A High-Throughput Microtiter Plate Based Method for the Determination of Peracetic Acid and Hydrogen Peroxide

PubMed Central

Putt, Karson S.; Pugh, Randall B.

2013-01-01

Peracetic acid is gaining usage in numerous industries who have found a myriad of uses for its antimicrobial activity. However, rapid high throughput quantitation methods for peracetic acid and hydrogen peroxide are lacking. Herein, we describe the development of a high-throughput microtiter plate based assay based upon the well known and trusted titration chemical reactions. The adaptation of these titration chemistries to rapid plate based absorbance methods for the sequential determination of hydrogen peroxide specifically and the total amount of peroxides present in solution are described. The results of these methods were compared to those of a standard titration and found to be in good agreement. Additionally, the utility of the developed method is demonstrated through the generation of degradation curves of both peracetic acid and hydrogen peroxide in a mixed solution. PMID:24260173

A noninvasive, direct real-time PCR method for sex determination in multiple avian species

USGS Publications Warehouse

Brubaker, Jessica L.; Karouna-Renier, Natalie K.; Chen, Yu; Jenko, Kathryn; Sprague, Daniel T.; Henry, Paula F.P.

2011-01-01

Polymerase chain reaction (PCR)-based methods to determine the sex of birds are well established and have seen few modifications since they were first introduced in the 1990s. Although these methods allowed for sex determination in species that were previously difficult to analyse, they were not conducive to high-throughput analysis because of the laboriousness of DNA extraction and gel electrophoresis. We developed a high-throughput real-time PCR-based method for analysis of sex in birds, which uses noninvasive sample collection and avoids DNA extraction and gel electrophoresis.

Development of rapid and sensitive high throughput pharmacologic assays for marine phycotoxins.

PubMed

Van Dolah, F M; Finley, E L; Haynes, B L; Doucette, G J; Moeller, P D; Ramsdell, J S

1994-01-01

The lack of rapid, high throughput assays is a major obstacle to many aspects of research on marine phycotoxins. Here we describe the application of microplate scintillation technology to develop high throughput assays for several classes of marine phycotoxin based on their differential pharmacologic actions. High throughput "drug discovery" format microplate receptor binding assays developed for brevetoxins/ciguatoxins and for domoic acid are described. Analysis for brevetoxins/ciguatoxins is carried out by binding competition with [3H] PbTx-3 for site 5 on the voltage dependent sodium channel in rat brain synaptosomes. Analysis of domoic acid is based on binding competition with [3H] kainic acid for the kainate/quisqualate glutamate receptor using frog brain synaptosomes. In addition, a high throughput microplate 45Ca flux assay for determination of maitotoxins is described. These microplate assays can be completed within 3 hours, have sensitivities of less than 1 ng, and can analyze dozens of samples simultaneously. The assays have been demonstrated to be useful for assessing algal toxicity and for assay-guided purification of toxins, and are applicable to the detection of biotoxins in seafood.

LOCATE: a mouse protein subcellular localization database

PubMed Central

Fink, J. Lynn; Aturaliya, Rajith N.; Davis, Melissa J.; Zhang, Fasheng; Hanson, Kelly; Teasdale, Melvena S.; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide; Teasdale, Rohan D.

2006-01-01

We present here LOCATE, a curated, web-accessible database that houses data describing the membrane organization and subcellular localization of proteins from the FANTOM3 Isoform Protein Sequence set. Membrane organization is predicted by the high-throughput, computational pipeline MemO. The subcellular locations of selected proteins from this set were determined by a high-throughput, immunofluorescence-based assay and by manually reviewing >1700 peer-reviewed publications. LOCATE represents the first effort to catalogue the experimentally verified subcellular location and membrane organization of mammalian proteins using a high-throughput approach and provides localization data for ∼40% of the mouse proteome. It is available at . PMID:16381849

Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings

PubMed Central

Alexander, Crispin G.; Wanner, Randy; Johnson, Christopher M.; Breitsprecher, Dennis; Winter, Gerhard; Duhr, Stefan; Baaske, Philipp; Ferguson, Neil

2014-01-01

Chemical denaturant titrations can be used to accurately determine protein stability. However, data acquisition is typically labour intensive, has low throughput and is difficult to automate. These factors, combined with high protein consumption, have limited the adoption of chemical denaturant titrations in commercial settings. Thermal denaturation assays can be automated, sometimes with very high throughput. However, thermal denaturation assays are incompatible with proteins that aggregate at high temperatures and large extrapolation of stability parameters to physiological temperatures can introduce significant uncertainties. We used capillary-based instruments to measure chemical denaturant titrations by intrinsic fluorescence and microscale thermophoresis. This allowed higher throughput, consumed several hundred-fold less protein than conventional, cuvette-based methods yet maintained the high quality of the conventional approaches. We also established efficient strategies for automated, direct determination of protein stability at a range of temperatures via chemical denaturation, which has utility for characterising stability for proteins that are difficult to purify in high yield. This approach may also have merit for proteins that irreversibly denature or aggregate in classical thermal denaturation assays. We also developed procedures for affinity ranking of protein–ligand interactions from ligand-induced changes in chemical denaturation data, and proved the principle for this by correctly ranking the affinity of previously unreported peptide–PDZ domain interactions. The increased throughput, automation and low protein consumption of protein stability determinations afforded by using capillary-based methods to measure denaturant titrations, can help to revolutionise protein research. We believe that the strategies reported are likely to find wide applications in academia, biotherapeutic formulation and drug discovery programmes. PMID:25262836

High-throughput analysis using non-depletive SPME: challenges and applications to the determination of free and total concentrations in small sample volumes.

PubMed

Boyacı, Ezel; Bojko, Barbara; Reyes-Garcés, Nathaly; Poole, Justen J; Gómez-Ríos, Germán Augusto; Teixeira, Alexandre; Nicol, Beate; Pawliszyn, Janusz

2018-01-18

In vitro high-throughput non-depletive quantitation of chemicals in biofluids is of growing interest in many areas. Some of the challenges facing researchers include the limited volume of biofluids, rapid and high-throughput sampling requirements, and the lack of reliable methods. Coupled to the above, growing interest in the monitoring of kinetics and dynamics of miniaturized biosystems has spurred the demand for development of novel and revolutionary methodologies for analysis of biofluids. The applicability of solid-phase microextraction (SPME) is investigated as a potential technology to fulfill the aforementioned requirements. As analytes with sufficient diversity in their physicochemical features, nicotine, N,N-Diethyl-meta-toluamide, and diclofenac were selected as test compounds for the study. The objective was to develop methodologies that would allow repeated non-depletive sampling from 96-well plates, using 100 µL of sample. Initially, thin film-SPME was investigated. Results revealed substantial depletion and consequent disruption in the system. Therefore, new ultra-thin coated fibers were developed. The applicability of this device to the described sampling scenario was tested by determining the protein binding of the analytes. Results showed good agreement with rapid equilibrium dialysis. The presented method allows high-throughput analysis using small volumes, enabling fast reliable free and total concentration determinations without disruption of system equilibrium.

Developing a gene biomarker at the tipping point of adaptive and adverse responses in human bronchial epithelial cells

EPA Science Inventory

Determining mechanism-based biomarkers that distinguish adaptive and adverse cellular processes is critical to understanding the health effects of environmental exposures. Shifting from in vivo, low-throughput toxicity studies to high-throughput screening (HTS) paradigms and risk...

The combination of gas-phase fluorophore technology and automation to enable high-throughput analysis of plant respiration.

PubMed

Scafaro, Andrew P; Negrini, A Clarissa A; O'Leary, Brendan; Rashid, F Azzahra Ahmad; Hayes, Lucy; Fan, Yuzhen; Zhang, You; Chochois, Vincent; Badger, Murray R; Millar, A Harvey; Atkin, Owen K

2017-01-01

Mitochondrial respiration in the dark ( R dark ) is a critical plant physiological process, and hence a reliable, efficient and high-throughput method of measuring variation in rates of R dark is essential for agronomic and ecological studies. However, currently methods used to measure R dark in plant tissues are typically low throughput. We assessed a high-throughput automated fluorophore system of detecting multiple O 2 consumption rates. The fluorophore technique was compared with O 2 -electrodes, infrared gas analysers (IRGA), and membrane inlet mass spectrometry, to determine accuracy and speed of detecting respiratory fluxes. The high-throughput fluorophore system provided stable measurements of R dark in detached leaf and root tissues over many hours. High-throughput potential was evident in that the fluorophore system was 10 to 26-fold faster per sample measurement than other conventional methods. The versatility of the technique was evident in its enabling: (1) rapid screening of R dark in 138 genotypes of wheat; and, (2) quantification of rarely-assessed whole-plant R dark through dissection and simultaneous measurements of above- and below-ground organs. Variation in absolute R dark was observed between techniques, likely due to variation in sample conditions (i.e. liquid vs. gas-phase, open vs. closed systems), indicating that comparisons between studies using different measuring apparatus may not be feasible. However, the high-throughput protocol we present provided similar values of R dark to the most commonly used IRGA instrument currently employed by plant scientists. Together with the greater than tenfold increase in sample processing speed, we conclude that the high-throughput protocol enables reliable, stable and reproducible measurements of R dark on multiple samples simultaneously, irrespective of plant or tissue type.

Rapid Catalyst Screening by a Continuous-Flow Microreactor Interfaced with Ultra High Pressure Liquid Chromatography

PubMed Central

Fang, Hui; Xiao, Qing; Wu, Fanghui; Floreancig, Paul E.; Weber, Stephen G.

2010-01-01

A high-throughput screening system for homogeneous catalyst discovery has been developed by integrating a continuous-flow capillary-based microreactor with ultra-high pressure liquid chromatography (UHPLC) for fast online analysis. Reactions are conducted in distinct and stable zones in a flow stream that allows for time and temperature regulation. UHPLC detection at high temperature allows high throughput online determination of substrate, product, and byproduct concentrations. We evaluated the efficacies of a series of soluble acid catalysts for an intramolecular Friedel-Crafts addition into an acyliminium ion intermediate within one day and with minimal material investment. The effects of catalyst loading, reaction time, and reaction temperature were also screened. This system exhibited high reproducibility for high-throughput catalyst screening and allowed several acid catalysts for the reaction to be identified. Major side products from the reactions were determined through off-line mass spectrometric detection. Er(OTf)3, the catalyst that showed optimal efficiency in the screening, was shown to be effective at promoting the cyclization reaction on a preparative scale. PMID:20666502

A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells.

PubMed

Massey, Andrew J

2018-01-01

Determining and understanding drug target engagement is critical for drug discovery. This can be challenging within living cells as selective readouts are often unavailable. Here we describe a novel method for measuring target engagement in living cells based on the principle of altered protein thermal stabilization / destabilization in response to ligand binding. This assay (HCIF-CETSA) utilizes high content, high throughput single cell immunofluorescent detection to determine target protein levels following heating of adherent cells in a 96 well plate format. We have used target engagement of Chk1 by potent small molecule inhibitors to validate the assay. Target engagement measured by this method was subsequently compared to target engagement measured by two alternative methods (autophosphorylation and CETSA). The HCIF-CETSA method appeared robust and a good correlation in target engagement measured by this method and CETSA for the selective Chk1 inhibitor V158411 was observed. However, these EC50 values were 23- and 12-fold greater than the autophosphorylation IC50. The described method is therefore a valuable advance in the CETSA method allowing the high throughput determination of target engagement in adherent cells.

High-throughput determination of biochemical oxygen demand (BOD) by a microplate-based biosensor.

PubMed

Pang, Hei-Leung; Kwok, Nga-Yan; Chan, Pak-Ho; Yeung, Chi-Hung; Lo, Waihung; Wong, Kwok-Yin

2007-06-01

The use of the conventional 5-day biochemical oxygen demand (BOD5) method in BOD determination is greatly hampered by its time-consuming sampling procedure and its technical difficulty in the handling of a large pool of wastewater samples. Thus, it is highly desirable to develop a fast and high-throughput biosensor for BOD measurements. This paper describes the construction of a microplate-based biosensor consisting of an organically modified silica (ORMOSIL) oxygen sensing film for high-throughput determination of BOD in wastewater. The ORMOSIL oxygen sensing film was prepared by reacting tetramethoxysilane with dimethyldimethoxysilane in the presence of the oxygen-sensitive dye tris(4,7-diphenyl-1,10-phenanthroline)ruthenium-(II) chloride. The silica composite formed a homogeneous, crack-free oxygen sensing film on polystyrene microtiter plates with high stability, and the embedded ruthenium dye interacted with the dissolved oxygen in wastewater according to the Stern-Volmer relation. The bacterium Stenotrophomonas maltophilia was loaded into the ORMOSIL/ PVA composite (deposited on the top of the oxygen sensing film) and used to metabolize the organic compounds in wastewater. This BOD biosensor was found to be able to determine the BOD values of wastewater samples within 20 min by monitoring the dissolved oxygen concentrations. Moreover, the BOD values determined by the BOD biosensor were in good agreement with those obtained by the conventional BOD5 method.

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

PubMed

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

2015-01-01

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

Low-dose fixed-target serial synchrotron crystallography.

PubMed

Owen, Robin L; Axford, Danny; Sherrell, Darren A; Kuo, Anling; Ernst, Oliver P; Schulz, Eike C; Miller, R J Dwayne; Mueller-Werkmeister, Henrike M

2017-04-01

The development of serial crystallography has been driven by the sample requirements imposed by X-ray free-electron lasers. Serial techniques are now being exploited at synchrotrons. Using a fixed-target approach to high-throughput serial sampling, it is demonstrated that high-quality data can be collected from myoglobin crystals, allowing room-temperature, low-dose structure determination. The combination of fixed-target arrays and a fast, accurate translation system allows high-throughput serial data collection at high hit rates and with low sample consumption.

High-throughput continuous hydrothermal synthesis of nanomaterials (part II): unveiling the as-prepared CexZryYzO2-δ phase diagram.

PubMed

Quesada-Cabrera, Raul; Weng, Xiaole; Hyett, Geoff; Clark, Robin J H; Wang, Xue Z; Darr, Jawwad A

2013-09-09

High-throughput continuous hydrothermal flow synthesis was used to manufacture 66 unique nanostructured oxide samples in the Ce-Zr-Y-O system. This synthesis approach resulted in a significant increase in throughput compared to that of conventional batch or continuous hydrothermal synthesis methods. The as-prepared library samples were placed into a wellplate for both automated high-throughput powder X-ray diffraction and Raman spectroscopy data collection, which allowed comprehensive structural characterization and phase mapping. The data suggested that a continuous cubic-like phase field connects all three Ce-Zr-O, Ce-Y-O, and Y-Zr-O binary systems together with a smooth and steady transition between the structures of neighboring compositions. The continuous hydrothermal process led to as-prepared crystallite sizes in the range of 2-7 nm (as determined by using the Scherrer equation).

Quantitative determination of free/bound atazanavir via high-throughput equilibrium dialysis and LC-MS/MS, and the application in ex vivo samples.

PubMed

Xu, Xiaohui Sophia; Rose, Anne; Demers, Roger; Eley, Timothy; Ryan, John; Stouffer, Bruce; Cojocaru, Laura; Arnold, Mark

2014-01-01

The determination of drug-protein binding is important in the pharmaceutical development process because of the impact of protein binding on both the pharmacokinetics and pharmacodynamics of drugs. Equilibrium dialysis is the preferred method to measure the free drug fraction because it is considered to be more accurate. The throughput of equilibrium dialysis has recently been improved by implementing a 96-well format plate. Results/methodology: This manuscript illustrates the successful application of a 96-well rapid equilibrium dialysis (RED) device in the determination of atazanavir plasma-protein binding. This RED method of measuring free fraction was successfully validated and then applied to the analysis of clinical plasma samples taken from HIV-infected pregnant women administered atazanavir. Combined with LC-MS/MS detection, the 96-well format equilibrium dialysis device was suitable for measuring the free and bound concentration of pharmaceutical molecules in a high-throughput mode.

Identifying Candidate Chemical-Disease Linkages ...

EPA Pesticide Factsheets

Presentation at meeting on Environmental and Epigenetic Determinants of IBD in New York, NY on identifying candidate chemical-disease linkages by using AOPs to identify molecular initiating events and using relevant high throughput assays to screen for candidate chemicals. This hazard information is combined with exposure models to inform risk assessment. Presentation at meeting on Environmental and Epigenetic Determinants of IBD in New York, NY on identifying candidate chemical-disease linkages by using AOPs to identify molecular initiating events and using relevant high throughput assays to screen for candidate chemicals. This hazard information is combined with exposure models to inform risk assessment.

High-throughput process development: determination of dynamic binding capacity using microtiter filter plates filled with chromatography resin.

PubMed

Bergander, Tryggve; Nilsson-Välimaa, Kristina; Oberg, Katarina; Lacki, Karol M

2008-01-01

Steadily increasing demand for more efficient and more affordable biomolecule-based therapies put a significant burden on biopharma companies to reduce the cost of R&D activities associated with introduction of a new drug to the market. Reducing the time required to develop a purification process would be one option to address the high cost issue. The reduction in time can be accomplished if more efficient methods/tools are available for process development work, including high-throughput techniques. This paper addresses the transitions from traditional column-based process development to a modern high-throughput approach utilizing microtiter filter plates filled with a well-defined volume of chromatography resin. The approach is based on implementing the well-known batch uptake principle into microtiter plate geometry. Two variants of the proposed approach, allowing for either qualitative or quantitative estimation of dynamic binding capacity as a function of residence time, are described. Examples of quantitative estimation of dynamic binding capacities of human polyclonal IgG on MabSelect SuRe and of qualitative estimation of dynamic binding capacity of amyloglucosidase on a prototype of Capto DEAE weak ion exchanger are given. The proposed high-throughput method for determination of dynamic binding capacity significantly reduces time and sample consumption as compared to a traditional method utilizing packed chromatography columns without sacrificing the accuracy of data obtained.

Formation of Linear Gradient of Antibiotics on Microfluidic Chips for High-throughput Antibiotic Susceptibility Testing

NASA Astrophysics Data System (ADS)

Kim, Seunggyu; Lee, Seokhun; Jeon, Jessie S.

2017-11-01

To determine the most effective antimicrobial treatments of infectious pathogen, high-throughput antibiotic susceptibility test (AST) is critically required. However, the conventional AST requires at least 16 hours to reach the minimum observable population. Therefore, we developed a microfluidic system that allows maintenance of linear antibiotic concentration and measurement of local bacterial density. Based on the Stokes-Einstein equation, the flow rate in the microchannel was optimized so that linearization was achieved within 10 minutes, taking into account the diffusion coefficient of each antibiotic in the agar gel. As a result, the minimum inhibitory concentration (MIC) of each antibiotic against P. aeruginosa could be immediately determined 6 hours after treatment of the linear antibiotic concentration. In conclusion, our system proved the efficacy of a high-throughput AST platform through MIC comparison with Clinical and Laboratory Standards Institute (CLSI) range of antibiotics. This work was supported by the Climate Change Research Hub (Grant No. N11170060) of the KAIST and by the Brain Korea 21 Plus project.

Crystal Symmetry Algorithms in a High-Throughput Framework for Materials

NASA Astrophysics Data System (ADS)

Taylor, Richard

The high-throughput framework AFLOW that has been developed and used successfully over the last decade is improved to include fully-integrated software for crystallographic symmetry characterization. The standards used in the symmetry algorithms conform with the conventions and prescriptions given in the International Tables of Crystallography (ITC). A standard cell choice with standard origin is selected, and the space group, point group, Bravais lattice, crystal system, lattice system, and representative symmetry operations are determined. Following the conventions of the ITC, the Wyckoff sites are also determined and their labels and site symmetry are provided. The symmetry code makes no assumptions on the input cell orientation, origin, or reduction and has been integrated in the AFLOW high-throughput framework for materials discovery by adding to the existing code base and making use of existing classes and functions. The software is written in object-oriented C++ for flexibility and reuse. A performance analysis and examination of the algorithms scaling with cell size and symmetry is also reported.

High Throughput Determination of Critical Human Dosing ...

EPA Pesticide Factsheets

High throughput toxicokinetics (HTTK) is a rapid approach that uses in vitro data to estimate TK for hundreds of environmental chemicals. Reverse dosimetry (i.e., reverse toxicokinetics or RTK) based on HTTK data converts high throughput in vitro toxicity screening (HTS) data into predicted human equivalent doses that can be linked with biologically relevant exposure scenarios. Thus, HTTK provides essential data for risk prioritization for thousands of chemicals that lack TK data. One critical HTTK parameter that can be measured in vitro is the unbound fraction of a chemical in plasma (Fub). However, for chemicals that bind strongly to plasma, Fub is below the limits of detection (LOD) for high throughput analytical chemistry, and therefore cannot be quantified. A novel method for quantifying Fub was implemented for 85 strategically selected chemicals: measurement of Fub was attempted at 10%, 30%, and 100% of physiological plasma concentrations using rapid equilibrium dialysis assays. Varying plasma concentrations instead of chemical concentrations makes high throughput analytical methodology more likely to be successful. Assays at 100% plasma concentration were unsuccessful for 34 chemicals. For 12 of these 34 chemicals, Fub could be quantified at 10% and/or 30% plasma concentrations; these results imply that the assay failure at 100% plasma concentration was caused by plasma protein binding for these chemicals. Assay failure for the remaining 22 chemicals may

X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization

PubMed Central

Schieferstein, Jeremy M.; Pawate, Ashtamurthy S.; Wan, Frank; Sheraden, Paige N.; Broecker, Jana; Ernst, Oliver P.; Gennis, Robert B.

2017-01-01

Elucidating and clarifying the function of membrane proteins ultimately requires atomic resolution structures as determined most commonly by X-ray crystallography. Many high impact membrane protein structures have resulted from advanced techniques such as in meso crystallization that present technical difficulties for the set-up and scale-out of high-throughput crystallization experiments. In prior work, we designed a novel, low-throughput X-ray transparent microfluidic device that automated the mixing of protein and lipid by diffusion for in meso crystallization trials. Here, we report X-ray transparent microfluidic devices for high-throughput crystallization screening and optimization that overcome the limitations of scale and demonstrate their application to the crystallization of several membrane proteins. Two complementary chips are presented: (1) a high-throughput screening chip to test 192 crystallization conditions in parallel using as little as 8 nl of membrane protein per well and (2) a crystallization optimization chip to rapidly optimize preliminary crystallization hits through fine-gradient re-screening. We screened three membrane proteins for new in meso crystallization conditions, identifying several preliminary hits that we tested for X-ray diffraction quality. Further, we identified and optimized the crystallization condition for a photosynthetic reaction center mutant and solved its structure to a resolution of 3.5 Å. PMID:28469762

Fast log P determination by ultra-high-pressure liquid chromatography coupled with UV and mass spectrometry detections.

PubMed

Henchoz, Yveline; Guillarme, Davy; Martel, Sophie; Rudaz, Serge; Veuthey, Jean-Luc; Carrupt, Pierre-Alain

2009-08-01

Ultra-high-pressure liquid chromatography (UHPLC) systems able to work with columns packed with sub-2 microm particles offer very fast methods to determine the lipophilicity of new chemical entities. The careful development of the most suitable experimental conditions presented here will help medicinal chemists for high-throughput screening (HTS) log P(oct) measurements. The approach was optimized using a well-balanced set of 38 model compounds and a series of 28 basic compounds such as beta-blockers, local anesthetics, piperazines, clonidine, and derivatives. Different organic modifiers and hybrid stationary phases packed with 1.7-microm particles were evaluated in isocratic as well as gradient modes, and the advantages and limitations of tested conditions pointed out. The UHPLC approach offered a significant enhancement over the classical HPLC methods, by a factor 50 in the lipophilicity determination throughput. The hyphenation of UHPLC with MS detection allowed a further increase in the throughput. Data and results reported herein prove that the UHPLC-MS method can represent a progress in the HTS-measurement of lipophilicity due to its speed (at least a factor of 500 with respect to HPLC approaches) and to an extended field of application.

Microreactor Cells for High-Throughput X-ray Absorption Spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beesley, Angela; Tsapatsaris, Nikolaos; Weiher, Norbert

2007-01-19

High-throughput experimentation has been applied to X-ray Absorption spectroscopy as a novel route for increasing research productivity in the catalysis community. Suitable instrumentation has been developed for the rapid determination of the local structure in the metal component of precursors for supported catalysts. An automated analytical workflow was implemented that is much faster than traditional individual spectrum analysis. It allows the generation of structural data in quasi-real time. We describe initial results obtained from the automated high throughput (HT) data reduction and analysis of a sample library implemented through the 96 well-plate industrial standard. The results show that a fullymore » automated HT-XAS technology based on existing industry standards is feasible and useful for the rapid elucidation of geometric and electronic structure of materials.« less

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

PubMed

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

2011-10-01

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hura, Greg L.; Menon, Angeli L.; Hammel, Michal

2009-07-20

We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes formore » 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.« less

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

20170308 - Higher Throughput Toxicokinetics to Allow ...

EPA Pesticide Factsheets

As part of "Ongoing EDSP Directions & Activities" I will present CSS research on high throughput toxicokinetics, including in vitro data and models to allow rapid determination of the real world doses that may cause endocrine disruption. This is a presentation as part of the U.S. Environmental Protection Agency – Japan Ministry of the Environment 12th Bilateral Meeting on Endocrine Disruption Test Methods Development.

Baculovirus expression system and method for high throughput expression of genetic material

DOEpatents

Clark, Robin; Davies, Anthony

2001-01-01

The present invention provides novel recombinant baculovirus expression systems for expressing foreign genetic material in a host cell. Such expression systems are readily adapted to an automated method for expression foreign genetic material in a high throughput manner. In other aspects, the present invention features a novel automated method for determining the function of foreign genetic material by transfecting the same into a host by way of the recombinant baculovirus expression systems according to the present invention.

A high-throughput headspace gas chromatographic technique for the determination of nitrite content in water samples.

PubMed

Zhang, Shu-Xin; Peng, Rong; Jiang, Ran; Chai, Xin-Sheng; Barnes, Donald G

2018-02-23

This paper reports on a high-throughput headspace gas chromatographic method (HS-GC) for the determination of nitrite content in water sample, based on GC measurement of cyclohexene produced from the reaction between nitrite and cyclamate in a closed vial. The method has a relative standard deviation of <3.5%; The differences between the results of the nitrite measurements obtained by this method and those of a reference method were less than 5.8% and the recoveries of the method were in the range of 94.8-102% (for a spiked nitrite content range from 0.002 to 0.03 mg/L). The limit of detection of the method was 0.46 μg L -1 . Due to an overlapping mode in the headspace auto-sampler system, the method can provide an automated and high-throughput nitrite analysis for the surface water samples. In short, the present HS-GC method is simple, accurate, and sensitive, and it is very suitable to be used in the batch sample testing. Copyright © 2018 Elsevier B.V. All rights reserved.

Microarray Detection of Duplex and Triplex DNA Binders with DNA-Modified Gold Nanoparticles

PubMed Central

Lytton-Jean, Abigail K. R.; Han, Min Su; Mirkin, Chad A.

2008-01-01

We have designed a chip-based assay, using microarray technology, for determining the relative binding affinities of duplex and triplex DNA binders. This assay combines the high discrimination capabilities afforded by DNA-modified Au nanoparticles with the high-throughput capabilities of DNA microarrays. The detection and screening of duplex DNA binders are important because these molecules, in many cases, are potential anticancer agents as well as toxins. Triplex DNA binders are also promising drug candidates. These molecules, in conjunction with triplex forming oligonucleotides, could potentially be used to achieve control of gene expression by interfering with transcription factors that bind to DNA. Therefore, the ability to screen for these molecules in a high-throughput fashion could dramatically improve the drug screening process. The assay reported here provides excellent discrimination between strong, intermediate, and weak duplex and triplex DNA binders in a high-throughput fashion. PMID:17614366

The promise and challenge of high-throughput sequencing of the antibody repertoire

PubMed Central

Georgiou, George; Ippolito, Gregory C; Beausang, John; Busse, Christian E; Wardemann, Hedda; Quake, Stephen R

2014-01-01

Efforts to determine the antibody repertoire encoded by B cells in the blood or lymphoid organs using high-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and are transforming our understanding of humoral immune responses. Information gained from high-throughput DNA sequencing of immunoglobulin genes (Ig-seq) can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development and to understand autoimmunity. Rapid progress in the development of experimental protocols and informatics analysis tools is helping to reduce sequencing artifacts, to achieve more precise quantification of clonal diversity and to extract the most pertinent biological information. That said, broader application of Ig-seq, especially in clinical settings, will require the development of a standardized experimental design framework that will enable the sharing and meta-analysis of sequencing data generated by different laboratories. PMID:24441474

Multi-step high-throughput conjugation platform for the development of antibody-drug conjugates.

PubMed

Andris, Sebastian; Wendeler, Michaela; Wang, Xiangyang; Hubbuch, Jürgen

2018-07-20

Antibody-drug conjugates (ADCs) form a rapidly growing class of biopharmaceuticals which attracts a lot of attention throughout the industry due to its high potential for cancer therapy. They combine the specificity of a monoclonal antibody (mAb) and the cell-killing capacity of highly cytotoxic small molecule drugs. Site-specific conjugation approaches involve a multi-step process for covalent linkage of antibody and drug via a linker. Despite the range of parameters that have to be investigated, high-throughput methods are scarcely used so far in ADC development. In this work an automated high-throughput platform for a site-specific multi-step conjugation process on a liquid-handling station is presented by use of a model conjugation system. A high-throughput solid-phase buffer exchange was successfully incorporated for reagent removal by utilization of a batch cation exchange step. To ensure accurate screening of conjugation parameters, an intermediate UV/Vis-based concentration determination was established including feedback to the process. For conjugate characterization, a high-throughput compatible reversed-phase chromatography method with a runtime of 7 min and no sample preparation was developed. Two case studies illustrate the efficient use for mapping the operating space of a conjugation process. Due to the degree of automation and parallelization, the platform is capable of significantly reducing process development efforts and material demands and shorten development timelines for antibody-drug conjugates. Copyright © 2018 Elsevier B.V. All rights reserved.

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

PubMed

Laurens, L M L; Wolfrum, E J

2013-12-18

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

High-Throughput Synthesis and Structure of Zeolite ZSM-43 with Two-Directional 8-Ring Channels.

PubMed

Willhammar, Tom; Su, Jie; Yun, Yifeng; Zou, Xiaodong; Afeworki, Mobae; Weston, Simon C; Vroman, Hilda B; Lonergan, William W; Strohmaier, Karl G

2017-08-07

The aluminosilicate zeolite ZSM-43 (where ZSM = Zeolite Socony Mobil) was first synthesized more than 3 decades ago, but its chemical structure remained unsolved because of its poor crystallinity and small crystal size. Here we present optimization of the ZSM-43 synthesis using a high-throughput approach and subsequent structure determination by the combination of electron crystallographic methods and powder X-ray diffraction. The synthesis required the use of a combination of both inorganic (Cs + and K + ) and organic (choline) structure-directing agents. High-throughput synthesis enabled a screening of the synthesis conditions, which made it possible to optimize the synthesis, despite its complexity, in order to obtain a material with significantly improved crystallinity. When both rotation electron diffraction and high-resolution transmission electron microscopy imaging techniques are applied, the structure of ZSM-43 could be determined. The structure of ZSM-43 is a new zeolite framework type and possesses a unique two-dimensional channel system limited by 8-ring channels. ZSM-43 is stable upon calcination, and sorption measurements show that the material is suitable for adsorption of carbon dioxide as well as methane.

AELAS: Automatic ELAStic property derivations via high-throughput first-principles computation

NASA Astrophysics Data System (ADS)

Zhang, S. H.; Zhang, R. F.

2017-11-01

The elastic properties are fundamental and important for crystalline materials as they relate to other mechanical properties, various thermodynamic qualities as well as some critical physical properties. However, a complete set of experimentally determined elastic properties is only available for a small subset of known materials, and an automatic scheme for the derivations of elastic properties that is adapted to high-throughput computation is much demanding. In this paper, we present the AELAS code, an automated program for calculating second-order elastic constants of both two-dimensional and three-dimensional single crystal materials with any symmetry, which is designed mainly for high-throughput first-principles computation. Other derivations of general elastic properties such as Young's, bulk and shear moduli as well as Poisson's ratio of polycrystal materials, Pugh ratio, Cauchy pressure, elastic anisotropy and elastic stability criterion, are also implemented in this code. The implementation of the code has been critically validated by a lot of evaluations and tests on a broad class of materials including two-dimensional and three-dimensional materials, providing its efficiency and capability for high-throughput screening of specific materials with targeted mechanical properties. Program Files doi:http://dx.doi.org/10.17632/f8fwg4j9tw.1 Licensing provisions: BSD 3-Clause Programming language: Fortran Nature of problem: To automate the calculations of second-order elastic constants and the derivations of other elastic properties for two-dimensional and three-dimensional materials with any symmetry via high-throughput first-principles computation. Solution method: The space-group number is firstly determined by the SPGLIB code [1] and the structure is then redefined to unit cell with IEEE-format [2]. Secondly, based on the determined space group number, a set of distortion modes is automatically specified and the distorted structure files are generated. Afterwards, the total energy for each distorted structure is calculated by the first-principles codes, e.g. VASP [3]. Finally, the second-order elastic constants are determined from the quadratic coefficients of the polynomial fitting of the energies vs strain relationships and other elastic properties are accordingly derived. References [1] http://atztogo.github.io/spglib/. [2] A. Meitzler, H.F. Tiersten, A.W. Warner, D. Berlincourt, G.A. Couqin, F.S. Welsh III, IEEE standard on piezoelectricity, Society, 1988. [3] G. Kresse, J. Furthmüller, Phys. Rev. B 54 (1996) 11169.

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing.

PubMed

Stiffler, Michael A; Subramanian, Subu K; Salinas, Victor H; Ranganathan, Rama

2016-07-03

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 β-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to β-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

A rapid and high-throughput microplate spectrophotometric method for field measurement of nitrate in seawater and freshwater.

PubMed

Wu, Jiapeng; Hong, Yiguo; Guan, Fengjie; Wang, Yan; Tan, Yehui; Yue, Weizhong; Wu, Meilin; Bin, Liying; Wang, Jiaping; Wen, Jiali

2016-02-01

The well-known zinc-cadmium reduction method is frequently used for determination of nitrate. However, this method is seldom to be applied on field research of nitrate due to the long time consuming and large sample volume demand. Here, we reported a modified zinc-cadmium reduction method (MZCRM) for measurement of nitrate at natural-abundance level in both seawater and freshwater. The main improvements of MZCRM include using small volume disposable tubes for reaction, a vortex apparatus for shaking to increase reduction rate, and a microplate reader for high-throughput spectrophotometric measurements. Considering salt effect, two salinity sections (5~10 psu and 20~35 psu) were set up for more accurate determination of nitrate in low and high salinity condition respectively. Under optimized experimental conditions, the reduction rates were stabilized on 72% and 63% on the salinity of 5 and 20 psu respectively. The lowest detection limit for nitrate was 0.5 μM and was linear up to 100 μM (RSDs was 4.8%). Environmental samples assay demonstrated that MZCRM was well consistent with conventional zinc-cadmium reduction method. In total, this modified method improved accuracy and efficiency of operations greatly, and would be realized a rapid and high-throughput determination of nitrate in field analysis of nitrate with low cost.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 bemore » used independently for detecting nitrite residues/contamination in environmental/food samples. 10 refs., 2 figs.« less

Optimization of wide-area ATM and local-area ethernet/FDDI network configurations for high-speed telemedicine communications employing NASA's ACTS.

PubMed

McDermott, W R; Tri, J L; Mitchell, M P; Levens, S P; Wondrow, M A; Huie, L M; Khandheria, B K; Gilbert, B K

1999-01-01

A high data rate terrestrial and satellite network was implemented to transfer medical images and data. This article describes the a optimization of the workstations and switching equipment incorporated into the network. Topics discussed in this article include tuning of the network software, the configuration of the Sun Microsystems workstations, the FORE Systems asynchronous transfer mode switches, as well as the throughput results of two telemedicine experiments undertaken by Mayo's physician staff. The technical staff was successful in achieving the data throughput needed by the telemedicine software; particularly important was the proper determination of peak throughput and TCP window sizes to ensure optimum use of the resources available on the Sun Microsystems and Hewlett Packard workstations.

High-throughput bioinformatics with the Cyrille2 pipeline system

PubMed Central

Fiers, Mark WEJ; van der Burgt, Ate; Datema, Erwin; de Groot, Joost CW; van Ham, Roeland CHJ

2008-01-01

Background Modern omics research involves the application of high-throughput technologies that generate vast volumes of data. These data need to be pre-processed, analyzed and integrated with existing knowledge through the use of diverse sets of software tools, models and databases. The analyses are often interdependent and chained together to form complex workflows or pipelines. Given the volume of the data used and the multitude of computational resources available, specialized pipeline software is required to make high-throughput analysis of large-scale omics datasets feasible. Results We have developed a generic pipeline system called Cyrille2. The system is modular in design and consists of three functionally distinct parts: 1) a web based, graphical user interface (GUI) that enables a pipeline operator to manage the system; 2) the Scheduler, which forms the functional core of the system and which tracks what data enters the system and determines what jobs must be scheduled for execution, and; 3) the Executor, which searches for scheduled jobs and executes these on a compute cluster. Conclusion The Cyrille2 system is an extensible, modular system, implementing the stated requirements. Cyrille2 enables easy creation and execution of high throughput, flexible bioinformatics pipelines. PMID:18269742

Comprehensive analysis of the T-cell receptor beta chain gene in rhesus monkey by high throughput sequencing

PubMed Central

Li, Zhoufang; Liu, Guangjie; Tong, Yin; Zhang, Meng; Xu, Ying; Qin, Li; Wang, Zhanhui; Chen, Xiaoping; He, Jiankui

2015-01-01

Profiling immune repertoires by high throughput sequencing enhances our understanding of immune system complexity and immune-related diseases in humans. Previously, cloning and Sanger sequencing identified limited numbers of T cell receptor (TCR) nucleotide sequences in rhesus monkeys, thus their full immune repertoire is unknown. We applied multiplex PCR and Illumina high throughput sequencing to study the TCRβ of rhesus monkeys. We identified 1.26 million TCRβ sequences corresponding to 643,570 unique TCRβ sequences and 270,557 unique complementarity-determining region 3 (CDR3) gene sequences. Precise measurements of CDR3 length distribution, CDR3 amino acid distribution, length distribution of N nucleotide of junctional region, and TCRV and TCRJ gene usage preferences were performed. A comprehensive profile of rhesus monkey immune repertoire might aid human infectious disease studies using rhesus monkeys. PMID:25961410

Isolating Viral and Host RNA Sequences from Archival Material and Production of cDNA Libraries for High-Throughput DNA Sequencing

PubMed Central

Xiao, Yongli; Sheng, Zong-Mei; Taubenberger, Jeffery K.

