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Sample records for mirexpress analyzing high-throughput

  1. High throughput assays for analyzing transcription factors.

    PubMed

    Li, Xianqiang; Jiang, Xin; Yaoi, Takuro

    2006-06-01

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

  2. High-throughput single-microparticle imaging flow analyzer

    PubMed Central

    Goda, Keisuke; Ayazi, Ali; Gossett, Daniel R.; Sadasivam, Jagannath; Lonappan, Cejo K.; Sollier, Elodie; Fard, Ali M.; Hur, Soojung Claire; Adam, Jost; Murray, Coleman; Wang, Chao; Brackbill, Nora; Di Carlo, Dino; Jalali, Bahram

    2012-01-01

    Optical microscopy is one of the most widely used diagnostic methods in scientific, industrial, and biomedical applications. However, while useful for detailed examination of a small number (< 10,000) of microscopic entities, conventional optical microscopy is incapable of statistically relevant screening of large populations (> 100,000,000) with high precision due to its low throughput and limited digital memory size. We present an automated flow-through single-particle optical microscope that overcomes this limitation by performing sensitive blur-free image acquisition and nonstop real-time image-recording and classification of microparticles during high-speed flow. This is made possible by integrating ultrafast optical imaging technology, self-focusing microfluidic technology, optoelectronic communication technology, and information technology. To show the system’s utility, we demonstrate high-throughput image-based screening of budding yeast and rare breast cancer cells in blood with an unprecedented throughput of 100,000 particles/s and a record false positive rate of one in a million. PMID:22753513

  3. Development of a high-throughput IMS-IMS-MS approach for analyzing mixtures of biomolecules

    PubMed Central

    Kurulugama, Ruwan T.; Valentine, Stephen J.; Sowell, Rena A.; Clemmer, David E.

    2008-01-01

    A high-throughput approach for biomolecule analysis is demonstrated for a mixture of peptides from tryptic digest of four proteins as well as a tryptic digests of human plasma. In this method a chip-based electrospray autosampler coupled to a hybrid ion mobility (IMS) mass spectrometer (MS) is utilized to achieve rapid sample analysis. This high-throughput measurement is realized by exploiting the direct infusion capability of the chip based electrospray with its rapid sample manipulating capability as well as a high sensitive IMS-MS with a recently developed IMS-IMS separation technique that can be multiplexed to provide greater throughput. From replicate IMS-MS runs of known mixtures, the average uncertainty of peak intensities is determined to be ±7% (relative standard deviation), and a detection limit in the low attomole range is established. The method is illustrated by analyzing 124 human plasma protein samples in duplicate, a measurement that required 16.5 hours. Current limitations as well as implications of the high-throughput approach for complex biological sample analysis are discussed. PMID:18590839

  4. Diversity and distribution of unicellular opisthokonts along the European coast analyzed using high-throughput sequencing

    PubMed Central

    del Campo, Javier; Mallo, Diego; Massana, Ramon; de Vargas, Colomban; Richards, Thomas A.; Ruiz-Trillo, Iñaki

    2015-01-01

    Summary The opisthokonts are one of the major super-groups of eukaryotes. It comprises two major clades: 1) the Metazoa and their unicellular relatives and 2) the Fungi and their unicellular relatives. There is, however, little knowledge of the role of opisthokont microbes in many natural environments, especially among non-metazoan and non-fungal opisthokonts. Here we begin to address this gap by analyzing high throughput 18S rDNA and 18S rRNA sequencing data from different European coastal sites, sampled at different size fractions and depths. In particular, we analyze the diversity and abundance of choanoflagellates, filastereans, ichthyosporeans, nucleariids, corallochytreans and their related lineages. Our results show the great diversity of choanoflagellates in coastal waters as well as a relevant role of the ichthyosporeans and the uncultured marine opisthokonts (MAOP). Furthermore, we describe a new lineage of marine fonticulids (MAFO) that appears to be abundant in sediments. Therefore, our work points to a greater potential ecological role for unicellular opisthokonts than previously appreciated in marine environments, both in water column and sediments, and also provides evidence of novel opisthokont phylogenetic lineages. This study highlights the importance of high throughput sequencing approaches to unravel the diversity and distribution of both known and novel eukaryotic lineages. PMID:25556908

  5. Compositional analysis: a valid approach to analyze microbiome high-throughput sequencing data.

    PubMed

    Gloor, Gregory B; Reid, Gregor

    2016-08-01

    A workshop held at the 2015 annual meeting of the Canadian Society of Microbiologists highlighted compositional data analysis methods and the importance of exploratory data analysis for the analysis of microbiome data sets generated by high-throughput DNA sequencing. A summary of the content of that workshop, a review of new methods of analysis, and information on the importance of careful analyses are presented herein. The workshop focussed on explaining the rationale behind the use of compositional data analysis, and a demonstration of these methods for the examination of 2 microbiome data sets. A clear understanding of bioinformatics methodologies and the type of data being analyzed is essential, given the growing number of studies uncovering the critical role of the microbiome in health and disease and the need to understand alterations to its composition and function following intervention with fecal transplant, probiotics, diet, and pharmaceutical agents. PMID:27314511

  6. Finding disease genes: a fast and flexible approach for analyzing high-throughput data

    PubMed Central

    Stewart, William C L; Drill, Esther N; Greenberg, David A

    2011-01-01

    Linkage disequilibrium (LD) is the non-random distribution of alleles across the genome, and it can create serious problems for modern linkage studies. In particular, computational feasibility is often obtained at the expense of power, precision, and/or accuracy. In our new approach, we combine linkage results over multiple marker subsets to provide fast, efficient, and robust analyses, without compromising power, precision, or accuracy. Allele frequencies and LD in the densely spaced markers are used to construct subsamples that are highly informative for linkage. We have tested our approach extensively, and implemented it in the software package EAGLET (Efficient Analysis of Genetic Linkage: Estimation and Testing). Relative to several commonly used methods we show that EAGLET has increased power to detect disease genes across a range of trait models, LD patterns, and family structures using both simulated and real data. In particular, when the underlying LD pattern is derived from real data, we find that EAGLET outperforms several commonly used linkage methods. In-depth analysis of family data, simulated with linkage and under the real-data derived LD pattern, showed that EAGLET had 78.1% power to detect a dominant disease with incomplete penetrance, whereas the method that uses one marker per cM had 69.7% power, and the cluster-based approach implemented in MERLIN had 76.7% power. In this same setting, EAGLET was three times faster than MERLIN, and it narrowed the MERLIN-based confidence interval for trait location by 29%. Overall, EAGLET gives researchers a fast, accurate, and powerful new tool for analyzing high-throughput linkage data, and large extended families are easily accommodated. PMID:21610749

  7. High throughput methods for analyzing transition metals in proteins on a microgram scale.

    PubMed

    Atanassova, Anelia; Högbom, Martin; Zamble, Deborah B

    2008-01-01

    Transition metals are among the most common ligands that contribute to the biochemical and physiological properties of proteins. In the course of structural proteomic projects, the detection of transition metal cofactors prior to the determination of a high-resolution structure is extremely beneficial. This information can be used to select tractable targets from the proteomic pipeline because the presence of a metal often improves protein stability and can be used to help solve the phasing problem in x-ray crystallography. Recombinant proteins are often purified with substoichiometric amounts of metal loaded, so additional metal may be needed to obtain the homogeneous protein solution crucial for structural analysis. Furthermore, identifying a metal cofactor provides a clue about the nature of the biological role of an unclassified protein and can be applied with structural data in the assignation of a putative function. Many of the existing methods for transition metal analysis of purified proteins have limitations, which include a requirement for a large quantity of protein or a reliance on equipment with a prohibitive cost.The authors have developed two simple high throughput methods for identifying metalloproteins on a microgram scale. Each of the techniques has distinct advantages and can be applied to address divergent experimental goals. The first method, based on simple luminescence and colorimetric reactions, is fast, cheap, and semiquantitative. The second method, which employs HPLC separation, is accurate and affords unambiguous metal identification. PMID:18542873

  8. High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer

    PubMed Central

    Reusch, Dietmar; Haberger, Markus; Kailich, Tobias; Heidenreich, Anna-Katharina; Kampe, Michael; Bulau, Patrick; Wuhrer, Manfred

    2014-01-01

    The Fc glycosylation of therapeutic antibodies is crucial for their effector functions and their behavior in pharmacokinetics and pharmacodynamics. To monitor the Fc glycosylation in bioprocess development and characterization, high-throughput techniques for glycosylation analysis are needed. Here, we describe the development of a largely automated high-throughput glycosylation profiling method with multiplexing capillary-gel-electrophoresis (CGE) with laser induced fluorescence (LIF) detection using a DNA analyzer. After PNGaseF digestion, the released glycans were labeled with 9-aminopyrene-1,3,6-trisulfonic acid (APTS) in 96-well plates, which was followed by the simultaneous analysis of up to 48 samples. The peak assignment was conducted by HILIC-UPLC-MS/MS of the APTS-labeled glycans combined with peak fractionation and subsequent CGE-LIF analysis of the MS-characterized fractions. Quantitative data evaluation of the various IgG glycans was performed automatically using an in-house developed software solution. The excellent method accuracy and repeatability of the test system was verified by comparison with two UPLC-based methods for glycan analysis. Finally, the practical value of the developed method was demonstrated by analyzing the antibody glycosylation profiles from fermentation broths after small scale protein A purification. PMID:24135630

  9. GeoChip 3.0: A High Throughput Tool for Analyzing Microbial Community, Composition, Structure, and Functional Activity

    SciTech Connect

    He, Zhili; Deng, Ye; Nostrand, Joy Van; Tu, Qichao; Xu, Meiying; Hemme, Chris; Wu, Liyou; Hazen, Terry; Zhou, Jizhong; Li, Xingyuan; Gentry, Terry; Yin, Yifeng; Liebich, Jost

    2010-05-17

    Microarray-based genomic technology has been widely used for microbial community analysis, and it is expected that microarray-based genomic technologies will revolutionize the analysis of microbial community structure, function and dynamics. A new generation of functional gene arrays (GeoChip 3.0) has been developed, with 27,812 probes covering 56,990 gene variants from 292 functional gene families involved in carbon, nitrogen, phosphorus and sulfur cycles, energy metabolism, antibiotic resistance, metal resistance, and organic contaminant degradation. Those probes were derived from 2,744, 140, and 262 species for bacteria, archaea, and fungi, respectively. GeoChip 3.0 has several other distinct features, such as a common oligo reference standard (CORS) for data normalization and comparison, a software package for data management and future updating, and the gyrB gene for phylogenetic analysis. Our computational evaluation of probe specificity indicated that all designed probes had a high specificity to their corresponding targets. Also, experimental analysis with synthesized oligonucleotides and genomic DNAs showed that only 0.0036percent-0.025percent false positive rates were observed, suggesting that the designed probes are highly specific under the experimental conditions examined. In addition, GeoChip 3.0 was applied to analyze soil microbial communities in a multifactor grassland ecosystem in Minnesota, USA, which demonstrated that the structure, composition, and potential activity of soil microbial communities significantly changed with the plant species diversity. All results indicate that GeoChip 3.0 is a high throughput powerful tool for studying microbial community functional structure, and linking microbial communities to ecosystem processes and functioning. To our knowledge, GeoChip 3.0 is the most comprehensive microarrays currently available for studying microbial communities associated with geobiochemical cycling, global climate change, bioenergy

  10. High-Throughput Proteomics

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

  11. High-Throughput Analysis of Methylmalonic Acid in Serum, Plasma, and Urine by LC-MS/MS. Method for Analyzing Isomers Without Chromatographic Separation.

    PubMed

    Kushnir, Mark M; Nelson, Gordon J; Frank, Elizabeth L; Rockwood, Alan L

    2016-01-01

    Measurement of methylmalonic acid (MMA) plays an important role in the diagnosis of vitamin B12 deficiency. Vitamin B12 is an essential cofactor for the enzymatic carbon rearrangement of methylmalonyl-CoA (MMA-CoA) to succinyl-CoA (SA-CoA), and the lack of vitamin B12 leads to elevated concentrations of MMA. Presence of succinic acid (SA) complicates the analysis because mass spectra of MMA and SA are indistinguishable, when analyzed in negative ion mode and the peaks are difficult to resolve chromatographically. We developed a method for the selective analysis of MMA that exploits the significant difference in fragmentation patterns of di-butyl derivatives of the isomers MMA and SA in a tandem mass spectrometer when analyzed in positive ion mode. Tandem mass spectra of di-butyl derivatives of MMA and SA are very distinct; this allows selective analysis of MMA in the presence of SA. The instrumental analysis is performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive ion mode, which is, in combination with selective extraction of acidic compounds, is highly selective for organic acids with multiple carboxyl groups (dicarboxylic, tricarboxylic, etc.). In this method organic acids with a single carboxyl group are virtually undetectable in the mass spectrometer; the only organic acid, other than MMA, that is detected by this method is its isomer, SA. Quantitative measurement of MMA in this method is performed using a deconvolution algorithm, which mathematically resolves the signal corresponding to MMA and does not require chromatographic resolution of the MMA and SA peaks. Because of its high selectivity, the method utilizes isocratic chromatographic separation; reconditioning and re-equilibration of the chromatographic column between injections is unnecessary. The above features of the method allow high-throughput analysis of MMA with analysis cycle time of 1 min. PMID:26602128

  12. Revealing editing and SNPs of microRNAs in colon tissues by analyzing high-throughput sequencing profiles of small RNAs

    PubMed Central

    2014-01-01

    Background Editing and mutations in microRNAs (miRNAs) can change the stability of pre-miRNAs and/or complementarities between miRNAs and their targets. Small RNA (sRNA) high-throughput sequencing (HTS) profiles contain miRNAs that are originated from mutated DNAs or are edited during their biogenesis procedures. It is largely unknown whether miRNAs are edited in colon tissues since existing studies mainly focused their attention on the editing of miRNAs in brain tissues. Results Through comprehensive analysis of four high-throughput sequencing profiles of normal and cancerous colon tissues, we identified 548 editing and/or SNPs in miRNAs that are significant in at least one of the sequencing profiles used. Our results show that the most abundant editing events of miRNAs in colon tissues are 3'-A and 3'-U. In addition to four known A-to-I editing sites previously reported in brain tissues, four novel A-to-I editing sites are also identified in colon tissues. Conclusions This suggests that A-to-I editing of miRNAs potentially is a commonly existing mechanism in different tissues to diversify the possible functional roles of miRNAs, but only a small portion of different miRNAs are edited by the A-to-I mechanism at a significant level. Our results suggest that there are other types of editing in miRNAs through unknown mechanisms. Furthermore, several SNPs in miRNAs are also identified. PMID:25521855

  13. High throughput continuous cryopump

    SciTech Connect

    Foster, C.A.

    1986-01-01

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

  14. High throughput optical scanner

    DOEpatents

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

    2001-01-01

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

  15. Diel growth cycle of isolated leaf discs analyzed with a novel, high-throughput three-dimensional imaging method is identical to that of intact leaves.

    PubMed

    Biskup, Bernhard; Scharr, Hanno; Fischbach, Andreas; Wiese-Klinkenberg, Anika; Schurr, Ulrich; Walter, Achim

    2009-03-01

    Dicot leaves grow with pronounced diel (24-h) cycles that are controlled by a complex network of factors. It is an open question to what extent leaf growth dynamics are controlled by long-range or by local signals. To address this question, we established a stereoscopic imaging system, GROWSCREEN 3D, which quantifies surface growth of isolated leaf discs floating on nutrient solution in wells of microtiter plates. A total of 458 leaf discs of tobacco (Nicotiana tabacum) were cut at different developmental stages, incubated, and analyzed for their relative growth rates. The camera system was automatically displaced across the array of leaf discs; visualization and camera displacement took about 12 s for each leaf disc, resulting in a time interval of 1.5 h for consecutive size analyses. Leaf discs showed a comparable diel leaf growth cycle as intact leaves but weaker peak growth activity. Hence, it can be concluded that the timing of leaf growth is regulated by local rather than by systemic control processes. This conclusion was supported by results from leaf discs of Arabidopsis (Arabidopsis thaliana) Landsberg erecta wild-type plants and starch-free1 mutants. At night, utilization of transitory starch leads to increased growth of Landsberg erecta wild-type discs compared with starch-free1 discs. Moreover, the decrease of leaf disc growth when exposed to different concentrations of glyphosate showed an immediate dose-dependent response. Our results demonstrate that a dynamic leaf disc growth analysis as we present it here is a promising approach to uncover the effects of internal and external cues on dicot leaf development. PMID:19168641

  16. High-throughput tetrad analysis.

    PubMed

    Ludlow, Catherine L; Scott, Adrian C; Cromie, Gareth A; Jeffery, Eric W; Sirr, Amy; May, Patrick; Lin, Jake; Gilbert, Teresa L; Hays, Michelle; Dudley, Aimée M

    2013-07-01

    Tetrad analysis has been a gold-standard genetic technique for several decades. Unfortunately, the need to manually isolate, disrupt and space tetrads has relegated its application to small-scale studies and limited its integration with high-throughput DNA sequencing technologies. We have developed a rapid, high-throughput method, called barcode-enabled sequencing of tetrads (BEST), that uses (i) a meiosis-specific GFP fusion protein to isolate tetrads by FACS and (ii) molecular barcodes that are read during genotyping to identify spores derived from the same tetrad. Maintaining tetrad information allows accurate inference of missing genetic markers and full genotypes of missing (and presumably nonviable) individuals. An individual researcher was able to isolate over 3,000 yeast tetrads in 3 h, an output equivalent to that of almost 1 month of manual dissection. BEST is transferable to other microorganisms for which meiotic mapping is significantly more laborious. PMID:23666411

  17. 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

  18. High-throughput continuous cryopump

    SciTech Connect

    Foster, C.A.

    1986-01-01

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

  19. 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

  20. 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.

  1. High-throughput Tetrad Analysis

    PubMed Central

    Ludlow, Catherine L.; Scott, Adrian C.; Cromie, Gareth A.; Jeffery, Eric W.; Sirr, Amy; May, Patrick; Lin, Jake; Gilbert, Teresa L.; Hays, Michelle; Dudley, Aimée M.

    2013-01-01

    Tetrad analysis has been a gold standard genetic technique for several decades. Unfortunately, the manual nature of the process has relegated its application to small-scale studies and limited its integration with rapidly evolving DNA sequencing technologies. We have developed a rapid, high-throughput method, called Barcode Enabled Sequencing of Tetrads (BEST), that replaces the manual processes of isolating, disrupting and spacing tetrads. BEST uses a meiosis-specific GFP fusion protein to isolate tetrads by fluorescence-activated cell sorting and molecular barcodes that are read during genotyping to identify spores derived from the same tetrad. Maintaining tetrad information allows accurate inference of missing genetic markers and full genotypes of missing (and presumably nonviable) individuals. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. We demonstrate the approach in Saccharomyces cerevisiae, but BEST is readily transferable to microorganisms in which meiotic mapping is significantly more laborious. PMID:23666411

  2. High-Throughput Analysis of Enzyme Activities

    SciTech Connect

    Guoxin Lu

    2007-12-01

    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

  3. Advances in High-Throughput Single-Cell Microtechnologies

    PubMed Central

    Weaver, Westbrook M.; Tseng, Peter; Kunze, Anja; Masaeli, Mahdohkht; Chung, Aram J.; Dudani, Jaideep S.; Kittur, Harsha; Kulkarni, Rajan P.; Di Carlo, Dino

    2013-01-01

    Micro-scale biological tools that have allowed probing of individual cells - from the genetic, to proteomic, to phenotypic level - have revealed important contributions of single cells to direct normal and diseased body processes. In analyzing single cells, sample heterogeneity between and within specific cell types drives the need for high-throughput and quantitative measurement of cellular parameters. In recent years, high-throughput single-cell analysis platforms have revealed rare genetic subpopulations in growing tumors, begun to uncover the mechanisms of antibiotic resistance in bacteria, and described the cell-to-cell variations in stem cell differentiation and immune cell response to activation by pathogens. This review surveys these recent technologies, presenting their strengths and contributions to the field, and identifies needs still unmet toward the development of high-throughput single-cell analysis tools to benefit life science research and clinical diagnostics. PMID:24484889

  4. High Throughput Sequence Analysis for Disease Resistance in Maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Preliminary results of a computational analysis of high throughput sequencing data from Zea mays and the fungus Aspergillus are reported. The Illumina Genome Analyzer was used to sequence RNA samples from two strains of Z. mays (Va35 and Mp313) collected over a time course as well as several specie...

  5. Application of a high-throughput analyzer in evaluating solid adsorbents for post-combustion carbon capture via multicomponent adsorption of CO2, N2, and H2O.

    PubMed

    Mason, Jarad A; McDonald, Thomas M; Bae, Tae-Hyun; Bachman, Jonathan E; Sumida, Kenji; Dutton, Justin J; Kaye, Steven S; Long, Jeffrey R

    2015-04-15

    Despite the large number of metal-organic frameworks that have been studied in the context of post-combustion carbon capture, adsorption equilibria of gas mixtures including CO2, N2, and H2O, which are the three biggest components of the flue gas emanating from a coal- or natural gas-fired power plant, have never been reported. Here, we disclose the design and validation of a high-throughput multicomponent adsorption instrument that can measure equilibrium adsorption isotherms for mixtures of gases at conditions that are representative of an actual flue gas from a power plant. This instrument is used to study 15 different metal-organic frameworks, zeolites, mesoporous silicas, and activated carbons representative of the broad range of solid adsorbents that have received attention for CO2 capture. While the multicomponent results presented in this work provide many interesting fundamental insights, only adsorbents functionalized with alkylamines are shown to have any significant CO2 capacity in the presence of N2 and H2O at equilibrium partial pressures similar to those expected in a carbon capture process. Most significantly, the amine-appended metal organic framework mmen-Mg2(dobpdc) (mmen = N,N'-dimethylethylenediamine, dobpdc (4-) = 4,4'-dioxido-3,3'-biphenyldicarboxylate) exhibits a record CO2 capacity of 4.2 ± 0.2 mmol/g (16 wt %) at 0.1 bar and 40 °C in the presence of a high partial pressure of H2O. PMID:25844924

  6. Application of a High-Throughput Analyzer in Evaluating Solid Adsorbents for Post-Combustion Carbon Capture via Multicomponent Adsorption of CO2, N-2, and H2O

    SciTech Connect

    Mason, JA; McDonald, TM; Bae, TH; Bachman, JE; Sumida, K; Dutton, JJ; Kaye, SS; Long, JR

    2015-04-15

    Despite the large number of metal-organic frameworks that have been studied in the context of post-combustion carbon capture, adsorption equilibria of gas mixtures including CO2, N-2, and H2O, which are the three biggest components of the flue gas emanating from a coal- or natural gas-fired power plant, have never been reported. Here, we disclose the design and validation of a high-throughput multicomponent adsorption instrument that can measure equilibrium adsorption isotherms for mixtures of gases at conditions that are representative of an actual flue gas from a power plant. This instrument is used to study 15 different metal-organic frameworks, zeolites, mesoporous silicas, and activated carbons representative of the broad range of solid adsorbents that have received attention for CO2 capture. While the multicomponent results presented in this work provide many interesting fundamental insights, only adsorbents functionalized with alkylamines are shown to have any significant CO2 capacity in the presence of N-2 and H2O at equilibrium partial pressures similar to those expected in a carbon capture process. Most significantly, the amine-appended metal organic framework mmen-Mg-2(dobpdc) (mmen = N,N'-dimethylethylenediamine, dobpdc (4-) = 4,4'-dioxido-3,3'-biphenyldicarboxylate) exhibits a record CO2 capacity of 4.2 +/- 0.2 mmol/g (16 wt %) at 0.1 bar and 40 degrees C in the presence of a high partial pressure of H2O.

  7. High-Throughput Optical Sensing Immunoassays on Smartphone.

    PubMed

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

    2016-08-16

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

  8. High throughput network for multiprocessor interconnections

    NASA Astrophysics Data System (ADS)

    Raatikainen, Pertti; Zidbeck, Juha

    1993-05-01

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

  9. High-throughput screening methods for nitrilases.

    PubMed

    Xue, Ya-Ping; Yang, Yue-Kai; Lv, Sheng-Zhi; Liu, Zhi-Qiang; Zheng, Yu-Guo

    2016-04-01

    Nitrilases have been widely acknowledged as important alternatives to chemical catalysts, as they have been proved to transform an immense variety of nitriles under mild conditions and often in a stereoselective or regioselective manner. In the discovery of new nitrilases to establish viable industrial processes, screening plays an important role in identifying which subset of candidates contains a nitrilase of interest from a collection of organisms, clone banks, or enzyme libraries. However, the traditional methods for evaluating the nitrilases are a time-consuming, laborious, and costly process and have been regarded as a bottleneck in developing these nitrilases as industrial biocatalysts. In the past few years, a number of high-throughput screening methods have been developed for rapid evaluation and identification of nitrilases. Here, we review the various methodologies developed for high-throughput screening of nitrilases and focus on their advantages and limitations. PMID:26894402

  10. A Novel High-Throughput Viscometer.

    PubMed

    Deshmukh, Suraj; Bishop, Matthew T; Dermody, Daniel; Dietsche, Laura; Kuo, Tzu-Chi; Mushrush, Melissa; Harris, Keith; Zieman, Jonathan; Morabito, Paul; Orvosh, Brian; Patrick, Don

    2016-07-11

    A novel, rapid, parallel, and high-throughput system for measuring viscosity of materials under different conditions of shear rate, temperature, time, etc., has been developed. This unique system utilizes the transient flow of a complex fluid through pipettes. This approach offers significant practical advantages over microfluidic-based devices for viscosity screening: no cleanup is required, the method is high throughput (<1 h for 100 samples), and only small sample volumes (<1 mL) are used. This paper details for the first time the experimental and modeling efforts to implement this mass- and pressure-based viscosity measurement concept as a robust viscosity estimation tool. This approach is very well-suited for viscosity measurements in high-throughput formulation workflows, as it is rapid and parallel and operates directly on samples in various microtiter plate formats. We present systematic experimental observations together with numerical and analytical modeling approaches to characterize instrument capabilities and limitations. The complex transient flow of fluids through these pipettes leads to data-rich pressure profiles. Numerical and analytical modeling is then used to extract viscosity and other rheological parameters from these pressure profiles. We have successfully utilized this viscosity screening tool for a multitude of complex fluids including oils, paints, solvents, and detergents. PMID:27259016

  11. High-Throughput Investigation of Delafossite materials

    NASA Astrophysics Data System (ADS)

    Haycock, Barry; Kylee Underwood, M.; Lekse, Jonathan; Matranga, Christopher; Lewis, James P.

    2013-03-01

    We present the application of high-throughput calculations to the intriguing problem of the forbidden optical transition in the CuGa1-xFexO2 delafossites, which is prototypical of many delafossite systems. When 5% or more of the Ga sites are replaced with Fe, there is a sudden shift to an optical band gap of 1.5eV from 2.5eV. Using high-throughput calculations and data mining techniques, we show the most likely positional configurations for x = 0.00 through x = 0.10 of the Fe atoms relative to one another. Implications of this result and applications of the techniques used are discussed, including the development of candidate materials via high-throughput analysis of constituent search-space. Funded by the National Science Foundation through NSF DMR 09-03225 and a subcontract from NETL (URS RES) for Work Activity 0004000.6.600.007.002.420.000.005 ARRA ICMI Project.

  12. 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...

  13. 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.

  14. 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

  15. 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.

  16. High throughput chemical munitions treatment system

    DOEpatents

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

    2011-11-01

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

  17. 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).

  18. 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?

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

    PubMed Central

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

    2014-01-01

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

  20. A High-Throughput Radiometric Kinase Assay.

    PubMed

    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 of 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

  1. High-throughput cellular RNA device engineering.

    PubMed

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

    2015-10-01

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

  2. High-Throughput Cell Toxicity Assays.

    PubMed

    Murray, David; McWilliams, Lisa; Wigglesworth, Mark

    2016-01-01

    Understanding compound-driven cell toxicity is vitally important for all drug discovery approaches. With high-throughput screening (HTS) being the key strategy to find hit and lead compounds for drug discovery projects in the pharmaceutical industry [1], an understanding of the cell toxicity profile of hit molecules from HTS activities is fundamentally important. Recently, there has been a resurgence of interest in phenotypic drug discovery and these cell-based assays are now being run in HTS labs on ever increasing numbers of compounds. As the use of cell assays increases the ability to measure toxicity of compounds on a large scale becomes increasingly important to ensure that false hits are not progressed and that compounds do not carry forward a toxic liability that may cause them to fail at later stages of a project. Here we describe methods employed in the AstraZeneca HTS laboratory to carry out very large scale cell toxicity screening. PMID:27317000

  3. 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

  4. 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

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2016-01-01

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

  7. 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

  8. 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.

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

    PubMed Central

    2015-01-01

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

  10. Data Analysis for High-Throughput RNAi Screening.

    PubMed

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

    2016-01-01

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

  11. High throughput sample processing and automated scoring.

    PubMed

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

    2014-01-01

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

  12. High-Throughput Screening in Primary Neurons

    PubMed Central

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

    2013-01-01

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

  13. High throughput sample processing and automated scoring

    PubMed Central

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

    2014-01-01

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

  14. Orthogonal NGS for High Throughput Clinical Diagnostics

    PubMed Central

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

    2016-01-01

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

  15. High-throughput screening in primary neurons.

    PubMed

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

    2012-01-01

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

  16. High-throughput rod-induced electrospinning

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  17. 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.

  18. 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. PMID:26928208

  19. 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.

  20. High-throughput characterization of protein–RNA interactions

    PubMed Central

    Cook, Kate B.; Hughes, Timothy R.

    2015-01-01

    RNA-binding proteins (RBPs) are important regulators of eukaryotic gene expression. Genomes typically encode dozens to hundreds of proteins containing RNA-binding domains, which collectively recognize diverse RNA sequences and structures. Recent advances in high-throughput methods for assaying the targets of RBPs in vitro and in vivo allow large-scale derivation of RNA-binding motifs as well as determination of RNA–protein interactions in living cells. In parallel, many computational methods have been developed to analyze and interpret these data. The interplay between RNA secondary structure and RBP binding has also been a growing theme. Integrating RNA–protein interaction data with observations of post-transcriptional regulation will enhance our understanding of the roles of these important proteins. PMID:25504152

  1. A Microfluidic Platform for High-Throughput Multiplexed Protein Quantitation

    PubMed Central

    Volpetti, Francesca; Garcia-Cordero, Jose; Maerkl, Sebastian J.

    2015-01-01

    We present a high-throughput microfluidic platform capable of quantitating up to 384 biomarkers in 4 distinct samples by immunoassay. The microfluidic device contains 384 unit cells, which can be individually programmed with pairs of capture and detection antibody. Samples are quantitated in each unit cell by four independent MITOMI detection areas, allowing four samples to be analyzed in parallel for a total of 1,536 assays per device. We show that the device can be pre-assembled and stored for weeks at elevated temperature and we performed proof-of-concept experiments simultaneously quantitating IL-6, IL-1β, TNF-α, PSA, and GFP. Finally, we show that the platform can be used to identify functional antibody combinations by screening 64 antibody combinations requiring up to 384 unique assays per device. PMID:25680117

  2. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

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

    2006-01-01

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

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

    PubMed

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

    2016-01-01

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

  4. High-throughput operando Raman-quadrupole mass spectrometer (QMS) system to screen catalytic systems.

    PubMed

    García-Casado, Manuel; Prieto, José; Vico-Ruiz, Emilio; Lozano-Diz, Enrique; Goberna-Selma, Consuelo; Bañares, Miguel A

    2014-01-01

    This paper describes the design and setup of a high-throughput Raman system for an array of eight parallel catalytic reactors during reaction conditions. The "operando" methodology combines in situ spectroscopy during catalytic reaction with a simultaneous activity measurement. The high-throughput operando Raman system, multi-operando, is a device that automates this operando methodology for several catalyst samples at the same time, all samples being in the same reaction conditions. We describe how the system is made, how Raman system positions and acquires spectra, and how each reactor outlet gas is selected and analyzed. PMID:24405956

  5. MIrExpress: A Database for Gene Coexpression Correlation in Immune Cells Based on Mutual Information and Pearson Correlation

    PubMed Central

    Wang, Luman; Mo, Qiaochu; Wang, Jianxin

    2015-01-01

    Most current gene coexpression databases support the analysis for linear correlation of gene pairs, but not nonlinear correlation of them, which hinders precisely evaluating the gene-gene coexpression strengths. Here, we report a new database, MIrExpress, which takes advantage of the information theory, as well as the Pearson linear correlation method, to measure the linear correlation, nonlinear correlation, and their hybrid of cell-specific gene coexpressions in immune cells. For a given gene pair or probe set pair input by web users, both mutual information (MI) and Pearson correlation coefficient (r) are calculated, and several corresponding values are reported to reflect their coexpression correlation nature, including MI and r values, their respective rank orderings, their rank comparison, and their hybrid correlation value. Furthermore, for a given gene, the top 10 most relevant genes to it are displayed with the MI, r, or their hybrid perspective, respectively. Currently, the database totally includes 16 human cell groups, involving 20,283 human genes. The expression data and the calculated correlation results from the database are interactively accessible on the web page and can be implemented for other related applications and researches. PMID:26881263

  6. MIrExpress: A Database for Gene Coexpression Correlation in Immune Cells Based on Mutual Information and Pearson Correlation.

    PubMed

    Wang, Luman; Mo, Qiaochu; Wang, Jianxin

    2015-01-01

    Most current gene coexpression databases support the analysis for linear correlation of gene pairs, but not nonlinear correlation of them, which hinders precisely evaluating the gene-gene coexpression strengths. Here, we report a new database, MIrExpress, which takes advantage of the information theory, as well as the Pearson linear correlation method, to measure the linear correlation, nonlinear correlation, and their hybrid of cell-specific gene coexpressions in immune cells. For a given gene pair or probe set pair input by web users, both mutual information (MI) and Pearson correlation coefficient (r) are calculated, and several corresponding values are reported to reflect their coexpression correlation nature, including MI and r values, their respective rank orderings, their rank comparison, and their hybrid correlation value. Furthermore, for a given gene, the top 10 most relevant genes to it are displayed with the MI, r, or their hybrid perspective, respectively. Currently, the database totally includes 16 human cell groups, involving 20,283 human genes. The expression data and the calculated correlation results from the database are interactively accessible on the web page and can be implemented for other related applications and researches. PMID:26881263

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

    NASA Astrophysics Data System (ADS)

    Tian, Peng

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

  8. 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...

  9. 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 ...

  10. 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, ...

  11. 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...

  12. High throughput growth and characterization of thin film materials

    NASA Astrophysics Data System (ADS)

    Mao, Samuel S.

    2013-09-01

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

  13. High-throughput sequencing of cytosine methylation in plant DNA

    PubMed Central

    2013-01-01

    Cytosine methylation is a significant and widespread regulatory factor in plant systems. Methods for the high-throughput sequencing of methylation have allowed a greatly improved characterisation of the methylome. Here we discuss currently available methods for generation and analysis of high-throughput sequencing of methylation data. We also discuss the results previously acquired through sequencing plant methylomes, and highlight remaining challenges in this field. PMID:23758782

  14. Environmental surveillance and monitoring--The next frontiers for high-throughput toxicology.

    PubMed

    Schroeder, Anthony L; Ankley, Gerald T; Houck, Keith A; Villeneuve, Daniel L

    2016-03-01

    High-throughput toxicity testing technologies along with the World Wide Web are revolutionizing both generation of and access to data regarding the biological activities that chemicals can elicit when they interact with specific proteins, genes, or other targets in the body of an organism. To date, however, most of the focus has been on the application of such data to assessment of individual chemicals. The authors suggest that environmental surveillance and monitoring represent the next frontiers for high-throughput toxicity testing. Resources already exist in curated databases of chemical-biological interactions, including highly standardized quantitative dose-response data generated from nascent high-throughput toxicity testing programs such as ToxCast and Tox21, to link chemicals detected through environmental analytical chemistry to known biological activities. The emergence of the adverse outcome pathway framework and the associated knowledge base for linking molecular-level or pathway-level perturbations of biological systems to adverse outcomes traditionally considered in risk assessment and regulatory decision-making through a series of measurable biological changes provides a critical link between activity and hazard. Furthermore, environmental samples can be directly analyzed via high-throughput toxicity testing platforms to provide an unprecedented breadth of biological activity characterization that integrates the effects of all compounds present in a mixture, whether known or not. Novel application of these chemical-biological interaction data provides an opportunity to transform scientific characterization of potential hazards associated with exposure to complex mixtures of environmental contaminants. PMID:26923854

  15. Translational informatics: enabling high-throughput research paradigms

    PubMed Central

    Embi, Peter J.; Sen, Chandan K.

