential to have reliable methods of detection and quantitation of these proteins. Traditionally, major milk proteins are quantified using liquid chromatography (LC and ultra violet detection method. However, as these protein variants co-elute to some degree, another dimension of separation is beneficial to accurately measure their amounts. Mass spectrometry (MS offers such a tool. In this study, we tested several RP-HPLC and MS parameters to optimise the analysis of intact bovine proteins from milk. From our tests, we developed an optimum method that includes a 20-28-40% phase B gradient with 0.02% TFA in both mobile phases, at 0.2 mL/min flow rate, using 75°C for the C8 column temperature, scanning every 3 sec over a 600-3000 m/z window. The optimisations were performed using external standards commercially purchased for which ionisation efficiency, linearity of calibration, LOD, LOQ, sensitivity, selectivity, precision, reproducibility, and mass accuracy were demonstrated. From the MS analysis, we can use extracted ion chromatograms (EICs of specific ion series of known proteins and integrate peaks at defined retention time (RT window for quantitation purposes. This optimum quantitative method was successfully applied to two bulk milk samples from different breeds, Holstein-Friesian and Jersey, to assess differences in protein variant levels.

  10. Quantitative high-throughput profiling of snake venom gland transcriptomes and proteomes (Ovophis okinavensis and Protobothrops flavoviridis)

    Science.gov (United States)

    2013-01-01

    Background Advances in DNA sequencing and proteomics have facilitated quantitative comparisons of snake venom composition. Most studies have employed one approach or the other. Here, both Illumina cDNA sequencing and LC/MS were used to compare the transcriptomes and proteomes of two pit vipers, Protobothrops flavoviridis and Ovophis okinavensis, which differ greatly in their biology. Results Sequencing of venom gland cDNA produced 104,830 transcripts. The Protobothrops transcriptome contained transcripts for 103 venom-related proteins, while the Ovophis transcriptome contained 95. In both, transcript abundances spanned six orders of magnitude. Mass spectrometry identified peptides from 100% of transcripts that occurred at higher than contaminant (e.g. human keratin) levels, including a number of proteins never before sequenced from snakes. These transcriptomes reveal fundamentally different envenomation strategies. Adult Protobothrops venom promotes hemorrhage, hypotension, incoagulable blood, and prey digestion, consistent with mammalian predation. Ovophis venom composition is less readily interpreted, owing to insufficient pharmacological data for venom serine and metalloproteases, which comprise more than 97.3% of Ovophis transcripts, but only 38.0% of Protobothrops transcripts. Ovophis venom apparently represents a hybrid strategy optimized for frogs and small mammals. Conclusions This study illustrates the power of cDNA sequencing combined with MS profiling. The former quantifies transcript composition, allowing detection of novel proteins, but cannot indicate which proteins are actually secreted, as does MS. We show, for the first time, that transcript and peptide abundances are correlated. This means that MS can be used for quantitative, non-invasive venom profiling, which will be beneficial for studies of endangered species. PMID:24224955

  11. Computer Vision for High-Throughput Quantitative Phenotyping: A Case Study of Grapevine Downy Mildew Sporulation and Leaf Trichomes.

    Science.gov (United States)

    Divilov, Konstantin; Wiesner-Hanks, Tyr; Barba, Paola; Cadle-Davidson, Lance; Reisch, Bruce I

    2017-09-28

    Quantitative phenotyping of downy mildew sporulation is frequently used in plant breeding and genetic studies, as well as in studies focused on pathogen biology such as chemical efficacy trials. In these scenarios, phenotyping a large number of genotypes or treatments can be advantageous but is often limited by time and cost. We present a novel computational pipeline dedicated to estimating the percent area of downy mildew sporulation from images of inoculated grapevine leaf discs in a manner that is time and cost efficient. The pipeline was tested on images from leaf disc assay experiments involving two F1 grapevine families, one that had glabrous leaves (Vitis rupestris B38 × 'Horizon' [RH]) and another that had leaf trichomes (Horizon × V. cinerea B9 [HC]). Correlations between computer vision and manual visual ratings reached 0.89 in the RH family and 0.43 in the HC family. Additionally, we were able to use the computer vision system prior to sporulation to measure the percent leaf trichome area. We estimate that an experienced rater scoring sporulation would spend at least 90% less time using the computer vision system compared with the manual visual method. This will allow more treatments to be phenotyped in order to better understand the genetic architecture of downy mildew resistance and of leaf trichome density. We anticipate that this computer vision system will find applications in other pathosystems or traits where responses can be imaged with sufficient contrast from the background.

  12. High-throughput quantitation of Fc-containing recombinant proteins in cell culture supernatant by fluorescence polarization spectroscopy.

    Science.gov (United States)

    Thompson, Ben; Clifford, Jerry; Jenns, Mike; Smith, Andrew; Field, Ray; Nayyar, Kalpana; James, David C

    2017-10-01

    Measurement of recombinant protein product titer critically underpins all biopharmaceutical manufacturing process development, as well as diverse research and discovery activity. Here, we describe a simple rapid (quantitation of recombinant immunoglobulin G and Fc-containing IgG derivatives in mammalian cell culture supernatant over a wide dynamic range of 2.5-80 mg/L, using microplate fluorescence polarization (FP) spectroscopy. The solution-phase FP assay is based on the detection of immunoglobulin Fc domain containing analyte binding to FITC-conjugated recombinant Protein G ligand to measure analyte concentration dependent changes in emitted FP. For ease of use and maximal shelf life, we showed that air-dried assay microplates containing pre-formulated ligand that is re-solubilized on addition of analyte containing solution did not affect assay performance, typically yielding an across plate coefficient of variation of Protein A HPLC and bio-interferometry) yielded a coefficient of determination >0.99 in each case. Copyright © 2017. Published by Elsevier Inc.

  13. Evaluation of Flow-Injection Tandem Mass Spectrometry for Rapid and High-Throughput Quantitative Determination of B-Vitamins in Nutritional Supplements

    Energy Technology Data Exchange (ETDEWEB)

    Bhandari, Deepak [ORNL; Van Berkel, Gary J [ORNL

    2012-01-01

    The use of flow-injection electrospray ionization tandem mass spectrometry for rapid and high-throughput mass spectral analysis of selected B-vitamins, viz. B1, B2, B3, B5, and B6, in nutritional formulations was demonstrated. A simple and rapid (~5 min) in-tube sample preparation was performed by adding extraction solvent to a powdered sample aliquot followed by agitation, centrifugation, and filtration to recover an extract for analysis. Automated flow injection introduced 1 L of the extracts directly into the mass spectrometer ion source without chromatographic separation. Sample-to-sample analysis time was 60 s representing significant improvement over conventional liquid chromatography approaches which typically require 25-45 min, and often require more significant sample preparation procedures. Quantitative capabilities of the flow-injection analysis were tested using the method of standard additions and NIST standard reference material (SRM 3280) multivitamin/multielement tablets. The quantity determined for each B-vitamin in SRM 3280 was within the statistical range provided for the respective certified values. The same sample preparation and analysis approach was also applied to two different commercial vitamin supplement tablets and proved to be successful in the quantification of the selected B-vitamins as evidenced by an agreement with the labels values and the results obtained using isotope dilution liquid chromatography/mass spectrometry.

  14. The Microbiome and Metabolites in Fermented Pu-erh Tea as Revealed by High-Throughput Sequencing and Quantitative Multiplex Metabolite Analysis.

    Directory of Open Access Journals (Sweden)

    Yongjie Zhang

    Full Text Available Pu-erh is a tea produced in Yunnan, China by microbial fermentation of fresh Camellia sinensis leaves by two processes, the traditional raw fermentation and the faster, ripened fermentation. We characterized fungal and bacterial communities in leaves and both Pu-erhs by high-throughput, rDNA-amplicon sequencing and we characterized the profile of bioactive extrolite mycotoxins in Pu-erh teas by quantitative liquid chromatography-tandem mass spectrometry. We identified 390 fungal and 629 bacterial OTUs from leaves and both Pu-erhs. Major findings are: 1 fungal diversity drops and bacterial diversity rises due to raw or ripened fermentation, 2 fungal and bacterial community composition changes significantly between fresh leaves and both raw and ripened Pu-erh, 3 aging causes significant changes in the microbial community of raw, but not ripened, Pu-erh, and, 4 ripened and well-aged raw Pu-erh have similar microbial communities that are distinct from those of young, raw Ph-erh tea. Twenty-five toxic metabolites, mainly of fungal origin, were detected, with patulin and asperglaucide dominating and at levels supporting the Chinese custom of discarding the first preparation of Pu-erh and using the wet tea to then brew a pot for consumption.

  15. Rapid and High-Throughput Detection and Quantitation of Radiation Biomarkers in Human and Nonhuman Primates by Differential Mobility Spectrometry-Mass Spectrometry.

    Science.gov (United States)

    Chen, Zhidan; Coy, Stephen L; Pannkuk, Evan L; Laiakis, Evagelia C; Hall, Adam B; Fornace, Albert J; Vouros, Paul

    2016-10-01

    Radiation exposure is an important public health issue due to a range of accidental and intentional threats. Prompt and effective large-scale screening and appropriate use of medical countermeasures (MCM) to mitigate radiation injury requires rapid methods for determining the radiation dose. In a number of studies, metabolomics has identified small-molecule biomarkers responding to the radiation dose. Differential mobility spectrometry-mass spectrometry (DMS-MS) has been used for similar compounds for high-throughput small-molecule detection and quantitation. In this study, we show that DMS-MS can detect and quantify two radiation biomarkers, trimethyl-L-lysine (TML) and hypoxanthine. Hypoxanthine is a human and nonhuman primate (NHP) radiation biomarker and metabolic intermediate, whereas TML is a radiation biomarker in humans but not in NHP, which is involved in carnitine synthesis. They have been analyzed by DMS-MS from urine samples after a simple strong cation exchange-solid phase extraction (SCX-SPE). The dramatic suppression of background and chemical noise provided by DMS-MS results in an approximately 10-fold reduction in time, including sample pretreatment time, compared with liquid chromatography-mass spectrometry (LC-MS). DMS-MS quantitation accuracy has been verified by validation testing for each biomarker. Human samples are not yet available, but for hypoxanthine, selected NHP urine samples (pre- and 7-d-post 10 Gy exposure) were analyzed, resulting in a mean change in concentration essentially identical to that obtained by LC-MS (fold-change 2.76 versus 2.59). These results confirm the potential of DMS-MS for field or clinical first-level rapid screening for radiation exposure. Graphical Abstract ᅟ.

  16. Field-Based High-Throughput Plant Phenotyping Reveals the Temporal Patterns of Quantitative Trait Loci Associated with Stress-Responsive Traits in Cotton.

    Science.gov (United States)

    Pauli, Duke; Andrade-Sanchez, Pedro; Carmo-Silva, A Elizabete; Gazave, Elodie; French, Andrew N; Heun, John; Hunsaker, Douglas J; Lipka, Alexander E; Setter, Tim L; Strand, Robert J; Thorp, Kelly R; Wang, Sam; White, Jeffrey W; Gore, Michael A

    2016-04-07

    The application of high-throughput plant phenotyping (HTPP) to continuously study plant populations under relevant growing conditions creates the possibility to more efficiently dissect the genetic basis of dynamic adaptive traits. Toward this end, we employed a field-based HTPP system that deployed sets of sensors to simultaneously measure canopy temperature, reflectance, and height on a cotton (Gossypium hirsutum L.) recombinant inbred line mapping population. The evaluation trials were conducted under well-watered and water-limited conditions in a replicated field experiment at a hot, arid location in central Arizona, with trait measurements taken at different times on multiple days across 2010-2012. Canopy temperature, normalized difference vegetation index (NDVI), height, and leaf area index (LAI) displayed moderate-to-high broad-sense heritabilities, as well as varied interactions among genotypes with water regime and time of day. Distinct temporal patterns of quantitative trait loci (QTL) expression were mostly observed for canopy temperature and NDVI, and varied across plant developmental stages. In addition, the strength of correlation between HTPP canopy traits and agronomic traits, such as lint yield, displayed a time-dependent relationship. We also found that the genomic position of some QTL controlling HTPP canopy traits were shared with those of QTL identified for agronomic and physiological traits. This work demonstrates the novel use of a field-based HTPP system to study the genetic basis of stress-adaptive traits in cotton, and these results have the potential to facilitate the development of stress-resilient cotton cultivars. Copyright © 2016 Pauli et al.

  17. Ultra-Sensitive, High-Resolution Liquid Chromatography Methods for the High-Throughput Quantitative Analysis of Bacterial Cell Wall Chemistry and Structure.

    Science.gov (United States)

    Alvarez, Laura; Hernandez, Sara B; de Pedro, Miguel A; Cava, Felipe

    2016-01-01

    High-performance liquid chromatography (HPLC) analysis has been critical for determining the structural and chemical complexity of the cell wall. However this method is very time consuming in terms of sample preparation and chromatographic separation. Here we describe (1) optimized methods for peptidoglycan isolation from both Gram-negative and Gram-positive bacteria that dramatically reduce the sample preparation time, and (2) the application of the fast and highly efficient ultra-performance liquid chromatography (UPLC) technology to muropeptide separation and quantification. The advances in both analytical instrumentation and stationary-phase chemistry have allowed for evolved protocols which cut run time from hours (2-3 h) to minutes (10-20 min), and sample demands by at least one order of magnitude. Furthermore, development of methods based on organic solvents permits in-line mass spectrometry (MS) of the UPLC-resolved muropeptides. Application of these technologies to high-throughput analysis will expedite the better understanding of the cell wall biology.

  18. Assessment of the DNA damaging potential of environmental chemicals using a quantitative high-throughput screening approach to measure p53 activation.

    Science.gov (United States)

    Witt, Kristine L; Hsieh, Jui-Hua; Smith-Roe, Stephanie L; Xia, Menghang; Huang, Ruili; Zhao, Jinghua; Auerbach, Scott S; Hur, Junguk; Tice, Raymond R

    2017-08-01

    Genotoxicity potential is a critical component of any comprehensive toxicological profile. Compounds that induce DNA or chromosomal damage often activate p53, a transcription factor essential to cell cycle regulation. Thus, within the US Tox21 Program, we screened a library of ∼10,000 (∼8,300 unique) environmental compounds and drugs for activation of the p53-signaling pathway using a quantitative high-throughput screening assay employing HCT-116 cells (p53(+/+) ) containing a stably integrated β-lactamase reporter gene under control of the p53 response element (p53RE). Cells were exposed (-S9) for 16 hr at 15 concentrations (generally 1.2 nM to 92 μM) three times, independently. Excluding compounds that failed analytical chemistry analysis or were suspected of inducing assay interference, 365 (4.7%) of 7,849 unique compounds were concluded to activate p53. As part of an in-depth characterization of our results, we first compared them with results from traditional in vitro genotoxicity assays (bacterial mutation, chromosomal aberration); ∼15% of known, direct-acting genotoxicants in our library activated the p53RE. Mining the Comparative Toxicogenomics Database revealed that these p53 actives were significantly associated with increased expression of p53 downstream genes involved in DNA damage responses. Furthermore, 53 chemical substructures associated with genotoxicity were enriched in certain classes of p53 actives, for example, anthracyclines (antineoplastics) and vinca alkaloids (tubulin disruptors). Interestingly, the tubulin disruptors manifested unusual nonmonotonic concentration response curves suggesting activity through a unique p53 regulatory mechanism. Through the analysis of our results, we aim to define a role for this assay as one component of a comprehensive toxicological characterization of large compound libraries. Environ. Mol. Mutagen. 58:494-507, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  19. High throughput protein production screening

    Science.gov (United States)

    Beernink, Peter T [Walnut Creek, CA; Coleman, Matthew A [Oakland, CA; Segelke, Brent W [San Ramon, CA

    2009-09-08

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

  20. A novel high-throughput method for supported liquid extraction of retinol and alpha-tocopherol from human serum and simultaneous quantitation by liquid chromatography tandem mass spectrometry.

    Science.gov (United States)

    Hinchliffe, Edward; Rudge, James; Reed, Paul

    2016-07-01

    Measurement of vitamin A (retinol) and E (alpha-tocopherol) in UK clinical laboratories is currently performed exclusively by high-performance liquid chromatography with ultraviolet detection. We investigated whether retinol and alpha-tocopherol could be measured simultaneously by liquid chromatography tandem mass spectrometry. Serum samples (100 μL) were extracted using Isolute + Supported Liquid Extraction plates. Chromatography was performed on a Phenomenex Kinetex Biphenyl 2.6 μm, 50 × 2.1 mm column, and liquid chromatography tandem mass spectrometry on a Waters Acquity TQD. Injection-to-injection time was 4.3 min. The assay was validated according to published guidelines. Patient samples were used to compare liquid chromatography tandem mass spectrometry and high-performance liquid chromatography with ultraviolet detection methods. For retinol and alpha-tocopherol, respectively, the assay was linear up to 6.0 and 80.0 μmol/L, and lower limit of quantification was 0.07 and 0.26 μmol/L. Intra and interassay imprecision were within desirable analytical specifications. Analysis of quality control material aligned to NIST SRM 968e, and relative spiked recovery from human serum, both yielded results within 15% of target values. Method comparison with high-performance liquid chromatography with ultraviolet detection methodology demonstrated a negative bias for retinol and alpha-tocopherol by the liquid chromatography tandem mass spectrometry method. Analysis of United Kingdom National External Quality Assurance Scheme samples yielded mean bias from the target value of +3.0% for retinol and -11.2% for alpha-tocopherol. We have developed a novel, high-throughput method for extraction of retinol and alpha-tocopherol from human serum followed by simultaneous quantitation by liquid chromatography tandem mass spectrometry. The method offers a rapid, sensitive, specific and cost-effective alternative to high-performance liquid chromatography with

  1. Investigation of DNA damage response and apoptotic gene methylation pattern in sporadic breast tumors using high throughput quantitative DNA methylation analysis technology

    Directory of Open Access Journals (Sweden)

    Prakash Neeraj

    2010-11-01

    Full Text Available Abstract Background- Sporadic breast cancer like many other cancers is proposed to be a manifestation of abnormal genetic and epigenetic changes. For the past decade our laboratory has identified genes involved in DNA damage response (DDR, apoptosis and immunesurvelliance pathways to influence sporadic breast cancer risk in north Indian population. Further to enhance our knowledge at the epigenetic level, we performed DNA methylation study involving 17 gene promoter regions belonging to DNA damage response (DDR and death receptor apoptotic pathway in 162 paired normal and cancerous breast tissues from 81 sporadic breast cancer patients, using a high throughput quantitative DNA methylation analysis technology. Results- The study identified five genes with statistically significant difference between normal and tumor tissues. Hypermethylation of DR5 (P = 0.001, DCR1 (P = 0.00001, DCR2 (P = 0.0000000005 and BRCA2 (P = 0.007 and hypomethylation of DR4 (P = 0.011 in sporadic breast tumor tissues suggested a weak/aberrant activation of the DDR/apoptotic pathway in breast tumorigenesis. Negative correlation was observed between methylation status and transcript expression levels for TRAIL, DR4, CASP8, ATM, CHEK2, BRCA1 and BRCA2 CpG sites. Categorization of the gene methylation with respect to the clinicopathological parameters showed an increase in aberrant methylation pattern in advanced tumors. These uncharacteristic methylation patterns corresponded with decreased death receptor apoptosis (P = 0.047 and DNA damage repair potential (P = 0.004 in advanced tumors. The observation of BRCA2 -26 G/A 5'UTR polymorphism concomitant with the presence of methylation in the promoter region was novel and emerged as a strong candidate for susceptibility to sporadic breast tumors. Conclusion- Our study indicates that methylation of DDR-apoptotic gene promoters in sporadic breast cancer is not a random phenomenon. Progressive epigenetic alterations in advancing

  2. 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).

  3. Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples.

    Science.gov (United States)

    Pulford, D J; Gias, E; Bueno, I M; McFadden, Amj

    2016-01-01

    To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis. Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay. The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R(2) = 0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6 × 10(4) and 3.3 × 10(6) genomes per

  4. Quantitative RT-PCR assay for high-throughput screening (HTS of drugs against the growth of Cryptosporidium parvum in vitro

    Directory of Open Access Journals (Sweden)

    Haili eZhang

    2015-09-01

    Full Text Available Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent. Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively. This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds.

  5. Modeling Steroidogenesis Disruption Using High-Throughput ...

    Science.gov (United States)

    Environmental chemicals can elicit endocrine disruption by altering steroid hormone biosynthesis and metabolism (steroidogenesis) causing adverse reproductive and developmental effects. Historically, a lack of assays resulted in few chemicals having been evaluated for effects on steroidogenesis. The steroidogenic pathway is a series of hydroxylation and dehydrogenation steps carried out by CYP450 and hydroxysteroid dehydrogenase enzymes, yet the only enzyme in the pathway for which a high-throughput screening (HTS) assay has been developed is aromatase (CYP19A1), responsible for the aromatization of androgens to estrogens. Recently, the ToxCast HTS program adapted the OECD validated H295R steroidogenesis assay using human adrenocortical carcinoma cells into a high-throughput model to quantitatively assess the concentration-dependent (0.003-100 µM) effects of chemicals on 10 steroid hormones including progestagens, androgens, estrogens and glucocorticoids. These results, in combination with two CYP19A1 inhibition assays, comprise a large dataset amenable to clustering approaches supporting the identification and characterization of putative mechanisms of action (pMOA) for steroidogenesis disruption. In total, 514 chemicals were tested in all CYP19A1 and steroidogenesis assays. 216 chemicals were identified as CYP19A1 inhibitors in at least one CYP19A1 assay. 208 of these chemicals also altered hormone levels in the H295R assay, suggesting 96% sensitivity in the

  6. High-throughput hyperdimensional vertebrate phenotyping.

    Science.gov (United States)

    Pardo-Martin, Carlos; Allalou, Amin; Medina, Jaime; Eimon, Peter M; Wählby, Carolina; Fatih Yanik, Mehmet

    2013-01-01

    Most gene mutations and biologically active molecules cause complex responses in animals that cannot be predicted by cell culture models. Yet animal studies remain too slow and their analyses are often limited to only a few readouts. Here we demonstrate high-throughput optical projection tomography with micrometre resolution and hyperdimensional screening of entire vertebrates in tens of seconds using a simple fluidic system. Hundreds of independent morphological features and complex phenotypes are automatically captured in three dimensions with unprecedented speed and detail in semitransparent zebrafish larvae. By clustering quantitative phenotypic signatures, we can detect and classify even subtle alterations in many biological processes simultaneously. We term our approach hyperdimensional in vivo phenotyping. To illustrate the power of hyperdimensional in vivo phenotyping, we have analysed the effects of several classes of teratogens on cartilage formation using 200 independent morphological measurements, and identified similarities and differences that correlate well with their known mechanisms of actions in mammals.

  7. Development of Transmission Raman Spectroscopy towards the in line, high throughput and non-destructive quantitative analysis of pharmaceutical solid oral dose.

    Science.gov (United States)

    Griffen, Julia A; Owen, Andrew W; Matousek, Pavel

    2015-01-07

    Transmission Raman spectroscopy (TRS) is a recently introduced analytical technique to pharmaceutical analysis permitting volumetric sampling by non-destructive means. Here we demonstrate experimentally, for the first time, the enhanced speed of quantification of pharmaceutical tablets by an order of magnitude compared with conventional TRS. This is achieved using an enhancing element, "photon diode", avoiding the loss of laser photons at laser coupling interface. The proof-of-concept experiments were performed on a complex mixture consisting of 5 components (3 APIs and 2 excipients) with nominal concentrations ranging between 0.4 and 89%. Acquisition times as short as 0.01 s were reached with satisfactory quantification accuracy for all the sample components. Results suggest that even faster sampling speeds would be achievable for components with stronger Raman scattering cross sections or with higher laser powers. This major improvement in speed of volumetric analysis enables high throughput deployment of TRS for in line quality control applications within the batch or continuous manufacturing process and facilitating non-destructive analysis of large fractions.

  8. High Throughput Architecture for High Performance NoC

    OpenAIRE

    Ghany, Mohamed A. Abd El; El-Moursy, Magdy A.; Ismail, Mohammed

    2010-01-01

    In this chapter, the high throughput NoC architecture is proposed to increase the throughput of the switch in NoC. The proposed architecture can also improve the latency of the network. The proposed high throughput interconnect architecture is applied on different NoC architectures. The architecture increases the throughput of the network by more than 38% while preserving the average latency. The area of high throughput NoC switch is decreased by 18% as compared to the area of BFT switch. The...

  9. High-throughput scoring of seed germination

    NARCIS (Netherlands)

    Ligterink, Wilco; Hilhorst, Henk W.M.

    2017-01-01

    High-throughput analysis of seed germination for phenotyping large genetic populations or mutant collections is very labor intensive and would highly benefit from an automated setup. Although very often used, the total germination percentage after a nominated period of time is not very

  10. Inflammatory breast cancer tumor emboli express high levels of anti-apoptotic proteins: use of a quantitative high content and high-throughput 3D IBC spheroid assay to identify targeting strategies

    Science.gov (United States)

    Tarpley, Michael; Vermeulen, Peter; Rypens, Charlotte; Van Laere, Steven; Williams, Kevin P.; Devi, Gayathri R.; Dewhirst, Mark W.

    2017-01-01

    Inflammatory breast cancer (IBC) is one of the most lethal breast cancer variants; with existing therapy, 5-yr survival rate is only 35%. Current barriers to successful treatment of IBC include frequent infiltration and the presence of tumor cell clusters, termed tumor emboli, within the breast parenchyma and lymphatics. Prior studies have identified the role of anti-apoptotic signaling, in particular hyperactivation of NFκB and its target genes, in IBC pathobiology and therapeutic resistance. The objectives of this study were to: (1) determine if IBC tumor emboli express anti-apoptotic proteins and (2) develop a high content, multiparametric assay to assess the morphology of the IBC 3D spheroids and to optimize a high throughput format to screen for compounds that can inhibit the formation of the IBC tumor clusters/embolic structures. Immunohistochemical analysis of IBC patient tumor samples with documented tumor emboli revealed high NFκB (p65) staining along with expression of XIAP, a potent anti-apoptotic protein known to interact with NFκB signaling in enhancing survival of malignant cells. Subsequently, the high content assay developed allowed for simultaneous imaging and morphometric analysis, including count and viability of spheroids derived from SUM149, rSUM149 and SUM190 cells and its application to evaluate XIAP and NFκB inhibitory agents. We demonstrate the efficacy of the off-patent drug disulfiram when chelated with copper, which we had previously reported to inhibit NFκB signaling, was highly effective in disrupting both IBC spheroids and emboli grown in vitro. Taken together, these results identify a high-throughput approach to target tumor spheroid formation for drug discovery. Finally, disulfiram is a safe and approved drug for management of alcohol abuse, warranting its evaluation for repurposing in IBC therapy. PMID:28460441

  11. Development and validation of a high throughput LC–MS/MS method for simultaneous quantitation of pioglitazone and telmisartan in rat plasma and its application to a pharmacokinetic study

    Directory of Open Access Journals (Sweden)

    Pinaki Sengupta

    2017-12-01

    Full Text Available Management of cardiovascular risk factors in diabetes demands special attention due to their co-existence. Pioglitazone (PIO and telmisartan (TLM combination can be beneficial in effective control of cardiovascular complication in diabetes. In this research, we developed and validated a high throughput LC–MS/MS method for simultaneous quantitation of PIO and TLM in rat plasma. This developed method is more sensitive and can quantitate the analytes in relatively shorter period of time compared to the previously reported methods for their individual quantification. Moreover, till date, there is no bioanalytical method available to simultaneously quantitate PIO and TLM in a single run. The method was validated according to the USFDA guidelines for bioanalytical method validation. A linear response of the analytes was observed over the range of 0.005–10 µg/mL with satisfactory precision and accuracy. Accuracy at four quality control levels was within 94.27%–106.10%. The intra- and inter-day precision ranged from 2.32%–10.14 and 5.02%–8.12%, respectively. The method was reproducible and sensitive enough to quantitate PIO and TLM in rat plasma samples of a preclinical pharmacokinetic study. Due to the potential of PIO-TLM combination to be therapeutically explored, this method is expected to have significant usefulness in future. Keywords: LC–MS/MS, Rat plasma, Pharmacokinetic applicability, Telmisartan, Pioglitazone, Pharmacokinetic application

  12. High-Throughput Toxicity Testing: New Strategies for ...

    Science.gov (United States)

    In recent years, the food industry has made progress in improving safety testing methods focused on microbial contaminants in order to promote food safety. However, food industry toxicologists must also assess the safety of food-relevant chemicals including pesticides, direct additives, and food contact substances. With the rapidly growing use of new food additives, as well as innovation in food contact substance development, an interest in exploring the use of high-throughput chemical safety testing approaches has emerged. Currently, the field of toxicology is undergoing a paradigm shift in how chemical hazards can be evaluated. Since there are tens of thousands of chemicals in use, many of which have little to no hazard information and there are limited resources (namely time and money) for testing these chemicals, it is necessary to prioritize which chemicals require further safety testing to better protect human health. Advances in biochemistry and computational toxicology have paved the way for animal-free (in vitro) high-throughput screening which can characterize chemical interactions with highly specific biological processes. Screening approaches are not novel; in fact, quantitative high-throughput screening (qHTS) methods that incorporate dose-response evaluation have been widely used in the pharmaceutical industry. For toxicological evaluation and prioritization, it is the throughput as well as the cost- and time-efficient nature of qHTS that makes it

  13. Economic consequences of high throughput maskless lithography

    Science.gov (United States)

    Hartley, John G.; Govindaraju, Lakshmi

    2005-11-01

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

  14. 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.

  15. A high throughput spectral image microscopy system

    Science.gov (United States)

    Gesley, M.; Puri, R.

    2018-01-01

    A high throughput spectral image microscopy system is configured for rapid detection of rare cells in large populations. To overcome flow cytometry rates and use of fluorophore tags, a system architecture integrates sample mechanical handling, signal processors, and optics in a non-confocal version of light absorption and scattering spectroscopic microscopy. Spectral images with native contrast do not require the use of exogeneous stain to render cells with submicron resolution. Structure may be characterized without restriction to cell clusters of differentiation.

  16. High Throughput PBTK: Open-Source Data and Tools for ...

    Science.gov (United States)

    Presentation on High Throughput PBTK at the PBK Modelling in Risk Assessment meeting in Ispra, Italy Presentation on High Throughput PBTK at the PBK Modelling in Risk Assessment meeting in Ispra, Italy

  17. Ultra-High-Throughput Analytical Strategy Based on UHPLC-DAD in Combination with Syringe Filtration for the Quantitation of Nine Synthetic Colorants in Beverages: Impacts of Syringe Membrane Types and Sample pH on Recovery.

    Science.gov (United States)

    Shin, Je Young; Jung, Mun Yhung

    2017-11-15

    An ultra-high-throughput approach based on ultra-high-performance liquid chromatography with diode array detection (UHPLC-DAD) in combination with simple syringe filtration was successfully developed and validated for the quantitation of nine synthetic colorants in beverages. The recoveries of the colorants from the beverages were found to be dramatically affected by the syringe filter membrane types and pH of the sample solution. The high recoveries of the nine colorants (92.7-105.9%) were achieved by syringe filtration with poly(vinylidene difluoride) membrane following the pH adjustment of sample solution at pH 7.0. The sample treatment procedure was very simple and took only 1 min. The fast chromatographic separation (1 min) of the nine synthetic colorants was achieved by UHPLC-DAD using a C18-core-shell column. This analytical approach (UHPLC-DAD combined with syringe filtration) took only approximately 3 min. The established method was ultrafast, sensitive, precise, accurate, and reliable. The method was successfully applied to rapidly determine the 9 colorants in 17 beverages.

  18. High-throughput and sensitive quantitation of plasma catecholamines by ultraperformance liquid chromatography-tandem mass spectrometry using a solid phase microwell extraction plate.

    Science.gov (United States)

    Dunand, Marielle; Gubian, Danilo; Stauffer, Maxime; Abid, Karim; Grouzmann, Eric

    2013-04-02

    Plasma catecholamines provide a reliable biomarker of sympathetic activity. The low circulating concentrations of catecholamines and analytical interferences require tedious sample preparation and long chromatographic runs to ensure their accurate quantification by HPLC with electrochemical detection. Published or commercially available methods relying on solid phase extraction technology lack sensitivity or require derivatization of catecholamine by hazardous reagents prior to tandem mass spectrometry (MS) analysis. Here, we manufactured a novel 96-well microplate device specifically designed to extract plasma catecholamines prior to their quantification by a new and highly sensitive ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Processing time, which included sample purification on activated aluminum oxide and elution, is less than 1 h per 96-well microplate. The UPLC-MS/MS analysis run time is 2.0 min per sample. This UPLC-MS/MS method does not require a derivatization step, reduces the turnaround time by 10-fold compared to conventional methods used for routine application, and allows catecholamine quantification in reduced plasma sample volumes (50-250 μL, e.g., from children and mice).

  19. 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).

  20. 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

  1. High throughput assays for analyzing transcription factors.

    Science.gov (United States)

    Li, Xianqiang; Jiang, Xin; Yaoi, Takuro

    2006-06-01

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

  2. High-Throughput Process Development for Biopharmaceuticals.

    Science.gov (United States)

    Shukla, Abhinav A; Rameez, Shahid; Wolfe, Leslie S; Oien, Nathan

    2017-11-14

    The ability to conduct multiple experiments in parallel significantly reduces the time that it takes to develop a manufacturing process for a biopharmaceutical. This is particularly significant before clinical entry, because process development and manufacturing are on the "critical path" for a drug candidate to enter clinical development. High-throughput process development (HTPD) methodologies can be similarly impactful during late-stage development, both for developing the final commercial process as well as for process characterization and scale-down validation activities that form a key component of the licensure filing package. This review examines the current state of the art for HTPD methodologies as they apply to cell culture, downstream purification, and analytical techniques. In addition, we provide a vision of how HTPD activities across all of these spaces can integrate to create a rapid process development engine that can accelerate biopharmaceutical drug development. Graphical Abstract.

  3. Applications of High Throughput Nucleotide Sequencing

    DEFF Research Database (Denmark)

    Waage, Johannes Eichler

    The recent advent of high throughput sequencing of nucleic acids (RNA and DNA) has vastly expanded research into the functional and structural biology of the genome of all living organisms (and even a few dead ones). With this enormous and exponential growth in biological data generation come...... equally large demands in data handling, analysis and interpretation, perhaps defining the modern challenge of the computational biologist of the post-genomic era. The first part of this thesis consists of a general introduction to the history, common terms and challenges of next generation sequencing......, focusing on oft encountered problems in data processing, such as quality assurance, mapping, normalization, visualization, and interpretation. Presented in the second part are scientific endeavors representing solutions to problems of two sub-genres of next generation sequencing. For the first flavor, RNA-sequencing...

  4. Applications of High Throughput Nucleotide Sequencing

    DEFF Research Database (Denmark)

    Waage, Johannes Eichler

    The recent advent of high throughput sequencing of nucleic acids (RNA and DNA) has vastly expanded research into the functional and structural biology of the genome of all living organisms (and even a few dead ones). With this enormous and exponential growth in biological data generation come...... equally large demands in data handling, analysis and interpretation, perhaps defining the modern challenge of the computational biologist of the post-genomic era. The first part of this thesis consists of a general introduction to the history, common terms and challenges of next generation sequencing......). For the second flavor, DNA-seq, a study presenting genome wide profiling of transcription factor CEBP/A in liver cells undergoing regeneration after partial hepatectomy (article IV) is included....

  5. High Throughput Spectroscopic Catalyst Screening via Surface Plasmon Spectroscopy

    Science.gov (United States)

    2015-07-15

    Final 3. DATES COVERED (From - To) 26-June-2014 to 25-March-2015 4. TITLE AND SUBTITLE High Throughput Catalyst Screening via Surface...TITLE AND SUBTITLE High Throughput Catalyst Screening via Surface Plasmon Spectroscopy 5a. CONTRACT NUMBER FA2386-14-1-4064 5b. GRANT NUMBER 5c...AOARD Grant 144064 FA2386-14-1-4064 “High Throughput Spectroscopic Catalyst Screening by Surface Plasmon Spectroscopy” Date July 15, 2015

  6. New Automated and High-Throughput Quantitative Analysis of Urinary Ketones by Multifiber Exchange-Solid Phase Microextraction Coupled to Fast Gas Chromatography/Negative Chemical-Electron Ionization/Mass Spectrometry

    Science.gov (United States)

    Pacenti, Marco; Dugheri, Stefano; Traldi, Pietro; Degli Esposti, Filippo; Perchiazzi, Nicola; Franchi, Elena; Calamante, Massimo; Kikic, Ireneo; Alessi, Paolo; Bonacchi, Alice; Salvadori, Edoardo; Arcangeli, Giulio; Cupelli, Vincenzo

    2010-01-01

    The present research is focused on automation, miniaturization, and system interaction with high throughput for multiple and specific Direct Immersion-Solid Phase Microextraction/Fast Gas Chromatography analysis of the urinary ketones. The specific Mass Spectrometry instrumentation, capable of supporting such the automated changeover from Negative Chemical to Electron Ionization mode, as well as the automation of the preparation procedure by new device called MultiFiber Exchange, through change of the fibers, allowed a friendly use of mass spectrometry apparatus with a number of advantages including reduced analyst time and greater reproducibility (2.01–5.32%). The detection limits for the seven ketones were less than 0.004 mg/L. For an innovative powerful meaning in high-throughput routine, the generality of the structurally informative Mass Spectrometry fragmentation patterns together with the chromatographic separation and software automation are also investigated. PMID:20628512

  7. Plant chip for high-throughput phenotyping of Arabidopsis.

    Science.gov (United States)

    Jiang, Huawei; Xu, Zhen; Aluru, Maneesha R; Dong, Liang

    2014-04-07

    We report on the development of a vertical and transparent microfluidic chip for high-throughput phenotyping of Arabidopsis thaliana plants. Multiple Arabidopsis seeds can be germinated and grown hydroponically over more than two weeks in the chip, thus enabling large-scale and quantitative monitoring of plant phenotypes. The novel vertical arrangement of this microfluidic device not only allows for normal gravitropic growth of the plants but also, more importantly, makes it convenient to continuously monitor phenotypic changes in plants at the whole organismal level, including seed germination and root and shoot growth (hypocotyls, cotyledons, and leaves), as well as at the cellular level. We also developed a hydrodynamic trapping method to automatically place single seeds into seed holding sites of the device and to avoid potential damage to seeds that might occur during manual loading. We demonstrated general utility of this microfluidic device by showing clear visible phenotypes of the immutans mutant of Arabidopsis, and we also showed changes occurring during plant-pathogen interactions at different developmental stages. Arabidopsis plants grown in the device maintained normal morphological and physiological behaviour, and distinct phenotypic variations consistent with a priori data were observed via high-resolution images taken in real time. Moreover, the timeline for different developmental stages for plants grown in this device was highly comparable to growth using a conventional agar plate method. This prototype plant chip technology is expected to lead to the establishment of a powerful experimental and cost-effective framework for high-throughput and precise plant phenotyping.

  8. 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...