2015-01-01

The vast majority of surgical biopsy and post-mortem tissue samples are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. As an example, the viral RNA genome of the 1918 pandemic influenza A virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Using the protocols described here, the full genome of the 1918 virus at high coverage was determined in one high-throughput sequencing run of a cDNA library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. This basic methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of infectious diseases. PMID:26344216

Identification of Inhibitors in Lignocellulosic Slurries and Determination of Their Effect on Hydrocarbon-Producing Microorganisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Shihui; Franden, Mary A; Yang, Qing

The aim of this work was to identify inhibitors in pretreated lignocellulosic slurries, evaluate high-throughput screening strategies, and investigate the impact of inhibitors on potential hydrocarbon-producing microorganisms. Compounds present in slurries that could inhibit microbial growth were identified through a detailed analysis of saccharified slurries by applying a combination of approaches of high-performance liquid chromatography, GC-MS, LC-DAD-MS, and ICP-MS. Several high-throughput assays were then evaluated to generate toxicity profiles. Our results demonstrated that Bioscreen C was useful for analyzing bacterial toxicity but not for yeast. AlamarBlue reduction assay can be a useful high-throughput assay for both bacterial and yeast strainsmore » as long as medium components do not interfere with fluorescence measurements. In addition, this work identified two major inhibitors (furfural and ammonium acetate) for three potential hydrocarbon-producing bacterial species that include Escherichia coli, Cupriavidus necator, and Rhodococcus opacus PD630, which are also the primary inhibitors for ethanologens. Here, this study was strived to establish a pipeline to quantify inhibitory compounds in biomass slurries and high-throughput approaches to investigate the effect of inhibitors on microbial biocatalysts, which can be applied for various biomass slurries or hydrolyzates generated through different pretreatment and enzymatic hydrolysis processes or different microbial candidates.« less

Identification of Inhibitors in Lignocellulosic Slurries and Determination of Their Effect on Hydrocarbon-Producing Microorganisms

DOE PAGES

Yang, Shihui; Franden, Mary A; Yang, Qing; ...

2018-04-04

The aim of this work was to identify inhibitors in pretreated lignocellulosic slurries, evaluate high-throughput screening strategies, and investigate the impact of inhibitors on potential hydrocarbon-producing microorganisms. Compounds present in slurries that could inhibit microbial growth were identified through a detailed analysis of saccharified slurries by applying a combination of approaches of high-performance liquid chromatography, GC-MS, LC-DAD-MS, and ICP-MS. Several high-throughput assays were then evaluated to generate toxicity profiles. Our results demonstrated that Bioscreen C was useful for analyzing bacterial toxicity but not for yeast. AlamarBlue reduction assay can be a useful high-throughput assay for both bacterial and yeast strainsmore » as long as medium components do not interfere with fluorescence measurements. In addition, this work identified two major inhibitors (furfural and ammonium acetate) for three potential hydrocarbon-producing bacterial species that include Escherichia coli, Cupriavidus necator, and Rhodococcus opacus PD630, which are also the primary inhibitors for ethanologens. Here, this study was strived to establish a pipeline to quantify inhibitory compounds in biomass slurries and high-throughput approaches to investigate the effect of inhibitors on microbial biocatalysts, which can be applied for various biomass slurries or hydrolyzates generated through different pretreatment and enzymatic hydrolysis processes or different microbial candidates.« less

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

PubMed Central

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

2016-01-01

Seed storage compounds are of crucial importance for human diet, feed and industrial uses. In oleo-proteaginous species like rapeseed, seed oil and protein are the qualitative determinants that conferred economic value to the harvested seed. To date, although the biosynthesis pathways of oil and storage protein are rather well-known, the factors that determine how these types of reserves are partitioned in seeds have to be identified. With the aim of implementing a quantitative genetics approach, requiring phenotyping of 100s of plants, our first objective was to establish near-infrared reflectance spectroscopic (NIRS) predictive equations in order to estimate oil, protein, carbon, and nitrogen content in Arabidopsis seed with high-throughput level. Our results demonstrated that NIRS is a powerful non-destructive, high-throughput method to assess the content of these four major components studied in Arabidopsis seed. With this tool in hand, we analyzed Arabidopsis natural variation for these four components and illustrated that they all displayed a wide range of variation. Finally, NIRS was used in order to map QTL for these four traits using seeds from the Arabidopsis thaliana Ct-1 × Col-0 recombinant inbred line population. Some QTL co-localized with QTL previously identified, but others mapped to chromosomal regions never identified so far for such traits. This paper illustrates the usefulness of NIRS predictive equations to perform accurate high-throughput phenotyping of Arabidopsis seed content, opening new perspectives in gene identification following QTL mapping and genome wide association studies. PMID:27891138

High-throughput measurement of rice tillers using a conveyor equipped with x-ray computed tomography

NASA Astrophysics Data System (ADS)

Yang, Wanneng; Xu, Xiaochun; Duan, Lingfeng; Luo, Qingming; Chen, Shangbin; Zeng, Shaoqun; Liu, Qian

2011-02-01

Tillering is one of the most important agronomic traits because the number of shoots per plant determines panicle number, a key component of grain yield. The conventional method of counting tillers is still manual. Under the condition of mass measurement, the accuracy and efficiency could be gradually degraded along with fatigue of experienced staff. Thus, manual measurement, including counting and recording, is not only time consuming but also lack objectivity. To automate this process, we developed a high-throughput facility, dubbed high-throughput system for measuring automatically rice tillers (H-SMART), for measuring rice tillers based on a conventional x-ray computed tomography (CT) system and industrial conveyor. Each pot-grown rice plant was delivered into the CT system for scanning via the conveyor equipment. A filtered back-projection algorithm was used to reconstruct the transverse section image of the rice culms. The number of tillers was then automatically extracted by image segmentation. To evaluate the accuracy of this system, three batches of rice at different growth stages (tillering, heading, or filling) were tested, yielding absolute mean absolute errors of 0.22, 0.36, and 0.36, respectively. Subsequently, the complete machine was used under industry conditions to estimate its efficiency, which was 4320 pots per continuous 24 h workday. Thus, the H-SMART could determine the number of tillers of pot-grown rice plants, providing three advantages over the manual tillering method: absence of human disturbance, automation, and high throughput. This facility expands the application of agricultural photonics in plant phenomics.

High-throughput measurement of rice tillers using a conveyor equipped with x-ray computed tomography.

PubMed

Yang, Wanneng; Xu, Xiaochun; Duan, Lingfeng; Luo, Qingming; Chen, Shangbin; Zeng, Shaoqun; Liu, Qian

2011-02-01

Tillering is one of the most important agronomic traits because the number of shoots per plant determines panicle number, a key component of grain yield. The conventional method of counting tillers is still manual. Under the condition of mass measurement, the accuracy and efficiency could be gradually degraded along with fatigue of experienced staff. Thus, manual measurement, including counting and recording, is not only time consuming but also lack objectivity. To automate this process, we developed a high-throughput facility, dubbed high-throughput system for measuring automatically rice tillers (H-SMART), for measuring rice tillers based on a conventional x-ray computed tomography (CT) system and industrial conveyor. Each pot-grown rice plant was delivered into the CT system for scanning via the conveyor equipment. A filtered back-projection algorithm was used to reconstruct the transverse section image of the rice culms. The number of tillers was then automatically extracted by image segmentation. To evaluate the accuracy of this system, three batches of rice at different growth stages (tillering, heading, or filling) were tested, yielding absolute mean absolute errors of 0.22, 0.36, and 0.36, respectively. Subsequently, the complete machine was used under industry conditions to estimate its efficiency, which was 4320 pots per continuous 24 h workday. Thus, the H-SMART could determine the number of tillers of pot-grown rice plants, providing three advantages over the manual tillering method: absence of human disturbance, automation, and high throughput. This facility expands the application of agricultural photonics in plant phenomics.

Comparative analysis of miRNA and mRNA abundance in determinate cucumber by high-throughput sequencing

USDA-ARS?s Scientific Manuscript database

Determinate cucumber is characterized with short vines, fewer nodes, and terminal flowers, which is a useful plant architecture for cucumbers in certain production systems. The genetic and molecular mechanisms of determinate growth habit is not well understood. In addition, environmental factors als...

High-throughput simultaneous determination of plasma water deuterium and 18-oxygen enrichment using a high-temperature conversion elemental analyzer with isotope ratio mass spectrometry.

PubMed

Richelle, M; Darimont, C; Piguet-Welsch, C; Fay, L B

2004-01-01

This paper presents a high-throughput method for the simultaneous determination of deuterium and oxygen-18 (18O) enrichment of water samples isolated from blood. This analytical method enables rapid and simple determination of these enrichments of microgram quantities of water. Water is converted into hydrogen and carbon monoxide gases by the use of a high-temperature conversion elemental analyzer (TC-EA), that are then transferred on-line into the isotope ratio mass spectrometer. Accuracy determined with the standard light Antartic precipitation (SLAP) and Greenland ice sheet precipitation (GISP) is reliable for deuterium and 18O enrichments. The range of linearity is from 0 up to 0.09 atom percent excess (APE, i.e. -78 up to 5725 delta per mil (dpm)) for deuterium enrichment and from 0 up to 0.17 APE (-11 up to 890 dpm) for 18O enrichment. Memory effects do exist but can be avoided by analyzing the biological samples in quintuplet. This method allows the determination of 1440 samples per week, i.e. 288 biological samples per week. Copyright 2004 John Wiley & Sons, Ltd.

High-Throughput rRNA Gene Sequencing Reveals High
and Complex Bacterial Diversity Associated with
Brazilian Coffee Bean Fermentation

PubMed Central

Vinícius de Melo, Gilberto

2018-01-01

Summary Coffee bean fermentation is a spontaneous, on-farm process involving the action of different microbial groups, including bacteria and fungi. In this study, high-throughput sequencing approach was employed to study the diversity and dynamics of bacteria associated with Brazilian coffee bean fermentation. The total DNA from fermenting coffee samples was extracted at different time points, and the 16S rRNA gene with segments around the V4 variable region was sequenced by Illumina high-throughput platform. Using this approach, the presence of over eighty bacterial genera was determined, many of which have been detected for the first time during coffee bean fermentation, including Fructobacillus, Pseudonocardia, Pedobacter, Sphingomonas and Hymenobacter. The presence of Fructobacillus suggests an influence of these bacteria on fructose metabolism during coffee fermentation. Temporal analysis showed a strong dominance of lactic acid bacteria with over 97% of read sequences at the end of fermentation, mainly represented by the Leuconostoc and Lactococcus. Metabolism of lactic acid bacteria was associated with the high formation of lactic acid during fermentation, as determined by HPLC analysis. The results reported in this study confirm the underestimation of bacterial diversity associated with coffee fermentation. New microbial groups reported in this study may be explored as functional starter cultures for on-farm coffee processing.

Novel method for the high-throughput processing of slides for the comet assay

PubMed Central

Karbaschi, Mahsa; Cooke, Marcus S.

2014-01-01

Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay's low sample throughput and laborious sample workup procedure are limiting factors to its application. “Scoring”, or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure. PMID:25425241

Novel method for the high-throughput processing of slides for the comet assay.

PubMed

Karbaschi, Mahsa; Cooke, Marcus S

2014-11-26

Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay's low sample throughput and laborious sample workup procedure are limiting factors to its application. "Scoring", or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure.

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.

Identifying Candidate Chemical-Disease Linkages (Environmental and Epigenetic Determinants of IBD)

EPA Science Inventory

Presentation at meeting on Environmental and Epigenetic Determinants of IBD in New York, NY on identifying candidate chemical-disease linkages by using AOPs to identify molecular initiating events and using relevant high throughput assays to screen for candidate chemicals. This h...

A high-throughput solid-phase extraction microchip combined with inductively coupled plasma-mass spectrometry for rapid determination of trace heavy metals in natural water.

PubMed

Shih, Tsung-Ting; Hsieh, Cheng-Chuan; Luo, Yu-Ting; Su, Yi-An; Chen, Ping-Hung; Chuang, Yu-Chen; Sun, Yuh-Chang

2016-04-15

Herein, a hyphenated system combining a high-throughput solid-phase extraction (htSPE) microchip with inductively coupled plasma-mass spectrometry (ICP-MS) for rapid determination of trace heavy metals was developed. Rather than performing multiple analyses in parallel for the enhancement of analytical throughput, we improved the processing speed for individual samples by increasing the operation flow rate during SPE procedures. To this end, an innovative device combining a micromixer and a multi-channeled extraction unit was designed. Furthermore, a programmable valve manifold was used to interface the developed microchip and ICP-MS instrumentation in order to fully automate the system, leading to a dramatic reduction in operation time and human error. Under the optimized operation conditions for the established system, detection limits of 1.64-42.54 ng L(-1) for the analyte ions were achieved. Validation procedures demonstrated that the developed method could be satisfactorily applied to the determination of trace heavy metals in natural water. Each analysis could be readily accomplished within just 186 s using the established system. This represents, to the best of our knowledge, an unprecedented speed for the analysis of trace heavy metal ions. Copyright © 2016 Elsevier B.V. All rights reserved.

A Rapid Method for the Determination of Fucoxanthin in Diatom

PubMed Central

Wang, Li-Juan; Fan, Yong; Parsons, Ronald L.; Hu, Guang-Rong; Zhang, Pei-Yu

2018-01-01

Fucoxanthin is a natural pigment found in microalgae, especially diatoms and Chrysophyta. Recently, it has been shown to have anti-inflammatory, anti-tumor, and anti-obesityactivity in humans. Phaeodactylum tricornutum is a diatom with high economic potential due to its high content of fucoxanthin and eicosapentaenoic acid. In order to improve fucoxanthin production, physical and chemical mutagenesis could be applied to generate mutants. An accurate and rapid method to assess the fucoxanthin content is a prerequisite for a high-throughput screen of mutants. In this work, the content of fucoxanthin in P. tricornutum was determined using spectrophotometry instead of high performance liquid chromatography (HPLC). This spectrophotometric method is easier and faster than liquid chromatography and the standard error was less than 5% when compared to the HPLC results. Also, this method can be applied to other diatoms, with standard errors of 3–14.6%. It provides a high throughput screening method for microalgae strains producing fucoxanthin. PMID:29361768

Strategic and Operational Plan for Integrating Transcriptomics ...

EPA Pesticide Factsheets

Plans for incorporating high throughput transcriptomics into the current high throughput screening activities at NCCT; the details are in the attached slide presentation presentation on plans for incorporating high throughput transcriptomics into the current high throughput screening activities at NCCT, given at the OECD meeting on June 23, 2016

High-Throughput Experimental Approach Capabilities | Materials Science |

Science.gov Websites

NREL High-Throughput Experimental Approach Capabilities High-Throughput Experimental Approach by yellow and is for materials in the upper right sector. NREL's high-throughput experimental ,Te) and oxysulfide sputtering Combi-5: Nitrides and oxynitride sputtering We also have several non

Life in the fast lane for protein crystallization and X-ray crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

2005-01-01

The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high-rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today's high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from the efforts of the Southeast Collaboratory for Structural Genomics (SECSG).

Life in the Fast Lane for Protein Crystallization and X-Ray Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

2004-01-01

The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today s high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from the efforts of the Southeast Collaboratory for Structural Genomics (SECSG).

Large-Scale Biomonitoring of Remote and Threatened Ecosystems via High-Throughput Sequencing

PubMed Central

Gibson, Joel F.; Shokralla, Shadi; Curry, Colin; Baird, Donald J.; Monk, Wendy A.; King, Ian; Hajibabaei, Mehrdad

2015-01-01

Biodiversity metrics are critical for assessment and monitoring of ecosystems threatened by anthropogenic stressors. Existing sorting and identification methods are too expensive and labour-intensive to be scaled up to meet management needs. Alternately, a high-throughput DNA sequencing approach could be used to determine biodiversity metrics from bulk environmental samples collected as part of a large-scale biomonitoring program. Here we show that both morphological and DNA sequence-based analyses are suitable for recovery of individual taxonomic richness, estimation of proportional abundance, and calculation of biodiversity metrics using a set of 24 benthic samples collected in the Peace-Athabasca Delta region of Canada. The high-throughput sequencing approach was able to recover all metrics with a higher degree of taxonomic resolution than morphological analysis. The reduced cost and increased capacity of DNA sequence-based approaches will finally allow environmental monitoring programs to operate at the geographical and temporal scale required by industrial and regulatory end-users. PMID:26488407

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

An Automated High-throughput Array Microscope for Cancer Cell Mechanics

NASA Astrophysics Data System (ADS)

Cribb, Jeremy A.; Osborne, Lukas D.; Beicker, Kellie; Psioda, Matthew; Chen, Jian; O'Brien, E. Timothy; Taylor, Russell M., II; Vicci, Leandra; Hsiao, Joe Ping-Lin; Shao, Chong; Falvo, Michael; Ibrahim, Joseph G.; Wood, Kris C.; Blobe, Gerard C.; Superfine, Richard

2016-06-01

Changes in cellular mechanical properties correlate with the progression of metastatic cancer along the epithelial-to-mesenchymal transition (EMT). Few high-throughput methodologies exist that measure cell compliance, which can be used to understand the impact of genetic alterations or to screen the efficacy of chemotherapeutic agents. We have developed a novel array high-throughput microscope (AHTM) system that combines the convenience of the standard 96-well plate with the ability to image cultured cells and membrane-bound microbeads in twelve independently-focusing channels simultaneously, visiting all wells in eight steps. We use the AHTM and passive bead rheology techniques to determine the relative compliance of human pancreatic ductal epithelial (HPDE) cells, h-TERT transformed HPDE cells (HPNE), and four gain-of-function constructs related to EMT. The AHTM found HPNE, H-ras, Myr-AKT, and Bcl2 transfected cells more compliant relative to controls, consistent with parallel tests using atomic force microscopy and invasion assays, proving the AHTM capable of screening for changes in mechanical phenotype.

Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.

PubMed

Ngo, Tony; Coleman, James L J; Smith, Nicola J

2015-01-01

Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system.

A Method of High Throughput Monitoring Crop Physiology Using Chlorophyll Fluorescence and Multispectral Imaging.

PubMed

Wang, Heng; Qian, Xiangjie; Zhang, Lan; Xu, Sailong; Li, Haifeng; Xia, Xiaojian; Dai, Liankui; Xu, Liang; Yu, Jingquan; Liu, Xu

2018-01-01

We present a high throughput crop physiology condition monitoring system and corresponding monitoring method. The monitoring system can perform large-area chlorophyll fluorescence imaging and multispectral imaging. The monitoring method can determine the crop current condition continuously and non-destructively. We choose chlorophyll fluorescence parameters and relative reflectance of multispectral as the indicators of crop physiological status. Using tomato as experiment subject, the typical crop physiological stress, such as drought, nutrition deficiency and plant disease can be distinguished by the monitoring method. Furthermore, we have studied the correlation between the physiological indicators and the degree of stress. Besides realizing the continuous monitoring of crop physiology, the monitoring system and method provide the possibility of machine automatic diagnosis of the plant physiology. Highlights: A newly designed high throughput crop physiology monitoring system and the corresponding monitoring method are described in this study. Different types of stress can induce distinct fluorescence and spectral characteristics, which can be used to evaluate the physiological status of plants.

High-throughput analysis of the protein sequence-stability landscape using a quantitative "yeast surface two-hybrid" system and fragment reconstitution

PubMed Central

Dutta, Sanjib; Koide, Akiko; Koide, Shohei

2008-01-01

Stability evaluation of many mutants can lead to a better understanding of the sequence determinants of a structural motif and of factors governing protein stability and protein evolution. The traditional biophysical analysis of protein stability is low throughput, limiting our ability to widely explore the sequence space in a quantitative manner. In this study, we have developed a high-throughput library screening method for quantifying stability changes, which is based on protein fragment reconstitution and yeast surface display. Our method exploits the thermodynamic linkage between protein stability and fragment reconstitution and the ability of the yeast surface display technique to quantitatively evaluate protein-protein interactions. The method was applied to a fibronectin type III (FN3) domain. Characterization of fragment reconstitution was facilitated by the co-expression of two FN3 fragments, thus establishing a "yeast surface two-hybrid" method. Importantly, our method does not rely on competition between clones and thus eliminates a common limitation of high-throughput selection methods in which the most stable variants are predominantly recovered. Thus, it allows for the isolation of sequences that exhibits a desired level of stability. We identified over one hundred unique sequences for a β-bulge motif, which was significantly more informative than natural sequences of the FN3 family in revealing the sequence determinants for the β-bulge. Our method provides a powerful means to rapidly assess stability of many variants, to systematically assess contribution of different factors to protein stability and to enhance protein stability. PMID:18674545

High throughput ion-channel pharmacology: planar-array-based voltage clamp.

PubMed

Kiss, Laszlo; Bennett, Paul B; Uebele, Victor N; Koblan, Kenneth S; Kane, Stefanie A; Neagle, Brad; Schroeder, Kirk

2003-02-01

Technological advances often drive major breakthroughs in biology. Examples include PCR, automated DNA sequencing, confocal/single photon microscopy, AFM, and voltage/patch-clamp methods. The patch-clamp method, first described nearly 30 years ago, was a major technical achievement that permitted voltage-clamp analysis (membrane potential control) of ion channels in most cells and revealed a role for channels in unimagined areas. Because of the high information content, voltage clamp is the best way to study ion-channel function; however, throughput is too low for drug screening. Here we describe a novel breakthrough planar-array-based HT patch-clamp technology developed by Essen Instruments capable of voltage-clamping thousands of cells per day. This technology provides greater than two orders of magnitude increase in throughput compared with the traditional voltage-clamp techniques. We have applied this method to study the hERG K(+) channel and to determine the pharmacological profile of QT prolonging drugs.

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.

PRELIMINARY ASSESSMENTS OF IN VITRO PHARMACOKINETIC DATA AND EXPOSURE INFORMATION FOR THE TOXCAST PHASE II CHEMICALS

EPA Science Inventory

Momentum has been growing in Toxicology to assess the utility of high-throughput screening (HTS) assays in the determination of chemical testing priorities. However, in vitro potencies determined in these assays do not consider in vivo bioavailability, clearance or exposure estim...

High-Throughput Continuous Hydrothermal Synthesis of Transparent Conducting Aluminum and Gallium Co-doped Zinc Oxides.

PubMed

Howard, Dougal P; Marchand, Peter; McCafferty, Liam; Carmalt, Claire J; Parkin, Ivan P; Darr, Jawwad A

2017-04-10

High-throughput continuous hydrothermal flow synthesis was used to generate a library of aluminum and gallium-codoped zinc oxide nanoparticles of specific atomic ratios. Resistivities of the materials were determined by Hall Effect measurements on heat-treated pressed discs and the results collated into a conductivity-composition map. Optimal resistivities of ∼9 × 10 -3 Ω cm were reproducibly achieved for several samples, for example, codoped ZnO with 2 at% Ga and 1 at% Al. The optimum sample on balance of performance and cost was deemed to be ZnO codoped with 3 at% Al and 1 at% Ga.

High-throughput ab-initio dilute solute diffusion database.

PubMed

Wu, Henry; Mayeshiba, Tam; Morgan, Dane

2016-07-19

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.

Development of Novel Random Network Theory-Based Approaches to Identify Network Interactions among Nitrifying Bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Cindy

2015-07-17

The interactions among different microbial populations in a community could play more important roles in determining ecosystem functioning than species numbers and their abundances, but very little is known about such network interactions at a community level. The goal of this project is to develop novel framework approaches and associated software tools to characterize the network interactions in microbial communities based on high throughput, large scale high-throughput metagenomics data and apply these approaches to understand the impacts of environmental changes (e.g., climate change, contamination) on network interactions among different nitrifying populations and associated microbial communities.

Selecting the most appropriate time points to profile in high-throughput studies

PubMed Central

Kleyman, Michael; Sefer, Emre; Nicola, Teodora; Espinoza, Celia; Chhabra, Divya; Hagood, James S; Kaminski, Naftali; Ambalavanan, Namasivayam; Bar-Joseph, Ziv

2017-01-01

Biological systems are increasingly being studied by high throughput profiling of molecular data over time. Determining the set of time points to sample in studies that profile several different types of molecular data is still challenging. Here we present the Time Point Selection (TPS) method that solves this combinatorial problem in a principled and practical way. TPS utilizes expression data from a small set of genes sampled at a high rate. As we show by applying TPS to study mouse lung development, the points selected by TPS can be used to reconstruct an accurate representation for the expression values of the non selected points. Further, even though the selection is only based on gene expression, these points are also appropriate for representing a much larger set of protein, miRNA and DNA methylation changes over time. TPS can thus serve as a key design strategy for high throughput time series experiments. Supporting Website: www.sb.cs.cmu.edu/TPS DOI: http://dx.doi.org/10.7554/eLife.18541.001 PMID:28124972

Human Papillomavirus Biology, Pathogenesis, and Potential for Drug Discovery: A Literature Review for HIV Nurse Clinical Scientists.

PubMed

Walhart, Tara

2015-01-01

Persistent oncogenic human papillomavirus (HPV) infection increases the probability that precancerous anal high-grade squamous intraepithelial lesions will progress to invasive anal cancer. Anal neoplasia associated with HPV disproportionately affects HIV-infected individuals, especially men who have sex with men. Prevention is limited to HPV vaccine recommendations, highlighting the need for new treatments. The purpose of this review is to provide HIV information to nurse clinical scientists about HPV-related cancer to highlight the connection between: (a) HPV biology and pathogenesis and (b) the development of drugs and novel therapeutic methods using high-throughput screening. PubMed and CINAHL were used to search the literature to determine HPV-related epidemiology, biology, and use of high-throughput screening for drug discovery. Several events in the HPV life cycle have the potential to be developed into biologic targets for drug discovery using the high-throughput screening technique, which has been successfully used to identify compounds to inhibit HPV infections. Copyright © 2015 Association of Nurses in AIDS Care. Published by Elsevier Inc. All rights reserved.

RhizoTubes as a new tool for high throughput imaging of plant root development and architecture: test, comparison with pot grown plants and validation.

PubMed

Jeudy, Christian; Adrian, Marielle; Baussard, Christophe; Bernard, Céline; Bernaud, Eric; Bourion, Virginie; Busset, Hughes; Cabrera-Bosquet, Llorenç; Cointault, Frédéric; Han, Simeng; Lamboeuf, Mickael; Moreau, Delphine; Pivato, Barbara; Prudent, Marion; Trouvelot, Sophie; Truong, Hoai Nam; Vernoud, Vanessa; Voisin, Anne-Sophie; Wipf, Daniel; Salon, Christophe

2016-01-01

In order to maintain high yields while saving water and preserving non-renewable resources and thus limiting the use of chemical fertilizer, it is crucial to select plants with more efficient root systems. This could be achieved through an optimization of both root architecture and root uptake ability and/or through the improvement of positive plant interactions with microorganisms in the rhizosphere. The development of devices suitable for high-throughput phenotyping of root structures remains a major bottleneck. Rhizotrons suitable for plant growth in controlled conditions and non-invasive image acquisition of plant shoot and root systems (RhizoTubes) are described. These RhizoTubes allow growing one to six plants simultaneously, having a maximum height of 1.1 m, up to 8 weeks, depending on plant species. Both shoot and root compartment can be imaged automatically and non-destructively throughout the experiment thanks to an imaging cabin (RhizoCab). RhizoCab contains robots and imaging equipment for obtaining high-resolution pictures of plant roots. Using this versatile experimental setup, we illustrate how some morphometric root traits can be determined for various species including model (Medicago truncatula), crops (Pisum sativum, Brassica napus, Vitis vinifera, Triticum aestivum) and weed (Vulpia myuros) species grown under non-limiting conditions or submitted to various abiotic and biotic constraints. The measurement of the root phenotypic traits using this system was compared to that obtained using "classic" growth conditions in pots. This integrated system, to include 1200 Rhizotubes, will allow high-throughput phenotyping of plant shoots and roots under various abiotic and biotic environmental conditions. Our system allows an easy visualization or extraction of roots and measurement of root traits for high-throughput or kinetic analyses. The utility of this system for studying root system architecture will greatly facilitate the identification of genetic and environmental determinants of key root traits involved in crop responses to stresses, including interactions with soil microorganisms.

Experiments and Analyses of Data Transfers Over Wide-Area Dedicated Connections

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rao, Nageswara S.; Liu, Qiang; Sen, Satyabrata

Dedicated wide-area network connections are increasingly employed in high-performance computing and big data scenarios. One might expect the performance and dynamics of data transfers over such connections to be easy to analyze due to the lack of competing traffic. However, non-linear transport dynamics and end-system complexities (e.g., multi-core hosts and distributed filesystems) can in fact make analysis surprisingly challenging. We present extensive measurements of memory-to-memory and disk-to-disk file transfers over 10 Gbps physical and emulated connections with 0–366 ms round trip times (RTTs). For memory-to-memory transfers, profiles of both TCP and UDT throughput as a function of RTT show concavemore » and convex regions; large buffer sizes and more parallel flows lead to wider concave regions, which are highly desirable. TCP and UDT both also display complex throughput dynamics, as indicated by their Poincare maps and Lyapunov exponents. For disk-to-disk transfers, we determine that high throughput can be achieved via a combination of parallel I/O threads, parallel network threads, and direct I/O mode. Our measurements also show that Lustre filesystems can be mounted over long-haul connections using LNet routers, although challenges remain in jointly optimizing file I/O and transport method parameters to achieve peak throughput.« less

Quality Control for Ambient Sampling of PCDD/PCDF from Open Combustion Sources

EPA Science Inventory

Both long duration (> 6 h) and high temperature (up to 139o C) sampling efforts were conducted using ambient air sampling methods to determine if either high volume throughput or higher than ambient sampling temperatures resulted in loss of target polychlorinated dibenzodioxins/d...

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

PubMed

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

2007-01-01

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

Meeting Materials for the November 28-29, 2017 Scientific Advisory Panel

EPA Pesticide Factsheets

Meeting Materials for the November 28-30, 2017 Scientific Advisory Panel on Continuing Development of Alternative High-Throughput Screens to Determine Endocrine Disruption, Focusing on Androgen Receptor, Steroidogenesis, and Thyroid Pathways.

Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pinzon, NM; Aukema, KG; Gralnick, JA

A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone productionmore » as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. IMPORTANCE In recent years, there has been renewed interest in advanced biofuel sources such as bacterial hydrocarbon production. Previous studies used solvent extraction of bacterial cultures followed by gas chromatography-mass spectrometry (GC-MS) to detect and quantify ketones and hydrocarbons (Beller HR, Goh EB, Keasling JD, Appl. Environ. Microbiol. 76: 1212-1223, 2010; Sukovich DJ, Seffernick JL, Richman JE, Gralnick JA, Wackett LP, Appl. Environ. Microbiol. 76: 3850-3862, 2010). While these analyses are powerful and accurate, their labor-intensive nature makes them intractable to high-throughput screening; therefore, methods for rapid identification of bacterial strains that are overproducing hydrocarbons are needed. The use of high-throughput evaluation of bacterial and algal hydrophobic molecule production via Nile red fluorescence from lipids and esters was extended in this study to include hydrocarbons and ketones. This work demonstrated accurate, high-throughput detection of high-level bacterial long-chain ketone and hydrocarbon production by screening for increased fluorescence of the hydrophobic dye Nile red.« less

High-throughput analysis of lipid hydroperoxides in edible oils and fats using the fluorescent reagent diphenyl-1-pyrenylphosphine.

PubMed

Santas, Jonathan; Guzmán, Yeimmy J; Guardiola, Francesc; Rafecas, Magdalena; Bou, Ricard

2014-11-01

A fluorometric method for the determination of hydroperoxides (HP) in edible oils and fats using the reagent diphenyl-1-pyrenylphosphine (DPPP) was developed and validated. Two solvent media containing 100% butanol or a mixture of chloroform/methanol (2:1, v/v) can be used to solubilise lipid samples. Regardless of the solvent used to solubilise the sample, the DPPP method was precise, accurate, sensitive and easy to perform. The HP content of 43 oil and fat samples was determined and the results were compared with those obtained by means of the AOCS Official Method for the determination of peroxide value (PV) and the ferrous oxidation-xylenol orange (FOX) method. The proposed method not only correlates well with the PV and FOX methods, but also presents some advantages such as requiring low sample and solvent amounts and being suitable for high-throughput sample analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

A bioinformatics roadmap for the human vaccines project.

PubMed

Scheuermann, Richard H; Sinkovits, Robert S; Schenkelberg, Theodore; Koff, Wayne C

2017-06-01

Biomedical research has become a data intensive science in which high throughput experimentation is producing comprehensive data about biological systems at an ever-increasing pace. The Human Vaccines Project is a new public-private partnership, with the goal of accelerating development of improved vaccines and immunotherapies for global infectious diseases and cancers by decoding the human immune system. To achieve its mission, the Project is developing a Bioinformatics Hub as an open-source, multidisciplinary effort with the overarching goal of providing an enabling infrastructure to support the data processing, analysis and knowledge extraction procedures required to translate high throughput, high complexity human immunology research data into biomedical knowledge, to determine the core principles driving specific and durable protective immune responses.

Bioanalytical high-throughput selected reaction monitoring-LC/MS determination of selected estrogen receptor modulators in human plasma: 2000 samples/day.

PubMed

Zweigenbaum, J; Henion, J

2000-06-01

The high-throughput determination of small molecules in biological matrixes has become an important part of drug discovery. This work shows that increased throughput LC/MS/MS techniques can be used for the analysis of selected estrogen receptor modulators in human plasma where more than 2000 samples may be analyzed in a 24-h period. The compounds used to demonstrate the high-throughput methodology include tamoxifen, raloxifene, 4-hydroxytamoxifen, nafoxidine, and idoxifene. Tamoxifen and raloxifene are used in both breast cancer therapy and osteoporosis and have shown prophylactic potential for the reduction of the risk of breast cancer. The described strategy provides LC/MS/MS separation and quantitation for each of the five test articles in control human plasma. The method includes sample preparation employing liquid-liquid extraction in the 96-well format, an LC separation of the five compounds in less than 30 s, and selected reaction monitoring detection from low nano- to microgram per milliter levels. Precision and accuracy are determined where each 96-well plate is considered a typical "tray" having calibration standards and quality control (QC) samples dispersed through each plate. A concept is introduced where 24 96-well plates analyzed in 1 day is considered a "grand tray", and the method is cross-validated with standards placed only at the beginning of the first plate and the end of the last plate. Using idoxifene-d5 as an internal standard, the results obtained for idoxifene and tamoxifen satisfy current bioanalytical method validation criteria on two separate days where 2112 and 2304 samples were run, respectively. Method validation included 24-h autosampler stability and one freeze-thaw cycle stability for the extracts. Idoxifene showed acceptable results with accuracy ranging from 0.3% for the high quality control (QC) to 15.4% for the low QC and precision of 3.6%-13.9% relative standard deviation. Tamoxifen showed accuracy ranging from 1.6% to 13.8% and precision from 7.8% to 15.2%. The linear dynamic range for these compounds was 3 orders of magnitude. The limit of quantification was 5 and 50 ng/ mL for tamoxifen and idoxifene, respectively. The other compounds in this study in general satisfy the more relaxed bioanalytical acceptance criteria for modern drug discovery. It is suggested that the quantification levels reported in this high-throughput analysis example are adequate for many drug discovery and related early pharmaceutical studies.

Robust high-throughput batch screening method in 384-well format with optical in-line resin quantification.

PubMed

Kittelmann, Jörg; Ottens, Marcel; Hubbuch, Jürgen

2015-04-15

High-throughput batch screening technologies have become an important tool in downstream process development. Although continuative miniaturization saves time and sample consumption, there is yet no screening process described in the 384-well microplate format. Several processes are established in the 96-well dimension to investigate protein-adsorbent interactions, utilizing between 6.8 and 50 μL resin per well. However, as sample consumption scales with resin volumes and throughput scales with experiments per microplate, they are limited in costs and saved time. In this work, a new method for in-well resin quantification by optical means, applicable in the 384-well format, and resin volumes as small as 0.1 μL is introduced. A HTS batch isotherm process is described, utilizing this new method in combination with optical sample volume quantification for screening of isotherm parameters in 384-well microplates. Results are qualified by confidence bounds determined by bootstrap analysis and a comprehensive Monte Carlo study of error propagation. This new approach opens the door to a variety of screening processes in the 384-well format on HTS stations, higher quality screening data and an increase in throughput. Copyright © 2015 Elsevier B.V. All rights reserved.

Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis.

PubMed

Pan, Yuchen; Sackmann, Eric K; Wypisniak, Karolina; Hornsby, Michael; Datwani, Sammy S; Herr, Amy E

2016-12-23

High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality - the binding affinity - is quantified through the dissociation constant (K D ) of each recombinant antibody and the target antigen. To characterize the K D of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The K D for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization.

Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis

PubMed Central

Pan, Yuchen; Sackmann, Eric K.; Wypisniak, Karolina; Hornsby, Michael; Datwani, Sammy S.; Herr, Amy E.

2016-01-01

High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality – the binding affinity – is quantified through the dissociation constant (KD) of each recombinant antibody and the target antigen. To characterize the KD of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The KD for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization. PMID:28008969

Influence of Protein Abundance on High-Throughput Protein-Protein Interaction Detection

DTIC Science & Technology

2009-06-05

the interaction data sets we determined, via comparisons with strict randomized simulations , the propensity for essential proteins to selectively...and analysis of high- quality PPI data sets. Materials and Methods We analyzed protein interaction networks for yeast and E. coli determined from Y2H...we reinvestigated the centrality-lethality rule, which implies that proteins having more interactions are more likely to be essential. From analysis

A spectrophotometric assay for fatty acid amide hydrolase suitable for high-throughput screening.

PubMed

De Bank, Paul A; Kendall, David A; Alexander, Stephen P H

2005-04-15

Signalling via the endocannabinoids anandamide and 2-arachidonylglycerol appears to be terminated largely through the action of the enzyme fatty acid amide hydrolase (FAAH). In this report, we describe a simple spectrophotometric assay to detect FAAH activity in vitro using the ability of the enzyme to hydrolyze oleamide and measuring the resultant production of ammonia with a NADH/NAD+-coupled enzyme reaction. This dual-enzyme assay was used to determine Km and Vmax values of 104 microM and 5.7 nmol/min/mgprotein, respectively, for rat liver FAAH-catalyzed oleamide hydrolysis. Inhibitor potency was determined with the resultant rank order of methyl arachidonyl fluorophosphonate>phenylmethylsulphonyl fluoride>anandamide. This assay system was also adapted for use in microtiter plates and its ability to detect a known inhibitor of FAAH demonstrated, highlighting its potential for use in high-throughput screening.

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

PubMed Central

Lane, Darius J. R.; Lawen, Alfons

2014-01-01

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture. PMID:24747535

A high-throughput technique for determining grain boundary character non-destructively in microstructures with through-thickness grains

DOE PAGES

Seita, Matteo; Volpi, Marco; Patala, Srikanth; ...

2016-06-24

Grain boundaries (GBs) govern many properties of polycrystalline materials. However, because of their structural variability, our knowledge of GB constitutive relations is still very limited. We present a novel method to characterise the complete crystallography of individual GBs non-destructively, with high-throughput, and using commercially available tools. This method combines electron diffraction, optical reflectance and numerical image analysis to determine all five crystallographic parameters of numerous GBs in samples with through-thickness grains. We demonstrate the technique by measuring the crystallographic character of about 1,000 individual GBs in aluminum in a single run. Our method enables cost- and time-effective assembly of crystallography–propertymore » databases for thousands of individual GBs. Furthermore, such databases are essential for identifying GB constitutive relations and for predicting GB-related behaviours of polycrystalline solids.« less

Applicability of discovery science approach to determine biological effects of mobile phone radiation.