    2009-01-01

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

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

    PubMed Central

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

    2010-01-01

    Motivation: The advent of high-throughput sequencing (HTS) technologies has made it affordable to sequence many individuals' genomes. Simultaneously the computational analysis of the large volumes of data generated by the new sequencing machines remains a challenge. While a plethora of tools are available to map the resulting reads to a reference genome, and to conduct primary analysis of the mappings, it is often necessary to visually examine the results and underlying data to confirm predictions and understand the functional effects, especially in the context of other datasets. Results: We introduce Savant, the Sequence Annotation, Visualization and ANalysis Tool, a desktop visualization and analysis browser for genomic data. Savant was developed for visualizing and analyzing HTS data, with special care taken to enable dynamic visualization in the presence of gigabases of genomic reads and references the size of the human genome. Savant supports the visualization of genome-based sequence, point, interval and continuous datasets, and multiple visualization modes that enable easy identification of genomic variants (including single nucleotide polymorphisms, structural and copy number variants), and functional genomic information (e.g. peaks in ChIP-seq data) in the context of genomic annotations. Availability: Savant is freely available at http://compbio.cs.toronto.edu/savant Contact: savant@cs.toronto.edu PMID:20562449

  17. Unconventional Architectures for High-Throughput Sciences

    SciTech Connect

    Nieplocha, Jarek; Marquez, Andres; Petrini, Fabrizio; Chavarría-Miranda, Daniel

    2007-06-15

    Science laboratories and sophisticated simulations are producing data of increasing volumes and complexities, and that’s posing significant challenges to current data infrastructures as terabytes to petabytes of data must be processed and analyzed. Traditional computing platforms, originally designed to support model-driven applications, are unable to meet the demands of the data-intensive scientific applications. Pacific Northwest National Laboratory (PNNL) research goes beyond “traditional supercomputing” applications to address emerging problems that need scalable, real-time solutions. The outcome is new unconventional architectures for data-intensive applications specifically designed to process the deluge of scientific data, including FPGAs, multithreaded architectures and IBM's Cell.

  18. 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.

  19. 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. PMID:21902639

  20. 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

  1. Workflow for High Throughput Screening of Gas Sensing Materials

    PubMed Central

    Koplin, Tobias J.; Siemons, Maike; Océn-Valéntin, César; Sanders, Daniel; Simon, Ulrich

    2006-01-01

    The workflow of a high throughput screening setup for the rapid identification of new and improved sensor materials is presented. The polyol method was applied to prepare nanoparticular metal oxides as base materials, which were functionalised by surface doping. Using multi-electrode substrates and high throughput impedance spectroscopy (HT-IS) a wide range of materials could be screened in a short time. Applying HT-IS in search of new selective gas sensing materials a NO2-tolerant NO sensing material with reduced sensitivities towards other test gases was identified based on iridium doped zinc oxide. Analogous behaviour was observed for iridium doped indium oxide.

  2. Advances in high throughput DNA sequence data compression.

    PubMed

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

    2016-06-01

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

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

    PubMed

    Tuntland, Micheal L; Fung, L W-M

    2016-10-01

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

  4. C. elegans in high-throughput drug discovery

    PubMed Central

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

    2014-01-01

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

  5. High throughput drug discovery with ESI-FTICR

    NASA Astrophysics Data System (ADS)

    Sannes-Lowery, Kristin A.; Cummins, Lendell L.; Chen, Shuo; Drader, Jared J.; Hofstadler, Steven A.

    2004-11-01

    Ribonucleic acids (RNA) are an attractive target for drug discovery since they play critical roles in cellular functions. Because small structured subdomains are known to mimic the behavior of the entire RNA, it is possible to design RNA drug targets that are amenable to interrogation by high performance mass spectrometry. We have developed a high throughput drug discovery platform that uses electrospray ionization Fourier transform ion cyclotron mass spectrometry to investigate ligand binding to structured RNA drug targets. This assay is called multitarget affinity/specificity screening (MASS). Using MASS, we show that it is possible to screen synthetic and natural product libraries in a high throughput and robust manner.

  6. Towards Chip Scale Liquid Chromatography and High Throughput Immunosensing

    SciTech Connect

    Ni, J.

    2000-09-21

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

  7. Aspirator Gun for High-Throughput Mosquito Bioassays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe an innovative aspirator gun designed to transfer anaesthetized mosquitoes directly into glass bioassay tubes. The gun has been used for thousands of transfers with extremely low associated mortality and is the central component of a high-throughput bioassay system. The gun is constructed...

  8. 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,...

  9. 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...

  10. Aspirator gun for high-throughput mosquito bioassays.

    PubMed

    Aldridge, Robert L; Wynn, W Wayne; Britch, Seth C; Linthicum, Kenneth J

    2012-03-01

    We describe an innovative aspirator gun designed to transfer individual anesthetized mosquitoes directly into glass bioassay tubes. The gun has been used for thousands of transfers with extremely low associated mortality and is the central component of a high-throughput bioassay system. The gun is constructed using readily obtainable materials and can be modified for a range of insects. PMID:22533090

  11. 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...

  12. 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...

  13. 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 ...

  14. 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...

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

    EPA Science Inventory

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

  16. GENO PROFILER: BATCH PROCESSING OF HIGH THROUGHPUT CAPILLARY FINGERPRINTING DATA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High-throughput fingerprinting techniques employing capillary electrophoresis place new demands on the editing of fingerprint files for the downstream contig assembly program, FPC. A cross-platform software application, GenoProfiler, was developed for automated editing of sized fingerprinting profil...

  17. 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...

  18. Bioinformatics of Cancer ncRNA in High Throughput Sequencing: Present State and Challenges

    PubMed Central

    Jorge, Natasha Andressa Nogueira; Ferreira, Carlos Gil; Passetti, Fabio

    2012-01-01

    The numerous genome sequencing projects produced unprecedented amount of data providing significant information to the discovery of novel non-coding RNA (ncRNA). Several ncRNAs have been described to control gene expression and display important role during cell differentiation and homeostasis. In the last decade, high throughput methods in conjunction with approaches in bioinformatics have been used to identify, classify, and evaluate the expression of hundreds of ncRNA in normal and pathological states, such as cancer. Patient outcomes have been already associated with differential expression of ncRNAs in normal and tumoral tissues, providing new insights in the development of innovative therapeutic strategies in oncology. In this review, we present and discuss bioinformatics advances in the development of computational approaches to analyze and discover ncRNA data in oncology using high throughput sequencing technologies. PMID:23251139

  19. MPIC: a high-throughput analytical method for multiple DNA targets.

    PubMed

    Guo, Jinchao; Yang, Litao; Chen, Lili; Morisset, Dany; Li, Xiang; Pan, Liangwen; Zhang, Dabing

    2011-03-01

    We describe the development of a novel combined approach for high-throughput analysis of multiple DNA targets based on multiplex Microdroplet PCR Implemented Capillary gel electrophoresis (MPIC), a two-step PCR amplification strategy. In the first step, the multiple target DNAs are preamplified using bipartite primers attached with universal tail sequences on their 5'-ends. Then, the preamplified templates are compartmentalized individually in the microdroplet of the PCR system, and multiple targets can be amplified in parallel, employing primers targeting their universal sequences. Subsequently, the resulting multiple products are analyzed by capillary gel electrophoresis (CGE). Using genetically modified organism (GMO) analysis as a model, 24 DNA targets can be simultaneously detected with a relative limit of detection of 0.1% (w/w) and absolute limit of detection of 39 target DNA copies. The described system provides a promising alternative for high-throughput analysis of multiple DNA targets. PMID:21291179

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

    PubMed

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

    2016-03-01

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

  1. A human cDNA library for high-throughput protein expression screening.

    PubMed

    Büssow, K; Nordhoff, E; Lübbert, C; Lehrach, H; Walter, G

    2000-04-01

    We have constructed a human fetal brain cDNA library in an Escherichia coli expression vector for high-throughput screening of recombinant human proteins. Using robot technology, the library was arrayed in microtiter plates and gridded onto high-density filter membranes. Putative expression clones were detected on the filters using an antibody against the N-terminal sequence RGS-His(6) of fusion proteins. Positive clones were rearrayed into a new sublibrary, and 96 randomly chosen clones were analyzed. Expression products were analyzed by SDS-PAGE, affinity purification, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and the determined protein masses were compared to masses predicted from DNA sequencing data. It was found that 66% of these clones contained inserts in a correct reading frame. Sixty-four percent of the correct reading frame clones comprised the complete coding sequence of a human protein. High-throughput microtiter plate methods were developed for protein expression, extraction, purification, and mass spectrometric analyses. An enzyme assay for glyceraldehyde-3-phosphate dehydrogenase activity in native extracts was adapted to the microtiter plate format. Our data indicate that high-throughput screening of an arrayed protein expression library is an economical way of generating large numbers of clones producing recombinant human proteins for structural and functional analyses. PMID:10777659

  2. High-throughput determination of structural phase diagram and constituent phases using GRENDEL.

    PubMed

    Kusne, A G; Keller, D; Anderson, A; Zaban, A; Takeuchi, I

    2015-11-01

    Advances in high-throughput materials fabrication and characterization techniques have resulted in faster rates of data collection and rapidly growing volumes of experimental data. To convert this mass of information into actionable knowledge of material process-structure-property relationships requires high-throughput data analysis techniques. This work explores the use of the Graph-based endmember extraction and labeling (GRENDEL) algorithm as a high-throughput method for analyzing structural data from combinatorial libraries, specifically, to determine phase diagrams and constituent phases from both x-ray diffraction and Raman spectral data. The GRENDEL algorithm utilizes a set of physical constraints to optimize results and provides a framework by which additional physics-based constraints can be easily incorporated. GRENDEL also permits the integration of database data as shown by the use of critically evaluated data from the Inorganic Crystal Structure Database in the x-ray diffraction data analysis. Also the Sunburst radial tree map is demonstrated as a tool to visualize material structure-property relationships found through graph based analysis. PMID:26469294

  3. High-throughput determination of structural phase diagram and constituent phases using GRENDEL

    NASA Astrophysics Data System (ADS)

    Kusne, A. G.; Keller, D.; Anderson, A.; Zaban, A.; Takeuchi, I.

    2015-11-01

    Advances in high-throughput materials fabrication and characterization techniques have resulted in faster rates of data collection and rapidly growing volumes of experimental data. To convert this mass of information into actionable knowledge of material process-structure-property relationships requires high-throughput data analysis techniques. This work explores the use of the Graph-based endmember extraction and labeling (GRENDEL) algorithm as a high-throughput method for analyzing structural data from combinatorial libraries, specifically, to determine phase diagrams and constituent phases from both x-ray diffraction and Raman spectral data. The GRENDEL algorithm utilizes a set of physical constraints to optimize results and provides a framework by which additional physics-based constraints can be easily incorporated. GRENDEL also permits the integration of database data as shown by the use of critically evaluated data from the Inorganic Crystal Structure Database in the x-ray diffraction data analysis. Also the Sunburst radial tree map is demonstrated as a tool to visualize material structure-property relationships found through graph based analysis.

  4. Optical tools for high-throughput screening of abrasion resistance of combinatorial libraries of organic coatings

    NASA Astrophysics Data System (ADS)

    Potyrailo, Radislav A.; Chisholm, Bret J.; Olson, Daniel R.; Brennan, Michael J.; Molaison, Chris A.

    2002-02-01

    Design, validation, and implementation of an optical spectroscopic system for high-throughput analysis of combinatorially developed protective organic coatings are reported. Our approach replaces labor-intensive coating evaluation steps with an automated system that rapidly analyzes 8x6 arrays of coating elements that are deposited on a plastic substrate. Each coating element of the library is 10 mm in diameter and 2 to 5 micrometers thick. Performance of coatings is evaluated with respect to their resistance to wear abrasion because this parameter is one of the primary considerations in end-use applications. Upon testing, the organic coatings undergo changes that are impossible to quantitatively predict using existing knowledge. Coatings are abraded using industry-accepted abrasion test methods at single-or multiple-abrasion conditions, followed by high- throughput analysis of abrasion-induced light scatter. The developed automated system is optimized for the analysis of diffusively scattered light that corresponds to 0 to 30% haze. System precision of 0.1 to 2.5% relative standard deviation provides capability for the reliable ranking of coatings performance. While the system was implemented for high-throughput screening of combinatorially developed organic protective coatings for automotive applications, it can be applied to a variety of other applications where materials ranking can be achieved using optical spectroscopic tools.

  5. Digital fragment analysis of short tandem repeats by high-throughput amplicon sequencing.

    PubMed

    Darby, Brian J; Erickson, Shay F; Hervey, Samuel D; Ellis-Felege, Susan N

    2016-07-01

    High-throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high-throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high-throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost-effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence-aware microsatellite data. PMID:27386092

  6. Demonstration of High-Throughput Water Isotopologue Measurements Using Cavity Ring-Down Spectroscopy

    NASA Astrophysics Data System (ADS)

    van Pelt, A. D.; Gupta, P.; Green, I.

    2009-12-01

    The ability to measure the δ18O and δD isotopic content of water has long relied on cumbersome methods that require well equipped laboratories, highly qualified technicians and frequently calibrated instruments. The advent of commercial analyzers based on Wavelength Scanned Cavity Ring-Down Spectroscopy (WS-CRDS) for isotopic water measurements has opened up new possibilities for mobile laboratory and field deployable isotopic instruments. For many laboratories, sample throughput has been a major bottleneck - either real-time sampling of stream flow or simply the number of samples gathered during a campaign can be a daunting challenge. It is not uncommon for users to have a huge backlog on the water samples that need to be analyzed within a short period of time. We present results of a new high throughput water analyzer based on WS-CRDS technology. This high throughput method comes with negligible impact on the precision and memory and absolutely no impact on the drift characteristics of the analyzer. In order to provide confidence in the data collected, even in the most challenging environments, there can be no comprise on the consistency or reproducibility of the instrument performance. The new high throughput isotopic water analyzer measures isotopologues of water with a typical precision of better than 0.15‰ for δ18O and better than 0.6‰ for δD and can execute over 380 injections per day. The analyzer has extremely low drift of < ±0.3‰ for δ18O and < ±0.9‰ for δD. This presentation demonstrates these capabilities of the high throughput isotopic water analyzer. This water isotope analyzer can be configured to analyze water vapor, liquid, or alternate between vapor and liquid. The alternating configuration enables the periodic recalibration of water vapor measurements using liquid water isotopic standards. The results of this study clearly demonstrate that the precision of the analyzer is very high and the memory and drift are exceptional even

  7. A high-throughput label-free nanoparticle analyser

    NASA Astrophysics Data System (ADS)

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

    2011-05-01

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

  8. 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).

  9. 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

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

    PubMed

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

    2013-11-01

    Fusion genes are hybrid genes that combine parts of two or more original genes. They can form as a result of chromosomal rearrangements or abnormal transcription, and have been shown to act as drivers of malignant transformation and progression in many human cancers. The biological significance of fusion genes together with their specificity to cancer cells has made them into excellent targets for molecular therapy. Fusion genes are also used as diagnostic and prognostic markers to confirm cancer diagnosis and monitor response to molecular therapies. High-throughput sequencing has enabled the systematic discovery of fusion genes in a wide variety of cancer types. In this review, we describe the history of fusion genes in cancer and the ways in which fusion genes form and affect cellular function. We also describe computational methodologies for detecting fusion genes from high-throughput sequencing experiments, and the most common sources of error that lead to false discovery of fusion genes. PMID:23376639

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

    PubMed

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

    2013-12-17

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

  12. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    Gale, Molly; Yan, Qin

    2015-01-01

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

  15. Perspectives on high-throughput technologies applied to protein crystallization.

    PubMed

    Saridakis, Emmanuel

    2012-07-01

    High-throughput crystallisation requires the rapid and accurate dispensing of protein and precipitating agent solutions at nanovolumes, but does not end there. The choice of the initial screens is very important, especially with respect to the availability of protein material. Data from previous crystallisation experiments that are scattered in the literature and only partially available in databases have to be analysed in efficient ways that will maximise their utility for designing new screens. A larger portion of crystallisation parameter space should be made accessible to screening, through the use of nucleants and seeding. Observation, assessment and scaling up of the crystallisation trials should be efficiently performed and, finally yet importantly, optimisation of conditions must also be adapted to the high-throughput environment. The above requirements are briefly addressed in the following paper. PMID:22489783

  16. 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

  17. 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

  18. 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

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

    PubMed

    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

  20. High Throughput siRNA Screening Using Reverse Transfection.

    PubMed

    von Schantz, Carina; Saarela, Jani

    2016-01-01

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

  1. 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

  2. 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

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

    PubMed Central

    2012-01-01

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

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

    PubMed

    Chiaraviglio, Lucius; Kirby, James E

    2015-12-01

    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

  5. High-Throughput Computational and Experimental Techniques in Structural Genomics

    PubMed Central

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

    2004-01-01

    Structural genomics has as its goal the provision of structural information for all possible ORF sequences through a combination of experimental and computational approaches. The access to genome sequences and cloning resources from an ever-widening array of organisms is driving high-throughput structural studies by the New York Structural Genomics Research Consortium. In this report, we outline the progress of the Consortium in establishing its pipeline for structural genomics, and some of the experimental and bioinformatics efforts leading to structural annotation of proteins. The Consortium has established a pipeline for structural biology studies, automated modeling of ORF sequences using solved (template) structures, and a novel high-throughput approach (metallomics) to examining the metal binding to purified protein targets. The Consortium has so far produced 493 purified proteins from >1077 expression vectors. A total of 95 have resulted in crystal structures, and 81 are deposited in the Protein Data Bank (PDB). Comparative modeling of these structures has generated >40,000 structural models. We also initiated a high-throughput metal analysis of the purified proteins; this has determined that 10%-15% of the targets contain a stoichiometric structural or catalytic transition metal atom. The progress of the structural genomics centers in the U.S. and around the world suggests that the goal of providing useful structural information on most all ORF domains will be realized. This projected resource will provide structural biology information important to understanding the function of most proteins of the cell. PMID:15489337

  6. High throughput screening of ferroelectric thin film libraries

    NASA Astrophysics Data System (ADS)

    Schroeter, Christian; Wessler, Berit; Schoenecker, Andreas; Keitel, Uwe; Eng, Lukas M.

    2006-12-01

    High throughput methods can significantly speed up the search for advanced materials in a multidimensional configuration space, hence keeping innovation cycles short. In the search for improved materials, high throughput methods are wanted to optimize composition and processing of promising systems, and to find candidate compounds. Such a method is described here which is applicable to the development of ferroelectric thin films. Libraries with samples of varying chemical composition were produced via the sol-gel route on structured and metallized silicon wafers. To determine the permittivity of the films, automated measurements of film thickness and capacity were established. Furthermore, ferroelectric hysterisis measurements were performed on samples with a particularly high permittivity. This high throughput route, which allows for synthesis and characterization of over hundred samples per day, was proved and tested by means of lead zirconate titanate as a standard material. It was possible to obtain films with remarkable high permittivity and low coercive field at optimal lead zirconate/lead titanate ratio and by compensating for lead loss during processing by finding the optimal lead excess added to the precursor solutions.

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

    PubMed

    Finak, Greg; Gottardo, Raphael

    2016-01-01

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

  8. High-Throughput Analysis of RNA Structure and Ribonucleoprotein Assembly

    PubMed Central

    McGinnis, Jennifer L.; Duncan, Caia D. S.; Weeks, Kevin M.

    2016-01-01

    RNA folds to form complex structures vital to many cellular functions. Proteins facilitate RNA folding at both the secondary and tertiary structure levels. An absolute prerequisite for understanding RNA folding and ribonucleoprotein (RNP) assembly reactions is a complete understanding of the RNA structure at each stage of the folding or assembly process. Here we provide a guide for comprehensive and high-throughput analysis of RNA secondary and tertiary structure using SHAPE and hydroxyl radical footprinting. As an example of the strong and sometimes surprising conclusions that can emerge from high-throughput analysis of RNA folding and RNP assembly, we summarize the structure of the bI3 group I intron RNA in four distinct states. Dramatic structural rearrangements occur in both secondary and tertiary structure as the RNA folds from the free state to the active, six-component, RNP complex. As high-throughput and high-resolution approaches are applied broadly to large protein-RNA complexes, other proteins previously viewed as making simple contributions to RNA folding are also likely to be found to exert multifaceted, long-range, cooperative, and non-additive effects on RNA folding. These protein-induced contributions add another level of control, and potential regulatory function, in RNP complexes. PMID:20946765

  9. High-throughput analysis of growth differences among phage strains.

    PubMed

    Turner, Paul E; Draghi, Jeremy A; Wilpiszeski, Regina

    2012-01-01

    Although methods such as spectrophotometry are useful for identifying growth differences among bacterial strains, it is currently difficult to similarly determine whether bacteriophage strains differ in growth using high throughput methods. Here we use automated spectrophotometry to develop an in vitro method for indirectly distinguishing fitness (growth) differences among virus strains, based on direct measures of their infected bacterial hosts. We used computer simulations of a mathematical model for phage growth to predict which features of bacterial growth curves were best associated with differences in growth among phage strains. We then tested these predictions using the in vitro method to confirm which of the inferred viral growth traits best reflected known fitness differences among genotypes of the RNA phage phi-6, when infecting a Pseudomonas syringae host. Results showed that the inferred phage trait of time-to-extinction (time required to drive bacterial density below detectable optical density) reliably correlated with genotype rankings based on absolute fitness (phage titer per ml). These data suggested that the high-throughput analysis was valuable for identifying growth differences among virus strains, and that the method may be especially useful for high throughput analyses of fitness differences among phage strains cultured and/or evolved in liquid (unstructured) environments. PMID:22101310

  10. High-throughput 2D root system phenotyping platform facilitates genetic analysis of root growth and development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 analyz...

  11. Evaluation of High-throughput Genotoxicity Assays Used in Profiling the US EPA ToxCast Chemicals

    EPA Science Inventory

    Three high-throughput screening (HTS) genotoxicity assays-GreenScreen HC GADD45a-GFP (Gentronix Ltd.), CellCiphr p53 (Cellumen Inc.) and CellSensor p53RE-bla (Invitrogen Corp.)-were used to analyze the collection of 320 predominantly pesticide active compounds being tested in Pha...

  12. High-throughput flow cytometric screening of combinatorial chemistry bead libraries for proteomics and drug discovery

    NASA Astrophysics Data System (ADS)

    Leary, James F.; Reece, Lisa M.; Yang, Xian-Bin; Gorenstein, David

    2005-04-01

    For proteomics drug discovery applications, combinatorial microbead thioaptamer libraries (one thioaptamer sequence per bead) are being created by split synthesis method, creating a "proteomics library" of protein capture beads which can be analyzed by high-throughput screening methods in this case, flow cytometry and cell sorting. Thioaptamers, oligonucleotides with thiophosphate backbone substitutions, function like antibodies in terms of recognizing specific protein sequences but have a number of advantages over antibody libraries. These proteomics beads can then be analyzed by high-speed flow cytometry and sorted to single-bead level depending on relative fluorescence brightness of fluorescently-labeled proteins, or for a specific protein from all of the molecules of cell subpopulations being analyzed. The thioaptamer sequences on a given bead showing high affinity for that protein can then be sequenced. Alternatively, the protein-capturing beads can be analyzed by MALDI-TOF mass spectrometry for analysis of the bound proteins. The beads can be thought of as equivalent to single-element positions of a proteomics chip arrays but with the advantage of being able to much more rapidly analyze hundreds of millions of possible amino acid sequences/epitopes on the basis of thioaptamer sequence affinities to select single sequences of interest. Additionally, those beads can be manipulated and isolated at the single bead level by high-throughput flow cytometry/cell sorting for subsequent sequencing of the thioaptamer sequences.

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

    SciTech Connect

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

    2006-05-28

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

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

    PubMed

    Zheng, S

    2016-01-01

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

  15. 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.

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

    PubMed

    Poisot, Timothée; Péquin, Bérangère; Gravel, Dominique

    2013-04-01

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

  17. 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).

  18. Creation of a small high-throughput screening facility.

    PubMed

    Flak, Tod

    2009-01-01

    The creation of a high-throughput screening facility within an organization is a difficult task, requiring a substantial investment of time, money, and organizational effort. Major issues to consider include the selection of equipment, the establishment of data analysis methodologies, and the formation of a group having the necessary competencies. If done properly, it is possible to build a screening system in incremental steps, adding new pieces of equipment and data analysis modules as the need grows. Based upon our experience with the creation of a small screening service, we present some guidelines to consider in planning a screening facility. PMID:19551356

  19. Orchestrating high-throughput genomic analysis with Bioconductor.

    PubMed

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

    2015-02-01

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

  20. 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. A probabilistic approach to high throughput drug discovery.

    PubMed

    Labute, Paul; Nilar, Shahul; Williams, Christopher

    2002-03-01

    A methodology is presented in which high throughput screening experimental data are used to construct a probabilistic QSAR model which is subsequently used to select building blocks for a virtual combinatorial library. The methodology is based upon statistical probability estimation and not regression. The methodology is applied to the construction of two focused virtual combinatorial libraries: one for cyclic GMP phosphodiesterase type V inhibitors and one for acyl-CoA:cholesterol O-acyltransferase inhibitors. The results suggest that the methodology is capable of selecting combinatorial substituents that lead to active compounds starting with binary (pass/fail) activity measurements. PMID:11966422

  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. PMID:25089276

  3. 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.

  4. Towards A Fully Automated High-Throughput Phototransfection System

    PubMed Central

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

    2010-01-01

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

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. Reconfigurable microfluidic dilution for high-throughput quantitative assays.

    PubMed

    Fan, Jinzhen; Li, Baoqing; Xing, Siyuan; Pan, Tingrui

    2015-06-21

    This paper reports a reconfigurable microfluidic dilution device for high-throughput quantitative assays, which can easily produce discrete logarithmic/binary concentration profiles ranging from 1 to 100-fold dilution in parallel from a fixed sample volume (e.g., 10 μL) without any assistance of continuous fluidic pump or robotic automation. The integrated dilution generation chip consists of switchable distribution and collection channels, metering reservoirs, reaction chambers, and pressure-activatable Laplace valves. Following the sequential loading of a sample, a diluent, and a detection reagent into their individual metering chambers, the top microfluidic layer can be reconfigured to collect the metered chemicals into the reaction chambers in parallel, where detection will be conducted. To facilitate mixing and reaction in the microchambers, two acoustic microstreaming actuation mechanisms have been investigated for easy integrability and accessibility. Furthermore, the microfluidic dilution generator has been characterized by both colorimetric and fluorescent means. A further demonstration of the generic usage of the quantitative dilution chip has utilized the commonly available bicinchoninic acid (BCA) assay to analyse the protein concentrations of human tissue extracts. In brief, the microfluidic dilution generator offers a high-throughput high-efficiency quantitative analytical alternative to conventional quantitative assay platforms, by simple manipulation of a minute amount of chemicals in a compact microfluidic device with minimal equipment requirement, which can serve as a facile tool for biochemical and biological analyses in regular laboratories, point-of-care settings and low-resource environments. PMID:25994379

  10. Computational analysis of high-throughput flow cytometry data

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2004-01-01

    The wealth of transcript information that has been made publicly available in recent years requires the development of high-throughput functional genomics and proteomics approaches for its analysis. Such approaches need suitable data integration procedures and a high level of automation in order to gain maximum benefit from the results generated. We have designed an automatic pipeline to analyse annotated open reading frames (ORFs) stemming from full-length cDNAs produced mainly by the German cDNA Consortium. The ORFs are cloned into expression vectors for use in large-scale assays such as the determination of subcellular protein localization or kinase reaction specificity. Additionally, all identified ORFs undergo exhaustive bioinformatic analysis such as similarity searches, protein domain architecture determination and prediction of physicochemical characteristics and secondary structure, using a wide variety of bioinformatic methods in combination with the most up-to-date public databases (e.g. PRINTS, BLOCKS, INTERPRO, PROSITE SWISSPROT). Data from experimental results and from the bioinformatic analysis are integrated and stored in a relational database (MS SQL-Server), which makes it possible for researchers to find answers to biological questions easily, thereby speeding up the selection of targets for further analysis. The designed pipeline constitutes a new automatic approach to obtaining and administrating relevant biological data from high-throughput investigations of cDNAs in order to systematically identify and characterize novel genes, as well as to comprehensively describe the function of the encoded proteins. PMID:14762202

  12. 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.

  13. 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.

  14. High-throughput assays for DNA gyrase and other topoisomerases.

    PubMed

    Maxwell, Anthony; Burton, Nicolas P; O'Hagan, Natasha

    2006-01-01

    We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format. PMID:16936317

  15. Empirical assessment of sequencing errors for high throughput pyrosequencing data

    PubMed Central

    2013-01-01

    Background Sequencing-by-synthesis technologies significantly improve over the Sanger method in terms of speed and cost per base. However, they still usually fail to compete in terms of read length and quality. Current high-throughput implementations of the pyrosequencing technique yield reads whose length approach those of the capillary electrophoresis method. A less obvious question is whether their quality is affected by platform-specific sequencing errors. Results We present an empirical study aimed at assessing the quality and characterising sequencing errors for high throughput pyrosequencing data. We have developed a procedure for extracting sequencing error data from genome assemblies and study their characteristics, in particular the length distribution of indel gaps and their relation to the sequence contexts where they occur. We used this procedure to analyse data from three prokaryotic genomes sequenced with the GS FLX technology. We also compared two models previously employed with success for peptide sequence alignment. Conclusions We observed an overall very low error rate in the analysed data, with indel errors being much more abundant than substitutions. We also observed a dependence between the length of the gaps and that of the homopolymer context where they occur. As with protein alignments, a power-law model seems to approximate the indel errors more accurately, although the results are not so conclusive as to justify a depart from the commonly used affine gap penalty scheme. In whichever case, however, our procedure can be used to estimate more realistic error model parameters. PMID:23339526

  16. Human transcriptome array for high-throughput clinical studies

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2016-08-01

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

  18. 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.

  19. Polymer Microarrays for High Throughput Discovery of Biomaterials

    PubMed Central

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

    2012-01-01

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

  20. 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.

  1. High-throughput characterization for solar fuels materials discovery

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  2. 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

  3. A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide.

    PubMed

    Motabar, Omid; Goldin, Ehud; Leister, William; Liu, Ke; Southall, Noel; Huang, Wenwei; Marugan, Juan J; Sidransky, Ellen; Zheng, Wei

    2012-01-01

    Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening. PMID:22033823

  4. A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide

    PubMed Central

    Motabar, Omid; Goldin, Ehud; Leister, William; Liu, Ke; Southall, Noel; Huang, Wenwei; Marugan, Juan J.; Sidransky, Ellen

    2012-01-01

    Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening. PMID:22033823

  5. Versatile protein biotinylation strategies for potential high-throughput proteomics.

    PubMed

    Lue, Rina Y P; Chen, Grace Y J; Hu, Yi; Zhu, Qing; Yao, Shao Q

    2004-02-01

    We present intein-mediated approaches for efficient biotinylation of proteins site-specifically. The reactive C-terminal thioester generated from intein-assisted protein splicing (either in vitro or in live cells) served as an attractive and exclusive site for attaching cysteine-containing biotin. Using these novel biotinylation strategies, we were able to efficiently biotinylate many proteins from different biological sources in a potentially high-throughput, high-content fashion. Some of these proteins were subsequently immobilized, in a very simple manner, onto different avidin-functionalized solid surfaces for applications such as protein microarray and surface plasmon resonance (SPR) spectroscopy, highlighting the numerous advantages of using biotin over other tags (e.g., GST, His-tag, etc.) as the method of choice in protein purification/immobilization. In addition, our intein-mediated strategies provided critical advantages over other protein biotinylation strategies in a number of ways. For the first time, we also successfully demonstrated that intein-mediated protein biotinylation proceeded adequately inside both bacterial and mammalian living cells, as well as in a cell-free protein synthesis system. Taken together, our results indicate the versatility of these intein-mediated strategies for potential high-throughput proteomics applications. They may also serve as useful tools for various biochemical and biophysical studies of proteins both in vitro and in vivo. PMID:14746473

  6. 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.

  7. 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

  8. Methods of high throughput biophysical characterization in biopharmaceutical development.

    PubMed

    Razinkov, Vladimir I; Treuheit, Michael J; Becker, Gerald W

    2013-03-01

    Discovery and successful development of biopharmaceutical products depend on a thorough characterization of the molecule both before and after formulation. Characterization of a formulated biotherapeutic, typically a protein or large peptide, requires a rigorous assessment of the molecule's physical stability. Stability of a biotherapeutic includes not only chemical stability, i.e., degradation of the molecule to form undesired modifications, but also structural stability, including the formation of aggregates. In this review, high throughput biophysical characterization techniques are described according to their specific applications during biopharmaceutical discovery, development and manufacturing. The methods presented here are classified according to these attributes, and include spectroscopic assays based on absorbance, polarization, intrinsic and extrinsic fluorescence, surface plasmon resonance instrumentation, calorimetric methods, dynamic and static light scattering techniques, several visible particle counting and sizing methods, new viscosity assay, based on light scattering and mass spectrometry. Several techniques presented here are already implemented in industry; but, many high throughput biophysical methods are still in the initial stages of implementation or even in the prototype stage. Each technique in this report is judged by the specific application of the method through the biopharmaceutical development process. PMID:22725690

  9. High-throughput proteomics and the fight against pathogens.

    PubMed

    Horvatić, Anita; Kuleš, Josipa; Guillemin, Nicolas; Galan, Asier; Mrljak, Vladimir; Bhide, Mangesh

    2016-07-19

    Pathogens pose a major threat to human and animal welfare. Understanding the interspecies host-pathogen protein-protein interactions could lead to the development of novel strategies to combat infectious diseases through the rapid development of new therapeutics. The first step in understanding the host-pathogen crosstalk is to identify interacting proteins in order to define crucial hot-spots in the host-pathogen interactome, such as the proposed pharmaceutical targets by means of high-throughput proteomic methodologies. In order to obtain holistic insight into the inter- and intra-species bimolecular interactions, apart from the proteomic approach, sophisticated in silico modeling is used to correlate the obtained large data sets with other omics data and clinical outcomes. Since the main focus in this area has been directed towards human medicine, it is time to extrapolate the existing expertise to a new emerging field: the 'systems veterinary medicine'. Therefore, this review addresses high-throughput mass spectrometry-based technology for monitoring protein-protein interactions in vitro and in vivo and discusses pathogen cultivation, model host cells and available bioinformatic tools employed in vaccine development. PMID:27227577

  10. 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

  11. High-throughput search for improved transparent conducting oxides

    NASA Astrophysics Data System (ADS)

    Miglio, Anna

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

  12. 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

  13. High-throughput GPU-based LDPC decoding

    NASA Astrophysics Data System (ADS)

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

    2010-08-01

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

  14. Droplet Electrospray Ionization Mass Spectrometry for High Throughput Screening for Enzyme Inhibitors

    PubMed Central

    2015-01-01

    High throughput screening (HTS) is important for identifying molecules with desired properties. Mass spectrometry (MS) is potentially powerful for label-free HTS due to its high sensitivity, speed, and resolution. Segmented flow, where samples are manipulated as droplets separated by an immiscible fluid, is an intriguing format for high throughput MS because it can be used to reliably and precisely manipulate nanoliter volumes and can be directly coupled to electrospray ionization (ESI) MS for rapid analysis. In this study, we describe a “MS Plate Reader” that couples standard multiwell plate HTS workflow to droplet ESI-MS. The MS plate reader can reformat 3072 samples from eight 384-well plates into nanoliter droplets segmented by an immiscible oil at 4.5 samples/s and sequentially analyze them by MS at 2 samples/s. Using the system, a label-free screen for cathepsin B modulators against 1280 chemicals was completed in 45 min with a high Z-factor (>0.72) and no false positives (24 of 24 hits confirmed). The assay revealed 11 structures not previously linked to cathepsin inhibition. For even larger scale screening, reformatting and analysis could be conducted simultaneously, which would enable more than 145 000 samples to be analyzed in 1 day. PMID:25137241

  15. MassCode Liquid Arrays as a Tool for Multiplexed High-Throughput Genetic Profiling

    PubMed Central

    Richmond, Gregory S.; Khine, Htet; Zhou, Tina T.; Ryan, Daniel E.; Brand, Tony; McBride, Mary T.; Killeen, Kevin

    2011-01-01

    Multiplexed detection assays that analyze a modest number of nucleic acid targets over large sample sets are emerging as the preferred testing approach in such applications as routine pathogen typing, outbreak monitoring, and diagnostics. However, very few DNA testing platforms have proven to offer a solution for mid-plexed analysis that is high-throughput, sensitive, and with a low cost per test. In this work, an enhanced genotyping method based on MassCode technology was devised and integrated as part of a high-throughput mid-plexing analytical system that facilitates robust qualitative differential detection of DNA targets. Samples are first analyzed using MassCode PCR (MC-PCR) performed with an array of primer sets encoded with unique mass tags. Lambda exonuclease and an array of MassCode probes are then contacted with MC-PCR products for further interrogation and target sequences are specifically identified. Primer and probe hybridizations occur in homogeneous solution, a clear advantage over micro- or nanoparticle suspension arrays. The two cognate tags coupled to resultant MassCode hybrids are detected in an automated process using a benchtop single quadrupole mass spectrometer. The prospective value of using MassCode probe arrays for multiplexed bioanalysis was demonstrated after developing a 14plex proof of concept assay designed to subtype a select panel of Salmonella enterica serogroups and serovars. This MassCode system is very flexible and test panels can be customized to include more, less, or different markers. PMID:21544191

  16. High-throughput microcavitation bubble induced cellular mechanotransduction

    NASA Astrophysics Data System (ADS)

    Compton, Jonathan Lee

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

  17. Ultra-Sensitive, High Throughput and Quantitative Proteomics Measurements

    SciTech Connect

    Jacobs, Jon M.; Monroe, Matthew E.; Qian, Weijun; Shen, Yufeng; Anderson, Gordon A.; Smith, Richard D.