  9. High-throughput ballistic injection nanorheology to measure cell mechanics

    Science.gov (United States)

    Wu, Pei-Hsun; Hale, Christopher M; Chen, Wei-Chiang; Lee, Jerry S H; Tseng, Yiider; Wirtz, Denis

    2015-01-01

    High-throughput ballistic injection nanorheology is a method for the quantitative study of cell mechanics. Cell mechanics are measured by ballistic injection of submicron particles into the cytoplasm of living cells and tracking the spontaneous displacement of the particles at high spatial resolution. The trajectories of the cytoplasm-embedded particles are transformed into mean-squared displacements, which are subsequently transformed into frequency-dependent viscoelastic moduli and time-dependent creep compliance of the cytoplasm. This method allows for the study of a wide range of cellular conditions, including cells inside a 3D matrix, cell subjected to shear flows and biochemical stimuli, and cells in a live animal. Ballistic injection lasts < 1 min and is followed by overnight incubation. Multiple particle tracking for one cell lasts < 1 min. Forty cells can be examined in < 1 h. PMID:22222790

  10. High Throughput Determinations of Critical Dosing Parameters (IVIVE workshop)

    Science.gov (United States)

    High throughput toxicokinetics (HTTK) is an approach that allows for rapid estimations of TK for hundreds of environmental chemicals. HTTK-based reverse dosimetry (i.e, reverse toxicokinetics or RTK) is used in order to convert high throughput in vitro toxicity screening (HTS) da...

  11. 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...

  12. 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.

  13. High-throughput crystallography for structural genomics.

    Science.gov (United States)

    Joachimiak, Andrzej

    2009-10-01

    Protein X-ray crystallography recently celebrated its 50th anniversary. The structures of myoglobin and hemoglobin determined by Kendrew and Perutz provided the first glimpses into the complex protein architecture and chemistry. Since then, the field of structural molecular biology has experienced extraordinary progress and now more than 55000 protein structures have been deposited into the Protein Data Bank. In the past decade many advances in macromolecular crystallography have been driven by world-wide structural genomics efforts. This was made possible because of third-generation synchrotron sources, structure phasing approaches using anomalous signal, and cryo-crystallography. Complementary progress in molecular biology, proteomics, hardware and software for crystallographic data collection, structure determination and refinement, computer science, databases, robotics and automation improved and accelerated many processes. These advancements provide the robust foundation for structural molecular biology and assure strong contribution to science in the future. In this report we focus mainly on reviewing structural genomics high-throughput X-ray crystallography technologies and their impact.

  14. High-throughput Crystallography for Structural Genomics

    Science.gov (United States)

    Joachimiak, Andrzej

    2009-01-01

    Protein X-ray crystallography recently celebrated its 50th anniversary. The structures of myoglobin and hemoglobin determined by Kendrew and Perutz provided the first glimpses into the complex protein architecture and chemistry. Since then, the field of structural molecular biology has experienced extraordinary progress and now over 53,000 proteins structures have been deposited into the Protein Data Bank. In the past decade many advances in macromolecular crystallography have been driven by world-wide structural genomics efforts. This was made possible because of third-generation synchrotron sources, structure phasing approaches using anomalous signal and cryo-crystallography. Complementary progress in molecular biology, proteomics, hardware and software for crystallographic data collection, structure determination and refinement, computer science, databases, robotics and automation improved and accelerated many processes. These advancements provide the robust foundation for structural molecular biology and assure strong contribution to science in the future. In this report we focus mainly on reviewing structural genomics high-throughput X-ray crystallography technologies and their impact. PMID:19765976

  15. Using high-throughput DNA sequencing, genetic fingerprinting, and quantitative PCR as tools for monitoring bloom-forming and toxigenic cyanobacteria in Upper Klamath Lake, Oregon, 2013 and 2014

    Science.gov (United States)

    Caldwell Eldridge, Sara L.; Driscoll, Conner; Dreher, Theo W.

    2017-06-05

    Monitoring the community structure and metabolic activities of cyanobacterial blooms in Upper Klamath Lake, Oregon, is critical to lake management because these blooms degrade water quality and produce toxic microcystins that are harmful to humans, domestic animals, and wildlife. Genetic tools, such as DNA fingerprinting by terminal restriction fragment length polymorphism (T-RFLP) analysis, high-throughput DNA sequencing (HTS), and real-time, quantitative polymerase chain reaction (qPCR), provide more sensitive and rapid assessments of bloom ecology than traditional techniques. The objectives of this study were (1) to characterize the microbial community at one site in Upper Klamath Lake and determine changes in the cyanobacterial community through time using T-RFLP and HTS in comparison with traditional light microscopy; (2) to determine relative abundances and changes in abundance over time of toxigenic Microcystis using qPCR; and (3) to determine relative abundances and changes in abundance over time of Aphanizomenon, Microcystis, and total cyanobacteria using qPCR. T-RFLP analysis of total cyanobacteria showed a dominance of only one or two distinct genotypes in samples from 2013, but results of HTS in 2013 and 2014 showed more variations in the bloom cycle that fit with the previous understanding of bloom dynamics in Upper Klamath Lake and indicated that potentially toxigenic Microcystis was more prevalent in 2014 than in years prior. The qPCR-estimated copy numbers of all target genes were higher in 2014 than in 2013, when microcystin concentrations also were higher. Total Microcystis density was shown with qPCR to be a better predictor of late-season increases in microcystin concentrations than the relative proportions of potentially toxigenic cells. In addition, qPCR targeting Aphanizomenon at one site in Upper Klamath Lake indicated a moderate bloom of this species (corresponding to chlorophyll a concentrations between approximately 75 and 200 micrograms

  16. High-throughput technology for novel SO2 oxidation catalysts

    Directory of Open Access Journals (Sweden)

    Jonas Loskyll, Klaus Stoewe and Wilhelm F Maier

    2011-01-01

    Full Text Available We review the state of the art and explain the need for better SO2 oxidation catalysts for the production of sulfuric acid. A high-throughput technology has been developed for the study of potential catalysts in the oxidation of SO2 to SO3. High-throughput methods are reviewed and the problems encountered with their adaptation to the corrosive conditions of SO2 oxidation are described. We show that while emissivity-corrected infrared thermography (ecIRT can be used for primary screening, it is prone to errors because of the large variations in the emissivity of the catalyst surface. UV-visible (UV-Vis spectrometry was selected instead as a reliable analysis method of monitoring the SO2 conversion. Installing plain sugar absorbents at reactor outlets proved valuable for the detection and quantitative removal of SO3 from the product gas before the UV-Vis analysis. We also overview some elements used for prescreening and those remaining after the screening of the first catalyst generations.

  17. Nanoliter high-throughput PCR for DNA and RNA profiling.

    Science.gov (United States)

    Brenan, Colin J H; Roberts, Douglas; Hurley, James

    2009-01-01

    The increasing emphasis in life science research on utilization of genetic and genomic information underlies the need for high-throughput technologies capable of analyzing the expression of multiple genes or the presence of informative single nucleotide polymorphisms (SNPs) in large-scale, population-based applications. Human disease research, disease diagnosis, personalized therapeutics, environmental monitoring, blood testing, and identification of genetic traits impacting agricultural practices, both in terms of food quality and production efficiency, are a few areas where such systems are in demand. This has stimulated the need for PCR technologies that preserves the intrinsic analytical benefits of PCR yet enables higher throughputs without increasing the time to answer, labor and reagent expenses and workflow complexity. An example of such a system based on a high-density array of nanoliter PCR assays is described here. Functionally equivalent to a microtiter plate, the nanoplate system makes possible up to 3,072 simultaneous end-point or real-time PCR measurements in a device, the size of a standard microscope slide. Methods for SNP genotyping with end-point TaqMan PCR assays and quantitative measurement of gene expression with SYBR Green I real-time PCR are outlined and illustrative data showing system performance is provided.

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

    Science.gov (United States)

    Fornari, Marco

    2015-03-01

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

  19. Resolution- and throughput-enhanced spectroscopy using a high-throughput computational slit

    Science.gov (United States)

    Kazemzadeh, Farnoud; Wong, Alexander

    2016-09-01

    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), a numerical technique 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 ~ 50% over the control spectral resolution and improvements in efficiency of > 2 times over the efficiency of the largest entrance aperture used in the study while producing highly accurate spectra.

  20. High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death

    Science.gov (United States)

    Human ATAD5 is an excellent biomarker for identifying genotoxic compounds because ATADS protein levels increase post-transcriptionally following exposure to a variety of DNA damaging agents. Here we report a novel quantitative high-throughput ATAD5-Iuciferase assay that can moni...

  1. High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry

    DEFF Research Database (Denmark)

    Ståhlman, Marcus; Ejsing, Christer S.; Tarasov, Kirill

    2009-01-01

    we describe a novel high-throughput shotgun lipidomic platform based on 96-well robot-assisted lipid extraction, automated sample infusion by mircofluidic-based nanoelectrospray ionization, and quantitative multiple precursor ion scanning analysis on a quadrupole time-of-flight mass spectrometer...

  2. Scanning droplet cell for high throughput electrochemical and photoelectrochemical measurements

    Science.gov (United States)

    Gregoire, John M.; Xiang, Chengxiang; Liu, Xiaonao; Marcin, Martin; Jin, Jian

    2013-02-01

    High throughput electrochemical techniques are widely applied in material discovery and optimization. For many applications, the most desirable electrochemical characterization requires a three-electrode cell under potentiostat control. In high throughput screening, a material library is explored by either employing an array of such cells, or rastering a single cell over the library. To attain this latter capability with unprecedented throughput, we have developed a highly integrated, compact scanning droplet cell that is optimized for rapid electrochemical and photoeletrochemical measurements. Using this cell, we screened a quaternary oxide library as (photo)electrocatalysts for the oxygen evolution (water splitting) reaction. High quality electrochemical measurements were carried out and key electrocatalytic properties were identified for each of 5456 samples with a throughput of 4 s per sample.

  3. 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...

  4. 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...

  5. 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...

  6. High-Throughput Analysis and Automation for Glycomics Studies

    NARCIS (Netherlands)

    Shubhakar, A.; Reiding, K.R.; Gardner, R.A.; Spencer, D.I.R.; Fernandes, D.L.; Wuhrer, M.

    2015-01-01

    This review covers advances in analytical technologies for high-throughput (HTP) glycomics. Our focus is on structural studies of glycoprotein glycosylation to support biopharmaceutical realization and the discovery of glycan biomarkers for human disease. For biopharmaceuticals, there is increasing

  7. Materiomics - High-Throughput Screening of Biomaterial Properties

    NARCIS (Netherlands)

    de Boer, Jan; van Blitterswijk, Clemens

    2013-01-01

    This complete, yet concise, guide introduces you to the rapidly developing field of high throughput screening of biomaterials: materiomics. Bringing together the key concepts and methodologies used to determine biomaterial properties, you will understand the adaptation and application of materomics

  8. 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...

  9. Applications of High Throughput Sequencing for Immunology and Clinical Diagnostics

    OpenAIRE

    Kim, Hyunsung John

    2014-01-01

    High throughput sequencing methods have fundamentally shifted the manner in which biological experiments are performed. In this dissertation, conventional and novel high throughput sequencing and bioinformatics methods are applied to immunology and diagnostics. In order to study rare subsets of cells, an RNA sequencing method was first optimized for use with minimal levels of RNA and cellular input. The optimized RNA sequencing method was then applied to study the transcriptional differences ...

  10. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. High Throughput Heuristics for Prioritizing Human Exposure to ...

    Science.gov (United States)

    The risk posed to human health by any of the thousands of untested anthropogenic chemicals in our environment is a function of both the potential hazard presented by the chemical, and the possibility of being exposed. Without the capacity to make quantitative, albeit uncertain, forecasts of exposure, the putative risk of adverse health effect from a chemical cannot be evaluated. We used Bayesian methodology to infer ranges of exposure intakes that are consistent with biomarkers of chemical exposures identified in urine samples from the U.S. population by the National Health and Nutrition Examination Survey (NHANES). We perform linear regression on inferred exposure for demographic subsets of NHANES demarked by age, gender, and weight using high throughput chemical descriptors gleaned from databases and chemical structure-based calculators. We find that five of these descriptors are capable of explaining roughly 50% of the variability across chemicals for all the demographic groups examined, including children aged 6-11. For the thousands of chemicals with no other source of information, this approach allows rapid and efficient prediction of average exposure intake of environmental chemicals. The methods described by this manuscript provide a highly improved methodology for HTS of human exposure to environmental chemicals. The manuscript includes a ranking of 7785 environmental chemicals with respect to potential human exposure, including most of the Tox21 in vit

  12. Performance of high-throughput DNA quantification methods

    Directory of Open Access Journals (Sweden)

    Chanock Stephen J

    2003-10-01

    Full Text Available Abstract Background The accuracy and precision of estimates of DNA concentration are critical factors for efficient use of DNA samples in high-throughput genotype and sequence analyses. We evaluated the performance of spectrophotometric (OD DNA quantification, and compared it to two fluorometric quantification methods, the PicoGreen® assay (PG, and a novel real-time quantitative genomic PCR assay (QG specific to a region at the human BRCA1 locus. Twenty-Two lymphoblastoid cell line DNA samples with an initial concentration of ~350 ng/uL were diluted to 20 ng/uL. DNA concentration was estimated by OD and further diluted to 5 ng/uL. The concentrations of multiple aliquots of the final dilution were measured by the OD, QG and PG methods. The effects of manual and robotic laboratory sample handling procedures on the estimates of DNA concentration were assessed using variance components analyses. Results The OD method was the DNA quantification method most concordant with the reference sample among the three methods evaluated. A large fraction of the total variance for all three methods (36.0–95.7% was explained by sample-to-sample variation, whereas the amount of variance attributable to sample handling was small (0.8–17.5%. Residual error (3.2–59.4%, corresponding to un-modelled factors, contributed a greater extent to the total variation than the sample handling procedures. Conclusion The application of a specific DNA quantification method to a particular molecular genetic laboratory protocol must take into account the accuracy and precision of the specific method, as well as the requirements of the experimental workflow with respect to sample volumes and throughput. While OD was the most concordant and precise DNA quantification method in this study, the information provided by the quantitative PCR assay regarding the suitability of DNA samples for PCR may be an essential factor for some protocols, despite the decreased concordance and

  13. High-throughput optical coherence tomography at 800 nm.

    Science.gov (United States)

    Goda, Keisuke; Fard, Ali; Malik, Omer; Fu, Gilbert; Quach, Alan; Jalali, Bahram

    2012-08-27

    We report high-throughput optical coherence tomography (OCT) that offers 1,000 times higher axial scan rate than conventional OCT in the 800 nm spectral range. This is made possible by employing photonic time-stretch for chirping a pulse train and transforming it into a passive swept source. We demonstrate a record high axial scan rate of 90.9 MHz. To show the utility of our method, we also demonstrate real-time observation of laser ablation dynamics. Our high-throughput OCT is expected to be useful for industrial applications where the speed of conventional OCT falls short.

  14. A novel high throughput method to investigate polymer dissolution.

    Science.gov (United States)

    Zhang, Ying; Mallapragada, Surya K; Narasimhan, Balaji

    2010-02-16

    The dissolution behavior of polystyrene (PS) in biodiesel was studied by developing a novel high throughput approach based on Fourier-transform infrared (FTIR) microscopy. A multiwell device for high throughput dissolution testing was fabricated using a photolithographic rapid prototyping method. The dissolution of PS films in each well was tracked by following the characteristic IR band of PS and the effect of PS molecular weight and temperature on the dissolution rate was simultaneously investigated. The results were validated with conventional gravimetric methods. The high throughput method can be extended to evaluate the dissolution profiles of a large number of samples, or to simultaneously investigate the effect of variables such as polydispersity, crystallinity, and mixed solvents. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. A robust high-throughput sample preparation and liquid chromatography/tandem mass spectrometry method for the quantitation of β-lyase metabolites of sulfur mustard as 1,1'-sulfonylbis-[2-(methylthio)ethane] in human urine.

    Science.gov (United States)

    Reddy, Muntha K; Nixon, Christopher; Wyatt, Shane A; Croley, Timothy R

    2013-05-30

    Sulfur mustard (HD) is a major chemical warfare agent threat to humans. Since World War I, several incidents of human exposure to sulfur mustard have been reported. In order to assist health professionals during an exposure event and support biological monitoring, a rapid analytical method is required to measure the exposure of humans to HD. The β-lyase metabolites of HD, 1-methylsulfinyl-2-[2-(methylthio)ethylsulfonyl]ethane (MSMTESE) and 1,1'-sulfonylbis[2-(methylsulfinyl)ethane] (SBMSE) were reduced to the single biomarker, 1,1'-sulfonylbis-[2-(methylthio)ethane] (SBMTE), using titanium(III) chloride. High-throughput sample preparation was performed on a Tecan Freedom EVO liquid handler and analysis was performed by electrospray ionization liquid chromatography and tandem mass spectrometry (LC/MS/MS) in the multiple-reaction monitoring mode. Each analytical run consisted of a matrix blank, calibration standards (0.1-100 ng/mL), low quality controls (QCs), 2.5 ng/mL, and high QCs, 25.0 ng/mL, of SBMTE in human urine. The method was validated with 20 analytical runs performed by four analysts. The mean calculated concentrations of the low and high QCs were 2.52 and 25.5 ng/mL with relative standard deviations of 3.6% and 2.3%, respectively. This semi-automated method has few manual transfer steps, thus minimizing common manual errors and saving time. Therefore, this method would be very helpful to responding laboratories in a large-scale exposure event related to HD. Copyright © 2013 John Wiley & Sons, Ltd.

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

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.|info:eu-repo/dai/nl/074334603; Folkers, G.E.|info:eu-repo/dai/nl/162277202

    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

  17. High throughput 16S rRNA gene amplicon sequencing

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Larsen, Poul; Jørgensen, Mads Koustrup

    S rRNA gene amplicon sequencing has been developed over the past few years and is now ready to use for more comprehensive studies related to plant operation and optimization thanks to short analysis time, low cost, high throughput, and high taxonomic resolution. In this study we show how 16S r...

  18. 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.

  19. High throughput materials research and development for lithium ion batteries

    Directory of Open Access Journals (Sweden)

    Parker Liu

    2017-09-01

    Full Text Available Development of next generation batteries requires a breakthrough in materials. Traditional one-by-one method, which is suitable for synthesizing large number of sing-composition material, is time-consuming and costly. High throughput and combinatorial experimentation, is an effective method to synthesize and characterize huge amount of materials over a broader compositional region in a short time, which enables to greatly speed up the discovery and optimization of materials with lower cost. In this work, high throughput and combinatorial materials synthesis technologies for lithium ion battery research are discussed, and our efforts on developing such instrumentations are introduced.

  20. High throughput calorimetry for evaluating enzymatic reactions generating phosphate.

    Science.gov (United States)

    Hoflack, Lieve; De Groeve, Manu; Desmet, Tom; Van Gerwen, Peter; Soetaert, Wim

    2010-05-01

    A calorimetric assay is described for the high-throughput screening of enzymes that produce inorganic phosphate. In the current example, cellobiose phosphorylase (EC 2.4.1.20) is tested for its ability to synthesise rare disaccharides. The generated phosphate is measured in a high-throughput calorimeter by coupling the reaction to pyruvate oxidase and catalase. This procedure allows for the simultaneous analysis of 48 reactions in microtiter plate format and has been validated by comparison with a colorimetric phosphate assay. The proposed assay has a coefficient of variation of 3.14% and is useful for screening enzyme libraries for enhanced activity and substrate libraries for enzyme promiscuity.

  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. INSIDIA: A FIJI Macro Delivering High-Throughput and High-Content Spheroid Invasion Analysis.

    Science.gov (United States)

    Moriconi, Chiara; Palmieri, Valentina; Di Santo, Riccardo; Tornillo, Giusy; Papi, Massimiliano; Pilkington, Geoff; De Spirito, Marco; Gumbleton, Mark

    2017-10-01

    Time-series image capture of in vitro 3D spheroidal cancer models embedded within an extracellular matrix affords examination of spheroid growth and cancer cell invasion. However, a customizable, comprehensive and open source solution for the quantitative analysis of such spheroid images is lacking. Here, the authors describe INSIDIA (INvasion SpheroID ImageJ Analysis), an open-source macro implemented as a customizable software algorithm running on the FIJI platform, that enables high-throughput high-content quantitative analysis of spheroid images (both bright-field gray and fluorescent images) with the output of a range of parameters defining the spheroid "tumor" core and its invasive characteristics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. High-throughput and high-sensitivity quantitative analysis of serum unsaturated fatty acids by chip-based nanoelectrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry: early stage diagnostic biomarkers of pancreatic cancer.

    Science.gov (United States)

    Zhang, Yaping; Qiu, Ling; Wang, Yanmin; Qin, Xuzhen; Li, Zhili

    2014-04-07

    In this study, Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) coupled with chip-based direct-infusion nanoelectrospray ionization source (CBDInanoESI) in a negative ion mode is first employed to evaluate the effect of serum and its corresponding supernatant matrixes on the recoveries of serum free fatty acids (FFAs) based on spike-and-recovery experimental strategy by adding analytes along with analog internal standard (IS). The recoveries between serum (69.8-115.6%) and the supernatant (73.6-99.0%) matrixes are almost identical. Multiple point internal standard calibration curves between the concentration ratios of individual fatty acids to ISs, (C(17:1) as IS of C(16:1), C(18:3), C(18:2), or C(18:1) or C(21:0) as IS of C(20:4) or C(22:6)) versus their corresponding intensity ratios were constructed for C(16:1), C(18:3), C(18:2), C(18:1), C(20:4) and C(22:6), respectively, with correlation coefficients of greater than 0.99, lower limits of detection between 0.3 and 1.8 nM, and intra- and inter-day precision (relative standard deviations <18%), along with the linear dynamic range of three orders of magnitude. Sequentially, this advanced analytical platform was applied to perform simultaneous quantitative and qualitative analysis of multiple targets, e.g., serum supernatant unsaturated FFAs from 361 participants including 95 patients with pancreatic cancer (PC), 61 patients with pancreatitis and 205 healthy controls. Experimental results indicate that the levels of C(18:1), C(18:2), C(18:3), C(20:4) and C(22:6), as well as the level ratios of C(18:2)/C(18:1) and C(18:3)/C(18:1) of the PC patients were significantly decreased compared with those of healthy controls and the patients with pancreatitis (p < 0.01). It is worth noting that the ratio of C(18:2)/C(18:1), polyunsaturated fatty acids (PUFAs) (C(18:2), C(18:3), C(20:4), and C(22:6)), panel a (C(16:1), C(18:3), C(18:2), C(20:4) and C(22:6)) and panel b (C(18:2)/C(18:1) and C(18:3)/C(18

  4. High throughput defect detection with multiple parallel electron beams

    NARCIS (Netherlands)

    Himbergen, H.M.P. van; Nijkerk, M.D.; Jager, P.W.H. de; Hosman, T.C.; Kruit, P.

    2007-01-01

    A new concept for high throughput defect detection with multiple parallel electron beams is described. As many as 30 000 beams can be placed on a footprint of a in.2, each beam having its own microcolumn and detection system without cross-talk. Based on the International Technology Roadmap for

  5. 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 s...

  6. High-throughput cloning and expression in recalcitrant bacteria

    NARCIS (Netherlands)

    Geertsma, Eric R.; Poolman, Bert

    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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

  9. High-throughput sequencing in mitochondrial DNA research.

    Science.gov (United States)

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

    2014-07-01

    Next-generation sequencing, also known as high-throughput sequencing, has greatly enhanced researchers' ability to conduct biomedical research on all levels. Mitochondrial research has also benefitted greatly from high-throughput sequencing; sequencing technology now allows for screening of all 16,569 base pairs of the mitochondrial genome simultaneously for SNPs and low level heteroplasmy and, in some cases, the estimation of mitochondrial DNA copy number. It is important to realize the full potential of high-throughput sequencing for the advancement of mitochondrial research. To this end, we review how high-throughput sequencing has impacted mitochondrial research in the categories of SNPs, low level heteroplasmy, copy number, and structural variants. We also discuss the different types of mitochondrial DNA sequencing and their pros and cons. Based on previous studies conducted by various groups, we provide strategies for processing mitochondrial DNA sequencing data, including assembly, variant calling, and quality control. Copyright © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

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

    Science.gov (United States)

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

  11. Chemometric Optimization Studies in Catalysis Employing High-Throughput Experimentation

    NARCIS (Netherlands)

    Pereira, S.R.M.

    2008-01-01

    The main topic of this thesis is the investigation of the synergies between High-Throughput Experimentation (HTE) and Chemometric Optimization methodologies in Catalysis research and of the use of such methodologies to maximize the advantages of using HTE methods. Several case studies were analysed

  12. High throughput phenotyping to accelerate crop breeding and monitoring of diseases in the field.

    Science.gov (United States)

    Shakoor, Nadia; Lee, Scott; Mockler, Todd C

    2017-08-01

    Effective implementation of technology that facilitates accurate and high-throughput screening of thousands of field-grown lines is critical for accelerating crop improvement and breeding strategies for higher yield and disease tolerance. Progress in the development of field-based high throughput phenotyping methods has advanced considerably in the last 10 years through technological progress in sensor development and high-performance computing. Here, we review recent advances in high throughput field phenotyping technologies designed to inform the genetics of quantitative traits, including crop yield and disease tolerance. Successful application of phenotyping platforms to advance crop breeding and identify and monitor disease requires: (1) high resolution of imaging and environmental sensors; (2) quality data products that facilitate computer vision, machine learning and GIS; (3) capacity infrastructure for data management and analysis; and (4) automated environmental data collection. Accelerated breeding for agriculturally relevant crop traits is key to the development of improved varieties and is critically dependent on high-resolution, high-throughput field-scale phenotyping technologies that can efficiently discriminate better performing lines within a larger population and across multiple environments. Copyright © 2017. Published by Elsevier Ltd.

  13. Computational and Statistical Methods for High-Throughput Mass Spectrometry-Based PTM Analysis.

    Science.gov (United States)

    Schwämmle, Veit; Vaudel, Marc

    2017-01-01

    Cell signaling and functions heavily rely on post-translational modifications (PTMs) of proteins. Their high-throughput characterization is thus of utmost interest for multiple biological and medical investigations. In combination with efficient enrichment methods, peptide mass spectrometry analysis allows the quantitative comparison of thousands of modified peptides over different conditions. However, the large and complex datasets produced pose multiple data interpretation challenges, ranging from spectral interpretation to statistical and multivariate analyses. Here, we present a typical workflow to interpret such data.

  14. Machine learning in computational biology to accelerate high-throughput protein expression

    DEFF Research Database (Denmark)

    Sastry, Anand; Monk, Jonathan M.; Tegel, Hanna

    2017-01-01

    over 40 000 unique human protein fragments have been expressed in E. coli. These datasets enable quantitative tracking of entire cellular proteomes and present new avenues for understanding molecular-level properties influencing expression and solubility. Results: Combining computational biology......Motivation: The Human Protein Atlas (HPA) enables the simultaneous characterization of thousands of proteins across various tissues to pinpoint their spatial location in the human body. This has been achieved through transcriptomics and high-throughput immunohistochemistry-based approaches, where...

  15. A high-throughput method for quantifying metabolically active yeast cells

    DEFF Research Database (Denmark)

    Nandy, Subir Kumar; Knudsen, Peter Boldsen; Rosenkjær, Alexander

    2015-01-01

    By redesigning the established methylene blue reduction test for bacteria and yeast, we present a cheap and efficient methodology for quantitative physiology of eukaryotic cells applicable for high-throughput systems. Validation of themethod in fermenters and highthroughput systems proved...... equivalent, displaying reduction curves that interrelated directly with CFU counts. For growth rate estimation, the methylene blue reduction test (MBRT) proved superior, since the discriminatory nature of the method allowed for the quantification of metabolically active cells only, excluding dead cells...

  16. Rapid 2,2'-bicinchoninic-based xylanase assay compatible with high throughput screening

    Science.gov (United States)

    William R. Kenealy; Thomas W. Jeffries

    2003-01-01

    High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2'-bicinchoninic-based protein reagent. Endo-1,4-â-D-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml−1. The assay is linear for sugar...

  17. Transcriptomic profiling and quantitative high-throughput (qHTS) drug screening of CDH1 deficient hereditary diffuse gastric cancer (HDGC) cells identify treatment leads for familial gastric cancer.

    Science.gov (United States)

    Chen, Ina; Mathews-Greiner, Lesley; Li, Dandan; Abisoye-Ogunniyan, Abisola; Ray, Satyajit; Bian, Yansong; Shukla, Vivek; Zhang, Xiaohu; Guha, Raj; Thomas, Craig; Gryder, Berkley; Zacharia, Athina; Beane, Joal D; Ravichandran, Sarangan; Ferrer, Marc; Rudloff, Udo

    2017-05-01

    Patients with hereditary diffuse gastric cancer (HDGC), a cancer predisposition syndrome associated with germline mutations of the CDH1 (E-cadherin) gene, have few effective treatment options. Despite marked differences in natural history, histopathology, and genetic profile to patients afflicted by sporadic gastric cancer, patients with HDGC receive, in large, identical systemic regimens. The lack of a robust preclinical in vitro system suitable for effective drug screening has been one of the obstacles to date which has hampered therapeutic advances in this rare disease. In order to identify therapeutic leads selective for the HDGC subtype of gastric cancer, we compared gene expression profiles and drug phenotype derived from an oncology library of 1912 compounds between gastric cancer cells established from a patient with metastatic HDGC harboring a c.1380delA CDH1 germline variant and sporadic gastric cancer cells. Unsupervised hierarchical cluster analysis shows select gene expression alterations in c.1380delA CDH1 SB.mhdgc-1 cells compared to a panel of sporadic gastric cancer cell lines with enrichment of ERK1-ERK2 (extracellular signal regulated kinase) and IP3 (inositol trisphosphate)/DAG (diacylglycerol) signaling as the top networks in c.1380delA SB.mhdgc-1 cells. Intracellular phosphatidylinositol intermediaries were increased upon direct measure in c.1380delA CDH1 SB.mhdgc-1 cells. Differential high-throughput drug screening of c.1380delA CDH1 SB.mhdgc-1 versus sporadic gastric cancer cells identified several compound classes with enriched activity in c.1380 CDH1 SB.mhdgc-1 cells including mTOR (Mammalian Target Of Rapamycin), MEK (Mitogen-Activated Protein Kinase), c-Src kinase, FAK (Focal Adhesion Kinase), PKC (Protein Kinase C), or TOPO2 (Topoisomerase II) inhibitors. Upon additional drug response testing, dual PI3K (Phosphatidylinositol 3-Kinase)/mTOR and topoisomerase 2A inhibitors displayed up to >100-fold increased activity in hereditary c.1380

  18. High-Throughput Microfluidics for the Screening of Yeast Libraries.

    Science.gov (United States)

    Huang, Mingtao; Joensson, Haakan N; Nielsen, Jens

    2018-01-01

    Cell factory development is critically important for efficient biological production of chemicals, biofuels, and pharmaceuticals. Many rounds of the Design-Build-Test-Learn cycles may be required before an engineered strain meeting specific metrics required for industrial application. The bioindustry prefer products in secreted form (secreted products or extracellular metabolites) as it can lower the cost of downstream processing, reduce metabolic burden to cell hosts, and allow necessary modification on the final products , such as biopharmaceuticals. Yet, products in secreted form result in the disconnection of phenotype from genotype, which may have limited throughput in the Test step for identification of desired variants from large libraries of mutant strains. In droplet microfluidic screening, single cells are encapsulated in individual droplet and enable high-throughput processing and sorting of single cells or clones. Encapsulation in droplets allows this technology to overcome the throughput limitations present in traditional methods for screening by extracellular phenotypes. In this chapter, we describe a protocol/guideline for high-throughput droplet microfluidics screening of yeast libraries for higher protein secretion . This protocol can be adapted to screening by a range of other extracellular products from yeast or other hosts.

  19. 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

  20. A high throughput droplet based electroporation system

    Science.gov (United States)

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

    2014-11-01

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

  1. 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).

  2. Validation of high throughput sequencing and microbial forensics applications

    OpenAIRE

    Budowle, Bruce; Connell, Nancy D.; Bielecka-Oder, Anna; Rita R Colwell; Corbett, Cindi R.; Fletcher, Jacqueline; Forsman, Mats; Kadavy, Dana R; Markotic, Alemka; Morse, Stephen A.; Murch, Randall S; Sajantila, Antti; Schemes, Sarah E; Ternus, Krista L; Turner, Stephen D

    2014-01-01

    Abstract High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results a...

  3. DNA from buccal swabs suitable for high-throughput SNP multiplex analysis.

    Science.gov (United States)

    McMichael, Gai L; Gibson, Catherine S; O'Callaghan, Michael E; Goldwater, Paul N; Dekker, Gustaaf A; Haan, Eric A; MacLennan, Alastair H

    2009-12-01

    We sought a convenient and reliable method for collection of genetic material that is inexpensive and noninvasive and suitable for self-collection and mailing and a compatible, commercial DNA extraction protocol to meet quantitative and qualitative requirements for high-throughput single nucleotide polymorphism (SNP) multiplex analysis on an automated platform. Buccal swabs were collected from 34 individuals as part of a pilot study to test commercially available buccal swabs and DNA extraction kits. DNA was quantified on a spectrofluorometer with Picogreen dsDNA prior to testing the DNA integrity with predesigned SNP multiplex assays. Based on the pilot study results, the Catch-All swabs and Isohelix buccal DNA isolation kit were selected for our high-throughput application and extended to a further 1140 samples as part of a large cohort study. The average DNA yield in the pilot study (n=34) was 1.94 microg +/- 0.54 with a 94% genotyping pass rate. For the high-throughput application (n=1140), the average DNA yield was 2.44 microg +/- 1.74 with a >or=93% genotyping pass rate. The Catch-All buccal swabs are a convenient and cost-effective alternative to blood sampling. Combined with the Isohelix buccal DNA isolation kit, they provided DNA of sufficient quantity and quality for high-throughput SNP multiplex analysis.

  4. 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.

  5. 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.

  6. High-throughput sequence alignment using Graphics Processing Units.

    Science.gov (United States)

    Schatz, Michael C; Trapnell, Cole; Delcher, Arthur L; Varshney, Amitabh

    2007-12-10

    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. 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. 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.

  7. 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; Toner, Mehmet; Warren, H. Shaw; Schoenfeld, David A.; Rahme, Laurence; McDonald-Smith, Grace P.; Hayden, Douglas; Mason, Philip; Fagan, Shawn; Yu, Yong-Ming; Cobb, J. Perren; Remick, Daniel G.; Mannick, John A.; Lederer, James A.; Gamelli, Richard L.; Silver, Geoffrey M.; West, Michael A.; Shapiro, Michael B.; Smith, Richard; Camp, David G.; Qian, Weijun; Tibshirani, Rob; Lowry, Stephen; Calvano, Steven; Chaudry, Irshad; Cohen, Mitchell; Moore, Ernest E.; Johnson, Jeffrey; Baker, Henry V.; Efron, Philip A.; Balis, Ulysses G. J.; Billiar, Timothy R.; Ochoa, Juan B.; Sperry, Jason L.; Miller-Graziano, Carol L.; De, Asit K.; Bankey, Paul E.; Herndon, David N.; Finnerty, Celeste C.; Jeschke, Marc G.; Minei, Joseph P.; Arnoldo, Brett D.; Hunt, John L.; Horton, Jureta; Cobb, J. Perren; Brownstein, Bernard; Freeman, Bradley; Nathens, Avery B.; Cuschieri, Joseph; Gibran, Nicole; Klein, Matthew; O'Keefe, Grant

    2011-01-01

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

  8. Computational analysis of high-throughput flow cytometry data.

    Science.gov (United States)

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

    2012-08-01

    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. 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. 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.

  9. 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

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

    Science.gov (United States)

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

    2016-07-29

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

  11. High-Throughput Thermodynamic Modeling and Uncertainty Quantification for ICME

    Science.gov (United States)

    Otis, Richard A.; Liu, Zi-Kui

    2017-05-01

    One foundational component of the integrated computational materials engineering (ICME) and Materials Genome Initiative is the computational thermodynamics based on the calculation of phase diagrams (CALPHAD) method. The CALPHAD method pioneered by Kaufman has enabled the development of thermodynamic, atomic mobility, and molar volume databases of individual phases in the full space of temperature, composition, and sometimes pressure for technologically important multicomponent engineering materials, along with sophisticated computational tools for using the databases. In this article, our recent efforts will be presented in terms of developing new computational tools for high-throughput modeling and uncertainty quantification based on high-throughput, first-principles calculations and the CALPHAD method along with their potential propagations to downstream ICME modeling and simulations.

  12. Trade-Off Analysis in High-Throughput Materials Exploration.

    Science.gov (United States)

    Volety, Kalpana K; Huyberechts, Guido P J

    2017-03-13

    This Research Article presents a strategy to identify the optimum compositions in metal alloys with certain desired properties in a high-throughput screening environment, using a multiobjective optimization approach. In addition to the identification of the optimum compositions in a primary screening, the strategy also allows pointing to regions in the compositional space where further exploration in a secondary screening could be carried out. The strategy for the primary screening is a combination of two multiobjective optimization approaches namely Pareto optimality and desirability functions. The experimental data used in the present study have been collected from over 200 different compositions belonging to four different alloy systems. The metal alloys (comprising Fe, Ti, Al, Nb, Hf, Zr) are synthesized and screened using high-throughput technologies. The advantages of such a kind of approach compared to the limitations of the traditional and comparatively simpler approaches like ranking and calculating figures of merit are discussed.

  13. 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...

  14. 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.

  15. High-throughput optical screening of cellular mechanotransduction

    OpenAIRE

    Compton, JL; Luo, JC; Ma, H.; Botvinick, E; Venugopalan, V

    2014-01-01

    We introduce an optical platform for rapid, high-throughput screening of exogenous molecules that affect cellular mechanotransduction. Our method initiates mechanotransduction in adherent cells using single laser-microbeam generated microcavitation bubbles without requiring flow chambers or microfluidics. These microcavitation bubbles expose adherent cells to a microtsunami, a transient microscale burst of hydrodynamic shear stress, which stimulates cells over areas approaching 1 mm2. We demo...

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

    CERN Multimedia

    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.

  17. High-throughput evaluation of synthetic metabolic pathways.

    Science.gov (United States)

    Klesmith, Justin R; Whitehead, Timothy A

    2016-03-01

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

  18. The high-throughput highway to computational materials design.

    Science.gov (United States)

    Curtarolo, Stefano; Hart, Gus L W; Nardelli, Marco Buongiorno; Mingo, Natalio; Sanvito, Stefano; Levy, Ohad

    2013-03-01

    High-throughput computational materials design is an emerging area of materials science. By combining advanced thermodynamic and electronic-structure methods with intelligent data mining and database construction, and exploiting the power of current supercomputer architectures, scientists generate, manage and analyse enormous data repositories for the discovery of novel materials. In this Review we provide a current snapshot of this rapidly evolving field, and highlight the challenges and opportunities that lie ahead.