PubMed

Leszczynski, Dariusz; Nylund, Reetta; Joenväärä, Sakari; Reivinen, Jukka

2004-02-01

We argue that the use of high-throughput screening techniques, although expensive and laborious, is justified and necessary in studies that examine biological effects of mobile phone radiation. The "case of hsp27 protein" presented here suggests that even proteins with only modestly altered (by exposure to mobile phone radiation) expression and activity might have an impact on cell physiology. However, this short communication does not attempt to present the full scientific evidence that is far too large to be presented in a single article and that is being prepared for publication in three separate research articles. Examples of the experimental evidence presented here were designed to show the flow of experimental process demonstrating that the use of high-throughput screening techniques might help in rapid identification of the responding proteins. This, in turn, can help in speeding up of the process of determining whether these changes might affect human health.*

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

EPA Pesticide Factsheets

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

Medium-throughput processing of whole mount in situ hybridisation experiments into gene expression domains.

PubMed

Crombach, Anton; Cicin-Sain, Damjan; Wotton, Karl R; Jaeger, Johannes

2012-01-01

Understanding the function and evolution of developmental regulatory networks requires the characterisation and quantification of spatio-temporal gene expression patterns across a range of systems and species. However, most high-throughput methods to measure the dynamics of gene expression do not preserve the detailed spatial information needed in this context. For this reason, quantification methods based on image bioinformatics have become increasingly important over the past few years. Most available approaches in this field either focus on the detailed and accurate quantification of a small set of gene expression patterns, or attempt high-throughput analysis of spatial expression through binary pattern extraction and large-scale analysis of the resulting datasets. Here we present a robust, "medium-throughput" pipeline to process in situ hybridisation patterns from embryos of different species of flies. It bridges the gap between high-resolution, and high-throughput image processing methods, enabling us to quantify graded expression patterns along the antero-posterior axis of the embryo in an efficient and straightforward manner. Our method is based on a robust enzymatic (colorimetric) in situ hybridisation protocol and rapid data acquisition through wide-field microscopy. Data processing consists of image segmentation, profile extraction, and determination of expression domain boundary positions using a spline approximation. It results in sets of measured boundaries sorted by gene and developmental time point, which are analysed in terms of expression variability or spatio-temporal dynamics. Our method yields integrated time series of spatial gene expression, which can be used to reverse-engineer developmental gene regulatory networks across species. It is easily adaptable to other processes and species, enabling the in silico reconstitution of gene regulatory networks in a wide range of developmental contexts.

High-throughput determination of vancomycin in human plasma by a cost-effective system of two-dimensional liquid chromatography.

PubMed

Sheng, Yanghao; Zhou, Boting

2017-05-26

Therapeutic drug monitoring (TDM) is one of the most important services of clinical laboratories. Two main techniques are commonly used: the immunoassay and chromatography method. We have developed a cost-effective system of two-dimensional liquid chromatography with ultraviolet detection (2D-LC-UV) for high-throughput determination of vancomycin in human plasma that combines the automation and low start-up costs of the immunoassay with the high selectivity and sensitivity of the liquid chromatography coupled with mass spectrometric detection without incurring their disadvantages, achieving high cost-effectiveness. This 2D-LC system offers a large volume injection to provide sufficient sensitivity and uses simulated gradient peak compression technology to control peak broadening and to improve peak shape. A middle column was added to reduce the analysis cycle time and make it suitable for high-throughput routine clinical assays. The analysis cycle time was 4min and the peak width was 0.8min. Compared with other chromatographic methods that have been developed, the analysis cycle time and peak width for vancomycin was reduced significantly. The lower limit of quantification was 0.20μg/mL for vancomycin, which is the same as certain LC-MS/MS methods that have been recently developed and validated. The method is rapid, automated, and low-cost and has high selectivity and sensitivity for the quantification of vancomycin in human plasma, thus making it well-suited for use in hospital clinical laboratories. Copyright © 2017 Elsevier B.V. All rights reserved.

Apparatus and methods for monitoring the concentrations of hazardous airborne substances, especially lead

DOEpatents

Zaromb, Solomon

2004-07-13

Air is sampled at a rate in excess of 100 L/min, preferably at 200-300 L/min, so as to collect therefrom a substantial fraction, i.e., at least 20%, preferably 60-100%, of airborne particulates. A substance of interest (analyte), such as lead, is rapidly solubilized from the the collected particulates into a sample of liquid extractant, and the concentration of the analyte in the extractant sample is determined. The high-rate air sampling and particulate collection may be effected with a high-throughput filter cartridge or with a recently developed portable high-throughput liquid-absorption air sampler. Rapid solubilization of lead is achieved by a liquid extractant comprising 0.1-1 M of acetic acid or acetate, preferably at a pH of 5 or less and preferably with inclusion of 1-10% of hydrogen peroxide. Rapid determination of the lead content in the liquid extractant may be effected with a colorimetric or an electroanalytical analyzer.

Using high-throughput barcode sequencing to efficiently map connectomes

PubMed Central

Peikon, Ian D.; Kebschull, Justus M.; Vagin, Vasily V.; Ravens, Diana I.; Sun, Yu-Chi; Brouzes, Eric; Corrêa, Ivan R.; Bressan, Dario

2017-01-01

Abstract The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision—a ‘connectome’—is based on imaging methods that are slow, labor-intensive and expensive. Here, we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence—an RNA ‘barcode’—which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and joined into a form suitable for sequencing. Although our failure to develop an efficient barcode joining scheme precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost. PMID:28449067

An automated, high-throughput plant phenotyping system using machine learning-based plant segmentation and image analysis.

PubMed

Lee, Unseok; Chang, Sungyul; Putra, Gian Anantrio; Kim, Hyoungseok; Kim, Dong Hwan

2018-01-01

A high-throughput plant phenotyping system automatically observes and grows many plant samples. Many plant sample images are acquired by the system to determine the characteristics of the plants (populations). Stable image acquisition and processing is very important to accurately determine the characteristics. However, hardware for acquiring plant images rapidly and stably, while minimizing plant stress, is lacking. Moreover, most software cannot adequately handle large-scale plant imaging. To address these problems, we developed a new, automated, high-throughput plant phenotyping system using simple and robust hardware, and an automated plant-imaging-analysis pipeline consisting of machine-learning-based plant segmentation. Our hardware acquires images reliably and quickly and minimizes plant stress. Furthermore, the images are processed automatically. In particular, large-scale plant-image datasets can be segmented precisely using a classifier developed using a superpixel-based machine-learning algorithm (Random Forest), and variations in plant parameters (such as area) over time can be assessed using the segmented images. We performed comparative evaluations to identify an appropriate learning algorithm for our proposed system, and tested three robust learning algorithms. We developed not only an automatic analysis pipeline but also a convenient means of plant-growth analysis that provides a learning data interface and visualization of plant growth trends. Thus, our system allows end-users such as plant biologists to analyze plant growth via large-scale plant image data easily.

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

Combinatorial electrochemical cell array for high throughput screening of micro-fuel-cells and metal/air batteries.

PubMed

Jiang, Rongzhong

2007-07-01

An electrochemical cell array was designed that contains a common air electrode and 16 microanodes for high throughput screening of both fuel cells (based on polymer electrolyte membrane) and metal/air batteries (based on liquid electrolyte). Electrode materials can easily be coated on the anodes of the electrochemical cell array and screened by switching a graphite probe from one cell to the others. The electrochemical cell array was used to study direct methanol fuel cells (DMFCs), including high throughput screening of electrode catalysts and determination of optimum operating conditions. For screening of DMFCs, there is about 6% relative standard deviation (percentage of standard deviation versus mean value) for discharge current from 10 to 20 mAcm(2). The electrochemical cell array was also used to study tin/air batteries. The effect of Cu content in the anode electrode on the discharge performance of the tin/air battery was investigated. The relative standard deviations for screening of metal/air battery (based on zinc/air) are 2.4%, 3.6%, and 5.1% for discharge current at 50, 100, and 150 mAcm(2), respectively.

Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

PubMed Central

Pinzon, Neissa M.; Aukema, Kelly G.; Gralnick, Jeffrey A.; Wackett, Lawrence P.

2011-01-01

ABSTRACT A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone production as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. PMID:21712420

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodland, Karin D.; Adkins, Joshua N.; Ansong, Charles

Capabilities in mass spectrometry are evolving rapidly, with recent improvements in sensitivity, data analysis, and most important, from the standpoint of this review, much higher throughput allowing analysis of many samples in a single day. This short review describes how these improvements in mass spectrometry can be used to dissect host-pathogen interactions using Salmonella as a model system. This approach enabled direct identification of the majority of annotated Salmonella proteins, quantitation of expression changes under various in vitro growth conditions, and new insights into virulence and expression of Salmonella proteins within host cell cells. One of the most significant findingsmore » is that a very high percentage of the all annotated genes (>20%) in Salmonella 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 these regulators in coordinating virulence expression. Overall high throughput mass spectrometry provides a new view of pathogen-host interactions emphasizing the protein products and defining how protein interactions determine the outcome of infection.« less

High-throughput protein analysis integrating bioinformatics and experimental assays

PubMed Central

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

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.

Application of ToxCast High-Throughput Screening and ...

EPA Pesticide Factsheets

Slide presentation at the SETAC annual meeting on High-Throughput Screening and Modeling Approaches to Identify Steroidogenesis Distruptors Slide presentation at the SETAC annual meeting on High-Throughput Screening and Modeling Approaches to Identify Steroidogenssis Distruptors

96-Well Plate Colorimetric Assay for K(sub i) Determination of (plusmn)-2-Benzylsuccinic Acid, an Inhibitor of Carboxypeptidase A

ERIC Educational Resources Information Center

Wentland, Mark P.; Raza, Shaan; Yingtong Gao

2004-01-01

An appropriate assay to determine the inhibition potency of carboxypeptidase A (CPA) in 96-well format to illustrate how high throughput screening is used in modern drug discovery to identify bioactive molecules is developed. Efforts in developing a colorimetric 96-well plate assay for determination of the K(sub i) for inhibition of CPA by…

High-throughput analysis of sulfatides in cerebrospinal fluid using automated extraction and UPLC-MS/MS.

PubMed

Blomqvist, Maria; Borén, Jan; Zetterberg, Henrik; Blennow, Kaj; Månsson, Jan-Eric; Ståhlman, Marcus

2017-07-01

Sulfatides (STs) are a group of glycosphingolipids that are highly expressed in brain. Due to their importance for normal brain function and their potential involvement in neurological diseases, development of accurate and sensitive methods for their determination is needed. Here we describe a high-throughput oriented and quantitative method for the determination of STs in cerebrospinal fluid (CSF). The STs were extracted using a fully automated liquid/liquid extraction method and quantified using ultra-performance liquid chromatography coupled to tandem mass spectrometry. With the high sensitivity of the developed method, quantification of 20 ST species from only 100 μl of CSF was performed. Validation of the method showed that the STs were extracted with high recovery (90%) and could be determined with low inter- and intra-day variation. Our method was applied to a patient cohort of subjects with an Alzheimer's disease biomarker profile. Although the total ST levels were unaltered compared with an age-matched control group, we show that the ratio of hydroxylated/nonhydroxylated STs was increased in the patient cohort. In conclusion, we believe that the fast, sensitive, and accurate method described in this study is a powerful new tool for the determination of STs in clinical as well as preclinical settings. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed

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

2014-05-01

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

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...

The Stanford Automated Mounter: Enabling High-Throughput Protein Crystal Screening at SSRL

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, C.A.; Cohen, A.E.

2009-05-26

The macromolecular crystallography experiment lends itself perfectly to high-throughput technologies. The initial steps including the expression, purification, and crystallization of protein crystals, along with some of the later steps involving data processing and structure determination have all been automated to the point where some of the last remaining bottlenecks in the process have been crystal mounting, crystal screening, and data collection. At the Stanford Synchrotron Radiation Laboratory, a National User Facility that provides extremely brilliant X-ray photon beams for use in materials science, environmental science, and structural biology research, the incorporation of advanced robotics has enabled crystals to be screenedmore » in a true high-throughput fashion, thus dramatically accelerating the final steps. Up to 288 frozen crystals can be mounted by the beamline robot (the Stanford Auto-Mounting System) and screened for diffraction quality in a matter of hours without intervention. The best quality crystals can then be remounted for the collection of complete X-ray diffraction data sets. Furthermore, the entire screening and data collection experiment can be controlled from the experimenter's home laboratory by means of advanced software tools that enable network-based control of the highly automated beamlines.« less

An Efficient Semi-supervised Learning Approach to Predict SH2 Domain Mediated Interactions.

PubMed

Kundu, Kousik; Backofen, Rolf

2017-01-01

Src homology 2 (SH2) domain is an important subclass of modular protein domains that plays an indispensable role in several biological processes in eukaryotes. SH2 domains specifically bind to the phosphotyrosine residue of their binding peptides to facilitate various molecular functions. For determining the subtle binding specificities of SH2 domains, it is very important to understand the intriguing mechanisms by which these domains recognize their target peptides in a complex cellular environment. There are several attempts have been made to predict SH2-peptide interactions using high-throughput data. However, these high-throughput data are often affected by a low signal to noise ratio. Furthermore, the prediction methods have several additional shortcomings, such as linearity problem, high computational complexity, etc. Thus, computational identification of SH2-peptide interactions using high-throughput data remains challenging. Here, we propose a machine learning approach based on an efficient semi-supervised learning technique for the prediction of 51 SH2 domain mediated interactions in the human proteome. In our study, we have successfully employed several strategies to tackle the major problems in computational identification of SH2-peptide interactions.

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

NASA Astrophysics Data System (ADS)

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

2015-12-01

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

Development of high-throughput and high sensitivity capillary gel electrophoresis platform method for Western, Eastern, and Venezuelan equine encephalitis (WEVEE) virus like particles (VLPs) purity determination and characterization.

PubMed

Gollapudi, Deepika; Wycuff, Diane L; Schwartz, Richard M; Cooper, Jonathan W; Cheng, K C

2017-10-01

In this paper, we describe development of a high-throughput, highly sensitive method based on Lab Chip CGE-SDS platform for purity determination and characterization of virus-like particle (VLP) vaccines. A capillary gel electrophoresis approach requiring about 41 s per sample for analysis and demonstrating sensitivity to protein initial concentrations as low as 20 μg/mL, this method has been used previously to evaluate monoclonal antibodies, but this application for lot release assay of VLPs using this platform is unique. The method was qualified and shown to be accurate for the quantitation of VLP purity. Assay repeatability was confirmed to be less than 2% relative standard deviation of the mean (% RSD) with interday precision less than 2% RSD. The assay can evaluate purified VLPs in a concentration range of 20-249 μg/mL for VEE and 20-250 μg/mL for EEE and WEE VLPs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Automated Transition State Theory Calculations for High-Throughput Kinetics.

PubMed

Bhoorasingh, Pierre L; Slakman, Belinda L; Seyedzadeh Khanshan, Fariba; Cain, Jason Y; West, Richard H

2017-09-21

A scarcity of known chemical kinetic parameters leads to the use of many reaction rate estimates, which are not always sufficiently accurate, in the construction of detailed kinetic models. To reduce the reliance on these estimates and improve the accuracy of predictive kinetic models, we have developed a high-throughput, fully automated, reaction rate calculation method, AutoTST. The algorithm integrates automated saddle-point geometry search methods and a canonical transition state theory kinetics calculator. The automatically calculated reaction rates compare favorably to existing estimated rates. Comparison against high level theoretical calculations show the new automated method performs better than rate estimates when the estimate is made by a poor analogy. The method will improve by accounting for internal rotor contributions and by improving methods to determine molecular symmetry.

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants*

PubMed Central

Carmona, Santiago J.; Nielsen, Morten; Schafer-Nielsen, Claus; Mucci, Juan; Altcheh, Jaime; Balouz, Virginia; Tekiel, Valeria; Frasch, Alberto C.; Campetella, Oscar; Buscaglia, Carlos A.; Agüero, Fernán

2015-01-01

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens. PMID:25922409

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants.

PubMed

Carmona, Santiago J; Nielsen, Morten; Schafer-Nielsen, Claus; Mucci, Juan; Altcheh, Jaime; Balouz, Virginia; Tekiel, Valeria; Frasch, Alberto C; Campetella, Oscar; Buscaglia, Carlos A; Agüero, Fernán

2015-07-01

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15 mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15 mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼ threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A high-throughput assay of NK cell activity in whole blood and its clinical application

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung

2014-03-14

Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate themore » status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.« less

High Throughput Transcriptomics: From screening to pathways

EPA Science Inventory

The EPA ToxCast effort has screened thousands of chemicals across hundreds of high-throughput in vitro screening assays. The project is now leveraging high-throughput transcriptomic (HTTr) technologies to substantially expand its coverage of biological pathways. The first HTTr sc...

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

NASA Astrophysics Data System (ADS)

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

2017-12-01

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

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

PubMed

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

2017-01-01

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

High Throughput Pharmacokinetics for Environmental Chemicals (FutureToxII)

EPA Science Inventory

Pharmacokinetic (PK) models are critical to determine whether chemical exposures produce potentially hazardous tissue concentrations. For bioactivity identified in vitro (e.g. ToxCast) – hazardous or not – PK models can forecast exposure thresholds, below which no significant bio...

High Throughput Experimental Materials Database

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zakutayev, Andriy; Perkins, John; Schwarting, Marcus

The mission of the High Throughput Experimental Materials Database (HTEM DB) is to enable discovery of new materials with useful properties by releasing large amounts of high-quality experimental data to public. The HTEM DB contains information about materials obtained from high-throughput experiments at the National Renewable Energy Laboratory (NREL).

20180311 - High Throughput Transcriptomics: From screening to pathways (SOT 2018)

EPA Science Inventory

The EPA ToxCast effort has screened thousands of chemicals across hundreds of high-throughput in vitro screening assays. The project is now leveraging high-throughput transcriptomic (HTTr) technologies to substantially expand its coverage of biological pathways. The first HTTr sc...

Evaluation of Sequencing Approaches for High-Throughput Transcriptomics - (BOSC)

EPA Science Inventory

Whole-genome in vitro transcriptomics has shown the capability to identify mechanisms of action and estimates of potency for chemical-mediated effects in a toxicological framework, but with limited throughput and high cost. The generation of high-throughput global gene expression...

Performance Evaluation of IEEE 802.11ah Networks With High-Throughput Bidirectional Traffic.

PubMed

Šljivo, Amina; Kerkhove, Dwight; Tian, Le; Famaey, Jeroen; Munteanu, Adrian; Moerman, Ingrid; Hoebeke, Jeroen; De Poorter, Eli

2018-01-23

So far, existing sub-GHz wireless communication technologies focused on low-bandwidth, long-range communication with large numbers of constrained devices. Although these characteristics are fine for many Internet of Things (IoT) applications, more demanding application requirements could not be met and legacy Internet technologies such as Transmission Control Protocol/Internet Protocol (TCP/IP) could not be used. This has changed with the advent of the new IEEE 802.11ah Wi-Fi standard, which is much more suitable for reliable bidirectional communication and high-throughput applications over a wide area (up to 1 km). The standard offers great possibilities for network performance optimization through a number of physical- and link-layer configurable features. However, given that the optimal configuration parameters depend on traffic patterns, the standard does not dictate how to determine them. Such a large number of configuration options can lead to sub-optimal or even incorrect configurations. Therefore, we investigated how two key mechanisms, Restricted Access Window (RAW) grouping and Traffic Indication Map (TIM) segmentation, influence scalability, throughput, latency and energy efficiency in the presence of bidirectional TCP/IP traffic. We considered both high-throughput video streaming traffic and large-scale reliable sensing traffic and investigated TCP behavior in both scenarios when the link layer introduces long delays. This article presents the relations between attainable throughput per station and attainable number of stations, as well as the influence of RAW, TIM and TCP parameters on both. We found that up to 20 continuously streaming IP-cameras can be reliably connected via IEEE 802.11ah with a maximum average data rate of 160 kbps, whereas 10 IP-cameras can achieve average data rates of up to 255 kbps over 200 m. Up to 6960 stations transmitting every 60 s can be connected over 1 km with no lost packets. The presented results enable the fine tuning of RAW and TIM parameters for throughput-demanding reliable applications (i.e., video streaming, firmware updates) on one hand, and very dense low-throughput reliable networks with bidirectional traffic on the other hand.

Performance Evaluation of IEEE 802.11ah Networks With High-Throughput Bidirectional Traffic

PubMed Central

Kerkhove, Dwight; Tian, Le; Munteanu, Adrian; De Poorter, Eli

2018-01-01

So far, existing sub-GHz wireless communication technologies focused on low-bandwidth, long-range communication with large numbers of constrained devices. Although these characteristics are fine for many Internet of Things (IoT) applications, more demanding application requirements could not be met and legacy Internet technologies such as Transmission Control Protocol/Internet Protocol (TCP/IP) could not be used. This has changed with the advent of the new IEEE 802.11ah Wi-Fi standard, which is much more suitable for reliable bidirectional communication and high-throughput applications over a wide area (up to 1 km). The standard offers great possibilities for network performance optimization through a number of physical- and link-layer configurable features. However, given that the optimal configuration parameters depend on traffic patterns, the standard does not dictate how to determine them. Such a large number of configuration options can lead to sub-optimal or even incorrect configurations. Therefore, we investigated how two key mechanisms, Restricted Access Window (RAW) grouping and Traffic Indication Map (TIM) segmentation, influence scalability, throughput, latency and energy efficiency in the presence of bidirectional TCP/IP traffic. We considered both high-throughput video streaming traffic and large-scale reliable sensing traffic and investigated TCP behavior in both scenarios when the link layer introduces long delays. This article presents the relations between attainable throughput per station and attainable number of stations, as well as the influence of RAW, TIM and TCP parameters on both. We found that up to 20 continuously streaming IP-cameras can be reliably connected via IEEE 802.11ah with a maximum average data rate of 160 kbps, whereas 10 IP-cameras can achieve average data rates of up to 255 kbps over 200 m. Up to 6960 stations transmitting every 60 s can be connected over 1 km with no lost packets. The presented results enable the fine tuning of RAW and TIM parameters for throughput-demanding reliable applications (i.e., video streaming, firmware updates) on one hand, and very dense low-throughput reliable networks with bidirectional traffic on the other hand. PMID:29360798

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...

Rapid determination of enantiomeric excess: a focus on optical approaches.

PubMed

Leung, Diana; Kang, Sung Ok; Anslyn, Eric V

2012-01-07

High-throughput screening (HTS) methods are becoming increasingly essential in discovering chiral catalysts or auxiliaries for asymmetric transformations due to the advent of parallel synthesis and combinatorial chemistry. Both parallel synthesis and combinatorial chemistry can lead to the exploration of a range of structural candidates and reaction conditions as a means to obtain the highest enantiomeric excess (ee) of a desired transformation. One current bottleneck in these approaches to asymmetric reactions is the determination of ee, which has led researchers to explore a wide range of HTS techniques. To be truly high-throughput, it has been proposed that a technique that can analyse a thousand or more samples per day is needed. Many of the current approaches to this goal are based on optical methods because they allow for a rapid determination of ee due to quick data collection and their parallel analysis capabilities. In this critical review these techniques are reviewed with a discussion of their respective advantages and drawbacks, and with a contrast to chromatographic methods (180 references). This journal is © The Royal Society of Chemistry 2012

Towards roll-to-roll manufacturing of polymer photonic devices

NASA Astrophysics Data System (ADS)

Subbaraman, Harish; Lin, Xiaohui; Ling, Tao; Guo, L. Jay; Chen, Ray T.

2014-03-01

Traditionally, polymer photonic devices are fabricated using clean-room processes such as photolithography, e-beam lithography, reactive ion etching (RIE) and lift-off methods etc, which leads to long fabrication time, low throughput and high cost. We have utilized a novel process for fabricating polymer photonic devices using a combination of imprinting and ink jet printing methods, which provides high throughput on a variety of rigid and flexible substrates with low cost. We discuss the manufacturing challenges that need to be overcome in order to realize true implementation of roll-to-roll manufacturing of flexible polymer photonic systems. Several metrology and instrumentation challenges involved such as availability of particulate-free high quality substrate, development and implementation of high-speed in-line and off-line inspection and diagnostic tools with adaptive control for patterned and unpatterned material films, development of reliable hardware, etc need to be addressed and overcome in order to realize a successful manufacturing process. Due to extreme resolution requirements compared to print media, the burden of software and hardware tools on the throughput also needs to be carefully determined. Moreover, the effect of web wander and variations in web speed need to accurately be determined in the design of the system hardware and software. In this paper, we show the realization of solutions for few challenges, and utilizing these solutions for developing a high-rate R2R dual stage ink-jet printer that can provide alignment accuracy of <10μm at a web speed of 5m/min. The development of a roll-to-roll manufacturing system for polymer photonic systems opens limitless possibilities for the deployment of high performance components in a variety of applications including communication, sensing, medicine, agriculture, energy, lighting etc.

High-throughput screening in two dimensions: binding intensity and off-rate on a peptide microarray.

PubMed

Greving, Matthew P; Belcher, Paul E; Cox, Conor D; Daniel, Douglas; Diehnelt, Chris W; Woodbury, Neal W

2010-07-01

We report a high-throughput two-dimensional microarray-based screen, incorporating both target binding intensity and off-rate, which can be used to analyze thousands of compounds in a single binding assay. Relative binding intensities and time-resolved dissociation are measured for labeled tumor necrosis factor alpha (TNF-alpha) bound to a peptide microarray. The time-resolved dissociation is fitted to a one-component exponential decay model, from which relative dissociation rates are determined for all peptides with binding intensities above background. We show that most peptides with the slowest off-rates on the microarray also have the slowest off-rates when measured by surface plasmon resonance (SPR). 2010 Elsevier Inc. All rights reserved.

High Throughput Determination of Ricinine Abrine and Alpha ...

EPA Pesticide Factsheets

Analytical Method This document provides the standard operating procedure for determination of ricinine (RIC), abrine (ABR), and α-amanitin (AMAN) in drinking water by isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS). This method is designed to support site-specific cleanup goals of environmental remediation activities following a homeland security incident involving one or a combination of these analytes.

High throughput nonparametric probability density estimation.

PubMed

Farmer, Jenny; Jacobs, Donald

2018-01-01

In high throughput applications, such as those found in bioinformatics and finance, it is important to determine accurate probability distribution functions despite only minimal information about data characteristics, and without using human subjectivity. Such an automated process for univariate data is implemented to achieve this goal by merging the maximum entropy method with single order statistics and maximum likelihood. The only required properties of the random variables are that they are continuous and that they are, or can be approximated as, independent and identically distributed. A quasi-log-likelihood function based on single order statistics for sampled uniform random data is used to empirically construct a sample size invariant universal scoring function. Then a probability density estimate is determined by iteratively improving trial cumulative distribution functions, where better estimates are quantified by the scoring function that identifies atypical fluctuations. This criterion resists under and over fitting data as an alternative to employing the Bayesian or Akaike information criterion. Multiple estimates for the probability density reflect uncertainties due to statistical fluctuations in random samples. Scaled quantile residual plots are also introduced as an effective diagnostic to visualize the quality of the estimated probability densities. Benchmark tests show that estimates for the probability density function (PDF) converge to the true PDF as sample size increases on particularly difficult test probability densities that include cases with discontinuities, multi-resolution scales, heavy tails, and singularities. These results indicate the method has general applicability for high throughput statistical inference.

High throughput nonparametric probability density estimation

PubMed Central

Farmer, Jenny

2018-01-01

In high throughput applications, such as those found in bioinformatics and finance, it is important to determine accurate probability distribution functions despite only minimal information about data characteristics, and without using human subjectivity. Such an automated process for univariate data is implemented to achieve this goal by merging the maximum entropy method with single order statistics and maximum likelihood. The only required properties of the random variables are that they are continuous and that they are, or can be approximated as, independent and identically distributed. A quasi-log-likelihood function based on single order statistics for sampled uniform random data is used to empirically construct a sample size invariant universal scoring function. Then a probability density estimate is determined by iteratively improving trial cumulative distribution functions, where better estimates are quantified by the scoring function that identifies atypical fluctuations. This criterion resists under and over fitting data as an alternative to employing the Bayesian or Akaike information criterion. Multiple estimates for the probability density reflect uncertainties due to statistical fluctuations in random samples. Scaled quantile residual plots are also introduced as an effective diagnostic to visualize the quality of the estimated probability densities. Benchmark tests show that estimates for the probability density function (PDF) converge to the true PDF as sample size increases on particularly difficult test probability densities that include cases with discontinuities, multi-resolution scales, heavy tails, and singularities. These results indicate the method has general applicability for high throughput statistical inference. PMID:29750803

HiCTMap: Detection and analysis of chromosome territory structure and position by high-throughput imaging.

PubMed

Jowhar, Ziad; Gudla, Prabhakar R; Shachar, Sigal; Wangsa, Darawalee; Russ, Jill L; Pegoraro, Gianluca; Ried, Thomas; Raznahan, Armin; Misteli, Tom

2018-06-01

The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues. Published by Elsevier Inc.

Spatial tuning of acoustofluidic pressure nodes by altering net sonic velocity enables high-throughput, efficient cell sorting

DOE PAGES

Jung, Seung-Yong; Notton, Timothy; Fong, Erika; ...

2015-01-07

Particle sorting using acoustofluidics has enormous potential but widespread adoption has been limited by complex device designs and low throughput. Here, we report high-throughput separation of particles and T lymphocytes (600 μL min -1) by altering the net sonic velocity to reposition acoustic pressure nodes in a simple two-channel device. Finally, the approach is generalizable to other microfluidic platforms for rapid, high-throughput analysis.

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

PubMed Central

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

2017-01-01

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

Development of a high-throughput in vitro assay using a novel Caco-2/rat hepatocyte system for the prediction of oral plasma area under the concentration versus time curve (AUC) in rats.

PubMed

Cheng, K-C; Li, Cheng; Hsieh, Yunsheng; Montgomery, Diana; Liu, Tongtong; White, Ronald E

2006-01-01

Previously, we have shown that a novel Caco-2/human hepatocyte system is a useful model for the prediction of oral bioavailability in humans. In this study, we attempted to use a similar system in a high-throughput screening mode for the selection of new compound entities (NCE) in drug discovery. A total of 72 compounds randomly selected from three different chemotypes were dosed orally in rats. In vivo plasma area under the concentration versus time curve (AUC) from 0-6 h of the parent compound was determined. The same compounds were also tested in the Caco-2/rat hepatocyte system. In vitro AUC from 0-3 h in the Caco-2 rat hepatocyte system was determined. The predictive usefulness of the Caco-2/rat hepatocyte system was evaluated by comparing the in vivo plasma AUC and the in vitro AUC. Linear regression analysis showed a reasonable correlation (R2 = 0.5) between the in vivo AUC and the in vitro AUC. Using 0.4 microM h in vivo AUC as a cut-off, compounds were categorized as either low or high AUC. The in vitro AUC successfully matched the corresponding in vivo category for sixty-three out of seventy-two compounds. The results presented in this study suggest that the Caco-2/rat hepatocyte system may be used as a high-throughput screen in drug discovery for pharmacokinetic behaviors of compounds in rats.

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

DOE PAGES

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

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

High-throughput screening (HTS) and modeling of the retinoid ...

EPA Pesticide Factsheets

Presentation at the Retinoids Review 2nd workshop in Brussels, Belgium on the application of high throughput screening and model to the retinoid system Presentation at the Retinoids Review 2nd workshop in Brussels, Belgium on the application of high throughput screening and model to the retinoid system

Evaluating High Throughput Toxicokinetics and Toxicodynamics for IVIVE (WC10)

EPA Science Inventory

High-throughput screening (HTS) generates in vitro data for characterizing potential chemical hazard. TK models are needed to allow in vitro to in vivo extrapolation (IVIVE) to real world situations. The U.S. EPA has created a public tool (R package “httk” for high throughput tox...

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

USDA-ARS?s Scientific Manuscript database

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...

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

EPA Science Inventory

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

A quantitative literature-curated gold standard for kinase-substrate pairs

PubMed Central

2011-01-01

We describe the Yeast Kinase Interaction Database (KID, http://www.moseslab.csb.utoronto.ca/KID/), which contains high- and low-throughput data relevant to phosphorylation events. KID includes 6,225 low-throughput and 21,990 high-throughput interactions, from greater than 35,000 experiments. By quantitatively integrating these data, we identified 517 high-confidence kinase-substrate pairs that we consider a gold standard. We show that this gold standard can be used to assess published high-throughput datasets, suggesting that it will enable similar rigorous assessments in the future. PMID:21492431

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

PubMed

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

2016-09-12

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

Outlook for Development of High-throughput Cryopreservation for Small-bodied Biomedical Model Fishes★

PubMed Central

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

2011-01-01

With the development of genomic research technologies, comparative genome studies among vertebrate species are becoming commonplace for human biomedical research. Fish offer unlimited versatility for biomedical research. Extensive studies are done using these fish models, yielding tens of thousands of specific strains and lines, and the number is increasing every day. Thus, high-throughput sperm cryopreservation is urgently needed to preserve these genetic resources. Although high-throughput processing has been widely applied for sperm cryopreservation in livestock for decades, application in biomedical model fishes is still in the concept-development stage because of the limited sample volumes and the biological characteristics of fish sperm. High-throughput processing in livestock was developed based on advances made in the laboratory and was scaled up for increased processing speed, capability for mass production, and uniformity and quality assurance. Cryopreserved germplasm combined with high-throughput processing constitutes an independent industry encompassing animal breeding, preservation of genetic diversity, and medical research. Currently, there is no specifically engineered system available for high-throughput of cryopreserved germplasm for aquatic species. This review is to discuss the concepts and needs for high-throughput technology for model fishes, propose approaches for technical development, and overview future directions of this approach. PMID:21440666

High-throughput accurate-wavelength lens-based visible spectrometer.

PubMed

Bell, Ronald E; Scotti, Filippo

2010-10-01

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

High-throughput measurements of biochemical responses using the plate::vision multimode 96 minilens array reader.

PubMed

Huang, Kuo-Sen; Mark, David; Gandenberger, Frank Ulrich

2006-01-01

The plate::vision is a high-throughput multimode reader capable of reading absorbance, fluorescence, fluorescence polarization, time-resolved fluorescence, and luminescence. Its performance has been shown to be quite comparable with other readers. When the reader is integrated into the plate::explorer, an ultrahigh-throughput screening system with event-driven software and parallel plate-handling devices, it becomes possible to run complicated assays with kinetic readouts in high-density microtiter plate formats for high-throughput screening. For the past 5 years, we have used the plate::vision and the plate::explorer to run screens and have generated more than 30 million data points. Their throughput, performance, and robustness have speeded up our drug discovery process greatly.

Sensitive high-throughput screening for the detection of reducing sugars.

PubMed

Mellitzer, Andrea; Glieder, Anton; Weis, Roland; Reisinger, Christoph; Flicker, Karlheinz

2012-01-01

The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Bruni, Renato

2014-01-01

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

Assessment of DNA damage in car spray painters exposed to organic solvents by the high-throughput comet assay.

PubMed

Londoño-Velasco, Elizabeth; Martínez-Perafán, Fabián; Carvajal-Varona, Silvio; García-Vallejo, Felipe; Hoyos-Giraldo, Luz Stella

2016-05-01

Occupational exposure as a painter is associated with DNA damage and development of cancer. Comet assay has been widely adopted as a sensitive and quantitative tool for DNA damage assessment at the individual cell level in populations exposed to genotoxics. The aim of this study was to assess the application of the high-throughput comet assay, to determine the DNA damage in car spray painters. The study population included 52 car spray painters and 52 unexposed subjects. A significant increase in the %TDNA median (p <  0.001) was observed in the exposed group in comparison to the unexposed group. Neither age (%TDNA: p =  0.913) nor time of exposure (%TDNA: p = 0.398) were significantly correlated with DNA damage. The car spray painters who consumed alcohol did not show a significant increase in DNA damage compared to nonalcohol consumers (p  > 0.05). The results showed an increase in DNA breaks in car spray painters exposed to organic solvents and paints; furthermore, they demonstrated the application of high-throughput comet assay in an occupational exposure study to genotoxic agents.

High-throughput screening of PLGA thin films utilizing hydrophobic fluorescent dyes for hydrophobic drug compounds.

PubMed

Steele, Terry W J; Huang, Charlotte L; Kumar, Saranya; Widjaja, Effendi; Chiang Boey, Freddy Yin; Loo, Joachim S C; Venkatraman, Subbu S

2011-10-01

Hydrophobic, antirestenotic drugs such as paclitaxel (PCTX) and rapamycin are often incorporated into thin film coatings for local delivery using implantable medical devices and polymers such as drug-eluting stents and balloons. Selecting the optimum coating formulation through screening the release profile of these drugs in thin films is time consuming and labor intensive. We describe here a high-throughput assay utilizing three model hydrophobic fluorescent compounds: fluorescein diacetate (FDAc), coumarin-6, and rhodamine 6G that were incorporated into poly(d,l-lactide-co-glycolide) (PLGA) and PLGA-polyethylene glycol films. Raman microscopy determined the hydrophobic fluorescent dye distribution within the PLGA thin films in comparison with that of PCTX. Their subsequent release was screened in a high-throughput assay and directly compared with HPLC quantification of PCTX release. It was observed that PCTX controlled-release kinetics could be mimicked by a hydrophobic dye that had similar octanol-water partition coefficient values and homogeneous dissolution in a PLGA matrix as the drug. In particular, FDAc was found to be the optimal hydrophobic dye at modeling the burst release as well as the total amount of PCTX released over a period of 30 days. Copyright © 2011 Wiley-Liss, Inc.

High-Throughput Sequencing Reveals Principles of Adeno-Associated Virus Serotype 2 Integration

PubMed Central

Janovitz, Tyler; Klein, Isaac A.; Oliveira, Thiago; Mukherjee, Piali; Nussenzweig, Michel C.; Sadelain, Michel

2013-01-01

Viral integrations are important in human biology, yet genome-wide integration profiles have not been determined for many viruses. Adeno-associated virus (AAV) infects most of the human population and is a prevalent gene therapy vector. AAV integrates into the human genome with preference for a single locus, termed AAVS1. However, the genome-wide integration of AAV has not been defined, and the principles underlying this recombination remain unclear. Using a novel high-throughput approach, integrant capture sequencing, nearly 12 million AAV junctions were recovered from a human cell line, providing five orders of magnitude more data than were previously available. Forty-five percent of integrations occurred near AAVS1, and several thousand novel integration hotspots were identified computationally. Most of these occurred in genes, with dozens of hotspots targeting known oncogenes. Viral replication protein binding sites (RBS) and transcriptional activity were major factors favoring integration. In a first for eukaryotic viruses, the data reveal a unique asymmetric integration profile with distinctive directional orientation of viral genomes. These studies provide a new understanding of AAV integration biology through the use of unbiased high-throughput data acquisition and bioinformatics. PMID:23720718

Automatic poisson peak harvesting for high throughput protein identification.