    2005-02-01

    We describe the broad basis and application of an approach for very high throughput, ultra-sensitive, and quantitative proteomic measurements based upon the use of ultra-high performance separations and mass spectrometry. An overview of the accurate mass and time (AMT) tag approach and a description of the incorporated data analysis pipeline necessary for efficient proteomic studies are presented. Adjunct technologies, including stable-isotope labeling methodologies and improvements in the utilization of LC-MS peak intensity information for quantitative purposes are discussed. Related areas include the use of automated sample handling for improving analysis reproducibility, methods for using information from the separation for more confident peptide peak identification, and the utilization of smaller diameter capillary columns having lower volumetric flow rates to increase electrospray ionization efficiency and allow for more predictable and quantitative results. The developments are illustrated in the context of studies of complex biological systems.

  18. High-throughput analysis of behavior for drug discovery.

    PubMed

    Alexandrov, Vadim; Brunner, Dani; Hanania, Taleen; Leahy, Emer

    2015-03-01

    Drug testing with traditional behavioral assays constitutes a major bottleneck in the development of novel therapies. PsychoGenics developed three comprehensive high-throughput systems, SmartCube(®), NeuroCube(®) and PhenoCube(®) systems, to increase the efficiency of the drug screening and phenotyping in rodents. These three systems capture different domains of behavior, namely, cognitive, motor, circadian, social, anxiety-like, gait and others, using custom-built computer vision software and machine learning algorithms for analysis. This review exemplifies the use of the three systems and explains how they can advance drug screening with their applications to phenotyping of disease models, drug screening, selection of lead candidates, behavior-driven lead optimization, and drug repurposing. PMID:25592319

  19. Proteomics equipped with multiplexing toward ultra high throughput.

    PubMed

    Kim, Min-Sik

    2015-01-01

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

  20. Characterizing immune repertoires by high throughput sequencing: strategies and applications

    PubMed Central

    Calis, Jorg J.A.; Rosenberg, Brad R.

    2014-01-01

    As the key cellular effectors of adaptive immunity, T and B lymphocytes utilize specialized receptors to recognize, respond to, and neutralize a diverse array of extrinsic threats. These receptors (immunoglobulins in B lymphocytes, T cell receptors in T lymphocytes) are incredibly variable, the products of specialized genetic diversification mechanisms that generate complex lymphocyte repertoires with extensive collections of antigen specificities. Recent advances in high throughput sequencing (HTS) technologies have transformed our ability to examine antigen receptor repertoires at single nucleotide, and more recently, single cell, resolution. Here we review current approaches to examining antigen receptor repertoires by HTS, and discuss inherent biological and technical challenges. We further describe emerging applications of this powerful methodology for exploring the adaptive immune system. PMID:25306219

  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. PMID:26319325

  2. High-throughput screening of microbial adaptation to environmental stress.

    PubMed

    Bélanger, Pier-Anne; Beaudin, Julie; Roy, Sébastien

    2011-05-01

    We developed a microwell plate, high-throughput, screening method aimed at quantitating the tolerance of a panel of Gram-positive and Gram-negative bacteria to metals (Frankia sp., Escherichia coli, Cupriavidus metallidurans, Rhizobium leguminosarum, and Streptomyces scabies). Microbial viability was quantified using MTS; a tetrazolium salt converted to a water-soluble formazan through microbial reduction. In this paper, we present the stepwise development of the method, highlighting the main elements underlying its reliability, and compare results obtained with literature. We conclude the method is well suited to efficiently screen bacteria, including those that are filamentous and slow-growing, without the need for large amounts of inoculum which may not always be available. The method allows testing of compound gradients with sufficient replicates to generate statistically robust results, and is transposable to other types of cell proliferation assays such as those for antimicrobial susceptibility, and chemoresistance. PMID:21315114

  3. High Throughput In Situ XAFS Screening of Catalysts

    SciTech Connect

    Tsapatsaris, Nikolaos; Beesley, Angela M.; Weiher, Norbert; Tatton, Helen; Schroeder, Sven L. M.; Dent, Andy J.; Mosselmans, Frederick J. W.; Tromp, Moniek; Russu, Sergio; Evans, John; Harvey, Ian; Hayama, Shu

    2007-02-02

    We outline and demonstrate the feasibility of high-throughput (HT) in situ XAFS for synchrotron radiation studies. An XAS data acquisition and control system for the analysis of dynamic materials libraries under control of temperature and gaseous environments has been developed. The system is compatible with the 96-well industry standard and coupled to multi-stream quadrupole mass spectrometry (QMS) analysis of reactor effluents. An automated analytical workflow generates data quickly compared to traditional individual spectrum acquisition and analyses them in quasi-real time using an HT data analysis tool based on IFFEFIT. The system was used for the automated characterization of a library of 91 catalyst precursors containing ternary combinations of Cu, Pt, and Au on {gamma}-Al2O3, and for the in situ characterization of Au catalysts supported on Al2O3 and TiO2.

  4. Design and implementation of high throughput screening assays.

    PubMed

    Macarrón, Ricardo; Hertzberg, Robert P

    2011-03-01

    High throughput screening (HTS) is at the core of the drug discovery process, and so it is critical to design and implement HTS assays in a comprehensive fashion involving scientists from the disciplines of biology, chemistry, engineering, and informatics. This requires careful analysis of many variables, starting with the choice of assay target and ending with the discovery of lead compounds. At every step in this process, there are decisions to be made that can greatly impact the outcome of the HTS effort, to the point of making it a success or a failure. Although specific guidelines should be established to insure that the screening assay reaches an acceptable level of quality, many choices require pragmatism and the ability to compromise opposing forces. PMID:20865348

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

    SciTech Connect

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

    1999-07-20

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

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

    PubMed Central

    Wu, Henry; Mayeshiba, Tam; Morgan, Dane

    2016-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2016-01-01

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

  9. 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.

  10. 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. PMID:24761741

  11. 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).

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

    PubMed Central

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

    2015-01-01

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

  13. High-throughput sequencing of immune repertoires in multiple sclerosis.

    PubMed

    Lossius, Andreas; Johansen, Jorunn N; Vartdal, Frode; Holmøy, Trygve

    2016-04-01

    T cells and B cells are crucial in the initiation and maintenance of multiple sclerosis (MS), and the activation of these cells is believed to be mediated through specific recognition of antigens by the T- and B-cell receptors. The antigen receptors are highly polymorphic due to recombination (T- and B-cell receptors) and mutation (B-cell receptors) of the encoding genes, which can therefore be used as fingerprints to track individual T- and B-cell clones. Such studies can shed light on mechanisms driving the immune responses and provide new insights into the pathogenesis. Here, we summarize studies that have explored the T- and B-cell receptor repertoires using earlier methodological approaches, and we focus on how high-throughput sequencing has provided new knowledge by surveying the immune repertoires in MS in even greater detail and with unprecedented depth. PMID:27081660

  14. Proposed high throughput electrorefining treatment for spent N- Reactor fuel

    SciTech Connect

    Gay, E.C.; Miller, W.E.; Laidler, J.J.

    1996-05-01

    A high-throughput electrorefining process is being adapted to treat spent N-Reactor fuel for ultimate disposal in a geologic repository. Anodic dissolution tests were made with unirradiated N-Reactor fuel to determine the type of fragmentation necessary to provide fuel segments suitable for this process. Based on these tests, a conceptual design was produced of a plant-scale electrorefiner. In this design, the diameter of an electrode assembly is about 1.07 m (42 in.). Three of these assemblies in an electrorefiner would accommodate a 3-metric-ton batch of N-Reactor fuel that would be processed at a rate of 42 kg of uranium per hour.

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

    PubMed

    Ramani, Vijay; Qiu, Ruolan; Shendure, Jay

    2015-09-01

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

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

    NASA Astrophysics Data System (ADS)

    MacBeath, Gavin; Schreiber, Stuart L.

    2000-09-01

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

  17. High Throughput Substrate Phage Display for Protease Profiling

    PubMed Central

    Ratnikov, Boris; Cieplak, Piotr; Smith, Jeffrey W.

    2012-01-01

    Summary The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imaging agents, and to initiate the design of active site inhibitors. Our group has characterized protease specificities of several matrix metalloproteinases using substrate phage display. Recently, we have adapted this method to a semiautomated platform that includes several high-throughput steps. The semiautomated platform allows one to obtain an order of magnitude more data, thus permitting precise comparisons among related proteases to define their functional distinctions. PMID:19377968

  18. High throughput substrate phage display for protease profiling.

    PubMed

    Ratnikov, Boris; Cieplak, Piotr; Smith, Jeffrey W

    2009-01-01

    The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imaging agents, and to initiate the design of active site inhibitors. Our group has characterized protease specificities of several matrix metalloproteinases using substrate phage display. Recently, we have adapted this method to a semiautomated platform that includes several high-throughput steps. The semiautomated platform allows one to obtain an order of magnitude more data, thus permitting precise comparisons among related proteases to define their functional distinctions. PMID:19377968

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-08-01

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

  2. Representation and classification for high-throughput data

    NASA Astrophysics Data System (ADS)

    Wessels, Lodewyk F. A.; Reinders, Marcel J. T.; van Welsem, Tibor; Nederlof, Petra M.

    2002-06-01

    Survival prediction and optimal treatment choice for cancer patients are dependent on correct disease classification. This classification can be improved significantly when high- throughput data such as microarray expression analysis is employed. These data sets usually suffer from the dimensionality problem: many features and few patients. Consequently, care must be taken when feature selection is performed and classifiers for disease classification are designed. In this paper we investigate several issues associated with this problem, including 1) data representation; 2) the type of classifier employed and 3) classifier construction, with specific emphasis on feature selection approaches. More specifically, 'filter' and 'wrapper' approaches for feature selection are studied. The different representations, selection criteria, classifiers and feature selection approaches are evaluated with regard to the effect on true classification performance. As test cases we employ a Comparative Genomic Hybridization breast cancer data sets and two publicly available gene expression data sets.

  3. Integrated control system environment for high-throughput tomography

    NASA Astrophysics Data System (ADS)

    Khokhriakov, Igor; Lottermoser, Lars; Gehrke, Rainer; Kracht, Thorsten; Wintersberger, Eugen; Kopmann, Andreas; Vogelgesang, Matthias; Beckmann, Felix

    2014-09-01

    A new control system for high-throughput experiments (X-Ray, Neutrons) is introduced in this article. The system consists of several software components which are required to make optimized use of the beamtime and to fulfill the demand to implement the new standardized data format established within the Helmholtz Association in Germany. The main components are: PreExperiment Data Collector; Status server; Data Format Server. Especially for tomography a concept for an online reconstruction based on GPU computing is presented. One of the main goals of the system is to collect data that extends standard experimental data, e.g. instrument's hardware state, preinvestigation data, experiment description data etc. The collected data is stored together with the experiment data in the permanent storage of the user. The stored data is then used for post processing and analysis of the experiment.

  4. High-throughput rheology in a microfluidic device

    NASA Astrophysics Data System (ADS)

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

    2011-11-01

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

  5. High-throughput screening of binary catalysts for oxygen electroreduction

    NASA Astrophysics Data System (ADS)

    Liu, Jing Hua; Jeon, Min Ku; Woo, Seong Ihl

    2006-01-01

    A series of Pt based and non-Pt catalysts for proton exchange membrane fuel cell (PEMFC) and direct methanol fuel cell (DMFC) have been evaluated towards oxygen reduction, by high-throughput optical screening. Fluorescein was first used as pH indicator for detecting pH change of the electrolyte in the vicinity of cathode caused by oxygen reduction. Arrays of catalyst spot comprised of binary catalysts and pure Pt were prepared by using robotic micro-dispenser. The analysis of fluorescence images has showed that some of Pt based catalysts including PtBi, PtCu, PtSe, PtTe and PtIr, as well as RuFe, as a non-Pt catalyst, exhibited higher activities and methanol tolerance than pure Pt. Moreover, acceptable stability of these catalysts at high potential in acid environment suits them to the requirements of cathode catalyst in PEMFC or DMFC.

  6. Biological Processes Discovered by High-Throughput Sequencing.

    PubMed

    Reon, Brian J; Dutta, Anindya

    2016-04-01

    Advances in DNA and RNA sequencing technologies have completely transformed the field of genomics. High-throughput sequencing (HTS) is now a widely used and accessible technology that allows scientists to sequence an entire transcriptome or genome in a timely and cost-effective manner. Application of HTS techniques has led to many key discoveries, including the identification of long noncoding RNAs, microDNAs, a family of small extrachromosomal circular DNA species, and tRNA-derived fragments, which are a group of small non-miRNAs that are derived from tRNAs. Furthermore, public sequencing repositories provide unique opportunities for laboratories to parse large sequencing databases to identify proteins and noncoding RNAs at a scale that was not possible a decade ago. Herein, we review how HTS has led to the discovery of novel nucleic acid species and uncovered new biological processes during the course. PMID:26828742

  7. 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

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

    PubMed

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

    2016-03-01

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

  9. High-Throughput Sequencing of Complete Mitochondrial Genomes.

    PubMed

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

    2016-01-01

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

  10. 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.

  11. High Throughput In Situ XAFS Screening of Catalysts

    NASA Astrophysics Data System (ADS)

    Tsapatsaris, Nikolaos; Beesley, Angela M.; Weiher, Norbert; Tatton, Helen; Dent, Andy J.; Mosselmans, Frederick J. W.; Tromp, Moniek; Russu, Sergio; Evans, John; Harvey, Ian; Hayama, Shu; Schroeder, Sven L. M.

    2007-02-01

    We outline and demonstrate the feasibility of high-throughput (HT) in situ XAFS for synchrotron radiation studies. An XAS data acquisition and control system for the analysis of dynamic materials libraries under control of temperature and gaseous environments has been developed. The system is compatible with the 96-well industry standard and coupled to multi-stream quadrupole mass spectrometry (QMS) analysis of reactor effluents. An automated analytical workflow generates data quickly compared to traditional individual spectrum acquisition and analyses them in quasi-real time using an HT data analysis tool based on IFFEFIT. The system was used for the automated characterization of a library of 91 catalyst precursors containing ternary combinations of Cu, Pt, and Au on γ-Al2O3, and for the in situ characterization of Au catalysts supported on Al2O3 and TiO2.

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

    PubMed

    Wu, Henry; Mayeshiba, Tam; Morgan, Dane

    2016-01-01

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

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

    PubMed Central

    Ramani, Vijay; Qiu, Ruolan; Shendure, Jay

    2015-01-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

    2010-01-01

    Background Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening. Results Here we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction. Conclusions This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay. See Commentary article: http://www.biomedcentral.com/1741-7007/8/148. PMID:21176202

  16. 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. PMID:27283099

  17. Quantitative high throughput analytics to support polysaccharide production process development.

    PubMed

    Noyes, Aaron; Godavarti, Ranga; Titchener-Hooker, Nigel; Coffman, Jonathan; Mukhopadhyay, Tarit

    2014-05-19

    The rapid development of purification processes for polysaccharide vaccines is constrained by a lack of analytical tools current technologies for the measurement of polysaccharide recovery and process-related impurity clearance are complex, time-consuming, and generally not amenable to high throughput process development (HTPD). HTPD is envisioned to be central to the improvement of existing polysaccharide manufacturing processes through the identification of critical process parameters that potentially impact the quality attributes of the vaccine and to the development of de novo processes for clinical candidates, across the spectrum of downstream processing. The availability of a fast and automated analytics platform will expand the scope, robustness, and evolution of Design of Experiment (DOE) studies. This paper details recent advances in improving the speed, throughput, and success of in-process analytics at the micro-scale. Two methods, based on modifications of existing procedures, are described for the rapid measurement of polysaccharide titre in microplates without the need for heating steps. A simplification of a commercial endotoxin assay is also described that features a single measurement at room temperature. These assays, along with existing assays for protein and nucleic acids are qualified for deployment in the high throughput screening of polysaccharide feedstreams. Assay accuracy, precision, robustness, interference, and ease of use are assessed and described. In combination, these assays are capable of measuring the product concentration and impurity profile of a microplate of 96 samples in less than one day. This body of work relies on the evaluation of a combination of commercially available and clinically relevant polysaccharides to ensure maximum versatility and reactivity of the final assay suite. Together, these advancements reduce overall process time by up to 30-fold and significantly reduce sample volume over current practices. The

  18. High-Throughput Genomics Enhances Tomato Breeding Efficiency

    PubMed Central

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

    2009-01-01

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

  19. A strategy for high-throughput screening of ligands suitable for molecular imprinting of proteins.

    PubMed

    Eppler, Stefan; Schröder, Tim; Friedle, Jürgen; Michl, Simone; Dangel, Werner; Mizaikoff, Boris

    2012-05-15

    For facilitating the identification of appropriate functionalities that may serve as a binding motif of functional monomers, a selection strategy based on high-throughput screening of the binding properties of readily available sorbent materials has been developed. Thereby, the affinity of such ligands to the protein of interest may be rapidly determined. From these studies, it is anticipated that ligand functionalities will be derived, which may lead to advanced selection and design of dedicated functional monomers suitable for decorating the surface of a scavenger material. Thus, specific binding of the target protein of interest should be enabled even in complex solutions such as e.g., biotechnologically relevant cell lysates. In the present contribution, an automated screening method for studying ligand interactions of selected sorbent materials with pepsin - a protein of the protease family - was developed. Aqueous buffer solutions containing pepsin at known constant concentration were pipetted through an array of miniaturized chromatographic solid phase extraction (SPE) columns containing a variety of sorbent materials, and the eluted solutions were analyzed by UV/vis spectroscopy. The established screening protocol was validated against resin materials of known interaction with pepsin. Finally, the developed screening strategy was adapted for a robot system enabling high-throughput screening for a wide variety of sorbent materials and ligand functionalities in a fully automated approach. The obtained results clearly indicate that the established screening routine provides valuable data for characterizing resin-immobilized ligands, and their affinity toward pepsin. PMID:22472529

  20. High-throughput sequencing of small RNAs and anatomical characteristics associated with leaf development in celery.

    PubMed

    Jia, Xiao-Ling; Li, Meng-Yao; Jiang, Qian; Xu, Zhi-Sheng; Wang, Feng; Xiong, Ai-Sheng

    2015-01-01

    MicroRNAs (miRNAs) exhibit diverse and important roles in plant growth, development, and stress responses and regulate gene expression at the post-transcriptional level. Knowledge about the diversity of miRNAs and their roles in leaf development in celery remains unknown. To elucidate the roles of miRNAs in celery leaf development, we identified leaf development-related miRNAs through high-throughput sequencing. Small RNA libraries were constructed using leaves from three stages (10, 20, and 30 cm) of celery cv.'Ventura' and then subjected to high-throughput sequencing and bioinformatics analysis. At Stage 1, Stage 2, and Stage 3 of 'Ventura', a total of 333, 329, and 344 conserved miRNAs (belonging to 35, 35, and 32 families, respectively) were identified. A total of 131 miRNAs were identified as novel in 'Ventura'. Potential miRNA target genes were predicted and annotated using the eggNOG, GO, and KEGG databases to explore gene functions. The abundance of five conserved miRNAs and their corresponding potential target genes were validated. Expression profiles of novel potential miRNAs were also detected. Anatomical characteristics of the leaf blades and petioles at three leaf stages were further analyzed. This study contributes to our understanding on the functions and molecular regulatory mechanisms of miRNAs in celery leaf development. PMID:26057455

  1. High-throughput SNP scoring with GAMMArrays: genomic analysis using multiplexed microsphere arrays

    NASA Astrophysics Data System (ADS)

    Green, Lance D.; Cai, Hong; Torney, David C.; Wood, Diane J.; Uribe-Romeo, Francisco J.; Kaderali, Lars; Nolan, John P.; White, P. S.

    2002-06-01

    We have developed a SNP scoring platform, yielding high throughput, inexpensive assays. The basic platform uses fluorescently labeled DNA fragments bound to microspheres, which are analyzed using flow cytometry. SNP scoring is performed using minisequencing primers and fluorescently labeled dideoxynucleotides. Furthermore, multiplexed microspheres make it possible to score hundreds of SNPs simultaneously. Multiplexing, coupled with high throughput rates allow inexpensive scoring of several million SNPs/day. GAMMArrays use universal tags that consist of computer designed, unique DNA tails. These are incorporated into each primer, and the reverse-component is attached to a discrete population of microspheres in a multiplexed set. This enables simultaneous minisequencing of many SNPs in solution, followed by capture onto the appropriate microsphere for multiplexed analysis by flow cytometry. We present results from multiplexed SNP analyses of bacterial pathogens, and human mtDNA variation. Analytes are performed on PCR amplicons, each containing numerous SNPs scored simultaneously. In addition, these assays easily integrate into conventional liquid handling automation, and require no unique instrumentation for setup and analysis. Very high signal-to-noise ratios, ease of setup, flexibility in format and scale, and low cost make these assays extremely versatile and valuable tools for a wide variety of SNP scoring applications.

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

    PubMed Central

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

    2015-01-01

    The booming nanotech industry has raised public concerns about the environmental health and safety impact of engineered nanomaterials (ENMs). High-throughput assays are needed to obtain toxicity data for the rapidly increasing number of ENMs. Here we present a suite of high-throughput methods to study nanotoxicity in intact animals using Caenorhabditis elegans as a model. At the population level, our system measures food consumption of thousands of animals to evaluate population fitness. At the organism level, our automated system analyzes hundreds of individual animals for body length, locomotion speed, and lifespan. To demonstrate the utility of our system, we applied this technology to test the toxicity of 20 nanomaterials under four concentrations. Only fullerene nanoparticles (nC60), fullerol, TiO2, and CeO2 showed little or no toxicity. Various degrees of toxicity were detected from different forms of carbon nanotubes, graphene, carbon black, Ag, and fumed SiO2 nanoparticles. Aminofullerene and UV irradiated nC60 also showed small but significant toxicity. We further investigated the effects of nanomaterial size, shape, surface chemistry, and exposure conditions on toxicity. Our data are publicly available at the open-access nanotoxicity database www.QuantWorm.org/nano. PMID:25611253

  3. A high-throughput RNA-seq approach to profile transcriptional responses

    PubMed Central

    Moyerbrailean, G. A.; Davis, G. O.; Harvey, C. T.; Watza, D.; Wen, X.; Pique-Regi, R.; Luca, F.

    2015-01-01

    In recent years RNA-seq protocols have been developed to investigate a variety of biological problems by measuring the abundance of different RNAs. Many study designs involve performing expensive preliminary studies to screen or optimize experimental conditions. Testing a large number of conditions in parallel may be more cost effective. For example, analyzing tissue/environment-specific gene expression generally implies screening a large number of cellular conditions and samples, without prior knowledge of which conditions are most informative (e.g., some cell types may not respond to certain treatments). To circumvent these challenges, we have established a new two-step high-throughput RNA-seq approach: the first step consists of gene expression screening of a large number of conditions, while the second step focuses on deep sequencing of the most relevant conditions (e.g., largest number of differentially expressed genes). This study design allows for a fast and economical screen in step one, with a more efficient allocation of resources for the deep sequencing of the most biologically relevant libraries in step two. We have applied this approach to study the response to 23 treatments in three lymphoblastoid cell lines demonstrating that it should also be useful for other high-throughput transcriptome profiling applications requiring iterative refinement or screening. PMID:26510397

  4. High throughput ab-intio modeling of proton transport in solid electrolytes

    NASA Astrophysics Data System (ADS)

    Balachandran, Janakiraman; Lin, Lianshan; Ganesh, Panchapakesan

    Solid oxide materials that can selectively transport protons have great potential for fuel cell applications. However several fundamental questions remain unanswered such as (a) How do the dopants organize at various dopant concentrations, (b) How spatial organization of dopants influence proton migration energy, (c) How disorder and strain in a material influence its ionic transport. In this work have developed an integrated high throughput framework to calculate proton transport properties by integrating open source packages (such as pymatgen, fireworks) The high throughput framework scales well on supercomputing clusters. We have used this framework to analyze over 100 perovskites compounds with over 12 different dopant atoms. These computational models enable us to obtain insights how the proton transport properties depend on host and dopant atoms. Further, we also perform ab-initio modeling to understand how dopants spatially organize at different dopant concentrations, and how this spatial organization affects proton conductivity. This analysis enabled us to obtain fundamental insights on why proton conductivity decreases in Y doped BaZrO3 at high dopant concentrations.

  5. A High Throughput Assay for Screening Host Restriction Factors and Antivirals Targeting Influenza A Virus

    PubMed Central

    Wang, Lingyan; Li, Wenjun; Li, Shitao

    2016-01-01

    Influenza A virus (IAV) is a human respiratory pathogen that causes seasonal epidemics and occasional global pandemics with devastating levels of morbidity and mortality. Currently approved treatments against influenza are losing effectiveness, as new viral strains are often refractory to conventional treatments. Thus, there is an urgent need to find new therapeutic targets with which to develop novel antiviral drugs. The common strategy to discover new drug targets and antivirals is high throughput screening. However, most current screenings for IAV rely on the engineered virus carrying a reporter, which prevents the application to newly emerging wild type flu viruses, such as 2009 pandemic H1N1 flu. Here we developed a simple and sensitive screening assay for wild type IAV by quantitatively analyzing viral protein levels using a Dot Blot Assay in combination with the LI-COR Imaging System (DBALIS). We first validated DBALIS in overexpression and RNAi assays, which are suitable methods for screening host factors regulating viral infection. More importantly, we also validated and initiated drug screening using DBALIS. A pilot compound screening identified a small molecule that inhibited IAV infection. Taken together, our method represents a reliable and convenient high throughput assay for screening novel host factors and antiviral compounds. PMID:27375580

  6. High-throughput diagnosis of potato cyst nematodes in soil samples.

    PubMed

    Reid, Alex; Evans, Fiona; Mulholland, Vincent; Cole, Yvonne; Pickup, Jon

    2015-01-01

    Potato cyst nematode (PCN) is a damaging soilborne pest of potatoes which can cause major crop losses. In 2010, a new European Union directive (2007/33/EC) on the control of PCN came into force. Under the new directive, seed potatoes can only be planted on land which has been found to be free from PCN infestation following an official soil test. A major consequence of the new directive was the introduction of a new harmonized soil sampling rate resulting in a threefold increase in the number of samples requiring testing. To manage this increase with the same staffing resources, we have replaced the traditional diagnostic methods. A system has been developed for the processing of soil samples, extraction of DNA from float material, and detection of PCN by high-throughput real-time PCR. Approximately 17,000 samples are analyzed each year using this method. This chapter describes the high-throughput processes for the production of float material from soil samples, DNA extraction from the entire float, and subsequent detection and identification of PCN within these samples. PMID:25981252

  7. Ribosomal Database Project: data and tools for high throughput rRNA analysis

    PubMed Central

    Cole, James R.; Wang, Qiong; Fish, Jordan A.; Chai, Benli; McGarrell, Donna M.; Sun, Yanni; Brown, C. Titus; Porras-Alfaro, Andrea; Kuske, Cheryl R.; Tiedje, James M.

    2014-01-01

    Ribosomal Database Project (RDP; http://rdp.cme.msu.edu/) provides the research community with aligned and annotated rRNA gene sequence data, along with tools to allow researchers to analyze their own rRNA gene sequences in the RDP framework. RDP data and tools are utilized in fields as diverse as human health, microbial ecology, environmental microbiology, nucleic acid chemistry, taxonomy and phylogenetics. In addition to aligned and annotated collections of bacterial and archaeal small subunit rRNA genes, RDP now includes a collection of fungal large subunit rRNA genes. RDP tools, including Classifier and Aligner, have been updated to work with this new fungal collection. The use of high-throughput sequencing to characterize environmental microbial populations has exploded in the past several years, and as sequence technologies have improved, the sizes of environmental datasets have increased. With release 11, RDP is providing an expanded set of tools to facilitate analysis of high-throughput data, including both single-stranded and paired-end reads. In addition, most tools are now available as open source packages for download and local use by researchers with high-volume needs or who would like to develop custom analysis pipelines. PMID:24288368

  8. 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.

  9. High-throughput microsomal stability assay for screening new chemical entities in drug discovery.

    PubMed

    Fonsi, Massimiliano; Orsale, Maria V; Monteagudo, Edith

    2008-10-01

    In this work, the authors present a novel, robotic, automated protocol for assessing a metabolic stability protocol assembled on a Hamilton platform and a new strategy for pooling samples (cassette analysis). To increase the high throughput of the liquid chromatography (LC) step, fast chromatography and automated liquid chromatography tandem mass spectrometry (LC/MS/MS) analytical methods were also developed, and a rapid data analysis system was generated that converts peak areas obtained by LC/MS/MS in intrinsic clearance values. All of the steps of the microsomal stability assay were carefully studied and optimized. Standard errors and confidence intervals of the measured clearances were also automatically generated in the process to allow an immediate evaluation of the significance of observed values. Methods based on pooling analysis of 2 and 4 different analytes were compared with a standard method without pooling. A simple statistical treatment was used to show their equivalence. The different protocols developed were analyzed in terms of the best compromise between accuracy and high-throughput capabilities. PMID:18812573

  10. 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.

  11. A High Throughput Assay for Screening Host Restriction Factors and Antivirals Targeting Influenza A Virus.

    PubMed

    Wang, Lingyan; Li, Wenjun; Li, Shitao

    2016-01-01

    Influenza A virus (IAV) is a human respiratory pathogen that causes seasonal epidemics and occasional global pandemics with devastating levels of morbidity and mortality. Currently approved treatments against influenza are losing effectiveness, as new viral strains are often refractory to conventional treatments. Thus, there is an urgent need to find new therapeutic targets with which to develop novel antiviral drugs. The common strategy to discover new drug targets and antivirals is high throughput screening. However, most current screenings for IAV rely on the engineered virus carrying a reporter, which prevents the application to newly emerging wild type flu viruses, such as 2009 pandemic H1N1 flu. Here we developed a simple and sensitive screening assay for wild type IAV by quantitatively analyzing viral protein levels using a Dot Blot Assay in combination with the LI-COR Imaging System (DBALIS). We first validated DBALIS in overexpression and RNAi assays, which are suitable methods for screening host factors regulating viral infection. More importantly, we also validated and initiated drug screening using DBALIS. A pilot compound screening identified a small molecule that inhibited IAV infection. Taken together, our method represents a reliable and convenient high throughput assay for screening novel host factors and antiviral compounds. PMID:27375580

  12. High-throughput assay and engineering of self-cleaving ribozymes by sequencing

    PubMed Central

    Kobori, Shungo; Nomura, Yoko; Miu, Anh; Yokobayashi, Yohei

    2015-01-01

    Self-cleaving ribozymes are found in all domains of life and are believed to play important roles in biology. Additionally, self-cleaving ribozymes have been the subject of extensive engineering efforts for applications in synthetic biology. These studies often involve laborious assays of multiple individual variants that are either designed rationally or discovered through selection or screening. However, these assays provide only a limited view of the large sequence space relevant to the ribozyme function. Here, we report a strategy that allows quantitative characterization of greater than 1000 ribozyme variants in a single experiment. We generated a library of predefined ribozyme variants that were converted to DNA and analyzed by high-throughput sequencing. By counting the number of cleaved and uncleaved reads of every variant in the library, we obtained a complete activity profile of the ribozyme pool which was used to both analyze and engineer allosteric ribozymes. PMID:25829176

  13. On-chip polarimetry for high-throughput screening of nanoliter and smaller sample volumes

    NASA Technical Reports Server (NTRS)

    Bornhop, Darryl J. (Inventor); Dotson, Stephen (Inventor); Bachmann, Brian O. (Inventor)

    2012-01-01

    A polarimetry technique for measuring optical activity that is particularly suited for high throughput screening employs a chip or substrate (22) having one or more microfluidic channels (26) formed therein. A polarized laser beam (14) is directed onto optically active samples that are disposed in the channels. The incident laser beam interacts with the optically active molecules in the sample, which slightly alter the polarization of the laser beam as it passes multiple times through the sample. Interference fringe patterns (28) are generated by the interaction of the laser beam with the sample and the channel walls. A photodetector (34) is positioned to receive the interference fringe patterns and generate an output signal that is input to a computer or other analyzer (38) for analyzing the signal and determining the rotation of plane polarized light by optically active material in the channel from polarization rotation calculations.

  14. Parallel tools in HEVC for high-throughput processing

    NASA Astrophysics Data System (ADS)

    Zhou, Minhua; Sze, Vivienne; Budagavi, Madhukar

    2012-10-01

    HEVC (High Efficiency Video Coding) is the next-generation video coding standard being jointly developed by the ITU-T VCEG and ISO/IEC MPEG JCT-VC team. In addition to the high coding efficiency, which is expected to provide 50% more bit-rate reduction when compared to H.264/AVC, HEVC has built-in parallel processing tools to address bitrate, pixel-rate and motion estimation (ME) throughput requirements. This paper describes how CABAC, which is also used in H.264/AVC, has been redesigned for improved throughput, and how parallel merge/skip and tiles, which are new tools introduced for HEVC, enable high-throughput processing. CABAC has data dependencies which make it difficult to parallelize and thus limit its throughput. The prediction error/residual, represented as quantized transform coefficients, accounts for the majority of the CABAC workload. Various improvements have been made to the context selection and scans in transform coefficient coding that enable CABAC in HEVC to potentially achieve higher throughput and increased coding gains relative to H.264/AVC. The merge/skip mode is a coding efficiency enhancement tool in HEVC; the parallel merge/skip breaks dependency between the regular and merge/skip ME, which provides flexibility for high throughput and high efficiency HEVC encoder designs. For ultra high definition (UHD) video, such as 4kx2k and 8kx4k resolutions, low-latency and real-time processing may be beyond the capability of a single core codec. Tiles are an effective tool which enables pixel-rate balancing among the cores to achieve parallel processing with a throughput scalable implementation of multi-core UHD video codec. With the evenly divided tiles, a multi-core video codec can be realized by simply replicating single core codec and adding a tile boundary processing core on top of that. These tools illustrate that accounting for implementation cost when designing video coding algorithms can enable higher processing speed and reduce

  15. Silicon microphysiometer for high-throughput drug screening

    NASA Astrophysics Data System (ADS)

    Verhaegen, Katarina; Baert, Christiaan; Puers, Bob; Sansen, Willy; Simaels, Jeannine; Van Driessche, Veerle; Hermans, Lou; Mertens, Robert P.

    1999-06-01

    We report on a micromachined silicon chip that is capable of providing a high-throughput functional assay based on calorimetry. A prototype twin microcalorimeter based on the Seebeck effect has been fabricated by IC technology and micromachined postprocessing techniques. A biocompatible liquid rubber membrane supports two identical 0.5 X 2 cm2 measurement chambers, situated at the cold and hot junction of a 666-junction aluminum/p+-polysilicon thermopile. The chambers can house up to 106 eukaryotic cells cultured to confluence. The advantage of the device over microcalorimeters on the market, is the integration of the measurement channels on chip, rendering microvolume reaction vessels, ranging from 10 to 600 (mu) l, in the closest possible contact with the thermopile sensor (no springs are needed). Power and temperature sensitivity of the sensor are 23 V/W and 130 mV/K, respectively. The small thermal inertia of the microchannels results in the short response time of 70 s, when filled with 50 (mu) l of water. Biological experiments were done with cultured kidney cells of Xenopus laevis (A6). The thermal equilibration time of the device is 45 min. Stimulation of transport mechanisms by reducing bath osmolality by 50% increased metabolism by 20%. Our results show that it is feasible to apply this large-area, small- volume whole-cell biosensor for drug discovery, where the binding assays that are commonly used to provide high- throughput need to be complemented with a functional assay. Solutions are brought onto the sensor by a simple pipette, making the use of an industrial microtiterplate dispenser feasible on a nx96-array of the microcalorimeter biosensor. Such an array of biosensors has been designed based on a new set of requirements as set forth by people in the field as this project moved on. The results obtained from the prototype large-area sensor were used to obtain an accurate model of the calorimeter, checked for by the simulation software ANSYS. At

  16. 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

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

    PubMed Central

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

    2011-01-01

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

  18. Protocol: High-throughput and quantitative assays of auxin and auxin precursors from minute tissue samples

    PubMed Central

    2012-01-01

    Background The plant hormone auxin, indole-3-acetic acid (IAA), plays important roles in plant growth and development. The signaling response to IAA is largely dependent on the local concentration of IAA, and this concentration is regulated by multiple mechanisms in plants. Therefore, the precise quantification of local IAA concentration provides insights into the regulation of IAA and its biological roles. Meanwhile, pathways and genes involved in IAA biosynthesis are not fully understood, so it is necessary to analyze the production of IAA at the metabolite level for unbiased studies of IAA biosynthesis. Results We have developed high-throughput methods to quantify plant endogenous IAA and its biosynthetic precursors including indole, tryptophan, indole-3-pyruvic acid (IPyA), and indole-3-butyric acid (IBA). The protocol starts with homogenizing plant tissues with stable-labeled internal standards added, followed by analyte purification using solid phase extraction (SPE) tips and analyte derivatization. The derivatized analytes are finally analyzed by selected reaction monitoring on a gas chromatograph-mass spectrometer (GC-MS/MS) to determine the precise abundance of analytes. The amount of plant tissue required for the assay is small (typically 2–10 mg fresh weight), and the use of SPE tips is simple and convenient, which allows preparation of large sets of samples within reasonable time periods. Conclusions The SPE tips and GC-MS/MS based method enables high-throughput and accurate quantification of IAA and its biosynthetic precursors from minute plant tissue samples. The protocol can be used for measurement of these endogenous compounds using isotope dilution, and it can also be applied to analyze IAA biosynthesis and biosynthetic pathways using stable isotope labeling. The method will potentially advance knowledge of the role and regulation of IAA. PMID:22883136

  19. 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

  20. Review of high-throughput techniques for detecting solid phase Transformation from material libraries produced by combinatorial methods

    NASA Technical Reports Server (NTRS)

    Lee, Jonathan A.