  19. Web-based visual analysis for high-throughput genomics.

    Science.gov (United States)

    Goecks, Jeremy; Eberhard, Carl; Too, Tomithy; Nekrutenko, Anton; Taylor, James

    2013-06-13

    Visualization plays an essential role in genomics research by making it possible to observe correlations and trends in large datasets as well as communicate findings to others. Visual analysis, which combines visualization with analysis tools to enable seamless use of both approaches for scientific investigation, offers a powerful method for performing complex genomic analyses. However, there are numerous challenges that arise when creating rich, interactive Web-based visualizations/visual analysis applications for high-throughput genomics. These challenges include managing data flow from Web server to Web browser, integrating analysis tools and visualizations, and sharing visualizations with colleagues. We have created a platform simplifies the creation of Web-based visualization/visual analysis applications for high-throughput genomics. This platform provides components that make it simple to efficiently query very large datasets, draw common representations of genomic data, integrate with analysis tools, and share or publish fully interactive visualizations. Using this platform, we have created a Circos-style genome-wide viewer, a generic scatter plot for correlation analysis, an interactive phylogenetic tree, a scalable genome browser for next-generation sequencing data, and an application for systematically exploring tool parameter spaces to find good parameter values. All visualizations are interactive and fully customizable. The platform is integrated with the Galaxy (http://galaxyproject.org) genomics workbench, making it easy to integrate new visual applications into Galaxy. Visualization and visual analysis play an important role in high-throughput genomics experiments, and approaches are needed to make it easier to create applications for these activities. Our framework provides a foundation for creating Web-based visualizations and integrating them into Galaxy. Finally, the visualizations we have created using the framework are useful tools for high-throughput

  20. Image Harvest: an open-source platform for high-throughput plant image processing and analysis

    Science.gov (United States)

    Knecht, Avi C.; Campbell, Malachy T.; Caprez, Adam; Swanson, David R.; Walia, Harkamal

    2016-01-01

    High-throughput plant phenotyping is an effective approach to bridge the genotype-to-phenotype gap in crops. Phenomics experiments typically result in large-scale image datasets, which are not amenable for processing on desktop computers, thus creating a bottleneck in the image-analysis pipeline. Here, we present an open-source, flexible image-analysis framework, called Image Harvest (IH), for processing images originating from high-throughput plant phenotyping platforms. Image Harvest is developed to perform parallel processing on computing grids and provides an integrated feature for metadata extraction from large-scale file organization. Moreover, the integration of IH with the Open Science Grid provides academic researchers with the computational resources required for processing large image datasets at no cost. Image Harvest also offers functionalities to extract digital traits from images to interpret plant architecture-related characteristics. To demonstrate the applications of these digital traits, a rice (Oryza sativa) diversity panel was phenotyped and genome-wide association mapping was performed using digital traits that are used to describe different plant ideotypes. Three major quantitative trait loci were identified on rice chromosomes 4 and 6, which co-localize with quantitative trait loci known to regulate agronomically important traits in rice. Image Harvest is an open-source software for high-throughput image processing that requires a minimal learning curve for plant biologists to analyzephenomics datasets. PMID:27141917

  1. High-throughput screening to identify selective inhibitors of microbial sulfate reduction (and beyond)

    Science.gov (United States)

    Carlson, H. K.; Coates, J. D.; Deutschbauer, A. M.

    2015-12-01

    The selective perturbation of complex microbial ecosystems to predictably influence outcomes in engineered and industrial environments remains a grand challenge for geomicrobiology. In some industrial ecosystems, such as oil reservoirs, sulfate reducing microorganisms (SRM) produce hydrogen sulfide which is toxic, explosive and corrosive. Current strategies to selectively inhibit sulfidogenesis are based on non-specific biocide treatments, bio-competitive exclusion by alternative electron acceptors or sulfate-analogs which are competitive inhibitors or futile/alternative substrates of the sulfate reduction pathway. Despite the economic cost of sulfidogenesis, there has been minimal exploration of the chemical space of possible inhibitory compounds, and very little work has quantitatively assessed the selectivity of putative souring treatments. We have developed a high-throughput screening strategy to target SRM, quantitatively ranked the selectivity and potency of hundreds of compounds and identified previously unrecognized SRM selective inhibitors and synergistic interactions between inhibitors. Once inhibitor selectivity is defined, high-throughput characterization of microbial community structure across compound gradients and identification of fitness determinants using isolate bar-coded transposon mutant libraries can give insights into the genetic mechanisms whereby compounds structure microbial communities. The high-throughput (HT) approach we present can be readily applied to target SRM in diverse environments and more broadly, could be used to identify and quantify the potency and selectivity of inhibitors of a variety of microbial metabolisms. Our findings and approach are relevant for engineering environmental ecosystems and also to understand the role of natural gradients in shaping microbial niche space.

  2. Image Harvest: an open-source platform for high-throughput plant image processing and analysis.

    Science.gov (United States)

    Knecht, Avi C; Campbell, Malachy T; Caprez, Adam; Swanson, David R; Walia, Harkamal

    2016-05-01

    High-throughput plant phenotyping is an effective approach to bridge the genotype-to-phenotype gap in crops. Phenomics experiments typically result in large-scale image datasets, which are not amenable for processing on desktop computers, thus creating a bottleneck in the image-analysis pipeline. Here, we present an open-source, flexible image-analysis framework, called Image Harvest (IH), for processing images originating from high-throughput plant phenotyping platforms. Image Harvest is developed to perform parallel processing on computing grids and provides an integrated feature for metadata extraction from large-scale file organization. Moreover, the integration of IH with the Open Science Grid provides academic researchers with the computational resources required for processing large image datasets at no cost. Image Harvest also offers functionalities to extract digital traits from images to interpret plant architecture-related characteristics. To demonstrate the applications of these digital traits, a rice (Oryza sativa) diversity panel was phenotyped and genome-wide association mapping was performed using digital traits that are used to describe different plant ideotypes. Three major quantitative trait loci were identified on rice chromosomes 4 and 6, which co-localize with quantitative trait loci known to regulate agronomically important traits in rice. Image Harvest is an open-source software for high-throughput image processing that requires a minimal learning curve for plant biologists to analyzephenomics datasets. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  3. Graph-based signal integration for high-throughput phenotyping.

    Science.gov (United States)

    Herskovic, Jorge R; Subramanian, Devika; Cohen, Trevor; Bozzo-Silva, Pamela A; Bearden, Charles F; Bernstam, Elmer V

    2012-01-01

    Electronic Health Records aggregated in Clinical Data Warehouses (CDWs) promise to revolutionize Comparative Effectiveness Research and suggest new avenues of research. However, the effectiveness of CDWs is diminished by the lack of properly labeled data. We present a novel approach that integrates knowledge from the CDW, the biomedical literature, and the Unified Medical Language System (UMLS) to perform high-throughput phenotyping. In this paper, we automatically construct a graphical knowledge model and then use it to phenotype breast cancer patients. We compare the performance of this approach to using MetaMap when labeling records. MetaMap's overall accuracy at identifying breast cancer patients was 51.1% (n=428); recall=85.4%, precision=26.2%, and F1=40.1%. Our unsupervised graph-based high-throughput phenotyping had accuracy of 84.1%; recall=46.3%, precision=61.2%, and F1=52.8%. We conclude that our approach is a promising alternative for unsupervised high-throughput phenotyping.

  4. 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.

  5. High-throughput computational and experimental techniques in structural genomics.

    Science.gov (United States)

    Chance, Mark R; Fiser, Andras; Sali, Andrej; Pieper, Ursula; Eswar, Narayanan; Xu, Guiping; Fajardo, J Eduardo; Radhakannan, Thirumuruhan; Marinkovic, Nebojsa

    2004-10-01

    Structural genomics has as its goal the provision of structural information for all possible ORF sequences through a combination of experimental and computational approaches. The access to genome sequences and cloning resources from an ever-widening array of organisms is driving high-throughput structural studies by the New York Structural Genomics Research Consortium. In this report, we outline the progress of the Consortium in establishing its pipeline for structural genomics, and some of the experimental and bioinformatics efforts leading to structural annotation of proteins. The Consortium has established a pipeline for structural biology studies, automated modeling of ORF sequences using solved (template) structures, and a novel high-throughput approach (metallomics) to examining the metal binding to purified protein targets. The Consortium has so far produced 493 purified proteins from >1077 expression vectors. A total of 95 have resulted in crystal structures, and 81 are deposited in the Protein Data Bank (PDB). Comparative modeling of these structures has generated >40,000 structural models. We also initiated a high-throughput metal analysis of the purified proteins; this has determined that 10%-15% of the targets contain a stoichiometric structural or catalytic transition metal atom. The progress of the structural genomics centers in the U.S. and around the world suggests that the goal of providing useful structural information on most all ORF domains will be realized. This projected resource will provide structural biology information important to understanding the function of most proteins of the cell.

  6. Microfluidic droplet-based PCR instrumentation for high-throughput gene expression profiling and biomarker discovery

    Directory of Open Access Journals (Sweden)

    Christopher J. Hayes

    2015-06-01

    Full Text Available PCR is a common and often indispensable technique used in medical and biological research labs for a variety of applications. Real-time quantitative PCR (RT-qPCR has become a definitive technique for quantitating differences in gene expression levels between samples. Yet, in spite of this importance, reliable methods to quantitate nucleic acid amounts in a higher throughput remain elusive. In the following paper, a unique design to quantify gene expression levels at the nanoscale in a continuous flow system is presented. Fully automated, high-throughput, low volume amplification of deoxynucleotides (DNA in a droplet based microfluidic system is described. Unlike some conventional qPCR instrumentation that use integrated fluidic circuits or plate arrays, the instrument performs qPCR in a continuous, micro-droplet flowing process with droplet generation, distinctive reagent mixing, thermal cycling and optical detection platforms all combined on one complete instrument. Detailed experimental profiling of reactions of less than 300 nl total volume is achieved using the platform demonstrating the dynamic range to be 4 order logs and consistent instrument sensitivity. Furthermore, reduced pipetting steps by as much as 90% and a unique degree of hands-free automation makes the analytical possibilities for this instrumentation far reaching. In conclusion, a discussion of the first demonstrations of this approach to perform novel, continuous high-throughput biological screens is presented. The results generated from the instrument, when compared with commercial instrumentation, demonstrate the instrument reliability and robustness to carry out further studies of clinical significance with added throughput and economic benefits.

  7. 76 FR 28990 - Ultra High Throughput Sequencing for Clinical Diagnostic Applications-Approaches To Assess...

    Science.gov (United States)

    2011-05-19

    ... Clinical Diagnostic Applications--Approaches To Assess Analytical Validity.'' The purpose of the public... approaches to assess analytical validity of ultra high throughput sequencing for clinical diagnostic... HUMAN SERVICES Food and Drug Administration Ultra High Throughput Sequencing for Clinical Diagnostic...

  8. Dimensioning storage and computing clusters for efficient High Throughput Computing

    CERN Multimedia

    CERN. Geneva

    2012-01-01

    Scientific experiments are producing huge amounts of data, and they continue increasing the size of their datasets and the total volume of data. These data are then processed by researchers belonging to large scientific collaborations, with the Large Hadron Collider being a good example. The focal point of Scientific Data Centres has shifted from coping efficiently with PetaByte scale storage to deliver quality data processing throughput. The dimensioning of the internal components in High Throughput Computing (HTC) data centers is of crucial importance to cope with all the activities demanded by the experiments, both the online (data acceptance) and the offline (data processing, simulation and user analysis). This requires a precise setup involving disk and tape storage services, a computing cluster and the internal networking to prevent bottlenecks, overloads and undesired slowness that lead to losses cpu cycles and batch jobs failures. In this paper we point out relevant features for running a successful s...

  9. Automated High-Throughput Root Phenotyping of Arabidopsis thaliana Under Nutrient Deficiency Conditions.

    Science.gov (United States)

    Satbhai, Santosh B; Göschl, Christian; Busch, Wolfgang

    2017-01-01

    The central question of genetics is how a genotype determines the phenotype of an organism. Genetic mapping approaches are a key for finding answers to this question. In particular, genome-wide association (GWA) studies have been rapidly adopted to study the architecture of complex quantitative traits. This was only possible due to the improvement of high-throughput and low-cost phenotyping methodologies. In this chapter we provide a detailed protocol for obtaining root trait data from the model species Arabidopsis thaliana using the semiautomated, high-throughput phenotyping pipeline BRAT (Busch-lab Root Analysis Toolchain) for early root growth under the stress condition of iron deficiency. Extracted root trait data can be directly used to perform GWA mapping using the freely accessible web application GWAPP to identify marker polymorphisms associated with the phenotype of interest.

  10. Development of New Sensing Materials Using Combinatorial and High-Throughput Experimentation

    Science.gov (United States)

    Potyrailo, Radislav A.; Mirsky, Vladimir M.

    New sensors with improved performance characteristics are needed for applications as diverse as bedside continuous monitoring, tracking of environmental pollutants, monitoring of food and water quality, monitoring of chemical processes, and safety in industrial, consumer, and automotive settings. Typical requirements in sensor improvement are selectivity, long-term stability, sensitivity, response time, reversibility, and reproducibility. Design of new sensing materials is the important cornerstone in the effort to develop new sensors. Often, sensing materials are too complex to predict their performance quantitatively in the design stage. Thus, combinatorial and high-throughput experimentation methodologies provide an opportunity to generate new required data to discover new sensing materials and/or to optimize existing material compositions. The goal of this chapter is to provide an overview of the key concepts of experimental development of sensing materials using combinatorial and high-throughput experimentation tools, and to promote additional fruitful interactions between computational scientists and experimentalists.

  11. High-throughput cell mechanical phenotyping for label-free titration assays of cytoskeletal modifications.

    Science.gov (United States)

    Golfier, Stefan; Rosendahl, Philipp; Mietke, Alexander; Herbig, Maik; Guck, Jochen; Otto, Oliver

    2017-08-01

    The mechanical fingerprint of cells is inherently linked to the structure of the cytoskeleton and can serve as a label-free marker for cell homeostasis or pathologic states. How cytoskeletal composition affects the physical response of cells to external loads has been intensively studied with a spectrum of techniques, yet quantitative and statistically powerful investigations in the form of titration assays are hampered by the low throughput of most available methods. In this study, we employ real-time deformability cytometry (RT-DC), a novel microfluidic tool to examine the effects of biochemically modified F-actin and microtubule stability and nuclear chromatin structure on cell deformation in a human leukemia cell line (HL60). The high throughput of our method facilitates extensive titration assays that allow for significance assessment of the observed effects and extraction of half-maximal concentrations for most of the applied reagents. We quantitatively show that integrity of the F-actin cortex and microtubule network dominate cell deformation on millisecond timescales probed with RT-DC. Drug-induced alterations in the nuclear chromatin structure were not found to consistently affect cell deformation. The sensitivity of the high-throughput cell mechanical measurements to the cytoskeletal modifications we present in this study opens up new possibilities for label-free dose-response assays of cytoskeletal modifications. © 2017 The Authors Cytoskeleton Published by Wiley Periodicals, Inc.

  12. Large scale library generation for high throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Erik Borgström

    Full Text Available BACKGROUND: Large efforts have recently been made to automate the sample preparation protocols for massively parallel sequencing in order to match the increasing instrument throughput. Still, the size selection through agarose gel electrophoresis separation is a labor-intensive bottleneck of these protocols. METHODOLOGY/PRINCIPAL FINDINGS: In this study a method for automatic library preparation and size selection on a liquid handling robot is presented. The method utilizes selective precipitation of certain sizes of DNA molecules on to paramagnetic beads for cleanup and selection after standard enzymatic reactions. CONCLUSIONS/SIGNIFICANCE: The method is used to generate libraries for de novo and re-sequencing on the Illumina HiSeq 2000 instrument with a throughput of 12 samples per instrument in approximately 4 hours. The resulting output data show quality scores and pass filter rates comparable to manually prepared samples. The sample size distribution can be adjusted for each application, and are suitable for all high throughput DNA processing protocols seeking to control size intervals.

  13. Achieving High Throughput for Data Transfer over ATM Networks

    Science.gov (United States)

    Johnson, Marjory J.; Townsend, Jeffrey N.

    1996-01-01

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

  14. Development of carbon plasma-coated multiwell plates for high-throughput mass spectrometric analysis of highly lipophilic fermentation products.

    Science.gov (United States)

    Heinig, Uwe; Scholz, Susanne; Dahm, Pia; Grabowy, Udo; Jennewein, Stefan

    2010-08-01

    Classical approaches to strain improvement and metabolic engineering rely on rapid qualitative and quantitative analyses of the metabolites of interest. As an analytical tool, mass spectrometry (MS) has proven to be efficient and nearly universally applicable for timely screening of metabolites. Furthermore, gas chromatography (GC)/MS- and liquid chromatography (LC)/MS-based metabolite screens can often be adapted to high-throughput formats. We recently engineered a Saccharomyces cerevisiae strain to produce taxa-4(5),11(12)-diene, the first pathway-committing biosynthetic intermediate for the anticancer drug Taxol, through the heterologous and homologous expression of several genes related to isoprenoid biosynthesis. To date, GC/MS- and LC/MS-based high-throughput methods have been inherently difficult to adapt to the screening of isoprenoid-producing microbial strains due to the need for extensive sample preparation of these often highly lipophilic compounds. In the current work, we examined different approaches to the high-throughput analysis of taxa-4(5),11(12)-diene biosynthesizing yeast strains in a 96-deep-well format. Carbon plasma coating of standard 96-deep-well polypropylene plates allowed us to circumvent the inherent solvent instability of commonly used deep-well plates. In addition, efficient adsorption of the target isoprenoid product by the coated plates allowed rapid and simple qualitative and quantitative analyses of the individual cultures. Copyright 2010 Elsevier Inc. All rights reserved.

  15. High throughput holographic imaging-in-flow for the analysis of a wide plankton size range.

    Science.gov (United States)

    Yourassowsky, Catherine; Dubois, Frank

    2014-03-24

    We developed a Digital Holographic Microscope (DHM) working with a partial coherent source specifically adapted to perform high throughput recording of holograms of plankton organisms in-flow, in a size range of 3 µm-300 µm, which is of importance for this kind of applications. This wide size range is achieved with the same flow cell and with the same microscope magnification. The DHM configuration combines a high magnification with a large field of view and provides high-resolution intensity and quantitative phase images refocusing on high sample flow rate. Specific algorithms were developed to detect and extract automatically the particles and organisms present in the samples in order to build holograms of each one that are used for holographic refocusing and quantitative phase contrast imaging. Experimental results are shown and discussed.

  16. High-throughput cultivation and screening platform for unicellular phototrophs.

    Science.gov (United States)

    Tillich, Ulrich M; Wolter, Nick; Schulze, Katja; Kramer, Dan; Brödel, Oliver; Frohme, Marcus

    2014-09-16

    High-throughput cultivation and screening methods allow a parallel, miniaturized and cost efficient processing of many samples. These methods however, have not been generally established for phototrophic organisms such as microalgae or cyanobacteria. In this work we describe and test high-throughput methods with the model organism Synechocystis sp. PCC6803. The required technical automation for these processes was achieved with a Tecan Freedom Evo 200 pipetting robot. The cultivation was performed in 2.2 ml deepwell microtiter plates within a cultivation chamber outfitted with programmable shaking conditions, variable illumination, variable temperature, and an adjustable CO2 atmosphere. Each microtiter-well within the chamber functions as a separate cultivation vessel with reproducible conditions. The automated measurement of various parameters such as growth, full absorption spectrum, chlorophyll concentration, MALDI-TOF-MS, as well as a novel vitality measurement protocol, have already been established and can be monitored during cultivation. Measurement of growth parameters can be used as inputs for the system to allow for periodic automatic dilutions and therefore a semi-continuous cultivation of hundreds of cultures in parallel. The system also allows the automatic generation of mid and long term backups of cultures to repeat experiments or to retrieve strains of interest. The presented platform allows for high-throughput cultivation and screening of Synechocystis sp. PCC6803. The platform should be usable for many phototrophic microorganisms as is, and be adaptable for even more. A variety of analyses are already established and the platform is easily expandable both in quality, i.e. with further parameters to screen for additional targets and in quantity, i.e. size or number of processed samples.

  17. Droplet microfluidic technology for single-cell high-throughput screening.

    Science.gov (United States)

    Brouzes, Eric; Medkova, Martina; Savenelli, Neal; Marran, Dave; Twardowski, Mariusz; Hutchison, J Brian; Rothberg, Jonathan M; Link, Darren R; Perrimon, Norbert; Samuels, Michael L

    2009-08-25

    We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses.

  18. High throughput inclusion body sizing: Nano particle tracking analysis.

    Science.gov (United States)

    Reichelt, Wieland N; Kaineder, Andreas; Brillmann, Markus; Neutsch, Lukas; Taschauer, Alexander; Lohninger, Hans; Herwig, Christoph

    2017-06-01

    The expression of pharmaceutical relevant proteins in Escherichia coli frequently triggers inclusion body (IB) formation caused by protein aggregation. In the scientific literature, substantial effort has been devoted to the quantification of IB size. However, particle-based methods used up to this point to analyze the physical properties of representative numbers of IBs lack sensitivity and/or orthogonal verification. Using high pressure freezing and automated freeze substitution for transmission electron microscopy (TEM) the cytosolic inclusion body structure was preserved within the cells. TEM imaging in combination with manual grey scale image segmentation allowed the quantification of relative areas covered by the inclusion body within the cytosol. As a high throughput method nano particle tracking analysis (NTA) enables one to derive the diameter of inclusion bodies in cell homogenate based on a measurement of the Brownian motion. The NTA analysis of fixated (glutaraldehyde) and non-fixated IBs suggests that high pressure homogenization annihilates the native physiological shape of IBs. Nevertheless, the ratio of particle counts of non-fixated and fixated samples could potentially serve as factor for particle stickiness. In this contribution, we establish image segmentation of TEM pictures as an orthogonal method to size biologic particles in the cytosol of cells. More importantly, NTA has been established as a particle-based, fast and high throughput method (1000-3000 particles), thus constituting a much more accurate and representative analysis than currently available methods. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. 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...... to be serially diluted before they can be used as assay plates. This process, however, leads to an explosion in the number of plates and samples to be tracked. Here, we present SAVANAH, the first tool to effectively manage molecular screening libraries across dilution series. It conveniently links (connects...

  20. High-throughput genomics enhances tomato breeding efficiency.

    Science.gov (United States)

    Barone, A; Di Matteo, A; Carputo, D; Frusciante, L

    2009-03-01

    Tomato (Solanum lycopersicum) is considered a model plant species for a group of economically important crops, such as potato, pepper, eggplant, since it exhibits a reduced genomic size (950 Mb), a short generation time, and routine transformation technologies. Moreover, it shares with the other Solanaceous plants the same haploid chromosome number and a high level of conserved genomic organization. Finally, many genomic and genetic resources are actually available for tomato, and the sequencing of its genome is in progress. These features make tomato an ideal species for theoretical studies and practical applications in the genomics field. The present review describes how structural genomics assist the selection of new varieties resistant to pathogens that cause damage to this crop. Many molecular markers highly linked to resistance genes and cloned resistance genes are available and could be used for a high-throughput screening of multiresistant varieties. Moreover, a new genomics-assisted breeding approach for improving fruit quality is presented and discussed. It relies on the identification of genetic mechanisms controlling the trait of interest through functional genomics tools. Following this approach, polymorphisms in major gene sequences responsible for variability in the expression of the trait under study are then exploited for tracking simultaneously favourable allele combinations in breeding programs using high-throughput genomic technologies. This aims at pyramiding in the genetic background of commercial cultivars alleles that increase their performances. In conclusion, tomato breeding strategies supported by advanced technologies are expected to target increased productivity and lower costs of improved genotypes even for complex traits.

  1. 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.

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

    Science.gov (United States)

    Poisot, Timothée; Péquin, Bérangère; Gravel, Dominique

    2013-04-01

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

  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 (pphenotyping traits.

  4. REDItools: high-throughput RNA editing detection made easy.

    Science.gov (United States)

    Picardi, Ernesto; Pesole, Graziano

    2013-07-15

    The reliable detection of RNA editing sites from massive sequencing data remains challenging and, although several methodologies have been proposed, no computational tools have been released to date. Here, we introduce REDItools a suite of python scripts to perform high-throughput investigation of RNA editing using next-generation sequencing data. REDItools are in python programming language and freely available at http://code.google.com/p/reditools/. ernesto.picardi@uniba.it or graziano.pesole@uniba.it Supplementary data are available at Bioinformatics online.

  5. High throughput platforms for structural genomics of integral membrane proteins.

    Science.gov (United States)

    Mancia, Filippo; Love, James

    2011-08-01

    Structural genomics approaches on integral membrane proteins have been postulated for over a decade, yet specific efforts are lagging years behind their soluble counterparts. Indeed, high throughput methodologies for production and characterization of prokaryotic integral membrane proteins are only now emerging, while large-scale efforts for eukaryotic ones are still in their infancy. Presented here is a review of recent literature on actively ongoing structural genomics of membrane protein initiatives, with a focus on those aimed at implementing interesting techniques aimed at increasing our rate of success for this class of macromolecules. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. 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 individual...... 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....

  7. Bifrost: Stream processing framework for high-throughput applications

    Science.gov (United States)

    Barsdell, Ben; Price, Daniel; Cranmer, Miles; Garsden, Hugh; Dowell, Jayce

    2017-11-01

    Bifrost is a stream processing framework that eases the development of high-throughput processing CPU/GPU pipelines. It is designed for digital signal processing (DSP) applications within radio astronomy. Bifrost uses a flexible ring buffer implementation that allows different signal processing blocks to be connected to form a pipeline. Each block may be assigned to a CPU core, and the ring buffers are used to transport data to and from blocks. Processing blocks may be run on either the CPU or GPU, and the ring buffer will take care of memory copies between the CPU and GPU spaces.

  8. 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.

  9. 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.

  10. A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants.

    Science.gov (United States)

    Yus, Eva; Yang, Jae-Seong; Sogues, Adrià; Serrano, Luis

    2017-08-28

    Quantitative analysis of the sequence determinants of transcription and translation regulation is relevant for systems and synthetic biology. To identify these determinants, researchers have developed different methods of screening random libraries using fluorescent reporters or antibiotic resistance genes. Here, we have implemented a generic approach called ELM-seq (expression level monitoring by DNA methylation) that overcomes the technical limitations of such classic reporters. ELM-seq uses DamID (Escherichia coli DNA adenine methylase as a reporter coupled with methylation-sensitive restriction enzyme digestion and high-throughput sequencing) to enable in vivo quantitative analyses of upstream regulatory sequences. Using the genome-reduced bacterium Mycoplasma pneumoniae, we show that ELM-seq has a large dynamic range and causes minimal toxicity. We use ELM-seq to determine key sequences (known and putatively novel) of promoter and untranslated regions that influence transcription and translation efficiency. Applying ELM-seq to other organisms will help us to further understand gene expression and guide synthetic biology.Quantitative analysis of how DNA sequence determines transcription and translation regulation is of interest to systems and synthetic biologists. Here the authors present ELM-seq, which uses Dam activity as reporter for high-throughput analysis of promoter and 5'-UTR regions.

  11. 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

  12. 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. © 2013 Published by Elsevier Ltd.

  13. A pocket device for high-throughput optofluidic holographic microscopy

    Science.gov (United States)

    Mandracchia, B.; Bianco, V.; Wang, Z.; Paturzo, M.; Bramanti, A.; Pioggia, G.; Ferraro, P.

    2017-06-01

    Here we introduce a compact holographic microscope embedded onboard a Lab-on-a-Chip (LoC) platform. A wavefront division interferometer is realized by writing a polymer grating onto the channel to extract a reference wave from the object wave impinging the LoC. A portion of the beam reaches the samples flowing along the channel path, carrying their information content to the recording device, while one of the diffraction orders from the grating acts as an off-axis reference wave. Polymeric micro-lenses are delivered forward the chip by Pyro-ElectroHydroDynamic (Pyro-EHD) inkjet printing techniques. Thus, all the required optical components are embedded onboard a pocket device, and fast, non-iterative, reconstruction algorithms can be used. We use our device in combination with a novel high-throughput technique, named Space-Time Digital Holography (STDH). STDH exploits the samples motion inside microfluidic channels to obtain a synthetic hologram, mapped in a hybrid space-time domain, and with intrinsic useful features. Indeed, a single Linear Sensor Array (LSA) is sufficient to build up a synthetic representation of the entire experiment (i.e. the STDH) with unlimited Field of View (FoV) along the scanning direction, independently from the magnification factor. The throughput of the imaging system is dramatically increased as STDH provides unlimited FoV, refocusable imaging of samples inside the liquid volume with no need for hologram stitching. To test our embedded STDH microscopy module, we counted, imaged and tracked in 3D with high-throughput red blood cells moving inside the channel volume under non ideal flow conditions.

  14. An Updated Protocol for High Throughput Plant Tissue Sectioning

    Directory of Open Access Journals (Sweden)

    Jonathan A. Atkinson

    2017-10-01

    Full Text Available Quantification of the tissue and cellular structure of plant material is essential for the study of a variety of plant sciences applications. Currently, many methods for sectioning plant material are either low throughput or involve free-hand sectioning which requires a significant amount of practice. Here, we present an updated method to provide rapid and high-quality cross sections, primarily of root tissue but which can also be readily applied to other tissues such as leaves or stems. To increase the throughput of traditional agarose embedding and sectioning, custom designed 3D printed molds were utilized to embed 5–15 roots in a block for sectioning in a single cut. A single fluorescent stain in combination with laser scanning confocal microscopy was used to obtain high quality images of thick sections. The provided CAD files allow production of the embedding molds described here from a number of online 3D printing services. Although originally developed for roots, this method provides rapid, high quality cross sections of many plant tissue types, making it suitable for use in forward genetic screens for differences in specific cell structures or developmental changes. To demonstrate the utility of the technique, the two parent lines of the wheat (Triticum aestivum Chinese Spring × Paragon doubled haploid mapping population were phenotyped for root anatomical differences. Significant differences in adventitious cross section area, stele area, xylem, phloem, metaxylem, and cortical cell file count were found.

  15. High-throughput search for improved transparent conducting oxides

    Science.gov (United States)

    Miglio, Anna

    High-throughput methodologies are a very useful computational tool to explore the space of binary and ternary oxides. We use these methods to search for new and improved transparent conducting oxides (TCOs). TCOs exhibit both visible transparency and good carrier mobility and underpin many energy and electronic applications (e.g. photovoltaics, transparent transistors). We find several potential new n-type and p-type TCOs with a low effective mass. Combining different ab initio approaches, we characterize candidate oxides by their effective mass (mobility), band gap (transparency) and dopability. We present several compounds, not considered previously as TCOs, and discuss the chemical rationale for their promising properties. This analysis is useful to formulate design strategies for future high mobility oxides and has led to follow-up studies including preliminary experimental characterization of a p-type TCO candidate with unexpected chemistry. G. Hautier, A. Miglio, D. Waroquiers, G.-M. Rignanese, and X. Gonze, ``How Does Chemistry Influence Electron Effective Mass in Oxides? A High-Throughput Computational Analysis'', Chem. Mater. 26, 5447 (2014). G. Hautier, A. Miglio, G. Ceder, G.-M. Rignanese, and X. Gonze, ``Identification and design principles of low hole effective mass p-type transparent conducting oxides'', Nature Commun. 4, 2292 (2013).

  16. Mass Spectrometry-based Assay for High Throughput and High Sensitivity Biomarker Verification

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Xuejiang; Tang, Keqi

    2017-06-14

    Searching for disease specific biomarkers has become a major undertaking in the biomedical research field as the effective diagnosis, prognosis and treatment of many complex human diseases are largely determined by the availability and the quality of the biomarkers. A successful biomarker as an indicator to a specific biological or pathological process is usually selected from a large group of candidates by a strict verification and validation process. To be clinically useful, the validated biomarkers must be detectable and quantifiable by the selected testing techniques in their related tissues or body fluids. Due to its easy accessibility, protein biomarkers would ideally be identified in blood plasma or serum. However, most disease related protein biomarkers in blood exist at very low concentrations (<1ng/mL) and are “masked” by many none significant species at orders of magnitude higher concentrations. The extreme requirements of measurement sensitivity, dynamic range and specificity make the method development extremely challenging. The current clinical protein biomarker measurement primarily relies on antibody based immunoassays, such as ELISA. Although the technique is sensitive and highly specific, the development of high quality protein antibody is both expensive and time consuming. The limited capability of assay multiplexing also makes the measurement an extremely low throughput one rendering it impractical when hundreds to thousands potential biomarkers need to be quantitatively measured across multiple samples. Mass spectrometry (MS)-based assays have recently shown to be a viable alternative for high throughput and quantitative candidate protein biomarker verification. Among them, the triple quadrupole MS based assay is the most promising one. When it is coupled with liquid chromatography (LC) separation and electrospray ionization (ESI) source, a triple quadrupole mass spectrometer operating in a special selected reaction monitoring (SRM) mode

  17. Carbohydrate chips for studying high-throughput carbohydrate-protein interactions.

    Science.gov (United States)

    Park, Sungjin; Lee, Myung-ryul; Pyo, Soon-Jin; Shin, Injae

    2004-04-21

    Carbohydrate-protein interactions play important biological roles in living organisms. For the most part, biophysical and biochemical methods have been used for studying these biomolecular interactions. Less attention has been given to the development of high-throughput methods to elucidate recognition events between carbohydrates and proteins. In the current effort to develop a novel high-throughput tool for monitoring carbohydrate-protein interactions, we prepared carbohydrate microarrays by immobilizing maleimide-linked carbohydrates on thiol-derivatized glass slides and carried out lectin binding experiments by using these microarrays. The results showed that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. In addition, binding affinities of lectins to carbohydrates were also quantitatively analyzed by determining IC(50) values of soluble carbohydrates with the carbohydrate microarrays. To fabricate carbohydrate chips that contained more diverse carbohydrate probes, solution-phase parallel and enzymatic glycosylations were performed. Three model disaccharides were in parallel synthesized in solution-phase and used as carbohydrate probes for the fabrication of carbohydrate chips. Three enzymatic glycosylations on glass slides were consecutively performed to generate carbohydrate microarrays that contained the complex oligosaccharide, sialyl Le(x). Overall, these works demonstrated that carbohydrate chips could be efficiently prepared by covalent immobilization of maleimide-linked carbohydrates on the thiol-coated glass slides and applied for the high-throughput analyses of carbohydrate-protein interactions.

  18. Machine learning in computational biology to accelerate high-throughput protein expression.

    Science.gov (United States)

    Sastry, Anand; Monk, Jonathan; Tegel, Hanna; Uhlen, Mathias; Palsson, Bernhard O; Rockberg, Johan; Brunk, Elizabeth

    2017-08-15

    The Human Protein Atlas (HPA) enables the simultaneous characterization of thousands of proteins across various tissues to pinpoint their spatial location in the human body. This has been achieved through transcriptomics and high-throughput immunohistochemistry-based approaches, where over 40 000 unique human protein fragments have been expressed in E. coli. These datasets enable quantitative tracking of entire cellular proteomes and present new avenues for understanding molecular-level properties influencing expression and solubility. Combining computational biology and machine learning identifies protein properties that hinder the HPA high-throughput antibody production pipeline. We predict protein expression and solubility with accuracies of 70% and 80%, respectively, based on a subset of key properties (aromaticity, hydropathy and isoelectric point). We guide the selection of protein fragments based on these characteristics to optimize high-throughput experimentation. We present the machine learning workflow as a series of IPython notebooks hosted on GitHub (https://github.com/SBRG/Protein_ML). The workflow can be used as a template for analysis of further expression and solubility datasets. ebrunk@ucsd.edu or johanr@biotech.kth.se. Supplementary data are available at Bioinformatics online.

  19. Arabidopsis Seed Content QTL Mapping Using High-Throughput Phenotyping: The Assets of Near Infrared Spectroscopy.

    Science.gov (United States)

    Jasinski, Sophie; Lécureuil, Alain; Durandet, Monique; Bernard-Moulin, Patrick; Guerche, Philippe

    2016-01-01

    Seed storage compounds are of crucial importance for human diet, feed and industrial uses. In oleo-proteaginous species like rapeseed, seed oil and protein are the qualitative determinants that conferred economic value to the harvested seed. To date, although the biosynthesis pathways of oil and storage protein are rather well-known, the factors that determine how these types of reserves are partitioned in seeds have to be identified. With the aim of implementing a quantitative genetics approach, requiring phenotyping of 100s of plants, our first objective was to establish near-infrared reflectance spectroscopic (NIRS) predictive equations in order to estimate oil, protein, carbon, and nitrogen content in Arabidopsis seed with high-throughput level. Our results demonstrated that NIRS is a powerful non-destructive, high-throughput method to assess the content of these four major components studied in Arabidopsis seed. With this tool in hand, we analyzed Arabidopsis natural variation for these four components and illustrated that they all displayed a wide range of variation. Finally, NIRS was used in order to map QTL for these four traits using seeds from the Arabidopsis thaliana Ct-1 × Col-0 recombinant inbred line population. Some QTL co-localized with QTL previously identified, but others mapped to chromosomal regions never identified so far for such traits. This paper illustrates the usefulness of NIRS predictive equations to perform accurate high-throughput phenotyping of Arabidopsis seed content, opening new perspectives in gene identification following QTL mapping and genome wide association studies.

  20. Arabidopsis seed content QTL mapping using high-throughput phenotyping: the assets of Near Infrared Spectroscopy

    Directory of Open Access Journals (Sweden)

    Sophie Jasinski

    2016-11-01

    Full Text Available Seed storage compounds are of crucial importance for human diet, feed and industrial uses. In oleo-proteaginous species like rapeseed, seed oil and protein are the qualitative determinants that conferred economic value to the harvested seed. To date, although the biosynthesis pathways of oil and storage protein are rather well known, the factors that determine how these types of reserves are partitioned in seeds have to be identified. With the aim of implementing a quantitative genetics approach, requiring phenotyping of hundreds of plants, our first objective was to establish near-infrared reflectance spectroscopic (NIRS predictive equations in order to estimate oil, protein, carbon and nitrogen content in Arabidopsis seed with high-throughput level. Our results demonstrated that NIRS is a powerful non-destructive, high-throughput method to assess the content of these four major components studied in Arabidopsis seed. With this tool in hand, we analysed Arabidopsis natural variation for these four components and illustrated that they all displayed a wide range of variation. Finally, NIRS was used in order to map QTL for these four traits using seeds from the Arabidopsis thaliana Ct-1 x Col-0 recombinant inbred line population. Some QTL co-localised with QTL previously identified, but others mapped to chromosomal regions never identified so far for such traits. This paper illustrates the usefulness of NIRS predictive equations to perform accurate high-throughput phenotyping of Arabidopsis seed content, opening new perspectives in gene identification following QTL mapping and Genome Wide Association Studies.

  1. High-throughput mapping of cell-wall polymers within and between plants using novel microarrays

    DEFF Research Database (Denmark)

    Moller, Isabel Eva; Sørensen, Iben; Bernal Giraldo, Adriana Jimena

    2007-01-01

    We describe here a methodology that enables the occurrence of cell-wall glycans to be systematically mapped throughout plants in a semi-quantitative high-throughput fashion. The technique (comprehensive microarray polymer profiling, or CoMPP) integrates the sequential extraction of glycans from...... analysis of mutant and wild-type plants, as demonstrated here for the Arabidopsis thaliana mutants fra8, mur1 and mur3. CoMPP was also applied to Physcomitrella patens cell walls and was validated by carbohydrate linkage analysis. These data provide new insights into the structure and functions of plant...

