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Sample records for high-throughput phenotypic characterization

  1. High-throughput mouse phenotyping.

    PubMed

    Gates, Hilary; Mallon, Ann-Marie; Brown, Steve D M

    2011-04-01

    Comprehensive phenotyping will be required to reveal the pleiotropic functions of a gene and to uncover the wider role of genetic loci within diverse biological systems. The challenge will be to devise phenotyping approaches to characterise the thousands of mutants that are being generated as part of international efforts to acquire a mutant for every gene in the mouse genome. In order to acquire robust datasets of broad based phenotypes from mouse mutants it is necessary to design and implement pipelines that incorporate standardised phenotyping platforms that are validated across diverse mouse genetics centres or mouse clinics. We describe here the rationale and methodology behind one phenotyping pipeline, EMPReSSslim, that was designed as part of the work of the EUMORPHIA and EUMODIC consortia, and which exemplifies some of the challenges facing large-scale phenotyping. EMPReSSslim captures a broad range of data on diverse biological systems, from biochemical to physiological amongst others. Data capture and dissemination is pivotal to the operation of large-scale phenotyping pipelines, including the definition of parameters integral to each phenotyping test and the associated ontological descriptions. EMPReSSslim data is displayed within the EuroPhenome database, where a variety of tools are available to allow the user to search for interesting biological or clinical phenotypes.

  2. A platform for high-throughput bioenergy production phenotype characterization in single cells

    PubMed Central

    Kelbauskas, Laimonas; Glenn, Honor; Anderson, Clifford; Messner, Jacob; Lee, Kristen B.; Song, Ganquan; Houkal, Jeff; Su, Fengyu; Zhang, Liqiang; Tian, Yanqing; Wang, Hong; Bussey, Kimberly; Johnson, Roger H.; Meldrum, Deirdre R.

    2017-01-01

    Driven by an increasing number of studies demonstrating its relevance to a broad variety of disease states, the bioenergy production phenotype has been widely characterized at the bulk sample level. Its cell-to-cell variability, a key player associated with cancer cell survival and recurrence, however, remains poorly understood due to ensemble averaging of the current approaches. We present a technology platform for performing oxygen consumption and extracellular acidification measurements of several hundreds to 1,000 individual cells per assay, while offering simultaneous analysis of cellular communication effects on the energy production phenotype. The platform comprises two major components: a tandem optical sensor for combined oxygen and pH detection, and a microwell device for isolation and analysis of single and few cells in hermetically sealed sub-nanoliter chambers. Our approach revealed subpopulations of cells with aberrant energy production profiles and enables determination of cellular response variability to electron transfer chain inhibitors and ion uncouplers. PMID:28349963

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed

    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

    2016-10-14

    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.

  7. High-throughput discovery of novel developmental phenotypes.

    PubMed

    Dickinson, Mary E; Flenniken, Ann M; Ji, Xiao; Teboul, Lydia; Wong, Michael D; White, Jacqueline K; Meehan, Terrence F; Weninger, Wolfgang J; Westerberg, Henrik; Adissu, Hibret; Baker, Candice N; Bower, Lynette; Brown, James M; Caddle, L Brianna; Chiani, Francesco; Clary, Dave; Cleak, James; Daly, Mark J; Denegre, James M; Doe, Brendan; Dolan, Mary E; Edie, Sarah M; Fuchs, Helmut; Gailus-Durner, Valerie; Galli, Antonella; Gambadoro, Alessia; Gallegos, Juan; Guo, Shiying; Horner, Neil R; Hsu, Chih-Wei; Johnson, Sara J; Kalaga, Sowmya; Keith, Lance C; Lanoue, Louise; Lawson, Thomas N; Lek, Monkol; Mark, Manuel; Marschall, Susan; Mason, Jeremy; McElwee, Melissa L; Newbigging, Susan; Nutter, Lauryl M J; Peterson, Kevin A; Ramirez-Solis, Ramiro; Rowland, Douglas J; Ryder, Edward; Samocha, Kaitlin E; Seavitt, John R; Selloum, Mohammed; Szoke-Kovacs, Zsombor; Tamura, Masaru; Trainor, Amanda G; Tudose, Ilinca; Wakana, Shigeharu; Warren, Jonathan; Wendling, Olivia; West, David B; Wong, Leeyean; Yoshiki, Atsushi; MacArthur, Daniel G; Tocchini-Valentini, Glauco P; Gao, Xiang; Flicek, Paul; Bradley, Allan; Skarnes, William C; Justice, Monica J; Parkinson, Helen E; Moore, Mark; Wells, Sara; Braun, Robert E; Svenson, Karen L; de Angelis, Martin Hrabe; Herault, Yann; Mohun, Tim; Mallon, Ann-Marie; Henkelman, R Mark; Brown, Steve D M; Adams, David J; Lloyd, K C Kent; McKerlie, Colin; Beaudet, Arthur L; Bućan, Maja; Murray, Stephen A

    2016-09-22

    Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.

  8. High-throughput discovery of novel developmental phenotypes

    PubMed Central

    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

  9. High-throughput phenotyping of root growth dynamics.

    PubMed

    Yazdanbakhsh, Nima; Fisahn, Joachim

    2012-01-01

    Plant organ phenotyping by noninvasive video imaging techniques provides a powerful tool to assess physiological traits, circadian and diurnal rhythms, and biomass production. In particular, growth of individual plant organs is known to exhibit a high plasticity and occurs as a result of the interaction between various endogenous and environmental processes. Thus, any investigation aiming to unravel mechanisms that determine plant or organ growth has to accurately control and document the environmental growth conditions. Here we describe challenges in establishing a recently developed plant root monitoring platform (PlaRoM) specially suited for noninvasive high-throughput plant growth analysis with highest emphasis on the detailed documentation of capture time, as well as light and temperature conditions. Furthermore, we discuss the experimental procedure for measuring root elongation kinetics and key points that must be considered in such measurements. PlaRoM consists of a robotized imaging platform enclosed in a custom designed phytochamber and a root extension profiling software application. This platform has been developed for multi-parallel recordings of root growth phenotypes of up to 50 individual seedlings over several days, with high spatial and temporal resolution. Two Petri dishes are mounted on a vertical sample stage in a custom designed phytochamber that provides exact temperature control. A computer-controlled positioning unit moves these Petri dishes in small increments and enables continuous screening of the surface under a binocular microscope. Detection of the root tip is achieved by applying thresholds on image pixel data and verifying the neighbourhood for each dark pixel. The growth parameters are visualized as position over time or growth rate over time graphs and averaged over consecutive days, light-dark periods and 24 h day periods. This setup enables the investigation of root extension profiles of different genotypes in various growth

  10. Histopathology reveals correlative and unique phenotypes in a high-throughput mouse phenotyping screen.

    PubMed

    Adissu, Hibret A; Estabel, Jeanne; Sunter, David; Tuck, Elizabeth; Hooks, Yvette; Carragher, Damian M; Clarke, Kay; Karp, Natasha A; Newbigging, Susan; Jones, Nora; Morikawa, Lily; White, Jacqueline K; McKerlie, Colin

    2014-05-01

    The Mouse Genetics Project (MGP) at the Wellcome Trust Sanger Institute aims to generate and phenotype over 800 genetically modified mouse lines over the next 5 years to gain a better understanding of mammalian gene function and provide an invaluable resource to the scientific community for follow-up studies. Phenotyping includes the generation of a standardized biobank of paraffin-embedded tissues for each mouse line, but histopathology is not routinely performed. In collaboration with the Pathology Core of the Centre for Modeling Human Disease (CMHD) we report the utility of histopathology in a high-throughput primary phenotyping screen. Histopathology was assessed in an unbiased selection of 50 mouse lines with (n=30) or without (n=20) clinical phenotypes detected by the standard MGP primary phenotyping screen. Our findings revealed that histopathology added correlating morphological data in 19 of 30 lines (63.3%) in which the primary screen detected a phenotype. In addition, seven of the 50 lines (14%) presented significant histopathology findings that were not associated with or predicted by the standard primary screen. Three of these seven lines had no clinical phenotype detected by the standard primary screen. Incidental and strain-associated background lesions were present in all mutant lines with good concordance to wild-type controls. These findings demonstrate the complementary and unique contribution of histopathology to high-throughput primary phenotyping of mutant mice.

  11. Salinity tolerance loci revealed in rice using high-throughput non-invasive phenotyping

    PubMed Central

    Al-Tamimi, Nadia; Brien, Chris; Oakey, Helena; Berger, Bettina; Saade, Stephanie; Ho, Yung Shwen; Schmöckel, Sandra M.; Tester, Mark; Negrão, Sónia

    2016-01-01

    High-throughput phenotyping produces multiple measurements over time, which require new methods of analyses that are flexible in their quantification of plant growth and transpiration, yet are computationally economic. Here we develop such analyses and apply this to a rice population genotyped with a 700k SNP high-density array. Two rice diversity panels, indica and aus, containing a total of 553 genotypes, are phenotyped in waterlogged conditions. Using cubic smoothing splines to estimate plant growth and transpiration, we identify four time intervals that characterize the early responses of rice to salinity. Relative growth rate, transpiration rate and transpiration use efficiency (TUE) are analysed using a new association model that takes into account the interaction between treatment (control and salt) and genetic marker. This model allows the identification of previously undetected loci affecting TUE on chromosome 11, providing insights into the early responses of rice to salinity, in particular into the effects of salinity on plant growth and transpiration. PMID:27853175

  12. High-Throughput Quantification of Phenotype Heterogeneity Using Statistical Features

    PubMed Central

    Chaddad, Ahmad; Tanougast, Camel

    2015-01-01

    Statistical features are widely used in radiology for tumor heterogeneity assessment using magnetic resonance (MR) imaging technique. In this paper, feature selection based on decision tree is examined to determine the relevant subset of glioblastoma (GBM) phenotypes in the statistical domain. To discriminate between active tumor (vAT) and edema/invasion (vE) phenotype, we selected the significant features using analysis of variance (ANOVA) with p value < 0.01. Then, we implemented the decision tree to define the optimal subset features of phenotype classifier. Naïve Bayes (NB), support vector machine (SVM), and decision tree (DT) classifier were considered to evaluate the performance of the feature based scheme in terms of its capability to discriminate vAT from vE. Whole nine features were statistically significant to classify the vAT from vE with p value < 0.01. Feature selection based on decision tree showed the best performance by the comparative study using full feature set. The feature selected showed that the two features Kurtosis and Skewness achieved a highest range value of 58.33–75.00% accuracy classifier and 73.88–92.50% AUC. This study demonstrated the ability of statistical features to provide a quantitative, individualized measurement of glioblastoma patient and assess the phenotype progression. PMID:26640485

  13. The Impact of Data Fragmentation on High-Throughput Clinical Phenotyping

    ERIC Educational Resources Information Center

    Wei, Weiqi

    2012-01-01

    Subject selection is essential and has become the rate-limiting step for harvesting knowledge to advance healthcare through clinical research. Present manual approaches inhibit researchers from conducting deep and broad studies and drawing confident conclusions. High-throughput clinical phenotyping (HTCP), a recently proposed approach, leverages…

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

    PubMed Central

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

    2016-01-01

    With the implementation of novel automated, high throughput methods and facilities in the last years, plant phenomics has developed into a highly interdisciplinary research domain integrating biology, engineering and bioinformatics. Here we present a dataset of a non-invasive high throughput plant phenotyping experiment, which uses image- and image analysis- based approaches to monitor the growth and development of 484 Arabidopsis thaliana plants (thale cress). The result is a comprehensive dataset of images and extracted phenotypical features. Such datasets require detailed documentation, standardized description of experimental metadata as well as sustainable data storage and publication in order to ensure the reproducibility of experiments, data reuse and comparability among the scientific community. Therefore the here presented dataset has been annotated using the standardized ISA-Tab format and considering the recently published recommendations for the semantical description of plant phenotyping experiments. PMID:27529152

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

    PubMed

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

    2016-08-16

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

  16. High-throughput screening of mouse gene knockouts identifies established and novel skeletal phenotypes

    PubMed Central

    Brommage, Robert; Liu, Jeff; Hansen, Gwenn M; Kirkpatrick, Laura L; Potter, David G; Sands, Arthur T; Zambrowicz, Brian; Powell, David R; Vogel, Peter

    2014-01-01

    Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening (HTS) data for 3 762 distinct global gene knockout (KO) mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type (WT) cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass: 23 previously reported genes (Calcr, Cebpb, Crtap, Dcstamp, Dkk1, Duoxa2, Enpp1, Fgf23, Kiss1/Kiss1r, Kl (Klotho), Lrp5, Mstn, Neo1, Npr2, Ostm1, Postn, Sfrp4, Slc30a5, Slc39a13, Sost, Sumf1, Src, Wnt10b), five novel genes extensively characterized (Cldn18, Fam20c, Lrrk1, Sgpl1, Wnt16), five novel genes with preliminary characterization (Agpat2, Rassf5, Slc10a7, Slc26a7, Slc30a10) and three novel undisclosed genes coding for potential osteoporosis drug targets. PMID:26273529

  17. High-throughput mouse phenotyping using non-rigid registration and robust principal component analysis

    NASA Astrophysics Data System (ADS)

    Xie, Zhongliu; Kitamoto, Asanobu; Tamura, Masaru; Shiroishi, Toshihiko; Gillies, Duncan

    2016-03-01

    Intensive international efforts are underway towards phenotyping the mouse genome, by knocking out each of its ≍25,000 genes one-by-one for comparative study. With vast amounts of data to analyze, the traditional method using time-consuming histological examination is clearly impractical, leading to an overwhelming demand for some high-throughput phenotyping framework, especially with the employment of biomedical image informatics to efficiently identify phenotypes concerning morphological abnormality. Existing work has either excessively relied on volumetric analytics which is insensitive to phenotypes associated with no severe volume variations, or tailored for specific defects and thus fails to serve a general phenotyping purpose. Furthermore, the prevailing requirement of an atlas for image segmentation in contrast to its limited availability further complicates the issue in practice. In this paper we propose a high-throughput general-purpose phenotyping framework that is able to efficiently perform batch-wise anomaly detection without prior knowledge of the phenotype and the need for atlas-based segmentation. Anomaly detection is centered on the combined use of group-wise non-rigid image registration and robust principal component analysis (RPCA) for feature extraction and decomposition.

  18. Exploring the elephant: histopathology in high-throughput phenotyping of mutant mice.

    PubMed

    Schofield, Paul N; Vogel, Peter; Gkoutos, Georgios V; Sundberg, John P

    2012-01-01

    Recent advances in gene knockout techniques and the in vivo analysis of mutant mice, together with the advent of large-scale projects for systematic mouse mutagenesis and genome-wide phenotyping, have allowed the creation of platforms for the most complete and systematic analysis of gene function ever undertaken in a vertebrate. The development of high-throughput phenotyping pipelines for these and other large-scale projects allows investigators to search and integrate large amounts of directly comparable phenotype data from many mutants, on a genomic scale, to help develop and test new hypotheses about the origins of disease and the normal functions of genes in the organism. Histopathology has a venerable history in the understanding of the pathobiology of human and animal disease, and presents complementary advantages and challenges to in vivo phenotyping. In this review, we present evidence for the unique contribution that histopathology can make to a large-scale phenotyping effort, using examples from past and current programmes at Lexicon Pharmaceuticals and The Jackson Laboratory, and critically assess the role of histopathology analysis in high-throughput phenotyping pipelines.

  19. Infra-red Thermography for High Throughput Field Phenotyping in Solanum tuberosum

    PubMed Central

    Prashar, Ankush; Yildiz, Jane; McNicol, James W.; Bryan, Glenn J.; Jones, Hamlyn G.

    2013-01-01

    The rapid development of genomic technology has made high throughput genotyping widely accessible but the associated high throughput phenotyping is now the major limiting factor in genetic analysis of traits. This paper evaluates the use of thermal imaging for the high throughput field phenotyping of Solanum tuberosum for differences in stomatal behaviour. A large multi-replicated trial of a potato mapping population was used to investigate the consistency in genotypic rankings across different trials and across measurements made at different times of day and on different days. The results confirmed a high degree of consistency between the genotypic rankings based on relative canopy temperature on different occasions. Genotype discrimination was enhanced both through normalising data by expressing genotype temperatures as differences from image means and through the enhanced replication obtained by using overlapping images. A Monte Carlo simulation approach was used to confirm the magnitude of genotypic differences that it is possible to discriminate. The results showed a clear negative association between canopy temperature and final tuber yield for this population, when grown under ample moisture supply. We have therefore established infrared thermography as an easy, rapid and non-destructive screening method for evaluating large population trials for genetic analysis. We also envisage this approach as having great potential for evaluating plant response to stress under field conditions. PMID:23762433

  20. High-throughput phenotyping of plant populations using a personal digital assistant.

    PubMed

    Vankudavath, Raju Naik; Bodanapu, Reddaiah; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2012-01-01

    During many biological experiments voluminous data is acquired, which can be best collected with -portable data acquisition devices and later analyzed with a personal computer (PC). Public domain software catering to data acquisition and analysis is currently limited. The necessity of phenotyping large plant populations led to the development of the application "PHENOME" to manage the data. PHENOME allows acquisition of phenotypic data using a personal digital assistant (PDA) with a built-in barcode scanner. The acquired data can be exported to a customized database on a PC for further analysis and cataloging. PHENOME can be used for a variety of applications, for example high-throughput phenotyping of a mutagenized or mapping population, or phenotyping of several individuals in one or more ecological niches.

  1. High-Throughput Phenotyping of Maize Leaf Physiological and Biochemical Traits Using Hyperspectral Reflectance1[OPEN

    PubMed Central

    Yendrek, Craig R.; Tomaz, Tiago; Montes, Christopher M.; Cao, Youyuan; Morse, Alison M.; Brown, Patrick J.; McIntyre, Lauren M.; Leakey, Andrew D.B.

    2017-01-01

    High-throughput, noninvasive field phenotyping has revealed genetic variation in crop morphological, developmental, and agronomic traits, but rapid measurements of the underlying physiological and biochemical traits are needed to fully understand genetic variation in plant-environment interactions. This study tested the application of leaf hyperspectral reflectance (λ = 500–2,400 nm) as a high-throughput phenotyping approach for rapid and accurate assessment of leaf photosynthetic and biochemical traits in maize (Zea mays). Leaf traits were measured with standard wet-laboratory and gas-exchange approaches alongside measurements of leaf reflectance. Partial least-squares regression was used to develop a measure of leaf chlorophyll content, nitrogen content, sucrose content, specific leaf area, maximum rate of phosphoenolpyruvate carboxylation, [CO2]-saturated rate of photosynthesis, and leaf oxygen radical absorbance capacity from leaf reflectance spectra. Partial least-squares regression models accurately predicted five out of seven traits and were more accurate than previously used simple spectral indices for leaf chlorophyll, nitrogen content, and specific leaf area. Correlations among leaf traits and statistical inferences about differences among genotypes and treatments were similar for measured and modeled data. The hyperspectral reflectance approach to phenotyping was dramatically faster than traditional measurements, enabling over 1,000 rows to be phenotyped during midday hours over just 2 to 4 d, and offers a nondestructive method to accurately assess physiological and biochemical trait responses to environmental stress. PMID:28049858

  2. EuroPhenome: a repository for high-throughput mouse phenotyping data.

    PubMed

    Morgan, Hugh; Beck, Tim; Blake, Andrew; Gates, Hilary; Adams, Niels; Debouzy, Guillaume; Leblanc, Sophie; Lengger, Christoph; Maier, Holger; Melvin, David; Meziane, Hamid; Richardson, Dave; Wells, Sara; White, Jacqui; Wood, Joe; de Angelis, Martin Hrabé; Brown, Steve D M; Hancock, John M; Mallon, Ann-Marie

    2010-01-01

    The broad aim of biomedical science in the postgenomic era is to link genomic and phenotype information to allow deeper understanding of the processes leading from genomic changes to altered phenotype and disease. The EuroPhenome project (http://www.EuroPhenome.org) is a comprehensive resource for raw and annotated high-throughput phenotyping data arising from projects such as EUMODIC. EUMODIC is gathering data from the EMPReSSslim pipeline (http://www.empress.har.mrc.ac.uk/) which is performed on inbred mouse strains and knock-out lines arising from the EUCOMM project. The EuroPhenome interface allows the user to access the data via the phenotype or genotype. It also allows the user to access the data in a variety of ways, including graphical display, statistical analysis and access to the raw data via web services. The raw phenotyping data captured in EuroPhenome is annotated by an annotation pipeline which automatically identifies statistically different mutants from the appropriate baseline and assigns ontology terms for that specific test. Mutant phenotypes can be quickly identified using two EuroPhenome tools: PhenoMap, a graphical representation of statistically relevant phenotypes, and mining for a mutant using ontology terms. To assist with data definition and cross-database comparisons, phenotype data is annotated using combinations of terms from biological ontologies.

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

    PubMed Central

    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. PMID:27891138

  4. High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis

    PubMed Central

    Thomas, Holly R.; Percival, Stefanie M.; Yoder, Bradley K.; Parant, John M.

    2014-01-01

    With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput

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

    PubMed

    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.

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

    PubMed Central

    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.

    2017-01-01

    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. PMID:28220807

  7. GiNA, an Efficient and High-Throughput Software for Horticultural Phenotyping

    PubMed Central

    Diaz-Garcia, Luis; Covarrubias-Pazaran, Giovanny; Schlautman, Brandon; Zalapa, Juan

    2016-01-01

    Traditional methods for trait phenotyping have been a bottleneck for research in many crop species due to their intensive labor, high cost, complex implementation, lack of reproducibility and propensity to subjective bias. Recently, multiple high-throughput phenotyping platforms have been developed, but most of them are expensive, species-dependent, complex to use, and available only for major crops. To overcome such limitations, we present the open-source software GiNA, which is a simple and free tool for measuring horticultural traits such as shape- and color-related parameters of fruits, vegetables, and seeds. GiNA is multiplatform software available in both R and MATLAB® programming languages and uses conventional images from digital cameras with minimal requirements. It can process up to 11 different horticultural morphological traits such as length, width, two-dimensional area, volume, projected skin, surface area, RGB color, among other parameters. Different validation tests produced highly consistent results under different lighting conditions and camera setups making GiNA a very reliable platform for high-throughput phenotyping. In addition, five-fold cross validation between manually generated and GiNA measurements for length and width in cranberry fruits were 0.97 and 0.92. In addition, the same strategy yielded prediction accuracies above 0.83 for color estimates produced from images of cranberries analyzed with GiNA compared to total anthocyanin content (TAcy) of the same fruits measured with the standard methodology of the industry. Our platform provides a scalable, easy-to-use and affordable tool for massive acquisition of phenotypic data of fruits, seeds, and vegetables. PMID:27529547

  8. Combining high-throughput phenotyping and genome-wide association studies to reveal natural genetic variation in rice

    PubMed Central

    Yang, Wanneng; Guo, Zilong; Huang, Chenglong; Duan, Lingfeng; Chen, Guoxing; Jiang, Ni; Fang, Wei; Feng, Hui; Xie, Weibo; Lian, Xingming; Wang, Gongwei; Luo, Qingming; Zhang, Qifa; Liu, Qian; Xiong, Lizhong

    2014-01-01

    Even as the study of plant genomics rapidly develops through the use of high-throughput sequencing techniques, traditional plant phenotyping lags far behind. Here we develop a high-throughput rice phenotyping facility (HRPF) to monitor 13 traditional agronomic traits and 2 newly defined traits during the rice growth period. Using genome-wide association studies (GWAS) of the 15 traits, we identify 141 associated loci, 25 of which contain known genes such as the Green Revolution semi-dwarf gene, SD1. Based on a performance evaluation of the HRPF and GWAS results, we demonstrate that high-throughput phenotyping has the potential to replace traditional phenotyping techniques and can provide valuable gene identification information. The combination of the multifunctional phenotyping tools HRPF and GWAS provides deep insights into the genetic architecture of important traits. PMID:25295980

  9. A Direct Comparison of Remote Sensing Approaches for High-Throughput Phenotyping in Plant Breeding.

    PubMed

    Tattaris, Maria; Reynolds, Matthew P; Chapman, Scott C

    2016-01-01

    Remote sensing (RS) of plant canopies permits non-intrusive, high-throughput monitoring of plant physiological characteristics. This study compared three RS approaches using a low flying UAV (unmanned aerial vehicle), with that of proximal sensing, and satellite-based imagery. Two physiological traits were considered, canopy temperature (CT) and a vegetation index (NDVI), to determine the most viable approaches for large scale crop genetic improvement. The UAV-based platform achieves plot-level resolution while measuring several hundred plots in one mission via high-resolution thermal and multispectral imagery measured at altitudes of 30-100 m. The satellite measures multispectral imagery from an altitude of 770 km. Information was compared with proximal measurements using IR thermometers and an NDVI sensor at a distance of 0.5-1 m above plots. For robust comparisons, CT and NDVI were assessed on panels of elite cultivars under irrigated and drought conditions, in different thermal regimes, and on un-adapted genetic resources under water deficit. Correlations between airborne data and yield/biomass at maturity were generally higher than equivalent proximal correlations. NDVI was derived from high-resolution satellite imagery for only larger sized plots (8.5 × 2.4 m) due to restricted pixel density. Results support use of UAV-based RS techniques for high-throughput phenotyping for both precision and efficiency.

  10. A Direct Comparison of Remote Sensing Approaches for High-Throughput Phenotyping in Plant Breeding

    PubMed Central

    Tattaris, Maria; Reynolds, Matthew P.; Chapman, Scott C.

    2016-01-01

    Remote sensing (RS) of plant canopies permits non-intrusive, high-throughput monitoring of plant physiological characteristics. This study compared three RS approaches using a low flying UAV (unmanned aerial vehicle), with that of proximal sensing, and satellite-based imagery. Two physiological traits were considered, canopy temperature (CT) and a vegetation index (NDVI), to determine the most viable approaches for large scale crop genetic improvement. The UAV-based platform achieves plot-level resolution while measuring several hundred plots in one mission via high-resolution thermal and multispectral imagery measured at altitudes of 30–100 m. The satellite measures multispectral imagery from an altitude of 770 km. Information was compared with proximal measurements using IR thermometers and an NDVI sensor at a distance of 0.5–1 m above plots. For robust comparisons, CT and NDVI were assessed on panels of elite cultivars under irrigated and drought conditions, in different thermal regimes, and on un-adapted genetic resources under water deficit. Correlations between airborne data and yield/biomass at maturity were generally higher than equivalent proximal correlations. NDVI was derived from high-resolution satellite imagery for only larger sized plots (8.5 × 2.4 m) due to restricted pixel density. Results support use of UAV-based RS techniques for high-throughput phenotyping for both precision and efficiency. PMID:27536304

  11. High Throughput Phenotypic Analysis of Mycobacterium tuberculosis and Mycobacterium bovis Strains' Metabolism Using Biolog Phenotype Microarrays

    PubMed Central

    Khatri, Bhagwati; Fielder, Mark; Jones, Gareth; Newell, William; Abu-Oun, Manal; Wheeler, Paul R.

    2013-01-01

    Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria and already has

  12. Towards high-throughput mouse embryonic phenotyping: a novel approach to classifying ventricular septal defects

    NASA Astrophysics Data System (ADS)

    Liang, Xi; Xie, Zhongliu; Tamura, Masaru; Shiroishi, Toshihiko; Kitamoto, Asanobu

    2015-03-01

    The goal of the International Mouse Phenotyping Consortium (IMPC, www.mousephenotype.org) is to study all the over 23,000 genes in the mouse by knocking them out one-by-one for comparative analysis. Large amounts of knockout mouse lines have been raised, leading to a strong demand for high-throughput phenotyping technologies. Traditional means via time-consuming histological examination is clearly unsuitable in this scenario. Biomedical imaging technologies such as CT and MRI therefore have started being used to develop more efficient phenotyping approaches. Existing work however primarily rests on volumetric analytics over anatomical structures to detect anomaly, yet this type of methods generally fail when features are subtle such as ventricular septal defects (VSD) in the heart, and meanwhile phenotypic assessment normally requires expert manual labor. This study proposes, to the best of our knowledge, the first automatic VSD diagnostic system for mouse embryos. Our algorithm starts with the creation of an atlas using wild-type mouse images, followed by registration of knockouts to the atlas to perform atlas-based segmentation on the heart and then ventricles, after which ventricle segmentation is further refined using a region growing technique. VSD classification is completed by checking the existence of an overlap between left and right ventricles. Our approach has been validated on a database of 14 mouse embryo images, and achieved an overall accuracy of 90.9%, with sensitivity of 66.7% and specificity of 100%.

  13. Novel Phenotypic Fluorescent Three-Dimensional Platforms for High-throughput Drug Screening and Personalized Chemotherapy.

    PubMed

    Fang, Changge; Avis, Ingalill; Salomon, David; Cuttitta, Frank

    2013-01-01

    We have developed novel phenotypic fluorescent three-dimensional co-culture platforms that efficiently and economically screen anti-angiogenic/anti-metastatic drugs on a high-throughput scale. Individual cell populations can be identified and isolated for protein/gene expression profiling studies and cellular movement/interactions can be tracked by time-lapse cinematography. More importantly, these platforms closely parallel the in vivo angiogenic and metastatic outcomes of a given tumor xenograft in the nude mouse model but, unlike in vivo models, our co-culture platforms produce comparable results in five to nine days. Potentially, by incorporating cancer patient biopsies, the co-culture platforms should greatly improve the effectiveness and efficiency of personalized chemotherapy.

  14. Vinobot and Vinoculer: Two Robotic Platforms for High-Throughput Field Phenotyping

    PubMed Central

    Shafiekhani, Ali; Kadam, Suhas; Fritschi, Felix B.; DeSouza, Guilherme N.

    2017-01-01

    In this paper, a new robotic architecture for plant phenotyping is being introduced. The architecture consists of two robotic platforms: an autonomous ground vehicle (Vinobot) and a mobile observation tower (Vinoculer). The ground vehicle collects data from individual plants, while the observation tower oversees an entire field, identifying specific plants for further inspection by the Vinobot. The advantage of this architecture is threefold: first, it allows the system to inspect large areas of a field at any time, during the day and night, while identifying specific regions affected by biotic and/or abiotic stresses; second, it provides high-throughput plant phenotyping in the field by either comprehensive or selective acquisition of accurate and detailed data from groups or individual plants; and third, it eliminates the need for expensive and cumbersome aerial vehicles or similarly expensive and confined field platforms. As the preliminary results from our algorithms for data collection and 3D image processing, as well as the data analysis and comparison with phenotype data collected by hand demonstrate, the proposed architecture is cost effective, reliable, versatile, and extendable. PMID:28124976

  15. Vinobot and Vinoculer: Two Robotic Platforms for High-Throughput Field Phenotyping.

    PubMed

    Shafiekhani, Ali; Kadam, Suhas; Fritschi, Felix B; DeSouza, Guilherme N

    2017-01-23

    In this paper, a new robotic architecture for plant phenotyping is being introduced. The architecture consists of two robotic platforms: an autonomous ground vehicle (Vinobot) and a mobile observation tower (Vinoculer). The ground vehicle collects data from individual plants, while the observation tower oversees an entire field, identifying specific plants for further inspection by the Vinobot. The advantage of this architecture is threefold: first, it allows the system to inspect large areas of a field at any time, during the day and night, while identifying specific regions affected by biotic and/or abiotic stresses; second, it provides high-throughput plant phenotyping in the field by either comprehensive or selective acquisition of accurate and detailed data from groups or individual plants; and third, it eliminates the need for expensive and cumbersome aerial vehicles or similarly expensive and confined field platforms. As the preliminary results from our algorithms for data collection and 3D image processing, as well as the data analysis and comparison with phenotype data collected by hand demonstrate, the proposed architecture is cost effective, reliable, versatile, and extendable.

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

    PubMed Central

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

    2015-01-01

    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. PMID

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  18. High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress

    PubMed Central

    Malik, Nasir; Shah, Sonia; Zhao, Jean; Class, Bradley; Aguisanda, Francis; Southall, Noel; Xia, Menghang; McKew, John C.; Rao, Mahendra

    2016-01-01

    Astrocytes are the predominant cell type in the nervous system and play a significant role in maintaining neuronal health and homeostasis. Recently, astrocyte dysfunction has been implicated in the pathogenesis of many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Astrocytes are thus an attractive new target for drug discovery for neurological disorders. Using astrocytes differentiated from human embryonic stem cells, we have developed an assay to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. This phenotypic oxidative stress assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, we identified a set of 22 that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress. Nine of these compounds were also found to be protective of induced pluripotent stem cell-differentiated astrocytes in a related assay. These compounds are thought to confer protection through hormesis, activating stress-response pathways and preconditioning astrocytes to handle subsequent exposure to hydrogen peroxide. In fact, four of these compounds were found to activate the antioxidant response element/nuclear factor-E2-related factor 2 pathway, a protective pathway induced by toxic insults. Our results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development. Significance Astrocytes play a key role in neurological diseases. Drug discovery efforts that target astrocytes can identify novel therapeutics. Human astrocytes are difficult to obtain and thus are challenging to use for high-throughput screening, which requires large numbers of cells. Using human embryonic stem cell

  19. RGB picture vegetation indexes for High-Throughput Phenotyping Platforms (HTPPs)

    NASA Astrophysics Data System (ADS)

    Kefauver, Shawn C.; El-Haddad, George; Vergara-Diaz, Omar; Araus, José Luis

    2015-10-01

    Extreme and abnormal weather events, as well as the more gradual meteorological changes associated with climate change, often coincide with not only increased abiotic risks (such as increases in temperature and decreases in precipitation), but also increased biotic risks due to environmental conditions that favor the rapid spread of crop pests and diseases. Durum wheat is by extension the most cultivated cereal in the south and east margins of the Mediterranean Basin. It is of strategic importance for Mediterranean agriculture to develop new varieties of durum wheat with greater production potential, better adaptation to increasingly adverse environmental conditions (drought) and better grain quality. Similarly, maize is the top staple crop for low-income populations in Sub-Saharan Africa and is currently suffering from the appearance of new diseases, which, together with increased abiotic stresses from climate change, are challenging the very sustainability of African societies. Current constraints in field phenotyping remain a major bottleneck for future breeding advances, but RGB-based High-Throughput Phenotyping Platforms (HTPPs) have shown promise for rapidly developing both disease-resistant and weather-resilient crops. RGB cameras have proven costeffective in studies assessing the effect of abiotic stresses, but have yet to be fully exploited to phenotype disease resistance. Recent analyses of durum wheat in Spain have shown RGB vegetation indexes to outperform multispectral indexes such as NDVI consistently in disease and yield prediction. Towards HTTP development for breeding maize disease resistance, some of the same RGB picture vegetation indexes outperformed NDVI (Normalized Difference Vegetation Index), with R2 values up to 0.65, compared to 0.56 for NDVI. . Specifically, hue, a*, u*, and Green Area (GA), as produced by FIJI and BreedPix open source software, performed similar to or better than NDVI in predicting yield and disease severity conditions

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

    PubMed

    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.

  1. Optimizing synchrotron microCT for high-throughput phenotyping of zebrafish

    NASA Astrophysics Data System (ADS)

    La Rivière, Patrick J.; Clark, Darin; Rojek, Alexandra; Vargas, Phillip; Xiao, Xianghui; DeCarlo, Francesco; Kindlmann, Gordon; Cheng, Keith

    2010-09-01

    We are creating a state-of-the-art 2D and 3D imaging atlas of zebrafish development. The atlas employs both 2D histology slides and 3D benchtop and synchrotron micro CT results. Through this atlas, we expect to document normal and abnormal organogenesis, to reveal new levels of structural detail, and to advance image informatics as a form of systems biology. The zebrafish has become a widely used model organism in biological and biomedical research for studies of vertebrate development and gene function. In this work, we will report on efforts to optimize synchrotron microCT imaging parameters for zebrafish at crucial developmental stages. The aim of these studies is to establish protocols for high-throughput phenotyping of normal, mutant and diseased zebrafish. We have developed staining and embedding protocols using different heavy metal stains (osmium tetroxide and uranyl acetate) and different embedding media (Embed 812 and glycol methacrylate). We have explored the use of edge subtraction and multi-energy techniques for contrast enhancement and we have examined the use of different sample-detector distances with unstained samples to explore and optimize phase-contrast enhancement effects. We will report principally on our efforts to optimize energy choice for single- and multi-energy studies as well as our efforts to optimize the degree of phase contrast enhancement.

  2. Protocols and programs for high-throughput growth and aging phenotyping in yeast.

    PubMed

    Jung, Paul P; Christian, Nils; Kay, Daniel P; Skupin, Alexander; Linster, Carole L

    2015-01-01

    In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting "Colony Forming Units". To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.

  3. Protocols and Programs for High-Throughput Growth and Aging Phenotyping in Yeast

    PubMed Central

    Jung, Paul P.; Christian, Nils; Kay, Daniel P.; Skupin, Alexander; Linster, Carole L.

    2015-01-01

    In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting “Colony Forming Units”. To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens. PMID:25822370

  4. A high throughput and sensitive method correlates neuronal disorder genotypes to Drosophila larvae crawling phenotypes.

    PubMed

    Jakubowski, Brandon R; Longoria, Rafael A; Shubeita, George T

    2012-01-01

    Drosophila melanogaster is widely used as a model system for development and disease. Due to the homology between Drosophila and human genes, as well as the tractable genetics of the fly, its use as a model for neurologic disorders, in particular, has been rising. Locomotive impairment is a commonly used diagnostic for screening and characterization of these models, yet a fast, sensitive and model-free method to compare behavior is lacking. Here, we present a high throughput method to quantify the crawling behavior of larvae. We use the mean squared displacement as well as the direction autocorrelation of the crawling larvae as descriptors of their motion. By tracking larvae from wild-type strains and models of the Fragile X mental retardation as well as Alzheimer disease, we show these mutants exhibit impaired crawling. We further show that the magnitude of impairment correlates with the severity of the mutation, demonstrating the sensitivity and the dynamic range of the method. Finally, we study larvae with altered expression of the shaggy gene, a homolog of Glycogen Synthase Kinase-3 (GSK-3), which has been implicated in Alzheimer disease. Surprisingly, we find that both increased and decreased expression of dGSK-3 lead to similar larval crawling impairment. These findings have implications for the use of GSK-3 inhibitors recently proposed for Alzheimer treatment.

  5. A high-throughput phenotypic screen identifies clofazimine as a potential treatment for cryptosporidiosis.

    PubMed

    Love, Melissa S; Beasley, Federico C; Jumani, Rajiv S; Wright, Timothy M; Chatterjee, Arnab K; Huston, Christopher D; Schultz, Peter G; McNamara, Case W

    2017-02-01

    Cryptosporidiosis has emerged as a leading cause of non-viral diarrhea in children under five years of age in the developing world, yet the current standard of care to treat Cryptosporidium infections, nitazoxanide, demonstrates limited and immune-dependent efficacy. Given the lack of treatments with universal efficacy, drug discovery efforts against cryptosporidiosis are necessary to find therapeutics more efficacious than the standard of care. To date, cryptosporidiosis drug discovery efforts have been limited to a few targeted mechanisms in the parasite and whole cell phenotypic screens against small, focused collections of compounds. Using a previous screen as a basis, we initiated the largest known drug discovery effort to identify novel anticryptosporidial agents. A high-content imaging assay for inhibitors of Cryptosporidium parvum proliferation within a human intestinal epithelial cell line was miniaturized and automated to enable high-throughput phenotypic screening against a large, diverse library of small molecules. A screen of 78,942 compounds identified 12 anticryptosporidial hits with sub-micromolar activity, including clofazimine, an FDA-approved drug for the treatment of leprosy, which demonstrated potent and selective in vitro activity (EC50 = 15 nM) against C. parvum. Clofazimine also displayed activity against C. hominis-the other most clinically-relevant species of Cryptosporidium. Importantly, clofazimine is known to accumulate within epithelial cells of the small intestine, the primary site of Cryptosporidium infection. In a mouse model of acute cryptosporidiosis, a once daily dosage regimen for three consecutive days or a single high dose resulted in reduction of oocyst shedding below the limit detectable by flow cytometry. Recently, a target product profile (TPP) for an anticryptosporidial compound was proposed by Huston et al. and highlights the need for a short dosing regimen (< 7 days) and formulations for children < 2 years. Clofazimine

  6. A high-throughput phenotypic screen identifies clofazimine as a potential treatment for cryptosporidiosis

    PubMed Central

    Jumani, Rajiv S.; Wright, Timothy M.; Chatterjee, Arnab K.; Huston, Christopher D.; Schultz, Peter G.; McNamara, Case W.

    2017-01-01

    Cryptosporidiosis has emerged as a leading cause of non-viral diarrhea in children under five years of age in the developing world, yet the current standard of care to treat Cryptosporidium infections, nitazoxanide, demonstrates limited and immune-dependent efficacy. Given the lack of treatments with universal efficacy, drug discovery efforts against cryptosporidiosis are necessary to find therapeutics more efficacious than the standard of care. To date, cryptosporidiosis drug discovery efforts have been limited to a few targeted mechanisms in the parasite and whole cell phenotypic screens against small, focused collections of compounds. Using a previous screen as a basis, we initiated the largest known drug discovery effort to identify novel anticryptosporidial agents. A high-content imaging assay for inhibitors of Cryptosporidium parvum proliferation within a human intestinal epithelial cell line was miniaturized and automated to enable high-throughput phenotypic screening against a large, diverse library of small molecules. A screen of 78,942 compounds identified 12 anticryptosporidial hits with sub-micromolar activity, including clofazimine, an FDA-approved drug for the treatment of leprosy, which demonstrated potent and selective in vitro activity (EC50 = 15 nM) against C. parvum. Clofazimine also displayed activity against C. hominis–the other most clinically-relevant species of Cryptosporidium. Importantly, clofazimine is known to accumulate within epithelial cells of the small intestine, the primary site of Cryptosporidium infection. In a mouse model of acute cryptosporidiosis, a once daily dosage regimen for three consecutive days or a single high dose resulted in reduction of oocyst shedding below the limit detectable by flow cytometry. Recently, a target product profile (TPP) for an anticryptosporidial compound was proposed by Huston et al. and highlights the need for a short dosing regimen (< 7 days) and formulations for children < 2 years

  7. Novel inhibitors of Mycobacterium tuberculosis GuaB2 identified by a target based high-throughput phenotypic screen

    PubMed Central

    Cox, Jonathan A. G.; Mugumbate, Grace; Del Peral, Laura Vela-Glez; Jankute, Monika; Abrahams, Katherine A.; Jervis, Peter; Jackenkroll, Stefan; Perez, Arancha; Alemparte, Carlos; Esquivias, Jorge; Lelièvre, Joël; Ramon, Fernando; Barros, David; Ballell, Lluis; Besra, Gurdyal S.

    2016-01-01

    High-throughput phenotypic screens have re-emerged as screening tools in antibiotic discovery. The advent of such technologies has rapidly accelerated the identification of ‘hit’ compounds. A pre-requisite to medicinal chemistry optimisation programmes required to improve the drug-like properties of a ‘hit’ molecule is identification of its mode of action. Herein, we have combined phenotypic screening with a biased target-specific screen. The inosine monophosphate dehydrogenase (IMPDH) protein GuaB2 has been identified as a drugable target in Mycobacterium tuberculosis, however previously identified compounds lack the desired characteristics necessary for further development into lead-like molecules. This study has identified 7 new chemical series from a high-throughput resistance-based phenotypic screen using Mycobacterium bovis BCG over-expressing GuaB2. Hit compounds were identified in a single shot high-throughput screen, validated by dose response and subjected to further biochemical analysis. The compounds were also assessed using molecular docking experiments, providing a platform for their further optimisation using medicinal chemistry. This work demonstrates the versatility and potential of GuaB2 as an anti-tubercular drug target. PMID:27982051

  8. Biophysics of cancer progression and high-throughput mechanical characterization of biomaterials

    NASA Astrophysics Data System (ADS)

    Osborne, Lukas Dylan

    Cancer metastasis involves a series of events known as the metastatic cascade. In this complex progression, cancer cells detach from the primary tumor, invade the surrounding stromal space, transmigrate the vascular system, and establish secondary tumors at distal sites. Specific mechanical phenotypes are likely adopted to enable cells to successfully navigate the mechanical environments encountered during metastasis. To examine the role of cell mechanics in cancer progression, I employed force-consistent biophysical and biochemical assays to characterize the mechanistic links between stiffness, stiffness response and cell invasion during the epithelial to mesenchymal transition (EMT). EMT is an essential physiological process, whose abnormal reactivation has been implicated in the detachment of cancer cells from epithelial tissue and their subsequent invasion into stromal tissue. I demonstrate that epithelial-state cells respond to force by evoking a stiffening response, and that after EMT, mesenchymal-state cells have reduced stiffness but also lose the ability to increase their stiffness in response to force. Using loss and gain of function studies, two proteins are established as functional connections between attenuated stiffness and stiffness response and the increased invasion capacity acquired after EMT. To enable larger scale assays to more fully explore the connection between biomechanics and cancer, I discuss the development of an automated array high throughput (AHT) microscope. The AHT system is shown to implement passive microbead rheology to accurately characterize the mechanical properties of biomaterials. Compared to manually performed mechanical characterizations, the AHT system executes experiments in two orders of magnitude less time. Finally, I use the AHT microscope to study the effect of gain of function oncogenic molecules on cell stiffness. I find evidence that our assay can identify alterations in cell stiffness due to constitutive

  9. Near infrared spectroscopy for high-throughput characterization of Shea tree (Vitellaria paradoxa) nut fat profiles.

    PubMed

    Davrieux, Fabrice; Allal, François; Piombo, Georges; Kelly, Bokary; Okulo, John B; Thiam, Massamba; Diallo, Ousmane B; Bouvet, Jean-Marc

    2010-07-14

    The Shea tree (Vitellaria paradoxa) is a major tree species in African agroforestry systems. Butter extracted from its nuts offers an opportunity for sustainable development in Sudanian countries and an attractive potential for the food and cosmetics industries. The purpose of this study was to develop near-infrared spectroscopy (NIRS) calibrations to characterize Shea nut fat profiles. Powders prepared from nuts collected from 624 trees in five African countries (Senegal, Mali, Burkina Faso, Ghana and Uganda) were analyzed for moisture content, fat content using solvent extraction, and fatty acid profiles using gas chromatography. Results confirmed the differences between East and West African Shea nut fat composition: eastern nuts had significantly higher fat and oleic acid contents. Near infrared reflectance spectra were recorded for each sample. Ten percent of the samples were randomly selected for validation and the remaining samples used for calibration. For each constituent, calibration equations were developed using modified partial least squares (MPLS) regression. The equation performances were evaluated using the ratio performance to deviation (RPD(p)) and R(p)(2) parameters, obtained by comparison of the validation set NIR predictions and corresponding laboratory values. Moisture (RPD(p) = 4.45; R(p)(2) = 0.95) and fat (RPD(p) = 5.6; R(p)(2) = 0.97) calibrations enabled accurate determination of these traits. NIR models for stearic (RPD(p) = 6.26; R(p)(2) = 0.98) and oleic (RPD(p) = 7.91; R(p)(2) = 0.99) acids were highly efficient and enabled sharp characterization of these two major Shea butter fatty acids. This study demonstrated the ability of near-infrared spectroscopy for high-throughput phenotyping of Shea nuts.

  10. High-throughput two-dimensional root system phenotyping platform facilitates genetic analysis of root growth and development.

    PubMed

    Clark, Randy T; Famoso, Adam N; Zhao, Keyan; Shaff, Jon E; Craft, Eric J; Bustamante, Carlos D; McCouch, Susan R; Aneshansley, Daniel J; Kochian, Leon V

    2013-02-01

    High-throughput phenotyping of root systems requires a combination of specialized techniques and adaptable plant growth, root imaging and software tools. A custom phenotyping platform was designed to capture images of whole root systems, and novel software tools were developed to process and analyse these images. The platform and its components are adaptable to a wide range root phenotyping studies using diverse growth systems (hydroponics, paper pouches, gel and soil) involving several plant species, including, but not limited to, rice, maize, sorghum, tomato and Arabidopsis. The RootReader2D software tool is free and publicly available and was designed with both user-guided and automated features that increase flexibility and enhance efficiency when measuring root growth traits from specific roots or entire root systems during large-scale phenotyping studies. To demonstrate the unique capabilities and high-throughput capacity of this phenotyping platform for studying root systems, genome-wide association studies on rice (Oryza sativa) and maize (Zea mays) root growth were performed and root traits related to aluminium (Al) tolerance were analysed on the parents of the maize nested association mapping (NAM) population.

  11. High-Throughput Synthesis and Characterization of BiMoVOX Materials

    NASA Astrophysics Data System (ADS)

    Russu, Sergio; Tromp, Moniek; Tsapatsaris, Nikolaos; Beesley, Angela M.; Schroeder, Sven L. M.; Weller, Mark T.; Evans, John

    2007-02-01

    The high throughput synthesis and characterization of a particular family of ceramic materials, bismuth molybdenum vanadium oxides (BiMoVOX), suitable as inorganic yellow pigments and low temperature oxidation catalysts, is described. Samples, synthesized by calcination and peroxo sol-gel methods, are characterized by X-ray powder diffraction, UV-visible and XAFS spectroscopy. A combined high-throughput XRD/XAFS study of a 54 samples array, with simultaneous refinement of data of both techniques, has been performed. Molybdenum doping of bismuth vanadate results in a phase transition from monoclinic BiVO4 to tetragonal Bi(V,Mo)O4, both of scheelite type. Both central metals, V5+ and Mo6+, remain in a tetrahedral coordination. UV/visible spectroscopy identifies a linear blue shift as a function of Mo6+ amount.

  12. High-throughput characterization of film thickness in thin film materials libraries by digital holographic microscopy.

    PubMed

    Lai, Yiu Wai; Krause, Michael; Savan, Alan; Thienhaus, Sigurd; Koukourakis, Nektarios; Hofmann, Martin R; Ludwig, Alfred

    2011-10-01

    A high-throughput characterization technique based on digital holography for mapping film thickness in thin-film materials libraries was developed. Digital holographic microscopy is used for fully automatic measurements of the thickness of patterned films with nanometer resolution. The method has several significant advantages over conventional stylus profilometry: it is contactless and fast, substrate bending is compensated, and the experimental setup is simple. Patterned films prepared by different combinatorial thin-film approaches were characterized to investigate and demonstrate this method. The results show that this technique is valuable for the quick, reliable and high-throughput determination of the film thickness distribution in combinatorial materials research. Importantly, it can also be applied to thin films that have been structured by shadow masking.