PubMed

Breen, E J; Hopwood, F G; Williams, K L; Wilkins, M R

2000-06-01

High throughput identification of proteins by peptide mass fingerprinting requires an efficient means of picking peaks from mass spectra. Here, we report the development of a peak harvester to automatically pick monoisotopic peaks from spectra generated on matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometers. The peak harvester uses advanced mathematical morphology and watershed algorithms to first process spectra to stick representations. Subsequently, Poisson modelling is applied to determine which peak in an isotopically resolved group represents the monoisotopic mass of a peptide. We illustrate the features of the peak harvester with mass spectra of standard peptides, digests of gel-separated bovine serum albumin, and with Escherictia coli proteins prepared by two-dimensional polyacrylamide gel electrophoresis. In all cases, the peak harvester proved effective in its ability to pick similar monoisotopic peaks as an experienced human operator, and also proved effective in the identification of monoisotopic masses in cases where isotopic distributions of peptides were overlapping. The peak harvester can be operated in an interactive mode, or can be completely automated and linked through to peptide mass fingerprinting protein identification tools to achieve high throughput automated protein identification.

Breast cancer diagnosis using spatial light interference microscopy

NASA Astrophysics Data System (ADS)

Majeed, Hassaan; Kandel, Mikhail E.; Han, Kevin; Luo, Zelun; Macias, Virgilia; Tangella, Krishnarao; Balla, Andre; Popescu, Gabriel

2015-11-01

The standard practice in histopathology of breast cancers is to examine a hematoxylin and eosin (H&E) stained tissue biopsy under a microscope to diagnose whether a lesion is benign or malignant. This determination is made based on a manual, qualitative inspection, making it subject to investigator bias and resulting in low throughput. Hence, a quantitative, label-free, and high-throughput diagnosis method is highly desirable. We present here preliminary results showing the potential of quantitative phase imaging for breast cancer screening and help with differential diagnosis. We generated phase maps of unstained breast tissue biopsies using spatial light interference microscopy (SLIM). As a first step toward quantitative diagnosis based on SLIM, we carried out a qualitative evaluation of our label-free images. These images were shown to two pathologists who classified each case as either benign or malignant. This diagnosis was then compared against the diagnosis of the two pathologists on corresponding H&E stained tissue images and the number of agreements were counted. The agreement between SLIM and H&E based diagnosis was 88% for the first pathologist and 87% for the second. Our results demonstrate the potential and promise of SLIM for quantitative, label-free, and high-throughput diagnosis.

Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

PubMed

Ryall, Karen A; Shin, Jimin; Yoo, Minjae; Hinz, Trista K; Kim, Jihye; Kang, Jaewoo; Heasley, Lynn E; Tan, Aik Choon

2015-12-01

Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. aikchoon.tan@ucdenver.edu. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Toxicokinetic and Dosimetry Modeling Tools for Exposure ...

EPA Pesticide Factsheets

New technologies and in vitro testing approaches have been valuable additions to risk assessments that have historically relied solely on in vivo test results. Compared to in vivo methods, in vitro high throughput screening (HTS) assays are less expensive, faster and can provide mechanistic insights on chemical action. However, extrapolating from in vitro chemical concentrations to target tissue or blood concentrations in vivo is fraught with uncertainties, and modeling is dependent upon pharmacokinetic variables not measured in in vitro assays. To address this need, new tools have been created for characterizing, simulating, and evaluating chemical toxicokinetics. Physiologically-based pharmacokinetic (PBPK) models provide estimates of chemical exposures that produce potentially hazardous tissue concentrations, while tissue microdosimetry PK models relate whole-body chemical exposures to cell-scale concentrations. These tools rely on high-throughput in vitro measurements, and successful methods exist for pharmaceutical compounds that determine PK from limited in vitro measurements and chemical structure-derived property predictions. These high throughput (HT) methods provide a more rapid and less resource–intensive alternative to traditional PK model development. We have augmented these in vitro data with chemical structure-based descriptors and mechanistic tissue partitioning models to construct HTPBPK models for over three hundred environmental and pharmace

TreeMAC: Localized TDMA MAC protocol for real-time high-data-rate sensor networks

USGS Publications Warehouse

Song, W.-Z.; Huang, R.; Shirazi, B.; Husent, R.L.

2009-01-01

Earlier sensor network MAC protocols focus on energy conservation in low-duty cycle applications, while some recent applications involve real-time high-data-rate signals. This motivates us to design an innovative localized TDMA MAC protocol to achieve high throughput and low congestion in data collection sensor networks, besides energy conservation. TreeMAC divides a time cycle into frames and frame into slots. Parent determines children's frame assigmnent based on their relative bandwidth demand, and each node calculates its own slot assignment based on its hop-count to the sink. This innovative 2-dimensional frame-slot assignment algorithm has the following nice theory properties. Firstly, given any node, at any time slot, there is at most one active sender in its neighborhood (includ ing itself). Secondly, the packet scheduling with TreelMAC is bufferless, which therefore minimizes the probability of network congestion. Thirdly, the data throughput to gateway is at least 1/3 of the optimum assuming reliable links. Our experiments on a 24 node test bed demonstrate that TreeMAC protocol significantly improves network throughput and energy efficiency, by comparing to the TinyOS's default CSMA MAC protocol and a recent TDMA MAC protocol Funneling-MAC[8]. ?? 2009 IEEE.

High-throughput flow alignment of barcoded hydrogel microparticles†

PubMed Central

Chapin, Stephen C.; Pregibon, Daniel C.

2010-01-01

Suspension (particle-based) arrays offer several advantages over conventional planar arrays in the detection and quantification of biomolecules, including the use of smaller sample volumes, more favorable probe-target binding kinetics, and rapid probe-set modification. We present a microfluidic system for the rapid alignment of multifunctional hydrogel microparticles designed to bear one or several biomolecule probe regions, as well as a graphical code to identify the embedded probes. Using high-speed imaging, we have developed and optimized a flow-through system that (1) allows for a high particle throughput, (2) ensures proper particle alignment for decoding and target quantification, and (3) can be reliably operated continuously without clogging. A tapered channel flanked by side focusing streams is used to orient the flexible, tablet-shaped particles into a well-ordered flow in the center of the channel. The effects of channel geometry, particle geometry, particle composition, particle loading density, and barcode design are explored to determine the best combination for eventual use in biological assays. Particles in the optimized system move at velocities of ~50 cm s−1 and with throughputs of ~40 particles s−1. Simple physical models and CFD simulations have been used to investigate flow behavior in the device. PMID:19823726

An enzyme-mediated protein-fragment complementation assay for substrate screening of sortase A.

PubMed

Li, Ning; Yu, Zheng; Ji, Qun; Sun, Jingying; Liu, Xiao; Du, Mingjuan; Zhang, Wei

2017-04-29

Enzyme-mediated protein conjugation has gained great attention recently due to the remarkable site-selectivity and mild reaction condition affected by the nature of enzyme. Among all sorts of enzymes reported, sortase A from Staphylococcus aureus (SaSrtA) is the most popular enzyme due to its selectivity and well-demonstrated applications. Position scanning has been widely applied to understand enzyme substrate specificity, but the low throughput of chemical synthesis of peptide substrates and analytical methods (HPLC, LC-ESI-MS) have been the major hurdle to fully decode enzyme substrate profile. We have developed a simple high-throughput substrate profiling method to reveal novel substrates of SaSrtA 7M, a widely used hyperactive peptide ligase, by modified protein-fragment complementation assay (PCA). A small library targeting the LPATG motif recognized by SaSrtA 7M was generated and screened against proteins carrying N-terminal glycine. Using this method, we have confirmed all currently known substrates of the enzyme, and moreover identified some previously unknown substrates with varying activities. The method provides an easy, fast and highly-sensitive way to determine substrate profile of a peptide ligase in a high-throughput manner. Copyright © 2017 Elsevier Inc. All rights reserved.

Using high-throughput barcode sequencing to efficiently map connectomes.

PubMed

Peikon, Ian D; Kebschull, Justus M; Vagin, Vasily V; Ravens, Diana I; Sun, Yu-Chi; Brouzes, Eric; Corrêa, Ivan R; Bressan, Dario; Zador, Anthony M

2017-07-07

The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision-a 'connectome'-is based on imaging methods that are slow, labor-intensive and expensive. Here, we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence-an RNA 'barcode'-which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and joined into a form suitable for sequencing. Although our failure to develop an efficient barcode joining scheme precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mining Human Biomonitoring Data to Identify Prevalent Chemical Mixtures (SOT abstract)

EPA Science Inventory

Through food, water, air, and consumer products, humans are exposed to tens of thousands of environmental chemicals, and most of these have not been evaluated to determine their potential toxicities. In recent years, high-throughput screening (HTS) methods have been developed tha...

Air Ground Data Link VHF Airline Communications and Reporting System (ACARS) Preliminary Test Report

DOT National Transportation Integrated Search

1995-02-01

An effort was conducted to determine actual ground-to-air, and air-to-ground : performance of the Airline Communications and Reporting system (ACARS), Very : High Frequency (VHF) Data Link System. Parameters of system throughput, error : rates, and a...

High-throughput PBPK and Microdosimetry: Cell-level Exposures in a Virtual Tissue Context (WC9)

EPA Science Inventory

Toxicokinetic (TK) models can determine whether chemical exposures produce potentially hazardous tissue concentrations. Tissue microdosimetry TK models relate whole-body chemical exposures to cell-scale concentrations. As a proof of concept, we approximated the micro-anatomic arc...

High-throughput Crystallography for Structural Genomics

PubMed Central

Joachimiak, Andrzej

2009-01-01

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

Rapid identification of areas of interest in thin film materials libraries by combining electrical, optical, X-ray diffraction, and mechanical high-throughput measurements: a case study for the system Ni-Al.

PubMed

Thienhaus, S; Naujoks, D; Pfetzing-Micklich, J; König, D; Ludwig, A

2014-12-08

The efficient identification of compositional areas of interest in thin film materials systems fabricated by combinatorial deposition methods is essential in combinatorial materials science. We use a combination of compositional screening by EDX together with high-throughput measurements of electrical and optical properties of thin film libraries to determine efficiently the areas of interest in a materials system. Areas of interest are compositions which show distinctive properties. The crystallinity of the thus determined areas is identified by X-ray diffraction. Additionally, by using automated nanoindentation across the materials library, mechanical data of the thin films can be obtained which complements the identification of areas of interest. The feasibility of this approach is demonstrated by using a Ni-Al thin film library as a reference system. The obtained results promise that this approach can be used for the case of ternary and higher order systems.

Real-time reliable determination of binding kinetics of DNA hybridization using a multi-channel graphene biosensor

NASA Astrophysics Data System (ADS)

Xu, Shicai; Zhan, Jian; Man, Baoyuan; Jiang, Shouzhen; Yue, Weiwei; Gao, Shoubao; Guo, Chengang; Liu, Hanping; Li, Zhenhua; Wang, Jihua; Zhou, Yaoqi

2017-03-01

Reliable determination of binding kinetics and affinity of DNA hybridization and single-base mismatches plays an essential role in systems biology, personalized and precision medicine. The standard tools are optical-based sensors that are difficult to operate in low cost and to miniaturize for high-throughput measurement. Biosensors based on nanowire field-effect transistors have been developed, but reliable and cost-effective fabrication remains a challenge. Here, we demonstrate that a graphene single-crystal domain patterned into multiple channels can measure time- and concentration-dependent DNA hybridization kinetics and affinity reliably and sensitively, with a detection limit of 10 pM for DNA. It can distinguish single-base mutations quantitatively in real time. An analytical model is developed to estimate probe density, efficiency of hybridization and the maximum sensor response. The results suggest a promising future for cost-effective, high-throughput screening of drug candidates, genetic variations and disease biomarkers by using an integrated, miniaturized, all-electrical multiplexed, graphene-based DNA array.

An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species

PubMed Central

Sitepu, I.R.; Ignatia, L.; Franz, A. K.; Wong, D. M.; Faulina, S.A.; Tsui, M.; Kanti, A.; Boundy-Mills, K.

2012-01-01

A rapid and inexpensive method for estimating lipid content of yeasts is needed for screening large numbers of yeasts samples. Nile red is a fluorescent lipophilic dye used for detection and quantification of intracellular lipid droplets in various biological system including algae, yeasts and filamentous fungi. However, a published assay for yeast is affected by variable diffusion across the cell membrane, and variation in the time required to reach maximal fluorescence emission. In this study, parameters that may influence the emission were varied to determine optimal assay conditions. An improved assay with a high-throughput capability was developed that includes the addition of dimethyl sulfoxide (DMSO) solvent to improve cell permeability, elimination of the washing step, the reduction of Nile red concentration, kinetic readings rather than single time-point reading, and utilization of a black 96-well microplate. The improved method was validated by comparison to gravimetric determination of lipid content of a broad variety of ascomycete and basidiomycete yeast species. PMID:22985718

A holistic high-throughput screening framework for biofuel feedstock assessment that characterises variations in soluble sugars and cell wall composition in Sorghum bicolor

PubMed Central

2013-01-01

Background A major hindrance to the development of high yielding biofuel feedstocks is the ability to rapidly assess large populations for fermentable sugar yields. Whilst recent advances have outlined methods for the rapid assessment of biomass saccharification efficiency, none take into account the total biomass, or the soluble sugar fraction of the plant. Here we present a holistic high-throughput methodology for assessing sweet Sorghum bicolor feedstocks at 10 days post-anthesis for total fermentable sugar yields including stalk biomass, soluble sugar concentrations, and cell wall saccharification efficiency. Results A mathematical method for assessing whole S. bicolor stalks using the fourth internode from the base of the plant proved to be an effective high-throughput strategy for assessing stalk biomass, soluble sugar concentrations, and cell wall composition and allowed calculation of total stalk fermentable sugars. A high-throughput method for measuring soluble sucrose, glucose, and fructose using partial least squares (PLS) modelling of juice Fourier transform infrared (FTIR) spectra was developed. The PLS prediction was shown to be highly accurate with each sugar attaining a coefficient of determination (R 2 ) of 0.99 with a root mean squared error of prediction (RMSEP) of 11.93, 5.52, and 3.23 mM for sucrose, glucose, and fructose, respectively, which constitutes an error of <4% in each case. The sugar PLS model correlated well with gas chromatography–mass spectrometry (GC-MS) and brix measures. Similarly, a high-throughput method for predicting enzymatic cell wall digestibility using PLS modelling of FTIR spectra obtained from S. bicolor bagasse was developed. The PLS prediction was shown to be accurate with an R 2 of 0.94 and RMSEP of 0.64 μg.mgDW-1.h-1. Conclusions This methodology has been demonstrated as an efficient and effective way to screen large biofuel feedstock populations for biomass, soluble sugar concentrations, and cell wall digestibility simultaneously allowing a total fermentable yield calculation. It unifies and simplifies previous screening methodologies to produce a holistic assessment of biofuel feedstock potential. PMID:24365407

Simultaneous determination of multiple angiotensin type 1 receptor antagonists and its application to high-throughput pharmacokinetic study

NASA Astrophysics Data System (ADS)

Zhu, Xiaoyan; Sun, Jianguo; Hao, Haiping; Wang, Guangji; Hu, Xiaoling; Lv, Hua; Gu, Shenghua; Wu, Xiaoming; Xu, Jinyi

2008-05-01

A rapid and sensitive high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) detection was developed for the simultaneous determination of multiple angiotensin type 1 receptor antagonists (AT1RAs) WX472, WX581, 1b and telmisartan in rat plasma for the purpose of high-throughout pharmacokinetic screening. The method was operated under selected reaction monitoring (SRM) mode in the positive ion mode. The analytes and the internal standard (pitavastatin) were extracted from 100 [mu]L rat plasma under acidic conditions by liquid-liquid extraction with ethyl acetate. The analytes and internal standard were baseline separated on a Gemini analytical column (3 [mu]m, 150 mm × 2.0 mm) with the adoption of a gradient elution using acetonitrile and 0.05% aqueous formic acid. The standard curves were linear in the concentration ranges of 4.5-900 ng/mL for WX472, 5-1000 ng/mL for WX581 and 0.5-100 ng/mL for 1b and telmisartan. Intra- and inter-batch precisions (R.S.D.%) were all within 15% and the method assessed a quite good accuracy (R.E.%). Recoveries were found to be >65% for all the compounds and no obvious matrix effects were found. This method has been successfully applied to the high-throughput pharmacokinetic screening study for both cassette dosing and cassette analysis of four compounds to rats. Significant drug-drug interactions were observed after cassette dosing. The study suggested that cassette analysis of pooled samples would be a better choice for the high-throughput pharmacokinetic screening of angiotensin type 1 receptor antagonists.

TCP Throughput Profiles Using Measurements over Dedicated Connections

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rao, Nageswara S.; Liu, Qiang; Sen, Satyabrata

Wide-area data transfers in high-performance computing infrastructures are increasingly being carried over dynamically provisioned dedicated network connections that provide high capacities with no competing traffic. We present extensive TCP throughput measurements and time traces over a suite of physical and emulated 10 Gbps connections with 0-366 ms round-trip times (RTTs). Contrary to the general expectation, they show significant statistical and temporal variations, in addition to the overall dependencies on the congestion control mechanism, buffer size, and the number of parallel streams. We analyze several throughput profiles that have highly desirable concave regions wherein the throughput decreases slowly with RTTs, inmore » stark contrast to the convex profiles predicted by various TCP analytical models. We present a generic throughput model that abstracts the ramp-up and sustainment phases of TCP flows, which provides insights into qualitative trends observed in measurements across TCP variants: (i) slow-start followed by well-sustained throughput leads to concave regions; (ii) large buffers and multiple parallel streams expand the concave regions in addition to improving the throughput; and (iii) stable throughput dynamics, indicated by a smoother Poincare map and smaller Lyapunov exponents, lead to wider concave regions. These measurements and analytical results together enable us to select a TCP variant and its parameters for a given connection to achieve high throughput with statistical guarantees.« less

Enhancing high throughput toxicology - development of putative adverse outcome pathways linking US EPA ToxCast screening targets to relevant apical hazards.

EPA Science Inventory

High throughput toxicology programs, such as ToxCast and Tox21, have provided biological effects data for thousands of chemicals at multiple concentrations. Compared to traditional, whole-organism approaches, high throughput assays are rapid and cost-effective, yet they generall...

Evaluation of High-Throughput Chemical Exposure Models via Analysis of Matched Environmental and Biological Media Measurements

EPA Science Inventory

The U.S. EPA, under its ExpoCast program, is developing high-throughput near-field modeling methods to estimate human chemical exposure and to provide real-world context to high-throughput screening (HTS) hazard data. These novel modeling methods include reverse methods to infer ...

The development of a general purpose ARM-based processing unit for the ATLAS TileCal sROD

NASA Astrophysics Data System (ADS)

Cox, M. A.; Reed, R.; Mellado, B.

2015-01-01

After Phase-II upgrades in 2022, the data output from the LHC ATLAS Tile Calorimeter will increase significantly. ARM processors are common in mobile devices due to their low cost, low energy consumption and high performance. It is proposed that a cost-effective, high data throughput Processing Unit (PU) can be developed by using several consumer ARM processors in a cluster configuration to allow aggregated processing performance and data throughput while maintaining minimal software design difficulty for the end-user. This PU could be used for a variety of high-level functions on the high-throughput raw data such as spectral analysis and histograms to detect possible issues in the detector at a low level. High-throughput I/O interfaces are not typical in consumer ARM System on Chips but high data throughput capabilities are feasible via the novel use of PCI-Express as the I/O interface to the ARM processors. An overview of the PU is given and the results for performance and throughput testing of four different ARM Cortex System on Chips are presented.

[Current applications of high-throughput DNA sequencing technology in antibody drug research].

PubMed

Yu, Xin; Liu, Qi-Gang; Wang, Ming-Rong

2012-03-01

Since the publication of a high-throughput DNA sequencing technology based on PCR reaction was carried out in oil emulsions in 2005, high-throughput DNA sequencing platforms have been evolved to a robust technology in sequencing genomes and diverse DNA libraries. Antibody libraries with vast numbers of members currently serve as a foundation of discovering novel antibody drugs, and high-throughput DNA sequencing technology makes it possible to rapidly identify functional antibody variants with desired properties. Herein we present a review of current applications of high-throughput DNA sequencing technology in the analysis of antibody library diversity, sequencing of CDR3 regions, identification of potent antibodies based on sequence frequency, discovery of functional genes, and combination with various display technologies, so as to provide an alternative approach of discovery and development of antibody drugs.

A paper-based microbial fuel cell array for rapid and high-throughput screening of electricity-producing bacteria.

PubMed

Choi, Gihoon; Hassett, Daniel J; Choi, Seokheun

2015-06-21

There is a large global effort to improve microbial fuel cell (MFC) techniques and advance their translational potential toward practical, real-world applications. Significant boosts in MFC performance can be achieved with the development of new techniques in synthetic biology that can regulate microbial metabolic pathways or control their gene expression. For these new directions, a high-throughput and rapid screening tool for microbial biopower production is needed. In this work, a 48-well, paper-based sensing platform was developed for the high-throughput and rapid characterization of the electricity-producing capability of microbes. 48 spatially distinct wells of a sensor array were prepared by patterning 48 hydrophilic reservoirs on paper with hydrophobic wax boundaries. This paper-based platform exploited the ability of paper to quickly wick fluid and promoted bacterial attachment to the anode pads, resulting in instant current generation upon loading of the bacterial inoculum. We validated the utility of our MFC array by studying how strategic genetic modifications impacted the electrochemical activity of various Pseudomonas aeruginosa mutant strains. Within just 20 minutes, we successfully determined the electricity generation capacity of eight isogenic mutants of P. aeruginosa. These efforts demonstrate that our MFC array displays highly comparable performance characteristics and identifies genes in P. aeruginosa that can trigger a higher power density.

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

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

PubMed

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

2018-06-01

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

Mass spectrometry-driven drug discovery for development of herbal medicine.

PubMed

Zhang, Aihua; Sun, Hui; Wang, Xijun

2018-05-01

Herbal medicine (HM) has made a major contribution to the drug discovery process with regard to identifying products compounds. Currently, more attention has been focused on drug discovery from natural compounds of HM. Despite the rapid advancement of modern analytical techniques, drug discovery is still a difficult and lengthy process. Fortunately, mass spectrometry (MS) can provide us with useful structural information for drug discovery, has been recognized as a sensitive, rapid, and high-throughput technology for advancing drug discovery from HM in the post-genomic era. It is essential to develop an efficient, high-quality, high-throughput screening method integrated with an MS platform for early screening of candidate drug molecules from natural products. We have developed a new chinmedomics strategy reliant on MS that is capable of capturing the candidate molecules, facilitating their identification of novel chemical structures in the early phase; chinmedomics-guided natural product discovery based on MS may provide an effective tool that addresses challenges in early screening of effective constituents of herbs against disease. This critical review covers the use of MS with related techniques and methodologies for natural product discovery, biomarker identification, and determination of mechanisms of action. It also highlights high-throughput chinmedomics screening methods suitable for lead compound discovery illustrated by recent successes. © 2016 Wiley Periodicals, Inc.

The development of a high-throughput measurement method of octanol/water distribution coefficient based on hollow fiber membrane solvent microextraction technique.

PubMed

Bao, James J; Liu, Xiaojing; Zhang, Yong; Li, Youxin

2014-09-15

This paper describes the development of a novel high-throughput hollow fiber membrane solvent microextraction technique for the simultaneous measurement of the octanol/water distribution coefficient (logD) for organic compounds such as drugs. The method is based on a designed system, which consists of a 96-well plate modified with 96 hollow fiber membrane tubes and a matching lid with 96 center holes and 96 side holes distributing in 96 grids. Each center hole was glued with a sealed on one end hollow fiber membrane tube, which is used to separate the aqueous phase from the octanol phase. A needle, such as microsyringe or automatic sampler, can be directly inserted into the membrane tube to deposit octanol as the accepted phase or take out the mixture of the octanol and the drug. Each side hole is filled with aqueous phase and could freely take in/out solvent as the donor phase from the outside of the hollow fiber membranes. The logD can be calculated by measuring the drug concentration in each phase after extraction equilibrium. After a comprehensive comparison, the polytetrafluoroethylene hollow fiber with the thickness of 210 μm, an extraction time of 300 min, a temperature of 25 °C and atmospheric pressure without stirring are selected for the high throughput measurement. The correlation coefficient of the linear fit of the logD values of five drugs determined by our system to reference values is 0.9954, showed a nice accurate. The -8.9% intra-day and -4.4% inter-day precision of logD for metronidazole indicates a good precision. In addition, the logD values of eight drugs were simultaneously and successfully measured, which indicated that the 96 throughput measure method of logD value was accurate, precise, reliable and useful for high throughput screening. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular Determinants of Hormone Refractory Prostate Cancer

DTIC Science & Technology

2017-07-01

Award Number: W81XWH-12-1-0062 TITLE: Molecular Determinants of Hormone Refractory Prostate Cancer PRINCIPAL INVESTIGATOR: Atish Choudhury...ABOVE ADDRESS. 1. REPORT DATE July 2017 2. REPORT TYPE Annual 3. DATES COVERED 1 July 2012 - 30 June 2017 4. TITLE AND SUBTITLE Molecular ...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT We have performed a high throughput, in vivo genetic screen to identify kinases that permit androgen

First report of Beet western yellows virus infecting Epiphyllum spp

USDA-ARS?s Scientific Manuscript database

Beet western yellow virus (BWYV) was identified from an orchid cactus (Epiphyllum spp.) hybrid without obvious symptoms by high-throughput sequencing. The nearly complete genomic sequence of 5,458 nucleotides of the virus was determined. The isolate has the highest nucleotide sequence identity (93%)...

Rapid sample preparation and fast GC-MS/MS for the analysis of pesticides and environmental contaminants in fish

USDA-ARS?s Scientific Manuscript database

A rapid high-throughput analytical method for the simultaneous determination of pesticides and environmental contaminants, including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and flame retardants (FRs) in fish was developed and ...

November 6, 2017, Virtual Meeting on the Charge Questions for the Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel (FIFRA SAP) Meeting on Endocrine Disruption

EPA Pesticide Factsheets

This virtual FIFRA SAP meeting will be discus questions on Continuing Development of Alternative High-Throughput Screens to Determine Endocrine Disruption, focusing on Androgen Receptor, Steroidogenesis, and Thyroid Pathways

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

High-throughput screening based on label-free detection of small molecule microarrays

NASA Astrophysics Data System (ADS)

Zhu, Chenggang; Fei, Yiyan; Zhu, Xiangdong

2017-02-01

Based on small-molecule microarrays (SMMs) and oblique-incidence reflectivity difference (OI-RD) scanner, we have developed a novel high-throughput drug preliminary screening platform based on label-free monitoring of direct interactions between target proteins and immobilized small molecules. The screening platform is especially attractive for screening compounds against targets of unknown function and/or structure that are not compatible with functional assay development. In this screening platform, OI-RD scanner serves as a label-free detection instrument which is able to monitor about 15,000 biomolecular interactions in a single experiment without the need to label any biomolecule. Besides, SMMs serves as a novel format for high-throughput screening by immobilization of tens of thousands of different compounds on a single phenyl-isocyanate functionalized glass slide. Based on the high-throughput screening platform, we sequentially screened five target proteins (purified target proteins or cell lysate containing target protein) in high-throughput and label-free mode. We found hits for respective target protein and the inhibition effects for some hits were confirmed by following functional assays. Compared to traditional high-throughput screening assay, the novel high-throughput screening platform has many advantages, including minimal sample consumption, minimal distortion of interactions through label-free detection, multi-target screening analysis, which has a great potential to be a complementary screening platform in the field of drug discovery.

Measuring molecular biomarkers in epidemiologic studies: laboratory techniques and biospecimen considerations.

PubMed

Erickson, Heidi S

2012-09-28

The future of personalized medicine depends on the ability to efficiently and rapidly elucidate a reliable set of disease-specific molecular biomarkers. High-throughput molecular biomarker analysis methods have been developed to identify disease risk, diagnostic, prognostic, and therapeutic targets in human clinical samples. Currently, high throughput screening allows us to analyze thousands of markers from one sample or one marker from thousands of samples and will eventually allow us to analyze thousands of markers from thousands of samples. Unfortunately, the inherent nature of current high throughput methodologies, clinical specimens, and cost of analysis is often prohibitive for extensive high throughput biomarker analysis. This review summarizes the current state of high throughput biomarker screening of clinical specimens applicable to genetic epidemiology and longitudinal population-based studies with a focus on considerations related to biospecimens, laboratory techniques, and sample pooling. Copyright © 2012 John Wiley & Sons, Ltd.

Challenges in NMR-based structural genomics

NASA Astrophysics Data System (ADS)

Sue, Shih-Che; Chang, Chi-Fon; Huang, Yao-Te; Chou, Ching-Yu; Huang, Tai-huang

2005-05-01

Understanding the functions of the vast number of proteins encoded in many genomes that have been completely sequenced recently is the main challenge for biologists in the post-genomics era. Since the function of a protein is determined by its exact three-dimensional structure it is paramount to determine the 3D structures of all proteins. This need has driven structural biologists to undertake the structural genomics project aimed at determining the structures of all known proteins. Several centers for structural genomics studies have been established throughout the world. Nuclear magnetic resonance (NMR) spectroscopy has played a major role in determining protein structures in atomic details and in a physiologically relevant solution state. Since the number of new genes being discovered daily far exceeds the number of structures determined by both NMR and X-ray crystallography, a high-throughput method for speeding up the process of protein structure determination is essential for the success of the structural genomics effort. In this article we will describe NMR methods currently being employed for protein structure determination. We will also describe methods under development which may drastically increase the throughput, as well as point out areas where opportunities exist for biophysicists to make significant contribution in this important field.

A Low-Cost, Reliable, High-Throughput System for Rodent Behavioral Phenotyping in a Home Cage Environment

PubMed Central

Parkison, Steven A.; Carlson, Jay D.; Chaudoin, Tammy R.; Hoke, Traci A.; Schenk, A. Katrin; Goulding, Evan H.; Pérez, Lance C.; Bonasera, Stephen J.

2016-01-01

Inexpensive, high-throughput, low maintenance systems for precise temporal and spatial measurement of mouse home cage behavior (including movement, feeding, and drinking) are required to evaluate products from large scale pharmaceutical design and genetic lesion programs. These measurements are also required to interpret results from more focused behavioral assays. We describe the design and validation of a highly-scalable, reliable mouse home cage behavioral monitoring system modeled on a previously described, one-of-a-kind system [1]. Mouse position was determined by solving static equilibrium equations describing the force and torques acting on the system strain gauges; feeding events were detected by a photobeam across the food hopper, and drinking events were detected by a capacitive lick sensor. Validation studies show excellent agreement between mouse position and drinking events measured by the system compared with video-based observation – a gold standard in neuroscience. PMID:23366406

Structural Genomics and Drug Discovery for Infectious Diseases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, W.F.

The application of structural genomics methods and approaches to proteins from organisms causing infectious diseases is making available the three dimensional structures of many proteins that are potential drug targets and laying the groundwork for structure aided drug discovery efforts. There are a number of structural genomics projects with a focus on pathogens that have been initiated worldwide. The Center for Structural Genomics of Infectious Diseases (CSGID) was recently established to apply state-of-the-art high throughput structural biology technologies to the characterization of proteins from the National Institute for Allergy and Infectious Diseases (NIAID) category A-C pathogens and organisms causing emerging,more » or re-emerging infectious diseases. The target selection process emphasizes potential biomedical benefits. Selected proteins include known drug targets and their homologs, essential enzymes, virulence factors and vaccine candidates. The Center also provides a structure determination service for the infectious disease scientific community. The ultimate goal is to generate a library of structures that are available to the scientific community and can serve as a starting point for further research and structure aided drug discovery for infectious diseases. To achieve this goal, the CSGID will determine protein crystal structures of 400 proteins and protein-ligand complexes using proven, rapid, highly integrated, and cost-effective methods for such determination, primarily by X-ray crystallography. High throughput crystallographic structure determination is greatly aided by frequent, convenient access to high-performance beamlines at third-generation synchrotron X-ray sources.« less

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

DOE PAGES

Rames, Matthew; Yu, Yadong; Ren, Gang

2014-08-15

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electronmore » microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high-resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography. Moreover, OpNS can be a high-throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.« less

Next Generation MUT-MAP, a High-Sensitivity High-Throughput Microfluidics Chip-Based Mutation Analysis Panel

PubMed Central

Patel, Rajesh; Tsan, Alison; Sumiyoshi, Teiko; Fu, Ling; Desai, Rupal; Schoenbrunner, Nancy; Myers, Thomas W.; Bauer, Keith; Smith, Edward; Raja, Rajiv

2014-01-01

Molecular profiling of tumor tissue to detect alterations, such as oncogenic mutations, plays a vital role in determining treatment options in oncology. Hence, there is an increasing need for a robust and high-throughput technology to detect oncogenic hotspot mutations. Although commercial assays are available to detect genetic alterations in single genes, only a limited amount of tissue is often available from patients, requiring multiplexing to allow for simultaneous detection of mutations in many genes using low DNA input. Even though next-generation sequencing (NGS) platforms provide powerful tools for this purpose, they face challenges such as high cost, large DNA input requirement, complex data analysis, and long turnaround times, limiting their use in clinical settings. We report the development of the next generation mutation multi-analyte panel (MUT-MAP), a high-throughput microfluidic, panel for detecting 120 somatic mutations across eleven genes of therapeutic interest (AKT1, BRAF, EGFR, FGFR3, FLT3, HRAS, KIT, KRAS, MET, NRAS, and PIK3CA) using allele-specific PCR (AS-PCR) and Taqman technology. This mutation panel requires as little as 2 ng of high quality DNA from fresh frozen or 100 ng of DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Mutation calls, including an automated data analysis process, have been implemented to run 88 samples per day. Validation of this platform using plasmids showed robust signal and low cross-reactivity in all of the newly added assays and mutation calls in cell line samples were found to be consistent with the Catalogue of Somatic Mutations in Cancer (COSMIC) database allowing for direct comparison of our platform to Sanger sequencing. High correlation with NGS when compared to the SuraSeq500 panel run on the Ion Torrent platform in a FFPE dilution experiment showed assay sensitivity down to 0.45%. This multiplexed mutation panel is a valuable tool for high-throughput biomarker discovery in personalized medicine and cancer drug development. PMID:24658394

Utilizing high-throughput bioassays associated with US EPA ToxCast Program to assess biological activity of environmental contaminants: A case study of chemical mixtures

EPA Science Inventory

Effects-based monitoring and surveillance is increasingly being utilized in conjunction with chemical monitoring to determine potential biological activity associated with environmental contaminants. Supervised approaches targeting specific chemical activity or molecular pathways...

Alfalfa virus S, a new species in the family Alphaflexiviridae

USDA-ARS?s Scientific Manuscript database

A new species of the family Alphaflexiviridae provisionally named alfalfa virus S (AVS) was discovered in alfalfa samples originating from Sudan. A complete nucleotide sequence of the viral genome consisting of 8,349 nucleotides excluding the 3’ poly(A) tail was determined by high throughput sequenc...

A space- and time-resolved single photon counting detector for fluorescence microscopy and spectroscopy

PubMed Central

Michalet, X.; Siegmund, O.H.W.; Vallerga, J.V.; Jelinsky, P.; Millaud, J.E.; Weiss, S.

2017-01-01

We have recently developed a wide-field photon-counting detector having high-temporal and high-spatial resolutions and capable of high-throughput (the H33D detector). Its design is based on a 25 mm diameter multi-alkali photocathode producing one photo electron per detected photon, which are then multiplied up to 107 times by a 3-microchannel plate stack. The resulting electron cloud is proximity focused on a cross delay line anode, which allows determining the incident photon position with high accuracy. The imaging and fluorescence lifetime measurement performances of the H33D detector installed on a standard epifluorescence microscope will be presented. We compare them to those of standard single-molecule detectors such as single-photon avalanche photodiode (SPAD) or electron-multiplying camera using model samples (fluorescent beads, quantum dots and live cells). Finally, we discuss the design and applications of future generation of H33D detectors for single-molecule imaging and high-throughput study of biomolecular interactions. PMID:29479130

40 CFR Table 9 to Subpart Eeee of... - Continuous Compliance With Operating Limits-High Throughput Transfer Racks

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 12 2010-07-01 2010-07-01 true Continuous Compliance With Operating Limits-High Throughput Transfer Racks 9 Table 9 to Subpart EEEE of Part 63 Protection of Environment...—Continuous Compliance With Operating Limits—High Throughput Transfer Racks As stated in §§ 63.2378(a) and (b...

A Dual-Mode Large-Arrayed CMOS ISFET Sensor for Accurate and High-Throughput pH Sensing in Biomedical Diagnosis.

PubMed

Huang, Xiwei; Yu, Hao; Liu, Xu; Jiang, Yu; Yan, Mei; Wu, Dongping

2015-09-01

The existing ISFET-based DNA sequencing detects hydrogen ions released during the polymerization of DNA strands on microbeads, which are scattered into microwell array above the ISFET sensor with unknown distribution. However, false pH detection happens at empty microwells due to crosstalk from neighboring microbeads. In this paper, a dual-mode CMOS ISFET sensor is proposed to have accurate pH detection toward DNA sequencing. Dual-mode sensing, optical and chemical modes, is realized by integrating a CMOS image sensor (CIS) with ISFET pH sensor, and is fabricated in a standard 0.18-μm CIS process. With accurate determination of microbead physical locations with CIS pixel by contact imaging, the dual-mode sensor can correlate local pH for one DNA slice at one location-determined microbead, which can result in improved pH detection accuracy. Moreover, toward a high-throughput DNA sequencing, a correlated-double-sampling readout that supports large array for both modes is deployed to reduce pixel-to-pixel nonuniformity such as threshold voltage mismatch. The proposed CMOS dual-mode sensor is experimentally examined to show a well correlated pH map and optical image for microbeads with a pH sensitivity of 26.2 mV/pH, a fixed pattern noise (FPN) reduction from 4% to 0.3%, and a readout speed of 1200 frames/s. A dual-mode CMOS ISFET sensor with suppressed FPN for accurate large-arrayed pH sensing is proposed and demonstrated with state-of-the-art measured results toward accurate and high-throughput DNA sequencing. The developed dual-mode CMOS ISFET sensor has great potential for future personal genome diagnostics with high accuracy and low cost.

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

PubMed

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

2016-10-01

Advancement of in silico tools would be enabled by the availability of data for metabolic reaction rates and intrinsic clearance (CLint) of a diverse compound structure data set by specific metabolic enzymes. Our goal is to measure CLint for a large set of compounds with each major human cytochrome P450 (P450) isozyme. To achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle a large volume of compound sets. The substrate depletion method [in vitro half-life (t1/2) method] was chosen to determine CLint The assay (384-well format) consisted of three parts: 1) a robotic system for incubation and sample cleanup; 2) two different integrated, ultraperformance liquid chromatography/mass spectrometry (UPLC/MS) platforms to determine the percent remaining of parent compound, and 3) an automated data analysis system. The CYP3A4 assay was evaluated using two long t1/2 compounds, carbamazepine and antipyrine (t1/2 > 30 minutes); one moderate t1/2 compound, ketoconazole (10 < t1/2 < 30 minutes); and two short t1/2 compounds, loperamide and buspirone (t½ < 10 minutes). Interday and intraday precision and accuracy of the assay were within acceptable range (∼12%) for the linear range observed. Using this assay, CYP3A4 CLint and t1/2 values for more than 3000 compounds were measured. This high-throughput, automated, and robust assay allows for rapid metabolic stability screening of large compound sets and enables advanced computational modeling for individual human P450 isozymes. U.S. Government work not protected by U.S. copyright.