  2. 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.

  3. Fusion genes and their discovery using high throughput sequencing.

    Science.gov (United States)

    Annala, M J; Parker, B C; Zhang, W; Nykter, M

    2013-11-01

    Fusion genes are hybrid genes that combine parts of two or more original genes. They can form as a result of chromosomal rearrangements or abnormal transcription, and have been shown to act as drivers of malignant transformation and progression in many human cancers. The biological significance of fusion genes together with their specificity to cancer cells has made them into excellent targets for molecular therapy. Fusion genes are also used as diagnostic and prognostic markers to confirm cancer diagnosis and monitor response to molecular therapies. High-throughput sequencing has enabled the systematic discovery of fusion genes in a wide variety of cancer types. In this review, we describe the history of fusion genes in cancer and the ways in which fusion genes form and affect cellular function. We also describe computational methodologies for detecting fusion genes from high-throughput sequencing experiments, and the most common sources of error that lead to false discovery of fusion genes. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  4. 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. PMID:24523863

  5. 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.

  6. A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

    Science.gov (United States)

    Todd, Douglas W; Philip, Rohit C; Niihori, Maki; Ringle, Ryan A; Coyle, Kelsey R; Zehri, Sobia F; Zabala, Leanne; Mudery, Jordan A; Francis, Ross H; Rodriguez, Jeffrey J; Jacob, Abraham

    2017-08-01

    Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

  7. A high-throughput, fully automated liquid/liquid extraction liquid chromatography/mass spectrometry method for the quantitation of a new investigational drug ABT-869 and its metabolite A-849529 in human plasma samples.

    Science.gov (United States)

    Rodila, Ramona C; Kim, Joseph C; Ji, Qin C; El-Shourbagy, Tawakol A

    2006-01-01

    ABT-869 is a novel ATP-competitive inhibitor for all the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine kinases (RTKs). It is one of the oncology drugs in development at Abbott Laboratories and has great potential for enhanced anti-tumor efficacy as well as activity in a broad range of human cancers. We report here an accurate, precise and rugged liquid chromatography/mass spectrometry (LC/MS/MS) assay for the quantitative measurement of ABT-869 and its acid metabolite A-849529. A fully automated 96-well liquid/liquid extraction method was achieved utilizing a Hamilton liquid handler. The only manual intervention required prior to LC/MS/MS injection is to transfer the 96-well plate to a drying rack to dry the extracts then transfer the plate back to the Hamilton for robotic reconstitution. The linear dynamic ranges were from 1.1 to 598.8 ng/mL for ABT-869 and from 1.1 to 605.8 ng/mL for A-849529. The coefficient of determination (r2) for all analytes was greater than 0.9995. For the drug ABT-869, the intra-assay coefficient of variance (CV) was between 0.4% and 3.7% and the inter-assay CV was between 0.9% and 2.8%. The inter-assay mean accuracy, expressed as percent of theoretical, was between 96.8% and 102.2%. For the metabolite A-849529, the intra-assay CV was between 0.5% and 5.1% and the inter-assay CV was between 0.8% and 4.9%. The inter-assay mean accuracy, expressed as percent of theoretical, was between 96.9% and 100.6%. Copyright 2006 John Wiley & Sons, Ltd.

  8. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. 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.

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

    Science.gov (United States)

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

    2016-02-01

    Advances in automated and high-throughput imaging technologies have resulted in a deluge of high-resolution images and sensor data of plants. However, extracting patterns and features from this large corpus of data requires the use of machine learning (ML) tools to enable data assimilation and feature identification for stress phenotyping. Four stages of the decision cycle in plant stress phenotyping and plant breeding activities where different ML approaches can be deployed are (i) identification, (ii) classification, (iii) quantification, and (iv) prediction (ICQP). We provide here a comprehensive overview and user-friendly taxonomy of ML tools to enable the plant community to correctly and easily apply the appropriate ML tools and best-practice guidelines for various biotic and abiotic stress traits. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. The Principals and Practice of Distributed High Throughput Computing

    CERN Multimedia

    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...

  12. High-throughput screening: update on practices and success.

    Science.gov (United States)

    Fox, Sandra; Farr-Jones, Shauna; Sopchak, Lynne; Boggs, Amy; Nicely, Helen Wang; Khoury, Richard; Biros, Michael

    2006-10-01

    High-throughput screening (HTS) has become an important part of drug discovery at most pharmaceutical and many biotechnology companies worldwide, and use of HTS technologies is expanding into new areas. Target validation, assay development, secondary screening, ADME/Tox, and lead optimization are among the areas in which there is an increasing use of HTS technologies. It is becoming fully integrated within drug discovery, both upstream and downstream, which includes increasing use of cell-based assays and high-content screening (HCS) technologies to achieve more physiologically relevant results and to find higher quality leads. In addition, HTS laboratories are continually evaluating new technologies as they struggle to increase their success rate for finding drug candidates. The material in this article is based on a 900-page HTS industry report involving 54 HTS directors representing 58 HTS laboratories and 34 suppliers.

  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.

    2017-02-01

    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. 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......, there are noteworthy differences in the less evolved species of algae as compared to land plants. The dynamic process orchestrating the deposition of these biopolymers both in algae and higher plants, is complex and highly heterogeneous, yet immensely important for the development and differentiation of the cell...... of green algae, during the development into land plants. Hence, there is a pressing need for rethinking the glycomic toolbox, by developing new and high-throughput (HTP) technology, in order to acquire information of the location and relative abundance of diverse cell wall polymers. In this dissertation...

  15. Ethoscopes: An open platform for high-throughput ethomics.

    Directory of Open Access Journals (Sweden)

    Quentin Geissmann

    2017-10-01

    Full Text Available Here, we present the use of ethoscopes, which are machines for high-throughput analysis of behavior in Drosophila and other animals. Ethoscopes provide a software and hardware solution that is reproducible and easily scalable. They perform, in real-time, tracking and profiling of behavior by using a supervised machine learning algorithm, are able to deliver behaviorally triggered stimuli to flies in a feedback-loop mode, and are highly customizable and open source. Ethoscopes can be built easily by using 3D printing technology and rely on Raspberry Pi microcomputers and Arduino boards to provide affordable and flexible hardware. All software and construction specifications are available at http://lab.gilest.ro/ethoscope.

  16. Ethoscopes: An open platform for high-throughput ethomics.

    Science.gov (United States)

    Geissmann, Quentin; Garcia Rodriguez, Luis; Beckwith, Esteban J; French, Alice S; Jamasb, Arian R; Gilestro, Giorgio F

    2017-10-01

    Here, we present the use of ethoscopes, which are machines for high-throughput analysis of behavior in Drosophila and other animals. Ethoscopes provide a software and hardware solution that is reproducible and easily scalable. They perform, in real-time, tracking and profiling of behavior by using a supervised machine learning algorithm, are able to deliver behaviorally triggered stimuli to flies in a feedback-loop mode, and are highly customizable and open source. Ethoscopes can be built easily by using 3D printing technology and rely on Raspberry Pi microcomputers and Arduino boards to provide affordable and flexible hardware. All software and construction specifications are available at http://lab.gilest.ro/ethoscope.

  17. Ethoscopes: An open platform for high-throughput ethomics

    Science.gov (United States)

    Geissmann, Quentin; Garcia Rodriguez, Luis; Beckwith, Esteban J.; French, Alice S.; Jamasb, Arian R.

    2017-01-01

    Here, we present the use of ethoscopes, which are machines for high-throughput analysis of behavior in Drosophila and other animals. Ethoscopes provide a software and hardware solution that is reproducible and easily scalable. They perform, in real-time, tracking and profiling of behavior by using a supervised machine learning algorithm, are able to deliver behaviorally triggered stimuli to flies in a feedback-loop mode, and are highly customizable and open source. Ethoscopes can be built easily by using 3D printing technology and rely on Raspberry Pi microcomputers and Arduino boards to provide affordable and flexible hardware. All software and construction specifications are available at http://lab.gilest.ro/ethoscope. PMID:29049280

  18. High-throughput drawing and testing of metallic glass nanostructures.

    Science.gov (United States)

    Hasan, Molla; Kumar, Golden

    2017-03-02

    Thermoplastic embossing of metallic glasses promises direct imprinting of metal nanostructures using templates. However, embossing high-aspect-ratio nanostructures faces unworkable flow resistance due to friction and non-wetting conditions at the template interface. Herein, we show that these inherent challenges of embossing can be reversed by thermoplastic drawing using templates. The flow resistance not only remains independent of wetting but also decreases with increasing feature aspect-ratio. Arrays of assembled nanotips, nanowires, and nanotubes with aspect-ratios exceeding 1000 can be produced through controlled elongation and fracture of metallic glass structures. In contrast to embossing, the drawing approach generates two sets of nanostructures upon final fracture; one set remains anchored to the metallic glass substrate while the second set is assembled on the template. This method can be readily adapted for high-throughput fabrication and testing of nanoscale tensile specimens, enabling rapid screening of size-effects in mechanical behavior.

  19. Statistically invalid classification of high throughput gene expression data

    Science.gov (United States)

    Barbash, Shahar; Soreq, Hermona

    2013-01-01

    Classification analysis based on high throughput data is a common feature in neuroscience and other fields of science, with a rapidly increasing impact on both basic biology and disease-related studies. The outcome of such classifications often serves to delineate novel biochemical mechanisms in health and disease states, identify new targets for therapeutic interference, and develop innovative diagnostic approaches. Given the importance of this type of studies, we screened 111 recently-published high-impact manuscripts involving classification analysis of gene expression, and found that 58 of them (53%) based their conclusions on a statistically invalid method which can lead to bias in a statistical sense (lower true classification accuracy then the reported classification accuracy). In this report we characterize the potential methodological error and its scope, investigate how it is influenced by different experimental parameters, and describe statistically valid methods for avoiding such classification mistakes. PMID:23346359

  20. High throughput sequencing of microRNAs in chicken somites.

    Science.gov (United States)

    Rathjen, Tina; Pais, Helio; Sweetman, Dylan; Moulton, Vincent; Munsterberg, Andrea; Dalmay, Tamas

    2009-05-06

    High throughput Solexa sequencing technology was applied to identify microRNAs in somites of developing chicken embryos. We obtained 651,273 reads, from which 340,415 were mapped to the chicken genome representing 1701 distinct sequences. Eighty-five of these were known microRNAs and 42 novel miRNA candidates were identified. Accumulation of 18 of 42 sequences was confirmed by Northern blot analysis. Ten of the 18 sequences are new variants of known miRNAs and eight short RNAs are novel miRNAs. Six of these eight have not been reported by other deep sequencing projects. One of the six new miRNAs is highly enriched in somite tissue suggesting that deep sequencing of other specific tissues has the potential to identify novel tissue specific miRNAs.

  1. 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.

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

    Science.gov (United States)

    Xin, Zhanguo; Chen, Junping

    2012-07-28

    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. 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. 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.

  3. High-throughput phenotyping of seminal root traits in wheat.

    Science.gov (United States)

    Richard, Cecile Ai; Hickey, Lee T; Fletcher, Susan; Jennings, Raeleen; Chenu, Karine; Christopher, Jack T

    2015-01-01

    Water availability is a major limiting factor for wheat (Triticum aestivum L.) production in rain-fed agricultural systems worldwide. Root system architecture has important functional implications for the timing and extent of soil water extraction, yet selection for root architectural traits in breeding programs has been limited by a lack of suitable phenotyping methods. The aim of this research was to develop low-cost high-throughput phenotyping methods to facilitate selection for desirable root architectural traits. Here, we report two methods, one using clear pots and the other using growth pouches, to assess the angle and the number of seminal roots in wheat seedlings- two proxy traits associated with the root architecture of mature wheat plants. Both methods revealed genetic variation for seminal root angle and number in the panel of 24 wheat cultivars. The clear pot method provided higher heritability and higher genetic correlations across experiments compared to the growth pouch method. In addition, the clear pot method was more efficient - requiring less time, space, and labour compared to the growth pouch method. Therefore the clear pot method was considered the most suitable for large-scale and high-throughput screening of seedling root characteristics in crop improvement programs. The clear-pot method could be easily integrated in breeding programs targeting drought tolerance to rapidly enrich breeding populations with desirable alleles. For instance, selection for narrow root angle and high number of seminal roots could lead to deeper root systems with higher branching at depth. Such root characteristics are highly desirable in wheat to cope with anticipated future climate conditions, particularly where crops rely heavily on stored soil moisture at depth, including some Australian, Indian, South American, and African cropping regions.

  4. High-Throughput Genomics Enhances Tomato Breeding Efficiency

    Science.gov (United States)

    Barone, A; Di Matteo, A; Carputo, D; Frusciante, L

    2009-01-01

    Tomato (Solanum lycopersicum) is considered a model plant species for a group of economically important crops, such as potato, pepper, eggplant, since it exhibits a reduced genomic size (950 Mb), a short generation time, and routine transformation technologies. Moreover, it shares with the other Solanaceous plants the same haploid chromosome number and a high level of conserved genomic organization. Finally, many genomic and genetic resources are actually available for tomato, and the sequencing of its genome is in progress. These features make tomato an ideal species for theoretical studies and practical applications in the genomics field. The present review describes how structural genomics assist the selection of new varieties resistant to pathogens that cause damage to this crop. Many molecular markers highly linked to resistance genes and cloned resistance genes are available and could be used for a high-throughput screening of multiresistant varieties. Moreover, a new genomics-assisted breeding approach for improving fruit quality is presented and discussed. It relies on the identification of genetic mechanisms controlling the trait of interest through functional genomics tools. Following this approach, polymorphisms in major gene sequences responsible for variability in the expression of the trait under study are then exploited for tracking simultaneously favourable allele combinations in breeding programs using high-throughput genomic technologies. This aims at pyramiding in the genetic background of commercial cultivars alleles that increase their performances. In conclusion, tomato breeding strategies supported by advanced technologies are expected to target increased productivity and lower costs of improved genotypes even for complex traits. PMID:19721805

  5. A homogeneous, high-throughput fluorescence anisotropy-based DNA supercoiling assay.

    Science.gov (United States)

    Shapiro, Adam; Jahic, Haris; Prasad, Swati; Ehmann, David; Thresher, Jason; Gao, Ning; Hajec, Laurel

    2010-10-01

    The degree of supercoiling of DNA is vital for cellular processes, such as replication and transcription. DNA topology is controlled by the action of DNA topoisomerase enzymes. Topoisomerases, because of their importance in cellular replication, are the targets of several anticancer and antibacterial drugs. In the search for new drugs targeting topoisomerases, a biochemical assay compatible with automated high-throughput screening (HTS) would be valuable. Gel electrophoresis is the standard method for measuring changes in the extent of supercoiling of plasmid DNA when acted upon by topoisomerases, but this is a low-throughput and laborious method. A medium-throughput method was described previously that quantitatively distinguishes relaxed and supercoiled plasmids by the difference in their abilities to form triplex structures with an immobilized oligonucleotide. In this article, the authors describe a homogeneous supercoiling assay based on triplex formation in which the oligonucleotide strand is labeled with a fluorescent dye and the readout is fluorescence anisotropy. The new assay requires no immobilization, filtration, or plate washing steps and is therefore well suited to HTS for inhibitors of topoisomerases. The utility of this assay is demonstrated with relaxation of supercoiled plasmid by Escherichia coli topoisomerase I, supercoiling of relaxed plasmid by E. coli DNA gyrase, and inhibition of gyrase by fluoroquinolones and nalidixic acid.

  6. Surrogate-assisted feature extraction for high-throughput phenotyping.

    Science.gov (United States)

    Yu, Sheng; Chakrabortty, Abhishek; Liao, Katherine P; Cai, Tianrun; Ananthakrishnan, Ashwin N; Gainer, Vivian S; Churchill, Susanne E; Szolovits, Peter; Murphy, Shawn N; Kohane, Isaac S; Cai, Tianxi

    2017-04-01

    Phenotyping algorithms are capable of accurately identifying patients with specific phenotypes from within electronic medical records systems. However, developing phenotyping algorithms in a scalable way remains a challenge due to the extensive human resources required. This paper introduces a high-throughput unsupervised feature selection method, which improves the robustness and scalability of electronic medical record phenotyping without compromising its accuracy. The proposed Surrogate-Assisted Feature Extraction (SAFE) method selects candidate features from a pool of comprehensive medical concepts found in publicly available knowledge sources. The target phenotype's International Classification of Diseases, Ninth Revision and natural language processing counts, acting as noisy surrogates to the gold-standard labels, are used to create silver-standard labels. Candidate features highly predictive of the silver-standard labels are selected as the final features. Algorithms were trained to identify patients with coronary artery disease, rheumatoid arthritis, Crohn's disease, and ulcerative colitis using various numbers of labels to compare the performance of features selected by SAFE, a previously published automated feature extraction for phenotyping procedure, and domain experts. The out-of-sample area under the receiver operating characteristic curve and F -score from SAFE algorithms were remarkably higher than those from the other two, especially at small label sizes. SAFE advances high-throughput phenotyping methods by automatically selecting a succinct set of informative features for algorithm training, which in turn reduces overfitting and the needed number of gold-standard labels. SAFE also potentially identifies important features missed by automated feature extraction for phenotyping or experts.

  7. 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

  8. 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 oomycete...... agricultural fields in Denmark, and 11 samples from carrot tissue with symptoms of Pythium infection. Sequence data from the Pythium and Phytophthora mock communities showed that our strategy successfully detected all included species. Taxonomic assignments of OTUs from 26 soil sample showed that 95...... the usefulness of the method not only in soil DNA but also in a plant DNA background. In conclusion, we demonstrate a successful approach for pyrosequencing of oomycete communities using ITS1 as the barcode sequence with well-known primers for oomycete DNA amplification....

  9. 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.

  10. High throughput parametric studies of the structure of complex nanomaterials

    Science.gov (United States)

    Tian, Peng

    The structure of nanoscale materials is difficult to study because crystallography, the gold-standard for structure studies, no longer works at the nanoscale. New tools are needed to study nanostructure. Furthermore, it is important to study the evolution of nanostructure of complex nanostructured materials as a function of various parameters such as temperature or other environmental variables. These are called parametric studies because an environmental parameter is being varied. This means that the new tools for studying nanostructure also need to be extended to work quickly and on large numbers of datasets. This thesis describes the development of new tools for high throughput studies of complex and nanostructured materials, and their application to study the structural evolution of bulk, and nanoparticles of, MnAs as a function of temperature. The tool for high throughput analysis of the bulk material was developed as part of this PhD thesis work and is called SrRietveld. A large part of making a new tool is to validate it and we did this for SrRietveld by carrying out a high-throughput study of uncertainties coming from the program using different ways of estimating the uncertainty. This tool was applied to study structural changes in MnAs as a function of temperature. We were also interested in studying different MnAs nanoparticles fabricated through different methods because of their applications in information storage. PDFgui, an existing tool for analyzing nanoparticles using Pair distribution function (PDF) refinement, was used in these cases. Comparing the results from the analysis by SrRietveld and PDFgui, we got more comprehensive structure information about MnAs. The layout of the thesis is as follows. First, the background knowledge about material structures is given. The conventional crystallographic analysis is introduced in both theoretical and practical ways. For high throughput study, the next-generation Rietveld analysis program: Sr

  11. High-Throughput Automation in Chemical Process Development.

    Science.gov (United States)

    Selekman, Joshua A; Qiu, Jun; Tran, Kristy; Stevens, Jason; Rosso, Victor; Simmons, Eric; Xiao, Yi; Janey, Jacob

    2017-06-07

    High-throughput (HT) techniques built upon laboratory automation technology and coupled to statistical experimental design and parallel experimentation have enabled the acceleration of chemical process development across multiple industries. HT technologies are often applied to interrogate wide, often multidimensional experimental spaces to inform the design and optimization of any number of unit operations that chemical engineers use in process development. In this review, we outline the evolution of HT technology and provide a comprehensive overview of how HT automation is used throughout different industries, with a particular focus on chemical and pharmaceutical process development. In addition, we highlight the common strategies of how HT automation is incorporated into routine development activities to maximize its impact in various academic and industrial settings.

  12. 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.

  13. High-Throughput Mass Spectrometry Applied to Structural Genomics

    Directory of Open Access Journals (Sweden)

    Rod Chalk

    2014-10-01

    Full Text Available Mass spectrometry (MS remains under-utilized for the analysis of expressed proteins because it is inaccessible to the non-specialist, and sample-turnaround from service labs is slow. Here, we describe 3.5 min Liquid-Chromatography (LC-MS and 16 min LC-MSMS methods which are tailored to validation and characterization of recombinant proteins in a high throughput structural biology pipeline. We illustrate the type and scope of MS data typically obtained from a 96-well expression and purification test for both soluble and integral membrane proteins (IMPs, and describe their utility in the selection of constructs for scale-up structural work, leading to cost and efficiency savings. We propose that value of MS data lies in how quickly it becomes available and that this can fundamentally change the way in which it is used.

  14. Applications of High-Throughput Nucleotide Sequencing (PhD)

    DEFF Research Database (Denmark)

    Waage, Johannes

    The recent advent of high throughput sequencing of nucleic acids (RNA and DNA) has vastly expanded research into the functional and structural biology of the genome of all living organisms (and even a few dead ones). With this enormous and exponential growth in biological data generation come...... equally large demands in data handling, analysis and interpretation, perhaps defining the modern challenge of the computational biologist of the post-genomic era. The first part of this thesis consists of a general introduction to the history, common terms and challenges of next generation sequencing......). For the second flavor, DNA-seq, a study presenting genome wide profiling of transcription factor CEBP/A in liver cells undergoing regeneration after partial hepatectomy (article IV) is included....

  15. Automated high-throughput behavioral analyses in zebrafish larvae.

    Science.gov (United States)

    Richendrfer, Holly; Créton, Robbert

    2013-07-04

    We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

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

    Science.gov (United States)

    Wu, Henry; Mayeshiba, Tam; Morgan, Dane

    2016-01-01

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

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

    DEFF Research Database (Denmark)

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

    RNAs with known killing effects as a model system to demonstrate that RPPA-based protein quantification can serve as substitute readout of cell viability, hereby reliably reflecting toxicity. In terms of automation, cell exposure, protein harvest, serial dilution and sample reformatting were performed using...... 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...

  18. A High-Throughput Antibody-Based Microarray Typing Platform

    Science.gov (United States)

    Andrew, Gehring; Charles, Barnett; Chu, Ted; DebRoy, Chitrita; D'Souza, Doris; Eaker, Shannon; Fratamico, Pina; Gillespie, Barbara; Hegde, Narasimha; Jones, Kevin; Lin, Jun; Oliver, Stephen; Paoli, George; Perera, Ashan; Uknalis, Joseph

    2013-01-01

    Many rapid methods have been developed for screening foods for the presence of pathogenic microorganisms. Rapid methods that have the additional ability to identify microorganisms via multiplexed immunological recognition have the potential for classification or typing of microbial contaminants thus facilitating epidemiological investigations that aim to identify outbreaks and trace back the contamination to its source. This manuscript introduces a novel, high throughput typing platform that employs microarrayed multiwell plate substrates and laser-induced fluorescence of the nucleic acid intercalating dye/stain SYBR Gold for detection of antibody-captured bacteria. The aim of this study was to use this platform for comparison of different sets of antibodies raised against the same pathogens as well as demonstrate its potential effectiveness for serotyping. To that end, two sets of antibodies raised against each of the “Big Six” non-O157 Shiga toxin-producing E. coli (STEC) as well as E. coli O157:H7 were array-printed into microtiter plates, and serial dilutions of the bacteria were added and subsequently detected. Though antibody specificity was not sufficient for the development of an STEC serotyping method, the STEC antibody sets performed reasonably well exhibiting that specificity increased at lower capture antibody concentrations or, conversely, at lower bacterial target concentrations. The favorable results indicated that with sufficiently selective and ideally concentrated sets of biorecognition elements (e.g., antibodies or aptamers), this high-throughput platform can be used to rapidly type microbial isolates derived from food samples within ca. 80 min of total assay time. It can also potentially be used to detect the pathogens from food enrichments and at least serve as a platform for testing antibodies. PMID:23645110

  19. 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.

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

    Science.gov (United States)

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

    2016-09-01

    Tape-lifting has since its introduction in the early 2000's become a well-established sampling method in forensic DNA analysis. Sampling is quick and straightforward while the following DNA extraction is more challenging due to the "stickiness", rigidity and size of the tape. We have developed, validated and implemented a simple and efficient direct lysis DNA extraction protocol for adhesive tapes that requires limited manual labour. The method uses Chelex beads and is applied with SceneSafe FAST tape. This direct lysis protocol provided higher mean DNA yields than PrepFiler Express BTA on Automate Express, although the differences were not significant when using clothes worn in a controlled fashion as reference material (p=0.13 and p=0.34 for T-shirts and button-down shirts, respectively). Through in-house validation we show that the method is fit-for-purpose for application in casework, as it provides high DNA yields and amplifiability, as well as good reproducibility and DNA extract stability. After implementation in casework, the proportion of extracts with DNA concentrations above 0.01ng/μL increased from 71% to 76%. Apart from providing higher DNA yields compared with the previous method, the introduction of the developed direct lysis protocol also reduced the amount of manual labour by half and doubled the potential throughput for tapes at the laboratory. Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions when the aim is to enable high-throughput DNA extraction of complex crime scene samples. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  1. A bioimage informatics platform for high-throughput embryo phenotyping.

    Science.gov (United States)

    Brown, James M; Horner, Neil R; Lawson, Thomas N; Fiegel, Tanja; Greenaway, Simon; Morgan, Hugh; Ring, Natalie; Santos, Luis; Sneddon, Duncan; Teboul, Lydia; Vibert, Jennifer; Yaikhom, Gagarine; Westerberg, Henrik; Mallon, Ann-Marie

    2018-01-01

    High-throughput phenotyping is a cornerstone of numerous functional genomics projects. In recent years, imaging screens have become increasingly important in understanding gene-phenotype relationships in studies of cells, tissues and whole organisms. Three-dimensional (3D) imaging has risen to prominence in the field of developmental biology for its ability to capture whole embryo morphology and gene expression, as exemplified by the International Mouse Phenotyping Consortium (IMPC). Large volumes of image data are being acquired by multiple institutions around the world that encompass a range of modalities, proprietary software and metadata. To facilitate robust downstream analysis, images and metadata must be standardized to account for these differences. As an open scientific enterprise, making the data readily accessible is essential so that members of biomedical and clinical research communities can study the images for themselves without the need for highly specialized software or technical expertise. In this article, we present a platform of software tools that facilitate the upload, analysis and dissemination of 3D images for the IMPC. Over 750 reconstructions from 80 embryonic lethal and subviable lines have been captured to date, all of which are openly accessible at mousephenotype.org. Although designed for the IMPC, all software is available under an open-source licence for others to use and develop further. Ongoing developments aim to increase throughput and improve the analysis and dissemination of image data. Furthermore, we aim to ensure that images are searchable so that users can locate relevant images associated with genes, phenotypes or human diseases of interest. © The Author 2016. Published by Oxford University Press.

  2. 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

  3. High-throughput detection, genotyping and quantification of the human papillomavirus using real-time PCR.

    Science.gov (United States)

    Micalessi, Isabel M; Boulet, Gaëlle A V; Bogers, Johannes J; Benoy, Ina H; Depuydt, Christophe E

    2011-12-20

    The establishment of the causal relationship between high-risk human papillomavirus (HR-HPV) infection and cervical cancer and its precursors has resulted in the development of HPV DNA detection systems. Currently, real-time PCR assays for the detection of HPV, such as the RealTime High Risk (HR) HPV assay (Abbott) and the cobas® 4800 HPV Test (Roche Molecular Diagnostics) are commercially available. However, none of them enables the detection and typing of all HR-HPV types in a clinical high-throughput setting. This paper describes the laboratory workflow and the validation of a type-specific real-time quantitative PCR (qPCR) assay for high-throughput HPV detection, genotyping and quantification. This assay is routinely applied in a liquid-based cytology screening setting (700 samples in 24 h) and was used in many epidemiological and clinical studies. The TaqMan-based qPCR assay enables the detection of 17 HPV genotypes and β-globin in seven multiplex reactions. These HPV types include all 12 high-risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59), three probably high-risk types (HPV53, 66 and 68), one low-risk type (HPV6) and one undetermined risk type (HPV67). An analytical sensitivity of ≤100 copies was obtained for all the HPV types. The analytical specificity of each primer pair was 100% and an intra- and inter-run variability of real-time PCR approach enables detection of 17 HPV types, identification of the HPV type and determination of the viral load in a single sensitive assay suitable for high-throughput screening.

  4. Emerging metrology for high-throughput nanomaterial genotoxicology.

    Science.gov (United States)

    Nelson, Bryant C; Wright, Christa W; Ibuki, Yuko; Moreno-Villanueva, Maria; Karlsson, Hanna L; Hendriks, Giel; Sims, Christopher M; Singh, Neenu; Doak, Shareen H

    2017-01-01

    The rapid development of the engineered nanomaterial (ENM) manufacturing industry has accelerated the incorporation of ENMs into a wide variety of consumer products across the globe. Unintentionally or not, some of these ENMs may be introduced into the environment or come into contact with humans or other organisms resulting in unexpected biological effects. It is thus prudent to have rapid and robust analytical metrology in place that can be used to critically assess and/or predict the cytotoxicity, as well as the potential genotoxicity of these ENMs. Many of the traditional genotoxicity test methods [e.g. unscheduled DNA synthesis assay, bacterial reverse mutation (Ames) test, etc.,] for determining the DNA damaging potential of chemical and biological compounds are not suitable for the evaluation of ENMs, due to a variety of methodological issues ranging from potential assay interferences to problems centered on low sample throughput. Recently, a number of sensitive, high-throughput genotoxicity assays/platforms (CometChip assay, flow cytometry/micronucleus assay, flow cytometry/γ-H2AX assay, automated 'Fluorimetric Detection of Alkaline DNA Unwinding' (FADU) assay, ToxTracker reporter assay) have been developed, based on substantial modifications and enhancements of traditional genotoxicity assays. These new assays have been used for the rapid measurement of DNA damage (strand breaks), chromosomal damage (micronuclei) and for detecting upregulated DNA damage signalling pathways resulting from ENM exposures. In this critical review, we describe and discuss the fundamental measurement principles and measurement endpoints of these new assays, as well as the modes of operation, analytical metrics and potential interferences, as applicable to ENM exposures. An unbiased discussion of the major technical advantages and limitations of each assay for evaluating and predicting the genotoxic potential of ENMs is also provided. Published by Oxford University Press on

  5. High-Throughput Printing Process for Flexible Electronics

    Science.gov (United States)

    Hyun, Woo Jin

    Printed electronics is an emerging field for manufacturing electronic devices with low cost and minimal material waste for a variety of applications including displays, distributed sensing, smart packaging, and energy management. Moreover, its compatibility with roll-to-roll production formats and flexible substrates is desirable for continuous, high-throughput production of flexible electronics. Despite the promise, however, the roll-to-roll production of printed electronics is quite challenging due to web movement hindering accurate ink registration and high-fidelity printing. In this talk, I will present a promising strategy for roll-to-roll production using a novel printing process that we term SCALE (Self-aligned Capillarity-Assisted Lithography for Electronics). By utilizing capillarity of liquid inks on nano/micro-structured substrates, the SCALE process facilitates high-resolution and self-aligned patterning of electrically functional inks with greatly improved printing tolerance. I will show the fabrication of key building blocks (e.g. transistor, resistor, capacitor) for electronic circuits using the SCALE process on plastics.

  6. 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...

  7. The complete automation of cell culture: improvements for high-throughput and high-content screening.

    Science.gov (United States)

    Jain, Shushant; Sondervan, David; Rizzu, Patrizia; Bochdanovits, Zoltan; Caminada, Daniel; Heutink, Peter

    2011-09-01

    Genomic approaches provide enormous amounts of raw data with regard to genetic variation, the diversity of RNA species, and protein complement. High-throughput (HT) and high-content (HC) cellular screens are ideally suited to contextualize the information gathered from other "omic" approaches into networks and can be used for the identification of therapeutic targets. Current methods used for HT-HC screens are laborious, time-consuming, and prone to human error. The authors thus developed an automated high-throughput system with an integrated fluorescent imager for HC screens called the AI.CELLHOST. The implementation of user-defined culturing and assay plate setup parameters allows parallel operation of multiple screens in diverse mammalian cell types. The authors demonstrate that such a system is able to successfully maintain different cell lines in culture for extended periods of time as well as significantly increasing throughput, accuracy, and reproducibility of HT and HC screens.

  8. 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

  9. High-throughput literature mining to support read-across ...

    Science.gov (United States)

    Building scientific confidence in the development and evaluation of read-across remains an ongoing challenge. Approaches include establishing systematic frameworks to identify sources of uncertainty and ways to address them. One source of uncertainty is related to characterizing biological similarity. Many research efforts are underway such as structuring mechanistic data in adverse outcome pathways and investigating the utility of high throughput (HT)/high content (HC) screening data. A largely untapped resource for read-across to date is the biomedical literature. This information has the potential to support read-across by facilitating the identification of valid source analogues with similar biological and toxicological profiles as well as providing the mechanistic understanding for any prediction made. A key challenge in using biomedical literature is to convert and translate its unstructured form into a computable format that can be linked to chemical structure. We developed a novel text-mining strategy to represent literature information for read across. Keywords were used to organize literature into toxicity signatures at the chemical level. These signatures were integrated with HT in vitro data and curated chemical structures. A rule-based algorithm assessed the strength of the literature relationship, providing a mechanism to rank and visualize the signature as literature ToxPIs (LitToxPIs). LitToxPIs were developed for over 6,000 chemicals for a varie

  10. 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.

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

    Science.gov (United States)

    List, Markus; Elnegaard, Marlene Pedersen; Schmidt, Steffen; Christiansen, Helle; Tan, Qihua; Mollenhauer, Jan; Baumbach, Jan

    2017-02-01

    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 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 to be serially diluted before they can be used as assay plates. This process, however, leads to an explosion in the number of plates and samples to be tracked. Here, we present SAVANAH, the first tool to effectively manage molecular screening libraries across dilution series. It conveniently links (connects) sample information from the library to experimental results from the assay plates. All results can be exported to the R statistical environment or piped into HiTSeekR ( http://hitseekr.compbio.sdu.dk ) for comprehensive follow-up analyses. In summary, SAVANAH supports the HTS community in managing and analyzing HTS experiments with an emphasis on serially diluted molecular libraries.

  12. High-Throughput Identification of Antimicrobial Peptides from Amphibious Mudskippers.

    Science.gov (United States)

    Yi, Yunhai; You, Xinxin; Bian, Chao; Chen, Shixi; Lv, Zhao; Qiu, Limei; Shi, Qiong

    2017-11-22

    Widespread existence of antimicrobial peptides (AMPs) has been reported in various animals with comprehensive biological activities, which is consistent with the important roles of AMPs as the first line of host defense system. However, no big-data-based analysis on AMPs from any fish species is available. In this study, we identified 507 AMP transcripts on the basis of our previously reported genomes and transcriptomes of two representative amphibious mudskippers, Boleophthalmus pectinirostris (BP) and Periophthalmus magnuspinnatus (PM). The former is predominantly aquatic with less time out of water, while the latter is primarily terrestrial with extended periods of time on land. Within these identified AMPs, 449 sequences are novel; 15 were reported in BP previously; 48 are identically overlapped between BP and PM; 94 were validated by mass spectrometry. Moreover, most AMPs presented differential tissue transcription patterns in the two mudskippers. Interestingly, we discovered two AMPs, hemoglobin β1 and amylin, with high inhibitions on Micrococcus luteus. In conclusion, our high-throughput screening strategy based on genomic and transcriptomic data opens an efficient pathway to discover new antimicrobial peptides for ongoing development of marine drugs.

  13. High-Throughput Identification of Antimicrobial Peptides from Amphibious Mudskippers

    Directory of Open Access Journals (Sweden)

    Yunhai Yi

    2017-11-01

    Full Text Available Widespread existence of antimicrobial peptides (AMPs has been reported in various animals with comprehensive biological activities, which is consistent with the important roles of AMPs as the first line of host defense system. However, no big-data-based analysis on AMPs from any fish species is available. In this study, we identified 507 AMP transcripts on the basis of our previously reported genomes and transcriptomes of two representative amphibious mudskippers, Boleophthalmus pectinirostris (BP and Periophthalmus magnuspinnatus (PM. The former is predominantly aquatic with less time out of water, while the latter is primarily terrestrial with extended periods of time on land. Within these identified AMPs, 449 sequences are novel; 15 were reported in BP previously; 48 are identically overlapped between BP and PM; 94 were validated by mass spectrometry. Moreover, most AMPs presented differential tissue transcription patterns in the two mudskippers. Interestingly, we discovered two AMPs, hemoglobin β1 and amylin, with high inhibitions on Micrococcus luteus. In conclusion, our high-throughput screening strategy based on genomic and transcriptomic data opens an efficient pathway to discover new antimicrobial peptides for ongoing development of marine drugs.

  14. 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.

  15. Validation of high throughput sequencing and microbial forensics applications.

    Science.gov (United States)

    Budowle, Bruce; Connell, Nancy D; Bielecka-Oder, Anna; Colwell, Rita R; Corbett, Cindi R; Fletcher, Jacqueline; Forsman, Mats; Kadavy, Dana R; Markotic, Alemka; Morse, Stephen A; Murch, Randall S; Sajantila, Antti; Schmedes, Sarah E; Ternus, Krista L; Turner, Stephen D; Minot, Samuel

    2014-01-01

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

  16. SNP-PHAGE – High throughput SNP discovery pipeline

    Directory of Open Access Journals (Sweden)

    Cregan Perry B

    2006-10-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs as defined here are single base sequence changes or short insertion/deletions between or within individuals of a given species. As a result of their abundance and the availability of high throughput analysis technologies SNP markers have begun to replace other traditional markers such as restriction fragment length polymorphisms (RFLPs, amplified fragment length polymorphisms (AFLPs and simple sequence repeats (SSRs or microsatellite markers for fine mapping and association studies in several species. For SNP discovery from chromatogram data, several bioinformatics programs have to be combined to generate an analysis pipeline. Results have to be stored in a relational database to facilitate interrogation through queries or to generate data for further analyses such as determination of linkage disequilibrium and identification of common haplotypes. Although these tasks are routinely performed by several groups, an integrated open source SNP discovery pipeline that can be easily adapted by new groups interested in SNP marker development is currently unavailable. Results We developed SNP-PHAGE (SNP discovery Pipeline with additional features for identification of common haplotypes within a sequence tagged site (Haplotype Analysis and GenBank (-dbSNP submissions. This tool was applied for analyzing sequence traces from diverse soybean genotypes to discover over 10,000 SNPs. This package was developed on UNIX/Linux platform, written in Perl and uses a MySQL database. Scripts to generate a user-friendly web interface are also provided with common queries for preliminary data analysis. A machine learning tool developed by this group for increasing the efficiency of SNP discovery is integrated as a part of this package as an optional feature. The SNP-PHAGE package is being made available open source at http://bfgl.anri.barc.usda.gov/ML/snp-phage/. Conclusion SNP-PHAGE provides a bioinformatics

  17. The Complete Automation of Cell Culture: Improvements for High-Throughput and High-Content Screening

    NARCIS (Netherlands)

    Jain, S.; Sondervan, D.; Rizzu, P.; Bochdanovits, Z.; Caminada, D.; Heutink, P.