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

    PubMed

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

    2007-01-01

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

  14. Image-Based High-Throughput Field Phenotyping of Crop Roots1[W][OPEN

    PubMed Central

    Bucksch, Alexander; Burridge, James; York, Larry M.; Das, Abhiram; Nord, Eric; Weitz, Joshua S.; Lynch, Jonathan P.

    2014-01-01

    Current plant phenotyping technologies to characterize agriculturally relevant traits have been primarily developed for use in laboratory and/or greenhouse conditions. In the case of root architectural traits, this limits phenotyping efforts, largely, to young plants grown in specialized containers and growth media. Hence, novel approaches are required to characterize mature root systems of older plants grown under actual soil conditions in the field. Imaging methods able to address the challenges associated with characterizing mature root systems are rare due, in part, to the greater complexity of mature root systems, including the larger size, overlap, and diversity of root components. Our imaging solution combines a field-imaging protocol and algorithmic approach to analyze mature root systems grown in the field. Via two case studies, we demonstrate how image analysis can be utilized to estimate localized root traits that reliably capture heritable architectural diversity as well as environmentally induced architectural variation of both monocot and dicot plants. In the first study, we show that our algorithms and traits (including 13 novel traits inaccessible to manual estimation) can differentiate nine maize (Zea mays) genotypes 8 weeks after planting. The second study focuses on a diversity panel of 188 cowpea (Vigna unguiculata) genotypes to identify which traits are sufficient to differentiate genotypes even when comparing plants whose harvesting date differs up to 14 d. Overall, we find that automatically derived traits can increase both the speed and reproducibility of the trait estimation pipeline under field conditions. PMID:25187526

  15. Microfluidic Impedance Flow Cytometry Enabling High-Throughput Single-Cell Electrical Property Characterization

    PubMed Central

    Chen, Jian; Xue, Chengcheng; Zhao, Yang; Chen, Deyong; Wu, Min-Hsien; Wang, Junbo

    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 and optical flow cytometry for single-cell analysis and (4) integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications. PMID:25938973

  16. FRET-based calcium imaging: a tool for high-throughput/content phenotypic drug screening in Alzheimer disease.

    PubMed

    Honarnejad, Kamran; Kirsch, Achim K; Daschner, Alexander; Szybinska, Aleksandra; Kuznicki, Jacek; Herms, Jochen

    2013-12-01

    Perturbed intracellular store calcium homeostasis is suggested to play a major role in the pathophysiology of Alzheimer disease (AD). A number of mechanisms have been suggested to underlie the impairment of endoplasmic reticulum calcium homeostasis associated with familial AD-linked presenilin 1 mutations (FAD-PS1). Without aiming at specifically targeting any of those pathophysiological mechanisms in particular, we rather performed a high-throughput phenotypic screen to identify compounds that can reverse the exaggerated agonist-evoked endoplasmic reticulum calcium release phenotype in HEK293 cells expressing FAD-PS1. For that purpose, we developed a fully automated high-throughput calcium imaging assay using a fluorescence resonance energy transfer-based calcium indicator at single-cell resolution. This novel robust assay offers a number of advantages compared with the conventional calcium measurement screening technologies. The assay was employed in a large-scale screen with a library of diverse compounds comprising 20,000 low-molecular-weight molecules, which resulted in the identification of 52 primary hits and 4 lead structures. In a secondary assay, several hits were found to alter the amyloid β (Aβ) production. In view of the recent failure of AD drug candidates identified by target-based approaches, such a phenotypic drug discovery paradigm may present an attractive alternative for the identification of novel AD therapeutics.

  17. A high throughput array microscope for the mechanical characterization of biomaterials

    NASA Astrophysics Data System (ADS)

    Cribb, Jeremy; Osborne, Lukas D.; Hsiao, Joe Ping-Lin; Vicci, Leandra; Meshram, Alok; O'Brien, E. Tim; Spero, Richard Chasen; Taylor, Russell; Superfine, Richard

    2015-02-01

    In the last decade, the emergence of high throughput screening has enabled the development of novel drug therapies and elucidated many complex cellular processes. Concurrently, the mechanobiology community has developed tools and methods to show that the dysregulation of biophysical properties and the biochemical mechanisms controlling those properties contribute significantly to many human diseases. Despite these advances, a complete understanding of the connection between biomechanics and disease will require advances in instrumentation that enable parallelized, high throughput assays capable of probing complex signaling pathways, studying biology in physiologically relevant conditions, and capturing specimen and mechanical heterogeneity. Traditional biophysical instruments are unable to meet this need. To address the challenge of large-scale, parallelized biophysical measurements, we have developed an automated array high-throughput microscope system that utilizes passive microbead diffusion to characterize mechanical properties of biomaterials. The instrument is capable of acquiring data on twelve-channels simultaneously, where each channel in the system can independently drive two-channel fluorescence imaging at up to 50 frames per second. We employ this system to measure the concentration-dependent apparent viscosity of hyaluronan, an essential polymer found in connective tissue and whose expression has been implicated in cancer progression.

  18. A high throughput array microscope for the mechanical characterization of biomaterials

    PubMed Central

    Cribb, Jeremy; Osborne, Lukas D.; Hsiao, Joe Ping-Lin; Vicci, Leandra; Meshram, Alok; O’Brien, E. Tim; Spero, Richard Chasen; Taylor, Russell; Superfine, Richard

    2015-01-01

    In the last decade, the emergence of high throughput screening has enabled the development of novel drug therapies and elucidated many complex cellular processes. Concurrently, the mechanobiology community has developed tools and methods to show that the dysregulation of biophysical properties and the biochemical mechanisms controlling those properties contribute significantly to many human diseases. Despite these advances, a complete understanding of the connection between biomechanics and disease will require advances in instrumentation that enable parallelized, high throughput assays capable of probing complex signaling pathways, studying biology in physiologically relevant conditions, and capturing specimen and mechanical heterogeneity. Traditional biophysical instruments are unable to meet this need. To address the challenge of large-scale, parallelized biophysical measurements, we have developed an automated array high-throughput microscope system that utilizes passive microbead diffusion to characterize mechanical properties of biomaterials. The instrument is capable of acquiring data on twelve-channels simultaneously, where each channel in the system can independently drive two-channel fluorescence imaging at up to 50 frames per second. We employ this system to measure the concentration-dependent apparent viscosity of hyaluronan, an essential polymer found in connective tissue and whose expression has been implicated in cancer progression. PMID:25725856

  19. Label-free high-throughput assays to screen and characterize novel lactate dehydrogenase inhibitors.

    PubMed

    Vanderporten, Erica; Frick, Lauren; Turincio, Rebecca; Thana, Peter; Lamarr, William; Liu, Yichin

    2013-10-15

    Catalytic turnover of pyruvate to lactate by lactate dehydrogenase (LDH) is critical in maintaining an intracellular nicotinamide adenine dinucleotide (NAD⁺) pool for continuous fueling of the glycolytic pathway. In this article, we describe two label-free high-throughput assays (a kinetic assay detecting the intrinsic reduced nicotinamide adenine dinucleotide (NADH) fluorescence and a mass spectrometric assay monitoring the conversion of pyruvate to lactate) that were designed to effectively identify LDH inhibitors, characterize their different mechanisms of action, and minimize potential false positives from a small molecule compound library screen. Using a fluorescence kinetic image-based reader capable of detecting NADH fluorescence in the ultra-high-throughput screening (uHTS) work flow, the enzyme activity was measured as the rate of NADH conversion to NAD⁺. Interference with NADH fluorescence by library compounds was readily identified during the primary screen. The mass spectrometric assay quantitated the lactate and pyruvate levels simultaneously. The multiple reaction monitoring mass spectrometric method accurately detected each of the two small organic acid molecules in the reaction mixture. With robust Z' scores of more than 0.7, these two high-throughput assays for LDH are both label free and complementary to each other in the HTS workflow by monitoring the activities of the compounds on each half of the LDH redox reaction.

  20. Construction and high-throughput phenotypic screening of Zymoseptoria tritici over-expression strains

    PubMed Central

    Cairns, T.C.; Sidhu, Y.S.; Chaudhari, Y.K.; Talbot, N.J.; Studholme, D.J.; Haynes, K.

    2015-01-01

    Targeted gene deletion has been instrumental in elucidating many aspects of Zymoseptoria tritici pathogenicity. Gene over-expression is a complementary approach that is amenable to rapid strain construction and high-throughput screening, which has not been exploited to analyze Z. tritici, largely due to a lack of available techniques. Here we exploit the Gateway® cloning technology for rapid construction of over-expression vectors and improved homologous integration efficiency of a Z. tritici Δku70 strain to build a pilot over-expression library encompassing 32 genes encoding putative DNA binding proteins, GTPases or kinases. We developed a protocol using a Rotor-HDA robot for rapid and reproducible cell pinning for high-throughput in vitro screening. This screen identified an over-expression strain that demonstrated a marked reduction in hyphal production relative to the isogenic progenitor. This study provides a protocol for rapid generation of Z. tritici over-expression libraries and a technique for functional genomic screening in this important pathogen. PMID:26092797

  1. Construction and high-throughput phenotypic screening ofZymoseptoria tritici over-expression strains.

    PubMed

    Cairns, T C; Sidhu, Y S; Chaudhari, Y K; Talbot, N J; Studholme, D J; Haynes, K

    2015-06-01

    Targeted gene deletion has been instrumental in elucidating many aspects of Zymoseptoria tritici pathogenicity. Gene over-expression is a complementary approach that is amenable to rapid strain construction and high-throughput screening, which has not been exploited to analyze Z. tritici, largely due to a lack of available techniques. Here we exploit the Gateway® cloning technology for rapid construction of over-expression vectors and improved homologous integration efficiency of a Z. tritici Δku70 strain to build a pilot over-expression library encompassing 32 genes encoding putative DNA binding proteins, GTPases or kinases. We developed a protocol using a Rotor-HDA robot for rapid and reproducible cell pinning for high-throughput in vitro screening. This screen identified an over-expression strain that demonstrated a marked reduction in hyphal production relative to the isogenic progenitor. This study provides a protocol for rapid generation of Z. tritici over-expression libraries and a technique for functional genomic screening in this important pathogen.

  2. Towards High-Throughput, Simultaneous Characterization of Thermal and Thermoelectric Properties

    NASA Astrophysics Data System (ADS)

    Miers, Collier Stephen

    The extension of thermoelectric generators to more general markets requires that the devices be affordable and practical (low $/Watt) to implement. A key challenge in this pursuit is the quick and accurate characterization of thermoelectric materials, which will allow researchers to tune and modify the material properties quickly. The goal of this thesis is to design and fabricate a high-throughput characterization system for the simultaneous characterization of thermal, electrical, and thermoelectric properties for device scale material samples. The measurement methodology presented in this thesis combines a custom designed measurement system created specifically for high-throughput testing with a novel device structure that permits simultaneous characterization of the material properties. The measurement system is based upon the 3o method for thermal conductivity measurements, with the addition of electrodes and voltage probes to measure the electrical conductivity and Seebeck coefficient. A device designed and optimized to permit the rapid characterization of thermoelectric materials is also presented. This structure is optimized to ensure 1D heat transfer within the sample, thus permitting rapid data analysis and fitting using a MATLAB script. Verification of the thermal portion of the system is presented using fused silica and sapphire materials for benchmarking. The fused silica samples yielded a thermal conductivity of 1.21 W/(m K), while a thermal conductivity of 31.2 W/(m K) was measured for the sapphire samples. The device and measurement system designed and developed in this thesis provide insight and serve as a foundation for the development of high throughput, simultaneous measurement platforms.

  3. High-Throughput Non-destructive Phenotyping of Traits that Contribute to Salinity Tolerance in Arabidopsis thaliana

    PubMed Central

    Awlia, Mariam; Nigro, Arianna; Fajkus, Jiří; Schmoeckel, Sandra M.; Negrão, Sónia; Santelia, Diana; Trtílek, Martin; Tester, Mark; Julkowska, Magdalena M.; Panzarová, Klára

    2016-01-01

    Reproducible and efficient high-throughput phenotyping approaches, combined with advances in genome sequencing, are facilitating the discovery of genes affecting plant performance. Salinity tolerance is a desirable trait that can be achieved through breeding, where most have aimed at selecting for plants that perform effective ion exclusion from the shoots. To determine overall plant performance under salt stress, it is helpful to investigate several plant traits collectively in one experimental setup. Hence, we developed a quantitative phenotyping protocol using a high-throughput phenotyping system, with RGB and chlorophyll fluorescence (ChlF) imaging, which captures the growth, morphology, color and photosynthetic performance of Arabidopsis thaliana plants in response to salt stress. We optimized our salt treatment by controlling the soil-water content prior to introducing salt stress. We investigated these traits over time in two accessions in soil at 150, 100, or 50 mM NaCl to find that the plants subjected to 100 mM NaCl showed the most prominent responses in the absence of symptoms of severe stress. In these plants, salt stress induced significant changes in rosette area and morphology, but less prominent changes in rosette coloring and photosystem II efficiency. Clustering of ChlF traits with plant growth of nine accessions maintained at 100 mM NaCl revealed that in the early stage of salt stress, salinity tolerance correlated with non-photochemical quenching processes and during the later stage, plant performance correlated with quantum yield. This integrative approach allows the simultaneous analysis of several phenotypic traits. In combination with various genetic resources, the phenotyping protocol described here is expected to increase our understanding of plant performance and stress responses, ultimately identifying genes that improve plant performance in salt stress conditions. PMID:27733855

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

    PubMed Central

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

    2015-01-01

    Caenorhabditis elegans postembryonic development consists of four discrete larval stages separated by molts. Typically, the speed of progression through these larval stages is investigated by visual inspection of the molting process. Here, we describe an automated method to monitor the timing of these discrete phases of C. elegans maturation, from the first larval stage through adulthood, using bioluminescence. The method was validated with a lin-42 mutant strain that shows delayed development relative to wild-type animals and with a daf-2 mutant that shows an extended second larval stage. This new method is inherently high-throughput and will finally allow dissecting the molecular machinery governing the speed of the developmental clock, which has so far been hampered by the lack of a method suitable for genetic screens. PMID:26294666

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

    PubMed

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

    2015-10-01

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

  6. Dynamic nuclear polarization NMR spectroscopy allows high-throughput characterization of microporous organic polymers.

    PubMed

    Blanc, Frédéric; Chong, Samantha Y; McDonald, Tom O; Adams, Dave J; Pawsey, Shane; Caporini, Marc A; Cooper, Andrew I

    2013-10-16

    Dynamic nuclear polarization (DNP) solid-state NMR was used to obtain natural abundance (13)C and (15)N CP MAS NMR spectra of microporous organic polymers with excellent signal-to-noise ratio, allowing for unprecedented details in the molecular structure to be determined for these complex polymer networks. Sensitivity enhancements larger than 10 were obtained with bis-nitroxide radical at 14.1 T and low temperature (∼105 K). This DNP MAS NMR approach allows efficient, high-throughput characterization of libraries of porous polymers prepared by combinatorial chemistry methods.

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

    PubMed Central

    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

  8. High-Throughput Phenotyping of Sorghum Plant Height Using an Unmanned Aerial Vehicle and Its Application to Genomic Prediction Modeling

    PubMed Central

    Watanabe, Kakeru; Guo, Wei; Arai, Keigo; Takanashi, Hideki; Kajiya-Kanegae, Hiromi; Kobayashi, Masaaki; Yano, Kentaro; Tokunaga, Tsuyoshi; Fujiwara, Toru; Tsutsumi, Nobuhiro; Iwata, Hiroyoshi

    2017-01-01

    Genomics-assisted breeding methods have been rapidly developed with novel technologies such as next-generation sequencing, genomic selection and genome-wide association study. However, phenotyping is still time consuming and is a serious bottleneck in genomics-assisted breeding. In this study, we established a high-throughput phenotyping system for sorghum plant height and its response to nitrogen availability; this system relies on the use of unmanned aerial vehicle (UAV) remote sensing with either an RGB or near-infrared, green and blue (NIR-GB) camera. We evaluated the potential of remote sensing to provide phenotype training data in a genomic prediction model. UAV remote sensing with the NIR-GB camera and the 50th percentile of digital surface model, which is an indicator of height, performed well. The correlation coefficient between plant height measured by UAV remote sensing (PHUAV) and plant height measured with a ruler (PHR) was 0.523. Because PHUAV was overestimated (probably because of the presence of taller plants on adjacent plots), the correlation coefficient between PHUAV and PHR was increased to 0.678 by using one of the two replications (that with the lower PHUAV value). Genomic prediction modeling performed well under the low-fertilization condition, probably because PHUAV overestimation was smaller under this condition due to a lower plant height. The predicted values of PHUAV and PHR were highly correlated with each other (r = 0.842). This result suggests that the genomic prediction models generated with PHUAV were almost identical and that the performance of UAV remote sensing was similar to that of traditional measurements in genomic prediction modeling. UAV remote sensing has a high potential to increase the throughput of phenotyping and decrease its cost. UAV remote sensing will be an important and indispensable tool for high-throughput genomics-assisted plant breeding.

  9. High-Throughput Phenotyping of Sorghum Plant Height Using an Unmanned Aerial Vehicle and Its Application to Genomic Prediction Modeling.

    PubMed

    Watanabe, Kakeru; Guo, Wei; Arai, Keigo; Takanashi, Hideki; Kajiya-Kanegae, Hiromi; Kobayashi, Masaaki; Yano, Kentaro; Tokunaga, Tsuyoshi; Fujiwara, Toru; Tsutsumi, Nobuhiro; Iwata, Hiroyoshi

    2017-01-01

    Genomics-assisted breeding methods have been rapidly developed with novel technologies such as next-generation sequencing, genomic selection and genome-wide association study. However, phenotyping is still time consuming and is a serious bottleneck in genomics-assisted breeding. In this study, we established a high-throughput phenotyping system for sorghum plant height and its response to nitrogen availability; this system relies on the use of unmanned aerial vehicle (UAV) remote sensing with either an RGB or near-infrared, green and blue (NIR-GB) camera. We evaluated the potential of remote sensing to provide phenotype training data in a genomic prediction model. UAV remote sensing with the NIR-GB camera and the 50th percentile of digital surface model, which is an indicator of height, performed well. The correlation coefficient between plant height measured by UAV remote sensing (PHUAV) and plant height measured with a ruler (PHR) was 0.523. Because PHUAV was overestimated (probably because of the presence of taller plants on adjacent plots), the correlation coefficient between PHUAV and PHR was increased to 0.678 by using one of the two replications (that with the lower PHUAV value). Genomic prediction modeling performed well under the low-fertilization condition, probably because PHUAV overestimation was smaller under this condition due to a lower plant height. The predicted values of PHUAV and PHR were highly correlated with each other (r = 0.842). This result suggests that the genomic prediction models generated with PHUAV were almost identical and that the performance of UAV remote sensing was similar to that of traditional measurements in genomic prediction modeling. UAV remote sensing has a high potential to increase the throughput of phenotyping and decrease its cost. UAV remote sensing will be an important and indispensable tool for high-throughput genomics-assisted plant breeding.

  10. Development and evaluation of a field-based high-throughput phenotyping platform

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Physiological and developmental traits that vary over time are difficult to phenotype under relevant growing conditions. In response to this challenge, we developed a novel system for phenotyping dynamic traits in the field. System performance was evaluated on a field experiment of 25 Pima cotton cu...

  11. Quantifying co-cultured cell phenotypes in high-throughput using pixel-based classification

    PubMed Central

    Logan, David J.; Shan, Jing; Bhatia, Sangeeta N.; Carpenter, Anne E.

    2016-01-01

    Biologists increasingly use co-culture systems in which two or more cell types are grown in cell culture together in order to better model cells’ native microenvironments. Co-cultures are often required for cell survival or proliferation, or to maintain physiological functioning in vitro. Having two cell types co-exist in culture, however, poses several challenges, including difficulties distinguishing the two populations during analysis using automated image analysis algorithms. We previously analyzed co-cultured primary human hepatocytes and mouse fibroblasts in a high-throughput image-based chemical screen, using a combination of segmentation, measurement, and subsequent machine learning to score each cell as hepatocyte or fibroblast. While this approach was successful in counting hepatocytes for primary screening, segmentation of the fibroblast nuclei was less accurate. Here, we present an improved approach that more accurately identifies both cell types. Pixel-based machine learning (using the software ilastik) is used to seed segmentation of each cell type individually (using the software CellProfiler). This streamlined and accurate workflow can be carried out using freely available and open source software. PMID:26687239

  12. A High Throughput Phenotypic Screen of Cytotoxic T Lymphocyte Lytic Granule Exocytosis Reveals Candidate Immunosuppressants

    PubMed Central

    Zhao, Ziyan; Haynes, Mark K.; Ursu, Oleg; Edwards, Bruce S.; Sklar, Larry A.; Zweifach, Adam

    2015-01-01

    We screened the NIH’s Molecular Libraries Small Molecule Repository for inhibitors of cytotoxic T lymphocyte (CTL) lytic granule exocytosis by measuring binding of an antibody in the extracellular solution to a lysosomal membrane protein (LAMP-1) that is transferred to the plasma membrane by exocytosis. We used TALL-104 human leukemic CTLs stimulated with soluble chemicals. Using high-throughput cluster cytometry to screen 364202 compounds in 1536-well plate format, identifying 2404 initial hits. 161 were confirmed on retesting, and dose-response measurements were performed. 75 of those compounds were obtained, and 48 were confirmed active. Experiments were conducted to determine the molecular mechanism of action (MMOA) of the active compounds. Fifteen blocked increases in intracellular calcium >50%. Seven blocked phosphorylation of ERK by upstream MAP kinase kinases >50%. One completely blocked the activity of the calcium-dependent phosphatase calcineurin. None blocked ERK catalytic activity. Eight blocked more than one pathway. For eight compounds, we were unable to determine an MMOA. The activity of one of these compounds was confirmed from powder resupply. We conclude that a screen based on antibody binding to CTLs is a good means of identifying novel candidate immunosuppressants with either known or unknown MMOA. PMID:25381253

  13. Characterizing ncRNAs in Human Pathogenic Protists Using High-Throughput Sequencing Technology

    PubMed Central

    Collins, Lesley Joan

    2011-01-01

    ncRNAs are key genes in many human diseases including cancer and viral infection, as well as providing critical functions in pathogenic organisms such as fungi, bacteria, viruses, and protists. Until now the identification and characterization of ncRNAs associated with disease has been slow or inaccurate requiring many years of testing to understand complicated RNA and protein gene relationships. High-throughput sequencing now offers the opportunity to characterize miRNAs, siRNAs, small nucleolar RNAs (snoRNAs), and long ncRNAs on a genomic scale, making it faster and easier to clarify how these ncRNAs contribute to the disease state. However, this technology is still relatively new, and ncRNA discovery is not an application of high priority for streamlined bioinformatics. Here we summarize background concepts and practical approaches for ncRNA analysis using high-throughput sequencing, and how it relates to understanding human disease. As a case study, we focus on the parasitic protists Giardia lamblia and Trichomonas vaginalis, where large evolutionary distance has meant difficulties in comparing ncRNAs with those from model eukaryotes. A combination of biological, computational, and sequencing approaches has enabled easier classification of ncRNA classes such as snoRNAs, but has also aided the identification of novel classes. It is hoped that a higher level of understanding of ncRNA expression and interaction may aid in the development of less harsh treatment for protist-based diseases. PMID:22303390

  14. A High-Throughput, Multi-Cell Phenotype Assay for the Identification of Novel Inhibitors of Chemotaxis/Migration.

    PubMed

    Liao, Xin-Hua; Meena, Netra Pal; Southall, Noel; Liu, Lunhua; Swaroop, Manju; Zhang, Arina Li; Xiang, Jan Jian; Parent, Carole A; Zheng, Wei; Kimmel, Alan R

    2016-03-09

    Chemotaxis and cell migration are fundamental, universal eukaryotic processes essential for biological functions such as embryogenesis, immunity, cell renewal, and wound healing, as well as for pathogenesis of many diseases including cancer metastasis and chronic inflammation. To identify novel chemotaxis inhibitors as probes for mechanistic studies and leads for development of new therapeutics, we developed a unique, unbiased phenotypic chemotaxis-dependent Dictyostelium aggregation assay for high-throughput screening using rapid, laser-scanning cytometry. Under defined conditions, individual Dictyostelium secrete chemoattractants, migrate, and aggregate. Chemotaxis is quantified by laser-scanning cytometry with a GFP marker expressed only in cells after chemotaxis/multi-cell aggregation. We applied the assay to screen 1,280 known compounds in a 1536-well plate format and identified two chemotaxis inhibitors. The chemotaxis inhibitory activities of both compounds were confirmed in both Dictyostelium and in human neutrophils in a directed EZ-TAXIscan chemotaxis assay. The compounds were also shown to inhibit migration of two human cancer cell lines in monolayer scratch assays. This test screen demonstrated that the miniaturized assay is extremely suited for high-throughput screening of very large libraries of small molecules to identify novel classes of chemotaxis/migratory inhibitors for drug development and research tools for targeting chemotactic pathways universal to humans and other systems.

  15. Lights, camera, action: high-throughput plant phenotyping is ready for a close-up

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Modern techniques for crop improvement rely on both DNA sequencing and accurate quantification of plant traits to identify genes and germplasm of interest. With rapid advances in DNA sequencing technologies, plant phenotyping is now a bottleneck in advancing crop yields [1,2]. Furthermore, the envir...

  16. Characterization of the mechanical properties of HL-1 cardiomyocytes with high throughput magnetic tweezers

    SciTech Connect

    Chen, La; Maybeck, Vanessa; Offenhäusser, Andreas; Krause, Hans-Joachim

    2015-08-03

    We characterized the mechanical properties of cardiomyocyte-like HL-1 cells using our recently developed multi-pole magnetic tweezers. With the optimized design, both high force and high throughput are achieved at the same time. Force up to 100 pN can be applied on a 1 μm diameter superparamagnetic bead in a workspace with 60 μm radius, which is encircled symmetrically by 3 sharp magnetic tips. By adjusting the coil currents, both the strength and direction of force can be controlled. The result shows that both viscosity and shear elastic modulus of HL-1 cells exhibit an approximately log-normal distribution. The cells became stiffer as they matured, consistent with a transition from proliferating cells to contractile muscle tissue. Moreover, the mechanical properties of HL-1 cells show high heterogeneity, which agrees well with their physiological structure.

  17. Methodology for High-Throughput Field Phenotyping of Canopy Temperature Using Airborne Thermography.

    PubMed

    Deery, David M; Rebetzke, Greg J; Jimenez-Berni, Jose A; James, Richard A; Condon, Anthony G; Bovill, William D; Hutchinson, Paul; Scarrow, Jamie; Davy, Robert; Furbank, Robert T

    2016-01-01

    Lower canopy temperature (CT), resulting from increased stomatal conductance, has been associated with increased yield in wheat. Historically, CT has been measured with hand-held infrared thermometers. Using the hand-held CT method on large field trials is problematic, mostly because measurements are confounded by temporal weather changes during the time required to measure all plots. The hand-held CT method is laborious and yet the resulting heritability low, thereby reducing confidence in selection in large scale breeding endeavors. We have developed a reliable and scalable crop phenotyping method for assessing CT in large field experiments. The method involves airborne thermography from a manned helicopter using a radiometrically-calibrated thermal camera. Thermal image data is acquired from large experiments in the order of seconds, thereby enabling simultaneous measurement of CT on potentially 1000s of plots. Effects of temporal weather variation when phenotyping large experiments using hand-held infrared thermometers are therefore reduced. The method is designed for cost-effective and large-scale use by the non-technical user and includes custom-developed software for data processing to obtain CT data on a single-plot basis for analysis. Broad-sense heritability was routinely >0.50, and as high as 0.79, for airborne thermography CT measured near anthesis on a wheat experiment comprising 768 plots of size 2 × 6 m. Image analysis based on the frequency distribution of temperature pixels to remove the possible influence of background soil did not improve broad-sense heritability. Total image acquisition and processing time was ca. 25 min and required only one person (excluding the helicopter pilot). The results indicate the potential to phenotype CT on large populations in genetics studies or for selection within a plant breeding program.

  18. Methodology for High-Throughput Field Phenotyping of Canopy Temperature Using Airborne Thermography

    PubMed Central

    Deery, David M.; Rebetzke, Greg J.; Jimenez-Berni, Jose A.; James, Richard A.; Condon, Anthony G.; Bovill, William D.; Hutchinson, Paul; Scarrow, Jamie; Davy, Robert; Furbank, Robert T.

    2016-01-01

    Lower canopy temperature (CT), resulting from increased stomatal conductance, has been associated with increased yield in wheat. Historically, CT has been measured with hand-held infrared thermometers. Using the hand-held CT method on large field trials is problematic, mostly because measurements are confounded by temporal weather changes during the time required to measure all plots. The hand-held CT method is laborious and yet the resulting heritability low, thereby reducing confidence in selection in large scale breeding endeavors. We have developed a reliable and scalable crop phenotyping method for assessing CT in large field experiments. The method involves airborne thermography from a manned helicopter using a radiometrically-calibrated thermal camera. Thermal image data is acquired from large experiments in the order of seconds, thereby enabling simultaneous measurement of CT on potentially 1000s of plots. Effects of temporal weather variation when phenotyping large experiments using hand-held infrared thermometers are therefore reduced. The method is designed for cost-effective and large-scale use by the non-technical user and includes custom-developed software for data processing to obtain CT data on a single-plot basis for analysis. Broad-sense heritability was routinely >0.50, and as high as 0.79, for airborne thermography CT measured near anthesis on a wheat experiment comprising 768 plots of size 2 × 6 m. Image analysis based on the frequency distribution of temperature pixels to remove the possible influence of background soil did not improve broad-sense heritability. Total image acquisition and processing time was ca. 25 min and required only one person (excluding the helicopter pilot). The results indicate the potential to phenotype CT on large populations in genetics studies or for selection within a plant breeding program. PMID:27999580

  19. High-throughput RAD-SNP genotyping for characterization of sugar beet genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High-throughput SNP genotyping provides a rapid way of developing resourceful set of markers for delineating the genetic architecture and for effective species discrimination. In the presented research, we demonstrate a set of 192 SNPs for effective genotyping in sugar beet using high-throughput mar...

  20. High-throughput phenotyping of uropathogenic E. coli isolates with Fourier transform infrared spectroscopy.

    PubMed

    AlRabiah, Haitham; Correa, Elon; Upton, Mathew; Goodacre, Royston

    2013-03-07

    Fourier transform infrared (FT-IR) spectroscopy is an established rapid whole-organism fingerprinting method that generates metabolic fingerprints from bacteria that reflect the phenotype of the microorganism under investigation. However, whilst FT-IR spectroscopy is fast (typically 10 s to 1 min per sample), the approaches for microbial sample preparation can be time consuming as plate culture or shake flasks are used for growth of the organism. We report a new approach that allows micro-cultivation of bacteria from low volumes (typically 200 μL) to be coupled with FT-IR spectroscopy. This approach is fast and easy to perform and gives equivalent data to the lengthier and more expensive shake flask cultivations (sample volume = 20 mL). With this micro-culture approach we also demonstrate high reproducibility of the metabolic fingerprints. The approach allowed separation of different isolates of Escherichia coli involved in urinary tract infection, including members of the globally disseminated ST131 clone, with respect to both genotype and resistance or otherwise to the antibiotic Ciprofloxacin.

  1. Unmanned Aerial Vehicles for High-Throughput Phenotyping and Agronomic Research.

    PubMed

    Shi, Yeyin; Thomasson, J Alex; Murray, Seth C; Pugh, N Ace; Rooney, William L; Shafian, Sanaz; Rajan, Nithya; Rouze, Gregory; Morgan, Cristine L S; Neely, Haly L; Rana, Aman; Bagavathiannan, Muthu V; Henrickson, James; Bowden, Ezekiel; Valasek, John; Olsenholler, Jeff; Bishop, Michael P; Sheridan, Ryan; Putman, Eric B; Popescu, Sorin; Burks, Travis; Cope, Dale; Ibrahim, Amir; McCutchen, Billy F; Baltensperger, David D; Avant, Robert V; Vidrine, Misty; Yang, Chenghai

    2016-01-01

    Advances in automation and data science have led agriculturists to seek real-time, high-quality, high-volume crop data to accelerate crop improvement through breeding and to optimize agronomic practices. Breeders have recently gained massive data-collection capability in genome sequencing of plants. Faster phenotypic trait data collection and analysis relative to genetic data leads to faster and better selections in crop improvement. Furthermore, faster and higher-resolution crop data collection leads to greater capability for scientists and growers to improve precision-agriculture practices on increasingly larger farms; e.g., site-specific application of water and nutrients. Unmanned aerial vehicles (UAVs) have recently gained traction as agricultural data collection systems. Using UAVs for agricultural remote sensing is an innovative technology that differs from traditional remote sensing in more ways than strictly higher-resolution images; it provides many new and unique possibilities, as well as new and unique challenges. Herein we report on processes and lessons learned from year 1-the summer 2015 and winter 2016 growing seasons-of a large multidisciplinary project evaluating UAV images across a range of breeding and agronomic research trials on a large research farm. Included are team and project planning, UAV and sensor selection and integration, and data collection and analysis workflow. The study involved many crops and both breeding plots and agronomic fields. The project's goal was to develop methods for UAVs to collect high-quality, high-volume crop data with fast turnaround time to field scientists. The project included five teams: Administration, Flight Operations, Sensors, Data Management, and Field Research. Four case studies involving multiple crops in breeding and agronomic applications add practical descriptive detail. Lessons learned include critical information on sensors, air vehicles, and configuration parameters for both. As the first and

  2. Unmanned Aerial Vehicles for High-Throughput Phenotyping and Agronomic Research

    PubMed Central

    Shi, Yeyin; Thomasson, J. Alex; Murray, Seth C.; Pugh, N. Ace; Rooney, William L.; Shafian, Sanaz; Rajan, Nithya; Rouze, Gregory; Morgan, Cristine L. S.; Neely, Haly L.; Rana, Aman; Bagavathiannan, Muthu V.; Henrickson, James; Bowden, Ezekiel; Valasek, John; Olsenholler, Jeff; Bishop, Michael P.; Sheridan, Ryan; Putman, Eric B.; Popescu, Sorin; Burks, Travis; Cope, Dale; Ibrahim, Amir; McCutchen, Billy F.; Baltensperger, David D.; Avant, Robert V.; Vidrine, Misty; Yang, Chenghai

    2016-01-01

    Advances in automation and data science have led agriculturists to seek real-time, high-quality, high-volume crop data to accelerate crop improvement through breeding and to optimize agronomic practices. Breeders have recently gained massive data-collection capability in genome sequencing of plants. Faster phenotypic trait data collection and analysis relative to genetic data leads to faster and better selections in crop improvement. Furthermore, faster and higher-resolution crop data collection leads to greater capability for scientists and growers to improve precision-agriculture practices on increasingly larger farms; e.g., site-specific application of water and nutrients. Unmanned aerial vehicles (UAVs) have recently gained traction as agricultural data collection systems. Using UAVs for agricultural remote sensing is an innovative technology that differs from traditional remote sensing in more ways than strictly higher-resolution images; it provides many new and unique possibilities, as well as new and unique challenges. Herein we report on processes and lessons learned from year 1—the summer 2015 and winter 2016 growing seasons–of a large multidisciplinary project evaluating UAV images across a range of breeding and agronomic research trials on a large research farm. Included are team and project planning, UAV and sensor selection and integration, and data collection and analysis workflow. The study involved many crops and both breeding plots and agronomic fields. The project’s goal was to develop methods for UAVs to collect high-quality, high-volume crop data with fast turnaround time to field scientists. The project included five teams: Administration, Flight Operations, Sensors, Data Management, and Field Research. Four case studies involving multiple crops in breeding and agronomic applications add practical descriptive detail. Lessons learned include critical information on sensors, air vehicles, and configuration parameters for both. As the first

  3. High-throughput development of amphiphile self-assembly materials: fast-tracking synthesis, characterization, formulation, application, and understanding.

    PubMed

    Mulet, Xavier; Conn, Charlotte E; Fong, Celesta; Kennedy, Danielle F; Moghaddam, Minoo J; Drummond, Calum J

    2013-07-16

    Amphiphile self-assembly materials, which contain both a hydrophilic and a hydrophobic domain, have great potential in high-throughput and combinatorial approaches to discovery and development. However, the materials chemistry community has not embraced these ideas to anywhere near the extent that the medicinal chemistry community has. While this situation is beginning to change, extracting the full potential of high-throughput approaches in the development of self-assembling materials will require further development in the synthesis, characterization, formulation, and application domains. One of the key factors that make small molecule amphiphiles prospective building blocks for next generation multifunctional materials is their ability to self-assemble into complex nanostructures through low-energy transformations. Scientists can potentially tune, control, and functionalize these structures, but only after establishing their inherent properties. Because both robotic materials handling and customized rapid characterization equipment are increasingly available, high-throughput solutions are now attainable. These address traditional development bottlenecks associated with self-assembling amphiphile materials, such as their structural characterization and the assessment of end-use functional performance. A high-throughput methodology can help streamline materials development workflows, in accord with existing high-throughput discovery pipelines such as those used by the pharmaceutical industry in drug discovery. Chemists have identified several areas that are amenable to a high-throughput approach for amphiphile self-assembly materials development. These allow an exploration of not only a large potential chemical, compositional, and structural space, but also material properties, formulation, and application variables. These areas of development include materials synthesis and preparation, formulation, characterization, and screening performance for the desired end

  4. Field-based high-throughput plant phenotyping reveals the temporal patterns of quantitative trait loci associated with stress-responsive traits in cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To dissect the genetic basis of dynamic adaptive traits under relevant growing conditions, we employed a field-based, high-throughput plant phenotyping (HTPP) system that deployed four sets of sensors to simultaneously measure canopy temperature, reflectance, and height on a cotton (Gossypium hirsut...

  5. High-Throughput Characterization of Porous Materials Using Graphics Processing Units

    SciTech Connect

    Kim, Jihan; Martin, Richard L.; Rübel, Oliver; Haranczyk, Maciej; Smit, Berend

    2012-05-08

    We have developed a high-throughput graphics processing units (GPU) code that can characterize a large database of crystalline porous materials. In our algorithm, the GPU is utilized to accelerate energy grid calculations where the grid values represent interactions (i.e., Lennard-Jones + Coulomb potentials) between gas molecules (i.e., CH$_{4}$ and CO$_{2}$) and material's framework atoms. Using a parallel flood fill CPU algorithm, inaccessible regions inside the framework structures are identified and blocked based on their energy profiles. Finally, we compute the Henry coefficients and heats of adsorption through statistical Widom insertion Monte Carlo moves in the domain restricted to the accessible space. The code offers significant speedup over a single core CPU code and allows us to characterize a set of porous materials at least an order of magnitude larger than ones considered in earlier studies. For structures selected from such a prescreening algorithm, full adsorption isotherms can be calculated by conducting multiple grand canonical Monte Carlo simulations concurrently within the GPU.

  6. High-throughput characterization of chemical-associated embryonic behavioral changes predicts teratogenic outcomes.

    PubMed

    Reif, David M; Truong, Lisa; Mandrell, David; Marvel, Skylar; Zhang, Guozhu; Tanguay, Robert L

    2016-06-01

    New strategies are needed to address the data gap between the bioactivity of chemicals in the environment versus existing hazard information. We address whether a high-throughput screening (HTS) system using a vertebrate organism (embryonic zebrafish) can characterize chemical-elicited behavioral responses at an early, 24 hours post-fertilization (hpf) stage that predict teratogenic consequences at a later developmental stage. The system was used to generate full concentration-response behavioral profiles at 24 hpf across 1060 ToxCast™ chemicals. Detailed, morphological evaluation of all individuals was performed as experimental follow-up at 5 days post-fertilization (dpf). Chemicals eliciting behavioral responses were also mapped against external HTS in vitro results to identify specific molecular targets and neurosignalling pathways. We found that, as an integrative measure of normal development, significant alterations in movement highlighted active chemicals representing several modes of action. These early behavioral responses were predictive for 17 specific developmental abnormalities and mortality measured at 5 dpf, often at lower (i.e., more potent) concentrations than those at which morphological effects were observed. Therefore, this system can provide rapid characterization of chemical-elicited behavioral responses at an early developmental stage that are predictive of observable adverse effects later in life.

  7. Toward a Low-Cost System for High-Throughput Image-Based Phenotyping of Root System Architecture

    NASA Astrophysics Data System (ADS)

    Davis, T. W.; Schneider, D. J.; Cheng, H.; Shaw, N.; Kochian, L. V.; Shaff, J. E.

    2015-12-01

    Root system architecture is being studied more closely for improved nutrient acquisition, stress tolerance and carbon sequestration by relating the genetic material that corresponds to preferential physical features. This information can help direct plant breeders in addressing the growing concerns regarding the global demand on crops and fossil fuels. To help support this incentive comes a need to make high-throughput image-based phenotyping of plant roots, at the individual plant scale, simpler and more affordable. Our goal is to create an affordable and portable product for simple image collection, processing and management that will extend root phenotyping to institutions with limited funding (e.g., in developing countries). Thus, a new integrated system has been developed using the Raspberry Pi single-board computer. Similar to other 3D-based imaging platforms, the system utilizes a stationary camera to photograph a rotating crop root system (e.g., rice, maize or sorghum) that is suspended either in a gel or on a mesh (for hydroponics). In contrast, the new design takes advantage of powerful open-source hardware and software to reduce the system costs, simplify the imaging process, and manage the large datasets produced by the high-resolution photographs. A newly designed graphical user interface (GUI) unifies the system controls (e.g., adjusting camera and motor settings and orchestrating the motor motion with image capture), making it easier to accommodate a variety of experiments. During each imaging session, integral metadata necessary for reproducing experiment results are collected (e.g., plant type and age, growing conditions and treatments, camera settings) using hierarchical data format files. These metadata are searchable within the GUI and can be selected and extracted for further analysis. The GUI also supports an image previewer that performs limited image processing (e.g., thresholding and cropping). Root skeletonization, 3D reconstruction and

  8. High-throughput characterization of virus-like particles by interlaced size-exclusion chromatography.

    PubMed

    Ladd Effio, Christopher; Oelmeier, Stefan A; Hubbuch, Jürgen

    2016-03-04

    The development and manufacturing of safe and effective vaccines relies essentially on the availability of robust and precise analytical techniques. Virus-like particles (VLPs) have emerged as an important and valuable class of vaccines for the containment of infectious diseases. VLPs are produced by recombinant protein expression followed by purification procedures to minimize the levels of process- and product-related impurities. The control of these impurities is necessary during process development and manufacturing. Especially monitoring of the VLP size distribution is important for the characterization of the final vaccine product. Currently used methods require long analysis times and tailor-made assays. In this work, we present a size-exclusion ultra-high performance liquid chromatography (SE-UHPLC) method to characterize VLPs and quantify aggregates within 3.1min per sample applying interlaced injections. Four analytical SEC columns were evaluated for the analysis of human B19 parvo-VLPs and murine polyoma-VLPs. The optimized method was successfully used for the characterization of five recombinant protein-based VLPs including human papillomavirus (HPV) VLPs, human enterovirus 71 (EV71) VLPs, and chimeric hepatitis B core antigen (HBcAg) VLPs pointing out the generic applicability of the assay. Measurements were supported by transmission electron microscopy and dynamic light scattering. It was demonstrated that the iSE-UHPLC method provides a rapid, precise and robust tool for the characterization of VLPs. Two case studies on purification tools for VLP aggregates and storage conditions of HPV VLPs highlight the relevance of the analytical method for high-throughput process development and process monitoring of virus-like particles.

  9. Strengths and Biases of High-Throughput Sequencing Data in the Characterization of Freshwater Ciliate Microbiomes.

    PubMed

    Boscaro, Vittorio; Rossi, Alessia; Vannini, Claudia; Verni, Franco; Fokin, Sergei I; Petroni, Giulio

    2016-12-28

    Molecular surveys of eukaryotic microbial communities employing high-throughput sequencing (HTS) techniques are rapidly supplanting traditional morphological approaches due to their larger data output and reduced bench work time. Here, we directly compare morphological and Illumina data obtained from the same samples, in an effort to characterize ciliate faunas from sediments in freshwater environments. We show how in silico processing affects the final outcome of our HTS analysis, providing evidence that quality filtering protocols strongly impact the number of predicted taxa, but not downstream conclusions such as biogeography patterns. We determine the abundance distribution of ciliates, showing that a small fraction of abundant taxa dominates read counts. At the same time, we advance reasons to believe that biases affecting HTS abundances may be significant enough to blur part of the underlying biological picture. We confirmed that the HTS approach detects many more taxa than morphological inspections, and highlight how the difference varies among taxonomic groups. Finally, we hypothesize that the two datasets actually correspond to different conceptions of "diversity," and consequently that neither is entirely superior to the other when investigating environmental protists.

  10. Fabrication and Characterization of Solid-state Nanopore Arrays for High Throughput DNA Sequencing

    PubMed Central

    Torre, Ruby dela; Larkin, Joseph; Singer, Alon; Meller, Amit

    2012-01-01

    We report the fabrication and characterization of uniformly-sized nanopore arrays, integrated into an optical detection system for high-throughput DNA sequencing applications. Nanopore arrays were fabricated using Focused Ion Beam milling followed by TiO2 coating using Atomic Layer Deposition (ALD). The TiO2 layer decreases the initial pore diameter down to sub-10nm range, compatible with the requirements for nanopore-based sequencing using optical readout. We find that the TiO2 layers produce a lower photoluminescence background as compared with the more broadly used Al2O3 coatings. The functionality of the nanopore array was demonstrated by the simultaneous optical detection of DNA-quantum dot-conjugates, which were electro-kinetically driven through the nanopores. Our optical scheme employs Total Internal Reflection Fluorescence (TIRF) microscopy to illuminate a wide area of the TiO2-coated membrane. A highly parallel system for observing DNA capture events in a uniformly-sized 6×6 nanopore array was experimentally realized. PMID:22948520

  11. Applications of High-Throughput Sequencing for In Vitro Selection and Characterization of Aptamers

    PubMed Central

    Nguyen Quang, Nam; Perret, Gérald; Ducongé, Frédéric

    2016-01-01

    Aptamers are identified through an iterative process of evolutionary selection starting from a random pool containing billions of sequences. Simultaneously to the amplification of high-affinity candidates, the diversity in the pool is exponentially reduced after several rounds of in vitro selection. Until now, cloning and Sanger sequencing of about 100 sequences was usually used to identify the enriched candidates. However, High-Throughput Sequencing (HTS) is now extensively used to replace such low throughput sequencing approaches. Providing a deeper analysis of the library, HTS is expected to accelerate the identification of aptamers as well as to identify aptamers with higher affinity. It is also expected that it can provide important information on the binding site of the aptamers. Nevertheless, HTS requires handling a large amount of data that is only possible through the development of new in silico methods. Here, this review presents these different strategies that have been recently developed to improve the identification and characterization of aptamers using HTS. PMID:27973417

  12. Characterization of Squamate Olfactory Receptor Genes and Their Transcripts by the High-Throughput Sequencing Approach

    PubMed Central

    Dehara, Yuki; Hashiguchi, Yasuyuki; Matsubara, Kazumi; Yanai, Tokuma; Kubo, Masahito; Kumazawa, Yoshinori

    2012-01-01

    The olfactory receptor (OR) genes represent the largest multigene family in the genome of terrestrial vertebrates. Here, the high-throughput next-generation sequencing (NGS) approach was applied to characterization of OR gene repertoires in the green anole lizard Anolis carolinensis and the Japanese four-lined ratsnake Elaphe quadrivirgata. Tagged polymerase chain reaction (PCR) products amplified from either genomic DNA or cDNA of the two species were used for parallel pyrosequencing, assembling, and screening for errors in PCR and pyrosequencing. Starting from the lizard genomic DNA, we accurately identified 56 of 136 OR genes that were identified from its draft genome sequence. These recovered genes were broadly distributed in the phylogenetic tree of vertebrate OR genes without severe biases toward particular OR families. Ninety-six OR genes were identified from the ratsnake genomic DNA, implying that the snake has more OR gene loci than the anole lizard in response to an increased need for the acuity of olfaction. This view is supported by the estimated number of OR genes in the Burmese python's draft genome (∼280), although squamates may generally have fewer OR genes than terrestrial mammals and amphibians. The OR gene repertoire of the python seems unique in that many class I OR genes are retained. The NGS approach also allowed us to identify candidates of highly expressed and silent OR gene copies in the lizard's olfactory epithelium. The approach will facilitate efficient and parallel characterization of considerable unbiased proportions of multigene family members and their transcripts from nonmodel organisms. PMID:22511035

  13. Synthesis and characterization of four new metal 5-phosphonoisophthalates discovered by high-throughput experimentation

    SciTech Connect

    Bauer, Sebastian

    2007-11-15

    A new ligand, 5-diethylphosphonoisophthalic acid ((HOOC){sub 2}C{sub 6}H{sub 3}-PO{sub 3}(C{sub 2}H{sub 5}){sub 2}, H{sub 2}Et{sub 2}L), for the hydrothermal synthesis of inorganic-organic hybrid compounds was prepared and characterized by NMR-spectroscopy. Its in situ hydrolysis leads to the corresponding 5-phosphonoisophthalic acid ((HOOC){sub 2}C{sub 6}H{sub 3}-PO{sub 3}H{sub 2}, H{sub 4}L). Applying high-throughput methods, different di- and trivalent metal salts for the synthesis of crystalline metal phosphonates based on H{sub 2}Et{sub 2}L have been screened. From the resulting discovery library, single-crystals of four new compounds, [Sm{sub 2}(H{sub 2}O){sub 4}(H(OOC){sub 2}C{sub 6}H{sub 3}-PO{sub 3}){sub 2}].2H{sub 2}O (1), [Cu{sub 3}(H{sub 2}O)(H(OOC){sub 2}C{sub 6}H{sub 3}-PO{sub 3}){sub 2}].2H{sub 2}O (2), Ca{sub 2}(H{sub 2}O)[H(OOC){sub 2}C{sub 6}H{sub 3}-PO{sub 3}H]{sub 2} (3), and Ba{sub 2}(H{sub 2}O){sub 3}(OOC){sub 2}C{sub 6}H{sub 3}-PO{sub 3} (4), have been isolated. The single-crystal structure determination of the title compounds shows H{sub 4}L to be a versatile ligand, exhibiting different types of coordination modes between the functional groups and the metal ions. A comparison of the structural features of the title compounds shows a varying degree of M-O-M connectivities. Thus, isolated metal-oxygen clusters (compounds 1 and 2), infinite M-O-M chains (compound 3), and infinite M-O-M layers (compound 4) are observed. The title compounds 1, 2, and 3 were further characterized by IR-spectroscopy, TG-, EDX-, and elemental chemical analysis. - Graphical abstract: Applying high-throughput methods, the new ligand 5-diethylphosphonoisophtalic acid, (HOOC){sub 2}C{sub 6}H{sub 3}-PO{sub 3}(C{sub 2}H{sub 5}){sub 2} (H{sub 2}Et{sub 2}L), was reacted with several di- and trivalent metal salts under hydrothermal conditions. Single-crystals of four new inorganic-organic hybrid compounds were isolated from the discovery library. The single

  14. Unbiased Characterization of Anopheles Mosquito Blood Meals by Targeted High-Throughput Sequencing

    PubMed Central

    Logue, Kyle; Keven, John Bosco; Cannon, Matthew V.; Reimer, Lisa; Siba, Peter; Walker, Edward D.; Zimmerman, Peter A.; Serre, David

    2016-01-01

    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. PMID:26963245

  15. Unbiased Characterization of Anopheles Mosquito Blood Meals by Targeted High-Throughput Sequencing.

    PubMed

    Logue, Kyle; Keven, John Bosco; Cannon, Matthew V; Reimer, Lisa; Siba, Peter; Walker, Edward D; Zimmerman, Peter A; Serre, David

    2016-03-01

    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.