    2005-01-01

    High-throughput measurement techniques are reviewed for solid phase transformation from materials produced by combinatorial methods, which are highly efficient concepts to fabricate large variety of material libraries with different compositional gradients on a single wafer. Combinatorial methods hold high potential for reducing the time and costs associated with the development of new materials, as compared to time-consuming and labor-intensive conventional methods that test large batches of material, one- composition at a time. These high-throughput techniques can be automated to rapidly capture and analyze data, using the entire material library on a single wafer, thereby accelerating the pace of materials discovery and knowledge generation for solid phase transformations. The review covers experimental techniques that are applicable to inorganic materials such as shape memory alloys, graded materials, metal hydrides, ferric materials, semiconductors and industrial alloys.

  1. Use of high-throughput targeted exome sequencing in genetic diagnosis of Chinese family with congenital cataract

    PubMed Central

    Ma, Ming-Fu; Li, Lian-Bing; Pei, Yun-Qi; Cheng, Zhi

    2016-01-01

    AIM To identify disease-causing mutation in a congenital cataract family using enrichment of targeted genes combined with next-generation sequencing. METHODS A total of 371 known genes related to inherited eye diseases of the proband was selected and captured, followed by high-throughput sequencing. The sequencing data were analyzed by established bioinformatics pipeline. Validation was performed by Sanger sequencing. RESULTS A recurrent heterozygous non-synonymous mutation c.130G>A (p.V44M) in the GJA3 gene was identified in the proband. The result was confirmed by Sanger sequencing. The mutation showed co-segregation with the disease phenotype in the family but was not detected in unaffected controls. CONCLUSION Targeted exome sequencing is a rapid, high-throughput and cost-efficient method for screening known genes and could be applied to the routine gene diagnosis of congenital cataract. PMID:27275416

  2. 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. PMID:27446699

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-01-01

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

  5. A high throughput screening for rarely transcribed differentially expressed genes.

    PubMed Central

    von Stein, O D; Thies, W G; Hofmann, M

    1997-01-01

    A novel method combining elements of suppression subtractive hybridization with high throughput differential screening permits the efficient and rapid cloning of rarely transcribed differentially expressed genes. The experimental strategy virtually excludes the possibility of isolating false positive clones. The potential of the method is demonstrated by the isolation of 625 differentially expressed cDNAs from the metastatic adenocarcinoma cell line Bsp73-ASML when subtracted from its non-metastatic counterpart Bsp73-1AS. Northern analysis of 72 randomly selected clones demonstrated that 68 were differentially expressed with respect to Bsp73-ASML, indicating a true positive rate of 94%. Additionally, a large proportion of these clones represented rare transcripts as determined by the exposure time required to detect a signal. Sequence data indicated that of the 625 clones obtained, 92 clones scored perfect or near perfect matches with already known genes. Two hundred and eighty one clones scored between 60 and 95% homology to known human and mouse genes, whereas 252 clones scored no match with any sequences in the public databases. The method we describe is ideally suited whenever subtle changes in gene expression profiles need to be determined. PMID:9185570

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

    SciTech Connect

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

    2012-06-01

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

  7. High-Throughput Screening Based Identification of Paramyxovirus Inhibitors

    PubMed Central

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

    2008-01-01

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

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

    PubMed

    Poddar, Rajesh; Kawai, Risa; Ölveczky, Bence P

    2013-01-01

    Addressing the neural mechanisms underlying complex learned behaviors requires training animals in well-controlled tasks, an often time-consuming and labor-intensive process that can severely limit the feasibility of such studies. To overcome this constraint, we developed a fully computer-controlled general purpose system for high-throughput training of rodents. By standardizing and automating the implementation of predefined training protocols within the animal's home-cage our system dramatically reduces the efforts involved in animal training while also removing human errors and biases from the process. We deployed this system to train rats in a variety of sensorimotor tasks, achieving learning rates comparable to existing, but more laborious, methods. By incrementally and systematically increasing the difficulty of the task over weeks of training, rats were able to master motor tasks that, in complexity and structure, resemble ones used in primate studies of motor sequence learning. By enabling fully automated training of rodents in a home-cage setting this low-cost and modular system increases the utility of rodents for studying the neural underpinnings of a variety of complex behaviors. PMID:24349451

  9. 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

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

    Kurt Barth

    2008-02-06

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

  12. High Throughput Profiling of Molecular Shapes in Crystals

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

  13. High-throughput optical screening of cellular mechanotransduction

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

  14. High-throughput screening of phosphodiesterase activity in living cells.

    PubMed

    Rich, Thomas C; Karpen, Jeffrey W

    2005-01-01

    Phosphodiesterases (PDEs) hydrolyze the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine 5'-monophosphate (cGMP) and play a crucial role in the termination and spatial segregation of cyclic nucleotide signals. Despite a wealth of molecular information, very little is known about how PDEs regulate cAMP and cGMP signals in living cells because conventional methods lack the necessary spatial and temporal resolution. We present here a sensitive optical method for monitoring cAMP levels and PDE activity near the membrane, using cyclic nucleotide-gated (CNG) ion channels as sensors. These channels are directly opened by the binding of cyclic nucleotides and allow cations to cross the membrane. The olfactory channel A subunit (CNGA2) has been genetically modified to improve its cAMP sensitivity and specificity. Channel activity is assessed by measuring Ca2+ influx using standard fluorometric techniques. In addition to studying PDEs in their native setting, the approach should be particularly useful in high-throughput screening assays to test for compounds that affect PDE activity, as well as the activities of the many G protein-coupled receptors that cause changes in intracellular cAMP. PMID:15988054

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

    NASA Astrophysics Data System (ADS)

    Liu, Mochi; Shaevitz, Joshua; Leifer, Andrew

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

  16. Salmonella Serotype Determination Utilizing High-Throughput Genome Sequencing Data

    PubMed Central

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

    2015-01-01

    Serotyping forms the basis of national and international surveillance networks for Salmonella, one of the most prevalent foodborne pathogens worldwide (1–3). Public health microbiology is currently being transformed by whole-genome sequencing (WGS), which opens the door to serotype determination using WGS data. SeqSero (www.denglab.info/SeqSero) is a novel Web-based tool for determining Salmonella serotypes using high-throughput genome sequencing data. SeqSero is based on curated databases of Salmonella serotype determinants (rfb gene cluster, fliC and fljB alleles) and is predicted to determine serotype rapidly and accurately for nearly the full spectrum of Salmonella serotypes (more than 2,300 serotypes), from both raw sequencing reads and genome assemblies. The performance of SeqSero was evaluated by testing (i) raw reads from genomes of 308 Salmonella isolates of known serotype; (ii) raw reads from genomes of 3,306 Salmonella isolates sequenced and made publicly available by GenomeTrakr, a U.S. national monitoring network operated by the Food and Drug Administration; and (iii) 354 other publicly available draft or complete Salmonella genomes. We also demonstrated Salmonella serotype determination from raw sequencing reads of fecal metagenomes from mice orally infected with this pathogen. SeqSero can help to maintain the well-established utility of Salmonella serotyping when integrated into a platform of WGS-based pathogen subtyping and characterization. PMID:25762776

  17. Automated Filtration-Based High-Throughput Plasmid Preparation System

    PubMed Central

    Itoh, Masayoshi; Kitsunai, Tokuji; Akiyama, Junichi; Shibata, Kazuhiro; Izawa, Masaki; Kawai, Jun; Tomaru, Yasuhiro; Carninci, Piero; Shibata, Yuko; Ozawa, Yasuhiro; Muramatsu, Masami; Okazaki, Yasushi; Hayashizaki, Yoshihide

    1999-01-01

    Current methods of plasmid preparation do not allow for large capacity automated processing. We have developed an automated high-throughput system that prepares plasmid DNA for large-scale sequencing. This system is based on our previously reported filtration method. In this method, cell harvesting, alkaline lysis, and plasmid purification occur in a single 96-well microtiter plate from which sequence-ready DNA samples are collected. The plates are designed to allow all reagents to be injected from above the wells and the spent reagents to be aspirated from below. This design has enabled us to build a linear process plasmid preparation system consisting of an automated filter plate stacker and a 21-stage automated plasmid preparator. The 96-well plates used are outfitted with glass-filters that trap Escherichia coli before the plates are stacked in the automated stacker. The plates move from the stacker to each of the 21 stages of the preparator. At specific stages, various reagents or chemicals are injected into the wells from above. Finally, the plates are collected in the second stacker. The optimal throughput of the preparator is 40,000 samples in 17.5 hr. Here, we describe a pilot experiment preparing 15,360 templates in 160 specially designed 96-well glass-filter plates. The prepared plasmids were subjected to restriction digestion, DNA sequencing, and transcriptional sequencing. PMID:10330126

  18. High throughput determination of glucan and xylan fractions in lignocelluloses.

    PubMed

    Selig, Michael J; Tucker, Melvin P; Law, Cody; Doeppke, Crissa; Himmel, Michael E; Decker, Stephen R

    2011-05-01

    The analysis of structural glucan and xylan in lignocellulose was scaled down from original two-stage sulfuric acid hydrolysis methods (Moore WE and Johnson DB 1967 Procedures for the chemical analysis of wood and wood products. U.S. Forest Products Laboratory, U.S. Department of Agriculture., Madison, WI) and integrated into a recently-developed, high throughput pretreatment and enzymatic saccharification system. Novel 96×1.8 ml-well Hastelloy reactor plates (128×86×51 mm) based on previously described 96-well pretreatment reactor plates were paired with custom aluminum filler plates (128×86×18 mm) for use in Symyx Powdernium solids dispensing systems. The incorporation of glucose oxidase and xylose dehydrogenase linked assays to speed post-hydrolysis sugar analysis dramatically reduced the time for analysis of large lignocellulosic sample sets. The current system permits the determination of the glucan and xylan content of 96 replicates (per reactor plate) in under 6 h and parallel plate processing increases the analysis throughput substantially. PMID:21287235

  19. High-throughput concept for tailoring switchable mirrors

    NASA Astrophysics Data System (ADS)

    Borgschulte, A.; Gremaud, R.; de Man, S.; Westerwaal, R. J.; Rector, J. H.; Dam, B.; Griessen, R.

    2006-11-01

    The optical properties, the switching kinetics and the lifetime of hydrogen switchable mirrors based on Mg-Ni alloys are determined with particular regard to the composition of the optically active metal-hydride layer in combination with the thickness of the catalytic capping layer. For this, a high-throughput experiment is introduced. The switching kinetics and the reversibility of switchable mirrors are strongly thickness dependent, though the details hinge on the fine structure of the clustered capping layer. Therefore, the kinetics is correlated with the surface structures of Pd on Mg yNi 1- y as investigated by scanning tunneling microscopy. The results are explained by the so-called strong metal-support interaction (SMSI) state, characterized by a complete encapsulation of the capping layer clusters by oxidized species originating from the support. The SMSI-effect is less important with increasing Pd-layer thickness, and is suppressed by a good wetting of the Pd-clusters on the optically active film. This explains the critical thickness for the catalyzed hydrogen uptake observed in many switchable mirror systems. Moreover, the degradation of the kinetics during cycling is found to depend on the Pd-layer thickness and on the gas environment. Only films, covered with at least 15 nm Pd, show small degradation caused by the SMSI-effect. The SMSI-effect is partly reversible: after changing the gas environment from hydrogen to oxygen, the oxide on the Pd-clusters can be partly removed.

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

    PubMed Central

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

    2015-01-01

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

  1. High Throughput Profiling of Molecular Shapes in Crystals.

    PubMed

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

    2016-01-01

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

  2. High-throughput analysis of protein-DNA binding affinity.

    PubMed

    Franco-Zorrilla, José M; Solano, Roberto

    2014-01-01

    Sequence-specific protein-DNA interactions mediate most regulatory processes underlying gene expression, such as transcriptional regulation by transcription factors (TFs) or chromatin organization. Current knowledge about DNA-binding specificities of TFs is based mostly on low- to medium-throughput methodologies that are time-consuming and often fail to identify DNA motifs recognized by a TF with lower affinity but retaining biological relevance. The use of protein-binding microarrays (PBMs) offers a high-throughput alternative for the identification of protein-DNA specificities. PBM consists in an array of pseudorandomized DNA sequences that are optimized to include all the possible 10- or 11-mer DNA sequences, allowing the determination of binding specificities of most eukaryotic TFs. PBMs that can be synthesized by several manufacturing companies as single-stranded DNA are converted into double-stranded in a simple primer extension reaction. The protein of interest fused to an epitope tag is then incubated onto the PBM, and specific DNA-protein complexes are revealed in a series of immunological reactions coupled to a fluorophore. After scanning and quantifying PBMs, specific DNA motifs recognized by the protein are identified with ready-to-use scripts, generating comprehensive but accessible information about the DNA-binding specificity of the protein. This chapter describes detailed procedures for preparation of double-stranded PBMs, incubation with recombinant protein, and detection of protein-DNA complexes. Finally, we outline some cues for evaluating the biological role of DNA motifs obtained in vitro. PMID:24057393

  3. High throughput cherry-picking of solvated samples.

    PubMed

    Schmitt, Robert; Traphagen, Linda; Hajduk, Phillip

    2010-07-01

    Advances in the design of automated compound storage systems have made it possible to store large collections of research compounds in individual single-use aliquots dissolved in dimethyl sulfoxide and rapidly retrieve a specific group off them. This 'cherry-picking' approach offers researchers the opportunity to request large numbers of compounds desired for testing without having to also retrieve all the other compounds stored on the same rack or plate. This makes it possible to meet the increasing demand for samples from High Throughput Screening and Therapeutic Area teams without adding staff to dispense from powder each time, without the constraints imposed by storing in solvated compounds in fixed-well 96- or 384-way plates, and without sacrificing sample quality or shelf life by storing at room temperature. We describe how this approach has been implemented at Abbott Laboratories' central compound repository to provide smaller amounts of more compounds faster and with high quality. In doing so, we have been able to better support the innovation of our Drug Discovery colleagues. PMID:20426754

  4. Probabilistic Assessment of High-Throughput Wireless Sensor Networks

    PubMed Central

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

    2016-01-01

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

  5. High-throughput optical screening of cellular mechanotransduction.

    PubMed

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

    2014-09-01

    We introduce an optical platform for rapid, high-throughput screening of exogenous molecules that affect cellular mechanotransduction. Our method initiates mechanotransduction in adherent cells using single laser-microbeam generated micro-cavitation bubbles (μCBs) without requiring flow chambers or microfluidics. These μCBs expose adherent cells to a microTsunami, a transient microscale burst of hydrodynamic shear stress, which stimulates cells over areas approaching 1mm(2). We demonstrate microTsunami-initiated mechanosignalling in primary human endothelial cells. This observed signalling is consistent with G-protein-coupled receptor stimulation resulting in Ca(2+) release by the endoplasmic reticulum. Moreover, we demonstrate the dose-dependent modulation of microTsunami-induced Ca(2+) signalling by introducing a known inhibitor to this pathway. The imaging of Ca(2+) signalling, and its modulation by exogenous molecules, demonstrates the capacity to initiate and assess cellular mechanosignalling in real-time. We utilize this capability to screen the effects of a set of small molecules on cellular mechanotransduction in 96-well plates using standard imaging cytometry. PMID:25309621

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

    PubMed

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

    2016-01-01

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

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

    PubMed

    Bray, Walter; Lokey, R Scott

    2016-01-01

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

  8. 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. PMID:25101166

  9. Viscoelasticity as a Biomarker for High-Throughput Flow Cytometry

    PubMed Central

    Sawetzki, Tobias; Eggleton, Charles D.; Desai, Sanjay A.; Marr, David W.M.

    2013-01-01

    The mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods. PMID:24268140

  10. Robust, high-throughput solution for blood group genotyping.

    PubMed

    Le Goff, Gaelle C; Brès, Jean-Charles; Rigal, Dominique; Blum, Loïc J; Marquette, Christophe A

    2010-07-15

    With the concomitant increase of blood transfusions and safety rules, there is a growing need to integrate high-throughput and multiparametric assays within blood qualification centers. Using a robust and automated solution, we describe a new method for extended blood group genotyping (HiFi-Blood 96) bringing together the throughput possibilities of complete automation and the microarray multiplexed analysis potential. Our approach provides a useful resource for upgrading blood qualification center facilities. A set of six single-nucleotide polymorphisms (SNPs) associated with clinically important blood group antigens (Kell, Kidd, Duffy, and MNS systems) were selected and the corresponding genotyping assays developed. A panel of 293 blood samples was used to validate the approach. The resulting genotypes were compared to phenotypes previously determined by standard serologic techniques, and excellent correlations were found for five SNPs out of six. For the Kell, Kidd, Duffy, and MNS3/MNS4 systems, high matching percentages of 100%, 98.9%, 97.7%, and 97.4% were obtained, respectively, whereas a concordance percentage of 83.3% only was attained for the MNS1/MNS2 polymorphism. PMID:20560530

  11. High-throughput optical screening of cellular mechanotransduction

    PubMed Central

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

    2014-01-01

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

  12. High-throughput automated refolding screening of inclusion bodies

    PubMed Central

    Vincentelli, Renaud; Canaan, Stéphane; Campanacci, Valérie; Valencia, Christel; Maurin, Damien; Frassinetti, Frédéric; Scappucini-Calvo, Loréna; Bourne, Yves; Cambillau, Christian; Bignon, Christophe

    2004-01-01

    One of the main stumbling blocks encountered when attempting to express foreign proteins in Escherichia coli is the occurrence of amorphous aggregates of misfolded proteins, called inclusion bodies (IB). Developing efficient protein native structure recovery procedures based on IB refolding is therefore an important challenge. Unfortunately, there is no “universal” refolding buffer: Experience shows that refolding buffer composition varies from one protein to another. In addition, the methods developed so far for finding a suitable refolding buffer suffer from a number of weaknesses. These include the small number of refolding formulations, which often leads to negative results, solubility assays incompatible with high-throughput, and experiment formatting not suitable for automation. To overcome these problems, it was proposed in the present study to address some of these limitations. This resulted in the first completely automated IB refolding screening procedure to be developed using a 96-well format. The 96 refolding buffers were obtained using a fractional factorial approach. The screening procedure is potentially applicable to any nonmembrane protein, and was validated with 24 proteins in the framework of two Structural Genomics projects. The tests used for this purpose included the use of quality control methods such as circular dichroism, dynamic light scattering, and crystallogenesis. Out of the 24 proteins, 17 remained soluble in at least one of the 96 refolding buffers, 15 passed large-scale purification tests, and five gave crystals. PMID:15388864

  13. A High-Throughput Screen for Antibiotic Drug Discovery

    PubMed Central

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

    2014-01-01

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

  14. Dimensioning storage and computing clusters for efficient high throughput computing

    NASA Astrophysics Data System (ADS)

    Accion, E.; Bria, A.; Bernabeu, G.; Caubet, M.; Delfino, M.; Espinal, X.; Merino, G.; Lopez, F.; Martinez, F.; Planas, E.

    2012-12-01

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

  15. A Fully Automated High-Throughput Training System for Rodents

    PubMed Central

    Poddar, Rajesh; Kawai, Risa; Ölveczky, Bence P.

    2013-01-01

    Addressing the neural mechanisms underlying complex learned behaviors requires training animals in well-controlled tasks, an often time-consuming and labor-intensive process that can severely limit the feasibility of such studies. To overcome this constraint, we developed a fully computer-controlled general purpose system for high-throughput training of rodents. By standardizing and automating the implementation of predefined training protocols within the animal’s home-cage our system dramatically reduces the efforts involved in animal training while also removing human errors and biases from the process. We deployed this system to train rats in a variety of sensorimotor tasks, achieving learning rates comparable to existing, but more laborious, methods. By incrementally and systematically increasing the difficulty of the task over weeks of training, rats were able to master motor tasks that, in complexity and structure, resemble ones used in primate studies of motor sequence learning. By enabling fully automated training of rodents in a home-cage setting this low-cost and modular system increases the utility of rodents for studying the neural underpinnings of a variety of complex behaviors. PMID:24349451

  16. 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. PMID:25914370

  17. Compound Management for Quantitative High-Throughput Screening

    PubMed Central

    Yasgar, Adam; Shinn, Paul; Jadhav, Ajit; Auld, Douglas; Michael, Sam; Zheng, Wei; Austin, Christopher P.; Inglese, James; Simeonov, Anton

    2008-01-01

    An efficient and versatile Compound Management operation is essential for the success of all downstream processes in high-throughput screening (HTS) and small molecule lead development. Staff, equipment, and processes need to be not only reliable, but remain flexible and prepared to incorporate paradigm changes. In the present report, we describe a system and associated processes which enable handling of compounds for both screening and follow-up purposes at the NIH Chemical Genomics Center (NCGC), a recently-established HTS and probe development center within the Molecular Libraries Initiative of the NIH Roadmap. Our screening process, termed quantitative HTS (qHTS), involves assaying the complete compound library, currently containing >200,000 members, at a series of dilutions to construct a full concentration-response profile. As such, Compound Management at the NCGC has been uniquely tasked to prepare, store, register, and track a vertically-developed plate dilution series (i.e., inter-plate titrations) in the 384-well format. These are compressed into a series of 1,536-well plates and are registered to track all subsequent plate storage. Here, we present details on the selection of equipment to enable automated, reliable and parallel compound manipulation in 384- and 1,536-well formats, protocols for preparation of inter-plate dilution series for qHTS, as well as qHTS-specific processes and issues. PMID:18496600

  18. High-Throughput RNA Interference Screening: Tricks of the Trade

    PubMed Central

    Nebane, N. Miranda; Coric, Tatjana; Whig, Kanupriya; McKellip, Sara; Woods, LaKeisha; Sosa, Melinda; Sheppard, Russell; Rasmussen, Lynn; Bjornsti, Mary-Ann; White, E. Lucile

    2016-01-01

    The process of validating an assay for high-throughput screening (HTS) involves identifying sources of variability and developing procedures that minimize the variability at each step in the protocol. The goal is to produce a robust and reproducible assay with good metrics. In all good cell-based assays, this means coefficient of variation (CV) values of less than 10% and a signal window of fivefold or greater. HTS assays are usually evaluated using Z′ factor, which incorporates both standard deviation and signal window. A Z′ factor value of 0.5 or higher is acceptable for HTS. We used a standard HTS validation procedure in developing small interfering RNA (siRNA) screening technology at the HTS center at Southern Research. Initially, our assay performance was similar to published screens, with CV values greater than 10% and Z′ factor values of 0.51 ± 0.16 (average ± standard deviation). After optimizing the siRNA assay, we got CV values averaging 7.2% and a robust Z′ factor value of 0.78 ± 0.06 (average ± standard deviation). We present an overview of the problems encountered in developing this whole-genome siRNA screening program at Southern Research and how equipment optimization led to improved data quality. PMID:23616418

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

    PubMed Central

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

    2015-01-01

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

  20. Microfluidic system for high throughput characterisation of echogenic particles.

    PubMed

    Rademeyer, Paul; Carugo, Dario; Lee, Jeong Yu; Stride, Eleanor

    2015-01-21

    Echogenic particles, such as microbubbles and volatile liquid micro/nano droplets, have shown considerable potential in a variety of clinical diagnostic and therapeutic applications. The accurate prediction of their response to ultrasound excitation is however extremely challenging, and this has hindered the optimisation of techniques such as quantitative ultrasound imaging and targeted drug delivery. Existing characterisation techniques, such as ultra-high speed microscopy provide important insights, but suffer from a number of limitations; most significantly difficulty in obtaining large data sets suitable for statistical analysis and the need to physically constrain the particles, thereby altering their dynamics. Here a microfluidic system is presented that overcomes these challenges to enable the measurement of single echogenic particle response to ultrasound excitation. A co-axial flow focusing device is used to direct a continuous stream of unconstrained particles through the combined focal region of an ultrasound transducer and a laser. Both the optical and acoustic scatter from individual particles are then simultaneously recorded. Calibration of the device and example results for different types of echogenic particle are presented, demonstrating a high throughput of up to 20 particles per second and the ability to resolve changes in particle radius down to 0.1 μm with an uncertainty of less than 3%. PMID:25367757

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

    PubMed

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

    2013-01-01

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

  2. High-Throughput Preparation of New Photoactive Nanocomposites.

    PubMed

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

    2016-06-01

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

  3. Towards Prebiotic Catalytic Amyloids Using High Throughput Screening

    PubMed Central

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

    2015-01-01

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

  4. High Throughput Profiling of Molecular Shapes in Crystals

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Feldman, Arkady

    2010-08-01

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

  6. Achieving High Throughput for Data Transfer over ATM Networks

    NASA Technical Reports Server (NTRS)

    Johnson, Marjory J.; Townsend, Jeffrey N.

    1996-01-01

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

  7. High-throughput analysis and protein engineering using microcapillary arrays.

    PubMed

    Chen, Bob; Lim, Sungwon; Kannan, Arvind; Alford, Spencer C; Sunden, Fanny; Herschlag, Daniel; Dimov, Ivan K; Baer, Thomas M; Cochran, Jennifer R

    2016-02-01

    We describe a multipurpose technology platform, termed μSCALE (microcapillary single-cell analysis and laser extraction), that enables massively parallel, quantitative biochemical and biophysical measurements on millions of protein variants expressed from yeast or bacteria. μSCALE spatially segregates single cells within a microcapillary array, enabling repeated imaging, cell growth and protein expression. We performed high-throughput analysis of cells and their protein products using a range of fluorescent assays, including binding-affinity measurements and dynamic enzymatic assays. A precise laser-based extraction method allows rapid recovery of live clones and their genetic material from microcapillaries for further study. With μSCALE, we discovered a new antibody against a clinical cancer target, evolved a fluorescent protein biosensor and engineered an enzyme to reduce its sensitivity to its inhibitor. These protein analysis and engineering applications each have unique assay requirements and different host organisms, highlighting the flexibility and technical capabilities of the μSCALE platform. PMID:26641932

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

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

    2006-10-15

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

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

    PubMed

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

    2016-08-15

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

  11. A high-throughput screening system for the evaluation of biomass-hydrolyzing glycoside hydrolases.

    PubMed

    Song, Letian; Laguerre, Sandrine; Dumon, Claire; Bozonnet, Sophie; O'Donohue, Michael J

    2010-11-01

    To implement a protein engineering strategy for the improvement of enzyme performance on biomass, a straightforward, robust high-throughput method was devised and tested with recombinant GH11 xylanase as acting on wheat straw. The method requires automated liquid handling equipment, but avoids the need for specialized milling and powder weighing devices and the use of labour intensive steps such as manual cutting of pipette tips. After expression in Escherichia coli cells grown in microtiter plates, recombinant xylanase was released into the culture medium and used directly for biomass hydrolysis. Reactions were monitored using a micro-3,5-dinitrosalicylic acid assay. The cumulative error of the method was less than 15%. To validate the method, randomly generated xylanase mutants were analyzed. This allowed the detection of one mutant, which produced a 74% increase in hydrolysis compared to the parental enzyme. Closer analysis revealed that this increase in activity was correlated with a twofold increase in xylanase expression. PMID:20579873

  12. Characterization of DNA-protein interactions using high-throughput sequencing data from pulldown experiments

    NASA Astrophysics Data System (ADS)

    Moreland, Blythe; Oman, Kenji; Curfman, John; Yan, Pearlly; Bundschuh, Ralf

    Methyl-binding domain (MBD) protein pulldown experiments have been a valuable tool in measuring the levels of methylated CpG dinucleotides. Due to the frequent use of this technique, high-throughput sequencing data sets are available that allow a detailed quantitative characterization of the underlying interaction between methylated DNA and MBD proteins. Analyzing such data sets, we first found that two such proteins cannot bind closer to each other than 2 bp, consistent with structural models of the DNA-protein interaction. Second, the large amount of sequencing data allowed us to find rather weak but nevertheless clearly statistically significant sequence preferences for several bases around the required CpG. These results demonstrate that pulldown sequencing is a high-precision tool in characterizing DNA-protein interactions. This material is based upon work supported by the National Science Foundation under Grant No. DMR-1410172.

  13. A high-throughput assay format for determination of nitrate reductase and nitrite reductase enzyme activities

    SciTech Connect

    McNally, N.; Liu, Xiang Yang; Choudary, P.V.

    1997-01-01

    The authors describe a microplate-based high-throughput procedure for rapid assay of the enzyme activities of nitrate reductase and nitrite reductase, using extremely small volumes of reagents. The new procedure offers the advantages of rapidity, small sample size-nanoliter volumes, low cost, and a dramatic increase in the throughput sample number that can be analyzed simultaneously. Additional advantages can be accessed by using microplate reader application software packages that permit assigning a group type to the wells, recording of the data on exportable data files and exercising the option of using the kinetic or endpoint reading modes. The assay can also be used independently for detecting nitrite residues/contamination in environmental/food samples. 10 refs., 2 figs.

  14. Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

    PubMed

    Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

    2014-01-01

    The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant. PMID:24744029

  15. Droplet-based microfluidics in drug discovery, transcriptomics and high-throughput molecular genetics.

    PubMed

    Shembekar, Nachiket; Chaipan, Chawaree; Utharala, Ramesh; Merten, Christoph A

    2016-04-12

    Droplet-based microfluidics enables assays to be carried out at very high throughput (up to thousands of samples per second) and enables researchers to work with very limited material, such as primary cells, patient's biopsies or expensive reagents. An additional strength of the technology is the possibility to perform large-scale genotypic or phenotypic screens at the single-cell level. Here we critically review the latest developments in antibody screening, drug discovery and highly multiplexed genomic applications such as targeted genetic workflows, single-cell RNAseq and single-cell ChIPseq. Starting with a comprehensive introduction for non-experts, we pinpoint current limitations, analyze how they might be overcome and give an outlook on exciting future applications. PMID:27025767

  16. Gold nanoparticle mediated laser transfection for high-throughput antisense applications

    NASA Astrophysics Data System (ADS)

    Kalies, S.; Heinemann, D.; Schomaker, M.; Birr, T.; Ripken, T.; Meyer, H.

    2013-06-01

    The delivery of antisense structures, like siRNA, is beneficial for new therapeutic approaches in regenerative sciences. Optical transfection techniques enable high spatial control combined with minimal invasive treatment of cells due to the use of short laser pulses. However, single cell laser transfection by a tightly focused laser beam, for example femtosecond laser transfection, has the major drawback of low throughput. Compared to this, high-throughput in laser transfection is possible by applying gold nanoparticles irradiated by a weakly focused laser beam scanning over the cell sample. Herein, we show the delivery of antisense molecules and demonstrate the minimal cytotoxicity of a method called gold nanoparticle mediated (GNOME) laser transfection. A 532 nm microchip laser in conjugation with 200 nm gold nanoparticles at a concentration of 0.5 μg/cm2 is used. In addition to antisense molecules, the uptake of dextrans of several sizes is analyzed.

  17. New Tools For Understanding Microbial Diversity Using High-throughput Sequence Data

    NASA Astrophysics Data System (ADS)

    Knight, R.; Hamady, M.; Liu, Z.; Lozupone, C.

    2007-12-01

    High-throughput sequencing techniques such as 454 are straining the limits of tools traditionally used to build trees, choose OTUs, and perform other essential sequencing tasks. We have developed a workflow for phylogenetic analysis of large-scale sequence data sets that combines existing tools, such as the Arb phylogeny package and the NAST multiple sequence alignment tool, with new methods for choosing and clustering OTUs and for performing phylogenetic community analysis with UniFrac. This talk discusses the cyberinfrastructure we are developing to support the human microbiome project, and the application of these workflows to analyze very large data sets that contrast the gut microbiota with a range of physical environments. These tools will ultimately help to define core and peripheral microbiomes in a range of environments, and will allow us to understand the physical and biotic factors that contribute most to differences in microbial diversity.

  18. High throughput kinetic Vibrio fischeri bioluminescence inhibition assay for study of toxic effects of nanoparticles.

    PubMed

    Mortimer, M; Kasemets, K; Heinlaan, M; Kurvet, I; Kahru, A

    2008-08-01

    Despite of the growing production and use of nanoparticles (NPs) in various applications, current regulations, including EC new chemical policy REACH, fail to address the environmental, health, and safety risks posed by NPs. This paper shows that kinetic Vibrio fischeri luminescence inhibition test--Flash Assay--that up to now was mainly used for toxicity analysis of solid and colored environmental samples (e.g. sediments, soil suspensions), is a powerful tool for screening the toxic properties of NPs. To demonstrate that Flash Assay (initially designed for a tube luminometer) can also be adapted to a microplate format for high throughput toxicity screening of NPs, altogether 11 chemicals were comparatively analyzed. The studied chemicals included bulk and nanosized CuO and ZnO, polyethylenimine (PEI) and polyamidoamine dendrimer generations 2 and 5 (PAMAM G2 and G5). The results showed that EC50 values of 30-min Flash Assay in tube and microplate formats were practically similar and correlated very well (log-logR2=0.98), classifying all analyzed chemicals, except nano CuO (that was more toxic in cuvette format), analogously when compared to the risk phrases of the EC Directive 93/67/EEC for ranking toxicity of chemicals for aquatic organisms. The 30-min EC50 values of nanoscale organic cationic polymers (PEI and dendrimers) ranged from 215 to 775 mg/l. Thirty-minute EC50 values of metal oxides varied largely, ranging from approximately 4 mg/l (bulk and nano ZnO) to approximately 100 mg/l (nano CuO) and approximately 4000 mg/l (bulk CuO). Thus, considering an excellent correlation between both formats, 96-well microplate Flash Assay can be successfully used for high throughput evaluation of harmful properties of chemicals (including organic and inorganic NPs) to bacteria. PMID:18400463

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-07-23

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

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

    PubMed

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

    2015-09-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

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

    PubMed

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

    2015-01-01

    The induction of anti-hypoxic stress enzymes and proteins has the potential to be a potent therapeutic strategy to prevent the progression of ischemic heart, kidney or brain diseases. To realize this idea, small chemical compounds, which mimic hypoxic conditions by activating the PHD-HIF-α system, have been developed. However, to date, none of these compounds were identified by monitoring the transcriptional activation of hypoxia-inducible factors (HIFs). Thus, to facilitate the discovery of potent inducers of HIF-α, we have developed an effective high-throughput screening (HTS) system to directly monitor the output of HIF-α transcription. We generated a HIF-α-dependent reporter system that responds to hypoxic stimuli in a concentration- and time-dependent manner. This system was developed through multiple optimization steps, resulting in the generation of a construct that consists of the secretion-type luciferase gene (Metridia luciferase, MLuc) under the transcriptional regulation of an enhancer containing 7 copies of 40-bp hypoxia responsive element (HRE) upstream of a mini-TATA promoter. This construct was stably integrated into the human neuroblastoma cell line, SK-N-BE(2)c, to generate a reporter system, named SKN:HRE-MLuc. To improve this system and to increase its suitability for the HTS platform, we incorporated the next generation luciferase, Nano luciferase (NLuc), whose longer half-life provides us with flexibility for the use of this reporter. We thus generated a stably transformed clone with NLuc, named SKN:HRE-NLuc, and found that it showed significantly improved reporter activity compared to SKN:HRE-MLuc. In this study, we have successfully developed the SKN:HRE-NLuc screening system as an efficient platform for future HTS. PMID:25746387

  4. High-throughput screening of solid-state catalyst libraries

    NASA Astrophysics Data System (ADS)

    Senkan, Selim M.