    2011-01-01

    Genomic approaches provide enormous amounts of raw data with regard to genetic variation, the diversity of RNA species, and protein complement. high-throughput (HT) and high-content (HC) cellular screens are ideally suited to contextualize the information gathered from other "omic" approaches into

  18. High-throughput ocular artifact reduction in multichannel electroencephalography (EEG) using component subspace projection.

    Science.gov (United States)

    Ma, Junshui; Bayram, Sevinç; Tao, Peining; Svetnik, Vladimir

    2011-03-15

    After a review of the ocular artifact reduction literature, a high-throughput method designed to reduce the ocular artifacts in multichannel continuous EEG recordings acquired at clinical EEG laboratories worldwide is proposed. The proposed method belongs to the category of component-based methods, and does not rely on any electrooculography (EOG) signals. Based on a concept that all ocular artifact components exist in a signal component subspace, the method can uniformly handle all types of ocular artifacts, including eye-blinks, saccades, and other eye movements, by automatically identifying ocular components from decomposed signal components. This study also proposes an improved strategy to objectively and quantitatively evaluate artifact reduction methods. The evaluation strategy uses real EEG signals to synthesize realistic simulated datasets with different amounts of ocular artifacts. The simulated datasets enable us to objectively demonstrate that the proposed method outperforms some existing methods when no high-quality EOG signals are available. Moreover, the results of the simulated datasets improve our understanding of the involved signal decomposition algorithms, and provide us with insights into the inconsistency regarding the performance of different methods in the literature. The proposed method was also applied to two independent clinical EEG datasets involving 28 volunteers and over 1000 EEG recordings. This effort further confirms that the proposed method can effectively reduce ocular artifacts in large clinical EEG datasets in a high-throughput fashion. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. High-throughput physiological phenotyping and screening system for the characterization of plant-environment interactions.

    Science.gov (United States)

    Halperin, Ofer; Gebremedhin, Alem; Wallach, Rony; Moshelion, Menachem

    2017-02-01

    We present a simple and effective high-throughput experimental platform for simultaneous and continuous monitoring of water relations in the soil-plant-atmosphere continuum of numerous plants under dynamic environmental conditions. This system provides a simultaneously measured, detailed physiological response profile for each plant in the array, over time periods ranging from a few minutes to the entire growing season, under normal, stress and recovery conditions and at any phenological stage. Three probes for each pot in the array and a specially designed algorithm enable detailed water-relations characterization of whole-plant transpiration, biomass gain, stomatal conductance and root flux. They also enable quantitative calculation of the whole plant water-use efficiency and relative water content at high resolution under dynamic soil and atmospheric conditions. The system has no moving parts and can fit into many growing environments. A screening of 65 introgression lines of a wild tomato species (Solanum pennellii) crossed with cultivated tomato (S. lycopersicum), using our system and conventional gas-exchange tools, confirmed the accuracy of the system as well as its diagnostic capabilities. The use of this high-throughput diagnostic screening method is discussed in light of the gaps in our understanding of the genetic regulation of whole-plant performance, particularly under abiotic stress. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  20. Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

    Science.gov (United States)

    Gaber, Rok; Majerle, Andreja; Jerala, Roman; Benčina, Mojca

    2013-01-01

    To effectively fight against the human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity. PMID:24287545

  1. SmartGrain: high-throughput phenotyping software for measuring seed shape through image analysis.

    Science.gov (United States)

    Tanabata, Takanari; Shibaya, Taeko; Hori, Kiyosumi; Ebana, Kaworu; Yano, Masahiro

    2012-12-01

    Seed shape and size are among the most important agronomic traits because they affect yield and market price. To obtain accurate seed size data, a large number of measurements are needed because there is little difference in size among seeds from one plant. To promote genetic analysis and selection for seed shape in plant breeding, efficient, reliable, high-throughput seed phenotyping methods are required. We developed SmartGrain software for high-throughput measurement of seed shape. This software uses a new image analysis method to reduce the time taken in the preparation of seeds and in image capture. Outlines of seeds are automatically recognized from digital images, and several shape parameters, such as seed length, width, area, and perimeter length, are calculated. To validate the software, we performed a quantitative trait locus (QTL) analysis for rice (Oryza sativa) seed shape using backcrossed inbred lines derived from a cross between japonica cultivars Koshihikari and Nipponbare, which showed small differences in seed shape. SmartGrain removed areas of awns and pedicels automatically, and several QTLs were detected for six shape parameters. The allelic effect of a QTL for seed length detected on chromosome 11 was confirmed in advanced backcross progeny; the cv Nipponbare allele increased seed length and, thus, seed weight. High-throughput measurement with SmartGrain reduced sampling error and made it possible to distinguish between lines with small differences in seed shape. SmartGrain could accurately recognize seed not only of rice but also of several other species, including Arabidopsis (Arabidopsis thaliana). The software is free to researchers.

  2. AHT-ChIP-seq: a completely automated robotic protocol for high-throughput chromatin immunoprecipitation.

    Science.gov (United States)

    Aldridge, Sarah; Watt, Stephen; Quail, Michael A; Rayner, Tim; Lukk, Margus; Bimson, Michael F; Gaffney, Daniel; Odom, Duncan T

    2013-11-07

    ChIP-seq is an established manually-performed method for identifying DNA-protein interactions genome-wide. Here, we describe a protocol for automated high-throughput (AHT) ChIP-seq. To demonstrate the quality of data obtained using AHT-ChIP-seq, we applied it to five proteins in mouse livers using a single 96-well plate, demonstrating an extremely high degree of qualitative and quantitative reproducibility among biological and technical replicates. We estimated the optimum and minimum recommended cell numbers required to perform AHT-ChIP-seq by running an additional plate using HepG2 and MCF7 cells. With this protocol, commercially available robotics can perform four hundred experiments in five days.

  3. 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

  4. 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....

  5. High-throughput and computational approaches for diagnostic and prognostic host tuberculosis biomarkers

    Directory of Open Access Journals (Sweden)

    January Weiner

    2017-03-01

    Full Text Available High-throughput techniques strive to identify new biomarkers that will be useful for the diagnosis, treatment, and prevention of tuberculosis (TB. However, their analysis and interpretation pose considerable challenges. Recent developments in the high-throughput detection of host biomarkers in TB are reported in this review.

  6. Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays (SOT)

    Science.gov (United States)

    Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput s...

  7. 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.

  8. A high-throughput Arabidopsis reverse genetics system.

    Science.gov (United States)

    Sessions, Allen; Burke, Ellen; Presting, Gernot; Aux, George; McElver, John; Patton, David; Dietrich, Bob; Ho, Patrick; Bacwaden, Johana; Ko, Cynthia; Clarke, Joseph D; Cotton, David; Bullis, David; Snell, Jennifer; Miguel, Trini; Hutchison, Don; Kimmerly, Bill; Mitzel, Theresa; Katagiri, Fumiaki; Glazebrook, Jane; Law, Marc; Goff, Stephen A

    2002-12-01

    A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.

  9. Use of High Throughput Screening Data in IARC Monograph ...

    Science.gov (United States)

    Purpose: Evaluation of carcinogenic mechanisms serves a critical role in IARC monograph evaluations, and can lead to “upgrade” or “downgrade” of the carcinogenicity conclusions based on human and animal evidence alone. Three recent IARC monograph Working Groups (110, 112, and 113) pioneered analysis of high throughput in vitro screening data from the U.S. Environmental Protection Agency’s ToxCast program in evaluations of carcinogenic mechanisms. Methods: For monograph 110, ToxCast assay data across multiple nuclear receptors were used to test the hypothesis that PFOA acts exclusively through the PPAR family of receptors, with activity profiles compared to several prototypical nuclear receptor-activating compounds. For monographs 112 and 113, ToxCast assays were systematically evaluated and used as an additional data stream in the overall evaluation of the mechanistic evidence. Specifically, ToxCast assays were mapped to 10 “key characteristics of carcinogens” recently identified by an IARC expert group, and chemicals’ bioactivity profiles were evaluated both in absolute terms (number of relevant assays positive for bioactivity) and relative terms (ranking with respect to other compounds evaluated by IARC, using the ToxPi methodology). Results: PFOA activates multiple nuclear receptors in addition to the PPAR family in the ToxCast assays. ToxCast assays offered substantial coverage for 5 of the 10 “key characteristics,” with the greates

  10. High-throughput optical screening of cellular mechanotransduction

    Science.gov (United States)

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

    2014-09-01

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

  11. Strategies for high-throughput gene cloning and expression.

    Science.gov (United States)

    Dieckman, L J; Hanly, W C; Collart, E R

    2006-01-01

    High-throughput approaches for gene cloning and expression require the development of new, nonstandard tools for use by molecular biologists and biochemists. We have developed and implemented a series of methods that enable the production of expression constructs in 96-well plate format. A screening process is described that facilitates the identification of bacterial clones expressing soluble protein. Application of the solubility screen then provides a plate map that identifies the location of wells containing clones producing soluble proteins. A series of semi-automated methods can then be applied for validation of solubility and production of freezer stocks for the protein production group. This process provides an 80% success rate for the identification of clones producing soluble protein and results in a significant decrease in the level of effort required for the labor-intensive components of validation and preparation of freezer stocks. This process is customized for large-scale structural genomics programs that rely on the production of large amounts of soluble proteins for crystallization trials.

  12. A high-throughput biliverdin assay using infrared fluorescence.

    Science.gov (United States)

    Berlec, Aleš; Štrukelj, Borut

    2014-07-01

    Biliverdin is an intermediate of heme degradation with an established role in veterinary clinical diagnostics of liver-related diseases. The need for chromatographic assays has so far prevented its wider use in diagnostic laboratories. The current report describes a simple, fast, high-throughput, and inexpensive assay, based on the interaction of biliverdin with infrared fluorescent protein (iRFP) that yields functional protein exhibiting infrared fluorescence. The assay is linear in the range of 0-10 µmol/l of biliverdin, has a limit of detection of 0.02 μmol/l, and has a limit of quantification of 0.03 µmol/l. The assay is accurate with relative error less than 0.15, and precise, with coefficient of variation less than 5% in the concentration range of 2-9 µmol/l of biliverdin. More than 95% of biliverdin was recovered from biological samples by simple dimethyl sulfoxide extraction. There was almost no interference by hemin, although bilirubin caused an increase in the biliverdin concentration, probably due to spontaneous oxidation of bilirubin to biliverdin. The newly developed biliverdin assay is appropriate for reliable quantification of large numbers of samples in veterinary medicine.

  13. Functional approach to high-throughput plant growth analysis

    Science.gov (United States)

    2013-01-01

    Method Taking advantage of the current rapid development in imaging systems and computer vision algorithms, we present HPGA, a high-throughput phenotyping platform for plant growth modeling and functional analysis, which produces better understanding of energy distribution in regards of the balance between growth and defense. HPGA has two components, PAE (Plant Area Estimation) and GMA (Growth Modeling and Analysis). In PAE, by taking the complex leaf overlap problem into consideration, the area of every plant is measured from top-view images in four steps. Given the abundant measurements obtained with PAE, in the second module GMA, a nonlinear growth model is applied to generate growth curves, followed by functional data analysis. Results Experimental results on model plant Arabidopsis thaliana show that, compared to an existing approach, HPGA reduces the error rate of measuring plant area by half. The application of HPGA on the cfq mutant plants under fluctuating light reveals the correlation between low photosynthetic rates and small plant area (compared to wild type), which raises a hypothesis that knocking out cfq changes the sensitivity of the energy distribution under fluctuating light conditions to repress leaf growth. Availability HPGA is available at http://www.msu.edu/~jinchen/HPGA. PMID:24565437

  14. Mouse eye enucleation for remote high-throughput phenotyping.

    Science.gov (United States)

    Mahajan, Vinit B; Skeie, Jessica M; Assefnia, Amir H; Mahajan, Maryann; Tsang, Stephen H

    2011-11-19

    The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.(1-5) Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. (6-9) An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

  15. 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.

  16. High-throughput membrane surface modification to control NOM fouling.

    Science.gov (United States)

    Zhou, Mingyan; Liu, Hongwei; Kilduff, James E; Langer, Robert; Anderson, Daniel G; Belfort, Georges

    2009-05-15

    A novel method for synthesis and screening of fouling-resistant membrane surfaces was developed by combining a high-throughput platform (HTP) approach together with photoinduced graft polymerization (PGP)forfacile modification of commercial poly(aryl sulfone) membranes. This method is an inexpensive, fast, simple, reproducible, and scalable approach to identify fouling-resistant surfaces appropriate for a specific feed. In this research, natural organic matter (NOM)-resistant surfaces were synthesized and indentified from a library of 66 monomers. Surfaces were prepared via graft polymerization onto poly(ether sulfone) (PES) membranes and were evaluated using an assay involving NOM adsorption, followed by pressure-driven-filtration. In this work new and previously tested low-fouling surfaces for NOM are identified, and their ability to mitigate NOM and protein (bovine serum albumin)fouling is compared. The best-performing monomers were the zwitterion [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, and diacetone acrylamide, a neutral monomer containing an amide group. Other excellent surfaces were synthesized from amides, amines, basic monomers, and long-chain poly(ethylene) glycols. Bench-scale studies conducted for selected monomers verified the scalability of HTP-PGP results. The results and the synthesis and screening method presented here offer new opportunities for choosing new membrane chemistries that minimize NOM fouling.

  17. 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.

  18. Savant: genome browser for high-throughput sequencing data.

    Science.gov (United States)

    Fiume, Marc; Williams, Vanessa; Brook, Andrew; Brudno, Michael

    2010-08-15

    The advent of high-throughput sequencing (HTS) technologies has made it affordable to sequence many individuals' genomes. Simultaneously the computational analysis of the large volumes of data generated by the new sequencing machines remains a challenge. While a plethora of tools are available to map the resulting reads to a reference genome, and to conduct primary analysis of the mappings, it is often necessary to visually examine the results and underlying data to confirm predictions and understand the functional effects, especially in the context of other datasets. We introduce Savant, the Sequence Annotation, Visualization and ANalysis Tool, a desktop visualization and analysis browser for genomic data. Savant was developed for visualizing and analyzing HTS data, with special care taken to enable dynamic visualization in the presence of gigabases of genomic reads and references the size of the human genome. Savant supports the visualization of genome-based sequence, point, interval and continuous datasets, and multiple visualization modes that enable easy identification of genomic variants (including single nucleotide polymorphisms, structural and copy number variants), and functional genomic information (e.g. peaks in ChIP-seq data) in the context of genomic annotations. Savant is freely available at http://compbio.cs.toronto.edu/savant.

  19. 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.

  20. 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.

  1. Generation of RNAi Libraries for High-Throughput Screens

    Directory of Open Access Journals (Sweden)

    Julie Clark

    2006-01-01

    Full Text Available The completion of the genome sequencing for several organisms has created a great demand for genomic tools that can systematically analyze the growing wealth of data. In contrast to the classical reverse genetics approach of creating specific knockout cell lines or animals that is time-consuming and expensive, RNA-mediated interference (RNAi has emerged as a fast, simple, and cost-effective technique for gene knockdown in large scale. Since its discovery as a gene silencing response to double-stranded RNA (dsRNA with homology to endogenous genes in Caenorhabditis elegans (C elegans, RNAi technology has been adapted to various high-throughput screens (HTS for genome-wide loss-of-function (LOF analysis. Biochemical insights into the endogenous mechanism of RNAi have led to advances in RNAi methodology including RNAi molecule synthesis, delivery, and sequence design. In this article, we will briefly review these various RNAi library designs and discuss the benefits and drawbacks of each library strategy.

  2. X-ray phase microtomography with a single grating for high-throughput investigations of biological tissue.

    Science.gov (United States)

    Zdora, Marie-Christine; Vila-Comamala, Joan; Schulz, Georg; Khimchenko, Anna; Hipp, Alexander; Cook, Andrew C; Dilg, Daniel; David, Christian; Grünzweig, Christian; Rau, Christoph; Thibault, Pierre; Zanette, Irene

    2017-02-01

    The high-throughput 3D visualisation of biological specimens is essential for studying diseases and developmental disorders. It requires imaging methods that deliver high-contrast, high-resolution volumetric information at short sample preparation and acquisition times. Here we show that X-ray phase-contrast tomography using a single grating can provide a powerful alternative to commonly employed techniques, such as high-resolution episcopic microscopy (HREM). We present the phase tomography of a mouse embryo in paraffin obtained with an X-ray single-grating interferometer at I13-2 Beamline at Diamond Light Source and discuss the results in comparison with HREM measurements. The excellent contrast and quantitative density information achieved non-destructively and without staining using a simple, robust setup make X-ray single-grating interferometry an optimum candidate for high-throughput imaging of biological specimens as an alternative for existing methods like HREM.

  3. High-throughput bubble screening method for combinatorial discovery of electrocatalysts for water splitting.

    Science.gov (United States)

    Xiang, Chengxiang; Suram, Santosh K; Haber, Joel A; Guevarra, Dan W; Soedarmadji, Ed; Jin, Jian; Gregoire, John M

    2014-02-10

    Combinatorial synthesis and screening for discovery of electrocatalysts has received increasing attention, particularly for energy-related technologies. High-throughput discovery strategies typically employ a fast, reliable initial screening technique that is able to identify active catalyst composition regions. Traditional electrochemical characterization via current-voltage measurements is inherently throughput-limited, as such measurements are most readily performed by serial screening. Parallel screening methods can yield much higher throughput and generally require the use of an indirect measurement of catalytic activity. In a water-splitting reaction, the change of local pH or the presence of oxygen and hydrogen in the solution can be utilized for parallel screening of active electrocatalysts. Previously reported techniques for measuring these signals typically function in a narrow pH range and are not suitable for both strong acidic and basic environments. A simple approach to screen the electrocatalytic activities by imaging the oxygen and hydrogen bubbles produced by the oxygen evolution reaction (OER) and hydrogen evolution reaction (HER) is reported here. A custom built electrochemical cell was employed to record the bubble evolution during the screening, where the testing materials were subject to desired electrochemical potentials. The transient of the bubble intensity obtained from the screening was quantitatively analyzed to yield a bubble figure of merit (FOM) that represents the reaction rate. Active catalysts in a pseudoternary material library, (Ni-Fe-Co)Ox, which contains 231 unique compositions, were identified in less than one minute using the bubble screening method. An independent, serial screening method on the same material library exhibited excellent agreement with the parallel bubble screening. This general approach is highly parallel and is independent of solution pH.

  4. Improving quantitative precision and throughput by reducing calibrator use in liquid chromatography-tandem mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Rule, Geoffrey S., E-mail: geoffrey.s.rule@aruplab.com [ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108 (United States); Rockwood, Alan L. [ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108 (United States); Department of Pathology, University of Utah School of Medicine, 2100 Jones Medical Research Bldg., Salt Lake City, UT 84132 (United States)

    2016-05-05

    To improve efficiency in our mass spectrometry laboratories we have made efforts to reduce the number of calibration standards utilized for quantitation over time. We often analyze three or more batches of 96 samples per day, on a single instrument, for a number of assays. With a conventional calibration scheme at six concentration levels this amounts to more than 5000 calibration points per year. Modern LC-tandem mass spectrometric instrumentation is extremely rugged however, and isotopically labelled internal standards are widely available. This made us consider whether alternative calibration strategies could be utilized to reduce the number of calibration standards analyzed while still retaining high precision and accurate quantitation. Here we demonstrate how, by utilizing a single calibration point in each sample batch, and using the resulting response factor (RF) to update an existing, historical response factor (HRF), we are able to obtain improved precision over a conventional multipoint calibration approach, as judged by quality control samples. The laboratory component of this study was conducted with an existing LC tandem mass spectrometric method for three androgen analytes in our production laboratory. Using examples from both simulated and laboratory data we illustrate several aspects of our single point alternative calibration strategy and compare it with a conventional, multipoint calibration approach. We conclude that both the cost and burden of preparing multiple calibration standards with every batch of samples can be reduced while at the same time maintaining, or even improving, analytical quality. - Highlights: • Use of a weighted single point calibration approach improves quantitative precision. • A weighted response factor approach incorporates historical calibration information. • Several scenarios are discussed with regard to their influence on quantitation.

  5. A high-throughput method for detection of DNA in chloroplasts using flow cytometry

    Directory of Open Access Journals (Sweden)

    Oldenburg Delene J

    2007-03-01

    Full Text Available Abstract Background The amount of DNA in the chloroplasts of some plant species has been shown recently to decline dramatically during leaf development. A high-throughput method of DNA detection in chloroplasts is now needed in order to facilitate the further investigation of this process using large numbers of tissue samples. Results The DNA-binding fluorophores 4',6-diamidino-2-phenylindole (DAPI, SYBR Green I (SG, SYTO 42, and SYTO 45 were assessed for their utility in flow cytometric analysis of DNA in Arabidopsis chloroplasts. Fluorescence microscopy and real-time quantitative PCR (qPCR were used to validate flow cytometry data. We found neither DAPI nor SYTO 45 suitable for flow cytometric analysis of chloroplast DNA (cpDNA content, but did find changes in cpDNA content during development by flow cytometry using SG and SYTO 42. The latter dye provided more sensitive detection, and the results were similar to those from the fluorescence microscopic analysis. Differences in SYTO 42 fluorescence were found to correlate with differences in cpDNA content as determined by qPCR using three primer sets widely spaced across the chloroplast genome, suggesting that the whole genome undergoes copy number reduction during development, rather than selective reduction/degradation of subgenomic regions. Conclusion Flow cytometric analysis of chloroplasts stained with SYTO 42 is a high-throughput method suitable for determining changes in cpDNA content during development and for sorting chloroplasts on the basis of DNA content.

  6. Sources of PCR-induced distortions in high-throughput sequencing data sets

    Science.gov (United States)

    Kebschull, Justus M.; Zador, Anthony M.

    2015-01-01

    PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error—bias, stochasticity, template switches and polymerase errors—on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules. PMID:26187991

  7. High-throughput dental biofilm growth analysis for multiparametric microenvironmental biochemical conditions using microfluidics.

    Science.gov (United States)

    Lam, Raymond H W; Cui, Xin; Guo, Weijin; Thorsen, Todd

    2016-04-26

    Dental biofilm formation is not only a precursor to tooth decay, but also induces more serious systematic health problems such as cardiovascular disease and diabetes. Understanding the conditions promoting colonization and subsequent biofilm development involving complex bacteria coaggregation is particularly important. In this paper, we report a high-throughput microfluidic 'artificial teeth' device offering controls of multiple microenvironmental factors (e.g. nutrients, growth factors, dissolved gases, and seeded cell populations) for quantitative characteristics of long-term dental bacteria growth and biofilm development. This 'artificial teeth' device contains multiple (up to 128) incubation chambers to perform parallel cultivation and analyses (e.g. biofilm thickness, viable-dead cell ratio, and spatial distribution of multiple bacterial species) of bacteria samples under a matrix of different combinations of microenvironmental factors, further revealing possible developmental mechanisms of dental biofilms. Specifically, we applied the 'artificial teeth' to investigate the growth of two key dental bacteria, Streptococci species and Fusobacterium nucleatum, in the biofilm under different dissolved gas conditions and sucrose concentrations. Together, this high-throughput microfluidic platform can provide extended applications for general biofilm research, including screening of the biofilm properties developing under combinations of specified growth parameters such as seeding bacteria populations, growth medium compositions, medium flow rates and dissolved gas levels.

  8. High-Throughput Phenotyping in Plant Stress Response: Methods and Potential Applications to Polyamine Field.

    Science.gov (United States)

    Marko, D; Briglia, N; Summerer, S; Petrozza, A; Cellini, F; Iannacone, R

    2018-01-01

    High-throughput phenotyping has opened whole new perspectives for crop improvement and better understanding of quantitative traits in plants. Generation of loss-of-function and gain-of-function plant mutants requires processing and imaging a large number of plants in order to determine unknown gene functions and phenotypic changes generated by genetic modifications or selection of new traits. The use of phenomics for the evaluation of transgenic lines contributed significantly to the identification of plants more tolerant to biotic/abiotic stresses and furthermore, helped in the identification of unknown gene functions. In this chapter we describe the High-throughput phenotyping (HTP) platform working in our facility, drawing the general protocol and showing some examples of data obtainable from the platform. Tomato transgenic plants over-expressing the arginine decarboxylase 2 gene, which is involved in the polyamine biosynthetic pathway, were analyzed through our HTP facility for their tolerance to abiotic stress and significant differences in water content and ability to recover after drought stress where highlighted. This demonstrates the applicability of this methodology to the plant polyamine field.

  9. Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of ADAMTS1 Inhibitors

    Directory of Open Access Journals (Sweden)

    Guanhua Du

    2011-12-01

    Full Text Available A disintegrin and metalloprotease with thrombospondin type I motifs-1 (ADAMTS1 plays a crucial role in inflammatory joint diseases and its inhibitors are potential candidates for anti-arthritis drugs. For the purposes of drug discovery, we reported the development and validation of fluorescence resonance energy transfer (FRET assay for high-throughput screening (HTS of the ADAMTS1 inhibitors. A FRET substrate was designed for a quantitative assay of ADAMTS1 activity and enzyme kinetics studies. The assay was developed into a 50-µL, 384-well assay format for high throughput screening of ADAMTS1 inhibitors with an overall Z’ factor of 0.89. ADAMTS1 inhibitors were screened against a diverse library of 40,960 total compounds with the established HTS system. Four structurally related hits, naturally occurring compounds, kuwanon P, kuwanon X, albafuran C and mulberrofuran J, extracted from the Chinese herb Morus alba L., were identified for further investigation. The results suggest that this FRET assay is an excellent tool, not only for measurement of ADAMTS1 activity but also for discovery of novel ADAMTS1 inhibitors with HTS.

  10. High-throughput direct measurement of magnetocaloric effect based on lock-in thermography technique

    Science.gov (United States)

    Hirayama, Yusuke; Iguchi, Ryo; Miao, Xue-Fei; Hono, Kazuhiro; Uchida, Ken-ichi

    2017-10-01

    We demonstrate a high-throughput direct measurement method for the magnetocaloric effect (MCE) by means of a lock-in thermography (LIT) technique. This method enables systematic measurements of the magnetic-field and operation-frequency dependences of the temperature change induced by the MCE. This is accomplished in a shorter time compared to conventional adiabatic temperature measurement methods. The direct measurement based on LIT is free from any possible miscalculations and errors arising from indirect measurements using thermodynamic relations. Importantly, the LIT technique makes simultaneous MCE measurements of multiple materials possible without increasing the measurement time, realizing high-throughput investigations of the MCE. By applying this method to Gd, we obtain the MCE-induced temperature change of 1.84 ± 0.11 K under a modulation field of 1.0 T and modulation frequency of 0.5 Hz at a temperature of 300.5 ± 0.5 K, offering evidence that the LIT method gives quantitative results.

  11. The Utilization of Formalin Fixed-Paraffin-Embedded Specimens in High Throughput Genomic Studies

    Directory of Open Access Journals (Sweden)

    Pan Zhang

    2017-01-01

    Full Text Available High throughput genomic assays empower us to study the entire human genome in short time with reasonable cost. Formalin fixed-paraffin-embedded (FFPE tissue processing remains the most economical approach for longitudinal tissue specimen storage. Therefore, the ability to apply high throughput genomic applications to FFPE specimens can expand clinical assays and discovery. Many studies have measured the accuracy and repeatability of data generated from FFPE specimens using high throughput genomic assays. Together, these studies demonstrate feasibility and provide crucial guidance for future studies using FFPE specimens. Here, we summarize the findings of these studies and discuss the limitations of high throughput data generated from FFPE specimens across several platforms that include microarray, high throughput sequencing, and NanoString.

  12. High-throughput flow cytometry data normalization for clinical trials.

    Science.gov (United States)

    Finak, Greg; Jiang, Wenxin; Krouse, Kevin; Wei, Chungwen; Sanz, Ignacio; Phippard, Deborah; Asare, Adam; De Rosa, Stephen C; Self, Steve; Gottardo, Raphael

    2014-03-01

    Flow cytometry datasets from clinical trials generate very large datasets and are usually highly standardized, focusing on endpoints that are well defined apriori. Staining variability of individual makers is not uncommon and complicates manual gating, requiring the analyst to adapt gates for each sample, which is unwieldy for large datasets. It can lead to unreliable measurements, especially if a template-gating approach is used without further correction to the gates. In this article, a computational framework is presented for normalizing the fluorescence intensity of multiple markers in specific cell populations across samples that is suitable for high-throughput processing of large clinical trial datasets. Previous approaches to normalization have been global and applied to all cells or data with debris removed. They provided no mechanism to handle specific cell subsets. This approach integrates tightly with the gating process so that normalization is performed during gating and is local to the specific cell subsets exhibiting variability. This improves peak alignment and the performance of the algorithm. The performance of this algorithm is demonstrated on two clinical trial datasets from the HIV Vaccine Trials Network (HVTN) and the Immune Tolerance Network (ITN). In the ITN data set we show that local normalization combined with template gating can account for sample-to-sample variability as effectively as manual gating. In the HVTN dataset, it is shown that local normalization mitigates false-positive vaccine response calls in an intracellular cytokine staining assay. In both datasets, local normalization performs better than global normalization. The normalization framework allows the use of template gates even in the presence of sample-to-sample staining variability, mitigates the subjectivity and bias of manual gating, and decreases the time necessary to analyze large datasets. © 2013 International Society for Advancement of Cytometry.

  13. 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.

  14. 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.

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

    Science.gov (United States)

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

    2014-06-01

    For approximately a decade, biophysical methods have been used to validate positive hits selected from high-throughput screening (HTS) campaigns with the goal to verify binding interactions using label-free assays. By applying label-free readouts, screen artifacts created by compound interference and fluorescence are discovered, enabling further characterization of the hits for their target specificity and selectivity. The use of several biophysical methods to extract this type of high-content information is required to prevent the promotion of false positives to the next level of hit validation and to select the best candidates for further chemical optimization. The typical technologies applied in this arena include dynamic light scattering, turbidometry, resonance waveguide, surface plasmon resonance, differential scanning fluorimetry, mass spectrometry, and others. Each technology can provide different types of information to enable the characterization of the binding interaction. Thus, these technologies can be incorporated in a hit-validation strategy not only according to the profile of chemical matter that is desired by the medicinal chemists, but also in a manner that is in agreement with the target protein's amenability to the screening format. Here, we present the results of screening strategies using biophysics with the objective to evaluate the approaches, discuss the advantages and challenges, and summarize the benefits in reference to lead discovery. In summary, the biophysics screens presented here demonstrated various hit rates from a list of ~2000 preselected, IC50-validated hits from HTS (an IC50 is the inhibitor concentration at which 50% inhibition of activity is observed). There are several lessons learned from these biophysical screens, which will be discussed in this article. © 2014 Society for Laboratory Automation and Screening.

  16. 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.

  17. High-throughput detection of ethanol-producing cyanobacteria in a microdroplet platform.

    Science.gov (United States)

    Abalde-Cela, Sara; Gould, Anna; Liu, Xin; Kazamia, Elena; Smith, Alison G; Abell, Chris

    2015-05-06

    Ethanol production by microorganisms is an important renewable energy source. Most processes involve fermentation of sugars from plant feedstock, but there is increasing interest in direct ethanol production by photosynthetic organisms. To facilitate this, a high-throughput screening technique for the detection of ethanol is required. Here, a method for the quantitative detection of ethanol in a microdroplet-based platform is described that can be used for screening cyanobacterial strains to identify those with the highest ethanol productivity levels. The detection of ethanol by enzymatic assay was optimized both in bulk and in microdroplets. In parallel, the encapsulation of engineered ethanol-producing cyanobacteria in microdroplets and their growth dynamics in microdroplet reservoirs were demonstrated. The combination of modular microdroplet operations including droplet generation for cyanobacteria encapsulation, droplet re-injection and pico-injection, and laser-induced fluorescence, were used to create this new platform to screen genetically engineered strains of cyanobacteria with different levels of ethanol production.

  18. Field-based high throughput phenotyping rapidly identifies genomic regions controlling yield components in rice.

    Science.gov (United States)

    Tanger, Paul; Klassen, Stephen; Mojica, Julius P; Lovell, John T; Moyers, Brook T; Baraoidan, Marietta; Naredo, Maria Elizabeth B; McNally, Kenneth L; Poland, Jesse; Bush, Daniel R; Leung, Hei; Leach, Jan E; McKay, John K

    2017-02-21

    To ensure food security in the face of population growth, decreasing water and land for agriculture, and increasing climate variability, crop yields must increase faster than the current rates. Increased yields will require implementing novel approaches in genetic discovery and breeding. Here we demonstrate the potential of field-based high throughput phenotyping (HTP) on a large recombinant population of rice to identify genetic variation underlying important traits. We find that detecting quantitative trait loci (QTL) with HTP phenotyping is as accurate and effective as traditional labor-intensive measures of flowering time, height, biomass, grain yield, and harvest index. Genetic mapping in this population, derived from a cross of an modern cultivar (IR64) with a landrace (Aswina), identified four alleles with negative effect on grain yield that are fixed in IR64, demonstrating the potential for HTP of large populations as a strategy for the second green revolution.

  19. High-Throughput Immunogenetics for Clinical and Research Applications in Immunohematology: Potential and Challenges

    NARCIS (Netherlands)

    Langerak, A.W.; Bruggemann, M.; Davi, F.; Darzentas, N.; Dongen, J.J. van; Gonzalez, D.; Cazzaniga, G.; Giudicelli, V.; Lefranc, M.P.; Giraud, M.; Macintyre, E.A.; Hummel, M.; Pott, C.; Groenen, P.J.T.A.; Stamatopoulos, K.

    2017-01-01

    Analysis and interpretation of Ig and TCR gene rearrangements in the conventional, low-throughput way have their limitations in terms of resolution, coverage, and biases. With the advent of high-throughput, next-generation sequencing (NGS) technologies, a deeper analysis of Ig and/or TCR (IG/TR)

  20. 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

    2015-02-01

    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.

  1. Missing call bias in high-throughput genotyping

    Directory of Open Access Journals (Sweden)

    Lin Rong

    2009-03-01

    Full Text Available Abstract Background The advent of high-throughput and cost-effective genotyping platforms made genome-wide association (GWA studies a reality. While the primary focus has been invested upon the improvement of reducing genotyping error, the problems associated with missing calls are largely overlooked. Results To probe into the effect of missing calls on GWAs, we demonstrated experimentally the prevalence and severity of the problem of missing call bias (MCB in four genotyping technologies (Affymetrix 500 K SNP array, SNPstream, TaqMan, and Illumina Beadlab. Subsequently, we showed theoretically that MCB leads to biased conclusions in the subsequent analyses, including estimation of allele/genotype frequencies, the measurement of HWE and association tests under various modes of inheritance relationships. We showed that MCB usually leads to power loss in association tests, and such power change is greater than what could be achieved by equivalent reduction of sample size unbiasedly. We also compared the bias in allele frequency estimation and in association tests introduced by MCB with those by genotyping errors. Our results illustrated that in most cases, the bias can be greatly reduced by increasing the call-rate at the cost of genotyping error rate. Conclusion The commonly used 'no-call' procedure for the observations of borderline quality should be modified. If the objective is to minimize the bias, the cut-off for call-rate and that for genotyping error rate should be properly coupled in GWA. We suggested that the ongoing QC cut-off for call-rate should be increased, while the cut-off for genotyping error rate can be reduced properly.

  2. A high-throughput microtiter plate based method for the determination of peracetic acid and hydrogen peroxide.

    Directory of Open Access Journals (Sweden)

    Karson S Putt

    Full Text Available Peracetic acid is gaining usage in numerous industries who have found a myriad of uses for its antimicrobial activity. However, rapid high throughput quantitation methods for peracetic acid and hydrogen peroxide are lacking. Herein, we describe the development of a high-throughput microtiter plate based assay based upon the well known and trusted titration chemical reactions. The adaptation of these titration chemistries to rapid plate based absorbance methods for the sequential determination of hydrogen peroxide specifically and the total amount of peroxides present in solution are described. The results of these methods were compared to those of a standard titration and found to be in good agreement. Additionally, the utility of the developed method is demonstrated through the generation of degradation curves of both peracetic acid and hydrogen peroxide in a mixed solution.

  3. High pressure inertial focusing for separation and concentration of bacteria at high throughput

    Science.gov (United States)

    Cruz, F. J.; Hjort, K.

    2017-11-01

    Inertial focusing is a phenomenon where particles migrate across streamlines in microchannels and focus at well-defined, size dependent equilibrium points of the cross section. It can be taken into advantage for focusing, separation and concentration of particles at high through-put and high efficiency. As particles decrease in size, smaller channels and higher pressures are needed. Hence, new designs are needed to decrease the pressure drop. In this work a novel design was adapted to focus and separate 1 µm from 3 µm spherical polystyrene particles. Also 0.5 µm spherical polystyrene particles were separated, although in a band instead of a single line. The ability to separate, concentrate and focus bacteria, its simplicity of use and high throughput make this technology a candidate for daily routines in laboratories and hospitals.