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

PubMed

Liu, Yun; Grossman, Jeffrey C

2014-12-10

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

Possibilities for serial femtosecond crystallography sample delivery at future light sourcesa)

PubMed Central

Chavas, L. M. G.; Gumprecht, L.; Chapman, H. N.

2015-01-01

Serial femtosecond crystallography (SFX) uses X-ray pulses from free-electron laser (FEL) sources that can outrun radiation damage and thereby overcome long-standing limits in the structure determination of macromolecular crystals. Intense X-ray FEL pulses of sufficiently short duration allow the collection of damage-free data at room temperature and give the opportunity to study irreversible time-resolved events. SFX may open the way to determine the structure of biological molecules that fail to crystallize readily into large well-diffracting crystals. Taking advantage of FELs with high pulse repetition rates could lead to short measurement times of just minutes. Automated delivery of sample suspensions for SFX experiments could potentially give rise to a much higher rate of obtaining complete measurements than at today's third generation synchrotron radiation facilities, as no crystal alignment or complex robotic motions are required. This capability will also open up extensive time-resolved structural studies. New challenges arise from the resulting high rate of data collection, and in providing reliable sample delivery. Various developments for fully automated high-throughput SFX experiments are being considered for evaluation, including new implementations for a reliable yet flexible sample environment setup. Here, we review the different methods developed so far that best achieve sample delivery for X-ray FEL experiments and present some considerations towards the goal of high-throughput structure determination with X-ray FELs. PMID:26798808

The toxicological application of transcriptomics and epigenomics in zebrafish and other teleosts.

PubMed

Williams, Tim D; Mirbahai, Leda; Chipman, J Kevin

2014-03-01

Zebrafish (Danio rerio) is one of a number of teleost fish species frequently employed in toxicology. Toxico-genomics determines global transcriptomic responses to chemical exposures and can predict their effects. It has been applied successfully within aquatic toxicology to assist in chemical testing, determination of mechanisms and environmental monitoring. Moreover, the related field of toxico-epigenomics, that determines chemical-induced changes in DNA methylation, histone modifications and micro-RNA expression, is emerging as a valuable contribution to understanding mechanisms of both adaptive and adverse responses. Zebrafish has proven a useful and convenient model species for both transcriptomic and epigenetic toxicological studies. Despite zebrafish's dominance in other areas of fish biology, alternative fish species are used extensively in toxico-genomics. The main reason for this is that environmental monitoring generally focuses on species native to the region of interest. We are starting to see advances in the integration of high-throughput screening, omics techniques and bioinformatics together with more traditional indicator endpoints that are relevant to regulators. Integration of such approaches with high-throughput testing of zebrafish embryos, leading to the discovery of adverse outcome pathways, promises to make a major contribution to ensuring the safety of chemicals in the environment.

Simultaneous Measurements of Auto-Immune and Infectious Disease Specific Antibodies Using a High Throughput Multiplexing Tool

PubMed Central

Asati, Atul; Kachurina, Olga; Kachurin, Anatoly

2012-01-01

Considering importance of ganglioside antibodies as biomarkers in various immune-mediated neuropathies and neurological disorders, we developed a high throughput multiplexing tool for the assessment of gangliosides-specific antibodies based on Biolpex/Luminex platform. In this report, we demonstrate that the ganglioside high throughput multiplexing tool is robust, highly specific and demonstrating ∼100-fold higher concentration sensitivity for IgG detection than ELISA. In addition to the ganglioside-coated array, the high throughput multiplexing tool contains beads coated with influenza hemagglutinins derived from H1N1 A/Brisbane/59/07 and H1N1 A/California/07/09 strains. Influenza beads provided an added advantage of simultaneous detection of ganglioside- and influenza-specific antibodies, a capacity important for the assay of both infectious antigen-specific and autoimmune antibodies following vaccination or disease. Taken together, these results support the potential adoption of the ganglioside high throughput multiplexing tool for measuring ganglioside antibodies in various neuropathic and neurological disorders. PMID:22952605

High-throughput sample adaptive offset hardware architecture for high-efficiency video coding

NASA Astrophysics Data System (ADS)

Zhou, Wei; Yan, Chang; Zhang, Jingzhi; Zhou, Xin

2018-03-01

A high-throughput hardware architecture for a sample adaptive offset (SAO) filter in the high-efficiency video coding video coding standard is presented. First, an implementation-friendly and simplified bitrate estimation method of rate-distortion cost calculation is proposed to reduce the computational complexity in the mode decision of SAO. Then, a high-throughput VLSI architecture for SAO is presented based on the proposed bitrate estimation method. Furthermore, multiparallel VLSI architecture for in-loop filters, which integrates both deblocking filter and SAO filter, is proposed. Six parallel strategies are applied in the proposed in-loop filters architecture to improve the system throughput and filtering speed. Experimental results show that the proposed in-loop filters architecture can achieve up to 48% higher throughput in comparison with prior work. The proposed architecture can reach a high-operating clock frequency of 297 MHz with TSMC 65-nm library and meet the real-time requirement of the in-loop filters for 8 K × 4 K video format at 132 fps.

Brain Connectivity as a DNA Sequencing Problem

NASA Astrophysics Data System (ADS)

Zador, Anthony

The mammalian cortex consists of millions or billions of neurons, each connected to thousands of other neurons. Traditional methods for determining the brain connectivity rely on microscopy to visualize neuronal connections, but such methods are slow, labor-intensive and often lack single neuron resolution. We have recently developed a new method, MAPseq, to recast the determination of brain wiring into a form that can exploit the tremendous recent advances in high-throughput DNA sequencing. DNA sequencing technology has outpaced even Moore's law, so that the cost of sequencing the human genome has dropped from a billion dollars in 2001 to below a thousand dollars today. MAPseq works by introducing random sequences of DNA-``barcodes''-to tag neurons uniquely. With MAPseq, we can determine the connectivity of over 50K single neurons in a single mouse cortex in about a week, an unprecedented throughput, ushering in the era of ``big data'' for brain wiring. We are now developing analytical tools and algorithms to make sense of these novel data sets.

toxoMine: an integrated omics data warehouse for Toxoplasma gondii systems biology research

PubMed Central

Rhee, David B.; Croken, Matthew McKnight; Shieh, Kevin R.; Sullivan, Julie; Micklem, Gos; Kim, Kami; Golden, Aaron

2015-01-01

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite that must monitor for changes in the host environment and respond accordingly; however, it is still not fully known which genetic or epigenetic factors are involved in regulating virulence traits of T. gondii. There are on-going efforts to elucidate the mechanisms regulating the stage transition process via the application of high-throughput epigenomics, genomics and proteomics techniques. Given the range of experimental conditions and the typical yield from such high-throughput techniques, a new challenge arises: how to effectively collect, organize and disseminate the generated data for subsequent data analysis. Here, we describe toxoMine, which provides a powerful interface to support sophisticated integrative exploration of high-throughput experimental data and metadata, providing researchers with a more tractable means toward understanding how genetic and/or epigenetic factors play a coordinated role in determining pathogenicity of T. gondii. As a data warehouse, toxoMine allows integration of high-throughput data sets with public T. gondii data. toxoMine is also able to execute complex queries involving multiple data sets with straightforward user interaction. Furthermore, toxoMine allows users to define their own parameters during the search process that gives users near-limitless search and query capabilities. The interoperability feature also allows users to query and examine data available in other InterMine systems, which would effectively augment the search scope beyond what is available to toxoMine. toxoMine complements the major community database ToxoDB by providing a data warehouse that enables more extensive integrative studies for T. gondii. Given all these factors, we believe it will become an indispensable resource to the greater infectious disease research community. Database URL: http://toxomine.org PMID:26130662

Analysis of high-throughput screening reveals the effect of surface topographies on cellular morphology.

PubMed

Hulsman, Marc; Hulshof, Frits; Unadkat, Hemant; Papenburg, Bernke J; Stamatialis, Dimitrios F; Truckenmüller, Roman; van Blitterswijk, Clemens; de Boer, Jan; Reinders, Marcel J T

2015-03-01

Surface topographies of materials considerably impact cellular behavior as they have been shown to affect cell growth, provide cell guidance, and even induce cell differentiation. Consequently, for successful application in tissue engineering, the contact interface of biomaterials needs to be optimized to induce the required cell behavior. However, a rational design of biomaterial surfaces is severely hampered because knowledge is lacking on the underlying biological mechanisms. Therefore, we previously developed a high-throughput screening device (TopoChip) that measures cell responses to large libraries of parameterized topographical material surfaces. Here, we introduce a computational analysis of high-throughput materiome data to capture the relationship between the surface topographies of materials and cellular morphology. We apply robust statistical techniques to find surface topographies that best promote a certain specified cellular response. By augmenting surface screening with data-driven modeling, we determine which properties of the surface topographies influence the morphological properties of the cells. With this information, we build models that predict the cellular response to surface topographies that have not yet been measured. We analyze cellular morphology on 2176 surfaces, and find that the surface topography significantly affects various cellular properties, including the roundness and size of the nucleus, as well as the perimeter and orientation of the cells. Our learned models capture and accurately predict these relationships and reveal a spectrum of topographies that induce various levels of cellular morphologies. Taken together, this novel approach of high-throughput screening of materials and subsequent analysis opens up possibilities for a rational design of biomaterial surfaces. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

TreeMAC: Localized TDMA MAC protocol for real-time high-data-rate sensor networks

USGS Publications Warehouse

Song, W.-Z.; Huang, R.; Shirazi, B.; LaHusen, R.

2009-01-01

Earlier sensor network MAC protocols focus on energy conservation in low-duty cycle applications, while some recent applications involve real-time high-data-rate signals. This motivates us to design an innovative localized TDMA MAC protocol to achieve high throughput and low congestion in data collection sensor networks, besides energy conservation. TreeMAC divides a time cycle into frames and each frame into slots. A parent node determines the children's frame assignment based on their relative bandwidth demand, and each node calculates its own slot assignment based on its hop-count to the sink. This innovative 2-dimensional frame-slot assignment algorithm has the following nice theory properties. First, given any node, at any time slot, there is at most one active sender in its neighborhood (including itself). Second, the packet scheduling with TreeMAC is bufferless, which therefore minimizes the probability of network congestion. Third, the data throughput to the gateway is at least 1/3 of the optimum assuming reliable links. Our experiments on a 24-node testbed show that TreeMAC protocol significantly improves network throughput, fairness, and energy efficiency compared to TinyOS's default CSMA MAC protocol and a recent TDMA MAC protocol Funneling-MAC. Partial results of this paper were published in Song, Huang, Shirazi and Lahusen [W.-Z. Song, R. Huang, B. Shirazi, and R. Lahusen, TreeMAC: Localized TDMA MAC protocol for high-throughput and fairness in sensor networks, in: The 7th Annual IEEE International Conference on Pervasive Computing and Communications, PerCom, March 2009]. Our new contributions include analyses of the performance of TreeMAC from various aspects. We also present more implementation detail and evaluate TreeMAC from other aspects. ?? 2009 Elsevier B.V.

A High Throughput Model of Post-Traumatic Osteoarthritis using Engineered Cartilage Tissue Analogs

PubMed Central

Mohanraj, Bhavana; Meloni, Gregory R.; Mauck, Robert L.; Dodge, George R.

2014-01-01

(1) Objective A number of in vitro models of post-traumatic osteoarthritis (PTOA) have been developed to study the effect of mechanical overload on the processes that regulate cartilage degeneration. While such frameworks are critical for the identification therapeutic targets, existing technologies are limited in their throughput capacity. Here, we validate a test platform for high-throughput mechanical injury incorporating engineered cartilage. (2) Method We utilized a high throughput mechanical testing platform to apply injurious compression to engineered cartilage and determined their strain and strain rate dependent responses to injury. Next, we validated this response by applying the same injury conditions to cartilage explants. Finally, we conducted a pilot screen of putative PTOA therapeutic compounds. (3) Results Engineered cartilage response to injury was strain dependent, with a 2-fold increase in GAG loss at 75% compared to 50% strain. Extensive cell death was observed adjacent to fissures, with membrane rupture corroborated by marked increases in LDH release. Testing of established PTOA therapeutics showed that pan-caspase inhibitor (ZVF) was effective at reducing cell death, while the amphiphilic polymer (P188) and the free-radical scavenger (NAC) reduced GAG loss as compared to injury alone. (4) Conclusions The injury response in this engineered cartilage model replicated key features of the response from cartilage explants, validating this system for application of physiologically relevant injurious compression. This study establishes a novel tool for the discovery of mechanisms governing cartilage injury, as well as a screening platform for the identification of new molecules for the treatment of PTOA. PMID:24999113

Prediction-based association control scheme in dense femtocell networks.

PubMed

Sung, Nak Woon; Pham, Ngoc-Thai; Huynh, Thong; Hwang, Won-Joo; You, Ilsun; Choo, Kim-Kwang Raymond

2017-01-01

The deployment of large number of femtocell base stations allows us to extend the coverage and efficiently utilize resources in a low cost manner. However, the small cell size of femtocell networks can result in frequent handovers to the mobile user, and consequently throughput degradation. Thus, in this paper, we propose predictive association control schemes to improve the system's effective throughput. Our design focuses on reducing handover frequency without impacting on throughput. The proposed schemes determine handover decisions that contribute most to the network throughput and are proper for distributed implementations. The simulation results show significant gains compared with existing methods in terms of handover frequency and network throughput perspective.

High throughput light absorber discovery, Part 1: An algorithm for automated tauc analysis

DOE PAGES

Suram, Santosh K.; Newhouse, Paul F.; Gregoire, John M.

2016-09-23

High-throughput experimentation provides efficient mapping of composition-property relationships, and its implementation for the discovery of optical materials enables advancements in solar energy and other technologies. In a high throughput pipeline, automated data processing algorithms are often required to match experimental throughput, and we present an automated Tauc analysis algorithm for estimating band gap energies from optical spectroscopy data. The algorithm mimics the judgment of an expert scientist, which is demonstrated through its application to a variety of high throughput spectroscopy data, including the identification of indirect or direct band gaps in Fe 2O 3, Cu 2V 2O 7, and BiVOmore » 4. Here, the applicability of the algorithm to estimate a range of band gap energies for various materials is demonstrated by a comparison of direct-allowed band gaps estimated by expert scientists and by automated algorithm for 60 optical spectra.« less

Ultra-High Throughput Synthesis of Nanoparticles with Homogeneous Size Distribution Using a Coaxial Turbulent Jet Mixer

PubMed Central

2015-01-01

High-throughput production of nanoparticles (NPs) with controlled quality is critical for their clinical translation into effective nanomedicines for diagnostics and therapeutics. Here we report a simple and versatile coaxial turbulent jet mixer that can synthesize a variety of NPs at high throughput up to 3 kg/d, while maintaining the advantages of homogeneity, reproducibility, and tunability that are normally accessible only in specialized microscale mixing devices. The device fabrication does not require specialized machining and is easy to operate. As one example, we show reproducible, high-throughput formulation of siRNA-polyelectrolyte polyplex NPs that exhibit effective gene knockdown but exhibit significant dependence on batch size when formulated using conventional methods. The coaxial turbulent jet mixer can accelerate the development of nanomedicines by providing a robust and versatile platform for preparation of NPs at throughputs suitable for in vivo studies, clinical trials, and industrial-scale production. PMID:24824296

In Vitro Toxicity Screening Technique for Volatile Substances ...

EPA Pesticide Factsheets

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However the challenge is that many of these chemicals are volatile and not amenable to HTS robotic liquid handling applications. We assembled an in vitro cell culture apparatus capable of screening volatile chemicals for toxicity with potential for miniaturization for high throughput. BEAS-2B lung cells were grown in an enclosed culture apparatus under air-liquid interface (ALI) conditions, and exposed to an array of xenobiotics in 5% CO2. Use of ALI conditions allows direct contact of cells with a gas xenobiotic, as well as release of endogenous gaseous molecules without interference by medium on the apical surface. To identify potential xenobiotic-induced perturbations in cell homeostasis, we monitored for alterations of endogenously-produced gaseous molecules in air directly above the cells, termed “headspace”. Alterations in specific endogenously-produced gaseous molecules (e.g., signaling molecules nitric oxide (NO) and carbon monoxide (CO) in headspace is indicative of xenobiotic-induced perturbations of specific cellular processes. Additionally, endogenously produced volatile organic compounds (VOCs) may be monitored in a nonspecific, discovery manner to determine whether cell processes are

High-Throughput Models for Exposure-Based Chemical ...

EPA Pesticide Factsheets

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 program to prioritize chemical inventories for potential hazard. Similar capabilities for estimating exposure potential would support rapid risk-based prioritization for chemicals with limited information; here, we propose a framework for high-throughput exposure assessment. To demonstrate application, an analysis was conducted that predicts human exposure potential for chemicals and estimates uncertainty in these predictions by comparison to biomonitoring data. We evaluated 1936 chemicals using far-field mass balance human exposure models (USEtox and RAIDAR) and an indicator for indoor and/or consumer use. These predictions were compared to exposures inferred by Bayesian analysis from urine concentrations for 82 chemicals reported in the National Health and Nutrition Examination Survey (NHANES). Joint regression on all factors provided a calibrated consensus prediction, the variance of which serves as an empirical determination of uncertainty for prioritization on absolute exposure potential. Information on use was found to be most predictive; generally, chemicals above the limit of detection in NHANES had consumer/indoor use. Coupled with hazard HTS, exposure HTS can place risk earlie

Membrane-on-a-chip: microstructured silicon/silicon-dioxide chips for high-throughput screening of membrane transport and viral membrane fusion.

PubMed

Kusters, Ilja; van Oijen, Antoine M; Driessen, Arnold J M

2014-04-22

Screening of transport processes across biological membranes is hindered by the challenge to establish fragile supported lipid bilayers and the difficulty to determine at which side of the membrane reactants reside. Here, we present a method for the generation of suspended lipid bilayers with physiological relevant lipid compositions on microstructured Si/SiO2 chips that allow for high-throughput screening of both membrane transport and viral membrane fusion. Simultaneous observation of hundreds of single-membrane channels yields statistical information revealing population heterogeneities of the pore assembly and conductance of the bacterial toxin α-hemolysin (αHL). The influence of lipid composition and ionic strength on αHL pore formation was investigated at the single-channel level, resolving features of the pore-assembly pathway. Pore formation is inhibited by a specific antibody, demonstrating the applicability of the platform for drug screening of bacterial toxins and cell-penetrating agents. Furthermore, fusion of H3N2 influenza viruses with suspended lipid bilayers can be observed directly using a specialized chip architecture. The presented micropore arrays are compatible with fluorescence readout from below using an air objective, thus allowing high-throughput screening of membrane transport in multiwell formats in analogy to plate readers.

Development of a high-throughput assay for rapid screening of butanologenic strains.

PubMed

Agu, Chidozie Victor; Lai, Stella M; Ujor, Victor; Biswas, Pradip K; Jones, Andy; Gopalan, Venkat; Ezeji, Thaddeus Chukwuemeka

2018-02-21

We report a Thermotoga hypogea (Th) alcohol dehydrogenase (ADH)-dependent spectrophotometric assay for quantifying the amount of butanol in growth media, an advance that will facilitate rapid high-throughput screening of hypo- and hyper-butanol-producing strains of solventogenic Clostridium species. While a colorimetric nitroblue tetrazolium chloride-based assay for quantitating butanol in acetone-butanol-ethanol (ABE) fermentation broth has been described previously, we determined that Saccharomyces cerevisiae (Sc) ADH used in this earlier study exhibits approximately 13-fold lower catalytic efficiency towards butanol than ethanol. Any Sc ADH-dependent assay for primary quantitation of butanol in an ethanol-butanol mixture is therefore subject to "ethanol interference". To circumvent this limitation and better facilitate identification of hyper-butanol-producing Clostridia, we searched the literature for native ADHs that preferentially utilize butanol over ethanol and identified Th ADH as a candidate. Indeed, recombinant Th ADH exhibited a 6-fold higher catalytic efficiency with butanol than ethanol, as measured using the reduction of NADP + to NADPH that accompanies alcohol oxidation. Moreover, the assay sensitivity was not affected by the presence of acetone, acetic acid or butyric acid (typical ABE fermentation products). We broadened the utility of our assay by adapting it to a high-throughput microtiter plate-based format, and piloted it successfully in an ongoing metabolic engineering initiative.

Model-Based Design of Long-Distance Tracer Transport Experiments in Plants.

PubMed

Bühler, Jonas; von Lieres, Eric; Huber, Gregor J

2018-01-01

Studies of long-distance transport of tracer isotopes in plants offer a high potential for functional phenotyping, but so far measurement time is a bottleneck because continuous time series of at least 1 h are required to obtain reliable estimates of transport properties. Hence, usual throughput values are between 0.5 and 1 samples h -1 . Here, we propose to increase sample throughput by introducing temporal gaps in the data acquisition of each plant sample and measuring multiple plants one after each other in a rotating scheme. In contrast to common time series analysis methods, mechanistic tracer transport models allow the analysis of interrupted time series. The uncertainties of the model parameter estimates are used as a measure of how much information was lost compared to complete time series. A case study was set up to systematically investigate different experimental schedules for different throughput scenarios ranging from 1 to 12 samples h -1 . Selected designs with only a small amount of data points were found to be sufficient for an adequate parameter estimation, implying that the presented approach enables a substantial increase of sample throughput. The presented general framework for automated generation and evaluation of experimental schedules allows the determination of a maximal sample throughput and the respective optimal measurement schedule depending on the required statistical reliability of data acquired by future experiments.

Spectrophotometric reading of EUCAST antifungal susceptibility testing of Aspergillus fumigatus.

PubMed

Meletiadis, J; Leth Mortensen, K; Verweij, P E; Mouton, J W; Arendrup, M C

2017-02-01

Given the increasing number of antifungal drugs and the emergence of resistant Aspergillus isolates, objective, automated and high-throughput antifungal susceptibility testing is important. The EUCAST E.Def 9.3 reference method for MIC determination of Aspergillus species relies on visual reading. Spectrophotometric reading was not adopted because of concern that non-uniform filamentous growth might lead to unreliable and non-reproducible results. We therefore evaluated spectrophotometric reading for the determination of MICs of antifungal azoles against Aspergillus fumigatus. Eighty-eight clinical isolates of A. fumigatus were tested against four medical azoles (posaconazole, voriconazole, itraconazole, isavuconazole) and one agricultural azole (tebuconazole) with EUCAST E.Def 9.3. The visually determined MICs (complete inhibition of growth) were compared with spectrophotometrically determined MICs and essential (±1 twofold dilution) and categorical (susceptible/intermediate/resistant or wild-type/non-wild-type) agreement was calculated. Spectrophotometric data were analysed with regression analysis using the E max model, and the effective concentration corresponding to 5% (EC 5 ) was estimated. Using the 5% cut-off, high essential (92%-97%) and categorical (93%-99%) agreement (<6% errors) was found between spectrophotometric and visual MICs. The EC 5 also correlated with the visually determined MICs with an essential agreement of 83%-96% and a categorical agreement of 90%-100% (<5% errors). Spectrophotometric determination of MICs of antifungal drugs may increase objectivity, and allow automation and high-throughput of EUCAST E.Def 9.3 antifungal susceptibility testing of Aspergillus species. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Characterization of aqueous two phase systems by combining lab-on-a-chip technology with robotic liquid handling stations.

PubMed

Amrhein, Sven; Schwab, Marie-Luise; Hoffmann, Marc; Hubbuch, Jürgen

2014-11-07

Over the last decade, the use of design of experiment approaches in combination with fully automated high throughput (HTP) compatible screenings supported by robotic liquid handling stations (LHS), adequate fast analytics and data processing has been developed in the biopharmaceutical industry into a strategy of high throughput process development (HTPD) resulting in lower experimental effort, sample reduction and an overall higher degree of process optimization. Apart from HTP technologies, lab-on-a-chip technology has experienced an enormous growth in the last years and allows further reduction of sample consumption. A combination of LHS and lab-on-a-chip technology is highly desirable and realized in the present work to characterize aqueous two phase systems with respect to tie lines. In particular, a new high throughput compatible approach for the characterization of aqueous two phase systems regarding tie lines by exploiting differences in phase densities is presented. Densities were measured by a standalone micro fluidic liquid density sensor, which was integrated into a liquid handling station by means of a developed generic Tip2World interface. This combination of liquid handling stations and lab-on-a-chip technology enables fast, fully automated, and highly accurate density measurements. The presented approach was used to determine the phase diagram of ATPSs composed of potassium phosphate (pH 7) and polyethylene glycol (PEG) with a molecular weight of 300, 400, 600 and 1000 Da respectively in the presence and in the absence of 3% (w/w) sodium chloride. Considering the whole ATPS characterization process, two complete ATPSs could be characterized within 24h, including four runs per ATPS for binodal curve determination (less than 45 min/run), and tie line determination (less than 45 min/run for ATPS preparation and 8h for density determination), which can be performed fully automated over night without requiring man power. The presented methodology provides a cost, time and material effective approach for characterization of ATPS phase diagram on base on highly accurate and comprehensive data. By this means the derived data opens the door for a more detailed description of ATPS towards generating mechanistic based models, since molecular approaches such as MD simulations or molecular descriptions along the line of QSAR heavily rely on accurate and comprehensive data. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization of matrix effects in developing rugged high-throughput LC-MS/MS methods for bioanalysis.

PubMed

Li, Fumin; Wang, Jun; Jenkins, Rand

2016-05-01

There is an ever-increasing demand for high-throughput LC-MS/MS bioanalytical assays to support drug discovery and development. Matrix effects of sofosbuvir (protonated) and paclitaxel (sodiated) were thoroughly evaluated using high-throughput chromatography (defined as having a run time ≤1 min) under 14 elution conditions with extracts from protein precipitation, liquid-liquid extraction and solid-phase extraction. A slight separation, in terms of retention time, between underlying matrix components and sofosbuvir/paclitaxel can greatly alleviate matrix effects. High-throughput chromatography, with proper optimization, can provide rapid and effective chromatographic separation under 1 min to alleviate matrix effects and enhance assay ruggedness for regulated bioanalysis.

Development of a high-throughput liquid state assay for lipase activity using natural substrates and rhodamine B.

PubMed

Zottig, Ximena; Meddeb-Mouelhi, Fatma; Beauregard, Marc

2016-03-01

A fluorescence-based assay for the determination of lipase activity using rhodamine B as an indicator, and natural substrates such as olive oil, is described. It is based on the use of a rhodamine B-natural substrate emulsion in liquid state, which is advantageous over agar plate assays. This high-throughput method is simple and rapid and can be automated, making it suitable for screening and metagenomics application. Reaction conditions such as pH and temperature can be varied and controlled. Using triolein or olive oil as a natural substrate allows monitoring of lipase activity in reaction conditions that are closer to those used in industrial settings. The described method is sensitive over a wide range of product concentrations and offers good reproducibility. Copyright © 2015 Elsevier Inc. All rights reserved.

High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

PubMed

Yu, Xiaobo; LaBaer, Joshua

2015-05-01

AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.

G protein-coupled receptor internalization assays in the high-content screening format.

PubMed

Haasen, Dorothea; Schnapp, Andreas; Valler, Martin J; Heilker, Ralf

2006-01-01

High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS format using a high-throughput, confocal cellular imaging device. GPCRs are the most successful group of therapeutic targets on the pharmaceutical market. Accordingly, the search for compounds that interfere with GPCR function in a specific and selective way is a major focus of the pharmaceutical industry today. This chapter describes methods for the ligand-induced internalization of GPCRs labeled previously with either a fluorophore-conjugated ligand or an antibody directed against an N-terminal tag of the GPCR. Both labeling techniques produce robust assay formats. Complementary to other functional GPCR drug discovery assays, internalization assays enable a pharmacological analysis of test compounds. We conclude that GPCR internalization assays represent a valuable medium/high-throughput screening format to determine the cellular activity of GPCR ligands.

High throughput secondary electron imaging of organic residues on a graphene surface

NASA Astrophysics Data System (ADS)

Zhou, Yangbo; O'Connell, Robert; Maguire, Pierce; Zhang, Hongzhou

2014-11-01

Surface organic residues inhibit the extraordinary electronic properties of graphene, hindering the development of graphene electronics. However, fundamental understanding of the residue morphology is still absent due to a lack of high-throughput and high-resolution surface characterization methods. Here, we demonstrate that secondary electron (SE) imaging in the scanning electron microscope (SEM) and helium ion microscope (HIM) can provide sub-nanometer information of a graphene surface and reveal the morphology of surface contaminants. Nanoscale polymethyl methacrylate (PMMA) residues are visible in the SE imaging, but their contrast, i.e. the apparent lateral dimension, varies with the imaging conditions. We have demonstrated a quantitative approach to readily obtain the physical size of the surface features regardless of the contrast variation. The fidelity of SE imaging is ultimately determined by the probe size of the primary beam. HIM is thus evaluated to be a superior SE imaging technique in terms of surface sensitivity and image fidelity. A highly efficient method to reveal the residues on a graphene surface has therefore been established.

Chicken skin virome analyzed by high-throughput sequencing shows a composition highly different from human skin.

PubMed

Denesvre, Caroline; Dumarest, Marine; Rémy, Sylvie; Gourichon, David; Eloit, Marc

2015-10-01

Recent studies show that human skin at homeostasis is a complex ecosystem whose virome include circular DNA viruses, especially papillomaviruses and polyomaviruses. To determine the chicken skin virome in comparison with human skin virome, a chicken swabs pool sample from fifteen indoor healthy chickens of five genetic backgrounds was examined for the presence of DNA viruses by high-throughput sequencing (HTS). The results indicate a predominance of herpesviruses from the Mardivirus genus, coming from either vaccinal origin or presumably asymptomatic infection. Despite the high sensitivity of the HTS method used herein to detect small circular DNA viruses, we did not detect any papillomaviruses, polyomaviruses, or circoviruses, indicating that these viruses may not be resident of the chicken skin. The results suggest that the turkey herpesvirus is a resident of chicken skin in vaccinated chickens. This study indicates major differences between the skin viromes of chickens and humans. The origin of this difference remains to be further studied in relation with skin physiology, environment, or virus population dynamics.

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

Carbohydrate Microarray Technology Applied to High-Throughput Mapping of Plant Cell Wall Glycans Using Comprehensive Microarray Polymer Profiling (CoMPP).

PubMed

Kračun, Stjepan Krešimir; Fangel, Jonatan Ulrik; Rydahl, Maja Gro; Pedersen, Henriette Lodberg; Vidal-Melgosa, Silvia; Willats, William George Tycho

2017-01-01

Cell walls are an important feature of plant cells and a major component of the plant glycome. They have both structural and physiological functions and are critical for plant growth and development. The diversity and complexity of these structures demand advanced high-throughput techniques to answer questions about their structure, functions and roles in both fundamental and applied scientific fields. Microarray technology provides both the high-throughput and the feasibility aspects required to meet that demand. In this chapter, some of the most recent microarray-based techniques relating to plant cell walls are described together with an overview of related contemporary techniques applied to carbohydrate microarrays and their general potential in glycoscience. A detailed experimental procedure for high-throughput mapping of plant cell wall glycans using the comprehensive microarray polymer profiling (CoMPP) technique is included in the chapter and provides a good example of both the robust and high-throughput nature of microarrays as well as their applicability to plant glycomics.

Identification of functional modules using network topology and high-throughput data.

PubMed

Ulitsky, Igor; Shamir, Ron

2007-01-26

With the advent of systems biology, biological knowledge is often represented today by networks. These include regulatory and metabolic networks, protein-protein interaction networks, and many others. At the same time, high-throughput genomics and proteomics techniques generate very large data sets, which require sophisticated computational analysis. Usually, separate and different analysis methodologies are applied to each of the two data types. An integrated investigation of network and high-throughput information together can improve the quality of the analysis by accounting simultaneously for topological network properties alongside intrinsic features of the high-throughput data. We describe a novel algorithmic framework for this challenge. We first transform the high-throughput data into similarity values, (e.g., by computing pairwise similarity of gene expression patterns from microarray data). Then, given a network of genes or proteins and similarity values between some of them, we seek connected sub-networks (or modules) that manifest high similarity. We develop algorithms for this problem and evaluate their performance on the osmotic shock response network in S. cerevisiae and on the human cell cycle network. We demonstrate that focused, biologically meaningful and relevant functional modules are obtained. In comparison with extant algorithms, our approach has higher sensitivity and higher specificity. We have demonstrated that our method can accurately identify functional modules. Hence, it carries the promise to be highly useful in analysis of high throughput data.

Stepping into the omics era: Opportunities and challenges for biomaterials science and engineering.

PubMed

Groen, Nathalie; Guvendiren, Murat; Rabitz, Herschel; Welsh, William J; Kohn, Joachim; de Boer, Jan

2016-04-01

The research paradigm in biomaterials science and engineering is evolving from using low-throughput and iterative experimental designs towards high-throughput experimental designs for materials optimization and the evaluation of materials properties. Computational science plays an important role in this transition. With the emergence of the omics approach in the biomaterials field, referred to as materiomics, high-throughput approaches hold the promise of tackling the complexity of materials and understanding correlations between material properties and their effects on complex biological systems. The intrinsic complexity of biological systems is an important factor that is often oversimplified when characterizing biological responses to materials and establishing property-activity relationships. Indeed, in vitro tests designed to predict in vivo performance of a given biomaterial are largely lacking as we are not able to capture the biological complexity of whole tissues in an in vitro model. In this opinion paper, we explain how we reached our opinion that converging genomics and materiomics into a new field would enable a significant acceleration of the development of new and improved medical devices. The use of computational modeling to correlate high-throughput gene expression profiling with high throughput combinatorial material design strategies would add power to the analysis of biological effects induced by material properties. We believe that this extra layer of complexity on top of high-throughput material experimentation is necessary to tackle the biological complexity and further advance the biomaterials field. In this opinion paper, we postulate that converging genomics and materiomics into a new field would enable a significant acceleration of the development of new and improved medical devices. The use of computational modeling to correlate high-throughput gene expression profiling with high throughput combinatorial material design strategies would add power to the analysis of biological effects induced by material properties. We believe that this extra layer of complexity on top of high-throughput material experimentation is necessary to tackle the biological complexity and further advance the biomaterials field. Copyright © 2016. Published by Elsevier Ltd.

High-throughput determination of urinary hexosamines for diagnosis of mucopolysaccharidoses by capillary electrophoresis and high-performance liquid chromatography.

PubMed

Coppa, Giovanni V; Galeotti, Fabio; Zampini, Lucia; Maccari, Francesca; Galeazzi, Tiziana; Padelia, Lucia; Santoro, Lucia; Gabrielli, Orazio; Volpi, Nicola

2011-04-01

Mucopolysaccharidoses (MPS) diagnosis is often delayed and irreversible organ damage can occur, making possible therapies less effective. This highlights the importance of early and accurate diagnosis. A high-throughput procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary galactosaminoglycans and glucosaminoglycans by capillary electrophoresis (CE) and HPLC has been performed and validated in subjects affected by various MPS including their mild and severe forms, Hurler and Hurler-Scheie, Hunter, Sanfilippo, Morquio, and Maroteaux-Lamy. Contrary to other analytical approaches, the present single analytical procedure, which is able to measure total abnormal amounts of urinary GAGs, high molecular mass, and related fragments, as well as specific hexosamines belonging to a group of GAGs, would be useful for possible application in their early diagnosis. After a rapid urine pretreatment, free hexosamines are generated by acidic hydrolysis, derivatized with 2-aminobenzoic acid and separated by CE/UV in ∼10min and reverse-phase (RP)-HPLC in fluorescence in ∼21min. The total content of hexosamines was found to be indicative of abnormal urinary excretion of GAGs in patients compared to the controls, and the galactosamine/glucosamine ratio was observed to be related to specific MPS syndromes in regard to both their mild and severe forms. As a consequence, important correlations between analytical response and clinical diagnosis and the severity of the disorders were observed. Furthermore, we can assume that the severity of the syndrome may be ascribed to the quantity of total GAGs, as high-molecular-mass polymers and fragments, accumulated in cells and directly excreted in the urine. Finally, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method is suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis, and their treatment. Copyright © 2010 Elsevier Inc. All rights reserved.

Microplate Bioassay for Determining Substrate Selectivity of "Candida rugosa" Lipase

ERIC Educational Resources Information Center

Wang, Shi-zhen; Fang, Bai-shan

2012-01-01

Substrate selectivity of "Candida rugosa" lipase was tested using "p"-nitrophenyl esters of increasing chain length (C[subscript 1], C[subscript 7], C[subscript 15]) using the high-throughput screening method. A fast and easy 96-well microplate bioassay was developed to help students learn and practice biotechnological specificity screen. The…

High-throughput biosensors for multiplexed foodborne pathogen detection

USDA-ARS?s Scientific Manuscript database

Incidental contamination of foods by harmful bacteria (such as E. coli and Salmonella) and the toxins that they produce is a serious threat to public health and the economy in the United States. The presence of such bacteri and toxins in foods must be rapidly determined at various stages of food pr...

Rapid high throughput amylose determination in freeze dried potato tuber samples

USDA-ARS?s Scientific Manuscript database

Approximately 80% of the fresh weight of a potato tuber is water; nearly all of the remaining dry matter is starch. Most of the starch (70%) is composed of amylopectin, while the remainder is amylose. The ratio between amylose and amylopectin is the most important property influencing the physical p...

On Selecting a Minimal Set of In Vitro Assays to Reliably Determine Estrogen Agonist Activity

EPA Science Inventory

The US EPA is charged with screening chemicals for their ability to be endocrine disruptors through interaction with the estrogen, androgen and thyroid axes. The agency is starting to explore the use of high-throughput in vitro assays to use in the Endocrine Disruptor Screening P...

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

PubMed Central

Jackson, Colin R.; Tyler, Heather L.; Millar, Justin J.

2013-01-01

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample. PMID:24121617

Determination of microbial extracellular enzyme activity in waters, soils, and sediments using high throughput microplate assays.

PubMed

Jackson, Colin R; Tyler, Heather L; Millar, Justin J

2013-10-01

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 metastablemore » 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.« less

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

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 12 2010-07-01 2010-07-01 true Operating Limits-High Throughput Transfer Racks 3 Table 3 to Subpart EEEE of Part 63 Protection of Environment ENVIRONMENTAL PROTECTION... Throughput Transfer Racks As stated in § 63.2346(e), you must comply with the operating limits for existing...

Compound Transfer by Acoustic Droplet Ejection Promotes Quality and Efficiency in Ultra-High-Throughput Screening Campaigns.