    1998-07-01

    Combinatorial synthesis methods allow the rapid preparation and processing of large libraries of solid-state materials. The use of these methods, together with the appropriate screening techniques, has recently led to the discovery of materials with promising superconducting, magnetoresistive, luminescent and dielectric properties. Solid-state catalysts, which play an increasingly important role in the chemical and oil industries, represent another class of material amenable to combinatorial synthesis. Yet typically, catalyst discovery still involves inefficient trial-and-error processes, because catalytic activity is inherently difficult to screen. In contrast to superconductivity, magnetoresistivity and dielectric properties, which can be tested by contact probes, or luminescence, which can be observed directly, the assessment of catalytic activity requires the unambiguous detection of a specific product molecule above a small catalyst site on a large library. Screening by in situ infrared thermography and microprobe sampling mass spectrometry, have been suggested, but the first method, while probing activity, provides no information on reaction products, whereas the second is difficult to implement because it requires the transport of minute gas samples from each library site to the detection system. Here I describe the use of laser-induced resonance-enhanced multiphoton ionization for sensitive, selective and high-throughput screening of a library of solid-state catalysts that activate the dehydrogenation of cyclohexane to benzene. I show that benzene, the product molecule, can be selectively photoionized in the vicinity of the catalytic sites, and that the detection of the resultant photoions by an array of microelectrodes provides information on the activity of individual sites. Adaptation of this technique for the screening of other catalytic reactions and larger libraries with smaller site size seems feasible, thus opening up the possibility of exploiting

  5. 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

  6. High-throughput process development: II. Membrane chromatography.

    PubMed

    Rathore, Anurag S; Muthukumar, Sampath

    2014-01-01

    Membrane chromatography is gradually emerging as an alternative to conventional column chromatography. It alleviates some of the major disadvantages associated with the latter including high pressure drop across the column bed and dependence on intra-particle diffusion for the transport of solute molecules to their binding sites within the pores of separation media. In the last decade, it has emerged as a method of choice for final polishing of biopharmaceuticals, in particular monoclonal antibody products. 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 a membrane chromatography step. It describes operation of a commercially available device (AcroPrep™ Advance filter plate with Mustang S membrane from Pall Corporation). This device is available in 96-well format with 7 μL membrane in each well. We 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 data is indeed very significant (regression coefficient 0.99). We think that this protocol will be of significant value to those involved in performing high-throughput process development of membrane chromatography. PMID:24648065

  7. 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

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

    PubMed Central

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

    2012-01-01

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

  9. A Robotic Platform for Quantitative High-Throughput Screening

    PubMed Central

    Michael, Sam; Auld, Douglas; Klumpp, Carleen; Jadhav, Ajit; Zheng, Wei; Thorne, Natasha; Austin, Christopher P.; Inglese, James

    2008-01-01

    Abstract High-throughput screening (HTS) is increasingly being adopted in academic institutions, where the decoupling of screening and drug development has led to unique challenges, as well as novel uses of instrumentation, assay formulations, and software tools. Advances in technology have made automated unattended screening in the 1,536-well plate format broadly accessible and have further facilitated the exploration of new technologies and approaches to screening. A case in point is our recently developed quantitative HTS (qHTS) paradigm, which tests each library compound at multiple concentrations to construct concentration-response curves (CRCs) generating a comprehensive data set for each assay. The practical implementation of qHTS for cell-based and biochemical assays across libraries of > 100,000 compounds (e.g., between 700,000 and 2,000,000 sample wells tested) requires maximal efficiency and miniaturization and the ability to easily accommodate many different assay formats and screening protocols. Here, we describe the design and utilization of a fully integrated and automated screening system for qHTS at the National Institutes of Health's Chemical Genomics Center. We report system productivity, reliability, and flexibility, as well as modifications made to increase throughput, add additional capabilities, and address limitations. The combination of this system and qHTS has led to the generation of over 6 million CRCs from > 120 assays in the last 3 years and is a technology that can be widely implemented to increase efficiency of screening and lead generation. PMID:19035846

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-09-01

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

  13. Scanning fluorescence detector for high-throughput DNA genotyping

    NASA Astrophysics Data System (ADS)

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

    1996-04-01

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

  14. 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.

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

    PubMed

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

    2007-08-01

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

  16. A High-Throughput Method for Lipophilicity Measurement

    PubMed Central

    Chen, Zhi; Weber, Stephen G.

    2008-01-01

    A high-throughput method has been developed for lipophilicity measurement. It measures the partition coefficient of a solute between a polymer phase and an aqueous phase (Ppw) in a 96-well format. The polymer is plasticized poly(vinyl chloride) (PVC), which is widely used as a material for clinical containers and ion-selective electrodes. The composition is 2:1 (w/w) dioctyl sebacate (DOS) and PVC. With 6 repeats, log Ppw values of 15 solutes have been determined in one 96-well microplate in 4 hours. A linear relationship between log Ppw and log Pow (octanol-water partition coefficient) values exists with a correlation coefficient of 0.979. The slope and intercept of the log Ppw vs. log Pow plot are statistically indistinguishable from 1 and 0, respectively. Similar to the HPLC method, by using the correlation line as a calibration curve, the measured log Ppw values can be used to predict log Pow. This protocol is faster than the HPLC method. Moreover, it is straightforward to extend the protocol to the determination of the distribution coefficient and pKa of charged solutes. We show that the log Ppw of the neutral form of racemic econazole is 4.83(±0.06), for the cationic form (presumably as a dihydrogen phosphate ion pair) 1.68(±0.04), and the pKa is 6.15(±0.04). This method has great flexibility as well, and is potentially fully automated. PMID:17263333

  17. 40 CFR Table 3 to Subpart Eeee of... - Operating Limits-High Throughput Transfer Racks

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 13 2012-07-01 2012-07-01 false Operating Limits-High Throughput Transfer Racks 3 Table 3 to Subpart EEEE of Part 63 Protection of Environment ENVIRONMENTAL PROTECTION...—High Throughput Transfer Racks As stated in § 63.2346(e), you must comply with the operating limits...

  18. 40 CFR Table 3 to Subpart Eeee of... - Operating Limits-High Throughput Transfer Racks

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 13 2013-07-01 2012-07-01 true Operating Limits-High Throughput Transfer Racks 3 Table 3 to Subpart EEEE of Part 63 Protection of Environment ENVIRONMENTAL PROTECTION...—High Throughput Transfer Racks As stated in § 63.2346(e), you must comply with the operating limits...

  19. 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...

  20. 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...

  1. Quantitative Assessment of RNA-Protein Interactions with High Throughput Sequencing - RNA Affinity Profiling (HiTS-RAP)

    PubMed Central

    Ozer, Abdullah; Tome, Jacob M.; Friedman, Robin C.; Gheba, Dan; Schroth, Gary P.; Lis, John T.

    2016-01-01

    Because RNA-protein interactions play a central role in a wide-array of biological processes, methods that enable a quantitative assessment of these interactions in a high-throughput manner are in great demand. Recently, we developed the High Throughput Sequencing-RNA Affinity Profiling (HiTS-RAP) assay, which couples sequencing on an Illumina GAIIx with the quantitative assessment of one or several proteins’ interactions with millions of different RNAs in a single experiment. We have successfully used HiTS-RAP to analyze interactions of EGFP and NELF-E proteins with their corresponding canonical and mutant RNA aptamers. Here, we provide a detailed protocol for HiTS-RAP, which can be completed in about a month (8 days hands-on time) including the preparation and testing of recombinant proteins and DNA templates, clustering DNA templates on a flowcell, high-throughput sequencing and protein binding with GAIIx, and finally data analysis. We also highlight aspects of HiTS-RAP that can be further improved and points of comparison between HiTS-RAP and two other recently developed methods, RNA-MaP and RBNS. A successful HiTS-RAP experiment provides the sequence and binding curves for approximately 200 million RNAs in a single experiment. PMID:26182240

  2. Development and Optimization of a Novel 384-Well Anti-Malarial Imaging Assay Validated for High-Throughput Screening

    PubMed Central

    Duffy, Sandra; Avery, Vicky M.

    2012-01-01

    With the increasing occurrence of drug resistance in the malaria parasite, Plasmodium falciparum, there is a great need for new and novel anti-malarial drugs. We have developed a 384-well, high-throughput imaging assay for the detection of new anti-malarial compounds, which was initially validated by screening a marine natural product library, and subsequently used to screen more than 3 million data points from a variety of compound sources. Founded on another fluorescence-based P. falciparum growth inhibition assay, the DNA-intercalating dye 4′,6-diamidino-2-phenylindole, was used to monitor changes in parasite number. Fluorescent images were acquired on the PerkinElmer Opera High Throughput confocal imaging system and analyzed with a spot detection algorithm using the Acapella data processing software. Further optimization of this assay sought to increase throughput, assay stability, and compatibility with our high-throughput screening equipment platforms. The assay typically yielded Z'-factor values of 0.5–0.6, with signal-to-noise ratios of 12. PMID:22232455

  3. Development of combinatorial chemistry methods for coatings: high-throughput screening of abrasion resistance of coatings libraries.

    PubMed

    Potyrailo, Radislav A; Chisholm, Bret J; Olson, Daniel R; Brennan, Michael J; Molaison, Chris A

    2002-10-01

    Design, validation, and implementation of an optical spectroscopic system for high-throughput analysis of combinatorially developed protective organic coatings are reported. Our approach replaces labor-intensive coating evaluation steps with an automated system that rapidly analyzes 8 x 6 arrays of coating elements that are discretely deposited on a single plastic substrate. Each coating element of the library is 10 mm in diameter and 2-5 microm thick. Performance of coatings is evaluated with respect to their resistance to wear abrasion because this parameter is one of the primary considerations in end-use applications. Upon testing, the coating materials undergo changes that are impossible to quantitatively predict using existing knowledge. Coatings are abraded using industry-accepted abrasion test methods at a single or multiple abrasion conditions followed by the high-throughput analysis of abrasion-induced light scatter. The developed automated system is optimized for the analysis of diffusively scattered light that corresponds to 0-30% haze. System precision of 0.1-2.5% relative standard deviation provides capability for the reliable ranking of coatings performance. Although the system was implemented for high-throughput screening of combinatorially developed organic protective coatings for automotive applications, it can be applied for a variety of other applications for which materials ranking can be achieved using optical spectroscopic tools. PMID:12380837

  4. High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.

    PubMed

    Inagaki, Soichi; Henry, Isabelle M; Lieberman, Meric C; Comai, Luca

    2015-01-01

    Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines. PMID:26445462

  5. High-throughput analysis of T-DNA location and structure using sequence capture

    SciTech Connect

    Inagaki, Soichi; Henry, Isabelle M.; Lieberman, Meric C.; Comai, Luca

    2015-10-07

    Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. As a result, our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.

  6. High-throughput analysis of T-DNA location and structure using sequence capture

    DOE PAGESBeta

    Inagaki, Soichi; Henry, Isabelle M.; Lieberman, Meric C.; Comai, Luca

    2015-10-07

    Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously,more » using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. As a result, our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.« less

  7. High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture

    PubMed Central

    Inagaki, Soichi; Henry, Isabelle M.; Lieberman, Meric C.; Comai, Luca

    2015-01-01

    Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines. PMID:26445462

  8. Chipster: user-friendly analysis software for microarray and other high-throughput data

    PubMed Central

    2011-01-01

    Background The growth of high-throughput technologies such as microarrays and next generation sequencing has been accompanied by active research in data analysis methodology, producing new analysis methods at a rapid pace. While most of the newly developed methods are freely available, their use requires substantial computational skills. In order to enable non-programming biologists to benefit from the method development in a timely manner, we have created the Chipster software. Results Chipster (http://chipster.csc.fi/) brings a powerful collection of data analysis methods within the reach of bioscientists via its intuitive graphical user interface. Users can analyze and integrate different data types such as gene expression, miRNA and aCGH. The analysis functionality is complemented with rich interactive visualizations, allowing users to select datapoints and create new gene lists based on these selections. Importantly, users can save the performed analysis steps as reusable, automatic workflows, which can also be shared with other users. Being a versatile and easily extendable platform, Chipster can be used for microarray, proteomics and sequencing data. In this article we describe its comprehensive collection of analysis and visualization tools for microarray data using three case studies. Conclusions Chipster is a user-friendly analysis software for high-throughput data. Its intuitive graphical user interface enables biologists to access a powerful collection of data analysis and integration tools, and to visualize data interactively. Users can collaborate by sharing analysis sessions and workflows. Chipster is open source, and the server installation package is freely available. PMID:21999641

  9. 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

  10. Development of an NMR microprobe procedure for high-throughput environmental metabolomics of Daphnia magna.

    PubMed

    Nagato, Edward G; Lankadurai, Brian P; Soong, Ronald; Simpson, André J; Simpson, Myrna J

    2015-09-01

    Nuclear magnetic resonance (NMR) is the primary platform used in high-throughput environmental metabolomics studies because its non-selectivity is well suited for non-targeted approaches. However, standard NMR probes may limit the use of NMR-based metabolomics for tiny organisms because of the sample volumes required for routine metabolic profiling. Because of this, keystone ecological species, such as the water flea Daphnia magna, are not commonly studied because of the analytical challenges associated with NMR-based approaches. Here, the use of a 1.7-mm NMR microprobe in analyzing tissue extracts from D. magna is tested. Three different extraction procedures (D2O-based buffer, Bligh and Dyer, and acetonitrile : methanol : water) were compared in terms of the yields and breadth of polar metabolites. The D2O buffer extraction yielded the most metabolites and resulted in the best reproducibility. Varying amounts of D. magna dry mass were extracted to optimize metabolite isolation from D. magna tissues. A ratio of 1-1.5-mg dry mass to 40 µl of extraction solvent provided excellent signal-to-noise and spectral resolution using (1)H NMR. The metabolite profile of a single daphnid was also investigated (approximately 0.2 mg). However, the signal-to-noise of the (1)H NMR was considerably lower, and while feasible for select applications would likely not be appropriate for high-throughput NMR-based metabolomics. Two-dimensional NMR experiments on D. magna extracts were also performed using the 1.7-mm NMR probe to confirm (1)H NMR metabolite assignments. This study provides an NMR-based analytical framework for future metabolomics studies that use D. magna in ecological and ecotoxicity studies. PMID:25891518

  11. Demonstration of submersible high-throughput microfluidic immunosensors for underwater explosives detection.

    PubMed

    Adams, André A; Charles, Paul T; Deschamps, Jeffrey R; Kusterbeck, Anne W

    2011-11-15

    Significant security threats posed by highly energetic nitroaromatic compounds in aquatic environments and the demilitarization and pending cleanup of areas previously used for munitions manufacture and storage represent a challenge for less expensive, faster, and more sensitive systems capable of analyzing groundwater and seawater samples for trace levels of explosive materials. Presented here is an inexpensive high throughput microfluidic immunosensor (HTMI) platform intended for the rapid, highly selective quantitation of nitroaromatic compounds in the field. Immunoaffinity and fluorescence detection schemes were implemented in tandem on a novel microfluidic device containing 39 parallel microchannels that were 500 μm tall, 250 μm wide, and 2.54 cm long with covalently tethered antibodies that was engineered for high-throughput high-volume sample processing. The devices were produced via a combination of high precision micromilling and hot embossing. Mass transfer limitations were found in conventional microsystems and were minimized due to higher surface area to volume ratios that exceeded those possessed by conventional microdevices and capillaries. Until now, these assays were limited to maximum total volume flow rates of ~1 mL/min due in part to kinetics and high head pressures of single microchannels. In the design demonstrated here, highly parallelized microchannels afforded up to a 100-fold increase in total volume flow rate while maintaining favorable kinetic constraints for efficient antigen-antibody interaction. The assay employed total volume throughput of up to 6 mL/min while yielding signal-to-noise ratios of >15 in all cases. In addition to samples being processed up to 60 times faster than in conventional displacement-based immunoassays, the current system was capable of quantitating 0.01 ng/mL TNT samples without implementing offline preconcentration, thereby, demonstrating the ability to improve sensitivity by as much as 2 orders of magnitude

  12. High-throughput full-automatic synchrotron-based tomographic microscopy

    SciTech Connect

    Mader, Kevin; Marone, Federica; Hintermüller, Christoph; Mikuljan, Gordan; Isenegger, Andreas; Stampanoni, Marco

    2011-08-16

    At the TOMCAT (TOmographic Microscopy and Coherent rAdiology experimenTs) beamline of the Swiss Light Source with an energy range of 8-45 keV and voxel size from 0.37 {micro}m to 7.4 {micro}m, full tomographic datasets are typically acquired in 5 to 10 min. To exploit the speed of the system and enable high-throughput studies to be performed in a fully automatic manner, a package of automation tools has been developed. The samples are automatically exchanged, aligned, moved to the correct region of interest, and scanned. This task is accomplished through the coordination of Python scripts, a robot-based sample-exchange system, sample positioning motors and a CCD camera. The tools are suited for any samples that can be mounted on a standard SEM stub, and require no specific environmental conditions. Up to 60 samples can be analyzed at a time without user intervention. The throughput of the system is dependent on resolution, energy and sample size, but rates of four samples per hour have been achieved with 0.74 {micro}m voxel size at 17.5 keV. The maximum intervention-free scanning time is theoretically unlimited, and in practice experiments have been running unattended as long as 53 h (the average beam time allocation at TOMCAT is 48 h per user). The system is the first fully automated high-throughput tomography station: mounting samples, finding regions of interest, scanning and reconstructing can be performed without user intervention. The system also includes many features which accelerate and simplify the process of tomographic microscopy.

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

    PubMed

    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

  14. Identification of Thyroid Hormone Receptor Active Compounds Using a Quantitative High-Throughput Screening Platform

    PubMed Central

    Freitas, Jaime; Miller, Nicole; Mengeling, Brenda J.; Xia, Menghang; Huang, Ruili; Houck, Keith; Rietjens, Ivonne M.C.M.; Furlow, J. David; Murk, Albertinka J.

    2014-01-01

    To adapt the use of GH3.TRE-Luc reporter gene cell line for a quantitative high-throughput screening (qHTS) platform, we miniaturized the reporter gene assay to a 1536-well plate format. 1280 chemicals from the Library of Pharmacologically Active Compounds (LOPAC) and the National Toxicology Program (NTP) 1408 compound collection were analyzed to identify potential thyroid hormone receptor (TR) agonists and antagonists. Of the 2688 compounds tested, eight scored as potential TR agonists when the positive hit cut-off was defined at ≥10% efficacy, relative to maximal triiodothyronine (T3) induction, and with only one of those compounds reaching ≥20% efficacy. One common class of compounds positive in the agonist assays were retinoids such as all-trans retinoic acid, which are likely acting via the retinoid-X receptor, the heterodimer partner with the TR. Five potential TR antagonists were identified, including the antiallergy drug tranilast and the anxiolytic drug SB 205384 but also some cytotoxic compounds like 5-fluorouracil. None of the inactive compounds were structurally related to T3, nor had been reported elsewhere to be thyroid hormone disruptors, so false negatives were not detected. None of the low potency (>100µM) TR agonists resembled T3 or T4, thus these may not bind directly in the ligand-binding pocket of the receptor. For TR agonists, in the qHTS, a hit cut-off of ≥20% efficacy at 100 µM may avoid identification of positives with low or no physiological relevance. The miniaturized GH3.TRE-Luc assay offers a promising addition to the in vitro test battery for endocrine disruption, and given the low percentage of compounds testing positive, its high-throughput nature is an important advantage for future toxicological screening. PMID:24772387

  15. High-throughput Saccharification assay for lignocellulosic materials.

    PubMed

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

    2011-01-01

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

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

    PubMed Central

    Sun, Yi; Chen, Jingqiu; Pruckmayr, Gregory; Baumgardner, James E; Eckmann, David M; Eckenhoff, Roderic G; Kelz, Max B

    2006-01-01

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

  17. High-throughput detection method for influenza virus.

    PubMed

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

    2012-01-01

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

  18. High-throughput Detection Method for Influenza Virus

    PubMed Central

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

    2012-01-01

    Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2. Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near

  19. FIM, a Novel FTIR-Based Imaging Method for High Throughput Locomotion Analysis

    PubMed Central

    Otto, Nils; Löpmeier, Tim; Valkov, Dimitar; Jiang, Xiaoyi; Klämbt, Christian

    2013-01-01

    We designed a novel imaging technique based on frustrated total internal reflection (FTIR) to obtain high resolution and high contrast movies. This FTIR-based Imaging Method (FIM) is suitable for a wide range of biological applications and a wide range of organisms. It operates at all wavelengths permitting the in vivo detection of fluorescent proteins. To demonstrate the benefits of FIM, we analyzed large groups of crawling Drosophila larvae. The number of analyzable locomotion tracks was increased by implementing a new software module capable of preserving larval identity during most collision events. This module is integrated in our new tracking program named FIMTrack which subsequently extracts a number of features required for the analysis of complex locomotion phenotypes. FIM enables high throughput screening for even subtle behavioral phenotypes. We tested this newly developed setup by analyzing locomotion deficits caused by the glial knockdown of several genes. Suppression of kinesin heavy chain (khc) or rab30 function led to contraction pattern or head sweeping defects, which escaped in previous analysis. Thus, FIM permits forward genetic screens aimed to unravel the neural basis of behavior. PMID:23349775

  20. SNiPloid: A Utility to Exploit High-Throughput SNP Data Derived from RNA-Seq in Allopolyploid Species.

    PubMed

    Peralta, Marine; Combes, Marie-Christine; Cenci, Alberto; Lashermes, Philippe; Dereeper, Alexis

    2013-01-01

    High-throughput sequencing is a common approach to discover SNP variants, especially in plant species. However, methods to analyze predicted SNPs are often optimized for diploid plant species whereas many crop species are allopolyploids and combine related but divergent subgenomes (homoeologous chromosome sets). We created a software tool, SNiPloid, that exploits and interprets putative SNPs in the context of allopolyploidy by comparing SNPs from an allopolyploid with those obtained in its modern-day diploid progenitors. SNiPloid can compare SNPs obtained from a sample to estimate the subgenome contribution to the transcriptome or SNPs obtained from two polyploid accessions to search for SNP divergence. PMID:24163691

  1. SNiPloid: A Utility to Exploit High-Throughput SNP Data Derived from RNA-Seq in Allopolyploid Species

    PubMed Central

    Peralta, Marine; Combes, Marie-Christine; Lashermes, Philippe; Dereeper, Alexis

    2013-01-01

    High-throughput sequencing is a common approach to discover SNP variants, especially in plant species. However, methods to analyze predicted SNPs are often optimized for diploid plant species whereas many crop species are allopolyploids and combine related but divergent subgenomes (homoeologous chromosome sets). We created a software tool, SNiPloid, that exploits and interprets putative SNPs in the context of allopolyploidy by comparing SNPs from an allopolyploid with those obtained in its modern-day diploid progenitors. SNiPloid can compare SNPs obtained from a sample to estimate the subgenome contribution to the transcriptome or SNPs obtained from two polyploid accessions to search for SNP divergence. PMID:24163691

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

    PubMed

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

    2016-08-15

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

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

    EPA Science Inventory

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

  4. High-throughput detection of prostate cancer in histological sections using probabilistic pairwise Markov models.

    PubMed

    Monaco, James P; Tomaszewski, John E; Feldman, Michael D; Hagemann, Ian; Moradi, Mehdi; Mousavi, Parvin; Boag, Alexander; Davidson, Chris; Abolmaesumi, Purang; Madabhushi, Anant

    2010-08-01

    In this paper we present a high-throughput system for detecting regions of carcinoma of the prostate (CaP) in HSs from radical prostatectomies (RPs) using probabilistic pairwise Markov models (PPMMs), a novel type of Markov random field (MRF). At diagnostic resolution a digitized HS can contain 80Kx70K pixels - far too many for current automated Gleason grading algorithms to process. However, grading can be separated into two distinct steps: (1) detecting cancerous regions and (2) then grading these regions. The detection step does not require diagnostic resolution and can be performed much more quickly. Thus, we introduce a CaP detection system capable of analyzing an entire digitized whole-mount HS (2x1.75cm(2)) in under three minutes (on a desktop computer) while achieving a CaP detection sensitivity and specificity of 0.87 and 0.90, respectively. We obtain this high-throughput by tailoring the system to analyze the HSs at low resolution (8microm per pixel). This motivates the following algorithm: (Step 1) glands are segmented, (Step 2) the segmented glands are classified as malignant or benign, and (Step 3) the malignant glands are consolidated into continuous regions. The classification of individual glands leverages two features: gland size and the tendency for proximate glands to share the same class. The latter feature describes a spatial dependency which we model using a Markov prior. Typically, Markov priors are expressed as the product of potential functions. Unfortunately, potential functions are mathematical abstractions, and constructing priors through their selection becomes an ad hoc procedure, resulting in simplistic models such as the Potts. Addressing this problem, we introduce PPMMs which formulate priors in terms of probability density functions, allowing the creation of more sophisticated models. To demonstrate the efficacy of our CaP detection system and assess the advantages of using a PPMM prior instead of the Potts, we alternately

  5. In silico identification of breast cancer genes by combined multiple high throughput analyses.

    PubMed

    Shen, Dejun; He, Jianbo; Chang, Helena R

    2005-02-01

    that the combined multiple high throughput analyses is an effective data mining strategy in cancer gene identification. This approach may improve the usage of public available genomic data through strategic data mining of high throughput analysis. PMID:15647832

  6. High-throughput detection and quantification of mitochondrial fusion through imaging flow cytometry.

    PubMed

    Nascimento, Aldo; Lannigan, Joanne; Kashatus, David

    2016-08-01

    Mitochondria are highly dynamic organelles whose fusion and fission play an increasingly important role in a number of both normal and pathological cellular functions. Despite the increased interest in mitochondrial dynamics, robust, and quantitative methods to analyze mitochondrial fusion and fission activity in intact cells have not been developed. The current state-of-the art method to measure mitochondrial fusion activity is the polyethylene glycol (PEG) fusion assay in which cells expressing distinct mitochondrially-targeted fluorescent proteins (FPs) are fused together and mitochondrial fusion activity is determined by the rate at which color mixing occurs. Although this assay is useful, cell-cell fusion events are rare, and finding the number of fused cells required to generate statistically rigorous data is both tedious and time-consuming. Furthermore, the data-collection methods available for fluorescence microscopy lead to inherent selection biases that are difficult to control for. To that end, we have developed an unbiased and high-throughput method to detect, image, and analyze fused cells using the Amnis ImagestreamX™ MKII. With IDEAS™ software, we developed algorithms for identifying the fused cells (two nuclei within a single cell), distinguishing them from cell aggregates. Additionally, using the fluorescence localization of the mitochondrially-targeted fluorescent proteins (YFP and DsRed), we applied a modified co-localization algorithm to identify those cells that had a high co-localization score indicating mitochondrial fusion activity. These algorithms were tested using negative controls (FPs associated with fusion deficient mitochondria) and positive controls (cells expressing both FPs in the same mitochondria). Once validated these algorithms could be applied to test samples to evaluate the degree of mitochondrial fusion in cells with various genetic mutations. Ultimately, this new method is the first robust, high-throughput way to

  7. High-Throughput Detection of Prostate Cancer in Histological Sections Using Probabilistic Pairwise Markov Models

    PubMed Central

    Monaco, James P.; Tomaszewski, John E.; Feldman, Michael D.; Hagemann, Ian; Moradi, Mehdi; Mousavi, Parvin; Boag, Alexander; Davidson, Chris; Abolmaesumi, Purang; Madabhushi, Anant

    2010-01-01

    In this paper we present a high-throughput system for detecting regions of carcinoma of the prostate (CaP) in HSs from radical prostatectomies (RPs) using probabilistic pairwise Markov models (PPMMs), a novel type of Markov random field (MRF). At diagnostic resolution a digitized HS can contain 80K×70K pixels — far too many for current automated Gleason grading algorithms to process. However, grading can be separated into two distinct steps: 1) detecting cancerous regions and 2) then grading these regions. The detection step does not require diagnostic resolution and can be performed much more quickly. Thus, we introduce a CaP detection system capable of analyzing an entire digitized whole-mount HS (2×1.75 cm2) in under three minutes (on a desktop computer) while achieving a CaP detection sensitivity and specificity of 0.87 and 0.90, respectively. We obtain this high-throughput by tailoring the system to analyze the HSs at low resolution (8 µm per pixel). This motivates the following algorithm: Step 1) glands are segmented, Step 2) the segmented glands are classified as malignant or benign, and Step 3) the malignant glands are consolidated into continuous regions. The classification of individual glands leverages two features: gland size and the tendency for proximate glands to share the same class. The latter feature describes a spatial dependency which we model using a Markov prior. Typically, Markov priors are expressed as the product of potential functions. Unfortunately, potential functions are mathematical abstractions, and constructing priors through their selection becomes an ad hoc procedure, resulting in simplistic models such as the Potts. Addressing this problem, we introduce PPMMs which formulate priors in terms of probability density functions, allowing the creation of more sophisticated models. To demonstrate the efficacy of our CaP detection system and assess the advantages of using a PPMM prior instead of the Potts, we alternately incorporate

  8. Integrating heterogeneous high-throughput data for meta-dimensional pharmacogenomics and disease-related studies.

    PubMed

    Holzinger, Emily R; Ritchie, Marylyn D

    2012-01-01

    The current paradigm of human genetics research is to analyze variation of a single data type (i.e., DNA sequence or RNA levels) to detect genes and pathways that underlie complex traits such as disease state or drug response. While these studies have detected thousands of variations that associate with hundreds of complex phenotypes, much of the estimated heritability, or trait variability due to genetic factors, remain unexplained. We may be able to account for a portion of the missing heritability if we incorporate a systems biology approach into these analyses. Rapid technological advances will make it possible for scientists to explore this hypothesis via the generation of high-throughput omics data - transcriptomic, proteomic and methylomic to name a few. Analyzing this 'meta-dimensional' data will require clever statistical techniques that allow for the integration of qualitative and quantitative predictor variables. For this article, we examine two major categories of approaches for integrated data analysis, give examples of their use in experimental and in silico datasets, and assess the limitations of each method. PMID:22256870

  9. Analysis of High-Throughput Flow Cytometry Data Using plateCore

    PubMed Central

    Strain, Errol; Hahne, Florian; Brinkman, Ryan R.; Haaland, Perry

    2009-01-01

    Flow cytometry (FCM) software packages from R/Bioconductor, such as flowCore and flowViz, serve as an open platform for development of new analysis tools and methods. We created plateCore, a new package that extends the functionality in these core packages to enable automated negative control-based gating and make the processing and analysis of plate-based data sets from high-throughput FCM screening experiments easier. plateCore was used to analyze data from a BD FACS CAP screening experiment where five Peripheral Blood Mononucleocyte Cell (PBMC) samples were assayed for 189 different human cell surface markers. This same data set was also manually analyzed by a cytometry expert using the FlowJo data analysis software package (TreeStar, USA). We show that the expression values for markers characterized using the automated approach in plateCore are in good agreement with those from FlowJo, and that using plateCore allows for more reproducible analyses of FCM screening data. PMID:19956418

  10. Optical design of a static LWIR Fourier-transform imaging spectrometer with high throughput

    NASA Astrophysics Data System (ADS)

    Wang, Hai-yang; Fu, Yan-peng; Zheng, Wei-jian; Liao, Ning-fang; Jin, Wei-qi

    2013-08-01

    A LWIR Fourier-transform imaging spectrometer based on the static Michelson interferometer with high throughput is presented. Advantages and disadvantages of some common structures of imaging spectrometer are analyzed. Some selection of optimum configurations for imaging spectrometer is proceeded. The interferogram is acquired over the whole field of the camera while the scene of interest scans the path difference range, and vignetting should be strongly limited while keeping the size of the interferometer as small as possible for manufacturability and practicability reasons. The key point is to put the entrance pupil of the imaging lens inside the interferometer. The design of optical system is proposed. The field of view(FOV) is 10°.The operating wavelength range is from 8 to 12μm, F number is 2 and the working temperature range is -20°C~40°C. Optical system with 100% cold shield efficiency is good adaptability to wide environment temperature change. The spectrometer system has low utilization of solar energy in the infrared band, so to ensure its transmittance, and it is necessary to use a small amount of lenses as possible, so here the method of the active electromechanical athermalisation just uses four lenses in the system. Modulation transfer function (MTF), aberrant and distortion etc of optical system are analyzed. The results show that an excellent performance and image performance are obtained despite the simple structure.

  11. Cheminformatics Aspects of High Throughput Screening: from Robots to Models: Symposium Summary

    PubMed Central

    Tseng, Y. Jane; Martin, Eric; Bologa, Cristian; Shelat, Anang A.

    2014-01-01

    The “Cheminformatics aspects of high throughput screening (HTS): from robots to models” symposium was part of the Computers in Chemistry (COMP) technical program at the American Chemical Society National Meeting in Denver, Colorado during the fall of 2011. This symposium brought together researchers from high throughput screening centersand molecular modelers from academia and industry to discuss the integration of currently available high throughput screening data and assays with computational analysis. The topics discussed at this symposium covered the data-infrastructure at various academic, hospital, and NIH-funded high throughput screening centers, the cheminformatics and molecular modeling methods used in real world examples to guide screening and hit-finding, and how academic and non-profit organizations can benefit from current high throughput screening cheminformatics resources. Specifically, this article also covers the remarks and discussions in the open panel discussion in thesymposium and summarizes the following talks on “Accurate Kinase virtual screening: biochemical, cellular and selectivity”, “Selective, privileged and promiscuous chemical patterns in high-throughput screening” and “Visualizing and exploring relationships among HTS hits using network graphs”. PMID:23636795

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

    PubMed Central

    JR, Luft; EH, Snell; GT, DeTitta

    2011-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

  14. Importance of high-throughput cell separation technologies for genomics/proteomics-based clinical diagnostics

    NASA Astrophysics Data System (ADS)

    Leary, James F.; Szaniszlo, Peter; Prow, Tarl W.; Reece, Lisa M.; Wang, Nan; Asmuth, David M.

    2002-06-01

    Gene expression microarray analyses of mixtures of cells approximate a weighted average of the gene expression profiles (GEPs) of each cell type according to its relative abundance in the overall cell sample being analyzed. If the targeted subpopulation of cells is in the minority, or the expected perturbations are marginal, then such changes will be masked by the GEP of the normal/unaffected cells. We show that the GEP of a minor cell subpopulation is often lost when that cell subpopulation is of a frequency less than 30 percent. The GEP is almost always masked by the other cell subpopulations when that frequency drops to 10 percent or less. Several methodologies can be employed to enrich the target cells submitted for microarray analyses. These include magnetic sorting and laser capture microdissection. However, high-throughput flow cytometry/cell sorting overcomes many restrictions of experimental enrichment conditions. This technology can also be used to sort smaller numbers of cells of specific cell subpopulations and subsequently amplify their mRNAs before microarray analyses. When purification techniques are applied to unfixed samples, the potential for changes in gene levels during the process of collection is an additional concern. High-throughput cell separation technologies are needed that can process the necessary number of cells expeditiously in order to avoid such uncontrolled changes in the target cells GEP. In cases where even the use of HTS yields only a small number of cells, the mRNAs (after reverse transcription to cDNA's) must be amplified to yield enough material for conventional microarray analyses. However, the problem of using microamplification PCR methods to expand the amount of cDNAs (from mRNAs) is that it is very difficult to amplify equally all of the mRNAs. Unequal amplification leads to a distorted gene expression profile on the microarray. Linear amplifications is difficult to achieve. Unfortunately, present-day gene-chips need to

  15. Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity

    PubMed Central

    Gedye, Craig A.; Hussain, Ali; Paterson, Joshua; Smrke, Alannah; Saini, Harleen; Sirskyj, Danylo; Pereira, Keira; Lobo, Nazleen; Stewart, Jocelyn; Go, Christopher; Ho, Jenny; Medrano, Mauricio; Hyatt, Elzbieta; Yuan, Julie; Lauriault, Stevan; Kondratyev, Maria; van den Beucken, Twan; Jewett, Michael; Dirks, Peter; Guidos, Cynthia J.; Danska, Jayne; Wang, Jean; Wouters, Bradly; Neel, Benjamin; Rottapel, Robert; Ailles, Laurie E.

    2014-01-01

    Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers. PMID:25170899

  16. Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies

    PubMed Central

    Gardner, Thomas J.; Bolovan-Fritts, Cynthia; Teng, Melissa W.; Redmann, Veronika; Kraus, Thomas A.; Sperling, Rhoda; Moran, Thomas; Britt, William; Weinberger, Leor S.