  16. Plant Phenotype Characterization System

    SciTech Connect

    Daniel W McDonald; Ronald B Michaels

    2005-09-09

    This report is the final scientific report for the DOE Inventions and Innovations Project: Plant Phenotype Characterization System, DE-FG36-04GO14334. The period of performance was September 30, 2004 through July 15, 2005. The project objective is to demonstrate the viability of a new scientific instrument concept for the study of plant root systems. The root systems of plants are thought to be important in plant yield and thus important to DOE goals in renewable energy sources. The scientific study and understanding of plant root systems is hampered by the difficulty in observing root activity and the inadequacy of existing root study instrumentation options. We have demonstrated a high throughput, non-invasive, high resolution technique for visualizing plant root systems in-situ. Our approach is based upon low-energy x-ray radiography and the use of containers and substrates (artificial soil) which are virtually transparent to x-rays. The system allows us to germinate and grow plant specimens in our containers and substrates and to generate x-ray images of the developing root system over time. The same plant can be imaged at different times in its development. The system can be used for root studies in plant physiology, plant morphology, plant breeding, plant functional genomics and plant genotype screening.

  17. Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae)

    PubMed Central

    2011-01-01

    Background Cucurbita pepo belongs to the Cucurbitaceae family. The "Zucchini" types rank among the highest-valued vegetables worldwide, and other C. pepo and related Cucurbita spp., are food staples and rich sources of fat and vitamins. A broad range of genomic tools are today available for other cucurbits that have become models for the study of different metabolic processes. However, these tools are still lacking in the Cucurbita genus, thus limiting gene discovery and the process of breeding. Results We report the generation of a total of 512,751 C. pepo EST sequences, using 454 GS FLX Titanium technology. ESTs were obtained from normalized cDNA libraries (root, leaves, and flower tissue) prepared using two varieties with contrasting phenotypes for plant, flowering and fruit traits, representing the two C. pepo subspecies: subsp. pepo cv. Zucchini and subsp. ovifera cv Scallop. De novo assembling was performed to generate a collection of 49,610 Cucurbita unigenes (average length of 626 bp) that represent the first transcriptome of the species. Over 60% of the unigenes were functionally annotated and assigned to one or more Gene Ontology terms. The distributions of Cucurbita unigenes followed similar tendencies than that reported for Arabidopsis or melon, suggesting that the dataset may represent the whole Cucurbita transcriptome. About 34% unigenes were detected to have known orthologs of Arabidopsis or melon, including genes potentially involved in disease resistance, flowering and fruit quality. Furthermore, a set of 1,882 unigenes with SSR motifs and 9,043 high confidence SNPs between Zucchini and Scallop were identified, of which 3,538 SNPs met criteria for use with high throughput genotyping platforms, and 144 could be detected as CAPS. A set of markers were validated, being 80% of them polymorphic in a set of variable C. pepo and C. moschata accessions. Conclusion We present the first broad survey of gene sequences and allelic variation in C. pepo, where

  18. Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

    PubMed Central

    Weisshaar, Marco; Cox, Robert; Morehouse, Zachary; Kumar Kyasa, Shiva; Yan, Dan; Oberacker, Phil; Mao, Shuli; Lowen, Anice C.; Natchus, Michael G.

    2016-01-01

    the first to apply a fully replication-competent third-generation IAV reporter strain to a large-scale high-throughput screen (HTS) drug discovery campaign, allowing multicycle infection and screening in physiologically relevant human respiratory cells. A large number of potential druggable targets was thus chemically interrogated, but mechanistic characterization, positive target identification, and resistance profiling demonstrated that three chemically promising and structurally distinct hit classes selected for further analysis all block HA-mediated membrane fusion. Viral escape from inhibition could be achieved through primary and secondary resistance mechanisms. In silico docking predicted compound binding to a microdomain located at the membrane-distal site of the prefusion HA stalk that was also previously suggested as a target site for chemically unrelated HA inhibitors. This study identifies an unexpected chemodominance of the HA stalk microdomain for small-molecule inhibitors in IAV inhibitor screening campaigns and highlights a novel mechanism of cooperative resistance to IAV entry blockers. PMID:27252534

  19. Dissecting the Phenotypic Components of Crop Plant Growth and Drought Responses Based on High-Throughput Image Analysis[W][OPEN

    PubMed Central

    Chen, Dijun; Neumann, Kerstin; Friedel, Swetlana; Kilian, Benjamin; Chen, Ming; Altmann, Thomas; Klukas, Christian

    2014-01-01

    Significantly improved crop varieties are urgently needed to feed the rapidly growing human population under changing climates. While genome sequence information and excellent genomic tools are in place for major crop species, the systematic quantification of phenotypic traits or components thereof in a high-throughput fashion remains an enormous challenge. In order to help bridge the genotype to phenotype gap, we developed a comprehensive framework for high-throughput phenotype data analysis in plants, which enables the extraction of an extensive list of phenotypic traits from nondestructive plant imaging over time. As a proof of concept, we investigated the phenotypic components of the drought responses of 18 different barley (Hordeum vulgare) cultivars during vegetative growth. We analyzed dynamic properties of trait expression over growth time based on 54 representative phenotypic features. The data are highly valuable to understand plant development and to further quantify growth and crop performance features. We tested various growth models to predict plant biomass accumulation and identified several relevant parameters that support biological interpretation of plant growth and stress tolerance. These image-based traits and model-derived parameters are promising for subsequent genetic mapping to uncover the genetic basis of complex agronomic traits. Taken together, we anticipate that the analytical framework and analysis results presented here will be useful to advance our views of phenotypic trait components underlying plant development and their responses to environmental cues. PMID:25501589

  20. Molecular Methods and Bioinformatic Tools for Adjuvant Characterization by High-Throughput Sequencing.

    PubMed

    Wiley, Steven R; Raman, Vanitha S

    2017-01-01

    Adjuvants in vaccine formulations are designed to enhance immune responses against a target antigen or pathogen. The ability of these vaccines to induce activation and differentiation of mature naïve B cells to produce pathogen-specific antibodies (immunoglobulins; Ig) helps guarantee long-lived humoral immunity. This process involves clonal expansion of antigen-specific B cells, genomic rearrangement of Ig heavy (IgH) and light (IgL) loci, somatic hypermutation (SHM), and clonal selection for affinity-matured antibody, resulting in a vast but directed repertoire of B cells expressing highly specific antibody proteins. High-throughput sequencing of the IgH and IgL complementary determining regions (CDRs) derived from various B cell populations provides an unprecedented way to observe dynamic responses of the humoral immune repertoire in response to vaccination. However, applying high-throughput sequencing (HTS) methodologies to multi-armed in vivo experiments requires careful coordination of sample preparation with downstream bioinformatics, particularly with regard to issues of quantitation, sequence fidelity, bar-coding, and multiplexing strategies. Here, we overview strategies of high-throughput sequencing and analysis of the adaptive immune complex loci applied to multi-armed, multiplexed experiments.

  1. Characterization and complete genome sequence of a panicovirus from Bermuda grass by high-throughput sequencing.

    PubMed

    Tahir, Muhammad N; Lockhart, Ben; Grinstead, Samuel; Mollov, Dimitre

    2017-04-01

    Bermuda grass samples were examined by transmission electron microscopy and 28-30 nm spherical virus particles were observed. Total RNA from these plants was subjected to high-throughput sequencing (HTS). The nearly full genome sequence of a panicovirus was identified from one HTS scaffold. Sanger sequencing was used to confirm the HTS results and complete the genome sequence of 4404 nt. This virus was provisionally named Bermuda grass latent virus (BGLV). Its predicted open reading frames follow the typical arrangement of the genus Panicovirus. Based on sequence comparisons and phylogenetic analyses BGLV differs from other viruses and therefore taxonomically it is a new member of the genus Panicovirus, family Tombusviridae.

  2. Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes.

    PubMed

    Greenough, Lucia; Schermerhorn, Kelly M; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E; Gardner, Andrew F

    2016-01-29

    Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3'-5' exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner.

  3. Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes

    PubMed Central

    Greenough, Lucia; Schermerhorn, Kelly M.; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E.; Gardner, Andrew F.

    2016-01-01

    Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3′-5′ exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. PMID:26365239

  4. High-throughput production and structural characterization of libraries of self-assembly lipidic cubic phase materials.

    PubMed

    Darmanin, Connie; Conn, Charlotte E; Newman, Janet; Mulet, Xavier; Seabrook, Shane A; Liang, Yi-Lynn; Hawley, Adrian; Kirby, Nigel; Varghese, Joseph N; Drummond, Calum J

    2012-04-09

    A protocol is presented for the high-throughput (HT) production of lyotropic liquid crystalline phases from libraries of lipids and lipid mixtures using standard liquid dispensing robotics, implementing methods that circumvent the problems traditionally associated with handling the highly viscous cubic phase. In addition, the ability to structurally characterize lipidic phases and assess functionality for membrane proteins contained within cubic phases, in a HT manner, is demonstrated. The techniques are combined and exemplified using the application of membrane protein crystallization within lipidic cubic phases.

  5. Characterization and high throughput analysis of metal hydrides for hydrogen storage

    NASA Astrophysics Data System (ADS)

    Barcelo, Steven James

    Efficient hydrogen storage is required for fuel cell vehicles to be competitive with those driven by internal combustion engines. Current methods of storage such as compressed gas and liquid hydrogen cannot meet this standard, so novel hydrogen storage materials such as metal hydrides are required. No simple metal hydride meets the required specifications. Research is required to find new materials or improve existing materials. This thesis describes the research practices necessary to achieve legitimate and repeatable results in laboratories across the world. Examples of experiments using these techniques are presented, such as a high throughput technique to optimize materials systems with up to three components such as calcium borohydride with titanium catalyst and magnesium hydride with nickel and aluminum as destabilizing elements and catalysts. Thin films composed of gradients of each material were deposited by sputtering, creating a single thin film sample covering all potential material combinations. Optical properties of the samples under hydrogen pressure were monitored to identify the regions with largest and fastest hydrogen uptake. In the Ca-B-Ti system, titanium did not sufficiently catalyze the borohydride formation reaction at low temperature. Substantial hydrogen uptake was shown in the Mg-Ni region of the Mg-Ni-Al films. Al did not participate in the reaction at low temperature. Further investigation of the role of catalysts and destabilizing elements in improving hydrogen storage performance through X-ray Absorption and Emission Spectroscopy measurements of the Mg-Ni system during hydrogenation is presented. Typical X-ray spectroscopy measurements use a synchrotron radiation source and require ultra high vacuum conditions. For these experiments we designed a chamber which can be inserted into a vacuum chamber allowing in situ measurements of a sample under hydrogen pressure, providing information on the role of Ni in hydrogen absorption of Mg

  6. High-throughput characterization of stresses in thin film materials libraries using Si cantilever array wafers and digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Lai, Y. W.; Hamann, S.; Ehmann, M.; Ludwig, A.

    2011-06-01

    We report the development of an advanced high-throughput stress characterization method for thin film materials libraries sputter-deposited on micro-machined cantilever arrays consisting of around 1500 cantilevers on 4-inch silicon-on-insulator wafers. A low-cost custom-designed digital holographic microscope (DHM) is employed to simultaneously monitor the thin film thickness, the surface topography and the curvature of each of the cantilevers before and after deposition. The variation in stress state across the thin film materials library is then calculated by Stoney's equation based on the obtained radii of curvature of the cantilevers and film thicknesses. DHM with nanometer-scale out-of-plane resolution allows stress measurements in a wide range, at least from several MPa to several GPa. By using an automatic x-y translation stage, the local stresses within a 4-inch materials library are mapped with high accuracy within 10 min. The speed of measurement is greatly improved compared with the prior laser scanning approach that needs more than an hour of measuring time. A high-throughput stress measurement of an as-deposited Fe-Pd-W materials library was evaluated for demonstration. The fast characterization method is expected to accelerate the development of (functional) thin films, e.g., (magnetic) shape memory materials, whose functionality is greatly stress dependent.

  7. High-throughput characterization of stresses in thin film materials libraries using Si cantilever array wafers and digital holographic microscopy.

    PubMed

    Lai, Y W; Hamann, S; Ehmann, M; Ludwig, A

    2011-06-01

    We report the development of an advanced high-throughput stress characterization method for thin film materials libraries sputter-deposited on micro-machined cantilever arrays consisting of around 1500 cantilevers on 4-inch silicon-on-insulator wafers. A low-cost custom-designed digital holographic microscope (DHM) is employed to simultaneously monitor the thin film thickness, the surface topography and the curvature of each of the cantilevers before and after deposition. The variation in stress state across the thin film materials library is then calculated by Stoney's equation based on the obtained radii of curvature of the cantilevers and film thicknesses. DHM with nanometer-scale out-of-plane resolution allows stress measurements in a wide range, at least from several MPa to several GPa. By using an automatic x-y translation stage, the local stresses within a 4-inch materials library are mapped with high accuracy within 10 min. The speed of measurement is greatly improved compared with the prior laser scanning approach that needs more than an hour of measuring time. A high-throughput stress measurement of an as-deposited Fe-Pd-W materials library was evaluated for demonstration. The fast characterization method is expected to accelerate the development of (functional) thin films, e.g., (magnetic) shape memory materials, whose functionality is greatly stress dependent.

  8. A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.

    PubMed

    Li, Xiaofeng; Suhar, Tom; Glass, Lateca; Rajaraman, Ganesh

    2014-03-03

    Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each.

  9. The development and implementation of high-throughput tools for discovery and characterization of proton exchange membranes

    NASA Astrophysics Data System (ADS)

    Reed, Keith Gregory

    The need for sustainable energy use has motivated the exploration of renewable alternative fuels and fuel conversion technology on a global scale. Fuel cells, which convert chemical energy directly into electrical energy with high efficiency and low emissions, provide a promising strategy for achieving energy sustainability. The current progress in fuel cell commercialization is mainly in portable and stationary applications, but fuel cell technology for transportation applications, which make up a substantial portion of the global energy market, have seen little commercial success. Proton exchange membrane fuel cells (PEMFCs) have high potential for addressing the future energy needs of the transportation energy sector. However, one of the prevailing limitations of the PEMFC is the availability of high-performance, cost-effective electrolyte materials. These materials may be realized in the near future by developing multifunctional polymer blends targeted at specific performance capabilities. Since the number of available polymer combinations and numerous processing variations provide an almost infinite source of PEMFC membrane candidates, efficient methods of discovering high-performance PEM materials are necessary. Combinatorial methods meet these needs using gradient or discrete techniques to capture process variations such as annealing temperature, thickness, and chemical composition into a single polymer sample that serves as a library of materials. To characterize these heterogeneous samples for fuel cell performance, specific high-throughput measurement techniques are necessary. In this work, a high-throughput mass transport assay (HT-MTA) has been developed to characterize water flux and permeability at multiple sample locations in parallel. The functionality of HT-MTA was evaluated using standard NafionRTM films and a model semi-interpenetrated polymer network with commercial polyvinylidine fluoride as the host matrix for a proprietary polyelectrolyte

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

    SciTech Connect

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

    2011-12-31

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

  11. A High-Throughput, Arbitrary-Waveform, MPI Spectrometer and Relaxometer for Comprehensive Magnetic Particle Optimization and Characterization

    NASA Astrophysics Data System (ADS)

    Tay, Zhi Wei; Goodwill, Patrick W.; Hensley, Daniel W.; Taylor, Laura A.; Zheng, Bo; Conolly, Steven M.

    2016-09-01

    Magnetic Particle Imaging (MPI) is a promising new tracer modality with zero attenuation deep in tissue, high contrast and sensitivity, and an excellent safety profile. However, the spatial resolution of MPI is limited to around 1 mm currently and urgently needs to be improved for clinical applications such as angiography and brain perfusion. Although MPI resolution is highly dependent on tracer characteristics and the drive waveforms, optimization is limited to a small subset of possible excitation strategies by current MPI hardware that only does sinusoidal drive waveforms at very few frequencies. To enable a more comprehensive and rapid optimization of drive waveforms for multiple metrics like resolution and signal strength simultaneously, we demonstrate the first untuned MPI spectrometer/relaxometer with unprecedented 400 kHz excitation bandwidth and capable of high-throughput acquisition of harmonic spectra (100 different drive-field frequencies in only 500 ms). It is also capable of arbitrary drive-field waveforms which have not been experimentally evaluated in MPI to date. Its high-throughput capability, frequency-agility and tabletop size makes this Arbitrary Waveform Relaxometer/Spectrometer (AWR) a convenient yet powerfully flexible tool for nanoparticle experts seeking to characterize magnetic particles and optimize MPI drive waveforms for in vitro biosensing and in vivo imaging with MPI.

  12. A High-Throughput, Arbitrary-Waveform, MPI Spectrometer and Relaxometer for Comprehensive Magnetic Particle Optimization and Characterization

    PubMed Central

    Tay, Zhi Wei; Goodwill, Patrick W.; Hensley, Daniel W.; Taylor, Laura A.; Zheng, Bo; Conolly, Steven M.

    2016-01-01

    Magnetic Particle Imaging (MPI) is a promising new tracer modality with zero attenuation deep in tissue, high contrast and sensitivity, and an excellent safety profile. However, the spatial resolution of MPI is limited to around 1 mm currently and urgently needs to be improved for clinical applications such as angiography and brain perfusion. Although MPI resolution is highly dependent on tracer characteristics and the drive waveforms, optimization is limited to a small subset of possible excitation strategies by current MPI hardware that only does sinusoidal drive waveforms at very few frequencies. To enable a more comprehensive and rapid optimization of drive waveforms for multiple metrics like resolution and signal strength simultaneously, we demonstrate the first untuned MPI spectrometer/relaxometer with unprecedented 400 kHz excitation bandwidth and capable of high-throughput acquisition of harmonic spectra (100 different drive-field frequencies in only 500 ms). It is also capable of arbitrary drive-field waveforms which have not been experimentally evaluated in MPI to date. Its high-throughput capability, frequency-agility and tabletop size makes this Arbitrary Waveform Relaxometer/Spectrometer (AWR) a convenient yet powerfully flexible tool for nanoparticle experts seeking to characterize magnetic particles and optimize MPI drive waveforms for in vitro biosensing and in vivo imaging with MPI. PMID:27686629

  13. A High-Throughput, Arbitrary-Waveform, MPI Spectrometer and Relaxometer for Comprehensive Magnetic Particle Optimization and Characterization.

    PubMed

    Tay, Zhi Wei; Goodwill, Patrick W; Hensley, Daniel W; Taylor, Laura A; Zheng, Bo; Conolly, Steven M

    2016-09-30

    Magnetic Particle Imaging (MPI) is a promising new tracer modality with zero attenuation deep in tissue, high contrast and sensitivity, and an excellent safety profile. However, the spatial resolution of MPI is limited to around 1 mm currently and urgently needs to be improved for clinical applications such as angiography and brain perfusion. Although MPI resolution is highly dependent on tracer characteristics and the drive waveforms, optimization is limited to a small subset of possible excitation strategies by current MPI hardware that only does sinusoidal drive waveforms at very few frequencies. To enable a more comprehensive and rapid optimization of drive waveforms for multiple metrics like resolution and signal strength simultaneously, we demonstrate the first untuned MPI spectrometer/relaxometer with unprecedented 400 kHz excitation bandwidth and capable of high-throughput acquisition of harmonic spectra (100 different drive-field frequencies in only 500 ms). It is also capable of arbitrary drive-field waveforms which have not been experimentally evaluated in MPI to date. Its high-throughput capability, frequency-agility and tabletop size makes this Arbitrary Waveform Relaxometer/Spectrometer (AWR) a convenient yet powerfully flexible tool for nanoparticle experts seeking to characterize magnetic particles and optimize MPI drive waveforms for in vitro biosensing and in vivo imaging with MPI.

  14. Canopy Temperature and Vegetation Indices from High-Throughput Phenotyping Improve Accuracy of Pedigree and Genomic Selection for Grain Yield in Wheat

    PubMed Central

    Rutkoski, Jessica; Poland, Jesse; Mondal, Suchismita; Autrique, Enrique; Pérez, Lorena González; Crossa, José; Reynolds, Matthew; Singh, Ravi

    2016-01-01

    Genomic selection can be applied prior to phenotyping, enabling shorter breeding cycles and greater rates of genetic gain relative to phenotypic selection. Traits measured using high-throughput phenotyping based on proximal or remote sensing could be useful for improving pedigree and genomic prediction model accuracies for traits not yet possible to phenotype directly. We tested if using aerial measurements of canopy temperature, and green and red normalized difference vegetation index as secondary traits in pedigree and genomic best linear unbiased prediction models could increase accuracy for grain yield in wheat, Triticum aestivum L., using 557 lines in five environments. Secondary traits on training and test sets, and grain yield on the training set were modeled as multivariate, and compared to univariate models with grain yield on the training set only. Cross validation accuracies were estimated within and across-environment, with and without replication, and with and without correcting for days to heading. We observed that, within environment, with unreplicated secondary trait data, and without correcting for days to heading, secondary traits increased accuracies for grain yield by 56% in pedigree, and 70% in genomic prediction models, on average. Secondary traits increased accuracy slightly more when replicated, and considerably less when models corrected for days to heading. In across-environment prediction, trends were similar but less consistent. These results show that secondary traits measured in high-throughput could be used in pedigree and genomic prediction to improve accuracy. This approach could improve selection in wheat during early stages if validated in early-generation breeding plots. PMID:27402362

  15. High-throughput screening of thin-film semiconductor material libraries II: characterization of Fe-W-O libraries.

    PubMed

    Meyer, Robert; Sliozberg, Kirill; Khare, Chinmay; Schuhmann, Wolfgang; Ludwig, Alfred

    2015-04-13

    Metal oxides are promising materials for solar water splitting. To identify suitable materials within the ternary system FeWO, thin-film material libraries with combined thickness and compositional gradients were synthesized by combinatorial reactive magnetron sputtering. These libraries (>1000 different samples) were investigated by means of structural and functional high-throughput characterization techniques to establish correlations between composition, crystallinity, morphology, thickness, and photocurrent density in the compositional range between (Fe6 W94 )Ox and (Fe61 W39 )Ox . In addition to the well-known phase WO3 , the binary phase W5 O14 and the ternary phase Fe2 O6 W show enhanced photoelectrochemical activity. The highest photocurrent density of 65 μA cm(-2) was achieved for the composition (Fe15 W85 )Ox , which contains the W5 O14 phase and has a thickness of 1060 nm.

  16. Polymer surface functionalities that control human embryoid body cell adhesion revealed by high throughput surface characterization of combinatorial material microarrays.

    PubMed

    Yang, Jing; Mei, Ying; Hook, Andrew L; Taylor, Michael; Urquhart, Andrew J; Bogatyrev, Said R; Langer, Robert; Anderson, Daniel G; Davies, Martyn C; Alexander, Morgan R

    2010-12-01

    High throughput materials discovery using combinatorial polymer microarrays to screen for new biomaterials with new and improved function is established as a powerful strategy. Here we combine this screening approach with high throughput surface characterization (HT-SC) to identify surface structure-function relationships. We explore how this combination can help to identify surface chemical moieties that control protein adsorption and subsequent cellular response. The adhesion of human embryoid body (hEB) cells to a large number (496) of different acrylate polymers synthesized in a microarray format is screened using a high throughput procedure. To determine the role of the polymer surface properties on hEB cell adhesion, detailed HT-SC of these acrylate polymers is carried out using time of flight secondary ion mass spectrometry (ToF SIMS), X-ray photoelectron spectroscopy (XPS), pico litre drop sessile water contact angle (WCA) measurement and atomic force microscopy (AFM). A structure-function relationship is identified between the ToF SIMS analysis of the surface chemistry after a fibronectin (Fn) pre-conditioning step and the cell adhesion to each spot using the multivariate analysis technique partial least squares (PLS) regression. Secondary ions indicative of the adsorbed Fn correlate with increased cell adhesion whereas glycol and other functionalities from the polymers are identified that reduce cell adhesion. Furthermore, a strong relationship between the ToF SIMS spectra of bare polymers and the cell adhesion to each spot is identified using PLS regression. This identifies a role for both the surface chemistry of the bare polymer and the pre-adsorbed Fn, as-represented in the ToF SIMS spectra, in controlling cellular adhesion. In contrast, no relationship is found between cell adhesion and wettability, surface roughness, elemental or functional surface composition. The correlation between ToF SIMS data of the surfaces and the cell adhesion demonstrates

  17. Autopilot: an online data acquisition control system for the enhanced high-throughput characterization of intact proteins.

    PubMed

    Durbin, Kenneth R; Fellers, Ryan T; Ntai, Ioanna; Kelleher, Neil L; Compton, Philip D

    2014-02-04

    The ability to study organisms by direct analysis of their proteomes without digestion via mass spectrometry has benefited greatly from recent advances in separation techniques, instrumentation, and bioinformatics. However, improvements to data acquisition logic have lagged in comparison. Past workflows for Top Down Proteomics (TDPs) have focused on high throughput at the expense of maximal protein coverage and characterization. This mode of data acquisition has led to enormous overlap in the identification of highly abundant proteins in subsequent LC-MS injections. Furthermore, a wealth of data is left underutilized by analyzing each newly targeted species as unique, rather than as part of a collection of fragmentation events on a distinct proteoform. Here, we present a major advance in software for acquisition of TDP data that incorporates a fully automated workflow able to detect intact masses, guide fragmentation to achieve maximal identification and characterization of intact protein species, and perform database search online to yield real-time protein identifications. On Pseudomonas aeruginosa, the software combines fragmentation events of the same precursor with previously obtained fragments to achieve improved characterization of the target form by an average of 42 orders of magnitude in confidence. When HCD fragmentation optimization was applied to intact proteins ions, there was an 18.5 order of magnitude gain in confidence. These improved metrics set the stage for increased proteome coverage and characterization of higher order organisms in the future for sharply improved control over MS instruments in a project- and lab-wide context.

  18. Autopilot: An Online Data Acquisition Control System for the Enhanced High-Throughput Characterization of Intact Proteins

    PubMed Central

    2015-01-01

    The ability to study organisms by direct analysis of their proteomes without digestion via mass spectrometry has benefited greatly from recent advances in separation techniques, instrumentation, and bioinformatics. However, improvements to data acquisition logic have lagged in comparison. Past workflows for Top Down Proteomics (TDPs) have focused on high throughput at the expense of maximal protein coverage and characterization. This mode of data acquisition has led to enormous overlap in the identification of highly abundant proteins in subsequent LC-MS injections. Furthermore, a wealth of data is left underutilized by analyzing each newly targeted species as unique, rather than as part of a collection of fragmentation events on a distinct proteoform. Here, we present a major advance in software for acquisition of TDP data that incorporates a fully automated workflow able to detect intact masses, guide fragmentation to achieve maximal identification and characterization of intact protein species, and perform database search online to yield real-time protein identifications. On Pseudomonas aeruginosa, the software combines fragmentation events of the same precursor with previously obtained fragments to achieve improved characterization of the target form by an average of 42 orders of magnitude in confidence. When HCD fragmentation optimization was applied to intact proteins ions, there was an 18.5 order of magnitude gain in confidence. These improved metrics set the stage for increased proteome coverage and characterization of higher order organisms in the future for sharply improved control over MS instruments in a project- and lab-wide context. PMID:24400813

  19. FASTER SCIENCE FOR BETTER DECISIONS: CHARACTERIZING ENVIRONMENTAL CONTAMINANT RISK FROM HIGH THROUGHPUT DATA

    EPA Science Inventory

    Tens of thousands of chemicals and other man-made contaminants exist in our environment, but only a fraction of these have been characterized for their potential risk to humans and there is widespread interest in closing this data gap in order to better manage contaminant risk. C...

  20. High-throughput ultrasensitive characterization of chemical, structural and plasmonic properties of EBL-fabricated single silver nanoparticles.

    PubMed

    Huang, Tao; Cao, Wei; Elsayed-Ali, Hani E; Xu, Xiao-Hong Nancy

    2012-01-21

    Electron beam lithography (EBL) has become a popular means to prepare a wide variety of nano-arrays for numerous studies and applications, including photonics and sensors. Their fabrications and characterizations are costly and time consuming, underscoring the importance of developing effective tools to rapidly study their physicochemical stabilities and properties over time. In this study, we characterized EBL-fabricated single silver nanoparticle (Ag NP) arrays over their 12-week exposure to ambient conditions using SEM/EDS, AFM and dark-field optical microscopy and spectroscopy (DFOMS). We found that chemical compositions, structural morphologies and plasmonic optical properties of single NPs altered drastically over the exposure. Single cuboid and triangular-prism Ag NPs degraded at rates of (0.74 ± 0.02) and (0.66 ± 0.02) per week, and their localized surface plasmon resonance (LSPR) spectra showed striking blue-shifts (171 ± 25 and 203 ± 35 nm) over the 12-week exposure, respectively. Plasmonic colors of single NPs changed distinctively from red to green over the 12-week exposure. The LSPR spectra of individual NPs in each array were acquired simultaneously and correlated specifically with their SEM and AFM images, demonstrating that DFOMS can serve as high-throughput, ultrasensitive and non-invasive means to characterize chemical, structural and optical properties of nano-arrays in situ in real time at single-NP resolution.

  1. Ultra-high frequency ultrasound biomicroscopy and high throughput cardiovascular phenotyping in a large scale mouse mutagenesis screen

    NASA Astrophysics Data System (ADS)

    Liu, Xiaoqin; Francis, Richard; Tobita, Kimimasa; Kim, Andy; Leatherbury, Linda; Lo, Cecilia W.

    2013-02-01

    Ultrasound biomicroscopy (UBM) is ideally suited for phenotyping fetal mice for congenital heart disease (CHD), as imaging can be carried out noninvasively to provide both hemodynamic and structural information essential for CHD diagnosis. Using the UBM (Vevo 2100; 40Hz) in conjunction with the clinical ultrasound system (Acuson Sequioa C512; 15Hz), we developed a two-step screening protocol to scan thousands fetuses derived from ENU mutagenized pedigrees. A wide spectrum of CHD was detected by the UBM, which were subsequently confirmed with follow-up necropsy and histopathology examination with episcopic fluorescence image capture. CHD observed included outflow anomalies, left/right heart obstructive lesions, septal/valvular defects and cardiac situs anomalies. Meanwhile, various extracardiac defects were found, such as polydactyly, craniofacial defects, exencephaly, omphalocele-cleft palate, most of which were associated with cardiac defects. Our analyses showed the UBM was better at assessing cardiac structure and blood flow profiles, while conventional ultrasound allowed higher throughput low-resolution screening. Our study showed the integration of conventional clinical ultrasound imaging with the UBM for fetal mouse cardiovascular phenotyping can maximize the detection and recovery of CHD mutants.

  2. Characterization of limes (Citrus aurantifolia) grown in Bhutan and Indonesia using high-throughput sequencing

    PubMed Central

    Penjor, Tshering; Mimura, Takashi; Matsumoto, Ryoji; Yamamoto, Masashi; Nagano, Yukio

    2014-01-01

    Lime [Citrus aurantifolia (Cristm.) Swingle] is a Citrus species that is a popular ingredient in many cuisines. Some citrus plants are known to originate in the area ranging from northeastern India to southwestern China. In the current study, we characterized and compared limes grown in Bhutan (n = 5 accessions) and Indonesia (n = 3 accessions). The limes were separated into two groups based on their morphology. Restriction site-associated DNA sequencing (RAD-seq) separated the eight accessions into two clusters. One cluster contained four accessions from Bhutan, whereas the other cluster contained one accession from Bhutan and the three accessions from Indonesia. This genetic classification supported the morphological classification of limes. The analysis suggests that the properties associated with asexual reproduction, and somatic homologous recombination, have contributed to the genetic diversification of limes. PMID:24781859

  3. A Class of Diacylglycerol Acyltransferase 1 Inhibitors Identified by a Combination of Phenotypic High-throughput Screening, Genomics, and Genetics.

    PubMed

    Tschapalda, Kirsten; Zhang, Ya-Qin; Liu, Li; Golovnina, Kseniya; Schlemper, Thomas; Eichmann, Thomas O; Lal-Nag, Madhu; Sreenivasan, Urmila; McLenithan, John; Ziegler, Slava; Sztalryd, Carole; Lass, Achim; Auld, Douglas; Oliver, Brian; Waldmann, Herbert; Li, Zhuyin; Shen, Min; Boxer, Matthew B; Beller, Mathias

    2016-06-01

    Excess lipid storage is an epidemic problem in human populations. Thus, the identification of small molecules to treat or prevent lipid storage-related metabolic complications is of great interest. Here we screened >320.000 compounds for their ability to prevent a cellular lipid accumulation phenotype. We used fly cells because the multifarious tools available for this organism should facilitate unraveling the mechanism-of-action of active small molecules. Of the several hundred lipid storage inhibitors identified in the primary screen we concentrated on three structurally diverse and potent compound classes active in cells of multiple species (including human) and negligible cytotoxicity. Together with Drosophila in vivo epistasis experiments, RNA-Seq expression profiles suggested that the target of one of the small molecules was diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in the production of triacylglycerols and prominent human drug target. We confirmed this prediction by biochemical and enzymatic activity tests.

  4. High-throughput behavioral phenotyping of drug and alcohol susceptibility traits in the expanded panel of BXD recombinant inbred strains

    SciTech Connect

    Philip, Vivek M; Ansah, T; Blaha, C,; Cook, Melloni N.; Hamre, Kristin M.; Lariviere, William R; Matthews, Douglas B; Goldowitz, Daniel; Chesler, Elissa J

    2010-01-01

    Genetic reference populations, particularly the BXD recombinant inbred strains, are a valuable resource for the discovery of the bio-molecular substrates and genetic drivers responsible for trait variation and co- ariation. This approach can be profitably applied in the analysis of susceptibility and mechanisms of drug and alcohol use disorders for which many predisposing behaviors may predict occurrence and manifestation of increased preference for these substances. Many of these traits are modeled by common mouse behavioral assays, facilitating the detection of patterns and sources of genetic co-regulation of predisposing phenotypes and substance consumption. Members of the Tennessee Mouse Genome Consortium have obtained behavioral phenotype data from 260 measures related to multiple behavioral assays across several domains: self-administration, response to, and withdrawal from cocaine, MDMA, morphine and alcohol; novelty seeking; behavioral despair and related neurological phenomena; pain sensitivity; stress sensitivity; anxiety; hyperactivity; and sleep/wake cycles. All traits have been measured in both sexes and the recently expanded panel of 69 additional BXD recombinant inbred strains (N=69). Sex differences and heritability estimates were obtained for each trait, and a comparison of early (N = 32) and recent BXD RI lines was performed. Primary data is publicly available for heritability, sex difference and genetic analyses using www.GeneNetwork.org. These analyses include QTL detection and genetic analysis of gene expression. Stored results from these analyses are available at http://ontologicaldiscovery.org for comparison to other genomic analysis results. Together with the results of related studies, these data form a public resource for integrative systems genetic analysis of neurobehavioral traits.

  5. Complex Reconstitution and Characterization by Combining Co-expression Techniques in Escherichia coli with High-Throughput.

    PubMed

    Vincentelli, Renaud; Romier, Christophe

    2016-01-01

    Single protein expression technologies have strongly benefited from the Structural Genomics initiatives that have introduced parallelization at the laboratory level. Specifically, the developments made in the wake of these initiatives have revitalized the use of Escherichia coli as major host for heterologous protein expression. In parallel to these improvements for single expression, technologies for complex reconstitution by co-expression in E. coli have been developed. Assessments of these co-expression technologies have highlighted the need for combinatorial experiments requiring automated protocols. These requirements can be fulfilled by adapting the high-throughput approaches that have been developed for single expression to the co-expression technologies. Yet, challenges are laying ahead that further need to be addressed and that are only starting to be taken into account in the case of single expression. These notably include the biophysical characterization of the samples at the small-scale level. Specifically, these approaches aim at discriminating the samples at an early stage of their production based on various biophysical criteria leading to cost-effectiveness and time-saving. This chapter addresses these various issues to provide the reader with a broad and comprehensive overview of complex reconstitution and characterization by co-expression in E. coli.

  6. A New Statistical Approach to Characterize Chemical-Elicited Behavioral Effects in High-Throughput Studies Using Zebrafish

    PubMed Central

    Zhang, Guozhu; Truong, Lisa; Tanguay, Robert L.

    2017-01-01

    Zebrafish have become an important alternative model for characterizing chemical bioactivity, partly due to the efficiency at which systematic, high-dimensional data can be generated. However, these new data present analytical challenges associated with scale and diversity. We developed a novel, robust statistical approach to characterize chemical-elicited effects in behavioral data from high-throughput screening (HTS) of all 1,060 Toxicity Forecaster (ToxCast™) chemicals across 5 concentrations at 120 hours post-fertilization (hpf). Taking advantage of the immense scale of data for a global view, we show that this new approach reduces bias introduced by extreme values yet allows for diverse response patterns that confound the application of traditional statistics. We have also shown that, as a summary measure of response for local tests of chemical-associated behavioral effects, it achieves a significant reduction in coefficient of variation compared to many traditional statistical modeling methods. This effective increase in signal-to-noise ratio augments statistical power and is observed across experimental periods (light/dark conditions) that display varied distributional response patterns. Finally, we integrated results with data from concomitant developmental endpoint measurements to show that appropriate statistical handling of HTS behavioral data can add important biological context that informs mechanistic hypotheses. PMID:28099482

  7. Distributed and Lumped Parameter Models for the Characterization of High Throughput Bioreactors.

    PubMed

    Iannetti, Laura; D'Urso, Giovanna; Conoscenti, Gioacchino; Cutrì, Elena; Tuan, Rocky S; Raimondi, Manuela T; Gottardi, Riccardo; Zunino, Paolo

    Next generation bioreactors are being developed to generate multiple human cell-based tissue analogs within the same fluidic system, to better recapitulate the complexity and interconnection of human physiology [1, 2]. The effective development of these devices requires a solid understanding of their interconnected fluidics, to predict the transport of nutrients and waste through the constructs and improve the design accordingly. In this work, we focus on a specific model of bioreactor, with multiple input/outputs, aimed at generating osteochondral constructs, i.e., a biphasic construct in which one side is cartilaginous in nature, while the other is osseous. We next develop a general computational approach to model the microfluidics of a multi-chamber, interconnected system that may be applied to human-on-chip devices. This objective requires overcoming several challenges at the level of computational modeling. The main one consists of addressing the multi-physics nature of the problem that combines free flow in channels with hindered flow in porous media. Fluid dynamics is also coupled with advection-diffusion-reaction equations that model the transport of biomolecules throughout the system and their interaction with living tissues and C constructs. Ultimately, we aim at providing a predictive approach useful for the general organ-on-chip community. To this end, we have developed a lumped parameter approach that allows us to analyze the behavior of multi-unit bioreactor systems with modest computational effort, provided that the behavior of a single unit can be fully characterized.

  8. Distributed and Lumped Parameter Models for the Characterization of High Throughput Bioreactors

    PubMed Central

    Conoscenti, Gioacchino; Cutrì, Elena; Tuan, Rocky S.; Raimondi, Manuela T.; Gottardi, Riccardo

    2016-01-01

    Next generation bioreactors are being developed to generate multiple human cell-based tissue analogs within the same fluidic system, to better recapitulate the complexity and interconnection of human physiology [1, 2]. The effective development of these devices requires a solid understanding of their interconnected fluidics, to predict the transport of nutrients and waste through the constructs and improve the design accordingly. In this work, we focus on a specific model of bioreactor, with multiple input/outputs, aimed at generating osteochondral constructs, i.e., a biphasic construct in which one side is cartilaginous in nature, while the other is osseous. We next develop a general computational approach to model the microfluidics of a multi-chamber, interconnected system that may be applied to human-on-chip devices. This objective requires overcoming several challenges at the level of computational modeling. The main one consists of addressing the multi-physics nature of the problem that combines free flow in channels with hindered flow in porous media. Fluid dynamics is also coupled with advection-diffusion-reaction equations that model the transport of biomolecules throughout the system and their interaction with living tissues and C constructs. Ultimately, we aim at providing a predictive approach useful for the general organ-on-chip community. To this end, we have developed a lumped parameter approach that allows us to analyze the behavior of multi-unit bioreactor systems with modest computational effort, provided that the behavior of a single unit can be fully characterized. PMID:27669413

  9. Scale-down characterization of post-centrifuge flocculation processes for high-throughput process development

    PubMed Central

    Espuny Garcia del Real, Georgina; Davies, Jim; Bracewell, Daniel G

    2014-01-01

    Abctract Flocculation unit operations are being revisited as a strategy to ease the burden posed on clarification and purification operations by the increasingly high cell density cultures used in the biopharmaceutical industry. The purpose of this study was to determine the key process parameters impacting flocculation scale-up and use this understanding to develop an automated ultra-scale down (USD) method for the rapid characterization of flocculation at the microliter scale. The conditions under which flocculation performance of a non-geometrically similar vessel three orders of magnitude larger can be mimicked by the USD platform are reported. Saccharomyces cerevisiae clarified homogenate was flocculated with poly(ethyleneimine) (PEI) to remove the residual solids remaining in the centrate. Flocculant addition time modulated flocculation performance depending on the predominant mixing time scale (i.e. macro-, meso- or micromixing). Particle growth and breakage was mimicked at the two flocculation scales by the average turbulent energy dissipation (εavg) and impeller tip speed (vtip) scale-up bases. The results obtained were used to develop an USD method. The USD method proposed uses constant εavg as the scale-up basis under a micromixing controlled regime. These conditions mimicked the STR flocculation performance within a ±5% error margin. Operation in the mesomixing regime led to particle size deviations between the flocculation scales of ≤50 %. These results, in addition to the microscopic observations made, demonstrate the USD system presented in this work can produce process-relevant flocculated material at the microliter scale under the correct operating conditions. PMID:24942244

  10. Comparative analysis and validation of the malachite green assay for the high throughput biochemical characterization of terpene synthases.

    PubMed

    Vardakou, Maria; Salmon, Melissa; Faraldos, Juan A; O'Maille, Paul E

    2014-01-01

    Terpenes are the largest group of natural products with important and diverse biological roles, while of tremendous economic value as fragrances, flavours and pharmaceutical agents. Class-I terpene synthases (TPSs), the dominant type of TPS enzymes, catalyze the conversion of prenyl diphosphates to often structurally diverse bioactive terpene hydrocarbons, and inorganic pyrophosphate (PPi). To measure their kinetic properties, current bio-analytical methods typically rely on the direct detection of hydrocarbon products by radioactivity measurements or gas chromatography-mass spectrometry (GC-MS). In this study we employed an established, rapid colorimetric assay, the pyrophosphate/malachite green assay (MG), as an alternative means for the biochemical characterization of class I TPSs activity.•We describe the adaptation of the MG assay for turnover and catalytic efficiency measurements of TPSs.•We validate the method by direct comparison with established assays. The agreement of k cat/K M among methods makes this adaptation optimal for rapid evaluation of TPSs.•We demonstrate the application of the MG assay for the high-throughput screening of TPS gene libraries.

  11. Development and Characterization of a High Throughput Screen to investigate the delayed Effects of Radiations Commonly Encountered in Space

    NASA Astrophysics Data System (ADS)

    Morgan, W. F.

    Astronauts based on the space station or on long-term space missions will be exposed to high Z radiations in the cosmic environment In order to evaluate the potentially deleterious effects of exposure to radiations commonly encountered in space we have developed and characterized a high throughput assay to detect mutation deletion events and or hyperrecombination in the progeny of exposed cells This assay is based on a plasmid vector containing a green fluorescence protein reporter construct We have shown that after stable transfection of the vector into human or hamster cells this construct can identify mutations specifically base changes and deletions as well as recombination events e g gene conversion or homologous recombination occurring as a result of exposure to ionizing radiation Our focus has been on those events occurring in the progeny of an irradiated cell that are potentially associated with radiation induced genomic instability rather than the more conventional assays that evaluate the direct immediate effects of radiation exposure Considerable time has been spent automating analysis of surviving colonies as a function of time after irradiation in order to determine when delayed instability is induced and the consequences of this delayed instability The assay is now automated permitting the evaluation of potentially rare events associated with low dose low dose rate radiations commonly encountered in space

  12. Effort versus Reward: Preparing Samples for Fungal Community Characterization in High-Throughput Sequencing Surveys of Soils

    PubMed Central

    Song, Zewei; Schlatter, Dan; Kennedy, Peter; Kinkel, Linda L.; Kistler, H. Corby; Nguyen, Nhu; Bates, Scott T.

    2015-01-01

    Next generation fungal amplicon sequencing is being used with increasing frequency to study fungal diversity in various ecosystems; however, the influence of sample preparation on the characterization of fungal community is poorly understood. We investigated the effects of four procedural modifications to library preparation for high-throughput sequencing (HTS). The following treatments were considered: 1) the amount of soil used in DNA extraction, 2) the inclusion of additional steps (freeze/thaw cycles, sonication, or hot water bath incubation) in the extraction procedure, 3) the amount of DNA template used in PCR, and 4) the effect of sample pooling, either physically or computationally. Soils from two different ecosystems in Minnesota, USA, one prairie and one forest site, were used to assess the generality of our results. The first three treatments did not significantly influence observed fungal OTU richness or community structure at either site. Physical pooling captured more OTU richness compared to individual samples, but total OTU richness at each site was highest when individual samples were computationally combined. We conclude that standard extraction kit protocols are well optimized for fungal HTS surveys, but because sample pooling can significantly influence OTU richness estimates, it is important to carefully consider the study aims when planning sampling procedures. PMID:25974078

  13. Identification and characterization of novel and conserved microRNAs in radish (Raphanus sativus L.) using high-throughput sequencing.

    PubMed

    Xu, Liang; Wang, Yan; Xu, Yuanyuan; Wang, Liangju; Zhai, Lulu; Zhu, Xianwen; Gong, Yiqin; Ye, Shan; Liu, Liwang

    2013-03-01

    MicroRNAs (miRNAs) are endogenous, non-coding, small RNAs that play significant regulatory roles in plant growth, development, and biotic and abiotic stress responses. To date, a great number of conserved and species-specific miRNAs have been identified in many important plant species such as Arabidopsis, rice and poplar. However, little is known about identification of miRNAs and their target genes in radish (Raphanus sativus L.). In the present study, a small RNA library from radish root was constructed and sequenced using the high-throughput Solexa sequencing. Through sequence alignment and secondary structure prediction, a total of 545 conserved miRNA families as well as 15 novel (with their miRNA* strand) and 64 potentially novel miRNAs were identified. Quantitative real-time PCR (qRT-PCR) analysis confirmed that both conserved and novel miRNAs were expressed in radish, and some of them were preferentially expressed in certain tissues. A total of 196 potential target genes were predicted for 42 novel radish miRNAs. Gene ontology (GO) analysis showed that most of the targets were involved in plant growth, development, metabolism and stress responses. This study represents a first large-scale identification and characterization of radish miRNAs and their potential target genes. These results could lead to the further identification of radish miRNAs and enhance our understanding of radish miRNA regulatory mechanisms in diverse biological and metabolic processes.

  14. High-throughput ultrasensitive characterization of chemical, structural and plasmonic properties of EBL-fabricated single silver nanoparticles

    NASA Astrophysics Data System (ADS)

    Huang, Tao; Cao, Wei; Elsayed-Ali, Hani E.; Xu, Xiao-Hong Nancy

    2012-01-01

    Electron beam lithography (EBL) has become a popular means to prepare a wide variety of nano-arrays for numerous studies and applications, including photonics and sensors. Their fabrications and characterizations are costly and time consuming, underscoring the importance of developing effective tools to rapidly study their physicochemical stabilities and properties over time. In this study, we characterized EBL-fabricated single silver nanoparticle (Ag NP) arrays over their 12-week exposure to ambient conditions using SEM/EDS, AFM and dark-field optical microscopy and spectroscopy (DFOMS). We found that chemical compositions, structural morphologies and plasmonic optical properties of single NPs altered drastically over the exposure. Single cuboid and triangular-prism Ag NPs degraded at rates of (0.74 +/- 0.02) and (0.66 +/- 0.02) per week, and their localized surface plasmon resonance (LSPR) spectra showed striking blue-shifts (171 +/- 25 and 203 +/- 35 nm) over the 12-week exposure, respectively. Plasmonic colors of single NPs changed distinctively from red to green over the 12-week exposure. The LSPR spectra of individual NPs in each array were acquired simultaneously and correlated specifically with their SEM and AFM images, demonstrating that DFOMS can serve as high-throughput, ultrasensitive and non-invasive means to characterize chemical, structural and optical properties of nano-arrays in situ in real time at single-NP resolution.Electron beam lithography (EBL) has become a popular means to prepare a wide variety of nano-arrays for numerous studies and applications, including photonics and sensors. Their fabrications and characterizations are costly and time consuming, underscoring the importance of developing effective tools to rapidly study their physicochemical stabilities and properties over time. In this study, we characterized EBL-fabricated single silver nanoparticle (Ag NP) arrays over their 12-week exposure to ambient conditions using SEM/EDS, AFM

  15. Automated High-Throughput Identification and Characterization of Clinically Important Bacteria and Fungi using Rapid Evaporative Ionization Mass Spectrometry.