  4. 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

  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. High-throughput metal susceptibility testing of microbial biofilms

    Directory of Open Access Journals (Sweden)

    Turner Raymond J

    2005-10-01

    Full Text Available Abstract Background Microbial biofilms exist all over the natural world, a distribution that is paralleled by metal cations and oxyanions. Despite this reality, very few studies have examined how biofilms withstand exposure to these toxic compounds. This article describes a batch culture technique for biofilm and planktonic cell metal susceptibility testing using the MBEC assay. This device is compatible with standard 96-well microtiter plate technology. As part of this method, a two part, metal specific neutralization protocol is summarized. This procedure minimizes residual biological toxicity arising from the carry-over of metals from challenge to recovery media. Neutralization consists of treating cultures with a chemical compound known to react with or to chelate the metal. Treated cultures are plated onto rich agar to allow metal complexes to diffuse into the recovery medium while bacteria remain on top to recover. Two difficulties associated with metal susceptibility testing were the focus of two applications of this technique. First, assays were calibrated to allow comparisons of the susceptibility of different organisms to metals. Second, the effects of exposure time and growth medium composition on the susceptibility of E. coli JM109 biofilms to metals were investigated. Results This high-throughput method generated 96-statistically equivalent biofilms in a single device and thus allowed for comparative and combinatorial experiments of media, microbial strains, exposure times and metals. By adjusting growth conditions, it was possible to examine biofilms of different microorganisms that had similar cell densities. In one example, Pseudomonas aeruginosa ATCC 27853 was up to 80 times more resistant to heavy metalloid oxyanions than Escherichia coli TG1. Further, biofilms were up to 133 times more tolerant to tellurite (TeO32- than corresponding planktonic cultures. Regardless of the growth medium, the tolerance of biofilm and planktonic

  7. High-throughput metal susceptibility testing of microbial biofilms

    Science.gov (United States)

    Harrison, Joe J; Turner, Raymond J; Ceri, Howard

    2005-01-01

    Background Microbial biofilms exist all over the natural world, a distribution that is paralleled by metal cations and oxyanions. Despite this reality, very few studies have examined how biofilms withstand exposure to these toxic compounds. This article describes a batch culture technique for biofilm and planktonic cell metal susceptibility testing using the MBEC assay. This device is compatible with standard 96-well microtiter plate technology. As part of this method, a two part, metal specific neutralization protocol is summarized. This procedure minimizes residual biological toxicity arising from the carry-over of metals from challenge to recovery media. Neutralization consists of treating cultures with a chemical compound known to react with or to chelate the metal. Treated cultures are plated onto rich agar to allow metal complexes to diffuse into the recovery medium while bacteria remain on top to recover. Two difficulties associated with metal susceptibility testing were the focus of two applications of this technique. First, assays were calibrated to allow comparisons of the susceptibility of different organisms to metals. Second, the effects of exposure time and growth medium composition on the susceptibility of E. coli JM109 biofilms to metals were investigated. Results This high-throughput method generated 96-statistically equivalent biofilms in a single device and thus allowed for comparative and combinatorial experiments of media, microbial strains, exposure times and metals. By adjusting growth conditions, it was possible to examine biofilms of different microorganisms that had similar cell densities. In one example, Pseudomonas aeruginosa ATCC 27853 was up to 80 times more resistant to heavy metalloid oxyanions than Escherichia coli TG1. Further, biofilms were up to 133 times more tolerant to tellurite (TeO32-) than corresponding planktonic cultures. Regardless of the growth medium, the tolerance of biofilm and planktonic cell E. coli JM109 to metals

  8. High-throughput quantification of antibiotic resistance genes from an urban wastewater treatment plant.

    Science.gov (United States)

    Karkman, Antti; Johnson, Timothy A; Lyra, Christina; Stedtfeld, Robert D; Tamminen, Manu; Tiedje, James M; Virta, Marko

    2016-03-01

    Antibiotic resistance among bacteria is a growing problem worldwide, and wastewater treatment plants have been considered as one of the major contributors to the dissemination of antibiotic resistance to the environment. There is a lack of comprehensive quantitative molecular data on extensive numbers of antibiotic resistance genes (ARGs) in different seasons with a sampling strategy that would cover both incoming and outgoing water together with the excess sludge that is removed from the process. In order to fill that gap we present a highly parallel quantitative analysis of ARGs and horizontal gene transfer potential over four seasons at an urban wastewater treatment plant using a high-throughput qPCR array. All analysed transposases and two-thirds of primer sets targeting ARGs were detected in the wastewater. The relative abundance of most of the genes was highest in influent and lower in effluent water and sludge. The resistance profiles of the samples cluster by sample location with a shift from raw influent through the final effluents and dried sludge to the sediments. Wastewater discharge enriched only a few genes, namely Tn25 type transposase gene and clinical class 1 integrons, in the sediment near the discharge pipe, but those enriched genes may indicate a potential for horizontal gene transfer. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. High-Throughput Phenotyping and QTL Mapping Reveals the Genetic Architecture of Maize Plant Growth.

    Science.gov (United States)

    Zhang, Xuehai; Huang, Chenglong; Wu, Di; Qiao, Feng; Li, Wenqiang; Duan, Lingfeng; Wang, Ke; Xiao, Yingjie; Chen, Guoxing; Liu, Qian; Xiong, Lizhong; Yang, Wanneng; Yan, Jianbing

    2017-03-01

    With increasing demand for novel traits in crop breeding, the plant research community faces the challenge of quantitatively analyzing the structure and function of large numbers of plants. A clear goal of high-throughput phenotyping is to bridge the gap between genomics and phenomics. In this study, we quantified 106 traits from a maize (Zea mays) recombinant inbred line population (n = 167) across 16 developmental stages using the automatic phenotyping platform. Quantitative trait locus (QTL) mapping with a high-density genetic linkage map, including 2,496 recombinant bins, was used to uncover the genetic basis of these complex agronomic traits, and 988 QTLs have been identified for all investigated traits, including three QTL hotspots. Biomass accumulation and final yield were predicted using a combination of dissected traits in the early growth stage. These results reveal the dynamic genetic architecture of maize plant growth and enhance ideotype-based maize breeding and prediction. © 2017 American Society of Plant Biologists. All Rights Reserved.

  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. High-throughput sequencing and graph-based cluster analysis facilitate microsatellite development from a highly complex genome.

    Science.gov (United States)

    Shah, Abhijeet B; Schielzeth, Holger; Albersmeier, Andreas; Kalinowski, Joern; Hoffman, Joseph I

    2016-08-01

    Despite recent advances in high-throughput sequencing, difficulties are often encountered when developing microsatellites for species with large and complex genomes. This probably reflects the close association in many species of microsatellites with cryptic repetitive elements. We therefore developed a novel approach for isolating polymorphic microsatellites from the club-legged grasshopper (Gomphocerus sibiricus), an emerging quantitative genetic and behavioral model system. Whole genome shotgun Illumina MiSeq sequencing was used to generate over three million 300 bp paired-end reads, of which 67.75% were grouped into 40,548 clusters within RepeatExplorer. Annotations of the top 468 clusters, which represent 60.5% of the reads, revealed homology to satellite DNA and a variety of transposable elements. Evaluating 96 primer pairs in eight wild-caught individuals, we found that primers mined from singleton reads were six times more likely to amplify a single polymorphic microsatellite locus than primers mined from clusters. Our study provides experimental evidence in support of the notion that microsatellites associated with repetitive elements are less likely to successfully amplify. It also reveals how advances in high-throughput sequencing and graph-based repetitive DNA analysis can be leveraged to isolate polymorphic microsatellites from complex genomes.

  12. Engineering High Affinity Protein-Protein Interactions Using a High-Throughput Microcapillary Array Platform.

    Science.gov (United States)

    Lim, Sungwon; Chen, Bob; Kariolis, Mihalis S; Dimov, Ivan K; Baer, Thomas M; Cochran, Jennifer R

    2017-02-17

    Affinity maturation of protein-protein interactions requires iterative rounds of protein library generation and high-throughput screening to identify variants that bind with increased affinity to a target of interest. We recently developed a multipurpose protein engineering platform, termed μSCALE (Microcapillary Single Cell Analysis and Laser Extraction). This technology enables high-throughput screening of libraries of millions of cell-expressing protein variants based on their binding properties or functional activity. Here, we demonstrate the first use of the μSCALE platform for affinity maturation of a protein-protein binding interaction. In this proof-of-concept study, we engineered an extracellular domain of the Axl receptor tyrosine kinase to bind tighter to its ligand Gas6. Within 2 weeks, two iterative rounds of library generation and screening resulted in engineered Axl variants with a 50-fold decrease in kinetic dissociation rate, highlighting the use of μSCALE as a new tool for directed evolution.

  13. Single-step enrichment by Ti4+-IMAC and label-free quantitation enables in-depth monitoring of phosphorylation dynamics with high reproducibility and temporal resolution

    NARCIS (Netherlands)

    de Graaf, Erik L|info:eu-repo/dai/nl/326659978; Giansanti, Piero|info:eu-repo/dai/nl/330827464; Altelaar, A F Maarten|info:eu-repo/dai/nl/304833517; Heck, Albert J R|info:eu-repo/dai/nl/105189332

    Quantitative phosphoproteomics workflows traditionally involve additional sample labeling and fractionation steps for accurate and in-depth analysis. Here we report a high-throughput, straightforward, and comprehensive label-free phosphoproteomics approach using the highly selective, reproducible,

  14. High-throughput phenotyping and genetic linkage of cortical bone microstructure in the mouse.

    Science.gov (United States)

    Mader, Kevin S; Donahue, Leah Rae; Müller, Ralph; Stampanoni, Marco

    2015-07-03

    Understanding cellular structure and organization, which plays an important role in biological systems ranging from mechanosensation to neural organization, is a complicated multifactorial problem depending on genetics, environmental factors, and stochastic processes. Isolating these factors necessitates the measurement and sensitive quantification of many samples in a reliable, high-throughput, unbiased manner. In this manuscript we present a pipelined approach using a fully automated framework based on Synchrotron-based X-ray Tomographic Microscopy (SRXTM) for performing a full 3D characterization of millions of substructures. We demonstrate the framework on a genetic study on the femur bones of in-bred mice. We measured 1300 femurs from a F2 cross experiment in mice without the growth hormone (which can confound many of the smaller structural differences between strains) and characterized more than 50 million osteocyte lacunae (cell-sized hollows in the bone). The results were then correlated with genetic markers in a process called quantitative trait localization (QTL). Our findings provide a mapping between regions of the genome (all 19 autosomes) and observable phenotypes which could explain between 8-40 % of the variance using between 2-10 loci for each trait. This map shows 4 areas of overlap with previous studies looking at bone strength and 3 areas not previously associated with bone. The mapping of microstructural phenotypes provides a starting point for both structure-function and genetic studies on murine bone structure and the specific loci can be investigated in more detail to identify single gene candidates which can then be translated to human investigations. The flexible infrastructure offers a full spectrum of shape, distribution, and connectivity metrics for cellular networks and can be adapted to a wide variety of materials ranging from plant roots to lung tissue in studies requiring high sample counts and sensitive metrics such as the drug

  15. Enzyme assays: high-throughput screening, genetic selection, and fingerprinting

    National Research Council Canada - National Science Library

    Reymond, Jean-Louis

    2006-01-01

    ... (Testing Many Substrates Toward Hydrolase) Comparison with Other Methods 26 Estimating and Measuring Selectivity 27 Estimating Selectivity without a Reference Compound 28 Quantitative Measure of Se...

  16. Assessment of network perturbation amplitudes by applying high-throughput data to causal biological networks

    Directory of Open Access Journals (Sweden)

    Martin Florian

    2012-05-01

    Full Text Available Abstract Background High-throughput measurement technologies produce data sets that have the potential to elucidate the biological impact of disease, drug treatment, and environmental agents on humans. The scientific community faces an ongoing challenge in the analysis of these rich data sources to more accurately characterize biological processes that have been perturbed at the mechanistic level. Here, a new approach is built on previous methodologies in which high-throughput data was interpreted using prior biological knowledge of cause and effect relationships. These relationships are structured into network models that describe specific biological processes, such as inflammatory signaling or cell cycle progression. This enables quantitative assessment of network perturbation in response to a given stimulus. Results Four complementary methods were devised to quantify treatment-induced activity changes in processes described by network models. In addition, companion statistics were developed to qualify significance and specificity of the results. This approach is called Network Perturbation Amplitude (NPA scoring because the amplitudes of treatment-induced perturbations are computed for biological network models. The NPA methods were tested on two transcriptomic data sets: normal human bronchial epithelial (NHBE cells treated with the pro-inflammatory signaling mediator TNFα, and HCT116 colon cancer cells treated with the CDK cell cycle inhibitor R547. Each data set was scored against network models representing different aspects of inflammatory signaling and cell cycle progression, and these scores were compared with independent measures of pathway activity in NHBE cells to verify the approach. The NPA scoring method successfully quantified the amplitude of TNFα-induced perturbation for each network model when compared against NF-κB nuclear localization and cell number. In addition, the degree and specificity to which CDK

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

    Science.gov (United States)

    Orth, Antony; Crozier, Kenneth

    2012-06-04

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

  18. 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...

  19. High-Throughput Approaches to Pinpoint Function within the Noncoding Genome.

    Science.gov (United States)

    Montalbano, Antonino; Canver, Matthew C; Sanjana, Neville E

    2017-10-05

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas nuclease system is a powerful tool for genome editing, and its simple programmability has enabled high-throughput genetic and epigenetic studies. These high-throughput approaches offer investigators a toolkit for functional interrogation of not only protein-coding genes but also noncoding DNA. Historically, noncoding DNA has lacked the detailed characterization that has been applied to protein-coding genes in large part because there has not been a robust set of methodologies for perturbing these regions. Although the majority of high-throughput CRISPR screens have focused on the coding genome to date, an increasing number of CRISPR screens targeting noncoding genomic regions continue to emerge. Here, we review high-throughput CRISPR-based approaches to uncover and understand functional elements within the noncoding genome and discuss practical aspects of noncoding library design and screen analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. 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.

  1. High-throughput phenotyping of multicellular organisms: finding the link between genotype and phenotype

    OpenAIRE

    Sozzani, Rosangela; Benfey, Philip N

    2011-01-01

    High-throughput phenotyping approaches (phenomics) are being combined with genome-wide genetic screens to identify alterations in phenotype that result from gene inactivation. Here we highlight promising technologies for 'phenome-scale' analyses in multicellular organisms.

  2. High-throughput phenotyping of multicellular organisms: finding the link between genotype and phenotype

    Science.gov (United States)

    2011-01-01

    High-throughput phenotyping approaches (phenomics) are being combined with genome-wide genetic screens to identify alterations in phenotype that result from gene inactivation. Here we highlight promising technologies for 'phenome-scale' analyses in multicellular organisms. PMID:21457493

  3. EMPeror: a tool for visualizing high-throughput microbial community data

    National Research Council Canada - National Science Library

    Vázquez-Baeza, Yoshiki; Pirrung, Meg; Gonzalez, Antonio; Knight, Rob

    2013-01-01

    As microbial ecologists take advantage of high-throughput sequencing technologies to describe microbial communities across ever-increasing numbers of samples, new analysis tools are required to relate...

  4. High-throughput system-wide engineering and screening for microbial biotechnology.

    Science.gov (United States)

    Vervoort, Yannick; Linares, Alicia Gutiérrez; Roncoroni, Miguel; Liu, Chengxun; Steensels, Jan; Verstrepen, Kevin J

    2017-08-01

    Genetic engineering and screening of large number of cells or populations is a crucial bottleneck in today's systems biology and applied (micro)biology. Instead of using standard methods in bottles, flasks or 96-well plates, scientists are increasingly relying on high-throughput strategies that miniaturize their experiments to the nanoliter and picoliter scale and the single-cell level. In this review, we summarize different high-throughput system-wide genome engineering and screening strategies for microbes. More specifically, we will emphasize the use of multiplex automated genome evolution (MAGE) and CRISPR/Cas systems for high-throughput genome engineering and the application of (lab-on-chip) nanoreactors for high-throughput single-cell or population screening. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. 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.

  6. An image analysis toolbox for high-throughput C. elegans assays.

    Science.gov (United States)

    Wählby, Carolina; Kamentsky, Lee; Liu, Zihan H; Riklin-Raviv, Tammy; Conery, Annie L; O'Rourke, Eyleen J; Sokolnicki, Katherine L; Visvikis, Orane; Ljosa, Vebjorn; Irazoqui, Javier E; Golland, Polina; Ruvkun, Gary; Ausubel, Frederick M; Carpenter, Anne E

    2012-04-22

    We present a toolbox for high-throughput screening of image-based Caenorhabditis elegans phenotypes. The image analysis algorithms measure morphological phenotypes in individual worms and are effective for a variety of assays and imaging systems. This WormToolbox is available through the open-source CellProfiler project and enables objective scoring of whole-worm high-throughput image-based assays of C. elegans for the study of diverse biological pathways that are relevant to human disease.

  7. Deep Recurrent Neural Network for Mobile Human Activity Recognition with High Throughput

    OpenAIRE

    Inoue, Masaya; Inoue, Sozo; Nishida, Takeshi

    2016-01-01

    In this paper, we propose a method of human activity recognition with high throughput from raw accelerometer data applying a deep recurrent neural network (DRNN), and investigate various architectures and its combination to find the best parameter values. The "high throughput" refers to short time at a time of recognition. We investigated various parameters and architectures of the DRNN by using the training dataset of 432 trials with 6 activity classes from 7 people. The maximum recognition ...

  8. Construction and analysis of high-density linkage map using high-throughput sequencing data.

    Directory of Open Access Journals (Sweden)

    Dongyuan Liu

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

  9. 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

    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...... 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....

  10. A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins

    Directory of Open Access Journals (Sweden)

    Andrew Gehring

    2014-06-01

    Full Text Available 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 and pooled horseradish peroxidase (HRP-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB, the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1–2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic and/or B-PER (a cell-disrupting, protein extraction reagent. Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.

  11. Genotyping by PCR and High-Throughput Sequencing of Commercial Probiotic Products Reveals Composition Biases.

    Directory of Open Access Journals (Sweden)

    Wesley Morovic

    2016-11-01

    Full Text Available Recent advances in microbiome research have brought renewed focus on beneficial bacteria, many of which are available in food and dietary supplements. Although probiotics have historically been defined as microorganisms that convey health benefits when ingested in sufficient viable amounts, this description now includes the stipulation well defined strains, encompassing definitive taxonomy for consumer consideration and regulatory oversight. Here, we evaluated 52 commercial dietary supplements covering a range of labeled species, and determined their content using plate counting, targeted genotyping. Additionally, strain identities were assessed using methods recently published by the United States Pharmacopeial Convention. We also determined the relative abundance of individual bacteria by high-throughput sequencing (HTS of the 16S rRNA sequence using paired-end 2x250bp Illumina MiSeq technology. Using multiple methods, we tested the hypothesis that products do contain the quantitative amount of labeled bacteria, and qualitative list of labeled microbial species. We found that 17 samples (33% were below label claim for CFU prior to their expiration dates. A multiplexed-PCR scheme showed that only 30/52 (58% of the products contained a correctly labeled classification, with issues encompassing incorrect taxonomy, missing species and un-labeled species. The HTS revealed that many blended products consisted predominantly of Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis. These results highlight the need for reliable methods to qualitatively determine the correct taxonomy and quantitatively ascertain the relative amounts of mixed microbial populations in commercial probiotic products.

  12. High-throughput phenotyping of plant resistance to aphids by automated video tracking.

    Science.gov (United States)

    Kloth, Karen J; Ten Broeke, Cindy Jm; Thoen, Manus Pm; Hanhart-van den Brink, Marianne; Wiegers, Gerrie L; Krips, Olga E; Noldus, Lucas Pjj; Dicke, Marcel; Jongsma, Maarten A

    2015-01-01

    Piercing-sucking insects are major vectors of plant viruses causing significant yield losses in crops. Functional genomics of plant resistance to these insects would greatly benefit from the availability of high-throughput, quantitative phenotyping methods. We have developed an automated video tracking platform that quantifies aphid feeding behaviour on leaf discs to assess the level of plant resistance. Through the analysis of aphid movement, the start and duration of plant penetrations by aphids were estimated. As a case study, video tracking confirmed the near-complete resistance of lettuce cultivar 'Corbana' against Nasonovia ribisnigri (Mosely), biotype Nr:0, and revealed quantitative resistance in Arabidopsis accession Co-2 against Myzus persicae (Sulzer). The video tracking platform was benchmarked against Electrical Penetration Graph (EPG) recordings and aphid population development assays. The use of leaf discs instead of intact plants reduced the intensity of the resistance effect in video tracking, but sufficiently replicated experiments resulted in similar conclusions as EPG recordings and aphid population assays. One video tracking platform could screen 100 samples in parallel. Automated video tracking can be used to screen large plant populations for resistance to aphids and other piercing-sucking insects.

  13. High-throughput protein precipitation and hydrophobic interaction chromatography: salt effects and thermodynamic interrelation.

    Science.gov (United States)

    Nfor, Beckley K; Hylkema, Nienke N; Wiedhaup, Koenraad R; Verhaert, Peter D E M; van der Wielen, Luuk A M; Ottens, Marcel

    2011-12-09

    Salt-induced protein precipitation and hydrophobic interaction chromatography (HIC) are two widely used methods for protein purification. In this study, salt effects in protein precipitation and HIC were investigated for a broad combination of proteins, salts and HIC resins. Interrelation between the critical thermodynamic salting out parameters in both techniques was equally investigated. Protein precipitation data were obtained by a high-throughput technique employing 96-well microtitre plates and robotic liquid handling technology. For the same protein-salt combinations, isocratic HIC experiments were performed using two or three different commercially available stationary phases-Phenyl Sepharose low sub, Butyl Sepharose and Resource Phenyl. In general, similar salt effects and deviations from the lyotropic series were observed in both separation methods, for example, the reverse Hofmeister effect reported for lysozyme below its isoelectric point and at low salt concentrations. The salting out constant could be expressed in terms of the preferential interaction parameter in protein precipitation, showing that the former is, in effect, the net result of preferential interaction of a protein with water molecules and salt ions in its vicinity. However, no general quantitative interrelation was found between salting out parameters or the number of released water molecules in protein precipitation and HIC. In other words, protein solubility and HIC retention factor could not be quantitatively interrelated, although for some proteins, regular trends were observed across the different resins and salt types. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. 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

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

    Science.gov (United States)

    Bashalkhanov, Stanislav; Rajora, Om P

    2008-08-01

    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. 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. 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.

  16. Direct multiplex sequencing (DMPS)--a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA

    National Research Council Canada - National Science Library

    Stiller, Mathias; Knapp, Michael; Stenzel, Udo; Hofreiter, Michael; Meyer, Matthias

    2009-01-01

    Although the emergence of high-throughput sequencing technologies has enabled whole-genome sequencing from extinct organisms, little progress has been made in accelerating targeted sequencing from highly degraded DNA...

  17. High-throughput sperm differential proteomics suggests that epigenetic alterations contribute to failed assisted reproduction.

    Science.gov (United States)

    Azpiazu, Rubén; Amaral, Alexandra; Castillo, Judit; Estanyol, Josep Maria; Guimerà, Marta; Ballescà, Josep Lluís; Balasch, Juan; Oliva, Rafael

    2014-06-01

    Are there quantitative alterations in the proteome of normozoospermic sperm samples that are able to complete IVF but whose female partner does not achieve pregnancy? Normozoospermic sperm samples with different IVF outcomes (pregnancy versus no pregnancy) differed in the levels of at least 66 proteins. The analysis of the proteome of sperm samples with distinct fertilization capacity using low-throughput proteomic techniques resulted in the detection of a few differential proteins. Current high-throughput mass spectrometry approaches allow the identification and quantification of a substantially higher number of proteins. This was a case-control study including 31 men with normozoospermic sperm and their partners who underwent IVF with successful fertilization recruited between 2007 and 2008. Normozoospermic sperm samples from 15 men whose female partners did not achieve pregnancy after IVF (no pregnancy) and 16 men from couples that did achieve pregnancy after IVF (pregnancy) were included in this study. To perform the differential proteomic experiments, 10 no pregnancy samples and 10 pregnancy samples were separately pooled and subsequently used for tandem mass tags (TMT) protein labelling, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry (LC-MS/MS) identification and peak intensity relative protein quantification. Bioinformatic analyses were performed using UniProt Knowledgebase, DAVID and Reactome. Individual samples (n = 5 no pregnancy samples; n = 6 pregnancy samples) and aliquots from the above TMT pools were used for western blotting. By using TMT labelling and LC-MS/MS, we have detected 31 proteins present at lower abundance (ratio no pregnancy/pregnancy 1.5) in the no pregnancy group. Bioinformatic analyses showed that the proteins with differing abundance are involved in chromatin assembly and lipoprotein metabolism (P values Economia y Competividad; FEDER BFU 2009-07118 and PI13/00699) and

  18. 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.

  19. SDR-ELISA: Ultrasensitive and high-throughput nucleic acid detection based on antibody-like DNA nanostructure.

    Science.gov (United States)

    Wen, Junlin; Chen, Junhua; Zhuang, Li; Zhou, Shungui

    2017-04-15

    An ultrasensitive and high-throughput nucleic acid detection system, termed as strand displacement reaction-enzyme linked immunosorbent assay (SDR-ELISA), has been developed on the basis of antibody-like DNA nanostructures. Three digoxigenin or biotin modified hairpin probes are utilized to construct antibody-like DNA nanostructures that feature affinity toward streptavidin and anti-digoxigenin antibody via isothermal target-triggered SDR amplification. These antibody-like nanostructures have been employed to conjugate horseradish-peroxidase-labeled anti-digoxigenin antibody with streptavidin that is immobilized on microliter plate wells for enzyme-linked colorimetric assay. The resulting SDR-ELISA system is ultrasensitive for target DNA with a low detection limit of 5 fM. Moreover, the SDR-ELISA system is capable of discriminating DNA sequences with single base mutations, and do so in a high-throughput manner by detection and quantification of up to 96 or 384 DNA samples in a single shot. This detection system is further applied to detect other DNA targets such as Shewanella oneidensis specific DNA sequence, which indicates the generality of proposed SDR-ELISA system. The integration of SDR amplification and convenient ELISA technique advances an intelligent strategy for ultrasensitive and high-throughput nucleic acid detection, which may be amenable for direct visual detection and quantification using an accompanying quantitative color chart. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. 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.

  1. High-Throughput Phase-Field Design of High-Energy-Density Polymer Nanocomposites.

    Science.gov (United States)

    Shen, Zhong-Hui; Wang, Jian-Jun; Lin, Yuanhua; Nan, Ce-Wen; Chen, Long-Qing; Shen, Yang

    2017-11-22

    Understanding the dielectric breakdown behavior of polymer nanocomposites is crucial to the design of high-energy-density dielectric materials with reliable performances. It is however challenging to predict the breakdown behavior due to the complicated factors involved in this highly nonequilibrium process. In this work, a comprehensive phase-field model is developed to investigate the breakdown behavior of polymer nanocomposites under electrostatic stimuli. It is found that the breakdown strength and path significantly depend on the microstructure of the nanocomposite. The predicted breakdown strengths for polymer nanocomposites with specific microstructures agree with existing experimental measurements. Using this phase-field model, a high throughput calculation is performed to seek the optimal microstructure. Based on the high-throughput calculation, a sandwich microstructure for PVDF-BaTiO3 nanocomposite is designed, where the upper and lower layers are filled with parallel nanosheets and the middle layer is filled with vertical nanofibers. It has an enhanced energy density of 2.44 times that of the pure PVDF polymer. The present work provides a computational approach for understanding the electrostatic breakdown, and it is expected to stimulate future experimental efforts on synthesizing polymer nanocomposites with novel microstructures to achieve high performances. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. High-Throughput Phenotyping and QTL Mapping Reveals the Genetic Architecture of Maize Plant Growth1[OPEN

    Science.gov (United States)

    Huang, Chenglong; Wu, Di; Qiao, Feng; Li, Wenqiang; Duan, Lingfeng; Wang, Ke; Xiao, Yingjie; Chen, Guoxing; Liu, Qian; Yang, Wanneng

    2017-01-01

    With increasing demand for novel traits in crop breeding, the plant research community faces the challenge of quantitatively analyzing the structure and function of large numbers of plants. A clear goal of high-throughput phenotyping is to bridge the gap between genomics and phenomics. In this study, we quantified 106 traits from a maize (Zea mays) recombinant inbred line population (n = 167) across 16 developmental stages using the automatic phenotyping platform. Quantitative trait locus (QTL) mapping with a high-density genetic linkage map, including 2,496 recombinant bins, was used to uncover the genetic basis of these complex agronomic traits, and 988 QTLs have been identified for all investigated traits, including three QTL hotspots. Biomass accumulation and final yield were predicted using a combination of dissected traits in the early growth stage. These results reveal the dynamic genetic architecture of maize plant growth and enhance ideotype-based maize breeding and prediction. PMID:28153923

  3. High Throughput Heuristics for Prioritizing Human Exposure to Environmental Chemicals

    Science.gov (United States)

    The risk posed to human health by any of the thousands of untested anthropogenic chemicals in our environment is a function of both the potential hazard presented by the chemical, and the possibility of being exposed. Without the capacity to make quantitative, albeit uncertain, f...

  4. Automated Counting of Airborne Asbestos Fibers by a High-Throughput Microscopy (HTM Method

    Directory of Open Access Journals (Sweden)

    Hwataik Han

    2011-07-01

    Full Text Available Inhalation of airborne asbestos causes serious health problems such as lung cancer and malignant mesothelioma. The phase-contrast microscopy (PCM method has been widely used for estimating airborne asbestos concentrations because it does not require complicated processes or high-priced equipment. However, the PCM method is time-consuming and laborious as it is manually performed off-site by an expert. We have developed a high-throughput microscopy (HTM method that can detect fibers distinguishable from other spherical particles in a sample slide by image processing both automatically and quantitatively. A set of parameters for processing and analysis of asbestos fiber images was adjusted for standard asbestos samples with known concentrations. We analyzed sample slides containing airborne asbestos fibers collected at 11 different workplaces following PCM and HTM methods, and found a reasonably good agreement in the asbestos concentration. Image acquisition synchronized with the movement of the robotic sample stages followed by an automated batch processing of a stack of sample images enabled us to count asbestos fibers with greatly reduced time and labors. HTM should be a potential alternative to conventional PCM, moving a step closer to realization of on-site monitoring of asbestos fibers in air.

  5. High-Throughput Metabolic Profiling of Soybean Leaves by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

    Science.gov (United States)

    Yilmaz, Ali; Rudolph, Heather L; Hurst, Jerod J; Wood, Troy D

    2016-01-19

    As a relatively recent research field, plant metabolomics has gained increasing interest in the past few years and has been applied to answer biological questions through large-scale qualitative and quantitative analyses of the plant metabolome. The combination of sensitivity and selectivity offered by mass spectrometry (MS) for measurement of many metabolites in a single shot makes it an indispensable platform in metabolomics. In this regard, Fourier-transform ion cyclotron resonance (FTICR) has the unique advantage of delivering high mass resolving power and mass accuracy simultaneously, making it ideal for the study of complex mixtures such as plant extracts. Here we optimize soybean leaf extraction methods compatible with high-throughput reproducible MS-based metabolomics. In addition, matrix-assisted laser desorption ionization (MALDI) and direct LDI of soybean leaves are compared for metabolite profiling. The extraction method combined with electrospray (ESI)-FTICR is supported by the significant reduction of chlorophyll and its related metabolites as the growing season moves from midsummer to the autumn harvest day. To our knowledge for the first time, the use of ESI-FTICR MS and MALDI-FTICR MS is described in a complementary manner with the aim of metabolic profiling of plant leaves that have been collected at different time points during the growing season.

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

    Science.gov (United States)

    Moy, Terence I.; Conery, Annie L.; 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 enhance survival of C. elegans infected with E. faecalis. The screen uses a robot to accurately dispense live, infected animals into 384-well plates, and automated microscopy and image analysis to generate quantitative, high content data. We identified 28 compounds and extracts that were not previously reported to have antimicrobial properties, including 6 structural classes that cure infected C. elegans animals but do not affect the growth of the pathogen in vitro, thus acting by a mechanism of action distinct from antibiotics currently in clinical use. Our versatile and robust screening system can be easily adapted for other whole animal assays to probe a broad range of biological processes. PMID:19572548

  7. MRT letter: Micro- to nanoscale sample collection for high throughput microscopy.

    Science.gov (United States)

    Cheong, Brandon Huey-Ping; Liew, Oi Wah; Ng, Tuck Wah

    2013-08-01

    In high throughput microscopy, it is often assumed that the objects under investigation are fixed spatially. In addition, it is also presumed that the objects are sufficiently populated, otherwise there will be need to search through vast tracks of field of views before any recording can be done. The ability to collect objects at one location in the hydrated state is thus desirable and this is a challenge when the density of target objects in a sample is very low. In this work, we report that the generation of a squeezing flow from a circular coverslip compressing on suspensions is able to collect particulate (microbeads, fluorescent nanobeads and live algal cells) and non-particulate (EGFP) objects at the rim region of the coverslip. With a coverslip of 13 mm diameter, volumes between 2 µL and 4 µL were found to completely fill the coverslip without breaching the rims. Sample compression speeds between 100 µm/s and 1000 µm/s did not have any effect on object collection outcomes. In effect, the simple placement of coverslips on top the drop of sample by hand without a motorized translator was found to produce similar collection outcomes. Quantitative measurements confirmed that all the objects investigated were displaced and relocated at the rim regions to a very high degree. Copyright © 2013 Wiley Periodicals, Inc.

  8. 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...

  9. 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.

  10. Software Switching for High Throughput Data Acquisition Networks

    CERN Document Server

    AUTHOR|(CDS)2089787; Lehmann Miotto, Giovanna

    The bursty many-to-one communication pattern, typical for data acquisition systems, is particularly demanding for commodity TCP/IP and Ethernet technologies. The problem arising from this pattern is widely known in the literature as \\emph{incast} and can be observed as TCP throughput collapse. It is a result of overloading the switch buffers, when a specific node in a network requests data from multiple sources. This will become even more demanding for future upgrades of the experiments at the Large Hadron Collider at CERN. It is questionable whether commodity TCP/IP and Ethernet technologies in their current form will be still able to effectively adapt to bursty traffic without losing packets due to the scarcity of buffers in the networking hardware. This thesis provides an analysis of TCP/IP performance in data acquisition networks and presents a novel approach to incast congestion in these networks based on software-based packet forwarding. Our first contribution lies in confirming the strong analogies bet...

  11. Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening

    Directory of Open Access Journals (Sweden)

    Santos Carla S

    2012-11-01

    Full Text Available Abstract Background Pine wilt disease (PWD, caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus, damages and kills pine trees and is causing serious economic damage worldwide. Although the ecological mechanism of infestation is well described, the plant’s molecular response to the pathogen is not well known. This is due mainly to the lack of genomic information and the complexity of the disease. High throughput sequencing is now an efficient approach for detecting the expression of genes in non-model organisms, thus providing valuable information in spite of the lack of the genome sequence. In an attempt to unravel genes potentially involved in the pine defense against the pathogen, we hereby report the high throughput comparative sequence analysis of infested and non-infested stems of Pinus pinaster (very susceptible to PWN and Pinus pinea (less susceptible to PWN. Results Four cDNA libraries from infested and non-infested stems of P. pinaster and P. pinea were sequenced in a full 454 GS FLX run, producing a total of 2,083,698 reads. The putative amino acid sequences encoded by the assembled transcripts were annotated according to Gene Ontology, to assign Pinus contigs into Biological Processes, Cellular Components and Molecular Functions categories. Most of the annotated transcripts corresponded to Picea genes-25.4-39.7%, whereas a smaller percentage, matched Pinus genes, 1.8-12.8%, probably a consequence of more public genomic information available for Picea than for Pinus. The comparative transcriptome analysis showed that when P. pinaster was infested with PWN, the genes malate dehydrogenase, ABA, water deficit stress related genes and PAR1 were highly expressed, while in PWN-infested P. pinea, the highly expressed genes were ricin B-related lectin, and genes belonging to the SNARE and high mobility group families. Quantitative PCR experiments confirmed the differential gene expression between the two pine species

  12. High-throughput phenotyping and genomic selection: the frontiers of crop breeding converge.

    Science.gov (United States)

    Cabrera-Bosquet, Llorenç; Crossa, José; von Zitzewitz, Jarislav; Serret, María Dolors; Araus, José Luis

    2012-05-01

    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. © 2012 Institute of Botany, Chinese Academy of Sciences.

  13. Preselection of shotgun clones by oligonucleotide fingerprinting: an efficient and high throughput strategy to reduce redundancy in large-scale sequencing projects

    National Research Council Canada - National Science Library

    Radelof, U; Hennig, S; Seranski, P; Steinfath, M; Ramser, J; Reinhardt, R; Poustka, A; Francis, F; Lehrach, H

    1998-01-01

    .... To reduce the overall effort and cost of those projects and to accelerate the sequencing throughput, we have developed an efficient, high throughput oligonucleotide fingerprinting protocol to select...

  14. Recent progress using high-throughput sequencing technologies in plant molecular breeding.

    Science.gov (United States)

    Gao, Qiang; Yue, Guidong; Li, Wenqi; Wang, Junyi; Xu, Jiaohui; Yin, Ye

    2012-04-01

    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, multi-faceted, 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. © 2012 Institute of Botany, Chinese Academy of Sciences.

  15. Repeated Assessment by High-Throughput Assay Demonstrates that Sperm DNA Methylation Levels Are Highly Reproducible

    Science.gov (United States)

    Cortessis, Victoria K.; Siegmund, Kimberly; Houshdaran, Sahar; Laird, Peter W.; Sokol, Rebecca Z.

    2011-01-01

    Objective To assess reliability of high-throughput assay of sperm DNA methylation. Design Observational study comparing DNA methylation of sperm isolated from three divided and twelve longitudinally collected semen samples. Setting Academic Medical Center Patients One man undergoing screening semen analysis during evaluation of the infertile couple and two healthy fertile male volunteers. Interventions Spermatozoa were separated from seminal plasma and somatic cells using gradient separation. DNA was extracted from spermatozoa, and DNA methylation was assessed at 1,505 DNA-sequence specific sites. Main Outcome Measures Repeatability of sperm DNA methylation measures, estimated by correlation coefficients. Results DNA methylation levels were highly correlated within matched sets of divided samples (all r≥0.97) and longitudinal samples (average r=0.97). Conclusions The described methodology reliably assesses methylation of sperm DNA at large numbers of sites. Methylation profiles were consistent over time. High-throughput assessment of sperm DNA methylation is a promising tool for studying the role of epigenetic state in male fertility. PMID:22035967

  16. 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. © 2016 Elsevier Inc. All rights reserved.

  17. Development of a high-throughput solution for crystallinity measurement using THz-Raman spectroscopy

    Science.gov (United States)

    Roy, Anjan; Fosse, Jean-Charles; Fernandes, Filipe; Ringwald, Alexandre; Ho, Lawrence

    2017-02-01

    Rapid identification and the quantitative analysis of crystalline content and the degree of crystallinity is important in pharmaceuticals and polymer manufacturing. Crystallinity affects the bioavailability of pharmaceutical molecules and there is a strong correlation between the performance of polymers and their degree of crystallinity. Low frequency/THz-Raman spectroscopy has enabled determination of crystalline content in materials as a complementary method to X-ray powder diffraction. By incorporating motion stages and microplates, we have extended the applicability of THz-Raman technology to high-throughput screening applications. We describe here a complete THz-Raman microplate reader, with integrated laser, optics, spectrograph and software that are necessary for detecting low-frequency Raman signals. In powder materials scattering is also affected by particle size and the presence of cavities, which lead to a lack of precision and repeatability in Raman intensity measurements. We address this problem by spatial averaging using specific stage motion patterns. This design facilitates rapid and precise measurement of low-frequency vibrational modes, differentiation of polymorphs and other structural characteristics for applications in pharmaceuticals, nano- and bio-materials and for the characterization of industrial polymers where XRPD is commonly used.