PubMed

Dawes, Timothy D; Turincio, Rebecca; Jones, Steven W; Rodriguez, Richard A; Gadiagellan, Dhireshan; Thana, Peter; Clark, Kevin R; Gustafson, Amy E; Orren, Linda; Liimatta, Marya; Gross, Daniel P; Maurer, Till; Beresini, Maureen H

2016-02-01

Acoustic droplet ejection (ADE) as a means of transferring library compounds has had a dramatic impact on the way in which high-throughput screening campaigns are conducted in many laboratories. Two Labcyte Echo ADE liquid handlers form the core of the compound transfer operation in our 1536-well based ultra-high-throughput screening (uHTS) system. Use of these instruments has promoted flexibility in compound formatting in addition to minimizing waste and eliminating compound carryover. We describe the use of ADE for the generation of assay-ready plates for primary screening as well as for follow-up dose-response evaluations. Custom software has enabled us to harness the information generated by the ADE instrumentation. Compound transfer via ADE also contributes to the screening process outside of the uHTS system. A second fully automated ADE-based system has been used to augment the capacity of the uHTS system as well as to permit efficient use of previously picked compound aliquots for secondary assay evaluations. Essential to the utility of ADE in the high-throughput screening process is the high quality of the resulting data. Examples of data generated at various stages of high-throughput screening campaigns are provided. Advantages and disadvantages of the use of ADE in high-throughput screening are discussed. © 2015 Society for Laboratory Automation and Screening.

Prediction-based association control scheme in dense femtocell networks

PubMed Central

Pham, Ngoc-Thai; Huynh, Thong; Hwang, Won-Joo; You, Ilsun; Choo, Kim-Kwang Raymond

2017-01-01

The deployment of large number of femtocell base stations allows us to extend the coverage and efficiently utilize resources in a low cost manner. However, the small cell size of femtocell networks can result in frequent handovers to the mobile user, and consequently throughput degradation. Thus, in this paper, we propose predictive association control schemes to improve the system’s effective throughput. Our design focuses on reducing handover frequency without impacting on throughput. The proposed schemes determine handover decisions that contribute most to the network throughput and are proper for distributed implementations. The simulation results show significant gains compared with existing methods in terms of handover frequency and network throughput perspective. PMID:28328992

Microwave assisted saponification (MAS) followed by on-line liquid chromatography (LC)-gas chromatography (GC) for high-throughput and high-sensitivity determination of mineral oil in different cereal-based foodstuffs.

PubMed

Moret, Sabrina; Scolaro, Marianna; Barp, Laura; Purcaro, Giorgia; Conte, Lanfranco S

2016-04-01

A high throughput, high-sensitivity procedure, involving simultaneous microwave-assisted extraction (MAS) and unsaponifiable extraction, followed by on-line liquid chromatography (LC)-gas chromatography (GC), has been optimised for rapid and efficient extraction and analytical determination of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) in cereal-based products of different composition. MAS has the advantage of eliminating fat before LC-GC analysis, allowing an increase in the amount of sample extract injected, and hence in sensitivity. The proposed method gave practically quantitative recoveries and good repeatability. Among the different cereal-based products analysed (dry semolina and egg pasta, bread, biscuits, and cakes), egg pasta packed in direct contact with recycled paperboard had on average the highest total MOSH level (15.9 mg kg(-1)), followed by cakes (10.4 mg kg(-1)) and bread (7.5 mg kg(-1)). About 50% of the pasta and bread samples and 20% of the biscuits and cake samples had detectable MOAH amounts. The highest concentrations were found in an egg pasta in direct contact with recycled paperboard (3.6 mg kg(-1)) and in a milk bread (3.6 mg kg(-1)). Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed

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

2016-08-12

This work describes the development of a model-based high-throughput design (MHD) tool for the operating space determination of a chromatographic cation-exchange protein purification process. Based on a previously developed thermodynamic mechanistic model, the MHD tool generates a large amount of system knowledge and thereby permits minimizing the required experimental workload. In particular, each new experiment is designed to generate information needed to help refine and improve the model. Unnecessary experiments that do not increase system knowledge are avoided. Instead of aspiring to a perfectly parameterized model, the goal of this design tool is to use early model parameter estimates to find interesting experimental spaces, and to refine the model parameter estimates with each new experiment until a satisfactory set of process parameters is found. The MHD tool is split into four sections: (1) prediction, high throughput experimentation using experiments in (2) diluted conditions and (3) robotic automated liquid handling workstations (robotic workstation), and (4) operating space determination and validation. (1) Protein and resin information, in conjunction with the thermodynamic model, is used to predict protein resin capacity. (2) The predicted model parameters are refined based on gradient experiments in diluted conditions. (3) Experiments on the robotic workstation are used to further refine the model parameters. (4) The refined model is used to determine operating parameter space that allows for satisfactory purification of the protein of interest on the HPLC scale. Each section of the MHD tool is used to define the adequate experimental procedures for the next section, thus avoiding any unnecessary experimental work. We used the MHD tool to design a polishing step for two proteins, a monoclonal antibody and a fusion protein, on two chromatographic resins, in order to demonstrate it has the ability to strongly accelerate the early phases of process development. Copyright © 2016 Elsevier B.V. All rights reserved.

An Automated High-Throughput System to Fractionate Plant Natural Products for Drug Discovery

PubMed Central

Tu, Ying; Jeffries, Cynthia; Ruan, Hong; Nelson, Cynthia; Smithson, David; Shelat, Anang A.; Brown, Kristin M.; Li, Xing-Cong; Hester, John P.; Smillie, Troy; Khan, Ikhlas A.; Walker, Larry; Guy, Kip; Yan, Bing

2010-01-01

The development of an automated, high-throughput fractionation procedure to prepare and analyze natural product libraries for drug discovery screening is described. Natural products obtained from plant materials worldwide were extracted and first prefractionated on polyamide solid-phase extraction cartridges to remove polyphenols, followed by high-throughput automated fractionation, drying, weighing, and reformatting for screening and storage. The analysis of fractions with UPLC coupled with MS, PDA and ELSD detectors provides information that facilitates characterization of compounds in active fractions. Screening of a portion of fractions yielded multiple assay-specific hits in several high-throughput cellular screening assays. This procedure modernizes the traditional natural product fractionation paradigm by seamlessly integrating automation, informatics, and multimodal analytical interrogation capabilities. PMID:20232897

Automated glycopeptide analysis—review of current state and future directions

PubMed Central

Dallas, David C.; Martin, William F.; Hua, Serenus

2013-01-01

Glycosylation of proteins is involved in immune defense, cell–cell adhesion, cellular recognition and pathogen binding and is one of the most common and complex post-translational modifications. Science is still struggling to assign detailed mechanisms and functions to this form of conjugation. Even the structural analysis of glycoproteins—glycoproteomics—remains in its infancy due to the scarcity of high-throughput analytical platforms capable of determining glycopeptide composition and structure, especially platforms for complex biological mixtures. Glycopeptide composition and structure can be determined with high mass-accuracy mass spectrometry, particularly when combined with chromatographic separation, but the sheer volume of generated data necessitates computational software for interpretation. This review discusses the current state of glycopeptide assignment software—advances made to date and issues that remain to be addressed. The various software and algorithms developed so far provide important insights into glycoproteomics. However, there is currently no freely available software that can analyze spectral data in batch and unambiguously determine glycopeptide compositions for N- and O-linked glycopeptides from relevant biological sources such as human milk and serum. Few programs are capable of aiding in structural determination of the glycan component. To significantly advance the field of glycoproteomics, analytical software and algorithms are required that: (i) solve for both N- and O-linked glycopeptide compositions, structures and glycosites in biological mixtures; (ii) are high-throughput and process data in batches; (iii) can interpret mass spectral data from a variety of sources and (iv) are open source and freely available. PMID:22843980

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gilbert, Andrew J.; Fast, James E.; Fulsom, Bryan G.

For many nuclear material safeguards inspections, spectroscopic gamma detectors are required which can achieve high event rates (in excess of 10^6 s^-1) while maintaining very good energy resolution for discrimination of neighboring gamma signatures in complex backgrounds. Such spectra can be useful for non-destructive assay (NDA) of spent nuclear fuel with long cooling times, which contains many potentially useful low-rate gamma lines, e.g., Cs-134, in the presence of a few dominating gamma lines, such as Cs-137. Detectors in use typically sacrifice energy resolution for count rate, e.g., LaBr3, or visa versa, e.g., CdZnTe. In contrast, we anticipate that beginning withmore » a detector with high energy resolution, e.g., high-purity germanium (HPGe), and adapting the data acquisition for high throughput will be able to achieve the goals of the ideal detector. In this work, we present quantification of Cs-134 and Cs-137 activities, useful for fuel burn-up quantification, in fuel that has been cooling for 22.3 years. A segmented, planar HPGe detector is used for this inspection, which has been adapted for a high-rate throughput in excess of 500k counts/s. Using a very-high-statistic spectrum of 2.4*10^11 counts, isotope activities can be determined with very low statistical uncertainty. However, it is determined that systematic uncertainties dominate in such a data set, e.g., the uncertainty in the pulse line shape. This spectrum offers a unique opportunity to quantify this uncertainty and subsequently determine required counting times for given precision on values of interest.« less

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

PubMed

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.

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

PubMed Central

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

2016-01-01

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

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

PubMed Central

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

2012-01-01

We present a toolbox for high-throughput screening of image-based Caenorhabditis elegans phenotypes. The image analysis algorithms measure morphological phenotypes in individual worms and are effective for a variety of assays and imaging systems. This WormToolbox is available via the open-source CellProfiler project and enables objective scoring of whole-animal high-throughput image-based assays of C. elegans for the study of diverse biological pathways relevant to human disease. PMID:22522656

High-throughput, image-based screening of pooled genetic variant libraries

PubMed Central

Emanuel, George; Moffitt, Jeffrey R.; Zhuang, Xiaowei

2018-01-01

Image-based, high-throughput screening of genetic perturbations will advance both biology and biotechnology. We report a high-throughput screening method that allows diverse genotypes and corresponding phenotypes to be imaged in numerous individual cells. We achieve genotyping by introducing barcoded genetic variants into cells and using massively multiplexed FISH to measure the barcodes. We demonstrated this method by screening mutants of the fluorescent protein YFAST, yielding brighter and more photostable YFAST variants. PMID:29083401

Experimental Design for Combinatorial and High Throughput Materials Development

NASA Astrophysics Data System (ADS)

Cawse, James N.

2002-12-01

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

A Novel High Throughput Assay for Anthelmintic Drug Screening and Resistance Diagnosis by Real-Time Monitoring of Parasite Motility

PubMed Central

Smout, Michael J.; Kotze, Andrew C.; McCarthy, James S.; Loukas, Alex

2010-01-01

Background Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods. Methodology/Principal Findings Here we describe a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC50 values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and –resistant isolates of H. contortus. Conclusions/Significance Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing. PMID:21103363

A Fusion Protein of the p53 Transaction Domain and the p53-Binding Domain of the Oncoprotein MdmX as an Efficient System for High-Throughput Screening of MdmX Inhibitors.

PubMed

Chen, Rong; Zhou, Jingjing; Qin, Lingyun; Chen, Yao; Huang, Yongqi; Liu, Huili; Su, Zhengding

2017-06-27

In nearly half of cancers, the anticancer activity of p53 protein is often impaired by the overexpressed oncoprotein Mdm2 and its homologue, MdmX, demanding efficient therapeutics to disrupt the aberrant p53-MdmX/Mdm2 interactions to restore the p53 activity. While many potent Mdm2-specific inhibitors have already undergone clinical investigations, searching for MdmX-specific inhibitors has become very attractive, requiring a more efficient screening strategy for evaluating potential scaffolds or leads. In this work, considering that the intrinsic fluorescence residue Trp23 in the p53 transaction domain (p53p) plays an important role in determining the p53-MdmX/Mdm2 interactions, we constructed a fusion protein to utilize this intrinsic fluorescence signal to monitor high-throughput screening of a compound library. The fusion protein was composed of the p53p followed by the N-terminal domain of MdmX (N-MdmX) through a flexible amino acid linker, while the whole fusion protein contained a sole intrinsic fluorescence probe. The fusion protein was then evaluated using fluorescence spectroscopy against model compounds. Our results revealed that the variation of the fluorescence signal was highly correlated with the concentration of the ligand within 65 μM. The fusion protein was further evaluated with respect to its feasibility for use in high-throughput screening using a model compound library, including controls. We found that the imidazo-indole scaffold was a bona fide scaffold for template-based design of MdmX inhibitors. Thus, the p53p-N-MdmX fusion protein we designed provides a convenient and efficient tool for high-throughput screening of new MdmX inhibitors. The strategy described in this work should be applicable for other protein targets to accelerate drug discovery.

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

Single-Molecule Flow Platform for the Quantification of Biomolecules Attached to Single Nanoparticles.

PubMed

Jung, Seung-Ryoung; Han, Rui; Sun, Wei; Jiang, Yifei; Fujimoto, Bryant S; Yu, Jiangbo; Kuo, Chun-Ting; Rong, Yu; Zhou, Xing-Hua; Chiu, Daniel T

2018-05-15

We describe here a flow platform for quantifying the number of biomolecules on individual fluorescent nanoparticles. The platform combines line-confocal fluorescence detection with near nanoscale channels (1-2 μm in width and height) to achieve high single-molecule detection sensitivity and throughput. The number of biomolecules present on each nanoparticle was determined by deconvolving the fluorescence intensity distribution of single-nanoparticle-biomolecule complexes with the intensity distribution of single biomolecules. We demonstrate this approach by quantifying the number of streptavidins on individual semiconducting polymer dots (Pdots); streptavidin was rendered fluorescent using biotin-Alexa647. This flow platform has high-throughput (hundreds to thousands of nanoparticles detected per second) and requires minute amounts of sample (∼5 μL at a dilute concentration of 10 pM). This measurement method is an additional tool for characterizing synthetic or biological nanoparticles.

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

NASA Technical Reports Server (NTRS)

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

Comparison of submerged and unsubmerged printing of ovarian cancer cells.

PubMed

Davidoff, Sherry N; Au, David; Smith, Samuel; Brooks, Amanda E; Brooks, Benjamin D

2015-01-01

A high-throughput cell based assay would greatly aid in the development and screening of ovarian cancer drug candidates. Previously, a three-dimensional microfluidic printer that is not only capable of controlling the location of cell deposition, but also of maintaining a liquid, nutrient rich environment to preserve cellular phenotype has been developed (Wasatch Microfluidics). In this study, we investigated the impact (i.e., viability, density, and phenotype) of depositing cells on a surface submerged in cell culture media. It was determined that submersion of the microfluidic print head in cell media did not alter the cell density, viability, or phenotype.. This article describes an in depth study detailing the impact of one of the fundamental components of a 3D microfluidic cell printer designed to mimic the in vivo cell environment. Development of such a tool holds promise as a high-throughput drug-screening platform for new cancer therapeutics.

A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis

PubMed Central

Timm, David M.; Chen, Jianbo; Sing, David; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Raphael, Robert M.; Dehghani, Mehdi; Rosenblatt, Kevin P.; Killian, T. C.; Tseng, Hubert; Souza, Glauco R.

2013-01-01

There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures. PMID:24141454

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gentry, T.; Schadt, C.; Zhou, J.

Microarray technology has the unparalleled potential tosimultaneously determine the dynamics and/or activities of most, if notall, of the microbial populations in complex environments such as soilsand sediments. Researchers have developed several types of arrays thatcharacterize the microbial populations in these samples based on theirphylogenetic relatedness or functional genomic content. Several recentstudies have used these microarrays to investigate ecological issues;however, most have only analyzed a limited number of samples withrelatively few experiments utilizing the full high-throughput potentialof microarray analysis. This is due in part to the unique analyticalchallenges that these samples present with regard to sensitivity,specificity, quantitation, and data analysis. Thismore » review discussesspecific applications of microarrays to microbial ecology research alongwith some of the latest studies addressing the difficulties encounteredduring analysis of complex microbial communities within environmentalsamples. With continued development, microarray technology may ultimatelyachieve its potential for comprehensive, high-throughput characterizationof microbial populations in near real-time.« less

A general protocol for creating high-throughput screening assays for reaction yield and enantiomeric excess applied to hydrobenzoin

PubMed Central

Shabbir, Shagufta H.; Regan, Clinton J.; Anslyn, Eric V.

2009-01-01

A general approach to high-throughput screening of enantiomeric excess (ee) and concentration was developed by using indicator displacement assays (IDAs), and the protocol was then applied to the vicinal diol hydrobenzoin. The method involves the sequential utilization of what we define herein as screening, training, and analysis plates. Several enantioselective boronic acid-based receptors were screened by using 96-well plates, both for their ability to discriminate the enantiomers of hydrobenzoin and to find their optimal pairing with indicators resulting in the largest optical responses. The best receptor/indicator combination was then used to train an artificial neural network to determine concentration and ee. To prove the practicality of the developed protocol, analysis plates were created containing true unknown samples of hydrobenzoin generated by established Sharpless asymmetric dihydroxylation reactions, and the best ligand was correctly identified. PMID:19332790

Molecular recognition and self-assembly special feature: A general protocol for creating high-throughput screening assays for reaction yield and enantiomeric excess applied to hydrobenzoin.

PubMed

Shabbir, Shagufta H; Regan, Clinton J; Anslyn, Eric V

2009-06-30

A general approach to high-throughput screening of enantiomeric excess (ee) and concentration was developed by using indicator displacement assays (IDAs), and the protocol was then applied to the vicinal diol hydrobenzoin. The method involves the sequential utilization of what we define herein as screening, training, and analysis plates. Several enantioselective boronic acid-based receptors were screened by using 96-well plates, both for their ability to discriminate the enantiomers of hydrobenzoin and to find their optimal pairing with indicators resulting in the largest optical responses. The best receptor/indicator combination was then used to train an artificial neural network to determine concentration and ee. To prove the practicality of the developed protocol, analysis plates were created containing true unknown samples of hydrobenzoin generated by established Sharpless asymmetric dihydroxylation reactions, and the best ligand was correctly identified.

High-Throughput Particle Uptake Analysis by Imaging Flow Cytometry

PubMed Central

Smirnov, Asya; Solga, Michael D.; Lannigan, Joanne; Criss, Alison K.

2017-01-01

Quantifying the efficiency of particle uptake by host cells is important in fields including infectious diseases, autoimmunity, cancer, developmental biology, and drug delivery. Here we present a protocol for high-throughput analysis of particle uptake using imaging flow cytometry, using the bacterium Neisseria gonorrhoeae attached and internalized to neutrophils as an example. Cells are exposed to fluorescently labeled bacteria, fixed, and stained with a bacteria-specific antibody of a different fluorophore. Thus in the absence of a permeabilizing agent, extracellular bacteria are double-labeled with two fluorophores while intracellular bacteria remain single-labeled. A spot count algorithm is used to determine the number of single- and double-labeled bacteria in individual cells, to calculate the percent of cells associated with bacteria, percent of cells with internalized bacteria, and percent of cell-associated bacteria that are internalized. These analyses quantify bacterial association and internalization across thousands of cells and can be applied to diverse experimental systems. PMID:28369762

High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq

PubMed Central

Kondrashova, Olga; Love, Clare J.; Lunke, Sebastian; Hsu, Arthur L.; Waring, Paul M.; Taylor, Graham R.

2015-01-01

Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity. PMID:26569395

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...

Rapid, Automated Determination of Elemental Compositions of Ions in Mass Spectra Obtained with an Open-Air Ion Source (2 of 2)

EPA Science Inventory

An inexpensive autosampler for a DART/TOFMS provides mass spectra from analytes absorbed on 76 cotton swab, wipe samples in 7.5 min. A field sample carrier simplifies sample collection and provides swabs nearly ready for analysis to the lab. Applications of the high throughput pr...

High-throughput protein concentration and buffer exchange: comparison of ultrafiltration and ammonium sulfate precipitation.

PubMed

Moore, Priscilla A; Kery, Vladimir

2009-01-01

High-throughput protein purification is a complex, multi-step process. There are several technical challenges in the course of this process that are not experienced when purifying a single protein. Among the most challenging are the high-throughput protein concentration and buffer exchange, which are not only labor-intensive but can also result in significant losses of purified proteins. We describe two methods of high-throughput protein concentration and buffer exchange: one using ammonium sulfate precipitation and one using micro-concentrating devices based on membrane ultrafiltration. We evaluated the efficiency of both methods on a set of 18 randomly selected purified proteins from Shewanella oneidensis. While both methods provide similar yield and efficiency, the ammonium sulfate precipitation is much less labor intensive and time consuming than the ultrafiltration.

Validation of high-throughput measurement system with microwave-assisted extraction, fully automated sample preparation device, and gas chromatography-electron capture detector for determination of polychlorinated biphenyls in whale blubber.

PubMed

Fujita, Hiroyuki; Honda, Katsuhisa; Hamada, Noriaki; Yasunaga, Genta; Fujise, Yoshihiro

2009-02-01

Validation of a high-throughput measurement system with microwave-assisted extraction (MAE), fully automated sample preparation device (SPD), and gas chromatography-electron capture detector (GC-ECD) for the determination of polychlorinated biphenyls (PCBs) in minke whale blubber was performed. PCB congeners accounting for > 95% of the total PCBs burden in blubber were efficiently extracted with a small volume (20 mL) of n-hexane using MAE due to simultaneous saponification and extraction. Further, the crude extract obtained by MAE was rapidly purified and automatically substituted to a small volume (1 mL) of toluene using SPD without using concentrators. Furthermore, the concentration of PCBs in the purified and concentrated solution was accurately determined by GC-ECD. Moreover, the result of accuracy test using a certified material (SRM 1588b; Cod liver oil) showed good agreement with the NIST certified concentration values. In addition, the method quantification limit of total-PCB in whale blubbers was 41 ng g(-1). This new measurement system for PCBs takes only four hours. Consequently, it indicated this method is the most suitable for the monitoring and screening of PCBs in the conservation of the marine ecosystem and safe distribution of foods.

High-Throughput Nanoindentation for Statistical and Spatial Property Determination

NASA Astrophysics Data System (ADS)

Hintsala, Eric D.; Hangen, Ude; Stauffer, Douglas D.

2018-04-01

Standard nanoindentation tests are "high throughput" compared to nearly all other mechanical tests, such as tension or compression. However, the typical rates of tens of tests per hour can be significantly improved. These higher testing rates enable otherwise impractical studies requiring several thousands of indents, such as high-resolution property mapping and detailed statistical studies. However, care must be taken to avoid systematic errors in the measurement, including choosing of the indentation depth/spacing to avoid overlap of plastic zones, pileup, and influence of neighboring microstructural features in the material being tested. Furthermore, since fast loading rates are required, the strain rate sensitivity must also be considered. A review of these effects is given, with the emphasis placed on making complimentary standard nanoindentation measurements to address these issues. Experimental applications of the technique, including mapping of welds, microstructures, and composites with varying length scales, along with studying the effect of surface roughness on nominally homogeneous specimens, will be presented.

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

Transparent Nanopore Cavity Arrays Enable Highly Parallelized Optical Studies of Single Membrane Proteins on Chip.

PubMed

Diederichs, Tim; Nguyen, Quoc Hung; Urban, Michael; Tampé, Robert; Tornow, Marc

2018-06-13

Membrane proteins involved in transport processes are key targets for pharmaceutical research and industry. Despite continuous improvements and new developments in the field of electrical readouts for the analysis of transport kinetics, a well-suited methodology for high-throughput characterization of single transporters with nonionic substrates and slow turnover rates is still lacking. Here, we report on a novel architecture of silicon chips with embedded nanopore microcavities, based on a silicon-on-insulator technology for high-throughput optical readouts. Arrays containing more than 14 000 inverted-pyramidal cavities of 50 femtoliter volumes and 80 nm circular pore openings were constructed via high-resolution electron-beam lithography in combination with reactive ion etching and anisotropic wet etching. These cavities feature both, an optically transparent bottom and top cap. Atomic force microscopy analysis reveals an overall extremely smooth chip surface, particularly in the vicinity of the nanopores, which exhibits well-defined edges. Our unprecedented transparent chip design provides parallel and independent fluorescent readout of both cavities and buffer reservoir for unbiased single-transporter recordings. Spreading of large unilamellar vesicles with efficiencies up to 96% created nanopore-supported lipid bilayers, which are stable for more than 1 day. A high lipid mobility in the supported membrane was determined by fluorescent recovery after photobleaching. Flux kinetics of α-hemolysin were characterized at single-pore resolution with a rate constant of 0.96 ± 0.06 × 10 -3 s -1 . Here, we deliver an ideal chip platform for pharmaceutical research, which features high parallelism and throughput, synergistically combined with single-transporter resolution.

Repurposing a Benchtop Centrifuge for High-Throughput Single-Molecule Force Spectroscopy.

PubMed

Yang, Darren; Wong, Wesley P

2018-01-01

We present high-throughput single-molecule manipulation using a benchtop centrifuge, overcoming limitations common in other single-molecule approaches such as high cost, low throughput, technical difficulty, and strict infrastructure requirements. An inexpensive and compact Centrifuge Force Microscope (CFM) adapted to a commercial centrifuge enables use by nonspecialists, and integration with DNA nanoswitches facilitates both reliable measurements and repeated molecular interrogation. Here, we provide detailed protocols for constructing the CFM, creating DNA nanoswitch samples, and carrying out single-molecule force measurements.

High throughput single cell counting in droplet-based microfluidics.

PubMed

Lu, Heng; Caen, Ouriel; Vrignon, Jeremy; Zonta, Eleonora; El Harrak, Zakaria; Nizard, Philippe; Baret, Jean-Christophe; Taly, Valérie

2017-05-02

Droplet-based microfluidics is extensively and increasingly used for high-throughput single-cell studies. However, the accuracy of the cell counting method directly impacts the robustness of such studies. We describe here a simple and precise method to accurately count a large number of adherent and non-adherent human cells as well as bacteria. Our microfluidic hemocytometer provides statistically relevant data on large populations of cells at a high-throughput, used to characterize cell encapsulation and cell viability during incubation in droplets.

High-Throughput Sequencing of Germline and Tumor From Men with Early-Onset Metastatic Prostate Cancer

DTIC Science & Technology

2016-12-01

AWARD NUMBER: W81XWH-13-1-0371 TITLE: High-Throughput Sequencing of Germline and Tumor From Men with Early- Onset Metastatic Prostate Cancer...DATES COVERED 30 Sep 2013 - 29 Sep 2016 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER High-Throughput Sequencing of Germline and Tumor From Men with...presenting with metastatic prostate cancer at a young age (before age 60 years). Whole exome sequencing identified a panel of germline variants that have

A high-throughput assay for enzymatic polyester hydrolysis activity by fluorimetric detection.

PubMed

Wei, Ren; Oeser, Thorsten; Billig, Susan; Zimmermann, Wolfgang

2012-12-01

A fluorimetric assay for the fast determination of the activity of polyester-hydrolyzing enzymes in a large number of samples has been developed. Terephthalic acid (TPA) is a main product of the enzymatic hydrolysis of polyethylene terephthalate (PET), a synthetic polyester. Terephthalate has been quantified following its conversion to the fluorescent 2-hydroxyterephthalate by an iron autoxidation-mediated generation of free hydroxyl radicals. The assay proved to be robust at different buffer concentrations, reaction times, pH values, and in the presence of proteins. A validation of the assay was performed by analyzing TPA formation from PET films and nanoparticles catalyzed by a polyester hydrolase from Thermobifida fusca KW3 in a 96-well microplate format. The results showed a close correlation (R(2) = 0.99) with those obtained by a considerably more tedious and time-consuming HPLC method, suggesting the aptness of the fluorimetric assay for a high-throughput screening for polyester hydrolases. The method described in this paper will facilitate the detection and development of biocatalysts for the modification and degradation of synthetic polymers. The fluorimetric assay can be used to quantify the amount of TPA obtained as the final degradation product of the enzymatic hydrolysis of PET. In a microplate format, this assay can be applied for the high-throughput screening of polyester hydrolases. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Citrulline-modified phage display: a novel high-throughput discovery approach for the identification of citrulline-containing ligands.

PubMed

Somers, Klaartje; Stinissen, Piet; Somers, Veerle

2011-06-01

Phage display is a high-throughput technology used to identify ligands for a given target. A drawback of the approach is the absence of PTMs in phage-displayed peptides. The applicability of phage display could be broadened considerably by the implementation of PTMs in this system. The aim of this study was to investigate the possible application of citrullination, a PTM of an arginine into a citrulline amino acid, in filamentous (M13) and lytic (T7) phage display. After in vitro citrullination of T7 and M13 phages, citrullination was confirmed and the infectivity of both citrullinated and non-citrullinated phage was compared by titer determination. We demonstrated the successful in vitro citrullination of T7 and M13 phage-displayed peptides. This in vitro modification did not affect the viability or infectivity of the T7 virions, a necessary prerequisite for the implementation of this approach in T7 phage display. For M13 phage, however, the infecting phage titer decreased five-fold upon citrullination, limiting the use of this modification in M13 phage display. In conclusion, in vitro citrullination can be applied in T7 phage display giving rise to a high-throughput and sensitive approach to identify citrulline-containing ligands by the use of the strengths of phage display technology. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Accelerating evaluation of converged lattice thermal conductivity

NASA Astrophysics Data System (ADS)

Qin, Guangzhao; Hu, Ming

2018-01-01

High-throughput computational materials design is an emerging area in materials science, which is based on the fast evaluation of physical-related properties. The lattice thermal conductivity (κ) is a key property of materials for enormous implications. However, the high-throughput evaluation of κ remains a challenge due to the large resources costs and time-consuming procedures. In this paper, we propose a concise strategy to efficiently accelerate the evaluation process of obtaining accurate and converged κ. The strategy is in the framework of phonon Boltzmann transport equation (BTE) coupled with first-principles calculations. Based on the analysis of harmonic interatomic force constants (IFCs), the large enough cutoff radius (rcutoff), a critical parameter involved in calculating the anharmonic IFCs, can be directly determined to get satisfactory results. Moreover, we find a simple way to largely ( 10 times) accelerate the computations by fast reconstructing the anharmonic IFCs in the convergence test of κ with respect to the rcutof, which finally confirms the chosen rcutoff is appropriate. Two-dimensional graphene and phosphorene along with bulk SnSe are presented to validate our approach, and the long-debate divergence problem of thermal conductivity in low-dimensional systems is studied. The quantitative strategy proposed herein can be a good candidate for fast evaluating the reliable κ and thus provides useful tool for high-throughput materials screening and design with targeted thermal transport properties.

Design and construction of a first-generation high-throughput integrated robotic molecular biology platform for bioenergy applications.

PubMed

Hughes, Stephen R; Butt, Tauseef R; Bartolett, Scott; Riedmuller, Steven B; Farrelly, Philip

2011-08-01

The molecular biological techniques for plasmid-based assembly and cloning of gene open reading frames are essential for elucidating the function of the proteins encoded by the genes. High-throughput integrated robotic molecular biology platforms that have the capacity to rapidly clone and express heterologous gene open reading frames in bacteria and yeast and to screen large numbers of expressed proteins for optimized function are an important technology for improving microbial strains for biofuel production. The process involves the production of full-length complementary DNA libraries as a source of plasmid-based clones to express the desired proteins in active form for determination of their functions. Proteins that were identified by high-throughput screening as having desired characteristics are overexpressed in microbes to enable them to perform functions that will allow more cost-effective and sustainable production of biofuels. Because the plasmid libraries are composed of several thousand unique genes, automation of the process is essential. This review describes the design and implementation of an automated integrated programmable robotic workcell capable of producing complementary DNA libraries, colony picking, isolating plasmid DNA, transforming yeast and bacteria, expressing protein, and performing appropriate functional assays. These operations will allow tailoring microbial strains to use renewable feedstocks for production of biofuels, bioderived chemicals, fertilizers, and other coproducts for profitable and sustainable biorefineries. Published by Elsevier Inc.

Determination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride by flow-injection analysis based on a specific condensation reaction between malonic acid and ethylenediamine.

PubMed

Seno, Kunihiko; Matumura, Kazuki; Oshita, Koji; Oshima, Mitsuko; Motomizu, Shoji

2009-03-01

A sensitive and rapid flow-injection analysis was developed for the determination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl), which was used for the formation of amide (peptide) and esters as a dehydration or condensation reagent. The EDC.HCl could be determined by the flow-injection analysis based on a specific condensation reaction between malonic acid and ethylenediamine in aquatic media. The reaction was accelerated at 60 degrees C, and the absorbance of the product was detected at 262 nm. The calibration graph of EDC.HCl showed good linearity in the range from 0 to 0.1% (0 to 0.0005 M), whose regression equation was y = 1.52 x 10(9)x (y, peak area; x, % concentration of EDC.HCl). The proposed method allowed high-throughput analysis; the sample throughput was 12 samples per hour. The limit of detection (LOD) and the relative standard deviation (RSD) were 2 x 10(-6) M and 1.0%, respectively. This reaction is proceeded in aqueous solution and specific for EDC.HCl.

Netest: A Tool to Measure the Maximum Burst Size, Available Bandwidth and Achievable Throughput

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Guojun; Tierney, Brian

2003-01-31

Distinguishing available bandwidth and achievable throughput is essential for improving network applications' performance. Achievable throughput is the throughput considering a number of factors such as network protocol, host speed, network path, and TCP buffer space, where as available bandwidth only considers the network path. Without understanding this difference, trying to improve network applications' performance is like ''blind men feeling the elephant'' [4]. In this paper, we define and distinguish bandwidth and throughput, and debate which part of each is achievable and which is available. Also, we introduce and discuss a new concept - Maximum Burst Size that is crucial tomore » the network performance and bandwidth sharing. A tool, netest, is introduced to help users to determine the available bandwidth, and provides information to achieve better throughput with fairness of sharing the available bandwidth, thus reducing misuse of the network.« less

Volume determination of irregularly-shaped quasi-spherical nanoparticles.

PubMed

Attota, Ravi Kiran; Liu, Eileen Cherry

2016-11-01

Nanoparticles (NPs) are widely used in diverse application areas, such as medicine, engineering, and cosmetics. The size (or volume) of NPs is one of the most important parameters for their successful application. It is relatively straightforward to determine the volume of regular NPs such as spheres and cubes from a one-dimensional or two-dimensional measurement. However, due to the three-dimensional nature of NPs, it is challenging to determine the proper physical size of many types of regularly and irregularly-shaped quasi-spherical NPs at high-throughput using a single tool. Here, we present a relatively simple method that determines a better volume estimate of NPs by combining measurements from their top-down projection areas and peak heights using two tools. The proposed method is significantly faster and more economical than the electron tomography method. We demonstrate the improved accuracy of the combined method over scanning electron microscopy (SEM) or atomic force microscopy (AFM) alone by using modeling, simulations, and measurements. This study also exposes the existence of inherent measurement biases for both SEM and AFM, which usually produce larger measured diameters with SEM than with AFM. However, in some cases SEM measured diameters appear to have less error compared to AFM measured diameters, especially for widely used IS-NPs such as of gold, and silver. The method provides a much needed, proper high-throughput volumetric measurement method useful for many applications. Graphical Abstract The combined method for volume determination of irregularly-shaped quasi-spherical nanoparticles.

Enrichment analysis in high-throughput genomics - accounting for dependency in the NULL.

PubMed

Gold, David L; Coombes, Kevin R; Wang, Jing; Mallick, Bani

2007-03-01

Translating the overwhelming amount of data generated in high-throughput genomics experiments into biologically meaningful evidence, which may for example point to a series of biomarkers or hint at a relevant pathway, is a matter of great interest in bioinformatics these days. Genes showing similar experimental profiles, it is hypothesized, share biological mechanisms that if understood could provide clues to the molecular processes leading to pathological events. It is the topic of further study to learn if or how a priori information about the known genes may serve to explain coexpression. One popular method of knowledge discovery in high-throughput genomics experiments, enrichment analysis (EA), seeks to infer if an interesting collection of genes is 'enriched' for a Consortium particular set of a priori Gene Ontology Consortium (GO) classes. For the purposes of statistical testing, the conventional methods offered in EA software implicitly assume independence between the GO classes. Genes may be annotated for more than one biological classification, and therefore the resulting test statistics of enrichment between GO classes can be highly dependent if the overlapping gene sets are relatively large. There is a need to formally determine if conventional EA results are robust to the independence assumption. We derive the exact null distribution for testing enrichment of GO classes by relaxing the independence assumption using well-known statistical theory. In applications with publicly available data sets, our test results are similar to the conventional approach which assumes independence. We argue that the independence assumption is not detrimental.

Field Evaluation of a High Throughput Loop Mediated Isothermal Amplification Test for the Detection of Asymptomatic Plasmodium Infections in Zanzibar

PubMed Central

Morris, Ulrika; Ding, Xavier C.; Jovel, Irina; Msellem, Mwinyi I.; Bergman, Daniel; Islam, Atiqul; Ali, Abdullah S.; Polley, Spencer; Gonzalez, Iveth J.; Mårtensson, Andreas; Björkman, Anders

2017-01-01

Background New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP) was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar. Methods HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a community-based survey in 2015. Results The PCR and HTP-LAMP determined malaria prevalences were 1.6% (95%CI 1.3–2.4) and 0.7% (95%CI 0.4–1.1), respectively. The sensitivity of HTP-LAMP compared to PCR was 40.8% (CI95% 27.0–55.8) and the specificity was 99.9% (CI95% 99.8–100). For the PCR positive samples, there was no statistically significant difference between the geometric mean parasite densities among the HTP-LAMP positive (2.5 p/μL, range 0.2–770) and HTP-LAMP negative (1.4 p/μL, range 0.1–7) samples (p = 0.088). Two lab technicians analysed up to 282 samples per day and the HTP-LAMP method was experienced as user friendly. Conclusions Although field applicable, this high throughput format of LAMP as used here was not sensitive enough to be recommended for detection of asymptomatic low-density infections in areas like Zanzibar, approaching malaria elimination. PMID:28095434

Field Evaluation of a High Throughput Loop Mediated Isothermal Amplification Test for the Detection of Asymptomatic Plasmodium Infections in Zanzibar.

PubMed

Aydin-Schmidt, Berit; Morris, Ulrika; Ding, Xavier C; Jovel, Irina; Msellem, Mwinyi I; Bergman, Daniel; Islam, Atiqul; Ali, Abdullah S; Polley, Spencer; Gonzalez, Iveth J; Mårtensson, Andreas; Björkman, Anders

2017-01-01

New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP) was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar. HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a community-based survey in 2015. The PCR and HTP-LAMP determined malaria prevalences were 1.6% (95%CI 1.3-2.4) and 0.7% (95%CI 0.4-1.1), respectively. The sensitivity of HTP-LAMP compared to PCR was 40.8% (CI95% 27.0-55.8) and the specificity was 99.9% (CI95% 99.8-100). For the PCR positive samples, there was no statistically significant difference between the geometric mean parasite densities among the HTP-LAMP positive (2.5 p/μL, range 0.2-770) and HTP-LAMP negative (1.4 p/μL, range 0.1-7) samples (p = 0.088). Two lab technicians analysed up to 282 samples per day and the HTP-LAMP method was experienced as user friendly. Although field applicable, this high throughput format of LAMP as used here was not sensitive enough to be recommended for detection of asymptomatic low-density infections in areas like Zanzibar, approaching malaria elimination.

Hospital economics of the hospitalist.