    PubMed

    Bolt, Frances; Cameron, Simon J S; Karancsi, Tamas; Simon, Daniel; Schaffer, Richard; Rickards, Tony; Hardiman, Kate; Burke, Adam; Bodai, Zsolt; Perdones-Montero, Alvaro; Rebec, Monica; Balog, Julia; Takats, Zoltan

    2016-10-04

    Rapid evaporative ionization mass spectrometry (REIMS) has been shown to quickly and accurately speciate microorganisms based upon their species-specific lipid profile. Previous work by members of this group showed that the use of a hand-held bipolar probe allowed REIMS to analyze microbial cultures directly from culture plates without any prior preparation. However, this method of analysis would likely be unsuitable for a high-throughput clinical microbiology laboratory. Here, we report the creation of a customized platform that enables automated, high-throughput REIMS analysis that requires minimal user input and operation and is suitable for use in clinical microbiology laboratories. The ability of this high-throughput platform to speciate clinically important microorganisms was tested through the analysis of 375 different clinical isolates collected from distinct patient samples from 25 microbial species. After optimization of our data analysis approach, we achieved substantially similar results between the two REIMS approaches. For hand-held bipolar probe REIMS, a speciation accuracy of 96.3% was achieved, whereas for high-throughput REIMS, an accuracy of 93.9% was achieved. Thus, high-throughput REIMS offers an alternative mass spectrometry based method for the rapid and accurate identification of clinically important microorganisms in clinical laboratories without any preanalysis preparative steps.

  16. A high-throughput comparative characterization of laser-induced soft tissue damage using 3D digital microscopy.

    PubMed

    Das, Debobrato; Reed, Stephanie; Klokkevold, Perry R; Wu, Benjamin M

    2013-02-01

    3D digital microscopy was used to develop a rapid alternative approach to quantify the effects of specific laser parameters on soft tissue ablation and charring in vitro without the use of conventional tissue processing techniques. Two diode lasers operating at 810 and 980 nm wavelengths were used to ablate three tissue types (bovine liver, turkey breast, and bovine muscle) at varying laser power (0.3, 1.0, and 2.0 W) and velocities (1-50 mm/s). Spectrophotometric analyses were performed on each tissue to determine tissue-specific absorption coefficients and were considered in creating wavelength-dependent energy attenuation models to evaluate minimum heat of tissue ablations. 3D surface contour profiles characterizing tissue damage revealed that ablation depth and tissue charring increased with laser power and decreased with lateral velocity independent of wavelength and tissue type. While bovine liver ablation and charring were statistically higher at 810 than 980 nm (p < 0.05), turkey breast and bovine muscle ablated and charred more at 980 than 810 nm (p < 0.05). Spectrophotometric analysis revealed that bovine liver tissue had a greater tissue-specific absorption coefficient at 810 than 980 nm, while turkey breast and bovine muscle had a larger absorption coefficient at 980 nm (p < 0.05). This rapid 3D microscopic analysis of robot-driven laser ablation yielded highly reproducible data that supported well-defined trends related to laser-tissue interactions and enabled high throughput characterization of many laser-tissue permutations. Since 3D microscopy quantifies entire lesions without altering the tissue specimens, conventional and immunohistologic techniques can be used, if desired, to further interrogate specific sections of the digitized lesions.

  17. High-Throughput Phenotyping of Wheat and Barley Plants Grown in Single or Few Rows in Small Plots Using Active and Passive Spectral Proximal Sensing

    PubMed Central

    Barmeier, Gero; Schmidhalter, Urs

    2016-01-01

    In the early stages of plant breeding, breeders evaluate a large number of varieties. Due to limited availability of seeds and space, plot sizes may range from one to four rows. Spectral proximal sensors can be used in place of labour-intensive methods to estimate specific plant traits. The aim of this study was to test the performance of active and passive sensing to assess single and multiple rows in a breeding nursery. A field trial with single cultivars of winter barley and winter wheat with four plot designs (single-row, wide double-row, three rows, and four rows) was conducted. A GreenSeeker RT100 and a passive bi-directional spectrometer were used to assess biomass fresh and dry weight, as well as aboveground nitrogen content and uptake. Generally, spectral passive sensing and active sensing performed comparably in both crops. Spectral passive sensing was enhanced by the availability of optimized ratio vegetation indices, as well as by an optimized field of view and by reduced distance dependence. Further improvements of both sensors in detecting the performance of plants in single rows can likely be obtained by optimization of sensor positioning or orientation. The results suggest that even in early selection cycles, enhanced high-throughput phenotyping might be able to assess plant performance within plots comprising single or multiple rows. This method has significant potential for advanced breeding. PMID:27827958

  18. The high-throughput phenotyping of the viscoelastic behavior of whole mouse intervertebral discs using a novel method of dynamic mechanical testing.

    PubMed

    Liu, Jennifer W; Abraham, Adam C; Tang, Simon Y

    2015-07-16

    Intervertebral disc (IVD) degeneration is highly correlated with lower back pain, and thus understanding the mechanisms of IVD degeneration is critical for the treatment of this disease. Utilizing mouse models to probe the mechanisms of degeneration is especially attractive due to the ease of manipulating mouse models and the availability of transgenics. Yet characterizing the mechanical behavior of mice IVDs remain challenging due to their minute size (approximately 540 μm in height and 1080 μm(2) in cross sectional area). We have thus developed a simple method to dynamically characterize the mechanical properties of intact mouse IVDs. The IVDs were dissected with the endplates intact, and dynamically compressed in the axial direction at 1% and 5% peak strains at 1 Hz. Utilizing this novel approach, we examined the effects of in vitro ribosylation and trypsin digestion for 24 or 72 h on the viscoelastic behavior of the whole murine IVD. Trypsin treatment resulted in a decrease of proteoglycans and loss of disc height, while ribosylation had no effect on structure or proteoglycan composition. The 72 h ribosylation group exhibited a stiffening of the disc, and both treatments significantly reduced viscous behavior of the IVDs, with the effects being more pronounced at 5% strain. Here we demonstrate a novel high-throughput method to mechanically characterize murine IVDs and detect strain-dependent differences in the elastic and the viscous behavior of the treated IVDs due to ribose and trypsin treatments.

  19. Identification and characterization of microRNAs in Eucheuma denticulatum by high-throughput sequencing and bioinformatics analysis.

    PubMed

    Gao, Fan; Nan, Fangru; Feng, Jia; Lv, Junping; Liu, Qi; Xie, Shulian

    2016-01-01

    Eucheuma denticulatum, an economically and industrially important red alga, is a valuable marine resource. Although microRNAs (miRNAs) play an essential role in gene post-transcriptional regulation, no research has been conducted to identify and characterize miRNAs in E. denticulatum. In this study, we identified 134 miRNAs (133 conserved miRNAs and one novel miRNA) from 2,997,135 small-RNA reads by high-throughput sequencing combined with bioinformatics analysis. BLAST searching against miRBase uncovered 126 potential miRNA families. A conservation and diversity analysis of predicted miRNA families in different plant species was performed by comparative alignment and homology searching. A total of 4 and 13 randomly selected miRNAs were respectively validated by northern blotting and stem-loop reverse transcription PCR, thereby demonstrating the reliability of the miRNA sequencing data. Altogether, 871 potential target genes were predicted using psRobot and TargetFinder. Target genes classification and enrichment were conducted based on Gene Ontology analysis. The functions of target gene products and associated metabolic pathways were predicted by Kyoto Encyclopedia of Genes and Genomes pathway analysis. A Cytoscape network was constructed to explore the interrelationships of miRNAs, miRNA-target genes and target genes. A large number of miRNAs with diverse target genes will play important roles for further understanding some essential biological processes in E. denticulatum. The uncovered information can serve as an important reference for the protection and utilization of this unique red alga in the future.

  20. Characterization of bacterial communities associating with larval development of Yesso Scallop ( Patinopecten yessoensisis Jay, 1857) by high-throughput sequencing

    NASA Astrophysics Data System (ADS)

    Sun, Xueying; Liu, Jichen; Li, Ming; Zhao, Xuewei; Liang, Jun; Sun, Pihai; Ma, Yuexin

    2016-12-01

    Bacterial community presumably plays an essential role in inhibiting pathogen colonization and maintaining the health of scallop larvae, but limiting data are available for Yesso scallop ( Patinopecten yessoensisis Jay, 1857) larval development stages. The aim of this study was to characterize and compare the bacterial communities associating with Yesso scallop larval development at fertilized egg S1, trochophora S2, D-shaped larvae S3, umbo larvae S4, and juvenile scallop S5 stages by Illumina high-throughput sequencing. Genomic DNA was extracted from the larvae and their associating bactera, and a gene segment covering V3-V4 region of 16S rRNA gene was amplified and sequenced using an Illumina Miseq sequencer. Overall, 106760 qualified sequences with an average length of 449 bp were obtained. Sequences were compared with those retrieved from 16S rRNA gene databases, and 4 phyla, 7 classes, 15 orders, 21 families, 31 genera were identified. Proteobacteria was predominant phylum, accounting for more than 99%, at all 5 larval development stages. At genus level, Pseudomonas was dominant at stages S1 (80.60%), S2 (87.77%) and S5 (68.71%), followed by Photobacterium (17.06%) and Aeromonas (1.64%) at stage S1, Serratia (6.94%), Stenotrophomonas (3.08%) and Acinetobacter (1.2%) at stage S2, Shewanella (25.95%) and Pseudoalteromonas (4.57%) at stage S5. Moreover, genus Pseudoalteromonas became dominant at stages S3 (44.85%) and S4 (56.02%), followed by Photobacterium (29.82%), Pseudomonas (11.86%), Aliivibrio (8.60%) and Shewanella (3.39%) at stage S3, Pseudomonas (18.16%), Aliivibrio (14.29%), Shewanella (4.11%), Psychromonas (4.04%) and Psychrobacter (1.81%) at stage S4. From the results, we concluded that the bacterial community changed significantly at different development stages of Yesso Scallop larvae.

  1. Experimental design-based functional mining and characterization of high-throughput sequencing data in the sequence read archive.

    PubMed

    Nakazato, Takeru; Ohta, Tazro; Bono, Hidemasa

    2013-01-01

    High-throughput sequencing technology, also called next-generation sequencing (NGS), has the potential to revolutionize the whole process of genome sequencing, transcriptomics, and epigenetics. Sequencing data is captured in a public primary data archive, the Sequence Read Archive (SRA). As of January 2013, data from more than 14,000 projects have been submitted to SRA, which is double that of the previous year. Researchers can download raw sequence data from SRA website to perform further analyses and to compare with their own data. However, it is extremely difficult to search entries and download raw sequences of interests with SRA because the data structure is complicated, and experimental conditions along with raw sequences are partly described in natural language. Additionally, some sequences are of inconsistent quality because anyone can submit sequencing data to SRA with no quality check. Therefore, as a criterion of data quality, we focused on SRA entries that were cited in journal articles. We extracted SRA IDs and PubMed IDs (PMIDs) from SRA and full-text versions of journal articles and retrieved 2748 SRA ID-PMID pairs. We constructed a publication list referring to SRA entries. Since, one of the main themes of -omics analyses is clarification of disease mechanisms, we also characterized SRA entries by disease keywords, according to the Medical Subject Headings (MeSH) extracted from articles assigned to each SRA entry. We obtained 989 SRA ID-MeSH disease term pairs, and constructed a disease list referring to SRA data. We previously developed feature profiles of diseases in a system called "Gendoo". We generated hyperlinks between diseases extracted from SRA and the feature profiles of it. The developed project, publication and disease lists resulting from this study are available at our web service, called "DBCLS SRA" (http://sra.dbcls.jp/). This service will improve accessibility to high-quality data from SRA.

  2. Identification and characterization of microRNAs in Eucheuma denticulatum by high-throughput sequencing and bioinformatics analysis

    PubMed Central

    Gao, Fan; Nan, Fangru; Feng, Jia; Lv, Junping; Liu, Qi; Xie, Shulian

    2016-01-01

    ABSTRACT Eucheuma denticulatum, an economically and industrially important red alga, is a valuable marine resource. Although microRNAs (miRNAs) play an essential role in gene post-transcriptional regulation, no research has been conducted to identify and characterize miRNAs in E. denticulatum. In this study, we identified 134 miRNAs (133 conserved miRNAs and one novel miRNA) from 2,997,135 small-RNA reads by high-throughput sequencing combined with bioinformatics analysis. BLAST searching against miRBase uncovered 126 potential miRNA families. A conservation and diversity analysis of predicted miRNA families in different plant species was performed by comparative alignment and homology searching. A total of 4 and 13 randomly selected miRNAs were respectively validated by northern blotting and stem-loop reverse transcription PCR, thereby demonstrating the reliability of the miRNA sequencing data. Altogether, 871 potential target genes were predicted using psRobot and TargetFinder. Target genes classification and enrichment were conducted based on Gene Ontology analysis. The functions of target gene products and associated metabolic pathways were predicted by Kyoto Encyclopedia of Genes and Genomes pathway analysis. A Cytoscape network was constructed to explore the interrelationships of miRNAs, miRNA-target genes and target genes. A large number of miRNAs with diverse target genes will play important roles for further understanding some essential biological processes in E. denticulatum. The uncovered information can serve as an important reference for the protection and utilization of this unique red alga in the future. PMID:26717154

  3. Molecular characterization of a new soybean-infecting member of the genus Nepovirus identified by high-throughput sequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete nucleotide sequence of a new soybean-infecting member of the Nepovirus genus (provisionally named Soybean latent spherical virus [SLSV]) was identified by high-throughput sequencing of RNAs from soybean leaf samples from North Dakota, USA. The sequences of RNAs 1 (8,190 nt) and 2 (5,788...

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

    PubMed Central

    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.

    2016-01-01

    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. PMID:26818078

  5. Miniaturized Analytical Platforms From Nanoparticle Components: Studies in the Construction, Characterization, and High-Throughput Usage of These Novel Architectures

    SciTech Connect

    Pris, Andrew David

    2003-01-01

    exhibiting a variety of surface chemistries and attempts to deconvolute general adsorption rules for their assembly on various substrates. Chapter 2 extends the usage of self-assembly of polymeric nanoparticles through a layer-by-layer deposition concept and photolithography methodologies to create analytical platforms with a vertical height controlled within the nanometer regime. This platform is then furthered in Chapter 3 by employing this integrated concept as a bio-recognition platform, with the extension of the method to a high-throughput screening system explored. Chapter 4 exploits two different types of nanoparticles, silica and gold, as multiplexed, self-assembled immunoassay sensors. This final research chapter is followed by a general summation and future prospectus section that concludes the dissertation.

  6. High-throughput phenotyping (HTP) identifies seedling root traits linked to variation in seed yield and nutrient capture in field-grown oilseed rape (Brassica napus L.)

    PubMed Central

    Thomas, C. L.; Graham, N. S.; Hayden, R.; Meacham, M. C.; Neugebauer, K.; Nightingale, M.; Dupuy, L. X.; Hammond, J. P.; White, P. J.; Broadley, M. R.

    2016-01-01

    Background and Aims Root traits can be selected for crop improvement. Techniques such as soil excavations can be used to screen root traits in the field, but are limited to genotypes that are well-adapted to field conditions. The aim of this study was to compare a low-cost, high-throughput root phenotyping (HTP) technique in a controlled environment with field performance, using oilseed rape (OSR; Brassica napus) varieties. Methods Primary root length (PRL), lateral root length and lateral root density (LRD) were measured on 14-d-old seedlings of elite OSR varieties (n = 32) using a ‘pouch and wick’ HTP system (∼40 replicates). Six field experiments were conducted using the same varieties at two UK sites each year for 3 years. Plants were excavated at the 6- to 8-leaf stage for general vigour assessments of roots and shoots in all six experiments, and final seed yield was determined. Leaves were sampled for mineral composition from one of the field experiments. Key Results Seedling PRL in the HTP system correlated with seed yield in four out of six (r = 0·50, 0·50, 0·33, 0·49; P < 0·05) and with emergence in three out of five (r = 0·59, 0·22, 0·49; P < 0·05) field experiments. Seedling LRD correlated positively with leaf concentrations of some minerals, e.g. calcium (r = 0·46; P < 0·01) and zinc (r = 0·58; P < 0·001), but did not correlate with emergence, general early vigour or yield in the field. Conclusions Associations between PRL and field performance are generally related to early vigour. These root traits might therefore be of limited additional selection value, given that vigour can be measured easily on shoots/canopies. In contrast, LRD cannot be assessed easily in the field and, if LRD can improve nutrient uptake, then it may be possible to use HTP systems to screen this trait in both elite and more genetically diverse, non-field-adapted OSR. PMID:27052342

  7. High-throughput manufacturing of size-tuned liposomes by a new microfluidics method using enhanced statistical tools for characterization.

    PubMed

    Kastner, Elisabeth; Kaur, Randip; Lowry, Deborah; Moghaddam, Behfar; Wilkinson, Alexander; Perrie, Yvonne

    2014-12-30

    Microfluidics has recently emerged as a new method of manufacturing liposomes, which allows for reproducible mixing in miliseconds on the nanoliter scale. Here we investigate microfluidics-based manufacturing of liposomes. The aim of these studies was to assess the parameters in a microfluidic process by varying the total flow rate (TFR) and the flow rate ratio (FRR) of the solvent and aqueous phases. Design of experiment and multivariate data analysis were used for increased process understanding and development of predictive and correlative models. High FRR lead to the bottom-up synthesis of liposomes, with a strong correlation with vesicle size, demonstrating the ability to in-process control liposomes size; the resulting liposome size correlated with the FRR in the microfluidics process, with liposomes of 50 nm being reproducibly manufactured. Furthermore, we demonstrate the potential of a high throughput manufacturing of liposomes using microfluidics with a four-fold increase in the volumetric flow rate, maintaining liposome characteristics. The efficacy of these liposomes was demonstrated in transfection studies and was modelled using predictive modeling. Mathematical modelling identified FRR as the key variable in the microfluidic process, with the highest impact on liposome size, polydispersity and transfection efficiency. This study demonstrates microfluidics as a robust and high-throughput method for the scalable and highly reproducible manufacture of size-controlled liposomes. Furthermore, the application of statistically based process control increases understanding and allows for the generation of a design-space for controlled particle characteristics.

  8. HighSSR: high-throughput SSR characterization and locus development from next-gen sequencing data

    PubMed Central

    Churbanov, Alexander; Ryan, Rachael; Hasan, Nabeeh; Bailey, Donovan; Chen, Haofeng; Milligan, Brook; Houde, Peter

    2012-01-01

    Motivation: Microsatellites are among the most useful genetic markers in population biology. High-throughput sequencing of microsatellite-enriched libraries dramatically expedites the traditional process of screening recombinant libraries for microsatellite markers. However, sorting through millions of reads to distill high-quality polymorphic markers requires special algorithms tailored to tolerate sequencing errors in locus reconstruction, distinguish paralogous loci, rarify raw reads originating from the same amplicon and sort out various artificial fragments resulting from recombination or concatenation of auxiliary adapters. Existing programs warrant improvement. Results: We describe a microsatellite prediction framework named HighSSR for microsatellite genotyping based on high-throughput sequencing. We demonstrate the utility of HighSSR in comparison to Roche gsAssembler on two Roche 454 GS FLX runs. The majority of the HighSSR-assembled loci were reliably mapped against model organism reference genomes. HighSSR demultiplexes pooled libraries, assesses locus polymorphism and implements Primer3 for the design of PCR primers flanking polymorphic microsatellite loci. As sequencing costs drop and permit the analysis of all project samples on next-generation platforms, this framework can also be used for direct simple sequence repeats genotyping. Availability: http://code.google.com/p/highssr/ Contact: alexander@big.ac.cn Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:22954626

  9. Toward development of an autonomous network of bacteria-based delivery systems (BacteriaBots): spatiotemporally high-throughput characterization of bacterial quorum-sensing response.

    PubMed

    Sahari, Ali; Traore, Mahama A; Stevens, Ann M; Scharf, Birgit E; Behkam, Bahareh

    2014-12-02

    Characterization of bacterial innate and engineered cooperative behavior, regulated through chemical signaling in a process known as quorum sensing, is critical to development of a myriad of bacteria-enabled systems including biohybrid drug delivery systems and biohybrid mobile sensor networks. Here, we demonstrate, for the first time, that microfluidic diffusive mixers can be used for spatiotemporally high-throughput characterization of bacterial quorum-sensing response. Using this batch characterization method, the quorum-sensing response in Escherichia coli MG1655, transformed with a truncated lux operon from Vibrio fischeri, in the presence of 1-100 nM exogenous acyl-homoserine lactone molecules has been quantified. This method provides a rapid and facile tool for high-throughput characterization of the quorum-sensing response of genetically modified bacteria in the presence of a wide concentration range of signaling molecules with a precision of ±0.5 nM. Furthermore, the quorum-sensing response of BacteriaBots has been characterized to determine if the results obtained from a large bacterial population can serve as a robust predictive tool for the small bacterial population attached to each BacteriaBot.

  10. Formulation, High Throughput In Vitro Screening and In Vivo Functional Characterization of Nanoemulsion-Based Intranasal Vaccine Adjuvants

    PubMed Central

    Wong, Pamela T.; Leroueil, Pascale R.; Smith, Douglas M.; Ciotti, Susan; Bielinska, Anna U.; Janczak, Katarzyna W.; Mullen, Catherine H.; Groom, Jeffrey V.; Taylor, Erin M.; Passmore, Crystal; Makidon, Paul E.; O’Konek, Jessica J.; Myc, Andrzej; Hamouda, Tarek; Baker, James R.

    2015-01-01

    Vaccine adjuvants have been reported to induce both mucosal and systemic immunity when applied to mucosal surfaces and this dual response appears important for protection against certain pathogens. Despite the potential advantages, however, no mucosal adjuvants are currently approved for human use. Evaluating compounds as mucosal adjuvants is a slow and costly process due to the need for lengthy animal immunogenicity studies. We have constructed a library of 112 intranasal adjuvant candidate formulations consisting of oil-in-water nanoemulsions that contain various cationic and nonionic surfactants. To facilitate adjuvant development we first evaluated this library in a series of high-throughput, in vitro assays for activities associated with innate and adaptive immune activation in vivo. These in vitro assays screened for the ability of the adjuvant to bind to mucin, induce cytotoxicity, facilitate antigen uptake in epithelial and dendritic cells, and activate cellular pathways. We then sought to determine how these parameters related to adjuvant activity in vivo. While the in vitro assays alone were not enough to predict the in vivo adjuvant activity completely, several interesting relationships were found with immune responses in mice. Furthermore, by varying the physicochemical properties of the surfactant components (charge, surfactant polar head size and hydrophobicity) and the surfactant blend ratio of the formulations, the strength and type of the immune response generated (TH1, TH2, TH17) could be modulated. These findings suggest the possibility of using high-throughput screens to aid in the design of custom adjuvants with unique immunological profiles to match specific mucosal vaccine applications. PMID:25962136

  11. Rhizoslides: paper-based growth system for non-destructive, high throughput phenotyping of root development by means of image analysis

    PubMed Central

    2014-01-01

    Rhizo’ offered the most rapid and precise evaluation of root lengths in diameter classes, but had weaknesses with respect to image segmentation and analysis of root system architecture. Conclusion A new technique has been established for non-destructive root growth studies and quantification of architectural traits beyond seedlings stages. However, automation of the scanning process and appropriate software remains the bottleneck for high throughput analysis. PMID:25093035

  12. A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells

    PubMed Central

    Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R.; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

    2016-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders. PMID:27739443

  13. A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells.

    PubMed

    Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

    2016-10-14

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders.

  14. Identification and Characterization of miRNAs in Chondrus crispus by High-Throughput Sequencing and Bioinformatics Analysis

    PubMed Central

    Gao, Fan; Nan, FangRu; Song, Wei; Feng, Jia; Lv, JunPing; Xie, ShuLian

    2016-01-01

    Chondrus crispus, an economically and medicinally important red alga, is a medicinally active substance and important for anti-tumor research. In this study, 117 C. crispus miRNAs (108 conserved and 9 novel) were identified from 2,416,181 small-RNA reads using high-throughput sequencing and bioinformatics methods. According to the BLAST search against the miRBase database, these miRNAs belonged to 110 miRNA families. Sequence alignment combined with homology searching revealed both the conservation and diversity of predicted potential miRNA families in different plant species. Four and 19 randomly selected miRNAs were validated by northern blotting and stem-loop quantitative real-time reverse transcription polymerase chain reaction detection, respectively. The validation rates (75% and 94.7%) demonstrated that most of the identified miRNAs could be credible. A total of 160 potential target genes were predicted and functionally annotated by Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis. We also analyzed the interrelationship of miRNAs, miRNA-target genes and target genes in C. crispus by constructing a Cytoscape network. The 117 miRNAs identified in our study should supply large quantities of information that will be important for red algae small RNA research. PMID:27193824

  15. Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing.

    PubMed

    Miyazaki, Nobuo; Kiyose, Norihiko; Akazawa, Yoko; Takashima, Mizuki; Hagihara, Yosihisa; Inoue, Naokazu; Matsuda, Tomonari; Ogawa, Ryu; Inoue, Seiya; Ito, Yuji

    2015-09-01

    The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning.

  16. Application of high-throughput mini-bioreactor system for systematic scale-down modeling, process characterization, and control strategy development.

    PubMed

    Janakiraman, Vijay; Kwiatkowski, Chris; Kshirsagar, Rashmi; Ryll, Thomas; Huang, Yao-Ming

    2015-01-01

    High-throughput systems and processes have typically been targeted for process development and optimization in the bioprocessing industry. For process characterization, bench scale bioreactors have been the system of choice. Due to the need for performing different process conditions for multiple process parameters, the process characterization studies typically span several months and are considered time and resource intensive. In this study, we have shown the application of a high-throughput mini-bioreactor system viz. the Advanced Microscale Bioreactor (ambr15(TM) ), to perform process characterization in less than a month and develop an input control strategy. As a pre-requisite to process characterization, a scale-down model was first developed in the ambr system (15 mL) using statistical multivariate analysis techniques that showed comparability with both manufacturing scale (15,000 L) and bench scale (5 L). Volumetric sparge rates were matched between ambr and manufacturing scale, and the ambr process matched the pCO2 profiles as well as several other process and product quality parameters. The scale-down model was used to perform the process characterization DoE study and product quality results were generated. Upon comparison with DoE data from the bench scale bioreactors, similar effects of process parameters on process yield and product quality were identified between the two systems. We used the ambr data for setting action limits for the critical controlled parameters (CCPs), which were comparable to those from bench scale bioreactor data. In other words, the current work shows that the ambr15(TM) system is capable of replacing the bench scale bioreactor system for routine process development and process characterization.

  17. Utilization of a high-throughput shoot imaging system to examine the dynamic phenotypic responses of a C4 cereal crop plant to nitrogen and water deficiency over time.

    PubMed

    Neilson, E H; Edwards, A M; Blomstedt, C K; Berger, B; Møller, B Lindberg; Gleadow, R M

    2015-04-01

    The use of high-throughput phenotyping systems and non-destructive imaging is widely regarded as a key technology allowing scientists and breeders to develop crops with the ability to perform well under diverse environmental conditions. However, many of these phenotyping studies have been optimized using the model plant Arabidopsis thaliana. In this study, The Plant Accelerator(®) at The University of Adelaide, Australia, was used to investigate the growth and phenotypic response of the important cereal crop, Sorghum bicolor L. Moench and related hybrids to water-limited conditions and different levels of fertilizer. Imaging in different spectral ranges was used to monitor plant composition, chlorophyll, and moisture content. Phenotypic image analysis accurately measured plant biomass. The data set obtained enabled the responses of the different sorghum varieties to the experimental treatments to be differentiated and modelled. Plant architectural instead of architecture elements were determined using imaging and found to correlate with an improved tolerance to stress, for example diurnal leaf curling and leaf area index. Analysis of colour images revealed that leaf 'greenness' correlated with foliar nitrogen and chlorophyll, while near infrared reflectance (NIR) analysis was a good predictor of water content and leaf thickness, and correlated with plant moisture content. It is shown that imaging sorghum using a high-throughput system can accurately identify and differentiate between growth and specific phenotypic traits. R scripts for robust, parsimonious models are provided to allow other users of phenomic imaging systems to extract useful data readily, and thus relieve a bottleneck in phenotypic screening of multiple genotypes of key crop plants.

  18. A novel high-throughput multi-parameter flow cytometry based method for monitoring and rapid characterization of microbiome dynamics in anaerobic systems.

    PubMed

    Dhoble, Abhishek S; Bekal, Sadia; Dolatowski, William; Yanz, Connor; Lambert, Kris N; Bhalerao, Kaustubh D

    2016-11-01

    A novel multidimensional flow cytometry based method has been demonstrated to monitor and rapidly characterize the dynamics of the complex anaerobic microbiome associated with perturbations in external environmental factors. While community fingerprinting provides an estimate of the meta genomic structure, flow cytometry provides a fingerprint of the community morphology including its autofluorescence spectrum in a high-throughput manner. Using anaerobic microbial consortia perturbed with the controlled addition of various carbon sources, it is possible to quantitatively discriminate between divergent microbiome analogous to community fingerprinting techniques using automated ribosomal intergenic spacer analysis (ARISA). The utility of flow cytometry based method has also been demonstrated in a fully functional industry scale anaerobic digester to distinguish between microbiome composition caused by varying hydraulic retention time (HRT). This approach exploits the rich multidimensional information from flow cytometry for rapid characterization of the dynamics of microbial communities.

  19. High-throughput proteomics

    NASA Astrophysics Data System (ADS)

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

    2001-04-01

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

  20. High throughput optical scanner

    SciTech Connect

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

    2001-01-01

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

  1. High-throughput sequencing of 16S rDNA amplicons characterizes bacterial composition in cerebrospinal fluid samples from patients with purulent meningitis.

    PubMed

    Liu, Aicui; Wang, Chao; Liang, Zhijuan; Zhou, Zhi-Wei; Wang, Lin; Ma, Qiaoli; Wang, Guowei; Zhou, Shu-Feng; Wang, Zhenhai

    2015-01-01

    Purulent meningitis (PM) is a severe infectious disease that is associated with high rates of morbidity and mortality. It has been recognized that bacterial infection is a major contributing factor to the pathogenesis of PM. However, there is a lack of information on the bacterial composition in PM, due to the low positive rate of cerebrospinal fluid bacterial culture. Herein, we aimed to discriminate and identify the main pathogens and bacterial composition in cerebrospinal fluid sample from PM patients using high-throughput sequencing approach. The cerebrospinal fluid samples were collected from 26 PM patients, and were determined as culture-negative samples. The polymerase chain reaction products of the hypervariable regions of 16S rDNA gene in these 26 samples of PM were sequenced using the 454 GS FLX system. The results showed that there were 71,440 pyrosequencing reads, of which, the predominant phyla were Proteobacteria and Firmicutes; and the predominant genera were Streptococcus, Acinetobacter, Pseudomonas, and Neisseria. The bacterial species in the cerebrospinal fluid were complex, with 61.5% of the samples presenting with mixed pathogens. A significant number of bacteria belonging to a known pathogenic potential was observed. The number of operational taxonomic units for individual samples ranged from six to 75 and there was a comparable difference in the species diversity that was calculated through alpha and beta diversity analysis. Collectively, the data show that high-throughput sequencing approach facilitates the characterization of the pathogens in cerebrospinal fluid and determine the abundance and the composition of bacteria in the cerebrospinal fluid samples of the PM patients, which may provide a better understanding of pathogens in PM and assist clinicians to make rational and effective therapeutic decisions.

  2. High-throughput sequencing of 16S rDNA amplicons characterizes bacterial composition in bronchoalveolar lavage fluid in patients with ventilator-associated pneumonia.

    PubMed

    Yang, Xiao-Jun; Wang, Yan-Bo; Zhou, Zhi-Wei; Wang, Guo-Wei; Wang, Xiao-Hong; Liu, Qing-Fu; Zhou, Shu-Feng; Wang, Zhen-Hai

    2015-01-01

    Ventilator-associated pneumonia (VAP) is a life-threatening disease that is associated with high rates of morbidity and likely mortality, placing a heavy burden on an individual and society. Currently available diagnostic and therapeutic approaches for VAP treatment are limited, and the prognosis of VAP is poor. The present study aimed to reveal and discriminate the identification of the full spectrum of the pathogens in patients with VAP using high-throughput sequencing approach and analyze the species richness and complexity via alpha and beta diversity analysis. The bronchoalveolar lavage fluid samples were collected from 27 patients with VAP in intensive care unit. The polymerase chain reaction products of the hypervariable regions of 16S rDNA gene in these 27 samples of VAP were sequenced using the 454 GS FLX system. A total of 103,856 pyrosequencing reads and 638 operational taxonomic units were obtained from these 27 samples. There were four dominant phyla, including Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. There were 90 different genera, of which 12 genera occurred in over ten different samples. The top five dominant genera were Streptococcus, Acinetobacter, Limnohabitans, Neisseria, and Corynebacterium, and the most widely distributed genera were Streptococcus, Limnohabitans, and Acinetobacter in these 27 samples. Of note, the mixed profile of causative pathogens was observed. Taken together, the results show that the high-throughput sequencing approach facilitates the characterization of the pathogens in bronchoalveolar lavage fluid samples and the determination of the profile for bacteria in the bronchoalveolar lavage fluid samples of the patients with VAP. This study can provide useful information of pathogens in VAP and assist clinicians to make rational and effective therapeutic decisions.

  3. Effort versus reward: preparing samples for fungal community characterization in high-throughput sequencing surveys of soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Next generation fungal amplicon sequencing is being used with increasing frequency to study fungal diversity in various ecosystems; however, the influence of sample preparation on the characterization of fungal community is poorly understood. We investigated the effects of four procedural modificati...

  4. Characterization of a Wheat Breeders' Array suitable for high-throughput SNP genotyping of global accessions of hexaploid bread wheat (Triticum aestivum).

    PubMed

    Allen, Alexandra M; Winfield, Mark O; Burridge, Amanda J; Downie, Rowena C; Benbow, Harriet R; Barker, Gary L A; Wilkinson, Paul A; Coghill, Jane; Waterfall, Christy; Davassi, Alessandro; Scopes, Geoff; Pirani, Ali; Webster, Teresa; Brew, Fiona; Bloor, Claire; Griffiths, Simon; Bentley, Alison R; Alda, Mark; Jack, Peter; Phillips, Andrew L; Edwards, Keith J

    2017-03-01

    Targeted selection and inbreeding have resulted in a lack of genetic diversity in elite hexaploid bread wheat accessions. Reduced diversity can be a limiting factor in the breeding of high yielding varieties and crucially can mean reduced resilience in the face of changing climate and resource pressures. Recent technological advances have enabled the development of molecular markers for use in the assessment and utilization of genetic diversity in hexaploid wheat. Starting with a large collection of 819 571 previously characterized wheat markers, here we describe the identification of 35 143 single nucleotide polymorphism-based markers, which are highly suited to the genotyping of elite hexaploid wheat accessions. To assess their suitability, the markers have been validated using a commercial high-density Affymetrix Axiom(®) genotyping array (the Wheat Breeders' Array), in a high-throughput 384 microplate configuration, to characterize a diverse global collection of wheat accessions including landraces and elite lines derived from commercial breeding communities. We demonstrate that the Wheat Breeders' Array is also suitable for generating high-density genetic maps of previously uncharacterized populations and for characterizing novel genetic diversity produced by mutagenesis. To facilitate the use of the array by the wheat community, the markers, the associated sequence and the genotype information have been made available through the interactive web site 'CerealsDB'.

  5. NIR and Py-mbms coupled with multivariate data analysis as a high-throughput biomass characterization technique: a review

    PubMed Central

    Xiao, Li; Wei, Hui; Himmel, Michael E.; Jameel, Hasan; Kelley, Stephen S.

    2014-01-01

    Optimizing the use of lignocellulosic biomass as the feedstock for renewable energy production is currently being developed globally. Biomass is a complex mixture of cellulose, hemicelluloses, lignins, extractives, and proteins; as well as inorganic salts. Cell wall compositional analysis for biomass characterization is laborious and time consuming. In order to characterize biomass fast and efficiently, several high through-put technologies have been successfully developed. Among them, near infrared spectroscopy (NIR) and pyrolysis-molecular beam mass spectrometry (Py-mbms) are complementary tools and capable of evaluating a large number of raw or modified biomass in a short period of time. NIR shows vibrations associated with specific chemical structures whereas Py-mbms depicts the full range of fragments from the decomposition of biomass. Both NIR vibrations and Py-mbms peaks are assigned to possible chemical functional groups and molecular structures. They provide complementary information of chemical insight of biomaterials. However, it is challenging to interpret the informative results because of the large amount of overlapping bands or decomposition fragments contained in the spectra. In order to improve the efficiency of data analysis, multivariate analysis tools have been adapted to define the significant correlations among data variables, so that the large number of bands/peaks could be replaced by a small number of reconstructed variables representing original variation. Reconstructed data variables are used for sample comparison (principal component analysis) and for building regression models (partial least square regression) between biomass chemical structures and properties of interests. In this review, the important biomass chemical structures measured by NIR and Py-mbms are summarized. The advantages and disadvantages of conventional data analysis methods and multivariate data analysis methods are introduced, compared and evaluated. This review

  6. NIR and Py-mbms coupled with multivariate data analysis as a high-throughput biomass characterization technique: a review.

    PubMed

    Xiao, Li; Wei, Hui; Himmel, Michael E; Jameel, Hasan; Kelley, Stephen S

    2014-01-01

    Optimizing the use of lignocellulosic biomass as the feedstock for renewable energy production is currently being developed globally. Biomass is a complex mixture of cellulose, hemicelluloses, lignins, extractives, and proteins; as well as inorganic salts. Cell wall compositional analysis for biomass characterization is laborious and time consuming. In order to characterize biomass fast and efficiently, several high through-put technologies have been successfully developed. Among them, near infrared spectroscopy (NIR) and pyrolysis-molecular beam mass spectrometry (Py-mbms) are complementary tools and capable of evaluating a large number of raw or modified biomass in a short period of time. NIR shows vibrations associated with specific chemical structures whereas Py-mbms depicts the full range of fragments from the decomposition of biomass. Both NIR vibrations and Py-mbms peaks are assigned to possible chemical functional groups and molecular structures. They provide complementary information of chemical insight of biomaterials. However, it is challenging to interpret the informative results because of the large amount of overlapping bands or decomposition fragments contained in the spectra. In order to improve the efficiency of data analysis, multivariate analysis tools have been adapted to define the significant correlations among data variables, so that the large number of bands/peaks could be replaced by a small number of reconstructed variables representing original variation. Reconstructed data variables are used for sample comparison (principal component analysis) and for building regression models (partial least square regression) between biomass chemical structures and properties of interests. In this review, the important biomass chemical structures measured by NIR and Py-mbms are summarized. The advantages and disadvantages of conventional data analysis methods and multivariate data analysis methods are introduced, compared and evaluated. This review

  7. Bulk combinatorial synthesis and high throughput characterization for rapid assessment of magnetic materials: Application of laser engineered net shaping (LENS)

    DOE PAGES

    Geng, J.; Nlebedim, I. C.; Besser, M. F.; ...

    2016-04-15

    A bulk combinatorial approach for synthesizing alloy libraries using laser engineered net shaping (LENS; i.e., 3D printing) was utilized to rapidly assess material systems for magnetic applications. The LENS system feeds powders in different ratios into a melt pool created by a laser to synthesize samples with bulk (millimeters) dimensions. By analyzing these libraries with autosampler differential scanning calorimeter/thermal gravimetric analysis and vibrating sample magnetometry, we are able to rapidly characterize the thermodynamic and magnetic properties of the libraries. Furthermore, the Fe-Co binary alloy was used as a model system and the results were compared with data in the literature.

  8. Bulk Combinatorial Synthesis and High Throughput Characterization for Rapid Assessment of Magnetic Materials: Application of Laser Engineered Net Shaping (LENS™)

    NASA Astrophysics Data System (ADS)

    Geng, J.; Nlebedim, I. C.; Besser, M. F.; Simsek, E.; Ott, R. T.

    2016-07-01

    A bulk combinatorial approach for synthesizing alloy libraries using laser engineered net shaping (LENS™; i.e., 3D printing) was utilized to rapidly assess material systems for magnetic applications. The LENS™ system feeds powders in different ratios into a melt pool created by a laser to synthesize samples with bulk (millimeters) dimensions. By analyzing these libraries with autosampler differential scanning calorimeter/thermal gravimetric analysis and vibrating sample magnetometry, we are able to rapidly characterize the thermodynamic and magnetic properties of the libraries. The Fe-Co binary alloy was used as a model system and the results were compared with data in the literature.

  9. High-throughput isolation and characterization of untagged membrane protein complexes: outer membrane complexes of Desulfovibrio vulgaris.

    PubMed

    Walian, Peter J; Allen, Simon; Shatsky, Maxim; Zeng, Lucy; Szakal, Evelin D; Liu, Haichuan; Hall, Steven C; Fisher, Susan J; Lam, Bonita R; Singer, Mary E; Geller, Jil T; Brenner, Steven E; Chandonia, John-Marc; Hazen, Terry C; Witkowska, H Ewa; Biggin, Mark D; Jap, Bing K

    2012-12-07

    Cell membranes represent the "front line" of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a "tagless" process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein-protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms.

  10. High-Throughput Cancer Cell Sphere Formation for Characterizing the Efficacy of Photo Dynamic Therapy in 3D Cell Cultures

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Lou, Xia; Zhang, Zhixiong; Ingram, Patrick; Yoon, Euisik

    2015-07-01

    Photodynamic therapy (PDT), wherein light sensitive non-toxic agents are locally and selectively activated using light, has emerged as an appealing alternative to traditional cancer chemotherapy. Yet to date, PDT efficacy has been mostly characterized using 2D cultures. Compared to 2D cultures, 3D sphere culture generates unique spatial distributions of nutrients and oxygen for the cells that better mimics the in-vivo conditions. Using a novel polyHEMA (non-adherent polymer) fabrication process, we developed a microfluidic sphere formation platform that can (1) generate 1,024 uniform (size variation <10%) cancer spheres within a 2 cm by 2 cm core area, (2) culture spheres for more than 2 weeks, and (3) allow the retrieval of spheres. Using the presented platform, we have successfully characterized the different responses in 2D and 3D cell culture to PDT. Furthermore, we investigated the treatment resistance effect in cancer cells induced by tumor associated fibroblasts (CAF). Although the CAFs can enhance the resistance to traditional chemotherapy agents, no significant difference in PDT was observed. The preliminary results suggest that the PDT can be an attractive alternative cancer therapy, which is less affected by the therapeutic resistance induced by cancer associated cells.

  11. High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris

    PubMed Central

    2012-01-01

    Cell membranes represent the “front line” of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a “tagless” process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein–protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms. PMID:23098413

  12. High-Throughput Cancer Cell Sphere Formation for Characterizing the Efficacy of Photo Dynamic Therapy in 3D Cell Cultures

    PubMed Central

    Chen, Yu-Chih; Lou, Xia; Zhang, Zhixiong; Ingram, Patrick; Yoon, Euisik

    2015-01-01

    Photodynamic therapy (PDT), wherein light sensitive non-toxic agents are locally and selectively activated using light, has emerged as an appealing alternative to traditional cancer chemotherapy. Yet to date, PDT efficacy has been mostly characterized using 2D cultures. Compared to 2D cultures, 3D sphere culture generates unique spatial distributions of nutrients and oxygen for the cells that better mimics the in-vivo conditions. Using a novel polyHEMA (non-adherent polymer) fabrication process, we developed a microfluidic sphere formation platform that can (1) generate 1,024 uniform (size variation <10%) cancer spheres within a 2 cm by 2 cm core area, (2) culture spheres for more than 2 weeks, and (3) allow the retrieval of spheres. Using the presented platform, we have successfully characterized the different responses in 2D and 3D cell culture to PDT. Furthermore, we investigated the treatment resistance effect in cancer cells induced by tumor associated fibroblasts (CAF). Although the CAFs can enhance the resistance to traditional chemotherapy agents, no significant difference in PDT was observed. The preliminary results suggest that the PDT can be an attractive alternative cancer therapy, which is less affected by the therapeutic resistance induced by cancer associated cells. PMID:26153550

  13. Unbiased high-throughput characterization of mussel transcriptomic responses to sublethal concentrations of the biotoxin okadaic acid

    PubMed Central

    Suarez-Ulloa, Victoria; Fernandez-Tajes, Juan; Aguiar-Pulido, Vanessa; Prego-Faraldo, M. Veronica; Florez-Barros, Fernanda; Sexto-Iglesias, Alexia; Mendez, Josefina

    2015-01-01

    Background. Harmful Algal Blooms (HABs) responsible for Diarrhetic Shellfish Poisoning (DSP) represent a major threat for human consumers of shellfish. The biotoxin Okadaic Acid (OA), a well-known phosphatase inhibitor and tumor promoter, is the primary cause of acute DSP intoxications. Although several studies have described the molecular effects of high OA concentrations on sentinel organisms (e.g., bivalve molluscs), the effect of prolonged exposures to low (sublethal) OA concentrations is still unknown. In order to fill this gap, this work combines Next-Generation sequencing and custom-made microarray technologies to develop an unbiased characterization of the transcriptomic response of mussels during early stages of a DSP bloom. Methods. Mussel specimens were exposed to a HAB episode simulating an early stage DSP bloom (200 cells/L of the dinoflagellate Prorocentrum lima for 24 h). The unbiased characterization of the transcriptomic responses triggered by OA was carried out using two complementary methods of cDNA library preparation: normalized and Suppression Subtractive Hybridization (SSH). Libraries were sequenced and read datasets were mapped to Gene Ontology and KEGG databases. A custom-made oligonucleotide microarray was developed based on these data, completing the expression analysis of digestive gland and gill tissues. Results. Our findings show that exposure to sublethal concentrations of OA is enough to induce gene expression modifications in the mussel Mytilus. Transcriptomic analyses revealed an increase in proteasomal activity, molecular transport, cell cycle regulation, energy production and immune activity in mussels. Oppositely, a number of transcripts hypothesized to be responsive to OA (notably the Serine/Threonine phosphatases PP1 and PP2A) failed to show substantial modifications. Both digestive gland and gill tissues responded similarly to OA, although expression modifications were more dramatic in the former, supporting the choice of

  14. Characterization of transcriptional networks in blood stem and progenitor cells using high-throughput single-cell gene expression analysis.

    PubMed

    Moignard, Victoria; Macaulay, Iain C; Swiers, Gemma; Buettner, Florian; Schütte, Judith; Calero-Nieto, Fernando J; Kinston, Sarah; Joshi, Anagha; Hannah, Rebecca; Theis, Fabian J; Jacobsen, Sten Eirik; de Bruijn, Marella F; Göttgens, Berthold

    2013-04-01

    Cellular decision-making is mediated by a complex interplay of external stimuli with the intracellular environment, in particular transcription factor regulatory networks. Here we have determined the expression of a network of 18 key haematopoietic transcription factors in 597 single primary blood stem and progenitor cells isolated from mouse bone marrow. We demonstrate that different stem/progenitor populations are characterized by distinctive transcription factor expression states, and through comprehensive bioinformatic analysis reveal positively and negatively correlated transcription factor pairings, including previously unrecognized relationships between Gata2, Gfi1 and Gfi1b. Validation using transcriptional and transgenic assays confirmed direct regulatory interactions consistent with a regulatory triad in immature blood stem cells, where Gata2 may function to modulate cross-inhibition between Gfi1 and Gfi1b. Single-cell expression profiling therefore identifies network states and allows reconstruction of network hierarchies involved in controlling stem cell fate choices, and provides a blueprint for studying both normal development and human disease.

  15. High-throughput characterization of Pt supported on thin film oxide material libraries applied in the oxygen reduction reaction.

    PubMed

    Schäfer, Dominik; Mardare, Cezarina; Savan, Alan; Sanchez, Miguel D; Mei, Bastian; Xia, Wei; Muhler, Martin; Ludwig, Alfred; Schuhmann, Wolfgang

    2011-03-15

    Thin film metal oxide material libraries were prepared by sputter deposition of nanoscale Ti/Nb precursor multilayers followed by ex situ oxidation. The metal composition was varied from 6 at.% Nb to 27 at.% Nb. Additionally, thin wedge-type layers of Pt with a nominal thickness gradient from 0 to 5 nm were sputter-deposited on top of the oxides. The materials libraries were characterized with respect to metallic film composition, oxide thickness, phases, electrical conductivity, Pt thickness, and electrochemical activity for the oxygen reduction reaction (ORR). Electrochemical investigations were carried out by cyclic voltammetry using an automated scanning droplet cell. For a nominal Pt thickness >1 nm, no significant dependence of the ORR activity on the Pt thickness or the substrate composition was observed. However, below that critical thickness, a strong decrease of the surface-normalized activity in terms of reduction currents and potentials was observed. For such thin Pt layers, the conductivity of the substrate seems to have a substantial impact on the catalytic activity. Results from X-ray photoelectron spectroscopy (XPS) measurements suggest that the critical Pt thickness coincides with the transition from a continuous Pt film into isolated particles at decreasing nominal Pt thickness. In the case of isolated Pt particles, the activity of Pt decisively depends on its ability to exchange electrons with the oxide layer, and hence, a dependence on the substrate conductivity is rationalized.

  16. Parallelization of a spatial random field characterization process using the Method of Anchored Distributions and the HTCondor high throughput computing system

    NASA Astrophysics Data System (ADS)

    Osorio-Murillo, C. A.; Over, M. W.; Frystacky, H.; Ames, D. P.; Rubin, Y.