    2013-01-01

    Infection by human cytomegalovirus (CMV) elicits a strong humoral immune response and robust anti-CMV antibody production. Diagnosis of virus infection can be carried out by using a variety of serological assays; however, quantification of serum antibodies against CMV may not present an accurate measure of a patient's ability to control a virus infection. CMV strains that express green fluorescent protein (GFP) fusion proteins can be used as screening tools for evaluating characteristics of CMV infection in vitro. In this study, we employed a CMV virus strain, AD169, that ectopically expresses a yellow fluorescent protein (YFP) fused to the immediate-early 2 (IE2) protein product (AD169IE2-YFP) to quantify a CMV infection in human cells. We created a high-throughput cell-based assay that requires minimal amounts of material and provides a platform for rapid analysis of the initial phase of virus infection, including virus attachment, fusion, and immediate-early viral gene expression. The AD169IE2-YFP cell infection system was utilized to develop a neutralization assay with a monoclonal antibody against the viral surface glycoprotein gH. The high-throughput assay was extended to measure the neutralization capacity of serum from CMV-positive subjects. These findings describe a sensitive and specific assay for the quantification of a key immunological response that plays a role in limiting CMV dissemination and transmission. Collectively, we have demonstrated that a robust high-throughput infection assay can analyze the early steps of the CMV life cycle and quantify the potency of biological reagents to attenuate a virus infection. PMID:23389931

  17. An automated shotgun lipidomics platform for high throughput, comprehensive, and quantitative analysis of blood plasma intact lipids

    PubMed Central

    Surma, Michal A; Herzog, Ronny; Vasilj, Andrej; Klose, Christian; Christinat, Nicolas; Morin-Rivron, Delphine; Simons, Kai; Masoodi, Mojgan; Sampaio, Julio L

    2015-01-01

    Blood plasma has gained protagonism in lipidomics studies due to its availability, uncomplicated collection and preparation, and informative readout of physiological status. At the same time, it is also technically challenging to analyze due to its complex lipid composition affected by many factors, which can hamper the throughput and/or lipidomics coverage. To tackle these issues, we developed a comprehensive, high throughput, and quantitative mass spectrometry-based shotgun lipidomics platform for blood plasma lipid analyses. The main hallmarks of this technology are (i) it is comprehensive, covering 22 quantifiable different lipid classes encompassing more than 200 lipid species; (ii) it is amenable to high-throughput, with less than 5 min acquisition time allowing the complete analysis of 200 plasma samples per day; (iii) it achieves absolute quantification, by inclusion of internal standards for every lipid class measured; (iv) it is highly reproducible, achieving an average coefficient of variation of <10% (intra-day), approx. 10% (inter-day), and approx. 15% (inter-site) for most lipid species; (v) it is easily transferable allowing the direct comparison of data acquired in different sites. Moreover, we thoroughly assessed the influence of blood stabilization with different anticoagulants and freeze-thaw cycles to exclude artifacts generated by sample preparation. Practical applications: This shotgun lipidomics platform can be implemented in different laboratories without compromising reproducibility, allowing multi-site studies and inter-laboratory comparisons. This possibility combined with the high-throughput, broad lipidomic coverage and absolute quantification are important aspects for clinical applications and biomarker research. PMID:26494980

  18. Insight into the transcriptome of Arthrobotrys conoides using high throughput sequencing.

    PubMed

    Ramesh, Pandit; Reena, Patel; Amitbikram, Mohapatra; Chaitanya, Joshi; Anju, Kunjadia

    2015-12-01

    Arthrobotrys conoides is a nematode-trapping fungus belonging to Orbiliales, Ascomycota group, and traps prey nematodes by means of adhesive network. Fungus has a potential to be used as a biocontrol agent against plant parasitic nematodes. In the present study, we characterized the transcriptome of A. conoides using high-throughput sequencing technology and characterized its virulence unigenes. Total 7,255 cDNA contigs with an average length of 425 bp were generated and 6184 (61.81%) transcripts were functionally annotated and characterized. Majority of unigenes were found analogous to the genes of plant pathogenic fungi. A total of 1749 transcripts were found to be orthologous with eukaryotic proteins of KOG database. Several carbohydrate active enzymes and peptidases were identified. We also analyzed classically and nonclassically secreted proteins and confirmed by BLASTP against fungal secretome database. A total of 916 contigs were analogous to 556 unique proteins of Pathogen Host Interaction (PHI) database. Further, we identified 91 unigenes homologous to the database of fungal virulence factor (DFVF). A total of 104 putative protein kinases coding transcripts were identified by BLASTP against KinBase database, which are major players in signaling pathways. This study provides a comprehensive look at the transcriptome of A. conoides and the identified unigenes might have a role in catching and killing prey nematodes by A. conoides. PMID:26301953

  19. A High Throughput Ambient Mass Spectrometric Approach to Species Identification and Classification from Chemical Fingerprint Signatures.

    PubMed

    Musah, Rabi A; Espinoza, Edgard O; Cody, Robert B; Lesiak, Ashton D; Christensen, Earl D; Moore, Hannah E; Maleknia, Simin; Drijfhout, Falko P

    2015-01-01

    A high throughput method for species identification and classification through chemometric processing of direct analysis in real time (DART) mass spectrometry-derived fingerprint signatures has been developed. The method entails introduction of samples to the open air space between the DART ion source and the mass spectrometer inlet, with the entire observed mass spectral fingerprint subjected to unsupervised hierarchical clustering processing. A range of both polar and non-polar chemotypes are instantaneously detected. The result is identification and species level classification based on the entire DART-MS spectrum. Here, we illustrate how the method can be used to: (1) distinguish between endangered woods regulated by the Convention for the International Trade of Endangered Flora and Fauna (CITES) treaty; (2) assess the origin and by extension the properties of biodiesel feedstocks; (3) determine insect species from analysis of puparial casings; (4) distinguish between psychoactive plants products; and (5) differentiate between Eucalyptus species. An advantage of the hierarchical clustering approach to processing of the DART-MS derived fingerprint is that it shows both similarities and differences between species based on their chemotypes. Furthermore, full knowledge of the identities of the constituents contained within the small molecule profile of analyzed samples is not required. PMID:26156000

  20. High-Throughput Quantification of Monofluoroacetate (1080) in Milk as a Response to an Extortion Threat.

    PubMed

    Cooney, Terry P; Varelis, Peter; Bendall, Justin G

    2016-02-01

    As a food defense measure against an extortion threat to poison infant formula with monofluoroacetate, a robust methodology for monofluoroacetate analysis in fluid milk and powdered dairy products was developed and optimized. Critical challenges posed by this situation required that the analytical methodology provide (i) high specificity, (ii) high throughput capable of analyzing thousands of samples of fluid milk per day, and (iii) trace-level detection of 1 ng/g or lower to achieve the maximum residue limit. Solid-phase extraction-purified acetone extracts of fluid milk were derivatized with aniline, and after ultrahigh-performance liquid chromatography using a Kinetex-C18 column packed with 1.3-μm shell particles, the resulting N-phenyl 2-fluoroacetamide could be determined by liquid chromatography-tandem mass spectrometry in a highly specific manner and with a limit of quantification of 0.5 ng/ml. By using 4-(4-chlorophenoxy)aniline as a derivatizing agent, the method could be extended to powdered dairy products with the same limit of quantification. Between January and July 2015, some 136,000 fluid milk samples were tested using this method. This analytical testing of fluid milk formed one element in a larger program of work by multiple agencies to ensure that consumers could continue to have confidence in the safety of New Zealand milk and dairy products. PMID:26818988

  1. Analysis of genetic variation and diversity of Rice stripe virus populations through high-throughput sequencing.

    PubMed

    Huang, Lingzhe; Li, Zefeng; Wu, Jianxiang; Xu, Yi; Yang, Xiuling; Fan, Longjiang; Fang, Rongxiang; Zhou, Xueping

    2015-01-01

    Plant RNA viruses often generate diverse populations in their host plants through error-prone replication and recombination. Recent studies on the genetic diversity of plant RNA viruses in various host plants have provided valuable information about RNA virus evolution and emergence of new diseases caused by RNA viruses. We analyzed and compared the genetic diversity of Rice stripe virus (RSV) populations in Oryza sativa (a natural host of RSV) and compared it with that of the RSV populations generated in an infection of Nicotiana benthamiana, an experimental host of RSV, using the high-throughput sequencing technology. From infected O. sativa and N. benthamiana plants, a total of 341 and 1675 site substitutions were identified in the RSV genome, respectively, and the average substitution ratio in these sites was 1.47 and 7.05 %, respectively, indicating that the RSV populations from infected N. benthamiana plant are more diverse than those from infected O. sativa plant. Our result gives a direct evidence that virus might allow higher genetic diversity for host adaptation. PMID:25852724

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

    PubMed

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

    2016-03-01

    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

  3. A High Throughput Ambient Mass Spectrometric Approach to Species Identification and Classification from Chemical Fingerprint Signatures

    NASA Astrophysics Data System (ADS)

    Musah, Rabi A.; Espinoza, Edgard O.; Cody, Robert B.; Lesiak, Ashton D.; Christensen, Earl D.; Moore, Hannah E.; Maleknia, Simin; Drijfhout, Falko P.

    2015-07-01

    A high throughput method for species identification and classification through chemometric processing of direct analysis in real time (DART) mass spectrometry-derived fingerprint signatures has been developed. The method entails introduction of samples to the open air space between the DART ion source and the mass spectrometer inlet, with the entire observed mass spectral fingerprint subjected to unsupervised hierarchical clustering processing. A range of both polar and non-polar chemotypes are instantaneously detected. The result is identification and species level classification based on the entire DART-MS spectrum. Here, we illustrate how the method can be used to: (1) distinguish between endangered woods regulated by the Convention for the International Trade of Endangered Flora and Fauna (CITES) treaty; (2) assess the origin and by extension the properties of biodiesel feedstocks; (3) determine insect species from analysis of puparial casings; (4) distinguish between psychoactive plants products; and (5) differentiate between Eucalyptus species. An advantage of the hierarchical clustering approach to processing of the DART-MS derived fingerprint is that it shows both similarities and differences between species based on their chemotypes. Furthermore, full knowledge of the identities of the constituents contained within the small molecule profile of analyzed samples is not required.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  5. A New Versatile Microarray-based Method for High Throughput Screening of Carbohydrate-active Enzymes*

    PubMed Central

    Vidal-Melgosa, Silvia; Pedersen, Henriette L.; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B.; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G. T.

    2015-01-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. PMID:25657012

  6. A new versatile microarray-based method for high throughput screening of carbohydrate-active enzymes.

    PubMed

    Vidal-Melgosa, Silvia; Pedersen, Henriette L; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G T

    2015-04-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. PMID:25657012

  7. iCanPlot: visual exploration of high-throughput omics data using interactive Canvas plotting.

    PubMed

    Sinha, Amit U; Armstrong, Scott A

    2012-01-01

    Increasing use of high throughput genomic scale assays requires effective visualization and analysis techniques to facilitate data interpretation. Moreover, existing tools often require programming skills, which discourages bench scientists from examining their own data. We have created iCanPlot, a compelling platform for visual data exploration based on the latest technologies. Using the recently adopted HTML5 Canvas element, we have developed a highly interactive tool to visualize tabular data and identify interesting patterns in an intuitive fashion without the need of any specialized computing skills. A module for geneset overlap analysis has been implemented on the Google App Engine platform: when the user selects a region of interest in the plot, the genes in the region are analyzed on the fly. The visualization and analysis are amalgamated for a seamless experience. Further, users can easily upload their data for analysis--which also makes it simple to share the analysis with collaborators. We illustrate the power of iCanPlot by showing an example of how it can be used to interpret histone modifications in the context of gene expression. PMID:22393367

  8. Rapid and accurate developmental stage recognition of C. elegans from high-throughput image data

    PubMed Central

    White, Amelia G.; Cipriani, Patricia G.; Kao, Huey-Ling; Lees, Brandon; Geiger, Davi; Sontag, Eduardo; Gunsalus, Kristin C.; Piano, Fabio

    2011-01-01

    We present a hierarchical principle for object recognition and its application to automatically classify developmental stages of C. elegans animals from a population of mixed stages. The object recognition machine consists of four hierarchical layers, each composed of units upon which evaluation functions output a label score, followed by a grouping mechanism that resolves ambiguities in the score by imposing local consistency constraints. Each layer then outputs groups of units, from which the units of the next layer are derived. Using this hierarchical principle, the machine builds up successively more sophisticated representations of the objects to be classified. The algorithm segments large and small objects, decomposes objects into parts, extracts features from these parts, and classifies them by SVM. We are using this system to analyze phenotypic data from C. elegans high-throughput genetic screens, and our system overcomes a previous bottleneck in image analysis by achieving near real-time scoring of image data. The system is in current use in a functioning C. elegans laboratory and has processed over two hundred thousand images for lab users. PMID:22053146

  9. Unlocking the Potential of High-Throughput Drug Combination Assays Using Acoustic Dispensing.

    PubMed

    Chan, Grace Ka Yan; Wilson, Stacy; Schmidt, Stephen; Moffat, John G

    2016-02-01

    Assessment of synergistic effects of drug combinations in vitro is a critical part of anticancer drug research. However, the complexities of dosing and analyzing two drugs over the appropriate range of doses have generally led to compromises in experimental design that restrict the quality and robustness of the data. In particular, the use of a single dose response of combined drugs, rather than a full two-way matrix of varying doses, has predominated in higher-throughput studies. Acoustic dispensing unlocks the potential of high-throughput dose matrix analysis. We have developed acoustic dispensing protocols that enable compound synergy assays in a 384-well format. This experimental design is considerably more efficient and flexible with respect to time, reagent usage, and labware than is achievable using traditional serial-dilution approaches. Data analysis tools integrated in Genedata Screener were used to efficiently deconvolute the combination compound mapping scheme and calculate compound potency and synergy metrics. We have applied this workflow to evaluate interactions among drugs targeting different nodes of the mitogen-activated protein kinase pathway in a panel of cancer cell lines. PMID:26160862

  10. A Data Analysis Pipeline Accounting for Artifacts in Tox21 Quantitative High-Throughput Screening Assays

    PubMed Central

    Hsieh, Jui-Hua; Sedykh, Alexander; Huang, Ruili; Xia, Menghang; Tice, Raymond R.

    2015-01-01

    A main goal of the U.S. Tox21 program is to profile a 10K-compound library for activity against a panel of stress-related and nuclear receptor signaling pathway assays using a quantitative high-throughput screening (qHTS) approach. However, assay artifacts, including nonreproducible signals and assay interference (e.g., autofluorescence), complicate compound activity interpretation. To address these issues, we have developed a data analysis pipeline that includes an updated signal noise–filtering/curation protocol and an assay interference flagging system. To better characterize various types of signals, we adopted a weighted version of the area under the curve (wAUC) to quantify the amount of activity across the tested concentration range in combination with the assay-dependent point-of-departure (POD) concentration. Based on the 32 Tox21 qHTS assays analyzed, we demonstrate that signal profiling using wAUC affords the best reproducibility (Pearson's r = 0.91) in comparison with the POD (0.82) only or the AC50 (i.e., half-maximal activity concentration, 0.81). Among the activity artifacts characterized, cytotoxicity is the major confounding factor; on average, about 8% of Tox21 compounds are affected, whereas autofluorescence affects less than 0.5%. To facilitate data evaluation, we implemented two graphical user interface applications, allowing users to rapidly evaluate the in vitro activity of Tox21 compounds. PMID:25904095

  11. AWV: high-throughput cross-array cross-wafer variation mapping

    NASA Astrophysics Data System (ADS)

    Yeo, Jeong-Ho; Lee, Byoung-Ho; Lee, Tae-Yong; Greenberg, Gadi; Meshulach, Doron; Ravid, Erez; Levi, Shimon; Kan, Kobi; Shabtay, Saar; Cohen, Yehuda; Rotlevi, Ofer

    2008-03-01

    Minute variations in advanced VLSI manufacturing processes are well known to significantly impact device performance and die yield. These variations drive the need for increased measurement sampling with a minimal impact on Fab productivity. Traditional discrete measurements such as CDSEM or OCD, provide, statistical information for process control and monitoring. Typically these measurements require a relatively long time and cover only a fraction of the wafer area. Across array across wafer variation mapping ( AWV) suggests a new approach for high throughput, full wafer process variation monitoring, using a DUV bright-field inspection tool. With this technique we present a full wafer scanning, visualizing the variation trends within a single die and across the wafer. The underlying principle of the AWV inspection method is to measure variations in the reflected light from periodic structures, under optimized illumination and collection conditions. Structural changes in the periodic array induce variations in the reflected light. This information is collected and analyzed in real time. In this paper we present AWV concept, measurements and simulation results. Experiments were performed using a DUV bright-field inspection tool (UVision (TM), Applied Materials) on a memory short loop experiment (SLE), Focus Exposure Matrix (FEM) and normal wafers. AWV and CDSEM results are presented to reflect CD variations within a memory array and across wafers.

  12. High-Throughput Quantitative Proteomic Analysis of Dengue Virus Type 2 Infected A549 Cells

    PubMed Central

    Chiu, Han-Chen; Hannemann, Holger; Heesom, Kate J.; Matthews, David A.; Davidson, Andrew D.

    2014-01-01

    Disease caused by dengue virus is a global health concern with up to 390 million individuals infected annually worldwide. There are no vaccines or antiviral compounds available to either prevent or treat dengue disease which may be fatal. To increase our understanding of the interaction of dengue virus with the host cell, we analyzed changes in the proteome of human A549 cells in response to dengue virus type 2 infection using stable isotope labelling in cell culture (SILAC) in combination with high-throughput mass spectrometry (MS). Mock and infected A549 cells were fractionated into nuclear and cytoplasmic extracts before analysis to identify proteins that redistribute between cellular compartments during infection and reduce the complexity of the analysis. We identified and quantified 3098 and 2115 proteins in the cytoplasmic and nuclear fractions respectively. Proteins that showed a significant alteration in amount during infection were examined using gene enrichment, pathway and network analysis tools. The analyses revealed that dengue virus infection modulated the amounts of proteins involved in the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection. PMID:24671231

  13. High Throughput Ambient Mass Spectrometric Approach to Species Identification and Classification from Chemical Fingerprint Signatures

    DOE PAGESBeta

    Musah, Rabi A.; Espinoza, Edgard O.; Cody, Robert B.; Lesiak, Ashton D.; Christensen, Earl D.; Moore, Hannah E.; Maleknia, Simin; Drijhout, Falko P.

    2015-07-09

    A high throughput method for species identification and classification through chemometric processing of direct analysis in real time (DART) mass spectrometry-derived fingerprint signatures has been developed. The method entails introduction of samples to the open air space between the DART ion source and the mass spectrometer inlet, with the entire observed mass spectral fingerprint subjected to unsupervised hierarchical clustering processing. Moreover, a range of both polar and non-polar chemotypes are instantaneously detected. The result is identification and species level classification based on the entire DART-MS spectrum. In this paper, we illustrate how the method can be used to: (1) distinguishmore » between endangered woods regulated by the Convention for the International Trade of Endangered Flora and Fauna (CITES) treaty; (2) assess the origin and by extension the properties of biodiesel feedstocks; (3) determine insect species from analysis of puparial casings; (4) distinguish between psychoactive plants products; and (5) differentiate between Eucalyptus species. An advantage of the hierarchical clustering approach to processing of the DART-MS derived fingerprint is that it shows both similarities and differences between species based on their chemotypes. Furthermore, full knowledge of the identities of the constituents contained within the small molecule profile of analyzed samples is not required.« less

  14. High Throughput Ambient Mass Spectrometric Approach to Species Identification and Classification from Chemical Fingerprint Signatures

    SciTech Connect

    Musah, Rabi A.; Espinoza, Edgard O.; Cody, Robert B.; Lesiak, Ashton D.; Christensen, Earl D.; Moore, Hannah E.; Maleknia, Simin; Drijhout, Falko P.

    2015-07-09

    A high throughput method for species identification and classification through chemometric processing of direct analysis in real time (DART) mass spectrometry-derived fingerprint signatures has been developed. The method entails introduction of samples to the open air space between the DART ion source and the mass spectrometer inlet, with the entire observed mass spectral fingerprint subjected to unsupervised hierarchical clustering processing. Moreover, a range of both polar and non-polar chemotypes are instantaneously detected. The result is identification and species level classification based on the entire DART-MS spectrum. In this paper, we illustrate how the method can be used to: (1) distinguish between endangered woods regulated by the Convention for the International Trade of Endangered Flora and Fauna (CITES) treaty; (2) assess the origin and by extension the properties of biodiesel feedstocks; (3) determine insect species from analysis of puparial casings; (4) distinguish between psychoactive plants products; and (5) differentiate between Eucalyptus species. An advantage of the hierarchical clustering approach to processing of the DART-MS derived fingerprint is that it shows both similarities and differences between species based on their chemotypes. Furthermore, full knowledge of the identities of the constituents contained within the small molecule profile of analyzed samples is not required.

  15. High-throughput liquid-absorption air-sampling apparatus and methods

    SciTech Connect

    2000-07-11

    A portable high-throughput liquid-absorption air sampler [PHTLAAS] has an asymmetric air inlet through which air is drawn upward by a small and light-weight centrifugal fan driven by a direct current motor that can be powered by a battery. The air inlet is so configured as to impart both rotational and downward components of motion to the sampled air near said inlet. The PHTLAAS comprises a glass tube of relatively small size through which air passes at a high rate in a swirling, highly turbulent motion, which facilitates rapid transfer of vapors and particulates to a liquid film covering the inner walls of the tube. The pressure drop through the glass tube is < 10 cm of water, usually < 5 cm of water. The sampler's collection efficiency is usually > 20% for vapors or airborne particulates in the 2--3 microns range and > 50% for particles larger than 4 microns. In conjunction with various analyzers, the PHTLAAS can serve to monitor a variety of hazardous or illicit airborne substances, such as lead-containing particulates, tritiated water vapor, biological aerosols, or traces of concealed drugs or explosives.

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  17. Information content and analysis methods for Multi-Modal High-Throughput Biomedical Data

    NASA Astrophysics Data System (ADS)

    Ray, Bisakha; Henaff, Mikael; Ma, Sisi; Efstathiadis, Efstratios; Peskin, Eric R.; Picone, Marco; Poli, Tito; Aliferis, Constantin F.; Statnikov, Alexander

    2014-03-01

    The spectrum of modern molecular high-throughput assaying includes diverse technologies such as microarray gene expression, miRNA expression, proteomics, DNA methylation, among many others. Now that these technologies have matured and become increasingly accessible, the next frontier is to collect ``multi-modal'' data for the same set of subjects and conduct integrative, multi-level analyses. While multi-modal data does contain distinct biological information that can be useful for answering complex biology questions, its value for predicting clinical phenotypes and contributions of each type of input remain unknown. We obtained 47 datasets/predictive tasks that in total span over 9 data modalities and executed analytic experiments for predicting various clinical phenotypes and outcomes. First, we analyzed each modality separately using uni-modal approaches based on several state-of-the-art supervised classification and feature selection methods. Then, we applied integrative multi-modal classification techniques. We have found that gene expression is the most predictively informative modality. Other modalities such as protein expression, miRNA expression, and DNA methylation also provide highly predictive results, which are often statistically comparable but not superior to gene expression data. Integrative multi-modal analyses generally do not increase predictive signal compared to gene expression data.

  18. High-throughput metabolic genotoxicity screening with a fluidic microwell chip and electrochemiluminescence†

    PubMed Central

    Wasalathanthri, Dhanuka P.; Malla, Spundana; Bist, Itti; Tang, Chi K.; Faria, Ronaldo C.; Rusling, James F.

    2014-01-01

    A high throughput electrochemiluminescent (ECL) chip was fabricated and integrated into a fluidic system for screening toxicity-related chemistry of drug and pollutant metabolites. The chip base is conductive pyrolytic graphite onto which are printed 64 microwells capable of holding one-µL droplets. Films combining DNA, metabolic enzymes and an ECL-generating ruthenium metallopolymer (RuIIPVP) are fabricated in these microwells. The system runs metabolic enzyme reactions, and subsequently detects DNA damage caused by reactive metabolites. The performance of the chip was tested by measuring DNA damage caused by metabolites of the well-known procarcinogen benzo[a]pyrene (B[a]P). Liver microsomes and cytochrome P450 (cyt P450) enzymes were used with and without epoxide hydrolase (EH), a conjugative enzyme required for multi-enzyme bioactivation of B[a]P. DNA adduct formation was confirmed by determining specific DNA-metabolite adducts using similar films of DNA/enzyme on magnetic bead biocolloid reactors, hydrolyzing the DNA, and analyzing by capillary liquid chromatography-mass spectrometry (CapLC-MS/MS). The fluidic chip was also used to measure IC50-values of inhibitors of cyt P450s. All results show good correlation with reported enzyme activity and inhibition assays. PMID:24113555

  19. Use of high-throughput mass spectrometry to elucidate host-pathogen interactions in Salmonella

    SciTech Connect

    Rodland, Karin D.; Adkins, Joshua N.; Ansong, Charles; Chowdhury, Saiful M.; Manes, Nathan P.; Shi, Liang; Yoon, Hyunjin; Smith, Richard D.; Heffron, Fred

    2008-12-01

    New improvements to mass spectrometry include increased sensitivity, improvements in analyzing the collected data, and most important, from the standpoint of this review, a much higher throughput allowing analysis of many samples in a single day. This short review describes how host-pathogen interactions can be dissected by mass spectrometry using Salmonella as a model system. The approach allowed direct identification of the majority of annotate Salmonella proteins, how expression changed under various in vitro growth conditions, and how this relates to virulence and expression within host cell cells. One of the most significant findings is that a very high percentage of the all annotated genes (>20%) are regulated post-transcriptionally. In addition, new and unexpected interactions have been identified for several Salmonella virulence regulators that involve protein-protein interactions suggesting additional functions of the regulator in coordinating virulence expression. Overall high throughput mass spectrometer provides a new view of pathogen-host interaction emphasizing the protein products and defining how protein interactions determine the outcome of infection.

  20. Information content and analysis methods for Multi-Modal High-Throughput Biomedical Data

    PubMed Central

    Ray, Bisakha; Henaff, Mikael; Ma, Sisi; Efstathiadis, Efstratios; Peskin, Eric R.; Picone, Marco; Poli, Tito; Aliferis, Constantin F.; Statnikov, Alexander

    2014-01-01

    The spectrum of modern molecular high-throughput assaying includes diverse technologies such as microarray gene expression, miRNA expression, proteomics, DNA methylation, among many others. Now that these technologies have matured and become increasingly accessible, the next frontier is to collect “multi-modal” data for the same set of subjects and conduct integrative, multi-level analyses. While multi-modal data does contain distinct biological information that can be useful for answering complex biology questions, its value for predicting clinical phenotypes and contributions of each type of input remain unknown. We obtained 47 datasets/predictive tasks that in total span over 9 data modalities and executed analytic experiments for predicting various clinical phenotypes and outcomes. First, we analyzed each modality separately using uni-modal approaches based on several state-of-the-art supervised classification and feature selection methods. Then, we applied integrative multi-modal classification techniques. We have found that gene expression is the most predictively informative modality. Other modalities such as protein expression, miRNA expression, and DNA methylation also provide highly predictive results, which are often statistically comparable but not superior to gene expression data. Integrative multi-modal analyses generally do not increase predictive signal compared to gene expression data. PMID:24651673

  1. High-throughput microfluidic device for single cell analysis using multiple integrated soft lithographic pumps.

    PubMed

    Patabadige, Damith E W; Mickleburgh, Tom; Ferris, Lorin; Brummer, Gage; Culbertson, Anne H; Culbertson, Christopher T

    2016-05-01

    The ability to accurately control fluid transport in microfluidic devices is key for developing high-throughput methods for single cell analysis. Making small, reproducible changes to flow rates, however, to optimize lysis and injection using pumps external to the microfluidic device are challenging and time-consuming. To improve the throughput and increase the number of cells analyzed, we have integrated previously reported micropumps into a microfluidic device that can increase the cell analysis rate to ∼1000 cells/h and operate for over an hour continuously. In order to increase the flow rates sufficiently to handle cells at a higher throughput, three sets of pumps were multiplexed. These pumps are simple, low-cost, durable, easy to fabricate, and biocompatible. They provide precise control of the flow rate up to 9.2 nL/s. These devices were used to automatically transport, lyse, and electrophoretically separate T-Lymphocyte cells loaded with Oregon green and 6-carboxyfluorescein. Peak overlap statistics predicted the number of fully resolved single-cell electropherograms seen. In addition, there was no change in the average fluorescent dye peak areas indicating that the cells remained intact and the dyes did not leak out of the cells over the 1 h analysis time. The cell lysate peak area distribution followed that expected of an asynchronous steady-state population of immortalized cells. PMID:26887846

  2. Novel high-throughput detection method to assess bacterial surfactant production.

    PubMed

    Burch, Adrien Y; Shimada, Briana K; Browne, Patrick J; Lindow, Steven E

    2010-08-01

    A novel biosurfactant detection assay was developed for the observation of surfactants on agar plates. By using an airbrush to apply a fine mist of oil droplets, surfactants can be observed instantaneously as halos around biosurfactant-producing colonies. This atomized oil assay can detect a wide range of different synthetic and bacterially produced surfactants. This method could detect much lower concentrations of many surfactants than a commonly used water drop collapse method. It is semiquantitative and therefore has broad applicability for uses such as high-throughput mutagenesis screens of biosurfactant-producing bacterial strains. The atomized oil assay was used to screen for mutants of the plant pathogen Pseudomonas syringae pv. syringae B728a that were altered in the production of biosurfactants. Transposon mutants displaying significantly altered surfactant halos were identified and further analyzed. All mutants identified displayed altered swarming motility, as would be expected of surfactant mutants. Additionally, measurements of the transcription of the syringafactin biosynthetic cluster in the mutants, the principal biosurfactant known to be produced by B728a, revealed novel regulators of this pathway. PMID:20562275

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

    SciTech Connect

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

    2015-04-13

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

  4. Analysis of Active Methylotrophic Communities: When DNA-SIP Meets High-Throughput Technologies.

    PubMed

    Taubert, Martin; Grob, Carolina; Howat, Alexandra M; Burns, Oliver J; Chen, Yin; Neufeld, Josh D; Murrell, J Colin

    2016-01-01

    Methylotrophs are microorganisms ubiquitous in the environment that can metabolize one-carbon (C1) compounds as carbon and/or energy sources. The activity of these prokaryotes impacts biogeochemical cycles within their respective habitats and can determine whether these habitats act as sources or sinks of C1 compounds. Due to the high importance of C1 compounds, not only in biogeochemical cycles, but also for climatic processes, it is vital to understand the contributions of these microorganisms to carbon cycling in different environments. One of the most challenging questions when investigating methylotrophs, but also in environmental microbiology in general, is which species contribute to the environmental processes of interest, or "who does what, where and when?" Metabolic labeling with C1 compounds substituted with (13)C, a technique called stable isotope probing, is a key method to trace carbon fluxes within methylotrophic communities. The incorporation of (13)C into the biomass of active methylotrophs leads to an increase in the molecular mass of their biomolecules. For DNA-based stable isotope probing (DNA-SIP), labeled and unlabeled DNA is separated by isopycnic ultracentrifugation. The ability to specifically analyze DNA of active methylotrophs from a complex background community by high-throughput sequencing techniques, i.e. targeted metagenomics, is the hallmark strength of DNA-SIP for elucidating ecosystem functioning, and a protocol is detailed in this chapter. PMID:26791507

  5. An Improved Method for High-throughput Discrimination and Enumeration of Sedimentary Cells Using Flow Cytometry

    NASA Astrophysics Data System (ADS)

    Morono, Y.; Kallmeyer, J.; Terada, T.; Inagaki, F.; IODP Expedition 329 Shipboard Science Party

    2011-12-01

    Detection and enumeration of microbial life in marine subsurface environments provides primary information on the extent and habitability of the Earth's biosphere. Flow cytometry (FCM) is a powerful tool for identifying and enumerating fluorescence-stained cells with high throughput, using fluorescent intensity, range of wavelength, and cell size. FCM is widely used in medical sciences and aquatic microbial ecology. However, mineral grains and difficulties in distinguishing between life cells and non-specific background fluorescence prevented FCM to be applied for counting microbial cells in sediment or rock samples. SYBR Green I-stained cells can be distinguished from non-biological background signals based on differences in their fluorescence spectra. Here we extended this technique to FCM analysis by modifying the cell detachment protocol using a density gradient method, and then standardized an FCM cell counting method for various types of marine subsurface sediments. Microbial cells in sediment samples could effectively be detached and analyzed discriminatively with FCM. The high capacity of FCM to count particles (up to 10,000 cells/sec) and its high sensitivity will provide information about microbial cell abundance at high spatial resolution and with unprecedented accuracy. This improved cell count method will be useful to evaluate samples with high depth resolution, including narrow geochemical and geological interfaces as potential specific microbial niches, and may even help to asses very low population densities at the fringe of the biosphere.

  6. High-throughput machining using high average power ultrashort pulse lasers and ultrafast polygon scanner

    NASA Astrophysics Data System (ADS)

    Schille, Joerg; Schneider, Lutz; Streek, André; Kloetzer, Sascha; Loeschner, Udo

    2016-03-01

    In this paper, high-throughput ultrashort pulse laser machining is investigated on various industrial grade metals (Aluminium, Copper, Stainless steel) and Al2O3 ceramic at unprecedented processing speeds. This is achieved by using a high pulse repetition frequency picosecond laser with maximum average output power of 270 W in conjunction with a unique, in-house developed two-axis polygon scanner. Initially, different concepts of polygon scanners are engineered and tested to find out the optimal architecture for ultrafast and precision laser beam scanning. Remarkable 1,000 m/s scan speed is achieved on the substrate, and thanks to the resulting low pulse overlap, thermal accumulation and plasma absorption effects are avoided at up to 20 MHz pulse repetition frequencies. In order to identify optimum processing conditions for efficient high-average power laser machining, the depths of cavities produced under varied parameter settings are analyzed and, from the results obtained, the characteristic removal values are specified. The maximum removal rate is achieved as high as 27.8 mm3/min for Aluminium, 21.4 mm3/min for Copper, 15.3 mm3/min for Stainless steel and 129.1 mm3/min for Al2O3 when full available laser power is irradiated at optimum pulse repetition frequency.

  7. High-throughput quantitative proteomic analysis of dengue virus type 2 infected A549 cells.

    PubMed

    Chiu, Han-Chen; Hannemann, Holger; Heesom, Kate J; Matthews, David A; Davidson, Andrew D

    2014-01-01

    Disease caused by dengue virus is a global health concern with up to 390 million individuals infected annually worldwide. There are no vaccines or antiviral compounds available to either prevent or treat dengue disease which may be fatal. To increase our understanding of the interaction of dengue virus with the host cell, we analyzed changes in the proteome of human A549 cells in response to dengue virus type 2 infection using stable isotope labelling in cell culture (SILAC) in combination with high-throughput mass spectrometry (MS). Mock and infected A549 cells were fractionated into nuclear and cytoplasmic extracts before analysis to identify proteins that redistribute between cellular compartments during infection and reduce the complexity of the analysis. We identified and quantified 3098 and 2115 proteins in the cytoplasmic and nuclear fractions respectively. Proteins that showed a significant alteration in amount during infection were examined using gene enrichment, pathway and network analysis tools. The analyses revealed that dengue virus infection modulated the amounts of proteins involved in the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection. PMID:24671231

  8. A Data Analysis Pipeline Accounting for Artifacts in Tox21 Quantitative High-Throughput Screening Assays.

    PubMed

    Hsieh, Jui-Hua; Sedykh, Alexander; Huang, Ruili; Xia, Menghang; Tice, Raymond R

    2015-08-01

    A main goal of the U.S. Tox21 program is to profile a 10K-compound library for activity against a panel of stress-related and nuclear receptor signaling pathway assays using a quantitative high-throughput screening (qHTS) approach. However, assay artifacts, including nonreproducible signals and assay interference (e.g., autofluorescence), complicate compound activity interpretation. To address these issues, we have developed a data analysis pipeline that includes an updated signal noise-filtering/curation protocol and an assay interference flagging system. To better characterize various types of signals, we adopted a weighted version of the area under the curve (wAUC) to quantify the amount of activity across the tested concentration range in combination with the assay-dependent point-of-departure (POD) concentration. Based on the 32 Tox21 qHTS assays analyzed, we demonstrate that signal profiling using wAUC affords the best reproducibility (Pearson's r = 0.91) in comparison with the POD (0.82) only or the AC(50) (i.e., half-maximal activity concentration, 0.81). Among the activity artifacts characterized, cytotoxicity is the major confounding factor; on average, about 8% of Tox21 compounds are affected, whereas autofluorescence affects less than 0.5%. To facilitate data evaluation, we implemented two graphical user interface applications, allowing users to rapidly evaluate the in vitro activity of Tox21 compounds. PMID:25904095

  9. Micro x-ray fluorescence as a high throughput screening method for metal chelating compounds

    NASA Astrophysics Data System (ADS)

    Minogue, Edel M.; Havrilla, George J.; Taylor, Tammy P.; Burrell, Anthony K.; Warner, Benjamin P.

    2005-06-01

    Micro X-ray Fluorescence (MXRF) has proven to be a powerful tool in the rapid and quantitative means of screening oliogpeptides. MXRF is a non-destructive method of analysis, which can detect elemental composition of a sample by measuring its characteristic X-ray emission wavelengths or energies. An effective high throughput screening technique is described for the rapid screening of bead-based libraries by MXRF in order to identify suitable chelating agents that will bind metals found in radioactive dispersive devices. It is a sensitive technique which in conjunction with the wide range of chemistry inherent in peptide libraries (e.g. varying charge, length, hydrophobicity, aromaticity etc.), provides a rapid and quantitative means for screening chelator-ion binding. The method involves the selection of a suitable library of ligands; in this case it is a bead-based library of peptides. The library is exposed to the cation of interest and immobilized on to a microarray. The array is then analyzed by MXRF enabling rapid identification of chelating agents. This enables the screening of approximately 27,500 sequences per day. Initial experiments carried out successfully identified sequences that are selective for Co under certain binding conditions. This involved the screening of 8,400 sequences in adverse environmental conditions containing possible interferences (e.g. Ca, Fe, Al, Cs, Ir), which could be encountered in our application.