PubMed

Gregory, Douglas; Baigelman, Walter; Wilson, Ira B

2003-06-01

To determine the economic impact on the hospital of a hospitalist program and to develop insights into the relative economic importance of variables such as reductions in mean length of stay and cost, improvements in throughput (patients discharged per unit time), payer methods of reimbursement, and the cost of the hospitalist program. The primary data source was Tufts-New England Medical Center in Boston. Patient demographics, utilization, cost, and revenue data were obtained from the hospital's cost accounting system and medical records. The hospitalist admitted and managed all patients during a six-week period on the general medical unit of Tufts-New England Medical Center. Reimbursement, cost, length of stay, and throughput outcomes during this period were contrasted with patients admitted to the unit in the same period in the prior year, in the preceding period, and in the following period. The hospitalist group compared with the control group demonstrated: length of stay reduced to 2.19 days from 3.45 days (p<.001); total hospital costs per admission reduced to 1,775 dollars from 2,332 dollars (p<.001); costs per day increased to 811 dollars from 679 dollars (p<.001); no differences for readmission within 30 days of discharge to extended care facilities. The hospital's expected incremental profitability with the hospitalist was -1.44 dollars per admission excluding incremental throughput effects, and it was most sensitive to changes in the ratio of per diem to case rate reimbursement. Incremental throughput with the hospitalist was estimated at 266 patients annually with an associated incremental profitability of 1.3 million dollars. Hospital interventions designed to reduce length of stay, such as the hospitalist, should be evaluated in terms of cost, throughput, and reimbursement effects. Excluding throughput effects, the hospitalist program was not economically viable due to the influence of per diem reimbursement. Throughput improvements occasioned by the hospitalist program with high baseline occupancy levels are substantial and tend to favor a hospitalist program.

High-throughput sequencing methods to study neuronal RNA-protein interactions.

PubMed

Ule, Jernej

2009-12-01

UV-cross-linking and RNase protection, combined with high-throughput sequencing, have provided global maps of RNA sites bound by individual proteins or ribosomes. Using a stringent purification protocol, UV-CLIP (UV-cross-linking and immunoprecipitation) was able to identify intronic and exonic sites bound by splicing regulators in mouse brain tissue. Ribosome profiling has been used to quantify ribosome density on budding yeast mRNAs under different environmental conditions. Post-transcriptional regulation in neurons requires high spatial and temporal precision, as is evident from the role of localized translational control in synaptic plasticity. It remains to be seen if the high-throughput methods can be applied quantitatively to study the dynamics of RNP (ribonucleoprotein) remodelling in specific neuronal populations during the neurodegenerative process. It is certain, however, that applications of new biochemical techniques followed by high-throughput sequencing will continue to provide important insights into the mechanisms of neuronal post-transcriptional regulation.

Evaluation of Compatibility of ToxCast High-Throughput/High-Content Screening Assays with Engineered Nanomaterials

EPA Science Inventory

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

SwellGel: an affinity chromatography technology for high-capacity and high-throughput purification of recombinant-tagged proteins.

PubMed

Draveling, C; Ren, L; Haney, P; Zeisse, D; Qoronfleh, M W

2001-07-01

The revolution in genomics and proteomics is having a profound impact on drug discovery. Today's protein scientist demands a faster, easier, more reliable way to purify proteins. A high capacity, high-throughput new technology has been developed in Perbio Sciences for affinity protein purification. This technology utilizes selected chromatography media that are dehydrated to form uniform aggregates. The SwellGel aggregates will instantly rehydrate upon addition of the protein sample, allowing purification and direct performance of multiple assays in a variety of formats. SwellGel technology has greater stability and is easier to handle than standard wet chromatography resins. The microplate format of this technology provides high-capacity, high-throughput features, recovering milligram quantities of protein suitable for high-throughput screening or biophysical/structural studies. Data will be presented applying SwellGel technology to recombinant 6x His-tagged protein and glutathione-S-transferase (GST) fusion protein purification. Copyright 2001 Academic Press.

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

NASA Astrophysics Data System (ADS)

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

2016-10-01

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

ToxCast Dashboard

EPA Pesticide Factsheets

The ToxCast Dashboard helps users examine high-throughput assay data to inform chemical safety decisions. To date, it has data on over 9,000 chemicals and information from more than 1,000 high-throughput assay endpoint components.

RapidTox Dashboard

EPA Pesticide Factsheets

The ToxCast Dashboard helps users examine high-throughput assay data to inform chemical safety decisions. To date, it has data on over 9,000 chemicals and information from more than 1,000 high-throughput assay endpoint components.

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

PubMed Central

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

2014-01-01

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

A high-quality annotated transcriptome of swine peripheral blood

USDA-ARS?s Scientific Manuscript database

Background: High throughput gene expression profiling assays of peripheral blood are widely used in biomedicine, as well as in animal genetics and physiology research. Accurate, comprehensive, and precise interpretation of such high throughput assays relies on well-characterized reference genomes an...

Compounds with species and cell type specific toxicity identified in a 2000 compound drug screen of neural stem cells and rat mixed cortical neurons.

PubMed

Malik, Nasir; Efthymiou, Anastasia G; Mather, Karly; Chester, Nathaniel; Wang, Xiantao; Nath, Avindra; Rao, Mahendra S; Steiner, Joseph P

2014-12-01

Human primary neural tissue is a vital component for the quick and simple determination of chemical compound neurotoxicity in vitro. In particular, such tissue would be ideal for high-throughput screens that can be used to identify novel neurotoxic or neurotherapeutic compounds. We have previously established a high-throughput screening platform using human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) and neurons. In this study, we conducted a 2000 compound screen with human NSCs and rat cortical cells to identify compounds that are selectively toxic to each group. Approximately 100 of the tested compounds showed specific toxicity to human NSCs. A secondary screen of a small subset of compounds from the primary screen on human iPSCs, NSC-derived neurons, and fetal astrocytes validated the results from >80% of these compounds with some showing cell specific toxicity. Amongst those compounds were several cardiac glycosides, all of which were selectively toxic to the human cells. As the screen was able to reliably identify neurotoxicants, many with species and cell-type specificity, this study demonstrates the feasibility of this NSC-driven platform for higher-throughput neurotoxicity screens. Published by Elsevier B.V.

LC-MS/MS determination of 2-(4-((2-(2S,5R)-2-Cyano-5-ethynyl-1-pyrrolidinyl)-2-oxoethylamino)-4-methyl-1-piperidinyl)-4-pyridinecarboxylic acid (ABT-279) in dog plasma with high-throughput protein precipitation sample preparation.

PubMed

Kim, Joseph; Flick, Jeanette; Reimer, Michael T; Rodila, Ramona; Wang, Perry G; Zhang, Jun; Ji, Qin C; El-Shourbagy, Tawakol A

2007-11-01

As an effective DPP-IV inhibitor, 2-(4-((2-(2S,5R)-2-Cyano-5-ethynyl-1-pyrrolidinyl)-2-oxoethylamino)-4-methyl-1-piperidinyl)-4-pyridinecarboxylic acid (ABT-279), is an investigational drug candidate under development at Abbott Laboratories for potential treatment of type 2 diabetes. In order to support the development of ABT-279, multiple analytical methods for an accurate, precise and selective concentration determination of ABT-279 in different matrices were developed and validated in accordance with the US Food and Drug Administration Guidance on Bioanalytical Method Validation. The analytical method for ABT-279 in dog plasma was validated in parallel to other validations for ABT-279 determination in different matrices. In order to shorten the sample preparation time and increase method precision, an automated multi-channel liquid handler was used to perform high-throughput protein precipitation and all other liquid transfers. The separation was performed through a Waters YMC ODS-AQ column (2.0 x 150 mm, 5 microm, 120 A) with a mobile phase of 20 mm ammonium acetate in 20% acetonitrile at a flow rate of 0.3 mL/min. Data collection started at 2.2 min and continued for 2.0 min. The validated linear dynamic range in dog plasma was between 3.05 and 2033.64 ng/mL using a 50 microL sample volume. The achieved r(2) coefficient of determination from three consecutive runs was between 0.998625 and 0.999085. The mean bias was between -4.1 and 4.3% for all calibration standards including lower limit of quantitation. The mean bias was between -8.0 and 0.4% for the quality control samples. The precision, expressed as a coefficient of variation (CV), was < or =4.1% for all levels of quality control samples. The validation results demonstrated that the high-throughput method was accurate, precise and selective for the determination of ABT-279 in dog plasma. The validated method was also employed to support two toxicology studies. The passing rate was 100% for all 49 runs from one validation study and two toxicology studies. Copyright (c) 2007 John Wiley & Sons, Ltd.

Accurate assessment of liver steatosis in animal models using a high throughput Raman fiber optic probe.

PubMed

Hewitt, Kevin C; Ghassemi Rad, Javad; McGregor, Hanna C; Brouwers, Erin; Sapp, Heidi; Short, Michael A; Fashir, Samia B; Zeng, Haishan; Alwayn, Ian P

2015-10-07

Due to the shortage of healthy donor organs, steatotic livers are commonly used for transplantation, placing patients at higher risk for graft dysfunction and lower survival rates. Raman Spectroscopy is a technique which has shown the ability to rapidly detect the vibration state of C-H bonds in triglycerides. The aim of this study is to determine whether conventional Raman spectroscopy can reliably detect and quantify fat in an animal model of liver steatosis. Mice and rats fed a methionine and choline-deficient (MCD) and control diets were sacrificed on one, two, three and four weeks' time points. A confocal Raman microscope, a commercial Raman (iRaman) fiber optic probe and a highly sensitive Raman fiber optic probe system, the latter utilizing a 785 nm excitation laser, were used to detect changes in the Raman spectra of steatotic mouse livers. Thin layer chromatography was used to assess the triglyceride content of liver specimens, and sections were scored blindly for fat content using histological examination. Principal component analysis (PCA) of Raman spectra was used to extract the principal components responsible for spectroscopic differences with MCD week (time on MCD diet). Confocal Raman microscopy revealed the presence of saturated fats in mice liver sections. A commercially available handheld Raman spectroscopy probe could not distinguish the presence of fat in the liver whereas our specially designed, high throughput Raman system could clearly distinguish lobe-specific changes in fat content. In the left lobe in particular, the Raman PC scores exhibited a significant correlation (R(2) = 0.96) with the gold standard, blinded scoring by histological examination. The specially designed, high throughput Raman system can be used for clinical purposes. Its application to the field of transplantation would enable surgeons to determine the hepatic fat content of the donor's liver in the field prior to proceeding with organ retrieval. Next steps include validating these results in a prospective analysis of human liver transplantation implant biopsies.

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision.

PubMed

Perreault, Andrea A; Venters, Bryan J

2016-12-23

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5' DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

Adapting the γ-H2AX assay for automated processing in human lymphocytes. 1. Technological aspects.

PubMed

Turner, Helen C; Brenner, David J; Chen, Youhua; Bertucci, Antonella; Zhang, Jian; Wang, Hongliang; Lyulko, Oleksandra V; Xu, Yanping; Shuryak, Igor; Schaefer, Julia; Simaan, Nabil; Randers-Pehrson, Gerhard; Yao, Y Lawrence; Amundson, Sally A; Garty, Guy

2011-03-01

The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstick-derived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparin-coated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes.

Industrializing electrophysiology: HT automated patch clamp on SyncroPatch® 96 using instant frozen cells.

PubMed

Polonchuk, Liudmila

2014-01-01

Patch-clamping is a powerful technique for investigating the ion channel function and regulation. However, its low throughput hampered profiling of large compound series in early drug development. Fortunately, automation has revolutionized the area of experimental electrophysiology over the past decade. Whereas the first automated patch-clamp instruments using the planar patch-clamp technology demonstrated rather a moderate throughput, few second-generation automated platforms recently launched by various companies have significantly increased ability to form a high number of high-resistance seals. Among them is SyncroPatch(®) 96 (Nanion Technologies GmbH, Munich, Germany), a fully automated giga-seal patch-clamp system with the highest throughput on the market. By recording from up to 96 cells simultaneously, the SyncroPatch(®) 96 allows to substantially increase throughput without compromising data quality. This chapter describes features of the innovative automated electrophysiology system and protocols used for a successful transfer of the established hERG assay to this high-throughput automated platform.

TaqMan 5′-Nuclease Human Immunodeficiency Virus Type 1 PCR Assay with Phage-Packaged Competitive Internal Control for High-Throughput Blood Donor Screening

PubMed Central

Drosten, C.; Seifried, E.; Roth, W. K.

2001-01-01

Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5′-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5′-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the gag genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5′-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a ≥95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1. PMID:11724836

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

PubMed Central

Chiaraviglio, Lucius; Kang, Yoon-Suk; Kirby, James E.

2016-01-01

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila. PMID:27911388

High-throughput determination of octanol/water partition coefficients using a shake-flask method and novel two-phase solvent system.

PubMed

Morikawa, Go; Suzuka, Chihiro; Shoji, Atsushi; Shibusawa, Yoichi; Yanagida, Akio

2016-01-05

A high-throughput method for determining the octanol/water partition coefficient (P(o/w)) of a large variety of compounds exhibiting a wide range in hydrophobicity was established. The method combines a simple shake-flask method with a novel two-phase solvent system comprising an acetonitrile-phosphate buffer (0.1 M, pH 7.4)-1-octanol (25:25:4, v/v/v; AN system). The AN system partition coefficients (K(AN)) of 51 standard compounds for which log P(o/w) (at pH 7.4; log D) values had been reported were determined by single two-phase partitioning in test tubes, followed by measurement of the solute concentration in both phases using an automatic flow injection-ultraviolet detection system. The log K(AN) values were closely related to reported log D values, and the relationship could be expressed by the following linear regression equation: log D=2.8630 log K(AN) -0.1497(n=51). The relationship reveals that log D values (+8 to -8) for a large variety of highly hydrophobic and/or hydrophilic compounds can be estimated indirectly from the narrow range of log K(AN) values (+3 to -3) determined using the present method. Furthermore, log K(AN) values for highly polar compounds for which no log D values have been reported, such as amino acids, peptides, proteins, nucleosides, and nucleotides, can be estimated using the present method. The wide-ranging log D values (+5.9 to -7.5) of these molecules were estimated for the first time from their log K(AN) values and the above regression equation. Copyright © 2015 Elsevier B.V. All rights reserved.

Lifetime Assessment of the NEXT Ion Thruster

NASA Technical Reports Server (NTRS)

VanNoord, Jonathan L.

2010-01-01

Ion thrusters are low thrust, high specific impulse devices with required operational lifetimes on the order of 10,000 to 100,000 hr. The NEXT ion thruster is the latest generation of ion thrusters under development. The NEXT ion thruster currently has a qualification level propellant throughput requirement of 450 kg of xenon, which corresponds to roughly 22,000 hr of operation at the highest throttling point. Currently, a NEXT engineering model ion thruster with prototype model ion optics is undergoing a long duration test to determine wear characteristics and establish propellant throughput capability. The NEXT thruster includes many improvements over previous generations of ion thrusters, but two of its component improvements have a larger effect on thruster lifetime. These include the ion optics with tighter tolerances, a masked region and better gap control, and the discharge cathode keeper material change to graphite. Data from the NEXT 2000 hr wear test, the NEXT long duration test, and further analysis is used to determine the expected lifetime of the NEXT ion thruster. This paper will review the predictions for all of the anticipated failure mechanisms. The mechanisms will include wear of the ion optics and cathode s orifice plate and keeper from the plasma, depletion of low work function material in each cathode s insert, and spalling of material in the discharge chamber leading to arcing. Based on the analysis of the NEXT ion thruster, the first failure mode for operation above a specific impulse of 2000 sec is expected to be the structural failure of the ion optics at 750 kg of propellant throughput, 1.7 times the qualification requirement. An assessment based on mission analyses for operation below a specific impulse of 2000 sec indicates that the NEXT thruster is capable of double the propellant throughput required by these missions.

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, ...

A New High-Throughput Approach to Genotype Ancient Human Gastrointestinal Parasites.

PubMed

Côté, Nathalie M L; Daligault, Julien; Pruvost, Mélanie; Bennett, E Andrew; Gorgé, Olivier; Guimaraes, Silvia; Capelli, Nicolas; Le Bailly, Matthieu; Geigl, Eva-Maria; Grange, Thierry

2016-01-01

Human gastrointestinal parasites are good indicators for hygienic conditions and health status of past and present individuals and communities. While microscopic analysis of eggs in sediments of archeological sites often allows their taxonomic identification, this method is rarely effective at the species level, and requires both the survival of intact eggs and their proper identification. Genotyping via PCR-based approaches has the potential to achieve a precise species-level taxonomic determination. However, so far it has mostly been applied to individual eggs isolated from archeological samples. To increase the throughput and taxonomic accuracy, as well as reduce costs of genotyping methods, we adapted a PCR-based approach coupled with next-generation sequencing to perform precise taxonomic identification of parasitic helminths directly from archeological sediments. Our study of twenty-five 100 to 7,200 year-old archeological samples proved this to be a powerful, reliable and efficient approach for species determination even in the absence of preserved eggs, either as a stand-alone method or as a complement to microscopic studies.

A Framework for Human Microbiome Research

DTIC Science & Technology

2012-06-14

determined that many compo- nents of data production and processing can contribute errors and artefacts. We investigated methods that avoid these errors and...protocol that ensured consistency in the high-throughput production . To maximize accuracy and consistency, protocols were evaluated primarily using a...future benefits, this resource may promote the development of novel prophylactic strategies such as the application of prebiotics and probiotics to

Phage typing or CRISPR typing for epidemiological surveillance of Salmonella Typhimurium?

PubMed

Mohammed, Manal

2017-11-07

Salmonella Typhimurium is the most dominant Salmonella serovar around the world. It is associated with foodborne gastroenteritis outbreaks but has recently been associated with invasive illness and deaths. Characterization of S. Typhimurium is therefore very crucial for epidemiological surveillance. Phage typing has been used for decades for subtyping of S. Typhimurium to determine the epidemiological relation among isolates. Recent studies however have suggested that high throughput clustered regular interspaced short palindromic repeats (CRISPR) typing has the potential to replace phage typing. This study aimed to determine the efficacy of high-throughput CRISPR typing over conventional phage typing in epidemiological surveillance and outbreak investigation of S. Typhimurium. In silico analysis of whole genome sequences (WGS) of well-documented phage types of S. Typhimurium reveals the presence of different CRISPR type among strains belong to the same phage type. Furthermore, different phage types of S. Typhimurium share identical CRISPR type. Interestingly, identical spacers were detected among outbreak and non-outbreak associated DT8 strains of S. Typhimurium. Therefore, CRISPR typing is not useful for the epidemiological surveillance and outbreak investigation of S. Typhimurium and phage typing, until it is replaced by WGS, is still the gold standard method for epidemiological surveillance of S. Typhimurium.

Quantification of locomotor activity in larval zebrafish: considerations for the design of high-throughput behavioral studies.

PubMed

Ingebretson, Justin J; Masino, Mark A

2013-01-01

High-throughput behavioral studies using larval zebrafish often assess locomotor activity to determine the effects of experimental perturbations. However, the results reported by different groups are difficult to compare because there is not a standardized experimental paradigm or measure of locomotor activity. To address this, we investigated the effects that several factors, including the stage of larval development and the physical dimensions (depth and diameter) of the behavioral arena, have on the locomotor activity produced by larval zebrafish. We provide evidence for differences in locomotor activity between larvae at different stages and when recorded in wells of different depths, but not in wells of different diameters. We also show that the variability for most properties of locomotor activity is less for older than younger larvae, which is consistent with previous reports. Finally, we show that conflicting interpretations of activity level can occur when activity is assessed with a single measure of locomotor activity. Thus, we conclude that although a combination of factors should be considered when designing behavioral experiments, the use of older larvae in deep wells will reduce the variability of locomotor activity, and that multiple properties of locomotor activity should be measured to determine activity level.

A microfluidic chip containing a molecularly imprinted polymer and a DNA aptamer for voltammetric determination of carbofuran.

PubMed

Li, Shuhuai; Li, Jianping; Luo, Jinhui; Xu, Zhi; Ma, Xionghui

2018-05-11

An electrochemical microfluidic chip is described for the determination of the insecticide carbofuran. It is making use of a molecularly imprinted film (MIP) and a DNA aptamer as dual recognition units. The analyte (carbofuran) is transported to the MIP and captured at the identification site in the channel. Then, carbofuran is eluted with carbinol-acetic acid and transported to the DNA aptamer on the testing position of the chip. It is captured again, this time by the aptamer, and detected by differential pulse voltammetry (DPV). The dual recognition (by aptamer and MIP) results in outstanding selectivity. Additionally, graphene oxide-supported gold nanoparticles (GO-AuNPs) were used to improve the sensitivity of electrochemical detector. DPV response is linear in the 0.2 to 50 nM carbofuran concentration range at a potential of -1.2 V, with a 67 pM detection limit. The method has attractive features such as its potential for high throughput, high degree of automation, and high integration. Conceivably, the method may be extended to other analytes for which appropriate MIPs and aptamers are available. Graphical abstract Schematic of an electrochemical microfluidic chip for carbofuran detection based on a molecularly imprinted film (MIP) and a DNA aptamer as dual recognition units. In the chip, targets were recognized by MIP and aptamer, respectively. It shows promising potential for the design of electrochemical devices with high throughput, high automation, and high integration.

GiNA, an efficient and high-throughput software for horticultural phenotyping

USDA-ARS?s Scientific Manuscript database

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,...

High-Throughput Method for Strontium Isotope Analysis by Multi-Collector-Inductively Coupled Plasma-Mass Spectrometer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wall, Andrew J.; Capo, Rosemary C.; Stewart, Brian W.

2016-09-22

This technical report presents the details of the Sr column configuration and the high-throughput Sr separation protocol. Data showing the performance of the method as well as the best practices for optimizing Sr isotope analysis by MC-ICP-MS is presented. Lastly, this report offers tools for data handling and data reduction of Sr isotope results from the Thermo Scientific Neptune software to assist in data quality assurance, which help avoid issues of data glut associated with high sample throughput rapid analysis.

High-Throughput Method for Strontium Isotope Analysis by Multi-Collector-Inductively Coupled Plasma-Mass Spectrometer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hakala, Jacqueline Alexandra

2016-11-22

This technical report presents the details of the Sr column configuration and the high-throughput Sr separation protocol. Data showing the performance of the method as well as the best practices for optimizing Sr isotope analysis by MC-ICP-MS is presented. Lastly, this report offers tools for data handling and data reduction of Sr isotope results from the Thermo Scientific Neptune software to assist in data quality assurance, which help avoid issues of data glut associated with high sample throughput rapid analysis.

A Memory Efficient Network Encryption Scheme

NASA Astrophysics Data System (ADS)

El-Fotouh, Mohamed Abo; Diepold, Klaus

In this paper, we studied the two widely used encryption schemes in network applications. Shortcomings have been found in both schemes, as these schemes consume either more memory to gain high throughput or low memory with low throughput. The need has aroused for a scheme that has low memory requirements and in the same time possesses high speed, as the number of the internet users increases each day. We used the SSM model [1], to construct an encryption scheme based on the AES. The proposed scheme possesses high throughput together with low memory requirements.

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

PubMed

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

2017-01-01

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

Combined Effect of Random Transmit Power Control and Inter-Path Interference Cancellation on DS-CDMA Packet Mobile Communications

NASA Astrophysics Data System (ADS)

Kudoh, Eisuke; Ito, Haruki; Wang, Zhisen; Adachi, Fumiyuki

In mobile communication systems, high speed packet data services are demanded. In the high speed data transmission, throughput degrades severely due to severe inter-path interference (IPI). Recently, we proposed a random transmit power control (TPC) to increase the uplink throughput of DS-CDMA packet mobile communications. In this paper, we apply IPI cancellation in addition to the random TPC. We derive the numerical expression of the received signal-to-interference plus noise power ratio (SINR) and introduce IPI cancellation factor. We also derive the numerical expression of system throughput when IPI is cancelled ideally to compare with the Monte Carlo numerically evaluated system throughput. Then we evaluate, by Monte-Carlo numerical computation method, the combined effect of random TPC and IPI cancellation on the uplink throughput of DS-CDMA packet mobile communications.

A High Throughput Method for Measuring Polycyclic Aromatic Hydrocarbons in Seafood Using QuEChERS Extraction and SBSE.

PubMed

Pfannkoch, Edward A; Stuff, John R; Whitecavage, Jacqueline A; Blevins, John M; Seely, Kathryn A; Moran, Jeffery H

2015-01-01

National Oceanic and Atmospheric Administration (NOAA) Method NMFS-NWFSC-59 2004 is currently used to quantitatively analyze seafood for polycyclic aromatic hydrocarbon (PAH) contamination, especially following events such as the Deepwater Horizon oil rig explosion that released millions of barrels of crude oil into the Gulf of Mexico. This method has limited throughput capacity; hence, alternative methods are necessary to meet analytical demands after such events. Stir bar sorptive extraction (SBSE) is an effective technique to extract trace PAHs in water and the quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction strategy effectively extracts PAHs from complex food matrices. This study uses SBSE to concentrate PAHs and eliminate matrix interference from QuEChERS extracts of seafood, specifically oysters, fish, and shrimp. This method provides acceptable recovery (65-138%) linear calibrations and is sensitive (LOD = 0.02 ppb, LOQ = 0.06 ppb) while providing higher throughput and maintaining equivalency between NOAA 2004 as determined by analysis of NIST SRM 1974b mussel tissue.

Human papillomavirus detection using the Abbott RealTime high-risk HPV tests compared with conventional nested PCR coupled to high-throughput sequencing of amplification products in cervical smear specimens from a Gabonese female population.

PubMed

Moussavou-Boundzanga, Pamela; Koumakpayi, Ismaël Hervé; Labouba, Ingrid; Leroy, Eric M; Belembaogo, Ernest; Berthet, Nicolas

2017-12-21

Cervical cancer is the fourth most common malignancy in women worldwide. However, screening with human papillomavirus (HPV) molecular tests holds promise for reducing cervical cancer incidence and mortality in low- and middle-income countries. The performance of the Abbott RealTime High-Risk HPV test (AbRT) was evaluated in 83 cervical smear specimens and compared with a conventional nested PCR coupled to high-throughput sequencing (HTS) to identify the amplicons. The AbRT assay detected at least one HPV genotype in 44.57% of women regardless of the grade of cervical abnormalities. Except for one case, good concordance was observed for the genotypes detected with the AbRT assay in the high-risk HPV category determined with HTS of the amplicon generated by conventional nested PCR. The AbRT test is an easy and reliable molecular tool and was as sensitive as conventional nested PCR in cervical smear specimens for detection HPVs associated with high-grade lesions. Moreover, sequencing amplicons using an HTS approach effectively identified the genotype of the hrHPV identified with the AbRT test.

A Machine Learned Classifier That Uses Gene Expression Data to Accurately Predict Estrogen Receptor Status

PubMed Central

Bastani, Meysam; Vos, Larissa; Asgarian, Nasimeh; Deschenes, Jean; Graham, Kathryn; Mackey, John; Greiner, Russell

2013-01-01

Background Selecting the appropriate treatment for breast cancer requires accurately determining the estrogen receptor (ER) status of the tumor. However, the standard for determining this status, immunohistochemical analysis of formalin-fixed paraffin embedded samples, suffers from numerous technical and reproducibility issues. Assessment of ER-status based on RNA expression can provide more objective, quantitative and reproducible test results. Methods To learn a parsimonious RNA-based classifier of hormone receptor status, we applied a machine learning tool to a training dataset of gene expression microarray data obtained from 176 frozen breast tumors, whose ER-status was determined by applying ASCO-CAP guidelines to standardized immunohistochemical testing of formalin fixed tumor. Results This produced a three-gene classifier that can predict the ER-status of a novel tumor, with a cross-validation accuracy of 93.17±2.44%. When applied to an independent validation set and to four other public databases, some on different platforms, this classifier obtained over 90% accuracy in each. In addition, we found that this prediction rule separated the patients' recurrence-free survival curves with a hazard ratio lower than the one based on the IHC analysis of ER-status. Conclusions Our efficient and parsimonious classifier lends itself to high throughput, highly accurate and low-cost RNA-based assessments of ER-status, suitable for routine high-throughput clinical use. This analytic method provides a proof-of-principle that may be applicable to developing effective RNA-based tests for other biomarkers and conditions. PMID:24312637

Two-dimensional Mathematical Model of Oil-bearing Materials in Extrusion-type Transportation over Rectangular Screw Core

NASA Astrophysics Data System (ADS)

Gukasyan, A. V.; Koshevoy, E. P.; Kosachev, V. S.

2018-05-01

A comparative analysis of alternative models for plastic flow in extrusive transportation of oil-bearing materials was conducted; the research was directed at determining the function describing the screw core throughput capacity of the press (extruder). Transition from a one-dimensional model to a two-dimensional model significantly improves the mathematical model and allows using two-dimensional rheological models determining the throughput of the screw core.

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 ...

Solar fuels photoanode materials discovery by integrating high-throughput theory and experiment

DOE PAGES

Yan, Qimin; Yu, Jie; Suram, Santosh K.; ...

2017-03-06

The limited number of known low-band-gap photoelectrocatalytic materials poses a significant challenge for the generation of chemical fuels from sunlight. Here, using high-throughput ab initio theory with experiments in an integrated workflow, we find eight ternary vanadate oxide photoanodes in the target band-gap range (1.2-2.8 eV). Detailed analysis of these vanadate compounds reveals the key role of VO 4 structural motifs and electronic band-edge character in efficient photoanodes, initiating a genome for such materials and paving the way for a broadly applicable high-throughput-discovery and materials-by-design feedback loop. Considerably expanding the number of known photoelectrocatalysts for water oxidation, our study establishesmore » ternary metal vanadates as a prolific class of photoanodematerials for generation of chemical fuels from sunlight and demonstrates our high-throughput theory-experiment pipeline as a prolific approach to materials discovery.« less

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

PubMed

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

2016-01-15

In the last decades, the basic techniques of microfluidics for the study of cells such as cell culture, cell separation, and cell lysis, have been well developed. Based on cell handling techniques, microfluidics has been widely applied in the field of PCR (Polymerase Chain Reaction), immunoassays, organ-on-chip, stem cell research, and analysis and identification of circulating tumor cells. As a major step in drug discovery, high-throughput screening allows rapid analysis of thousands of chemical, biochemical, genetic or pharmacological tests in parallel. In this review, we summarize the application of microfluidics in cell-based high throughput screening. The screening methods mentioned in this paper include approaches using the perfusion flow mode, the droplet mode, and the microarray mode. We also discuss the future development of microfluidic based high throughput screening platform for drug discovery. Copyright © 2015 Elsevier B.V. All rights reserved.

Solar fuels photoanode materials discovery by integrating high-throughput theory and experiment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Qimin; Yu, Jie; Suram, Santosh K.

The limited number of known low-band-gap photoelectrocatalytic materials poses a significant challenge for the generation of chemical fuels from sunlight. Here, using high-throughput ab initio theory with experiments in an integrated workflow, we find eight ternary vanadate oxide photoanodes in the target band-gap range (1.2-2.8 eV). Detailed analysis of these vanadate compounds reveals the key role of VO 4 structural motifs and electronic band-edge character in efficient photoanodes, initiating a genome for such materials and paving the way for a broadly applicable high-throughput-discovery and materials-by-design feedback loop. Considerably expanding the number of known photoelectrocatalysts for water oxidation, our study establishesmore » ternary metal vanadates as a prolific class of photoanodematerials for generation of chemical fuels from sunlight and demonstrates our high-throughput theory-experiment pipeline as a prolific approach to materials discovery.« less

Fulfilling the promise of the materials genome initiative with high-throughput experimental methodologies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Green, Martin L.; Choi, C. L.; Hattrick-Simpers, J. R.

The Materials Genome Initiative, a national effort to introduce new materials into the market faster and at lower cost, has made significant progress in computational simulation and modeling of materials. To build on this progress, a large amount of experimental data for validating these models, and informing more sophisticated ones, will be required. High-throughput experimentation generates large volumes of experimental data using combinatorial materials synthesis and rapid measurement techniques, making it an ideal experimental complement to bring the Materials Genome Initiative vision to fruition. This paper reviews the state-of-the-art results, opportunities, and challenges in high-throughput experimentation for materials design. Asmore » a result, a major conclusion is that an effort to deploy a federated network of high-throughput experimental (synthesis and characterization) tools, which are integrated with a modern materials data infrastructure, is needed.« less

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

BiQ Analyzer HT: locus-specific analysis of DNA methylation by high-throughput bisulfite sequencing

PubMed Central

Lutsik, Pavlo; Feuerbach, Lars; Arand, Julia; Lengauer, Thomas; Walter, Jörn; Bock, Christoph

2011-01-01

Bisulfite sequencing is a widely used method for measuring DNA methylation in eukaryotic genomes. The assay provides single-base pair resolution and, given sufficient sequencing depth, its quantitative accuracy is excellent. High-throughput sequencing of bisulfite-converted DNA can be applied either genome wide or targeted to a defined set of genomic loci (e.g. using locus-specific PCR primers or DNA capture probes). Here, we describe BiQ Analyzer HT (http://biq-analyzer-ht.bioinf.mpi-inf.mpg.de/), a user-friendly software tool that supports locus-specific analysis and visualization of high-throughput bisulfite sequencing data. The software facilitates the shift from time-consuming clonal bisulfite sequencing to the more quantitative and cost-efficient use of high-throughput sequencing for studying locus-specific DNA methylation patterns. In addition, it is useful for locus-specific visualization of genome-wide bisulfite sequencing data. PMID:21565797

A Self-Reporting Photocatalyst for Online Fluorescence Monitoring of High Throughput RAFT Polymerization.

PubMed

Yeow, Jonathan; Joshi, Sanket; Chapman, Robert; Boyer, Cyrille Andre Jean Marie

2018-04-25

Translating controlled/living radical polymerization (CLRP) from batch to the high throughput production of polymer libraries presents several challenges in terms of both polymer synthesis and characterization. Although recently there have been significant advances in the field of low volume, high throughput CLRP, techniques able to simultaneously monitor multiple polymerizations in an "online" manner have not yet been developed. Here, we report our discovery that 5,10,15,20-tetraphenyl-21H,23H-porphine zinc (ZnTPP) is a self-reporting photocatalyst that can mediate PET-RAFT polymerization as well as report on monomer conversion via changes in its fluorescence properties. This enables the use of a microplate reader to conduct high throughput "online" monitoring of PET-RAFT polymerizations performed directly in 384-well, low volume microtiter plates. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fulfilling the promise of the materials genome initiative with high-throughput experimental methodologies

DOE PAGES

Green, Martin L.; Choi, C. L.; Hattrick-Simpers, J. R.; ...

2017-03-28

The Materials Genome Initiative, a national effort to introduce new materials into the market faster and at lower cost, has made significant progress in computational simulation and modeling of materials. To build on this progress, a large amount of experimental data for validating these models, and informing more sophisticated ones, will be required. High-throughput experimentation generates large volumes of experimental data using combinatorial materials synthesis and rapid measurement techniques, making it an ideal experimental complement to bring the Materials Genome Initiative vision to fruition. This paper reviews the state-of-the-art results, opportunities, and challenges in high-throughput experimentation for materials design. Asmore » a result, a major conclusion is that an effort to deploy a federated network of high-throughput experimental (synthesis and characterization) tools, which are integrated with a modern materials data infrastructure, is needed.« less

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...

Stepping into the omics era: Opportunities and challenges for biomaterials science and engineering☆

PubMed Central

Rabitz, Herschel; Welsh, William J.; Kohn, Joachim; de Boer, Jan

2016-01-01

The research paradigm in biomaterials science and engineering is evolving from using low-throughput and iterative experimental designs towards high-throughput experimental designs for materials optimization and the evaluation of materials properties. Computational science plays an important role in this transition. With the emergence of the omics approach in the biomaterials field, referred to as materiomics, high-throughput approaches hold the promise of tackling the complexity of materials and understanding correlations between material properties and their effects on complex biological systems. The intrinsic complexity of biological systems is an important factor that is often oversimplified when characterizing biological responses to materials and establishing property-activity relationships. Indeed, in vitro tests designed to predict in vivo performance of a given biomaterial are largely lacking as we are not able to capture the biological complexity of whole tissues in an in vitro model. In this opinion paper, we explain how we reached our opinion that converging genomics and materiomics into a new field would enable a significant acceleration of the development of new and improved medical devices. The use of computational modeling to correlate high-throughput gene expression profiling with high throughput combinatorial material design strategies would add power to the analysis of biological effects induced by material properties. We believe that this extra layer of complexity on top of high-throughput material experimentation is necessary to tackle the biological complexity and further advance the biomaterials field. PMID:26876875

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens

PubMed Central

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-01-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue® mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents. PMID:22735701

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens.

PubMed

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-08-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents.

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

PubMed

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

2016-01-01

Many labs have conventional calorimeters where denaturation and binding experiments are setup and run one at a time. While these systems are highly informative to biopolymer folding and ligand interaction, they require considerable manual intervention for cleaning and setup. As such, the throughput for such setups is limited typically to a few runs a day. With a large number of experimental parameters to explore including different buffers, macromolecule concentrations, temperatures, ligands, mutants, controls, replicates, and instrument tests, the need for high-throughput automated calorimeters is on the rise. Lower sample volume requirements and reduced user intervention time compared to the manual instruments have improved turnover of calorimetry experiments in a high-throughput format where 25 or more runs can be conducted per day. The cost and efforts to maintain high-throughput equipment typically demands that these instruments be housed in a multiuser core facility. We describe here the steps taken to successfully start and run an automated biological calorimetry facility at Pennsylvania State University. Scientists from various departments at Penn State including Chemistry, Biochemistry and Molecular Biology, Bioengineering, Biology, Food Science, and Chemical Engineering are benefiting from this core facility. Samples studied include proteins, nucleic acids, sugars, lipids, synthetic polymers, small molecules, natural products, and virus capsids. This facility has led to higher throughput of data, which has been leveraged into grant support, attracting new faculty hire and has led to some exciting publications. © 2016 Elsevier Inc. All rights reserved.

Saccharomyces cerevisiae as a platform for assessing sphingolipid lipid kinase inhibitors

PubMed Central

Agah, Sayeh; Mendelson, Anna J.; Eletu, Oluwafunmilayo T.; Barkey-Bircann, Peter; Gesualdi, James

2018-01-01

Successful medicinal chemistry campaigns to discover and optimize sphingosine kinase inhibitors require a robust assay for screening chemical libraries and for determining rank order potencies. Existing assays for these enzymes are laborious, expensive and/or low throughput. The toxicity of excessive levels of phosphorylated sphingoid bases for the budding yeast, Saccharomyces cerevisiae, affords an assay wherein inhibitors added to the culture media rescue growth in a dose-dependent fashion. Herein, we describe our adaptation of a simple, inexpensive, and high throughput assay for assessing inhibitors of sphingosine kinase types 1 and 2 as well as ceramide kinase and for testing enzymatic activity of sphingosine kinase type 2 mutants. The assay was validated using recombinant enzymes and generally agrees with the rank order of potencies of existing inhibitors. PMID:29672528

Expedient Caution: Approximating Exposure and Dosimetry to Understand Chemical Risk (OSU EMT Research Day keynote presentation)

EPA Science Inventory

I describe research on high throughput exposure and toxicokinetics. These tools provide context for data generated by high throughput toxicity screening to allow risk-based prioritization of thousands of chemicals.

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...

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...