    2013-12-01

    A new software application called MAD# has been coupled with the HTCondor high throughput computing system to aid scientists and educators with the characterization of spatial random fields and enable understanding the spatial distribution of parameters used in hydrogeologic and related modeling. MAD# is an open source desktop software application used to characterize spatial random fields using direct and indirect information through Bayesian inverse modeling technique called the Method of Anchored Distributions (MAD). MAD relates indirect information with a target spatial random field via a forward simulation model. MAD# executes inverse process running the forward model multiple times to transfer information from indirect information to the target variable. MAD# uses two parallelization profiles according to computational resources available: one computer with multiple cores and multiple computers - multiple cores through HTCondor. HTCondor is a system that manages a cluster of desktop computers for submits serial or parallel jobs using scheduling policies, resources monitoring, job queuing mechanism. This poster will show how MAD# reduces the time execution of the characterization of random fields using these two parallel approaches in different case studies. A test of the approach was conducted using 1D problem with 400 cells to characterize saturated conductivity, residual water content, and shape parameters of the Mualem-van Genuchten model in four materials via the HYDRUS model. The number of simulations evaluated in the inversion was 10 million. Using the one computer approach (eight cores) were evaluated 100,000 simulations in 12 hours (10 million - 1200 hours approximately). In the evaluation on HTCondor, 32 desktop computers (132 cores) were used, with a processing time of 60 hours non-continuous in five days. HTCondor reduced the processing time for uncertainty characterization by a factor of 20 (1200 hours reduced to 60 hours.)

  17. High-throughput sequencing-based microbial characterization of size fractionated biomass in an anoxic anammox reactor for low-strength wastewater at low temperatures.

    PubMed

    Liu, Wenru; Yang, Dianhai; Chen, Wenjing; Gu, Xiao

    2017-05-01

    The microbial characterization of three size-fractionated sludge obtained from a suspended-growth anoxic anammox reactor treating low-strength wastewater at low temperatures were investigated by using high-throughput sequencing. Particularly, the spatial variability in relative abundance of microorganisms involved in nitrogen metabolism were analyzed in detail. Results showed that population segregation did occur in the reactor. It was found, for the first time, that the genus Nitrotoga was enriched only in large granules (>400μm). Three anammox genus including Candidatus Jettenia, Brocadia and Kuenenia were detected. Among them, Candidatus Brocadia and Kuenenia preferred to grow in large-sized granules (>400μm), whereas Candidatus Jettenia dominated in small- and moderate-sized sludge (<400μm). The members of genus Candidatus Jettenia appeared to play the vital role in nitrogen removal, since sludge with diameters smaller than 400μm accounted for 81.55% of the total biomass. However, further studies are required to identify the activity of different-size sludge.

  18. High-throughput 96-well solvent mediated sonic blending synthesis and on-plate solid/solution stability characterization of pharmaceutical cocrystals.

    PubMed

    Luu, Van; Jona, Janan; Stanton, Mary K; Peterson, Matthew L; Morrison, Henry G; Nagapudi, Karthik; Tan, Helming

    2013-01-30

    A 96-well high-throughput cocrystal screening workflow has been developed consisting of solvent-mediated sonic blending synthesis and on-plate solid/solution stability characterization by XRPD. A strategy of cocrystallization screening in selected blend solvents including water mixtures is proposed to not only manipulate solubility of the cocrystal components but also differentiate physical stability of the cocrystal products. Caffeine-oxalic acid and theophylline-oxalic acid cocrystals were prepared and evaluated in relation to saturation levels of the cocrystal components and stability of the cocrystal products in anhydrous and hydrous solvents. AMG 517 was screened with a number of coformers, and solid/solution stability of the resulting cocrystals on the 96-well plate was investigated. A stability trend was observed and confirmed that cocrystals comprised of lower aqueous solubility coformers tended to be more stable in water. Furthermore, cocrystals which could be isolated under hydrous solvent blending condition exhibited superior physical stability to those which could only be obtained under anhydrous condition. This integrated HTS workflow provides an efficient route in an API-sparing approach to screen and identify cocrystal candidates with proper solubility and solid/solution stability properties.

  19. Enzymatic Characterization of ER Stress-Dependent Kinase, PERK, and Development of a High-Throughput Assay for Identification of PERK Inhibitors.

    PubMed

    Pytel, Dariusz; Seyb, Kathleen; Liu, Min; Ray, Soumya S; Concannon, John; Huang, Mickey; Cuny, Gregory D; Diehl, J Alan; Glicksman, Marcie A

    2014-08-01

    PERK is serine/threonine kinase localized to the endoplasmic reticulum (ER) membrane. PERK is activated and contributes to cell survival in response to a variety of physiological stresses that affect protein quality control in the ER, such as hypoxia, glucose depravation, increased lipid biosynthesis, and increased protein translation. Pro-survival functions of PERK are triggered by such stresses, suggesting that development of small-molecule inhibitors of PERK may be efficacious in a variety of disease scenarios. Hence, we have conducted a detailed enzymatic characterization of the PERK kinase to develop a high-throughput-screening assay (HTS) that will permit the identification of small-molecule PERK inhibitors. In addition to establishing the K(m) of PERK for both its primary substrate, eIF2α, and for adenosine triphosphate, further mechanistic studies revealed that PERK targets its substrate via either a random/steady-state ordered mechanism. For HTS, we developed a time-resolved fluorescence resonance energy transfer-based assay that yielded a robust Z' factor and percent coefficient of variation value, enabling the successful screening of 79,552 compounds. This approach yielded one compound that exhibited good in vitro and cellular activity. These results demonstrate the validity of this screen and represent starting points for drug discovery efforts.

  20. A Comprehensive Approach to Assess Arabidopsis Survival Phenotype in Water-Limited Condition Using a Non-invasive High-Throughput Phenomics Platform

    PubMed Central

    Vello, Emilio; Tomita, Akiko; Diallo, Amadou Oury; Bureau, Thomas E.

    2015-01-01

    With the rapid rise in global population and the challenges caused by climate changes, the maximization of plant productivity and the development of sustainable agriculture strategies are vital for food security. One of the resources more affected in this new environment will be the limitation of water. In this study, we describe the use of non-invasive technologies exploiting sensors for visible, fluorescent, and near-infrared lights to accurately screen survival phenotypes in Arabidopsis thaliana exposed to water-limited conditions. We implemented two drought protocols and a robust analysis methodology that enabled us to clearly assess the wilting or dryness status of the plants at different time points using a phenomics platform. In conclusion, our approach has shown to be very accurate and suitable for experiments where hundred of samples have to be screened making a manual evaluation unthinkable. This approach can be used not only in functional genomics studies but also in agricultural applications. PMID:26697051

  1. LeasyScan: a novel concept combining 3D imaging and lysimetry for high-throughput phenotyping of traits controlling plant water budget.

    PubMed

    Vadez, Vincent; Kholová, Jana; Hummel, Grégoire; Zhokhavets, Uladzimir; Gupta, S K; Hash, C Tom

    2015-09-01

    In this paper, we describe the thought process and initial data behind the development of an imaging platform (LeasyScan) combined with lysimetric capacity, to assess canopy traits affecting water use (leaf area, leaf area index, transpiration). LeasyScan is based on a novel 3D scanning technique to capture leaf area development continuously, a scanner-to-plant concept to increase imaging throughput and analytical scales to combine gravimetric transpiration measurements. The paper presents how the technology functions, how data are visualised via a web-based interface and how data extraction and analysis is interfaced through 'R' libraries. Close agreement between scanned and observed leaf area data of individual plants in different crops was found (R(2) between 0.86 and 0.94). Similar agreement was found when comparing scanned and observed area of plants cultivated at densities reflecting field conditions (R(2) between 0.80 and 0.96). An example in monitoring plant transpiration by the analytical scales is presented. The last section illustrates some of the early ongoing applications of the platform to target key phenotypes: (i) the comparison of the leaf area development pattern of fine mapping recombinants of pearl millet; (ii) the leaf area development pattern of pearl millet breeding material targeted to different agro-ecological zones; (iii) the assessment of the transpiration response to high VPD in sorghum and pearl millet. This new platform has the potential to phenotype for traits controlling plant water use at a high rate and precision, of critical importance for drought adaptation, and creates an opportunity to harness their genetics for the breeding of improved varieties.

  2. High Throughput Plasma Water Treatment

    NASA Astrophysics Data System (ADS)

    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. A novel imaging-based high-throughput screening approach to anti-angiogenic drug discovery.

    PubMed

    Evensen, Lasse; Micklem, David R; Link, Wolfgang; Lorens, James B

    2010-01-01

    The successful progression to the clinic of angiogenesis inhibitors for cancer treatment has spurred interest in developing new classes of anti-angiogenic compounds. The resulting surge in available candidate therapeutics highlights the need for robust, high-throughput angiogenesis screening systems that adequately capture the complexity of new vessel formation while providing quantitative evaluation of the potency of these agents. Available in vitro angiogenesis assays are either cumbersome, impeding adaptation to high-throughput screening formats, or inadequately model the complex multistep process of new vessel formation. We therefore developed an organotypic endothelial-mural cell co-culture assay system that reflects several facets of angiogenesis while remaining compatible with high-throughput/high-content image screening. Co-culture of primary human endothelial cells (EC) and vascular smooth muscle cells (vSMC) results in assembly of a network of tubular endothelial structures enveloped with vascular basement membrane proteins, thus, comprising the three main components of blood vessels. Initially, EC are dependent on vSMC-derived VEGF and sensitive to clinical anti-angiogenic therapeutics. A subsequent phenotypic VEGF-switch renders EC networks resistant to anti-VEGF therapeutics, demarcating a mature vascular phenotype. Conversely, mature EC networks remain sensitive to vascular disrupting agents. Therefore, candidate anti-angiogenic compounds can be interrogated for their relative potency on immature and mature networks and classified as either vascular normalizing or vascular disrupting agents. Here, we demonstrate that the EC-vSMC co-culture assay represents a robust high-content imaging high-throughput screening system for identification of novel anti-angiogenic agents. A pilot high-throughput screening campaign was used to define informative imaging parameters and develop a follow-up dose-response scheme for hit characterization. High-throughput

  4. Characterization of the indigenous microflora in raw and pasteurized buffalo milk during storage at refrigeration temperature by high-throughput sequencing.

    PubMed

    Li, Ling; Renye, John A; Feng, Ling; Zeng, Qingkun; Tang, Yan; Huang, Li; Ren, Daxi; Yang, Pan

    2016-09-01

    The effect of refrigeration on bacterial communities within raw and pasteurized buffalo milk was studied using high-throughput sequencing. High-quality samples of raw buffalo milk were obtained from 3 dairy farms in the Guangxi province in southern China. Five liters of each milk sample were pasteurized (72°C; 15 s); and both raw and pasteurized milks were stored at refrigeration temperature (1-4°C) for various times with their microbial communities characterized using the Illumina Miseq platform (Novogene, Beijing, China). Results showed that both raw and pasteurized milks contained a diverse microbial population and that the populations changed over time during storage. In raw buffalo milk, Lactococcus and Streptococcus dominated the population within the first 24h; however, when stored for up to 72h the dominant bacteria were members of the Pseudomonas and Acinetobacter genera, totaling more than 60% of the community. In pasteurized buffalo milk, the microbial population shifted from a Lactococcus-dominated community (7d), to one containing more than 84% Paenibacillus by 21d of storage. To increase the shelf-life of buffalo milk and its products, raw milk needs to be refrigerated immediately after milking and throughout transport, and should be monitored for the presence of Paenibacillus. Results from this study suggest pasteurization should be performed within 24h of raw milk collection, when the number of psychrotrophic bacteria are low; however, as Paenibacillus spores are resistant to pasteurization, additional antimicrobial treatments may be required to extend shelf-life. The findings from this study are expected to aid in improving the quality and safety of raw and pasteurized buffalo milk.

  5. Characterization of the indigenous microflora in raw and pasteurized buffalo milk during storage at refrigeration temperature by high-throughput sequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of refrigeration on bacterial communities within raw and pasteurized buffalo milk was studied using high-throughput sequencing. High quality samples of raw buffalo milk were obtained from five dairy farms in the Guangxi province of China. A sample of each milk was pasteurized, and both r...

  6. High-throughput characterization of sediment organic matter by pyrolysis-gas chromatography/mass spectrometry and multivariate curve resolution: A promising analytical tool in (paleo)limnology.

    PubMed

    Tolu, Julie; Gerber, Lorenz; Boily, Jean-François; Bindler, Richard

    2015-06-23

    Molecular-level chemical information about organic matter (OM) in sediments helps to establish the sources of OM and the prevalent degradation/diagenetic processes, both essential for understanding the cycling of carbon (C) and of the elements associated with OM (toxic trace metals and nutrients) in lake ecosystems. Ideally, analytical methods for characterizing OM should allow high sample throughput, consume small amounts of sample and yield relevant chemical information, which are essential for multidisciplinary, high-temporal resolution and/or large spatial scale investigations. We have developed a high-throughput analytical method based on pyrolysis-gas chromatography/mass spectrometry and automated data processing to characterize sedimentary OM in sediments. Our method consumes 200 μg of freeze-dried and ground sediment sample. Pyrolysis was performed at 450°C, which was found to avoid degradation of specific biomarkers (e.g., lignin compounds, fresh carbohydrates/cellulose) compared to 650°C, which is in the range of temperatures commonly applied for environmental samples. The optimization was conducted using the top ten sediment samples of an annually resolved sediment record (containing 16-18% and 1.3-1.9% of total carbon and nitrogen, respectively). Several hundred pyrolytic compound peaks were detected of which over 200 were identified, which represent different classes of organic compounds (i.e., n-alkanes, n-alkenes, 2-ketones, carboxylic acids, carbohydrates, proteins, other N compounds, (methoxy)phenols, (poly)aromatics, chlorophyll and steroids/hopanoids). Technical reproducibility measured as relative standard deviation of the identified peaks in triplicate analyses was 5.5±4.3%, with 90% of the RSD values within 10% and 98% within 15%. Finally, a multivariate calibration model was calculated between the pyrolytic degradation compounds and the sediment depth (i.e., sediment age), which is a function of degradation processes and changes in OM

  7. High throughput protein production screening

    DOEpatents

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

    2009-09-08

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

  8. High-Throughput Sequencing Technologies

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Morgan, R E; Westwood, N J

    2004-01-01

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

  10. Global phenotypic characterization of bacteria

    PubMed Central

    Bochner, Barry R

    2009-01-01

    The measure of the quality of a systems biology model is how well it can reproduce and predict the behaviors of a biological system such as a microbial cell. In recent years, these models have been built up in layers, and each layer has been growing in sophistication and accuracy in parallel with a global data set to challenge and validate the models in predicting the content or activities of genes (genomics), proteins (proteomics), metabolites (metabolomics), and ultimately cell phenotypes (phenomics). This review focuses on the latter, the phenotypes of microbial cells. The development of Phenotype MicroArrays, which attempt to give a global view of cellular phenotypes, is described. In addition to their use in fleshing out and validating systems biology models, there are many other uses of this global phenotyping technology in basic and applied microbiology research, which are also described. PMID:19054113

  11. A high throughput in situ hybridization method to characterize mRNA expression patterns in the fetal mouse lower urogenital tract.

    PubMed

    Abler, Lisa L; Mehta, Vatsal; Keil, Kimberly P; Joshi, Pinak S; Flucus, Chelsea-Leigh; Hardin, Heather A; Schmitz, Christopher T; Vezina, Chad M

    2011-08-19

    Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions(1,2). Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer. The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma(3). This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals(4). The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators. Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In

  12. High-throughput electrophysiology with Xenopus oocytes

    PubMed Central

    Papke, Roger L.; Smith-Maxwell, Cathy

    2010-01-01

    Voltage-clamp techniques are typically used to study the plasma membrane proteins, such as ion channels and transporters that control bioelectrical signals. Many of these proteins have been cloned and can now be studied as potential targets for drug development. The two approaches most commonly used for heterologous expression of cloned ion channels and transporters involve either transfection of the genes into small cells grown in tissue culture or the injection of the genetic material into larger cells. The standard large cells used for the expression of cloned cDNA or synthetic RNA are the egg progenitor cells (oocytes) of the African frog, Xenopus laevis. Until recently, cellular electrophysiology was performed manually, one cell at a time by a single operator. However, methods of high-throughput electrophysiology have been developed which are automated and permit data acquisition and analysis from multiple cells in parallel. These methods are breaking a bottleneck in drug discovery, useful in some cases for primary screening as well as for thorough characterization of new drugs. Increasing throughput of high-quality functional data greatly augments the efficiency of academic research and pharmaceutical drug development. Some examples of studies that benefit most from high-throughput electrophysiology include pharmaceutical screening of targeted compound libraries, secondary screening of identified compounds for subtype selectivity, screening mutants of ligand-gated channels for changes in receptor function, scanning mutagenesis of protein segments, and mutant-cycle analysis. We describe here the main features and potential applications of OpusXpress, an efficient commercially available system for automated recording from Xenopus oocytes. We show some types of data that have been gathered by this system and review realized and potential applications. PMID:19149490

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

    EPA Science Inventory

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

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

    PubMed

    Kinser, Holly E; Pincus, Zachary

    2016-06-03

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

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

    EPA Science Inventory

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

  16. Competition experiments coupled with high-throughput analyses for functional genomics studies in yeast.

    PubMed

    Delneri, Daniela

    2011-01-01

    Competition experiments are an effective way to provide a measurement of the fitness of yeast strains. The availability of the Saccharomyces cerevisiae yeast knock-out (YKO) deletion collection allows scientists to retrieve fitness data for the ~6,000 S. cerevisiae genes at the same time in a given environment. The molecular barcodes, characterizing each yeast mutant, serve as strain identifiers, which can be detected in a single microarray analysis. Competition experiments in continuous culture using chemically defined media allow a more specific discrimination of the strains based on their fitness profile. With this high-throughput approach, a series of genes that, when one allele is missing, result in either defective (haplo-insufficient) or favored (haplo-proficient) growth phenotype have been discovered, for each nutrient-limiting condition tested. While haplo-insufficient genes seemed to overlap largely across all the media used, the haplo-proficient ones seem to be more environment specific. For example, genes involved in the protein secretion pathway were highly haplo-insufficient in all the contexts, whereas most of the genes encoding for proteasome components showed a haplo-proficient phenotype specific to nitrogen-limiting conditions. In this chapter, the method used for implementation of competition experiments for high-throughput studies in yeast is presented.

  17. Identification and Characterization of MicroRNAs from Longitudinal Muscle and Respiratory Tree in Sea Cucumber (Apostichopus japonicus) Using High-Throughput Sequencing.

    PubMed

    Wang, Hongdi; Liu, Shikai; Cui, Jun; Li, Chengze; Hu, Yucai; Zhou, Wei; Chang, Yaqing; Qiu, Xuemei; Liu, Zhanjiang; Wang, Xiuli

    2015-01-01

    MicroRNAs (miRNAs), as a family of non-coding small RNAs, play important roles in the post-transcriptional regulation of gene expression. Sea cucumber (Apostichopus japonicus) is an important economic species which is widely cultured in East Asia. The longitudinal muscle (LTM) and respiratory tree (RPT) are two important tissues in sea cucumber, playing important roles such as respiration and movement. In this study, we identified and characterized miRNAs in the LTM and RPT of sea cucumber (Apostichopus japonicus) using Illumina HiSeq 2000 platform. A total of 314 and 221 conserved miRNAs were identified in LTM and RPT, respectively. In addition, 27 and 34 novel miRNAs were identified in the LTM and RPT, respectively. A set of 58 miRNAs were identified to be differentially expressed between LTM and RPT. Among them, 9 miRNAs (miR-31a-3p, miR-738, miR-1692, let-7a, miR-72a, miR-100b-5p, miR-31b-5p, miR-429-3p, and miR-2008) in RPT and 7 miRNAs (miR-127, miR-340, miR-381, miR-3543, miR-434-5p, miR-136-3p, and miR-300-3p) in LTM were differentially expressed with foldchange value being greater than 10. A total of 14,207 and 12,174 target genes of these miRNAs were predicted, respectively. Functional analysis of these target genes of miRNAs were performed by GO analysis and pathway analysis. This result provided in this work will be useful for understanding biological characteristics of the LTM and RPT of sea cucumber and assisting molecular breeding of sea cucumber for aquaculture.

  18. Identification and Characterization of MicroRNAs from Longitudinal Muscle and Respiratory Tree in Sea Cucumber (Apostichopus japonicus) Using High-Throughput Sequencing

    PubMed Central

    Li, Chengze; Hu, Yucai; Zhou, Wei; Chang, Yaqing; Qiu, Xuemei; Liu, Zhanjiang; Wang, Xiuli

    2015-01-01

    MicroRNAs (miRNAs), as a family of non-coding small RNAs, play important roles in the post-transcriptional regulation of gene expression. Sea cucumber (Apostichopus japonicus) is an important economic species which is widely cultured in East Asia. The longitudinal muscle (LTM) and respiratory tree (RPT) are two important tissues in sea cucumber, playing important roles such as respiration and movement. In this study, we identified and characterized miRNAs in the LTM and RPT of sea cucumber (Apostichopus japonicus) using Illumina HiSeq 2000 platform. A total of 314 and 221 conserved miRNAs were identified in LTM and RPT, respectively. In addition, 27 and 34 novel miRNAs were identified in the LTM and RPT, respectively. A set of 58 miRNAs were identified to be differentially expressed between LTM and RPT. Among them, 9 miRNAs (miR-31a-3p, miR-738, miR-1692, let-7a, miR-72a, miR-100b-5p, miR-31b-5p, miR-429-3p, and miR-2008) in RPT and 7 miRNAs (miR-127, miR-340, miR-381, miR-3543, miR-434-5p, miR-136-3p, and miR-300-3p) in LTM were differentially expressed with foldchange value being greater than 10. A total of 14,207 and 12,174 target genes of these miRNAs were predicted, respectively. Functional analysis of these target genes of miRNAs were performed by GO analysis and pathway analysis. This result provided in this work will be useful for understanding biological characteristics of the LTM and RPT of sea cucumber and assisting molecular breeding of sea cucumber for aquaculture. PMID:26244987

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

    PubMed Central

    Chiaraviglio, Lucius

    2015-01-01

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

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

    PubMed

    Kassner, Paul D

    2008-03-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

    Anderson, Christopher N.; Naulleau, Patrick P.

    2007-07-30

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

  3. High-throughput computing in the sciences.

    PubMed

    Morgan, Mark; Grimshaw, Andrew

    2009-01-01

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

  4. Chromatography process development in the quality by design paradigm I: Establishing a high-throughput process development platform as a tool for estimating "characterization space" for an ion exchange chromatography step.

    PubMed

    Bhambure, R; Rathore, A S

    2013-01-01

    This article describes the development of a high-throughput process development (HTPD) platform for developing chromatography steps. An assessment of the platform as a tool for establishing the "characterization space" for an ion exchange chromatography step has been performed by using design of experiments. Case studies involving use of a biotech therapeutic, granulocyte colony-stimulating factor have been used to demonstrate the performance of the platform. We discuss the various challenges that arise when working at such small volumes along with the solutions that we propose to alleviate these challenges to make the HTPD data suitable for empirical modeling. Further, we have also validated the scalability of this platform by comparing the results from the HTPD platform (2 and 6 μL resin volumes) against those obtained at the traditional laboratory scale (resin volume, 0.5 mL). We find that after integration of the proposed correction factors, the HTPD platform is capable of performing the process optimization studies at 170-fold higher productivity. The platform is capable of providing semi-quantitative assessment of the effects of the various input parameters under consideration. We think that platform such as the one presented is an excellent tool for examining the "characterization space" and reducing the extensive experimentation at the traditional lab scale that is otherwise required for establishing the "design space." Thus, this platform will specifically aid in successful implementation of quality by design in biotech process development. This is especially significant in view of the constraints with respect to time and resources that the biopharma industry faces today.

  5. Characterizing visible and invisible cell wall mutant phenotypes.

    PubMed

    Carpita, Nicholas C; McCann, Maureen C

    2015-07-01

    About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with 'invisible' phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basis of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall.

  6. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples

    PubMed Central

    Ackerman, Margaret E.; Moldt, Brian; Wyatt, Richard T; Dugast, Anne-Sophie; McAndrew, Elizabeth; Tsoukas, Stephen; Jost, Stephanie; Berger, Christoph T.; Sciaranghella, Gaia; Liu, Qingquan; Irvine, Darrell J; Burton, Dennis R.; Alter, Galit

    2011-01-01

    Phagocytosis can be induced via the engagement of Fcγ receptors by antibody-opsonized material. Furthermore, the efficiency of antibody-induced effector functions has been shown to be dramatically modulated by changes in antibody glycosylation. Because infection can modulate antibody glycans, which in turn modulate antibody functions, assays capable of determining the induction of effector functions rather than neutralization or titer provide a valuable opportunity to more fully characterize the quality of the adaptive immune response. Here we describe a robust and high-throughput flow cytometric assay to define the phagocytic activity of antigen-specific antibodies from clinical samples. This assay employs a monocytic cell line that expresses numerous Fc receptors: including inhibitory and activating, and high and low affinity receptors—allowing complex phenotypes to be studied. We demonstrate the adaptability of this high-throughput, flow-based assay to measure antigen-specific antibody-mediated phagocytosis against an array of viruses, including influenza, HIV, and dengue. The phagocytosis assay format further allows for simultaneous analysis of cytokine release, as well as determination of the role of specific Fcγ-receptor subtypes, making it a highly useful system for parsing differences in the ability of clinical and vaccine induced antibody samples to recruit this critical effector function. PMID:21192942

  7. High Throughput Computing Impact on Meta Genomics (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

    ScienceCinema

    Gore, Brooklin [Morgridge Institute for Research

    2016-07-12

    This presentation includes a brief background on High Throughput Computing, correlating gene transcription factors, optical mapping, genotype to phenotype mapping via QTL analysis, and current work on next gen sequencing.

  8. MEGARes: an antimicrobial resistance database for high throughput sequencing

    PubMed Central

    Lakin, Steven M.; Dean, Chris; Noyes, Noelle R.; Dettenwanger, Adam; Ross, Anne Spencer; Doster, Enrique; Rovira, Pablo; Abdo, Zaid; Jones, Kenneth L.; Ruiz, Jaime; Belk, Keith E.; Morley, Paul S.; Boucher, Christina

    2017-01-01

    Antimicrobial resistance has become an imminent concern for public health. As methods for detection and characterization of antimicrobial resistance move from targeted culture and polymerase chain reaction to high throughput metagenomics, appropriate resources for the analysis of large-scale data are required. Currently, antimicrobial resistance databases are tailored to smaller-scale, functional profiling of genes using highly descriptive annotations. Such characteristics do not facilitate the analysis of large-scale, ecological sequence datasets such as those produced with the use of metagenomics for surveillance. In order to overcome these limitations, we present MEGARes (https://megares.meglab.org), a hand-curated antimicrobial resistance database and annotation structure that provides a foundation for the development of high throughput acyclical classifiers and hierarchical statistical analysis of big data. MEGARes can be browsed as a stand-alone resource through the website or can be easily integrated into sequence analysis pipelines through download. Also via the website, we provide documentation for AmrPlusPlus, a user-friendly Galaxy pipeline for the analysis of high throughput sequencing data that is pre-packaged for use with the MEGARes database. PMID:27899569

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

    PubMed

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

    2014-02-01

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

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

    EPA Pesticide Factsheets

    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

  11. MEGARes: an antimicrobial resistance database for high throughput sequencing.

    PubMed

    Lakin, Steven M; Dean, Chris; Noyes, Noelle R; Dettenwanger, Adam; Ross, Anne Spencer; Doster, Enrique; Rovira, Pablo; Abdo, Zaid; Jones, Kenneth L; Ruiz, Jaime; Belk, Keith E; Morley, Paul S; Boucher, Christina

    2017-01-04

    Antimicrobial resistance has become an imminent concern for public health. As methods for detection and characterization of antimicrobial resistance move from targeted culture and polymerase chain reaction to high throughput metagenomics, appropriate resources for the analysis of large-scale data are required. Currently, antimicrobial resistance databases are tailored to smaller-scale, functional profiling of genes using highly descriptive annotations. Such characteristics do not facilitate the analysis of large-scale, ecological sequence datasets such as those produced with the use of metagenomics for surveillance. In order to overcome these limitations, we present MEGARes (https://megares.meglab.org), a hand-curated antimicrobial resistance database and annotation structure that provides a foundation for the development of high throughput acyclical classifiers and hierarchical statistical analysis of big data. MEGARes can be browsed as a stand-alone resource through the website or can be easily integrated into sequence analysis pipelines through download. Also via the website, we provide documentation for AmrPlusPlus, a user-friendly Galaxy pipeline for the analysis of high throughput sequencing data that is pre-packaged for use with the MEGARes database.

  12. Microfabricated high-throughput electronic particle detector.

    PubMed

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

    2007-10-01

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

  13. High-throughput TILLING for functional genomics.

    PubMed

    Till, Bradley J; Colbert, Trenton; Tompa, Rachel; Enns, Linda C; Codomo, Christine A; Johnson, Jessica E; Reynolds, Steven H; Henikoff, Jorja G; Greene, Elizabeth A; Steine, Michael N; Comai, Luca; Henikoff, Steven

    2003-01-01

    Targeting-induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. Here, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.

  14. High-throughput TILLING for Arabidopsis.

    PubMed

    Till, Bradley J; Colbert, Trenton; Codomo, Christine; Enns, Linda; Johnson, Jessica; Reynolds, Steven H; Henikoff, Jorja G; Greene, Elizabeth A; Steine, Michael N; Comai, Luca; Henikoff, Steven

    2006-01-01

    Targeting induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. In this chapter, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.

  15. High-throughput in vivo vertebrate screening

    PubMed Central

    Pardo-Martin, Carlos; Chang, Tsung-Yao; Koo, Bryan Kyo; Gilleland, Cody L.; Wasserman, Steven C.; Yanik, Mehmet Fatih

    2010-01-01

    We demonstrate a high-throughput platform for cellular-resolution in vivo pharmaceutical and genetic screens on zebrafish larvae. The system automatically loads animals from reservoirs or multiwell plates, and positions and orients them for high-speed confocal imaging and laser manipulation of both superficial and deep organs within 19 seconds without damage. We show small-scale test screening of retinal axon guidance mutants and neuronal regeneration assays in combination with femtosecond laser microsurgery. PMID:20639868

  16. High-throughput neuro-imaging informatics.

    PubMed

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

    2013-01-01

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

  17. High-throughput neuro-imaging informatics

    PubMed Central

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

    2013-01-01

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

  18. Computational analysis of high-throughput flow cytometry data

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2003-12-01

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

  20. A High-Throughput Strategy for Dissecting Mammalian Genetic Interactions

    PubMed Central

    Stockman, Victoria B.; Ghamsari, Lila; Lasso, Gorka; Honig, Barry

    2016-01-01

    Comprehensive delineation of complex cellular networks requires high-throughput interrogation of genetic interactions. To address this challenge, we describe the development of a multiplex combinatorial strategy to assess pairwise genetic interactions using CRISPR-Cas9 genome editing and next-generation sequencing. We characterize the performance of combinatorial genome editing and analysis using different promoter and gRNA designs and identified regions of the chimeric RNA that are compatible with next-generation sequencing preparation and quantification. This approach is an important step towards elucidating genetic networks relevant to human diseases and the development of more efficient Cas9-based therapeutics. PMID:27936040

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

    EPA Science Inventory

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

  2. Automated High Throughput Drug Target Crystallography

    SciTech Connect

    Rupp, B

    2005-02-18

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

  3. Clustering of High Throughput Gene Expression Data

    PubMed Central

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

    2012-01-01

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

  4. High throughput chemical munitions treatment system

    DOEpatents

    Haroldsen, Brent L [Manteca, CA; Stofleth, Jerome H [Albuquerque, NM; Didlake, Jr., John E.; Wu, Benjamin C-P [San Ramon, CA

    2011-11-01

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

  5. High throughput screening technologies for ion channels

    PubMed Central

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

    2016-01-01

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

  6. A genome-enabled, high-throughput, and multiplexed fingerprinting platform for strawberry (Fragaria L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Strawberry (Fragaria L.) genotypes bear remarkable phenotypic similarity, even across ploidy levels. Additionally, breeding programs seek to introgress alleles from wild germplasm, so objective molecular description of genetic variation has great value. In this report, a high-throughput, robust prot...

  7. Preliminary High-Throughput Metagenome Assembly

    SciTech Connect

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

    2007-03-26

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

  8. Economic consequences of high throughput maskless lithography

    NASA Astrophysics Data System (ADS)

    Hartley, John G.; Govindaraju, Lakshmi

    2005-11-01

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

  9. High-Throughput Analysis of Enzyme Activities

    SciTech Connect

    Lu, Guoxin

    2007-01-01

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

  10. High throughput phenotyping using an unmanned aerial vehicle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Field trials are expensive and labor-intensive to carry out. Strategies to maximize data collection from these trials will improve research efficiencies. We have purchased a small unmanned aerial vehicle (AEV) to collect digital images from field plots. The AEV is remote-controlled and can be guided...

  11. A high-throughput neutron spectrometer

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  12. A high-throughput radiometric kinase assay

    PubMed Central

    Duong-Ly, Krisna C.; Peterson, Jeffrey R.

    2016-01-01

    Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest. Screening of more focused compound libraries may also be conducted in the later stages of inhibitor development to improve potency and optimize selectivity. The dot blot kinase assay is a robust, high-throughput kinase assay that can be used to screen a number of small molecule compounds against one kinase of interest or several kinases. Here, a protocol for a dot blot kinase assay used for measuring insulin receptor kinase activity is presented. This protocol can be readily adapted for use with other protein kinases. PMID:26501904

  13. Sequential stopping for high-throughput experiments.

    PubMed

    Rossell, David; Müller, Peter

    2013-01-01

    In high-throughput experiments, the sample size is typically chosen informally. Most formal sample-size calculations depend critically on prior knowledge. We propose a sequential strategy that, by updating knowledge when new data are available, depends less critically on prior assumptions. Experiments are stopped or continued based on the potential benefits in obtaining additional data. The underlying decision-theoretic framework guarantees the design to proceed in a coherent fashion. We propose intuitively appealing, easy-to-implement utility functions. As in most sequential design problems, an exact solution is prohibitive. We propose a simulation-based approximation that uses decision boundaries. We apply the method to RNA-seq, microarray, and reverse-phase protein array studies and show its potential advantages. The approach has been added to the Bioconductor package gaga.

  14. Origin and evolution of high throughput screening

    PubMed Central

    Pereira, D A; Williams, J A

    2007-01-01

    This article reviews the origin and evolution of high throughput screening (HTS) through the experience of an individual pharmaceutical company, revealing some of the mysteries of the early stages of drug discovery to the wider pharmacology audience. HTS in this company (Pfizer, Groton, USA) had its origin in natural products screening in 1986, by substituting fermentation broths with dimethyl sulphoxide solutions of synthetic compounds, using 96-well plates and reduced assay volumes of 50-100μl. A nominal 30mM source compound concentration provided high μM assay concentrations. Starting at 800 compounds each week, the process reached a steady state of 7200 compounds per week by 1989. Screening in the Applied Biotechnology and Screening Group was centralized with screens operating in lock-step to maximize efficiency. Initial screens were full files run in triplicate. Autoradiography and image analysis were introduced for 125I receptor ligand screens. Reverse transcriptase (RT) coupled with quantitative PCR and multiplexing addressed several targets in a single assay. By 1992 HTS produced ‘hits' as starting matter for approximately 40% of the Discovery portfolio. In 1995, the HTS methodology was expanded to include ADMET targets. ADME targets required each compound to be physically detected leading to the development of automated high throughput LC-MS. In 1996, 90 compounds/week were screened in microsomal, protein binding and serum stability assays. Subsequently, the mutagenic Ames assay was adapted to a 96-well plate liquid assay and novel algorithms permitted automated image analysis of the micronucleus assay. By 1999 ADME HTS was fully integrated into the discovery cycle. PMID:17603542

  15. High-Throughput Methods for Electron Crystallography

    PubMed Central

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

    2013-01-01

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

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

    SciTech Connect

    Havrilla, George J.; Miller, Thomasin C.

    2005-06-15

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

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

    NASA Astrophysics Data System (ADS)

    Havrilla, George J.; Miller, Thomasin C.

    2005-06-01

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

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

    PubMed

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

    2013-02-01

    Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in <4 h (corresponding to >3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K (D)) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.

  19. High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor

    PubMed Central

    Groot-Kormelink, Paul J.; Ferrand, Sandrine; Kelley, Nicholas; Bill, Anke; Freuler, Felix; Imbert, Pierre-Eloi; Marelli, Anthony; Gerwin, Nicole; Sivilotti, Lucia G.; Miraglia, Loren; Orth, Anthony P.; Oakeley, Edward J.; Schopfer, Ulrich; Siehler, Sandra

    2016-01-01

    High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype β1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation. PMID:27649498

  20. EDITORIAL: Combinatorial and High-Throughput Materials Research

    NASA Astrophysics Data System (ADS)

    Potyrailo, Radislav A.; Takeuchi, Ichiro

    2005-01-01

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

  1. Bulk Segregant Analysis by High-Throughput Sequencing Reveals a Novel Xylose Utilization Gene from Saccharomyces cerevisiae

    PubMed Central

    Wenger, Jared W.; Schwartz, Katja; Sherlock, Gavin

    2010-01-01

    Fermentation of xylose is a fundamental requirement for the efficient production of ethanol from lignocellulosic biomass sources. Although they aggressively ferment hexoses, it has long been thought that native Saccharomyces cerevisiae strains cannot grow fermentatively or non-fermentatively on xylose. Population surveys have uncovered a few naturally occurring strains that are weakly xylose-positive, and some S. cerevisiae have been genetically engineered to ferment xylose, but no strain, either natural or engineered, has yet been reported to ferment xylose as efficiently as glucose. Here, we used a medium-throughput screen to identify Saccharomyces strains that can increase in optical density when xylose is presented as the sole carbon source. We identified 38 strains that have this xylose utilization phenotype, including strains of S. cerevisiae, other sensu stricto members, and hybrids between them. All the S. cerevisiae xylose-utilizing strains we identified are wine yeasts, and for those that could produce meiotic progeny, the xylose phenotype segregates as a single gene trait. We mapped this gene by Bulk Segregant Analysis (BSA) using tiling microarrays and high-throughput sequencing. The gene is a putative xylitol dehydrogenase, which we name XDH1, and is located in the subtelomeric region of the right end of chromosome XV in a region not present in the S288c reference genome. We further characterized the xylose phenotype by performing gene expression microarrays and by genetically dissecting the endogenous Saccharomyces xylose pathway. We have demonstrated that natural S. cerevisiae yeasts are capable of utilizing xylose as the sole carbon source, characterized the genetic basis for this trait as well as the endogenous xylose utilization pathway, and demonstrated the feasibility of BSA using high-throughput sequencing. PMID:20485559

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

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

  3. AOPs and Biomarkers: Bridging High Throughput Screening ...

    EPA Pesticide Factsheets

    As high throughput screening (HTS) plays a larger role in toxicity testing, camputational toxicology has emerged as a critical component in interpreting the large volume of data produced. Computational models designed to quantify potential adverse effects based on HTS data will benefit from additional data sources that connect the magnitude of perturbation from the in vitro system to a level of concern at the organism or population level. The adverse outcome pathway (AOP) concept provides an ideal framework for combining these complementary data. Recent international efforts under the auspices of the Organization for Economic Co-operation and Development (OECD) have resulted in an AOP wiki designed to house formal descriptions of AOPs suitable for use in regulatory decision making. Recent efforts have built upon this to include an ontology describing the AOP with linkages to biological pathways, physiological terminology, and taxonomic applicability domains. Incorporation of an AOP network tool developed by the U.S. Army Corps of Engineers also allows consideration of cumulative risk from chemical and non-chemical stressors. Biomarkers are an important complement to formal AOP descriptions, particularly when dealing with susceptible subpopulations or lifestages in human health risk assessment. To address the issue of nonchemical stressors than may modify effects of criteria air pollutants, a novel method was used to integrate blood gene expression data with hema

  4. High-Throughput Enzyme Kinetics Using Microarrays

    SciTech Connect

    Guoxin Lu; Edward S. Yeung

    2007-11-01

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

  5. New High Throughput Methods to Estimate Chemical ...

    EPA Pesticide Factsheets

    EPA has made many recent advances in high throughput bioactivity testing. However, concurrent advances in rapid, quantitative prediction of human and ecological exposures have been lacking, despite the clear importance of both measures for a risk-based approach to prioritizing and screening chemicals. A recent report by the National Research Council of the National Academies, Exposure Science in the 21st Century: A Vision and a Strategy (NRC 2012) laid out a number of applications in chemical evaluation of both toxicity and risk in critical need of quantitative exposure predictions, including screening and prioritization of chemicals for targeted toxicity testing, focused exposure assessments or monitoring studies, and quantification of population vulnerability. Despite these significant needs, for the majority of chemicals (e.g. non-pesticide environmental compounds) there are no or limited estimates of exposure. For example, exposure estimates exist for only 7% of the ToxCast Phase II chemical list. In addition, the data required for generating exposure estimates for large numbers of chemicals is severely lacking (Egeghy et al. 2012). This SAP reviewed the use of EPA's ExpoCast model to rapidly estimate potential chemical exposures for prioritization and screening purposes. The focus was on bounded chemical exposure values for people and the environment for the Endocrine Disruptor Screening Program (EDSP) Universe of Chemicals. In addition to exposure, the SAP

  6. High-throughput plastic microlenses fabricated using microinjection molding techniques

    NASA Astrophysics Data System (ADS)

    Appasamy, Sreeram; Li, Weizhuo; Lee, Se Hwan; Boyd, Joseph T.; Ahn, Chong H.

    2005-12-01

    A novel fabrication scheme to develop high-throughput plastic microlenses using injection-molding techniques is realized. The initial microlens mold is fabricated using the well-known reflow technique. The reflow process is optimized to obtain reliable and repeatable microlens patterns. The master mold insert for the injection-molding process is fabricated using metal electroforming. The electroplating process is optimized for obtaining a low stress electroform. Two new plastic materials, cyclo olefin copolymer (COC) and Poly IR 2 are introduced in this work for fabricating microlenses. The plastic microlenses have been characterized for their focal lengths that range from 200 µm to 1.9 mm. This technique enables high-volume production of plastic microlenses with cycle times for a single chip being of the order of 60 s.

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

    PubMed Central

    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

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

    PubMed

    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.

  9. Characterizing the ADHD Phenotype for Genetic Studies

    ERIC Educational Resources Information Center

    Stevenson, Jim; Asherson, Phil; Hay, David; Levy, Florence; Swanson, Jim; Thapar, Anita; Willcutt, Erik

    2005-01-01

    The genetic study of ADHD has made considerable progress. Further developments in the field will be reliant in part on identifying the most appropriate phenotypes for genetic analysis. The use of both categorical and dimensional measures of symptoms related to ADHD has been productive. The use of multiple reporters is a valuable feature of the…

  10. High-throughput screening of a Corynebacterium glutamicum mutant library on genomic and metabolic level.

    PubMed

    Reimer, Lorenz C; Spura, Jana; Schmidt-Hohagen, Kerstin; Schomburg, Dietmar

    2014-01-01

    Due to impressive achievements in genomic research, the number of genome sequences has risen quickly, followed by an increasing number of genes with unknown or hypothetical function. This strongly calls for development of high-throughput methods in the fields of transcriptomics, proteomics and metabolomics. Of these platforms, metabolic profiling has the strongest correlation with the phenotype. We previously published a high-throughput metabolic profiling method for C. glutamicum as well as the automatic GC/MS processing software MetaboliteDetector. Here, we added a high-throughput transposon insertion determination for our C. glutamicum mutant library. The combination of these methods allows the parallel analysis of genotype/phenotype correlations for a large number of mutants. In a pilot project we analyzed the insertion points of 722 transposon mutants and found that 36% of the affected genes have unknown functions. This underlines the need for further information gathered by high-throughput techniques. We therefore measured the metabolic profiles of 258 randomly chosen mutants. The MetaboliteDetector software processed this large amount of GC/MS data within a few hours with a low relative error of 11.5% for technical replicates. Pairwise correlation analysis of metabolites over all genotypes showed dependencies of known and unknown metabolites. For a first insight into this large data set, a screening for interesting mutants was done by a pattern search, focusing on mutants with changes in specific pathways. We show that our transposon mutant library is not biased with respect to insertion points. A comparison of the results for specific mutants with previously published metabolic results on a deletion mutant of the same gene confirmed the concept of high-throughput metabolic profiling. Altogether the described method could be applied to whole mutant libraries and thereby help to gain comprehensive information about genes with unknown, hypothetical and known

  11. High throughput in vivo functional validation of candidate congenital heart disease genes in Drosophila.

    PubMed

    Zhu, Jun-Yi; Fu, Yulong; Nettleton, Margaret; Richman, Adam; Han, Zhe

    2017-01-20

    Genomic sequencing has implicated large numbers of genes and de novo mutations as potential disease risk factors. A high throughput in vivo model system is needed to validate gene associations with pathology. We developed a Drosophila-based functional system to screen candidate disease genes identified from Congenital Heart Disease (CHD) patients. 134 genes were tested in the Drosophila heart using RNAi-based gene silencing. Quantitative analyses of multiple cardiac phenotypes demonstrated essential structural, functional, and developmental roles for more than 70 genes, including a subgroup encoding histone H3K4 modifying proteins. We also demonstrated the use of Drosophila to evaluate cardiac phenotypes resulting from specific, patient-derived alleles of candidate disease genes. We describe the first high throughput in vivo validation system to screen candidate disease genes identified from patients. This approach has the potential to facilitate development of precision medicine approaches for CHD and other diseases associated with genetic factors.

  12. High-Throughput Sequencing, a Versatile Weapon to Support Genome-Based Diagnosis in Infectious Diseases: Applications to Clinical Bacteriology

    PubMed Central

    Caboche, Ségolène; Audebert, Christophe; Hot, David

    2014-01-01

    The recent progresses of high-throughput sequencing (HTS) technologies enable easy and cost-reduced access to whole genome sequencing (WGS) or re-sequencing. HTS associated with adapted, automatic and fast bioinformatics solutions for sequencing applications promises an accurate and timely identification and characterization of pathogenic agents. Many studies have demonstrated that data obtained from HTS analysis have allowed genome-based diagnosis, which has been consistent with phenotypic observations. These proofs of concept are probably the first steps toward the future of clinical microbiology. From concept to routine use, many parameters need to be considered to promote HTS as a powerful tool to help physicians and clinicians in microbiological investigations. This review highlights the milestones to be completed toward this purpose. PMID:25437800

  13. High-throughput identification of protein localization dependency networks.

    PubMed

    Christen, Beat; Fero, Michael J; Hillson, Nathan J; Bowman, Grant; Hong, Sun-Hae; Shapiro, Lucy; McAdams, Harley H

    2010-03-09

    Bacterial cells are highly organized with many protein complexes and DNA loci dynamically positioned to distinct subcellular sites over the course of a cell cycle. Such dynamic protein localization is essential for polar organelle development, establishment of asymmetry, and chromosome replication during the Caulobacter crescentus cell cycle. We used a fluorescence microscopy screen optimized for high-throughput to find strains with anomalous temporal or spatial protein localization patterns in transposon-generated mutant libraries. Automated image acquisition and analysis allowed us to identify genes that affect the localization of two polar cell cycle histidine kinases, PleC and DivJ, and the pole-specific pili protein CpaE, each tagged with a different fluorescent marker in a single strain. Four metrics characterizing the observed localization patterns of each of the three labeled proteins were extracted for hundreds of cell images from each of 854 mapped mutant strains. Using cluster analysis of the resulting set of 12-element vectors for each of these strains, we identified 52 strains with mutations that affected the localization pattern of the three tagged proteins. This information, combined with quantitative localization data from epitasis experiments, also identified all previously known proteins affecting such localization. These studies provide insights into factors affecting the PleC/DivJ localization network and into regulatory links between the localization of the pili assembly protein CpaE and the kinase localization pathway. Our high-throughput screening methodology can be adapted readily to any sequenced bacterial species, opening the potential for databases of localization regulatory networks across species, and investigation of localization network phylogenies.

  14. Phenotype MicroArrays as a complementary tool to next generation sequencing for characterization of tree endophytes

    PubMed Central

    Blumenstein, Kathrin; Macaya-Sanz, David; Martín, Juan A.; Albrectsen, Benedicte R.; Witzell, Johanna

    2015-01-01

    There is an increasing need to calibrate microbial community profiles obtained through next generation sequencing (NGS) with relevant taxonomic identities of the microbes, and to further associate these identities with phenotypic attributes. Phenotype MicroArray (PM) techniques provide a semi-high throughput assay for characterization and monitoring the microbial cellular phenotypes. Here, we present detailed descriptions of two different PM protocols used in our recent studies on fungal endophytes of forest trees, and highlight the benefits and limitations of this technique. We found that the PM approach enables effective screening of substrate utilization by endophytes. However, the technical limitations are multifaceted and the interpretation of the PM data challenging. For the best result, we recommend that the growth conditions for the fungi are carefully standardized. In addition, rigorous replication and control strategies should be employed whether using pre-configured, commercial microwell-plates or in-house designed PM plates for targeted substrate analyses. With these precautions, the PM technique is a valuable tool to characterize the metabolic capabilities of individual endophyte isolates, or successional endophyte communities identified by NGS, allowing a functional interpretation of the taxonomic data. Thus, PM approaches can provide valuable complementary information for NGS studies of fungal endophytes in forest trees. PMID:26441951

  15. High-Throughput Models for Exposure-Based Chemical Prioritization in the ExpoCast Project

    EPA Science Inventory

    The United States Environmental Protection Agency (U.S. EPA) must characterize potential risks to human health and the environment associated with manufacture and use of thousands of chemicals. High-throughput screening (HTS) for biological activity allows the ToxCast research pr...

  16. High-Throughput Simulation of Environmental Chemical Fate for Exposure Prioritization (Annual Meeting of ISES)

    EPA Science Inventory

    The U.S. EPA must consider thousands of chemicals when allocating resources to assess risk in human populations and the environment. High-throughput screening assays to characterize biological activity in vitro are being implemented in the ToxCastTM program to rapidly characteri...