  10. High-throughput liquid-absorption air-sampling apparatus and methods

    DOEpatents

    Zaromb, Solomon

    2000-01-01

    A portable high-throughput liquid-absorption air sampler [PHTLAAS] has an asymmetric air inlet through which air is drawn upward by a small and light-weight centrifugal fan driven by a direct current motor that can be powered by a battery. The air inlet is so configured as to impart both rotational and downward components of motion to the sampled air near said inlet. The PHTLAAS comprises a glass tube of relatively small size through which air passes at a high rate in a swirling, highly turbulent motion, which facilitates rapid transfer of vapors and particulates to a liquid film covering the inner walls of the tube. The pressure drop through the glass tube is <10 cm of water, usually <5 cm of water. The sampler's collection efficiency is usually >20% for vapors or airborne particulates in the 2-3.mu. range and >50% for particles larger than 4.mu.. In conjunction with various analyzers, the PHTLAAS can serve to monitor a variety of hazardous or illicit airborne substances, such as lead-containing particulates, tritiated water vapor, biological aerosols, or traces of concealed drugs or explosives.

  11. Advanced and High-Throughput Method for Mitochondrial Bioenergetics Evaluation in Neurotrauma.

    PubMed

    Pandya, Jignesh D; Sullivan, Patrick G; Leung, Lai Yee; Tortella, Frank C; Shear, Deborah A; Deng-Bryant, Ying

    2016-01-01

    Mitochondrial dysfunction is one of the key posttraumatic neuropathological events observed in various experimental models of traumatic brain injury (TBI). The extent of mitochondrial dysfunction has been associated with the severity and time course of secondary injury following brain trauma. Critically, several mitochondrial targeting preclinical drugs used in experimental TBI models have shown improved mitochondrial bioenergetics, together with cortical tissue sparing and cognitive behavioral outcome. Mitochondria, being a central regulator of cellular metabolic pathways and energy producer of cells, are of a great interest for researchers aiming to adopt cutting-edge methodology for mitochondrial bioenergetics assessment. The traditional way of mitochondrial bioenergetics analysis utilizing a Clark-type oxygen electrode (aka. oxytherm) is time-consuming and labor-intensive. In the present chapter, we describe an advanced and high-throughput method for mitochondrial bioenergetics assessments utilizing the Seahorse Biosciences XF(e)24 Flux Analyzer. This allows for simultaneous measurement of multiple samples with higher efficiency than the oxytherm procedure. This chapter provides helpful guidelines for conducting mitochondrial isolation and studying mitochondrial bioenergetics in brain tissue homogenates following experimental TBI. PMID:27604740

  12. WholePathwayScope: a comprehensive pathway-based analysis tool for high-throughput data

    PubMed Central

    Yi, Ming; Horton, Jay D; Cohen, Jonathan C; Hobbs, Helen H; Stephens, Robert M

    2006-01-01

    Background Analysis of High Throughput (HTP) Data such as microarray and proteomics data has provided a powerful methodology to study patterns of gene regulation at genome scale. A major unresolved problem in the post-genomic era is to assemble the large amounts of data generated into a meaningful biological context. We have developed a comprehensive software tool, WholePathwayScope (WPS), for deriving biological insights from analysis of HTP data. Result WPS extracts gene lists with shared biological themes through color cue templates. WPS statistically evaluates global functional category enrichment of gene lists and pathway-level pattern enrichment of data. WPS incorporates well-known biological pathways from KEGG (Kyoto Encyclopedia of Genes and Genomes) and Biocarta, GO (Gene Ontology) terms as well as user-defined pathways or relevant gene clusters or groups, and explores gene-term relationships within the derived gene-term association networks (GTANs). WPS simultaneously compares multiple datasets within biological contexts either as pathways or as association networks. WPS also integrates Genetic Association Database and Partial MedGene Database for disease-association information. We have used this program to analyze and compare microarray and proteomics datasets derived from a variety of biological systems. Application examples demonstrated the capacity of WPS to significantly facilitate the analysis of HTP data for integrative discovery. Conclusion This tool represents a pathway-based platform for discovery integration to maximize analysis power. The tool is freely available at . PMID:16423281

  13. A High Throughput Ambient Mass Spectrometric Approach to Species Identification and Classification from Chemical Fingerprint Signatures

    PubMed Central

    Musah, Rabi A.; Espinoza, Edgard O.; Cody, Robert B.; Lesiak, Ashton D.; Christensen, Earl D.; Moore, Hannah E.; Maleknia, Simin; Drijfhout, Falko P.

    2015-01-01

    A high throughput method for species identification and classification through chemometric processing of direct analysis in real time (DART) mass spectrometry-derived fingerprint signatures has been developed. The method entails introduction of samples to the open air space between the DART ion source and the mass spectrometer inlet, with the entire observed mass spectral fingerprint subjected to unsupervised hierarchical clustering processing. A range of both polar and non-polar chemotypes are instantaneously detected. The result is identification and species level classification based on the entire DART-MS spectrum. Here, we illustrate how the method can be used to: (1) distinguish between endangered woods regulated by the Convention for the International Trade of Endangered Flora and Fauna (CITES) treaty; (2) assess the origin and by extension the properties of biodiesel feedstocks; (3) determine insect species from analysis of puparial casings; (4) distinguish between psychoactive plants products; and (5) differentiate between Eucalyptus species. An advantage of the hierarchical clustering approach to processing of the DART-MS derived fingerprint is that it shows both similarities and differences between species based on their chemotypes. Furthermore, full knowledge of the identities of the constituents contained within the small molecule profile of analyzed samples is not required. PMID:26156000

  14. Perchlorate reduction by hydrogen autotrophic bacteria and microbial community analysis using high-throughput sequencing.

    PubMed

    Wan, Dongjin; Liu, Yongde; Niu, Zhenhua; Xiao, Shuhu; Li, Daorong

    2016-02-01

    Hydrogen autotrophic reduction of perchlorate have advantages of high removal efficiency and harmless to drinking water. But so far the reported information about the microbial community structure was comparatively limited, changes in the biodiversity and the dominant bacteria during acclimation process required detailed study. In this study, perchlorate-reducing hydrogen autotrophic bacteria were acclimated by hydrogen aeration from activated sludge. For the first time, high-throughput sequencing was applied to analyze changes in biodiversity and the dominant bacteria during acclimation process. The Michaelis-Menten model described the perchlorate reduction kinetics well. Model parameters q(max) and K(s) were 2.521-3.245 (mg ClO4(-)/gVSS h) and 5.44-8.23 (mg/l), respectively. Microbial perchlorate reduction occurred across at pH range 5.0-11.0; removal was highest at pH 9.0. The enriched mixed bacteria could use perchlorate, nitrate and sulfate as electron accepter, and the sequence of preference was: NO3(-) > ClO4(-) > SO4(2-). Compared to the feed culture, biodiversity decreased greatly during acclimation process, the microbial community structure gradually stabilized after 9 acclimation cycles. The Thauera genus related to Rhodocyclales was the dominated perchlorate reducing bacteria (PRB) in the mixed culture. PMID:26714962

  15. A Microliter-Scale High-throughput Screening System with Quantum-Dot Nanoprobes for Amyloid-β Aggregation Inhibitors

    PubMed Central

    Ishigaki, Yukako; Tanaka, Hiroyuki; Akama, Hiroaki; Ogara, Toshiki; Uwai, Koji; Tokuraku, Kiyotaka

    2013-01-01

    The aggregation of amyloid β protein (Aβ) is a key step in the pathogenesis of Alzheimer’s disease (AD), and therefore inhibitory substances for Aβ aggregation may have preventive and/or therapeutic potential for AD. Here we report a novel microliter-scale high-throughput screening system for Aβ aggregation inhibitors based on fluorescence microscopy-imaging technology with quantum-dot Nanoprobes. This screening system could be analyzed with a 5-µl sample volume when a 1536-well plate was used, and the inhibitory activity could be estimated as half-maximal effective concentrations (EC50). We attempted to comprehensively screen Aβ aggregation inhibitors from 52 spices using this system to assess whether this novel screening system is actually useful for screening inhibitors. Screening results indicate that approximately 90% of the ethanolic extracts from the spices showed inhibitory activity for Aβ aggregation. Interestingly, spices belonging to the Lamiaceae, the mint family, showed significantly higher activity than the average of tested spices. Furthermore, we tried to isolate the main inhibitory compound from Saturejahortensis, summer savory, a member of the Lamiaceae, using this system, and revealed that the main active compound was rosmarinic acid. These results demonstrate that this novel microliter-scale high-throughput screening system could be applied to the actual screening of Aβ aggregation inhibitors. Since this system can analyze at a microscopic scale, it is likely that further minimization of the system would easily be possible such as protein microarray technology. PMID:23991168

  16. SeqFold: genome-scale reconstruction of RNA secondary structure integrating high-throughput sequencing data.

    PubMed

    Ouyang, Zhengqing; Snyder, Michael P; Chang, Howard Y

    2013-02-01

    We present an integrative approach, SeqFold, that combines high-throughput RNA structure profiling data with computational prediction for genome-scale reconstruction of RNA secondary structures. SeqFold transforms experimental RNA structure information into a structure preference profile (SPP) and uses it to select stable RNA structure candidates representing the structure ensemble. Under a high-dimensional classification framework, SeqFold efficiently matches a given SPP to the most likely cluster of structures sampled from the Boltzmann-weighted ensemble. SeqFold is able to incorporate diverse types of RNA structure profiling data, including parallel analysis of RNA structure (PARS), selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), fragmentation sequencing (FragSeq) data generated by deep sequencing, and conventional SHAPE data. Using the known structures of a wide range of mRNAs and noncoding RNAs as benchmarks, we demonstrate that SeqFold outperforms or matches existing approaches in accuracy and is more robust to noise in experimental data. Application of SeqFold to reconstruct the secondary structures of the yeast transcriptome reveals the diverse impact of RNA secondary structure on gene regulation, including translation efficiency, transcription initiation, and protein-RNA interactions. SeqFold can be easily adapted to incorporate any new types of high-throughput RNA structure profiling data and is widely applicable to analyze RNA structures in any transcriptome. PMID:23064747

  17. High-Throughput Determination of Octanol-Water Partition Coefficients by Ultrasound-Assisted Liquid-Phase Microextraction.

    PubMed

    Guo, Yugao; Ma, Shuo; Guan, Shan; Zhang, Yan; Pan, Jie

    2015-09-01

    A method of high-throughput determination, which is based on ultrasound-assisted liquid-phase microextraction, was developed to measure directly the partition coefficients of n-octanol-water. In ultrasound-assisted liquid-phase microextraction, ultrasonic energy can facilitate the mass transfer process of six or more microextractors simultaneously. Therefore, high-throughput determination of n-octanol-water partition coefficients can be performed favorably, and the equilibrium time of each microextractor can be decreased effectively. Several experimental parameters including ultrasonic power and frequency, centrifugation conditions, extractant volume and sample concentration were analyzed and optimized at 25°C. Under the optimum conditions, it only takes 2 min to reach extraction equilibrium, and the solutions of sample can be separated by centrifugation in 4 min. After centrifugation, the concentrations in n-octanol phases are analyzed with gas chromatography. The method was further evaluated with eight reference compounds and the findings demonstrated that this method is suitable to determine the partition coefficients of organic compounds accurately and quickly. Next, the method was exploited to measure the partition coefficients of n-octanol-water containing 20 organic compounds, which cover the [Formula: see text] values from 0.05 to 4.36, with comparatively low relative standard deviation (RSD) directly. The results of this study illustrated that the RSD (n = 6) was under 3%. PMID:25583971

  18. In situ near-infrared (NIR) versus high-throughput mid-infrared (MIR) spectroscopy to monitor biopharmaceutical production.

    PubMed

    Sales, Kevin C; Rosa, Filipa; Sampaio, Pedro N; Fonseca, Luís P; Lopes, Marta B; Calado, Cecília R C

    2015-06-01

    protocols, due to possible space constraints or high costs associated with the use of multi-fiber optic probes for multi-bioreactors. In this case, MIR could be conducted in a high-throughput manner, analyzing hundreds of culture samples in a rapid and automatic mode. PMID:25955848

  19. High throughput chromatography strategies for potential use in the formal process characterization of a monoclonal antibody.

    PubMed

    Petroff, Matthew G; Bao, Haiying; Welsh, John P; van Beuningen-de Vaan, Miranda; Pollard, Jennifer M; Roush, David J; Kandula, Sunitha; Machielsen, Peter; Tugcu, Nihal; Linden, Thomas O

    2016-06-01

    High throughput experimental strategies are central to the rapid optimization of biologics purification processes. In this work, we extend common high throughput technologies towards the characterization of a multi-column chromatography process for a monoclonal antibody (mAb). Scale-down strategies were first evaluated by comparing breakthrough, retention, and performance (yields and clearance of aggregates and host cell protein) across miniature and lab scale columns. The process operating space was then evaluated using several integrated formats, with batch experimentation to define process testing ranges, miniature columns to evaluate the operating space, and comparison to traditional scale columns to establish scale-up correlations and verify the determined operating space. When compared to an independent characterization study at traditional lab column scale, the high throughput approach identified the same control parameters and similar process sensitivity. Importantly, the high throughput approach significantly decreased time and material needs while improving prediction robustness. Miniature columns and manufacturing scale centerpoint data comparisons support the validity of this approach, making the high throughput strategy an attractive and appropriate scale-down tool for the formal characterization of biotherapeutic processes in the future if regulatory acceptance of the miniature column data can be achieved. Biotechnol. Bioeng. 2016;113: 1273-1283. © 2015 Wiley Periodicals, Inc. PMID:26639315

  20. Compressed data organization for high throughput parallel entropy coding

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  1. AxonQuant: A Microfluidic Chamber Culture-Coupled Algorithm That Allows High-Throughput Quantification of Axonal Damage

    PubMed Central

    Li, Yang; Yang, Mengxue; Huang, Zhuo; Chen, Xiaoping; Maloney, Michael T.; Zhu, Li; Liu, Jianghong; Yang, Yanmin; Du, Sidan; Jiang, Xingyu; Wu, Jane Y.

    2014-01-01

    Published methods for imaging and quantitatively analyzing morphological changes in neuronal axons have serious limitations because of their small sample sizes, and their time-consuming and nonobjective nature. Here we present an improved microfluidic chamber design suitable for fast and high-throughput imaging of neuronal axons. We developed the Axon-Quant algorithm, which is suitable for automatic processing of axonal imaging data. This microfluidic chamber-coupled algorithm allows calculation of an ‘axonal continuity index’ that quantitatively measures axonal health status in a manner independent of neuronal or axonal density. This method allows quantitative analysis of axonal morphology in an automatic and nonbiased manner. Our method will facilitate large-scale high-throughput screening for genes or therapeutic compounds for neurodegenerative diseases involving axonal damage. When combined with imaging technologies utilizing different gene markers, this method will provide new insights into the mechanistic basis for axon degeneration. Our microfluidic chamber culture-coupled AxonQuant algorithm will be widely useful for studying axonal biology and neurodegenerative disorders. PMID:24603552

  2. A Quality Assurance Initiative for Commercial-Scale Production in High-Throughput Cryopreservation of Blue Catfish Sperm

    PubMed Central

    Hu, E; Liao, T. W.; Tiersch, T. R.

    2013-01-01

    Cryopreservation of fish sperm has been studied for decades at a laboratory (research) scale. However, high-throughput cryopreservation of fish sperm has recently been developed to enable industrial-scale production. This study treated blue catfish (Ictalurus furcatus) sperm high-throughput cryopreservation as a manufacturing production line and initiated quality assurance plan development. The main objectives were to identify: 1) the main production quality characteristics; 2) the process features for quality assurance; 3) the internal quality characteristics and their specification designs; 4) the quality control and process capability evaluation methods, and 5) the directions for further improvements and applications. The essential product quality characteristics were identified as fertility-related characteristics. Specification design which established the tolerance levels according to demand and process constraints was performed based on these quality characteristics. Meanwhile, to ensure integrity throughout the process, internal quality characteristics (characteristics at each quality control point within process) that could affect fertility-related quality characteristics were defined with specifications. Due to the process feature of 100% inspection (quality inspection of every fish), a specific calculation method, use of cumulative sum (CUSUM) control charts, was applied to monitor each quality characteristic. An index of overall process evaluation, process capacity, was analyzed based on in-control process and the designed specifications, which further integrates the quality assurance plan. With the established quality assurance plan, the process could operate stably and quality of products would be reliable. PMID:23872356

  3. Crosstalk between adjacent nanopores in a solid-state membrane array for multi-analyte high-throughput biomolecule detection

    NASA Astrophysics Data System (ADS)

    Raza, Muhammad Usman; Saleem, Sajid; Ali, Waqas; Iqbal, Samir M.

    2016-08-01

    Single nanopores are used to detect a variety of biological molecules. The modulations in ionic current under applied bias across the nanopore contain important information about translocating species, thus providing single analyte detection. These systems are, however, challenged in practical situations where multiple analytes have to be detected at high throughput. This paper presents the analysis of a multi-nanopore system that can be used for the detection of analytes with high throughput. As a scalable model, two nanopores were simulated in a single solid-state membrane. The interactions of the electric fields at the mouths of the individual nanopores were analyzed. The data elucidated the electrostatic properties of the nanopores from a single membrane and provided a framework to calculate the -3 dB distance, akin to the Debye length, from one nanopore to the other. This distance was the minimum distance between the adjacent nanopores such that their individual electric fields did not significantly interact with one another. The results can help in the optimal experimental design to construct solid-state nanopore arrays for any given nanopore size and applied bias.

  4. Targeted Biomarker Discovery by High Throughput Glycosylation Profiling of Human Plasma Alpha1-Antitrypsin and Immunoglobulin A

    PubMed Central

    Ruhaak, L. Renee; Koeleman, Carolien A. M.; Uh, Hae-Won; Stam, Jord C.; van Heemst, Diana; Maier, Andrea B.; Houwing-Duistermaat, Jeanine J.; Hensbergen, Paul J.; Slagboom, P. Eline; Deelder, André M.; Wuhrer, Manfred

    2013-01-01

    Protein N-glycosylation patterns are known to show vast genetic as well as physiological and pathological variation and represent a large pool of potential biomarkers. Large-scale studies are needed for the identification and validation of biomarkers, and the analytical techniques required have recently been developed. Such methods have up to now mainly been applied to complex mixtures of glycoproteins in biofluids (e.g. plasma). Here, we analyzed N-glycosylation profiles of alpha1-antitrypsin (AAT) and immunoglobulin A (IgA) enriched fractions by 96-well microtitration plate based high-throughput immuno-affinity capturing and N-glycan analysis using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). Human plasma samples were from the Leiden Longevity Study comprising 2415 participants of different chronological and biological ages. Glycosylation patterns of AAT enriched fractions were found to be associated with chronological (calendar) age and they differed between females and males. Moreover, several glycans in the AAT enriched fraction were associated with physiological parameters marking cardiovascular and metabolic diseases. Pronounced differences were found between males and females in the glycosylation profiles of IgA enriched fractions. Our results demonstrate that large-scale immuno-affinity capturing of proteins from human plasma using a bead-based method combined with high-throughput N-glycan analysis is a powerful tool for the discovery of glycosylation-based biomarker candidates. PMID:24039863

  5. The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond.

    PubMed

    Haslam, Carl; Hellicar, John; Dunn, Adrian; Fuetterer, Arne; Hardy, Neil; Marshall, Peter; Paape, Rainer; Pemberton, Michelle; Resemannand, Anja; Leveridge, Melanie

    2016-02-01

    Mass spectrometry (MS) offers a label-free, direct-detection method, in contrast to fluorescent or colorimetric methodologies. Over recent years, solid-phase extraction-based techniques, such as the Agilent RapidFire system, have emerged that are capable of analyzing samples in <10 s. While dramatically faster than liquid chromatography-coupled MS, an analysis time of 8-10 s is still considered relatively slow for full-diversity high-throughput screening (HTS). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) offers an alternative for high-throughput MS detection. However, sample preparation and deposition onto the MALDI target, as well as interference from matrix ions, have been considered limitations for the use of MALDI for screening assays. Here we describe the development and validation of assays for both small-molecule and peptide analytes using MALDI-TOF coupled with nanoliter liquid handling. Using the JMJD2c histone demethylase and acetylcholinesterase as model systems, we have generated robust data in a 1536 format and also increased sample deposition to 6144 samples per target. Using these methods, we demonstrate that this technology can deliver fast sample analysis time with low sample volume, and data comparable to that of current RapidFire assays. PMID:26428484

  6. Simulation modeling of high-throughput cryopreservation of aquatic germplasm: a case study of blue catfish sperm processing

    PubMed Central

    Hu, E; Liao, T. W.; Tiersch, T. R.

    2013-01-01

    Emerging commercial-level technology for aquatic sperm cryopreservation has not been modeled by computer simulation. Commercially available software (ARENA, Rockwell Automation, Inc. Milwaukee, WI) was applied to simulate high-throughput sperm cryopreservation of blue catfish (Ictalurus furcatus) based on existing processing capabilities. The goal was to develop a simulation model suitable for production planning and decision making. The objectives were to: 1) predict the maximum output for 8-hr workday; 2) analyze the bottlenecks within the process, and 3) estimate operational costs when run for daily maximum output. High-throughput cryopreservation was divided into six major steps modeled with time, resources and logic structures. The modeled production processed 18 fish and produced 1164 ± 33 (mean ± SD) 0.5-ml straws containing one billion cryopreserved sperm. Two such production lines could support all hybrid catfish production in the US and 15 such lines could support the entire channel catfish industry if it were to adopt artificial spawning techniques. Evaluations were made to improve efficiency, such as increasing scale, optimizing resources, and eliminating underutilized equipment. This model can serve as a template for other aquatic species and assist decision making in industrial application of aquatic germplasm in aquaculture, stock enhancement, conservation, and biomedical model fishes. PMID:25580079

  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. PMID:25180465

  8. Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts.

    PubMed

    Bae, Seunghee; An, In-Sook; An, Sungkwan

    2015-09-01

    Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fibroblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identification of putative materials regulating DNA repair in skin cells. First, we established a modified lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fibroblast WS-1 cells were infected with the modified lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The first step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity after pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain effective reagents identified in the first step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fibroblasts. PMID:26431110

  9. High-throughput Phenotyping of Lung Cancer Somatic Mutations.

    PubMed

    Berger, Alice H; Brooks, Angela N; Wu, Xiaoyun; Shrestha, Yashaswi; Chouinard, Candace; Piccioni, Federica; Bagul, Mukta; Kamburov, Atanas; Imielinski, Marcin; Hogstrom, Larson; Zhu, Cong; Yang, Xiaoping; Pantel, Sasha; Sakai, Ryo; Watson, Jacqueline; Kaplan, Nathan; Campbell, Joshua D; Singh, Shantanu; Root, David E; Narayan, Rajiv; Natoli, Ted; Lahr, David L; Tirosh, Itay; Tamayo, Pablo; Getz, Gad; Wong, Bang; Doench, John; Subramanian, Aravind; Golub, Todd R; Meyerson, Matthew; Boehm, Jesse S

    2016-08-01

    Recent genome sequencing efforts have identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood. Here we characterize 194 somatic mutations identified in primary lung adenocarcinomas. We present an expression-based variant-impact phenotyping (eVIP) method that uses gene expression changes to distinguish impactful from neutral somatic mutations. eVIP identified 69% of mutations analyzed as impactful and 31% as functionally neutral. A subset of the impactful mutations induces xenograft tumor formation in mice and/or confers resistance to cellular EGFR inhibition. Among these impactful variants are rare somatic, clinically actionable variants including EGFR S645C, ARAF S214C and S214F, ERBB2 S418T, and multiple BRAF variants, demonstrating that rare mutations can be functionally important in cancer. PMID:27478040

  10. High-Throughput Microfluidic Device for Rare Cell Isolation

    PubMed Central

    Yang, Daniel; Leong, Serena; Lei, Andy; Sohn, Lydia L.

    2016-01-01

    Enumerating and analyzing circulating tumor cells (CTCs)—cells that have been shed from primary solid tumors—can potentially be used to determine patient prognosis and track the progression of disease. There is a great challenge to create an effective platform that can isolate these cells, as they are extremely rare: only 1-10 CTCs are present in a 7.5mL of a cancer patient's peripheral blood. We have developed a novel microfluidic system that can isolate CTC populations label free. Our system consists of a multistage separator that employs inertial migration to sort cells based on size. We demonstrate the feasibility of our device by sorting colloids that are comparable in size to red blood cells (RBCs) and CTCs. PMID:26937065

  11. High-throughput cell analysis using multiplexed array technologies.

    PubMed

    Beske, Oren E; Goldbard, Simon

    2002-09-15

    The desire for more biologically relevant data from primary screening has resulted in a dramatic increase of cell-based assays in HTS labs. Consequently, new cell-array technologies are being developed to increase the quality and quantity of data emerging from such screens. These technologies take the form of both positional and non-positional formats, each with their own advantages. Notably, screens using these technologies generate databases of high-quality data that can be analyzed in ways currently not possible. The power of cell-based assays combined with new array and analytical technologies will enable the condensation of serial drug discovery processes, thereby decreasing the time and cost of taking a hit compound into clinical trials. Here, we compare array strategies being developed towards the goal of integrating multiplexed cell-based assays into HTS. PMID:12546879

  12. High-throughput microfluidic device for rare cell isolation

    NASA Astrophysics Data System (ADS)

    Yang, Daniel; Leong, Serena; Lei, Andy; Sohn, Lydia L.

    2015-06-01

    Enumerating and analyzing circulating tumor cells (CTCs)—cells that have been shed from primary solid tumors—can potentially be used to determine patient prognosis and track the progression of disease. There is a great challenge to create an effective platform that can isolate these cells, as they are extremely rare: only 1-10 CTCs are present in a 7.5mL of a cancer patient's peripheral blood. We have developed a novel microfluidic system that can isolate CTC populations label free. Our system consists of a multistage separator that employs inertial migration to sort cells based on size. We demonstrate the feasibility of our device by sorting colloids that are comparable in size to red blood cells (RBCs) and CTCs.

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

    PubMed

    Almada, Pedro; Culley, Siân; Henriques, Ricardo

    2015-10-15

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

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

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

  18. Development and Validation of an Automated High-Throughput System for Zebrafish In Vivo Screenings

    PubMed Central

    Virto, Juan M.; Holgado, Olaia; Diez, Maria; Izpisua Belmonte, Juan Carlos; Callol-Massot, Carles

    2012-01-01

    The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism. PMID:22615792

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

  20. High-throughput imaging: Focusing in on drug discovery in 3D.

    PubMed

    Li, Linfeng; Zhou, Qiong; Voss, Ty C; Quick, Kevin L; LaBarbera, Daniel V

    2016-03-01

    3D organotypic culture models such as organoids and multicellular tumor spheroids (MCTS) are becoming more widely used for drug discovery and toxicology screening. As a result, 3D culture technologies adapted for high-throughput screening formats are prevalent. While a multitude of assays have been reported and validated for high-throughput imaging (HTI) and high-content screening (HCS) for novel drug discovery and toxicology, limited HTI/HCS with large compound libraries have been reported. Nonetheless, 3D HTI instrumentation technology is advancing and this technology is now on the verge of allowing for 3D HCS of thousands of samples. This review focuses on the state-of-the-art high-throughput imaging systems, including hardware and software, and recent literature examples of 3D organotypic culture models employing this technology for drug discovery and toxicology screening. PMID:26608110

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-05-19

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

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

    PubMed

    Iles, LaKesla R; Bartholomeusz, Geoffrey A

    2016-01-01

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

  4. BioAssay Ontology (BAO): a semantic description of bioassays and high-throughput screening results

    PubMed Central

    2011-01-01

    Background High-throughput screening (HTS) is one of the main strategies to identify novel entry points for the development of small molecule chemical probes and drugs and is now commonly accessible to public sector research. Large amounts of data generated in HTS campaigns are submitted to public repositories such as PubChem, which is growing at an exponential rate. The diversity and quantity of available HTS assays and screening results pose enormous challenges to organizing, standardizing, integrating, and analyzing the datasets and thus to maximize the scientific and ultimately the public health impact of the huge investments made to implement public sector HTS capabilities. Novel approaches to organize, standardize and access HTS data are required to address these challenges. Results We developed the first ontology to describe HTS experiments and screening results using expressive description logic. The BioAssay Ontology (BAO) serves as a foundation for the standardization of HTS assays and data and as a semantic knowledge model. In this paper we show important examples of formalizing HTS domain knowledge and we point out the advantages of this approach. The ontology is available online at the NCBO bioportal http://bioportal.bioontology.org/ontologies/44531. Conclusions After a large manual curation effort, we loaded BAO-mapped data triples into a RDF database store and used a reasoner in several case studies to demonstrate the benefits of formalized domain knowledge representation in BAO. The examples illustrate semantic querying capabilities where BAO enables the retrieval of inferred search results that are relevant to a given query, but are not explicitly defined. BAO thus opens new functionality for annotating, querying, and analyzing HTS datasets and the potential for discovering new knowledge by means of inference. PMID:21702939

  5. EpiRegNet: constructing epigenetic regulatory network from high throughput gene expression data for humans.

    PubMed

    Wang, Lily Yan; Wang, Panwen; Li, Mulin Jun; Qin, Jing; Wang, Xiaowo; Zhang, Michael Q; Wang, Junwen

    2011-12-01

    The advances of high throughput profiling methods, such as microarray gene profiling and RNA-seq, have enabled researchers to identify thousands of differentially expressed genes under a certain perturbation. Much work has been done to understand the genetic factors that contribute to the expression changes by searching the over-represented regulatory motifs in the promoter regions of these genes. However, the changes could also be caused by epigenetic regulation, especially histone modifications, and no web server has been constructed to study the epigenetic factors responsible for gene expression changes. Here, we pre-sent a web tool for this purpose. Provided with different categories of genes (e.g., up-regulated, down-regulated or unchanged genes), the server will find epigenetic factors responsible for the difference among the categories and construct an epigenetic regulatory network. Furthermore, it will perform co-localization analyses between these epigenetic factors and transcription factors, which were collected from large scale experimental ChIP-seq or computational predicted data. In addition, for users who want to analyze dynamic change of a histone modification mark under different cell conditions, the server will find direct and indirect target genes of this mark by integrative analysis of experimental data and computational prediction, and present a regulatory network around this mark. Both networks can be visualized by a user friendly interface and the data are downloadable in batch. The server currently supports 12 cell types in human, including ESC and CD4+ T cells, and will expand as more public data are available. It also allows user to create a self-defined cell type, upload and analyze multiple ChIP-seq data. It is freely available to academic users at http://jjwanglab.org/EpiRegNet. PMID:22139581

  6. Determination of urine cofilin-1 level in acute kidney injury using a high-throughput localized surface plasmon-coupled fluorescence biosensor

    NASA Astrophysics Data System (ADS)

    Chang, Ying-Feng; Chao, Cheng-Han; Lin, Lih-Yuan; Tsai, Cheng-Han; Chou, Chien; Lee, Yi-Jang

    2014-01-01

    The actin-depolymerizing factor (ADF)/cofilin protein family has been reported to be associated with ischemia-induced renal disorders. We examine whether cofilin-1 is associated with acute kidney injury (AKI) using human urine samples. We exploited a 96-well based high-throughput biosensor that uses gold nanoparticles and a sandwich immunoassay to detect the urine cofilin-1 level of AKI patients. The mean urine cofilin-1 level of the AKI patients (n=37 from 47 cases analyzed) was twofold higher than that of healthy adults (n=21 from 29 cases analyzed). The receiver operating characteristic (ROC) curve showed that cofilin-1 was acceptable for discriminating AKI patients from healthy adults. However, an increase of the sample size is required to conclude the importance of urine cofilin-1 on AKI diagnosis, and the high-throughput ultrasensitive biosensor used in this study would greatly accelerate the measurement of urine cofilin-1 in an increased sample size.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  8. Self-Assembled Cell Microarray (SAMcell) for High-Throughput RNAi Screening.

    PubMed

    Zhang, Hanshuo; Li, Juan

    2016-01-01

    RNAi has now become a valuable research tool for cell-based high-throughput screening. However, traditional RNAi high-throughput methods are based on multi-well plates, relying on expensive instruments and complicated operations. In this chapter, we describe a method termed self-assembled cell microarray (SAMcell), which integrates micro-fabrication, reverse transfection, and RNAi technologies and allows for cell behavior investigations to be performed directly on the cell chip. This method has been successfully employed to perform large-scale functional screening assays to identify gene modulators of cell migration, cell proliferation, and cellular apoptosis. PMID:27581287

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

    SciTech Connect

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

    2004-06-01

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

  10. Express primer tool for high-throughput gene cloning and expression.

    SciTech Connect

    Yoon, J. R.; Laible, P. D.; Gu, M.; Scott, H. N.; Collart, F. R.; Biosciences Division

    2002-12-01

    High-throughput approaches for gene cloning and expression require the development of new nonstandard tools for molecular biologists and biochemists. We introduce a Web-based tool to design primers specifically for the generation of expression clones for both laboratory-scale and high-throughput projects. The application is designed not only to allow the user complete flexibility to specify primer design parameters but also to minimize the amount of manual intervention needed to generate a large number of primers for the simultaneous amplification of multiple target genes.

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

    PubMed Central

    Fowler, Tyler L.; Bailey, Alison M.; Bednarz, Bryan P.; Kimple, Randall J.

    2015-01-01

    Assessment of γH2AX expression for studying DNA double-strand break formation is often performed by manual counting of foci using immunofluorescence microscopy, an approach that is laborious and subject to significant foci selection bias. Here we present a novel high-throughput method for detecting DNA double-strand breaks using automated image cytometry assessment of cell average γH2AX immunofluorescence. Our technique provides an expedient, high-throughput, objective, and cost-effective method for γH2AX analysis. PMID:25605579

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

    PubMed

    Fowler, Tyler L; Bailey, Alison M; Bednarz, Bryan P; Kimple, Randall J

    2015-01-01

    Assessment of γH2AX expression for studying DNA double-strand break formation is often performed by manual counting of foci using immunofluorescence microscopy, an approach that is laborious and subject to significant foci selection bias. Here we present a novel high-throughput method for detecting DNA double-strand breaks using automated image cytometry assessment of cell average γH2AX immunofluorescence. Our technique provides an expedient, high-throughput, objective, and cost-effective method for γH2AX analysis. PMID:25605579

  13. High throughput materials research and development: a growing effort at NIST

    NASA Astrophysics Data System (ADS)

    Hewes, John D.; Bendersky, Leonid A.

    2002-04-01

    The US private sector has indicated that the National Institute of Standards and Technology (NIST) has a role to play in facilitating the implementation of high throughput experimentation, e.g. using combinatorial methods. The Advanced Technology Program (ATP) is currently funding research projects in high throughput discovery of catalysts, electronic materials, and polymer coatings internally and externally to NIST. The NIST Measurement and Standards Laboratories (MSL) is actively pursuing research in electronic materials and organic polymers. This presentation presents details on these efforts at NIST.

  14. Accelerating Virtual High-Throughput Ligand Docking: current technology and case study on a petascale supercomputer

    PubMed Central

    Ellingson, Sally R.; Dakshanamurthy, Sivanesan; Brown, Milton; Smith, Jeremy C.

    2013-01-01

    In this paper we give the current state of high-throughput virtual screening. We describe a case study of using a task-parallel MPI (Message Passing Interface) version of Autodock4 [1], [2] to run a virtual high-throughput screen of one-million compounds on the Jaguar Cray XK6 Supercomputer at Oak Ridge National Laboratory. We include a description of scripts developed to increase the efficiency of the predocking file preparation and postdocking analysis. A detailed tutorial, scripts, and source code for this MPI version of Autodock4 are available online at http://www.bio.utk.edu/baudrylab/autodockmpi.htm. PMID:24729746

  15. 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.

  16. Preparation of cDNA libraries for high-throughput RNA sequencing analysis of RNA 5′ ends

    PubMed Central

    Vvedenskaya, Irina O.; Goldman, Seth R.; Nickels, Bryce E.

    2015-01-01

    Summary We provide a detailed protocol for preparing cDNA libraries suitable for high throughput sequencing that are derived specifically from the 5′ ends of RNA (5′ specific RNA-seq). The protocol describes how cDNA libraries for 5′ specific RNA-seq can be tailored to analyze specific classes of RNAs based upon the phosphorylation status of the 5′ end. Thus, the analysis of cDNA libraries generated by these methods provides information regarding both the sequence and phosphorylation status of the 5′ ends of RNAs. 5′ specific RNA-seq can be used to analyze transcription initiation and post-transcriptional processing of RNAs with single base pair resolution on a genome-wide level. PMID:25665566

  17. An LC-IMS-MS Platform Providing Increased Dynamic Range for High-Throughput Proteomic Studies

    PubMed Central

    Baker, Erin Shammel; Livesay, Eric A.; Orton, Daniel J.; Moore, Ronald J.; Danielson, William F.; Prior, David C.; Ibrahim, Yehia M.; LaMarche, Brian L.; Mayampurath, Anoop M.; Schepmoes, Athena A.; Hopkins, Derek F.; Tang, Keqi; Smith, Richard D.; Belov, Mikhail E.