  18. A tool named Iris for versatile high-throughput phenotyping in microorganisms.

    Science.gov (United States)

    Kritikos, George; Banzhaf, Manuel; Herrera-Dominguez, Lucia; Koumoutsi, Alexandra; Wartel, Morgane; Zietek, Matylda; Typas, Athanasios

    2017-02-17

    Advances in our ability to systematically introduce and track controlled genetic variance in microorganisms have, in the past decade, fuelled high-throughput reverse genetics approaches. When coupled to quantitative readouts, such approaches are extremely powerful at elucidating gene function and providing insights into the underlying pathways and the overall cellular network organization. Yet, until now, all efforts to quantify microbial macroscopic phenotypes have been restricted to monitoring growth in a small number of model microorganisms. We have developed an image analysis software named Iris, which allows for systematic exploration of a number of orthogonal-to-growth processes, including biofilm formation, colony morphogenesis, envelope biogenesis, sporulation and reporter activity. In addition, Iris provides more sensitive growth measurements than currently available software and is compatible with a variety of different microorganisms, as well as with endpoint or kinetic data. We used Iris to reanalyse existing chemical genomics data in Escherichia coli and to perform proof-of-principle screens on colony biofilm formation and morphogenesis of different bacterial species and the pathogenic fungus Candida albicans. We thereby recapitulated existing knowledge but also identified a plethora of additional genes and pathways involved in both processes.

  19. Application of Titration-Based Screening for the Rapid Pilot Testing of High-Throughput Assays.

    Science.gov (United States)

    Zhang, Ji-Hu; Kang, Zhao B; Ardayfio, Ophelia; Ho, Pei-i; Smith, Thomas; Wallace, Iain; Bowes, Scott; Hill, W Adam; Auld, Douglas S

    2014-06-01

    Pilot testing of an assay intended for high-throughput screening (HTS) with small compound sets is a necessary but often time-consuming step in the validation of an assay protocol. When the initial testing concentration is less than optimal, this can involve iterative testing at different concentrations to further evaluate the pilot outcome, which can be even more time-consuming. Quantitative HTS (qHTS) enables flexible and rapid collection of assay performance statistics, hits at different concentrations, and concentration-response curves in a single experiment. Here we describe the qHTS process for pilot testing in which eight-point concentration-response curves are produced using an interplate asymmetric dilution protocol in which the first four concentrations are used to represent the range of typical HTS screening concentrations and the last four concentrations are added for robust curve fitting to determine potency/efficacy values. We also describe how these data can be analyzed to predict the frequency of false-positives, false-negatives, hit rates, and confirmation rates for the HTS process as a function of screening concentration. By taking into account the compound pharmacology, this pilot-testing paradigm enables rapid assessment of the assay performance and choosing the optimal concentration for the large-scale HTS in one experiment. © 2013 Society for Laboratory Automation and Screening.

  20. Benchmarking Ligand-Based Virtual High-Throughput Screening with the PubChem Database

    Directory of Open Access Journals (Sweden)

    Mariusz Butkiewicz

    2013-01-01

    Full Text Available With the rapidly increasing availability of High-Throughput Screening (HTS data in the public domain, such as the PubChem database, methods for ligand-based computer-aided drug discovery (LB-CADD have the potential to accelerate and reduce the cost of probe development and drug discovery efforts in academia. We assemble nine data sets from realistic HTS campaigns representing major families of drug target proteins for benchmarking LB-CADD methods. Each data set is public domain through PubChem and carefully collated through confirmation screens validating active compounds. These data sets provide the foundation for benchmarking a new cheminformatics framework BCL::ChemInfo, which is freely available for non-commercial use. Quantitative structure activity relationship (QSAR models are built using Artificial Neural Networks (ANNs, Support Vector Machines (SVMs, Decision Trees (DTs, and Kohonen networks (KNs. Problem-specific descriptor optimization protocols are assessed including Sequential Feature Forward Selection (SFFS and various information content measures. Measures of predictive power and confidence are evaluated through cross-validation, and a consensus prediction scheme is tested that combines orthogonal machine learning algorithms into a single predictor. Enrichments ranging from 15 to 101 for a TPR cutoff of 25% are observed.

  1. High-throughput analysis of lectin-oligosaccharide interactions by automated frontal affinity chromatography.

    Science.gov (United States)

    Nakamura-Tsuruta, Sachiko; Uchiyama, Noboru; Hirabayashi, Jun

    2006-01-01

    Frontal affinity chromatography (FAC) is a quantitative method that enables sensitive and reproducible measurements of interactions between lectins and oligosaccharides. The method is suitable even for the measurement of low-affinity interactions and is based on a simple procedure and a clear principle. To achieve high-throughput and efficient analysis, an automated FAC system was developed. The system designated FAC-1 consists of two isocratic pumps, an autosampler, and a couple of miniature columns (bed volume, 31.4 microl) connected in parallel to either a fluorescence or an ultraviolet detector. By use of this parallel-column system, the time required for each analysis was reduced substantially. Under the established conditions, fewer than 10 hrs are required for 100 interaction analyses, consuming as little as 1 pmol pyridylaminated oligosaccharide for each analysis. This strategy for FAC should contribute to the construction of a lectin-oligosaccharide interaction database essential for future glycomics. Overall features and practical protocols for interaction analyses using FAC-1 are described.

  2. A High-Throughput Process for the Solid-Phase Purification of Synthetic DNA Sequences.

    Science.gov (United States)

    Grajkowski, Andrzej; Cieślak, Jacek; Beaucage, Serge L

    2017-06-19

    An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5'-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5'-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid-phase capture of the nucleobase- and phosphate-deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full-length DNA sequences are first washed away from the capture support; the solid-phase purified DNA sequences are then released from this support upon reaction with tetra-n-butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid-phase-purified DNA sequences exceeds 98%. The simulated high-throughput and scalability features of the solid-phase purification process are demonstrated without sacrificing purity of the DNA sequences. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  3. High throughput gene expression profiling: a molecular approach to integrative physiology

    Science.gov (United States)

    Liang, Mingyu; Cowley, Allen W; Greene, Andrew S

    2004-01-01

    Integrative physiology emphasizes the importance of understanding multiple pathways with overlapping, complementary, or opposing effects and their interactions in the context of intact organisms. The DNA microarray technology, the most commonly used method for high-throughput gene expression profiling, has been touted as an integrative tool that provides insights into regulatory pathways. However, the physiology community has been slow in acceptance of these techniques because of early failure in generating useful data and the lack of a cohesive theoretical framework in which experiments can be analysed. With recent advances in both technology and analysis, we propose a concept of multidimensional integration of physiology that incorporates data generated by DNA microarray and other functional, genomic, and proteomic approaches to achieve a truly integrative understanding of physiology. Analysis of several studies performed in simpler organisms or in mammalian model animals supports the feasibility of such multidimensional integration and demonstrates the power of DNA microarray as an indispensable molecular tool for such integration. Evaluation of DNA microarray techniques indicates that these techniques, despite limitations, have advanced to a point where the question-driven profiling research has become a feasible complement to the conventional, hypothesis-driven research. With a keen sense of homeostasis, global regulation, and quantitative analysis, integrative physiologists are uniquely positioned to apply these techniques to enhance the understanding of complex physiological functions. PMID:14678487

  4. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  6. High-throughput high-resolution class I HLA genotyping in East Africa.

    Directory of Open Access Journals (Sweden)

    Rebecca N Koehler

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

  7. A high-throughput media design approach for high performance mammalian fed-batch cultures.

    Science.gov (United States)

    Rouiller, Yolande; Périlleux, Arnaud; Collet, Natacha; Jordan, Martin; Stettler, Matthieu; Broly, Hervé

    2013-01-01

    An innovative high-throughput medium development method based on media blending was successfully used to improve the performance of a Chinese hamster ovary fed-batch medium in shaking 96-deepwell plates. Starting from a proprietary chemically-defined medium, 16 formulations testing 43 of 47 components at 3 different levels were designed. Media blending was performed following a custom-made mixture design of experiments considering binary blends, resulting in 376 different blends that were tested during both cell expansion and fed-batch production phases in one single experiment. Three approaches were chosen to provide the best output of the large amount of data obtained. A simple ranking of conditions was first used as a quick approach to select new formulations with promising features. Then, prediction of the best mixes was done to maximize both growth and titer using the Design Expert software. Finally, a multivariate analysis enabled identification of individual potential critical components for further optimization. Applying this high-throughput method on a fed-batch, rather than on a simple batch, process opens new perspectives for medium and feed development that enables identification of an optimized process in a short time frame.

  8. Microbial metal resistance and metabolism across dynamic landscapes: high-throughput environmental microbiology [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Hans Carlson

    2017-06-01

    Full Text Available Multidimensional gradients of inorganic compounds influence microbial activity in diverse pristine and anthropogenically perturbed environments. Here, we suggest that high-throughput cultivation and genetics can be systematically applied to generate quantitative models linking gene function, microbial community activity, and geochemical parameters. Metal resistance determinants represent a uniquely universal set of parameters around which to study and evaluate microbial fitness because they represent a record of the environment in which all microbial life evolved. By cultivating microbial isolates and enrichments in laboratory gradients of inorganic ions, we can generate quantitative predictions of limits on microbial range in the environment, obtain more accurate gene annotations, and identify useful strategies for predicting and engineering the trajectory of natural ecosystems.

  9. The sva package for removing batch effects and other unwanted variation in high-throughput experiments.

    Science.gov (United States)

    Leek, Jeffrey T; Johnson, W Evan; Parker, Hilary S; Jaffe, Andrew E; Storey, John D

    2012-03-15

    Heterogeneity and latent variables are now widely recognized as major sources of bias and variability in high-throughput experiments. The most well-known source of latent variation in genomic experiments are batch effects-when samples are processed on different days, in different groups or by different people. However, there are also a large number of other variables that may have a major impact on high-throughput measurements. Here we describe the sva package for identifying, estimating and removing unwanted sources of variation in high-throughput experiments. The sva package supports surrogate variable estimation with the sva function, direct adjustment for known batch effects with the ComBat function and adjustment for batch and latent variables in prediction problems with the fsva function.

  10. Plant phenomics and high-throughput phenotyping: accelerating rice functional genomics using multidisciplinary technologies.

    Science.gov (United States)

    Yang, Wanneng; Duan, Lingfeng; Chen, Guoxing; Xiong, Lizhong; Liu, Qian

    2013-05-01

    The functional analysis of the rice genome has entered into a high-throughput stage, and a project named RICE2020 has been proposed to determine the function of every gene in the rice genome by the year 2020. However, as compared with the robustness of genetic techniques, the evaluation of rice phenotypic traits is still performed manually, and the process is subjective, inefficient, destructive and error-prone. To overcome these limitations and help rice phenomics more closely parallel rice genomics, reliable, automatic, multifunctional, and high-throughput phenotyping platforms should be developed. In this article, we discuss the key plant phenotyping technologies, particularly photonics-based technologies, and then introduce their current applications in rice (wheat or barley) phenomics. We also note the major challenges in rice phenomics and are confident that these reliable high-throughput phenotyping tools will give plant scientists new perspectives on the information encoded in the rice genome. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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

  12. 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.

  13. 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.

  14. High throughput discovery of families of high activity WGS catalysts: part I--history and methodology.

    Science.gov (United States)

    Yaccato, Karin; Carhart, Ray; Hagemeyer, Alfred; Herrmann, Michael; Lesik, Andreas; Strasser, Peter; Volpe, Anthony; Turner, Howard; Weinberg, Henry; Grasselli, Robert K; Brooks, Christopher J; Pigos, John M

    2010-05-01

    State-of-art water gas shift catalysts (FeCr for high temperature shift and CuZn for low temperature shift) are not active enough to be used in fuel processors for the production of hydrogen from hydrocarbon fuels for fuel cells. The need for drastically lower catalyst volumes has triggered a search for novel WGS catalysts that are an order of magnitude more active than current systems. Novel catalytic materials for the high, medium and low temperature water gas shift reactions have been discovered by application of combinatorial methodologies. Catalyst libraries were synthesized on 4 inch wafers in 16 x 16 arrays and screened in a high throughput scanning mass spectrometer in the temperature range 200 degrees C to 400 degrees C. More than 200 wafers were screened under various conditions and more than 250,000 experiments were conducted to comprehensively examine catalyst performance for various binary, ternary and higher-order compositions.

  15. 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.

  16. Complementing high-throughput X-ray powder diffraction data with quantum-chemical calculations

    DEFF Research Database (Denmark)

    Naelapaa, Kaisa; van de Streek, Jacco; Rantanen, Jukka

    2012-01-01

    High-throughput crystallisation and characterisation platforms provide an efficient means to carry out solid-form screening during the pre-formulation phase. To determine the crystal structures of identified new solid phases, however, usually requires independent crystallisation trials to produce...... obtained only during high-energy processing such as spray drying or milling....

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

    Science.gov (United States)

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

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

    Science.gov (United States)

    Liu, Yun; Grossman, Jeffrey C

    2014-12-10

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

  19. Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

    Science.gov (United States)

    Ioanna Skilitsi, Anastasia; Turko, Timothé; Cianfarani, Damien; Barre, Sophie; Uhring, Wilfried; Hassiepen, Ulrich; Léonard, Jérémie

    2017-09-01

    Time-resolved fluorescence detection for robust sensing of biomolecular interactions is developed by implementing time-correlated single photon counting in high-throughput conditions. Droplet microfluidics is used as a promising platform for the very fast handling of low-volume samples. We illustrate the potential of this very sensitive and cost-effective technology in the context of an enzymatic activity assay based on fluorescently-labeled biomolecules. Fluorescence lifetime detection by time-correlated single photon counting is shown to enable reliable discrimination between positive and negative control samples at a throughput as high as several hundred samples per second.

  20. High-Throughput 3D Tumor Culture in a Recyclable Microfluidic Platform.

    Science.gov (United States)

    Liu, Wenming; Wang, Jinyi

    2017-01-01

    Three-dimensional (3D) tumor culture miniaturized platforms are of importance to biomimetic model construction and pathophysiological studies. Controllable and high-throughput production of 3D tumors is desirable to make cell-based manipulation dynamic and efficient at micro-scale. Moreover, the 3D culture platform being reusable is convenient to research scholars. In this chapter, we describe a dynamically controlled 3D tumor manipulation and culture method using pneumatic microstructure-based microfluidics, which has potential applications in the fields of tissue engineering, tumor biology, and clinical medicine in a high-throughput way.

  1. Applications of high-throughput plant phenotyping to study nutrient use efficiency.

    Science.gov (United States)

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

    2013-01-01

    Remote sensing and spectral reflectance measurements of plants has long been used to assess the growth and nutrient status of plants in a noninvasive manner. With improved imaging and computer technologies, these approaches can now be used at high-throughput for more extensive physiological and genetic studies. Here, we present an example of how high-throughput imaging can be used to study the growth of plants exposed to different nutrient levels. In addition, the color of the leaves can be used to estimate leaf chlorophyll and nitrogen status of the plant.

  2. A platform for high-throughput screening of DNA-encoded catalyst libraries in organic solvents.

    Science.gov (United States)

    Hook, K Delaney; Chambers, John T; Hili, Ryan

    2017-10-01

    We have developed a novel high-throughput screening platform for the discovery of small-molecules catalysts for bond-forming reactions. The method employs an in vitro selection for bond-formation using amphiphilic DNA-encoded small molecules charged with reaction substrate, which enables selections to be conducted in a variety of organic or aqueous solvents. Using the amine-catalysed aldol reaction as a catalytic model and high-throughput DNA sequencing as a selection read-out, we demonstrate the 1200-fold enrichment of a known aldol catalyst from a library of 16.7-million uncompetitive library members.

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

    Directory of Open Access Journals (Sweden)

    Jason R. Hattrick-Simpers

    2016-05-01

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

  4. Macro-to-micro structural proteomics: native source proteins for high-throughput crystallization.

    Science.gov (United States)

    Totir, Monica; Echols, Nathaniel; Nanao, Max; Gee, Christine L; Moskaleva, Alisa; Gradia, Scott; Iavarone, Anthony T; Berger, James M; May, Andrew P; Zubieta, Chloe; Alber, Tom

    2012-01-01

    Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein crystallography applications. The Escherichia coli proteome was fractionated, purified, crystallized, and structurally characterized. Macro-scale fermentation and fractionation were used to subdivide the soluble proteome into 408 unique fractions of which 295 fractions yielded crystals in microfluidic crystallization chips. Of the 295 crystals, 152 were selected for optimization, diffraction screening, and data collection. Twenty-three structures were determined, four of which were novel. This study demonstrates the utility of native source proteins for high-throughput crystallography.

  5. HTP-NLP: A New NLP System for High Throughput Phenotyping.

    Science.gov (United States)

    Schlegel, Daniel R; Crowner, Chris; Lehoullier, Frank; Elkin, Peter L

    2017-01-01

    Secondary use of clinical data for research requires a method to quickly process the data so that researchers can quickly extract cohorts. We present two advances in the High Throughput Phenotyping NLP system which support the aim of truly high throughput processing of clinical data, inspired by a characterization of the linguistic properties of such data. Semantic indexing to store and generalize partially-processed results and the use of compositional expressions for ungrammatical text are discussed, along with a set of initial timing results for the system.

  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...... 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....

  7. 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.

  8. Using manifold embedding for assessing and predicting protein interactions from high-throughput experimental data.

    Science.gov (United States)

    You, Zhu-Hong; Lei, Ying-Ke; Gui, Jie; Huang, De-Shuang; Zhou, Xiaobo

    2010-11-01

    High-throughput protein interaction data, with ever-increasing volume, are becoming the foundation of many biological discoveries, and thus high-quality protein-protein interaction (PPI) maps are critical for a deeper understanding of cellular processes. However, the unreliability and paucity of current available PPI data are key obstacles to the subsequent quantitative studies. It is therefore highly desirable to develop an approach to deal with these issues from the computational perspective. Most previous works for assessing and predicting protein interactions either need supporting evidences from multiple information resources or are severely impacted by the sparseness of PPI networks. We developed a robust manifold embedding technique for assessing the reliability of interactions and predicting new interactions, which purely utilizes the topological information of PPI networks and can work on a sparse input protein interactome without requiring additional information types. After transforming a given PPI network into a low-dimensional metric space using manifold embedding based on isometric feature mapping (ISOMAP), the problem of assessing and predicting protein interactions is recasted into the form of measuring similarity between points of its metric space. Then a reliability index, a likelihood indicating the interaction of two proteins, is assigned to each protein pair in the PPI networks based on the similarity between the points in the embedded space. Validation of the proposed method is performed with extensive experiments on densely connected and sparse PPI network of yeast, respectively. Results demonstrate that the interactions ranked top by our method have high-functional homogeneity and localization coherence, especially our method is very efficient for large sparse PPI network with which the traditional algorithms fail. Therefore, the proposed algorithm is a much more promising method to detect both false positive and false negative interactions

  9. Liquid Phase Multiplex High-Throughput Screening of Metagenomic Libraries Using p-Nitrophenyl-Linked Substrates for Accessory Lignocellulosic Enzymes.

    Science.gov (United States)

    Smart, Mariette; Huddy, Robert J; Cowan, Don A; Trindade, Marla

    2017-01-01

    To access the genetic potential contained in large metagenomic libraries, suitable high-throughput functional screening methods are required. Here we describe a high-throughput screening approach which enables the rapid identification of metagenomic library clones expressing functional accessory lignocellulosic enzymes. The high-throughput nature of this method hinges on the multiplexing of both the E. coli metagenomic library clones and the colorimetric p-nitrophenyl linked substrates which allows for the simultaneous screening for β-glucosidases, β-xylosidases, and α-L-arabinofuranosidases. This method is readily automated and compatible with high-throughput robotic screening systems.

  10. 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

  11. 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.

  12. High pressure inertial focusing for separating and concentrating bacteria at high throughput

    Science.gov (United States)

    Cruz, J.; Hooshmand Zadeh, S.; Graells, T.; Andersson, M.; Malmström, J.; Wu, Z. G.; Hjort, K.

    2017-08-01

    Inertial focusing is a promising microfluidic technology for concentration and separation of particles by size. However, there is a strong correlation of increased pressure with decreased particle size. Theory and experimental results for larger particles were used to scale down the phenomenon and find the conditions that focus 1 µm particles. High pressure experiments in robust glass chips were used to demonstrate the alignment. We show how the technique works for 1 µm spherical polystyrene particles and for Escherichia coli, not being harmful for the bacteria at 50 µl min-1. The potential to focus bacteria, simplicity of use and high throughput make this technology interesting for healthcare applications, where concentration and purification of a sample may be required as an initial step.

  13. HiCTMap: Detection and analysis of chromosome territory structure and position by high-throughput imaging.

    Science.gov (United States)

    Jowhar, Ziad; Gudla, Prabhakar R; Shachar, Sigal; Wangsa, Darawalee; Russ, Jill L; Pegoraro, Gianluca; Ried, Thomas; Raznahan, Armin; Misteli, Tom

    2018-02-10

    The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues. Published by Elsevier Inc.

  14. Cell surface profiling using high-throughput flow cytometry: a platform for biomarker discovery and analysis of cellular heterogeneity.

    Directory of Open Access Journals (Sweden)

    Craig A Gedye

    Full Text Available Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell

  15. Validation and implementation of a novel high-throughput behavioral phenotyping instrument for mice

    Science.gov (United States)

    Brodkin, Jesse; Frank, Dana; Grippo, Ryan; Hausfater, Michal; Gulinello, Maria; Achterholt, Nils; Gutzen, Christian

    2015-01-01

    Background Behavioral assessment of mutant mouse models and novel candidate drugs is a slow and labor intensive process. This limitation produces a significant impediment to CNS drug discovery. New method By combining video and vibration analysis we created an automated system that provides the most detailed description of mouse behavior available. Our system (The Behavioral Spectrometer) allowed for the rapid assessment of behavioral abnormalities in the BTBR model of Autism, the restraint model of stress and the irritant model of inflammatory pain. Results We found that each model produced a unique alteration of the spectrum of behavior emitted by the mice. BTBR mice engaged in more grooming and less rearing behaviors. Prior restraint stress produced dramatic increases in grooming activity at the expense of locomotor behavior. Pain produced profound decreases in emitted behavior that were reversible with analgesic treatment. Comparison with existing method(s) We evaluated our system through a direct comparison on the same subjects with the current “gold standard” of human observation of video recordings. Using the same mice evaluated over the same range of behaviors, the Behavioral Spectrometer produced a quantitative categorization of behavior that was highly correlated with the scores produced by trained human observers (r=0.97). Conclusions Our results show that this new system is a highly valid and sensitive method to characterize behavioral effects in mice. As a fully automated and easily scalable instrument the Behavioral Spectrometer represents a high-throughput behavioral tool that reduces the time and labor involved in behavioral research. PMID:24384067

  16. High-throughput time-stretch microscopy with morphological and chemical specificity

    Science.gov (United States)

    Lei, Cheng; Ugawa, Masashi; Nozawa, Taisuke; Ideguchi, Takuro; Di Carlo, Dino; Ota, Sadao; Ozeki, Yasuyuki; Goda, Keisuke

    2016-03-01

    Particle analysis is an effective method in analytical chemistry for sizing and counting microparticles such as emulsions, colloids, and biological cells. However, conventional methods for particle analysis, which fall into two extreme categories, have severe limitations. Sieving and Coulter counting are capable of analyzing particles with high throughput, but due to their lack of detailed information such as morphological and chemical characteristics, they can only provide statistical results with low specificity. On the other hand, CCD or CMOS image sensors can be used to analyze individual microparticles with high content, but due to their slow charge download, the frame rate (hence, the throughput) is significantly limited. Here by integrating a time-stretch optical microscope with a three-color fluorescent analyzer on top of an inertial-focusing microfluidic device, we demonstrate an optofluidic particle analyzer with a sub-micrometer spatial resolution down to 780 nm and a high throughput of 10,000 particles/s. In addition to its morphological specificity, the particle analyzer provides chemical specificity to identify chemical expressions of particles via fluorescence detection. Our results indicate that we can identify different species of microparticles with high specificity without sacrificing throughput. Our method holds promise for high-precision statistical particle analysis in chemical industry and pharmaceutics.

  17. Predicting gene function through systematic analysis and quality assessment of high-throughput data.

    Science.gov (United States)

    Kemmeren, Patrick; Kockelkorn, Thessa T J P; Bijma, Theo; Donders, Rogier; Holstege, Frank C P

    2005-04-15

    Determining gene function is an important challenge arising from the availability of whole genome sequences. Until recently, approaches based on sequence homology were the only high-throughput method for predicting gene function. Use of high-throughput generated experimental data sets for determining gene function has been limited for several reasons. Here a new approach is presented for integration of high-throughput data sets, leading to prediction of function based on relationships supported by multiple types and sources of data. This is achieved with a database containing 125 different high-throughput data sets describing phenotypes, cellular localizations, protein interactions and mRNA expression levels from Saccharomyces cerevisiae, using a bit-vector representation and information content-based ranking. The approach takes characteristic and qualitative differences between the data sets into account, is highly flexible, efficient and scalable. Database queries result in predictions for 543 uncharacterized genes, based on multiple functional relationships each supported by at least three types of experimental data. Some of these are experimentally verified, further demonstrating their reliability. The results also generate insights into the relative merits of different data types and provide a coherent framework for functional genomic datamining. Free availability over the Internet. f.c.p.holstege@med.uu.nl http://www.genomics.med.uu.nl/pub/pk/comb_gen_network.

  18. 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 by develop......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....

  19. High-Throughput Cancer Cell Sphere Formation for 3D Cell Culture.

    Science.gov (United States)

    Chen, Yu-Chih; Yoon, Euisik

    2017-01-01

    Three-dimensional (3D) cell culture is critical in studying cancer pathology and drug response. Though 3D cancer sphere culture can be performed in low-adherent dishes or well plates, the unregulated cell aggregation may skew the results. On contrary, microfluidic 3D culture can allow precise control of cell microenvironments, and provide higher throughput by orders of magnitude. In this chapter, we will look into engineering innovations in a microfluidic platform for high-throughput cancer cell sphere formation and review the implementation methods in detail.

  20. Lights, camera, action: high-throughput plant phenotyping is ready for a close-up.

    Science.gov (United States)

    Fahlgren, Noah; Gehan, Malia A; Baxter, Ivan

    2015-04-01

    Anticipated population growth, shifting demographics, and environmental variability over the next century are expected to threaten global food security. In the face of these challenges, crop yield for food and fuel must be maintained and improved using fewer input resources. In recent years, genetic tools for profiling crop germplasm has benefited from rapid advances in DNA sequencing, and now similar advances are needed to improve the throughput of plant phenotyping. We highlight recent developments in high-throughput plant phenotyping using robotic-assisted imaging platforms and computer vision-assisted analysis tools. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Generating Mouse Models Using Zygote Electroporation of Nucleases (ZEN) Technology with High Efficiency and Throughput.

    Science.gov (United States)

    Wang, Wenbo; Zhang, Yingfan; Wang, Haoyi

    2017-01-01

    Mouse models with genetic modifications are widely used in biology and biomedical research. Although the application of CRISPR-Cas9 system greatly accelerated the process of generating genetically modified mice, the delivery method depending on manual injection of the components into the embryos remains a bottleneck, as it is laborious, low throughput, and technically demanding. To overcome this limitation, we invented and optimized the ZEN (Zygote electroporation of nucleases) technology to deliver CRISPR-Cas9 reagents via electroporation. Using ZEN, we were able to generate genetically modified mouse models with high efficiency and throughput. Here, we describe the protocol in great detail.

  2. 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.

  3. HTSSIP: An R package for analysis of high throughput sequencing data from nucleic acid stable isotope probing (SIP) experiments.

    Science.gov (United States)

    Youngblut, Nicholas D; Barnett, Samuel E; Buckley, Daniel H

    2018-01-01

    Combining high throughput sequencing with stable isotope probing (HTS-SIP) is a powerful method for mapping in situ metabolic processes to thousands of microbial taxa. However, accurately mapping metabolic processes to taxa is complex and challenging. Multiple HTS-SIP data analysis methods have been developed, including high-resolution stable isotope probing (HR-SIP), multi-window high-resolution stable isotope probing (MW-HR-SIP), quantitative stable isotope probing (qSIP), and ΔBD. Currently, there is no publicly available software designed specifically for analyzing HTS-SIP data. To address this shortfall, we have developed the HTSSIP R package, an open-source, cross-platform toolset for conducting HTS-SIP analyses in a straightforward and easily reproducible manner. The HTSSIP package, along with full documentation and examples, is available from CRAN at https://cran.r-project.org/web/packages/HTSSIP/index.html and Github at https://github.com/buckleylab/HTSSIP.

  4. Holographic memory module with ultra-high capacity and throughput

    Energy Technology Data Exchange (ETDEWEB)

    Vladimir A. Markov, Ph.D.

    2000-06-04

    High capacity, high transfer rate, random access memory systems are needed to archive and distribute the tremendous volume of digital information being generated, for example, the human genome mapping and online libraries. The development of multi-gigabit per second networks underscores the need for next-generation archival memory systems. During Phase I we conducted the theoretical analysis and accomplished experimental tests that validated the key aspects of the ultra-high density holographic data storage module with high transfer rate. We also inspected the secure nature of the encoding method and estimated the performance of full-scale system. Two basic architectures were considered, allowing for reversible compact solid-state configuration with limited capacity, and very large capacity write once read many memory system.

  5. 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

  6. 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...

  7. 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

  8. Microfluidic Impedance Flow Cytometry Enabling High-Throughput Single-Cell Electrical Property Characterization

    OpenAIRE

    Jian Chen; Chengcheng Xue; Yang Zhao; Deyong Chen; Min-Hsien Wu; Junbo Wang

    2015-01-01

    This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) microfluidic impedance flow cytometry with enhanced sensitivity; (3) microfluidic impedance ...

  9. High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots.

    Directory of Open Access Journals (Sweden)

    Guangbo Liu

    Full Text Available Saccharomyces cerevisiae (budding yeast is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids or genome mutation (e.g., gene mutation, deletion, epitope tagging is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.

  10. The Power of High-Throughput Experimentation in Homogeneous Catalysis Research for Fine Chemicals

    NARCIS (Netherlands)

    Vries, Johannes G. de; Vries, André H.M. de

    2003-01-01

    The use of high-throughput experimentation (HTE) in homogeneous catalysis research for the production of fine chemicals is an important breakthrough. Whereas in the past stoichiometric chemistry was often preferred because of time-to-market constraints, HTE allows catalytic solutions to be found

  11. Functional characterisation of human glycine receptors in a fluorescence-based high throughput screening assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.

    2005-01-01

    receptors in this assay were found to be in good agreement with those from electrophysiology studies of the receptors expressed in Xenopus oocytes or mammalian cell lines. Hence, this high throughput screening assay will be of great use in future pharmacological studies of glycine receptors, particular...

  12. tcpl: The ToxCast Pipeline for High-Throughput Screening Data

    Science.gov (United States)

    Motivation: The large and diverse high-throughput chemical screening efforts carried out by the US EPAToxCast program requires an efficient, transparent, and reproducible data pipeline.Summary: The tcpl R package and its associated MySQL database provide a generalized platform fo...

  13. Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis

    NARCIS (Netherlands)

    Au, K.; Folkers, G.E.; Kaptein, R.

    2006-01-01

    A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based

  14. 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...

  15. 20170913 - Retrofit Strategies for Incorporating Xenobiotic Metabolism into High Throughput Screening Assays (EMGS)

    Science.gov (United States)

    The US EPA’s ToxCast program is designed to assess chemical perturbations of molecular and cellular endpoints using a variety of high-throughput screening (HTS) assays. However, existing HTS assays have limited or no xenobiotic metabolism which could lead to a mischaracteri...

  16. 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...

  17. HTPheno: an image analysis pipeline for high-throughput plant phenotyping.

    Science.gov (United States)

    Hartmann, Anja; Czauderna, Tobias; Hoffmann, Roberto; Stein, Nils; Schreiber, Falk

    2011-05-12

    In the last few years high-throughput analysis methods have become state-of-the-art in the life sciences. One of the latest developments is automated greenhouse systems for high-throughput plant phenotyping. Such systems allow the non-destructive screening of plants over a period of time by means of image acquisition techniques. During such screening different images of each plant are recorded and must be analysed by applying sophisticated image analysis algorithms. This paper presents an image analysis pipeline (HTPheno) for high-throughput plant phenotyping. HTPheno is implemented as a plugin for ImageJ, an open source image processing software. It provides the possibility to analyse colour images of plants which are taken in two different views (top view and side view) during a screening. Within the analysis different phenotypical parameters for each plant such as height, width and projected shoot area of the plants are calculated for the duration of the screening. HTPheno is applied to analyse two barley cultivars. HTPheno, an open source image analysis pipeline, supplies a flexible and adaptable ImageJ plugin which can be used for automated image analysis in high-throughput plant phenotyping and therefore to derive new biological insights, such as determination of fitness.

  18. HTPheno: An image analysis pipeline for high-throughput plant phenotyping

    Directory of Open Access Journals (Sweden)

    Stein Nils

    2011-05-01

    Full Text Available Abstract Background In the last few years high-throughput analysis methods have become state-of-the-art in the life sciences. One of the latest developments is automated greenhouse systems for high-throughput plant phenotyping. Such systems allow the non-destructive screening of plants over a period of time by means of image acquisition techniques. During such screening different images of each plant are recorded and must be analysed by applying sophisticated image analysis algorithms. Results This paper presents an image analysis pipeline (HTPheno for high-throughput plant phenotyping. HTPheno is implemented as a plugin for ImageJ, an open source image processing software. It provides the possibility to analyse colour images of plants which are taken in two different views (top view and side view during a screening. Within the analysis different phenotypical parameters for each plant such as height, width and projected shoot area of the plants are calculated for the duration of the screening. HTPheno is applied to analyse two barley cultivars. Conclusions HTPheno, an open source image analysis pipeline, supplies a flexible and adaptable ImageJ plugin which can be used for automated image analysis in high-throughput plant phenotyping and therefore to derive new biological insights, such as determination of fitness.

  19. 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

  20. ESSENTIALS: Software for Rapid Analysis of High Throughput Transposon Insertion Sequencing Data.

    NARCIS (Netherlands)

    Zomer, A.L.; Burghout, P.J.; Bootsma, H.J.; Hermans, P.W.M.; Hijum, S.A.F.T. van

    2012-01-01

    High-throughput analysis of genome-wide random transposon mutant libraries is a powerful tool for (conditional) essential gene discovery. Recently, several next-generation sequencing approaches, e.g. Tn-seq/INseq, HITS and TraDIS, have been developed that accurately map the site of transposon

  1. Development of a thyroperoxidase inhibition assay for high-throughput screening

    Science.gov (United States)

    High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluores...

  2. Evaluation of Simple and Inexpensive High-Throughput Methods for Phytic Acid Determination

    DEFF Research Database (Denmark)

    Raboy, Victor; Johnson, Amy; Bilyeu, Kristin

    2017-01-01

    High-throughput/low-cost/low-tech methods for phytic acid determination that are sufficiently accurate and reproducible would be of value in plant genetics, crop breeding and in the food and feed industries. Variants of two candidate methods, those described by Vaintraub and Lapteva (Anal Biochem...

  3. Virtual high screening throughput and design of 14α-lanosterol ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-06

    Jul 6, 2009 ... Hildebert B. Maurice1*, Esther Tuarira1 and Kennedy Mwambete2. 1School of Pharmaceutical Sciences, Institute of Allied Health Sciences, Muhimbili University of ... high throughput screening (Guardiola-Diaz et al.,. 2001). It is therefore logical to think that developing inhi- bitors against the mycobacterial ...

  4. Increasing ecological inference from high throughput sequencing of fungi in the environment through a tagging approach

    Science.gov (United States)

    D. Lee Taylor; Michael G. Booth; Jack W. McFarland; Ian C. Herriott; Niall J. Lennon; Chad Nusbaum; Thomas G. Marr

    2008-01-01

    High throughput sequencing methods are widely used in analyses of microbial diversity but are generally applied to small numbers of samples, which precludes charaterization of patterns of microbial diversity across space and time. We have designed a primer-tagging approach that allows pooling and subsequent sorting of numerous samples, which is directed to...

  5. The protein crystallography beamline BW6 at DORIS - automatic operation and high-throughput data collection

    CERN Document Server

    Blume, H; Bourenkov, G P; Kosciesza, D; Bartunik, H D

    2001-01-01

    The wiggler beamline BW6 at DORIS has been optimized for de-novo solution of protein structures on the basis of MAD phasing. Facilities for automatic data collection, rapid data transfer and storage, and online processing have been developed which provide adequate conditions for high-throughput applications, e.g., in structural genomics.

  6. A high throughput platform for understanding the influence of excipients on physical and chemical stability

    DEFF Research Database (Denmark)

    Raijada, Dhara; Cornett, Claus; Rantanen, Jukka

    2013-01-01

    The present study puts forward a miniaturized high-throughput platform to understand influence of excipient selection and processing on the stability of a given drug compound. Four model drugs (sodium naproxen, theophylline, amlodipine besylate and nitrofurantoin) and ten different excipients were...

  7. 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...

  8. High-throughput siRNA screening applied to the ubiquitin-proteasome system

    DEFF Research Database (Denmark)

    Poulsen, Esben Guldahl; Nielsen, Sofie V.; Pietras, Elin J.

    2016-01-01

    that are not genetically tractable as, for instance, a yeast model system. Here, we describe a method relying on high-throughput cellular imaging of cells transfected with a targeted siRNA library to screen for components involved in degradation of a protein of interest. This method is a rapid and cost-effective tool...

  9. High-throughput verification of transcriptional starting sites by Deep-RACE

    DEFF Research Database (Denmark)

    Olivarius, Signe; Plessy, Charles; Carninci, Piero

    2009-01-01

    We present a high-throughput method for investigating the transcriptional starting sites of genes of interest, which we named Deep-RACE (Deep–rapid amplification of cDNA ends). Taking advantage of the latest sequencing technology, it allows the parallel analysis of multiple genes and is free...

  10. High throughput generated micro-aggregates of chondrocytes stimulate cartilage formation in vitro and in vivo

    NARCIS (Netherlands)

    Moreira Teixeira, Liliana; Leijten, Jeroen Christianus Hermanus; Sobral, J.; Jin, R.; van Apeldoorn, Aart A.; Feijen, Jan; van Blitterswijk, Clemens; Dijkstra, Pieter J.; Karperien, Hermanus Bernardus Johannes

    2012-01-01

    Cell-based cartilage repair strategies such as matrix-induced autologous chondrocyte implantation (MACI) could be improved by enhancing cell performance. We hypothesised that micro-aggregates of chondrocytes generated in high-throughput prior to implantation in a defect could stimulate cartilaginous

  11. 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...

  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

  13. 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

    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...

  14. High throughput system for magnetic manipulation of cells, polymers, and biomaterials

    Science.gov (United States)

    Spero, Richard Chasen; Vicci, Leandra; Cribb, Jeremy; Bober, David; Swaminathan, Vinay; O’Brien, E. Timothy; Rogers, Stephen L.; Superfine, R.