  17. High-Throughput Simulation of Environmental Chemical Fate for Exposure Prioritization

    EPA Science Inventory

    The U.S. EPA must consider lists of hundreds to thousands of chemicals when allocating resources to identify risk in human populations and the environment. High-throughput screening assays to characterize biological activity in vitro have allowed the ToxCastTM program to identify...

  18. Predictive Model of Rat Reproductive Toxicity from ToxCast High Throughput Screening

    EPA Science Inventory

    The EPA ToxCast research program uses high throughput screening for bioactivity profiling and predicting the toxicity of large numbers of chemicals. ToxCast Phase‐I tested 309 well‐characterized chemicals in over 500 assays for a wide range of molecular targets and cellular respo...

  19. Quantifying the Onset and Progression of Plant Senescence by Color Image Analysis for High Throughput Applications

    PubMed Central

    Cai, Jinhai; Okamoto, Mamoru; Atieno, Judith; Sutton, Tim; Li, Yongle; Miklavcic, Stanley J.

    2016-01-01

    Leaf senescence, an indicator of plant age and ill health, is an important phenotypic trait for the assessment of a plant’s response to stress. Manual inspection of senescence, however, is time consuming, inaccurate and subjective. In this paper we propose an objective evaluation of plant senescence by color image analysis for use in a high throughput plant phenotyping pipeline. As high throughput phenotyping platforms are designed to capture whole-of-plant features, camera lenses and camera settings are inappropriate for the capture of fine detail. Specifically, plant colors in images may not represent true plant colors, leading to errors in senescence estimation. Our algorithm features a color distortion correction and image restoration step prior to a senescence analysis. We apply our algorithm to two time series of images of wheat and chickpea plants to quantify the onset and progression of senescence. We compare our results with senescence scores resulting from manual inspection. We demonstrate that our procedure is able to process images in an automated way for an accurate estimation of plant senescence even from color distorted and blurred images obtained under high throughput conditions. PMID:27348807

  20. High-throughput biochemical fingerprinting of Saccharomyces cerevisiae by Fourier transform infrared spectroscopy.

    PubMed

    Kohler, Achim; Böcker, Ulrike; Shapaval, Volha; Forsmark, Annabelle; Andersson, Mats; Warringer, Jonas; Martens, Harald; Omholt, Stig W; Blomberg, Anders

    2015-01-01

    Single-channel optical density measurements of population growth are the dominant large scale phenotyping methodology for bridging the gene-function gap in yeast. However, a substantial amount of the genetic variation induced by single allele, single gene or double gene knock-out technologies fail to manifest in detectable growth phenotypes under conditions readily testable in the laboratory. Thus, new high-throughput phenotyping technologies capable of providing information about molecular level consequences of genetic variation are sorely needed. Here we report a protocol for high-throughput Fourier transform infrared spectroscopy (FTIR) measuring biochemical fingerprints of yeast strains. It includes high-throughput cultivation for FTIR spectroscopy, FTIR measurements and spectral pre-treatment to increase measurement accuracy. We demonstrate its capacity to distinguish not only yeast genera, species and populations, but also strains that differ only by a single gene, its excellent signal-to-noise ratio and its relative robustness to measurement bias. Finally, we illustrated its applicability by determining the FTIR signatures of all viable Saccharomyces cerevisiae single gene knock-outs corresponding to lipid biosynthesis genes. Many of the examined knock-out strains showed distinct, highly reproducible FTIR phenotypes despite having no detectable growth phenotype. These phenotypes were confirmed by conventional lipid analysis and could be linked to specific changes in lipid composition. We conclude that the introduced protocol is robust to noise and bias, possible to apply on a very large scale, and capable of generating biologically meaningful biochemical fingerprints that are strain specific, even when strains lack detectable growth phenotypes. Thus, it has a substantial potential for application in the molecular functionalization of the yeast genome.

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

    PubMed

    Alonso-Padilla, Julio; Rodríguez, Ana

    2014-12-01

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

  2. Functional approach to high-throughput plant growth analysis

    PubMed Central

    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

  3. The overshoot and phenotypic equilibrium in characterizing cancer dynamics of reversible phenotypic plasticity.

    PubMed

    Chen, Xiufang; Wang, Yue; Feng, Tianquan; Yi, Ming; Zhang, Xingan; Zhou, Da

    2016-02-07

    The paradigm of phenotypic plasticity indicates reversible relations of different cancer cell phenotypes, which extends the cellular hierarchy proposed by the classical cancer stem cell (CSC) theory. Since it is still questionable if the phenotypic plasticity is a crucial improvement to the hierarchical model or just a minor extension to it, it is worthwhile to explore the dynamic behavior characterizing the reversible phenotypic plasticity. In this study we compare the hierarchical model and the reversible model in predicting the cell-state dynamics observed in biological experiments. Our results show that the hierarchical model shows significant disadvantages over the reversible model in describing both long-term stability (phenotypic equilibrium) and short-term transient dynamics (overshoot) in cancer cell populations. In a very specific case in which the total growth of population due to each cell type is identical, the hierarchical model predicts neither phenotypic equilibrium nor overshoot, whereas the reversible model succeeds in predicting both of them. Even though the performance of the hierarchical model can be improved by relaxing the specific assumption, its prediction to the phenotypic equilibrium strongly depends on a precondition that may be unrealistic in biological experiments. Moreover, it still does not show as rich dynamics as the reversible model in capturing the overshoots of both CSCs and non-CSCs. By comparison, it is more likely for the reversible model to correctly predict the stability of the phenotypic mixture and various types of overshoot behavior.

  4. Discovery of New Compounds Active against Plasmodium falciparum by High Throughput Screening of Microbial Natural Products.

    PubMed

    Pérez-Moreno, Guiomar; Cantizani, Juan; Sánchez-Carrasco, Paula; Ruiz-Pérez, Luis Miguel; Martín, Jesús; El Aouad, Noureddine; Pérez-Victoria, Ignacio; Tormo, José Rubén; González-Menendez, Víctor; González, Ignacio; de Pedro, Nuria; Reyes, Fernando; Genilloud, Olga; Vicente, Francisca; González-Pacanowska, Dolores

    2016-01-01

    Due to the low structural diversity within the set of antimalarial drugs currently available in the clinic and the increasing number of cases of resistance, there is an urgent need to find new compounds with novel modes of action to treat the disease. Microbial natural products are characterized by their large diversity provided in terms of the chemical complexity of the compounds and the novelty of structures. Microbial natural products extracts have been underexplored in the search for new antiparasitic drugs and even more so in the discovery of new antimalarials. Our objective was to find new druggable natural products with antimalarial properties from the MEDINA natural products collection, one of the largest natural product libraries harboring more than 130,000 microbial extracts. In this work, we describe the optimization process and the results of a phenotypic high throughput screen (HTS) based on measurements of Plasmodium lactate dehydrogenase. A subset of more than 20,000 extracts from the MEDINA microbial products collection has been explored, leading to the discovery of 3 new compounds with antimalarial activity. In addition, we report on the novel antiplasmodial activity of 4 previously described natural products.

  5. High throughput imaging and analysis for biological interpretation of agricultural plants and environmental interaction

    NASA Astrophysics Data System (ADS)

    Hong, Hyundae; Benac, Jasenka; Riggsbee, Daniel; Koutsky, Keith

    2014-03-01

    High throughput (HT) phenotyping of crops is essential to increase yield in environments deteriorated by climate change. The controlled environment of a greenhouse offers an ideal platform to study the genotype to phenotype linkages for crop screening. Advanced imaging technologies are used to study plants' responses to resource limitations such as water and nutrient deficiency. Advanced imaging technologies coupled with automation make HT phenotyping in the greenhouse not only feasible, but practical. Monsanto has a state of the art automated greenhouse (AGH) facility. Handling of the soil, pots water and nutrients are all completely automated. Images of the plants are acquired by multiple hyperspectral and broadband cameras. The hyperspectral cameras cover wavelengths from visible light through short wave infra-red (SWIR). Inhouse developed software analyzes the images to measure plant morphological and biochemical properties. We measure phenotypic metrics like plant area, height, and width as well as biomass. Hyperspectral imaging allows us to measure biochemcical metrics such as chlorophyll, anthocyanin, and foliar water content. The last 4 years of AGH operations on crops like corn, soybean, and cotton have demonstrated successful application of imaging and analysis technologies for high throughput plant phenotyping. Using HT phenotyping, scientists have been showing strong correlations to environmental conditions, such as water and nutrient deficits, as well as the ability to tease apart distinct differences in the genetic backgrounds of crops.

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

    PubMed

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

    2013-06-07

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

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

    PubMed Central

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

    2004-01-01

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

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  11. HIGH THROUGHPUT ASSESSMENTS OF CONVENTIONAL AND ALTERNATIVE COMPOUNDS

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  13. High-Throughput Metagenomic Technologies for Complex Microbial Community Analysis: Open and Closed Formats

    PubMed Central

    He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G.; Alvarez-Cohen, Lisa

    2015-01-01

    ABSTRACT   Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied “open-format” and “closed-format” detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions. PMID:25626903

  14. High-throughput metagenomic technologies for complex microbial community analysis. Open and closed formats

    SciTech Connect

    Zhou, Jizhong; He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G.; Alvarez-Cohen, Lisa

    2015-01-27

    Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied “open-format” and “closed-format” detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions.

  15. A note on statistical repeatability and study design for high-throughput assays.

    PubMed

    Nicholson, George; Holmes, Chris

    2017-02-28

    Characterizing the technical precision of measurements is a necessary stage in the planning of experiments and in the formal sample size calculation for optimal design. Instruments that measure multiple analytes simultaneously, such as in high-throughput assays arising in biomedical research, pose particular challenges from a statistical perspective. The current most popular method for assessing precision of high-throughput assays is by scatterplotting data from technical replicates. Here, we question the statistical rationale of this approach from both an empirical and theoretical perspective, illustrating our discussion using four example data sets from different genomic platforms. We demonstrate that such scatterplots convey little statistical information of relevance and are potentially highly misleading. We present an alternative framework for assessing the precision of high-throughput assays and planning biomedical experiments. Our methods are based on repeatability-a long-established statistical quantity also known as the intraclass correlation coefficient. We provide guidance and software for estimation and visualization of repeatability of high-throughput assays, and for its incorporation into study design. © 2016 The Authors. Statistics in Medicine Published by John Wiley & Sons Ltd.

  16. Applications of high throughput (combinatorial) methodologies to electronic, magnetic, optical, and energy-related materials

    NASA Astrophysics Data System (ADS)

    Green, Martin L.; Takeuchi, Ichiro; Hattrick-Simpers, Jason R.

    2013-06-01

    High throughput (combinatorial) materials science methodology is a relatively new research paradigm that offers the promise of rapid and efficient materials screening, optimization, and discovery. The paradigm started in the pharmaceutical industry but was rapidly adopted to accelerate materials research in a wide variety of areas. High throughput experiments are characterized by synthesis of a "library" sample that contains the materials variation of interest (typically composition), and rapid and localized measurement schemes that result in massive data sets. Because the data are collected at the same time on the same "library" sample, they can be highly uniform with respect to fixed processing parameters. This article critically reviews the literature pertaining to applications of combinatorial materials science for electronic, magnetic, optical, and energy-related materials. It is expected that high throughput methodologies will facilitate commercialization of novel materials for these critically important applications. Despite the overwhelming evidence presented in this paper that high throughput studies can effectively inform commercial practice, in our perception, it remains an underutilized research and development tool. Part of this perception may be due to the inaccessibility of proprietary industrial research and development practices, but clearly the initial cost and availability of high throughput laboratory equipment plays a role. Combinatorial materials science has traditionally been focused on materials discovery, screening, and optimization to combat the extremely high cost and long development times for new materials and their introduction into commerce. Going forward, combinatorial materials science will also be driven by other needs such as materials substitution and experimental verification of materials properties predicted by modeling and simulation, which have recently received much attention with the advent of the Materials Genome

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

    PubMed

    Rathore, Anurag S; Bhambure, Rahul

    2014-01-01

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

  18. High throughput optoelectronic smart pixel systems using diffractive optics

    NASA Astrophysics Data System (ADS)

    Chen, Chih-Hao

    1999-12-01

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

  19. High-throughput neuroimaging-genetics computational infrastructure.

    PubMed

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

    2014-01-01

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

  20. High-throughput neuroimaging-genetics computational infrastructure

    PubMed Central

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

    2014-01-01

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

  1. Three-Dimensional High-Throughput Cell Encapsulation Platform to Study Changes in Cell-Matrix Interactions.

    PubMed

    Mabry, Kelly M; Schroeder, Megan E; Payne, Samuel Z; Anseth, Kristi S

    2016-08-31

    In their native extracellular microenvironment, cells respond to a complex array of biochemical and mechanical cues that can vary in both time and space. High-throughput methods that allow characterization of cell-laden matrices are valuable tools to screen through many combinations of variables, ultimately helping to evolve and test hypotheses related to cell-ECM signaling. Here, we developed a platform for high-throughput encapsulation of cells in peptide-functionalized poly(ethylene glycol) hydrogels. Hydrogels were synthesized using a thiol-ene, photoclick reaction, which allowed the cell matrix environment to be modified in real time. Matrix signals were dynamically altered by in situ tethering of RGDS (0-1.5 mM), a fibronectin-derived adhesive peptide that induced more elongation than RLD or IKVAV, and/or by increasing the matrix modulus (1 to 6 kPa). This method was demonstrated with aortic valvular interstitial cells (VICs), a population of cells responsible for the pathological fibrosis and matrix remodeling that leads to aortic stenosis. VIC response to cell-matrix interactions was characterized by quantifying cell morphology and the fraction of cells exhibiting α-smooth muscle actin (αSMA) stress fibers, a hallmark of the myofibroblast phenotype. VICs elongated in response to RGDS addition, with a dramatic change in morphology within 24 h. Myofibroblast activation was also dependent on RGDS addition, with VICs exhibiting high activation (16-24%) in 1 kPa gels with RGDS. Response to RGDS was path-dependent, with the amount of time exposed to the adhesive ligand important in determining VIC morphology and activation. Although VIC aspect ratios were dependent on the amount of time spent in a stiff vs soft gel, low levels of VIC activation (≤4%) were observed in any gels cultured in higher modulus (6 kPa vs 1 kPa) microenvironments.

  2. Validation of high throughput sequencing and microbial forensics applications

    PubMed Central

    2014-01-01

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

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

    PubMed

    Prummer, Michael

    2012-04-01

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

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

    PubMed

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

    2015-09-01

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

  5. The JCSG high-throughput structural biology pipeline

    PubMed Central

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

    2010-01-01

    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. PMID:20944202

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

    PubMed

    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.

  7. Tissue Doppler characterization of cardiac phenotype in mouse.

    PubMed

    Fayssoil, Abdallah

    2009-10-01

    Mice allow biologists to study various genes playing a role in cardiac function and pathophysiological situations. Echocardiography is a non-invasive tool for assessing cardiac phenotype. Because of load dependence of conventional parameters (left ventricular shortening fraction, left ventricular ejection fraction and mitral pulsed Doppler), we have to perform Doppler tissular velocity imaging and strain imaging for the characterization of cardiomyopathies mice models.

  8. Phenotypic Characterization of a Diversity Panel of Tomato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    At the USDA, ARS Plant Genetic Resources Unit (PGRU) we have phenotypically characterized more than 2,000 accessions of tomato (Solanum lycopersicum L.) for which data are publically available on the National Plant Germplasm System (NPGS) Germplasm Resources Information Network (GRIN) (http://www.ar...

  9. Primary cells and stem cells in drug discovery: emerging tools for high-throughput screening.

    PubMed

    Eglen, Richard; Reisine, Terry

    2011-04-01

    Many drug discovery screening programs employ immortalized cells, recombinantly engineered to express a defined molecular target. Several technologies are now emerging that render it feasible to employ more physiologically, and clinically relevant, cell phenotypes. Consequently, numerous approaches use primary cells, which retain many functions seen in vivo, as well as endogenously expressing the target of interest. Furthermore, stem cells, of either embryonic or adult origin, as well as those derived from differentiated cells, are now finding a place in drug discovery. Collectively, these cells are expanding the utility of authentic human cells, either as screening tools or as therapeutics, as well as providing cells derived directly from patients. Nonetheless, the growing use of phenotypically relevant cells (including primary cells or stem cells) is not without technical difficulties, particularly when their envisioned use lies in high-throughput screening (HTS) protocols. In particular, the limited availability of homogeneous primary or stem cell populations for HTS mandates that novel technologies be developed to accelerate their adoption. These technologies include detection of responses with very few cells as well as protocols to generate cell lines in abundant, homogeneous populations. In parallel, the growing use of changes in cell phenotype as the assay readout is driving greater use of high-throughput imaging techniques in screening. Taken together, the greater availability of novel primary and stem cell phenotypes as well as new detection technologies is heralding a new era of cellular screening. This convergence offers unique opportunities to identify drug candidates for disorders at which few therapeutics are presently available.

  10. Experimental Design for Combinatorial and High Throughput Materials Development

    NASA Astrophysics Data System (ADS)

    Cawse, James N.

    2002-12-01

    In the past decade, combinatorial and high throughput experimental methods have revolutionized the pharmaceutical industry, allowing researchers to conduct more experiments in a week than was previously possible in a year. Now high throughput experimentation is rapidly spreading from its origins in the pharmaceutical world to larger industrial research establishments such as GE and DuPont, and even to smaller companies and universities. Consequently, researchers need to know the kinds of problems, desired outcomes, and appropriate patterns for these new strategies. Editor James Cawse's far-reaching study identifies and applies, with specific examples, these important new principles and techniques. Experimental Design for Combinatorial and High Throughput Materials Development progresses from methods that are now standard, such as gradient arrays, to mathematical developments that are breaking new ground. The former will be particularly useful to researchers entering the field, while the latter should inspire and challenge advanced practitioners. The book's contents are contributed by leading researchers in their respective fields. Chapters include: -High Throughput Synthetic Approaches for the Investigation of Inorganic Phase Space -Combinatorial Mapping of Polymer Blends Phase Behavior -Split-Plot Designs -Artificial Neural Networks in Catalyst Development -The Monte Carlo Approach to Library Design and Redesign This book also contains over 200 useful charts and drawings. Industrial chemists, chemical engineers, materials scientists, and physicists working in combinatorial and high throughput chemistry will find James Cawse's study to be an invaluable resource.

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

    PubMed Central

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

    2015-01-01

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

  12. Adapting Cell-Based Assays to the High Throughput Screening Platform: Problems Encountered and Lessons Learned.

    PubMed

    Maddox, Clinton B; Rasmussen, Lynn; White, E Lucile

    2008-06-01

    In recent years, cell-based phenotypic assays have emerged as an effective and robust addition to the array of assay technologies available for drug discovery in the high throughput screening arena. Previously, biochemical target-based assays have been the technology of choice. With the emergence of stem cells as a basis for a new screening technology, it is important to keep in mind the lessons that have been learned from the adaptation of existing stable cell lines onto the high throughput screening drug discovery platform, with special consideration being given to assay miniaturization, liquid handling complications and instrument-introduced artifacts. We present an overview of the problems encountered with the implementation of multiple cell-based assays at the High Throughput Screening Center at Southern Research Institute as well as empirically defined effective solutions to these problems. These include examples of artifacts induced by temperature differences throughout the screening campaign, cell plating conditions including the effect of room temperature incubation on assay consistency, DMSO carry-over, and incubator induced artifacts.

  13. Rapid and high-throughput construction of microbial cell-factories with regulatory noncoding RNAs.

    PubMed

    Chaudhary, Amit Kumar; Na, Dokyun; Lee, Eun Yeol

    2015-11-01

    Due to global crises such as pollution and depletion of fossil fuels, sustainable technologies based on microbial cell-factories have been garnering great interest as an alternative to chemical factories. The development of microbial cell-factories is imperative in cutting down the overall manufacturing cost. Thus, diverse metabolic engineering strategies and engineering tools have been established to obtain a preferred genotype and phenotype displaying superior productivity. However, these tools are limited to only a handful of genes with permanent modification of a genome and significant labor costs, and this is one of the bottlenecks associated with biofactory construction. Therefore, a groundbreaking rapid and high-throughput engineering tool is needed for efficient construction of microbial cell-factories. During the last decade, copious small noncoding RNAs (ncRNAs) have been discovered in bacteria. These are involved in substantial regulatory roles like transcriptional and post-transcriptional gene regulation by modulating mRNA elongation, stability, or translational efficiency. Because of their vulnerability, ncRNAs can be used as another layer of conditional control over gene expression without modifying chromosomal sequences, and hence would be a promising high-throughput tool for metabolic engineering. Here, we review successful design principles and applications of ncRNAs for high-throughput metabolic engineering or physiological studies of diverse industrially important microorganisms.

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

    PubMed

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

    2015-07-28

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

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

    PubMed

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

    2014-06-01

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

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

    PubMed

    Liu, Wenshan; Jiang, Rongrong

    2015-03-01

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

  17. Phenotypic Characterization of Toxic Compound Effects on Liver Spheroids Derived from iPSC Using Confocal Imaging and Three-Dimensional Image Analysis.

    PubMed

    Sirenko, Oksana; Hancock, Michael K; Hesley, Jayne; Hong, Dihui; Cohen, Avrum; Gentry, Jason; Carlson, Coby B; Mann, David A

    2016-09-01

    Cell models are becoming more complex to better mimic the in vivo environment and provide greater predictivity for compound efficacy and toxicity. There is an increasing interest in exploring the use of three-dimensional (3D) spheroids for modeling developmental and tissue biology with the goal of accelerating translational research in these areas. Accordingly, the development of high-throughput quantitative assays using 3D cultures is an active area of investigation. In this study, we have developed and optimized methods for the formation of 3D liver spheroids derived from human iPS cells and used those for toxicity assessment. We used confocal imaging and 3D image analysis to characterize cellular information from a 3D matrix to enable a multi-parametric comparison of different spheroid phenotypes. The assay enables characterization of compound toxicities by spheroid size (volume) and shape, cell number and spatial distribution, nuclear characterization, number and distribution of cells expressing viability, apoptosis, mitochondrial potential, and viability marker intensities. In addition, changes in the content of live, dead, and apoptotic cells as a consequence of compound exposure were characterized. We tested 48 compounds and compared induced pluripotent stem cell (iPSC)-derived hepatocytes and HepG2 cells in both two-dimensional (2D) and 3D cultures. We observed significant differences in the pharmacological effects of compounds across the two cell types and between the different culture conditions. Our results indicate that a phenotypic assay using 3D model systems formed with human iPSC-derived hepatocytes is suitable for high-throughput screening and can be used for hepatotoxicity assessment in vitro.

  18. Phenotypic Characterization of Toxic Compound Effects on Liver Spheroids Derived from iPSC Using Confocal Imaging and Three-Dimensional Image Analysis

    PubMed Central

    Hancock, Michael K.; Hesley, Jayne; Hong, Dihui; Cohen, Avrum; Gentry, Jason; Carlson, Coby B.

    2016-01-01

    Abstract Cell models are becoming more complex to better mimic the in vivo environment and provide greater predictivity for compound efficacy and toxicity. There is an increasing interest in exploring the use of three-dimensional (3D) spheroids for modeling developmental and tissue biology with the goal of accelerating translational research in these areas. Accordingly, the development of high-throughput quantitative assays using 3D cultures is an active area of investigation. In this study, we have developed and optimized methods for the formation of 3D liver spheroids derived from human iPS cells and used those for toxicity assessment. We used confocal imaging and 3D image analysis to characterize cellular information from a 3D matrix to enable a multi-parametric comparison of different spheroid phenotypes. The assay enables characterization of compound toxicities by spheroid size (volume) and shape, cell number and spatial distribution, nuclear characterization, number and distribution of cells expressing viability, apoptosis, mitochondrial potential, and viability marker intensities. In addition, changes in the content of live, dead, and apoptotic cells as a consequence of compound exposure were characterized. We tested 48 compounds and compared induced pluripotent stem cell (iPSC)-derived hepatocytes and HepG2 cells in both two-dimensional (2D) and 3D cultures. We observed significant differences in the pharmacological effects of compounds across the two cell types and between the different culture conditions. Our results indicate that a phenotypic assay using 3D model systems formed with human iPSC-derived hepatocytes is suitable for high-throughput screening and can be used for hepatotoxicity assessment in vitro. PMID:27494736

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

    PubMed

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

    2014-05-01

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

  20. GBM heterogeneity characterization by radiomic analysis of phenotype anatomical planes

    NASA Astrophysics Data System (ADS)

    Chaddad, Ahmad; Desrosiers, Christian; Toews, Matthew

    2016-03-01

    Glioblastoma multiforme (GBM) is the most common malignant primary tumor of the central nervous system, characterized among other traits by rapid metastatis. Three tissue phenotypes closely associated with GBMs, namely, necrosis (N), contrast enhancement (CE), and edema/invasion (E), exhibit characteristic patterns of texture heterogeneity in magnetic resonance images (MRI). In this study, we propose a novel model to characterize GBM tissue phenotypes using gray level co-occurrence matrices (GLCM) in three anatomical planes. The GLCM encodes local image patches in terms of informative, orientation-invariant texture descriptors, which are used here to sub-classify GBM tissue phenotypes. Experiments demonstrate the model on MRI data of 41 GBM patients, obtained from the cancer genome atlas (TCGA). Intensity-based automatic image registration is applied to align corresponding pairs of fixed T1˗weighted (T1˗WI) post-contrast and fluid attenuated inversion recovery (FLAIR) images. GBM tissue regions are then segmented using the 3D Slicer tool. Texture features are computed from 12 quantifier functions operating on GLCM descriptors, that are generated from MRI intensities within segmented GBM tissue regions. Various classifier models are used to evaluate the effectiveness of texture features for discriminating between GBM phenotypes. Results based on T1-WI scans showed a phenotype classification accuracy of over 88.14%, a sensitivity of 85.37% and a specificity of 96.1%, using the linear discriminant analysis (LDA) classifier. This model has the potential to provide important characteristics of tumors, which can be used for the sub-classification of GBM phenotypes.

  1. Advances in high throughput DNA sequence data compression.

    PubMed

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

    2016-06-01

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

  2. Droplet microfluidics for high-throughput biological assays.

    PubMed

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

    2012-06-21

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

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

    PubMed Central

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

    2015-01-01

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

  4. Implementation of high throughput experimentation techniques for kinetic reaction testing.

    PubMed

    Nagy, Anton J

    2012-02-01

    Successful implementation of High throughput Experimentation (EE) tools has resulted in their increased acceptance as essential tools in chemical, petrochemical and polymer R&D laboratories. This article provides a number of concrete examples of EE systems, which have been designed and successfully implemented in studies, which focus on deriving reaction kinetic data. The implementation of high throughput EE tools for performing kinetic studies of both catalytic and non-catalytic systems results in a significantly faster acquisition of high-quality kinetic modeling data, required to quantitatively predict the behavior of complex, multistep reactions.

  5. Perspective: Data infrastructure for high throughput materials discovery

    NASA Astrophysics Data System (ADS)

    Pfeif, E. A.; Kroenlein, K.

    2016-05-01

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

  6. High-throughput synchrotron X-ray diffraction for combinatorial phase mapping.

    PubMed

    Gregoire, J M; Van Campen, D G; Miller, C E; Jones, R J R; Suram, S K; Mehta, A

    2014-11-01

    Discovery of new materials drives the deployment of new technologies. Complex technological requirements demand precisely tailored material functionalities, and materials scientists are driven to search for these new materials in compositionally complex and often non-equilibrium spaces containing three, four or more elements. The phase behavior of these high-order composition spaces is mostly unknown and unexplored. High-throughput methods can offer strategies for efficiently searching complex and multi-dimensional material genomes for these much needed new materials and can also suggest a processing pathway for synthesizing them. However, high-throughput structural characterization is still relatively under-developed for rapid material discovery. Here, a synchrotron X-ray diffraction and fluorescence experiment for rapid measurement of both X-ray powder patterns and compositions for an array of samples in a material library is presented. The experiment is capable of measuring more than 5000 samples per day, as demonstrated by the acquisition of high-quality powder patterns in a bismuth-vanadium-iron oxide composition library. A detailed discussion of the scattering geometry and its ability to be tailored for different material systems is provided, with specific attention given to the characterization of fiber textured thin films. The described prototype facility is capable of meeting the structural characterization needs for the first generation of high-throughput material genomic searches.

  7. The FlyCatwalk: A High-Throughput Feature-Based Sorting System for Artificial Selection in Drosophila

    PubMed Central

    Medici, Vasco; Vonesch, Sibylle Chantal; Fry, Steven N.; Hafen, Ernst

    2015-01-01

    Experimental evolution is a powerful tool for investigating complex traits. Artificial selection can be applied for a specific trait and the resulting phenotypically divergent populations pool-sequenced to identify alleles that occur at substantially different frequencies in the extreme populations. To maximize the proportion of loci that are causal to the phenotype among all enriched loci, population size and number of replicates need to be high. These requirements have, in fact, limited evolution studies in higher organisms, where the time investment required for phenotyping is often prohibitive for large-scale studies. Animal size is a highly multigenic trait that remains poorly understood, and an experimental evolution approach may thus aid in gaining new insights into the genetic basis of this trait. To this end, we developed the FlyCatwalk, a fully automated, high-throughput system to sort live fruit flies (Drosophila melanogaster) based on morphometric traits. With the FlyCatwalk, we can detect gender and quantify body and wing morphology parameters at a four-old higher throughput compared with manual processing. The phenotyping results acquired using the FlyCatwalk correlate well with those obtained using the standard manual procedure. We demonstrate that an automated, high-throughput, feature-based sorting system is able to avoid previous limitations in population size and replicate numbers. Our approach can likewise be applied for a variety of traits and experimental settings that require high-throughput phenotyping. PMID:25556112

  8. The FlyCatwalk: a high-throughput feature-based sorting system for artificial selection in Drosophila.

    PubMed

    Medici, Vasco; Vonesch, Sibylle Chantal; Fry, Steven N; Hafen, Ernst

    2015-01-02

    Experimental evolution is a powerful tool for investigating complex traits. Artificial selection can be applied for a specific trait and the resulting phenotypically divergent populations pool-sequenced to identify alleles that occur at substantially different frequencies in the extreme populations. To maximize the proportion of loci that are causal to the phenotype among all enriched loci, population size and number of replicates need to be high. These requirements have, in fact, limited evolution studies in higher organisms, where the time investment required for phenotyping is often prohibitive for large-scale studies. Animal size is a highly multigenic trait that remains poorly understood, and an experimental evolution approach may thus aid in gaining new insights into the genetic basis of this trait. To this end, we developed the FlyCatwalk, a fully automated, high-throughput system to sort live fruit flies (Drosophila melanogaster) based on morphometric traits. With the FlyCatwalk, we can detect gender and quantify body and wing morphology parameters at a four-old higher throughput compared with manual processing. The phenotyping results acquired using the FlyCatwalk correlate well with those obtained using the standard manual procedure. We demonstrate that an automated, high-throughput, feature-based sorting system is able to avoid previous limitations in population size and replicate numbers. Our approach can likewise be applied for a variety of traits and experimental settings that require high-throughput phenotyping.

  9. Vacuum Seed Sowing Manifold: a novel device for high-throughput sowing of Arabidopsis seeds

    PubMed Central

    2013-01-01

    The small size of Arabidopsis provides both opportunities and difficulties for laboratory research. Large numbers of plants can be grown in a relatively small area making it easy to observe and investigate interesting phenotypes. Conversely, their small size can also make it difficult to obtain large quantities of tissue for investigation using modern molecular techniques. Sowing large numbers of their seed can overcome this; however, their small seed size makes this difficult. Here we present the Vacuum Seed Sowing Manifold (VSSM), a simple device that can be printed using a 3D printer and provides a new high throughput method to sow large numbers of seeds at a range of densities. PMID:24148867

  10. Glycomics meets genomics, epigenomics and other high throughput omics for system biology studies.

    PubMed

    Zoldoš, Vlatka; Horvat, Tomislav; Lauc, Gordan

    2013-02-01

    Majority of eukaryotic proteins are glycosylated and their glycan moieties have numerous important structural, functional and regulatory roles. Because of structural complexity of glycans and technological limitations glycomics, and particularly glycoproteomics was not able to follow rapid progress in genomics and proteomics over last 30 years. However, the field of glycan has been progressing rapidly and first large-scale studies of the glycome have been completed recently. These studies have revealed significant differences in glycome composition between individuals, which may contribute to the human phenotypic variability. The current state-of-the-art in high-throughput glycomics and its integration with genomics, epigenomics and lipidomics is reviewed in this article.

  11. Automated Segmentation and Classification of High Throughput Yeast Assay Spots

    PubMed Central

    Jafari-Khouzani, Kourosh; Soltanian-Zadeh, Hamid; Fotouhi, Farshad; Parrish, Jodi R.; Finley, Russell L.

    2009-01-01

    Several technologies for characterizing genes and proteins from humans and other organisms use yeast growth or color development as read outs. The yeast two-hybrid assay, for example, detects protein-protein interactions by measuring the growth of yeast on a specific solid medium, or the ability of the yeast to change color when grown on a medium containing a chromogenic substrate. Current systems for analyzing the results of these types of assays rely on subjective and inefficient scoring of growth or color by human experts. Here an image analysis system is described for scoring yeast growth and color development in high throughput biological assays. The goal is to locate the spots and score them in color images of two types of plates named “X-Gal” and “growth assay” plates, with uniformly placed spots (cell areas) on each plate (both plates in one image). The scoring system relies on color for the X-Gal spots, and texture properties for the growth assay spots. A maximum likelihood projection-based segmentation is developed to automatically locate spots of yeast on each plate. Then color histogram and wavelet texture features are extracted for scoring using an optimal linear transformation. Finally an artificial neural network is used to score the X-Gal and growth assay spots using the extracted features. The performance of the system is evaluated using spots of 60 images. After training the networks using training and validation sets, the system was assessed on the test set. The overall accuracies of 95.4% and 88.2% are achieved respectively for scoring the X-Gal and growth assay spots. PMID:17948730

  12. High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC).

    PubMed

    Camilo, Cesar M; Polikarpov, Igor

    2014-07-01

    Recent advances in DNA sequencing techniques have led to an explosion in the amount of available genome sequencing data and this provided an inexhaustible source of uncharacterized glycoside hydrolases (GH) to be studied both structurally and enzymatically. Ligation-Independent Cloning (LIC), an interesting alternative to traditional, restriction enzyme-based cloning, and commercial recombinatorial cloning, was adopted and optimized successfully for a high throughput cloning, expression and purification pipeline. Using this platform, 130 genes encoding mainly uncharacterized glycoside hydrolases from 13 different organisms were cloned and submitted to a semi-automated protein expression and solubility screening in Escherichia coli, resulting in 73 soluble targets. The high throughput approach proved to be a powerful tool for production of recombinant glycoside hydrolases for further structural and biochemical characterization and confirmed that thioredoxin fusion tag (TRX) is a better choice to increase solubility of recombinant glycoside hydrolases expressed in E. coli, when compared to His-tag alone.

  13. High-throughput syntheses of iron phosphite open frameworks in ionic liquids

    NASA Astrophysics Data System (ADS)

    Wang, Zhixiu; Mu, Ying; Wang, Yilin; Bing, Qiming; Su, Tan; Liu, Jingyao

    2017-02-01

    Three open-framework iron phosphites: Feп5(NH4)2(HPO3)6 (1), Feп2Fe♯(NH4)(HPO3)4 (2) and Fe♯2(HPO3)3 (3) have been synthesized under ionothermal conditions. How the different synthesis parameters, such as the gel concentrations, synthetic times, reaction temperatures and solvents affect the products have been monitored by using high-throughput approaches. Within each type of experiment, relevant products have been investigated. The optimal reaction conditions are obtained from a series of experiments by high-throughput approaches. All the structures are determined by single-crystal X-ray diffraction analysis and further characterized by PXRD, TGA and FTIR analyses. Magnetic study reveals that those three compounds show interesting magnetic behavior at low temperature.

  14. From Classical to High Throughput Screening Methods for Feruloyl Esterases: A Review.

    PubMed

    Ramírez-Velasco, Lorena; Armendáriz-Ruiz, Mariana; Rodríguez-González, Jorge Alberto; Müller-Santos, Marcelo; Asaff-Torres, Ali; Mateos-Díaz, Juan Carlos

    2016-01-01

    Feruloyl esterases (FAEs) are a diverse group of hydrolases widely distributed in plants and microorganisms which catalyzes the cleavage and formation of ester bonds between plant cell wall polysaccharides and phenolic acids. FAEs have gained importance in biofuel, medicine and food industries due to their capability of acting on a large range of substrates for cleaving ester bonds and synthesizing highadded value molecules through esterification and transesterification reactions. During the past two decades extensive studies have been carried out on the production, characterization and classification of FAEs, however only a few reports of suitable High Throughput Screening assays for this kind of enzymes have been reported. This review is focused on a concise but complete revision of classical to High Throughput Screening methods for FAEs, highlighting its advantages and disadvantages, and finally suggesting future perspectives for this important research field.

  15. Microbiome Heterogeneity Characterizing Intestinal Tissue and Inflammatory Bowel Disease Phenotype.

    PubMed

    Tyler, Andrea D; Kirsch, Richard; Milgrom, Raquel; Stempak, Joanne M; Kabakchiev, Boyko; Silverberg, Mark S

    2016-04-01

    Inflammatory bowel disease has been associated with differential abundance of numerous organisms when compared to healthy controls (HCs); however, few studies have investigated variability in the microbiome across intestinal locations and how this variability might be related to disease location and phenotype. In this study, we have analyzed the microbiome of a large cohort of individuals recruited at Mount Sinai Hospital in Toronto, Canada. Biopsies were taken from subjects with Crohn's disease, ulcerative colitis, and HC, and also individuals having undergone ileal pouch-anal anastomosis for treatment of ulcerative colitis or familial adenomatous polyposis. Microbial 16S rRNA was sequenced using the Illumina MiSeq platform. We observed a great deal of variability in the microbiome characterizing different sampling locations. Samples from pouch and afferent limb were comparable in microbial composition. When comparing sigmoid and terminal ileum samples, more differences were observed. The greatest number of differentially abundant microbes was observed when comparing either pouch or afferent limb samples to sigmoid or terminal ileum. Despite these differences, we were able to observe modest microbial variability between inflammatory bowel disease phenotypes and HCs, even when controlling for sampling location and additional experimental factors. Most detected associations were observed between HCs and Crohn's disease, with decreases in specific genera in the families Ruminococcaceae and Lachnospiraceae characterizing tissue samples from individuals with Crohn's disease. This study highlights important considerations when analyzing the composition of the microbiome and also provides useful insight into differences in the microbiome characterizing these seemingly related phenotypes.

  16. New High Throughput Methods to Estimate Chemical Exposure

    EPA Science Inventory

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

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  1. Evaluating and Refining High Throughput Tools for Toxicokinetics

    EPA Science Inventory

    This poster summarizes efforts of the Chemical Safety for Sustainability's Rapid Exposure and Dosimetry (RED) team to facilitate the development and refinement of toxicokinetics (TK) tools to be used in conjunction with the high throughput toxicity testing data generated as a par...

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  4. Genomic and Phenotypic Characterization of Yeast Biosensor for Deep-space Radiation

    NASA Technical Reports Server (NTRS)

    Marina, Diana B.; Santa Maria, Sergio; Bhattacharya, Sharmila

    2016-01-01

    without losing radiation sensitivity. We employed Next-Generation Sequencing technology to better understand this phenotypic variation. Current effort is focusing on the analysis of high-throughput sequencing data to look for genomic changes in these reisolated clones compared to their original isolate.

  5. Dynamic analysis of MAPK signaling using a high-throughput microfluidic single-cell imaging platform.

    PubMed

    Taylor, R J; Falconnet, D; Niemistö, A; Ramsey, S A; Prinz, S; Shmulevich, I; Galitski, T; Hansen, C L

    2009-03-10

    Cells have evolved biomolecular networks that process and respond to changing chemical environments. Understanding how complex protein interactions give rise to emergent network properties requires time-resolved analysis of cellular response under a large number of genetic perturbations and chemical environments. To date, the lack of technologies for scalable cell analysis under well-controlled and time-varying conditions has made such global studies either impossible or impractical. To address this need, we have developed a high-throughput microfluidic imaging platform for single-cell studies of network response under hundreds of combined genetic perturbations and time-varying stimulant sequences. Our platform combines programmable on-chip mixing and perfusion with high-throughput image acquisition and processing to perform 256 simultaneous time-lapse live-cell imaging experiments. Nonadherent cells are captured in an array of 2,048 microfluidic cell traps to allow for the imaging of eight different genotypes over 12 h and in response to 32 unique sequences of stimulation, generating a total of 49,000 images per run. Using 12 devices, we carried out >3,000 live-cell imaging experiments to investigate the mating pheromone response in Saccharomyces cerevisiae under combined genetic perturbations and changing environmental conditions. Comprehensive analysis of 11 deletion mutants reveals both distinct thresholds for morphological switching and new dynamic phenotypes that are not observed in static conditions. For example, kss1Delta, fus3Delta, msg5Delta, and ptp2Delta mutants exhibit distinctive stimulus-frequency-dependent signaling phenotypes, implicating their role in filtering and network memory. The combination of parallel microfluidic control with high-throughput imaging provides a powerful tool for systems-level studies of single-cell decision making.

  6. Phenotypic and genetic characterization of a novel phenotype in pigs characterized by juvenile hairlessness and age dependent emphysema

    PubMed Central

    Bruun, Camilla S; Jørgensen, Claus B; Bay, Lene; Cirera, Susanna; Jensen, Henrik E; Leifsson, Páll S; Nielsen, Jens; Christensen, Knud; Fredholm, Merete

    2008-01-01

    Background A pig phenotype characterized by juvenile hairlessness, thin skin and age dependent lung emphysema has been discovered in a Danish pig herd. The trait shows autosomal co-dominant inheritance with all three genotypes distinguishable. Since the phenotype shows resemblance to the integrin β6 -/- knockout phenotype seen in mice, the two genes encoding the two subunits of integrin αvβ6, i.e. ITGB6 and ITGAV, were considered candidate genes for this trait. Results The mutated pig phenotype is characterized by hairlessness until puberty, thin skin with few hair follicles and absence of musculi arrectores pili, and at puberty or later localized areas of emphysema are seen in the lungs. Comparative mapping predicted that the porcine ITGB6 andITGAV orthologs map to SSC15. In an experimental family (n = 113), showing segregation of the trait, the candidate region was confirmed by linkage analysis with four microsatellite markers. Mapping of the porcine ITGB6 and ITGAV in the IMpRH radiation hybrid panel confirmed the comparative mapping information. Sequencing of the ITGB6 and ITGAV coding sequences from affected and normal pigs revealed no evidence of a causative mutation, but alternative splicing of the ITGB6 pre-mRNA was detected. For both ITGB6 and ITGAV quantitative PCR revealed no significant difference in the expression levels in normal and affected animals. In a western blot, ITGB6 was detected in lung protein samples of all three genotypes. This result was supported by flow cytometric analyses which showed comparable reactions of kidney cells from affected and normal pigs with an integrin αvβ6 monoclonal antibody. Also, immunohistochemical staining of lung tissue with an integrin β6 antibody showed immunoreaction in both normal and affected pigs. Conclusion A phenotype resembling the integrin β6 -/- knockout phenotype seen in mice has been characterized in the pig. The candidate region on SSC15 has been confirmed by linkage analysis but molecular

  7. Agrosuppression: a bioassay for the hypersensitive response suited to high-throughput screening.

    PubMed

    Kamoun, Sophien; Hamada, Walid; Huitema, Edgar

    2003-01-01

    We describe a novel method, agrosuppression, that addresses the need for an assay of the hypersensitive response (HR) in intact plants that is rapid and adapted to high-throughput functional screening of plant and pathogen genes. The agrosuppression assay is based on inoculation of intact plants with a mixture of Agrobacterium tumefaciens strains carrying (i) a binary plasmid with one or more candidate HR-inducing genes and (ii) a tumor-inducing (oncogenic) T-DNA. In the absence of HR induction, tumor formation is initiated, resulting in a typical crown gall phenotype. However, upon induction of the HR, tumor formation by the oncogenic T-DNA is suppressed, resulting in a phenotype that can be readily scored. We tested and optimized agrosuppression in Nicotiana benthamiana using the inf1 elicitin gene from the oomycete pathogen Phytophthora infestans, which specifically induces the HR in Nicotiana spp., and the gene-for-gene pair Avr9/Cf-9 from the fungal pathogen Cladosporium fulvum and Lycopersicon pimpinellifolium (currant tomato), respectively. Agrosuppression protocols that can be rapidly performed using simple mechanical wounding of petioles of intact N. benthamiana plants were developed and appeared particularly adapted to intensive high-throughput screening. This assay promises to greatly facilitate the cloning of novel plant R genes and pathogen Avr genes and to accelerate functional analyses and structure-function studies of these genes.

  8. High throughput in vivo functional validation of candidate congenital heart disease genes in Drosophila

    PubMed Central

    Zhu, Jun-yi; Fu, Yulong; Nettleton, Margaret; Richman, Adam; Han, Zhe

    2017-01-01

    Genomic sequencing has implicated large numbers of genes and de novo mutations as potential disease risk factors. A high throughput in vivo model system is needed to validate gene associations with pathology. We developed a Drosophila-based functional system to screen candidate disease genes identified from Congenital Heart Disease (CHD) patients. 134 genes were tested in the Drosophila heart using RNAi-based gene silencing. Quantitative analyses of multiple cardiac phenotypes demonstrated essential structural, functional, and developmental roles for more than 70 genes, including a subgroup encoding histone H3K4 modifying proteins. We also demonstrated the use of Drosophila to evaluate cardiac phenotypes resulting from specific, patient-derived alleles of candidate disease genes. We describe the first high throughput in vivo validation system to screen candidate disease genes identified from patients. This approach has the potential to facilitate development of precision medicine approaches for CHD and other diseases associated with genetic factors. DOI: http://dx.doi.org/10.7554/eLife.22617.001 PMID:28084990

  9. Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.

    PubMed

    El-Mowafi, S A; Sineva, E; Alumasa, J N; Nicoloff, H; Tomsho, J W; Ades, S E; Keiler, K C

    2015-01-01

    Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σ(E) is critical for maintenance of the cell envelope. Because σ(E) is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σ(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a σ(E) activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σ(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σ(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σ(E), inhibits RNA polymerase holoenzyme formation, and inhibits σ(E)-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σ(E) pathway.

  10. Digital fragment analysis of short tandem repeats by high-throughput amplicon sequencing.

    PubMed

    Darby, Brian J; Erickson, Shay F; Hervey, Samuel D; Ellis-Felege, Susan N

    2016-07-01

    High-throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high-throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high-throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost-effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence-aware microsatellite data.

  11. Adaptation and validation of DNA synthesis detection by fluorescent dye derivatization for high-throughput screening.

    PubMed

    Ranall, Max V; Gabrielli, Brian G; Gonda, Thomas J

    2010-05-01

    Cellular proliferation is fundamental to organism development, tissue renewal, and diverse disease states such as cancer. In vitro measurement of proliferation by high-throughput screening allows rapid characterization of the effects of small-molecule or genetic treatments on primary and established cell lines. Current assays that directly measure the cell cycle are not amenable to high-throughput processing and analysis. Here we report the adaptation of the chemical method for detecting DNA synthesis by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into both high-throughput liquid handling and high-content imaging analysis. We demonstrate that chemical detection of EdU incorporation is effective for high-resolution analysis and quantitation of DNA synthesis by high-content imaging. To validate this assay platform we used treatments of MCF10A cells with media supplements and pharmacological inhibitors that are known to affect cell proliferation. Treatments with specific kinase inhibitors indicate that EGF and serum stimulation employs both the mitogen extracellular kinase (MEK)/extracellular-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K)/AKT signaling networks. As described here, this method is fast, reliable, and inexpensive and yields robust data that can be easily interpreted.

  12. Ice-cap. A high-throughput method for capturing plant tissue samples for genotype analysis.

    PubMed

    Krysan, Patrick

    2004-07-01

    High-throughput genotype screening is rapidly becoming a standard research tool in the post-genomic era. A major bottleneck currently exists, however, that limits the utility of this approach in the plant sciences. The rate-limiting step in current high-throughput pipelines is that tissue samples from living plants must be collected manually, one plant at a time. In this article I describe a novel method for harvesting tissue samples from living seedlings that eliminates this bottleneck. The method has been named Ice-Cap to reflect the fact that ice is used to capture the tissue samples. The planting of seeds, growth of seedlings, and harvesting of tissue are all performed in a 96-well format. I demonstrate the utility of this system by using tissue harvested by Ice-Cap to genotype a population of Arabidopsis seedlings that is segregating a previously characterized mutation. Because the harvesting of tissue is performed in a nondestructive manner, plants with the desired genotype can be transferred to soil and grown to maturity. I also show that Ice-Cap can be used to analyze genomic DNA from rice (Oryza sativa) seedlings. It is expected that this method will be applicable to high-throughput screening with many different plant species, making it a useful technology for performing marker assisted selection.

  13. Automated disposable small scale reactor for high throughput bioprocess development: a proof of concept study.

    PubMed

    Bareither, Rachel; Bargh, Neil; Oakeshott, Robert; Watts, Kathryn; Pollard, David

    2013-12-01

    The acceleration of bioprocess development for biologics and vaccines can be enabled by automated high throughput technologies. This will alleviate the significant resource burden from the multi-factorial statistical experimentation required for controlling product quality attributes of complex biologics. Recent technology advances have improved clone evaluation and screening, but have struggled to combine the scale down criteria required for both high cell density cell culture and microbial processes, with sufficient automation and disposable technologies to accelerate process development. This article describes the proof of concept evaluations of an automated disposable small scale reactor for high throughput upstream process development. Characterization studies established the small scale stirred tank disposable 250 mL reactor as similar to those of lab and pilot scale. The reactor generated equivalent process performance for industrial biologics processes for therapeutic protein and monoclonal antibody production using CHO cell culture, Pichia pastoris and E. coli. This included similar growth, cell viability, product titer, and product quality. The technology was shown to be robust across multiple runs and met the requirements for the ability to run high cell density processes (>400 g/L wet cell weight) with exponential feeds and sophisticated event triggered processes. Combining this reactor into an automated array of reactors will ultimately be part of a high throughput process development strategy. This will combine upstream, small scale purification with rapid analytics that will dramatically shorten timelines and costs of developing biological processes.