    2010-01-01

    A high-throughput approach and platform using 15 minute reversed-phase capillary liquid chromatography (RPLC) separations in conjunction with ion mobility spectrometry-mass spectrometry (IMS-MS) measurements was evaluated for the rapid analysis of complex proteomics samples. To test the separation quality of the short LC gradient, a sample was prepared by spiking twenty reference peptides at varying concentrations from 1 ng/mL to 10 µg/mL into a tryptic digest of mouse blood plasma and analyzed with both a LC-Linear Ion Trap Fourier Transform (FT) MS and LC-IMS-TOF MS. The LC-FT MS detected thirteen out of the twenty spiked peptides that had concentrations ≥100 ng/mL. In contrast, the drift time selected mass spectra from the LC-IMS-TOF MS analyses yielded identifications for nineteen of the twenty peptides with all spiking levels present. The greater dynamic range of the LC-IMS-TOF MS system could be attributed to two factors. First, the LC-IMS-TOF MS system enabled drift time separation of the low concentration spiked peptides from the high concentration mouse peptide matrix components, reducing signal interference and background, and allowing species to be resolved that would otherwise be obscured by other components. Second, the automatic gain control (AGC) in the linear ion trap of the hybrid FT MS instrument limits the number of ions that are accumulated to reduce space charge effects and achieve high measurement accuracy, but in turn limits the achievable dynamic range compared to the IMS-TOF instrument. PMID:20000344

  18. High-Throughput Sequencing—The Key to Rapid Biodiversity Assessment of Marine Metazoa?

    PubMed Central

    Mohrbeck, Inga; Raupach, Michael J.; Martínez Arbizu, Pedro; Knebelsberger, Thomas; Laakmann, Silke

    2015-01-01

    The applications of traditional morphological and molecular methods for species identification are greatly restricted by processing speed and on a regional or greater scale are generally considered unfeasible. In this context, high-throughput sequencing, or metagenetics, has been proposed as an efficient tool to document biodiversity. Here we evaluated the effectiveness of 454 pyrosequencing in marine metazoan community analysis using the 18S rDNA: V1-V2 region. Multiplex pyrosequencing of the V1-V2 region was used to analyze two pooled samples of DNA, one comprising 118 and the other 37 morphologically identified species, and one natural sample taken directly from a North Sea zooplankton community. A DNA reference library comprising all species represented in the pooled samples was created by Sanger sequencing, and this was then used to determine the optimal similarity threshold for species delineation. The optimal threshold was found at 99% species similarity, with 85% identification success. Pyrosequencing was able to identify between fewer species: 67% and 78% of the species in the two pooled samples. Also, a large number of sequences for three species that were not included in the pooled samples were amplified by pyrosequencing, suggesting preferential amplification of some genotypes and the sensitivity of this approach to even low levels of contamination. Conversely, metagenetic analysis of the natural zooplankton sample identified many more species (particularly gelatinous zooplankton and meroplankton) than morphological analysis of a formalin-fixed sample from the same sampling site, suggesting an increased level of taxonomic resolution with pyrosequencing. The study demonstrated that, based on the V1-V2 region, 454 sequencing does not provide accurate species differentiation and reliable taxonomic classification, as it is required in most biodiversity monitoring. The analysis of artificially prepared samples indicated that species detection in

  19. 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.

  20. Gaussian process test for high-throughput sequencing time series: application to experimental evolution

    PubMed Central

    Topa, Hande; Jónás, Ágnes; Kofler, Robert; Kosiol, Carolin; Honkela, Antti

    2015-01-01

    Motivation: Recent advances in high-throughput sequencing (HTS) have made it possible to monitor genomes in great detail. New experiments not only use HTS to measure genomic features at one time point but also monitor them changing over time with the aim of identifying significant changes in their abundance. In population genetics, for example, allele frequencies are monitored over time to detect significant frequency changes that indicate selection pressures. Previous attempts at analyzing data from HTS experiments have been limited as they could not simultaneously include data at intermediate time points, replicate experiments and sources of uncertainty specific to HTS such as sequencing depth. Results: We present the beta-binomial Gaussian process model for ranking features with significant non-random variation in abundance over time. The features are assumed to represent proportions, such as proportion of an alternative allele in a population. We use the beta-binomial model to capture the uncertainty arising from finite sequencing depth and combine it with a Gaussian process model over the time series. In simulations that mimic the features of experimental evolution data, the proposed method clearly outperforms classical testing in average precision of finding selected alleles. We also present simulations exploring different experimental design choices and results on real data from Drosophila experimental evolution experiment in temperature adaptation. Availability and implementation: R software implementing the test is available at https://github.com/handetopa/BBGP. Contact: hande.topa@aalto.fi, agnes.jonas@vetmeduni.ac.at, carolin.kosiol@vetmeduni.ac.at, antti.honkela@hiit.fi Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25614471

  1. Soft inertial microfluidics for high throughput separation of bacteria from human blood cells

    SciTech Connect

    Wu, Zhigang; Willing, Ben; Bjerketorp, Joakim; Jansson, Janet K.; Hjort, Klas

    2009-01-05

    We developed a new approach to separate bacteria from human blood cells based on soft inertial force induced migration with flow defined curved and focused sample flow inside a microfluidic device. This approach relies on a combination of an asymmetrical sheath flow and proper channel geometry to generate a soft inertial force on the sample fluid in the curved and focused sample flow segment to deflect larger particles away while the smaller ones are kept on or near the original flow streamline. The curved and focused sample flow and inertial effect were visualized and verified using a fluorescent dye primed in the device. First the particle behavior was studied in detail using 9.9 and 1.0 {micro}m particles with a polymer-based prototype. The prototype device is compact with an active size of 3 mm{sup 2}. The soft inertial effect and deflection distance were proportional to the fluid Reynolds number (Re) and particle Reynolds number (Re{sub p}), respectively. We successfully demonstrated separation of bacteria (Escherichia coli) from human red blood cells at high cell concentrations (above 10{sup 8}/mL), using a sample flow rate of up to 18 {micro}L/min. This resulted in at least a 300-fold enrichment of bacteria at a wide range of flow rates with a controlled flow spreading. The separated cells were proven to be viable. Proteins from fractions before and after cell separation were analyzed by gel electrophoresis and staining to verify the removal of red blood cell proteins from the bacterial cell fraction. This novel microfluidic process is robust, reproducible, simple to perform, and has a high throughput compared to other cell sorting systems. Microfluidic systems based on these principles could easily be manufactured for clinical laboratory and biomedical applications.

  2. Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis.

    PubMed

    Rühmann, Broder; Schmid, Jochen; Sieber, Volker

    2016-01-01

    Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge

  3. An LC-IMS-MS Platform Providing Increased Dynamic Range for High-Throughput Proteomic Studies

    SciTech Connect

    Baker, Erin Shammel; Livesay, Eric A.; Orton, Daniel J.; Moore, Ronald J.; Danielson, William F.; Prior, David C.; Ibrahim, Yehia M.; Lamarche, Brian L.; Mayampurath, Anoop M.; Schepmoes, Athena A.; Hopkins, Derek F.; Tang, Keqi; Smith, Richard D.; Belov, Mikhail E.

    2010-02-05

    A high-throughput approach and platform using 15 minute reversed-phase capillary liquid chromatography (RPLC) separations in conjunction with ion mobility spectrometry-mass spectrometry (IMS-MS) measurements was evaluated for the rapid analysis of complex proteomics samples. To test the separation quality of the short LC gradient, a sample was prepared by spiking twenty reference peptides at varying concentrations from 1 ng/mL to 10 µg/mL into a tryptic digest of mouse blood plasma and analyzed with both a LC-Linear Ion Trap Fourier Transform (FT) MS and LC-IMS-TOF MS. The LC-FT MS detected thirteen out of the twenty spiked peptides that had concentrations ≥100 ng/mL. In contrast, the drift time selected mass spectra from the LC-IMS-TOF MS analyses yielded identifications for nineteen of the twenty peptides with all spiking level present. The greater dynamic range of the LC-IMS-TOF MS system could be attributed to two factors. First, the LC-IMS-TOF MS system enabled drift time separation of the low concentration spiked peptides from the high concentration mouse peptide matrix components, reducing signal interference and background, and allowing species to be resolved that would otherwise be obscured by other components. Second, the automatic gain control (AGC) in the linear ion trap of the hybrid FT MS instrument limits the number of ions that are accumulated to reduce space charge effects, but in turn limits the achievable dynamic range compared to the TOF detector.

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

    PubMed

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

    2013-01-01

    : The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening. PMID:23938087

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

    PubMed Central

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

    2011-01-01

    High-throughput X-ray absorption spectroscopy was used to measure transition metal content based on quantitative detection of X-ray fluorescence signals for 3879 purified proteins from several hundred different protein families generated by the New York SGX Research Center for Structural Genomics. Approximately 9% of the proteins analyzed showed the presence of transition metal atoms (Zn, Cu, Ni, Co, Fe, or Mn) in stoichiometric amounts. The method is highly automated and highly reliable based on comparison of the results to crystal structure data derived from the same protein set. To leverage the experimental metalloprotein annotations, we used a sequence-based de novo prediction method, MetalDetector, to identify Cys and His residues that bind to transition metals for the redundancy reduced subset of 2411 sequences sharing <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. PMID:21482623

  6. Gaussian graphical modeling reconstructs pathway reactions from high-throughput metabolomics data

    PubMed Central

    2011-01-01

    Background With the advent of high-throughput targeted metabolic profiling techniques, the question of how to interpret and analyze the resulting vast amount of data becomes more and more important. In this work we address the reconstruction of metabolic reactions from cross-sectional metabolomics data, that is without the requirement for time-resolved measurements or specific system perturbations. Previous studies in this area mainly focused on Pearson correlation coefficients, which however are generally incapable of distinguishing between direct and indirect metabolic interactions. Results In our new approach we propose the application of a Gaussian graphical model (GGM), an undirected probabilistic graphical model estimating the conditional dependence between variables. GGMs are based on partial correlation coefficients, that is pairwise Pearson correlation coefficients conditioned against the correlation with all other metabolites. We first demonstrate the general validity of the method and its advantages over regular correlation networks with computer-simulated reaction systems. Then we estimate a GGM on data from a large human population cohort, covering 1020 fasting blood serum samples with 151 quantified metabolites. The GGM is much sparser than the correlation network, shows a modular structure with respect to metabolite classes, and is stable to the choice of samples in the data set. On the example of human fatty acid metabolism, we demonstrate for the first time that high partial correlation coefficients generally correspond to known metabolic reactions. This feature is evaluated both manually by investigating specific pairs of high-scoring metabolites, and then systematically on a literature-curated model of fatty acid synthesis and degradation. Our method detects many known reactions along with possibly novel pathway interactions, representing candidates for further experimental examination. Conclusions In summary, we demonstrate strong signatures of

  7. High throughput determination of cleaning solutions to prevent the fouling of an anion exchange resin.

    PubMed

    Elich, Thomas; Iskra, Timothy; Daniels, William; Morrison, Christopher J

    2016-06-01

    Effective cleaning of chromatography resin is required to prevent fouling and maximize the number of processing cycles which can be achieved. Optimization of resin cleaning procedures, however, can lead to prohibitive material, labor, and time requirements, even when using milliliter scale chromatography columns. In this work, high throughput (HT) techniques were used to evaluate cleaning agents for a monoclonal antibody (mAb) polishing step utilizing Fractogel(®) EMD TMAE HiCap (M) anion exchange (AEX) resin. For this particular mAb feed stream, the AEX resin could not be fully restored with traditional NaCl and NaOH cleaning solutions, resulting in a loss of impurity capacity with resin cycling. Miniaturized microliter scale chromatography columns and an automated liquid handling system (LHS) were employed to evaluate various experimental cleaning conditions. Cleaning agents were monitored for their ability to maintain resin impurity capacity over multiple processing cycles by analyzing the flowthrough material for turbidity and high molecular weight (HMW) content. HT experiments indicated that a 167 mM acetic acid strip solution followed by a 0.5 M NaOH, 2 M NaCl sanitization provided approximately 90% cleaning improvement over solutions containing solely NaCl and/or NaOH. Results from the microliter scale HT experiments were confirmed in subsequent evaluations at the milliliter scale. These results identify cleaning agents which may restore resin performance for applications involving fouling species in ion exchange systems. In addition, this work demonstrates the use of miniaturized columns operated with an automated LHS for HT evaluation of chromatographic cleaning procedures, effectively decreasing material requirements while simultaneously increasing throughput. Biotechnol. Bioeng. 2016;113: 1251-1259. © 2015 Wiley Periodicals, Inc. PMID:26552005

  8. ZebraZoom: an automated program for high-throughput behavioral analysis and categorization

    PubMed Central

    Mirat, Olivier; Sternberg, Jenna R.; Severi, Kristen E.; Wyart, Claire

    2013-01-01

    The zebrafish larva stands out as an emergent model organism for translational studies involving gene or drug screening thanks to its size, genetics, and permeability. At the larval stage, locomotion occurs in short episodes punctuated by periods of rest. Although phenotyping behavior is a key component of large-scale screens, it has not yet been automated in this model system. We developed ZebraZoom, a program to automatically track larvae and identify maneuvers for many animals performing discrete movements. Our program detects each episodic movement and extracts large-scale statistics on motor patterns to produce a quantification of the locomotor repertoire. We used ZebraZoom to identify motor defects induced by a glycinergic receptor antagonist. The analysis of the blind mutant atoh7 revealed small locomotor defects associated with the mutation. Using multiclass supervised machine learning, ZebraZoom categorized all episodes of movement for each larva into one of three possible maneuvers: slow forward swim, routine turn, and escape. ZebraZoom reached 91% accuracy for categorization of stereotypical maneuvers that four independent experimenters unanimously identified. For all maneuvers in the data set, ZebraZoom agreed with four experimenters in 73.2–82.5% of cases. We modeled the series of maneuvers performed by larvae as Markov chains and observed that larvae often repeated the same maneuvers within a group. When analyzing subsequent maneuvers performed by different larvae, we found that larva–larva interactions occurred as series of escapes. Overall, ZebraZoom reached the level of precision found in manual analysis but accomplished tasks in a high-throughput format necessary for large screens. PMID:23781175

  9. A high-throughput diagnostic method for measuring human exposure to organophosphorus nerve agents.

    PubMed

    Knaack, Jennifer S; Zhou, Yingtao; Abney, Carter W; Jacob, Justin T; Prezioso, Samantha M; Hardy, Katelyn; Lemire, Sharon W; Thomas, Jerry; Johnson, Rudolph C

    2012-11-01

    An automated high-throughput immunomagnetic separation (IMS) method for diagnosing exposure to the organophosphorus nerve agents (OPNAs) sarin (GB), cyclohexylsarin (GF), VX, and Russian VX (RVX) was developed to increase sample processing capacity for emergency response applications. Diagnosis of exposure to OPNAs was based on the formation of OPNA adducts to butyrylcholinesterase (BuChE). Data reported with this method represent a ratio of the agent-specific BuChE adduct concentration, relative to the total BuChE peptide concentration that provides a nonactivity measurement expressed as percent adducted. All magnetic bead transfer steps and washes were performed using instrumentation in a 96-well format allowing for simultaneous extraction of 86 clinical samples plus reference materials. Automating extractions increased sample throughput 50-fold, as compared to a previously reported manual method. The limits of detection, determined using synthetic peptides, were 1 ng/mL for unadducted BuChE and GB-, GF-, VX-, and RVX-adducted BuChE. The automated method was characterized using unexposed serum and serum pools exposed to GB, GF, VX, or RVX. Variation for the measurement of percent adducted was <12% for all characterized quality control serum pools. Twenty-six (26) serum samples from individuals asymptomatic for cholinesterase inhibitor exposure were analyzed using this method, and no background levels of OPNA exposure were observed. Unexposed BuChE serum concentrations measured using this method ranged from 2.8 μg/mL to 10.6 μg/mL, with an average concentration of 6.4 μg/mL. PMID:23083472

  10. Identification of MicroRNAs from Eugenia uniflora by High-Throughput Sequencing and Bioinformatics Analysis

    PubMed Central

    Guzman, Frank; Almerão, Mauricio P.; Körbes, Ana P.; Loss-Morais, Guilherme; Margis, Rogerio

    2012-01-01

    Background microRNAs or miRNAs are small non-coding regulatory RNAs that play important functions in the regulation of gene expression at the post-transcriptional level by targeting mRNAs for degradation or inhibiting protein translation. Eugenia uniflora is a plant native to tropical America with pharmacological and ecological importance, and there have been no previous studies concerning its gene expression and regulation. To date, no miRNAs have been reported in Myrtaceae species. Results Small RNA and RNA-seq libraries were constructed to identify miRNAs and pre-miRNAs in Eugenia uniflora. Solexa technology was used to perform high throughput sequencing of the library, and the data obtained were analyzed using bioinformatics tools. From 14,489,131 small RNA clean reads, we obtained 1,852,722 mature miRNA sequences representing 45 conserved families that have been identified in other plant species. Further analysis using contigs assembled from RNA-seq allowed the prediction of secondary structures of 25 known and 17 novel pre-miRNAs. The expression of twenty-seven identified miRNAs was also validated using RT-PCR assays. Potential targets were predicted for the most abundant mature miRNAs in the identified pre-miRNAs based on sequence homology. Conclusions This study is the first large scale identification of miRNAs and their potential targets from a species of the Myrtaceae family without genomic sequence resources. Our study provides more information about the evolutionary conservation of the regulatory network of miRNAs in plants and highlights species-specific miRNAs. PMID:23166775

  11. Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

    PubMed Central

    Rühmann, Broder; Schmid, Jochen; Sieber, Volker

    2016-01-01

    Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge

  12. A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers.

    PubMed

    Avonto, Cristina; Chittiboyina, Amar G; Rua, Diego; Khan, Ikhlas A

    2015-12-01

    Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles after incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, 'HTS-DCYA assay', is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time. PMID:26455772

  13. High-Throughput Sequencing-The Key to Rapid Biodiversity Assessment of Marine Metazoa?

    PubMed

    Mohrbeck, Inga; Raupach, Michael J; Martínez Arbizu, Pedro; Knebelsberger, Thomas; Laakmann, Silke

    2015-01-01

    The applications of traditional morphological and molecular methods for species identification are greatly restricted by processing speed and on a regional or greater scale are generally considered unfeasible. In this context, high-throughput sequencing, or metagenetics, has been proposed as an efficient tool to document biodiversity. Here we evaluated the effectiveness of 454 pyrosequencing in marine metazoan community analysis using the 18S rDNA: V1-V2 region. Multiplex pyrosequencing of the V1-V2 region was used to analyze two pooled samples of DNA, one comprising 118 and the other 37 morphologically identified species, and one natural sample taken directly from a North Sea zooplankton community. A DNA reference library comprising all species represented in the pooled samples was created by Sanger sequencing, and this was then used to determine the optimal similarity threshold for species delineation. The optimal threshold was found at 99% species similarity, with 85% identification success. Pyrosequencing was able to identify between fewer species: 67% and 78% of the species in the two pooled samples. Also, a large number of sequences for three species that were not included in the pooled samples were amplified by pyrosequencing, suggesting preferential amplification of some genotypes and the sensitivity of this approach to even low levels of contamination. Conversely, metagenetic analysis of the natural zooplankton sample identified many more species (particularly gelatinous zooplankton and meroplankton) than morphological analysis of a formalin-fixed sample from the same sampling site, suggesting an increased level of taxonomic resolution with pyrosequencing. The study demonstrated that, based on the V1-V2 region, 454 sequencing does not provide accurate species differentiation and reliable taxonomic classification, as it is required in most biodiversity monitoring. The analysis of artificially prepared samples indicated that species detection in

  14. A high-throughput, precipitating colorimetric sandwich ELISA microarray for shiga toxins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies)...

  15. A novel high-throughput irradiator for in vitro radiation sensitivity bioassays

    NASA Astrophysics Data System (ADS)

    Fowler, Tyler L.

    Given the emphasis on more personalized radiation therapy there is an ongoing and compelling need to develop high-throughput screening tools to further examine the biological effects of ionizing radiation on cells, tissues and organ systems in either the research or clinical setting. Conventional x-ray irradiators are designed to provide maximum versatility to radiobiology researchers, typically accommodating small animals, tissue or blood samples, and cellular applications. This added versatility often impedes the overall sensitivity and specificity of an experiment resulting in a trade-off between the number of absorbed doses (or dose rates) and biological endpoints that can be investigated in vitro in a reasonable amount of time. Therefore, modern irradiator designs are incompatible with current high-throughput bioassay technologies. Furthermore, important dosimetry and calibration characteristics (i.e. dose build-up region, beam attenuation, and beam scatter) of these irradiators are typically unknown to the end user, which can lead to significant deviation between delivered dose and intended dose to cells that adversely impact experimental results. Therefore, the overarching goal of this research is to design and develop a robust and fully automated high-throughput irradiator for in vitro radiation sensitivity investigations. Additionally, in vitro biological validation of this system was performed by assessing intracellular reactive oxygen species production, physical DNA double strand breaks, and activation of cellular DNA repair mechanisms. Finally, the high-throughput irradiator was used to investigate autophagic flux, a cellular adaptive response, as a potential biomarker of radiation sensitivity.

  16. What Is High-Throughput Virtual Screening? A Perspective from Organic Materials Discovery

    NASA Astrophysics Data System (ADS)

    Pyzer-Knapp, Edward O.; Suh, Changwon; Gómez-Bombarelli, Rafael; Aguilera-Iparraguirre, Jorge; Aspuru-Guzik, Alán

    2015-07-01

    A philosophy for defining what constitutes a virtual high-throughput screen is discussed, and the choices that influence decisions at each stage of the computational funnel are investigated, including an in-depth discussion of the generation of molecular libraries. Additionally, we provide advice on the storing, analysis, and visualization of data on the basis of extensive experience in our research group.

  17. High-throughput imaging of heterogeneous cell organelles with an X-ray laser

    SciTech Connect

    Hantke, Max, F.

    2014-11-17

    Preprocessed detector images that were used for the paper "High-throughput imaging of heterogeneous cell organelles with an X-ray laser". The CXI file contains the entire recorded data - including both hits and blanks. It also includes down-sampled images and LCLS machine parameters. Additionally, the Cheetah configuration file is attached that was used to create the pre-processed data.

  18. High-throughput screening assays for antibacterial and antifungal activities of Lactobacillus species.

    PubMed

    Inglin, Raffael C; Stevens, Marc J A; Meile, Lukas; Lacroix, Christophe; Meile, Leo

    2015-07-01

    We describe high-throughput screening techniques to rapidly detect either antimicrobial activity, using an agar-well diffusion assay in microtiter plates, or antifungal activity using an agar-spot assay in 24-well plates. 504 Lactobacillus isolates were screened with minimal laboratory equipment and screening rates of 2000-5000 individual antimicrobial interactions. PMID:25937247

  19. High throughput system for magnetic manipulation of cells, polymers, and biomaterials.

    PubMed

    Spero, Richard Chasen; Vicci, Leandra; Cribb, Jeremy; Bober, David; Swaminathan, Vinay; O'Brien, E Timothy; Rogers, Stephen L; Superfine, R

    2008-08-01

    In the past decade, high throughput screening (HTS) has changed the way biochemical assays are performed, but manipulation and mechanical measurement of micro- and nanoscale systems have not benefited from this trend. Techniques using microbeads (particles approximately 0.1-10 mum) show promise for enabling high throughput mechanical measurements of microscopic systems. We demonstrate instrumentation to magnetically drive microbeads in a biocompatible, multiwell magnetic force system. It is based on commercial HTS standards and is scalable to 96 wells. Cells can be cultured in this magnetic high throughput system (MHTS). The MHTS can apply independently controlled forces to 16 specimen wells. Force calibrations demonstrate forces in excess of 1 nN, predicted force saturation as a function of pole material, and powerlaw dependence of F approximately r(-2.7+/-0.1). We employ this system to measure the stiffness of SR2+ Drosophila cells. MHTS technology is a key step toward a high throughput screening system for micro- and nanoscale biophysical experiments. PMID:19044357

  20. Quantitative in vitro-to-in vivo extrapolation in a high-throughput environment.

    PubMed

    Wetmore, Barbara A

    2015-06-01

    High-throughput in vitro toxicity screening provides an efficient way to identify potential biological targets for environmental and industrial chemicals while conserving limited testing resources. However, reliance on the nominal chemical concentrations in these in vitro assays as an indicator of bioactivity may misrepresent potential in vivo effects of these chemicals due to differences in clearance, protein binding, bioavailability, and other pharmacokinetic factors. Development of high-throughput in vitro hepatic clearance and protein binding assays and refinement of quantitative in vitro-to-in vivo extrapolation (QIVIVE) methods have provided key tools to predict xenobiotic steady state pharmacokinetics. Using a process known as reverse dosimetry, knowledge of the chemical steady state behavior can be incorporated with HTS data to determine the external in vivo oral exposure needed to achieve internal blood concentrations equivalent to those eliciting bioactivity in the assays. These daily oral doses, known as oral equivalents, can be compared to chronic human exposure estimates to assess whether in vitro bioactivity would be expected at the dose-equivalent level of human exposure. This review will describe the use of QIVIVE methods in a high-throughput environment and the promise they hold in shaping chemical testing priorities and, potentially, high-throughput risk assessment strategies. PMID:24907440

  1. A LOW-COST HIGH THROUGHPUT POLYACRYLAMIDE GEL ELECTROPHORESIS SYSTEM FOR GENOTYPING WITH MICROSATELLITE DNA MARKERS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microsatellite DNA markers are widely used in genetic research but the genotyping cost with this marker system is high and the throughput is limited with conventional methods. The objective of this paper is to introduce a low-cost, high-throughput system developed in our laboratories for the detect...

  2. High-throughput synthesis and characterization of nanocrystalline porphyrinic zirconium metal-organic frameworks.

    PubMed

    Kelty, M L; Morris, W; Gallagher, A T; Anderson, J S; Brown, K A; Mirkin, C A; Harris, T D

    2016-06-14

    We describe and employ a high-throughput screening method to accelerate the synthesis and identification of pure-phase, nanocrystalline metal-organic frameworks (MOFs). We demonstrate the efficacy of this method through its application to a series of porphyrinic zirconium MOFs, resulting in the isolation of MOF-525, MOF-545, and PCN-223 on the nanoscale. PMID:27247981

  3. High-throughput SNP genotyping with the GoldenGate assay in maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single nucleotide polymorphisms (SNPs) are abundant and evenly distributed, and they have become an ideal marker system for genetic research in many organisms. Several high throughput platforms have been developed that allow genotyping thousands to a million markers in parallel. In this study, a cus...

  4. Quantitative high-throughput population dynamics in continuous-culture by automated microscopy.

    PubMed

    Merritt, Jason; Kuehn, Seppe

    2016-01-01

    We present a high-throughput method to measure abundance dynamics in microbial communities sustained in continuous-culture. Our method uses custom epi-fluorescence microscopes to automatically image single cells drawn from a continuously-cultured population while precisely controlling culture conditions. For clonal populations of Escherichia coli our instrument reveals history-dependent resilience and growth rate dependent aggregation. PMID:27616752

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  7. 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...

  8. High-Throughput Dietary Exposure Predictions for Chemical Migrants from Food Packaging Materials

    EPA Science Inventory

    United States Environmental Protection Agency researchers have developed a Stochastic Human Exposure and Dose Simulation High -Throughput (SHEDS-HT) model for use in prioritization of chemicals under the ExpoCast program. In this research, new methods were implemented in SHEDS-HT...

  9. High-throughput genotyping of hop (Humulus lupulus L.) utilising diversity arrays technology (DArT)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Implementation of molecular methods in hop breeding is dependent on the availability of sizeable numbers of polymorphic markers and a comprehensive understanding of genetic variation. Diversity Arrays Technology (DArT) is a high-throughput cost-effective method for the discovery of large numbers of...

  10. An Improved, high-throughput method for detection of bluetongue virus RNA in Culicodes midges

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new rapid (less than 6 h from insect-to-results) high-throughput assay is reported that is sensitive and specific for detecting BTV RNA in Culicoides biting midges. Homogenization and extraction of nucleic acids from individual Culicoides specimens were performed in a 96-well plate format using sp...

  11. Estimating Toxicity Pathway Activating Doses for High Throughput Chemical Risk Assessments

    EPA Science Inventory

    Estimating a Toxicity Pathway Activating Dose (TPAD) from in vitro assays as an analog to a reference dose (RfD) derived from in vivo toxicity tests would facilitate high throughput risk assessments of thousands of data-poor environmental chemicals. Estimating a TPAD requires def...

  12. Chromosomal microarray analysis, or comparative genomic hybridization: A high throughput approach

    PubMed Central

    Haeri, Mohammad; Gelowani, Violet; Beaudet, Arthur L.

    2015-01-01

    Pathological copy number variants (CNVs) and point mutations are major genetic causes of hundreds of disorders. Comparative genomic hybridization (CGH) also known as chromosomal microarray analysis (CMA) is the best available tool to detect copy number variations in chromosomal make up. We have optimized several different protocols and introduce a high-throughput approach to perform a cost-effective, fast, high-throughput and high-quality CMA. We managed to reach to high quality arrays with 17 ± 0.04 (mean ± SD, n = 90) Derivative Log Ratio (DLR) spread, a measure of array quality (<0.20 considered as excellent) for our arrays. High-throughput and high-quality arrays are gaining more attention and the current manuscript is a step forward to this increasing demand.•This manuscript introduces a low cost, fast, efficient, high throughput and high-quality aCGH protocol;•This protocol provides specific instructions and crucial detail for processing up to 24 slides which is equal to 48, 96, or 192 arrays by only one person in one day;•This manuscript is accompanied with a step-by-step video. PMID:26862485

  13. Perspectives on Validation of High-Throughput Assays Supporting 21st Century Toxicity Testing

    EPA Science Inventory

    In vitro high-throughput screening (HTS) assays are seeing increasing use in toxicity testing. HTS assays can simultaneously test many chemicals but have seen limited use in the regulatory arena, in part because of the need to undergo rigorous, time-consuming formal validation. ...

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

    EPA Science Inventory

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

  15. Evaluation of food-relevant chemicals in the ToxCast high-throughput screening program

    EPA Science Inventory

    There are thousands of chemicals that are directly added to or come in contact with food, many of which have undergone little to no toxicological evaluation. The ToxCast high-throughput screening (HTS) program has evaluated over 1,800 chemicals in concentration-response across ~8...

  16. High Throughput Prioritization for Integrated Toxicity Testing Based on ToxCast Chemical Profiling

    EPA Science Inventory

    The rational prioritization of chemicals for integrated toxicity testing is a central goal of the U.S. EPA’s ToxCast™ program (http://epa.gov/ncct/toxcast/). ToxCast includes a wide-ranging battery of over 500 in vitro high-throughput screening assays which in Phase I was used to...

  17. High-throughput procedure for single pollen grain collection and polymerase chain reaction in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single pollen grain PCR has succeeded in several species, however only a limited number (up to 60) pollen grains were involved due to difficulties in pollen isolation and lysis. This has limited its application in genetic analysis and mapping studies in plants. A high-throughput procedure for dete...

  18. High throughput system for magnetic manipulation of cells, polymers, and biomaterials

    PubMed Central

    Spero, Richard Chasen; Vicci, Leandra; Cribb, Jeremy; Bober, David; Swaminathan, Vinay; O’Brien, E. Timothy; Rogers, Stephen L.; Superfine, R.

    2008-01-01

    In the past decade, high throughput screening (HTS) has changed the way biochemical assays are performed, but manipulation and mechanical measurement of micro- and nanoscale systems have not benefited from this trend. Techniques using microbeads (particles ∼0.1–10 μm) show promise for enabling high throughput mechanical measurements of microscopic systems. We demonstrate instrumentation to magnetically drive microbeads in a biocompatible, multiwell magnetic force system. It is based on commercial HTS standards and is scalable to 96 wells. Cells can be cultured in this magnetic high throughput system (MHTS). The MHTS can apply independently controlled forces to 16 specimen wells. Force calibrations demonstrate forces in excess of 1 nN, predicted force saturation as a function of pole material, and powerlaw dependence of F∼r−2.7±0.1. We employ this system to measure the stiffness of SR2+ Drosophila cells. MHTS technology is a key step toward a high throughput screening system for micro- and nanoscale biophysical experiments. PMID:19044357

  19. Use of High-Throughput Testing and Approaches for Evaluating Chemical Risk-Relevance to Humans

    EPA Science Inventory

    ToxCast is profiling the bioactivity of thousands of chemicals based on high-throughput screening (HTS) and computational models that integrate knowledge of biological systems and in vivo toxicities. Many of these assays probe signaling pathways and cellular processes critical to...

  20. 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...

  1. PLASMA PROTEIN PROFILING AS A HIGH THROUGHPUT TOOL FOR CHEMICAL SCREENING USING A SMALL FISH MODEL

    EPA Science Inventory

    Hudson, R. Tod, Michael J. Hemmer, Kimberly A. Salinas, Sherry S. Wilkinson, James Watts, James T. Winstead, Peggy S. Harris, Amy Kirkpatrick and Calvin C. Walker. In press. Plasma Protein Profiling as a High Throughput Tool for Chemical Screening Using a Small Fish Model (Abstra...

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

    EPA Science Inventory

    As high throughput screening (HTS) plays a larger role in toxicity testing, camputational toxicology has emerged as a critical component in interpreting the large volume of data produced. Computational models designed to quantify potential adverse effects based on HTS data will b...

  3. Thawing the Landscape of the Era of High Throughput: Signs of Spring?

    PubMed

    Brady, Donald W

    2015-09-01

    In his latest book, Dr. Kenneth Ludmerer examines the history of graduate medical education (GME) in the United States, including its "era of high throughput" during which residents admitted more patients for shorter periods of time as hospitals focused on decreasing length of stay secondary to prospective payment reform. The author of this Commentary considers the implications of the era of high throughput and how the U.S. health care system must change to address its lasting effects.The era of high throughput initially had incomplete penetrance across the health care system landscape and a variable effect on GME. Trainees were variably aware of the financial forces bearing down on the health care system. Over time, the pervasiveness of the financial pressures and managed care became more complete, and the ubiquity of information through the Internet and social media ensured that residents became more acutely aware of how the changes to the health care system were affecting their education. There is now an opportunity for GME to be the nidus for ushering in an era of cost consciousness focused on patient needs and higher-quality GME rather than on the financial pressures that characterized the era of high throughput. PMID:26164641

  4. High-throughput Raman chemical imaging for rapid evaluation of food safety and quality

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High-throughput macro-scale Raman chemical imaging was realized on a newly developed line-scan hyperspectral system. The system utilizes a custom-designed 785 nm line laser with maximum power of 5 W as an excitation source. A 24 cm × 1 mm excitation line is normally projected on the sample surface u...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Improved high-throughput bioassay for Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As we gain more information through functional genomic studies of Rhyzopertha dominica (F.), we need a high throughput bioassay system to screen potential biopesticides. R. dominica is an internal feeder during immature stages and presents unique challenges with traditional bioassay methods. Our pri...

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

    SciTech Connect

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

    2011-09-16

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

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

    EPA Science Inventory

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  10. Incorporating Human Dosimetry and Exposure into High-Throughput In Vitro Toxicity Screening

    EPA Science Inventory

    Many chemicals in commerce today have undergone limited or no safety testing. To reduce the number of untested chemicals and prioritize limited testing resources, several governmental programs are using high-throughput in vitro screens for assessing chemical effects across multip...

  11. High-Throughput Exposure Potential Prioritization for ToxCast Chemicals

    EPA Science Inventory

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

  12. 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…

  13. 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...

  14. High-Throughput Toxicokinetics (HTTK) R package (CompTox CoP presentation)

    EPA Science Inventory

    Toxicokinetics (TK) provides a bridge between HTS and HTE by predicting tissue concentrations due to exposure, but traditional TK methods are resource intensive. Relatively high throughput TK (HTTK) methods have been used by the pharmaceutical industry to determine range of effic...

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

    SciTech Connect

    Liu, Y; Grossman, JC

    2014-12-01

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

  16. Environmental surveillance and monitoring. The next frontiers for high-throughput toxicology

    EPA Science Inventory

    High throughput toxicity testing (HTT) technologies along with the world-wide web are revolutionizing both generation and access to data regarding the bioactivities that chemicals can elicit when they interact with specific proteins, genes, or other targets in the body of an orga...

  17. HIGH-THROUGHPUT IDENTIFICATION OF CATALYTIC REDOX-ACTIVE CYSTEINE RESIDUES

    EPA Science Inventory

    Cysteine (Cys) residues often play critical roles in proteins; however, identification of their specific functions has been limited to case-by-case experimental approaches. We developed a procedure for high-throughput identification of catalytic redox-active Cys in proteins by se...

  18. Development of a thyroperoxidase inhibition assay for high-throughput screening

    EPA Science Inventory

    High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluores...

  19. A High-Throughput MALDI-TOF Mass Spectrometry-Based Assay of Chitinase Activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A high-throughput MALDI-TOF mass spectrometric assay is described for assay of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates, and is readily achievable on a microliter scale (2 µL total volume, containing 2 µg of substrate and 1 ng of protein). The speed a...

  20. A click chemistry-based microRNA maturation assay optimized for high-throughput screening.

    PubMed

    Lorenz, Daniel A; Garner, Amanda L

    2016-07-01

    Catalytic enzyme-linked click-chemistry assays (cat-ELCCA) are an emerging class of biochemical assay. Herein we report on expanding the toolkit of cat-ELCCA to include the kinetically superior inverse-electron demand Diels-Alder (IEDDA) reaction. The result is a technology with improved sensitivity and reproducibility, enabling automated high-throughput screening. PMID:27284591