    2008-01-01

    In the past decade, high throughput screening (HTS) has changed the way biochemical assays are performed, but manipulation and mechanical measurement of micro- and nanoscale systems have not benefited from this trend. Techniques using microbeads (particles ∼0.1–10 μm) show promise for enabling high throughput mechanical measurements of microscopic systems. We demonstrate instrumentation to magnetically drive microbeads in a biocompatible, multiwell magnetic force system. It is based on commercial HTS standards and is scalable to 96 wells. Cells can be cultured in this magnetic high throughput system (MHTS). The MHTS can apply independently controlled forces to 16 specimen wells. Force calibrations demonstrate forces in excess of 1 nN, predicted force saturation as a function of pole material, and powerlaw dependence of F∼r−2.7±0.1. We employ this system to measure the stiffness of SR2+ Drosophila cells. MHTS technology is a key step toward a high throughput screening system for micro- and nanoscale biophysical experiments. PMID:19044357

  15. The Impact of Data Fragmentation on High-Throughput Clinical Phenotyping

    Science.gov (United States)

    Wei, Weiqi

    2012-01-01

    Subject selection is essential and has become the rate-limiting step for harvesting knowledge to advance healthcare through clinical research. Present manual approaches inhibit researchers from conducting deep and broad studies and drawing confident conclusions. High-throughput clinical phenotyping (HTCP), a recently proposed approach, leverages…

  16. 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

  17. High-throughput assessment of context-dependent effects of chromatin proteins

    NARCIS (Netherlands)

    Brueckner, L. (Laura); Van Arensbergen, J. (Joris); Akhtar, W. (Waseem); L. Pagie (Ludo); B. van Steensel (Bas)

    2016-01-01

    textabstractBackground: Chromatin proteins control gene activity in a concerted manner. We developed a high-throughput assay to study the effects of the local chromatin environment on the regulatory activity of a protein of interest. The assay combines a previously reported multiplexing strategy

  18. High-throughput phenotyping to detect drought tolerance QTL in wild barley introgression lines

    KAUST Repository

    Honsdorf, Nora

    2014-05-13

    Drought is one of the most severe stresses, endangering crop yields worldwide. In order to select drought tolerant genotypes, access to exotic germplasm and efficient phenotyping protocols are needed. In this study the high-throughput phenotyping platform "The Plant Accelerator", Adelaide, Australia, was used to screen a set of 47 juvenile (six week old) wild barley introgression lines (S42ILs) for drought stress responses. The kinetics of growth development was evaluated under early drought stress and well watered treatments. High correlation (r = 0.98) between image based biomass estimates and actual biomass was demonstrated, and the suitability of the system to accurately and non-destructively estimate biomass was validated. Subsequently, quantitative trait loci (QTL) were located, which contributed to the genetic control of growth under drought stress. In total, 44 QTL for eleven out of 14 investigated traits were mapped, which for example controlled growth rate and water use efficiency. The correspondence of those QTL with QTL previously identified in field trials is shown. For instance, six out of eight QTL controlling plant height were also found in previous field and glasshouse studies with the same introgression lines. This indicates that phenotyping juvenile plants may assist in predicting adult plant performance. In addition, favorable wild barley alleles for growth and biomass parameters were detected, for instance, a QTL that increased biomass by approximately 36%. In particular, introgression line S42IL-121 revealed improved growth under drought stress compared to the control Scarlett. The introgression line showed a similar behavior in previous field experiments, indicating that S42IL-121 may be an attractive donor for breeding of drought tolerant barley cultivars. © 2014 Honsdorf et al.

  19. 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.

  20. High-throughput phenotyping to detect drought tolerance QTL in wild barley introgression lines.

    Directory of Open Access Journals (Sweden)

    Nora Honsdorf

    Full Text Available Drought is one of the most severe stresses, endangering crop yields worldwide. In order to select drought tolerant genotypes, access to exotic germplasm and efficient phenotyping protocols are needed. In this study the high-throughput phenotyping platform "The Plant Accelerator", Adelaide, Australia, was used to screen a set of 47 juvenile (six week old wild barley introgression lines (S42ILs for drought stress responses. The kinetics of growth development was evaluated under early drought stress and well watered treatments. High correlation (r=0.98 between image based biomass estimates and actual biomass was demonstrated, and the suitability of the system to accurately and non-destructively estimate biomass was validated. Subsequently, quantitative trait loci (QTL were located, which contributed to the genetic control of growth under drought stress. In total, 44 QTL for eleven out of 14 investigated traits were mapped, which for example controlled growth rate and water use efficiency. The correspondence of those QTL with QTL previously identified in field trials is shown. For instance, six out of eight QTL controlling plant height were also found in previous field and glasshouse studies with the same introgression lines. This indicates that phenotyping juvenile plants may assist in predicting adult plant performance. In addition, favorable wild barley alleles for growth and biomass parameters were detected, for instance, a QTL that increased biomass by approximately 36%. In particular, introgression line S42IL-121 revealed improved growth under drought stress compared to the control Scarlett. The introgression line showed a similar behavior in previous field experiments, indicating that S42IL-121 may be an attractive donor for breeding of drought tolerant barley cultivars.

  1. High-throughput phenotyping to detect drought tolerance QTL in wild barley introgression lines.

    Science.gov (United States)

    Honsdorf, Nora; March, Timothy John; Berger, Bettina; Tester, Mark; Pillen, Klaus

    2014-01-01

    Drought is one of the most severe stresses, endangering crop yields worldwide. In order to select drought tolerant genotypes, access to exotic germplasm and efficient phenotyping protocols are needed. In this study the high-throughput phenotyping platform "The Plant Accelerator", Adelaide, Australia, was used to screen a set of 47 juvenile (six week old) wild barley introgression lines (S42ILs) for drought stress responses. The kinetics of growth development was evaluated under early drought stress and well watered treatments. High correlation (r=0.98) between image based biomass estimates and actual biomass was demonstrated, and the suitability of the system to accurately and non-destructively estimate biomass was validated. Subsequently, quantitative trait loci (QTL) were located, which contributed to the genetic control of growth under drought stress. In total, 44 QTL for eleven out of 14 investigated traits were mapped, which for example controlled growth rate and water use efficiency. The correspondence of those QTL with QTL previously identified in field trials is shown. For instance, six out of eight QTL controlling plant height were also found in previous field and glasshouse studies with the same introgression lines. This indicates that phenotyping juvenile plants may assist in predicting adult plant performance. In addition, favorable wild barley alleles for growth and biomass parameters were detected, for instance, a QTL that increased biomass by approximately 36%. In particular, introgression line S42IL-121 revealed improved growth under drought stress compared to the control Scarlett. The introgression line showed a similar behavior in previous field experiments, indicating that S42IL-121 may be an attractive donor for breeding of drought tolerant barley cultivars.

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

    Science.gov (United States)

    2013-01-01

    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. PMID:23938087

  3. Establishment of integrated protocols for automated high throughput kinetic chlorophyll fluorescence analyses.

    Science.gov (United States)

    Tschiersch, Henning; Junker, Astrid; Meyer, Rhonda C; Altmann, Thomas

    2017-01-01

    Automated plant phenotyping has been established as a powerful new tool in studying plant growth, development and response to various types of biotic or abiotic stressors. Respective facilities mainly apply non-invasive imaging based methods, which enable the continuous quantification of the dynamics of plant growth and physiology during developmental progression. However, especially for plants of larger size, integrative, automated and high throughput measurements of complex physiological parameters such as photosystem II efficiency determined through kinetic chlorophyll fluorescence analysis remain a challenge. We present the technical installations and the establishment of experimental procedures that allow the integrated high throughput imaging of all commonly determined PSII parameters for small and large plants using kinetic chlorophyll fluorescence imaging systems (FluorCam, PSI) integrated into automated phenotyping facilities (Scanalyzer, LemnaTec). Besides determination of the maximum PSII efficiency, we focused on implementation of high throughput amenable protocols recording PSII operating efficiency (ΦPSII). Using the presented setup, this parameter is shown to be reproducibly measured in differently sized plants despite the corresponding variation in distance between plants and light source that caused small differences in incident light intensity. Values of ΦPSII obtained with the automated chlorophyll fluorescence imaging setup correlated very well with conventionally determined data using a spot-measuring chlorophyll fluorometer. The established high throughput operating protocols enable the screening of up to 1080 small and 184 large plants per hour, respectively. The application of the implemented high throughput protocols is demonstrated in screening experiments performed with large Arabidopsis and maize populations assessing natural variation in PSII efficiency. The incorporation of imaging systems suitable for kinetic chlorophyll

  4. High Performance Computing Modernization Program Kerberos Throughput Test Report

    Science.gov (United States)

    2017-10-26

    ORGANIZATION REPORT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 10. SPONSOR / MONITOR’S ACRONYM(S)9. SPONSORING / MONITORING AGENCY...the high computing power of the main supercomputer. Each supercomputer is different in node architecture as well as hardware specifications. 2

  5. 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.

  6. CrossCheck: an open-source web tool for high-throughput screen data analysis.

    Science.gov (United States)

    Najafov, Jamil; Najafov, Ayaz

    2017-07-19

    Modern high-throughput screening methods allow researchers to generate large datasets that potentially contain important biological information. However, oftentimes, picking relevant hits from such screens and generating testable hypotheses requires training in bioinformatics and the skills to efficiently perform database mining. There are currently no tools available to general public that allow users to cross-reference their screen datasets with published screen datasets. To this end, we developed CrossCheck, an online platform for high-throughput screen data analysis. CrossCheck is a centralized database that allows effortless comparison of the user-entered list of gene symbols with 16,231 published datasets. These datasets include published data from genome-wide RNAi and CRISPR screens, interactome proteomics and phosphoproteomics screens, cancer mutation databases, low-throughput studies of major cell signaling mediators, such as kinases, E3 ubiquitin ligases and phosphatases, and gene ontological information. Moreover, CrossCheck includes a novel database of predicted protein kinase substrates, which was developed using proteome-wide consensus motif searches. CrossCheck dramatically simplifies high-throughput screen data analysis and enables researchers to dig deep into the published literature and streamline data-driven hypothesis generation. CrossCheck is freely accessible as a web-based application at http://proteinguru.com/crosscheck.

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

    Science.gov (United States)

    Walker, Steven L; Ariga, Junko; Mathias, Jonathan R; Coothankandaswamy, Veena; Xie, Xiayang; Distel, Martin; Köster, Reinhard W; Parsons, Michael J; Bhalla, Kapil N; Saxena, Meera T; Mumm, Jeff S

    2012-01-01

    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.

  8. Nanostructured biosensing platform-shadow edge lithography for high-throughput nanofabrication.

    Science.gov (United States)

    Bai, John G; Yeo, Woon-Hong; Chung, Jae-Hyun

    2009-02-07

    One of the critical challenges in nanostructured biosensors is to manufacture an addressable array of nanopatterns at low cost. The addressable array (1) provides multiplexing for biomolecule detection and (2) enables direct detection of biomolecules without labeling and amplification. To fabricate such an array of nanostructures, current nanolithography methods are limited by the lack of either high throughput or high resolution. This paper presents a high-resolution and high-throughput nanolithography method using the compensated shadow effect in high-vacuum evaporation. The approach enables the fabrication of uniform nanogaps down to 20 nm in width across a 100 mm silicon wafer. The nanogap pattern is used as a template for the routine fabrication of zero-, one-, and two-dimensional nanostructures with a high yield. The method can facilitate the fabrication of nanostructured biosensors on a wafer scale at a low manufacturing cost.

  9. Versatile High Throughput Microarray Analysis for Marine Glycobiology

    DEFF Research Database (Denmark)

    Asunción Salmeán, Armando

    Algal cell walls are a type of extracellular matrix mainly made of polysaccharides, highly diverse, complex and heterogeneous. They possess unique and original polymers in their composition including several polysaccharides with industrial relevance such as agar, agarose, carrageenans (red algae...... decaying tissue as a nutrient source. We successfully optimized the method and characterized four new carbohydrate-recognizing proteins (two carrageenan binders, one arabynoxylan binder and one xyloglucan binder), which may be used as analytical probes for polysaccharide research. Finally, we also wanted...

  10. Using Mendelian inheritance to improve high-throughput SNP discovery.

    Science.gov (United States)

    Chen, Nancy; Van Hout, Cristopher V; Gottipati, Srikanth; Clark, Andrew G

    2014-11-01

    Restriction site-associated DNA sequencing or genotyping-by-sequencing (GBS) approaches allow for rapid and cost-effective discovery and genotyping of thousands of single-nucleotide polymorphisms (SNPs) in multiple individuals. However, rigorous quality control practices are needed to avoid high levels of error and bias with these reduced representation methods. We developed a formal statistical framework for filtering spurious loci, using Mendelian inheritance patterns in nuclear families, that accommodates variable-quality genotype calls and missing data--both rampant issues with GBS data--and for identifying sex-linked SNPs. Simulations predict excellent performance of both the Mendelian filter and the sex-linkage assignment under a variety of conditions. We further evaluate our method by applying it to real GBS data and validating a subset of high-quality SNPs. These results demonstrate that our metric of Mendelian inheritance is a powerful quality filter for GBS loci that is complementary to standard coverage and Hardy-Weinberg filters. The described method, implemented in the software MendelChecker, will improve quality control during SNP discovery in nonmodel as well as model organisms. Copyright © 2014 by the Genetics Society of America.

  11. 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.

  12. High throughput selection of effective serodiagnostics for Trypanosoma cruzi infection.

    Directory of Open Access Journals (Sweden)

    Gretchen Cooley

    2008-10-01

    Full Text Available Diagnosis of Trypanosoma cruzi infection by direct pathogen detection is complicated by the low parasite burden in subjects persistently infected with this agent of human Chagas disease. Determination of infection status by serological analysis has also been faulty, largely due to the lack of well-characterized parasite reagents for the detection of anti-parasite antibodies.In this study, we screened more than 400 recombinant proteins of T. cruzi, including randomly selected and those known to be highly expressed in the parasite stages present in mammalian hosts, for the ability to detect anti-parasite antibodies in the sera of subjects with confirmed or suspected T. cruzi infection.A set of 16 protein groups were identified and incorporated into a multiplex bead array format which detected 100% of >100 confirmed positive sera and also documented consistent, strong and broad responses in samples undetected or discordant using conventional serologic tests. Each serum had a distinct but highly stable reaction pattern. This diagnostic panel was also useful for monitoring drug treatment efficacy in chronic Chagas disease.These results substantially extend the variety and quality of diagnostic targets for Chagas disease and offer a useful tool for determining treatment success or failure.

  13. 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.; Bucan, Maja; Murray, Stephen A.

    2016-01-01

    Approximately one third of all mammalian genes are essential for life. Phenotypes resulting from mouse knockouts of these genes have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5000 knockout mouse lines, we have identified 410 lethal genes during the production of the first 1751 unique gene knockouts. Using a standardised phenotyping platform that incorporates high-resolution 3D imaging, we identified novel 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 identified in our screen, thus providing a novel dataset that facilitates prioritization and validation of mutations identified in clinical sequencing efforts. PMID:27626380

  14. Single DNA molecule patterning for high-throughput epigenetic mapping.

    Science.gov (United States)

    Cerf, Aline; Cipriany, Benjamin R; Benítez, Jaime J; Craighead, Harold G

    2011-11-01

    We present a method for profiling the 5-methyl cytosine distribution on single DNA molecules. Our method combines soft-lithography and molecular elongation to form ordered arrays estimated to contain more than 250 000 individual DNA molecules immobilized on a solid substrate. The methylation state of the DNA is detected and mapped by binding of fluorescently labeled methyl-CpG binding domain peptides to the elongated dsDNA molecules and imaging of their distribution. The stretched molecules are fixed in their extended configuration by adsorption onto the substrate so analysis can be performed with high spatial resolution and signal averaging. We further prove this technique allows imaging of DNA molecules with different methylation states.

  15. High-Throughput Raman Spectroscopy Screening of Excipients for the Stabilization of Amorphous Drugs.

    Science.gov (United States)

    Chen, Xiaoyun; Stoneburner, Kris; Ladika, Mladen; Kuo, Tzu-Chi; Kalantar, Thomas H

    2015-11-01

    Low aqueous solubility of active pharmaceutical ingredients (APIs) is an enduring problem in pharmaceutical development, and it is becoming increasingly prevalent among new drug candidates. It is estimated that about 40% of drugs in the development pipeline and approximately 60% of the drugs coming directly from discovery suffer from poor aqueous solubility and slow dissolution, thereby reducing their bioavailability and efficacy and thus preventing their commercialization. It is well known that utilizing the amorphous form of a drug can be a useful approach to improve the dissolution rate and solubility of poorly water-soluble APIs. Amorphous compounds are thermodynamically unstable, but they can be stabilized by combining them with a carrier polymer (excipient) to form a solid dispersion. High-throughput Raman spectroscopy was used in this study to identify excipients that promote formation and stabilization of the amorphous drug form in solid dispersions. Four model APIs were used as poorly soluble drug candidates: ketoprofen, danazol, griseofulvin, and probucol. The Raman signals of excipients were generally negligible, and therefore Raman bands from the drugs were used with minimal spectral pre-processing. By comparing Raman spectra collected from the APIs in the crystalline and molten state, appropriate spectral features and regions were identified for the development of semi-quantitative methods to determine the amorphous content for each API. It is demonstrated that methods based on peak intensity ratio, peak width, peak distance, and classical least squares can all be effective methods for the screening of excipients. Interesting excipient-dependent phase transformation behavior was also observed for probucol.

  16. Unbiased Characterization of Anopheles Mosquito Blood Meals by Targeted High-Throughput Sequencing.

    Directory of Open Access Journals (Sweden)

    Kyle Logue

    2016-03-01

    Full Text Available Understanding mosquito host choice is important for assessing vector competence or identifying disease reservoirs. Unfortunately, the availability of an unbiased method for comprehensively evaluating the composition of insect blood meals is very limited, as most current molecular assays only test for the presence of a few pre-selected species. These approaches also have limited ability to identify the presence of multiple mammalian hosts in a single blood meal. Here, we describe a novel high-throughput sequencing method that enables analysis of 96 mosquitoes simultaneously and provides a comprehensive and quantitative perspective on the composition of each blood meal. We validated in silico that universal primers targeting the mammalian mitochondrial 16S ribosomal RNA genes (16S rRNA should amplify more than 95% of the mammalian 16S rRNA sequences present in the NCBI nucleotide database. We applied this method to 442 female Anopheles punctulatus s. l. mosquitoes collected in Papua New Guinea (PNG. While human (52.9%, dog (15.8% and pig (29.2% were the most common hosts identified in our study, we also detected DNA from mice, one marsupial species and two bat species. Our analyses also revealed that 16.3% of the mosquitoes fed on more than one host. Analysis of the human mitochondrial hypervariable region I in 102 human blood meals showed that 5 (4.9% of the mosquitoes unambiguously fed on more than one person. Overall, analysis of PNG mosquitoes illustrates the potential of this approach to identify unsuspected hosts and characterize mixed blood meals, and shows how this approach can be adapted to evaluate inter-individual variations among human blood meals. Furthermore, this approach can be applied to any disease-transmitting arthropod and can be easily customized to investigate non-mammalian host sources.

  17. WormSizer: high-throughput analysis of nematode size and shape.

    Directory of Open Access Journals (Sweden)

    Brad T Moore

    Full Text Available The fundamental phenotypes of growth rate, size and morphology are the result of complex interactions between genotype and environment. We developed a high-throughput software application, WormSizer, which computes size and shape of nematodes from brightfield images. Existing methods for estimating volume either coarsely model the nematode as a cylinder or assume the worm shape or opacity is invariant. Our estimate is more robust to changes in morphology or optical density as it only assumes radial symmetry. This open source software is written as a plugin for the well-known image-processing framework Fiji/ImageJ. It may therefore be extended easily. We evaluated the technical performance of this framework, and we used it to analyze growth and shape of several canonical Caenorhabditis elegans mutants in a developmental time series. We confirm quantitatively that a Dumpy (Dpy mutant is short and fat and that a Long (Lon mutant is long and thin. We show that daf-2 insulin-like receptor mutants are larger than wild-type upon hatching but grow slow, and WormSizer can distinguish dauer larvae from normal larvae. We also show that a Small (Sma mutant is actually smaller than wild-type at all stages of larval development. WormSizer works with Uncoordinated (Unc and Roller (Rol mutants as well, indicating that it can be used with mutants despite behavioral phenotypes. We used our complete data set to perform a power analysis, giving users a sense of how many images are needed to detect different effect sizes. Our analysis confirms and extends on existing phenotypic characterization of well-characterized mutants, demonstrating the utility and robustness of WormSizer.

  18. High throughput whole rumen metagenome profiling using untargeted massively parallel sequencing

    Directory of Open Access Journals (Sweden)

    Ross Elizabeth M

    2012-07-01

    Full Text Available Abstract Background Variation of microorganism communities in the rumen of cattle (Bos taurus is of great interest because of possible links to economically or environmentally important traits, such as feed conversion efficiency or methane emission levels. The resolution of studies investigating this variation may be improved by utilizing untargeted massively parallel sequencing (MPS, that is, sequencing without targeted amplification of genes. The objective of this study was to develop a method which used MPS to generate “rumen metagenome profiles”, and to investigate if these profiles were repeatable among samples taken from the same cow. Given faecal samples are much easier to obtain than rumen fluid samples; we also investigated whether rumen metagenome profiles were predictive of faecal metagenome profiles. Results Rather than focusing on individual organisms within the rumen, our method used MPS data to generate quantitative rumen micro-biome profiles, regardless of taxonomic classifications. The method requires a previously assembled reference metagenome. A number of such reference metagenomes were considered, including two rumen derived metagenomes, a human faecal microflora metagenome and a reference metagenome made up of publically available prokaryote sequences. Sequence reads from each test sample were aligned to these references. The “rumen metagenome profile” was generated from the number of the reads that aligned to each contig in the database. We used this method to test the hypothesis that rumen fluid microbial community profiles vary more between cows than within multiple samples from the same cow. Rumen fluid samples were taken from three cows, at three locations within the rumen. DNA from the samples was sequenced on the Illumina GAIIx. When the reads were aligned to a rumen metagenome reference, the rumen metagenome profiles were repeatable (P  Conclusions We have presented a simple and high throughput method of

  19. High-Throughput Mutational Analysis of a Twister Ribozyme.

    Science.gov (United States)

    Kobori, Shungo; Yokobayashi, Yohei

    2016-08-22

    Recent discoveries of new classes of self-cleaving ribozymes in diverse organisms have triggered renewed interest in the chemistry and biology of ribozymes. Functional analysis and engineering of ribozymes often involve performing biochemical assays on multiple ribozyme mutants. However, because each ribozyme mutant must be individually prepared and assayed, the number and variety of mutants that can be studied are severely limited. All of the single and double mutants of a twister ribozyme (a total of 10 296 mutants) were generated and assayed for their self-cleaving activity by exploiting deep sequencing to count the numbers of cleaved and uncleaved sequences for every mutant. Interestingly, we found that the ribozyme is highly robust against mutations such that 71 % and 30 % of all single and double mutants, respectively, retain detectable activity under the assay conditions. It was also observed that the structural elements that comprise the ribozyme exhibit distinct sensitivity to mutations. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  1. 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.

  2. Exploiting the multiplexing capabilities of tandem mass tags for high-throughput estimation of cellular protein abundances by mass spectrometry.

    Science.gov (United States)

    Ahrné, Erik; Martinez-Segura, Amalia; Syed, Afzal Pasha; Vina-Vilaseca, Arnau; Gruber, Andreas J; Marguerat, Samuel; Schmidt, Alexander

    2015-09-01

    The generation of dynamic models of biological processes critically depends on the determination of precise cellular concentrations of biomolecules. Measurements of system-wide absolute protein levels are particularly valuable information in systems biology. Recently, mass spectrometry based proteomics approaches have been developed to estimate protein concentrations on a proteome-wide scale. However, for very complex proteomes, fractionation steps are required, increasing samples number and instrument analysis time. As a result, the number of full proteomes that can be routinely analyzed is limited. Here we combined absolute quantification strategies with the multiplexing capabilities of isobaric tandem mass tags to determine cellular protein abundances in a high throughput and proteome-wide scale even for highly complex biological systems, such as a whole human cell line. We generated two independent data sets to demonstrate the power of the approach regarding sample throughput, dynamic range, quantitative precision and accuracy as well as proteome coverage in comparison to existing mass spectrometry based strategies. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. High-throughput RNA structure probing reveals critical folding events during early 60S ribosome assembly in yeast.

    Science.gov (United States)

    Burlacu, Elena; Lackmann, Fredrik; Aguilar, Lisbeth-Carolina; Belikov, Sergey; Nues, Rob van; Trahan, Christian; Hector, Ralph D; Dominelli-Whiteley, Nicholas; Cockroft, Scott L; Wieslander, Lars; Oeffinger, Marlene; Granneman, Sander

    2017-09-28

    While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a high-throughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein-rRNA interactions, providing intriguing insights into the temporal order of protein-rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.Ribosome biogenesis is a dynamic process that involves the ordered assembly of ribosomal proteins and numerous RNA structural rearrangements. Here the authors apply ChemModSeq, a high-throughput RNA structure probing method, to quantitatively measure changes in RNA flexibility during the nucleolar stages of 60S assembly in yeast.

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

    Directory of Open Access Journals (Sweden)

    Anand Srinivasan

    2011-04-01

    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.

  5. High Throughput Sequencing of Extracellular RNA from Human Plasma.

    Directory of Open Access Journals (Sweden)

    Kirsty M Danielson

    Full Text Available The presence and relative stability of extracellular RNAs (exRNAs in biofluids has led to an emerging recognition of their promise as 'liquid biopsies' for diseases. Most prior studies on discovery of exRNAs as disease-specific biomarkers have focused on microRNAs (miRNAs using technologies such as qRT-PCR and microarrays. The recent application of next-generation sequencing to discovery of exRNA biomarkers has revealed the presence of potential novel miRNAs as well as other RNA species such as tRNAs, snoRNAs, piRNAs and lncRNAs in biofluids. At the same time, the use of RNA sequencing for biofluids poses unique challenges, including low amounts of input RNAs, the presence of exRNAs in different compartments with varying degrees of vulnerability to isolation techniques, and the high abundance of specific RNA species (thereby limiting the sensitivity of detection of less abundant species. Moreover, discovery in human diseases often relies on archival biospecimens of varying age and limiting amounts of samples. In this study, we have tested RNA isolation methods to optimize profiling exRNAs by RNA sequencing in individuals without any known diseases. Our findings are consistent with other recent studies that detect microRNAs and ribosomal RNAs as the major exRNA species in plasma. Similar to other recent studies, we found that the landscape of biofluid microRNA transcriptome is dominated by several abundant microRNAs that appear to comprise conserved extracellular miRNAs. There is reasonable correlation of sets of conserved miRNAs across biological replicates, and even across other data sets obtained at different investigative sites. Conversely, the detection of less abundant miRNAs is far more dependent on the exact methodology of RNA isolation and profiling. This study highlights the challenges in detecting and quantifying less abundant plasma miRNAs in health and disease using RNA sequencing platforms.

  6. A high-throughput, multi-channel photon-counting detector with picosecond timing

    Science.gov (United States)

    Lapington, J. S.; Fraser, G. W.; Miller, G. M.; Ashton, T. J. R.; Jarron, P.; Despeisse, M.; Powolny, F.; Howorth, J.; Milnes, J.

    2009-06-01

    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 microchannel plate devices with very high time resolution, and high-speed multi-channel ASIC electronics developed for the LHC at CERN, provides the necessary building blocks for a high-throughput detector system with up to 1024 parallel counting channels and 20 ps time resolution. We describe the detector and electronic design, discuss the current status of the HiContent project and present the results from a 64-channel prototype system. In the absence of an operational detector, we present measurements of the electronics performance using a pulse generator to simulate detector events. Event timing results from the NINO high-speed front-end ASIC captured using a fast digital oscilloscope are compared with data taken with the proposed electronic configuration which uses the multi-channel HPTDC timing ASIC.

  7. Design of novel solar thermal fuels with high-throughput ab initio simulations

    Science.gov (United States)

    Liu, Yun; Grossman, Jeffrey

    2014-03-01

    Solar thermal fuels (STF) store the energy of sunlight, which can then be released later in the form of heat, offering an emission-free and renewable solution for both solar energy conversion and storage. However, this approach is currently limited by the lack of low-cost materials with high energy density and high stability. Previously we have predicted a new class of functional materials that have the potential to address these challenges. Recently, we have developed an ab initio high-throughput computational approach to accelerate the design process and allow for searches over a broad class of materials. The high-throughput screening algorithm we have developed can run through large numbers of molecules composed of earth-abundant elements, and identifies possible metastable structures of a given material. Corresponding isomerization enthalpies associated with the metastable structures are then computed. Using this high-throughput simulation approach, we have discovered molecular structures with high isomerization enthalpies that have the potential to be new candidates for high-energy density STF. We have also discovered physical design principles to guide further STF materials design through the correlation between isomerization enthalpy and structural properties.

  8. Predictions versus high-throughput experiments in T-cell epitope discovery: competition or synergy?

    DEFF Research Database (Denmark)

    Lundegaard, Claus; Lund, Ole; Nielsen, Morten

    2012-01-01

    Prediction methods as well as experimental methods for T-cell epitope discovery have developed significantly in recent years. High-throughput experimental methods have made it possible to perform full-length protein scans for epitopes restricted to a limited number of MHC alleles. The high costs...... discovery. We expect prediction methods as well as experimental validation methods to continue to develop and that we will soon see clinical trials of products whose development has been guided by prediction methods....

  9. Deep Mutational Scanning: Library Construction, Functional Selection, and High-Throughput Sequencing.

    Science.gov (United States)

    Starita, Lea M; Fields, Stanley

    2015-08-03

    Deep mutational scanning is a highly parallel method that uses high-throughput sequencing to track changes in >10(5) protein variants before and after selection to measure the effects of mutations on protein function. Here we outline the stages of a deep mutational scanning experiment, focusing on the construction of libraries of protein sequence variants and the preparation of Illumina sequencing libraries. © 2015 Cold Spring Harbor Laboratory Press.

  10. A High-Throughput, Adaptive FFT Architecture for FPGA-Based Space-Borne Data Processors

    Science.gov (United States)

    Nguyen, Kayla; Zheng, Jason; He, Yutao; Shah, Biren

    2010-01-01

    Historically, computationally-intensive data processing for space-borne instruments has heavily relied on ground-based computing resources. But with recent advances in functional densities of Field-Programmable Gate-Arrays (FPGAs), there has been an increasing desire to shift more processing on-board; therefore relaxing the downlink data bandwidth requirements. Fast Fourier Transforms (FFTs) are commonly used building blocks for data processing applications, with a growing need to increase the FFT block size. Many existing FFT architectures have mainly emphasized on low power consumption or resource usage; but as the block size of the FFT grows, the throughput is often compromised first. In addition to power and resource constraints, space-borne digital systems are also limited to a small set of space-qualified memory elements, which typically lag behind the commercially available counterparts in capacity and bandwidth. The bandwidth limitation of the external memory creates a bottleneck for a large, high-throughput FFT design with large block size. In this paper, we present the Multi-Pass Wide Kernel FFT (MPWK-FFT) architecture for a moderately large block size (32K) with considerations to power consumption and resource usage, as well as throughput. We will also show that the architecture can be easily adapted for different FFT block sizes with different throughput and power requirements. The result is completely contained within an FPGA without relying on external memories. Implementation results are summarized.

  11. An Air-Well sparging minifermenter system for high-throughput protein production.

    Science.gov (United States)

    Deantonio, Cecilia; Sedini, Valentina; Cesaro, Patrizia; Quasso, Fabio; Cotella, Diego; Persichetti, Francesca; Santoro, Claudio; Sblattero, Daniele

    2014-09-14

    Over the last few years High-Throughput Protein Production (HTPP) has played a crucial role for functional proteomics. High-quality, high yield and fast recombinant protein production are critical for new HTPP technologies. Escherichia coli is usually the expression system of choice in protein production thanks to its fast growth, ease of handling and high yields of protein produced. Even though shake-flask cultures are widely used, there is an increasing need for easy to handle, lab scale, high throughput systems. In this article we described a novel minifermenter system suitable for HTPP. The Air-Well minifermenter system is made by a homogeneous air sparging device that includes an air diffusion system, and a stainless steel 96 needle plate integrated with a 96 deep well plate where cultures take place. This system provides aeration to achieve higher optical density growth compared to classical shaking growth without the decrease in pH value and bacterial viability. Moreover the yield of recombinant protein is up to 3-fold higher with a considerable improvement in the amount of full length proteins. High throughput production of hundreds of proteins in parallel can be obtained sparging air in a continuous and controlled manner. The system used is modular and can be easily modified and scaled up to meet the demands for HTPP.

  12. A Novel High-Throughput Approach to Measure Hydroxyl Radicals Induced by Airborne Particulate Matter

    Directory of Open Access Journals (Sweden)

    Yeongkwon Son

    2015-10-01

    Full Text Available Oxidative stress is one of the key mechanisms linking ambient particulate matter (PM exposure with various adverse health effects. The oxidative potential of PM has been used to characterize the ability of PM induced oxidative stress. Hydroxyl radical (•OH is the most destructive radical produced by PM. However, there is currently no high-throughput approach which can rapidly measure PM-induced •OH for a large number of samples with an automated system. This study evaluated four existing molecular probes (disodium terephthalate, 3′-p-(aminophenylfluorescein, coumarin-3-carboxylic acid, and sodium benzoate for their applicability to measure •OH induced by PM in a high-throughput cell-free system using fluorescence techniques, based on both our experiments and on an assessment of the physicochemical properties of the probes reported in the literature. Disodium terephthalate (TPT was the most applicable molecular probe to measure •OH induced by PM, due to its high solubility, high stability of the corresponding fluorescent product (i.e., 2-hydroxyterephthalic acid, high yield compared with the other molecular probes, and stable fluorescence intensity in a wide range of pH environments. TPT was applied in a high-throughput format to measure PM (NIST 1648a-induced •OH, in phosphate buffered saline. The formed fluorescent product was measured at designated time points up to 2 h. The fluorescent product of TPT had a detection limit of 17.59 nM. The soluble fraction of PM contributed approximately 76.9% of the •OH induced by total PM, and the soluble metal ions of PM contributed 57.4% of the overall •OH formation. This study provides a promising cost-effective high-throughput method to measure •OH induced by PM on a routine basis.

  13. Accurate Classification of Protein Subcellular Localization from High-Throughput Microscopy Images Using Deep Learning

    Directory of Open Access Journals (Sweden)

    Tanel Pärnamaa

    2017-05-01

    Full Text Available High-throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently-tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held-out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high-throughput microscopy.

  14. The promise and challenge of high-throughput sequencing of the antibody repertoire

    Science.gov (United States)

    Georgiou, George; Ippolito, Gregory C; Beausang, John; Busse, Christian E; Wardemann, Hedda; Quake, Stephen R

    2014-01-01

    Efforts to determine the antibody repertoire encoded by B cells in the blood or lymphoid organs using high-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and are transforming our understanding of humoral immune responses. Information gained from high-throughput DNA sequencing of immunoglobulin genes (Ig-seq) can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development and to understand autoimmunity. Rapid progress in the development of experimental protocols and informatics analysis tools is helping to reduce sequencing artifacts, to achieve more precise quantification of clonal diversity and to extract the most pertinent biological information. That said, broader application of Ig-seq, especially in clinical settings, will require the development of a standardized experimental design framework that will enable the sharing and meta-analysis of sequencing data generated by different laboratories. PMID:24441474

  15. tcpl: the ToxCast pipeline for high-throughput screening data.

    Science.gov (United States)

    Filer, Dayne L; Kothiya, Parth; Setzer, R Woodrow; Judson, Richard S; Martin, Matthew T

    2017-02-15

    Large high-throughput screening (HTS) efforts are widely used in drug development and chemical toxicity screening. Wide use and integration of these data can benefit from an efficient, transparent and reproducible data pipeline. Summary: The tcpl R package and its associated MySQL database provide a generalized platform for efficiently storing, normalizing and dose-response modeling of large high-throughput and high-content chemical screening data. The novel dose-response modeling algorithm has been tested against millions of diverse dose-response series, and robustly fits data with outliers and cytotoxicity-related signal loss. tcpl is freely available on the Comprehensive R Archive Network under the GPL-2 license. martin.matt@epa.gov.

  16. High-throughput clone screening followed by protein expression cross-check: A visual assay platform.

    Science.gov (United States)

    Bose, Partha Pratim; Kumar, Prakash

    2017-01-01

    In high-throughput biotechnology and structural biology, molecular cloning is an essential prerequisite for attaining high yields of recombinant protein. However, a rapid, cost-effective, easy clone screening protocol is still required to identify colonies with desired insert along with a cross check method to certify the expression of the desired protein as the end product. We report an easy, fast, sensitive and cheap visual clone screening and protein expression cross check protocol employing gold nanoparticle based plasmonic detection phenomenon. This is a non-gel, non-PCR based visual detection technique, which can be used as simultaneous high throughput clone screening followed by the determination of expression of desired protein. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing

    Science.gov (United States)

    Woodruff, Kristina; Maerkl, Sebastian J.

    2016-01-01

    Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies. PMID:27030663

  18. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit

    National Research Council Canada - National Science Library

    Pronk, Sander; Páll, Szilárd; Schulz, Roland; Larsson, Per; Bjelkmar, Pär; Apostolov, Rossen; Shirts, Michael R; Smith, Jeremy C; Kasson, Peter M; van der Spoel, David; Hess, Berk; Lindahl, Erik

    2013-01-01

    Molecular simulation has historically been a low-throughput technique, but faster computers and increasing amounts of genomic and structural data are changing this by enabling large-scale automated...

  19. Low-Cost, High-Throughput Sequencing of DNA Assemblies Using a Highly Multiplexed Nextera Process.

    Science.gov (United States)

    Shapland, Elaine B; Holmes, Victor; Reeves, Christopher D; Sorokin, Elena; Durot, Maxime; Platt, Darren; Allen, Christopher; Dean, Jed; Serber, Zach; Newman, Jack; Chandran, Sunil

    2015-07-17

    In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrial-scale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15× coverage), which is a 20-fold reduction over using Sanger sequencing (2× coverage). We reduced the volume of the Nextera tagmentation reaction by 100-fold and developed an automated workflow to prepare thousands of samples for sequencing. We also developed software to track the samples and associated sequence data and to rapidly identify correctly assembled constructs having the fewest defects. As DNA synthesis and assembly become a centralized commodity, this NGS quality control (QC) process will be essential to groups operating high-throughput pipelines for DNA construction.

  20. High throughput integrated thermal characterization with non-contact optical calorimetry

    Science.gov (United States)

    Hou, Sichao; Huo, Ruiqing; Su, Ming

    2017-10-01

    Commonly used thermal analysis tools such as calorimeter and thermal conductivity meter are separated instruments and limited by low throughput, where only one sample is examined each time. This work reports an infrared based optical calorimetry with its theoretical foundation, which is able to provide an integrated solution to characterize thermal properties of materials with high throughput. By taking time domain temperature information of spatially distributed samples, this method allows a single device (infrared camera) to determine the thermal properties of both phase change systems (melting temperature and latent heat of fusion) and non-phase change systems (thermal conductivity and heat capacity). This method further allows these thermal properties of multiple samples to be determined rapidly, remotely, and simultaneously. In this proof-of-concept experiment, the thermal properties of a panel of 16 samples including melting temperatures, latent heats of fusion, heat capacities, and thermal conductivities have been determined in 2 min with high accuracy. Given the high thermal, spatial, and temporal resolutions of the advanced infrared camera, this method has the potential to revolutionize the thermal characterization of materials by providing an integrated solution with high throughput, high sensitivity, and short analysis time.