  14. Microfluidic-Enabled Print-to-Screen Platform for High-Throughput Screening of Combinatorial Chemotherapy.

    PubMed

    Ding, Yuzhe; Li, Jiannan; Xiao, Wenwu; Xiao, Kai; Lee, Joyce; Bhardwaj, Urvashi; Zhu, Zijie; Digiglio, Philip; Yang, Gaomai; Lam, Kit S; Pan, Tingrui

    2015-10-20

    Since the 1960s, combination chemotherapy has been widely utilized as a standard method to treat cancer. However, because of the potentially enormous number of drug candidates and combinations, conventional identification methods of the effective drug combinations are usually associated with significantly high operational costs, low throughput screening, laborious and time-consuming procedures, and ethical concerns. In this paper, we present a low-cost, high-efficiency microfluidic print-to-screen (P2S) platform, which integrates combinatorial screening with biomolecular printing for high-throughput screening of anticancer drug combinations. This P2S platform provides several distinct advantages and features, including automatic combinatorial printing, high-throughput parallel drug screening, modular disposable cartridge, and biocompatibility, which can potentially speed up the entire discovery cycle of potent drug combinations. Microfluidic impact printing utilizing plug-and-play microfluidic cartridges is experimentally characterized with controllable droplet volume and accurate positioning. Furthermore, the combinatorial print-to-screen assay is demonstrated in a proof-of-concept biological experiment which can identify the positive hits among the entire drug combination library in a parallel and rapid manner. Overall, this microfluidic print-to-screen platform offers a simple, low-cost, high-efficiency solution for high-throughput large-scale combinatorial screening and can be applicable for various emerging applications in drug cocktail discovery.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. High throughput and multiplex localization of proteins and cells for in situ micropatterning using pneumatic microfluidics.

    PubMed

    Wang, Jian-Chun; Liu, Wenming; Tu, Qin; Ma, Chao; Zhao, Lei; Wang, Yaolei; Ouyang, Jia; Pang, Long; Wang, Jinyi

    2015-02-07

    Micropatterning technologies are emerging as an enabling tool for various microfluidic-based applications in life sciences. However, the high throughput and multiplex localization of multiple bio-components in a microfluidic device has not yet been well established. In this paper, we describe a simple and in situ micropatterning method using an integrated microfluidic device with pneumatic microstructures (PμSs) for highly controllable immobilization of both proteins and cells in a high throughput, geometry-dynamic, and multi-patterning way. The precise Pluronic F127 passivation of a microchamber surface except the PμS-blocked regions was performed and characterized, and the spatial dynamics and consistency of both the PμSs and protein/cell micropatterning were optically evaluated and quantitatively demonstrated too. Furthermore, a systematic investigation of PμS-assisted micropatterning in microfluidics was carried out. The feature of high throughput and spatial control of micropatterning can be simply realized by using the well-designed PμS arrays. Meanwhile, the co-micropatterning of different proteins (bovine serum albumin and chicken egg albumin) and cells (human umbilical vein endothelial cells and human hepatocellular carcinoma cells) in a microfluidic device was successfully accomplished with the orderly serial manipulation of PμS groups. We demonstrate that PμS-assisted micropatterning can be applied as a convenient microfluidic component for large-scale and diversified protein/cell patterning and manipulation, which could be useful for cell-based tissue organization, high-throughput imaging, protein-related interactions and immunoassays.

  17. A Novel High-Throughput Approach to Measure Hydroxyl Radicals Induced by Airborne Particulate Matter

    PubMed Central

    Son, Yeongkwon; Mishin, Vladimir; Welsh, William; Lu, Shou-En; Laskin, Jeffrey D.; Kipen, Howard; Meng, Qingyu

    2015-01-01

    Oxidative stress is one of the key mechanisms linking ambient particulate matter (PM) exposure with various adverse health effects. The oxidative potential of PM has been used to characterize the ability of PM induced oxidative stress. Hydroxyl radical (•OH) is the most destructive radical produced by PM. However, there is currently no high-throughput approach which can rapidly measure PM-induced •OH for a large number of samples with an automated system. This study evaluated four existing molecular probes (disodium terephthalate, 3′-p-(aminophenyl)fluorescein, coumarin-3-carboxylic acid, and sodium benzoate) for their applicability to measure •OH induced by PM in a high-throughput cell-free system using fluorescence techniques, based on both our experiments and on an assessment of the physicochemical properties of the probes reported in the literature. Disodium terephthalate (TPT) was the most applicable molecular probe to measure •OH induced by PM, due to its high solubility, high stability of the corresponding fluorescent product (i.e., 2-hydroxyterephthalic acid), high yield compared with the other molecular probes, and stable fluorescence intensity in a wide range of pH environments. TPT was applied in a high-throughput format to measure PM (NIST 1648a)-induced •OH, in phosphate buffered saline. The formed fluorescent product was measured at designated time points up to 2 h. The fluorescent product of TPT had a detection limit of 17.59 nM. The soluble fraction of PM contributed approximately 76.9% of the •OH induced by total PM, and the soluble metal ions of PM contributed 57.4% of the overall •OH formation. This study provides a promising cost-effective high-throughput method to measure •OH induced by PM on a routine basis. PMID:26516887

  18. High-throughput gated photon counter with two detection windows programmable down to 70 ps width

    SciTech Connect

    Boso, Gianluca; Tosi, Alberto Zappa, Franco; Mora, Alberto Dalla

    2014-01-15

    We present the design and characterization of a high-throughput gated photon counter able to count electrical pulses occurring within two well-defined and programmable detection windows. We extensively characterized and validated this instrument up to 100 Mcounts/s and with detection window width down to 70 ps. This instrument is suitable for many applications and proves to be a cost-effective and compact alternative to time-correlated single-photon counting equipment, thanks to its easy configurability, user-friendly interface, and fully adjustable settings via a Universal Serial Bus (USB) link to a remote computer.

  19. Optimization of Fluorescence Assay of Cellular Manganese Status for High Throughput Screening

    PubMed Central

    Kumar, Kevin K.; Aboud, Asad A.; Patel, Devin K.; Aschner, Michael; Bowman, Aaron B.

    2013-01-01

    The advent of high throughput screening (HTS) technology permits identification of compounds that influence various cellular phenotypes. However, screening for small molecule chemical modifiers of neurotoxicants has been limited by the scalability of existing phenotyping assays. Furthermore, the adaptation of existing cellular assays to HTS format requires substantial modification of experimental parameters and analysis methodology to meet the necessary statistical requirements. Here we describe the successful optimization of the Cellular Fura-2 Manganese Extraction Assay (CFMEA) for HTS. By optimizing cellular density, manganese (Mn) exposure conditions, and extraction parameters, the sensitivity and dynamic range of the fura-2 Mn response was enhanced to permit detection of positive and negative modulators of cellular manganese status. Finally, we quantify and report strategies to control sources of intra-and inter-plate variability by batch level and plate-geometric level analysis. Our goal is to enable HTS with the CFMEA to identify novel modulators of Mn transport. PMID:23169769

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2008-12-24

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

  2. High throughput screening of starch structures using carbohydrate microarrays

    PubMed Central

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

    2016-01-01

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

  3. High-throughput theoretical design of lithium battery materials

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

  4. Direct assembling methodologies for high-throughput bioscreening

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    Bischof, Leanne; Převorovský, Martin; Rallis, Charalampos; Jeffares, Daniel C; Arzhaeva, Yulia; Bähler, Jürg

    2016-10-01

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

  6. A System for Performing High Throughput Assays of Synaptic Function

    PubMed Central

    Hempel, Chris M.; Sivula, Michael; Levenson, Jonathan M.; Rose, David M.; Li, Bing; Sirianni, Ana C.; Xia, Eva; Ryan, Timothy A.; Gerber, David J.; Cottrell, Jeffrey R.

    2011-01-01

    Unbiased, high-throughput screening has proven invaluable for dissecting complex biological processes. Application of this general approach to synaptic function would have a major impact on neuroscience research and drug discovery. However, existing techniques for studying synaptic physiology are labor intensive and low-throughput. Here, we describe a new high-throughput technology for performing assays of synaptic function in primary neurons cultured in microtiter plates. We show that this system can perform 96 synaptic vesicle cycling assays in parallel with high sensitivity, precision, uniformity, and reproducibility and can detect modulators of presynaptic function. By screening libraries of pharmacologically defined compounds on rat forebrain cultures, we have used this system to identify novel effects of compounds on specific aspects of presynaptic function. As a system for unbiased compound as well as genomic screening, this technology has significant applications for basic neuroscience research and for the discovery of novel, mechanism-based treatments for central nervous system disorders. PMID:21998743

  7. High-throughput evaluation of synthetic metabolic pathways

    PubMed Central

    Klesmith, Justin R.; Whitehead, Timothy A.

    2016-01-01

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

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

    PubMed

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

    2016-06-09

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

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

    PubMed Central

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

    2016-01-01

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

  10. A Functional High-Throughput Assay of Myelination in Vitro

    DTIC Science & Technology

    2014-07-01

    Human induced pluripotent stem cells , hydrogels, 3D culture, electrophysiology, high-throughput assay 16. SECURITY CLASSIFICATION OF: 17...clear that four of the seven human astrocyte cell lines (HA #1, 2, 3, and 7) show very large amounts of neuronal differentiation when using epigenetic...derived.   5    Fig. 1: Spontaneous differentiation toward neuronal lineage of iPS cells derived from human astrocytes. Left: phase contrast

  11. Rapid Methods for High-Throughput Detection of Sulfoxides▿

    PubMed Central

    Shainsky, Janna; Derry, Netta-Lee; Leichtmann-Bardoogo, Yael; Wood, Thomas K.; Fishman, Ayelet

    2009-01-01

    Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene ortho-monooxygenase in a saturation mutagenesis library, using methyl-p-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment. PMID:19465532

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

    PubMed

    Zimmermann, Hartmut F; Rieth, Jochen

    2007-03-01

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

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

    PubMed

    Knapp, Michael; Stiller, Mathias; Meyer, Matthias

    2012-01-01

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

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

    PubMed

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

    2012-05-01

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

  15. Novel High-throughput Approach for Purification of Infectious Virions

    PubMed Central

    James, Kevin T.; Cooney, Brad; Agopsowicz, Kate; Trevors, Mary Ann; Mohamed, Adil; Stoltz, Don; Hitt, Mary; Shmulevitz, Maya

    2016-01-01

    Viruses are extensively studied as pathogens and exploited as molecular tools and therapeutic agents. Existing methods to purify viruses such as gradient ultracentrifugation or chromatography have limitations, for example demand for technical expertise or specialized equipment, high time consumption, and restricted capacity. Our laboratory explores mutations in oncolytic reovirus that could improve oncolytic activity, and makes routine use of numerous virus variants, genome reassortants, and reverse engineered mutants. Our research pace was limited by the lack of high-throughput virus purification methods that efficiently remove confounding cellular contaminants such as cytokines and proteases. To overcome this shortcoming, we evaluated a commercially available resin (Capto Core 700) that captures molecules smaller than 700 kDa. Capto. Core 700 chromatography produced virion purity and infectivity indistinguishable from CsCl density gradient ultracentrifugation as determined by electron microscopy, gel electrophoresis analysis and plaque titration. Capto Core 700 resin was then effectively adapted to a rapid in-slurry pull-out approach for high-throughput purification of reovirus and adenovirus. The in-slurry purification approach offered substantially increased virus purity over crude cell lysates, media, or high-spin preparations and would be especially useful for high-throughput virus screening applications where density gradient ultracentrifugation is not feasible. PMID:27827454

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

    PubMed

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

    2009-01-01

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

  17. A microdroplet dilutor for high-throughput screening

    NASA Astrophysics Data System (ADS)

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

    2011-06-01

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

  18. A microdroplet dilutor for high-throughput screening.

    PubMed

    Niu, Xize; Gielen, Fabrice; Edel, Joshua B; deMello, Andrew J

    2011-06-01

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

  19. High throughput biotechnology in traditional fermented food industry.

    PubMed

    Yang, Yong; Xu, Rong-man; Song, Jia; Wang, Wei-min

    2010-11-01

    Traditional fermented food is not only the staple food for most of developing countries but also the key healthy food for developed countries. As the healthy function of these foods are gradually discovered, more and more high throughput biotechnologies are being used to promote the old and new industry. As a result, the microflora, manufacturing processes and product healthy function of these foods were pushed forward either in the respect of profundity or extensiveness nowadays. The application and progress of the high throughput biotechnologies into traditional fermented food industries were different from each other, which was reviewed and detailed by the catalogues of fermented milk products (yogurt, cheese), fermented sausages, fermented vegetables (kimchi, sauerkraut), fermented cereals (sourdough) and fermented beans (tempeh, natto). Given the further promotion by high throughput biotechnologies, the middle and/or down-stream process of traditional fermented foods would be optimized and the process of industrialization of local traditional fermented food having many functional factors but in small quantity would be accelerated. The article presents some promising patents on traditional fermented food industry.

  20. Blood group genotyping: from patient to high-throughput donor screening.

    PubMed

    Veldhuisen, B; van der Schoot, C E; de Haas, M

    2009-10-01

    Blood group antigens, present on the cell membrane of red blood cells and platelets, can be defined either serologically or predicted based on the genotypes of genes encoding for blood group antigens. At present, the molecular basis of many antigens of the 30 blood group systems and 17 human platelet antigens is known. In many laboratories, blood group genotyping assays are routinely used for diagnostics in cases where patient red cells cannot be used for serological typing due to the presence of auto-antibodies or after recent transfusions. In addition, DNA genotyping is used to support (un)-expected serological findings. Fetal genotyping is routinely performed when there is a risk of alloimmune-mediated red cell or platelet destruction. In case of patient blood group antigen typing, it is important that a genotyping result is quickly available to support the selection of donor blood, and high-throughput of the genotyping method is not a prerequisite. In addition, genotyping of blood donors will be extremely useful to obtain donor blood with rare phenotypes, for example lacking a high-frequency antigen, and to obtain a fully typed donor database to be used for a better matching between recipient and donor to prevent adverse transfusion reactions. Serological typing of large cohorts of donors is a labour-intensive and expensive exercise and hampered by the lack of sufficient amounts of approved typing reagents for all blood group systems of interest. Currently, high-throughput genotyping based on DNA micro-arrays is a very feasible method to obtain a large pool of well-typed blood donors. Several systems for high-throughput blood group genotyping are developed and will be discussed in this review.

  1. 'PACLIMS': A component LIM system for high-throughput functional genomic analysis

    PubMed Central

    Donofrio, Nicole; Rajagopalon, Ravi; Brown, Douglas; Diener, Stephen; Windham, Donald; Nolin, Shelly; Floyd, Anna; Mitchell, Thomas; Galadima, Natalia; Tucker, Sara; Orbach, Marc J; Patel, Gayatri; Farman, Mark; Pampanwar, Vishal; Soderlund, Cari; Lee, Yong-Hwan; Dean, Ralph A

    2005-01-01

    Background Recent advances in sequencing techniques leading to cost reduction have resulted in the generation of a growing number of sequenced eukaryotic genomes. Computational tools greatly assist in defining open reading frames and assigning tentative annotations. However, gene functions cannot be asserted without biological support through, among other things, mutational analysis. In taking a genome-wide approach to functionally annotate an entire organism, in this application the ~11,000 predicted genes in the rice blast fungus (Magnaporthe grisea), an effective platform for tracking and storing both the biological materials created and the data produced across several participating institutions was required. Results The platform designed, named PACLIMS, was built to support our high throughput pipeline for generating 50,000 random insertion mutants of Magnaporthe grisea. To be a useful tool for materials and data tracking and storage, PACLIMS was designed to be simple to use, modifiable to accommodate refinement of research protocols, and cost-efficient. Data entry into PACLIMS was simplified through the use of barcodes and scanners, thus reducing the potential human error, time constraints, and labor. This platform was designed in concert with our experimental protocol so that it leads the researchers through each step of the process from mutant generation through phenotypic assays, thus ensuring that every mutant produced is handled in an identical manner and all necessary data is captured. Conclusion Many sequenced eukaryotes have reached the point where computational analyses are no longer sufficient and require biological support for their predicted genes. Consequently, there is an increasing need for platforms that support high throughput genome-wide mutational analyses. While PACLIMS was designed specifically for this project, the source and ideas present in its implementation can be used as a model for other high throughput mutational endeavors. PMID

  2. High-throughput imaging and analysis of root system architecture in Brachypodium distachyon under differential nutrient availability

    PubMed Central

    Ingram, Paul A.; Zhu, Jinming; Shariff, Aabid; Davis, Ian W.; Benfey, Philip N.; Elich, Tedd

    2012-01-01

    Nitrogen (N) and phosphorus (P) deficiency are primary constraints for plant productivity, and root system architecture (RSA) plays a vital role in the acquisition of these nutrients. The genetic determinants of RSA are poorly understood, primarily owing to the complexity of crop genomes and the lack of sufficient RSA phenotyping methods. The objective of this study was to characterize the RSA of two Brachypodium distachyon accessions under different nutrient availability. To do so, we used a high-throughput plant growth and imaging platform, and developed software that quantified 19 different RSA traits. We found significant differences in RSA between two Brachypodium accessions grown on nutrient-rich, low-N and low-P conditions. More specifically, one accession maintained axile root growth under low N, while the other accession maintained lateral root growth under low P. These traits resemble the RSA of crops adapted to low-N and -P conditions, respectively. Furthermore, we found that a number of these traits were highly heritable. This work lays the foundation for future identification of important genetic components of RSA traits under nutrient limitation using a mapping population derived from these two accessions. PMID:22527399

  3. High-throughput imaging and analysis of root system architecture in Brachypodium distachyon under differential nutrient availability.

    PubMed

    Ingram, Paul A; Zhu, Jinming; Shariff, Aabid; Davis, Ian W; Benfey, Philip N; Elich, Tedd

    2012-06-05

    Nitrogen (N) and phosphorus (P) deficiency are primary constraints for plant productivity, and root system architecture (RSA) plays a vital role in the acquisition of these nutrients. The genetic determinants of RSA are poorly understood, primarily owing to the complexity of crop genomes and the lack of sufficient RSA phenotyping methods. The objective of this study was to characterize the RSA of two Brachypodium distachyon accessions under different nutrient availability. To do so, we used a high-throughput plant growth and imaging platform, and developed software that quantified 19 different RSA traits. We found significant differences in RSA between two Brachypodium accessions grown on nutrient-rich, low-N and low-P conditions. More specifically, one accession maintained axile root growth under low N, while the other accession maintained lateral root growth under low P. These traits resemble the RSA of crops adapted to low-N and -P conditions, respectively. Furthermore, we found that a number of these traits were highly heritable. This work lays the foundation for future identification of important genetic components of RSA traits under nutrient limitation using a mapping population derived from these two accessions.

  4. Plant phenomics and the need for physiological phenotyping across scales to narrow the genotype-to-phenotype knowledge gap.

    PubMed

    Großkinsky, Dominik K; Svensgaard, Jesper; Christensen, Svend; Roitsch, Thomas

    2015-09-01

    Plants are affected by complex genome×environment×management interactions which determine phenotypic plasticity as a result of the variability of genetic components. Whereas great advances have been made in the cost-efficient and high-throughput analyses of genetic information and non-invasive phenotyping, the large-scale analyses of the underlying physiological mechanisms lag behind. The external phenotype is determined by the sum of the complex interactions of metabolic pathways and intracellular regulatory networks that is reflected in an internal, physiological, and biochemical phenotype. These various scales of dynamic physiological responses need to be considered, and genotyping and external phenotyping should be linked to the physiology at the cellular and tissue level. A high-dimensional physiological phenotyping across scales is needed that integrates the precise characterization of the internal phenotype into high-throughput phenotyping of whole plants and canopies. By this means, complex traits can be broken down into individual components of physiological traits. Since the higher resolution of physiological phenotyping by 'wet chemistry' is inherently limited in throughput, high-throughput non-invasive phenotyping needs to be validated and verified across scales to be used as proxy for the underlying processes. Armed with this interdisciplinary and multidimensional phenomics approach, plant physiology, non-invasive phenotyping, and functional genomics will complement each other, ultimately enabling the in silico assessment of responses under defined environments with advanced crop models. This will allow generation of robust physiological predictors also for complex traits to bridge the knowledge gap between genotype and phenotype for applications in breeding, precision farming, and basic research.

  5. High-Throughput, Motility-Based Sorter for Microswimmers and Gene Discovery Platform

    NASA Astrophysics Data System (ADS)

    Yuan, Jinzhou; Raizen, David; Bau, Haim

    2015-11-01

    Animal motility varies with genotype, disease progression, aging, and environmental conditions. In many studies, it is desirable to carry out high throughput motility-based sorting to isolate rare animals for, among other things, forward genetic screens to identify genetic pathways that regulate phenotypes of interest. Many commonly used screening processes are labor-intensive, lack sensitivity, and require extensive investigator training. Here, we describe a sensitive, high throughput, automated, motility-based method for sorting nematodes. Our method was implemented in a simple microfluidic device capable of sorting many thousands of animals per hour per module, and is amenable to parallelism. The device successfully enriched for known C. elegans motility mutants. Furthermore, using this device, we isolated low-abundance mutants capable of suppressing the somnogenic effects of the flp-13 gene, which regulates sleep-like quiescence in C. elegans. Subsequent genomic sequencing led to the identification of a flp-13-suppressor gene. This research was supported, in part, by NIH NIA Grant 5R03AG042690-02.

  6. An Automated High-throughput Array Microscope for Cancer Cell Mechanics

    PubMed Central

    Cribb, Jeremy A.; Osborne, Lukas D.; Beicker, Kellie; Psioda, Matthew; Chen, Jian; O’Brien, E. Timothy; Taylor II, Russell M.; Vicci, Leandra; Hsiao, Joe Ping-Lin; Shao, Chong; Falvo, Michael; Ibrahim, Joseph G.; Wood, Kris C.; Blobe, Gerard C.; Superfine, Richard

    2016-01-01

    Changes in cellular mechanical properties correlate with the progression of metastatic cancer along the epithelial-to-mesenchymal transition (EMT). Few high-throughput methodologies exist that measure cell compliance, which can be used to understand the impact of genetic alterations or to screen the efficacy of chemotherapeutic agents. We have developed a novel array high-throughput microscope (AHTM) system that combines the convenience of the standard 96-well plate with the ability to image cultured cells and membrane-bound microbeads in twelve independently-focusing channels simultaneously, visiting all wells in eight steps. We use the AHTM and passive bead rheology techniques to determine the relative compliance of human pancreatic ductal epithelial (HPDE) cells, h-TERT transformed HPDE cells (HPNE), and four gain-of-function constructs related to EMT. The AHTM found HPNE, H-ras, Myr-AKT, and Bcl2 transfected cells more compliant relative to controls, consistent with parallel tests using atomic force microscopy and invasion assays, proving the AHTM capable of screening for changes in mechanical phenotype. PMID:27265611

  7. An Automated High-throughput Array Microscope for Cancer Cell Mechanics

    NASA Astrophysics Data System (ADS)

    Cribb, Jeremy A.; Osborne, Lukas D.; Beicker, Kellie; Psioda, Matthew; Chen, Jian; O’Brien, E. Timothy; Taylor, Russell M., II; Vicci, Leandra; Hsiao, Joe Ping-Lin; Shao, Chong; Falvo, Michael; Ibrahim, Joseph G.; Wood, Kris C.; Blobe, Gerard C.; Superfine, Richard

    2016-06-01

    Changes in cellular mechanical properties correlate with the progression of metastatic cancer along the epithelial-to-mesenchymal transition (EMT). Few high-throughput methodologies exist that measure cell compliance, which can be used to understand the impact of genetic alterations or to screen the efficacy of chemotherapeutic agents. We have developed a novel array high-throughput microscope (AHTM) system that combines the convenience of the standard 96-well plate with the ability to image cultured cells and membrane-bound microbeads in twelve independently-focusing channels simultaneously, visiting all wells in eight steps. We use the AHTM and passive bead rheology techniques to determine the relative compliance of human pancreatic ductal epithelial (HPDE) cells, h-TERT transformed HPDE cells (HPNE), and four gain-of-function constructs related to EMT. The AHTM found HPNE, H-ras, Myr-AKT, and Bcl2 transfected cells more compliant relative to controls, consistent with parallel tests using atomic force microscopy and invasion assays, proving the AHTM capable of screening for changes in mechanical phenotype.

  8. Pipeline for illumination correction of images for high-throughput microscopy.

    PubMed

    Singh, S; Bray, M-A; Jones, T R; Carpenter, A E

    2014-12-01

    The presence of systematic noise in images in high-throughput microscopy experiments can significantly impact the accuracy of downstream results. Among the most common sources of systematic noise is non-homogeneous illumination across the image field. This often adds an unacceptable level of noise, obscures true quantitative differences and precludes biological experiments that rely on accurate fluorescence intensity measurements. In this paper, we seek to quantify the improvement in the quality of high-content screen readouts due to software-based illumination correction. We present a straightforward illumination correction pipeline that has been used by our group across many experiments. We test the pipeline on real-world high-throughput image sets and evaluate the performance of the pipeline at two levels: (a) Z'-factor to evaluate the effect of the image correction on a univariate readout, representative of a typical high-content screen, and (b) classification accuracy on phenotypic signatures derived from the images, representative of an experiment involving more complex data mining. We find that applying the proposed post-hoc correction method improves performance in both experiments, even when illumination correction has already been applied using software associated with the instrument. To facilitate the ready application and future development of illumination correction methods, we have made our complete test data sets as well as open-source image analysis pipelines publicly available. This software-based solution has the potential to improve outcomes for a wide-variety of image-based HTS experiments.

  9. High-throughput metagenomic technologies for complex microbial community analysis. Open and closed formats

    DOE PAGES

    Zhou, Jizhong; He, Zhili; Yang, Yunfeng; ...

    2015-01-27

    Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied “open-format” and “closed-format” detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications andmore » focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions.« less

  10. A High-Throughput Screen Reveals New Small-Molecule Activators and Inhibitors of Pantothenate Kinases

    PubMed Central

    2016-01-01

    Pantothenate kinase (PanK) is a regulatory enzyme that controls coenzyme A (CoA) biosynthesis. The association of PanK with neurodegeneration and diabetes suggests that chemical modifiers of PanK activity may be useful therapeutics. We performed a high throughput screen of >520000 compounds from the St. Jude compound library and identified new potent PanK inhibitors and activators with chemically tractable scaffolds. The HTS identified PanK inhibitors exemplified by the detailed characterization of a tricyclic compound (7) and a preliminary SAR. Biophysical studies reveal that the PanK inhibitor acts by binding to the ATP–enzyme complex. PMID:25569308

  11. Accurate high-throughput identification of parallel G-quadruplex topology by a new tetraaryl-substituted imidazole.

    PubMed

    Hu, Ming-Hao; Chen, Shuo-Bin; Wang, Yu-Qing; Zeng, You-Mei; Ou, Tian-Miao; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu; Tan, Jia-Heng

    2016-09-15

    G-quadruplex nucleic acids are four-stranded DNA or RNA secondary structures that are formed in guanine-rich sequences. These structures exhibit extensive structural polymorphism and play a pivotal role in the control of a variety of cellular processes. To date, diverse approaches for high-throughput identification of G-quadruplex structures have been successfully developed, but high-throughput methods for further characterization of their topologies are still lacking. In this study, we report a new tetra-arylimidazole probe psIZCM-1, which was found to display significant and distinctive changes in both the absorption and the fluorescence spectra in the presence of parallel G-quadruplexes but show insignificant changes upon interactions with anti-parallel G-quadruplexes or other non-quadruplex oligonucleotides. In view of this dual-output feature, we used psIZCM-1 to identify the parallel G-quadruplexes from a large set of 314 oligonucleotides (including 300 G-quadruplex-forming oligonucleotides and 14 non-quadruplex oligonucleotides) via a microplate reader and accordingly established a high-throughput method for the characterization of parallel G-quadruplex topologies. The accuracy of this method was greater than 95%, which was much higher than that of the commercial probe NMM. To make the approach more practical, we further combined psIZCM-1 with another G-quadruplex probe IZCM-7 to realize the high-throughput classification of parallel, anti-parallel G-quadruplexes and non-quadruplex structures.

  12. Characterizing the Phenotype of Advanced Pelvic Organ Prolapse

    PubMed Central

    Levin, Pamela J.; Visco, Anthony G.; Shah, Svati H.; Fulton, Rebekah G.; Wu, Jennifer M.

    2012-01-01

    Objective Genetic studies require a clearly defined phenotype to reach valid conclusions. Our aim was to characterize the phenotype of advanced prolapse by comparing women with stage III to IV prolapse with controls without prolapse. Methods Based on the pelvic organ prolapse quantification examination, women with stage 0 to stage I prolapse (controls) and those with stage III to stage IV prolapse (cases) were prospectively recruited as part of a genetic epidemiologic study. Data regarding sociodemographics; medical, obstetric, and surgical history; family history; and body mass index were obtained by a questionnaire administered by a trained coordinator and abstracted from electronic medical records. Results There were 275 case patients with advanced prolapse and 206 controls with stage 0 to stage I prolapse. Based on our recruitment strategy, the women were younger than the controls (64.7±10.1 vs 68.6±10.4 years; P<0.001); cases were also more likely to have had one or more vaginal deliveries (96.0% vs 82.0%; P<0.001). There were no differences in race, body mass index, and constipation. Regarding family history, cases were more likely to report that either their mother and/or sister(s) had prolapse (44.8% vs 16.9%, P<0.001). In a logistic regression model, vaginal parity (odds ratio, 4.05; 95% confidence interval, 1.67–9.85) and family history of prolapse (odds ratio, 3.74; 95% confidence interval, 2.16–6.46) remained significantly associated with advanced prolapse. Conclusions Vaginal parity and a family history of prolapse are more common in women with advanced prolapse compared to those without prolapse. These characteristics are important in phenotyping advanced prolapse, suggesting that these data should be collected in future genetic epidemiologic studies. PMID:22983275

  13. Cell-based screening using high-throughput flow cytometry.

    PubMed

    Black, Christopher B; Duensing, Thomas D; Trinkle, Linda S; Dunlay, R Terry

    2011-02-01

    This review describes the use of high-throughput flow cytometry for performing multiplexed cell-based and bead-based screens. With the many advances in cell-based analysis and screening, flow cytometry has historically been underutilized as a screening tool largely due to the limitations in handling large numbers of samples. However, there has been a resurgence in the use of flow cytometry due to a combination of innovations around instrumentation and a growing need for cell-based and bead-based applications. The HTFC™ Screening System (IntelliCyt Corporation, Albuquerque, NM) is a novel flow cytometry-based screening platform that incorporates a fast sample-loading technology, HyperCyt®, with a two-laser, six-parameter flow cytometer and powerful data analysis capabilities. The system is capable of running multiplexed screening assays at speeds of up to 40 wells per minute, enabling the processing of a 96- and 384-well plates in as little as 3 and 12 min, respectively. Embedded in the system is HyperView®, a data analysis software package that allows rapid identification of hits from multiplexed high-throughput flow cytometry screening campaigns. In addition, the software is incorporated into a server-based data management platform that enables seamless data accessibility and collaboration across multiple sites. High-throughput flow cytometry using the HyperCyt technology has been applied to numerous assay areas and screening campaigns, including efflux transporters, whole cell and receptor binding assays, functional G-protein-coupled receptor screening, in vitro toxicology, and antibody screening.

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

    NASA Astrophysics Data System (ADS)

    Cooper, Khershed P.

    2013-09-01

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

  15. High-Throughput Sequencing: A Roadmap Toward Community Ecology

    PubMed Central

    Poisot, Timothée; Péquin, Bérangère; Gravel, Dominique

    2013-01-01

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

  16. Genomic outlier detection in high-throughput data analysis.

    PubMed

    Ghosh, Debashis

    2013-01-01

    In the analysis of high-throughput data, a very common goal is the detection of genes or of differential expression between two groups or classes. A recent finding from the scientific literature in prostate cancer demonstrates that by searching for a different pattern of differential expression, new candidate oncogenes might be found. In this chapter, we discuss the statistical problem, termed oncogene outlier detection, and discuss a variety of proposals to this problem. A statistical model in the multiclass situation is described; links with multiple testing concepts are established. Some new nonparametric procedures are described and compared to existing methods using simulation studies.

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

    DOEpatents

    Davidson, James C.; Balch, Joseph W.

    2000-01-01

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

  18. Adaptive Sampling for High Throughput Data Using Similarity Measures

    SciTech Connect

    Bulaevskaya, V.; Sales, A. P.

    2015-05-06

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

  19. Orchestrating high-throughput genomic analysis with Bioconductor

    PubMed Central

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

    2015-01-01

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

  20. High throughput computing: a solution for scientific analysis

    USGS Publications Warehouse

    O'Donnell, M.

    2011-01-01

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

  1. Live Cell Optical Sensing for High Throughput Applications

    NASA Astrophysics Data System (ADS)

    Fang, Ye

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

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

    PubMed

    Upadhyay, Santosh Kumar; Sharma, Shailesh

    2014-01-01

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

  3. High-throughput expression in microplate format in Saccharomyces cerevisiae.

    PubMed

    Holz, Caterina; Lang, Christine

    2004-01-01

    We have developed a high-throughput technology that allows parallel expression, purification, and analysis of large numbers of cloned cDNAs in the yeast Saccharomyces cerevisiae. The technology is based on a vector for intracellular protein expression under control of the inducible CUP1 promoter, where the gene products are fused to specific peptide sequences. These N-terminal and C-terminal epitope tags allow the immunological identification and purification of the gene products independent of the protein produced. By introducing the method of recombinational cloning we avoid time-consuming re-cloning steps and enable the easy switching between different expression vectors and host systems.

  4. Analysis of High-Throughput ELISA Microarray Data

    SciTech Connect

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

    2011-02-23

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

  5. Developing soluble polymers for high-throughput synthetic chemistry.

    PubMed

    Spanka, Carsten; Wentworth, Paul; Janda, Kim D

    2002-05-01

    Soluble polymers have emerged as viable alternatives to resin supports across the broad spectrum of high-throughput organic chemistry. As the application of these supports become more widespread, issues such as broad-spectrum solubility and loading are becoming limiting factors and therefore new polymers are required to overcome such limitations. This article details the approach made within our group to new soluble polymer supports and specifically focuses on parallel libraries of block copolymers, de novo poly(styrene-co-chloromethylstyrene), PEG- stealth stars, and substituted poly(norbornylene)s.

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

    SciTech Connect

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

    2007-01-03

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

  7. High-throughput quantitative real-time PCR.

    PubMed

    Arany, Zoltan P

    2008-07-01

    Recent technical advances in quantitative real-time PCR (qRT-PCR) have allowed for extensive miniaturization, thereby rendering the technique amenable to high-throughput assays. Large numbers of different nucleic acids can now rapidly be measured quantitatively. Many investigations can benefit from this approach, including determination of gene expression in hundreds of samples, determination of hundreds of genes in a few samples, or even quantification of nucleic acids other than mRNA. A simple technique is described here to quantify 1880 transcripts of choice from any number of starting RNA samples.

  8. High-throughput label-free image cytometry and image-based classification of live Euglena gracilis.

    PubMed

    Lei, Cheng; Ito, Takuro; Ugawa, Masashi; Nozawa, Taisuke; Iwata, Osamu; Maki, Masanori; Okada, Genki; Kobayashi, Hirofumi; Sun, Xinlei; Tiamsak, Pimsiri; Tsumura, Norimichi; Suzuki, Kengo; Di Carlo, Dino; Ozeki, Yasuyuki; Goda, Keisuke

    2016-07-01

    We demonstrate high-throughput label-free single-cell image cytometry and image-based classification of Euglena gracilis (a microalgal species) under different culture conditions. We perform it with our high-throughput optofluidic image cytometer composed of a time-stretch microscope with 780-nm resolution and 75-Hz line rate, and an inertial-focusing microfluidic device. By analyzing a large number of single-cell images from the image cytometer, we identify differences in morphological and intracellular phenotypes between E. gracilis cell groups and statistically classify them under various culture conditions including nitrogen deficiency for lipid induction. Our method holds promise for real-time evaluation of culture techniques for E. gracilis and possibly other microalgae in a non-invasive manner.

  9. High-throughput label-free image cytometry and image-based classification of live Euglena gracilis

    PubMed Central

    Lei, Cheng; Ito, Takuro; Ugawa, Masashi; Nozawa, Taisuke; Iwata, Osamu; Maki, Masanori; Okada, Genki; Kobayashi, Hirofumi; Sun, Xinlei; Tiamsak, Pimsiri; Tsumura, Norimichi; Suzuki, Kengo; Di Carlo, Dino; Ozeki, Yasuyuki; Goda, Keisuke

    2016-01-01

    We demonstrate high-throughput label-free single-cell image cytometry and image-based classification of Euglena gracilis (a microalgal species) under different culture conditions. We perform it with our high-throughput optofluidic image cytometer composed of a time-stretch microscope with 780-nm resolution and 75-Hz line rate, and an inertial-focusing microfluidic device. By analyzing a large number of single-cell images from the image cytometer, we identify differences in morphological and intracellular phenotypes between E. gracilis cell groups and statistically classify them under various culture conditions including nitrogen deficiency for lipid induction. Our method holds promise for real-time evaluation of culture techniques for E. gracilis and possibly other microalgae in a non-invasive manner. PMID:27446699

  10. DevStaR: high-throughput quantification of C. elegans developmental stages.

    PubMed

    White, Amelia G; Lees, Brandon; Kao, Huey-Ling; Cipriani, P Giselle; Munarriz, Eliana; Paaby, Annalise B; Erickson, Katherine; Guzman, Sherly; Rattanakorn, Kirk; Sontag, Eduardo; Geiger, Davi; Gunsalus, Kristin C; Piano, Fabio

    2013-10-01

    We present DevStaR, an automated computer vision and machine learning system that provides rapid, accurate, and quantitative measurements of C. elegans embryonic viability in high-throughput (HTP) applications. A leading genetic model organism for the study of animal development and behavior, C. elegans is particularly amenable to HTP functional genomic analysis due to its small size and ease of cultivation, but the lack of efficient and quantitative methods to score phenotypes has become a major bottleneck. DevStaR addresses this challenge using a novel hierarchical object recognition machine that rapidly segments, classifies, and counts animals at each developmental stage in images of mixed-stage populations of C. elegans. Here, we describe the algorithmic design of the DevStaR system and demonstrate its performance in scoring image data acquired in HTP screens.

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

    PubMed

    Eglen, Richard M; Randle, David H

    2015-06-01

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

  12. Resonant waveguide grating imagers for single cell analysis and high throughput screening

    NASA Astrophysics Data System (ADS)

    Fang, Ye

    2015-08-01

    Resonant waveguide grating (RWG) systems illuminate an array of diffractive nanograting waveguide structures in microtiter plate to establish evanescent wave for measuring tiny changes in local refractive index arising from the dynamic mass redistribution of living cells upon stimulation. Whole-plate RWG imager enables high-throughput profiling and screening of drugs. Microfluidics RWG imager not only manifests distinct receptor signaling waves, but also differentiates long-acting agonism and antagonism. Spatially resolved RWG imager allows for single cell analysis including receptor signaling heterogeneity and the invasion of cancer cells in a spheroidal structure through 3-dimensional extracellular matrix. High frequency RWG imager permits real-time detection of drug-induced cardiotoxicity. The wide coverage in target, pathway, assay, and cell phenotype has made RWG systems powerful tool in both basic research and early drug discovery process.

  13. High-throughput generation, optimization and analysis of genome-scale metabolic models.

    SciTech Connect

    Henry, C. S.; DeJongh, M.; Best, A. A.; Frybarger, P. M.; Linsay, B.; Stevens, R. L.

    2010-09-01

    Genome-scale metabolic models have proven to be valuable for predicting organism phenotypes from genotypes. Yet efforts to develop new models are failing to keep pace with genome sequencing. To address this problem, we introduce the Model SEED, a web-based resource for high-throughput generation, optimization and analysis of genome-scale metabolic models. The Model SEED integrates existing methods and introduces techniques to automate nearly every step of this process, taking {approx}48 h to reconstruct a metabolic model from an assembled genome sequence. We apply this resource to generate 130 genome-scale metabolic models representing a taxonomically diverse set of bacteria. Twenty-two of the models were validated against available gene essentiality and Biolog data, with the average model accuracy determined to be 66% before optimization and 87% after optimization.

  14. High-throughput technology for novel SO2 oxidation catalysts

    NASA Astrophysics Data System (ADS)

    Loskyll, Jonas; Stoewe, Klaus; Maier, Wilhelm F.

    2011-10-01

    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.

  15. A Microchip for High-throughput Axon Growth Drug Screening

    PubMed Central

    Kim, Hyun Soo; Jeong, Sehoon; Koo, Chiwan; Han, Arum; Park, Jaewon

    2016-01-01

    It has been recently known that not only the presence of inhibitory molecules associated with myelin but also the reduced growth capability of the axons limit mature central nervous system (CNS) axonal regeneration after injury. Conventional axon growth studies are typically conducted using multi-well cell culture plates that are very challenging to investigate localized effects of drugs and limited to low throughput. Unfortunately, there is currently no other in vitro tools that allow investigating localized axonal responses to biomolecules in high-throughput for screening potential drugs that might promote axonal growth. We have developed a compartmentalized neuron culture platform enabling localized biomolecular treatments in parallel to axons that are physically and fluidically isolated from their neuronal somata. The 24 axon compartments in the developed platform are designed to perform four sets of six different localized biomolecular treatments simultaneously on a single device. In addition, the novel microfluidic configuration allows culture medium of 24 axon compartments to be replenished altogether by a single aspiration process, making high-throughput drug screening a reality. PMID:27928514

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

    PubMed

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

    2014-06-27

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

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

    NASA Astrophysics Data System (ADS)

    Fornari, Marco

    2015-03-01

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

  18. A High Throughput Mechanical Screening Device for Cartilage Tissue Engineering

    PubMed Central

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

    2014-01-01

    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. PMID:24275442

  19. A medium or high throughput protein refolding assay.

    PubMed

    Cowieson, Nathan P; Wensley, Beth; Robin, Gautier; Guncar, Gregor; Forwood, Jade; Hume, David A; Kobe, Bostjan; Martin, Jennifer L

    2008-01-01

    Expression of insoluble protein in E. coli is a major bottleneck of high throughput structural biology projects. Refolding proteins into native conformations from inclusion bodies could significantly increase the number of protein targets that can be taken on to structural studies. This chapter presents a simple assay for screening insoluble protein targets and identifying those that are most amenable to refolding. The assay is based on the observation that when proteins are refolded while bound to metal affinity resin, misfolded proteins are generally not eluted by imidazole. This difference is exploited here to distinguish between folded and misfolded proteins. Two implementations of the assay are described. The assay fits well into a standard high throughput structural biology pipeline, because it begins with the inclusion body preparations that are a byproduct of small-scale, automated expression and purification trials and does not require additional facilities. Two formats of the assay are described, a manual assay that is useful for screening small numbers of targets, and an automated implementation that is useful for large numbers of targets.

  20. Image quantification of high-throughput tissue microarray

    NASA Astrophysics Data System (ADS)

    Wu, Jiahua; Dong, Junyu; Zhou, Huiyu

    2006-03-01

    Tissue microarray (TMA) technology allows rapid visualization of molecular targets in thousands of tissue specimens at a time and provides valuable information on expression of proteins within tissues at a cellular and sub-cellular level. TMA technology overcomes the bottleneck of traditional tissue analysis and allows it to catch up with the rapid advances in lead discovery. Studies using TMA on immunohistochemistry (IHC) can produce a large amount of images for interpretation within a very short time. Manual interpretation does not allow accurate quantitative analysis of staining to be undertaken. Automatic image capture and analysis has been shown to be superior to manual interpretation. The aims of this work is to develop a truly high-throughput and fully automated image capture and analysis system. We develop a robust colour segmentation algorithm using hue-saturation-intensity (HSI) colour space to provide quantification of signal intensity and partitioning of staining on high-throughput TMA. Initial segmentation results and quantification data have been achieved on 16,000 TMA colour images over 23 different tissue types.

  1. High-throughput technology for novel SO2 oxidation catalysts

    PubMed Central

    Loskyll, Jonas; Stoewe, Klaus; Maier, Wilhelm F

    2011-01-01

    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. PMID:27877427

  2. A High-Throughput Cidality Screen for Mycobacterium Tuberculosis

    PubMed Central

    Kaur, Parvinder; Ghosh, Anirban; Krishnamurthy, Ramya Vadageri; Bhattacharjee, Deepa Gagwani; Achar, Vijayashree; Datta, Santanu; Narayanan, Shridhar; Anbarasu, Anand; Ramaiah, Sudha

    2015-01-01

    Exposure to Mycobacterium tuberculosis (Mtb) aerosols is a major threat to tuberculosis (TB) researchers, even in bio-safety level-3 (BSL-3) facilities. Automation and high-throughput screens (HTS) in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well ‘spot-assay’ for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm) and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets). The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808). An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA) or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process. PMID:25693161

  3. Compression of Structured High-Throughput Sequencing Data

    PubMed Central

    Campagne, Fabien; Dorff, Kevin C.; Chambwe, Nyasha; Robinson, James T.; Mesirov, Jill P.

    2013-01-01

    Large biological datasets are being produced at a rapid pace and create substantial storage challenges, particularly in the domain of high-throughput sequencing (HTS). Most approaches currently used to store HTS data are either unable to quickly adapt to the requirements of new sequencing or analysis methods (because they do not support schema evolution), or fail to provide state of the art compression of the datasets. We have devised new approaches to store HTS data that support seamless data schema evolution and compress datasets substantially better than existing approaches. Building on these new approaches, we discuss and demonstrate how a multi-tier data organization can dramatically reduce the storage, computational and network burden of collecting, analyzing, and archiving large sequencing datasets. For instance, we show that spliced RNA-Seq alignments can be stored in less than 4% the size of a BAM file with perfect data fidelity. Compared to the previous compression state of the art, these methods reduce dataset size more than 40% when storing exome, gene expression or DNA methylation datasets. The approaches have been integrated in a comprehensive suite of software tools (http://goby.campagnelab.org) that support common analyses for a range of high-throughput sequencing assays. PMID:24260313

  4. High-throughput gene mapping in Caenorhabditis elegans.

    PubMed

    Swan, Kathryn A; Curtis, Damian E; McKusick, Kathleen B; Voinov, Alexander V; Mapa, Felipa A; Cancilla, Michael R

    2002-07-01

    Positional cloning of mutations in model genetic systems is a powerful method for the identification of targets of medical and agricultural importance. To facilitate the high-throughput mapping of mutations in Caenorhabditis elegans, we have identified a further 9602 putative new single nucleotide polymorphisms (SNPs) between two C. elegans strains, Bristol N2 and the Hawaiian mapping strain CB4856, by sequencing inserts from a CB4856 genomic DNA library and using an informatics pipeline to compare sequences with the canonical N2 genomic sequence. When combined with data from other laboratories, our marker set of 17,189 SNPs provides even coverage of the complete worm genome. To date, we have confirmed >1099 evenly spaced SNPs (one every 91 +/- 56 kb) across the six chromosomes and validated the utility of our SNP marker set and new fluorescence polarization-based genotyping methods for systematic and high-throughput identification of genes in C. elegans by cloning several proprietary genes. We illustrate our approach by recombination mapping and confirmation of the mutation in the cloned gene, dpy-18.

  5. Iterative ACORN as a high throughput tool in structural genomics.

    PubMed

    Selvanayagam, S; Velmurugan, D; Yamane, T

    2006-08-01

    High throughput macromolecular structure determination is very essential in structural genomics as the available number of sequence information far exceeds the number of available 3D structures. ACORN, a freely available resource in the CCP4 suite of programs is a comprehensive and efficient program for phasing in the determination of protein structures, when atomic resolution data are available. ACORN with the automatic model-building program ARP/wARP and refinement program REFMAC is a suitable combination for the high throughput structural genomics. ACORN can also be run with secondary structural elements like helices and sheets as inputs with high resolution data. In situations, where ACORN phasing is not sufficient for building the protein model, the fragments (incomplete model/dummy atoms) can again be used as a starting input. Iterative ACORN is proved to work efficiently in the subsequent model building stages in congerin (PDB-ID: lis3) and catalase (PDB-ID: 1gwe) for which models are available.

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

    PubMed Central

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

    2016-01-01

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

  7. High throughput SNP detection system based on magnetic nanoparticles separation.

    PubMed

    Liu, Bin; Jia, Yingying; Ma, Man; Li, Zhiyang; Liu, Hongna; Li, Song; Deng, Yan; Zhang, Liming; Lu, Zhuoxuan; Wang, Wei; He, Nongyue

    2013-02-01

    Single-nucleotide polymorphism (SNP) was one-base variations in DNA sequence that can often be helpful to find genes associations for hereditary disease, communicable disease and so on. We developed a high throughput SNP detection system based on magnetic nanoparticles (MNPs) separation and dual-color hybridization or single base extension. This system includes a magnetic separation unit for sample separation, three high precision robot arms for pipetting and microtiter plate transferring respectively, an accurate temperature control unit for PCR and DNA hybridization and a high accurate and sensitive optical signal detection unit for fluorescence detection. The cyclooxygenase-2 gene promoter region--65G > C polymorphism locus SNP genotyping experiment for 48 samples from the northern Jiangsu area has been done to verify that if this system can simplify manual operation of the researchers, save time and improve efficiency in SNP genotyping experiments. It can realize sample preparation, target sequence amplification, signal detection and data analysis automatically and can be used in clinical molecule diagnosis and high throughput fluorescence immunological detection and so on.

  8. High-throughput screening to enhance oncolytic virus immunotherapy

    PubMed Central

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

    2016-01-01

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

  9. Discriminative motif analysis of high-throughput dataset

    PubMed Central

    Yao, Zizhen; MacQuarrie, Kyle L.; Fong, Abraham P.; Tapscott, Stephen J.; Ruzzo, Walter L.; Gentleman, Robert C.

    2014-01-01

    Motivation: High-throughput ChIP-seq studies typically identify thousands of peaks for a single transcription factor (TF). It is common for traditional motif discovery tools to predict motifs that are statistically significant against a naïve background distribution but are of questionable biological relevance. Results: We describe a simple yet effective algorithm for discovering differential motifs between two sequence datasets that is effective in eliminating systematic biases and scalable to large datasets. Tested on 207 ENCODE ChIP-seq datasets, our method identifies correct motifs in 78% of the datasets with known motifs, demonstrating improvement in both accuracy and efficiency compared with DREME, another state-of-art discriminative motif discovery tool. More interestingly, on the remaining more challenging datasets, we identify common technical or biological factors that compromise the motif search results and use advanced features of our tool to control for these factors. We also present case studies demonstrating the ability of our method to detect single base pair differences in DNA specificity of two similar TFs. Lastly, we demonstrate discovery of key TF motifs involved in tissue specification by examination of high-throughput DNase accessibility data. Availability: The motifRG package is publically available via the bioconductor repository. Contact: yzizhen@fhcrc.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24162561

  10. Genotype-Frequency Estimation from High-Throughput Sequencing Data.

    PubMed

    Maruki, Takahiro; Lynch, Michael

    2015-10-01

    Rapidly improving high-throughput sequencing technologies provide unprecedented opportunities for carrying out population-genomic studies with various organisms. To take full advantage of these methods, it is essential to correctly estimate allele and genotype frequencies, and here we present a maximum-likelihood method that accomplishes these tasks. The proposed method fully accounts for uncertainties resulting from sequencing errors and biparental chromosome sampling and yields essentially unbiased estimates with minimal sampling variances with moderately high depths of coverage regardless of a mating system and structure of the population. Moreover, we have developed statistical tests for examining the significance of polymorphisms and their genotypic deviations from Hardy-Weinberg equilibrium. We examine the performance of the proposed method by computer simulations and apply it to low-coverage human data generated by high-throughput sequencing. The results show that the proposed method improves our ability to carry out population-genomic analyses in important ways. The software package of the proposed method is freely available from https://github.com/Takahiro-Maruki/Package-GFE.

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

    PubMed

    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.

  12. High throughput, quantitative analysis of human osteoclast differentiation and activity.

    PubMed

    Diepenhorst, Natalie A; Nowell, Cameron J; Rueda, Patricia; Henriksen, Kim; Pierce, Tracie; Cook, Anna E; Pastoureau, Philippe; Sabatini, Massimo; Charman, William N; Christopoulos, Arthur; Summers, Roger J; Sexton, Patrick M; Langmead, Christopher J

    2017-02-15

    Osteoclasts are multinuclear cells that degrade bone under both physiological and pathophysiological conditions. Osteoclasts are therefore a major target of osteoporosis therapeutics aimed at preserving bone. Consequently, analytical methods for osteoclast activity are useful for the development of novel biomarkers and/or pharmacological agents for the treatment of osteoporosis. The nucleation state of an osteoclast is indicative of its maturation and activity. To date, activity is routinely measured at the population level with only approximate consideration of the nucleation state (an 'osteoclast population' is typically defined as cells with ≥3 nuclei). Using a fluorescent substrate for tartrate-resistant acid phosphatase (TRAP), a routinely used marker of osteoclast activity, we developed a multi-labelled imaging method for quantitative measurement of osteoclast TRAP activity at the single cell level. Automated image analysis enables interrogation of large osteoclast populations in a high throughput manner using open source software. Using this methodology, we investigated the effects of receptor activator of nuclear factor kappa-B ligand (RANK-L) on osteoclast maturation and activity and demonstrated that TRAP activity directly correlates with osteoclast maturity (i.e. nuclei number). This method can be applied to high throughput screening of osteoclast-targeting compounds to determine changes in maturation and activity.

  13. Piezo-thermal Probe Array for High Throughput Applications

    PubMed Central

    Gaitas, Angelo; French, Paddy

    2012-01-01

    Microcantilevers are used in a number of applications including atomic-force microscopy (AFM). In this work, deflection-sensing elements along with heating elements are integrated onto micromachined cantilever arrays to increase sensitivity, and reduce complexity and cost. An array of probes with 5–10 nm gold ultrathin film sensors on silicon substrates for high throughput scanning probe microscopy is developed. The deflection sensitivity is 0.2 ppm/nm. Plots of the change in resistance of the sensing element with displacement are used to calibrate the probes and determine probe contact with the substrate. Topographical scans demonstrate high throughput and nanometer resolution. The heating elements are calibrated and the thermal coefficient of resistance (TCR) is 655 ppm/K. The melting temperature of a material is measured by locally heating the material with the heating element of the cantilever while monitoring the bending with the deflection sensing element. The melting point value measured with this method is in close agreement with the reported value in literature. PMID:23641125

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

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

  15. High-throughput single-cell analysis for the proteomic dynamics study of the yeast osmotic stress response

    PubMed Central

    Zhang, Rongfei; Yuan, Haiyu; Wang, Shujing; Ouyang, Qi; Chen, Yong; Hao, Nan; Luo, Chunxiong

    2017-01-01

    Motorized fluorescence microscopy combined with high-throughput microfluidic chips is a powerful method to obtain information about different biological processes in cell biology studies. Generally, to observe different strains under different environments, high-throughput microfluidic chips require complex preparatory work. In this study, we designed a novel and easily operated high-throughput microfluidic system to observe 96 different GFP-tagged yeast strains in one switchable culture condition or 24 different GFP-tagged yeast strains in four parallel switchable culture conditions. A multi-pipette is the only additional equipment required for high-throughput patterning of cells in the chip. Only eight connections are needed to control 96 conditions. Using these devices, the proteomic dynamics of the yeast stress response pathway were carefully studied based on single-cell data. A new method to characterize the proteomic dynamics using a single cell’s data is proposed and compared to previous methods, and the new technique should be useful for studying underlying control networks. Our method provides an easy and systematic way to study signaling pathways at the single-cell level. PMID:28181485

  16. High-throughput single-cell analysis for the proteomic dynamics study of the yeast osmotic stress response.

    PubMed

    Zhang, Rongfei; Yuan, Haiyu; Wang, Shujing; Ouyang, Qi; Chen, Yong; Hao, Nan; Luo, Chunxiong

    2017-02-09

    Motorized fluorescence microscopy combined with high-throughput microfluidic chips is a powerful method to obtain information about different biological processes in cell biology studies. Generally, to observe different strains under different environments, high-throughput microfluidic chips require complex preparatory work. In this study, we designed a novel and easily operated high-throughput microfluidic system to observe 96 different GFP-tagged yeast strains in one switchable culture condition or 24 different GFP-tagged yeast strains in four parallel switchable culture conditions. A multi-pipette is the only additional equipment required for high-throughput patterning of cells in the chip. Only eight connections are needed to control 96 conditions. Using these devices, the proteomic dynamics of the yeast stress response pathway were carefully studied based on single-cell data. A new method to characterize the proteomic dynamics using a single cell's data is proposed and compared to previous methods, and the new technique should be useful for studying underlying control networks. Our method provides an easy and systematic way to study signaling pathways at the single-cell level.

  17. Genomic and phenotypic characterization of the species Acinetobacter venetianus

    PubMed Central

    Fondi, Marco; Maida, Isabel; Perrin, Elena; Orlandini, Valerio; La Torre, Laura; Bosi, Emanuele; Negroni, Andrea; Zanaroli, Giulio; Fava, Fabio; Decorosi, Francesca; Giovannetti, Luciana; Viti, Carlo; Vaneechoutte, Mario; Dijkshoorn, Lenie; Fani, Renato

    2016-01-01

    Crude oil is a complex mixture of hydrocarbons and other organic compounds that can produce serious environmental problems and whose removal is highly demanding in terms of human and technological resources. The potential use of microbes as bioremediation agents is one of the most promising fields in this area. Members of the species Acinetobacter venetianus have been previously characterized for their capability to degrade n-alkanes and thus may represent interesting model systems to implement this process. Although a preliminary experimental characterization of the overall hydrocarbon degradation capability has been performed for five of them, to date, the genetic/genomic features underlying such molecular processes have not been identified. Here we have integrated genomic and phenotypic information for six A. venetianus strains, i.e. VE-C3, RAG-1T, LUH 13518, LUH 7437, LUH 5627 and LUH 8758. Besides providing a thorough description of the A. venetianus species, these data were exploited to infer the genetic features (presence/absence patterns of genes) and the short-term evolutionary events possibly responsible for the variability in n-alkane degradation efficiency of these strains, including the mechanisms of interaction with the fuel droplet and the subsequent catabolism of this pollutant. PMID:26902269

  18. Genomic and phenotypic characterization of the species Acinetobacter venetianus.

    PubMed

    Fondi, Marco; Maida, Isabel; Perrin, Elena; Orlandini, Valerio; La Torre, Laura; Bosi, Emanuele; Negroni, Andrea; Zanaroli, Giulio; Fava, Fabio; Decorosi, Francesca; Giovannetti, Luciana; Viti, Carlo; Vaneechoutte, Mario; Dijkshoorn, Lenie; Fani, Renato

    2016-02-23

    Crude oil is a complex mixture of hydrocarbons and other organic compounds that can produce serious environmental problems and whose removal is highly demanding in terms of human and technological resources. The potential use of microbes as bioremediation agents is one of the most promising fields in this area. Members of the species Acinetobacter venetianus have been previously characterized for their capability to degrade n-alkanes and thus may represent interesting model systems to implement this process. Although a preliminary experimental characterization of the overall hydrocarbon degradation capability has been performed for five of them, to date, the genetic/genomic features underlying such molecular processes have not been identified. Here we have integrated genomic and phenotypic information for six A. venetianus strains, i.e. VE-C3, RAG-1(T), LUH 13518, LUH 7437, LUH 5627 and LUH 8758. Besides providing a thorough description of the A. venetianus species, these data were exploited to infer the genetic features (presence/absence patterns of genes) and the short-term evolutionary events possibly responsible for the variability in n-alkane degradation efficiency of these strains, including the mechanisms of interaction with the fuel droplet and the subsequent catabolism of this pollutant.

  19. Phenotypic characterization of ESBL producing Enterobacter cloacae among children

    PubMed Central

    Amin, Hafsa; Zafar, Aizza; Ejaz, Hasan; Jameel, Noor-ul-Ain

    2013-01-01

    Objective: The emergence of ESBL producing Enterobacter cloacae in clinical isolates is posing a serious threat for treating nosocomial infections. The aim of the study was to determine the frequency of extended spectrum β-lactamase (ESBL) producing Enterobacter cloacae and to compare the phenotypic methods used for the characterization of ESBL producing strains. Methodology: This cross sectional observational study was conducted during April 2011 to March 2012 at Microbiology department of The Children’s Hospital and Institute of Child Health, Lahore. A total number of 20,257 various clinical samples were analyzed during the study period. Enterobacter cloacae were identified using API 20E system and ESBL detection was carried out using double-disk synergy test (DDST) and CLSI confirmatory test. Results: Enterobacter cloacae were isolated from 221 samples, out of which 33 (14.93%) were ESBL producers and 188 (85.07%) were non-ESBL producers. The gender distribution of ESBL producing Enterobacter cloacae was 21 (63.6%) in males and 12 (36.4%) in females. Highest frequency (63%) of ESBL producing Enterobacter cloacae was detected in blood samples. Comparison of DDST and CLSI confirmatory test showed that 25 (75.75%) isolates were characterized by DDST and 33 (100%) using CLSI confirmatory test. Conclusion: The present study shows moderately high frequency of ESBL producing Enterobacter cloacae among children. DDST was found to be less efficient in ESBL detection as compared to CLSI confirmatory test. PMID:24353527

  20. Three-Dimensional Cell Culture Microarray for High-Throughput Studies of Stem Cell Fate

    PubMed Central

    Fernandes, Tiago G.; Kwon, Seok-Joon; Bale, Shyam Sundhar; Lee, Moo-Yeal; Diogo, Maria Margarida; Clark, Douglas S.; Cabral, Joaquim M.S.

    2017-01-01

    We have developed a novel three-dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high-throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox-1, whose levels were also measured in situ using a GFP reporter system. In addition, the high-throughput capacity of the platform was tested using a dual-slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor-4 (FGF-4) on the pluripotency of mouse ES cells. This high-throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses. PMID:20069558

  1. The RABiT: High Throughput Technology for Assessing Global DSB Repair

    PubMed Central

    Turner, H.C.; Sharma, P.; Perrier, J.R.; Bertucci, A.; Smilenov, L.; Johnson, Gary; Taveras, M.; Brenner, D.J.; Garty, G.

    2014-01-01

    At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry we have developed a Rapid Automated Biodosimetry Tool (RABiT); this is a completely automated, ultra-high throughput robotically-based biodosimetry workstation designed for use following a large scale radiological event, to perform radiation biodosimetry measurements based on a fingerstick blood sample. High throughput is achieved through purpose built robotics, sample handling in filter-bottomed multi-well plates and innovations in high speed imaging and analysis. Currently, we are adapting the RABiT technologies for use in laboratory settings, for applications in epidemiological and clinical studies. Our overall goal is to extend the RABiT system to directly measure the kinetics of DNA repair proteins. The design of the kinetic/time dependent studies is based on repeated, automated sampling of lymphocytes from a central reservoir of cells housed in the RABiT incubator as a function of time after the irradiation challenge. In the present study, we have characterized the DNA repair kinetics of the following repair proteins: γ-H2AX, 53-BP1, ATM kinase, MDC1 at multiple times (0.5, 2, 4, 7, 24 hours) after irradiation with 4 Gy γ rays. In order to provide a consistent dose exposure at time zero, we have developed an automated capillary irradiator to introduce DNA DSBs into fingerstick-size blood samples within the RABiT. To demonstrate the scalability of the laboratory-based RABiT system, we have initiated a population study using γ-H2AX as a biomarker. PMID:24477408

  2. Minireactor-based high-throughput temperature profiling for the optimization of microbial and enzymatic processes

    PubMed Central

    2014-01-01

    Background Bioprocesses depend on a number of different operating parameters and temperature is one of the most important ones. Unfortunately, systems for rapid determination of temperature dependent reaction kinetics are rare. Obviously, there is a need for a high-throughput screening procedure of temperature dependent process behavior. Even though, well equipped micro-bioreactors are a promising approach sufficient temperature control is quite challenging and rather complex. Results In this work a unique system is presented combining an optical on-line monitoring device with a customized temperature control unit for 96 well microtiter plates. By exposing microtiter plates to specific temperature profiles, high-throughput temperature optimization for microbial and enzymatic systems in a micro-scale of 200 μL is realized. For single well resolved temperature measurement fluorescence thermometry was used, combining the fluorescent dyes Rhodamin B and Rhodamin 110. The real time monitoring of the microbial and enzymatic reactions provides extensive data output. To evaluate this novel system the temperature optima for Escherichia coli and Kluyveromyces lactis regarding growth and recombinant protein production were determined. Furthermore, the commercial cellulase mixture Celluclast as a representative for enzymes was investigated applying a fluorescent activity assay. Conclusion Microtiter plate-based high-throughput temperature profiling is a convenient tool for characterizing temperature dependent reaction processes. It allows the evaluation of numerous conditions, e.g. microorganisms, enzymes, media, and others, in a short time. The simple temperature control combined with a commercial on-line monitoring device makes it a user friendly system. PMID:25126113

  3. High throughput screen identifies small molecule inhibitors specific for Mycobacterium tuberculosis phosphoserine phosphatase.

    PubMed

    Arora, Garima; Tiwari, Prabhakar; Mandal, Rahul Shubhra; Gupta, Arpit; Sharma, Deepak; Saha, Sudipto; Singh, Ramandeep

    2014-09-05

    The emergence of drug-resistant strains of Mycobacterium tuberculosis makes identification and validation of newer drug targets a global priority. Phosphoserine phosphatase (PSP), a key essential metabolic enzyme involved in conversion of O-phospho-l-serine to l-serine, was characterized in this study. The M. tuberculosis genome harbors all enzymes involved in l-serine biosynthesis including two PSP homologs: Rv0505c (SerB1) and Rv3042c (SerB2). In the present study, we have biochemically characterized SerB2 enzyme and developed malachite green-based high throughput assay system to identify SerB2 inhibitors. We have identified 10 compounds that were structurally different from known PSP inhibitors, and few of these scaffolds were highly specific in their ability to inhibit SerB2 enzyme, were noncytotoxic against mammalian cell lines, and inhibited M. tuberculosis growth in vitro. Surface plasmon resonance experiments demonstrated the relative binding for these inhibitors. The two best hits identified in our screen, clorobiocin and rosaniline, were bactericidal in activity and killed intracellular bacteria in a dose-dependent manner. We have also identified amino acid residues critical for these SerB2-small molecule interactions. This is the first study where we validate that M. tuberculosis SerB2 is a druggable and suitable target to pursue for further high throughput assay system screening.

  4. Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis.

    PubMed

    Pan, Yuchen; Sackmann, Eric K; Wypisniak, Karolina; Hornsby, Michael; Datwani, Sammy S; Herr, Amy E

    2016-12-23

    High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality - the binding affinity - is quantified through the dissociation constant (KD) of each recombinant antibody and the target antigen. To characterize the KD of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The KD for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization.

  5. High Throughput Screen Identifies Small Molecule Inhibitors Specific for Mycobacterium tuberculosis Phosphoserine Phosphatase*

    PubMed Central

    Arora, Garima; Tiwari, Prabhakar; Mandal, Rahul Shubhra; Gupta, Arpit; Sharma, Deepak; Saha, Sudipto; Singh, Ramandeep

    2014-01-01

    The emergence of drug-resistant strains of Mycobacterium tuberculosis makes identification and validation of newer drug targets a global priority. Phosphoserine phosphatase (PSP), a key essential metabolic enzyme involved in conversion of O-phospho-l-serine to l-serine, was characterized in this study. The M. tuberculosis genome harbors all enzymes involved in l-serine biosynthesis including two PSP homologs: Rv0505c (SerB1) and Rv3042c (SerB2). In the present study, we have biochemically characterized SerB2 enzyme and developed malachite green-based high throughput assay system to identify SerB2 inhibitors. We have identified 10 compounds that were structurally different from known PSP inhibitors, and few of these scaffolds were highly specific in their ability to inhibit SerB2 enzyme, were noncytotoxic against mammalian cell lines, and inhibited M. tuberculosis growth in vitro. Surface plasmon resonance experiments demonstrated the relative binding for these inhibitors. The two best hits identified in our screen, clorobiocin and rosaniline, were bactericidal in activity and killed intracellular bacteria in a dose-dependent manner. We have also identified amino acid residues critical for these SerB2-small molecule interactions. This is the first study where we validate that M. tuberculosis SerB2 is a druggable and suitable target to pursue for further high throughput assay system screening. PMID:25037224

  6. Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis

    PubMed Central

    Pan, Yuchen; Sackmann, Eric K.; Wypisniak, Karolina; Hornsby, Michael; Datwani, Sammy S.; Herr, Amy E.

    2016-01-01

    High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality – the binding affinity – is quantified through the dissociation constant (KD) of each recombinant antibody and the target antigen. To characterize the KD of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The KD for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization. PMID:28008969

  7. High-throughput measurements of thermochromic behavior in V(1-x)Nb(x)O(2) combinatorial thin film libraries.

    PubMed

    Barron, S C; Gorham, J M; Patel, M P; Green, M L

    2014-10-13

    We describe a high-throughput characterization of near-infrared thermochromism in V1-xNbxO2 combinatorial thin film libraries. The oxide thin film library was prepared with a VO2 crystal structure and a continuous gradient in composition with Nb concentrations in the range of less than 1% to 45%. The thermochromic phase transition from monoclinic to tetragonal was characterized by the accompanying change in near-infrared reflectance. With increasing Nb substitution, the transition temperature was depressed from 65 to 35 °C, as desirable for smart window applications. However, the magnitude of the reflectance change across the thermochromic transition was also reduced with increasing Nb film content. Data collection, handling, and analysis supporting thermochromic characterization were fully automated to achieve high throughput. Using this system, in 14 h, temperature-dependent infrared reflectances were measured at 165 arbitrary locations on a thin film combinatorial library; these measurements were analyzed for thermochromic transitions in minutes.

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

    PubMed

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

    2012-09-06

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

  9. Phenotypic and genotypic characterization of peptidoglycan hydrolases of Lactobacillus sakei

    PubMed Central

    Najjari, Afef; Amairi, Houda; Chaillou, Stéphane; Mora, Diego; Boudabous, Abdellatif; Zagorec, Monique; Ouzari, Hadda

    2015-01-01

    Lactobacillus sakei, a lactic acid bacterium naturally found in fresh meat and sea products, is considered to be one of the most important bacterial species involved in meat fermentation and bio-preservation. Several enzymes of Lb. sakei species contributing to microbial safeguarding and organoleptic properties of fermented-meat were studied. However, the specific autolytic mechanisms and associated enzymes involved in Lb. sakei are not well understood. The autolytic phenotype of 22 Lb. sakei strains isolated from Tunisian meat and seafood products was evaluated under starvation conditions, at pH 6.5 and 8.5, and in the presence of different carbon sources. A higher autolytic rate was observed when cells were grown in the presence of glucose and incubated at pH 6.5. Almost all strains showed high resistance to mutanolysin, indicating a minor role of muramidases in Lb. sakei cell lysis. Using Micrococcus lysodeikticus cells as a substrate in activity gels zymogram, peptidoglycan hydrolase (PGH) patterns for all strains was characterized by two lytic bands of ∼80 (B1) and ∼70 kDa (B2), except for strain BMG.167 which harbored two activity signals at a lower MW. Lytic activity was retained in high salt and in acid/basic conditions and was active toward cells of Lb. sakei, Listeria monocytogenes, Listeria ivanovii and Listeria innocua. Analysis of five putative PGH genes found in the Lb. sakei 23 K model strain genome, indicated that one gene, lsa1437, could encode a PGH (N-acetylmuramoyl-L-alanine amidase) containing B1 and B2 as isoforms. According to this hypothesis, strain BMG.167 showed an allelic version of lsa1437 gene deleted of one of the five LysM domains, leading to a reduction in the MW of lytic bands and the high autolytic rate of this strain. Characterization of autolytic phenotype of Lb. sakei should expand the knowledge of their role in fermentation processes where they represent the dominant species. PMID:26843981

  10. Phenotypic and genotypic characterization of peptidoglycan hydrolases of Lactobacillus sakei.

    PubMed

    Najjari, Afef; Amairi, Houda; Chaillou, Stéphane; Mora, Diego; Boudabous, Abdellatif; Zagorec, Monique; Ouzari, Hadda

    2016-01-01

    Lactobacillus sakei, a lactic acid bacterium naturally found in fresh meat and sea products, is considered to be one of the most important bacterial species involved in meat fermentation and bio-preservation. Several enzymes of Lb. sakei species contributing to microbial safeguarding and organoleptic properties of fermented-meat were studied. However, the specific autolytic mechanisms and associated enzymes involved in Lb. sakei are not well understood. The autolytic phenotype of 22 Lb. sakei strains isolated from Tunisian meat and seafood products was evaluated under starvation conditions, at pH 6.5 and 8.5, and in the presence of different carbon sources. A higher autolytic rate was observed when cells were grown in the presence of glucose and incubated at pH 6.5. Almost all strains showed high resistance to mutanolysin, indicating a minor role of muramidases in Lb. sakei cell lysis. Using Micrococcus lysodeikticus cells as a substrate in activity gels zymogram, peptidoglycan hydrolase (PGH) patterns for all strains was characterized by two lytic bands of ∼80 (B1) and ∼70 kDa (B2), except for strain BMG.167 which harbored two activity signals at a lower MW. Lytic activity was retained in high salt and in acid/basic conditions and was active toward cells of Lb. sakei, Listeria monocytogenes, Listeria ivanovii and Listeria innocua. Analysis of five putative PGH genes found in the Lb. sakei 23 K model strain genome, indicated that one gene, lsa1437, could encode a PGH (N-acetylmuramoyl-L-alanine amidase) containing B1 and B2 as isoforms. According to this hypothesis, strain BMG.167 showed an allelic version of lsa1437 gene deleted of one of the five LysM domains, leading to a reduction in the MW of lytic bands and the high autolytic rate of this strain. Characterization of autolytic phenotype of Lb. sakei should expand the knowledge of their role in fermentation processes where they represent the dominant species.

  11. A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood

    PubMed Central

    Seeber, Stefan; Ros, Francesca; Thorey, Irmgard; Tiefenthaler, Georg; Kaluza, Klaus; Lifke, Valeria; Fischer, Jens André Alexander; Klostermann, Stefan; Endl, Josef; Kopetzki, Erhard; Pashine, Achal; Siewe, Basile; Kaluza, Brigitte; Platzer, Josef; Offner, Sonja

    2014-01-01

    We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits) yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal. PMID:24503933

  12. A novel multiplex cell viability assay for high-throughput RNAi screening.

    PubMed

    Gilbert, Daniel F; Erdmann, Gerrit; Zhang, Xian; Fritzsche, Anja; Demir, Kubilay; Jaedicke, Andreas; Muehlenberg, Katja; Wanker, Erich E; Boutros, Michael

    2011-01-01

    Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.

  13. A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells

    PubMed Central

    Bassett, Andrew R.; Kong, Lesheng; Liu, Ji-Long

    2015-01-01

    The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines, enabling screening for cellular phenotypes resulting from genetic aberrations. Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi. This is in part due to the lower degree of redundancy between genes in this organism, whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes. The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques, but allows analysis over longer periods of time which can be critical for certain phenotypes. In this study, we have designed and built a genome-wide CRISPR library covering 13,501 genes, among which 8989 genes are targeted by three or more independent single guide RNAs (sgRNAs). Moreover, we describe strategies to monitor the population of guide RNAs by high throughput sequencing (HTS). We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes, and as a source of guide RNA designs for future studies. PMID:26165496

  14. A genome-wide CRISPR library for high-throughput genetic screening in Drosophila cells.

    PubMed

    Bassett, Andrew R; Kong, Lesheng; Liu, Ji-Long

    2015-06-20

    The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines, enabling screening for cellular phenotypes resulting from genetic aberrations. Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi. This is in part due to the lower degree of redundancy between genes in this organism, whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes. The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques, but allows analysis over longer periods of time which can be critical for certain phenotypes. In this study, we have designed and built a genome-wide CRISPR library covering 13,501 genes, among which 8989 genes are targeted by three or more independent single guide RNAs (sgRNAs). Moreover, we describe strategies to monitor the population of guide RNAs by high throughput sequencing (HTS). We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes, and as a source of guide RNA designs for future studies.

  15. Native mass spectrometry: towards high-throughput structural proteomics.

    PubMed

    Kondrat, Frances D L; Struwe, Weston B; Benesch, Justin L P

    2015-01-01

    Native mass spectrometry (MS) has become a sensitive method for structural proteomics, allowing practitioners to gain insight into protein self-assembly, including stoichiometry and three-dimensional architecture, as well as complementary thermodynamic and kinetic aspects. Although MS is typically performed in vacuum, a body of literature has described how native solution-state structure is largely retained on the timescale of the experiment. Native MS offers the benefit that it requires substantially smaller quantities of a sample than traditional structural techniques such as NMR and X-ray crystallography, and is therefore well suited to high-throughput studies. Here we first describe the native MS approach and outline the structural proteomic data that it can deliver. We then provide practical details of experiments to examine the structural and dynamic properties of protein assemblies, highlighting potential pitfalls as well as principles of best practice.

  16. Interactive Visual Analysis of High Throughput Text Streams

    SciTech Connect

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

    2012-01-01

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

  17. Quantitative High-throughput Luciferase Screening in Identifying CAR Modulators

    PubMed Central

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

    2017-01-01

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

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

    PubMed

    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. Microfluidic cell chips for high-throughput drug screening.

    PubMed

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

    2016-05-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

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

  1. Towards high throughput screening of nanoparticle flotation collectors.

    PubMed

    Abarca, Carla; Yang, Songtao; Pelton, Robert H

    2015-12-15

    To function as flotation collectors for mineral processing, polymeric nanoparticles require a delicate balance of surface properties to give mineral-specific deposition and colloidal stability in high ionic strength alkaline media, while remaining sufficiently hydrophobic to promote flotation. Combinatorial nanoparticle surface modification, in conjunction with high throughput screening, is a promising approach for nanoparticle development. However, efficient automated screening assays are required to reject ineffective particles without having to undergo time consuming flotation testing. Herein we demonstrate that determining critical coagulation concentrations of sodium carbonate in combination with measuring the advancing water contact angle of nanoparticle-saturated glass surfaces can be used to screen ineffective nanoparticles. Finally, none of our first nanoparticle library based on poly(ethylene glycol) methyl ether methacrylate (PEG-methacrylate) were effective flotation collectors because the nanoparticles were too hydrophilic.

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

    PubMed

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

    2016-03-01

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

  3. Resolving postglacial phylogeography using high-throughput sequencing

    PubMed Central

    Emerson, Kevin J.; Merz, Clayton R.; Catchen, Julian M.; Hohenlohe, Paul A.; Cresko, William A.; Bradshaw, William E.; Holzapfel, Christina M.

    2010-01-01

    The distinction between model and nonmodel organisms is becoming increasingly blurred. High-throughput, second-generation sequencing approaches are being applied to organisms based on their interesting ecological, physiological, developmental, or evolutionary properties and not on the depth of genetic information available for them. Here, we illustrate this point using a low-cost, efficient technique to determine the fine-scale phylogenetic relationships among recently diverged populations in a species. This application of restriction site-associated DNA tags (RAD tags) reveals previously unresolved genetic structure and direction of evolution in the pitcher plant mosquito, Wyeomyia smithii, from a southern Appalachian Mountain refugium following recession of the Laurentide Ice Sheet at 22,000–19,000 B.P. The RAD tag method can be used to identify detailed patterns of phylogeography in any organism regardless of existing genomic data, and, more broadly, to identify incipient speciation and genome-wide variation in natural populations in general. PMID:20798348

  4. High-throughput electronic biology: mining information for drug discovery.

    PubMed

    Loging, William; Harland, Lee; Williams-Jones, Bryn

    2007-03-01

    The vast range of in silico resources that are available in life sciences research hold much promise towards aiding the drug discovery process. To fully realize this opportunity, computational scientists must consider the practical issues of data integration and identify how best to apply these resources scientifically. In this article we describe in silico approaches that are driven towards the identification of testable laboratory hypotheses; we also address common challenges in the field. We focus on flexible, high-throughput techniques, which may be initiated independently of 'wet-lab' experimentation, and which may be applied to multiple disease areas. The utility of these approaches in drug discovery highlights the contribution that in silico techniques can make and emphasizes the need for collaboration between the areas of disease research and computational science.

  5. High throughput x-ray optics: an overview.

    PubMed

    Gorenstein, P

    1988-04-15

    Several x-ray astronomy missions of the 1990s will contain focusing telescopes with significantly more collecting power than the Einstein Observatory. There is increasing emphasis on spectroscopy. ESA's XMM with 10(4) cm(2) of effective area will be the largest. A high throughput facility with over 10(5) cm(2) of effective area and 20-sec of arc angular resolution is needed ultimately for various scientific studies such as high resolution spectroscopic observations of QSOs. At least one of the following techniques currently being developed for fabricating x-ray telescopes including automated figuring of flats as parabolic reflectors, replication of cylindrical shells, and the alignment of thin lacquer-coated conical foils is likely to permit the construction of modular arrays of telescopes with the area and angular resolution required.

  6. Muscle plasticity and high throughput gene expression studies.

    PubMed

    Reggiani, Carlo; Kronnie, Geertruuy Te

    2004-01-01

    Changes in gene expression are known to contribute to muscle plasticity. Until recently most studies have described differences of one or few genes at a time, in the last few years, however, the development of new technology of high throughput mRNA expression analysis has allowed the study of a large part if not all transcripts in the same experiment. Knowledge on any muscle adaptive response has already gained from the application of this novel approach, but the most important new findings have come from studies on muscle atrophy. A new and unexpected groups of genes, which increase their expression during atrophy and are, therefore, designated as atrogins, have been discovered. In spite of the impressive power of the new technology many problems are still to be resolved to optimize the experimental design and to extract all information which are provided by the outcome of the global mRNA assessment.

  7. Macromolecular Crystallography conventional and high-throughput methods

    SciTech Connect

    Wasserman, Stephen R.; Smith, David W.; D'Amico, Kevin L.; Koss, John W.; Morisco, Laura L.; Burley, Stephen K.

    2007-09-27

    High-throughput data collection requires the seamless interoperation of various hardware components. User-supplied descriptions of protein crystals must also be directly linked with the diffraction data. Such linkages can be achieved efficiently with computer databases. A database that tracks production of the protein samples, crystallization, and diffraction from the resultant crystals serves as the glue that holds the entire gene-to-structure process together. This chapter begins by discussing data collection processes and hardware. It then illustrates how a well-constructed database ensures information flow through the steps of data acquisition. Such a database allows synchrotron beamline measurements to be directly and efficiently integrated into the process of protein crystallographic structure determination.

  8. Numerical techniques for high-throughput reflectance interference biosensing

    NASA Astrophysics Data System (ADS)

    Sevenler, Derin; Ünlü, M. Selim

    2016-06-01

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

  9. Microfluidics for High-Throughput Quantitative Studies of Early Development.

    PubMed

    Levario, Thomas J; Lim, Bomyi; Shvartsman, Stanislav Y; Lu, Hang

    2016-07-11

    Developmental biology has traditionally relied on qualitative analyses; recently, however, as in other fields of biology, researchers have become increasingly interested in acquiring quantitative knowledge about embryogenesis. Advances in fluorescence microscopy are enabling high-content imaging in live specimens. At the same time, microfluidics and automation technologies are increasing experimental throughput for studies of multicellular models of development. Furthermore, computer vision methods for processing and analyzing bioimage data are now leading the way toward quantitative biology. Here, we review advances in the areas of fluorescence microscopy, microfluidics, and data analysis that are instrumental to performing high-content, high-throughput studies in biology and specifically in development. We discuss a case study of how these techniques have allowed quantitative analysis and modeling of pattern formation in the Drosophila embryo.

  10. Microgradient-heaters as tools for high-throughput experimentation.

    PubMed

    Meyer, Robert; Hamann, Sven; Ehmann, Michael; Thienhaus, Sigurd; Jaeger, Stefanie; Thiede, Tobias; Devi, Anjana; Fischer, Roland A; Ludwig, Alfred

    2012-10-08

    A microgradient-heater (MGH) was developed, and its feasibility as a tool for high-throughput materials science experimentation was tested. The MGH is derived from microhot plate (MHP) systems and allows combinatorial thermal processing on the micronano scale. The temperature gradient is adjustable by the substrate material. For an Au-coated MGH membrane a temperature drop from 605 to 100 °C was measured over a distance of 965 μm, resulting in an average temperature change of 0.52 K/μm. As a proof of principle, we demonstrate the feasibility of MGHs on the example of a chemical vapor deposition (CVD) process. The achieved results show discontinuous changes in surface morphology within a continuous TiO2 film. Furthermore the MGH can be used to get insights into the energetic relations of film growth processes, giving it the potential for microcalorimetry measurements.

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

    PubMed

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

    2007-03-09

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

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

    PubMed

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

    2017-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

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

    PubMed

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

    2013-12-01

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

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

    PubMed

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

    2013-09-17

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

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

    NASA Astrophysics Data System (ADS)

    Wu, Henry; Mayeshiba, Tam; Morgan, Dane

    2016-07-01

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

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

    PubMed

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

    2016-07-15

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

  18. Phenotypic and functional characterization of Bacillus anthracis biofilms.

    PubMed

    Lee, Keehoon; Costerton, J W; Ravel, Jacques; Auerbach, Raymond K; Wagner, David M; Keim, Paul; Leid, Jeff G

    2007-06-01

    Biofilms, communities of micro-organisms attached to a surface, are responsible for many chronic diseases and are often associated with environmental reservoirs or lifestyles. Bacillus anthracis is a Gram-positive, endospore-forming bacterium and is the aetiological agent of pulmonary, gastrointestinal and cutaneous anthrax. Anthrax infections are part of the natural lifecycle of many ruminants in North America, including cattle and bison, and B. anthracis is thought to be a central part of this ecosystem. However, in endemic areas in which humans and livestock interact, chronic cases of cutaneous anthrax are commonly reported. This suggests that biofilms of B. anthracis exist in the environment and are part of the ecology associated with its lifecycle. Currently, there are few data that account for the importance of the biofilm mode of life in B. anthracis, yet biofilms have been characterized in other pathogenic and non-pathogenic Bacillus species, including Bacillus cereus and Bacillus subtilis, respectively. This study investigated the phenotypic and functional role of biofilms in B. anthracis. The results demonstrate that B. anthracis readily forms biofilms which are inherently resistant to commonly prescribed antibiotics, and that antibiotic resistance is not solely the function of sporulation.

  19. Phenotypic and Genotypic Characterization of Bacteriocinogenic Enterococci Against Clostridium botulinum.

    PubMed

    Shehata, Awad A; Tarabees, Reda; Basiouni, Shereen; Gamil, Mahmoud; Kamal, Ahmed S; Krüger, Monika

    2016-12-02

    The present study aimed to characterize Enterococcus faecalis (n = -6) and Enterococcus faecium (n = 1) isolated from healthy chickens to find a novel perspective probiotic candidate that antagonize Clostridium botulinum types A, B, D, and E. The isolated enterococci were characterized based on phenotypic properties, PCR, and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF). The virulence determinants including hemolytic activity on blood agar, gelatinase activity, sensitivity to vancomycin, and presence of gelatinase (gelE) and enterococcal surface protein (esp) virulence genes were investigated. Also, the presence of enterocin structural genes enterocin A, enterocin B, enterocin P, enterocin L50A/B, bacteriocin 31, enterocin AS48, enterocin 1071A/1071B, and enterocin 96 were assessed using PCR. Lastly, the antagonistic effect of the selected Enterococcus spp. on the growth of C. botulinum types A, B, D, and E was studied. The obtained results showed that four out of six E. faecalis and one E. faecium proved to be free from the tested virulence markers. All tested enterococci strains exhibited more than one of the tested enterocin. Interestingly, E. faecalis and E. faecium significantly restrained the growth of C. botulinum types A, B, D, and E. In conclusion, although, the data presented showed that bacteriocinogenic Enterococcus strains lacking of virulence determinants could be potentially used as a probiotic candidate against C. botulinum in vitro; however, further investigations are still urgently required to verify the beneficial effects of the tested Enterococcus spp. in vivo.

  20. High-throughput in situ cell electroporation microsystem for parallel delivery of single guide RNAs into mammalian cells

    PubMed Central

    Bian, Shengtai; Zhou, Yicen; Hu, Yawei; Cheng, Jing; Chen, Xiaofang; Xu, Youchun; Liu, Peng

    2017-01-01

    Arrayed genetic screens mediated by the CRISPR/Cas9 technology with single guide RNA (sgRNA) libraries demand a high-throughput platform capable of transfecting diverse cell types at a high efficiency in a genome-wide scale for detection and analysis of sophisticated cellular phenotypes. Here we developed a high-throughput in situ cell electroporation (HiCEP) microsystem which leveraged the superhydrophobic feature of the microwell array to achieve individually controlled conditions in each microwell and coupled an interdigital electrode array chip with the microwells in a modular-based scheme for highly efficient delivery of exogenous molecules into cells. Two plasmids encoding enhanced green and red fluorescent proteins (EGFP and ERFP), respectively, were successfully electroporated into attached HeLa cells on a 169-microwell array chip with transfection efficiencies of 71.6 ± 11.4% and 62.9 ± 2.7%, and a cell viability above 95%. We also successfully conducted selective electroporation of sgRNA into 293T cells expressing the Cas9 nuclease in a high-throughput manner and observed the four-fold increase of the GFP intensities due to the repair of the protein coding sequences mediated by the CRISPR/Cas9 system. This study proved that this HiCEP system has the great potential to be used for arrayed functional screens with genome-wide CRISPR libraries on hard-to-transfect cells in the future. PMID:28211892

  1. High-throughput and clogging-free microfluidic filtration platform for on-chip cell separation from undiluted whole blood

    PubMed Central

    Cheng, Yinuo; Ye, Xiongying; Ma, Zengshuai; Xie, Shuai; Wang, Wenhui

    2016-01-01

    Rapid separation of white blood cells from whole blood sample is often required for their subsequent analyses of functions and phenotypes, and many advances have been made in this field. However, most current microfiltration-based cell separation microfluidic chips still suffer from low-throughput and membrane clogging. This paper reports on a high-throughput and clogging-free microfluidic filtration platform, which features with an integrated bidirectional micropump and commercially available polycarbonate microporous membranes. The integrated bidirectional micropump enables the fluid to flush micropores back and forth, effectively avoiding membrane clogging. The microporous membrane allows red blood cells passing through high-density pores in a cross-flow mixed with dead-end filtration mode. All the separation processes, including blood and buffer loading, separation, and sample collection, are automatically controlled for easy operation and high throughput. Both microbead mixture and undiluted whole blood sample are separated by the platform effectively. In particular, for white blood cell separation, the chip recovered 72.1% white blood cells with an over 232-fold enrichment ratio at a throughput as high as 37.5 μl/min. This high-throughput, clogging-free, and highly integrated platform holds great promise for point-of-care blood pretreatment, analysis, and diagnosis applications. PMID:26909124

  2. High-throughput and clogging-free microfluidic filtration platform for on-chip cell separation from undiluted whole blood.

    PubMed

    Cheng, Yinuo; Ye, Xiongying; Ma, Zengshuai; Xie, Shuai; Wang, Wenhui

    2016-01-01

    Rapid separation of white blood cells from whole blood sample is often required for their subsequent analyses of functions and phenotypes, and many advances have been made in this field. However, most current microfiltration-based cell separation microfluidic chips still suffer from low-throughput and membrane clogging. This paper reports on a high-throughput and clogging-free microfluidic filtration platform, which features with an integrated bidirectional micropump and commercially available polycarbonate microporous membranes. The integrated bidirectional micropump enables the fluid to flush micropores back and forth, effectively avoiding membrane clogging. The microporous membrane allows red blood cells passing through high-density pores in a cross-flow mixed with dead-end filtration mode. All the separation processes, including blood and buffer loading, separation, and sample collection, are automatically controlled for easy operation and high throughput. Both microbead mixture and undiluted whole blood sample are separated by the platform effectively. In particular, for white blood cell separation, the chip recovered 72.1% white blood cells with an over 232-fold enrichment ratio at a throughput as high as 37.5 μl/min. This high-throughput, clogging-free, and highly integrated platform holds great promise for point-of-care blood pretreatment, analysis, and diagnosis applications.

  3. High-Throughput Electrophoretic Mobility Shift Assays for Quantitative Analysis of Molecular Binding Reactions

    PubMed Central

    2015-01-01

    We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for identification and characterization of molecular binding reactions. A photopatterned free-standing polyacrylamide gel array comprised of 8 mm-scale polyacrylamide gel strips acts as a chassis for 96 concurrent EMSAs. The high-throughput EMSAs was employed to assess binding of the Vc2 cyclic-di-GMP riboswitch to its ligand. In optimizing the riboswitch EMSAs on the free-standing polyacrylamide gel array, three design considerations were made: minimizing sample injection dispersion, mitigating evaporation from the open free-standing polyacrylamide gel structures during electrophoresis, and controlling unit-to-unit variation across the large-format free-standing polyacrylamide gel array. Optimized electrophoretic mobility shift conditions allowed for 10% difference in mobility shift baseline resolution within 3 min. The powerful 96-plex EMSAs increased the throughput to ∼10 data/min, notably more efficient than either conventional slab EMSAs (∼0.01 data/min) or even microchannel based microfluidic EMSAs (∼0.3 data/min). The free-standing polyacrylamide gel EMSAs yielded reliable quantification of molecular binding and associated mobility shifts for a riboswitch–ligand interaction, thus demonstrating a screening assay platform suitable for riboswitches and potentially a wide range of RNA and other macromolecular targets. PMID:25233437

  4. MEDIUM- AND HIGH-THROUGHPUT SCREENING OF NEUROTOXICANTS USING C. ELEGANS

    PubMed Central

    Boyd, Windy A.; Smith, Marjolein V.; Kissling, Grace E.; Freedman, Jonathan H.

    2009-01-01

    The U.S. National Toxicology Program, the U.S. Environmental Protection Agency, and other national and international agencies are committing significant resources towards the development of alternative species to be used as replacements for mammalian models in toxicological studies. Caenorhabditis elegans is a well-characterized soil nematode that is becoming a useful model in the assessment of neurotoxicants. To determine the effects of potential neurotoxicants on C. elegans, four medium-throughput (feeding, growth, reproduction and locomotion) and two high-throughput (growth and reproduction) assays have been developed. Three of these assays use the COPAS Biosort, a flow cytometer capable of rapidly measuring thousands of nematodes in minutes. Medium-throughput feeding, growth, and reproduction assays were used to assess the toxicity of eight suspected neurotoxicants. For several of the neurotoxicants examined, significant effects were observed at similar concentrations between assays. High-throughput reproduction and growth assays were used to estimate the toxicity of thousands of chemicals in two libraries. These assays will prove useful in evaluating the role of alternative toxicological models in tiered toxicity testing of thousands of chemicals. PMID:19166924

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

    PubMed

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

    2010-05-12

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

  6. Evaluation of a Pooled Strategy for High-Throughput Sequencing of Cosmid Clones from Metagenomic Libraries

    PubMed Central

    Lam, Kathy N.; Hall, Michael W.; Engel, Katja; Vey, Gregory; Cheng, Jiujun; Neufeld, Josh D.; Charles, Trevor C.

    2014-01-01

    High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones. PMID:24911009

  7. Novel High-Throughput Screening Approach for Functional Metal/Oxide Interfaces.

    PubMed

    Neufeld, Ofer; Toroker, Maytal Caspary

    2016-04-12

    Metal/oxide interfaces have long been studied for their fundamental importance in material microstructure as well as their broad applicability in electronic devices. However, the challenge involved in characterizing the relation between structure and electron transport of a large number of metal/oxide combinations inhibits the search for interfaces with improved functionality. Therefore, we develop a novel high-throughput screening approach that combines computational and theoretical techniques. We use a Density Functional Theory + U (DFT+U) quantum mechanical formalism to produce effective Schrödinger equations, which are solved by wave packet propagation to simulate charge transport across the metal/oxide interface. We demonstrate this method on α-Fe2O3/Mt interfaces, for Mt = Ag, Al, Au, Ir, Pd, or Pt metals. We use this novel method to screen for binary alloys of these metals at the α-Fe2O3/Mt interface and perform a successful validation test of the methodology. Finally, we correlate the interface potential energy and the charge transport permeability through the interface. Counterintuitively, among the interfaces studied, we find that higher mismatch interfaces have better charge transport permeability. We anticipate that this method will be useful as a computationally tractable strategy to perform high-throughput screening for new metal/oxide interfaces.

  8. Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

    PubMed

    Lam, Kathy N; Hall, Michael W; Engel, Katja; Vey, Gregory; Cheng, Jiujun; Neufeld, Josh D; Charles, Trevor C

    2014-01-01

    High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.

  9. Representing high throughput expression profiles via perturbation barcodes reveals compound targets.

    PubMed

    Filzen, Tracey M; Kutchukian, Peter S; Hermes, Jeffrey D; Li, Jing; Tudor, Matthew

    2017-02-01

    High throughput mRNA expression profiling can be used to characterize the response of cell culture models to perturbations such as pharmacologic modulators and genetic perturbations. As profiling campaigns expand in scope, it is important to homogenize, summarize, and analyze the resulting data in a manner that captures significant biological signals in spite of various noise sources such as batch effects and stochastic variation. We used the L1000 platform for large-scale profiling of 978 representative genes across thousands of compound treatments. Here, a method is described that uses deep learning techniques to convert the expression changes of the landmark genes into a perturbation barcode that reveals important features of the underlying data, performing better than the raw data in revealing important biological insights. The barcode captures compound structure and target information, and predicts a compound's high throughput screening promiscuity, to a higher degree than the original data measurements, indicating that the approach uncovers underlying factors of the expression data that are otherwise entangled or masked by noise. Furthermore, we demonstrate that visualizations derived from the perturbation barcode can be used to more sensitively assign functions to unknown compounds through a guilt-by-association approach, which we use to predict and experimentally validate the activity of compounds on the MAPK pathway. The demonstrated application of deep metric learning to large-scale chemical genetics projects highlights the utility of this and related approaches to the extraction of insights and testable hypotheses from big, sometimes noisy data.

  10. mQC: A Heuristic Quality-Control Metric for High-Throughput Drug Combination Screening

    PubMed Central

    Chen, Lu; Wilson, Kelli; Goldlust, Ian; Mott, Bryan T.; Eastman, Richard; Davis, Mindy I.; Zhang, Xiaohu; McKnight, Crystal; Klumpp-Thomas, Carleen; Shinn, Paul; Simmons, John; Gormally, Mike; Michael, Sam; Thomas, Craig J.; Ferrer, Marc; Guha, Rajarshi

    2016-01-01

    Quality control (QC) metrics are critical in high throughput screening (HTS) platforms to ensure reliability and confidence in assay data and downstream analyses. Most reported HTS QC metrics are designed for plate level or single well level analysis. With the advent of high throughput combination screening there is a need for QC metrics that quantify the quality of combination response matrices. We introduce a predictive, interpretable, matrix-level QC metric, mQC, based on a mix of data-derived and heuristic features. mQC accurately reproduces the expert assessment of combination response quality and correctly identifies unreliable response matrices that can lead to erroneous or misleading characterization of synergy. When combined with the plate-level QC metric, Z’, mQC provides a more appropriate determination of the quality of a drug combination screen. Retrospective analysis on a number of completed combination screens further shows that mQC is able to identify problematic screens whereas plate-level QC was not able to. In conclusion, our data indicates that mQC is a reliable QC filter that can be used to identify problematic drug combinations matrices and prevent further analysis on erroneously active combinations as well as for troubleshooting failed screens. The R source code of mQC is available at http://matrix.ncats.nih.gov/mQC. PMID:27883049

  11. High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

    PubMed Central

    Willats, William G. T.

    2016-01-01

    Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths. PMID:27684747

  12. High-throughput continuous flow femtosecond laser-assisted cell optoporation and transfection.

    PubMed

    Breunig, Hans Georg; Uchugonova, Aisada; Batista, Ana; König, Karsten

    2014-12-01

    We present a femtosecond-laser based nanoprocessing system for transient optical cell membrane poration to allow the introduction of foreign molecules into the interior of a cell with very high throughput. In the setup, cells flow throug