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

ScienceCinema

Gore, Brooklin

2018-02-01

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

Identifying drought adaptive traits in upland cotton using a proximal sensing cart for high-throughput phenotyping

USDA-ARS?s Scientific Manuscript database

Field-based high-throughput phenotyping is an emerging approach to characterize difficult, time-sensitive plant traits in relevant growing conditions. Proximal sensing carts have been developed as an alternative platform to more costly high-clearance tractors for phenotyping dynamic traits in the fi...

High-throughput profiling and analysis of plant responses over time to abiotic stress

USDA-ARS?s Scientific Manuscript database

Energy sorghum (Sorghum bicolor (L.) Moench) is a rapidly growing, high-biomass, annual crop prized for abiotic stress tolerance. Measuring genotype-by-environment (G x E) interactions remains a progress bottleneck. High throughput phenotyping within controlled environments has been proposed as a po...

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...

A high-throughput multiplex method adapted for GMO detection.

PubMed

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

2008-12-24

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

Geospatial analytics to evaluate point-of-dispensing sites for mass immunizations in Allegheny County, Pennsylvania.

PubMed

Everett, Kibri H; Potter, Margaret A; Wheaton, William D; Gleason, Sherrianne M; Brown, Shawn T; Lee, Bruce Y

2013-01-01

Public health agencies use mass immunization locations to quickly administer vaccines to protect a population against an epidemic. The selection of such locations is frequently determined by available staffing levels and in some places, not all potential sites can be opened, often because of a lack of resources. Public health agencies need assistance in determining which n sites are the prime ones to open given available staff to minimize travel time and travel distance for those in the population who need to get to a site to receive treatment. Employ geospatial analytical methods to identify the prime n locations from a predetermined set of potential locations (eg, schools) and determine which locations may not be able to achieve the throughput necessary to reach the herd immunity threshold based on varying R0 values. Spatial location-allocation algorithms were used to select the ideal n mass vaccination locations. Allegheny County, Pennsylvania, served as the study area. The most favorable sites were selected and the number of individuals required to be vaccinated to achieve the herd immunity threshold for a given R0, ranging from 1.5 to 7, was determined. Locations that did not meet the Centers for Disease Control and Prevention throughput recommendation for smallpox were identified. At R0 = 1.5, all mass immunization locations met the required throughput to achieve the herd immunity threshold within 5 days. As R0s increased from 2 to 7, an increasing number of sites were inadequate to meet throughput requirements. Identifying the top n sites and categorizing those with throughput challenges allows health departments to adjust staffing, shift length, or the number of sites. This method has the potential to be expanded to select immunization locations under a number of additional scenarios.

RIPiT-Seq: A high-throughput approach for footprinting RNA:protein complexes

PubMed Central

Singh, Guramrit; Ricci, Emiliano P.; Moore, Melissa J.

2013-01-01

Development of high-throughput approaches to map the RNA interaction sites of individual RNA binding proteins (RBPs) transcriptome-wide is rapidly transforming our understanding of post-transcriptional gene regulatory mechanisms. Here we describe a ribonucleoprotein (RNP) footprinting approach we recently developed for identifying occupancy sites of both individual RBPs and multi-subunit RNP complexes. RNA:protein immunoprecipitation in tandem (RIPiT) yields highly specific RNA footprints of cellular RNPs isolated via two sequential purifications; the resulting RNA footprints can then be identified by high-throughput sequencing (Seq). RIPiT-Seq is broadly applicable to all RBPs regardless of their RNA binding mode and thus provides a means to map the RNA binding sites of RBPs with poor inherent ultraviolet (UV) crosslinkability. Further, among current high-throughput approaches, RIPiT has the unique capacity to differentiate binding sites of RNPs with overlapping protein composition. It is therefore particularly suited for studying dynamic RNP assemblages whose composition evolves as gene expression proceeds. PMID:24096052

Development and validation of a 48-target analytical method for high-throughput monitoring of genetically modified organisms.

PubMed

Li, Xiaofei; Wu, Yuhua; Li, Jun; Li, Yunjing; Long, Likun; Li, Feiwu; Wu, Gang

2015-01-05

The rapid increase in the number of genetically modified (GM) varieties has led to a demand for high-throughput methods to detect genetically modified organisms (GMOs). We describe a new dynamic array-based high throughput method to simultaneously detect 48 targets in 48 samples on a Fludigm system. The test targets included species-specific genes, common screening elements, most of the Chinese-approved GM events, and several unapproved events. The 48 TaqMan assays successfully amplified products from both single-event samples and complex samples with a GMO DNA amount of 0.05 ng, and displayed high specificity. To improve the sensitivity of detection, a preamplification step for 48 pooled targets was added to enrich the amount of template before performing dynamic chip assays. This dynamic chip-based method allowed the synchronous high-throughput detection of multiple targets in multiple samples. Thus, it represents an efficient, qualitative method for GMO multi-detection.

Development and Validation of A 48-Target Analytical Method for High-throughput Monitoring of Genetically Modified Organisms

PubMed Central

Li, Xiaofei; Wu, Yuhua; Li, Jun; Li, Yunjing; Long, Likun; Li, Feiwu; Wu, Gang

2015-01-01

The rapid increase in the number of genetically modified (GM) varieties has led to a demand for high-throughput methods to detect genetically modified organisms (GMOs). We describe a new dynamic array-based high throughput method to simultaneously detect 48 targets in 48 samples on a Fludigm system. The test targets included species-specific genes, common screening elements, most of the Chinese-approved GM events, and several unapproved events. The 48 TaqMan assays successfully amplified products from both single-event samples and complex samples with a GMO DNA amount of 0.05 ng, and displayed high specificity. To improve the sensitivity of detection, a preamplification step for 48 pooled targets was added to enrich the amount of template before performing dynamic chip assays. This dynamic chip-based method allowed the synchronous high-throughput detection of multiple targets in multiple samples. Thus, it represents an efficient, qualitative method for GMO multi-detection. PMID:25556930

High Throughput Determination of VX in Drinking Water by ...

EPA Pesticide Factsheets

Methods Report This document provides the standard operating procedure for determination of the chemical warfare agent VX (O-Ethyl S-2-Diisopropylamino-Ethyl Methylphosphonothioate) in drinking water by isotope dilution liquid chromatography tandem mass spectrometer (LC/MS/MS). This method was adapted from one that was initially developed by the Centers for Disease Control and Prevention, in the National Center for Environmental Health for the determination and quantitation of VX in aqueous matrices. This method is designed to support site-specific cleanup goals of environmental remediation activities following a homeland security incident involving this analyte.

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 infections has been a long-standing quest in the HIV community. Over the past 15 years, these assays have traditionally measured various HIV-specific antibodies, but recent technological advancements have expanded the diversity of proposed accurate, user-friendly, and financially viable tools. Here we designed a high-throughput genomic HIV incidence assay based on the signature imprinted in the HIV gene sequence population. By combining next-generation sequencing techniques with bioinformatics analysis, we demonstrated that genomic fingerprints are capable of distinguishing recently infected patients from chronically infected patients with high precision. Our high-throughput platform is expected to allow us to process many patients' samples from a single experiment, permitting the assay to be cost-effective for routine surveillance. PMID:24371062

A high-throughput label-free nanoparticle analyser.

PubMed

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.

High throughput automated microbial bioreactor system used for clone selection and rapid scale‐down process optimization

PubMed Central

Velez‐Suberbie, M. Lourdes; Betts, John P. J.; Walker, Kelly L.; Robinson, Colin; Zoro, Barney

2017-01-01

High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed‐batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled‐up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale‐up. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:58–68, 2018 PMID:28748655

High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells

PubMed Central

Gerby, Bastien; Veiga, Diogo F.T.; Krosl, Jana; Nourreddine, Sami; Ouellette, Julianne; Haman, André; Lavoie, Geneviève; Fares, Iman; Tremblay, Mathieu; Litalien, Véronique; Ottoni, Elizabeth; Geoffrion, Dominique; Maddox, Paul S.; Chagraoui, Jalila; Hébert, Josée; Sauvageau, Guy; Kwok, Benjamin H.; Roux, Philippe P.

2016-01-01

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non–cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy. PMID:27797342

High-throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass.

PubMed

Chundawat, Shishir P S; Balan, Venkatesh; Dale, Bruce E

2008-04-15

Several factors will influence the viability of a biochemical platform for manufacturing lignocellulosic based fuels and chemicals, for example, genetically engineering energy crops, reducing pre-treatment severity, and minimizing enzyme loading. Past research on biomass conversion has focused largely on acid based pre-treatment technologies that fractionate lignin and hemicellulose from cellulose. However, for alkaline based (e.g., AFEX) and other lower severity pre-treatments it becomes critical to co-hydrolyze cellulose and hemicellulose using an optimized enzyme cocktail. Lignocellulosics are appropriate substrates to assess hydrolytic activity of enzyme mixtures compared to conventional unrealistic substrates (e.g., filter paper, chromogenic, and fluorigenic compounds) for studying synergistic hydrolysis. However, there are few, if any, high-throughput lignocellulosic digestibility analytical platforms for optimizing biomass conversion. The 96-well Biomass Conversion Research Lab (BCRL) microplate method is a high-throughput assay to study digestibility of lignocellulosic biomass as a function of biomass composition, pre-treatment severity, and enzyme composition. The most suitable method for delivering milled biomass to the microplate was through multi-pipetting slurry suspensions. A rapid bio-enzymatic, spectrophotometric assay was used to determine fermentable sugars. The entire procedure was automated using a robotic pipetting workstation. Several parameters that affect hydrolysis in the microplate were studied and optimized (i.e., particle size reduction, slurry solids concentration, glucan loading, mass transfer issues, and time period for hydrolysis). The microplate method was optimized for crystalline cellulose (Avicel) and ammonia fiber expansion (AFEX) pre-treated corn stover. Copyright 2008 Wiley Periodicals, Inc.

Metabolomics Approach for Toxicity Screening of Volatile Substances

EPA Science Inventory

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However, the ch...

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

EPA Science Inventory

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

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...

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...

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

NASA Astrophysics Data System (ADS)

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

2017-09-01

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

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy.

PubMed

Allan, Kristina J; Mahoney, Douglas J; Baird, Stephen D; Lefebvre, Charles A; Stojdl, David F

2018-04-03

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots.

PubMed

Liu, Guangbo; Lanham, Clayton; Buchan, J Ross; Kaplan, Matthew E

2017-01-01

Saccharomyces cerevisiae (budding yeast) is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc) transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids) or genome mutation (e.g., gene mutation, deletion, epitope tagging) is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.

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

EPA Pesticide Factsheets

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

Fast mass spectrometry-based enantiomeric excess determination of proteinogenic amino acids.

PubMed

Fleischer, Heidi; Thurow, Kerstin

2013-03-01

A rapid determination of the enantiomeric excess of proteinogenic amino acids is of great importance in various fields of chemical and biologic research and industries. Owing to their different biologic effects, enantiomers are interesting research subjects in drug development for the design of new and more efficient pharmaceuticals. Usually, the enantiomeric composition of amino acids is determined by conventional analytical methods such as liquid or gas chromatography or capillary electrophoresis. These analytical techniques do not fulfill the requirements of high-throughput screening due to their relative long analysis times. The method presented allows a fast analysis of chiral amino acids without previous time consuming chromatographic separation. The analytical measurements base on parallel kinetic resolution with pseudoenantiomeric mass tagged auxiliaries and were carried out by mass spectrometry with electrospray ionization. All 19 chiral proteinogenic amino acids were tested and Pro, Ser, Trp, His, and Glu were selected as model substrates for verification measurements. The enantiomeric excesses of amino acids with non-polar and aliphatic side chains as well as Trp and Phe (aromatic side chains) were determined with maximum deviations of the expected value less than or equal to 10ee%. Ser, Cys, His, Glu, and Asp were determined with deviations lower or equal to 14ee% and the enantiomeric excess of Tyr were calculated with 17ee% deviation. The total screening process is fully automated from the sample pretreatment to the data processing. The method presented enables fast measurement times about 1.38 min per sample and is applicable in the scope of high-throughput screenings.

Validation of high throughput screening of human sera for detection of anti-PA IgG by Enzyme-Linked Immunosorbent Assay (ELISA) as an emergency response to an anthrax incident

PubMed Central

Semenova, Vera A.; Steward-Clark, Evelene; Maniatis, Panagiotis; Epperson, Monica; Sabnis, Amit; Schiffer, Jarad

2017-01-01

To improve surge testing capability for a response to a release of Bacillus anthracis, the CDC anti-Protective Antigen (PA) IgG Enzyme-Linked Immunosorbent Assay (ELISA) was re-designed into a high throughput screening format. The following assay performance parameters were evaluated: goodness of fit (measured as the mean reference standard r2), accuracy (measured as percent error), precision (measured as coefficient of variance (CV)), lower limit of detection (LLOD), lower limit of quantification (LLOQ), dilutional linearity, diagnostic sensitivity (DSN) and diagnostic specificity (DSP). The paired sets of data for each sample were evaluated by Concordance Correlation Coefficient (CCC) analysis. The goodness of fit was 0.999; percent error between the expected and observed concentration for each sample ranged from −4.6% to 14.4%. The coefficient of variance ranged from 9.0% to 21.2%. The assay LLOQ was 2.6 μg/mL. The regression analysis results for dilutional linearity data were r2 = 0.952, slope = 1.02 and intercept = −0.03. CCC between assays was 0.974 for the median concentration of serum samples. The accuracy and precision components of CCC were 0.997 and 0.977, respectively. This high throughput screening assay is precise, accurate, sensitive and specific. Anti-PA IgG concentrations determined using two different assays proved high levels of agreement. The method will improve surge testing capability 18-fold from 4 to 72 sera per assay plate. PMID:27814939

Validation of high throughput screening of human sera for detection of anti-PA IgG by Enzyme-Linked Immunosorbent Assay (ELISA) as an emergency response to an anthrax incident.

PubMed

Semenova, Vera A; Steward-Clark, Evelene; Maniatis, Panagiotis; Epperson, Monica; Sabnis, Amit; Schiffer, Jarad

2017-01-01

To improve surge testing capability for a response to a release of Bacillus anthracis, the CDC anti-Protective Antigen (PA) IgG Enzyme-Linked Immunosorbent Assay (ELISA) was re-designed into a high throughput screening format. The following assay performance parameters were evaluated: goodness of fit (measured as the mean reference standard r 2 ), accuracy (measured as percent error), precision (measured as coefficient of variance (CV)), lower limit of detection (LLOD), lower limit of quantification (LLOQ), dilutional linearity, diagnostic sensitivity (DSN) and diagnostic specificity (DSP). The paired sets of data for each sample were evaluated by Concordance Correlation Coefficient (CCC) analysis. The goodness of fit was 0.999; percent error between the expected and observed concentration for each sample ranged from -4.6% to 14.4%. The coefficient of variance ranged from 9.0% to 21.2%. The assay LLOQ was 2.6 μg/mL. The regression analysis results for dilutional linearity data were r 2  = 0.952, slope = 1.02 and intercept = -0.03. CCC between assays was 0.974 for the median concentration of serum samples. The accuracy and precision components of CCC were 0.997 and 0.977, respectively. This high throughput screening assay is precise, accurate, sensitive and specific. Anti-PA IgG concentrations determined using two different assays proved high levels of agreement. The method will improve surge testing capability 18-fold from 4 to 72 sera per assay plate. Published by Elsevier Ltd.

Transfection of Sclerotinia sclerotiorum with in vitro transcripts of a naturally occurring interspecific recombinant of Sclerotinia sclerotiorum hypovirus 2 significantly reduces virulence of the fungus

USDA-ARS?s Scientific Manuscript database

A recombinant strain of Sclerotinia sclerotiorum hypovirus 2 (SsHV2) was identified from a North American Sclerotinia sclerotiorum isolate (#328) from lettuce (Lactuca sativa L.) by high-throughput sequencing of total RNA. The 5’ and 3’ terminal regions of the genome were determined by rapid amplifi...

High-throughput T-cell receptor sequencing across chronic liver diseases reveals distinct disease-associated repertoires.

PubMed

Liaskou, Evaggelia; Klemsdal Henriksen, Eva Kristine; Holm, Kristian; Kaveh, Fatemeh; Hamm, David; Fear, Janine; Viken, Marte K; Hov, Johannes Roksund; Melum, Espen; Robins, Harlan; Olweus, Johanna; Karlsen, Tom H; Hirschfield, Gideon M

2016-05-01

Hepatic T-cell infiltrates and a strong genetic human leukocyte antigen association represent characteristic features of various immune-mediated liver diseases. Conceptually the presence of disease-associated antigens is predicted to be reflected in T-cell receptor (TCR) repertoires. Here, we aimed to determine if disease-associated TCRs could be identified in the nonviral chronic liver diseases primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and alcoholic liver disease (ALD). We performed high-throughput sequencing of the TCRβ chain complementarity-determining region 3 of liver-infiltrating T cells from PSC (n = 20), PBC (n = 10), and ALD (n = 10) patients, alongside genomic human leukocyte antigen typing. The frequency of TCRβ nucleotide sequences was significantly higher in PSC samples (2.53 ± 0.80, mean ± standard error of the mean) compared to PBC samples (1.13 ± 0.17, P < 0.0001) and ALD samples (0.62 ± 0.10, P < 0.0001). An average clonotype overlap of 0.85% was detected among PSC samples, significantly higher compared to the average overlap of 0.77% seen within the PBC (P = 0.024) and ALD groups (0.40%, P < 0.0001). From eight to 42 clonotypes were uniquely detected in each of the three disease groups (≥30% of the respective patient samples). Multiple, unique sequences using different variable family genes encoded the same amino acid clonotypes, providing additional support for antigen-driven selection. In PSC and PBC, disease-associated clonotypes were detected among patients with human leukocyte antigen susceptibility alleles. We demonstrate liver-infiltrating disease-associated clonotypes in all three diseases evaluated, and evidence for antigen-driven clonal expansions. Our findings indicate that differential TCR signatures, as determined by high-throughput sequencing, may represent an imprint of distinctive antigenic repertoires present in the different chronic liver diseases; this thereby opens up the prospect of studying disease-relevant T cells in order to better understand and treat liver disease. © 2015 by the American Association for the Study of Liver Diseases.

Hazardous waste incinerators under waste uncertainty: balancing and throughput maximization via heat recuperation.

PubMed

Tsiliyannis, Christos Aristeides

2013-09-01

Hazardous waste incinerators (HWIs) differ substantially from thermal power facilities, since instead of maximizing energy production with the minimum amount of fuel, they aim at maximizing throughput. Variations in quantity or composition of received waste loads may significantly diminish HWI throughput (the decisive profit factor), from its nominal design value. A novel formulation of combustion balance is presented, based on linear operators, which isolates the wastefeed vector from the invariant combustion stoichiometry kernel. Explicit expressions for the throughput are obtained, in terms of incinerator temperature, fluegas heat recuperation ratio and design parameters, for an arbitrary number of wastes, based on fundamental principles (mass and enthalpy balances). The impact of waste variations, of recuperation ratio and of furnace temperature is explicitly determined. It is shown that in the presence of waste uncertainty, the throughput may be a decreasing or increasing function of incinerator temperature and recuperation ratio, depending on the sign of a dimensionless parameter related only to the uncertain wastes. The dimensionless parameter is proposed as a sharp a' priori waste 'fingerprint', determining the necessary increase or decrease of manipulated variables (recuperation ratio, excess air, auxiliary fuel feed rate, auxiliary air flow) in order to balance the HWI and maximize throughput under uncertainty in received wastes. A 10-step procedure is proposed for direct application subject to process capacity constraints. The results may be useful for efficient HWI operation and for preparing hazardous waste blends. Copyright © 2013 Elsevier Ltd. All rights reserved.

Lead discovery for mammalian elongation of long chain fatty acids family 6 using a combination of high-throughput fluorescent-based assay and RapidFire mass spectrometry assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takamiya, Mari; Discovery Technology Laboratories, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Kawagishi, Toda-shi, Saitama; Sakurai, Masaaki

A high-throughput RapidFire mass spectrometry assay is described for elongation of very long-chain fatty acids family 6 (Elovl6). Elovl6 is a microsomal enzyme that regulates the elongation of C12-16 saturated and monounsaturated fatty acids. Elovl6 may be a new therapeutic target for fat metabolism disorders such as obesity, type 2 diabetes, and nonalcoholic steatohepatitis. To identify new Elovl6 inhibitors, we developed a high-throughput fluorescence screening assay in 1536-well format. However, a number of false positives caused by fluorescent interference have been identified. To pick up the real active compounds among the primary hits from the fluorescence assay, we developed amore » RapidFire mass spectrometry assay and a conventional radioisotope assay. These assays have the advantage of detecting the main products directly without using fluorescent-labeled substrates. As a result, 276 compounds (30%) of the primary hits (921 compounds) in a fluorescence ultra-high-throughput screening method were identified as common active compounds in these two assays. It is concluded that both methods are very effective to eliminate false positives. Compared with the radioisotope method using an expensive {sup 14}C-labeled substrate, the RapidFire mass spectrometry method using unlabeled substrates is a high-accuracy, high-throughput method. In addition, some of the hit compounds selected from the screening inhibited cellular fatty acid elongation in HEK293 cells expressing Elovl6 transiently. This result suggests that these compounds may be promising lead candidates for therapeutic drugs. Ultra-high-throughput fluorescence screening followed by a RapidFire mass spectrometry assay was a suitable strategy for lead discovery against Elovl6. - Highlights: • A novel assay for elongation of very-long-chain fatty acids 6 (Elovl6) is proposed. • RapidFire mass spectrometry (RF-MS) assay is useful to select real screening hits. • RF-MS assay is proved to be beneficial because of its high-throughput and accuracy. • A combination of fluorescent and RF-MS assays is effective for Elovl6 inhibitors.« less

Using space and time to encode vibrotactile information: toward an estimate of the skin's achievable throughput.

PubMed

Novich, Scott D; Eagleman, David M

2015-10-01

Touch receptors in the skin can relay various forms of abstract information, such as words (Braille), haptic feedback (cell phones, game controllers, feedback for prosthetic control), and basic visual information such as edges and shape (sensory substitution devices). The skin can support such applications with ease: They are all low bandwidth and do not require a fine temporal acuity. But what of high-throughput applications? We use sound-to-touch conversion as a motivating example, though others abound (e.g., vision, stock market data). In the past, vibrotactile hearing aids have demonstrated improvement in speech perceptions in the deaf. However, a sound-to-touch sensory substitution device that works with high efficacy and without the aid of lipreading has yet to be developed. Is this because skin simply does not have the capacity to effectively relay high-throughput streams such as sound? Or is this because the spatial and temporal properties of skin have not been leveraged to full advantage? Here, we begin to address these questions with two experiments. First, we seek to determine the best method of relaying information through the skin using an identification task on the lower back. We find that vibrotactile patterns encoding information in both space and time yield the best overall information transfer estimate. Patterns encoded in space and time or "intensity" (the coupled coding of vibration frequency and force) both far exceed performance of only spatially encoded patterns. Next, we determine the vibrotactile two-tacton resolution on the lower back-the distance necessary for resolving two vibrotactile patterns. We find that our vibratory motors conservatively require at least 6 cm of separation to resolve two independent tactile patterns (>80 % correct), regardless of stimulus type (e.g., spatiotemporal "sweeps" versus single vibratory pulses). Six centimeter is a greater distance than the inter-motor distances used in Experiment 1 (2.5 cm), which explains the poor identification performance of spatially encoded patterns. Hence, when using an array of vibrational motors, spatiotemporal sweeps can overcome the limitations of vibrotactile two-tacton resolution. The results provide the first steps toward obtaining a realistic estimate of the skin's achievable throughput, illustrating the best ways to encode data to the skin (using as many dimensions as possible) and how far such interfaces would need to be separated if using multiple arrays in parallel.

High-throughput linear optical stretcher for mechanical characterization of blood cells.

PubMed

Roth, Kevin B; Neeves, Keith B; Squier, Jeff; Marr, David W M

2016-04-01

This study describes a linear optical stretcher as a high-throughput mechanical property cytometer. Custom, inexpensive, and scalable optics image a linear diode bar source into a microfluidic channel, where cells are hydrodynamically focused into the optical stretcher. Upon entering the stretching region, antipodal optical forces generated by the refraction of tightly focused laser light at the cell membrane deform each cell in flow. Each cell relaxes as it flows out of the trap and is compared to the stretched state to determine deformation. The deformation response of untreated red blood cells and neutrophils were compared to chemically treated cells. Statistically significant differences were observed between normal, diamide-treated, and glutaraldehyde-treated red blood cells, as well as between normal and cytochalasin D-treated neutrophils. Based on the behavior of the pure, untreated populations of red cells and neutrophils, a mixed population of these cells was tested and the discrete populations were identified by deformability. © 2015 International Society for Advancement of Cytometry. © 2015 International Society for Advancement of Cytometry.

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

PubMed Central

Harrison, Andrew; Binder, Hans; Buhot, Arnaud; Burden, Conrad J.; Carlon, Enrico; Gibas, Cynthia; Gamble, Lara J.; Halperin, Avraham; Hooyberghs, Jef; Kreil, David P.; Levicky, Rastislav; Noble, Peter A.; Ott, Albrecht; Pettitt, B. Montgomery; Tautz, Diethard; Pozhitkov, Alexander E.

2013-01-01

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized. PMID:23307556

Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology.

PubMed

Duarte, José M; Barbier, Içvara; Schaerli, Yolanda

2017-11-17

Synthetic biologists increasingly rely on directed evolution to optimize engineered biological systems. Applying an appropriate screening or selection method for identifying the potentially rare library members with the desired properties is a crucial step for success in these experiments. Special challenges include substantial cell-to-cell variability and the requirement to check multiple states (e.g., being ON or OFF depending on the input). Here, we present a high-throughput screening method that addresses these challenges. First, we encapsulate single bacteria into microfluidic agarose gel beads. After incubation, they harbor monoclonal bacterial microcolonies (e.g., expressing a synthetic construct) and can be sorted according their fluorescence by fluorescence activated cell sorting (FACS). We determine enrichment rates and demonstrate that we can measure the average fluorescent signals of microcolonies containing phenotypically heterogeneous cells, obviating the problem of cell-to-cell variability. Finally, we apply this method to sort a pBAD promoter library at ON and OFF states.

APG: an Active Protein-Gene network model to quantify regulatory signals in complex biological systems.

PubMed

Wang, Jiguang; Sun, Yidan; Zheng, Si; Zhang, Xiang-Sun; Zhou, Huarong; Chen, Luonan

2013-01-01

Synergistic interactions among transcription factors (TFs) and their cofactors collectively determine gene expression in complex biological systems. In this work, we develop a novel graphical model, called Active Protein-Gene (APG) network model, to quantify regulatory signals of transcription in complex biomolecular networks through integrating both TF upstream-regulation and downstream-regulation high-throughput data. Firstly, we theoretically and computationally demonstrate the effectiveness of APG by comparing with the traditional strategy based only on TF downstream-regulation information. We then apply this model to study spontaneous type 2 diabetic Goto-Kakizaki (GK) and Wistar control rats. Our biological experiments validate the theoretical results. In particular, SP1 is found to be a hidden TF with changed regulatory activity, and the loss of SP1 activity contributes to the increased glucose production during diabetes development. APG model provides theoretical basis to quantitatively elucidate transcriptional regulation by modelling TF combinatorial interactions and exploiting multilevel high-throughput information.

APG: an Active Protein-Gene Network Model to Quantify Regulatory Signals in Complex Biological Systems

PubMed Central

Wang, Jiguang; Sun, Yidan; Zheng, Si; Zhang, Xiang-Sun; Zhou, Huarong; Chen, Luonan

2013-01-01

Synergistic interactions among transcription factors (TFs) and their cofactors collectively determine gene expression in complex biological systems. In this work, we develop a novel graphical model, called Active Protein-Gene (APG) network model, to quantify regulatory signals of transcription in complex biomolecular networks through integrating both TF upstream-regulation and downstream-regulation high-throughput data. Firstly, we theoretically and computationally demonstrate the effectiveness of APG by comparing with the traditional strategy based only on TF downstream-regulation information. We then apply this model to study spontaneous type 2 diabetic Goto-Kakizaki (GK) and Wistar control rats. Our biological experiments validate the theoretical results. In particular, SP1 is found to be a hidden TF with changed regulatory activity, and the loss of SP1 activity contributes to the increased glucose production during diabetes development. APG model provides theoretical basis to quantitatively elucidate transcriptional regulation by modelling TF combinatorial interactions and exploiting multilevel high-throughput information. PMID:23346354

High throughput exploration of process-property linkages in Al-6061 using instrumented spherical microindentation and microstructurally graded samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weaver, Jordan S.; Khosravani, Ali; Castillo, Andrew

Recent spherical nanoindentation protocols have proven robust at capturing the local elastic-plastic response of polycrystalline metal samples at length scales much smaller than the grain size. In this work, we extend these protocols to length scales that include multiple grains to recover microindentation stress-strain curves. These new protocols are first established in this paper and then demonstrated for Al-6061 by comparing the measured indentation stress-strain curves with the corresponding measurements from uniaxial tension tests. More specifically, the scaling factors between the uniaxial yield strength and the indentation yield strength was determined to be about 1.9, which is significantly lower thanmore » the value of 2.8 used commonly in literature. Furthermore, the reasons for this difference are discussed. Second, the benefits of these new protocols in facilitating high throughput exploration of process-property relationships are demonstrated through a simple case study.« less

A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity.

PubMed

O'Brien-Simpson, Neil M; Pantarat, Namfon; Attard, Troy J; Walsh, Katrina A; Reynolds, Eric C

2016-01-01

We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy.

The ESFRI Instruct Core Centre Frankfurt: automated high-throughput crystallization suited for membrane proteins and more.

PubMed

Thielmann, Yvonne; Koepke, Juergen; Michel, Hartmut

2012-06-01

Structure determination of membrane proteins and membrane protein complexes is still a very challenging field. To facilitate the work on membrane proteins the Core Centre follows a strategy that comprises four labs of protein analytics and crystal handling, covering mass spectrometry, calorimetry, crystallization and X-ray diffraction. This general workflow is presented and a capacity of 20% of the operating time of all systems is provided to the European structural biology community within the ESFRI Instruct program. A description of the crystallization service offered at the Core Centre is given with detailed information on screening strategy, screens used and changes to adapt high throughput for membrane proteins. Our aim is to constantly develop the Core Centre towards the usage of more efficient methods. This strategy might also include the ability to automate all steps from crystallization trials to crystal screening; here we look ahead how this aim might be realized at the Core Centre.

ImmuneDB: a system for the analysis and exploration of high-throughput adaptive immune receptor sequencing data.

PubMed

Rosenfeld, Aaron M; Meng, Wenzhao; Luning Prak, Eline T; Hershberg, Uri

2017-01-15

As high-throughput sequencing of B cells becomes more common, the need for tools to analyze the large quantity of data also increases. This article introduces ImmuneDB, a system for analyzing vast amounts of heavy chain variable region sequences and exploring the resulting data. It can take as input raw FASTA/FASTQ data, identify genes, determine clones, construct lineages, as well as provide information such as selection pressure and mutation analysis. It uses an industry leading database, MySQL, to provide fast analysis and avoid the complexities of using error prone flat-files. ImmuneDB is freely available at http://immunedb.comA demo of the ImmuneDB web interface is available at: http://immunedb.com/demo CONTACT: Uh25@drexel.eduSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

High throughput exploration of process-property linkages in Al-6061 using instrumented spherical microindentation and microstructurally graded samples

DOE PAGES

Weaver, Jordan S.; Khosravani, Ali; Castillo, Andrew; ...

2016-06-14

Recent spherical nanoindentation protocols have proven robust at capturing the local elastic-plastic response of polycrystalline metal samples at length scales much smaller than the grain size. In this work, we extend these protocols to length scales that include multiple grains to recover microindentation stress-strain curves. These new protocols are first established in this paper and then demonstrated for Al-6061 by comparing the measured indentation stress-strain curves with the corresponding measurements from uniaxial tension tests. More specifically, the scaling factors between the uniaxial yield strength and the indentation yield strength was determined to be about 1.9, which is significantly lower thanmore » the value of 2.8 used commonly in literature. Furthermore, the reasons for this difference are discussed. Second, the benefits of these new protocols in facilitating high throughput exploration of process-property relationships are demonstrated through a simple case study.« less

A High-Throughput Arabidopsis Reverse Genetics System

PubMed Central

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

2002-01-01

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from ∼100,000 transformed lines. A total of 85,108 TAIL-PCR products from 52,964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org. PMID:12468722

Identification of microRNAs and their targets in Finger millet by high throughput sequencing.

PubMed

Usha, S; Jyothi, M N; Sharadamma, N; Dixit, Rekha; Devaraj, V R; Nagesh Babu, R

2015-12-15

MicroRNAs are short non-coding RNAs which play an important role in regulating gene expression by mRNA cleavage or by translational repression. The majority of identified miRNAs were evolutionarily conserved; however, others expressed in a species-specific manner. Finger millet is an important cereal crop; nonetheless, no practical information is available on microRNAs to date. In this study, we have identified 95 conserved microRNAs belonging to 39 families and 3 novel microRNAs by high throughput sequencing. For the identified conserved and novel miRNAs a total of 507 targets were predicted. 11 miRNAs were validated and tissue specificity was determined by stem loop RT-qPCR, Northern blot. GO analyses revealed targets of miRNA were involved in wide range of regulatory functions. This study implies large number of known and novel miRNAs found in Finger millet which may play important role in growth and development. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantifying domain-ligand affinities and specificities by high-throughput holdup assay

PubMed Central

Vincentelli, Renaud; Luck, Katja; Poirson, Juline; Polanowska, Jolanta; Abdat, Julie; Blémont, Marilyne; Turchetto, Jeremy; Iv, François; Ricquier, Kevin; Straub, Marie-Laure; Forster, Anne; Cassonnet, Patricia; Borg, Jean-Paul; Jacob, Yves; Masson, Murielle; Nominé, Yves; Reboul, Jérôme; Wolff, Nicolas; Charbonnier, Sebastian; Travé, Gilles

2015-01-01

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this aim, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to a thousand domain-motif equilibrium binding affinities per day. Extracts of overexpressed domains are incubated with peptide-coated resins and subjected to filtration. Binding affinities are deduced from microfluidic capillary electrophoresis of flow-throughs. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from Human Papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human PDZome. We obtained exquisite sequence-dependent binding profiles, describing quantitatively the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has a wide potential for quantifying the specificities of interactomes. PMID:26053890

Combinatorial Strategies for the Development of Bulk Metallic Glasses

NASA Astrophysics Data System (ADS)

Ding, Shiyan

The systematic identification of multi-component alloys out of the vast composition space is still a daunting task, especially in the development of bulk metallic glasses that are typically based on three or more elements. In order to address this challenge, combinatorial approaches have been proposed. However, previous attempts have not successfully coupled the synthesis of combinatorial libraries with high-throughput characterization methods. The goal of my dissertation is to develop efficient high-throughput characterization methods, optimized to identify glass formers systematically. Here, two innovative approaches have been invented. One is to measure the nucleation temperature in parallel for up-to 800 compositions. The composition with the lowest nucleation temperature has a reasonable agreement with the best-known glass forming composition. In addition, the thermoplastic formability of a metallic glass forming system is determined through blow molding a compositional library. Our results reveal that the composition with the largest thermoplastic deformation correlates well with the best-known formability composition. I have demonstrated both methods as powerful tools to develop new bulk metallic glasses.

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

A real-time high-throughput fluorescence assay for sphingosine kinases

PubMed Central

Lima, Santiago; Milstien, Sheldon; Spiegel, Sarah

2014-01-01

Sphingosine kinases (SphKs), of which there are two isoforms, SphK1 and SphK2, have been implicated in regulation of many important cellular processes. We have developed an assay for monitoring SphK1 and SphK2 activity in real time without the need for organic partitioning of products, radioactive materials, or specialized equipment. The assay conveniently follows SphK-dependent changes in 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled sphingosine (Sph) fluorescence and can be easily performed in 384-well plate format with small reaction volumes. We present data showing dose-proportional responses to enzyme, substrate, and inhibitor concentrations. The SphK1 and SphK2 binding affinities for NBD-Sph and the IC50 values of inhibitors determined were consistent with those reported with other methods. Because of the versatility and simplicity of the assay, it should facilitate the routine characterization of inhibitors and SphK mutants and can be readily used for compound library screening in high-throughput format. PMID:24792926

QoS support for end users of I/O-intensive applications using shared storage systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davis, Marion Kei; Zhang, Xuechen; Jiang, Song

2011-01-19

I/O-intensive applications are becoming increasingly common on today's high-performance computing systems. While performance of compute-bound applications can be effectively guaranteed with techniques such as space sharing or QoS-aware process scheduling, it remains a challenge to meet QoS requirements for end users of I/O-intensive applications using shared storage systems because it is difficult to differentiate I/O services for different applications with individual quality requirements. Furthermore, it is difficult for end users to accurately specify performance goals to the storage system using I/O-related metrics such as request latency or throughput. As access patterns, request rates, and the system workload change in time,more » a fixed I/O performance goal, such as bounds on throughput or latency, can be expensive to achieve and may not lead to a meaningful performance guarantees such as bounded program execution time. We propose a scheme supporting end-users QoS goals, specified in terms of program execution time, in shared storage environments. We automatically translate the users performance goals into instantaneous I/O throughput bounds using a machine learning technique, and use dynamically determined service time windows to efficiently meet the throughput bounds. We have implemented this scheme in the PVFS2 parallel file system and have conducted an extensive evaluation. Our results show that this scheme can satisfy realistic end-user QoS requirements by making highly efficient use of the I/O resources. The scheme seeks to balance programs attainment of QoS requirements, and saves as much of the remaining I/O capacity as possible for best-effort programs.« less

High-throughput Screening of ToxCast" Phase I Chemicals in an Embryonic Stem Cell Assay Reveals Potential Disruption of a Critical Developmental Signaling Pathway

EPA Science Inventory

Little is known about the developmental toxicity of the expansive chemical landscape in existence today. Significant efforts are being made to apply novel methods to predict developmental activity of chemicals utilizing high-throughput screening (HTS) and high-content screening (...

Comparison of Human Induced Pluripotent Stem Cell-Derived Neurons and Rat Primary CorticalNeurons as In Vitro Models of Neurite Outgrowth

EPA Science Inventory

High-throughput assays that can quantify chemical-induced changes at the cellular and molecular level have been recommended for use in chemical safety assessment. High-throughput, high content imaging assays for the key cellular events of neurodevelopment have been proposed to ra...

Evaluation of sequencing approaches for high-throughput toxicogenomics (SOT)

EPA Science Inventory

Whole-genome in vitro transcriptomics has shown the capability to identify mechanisms of action and estimates of potency for chemical-mediated effects in a toxicological framework, but with limited throughput and high cost. We present the evaluation of three toxicogenomics platfo...

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...

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...

Atlanta I-85 HOV-to-HOT conversion : analysis of vehicle and person throughput.

DOT National Transportation Integrated Search

2013-10-01

This report summarizes the vehicle and person throughput analysis for the High Occupancy Vehicle to High Occupancy Toll Lane : conversion in Atlanta, GA, undertaken by the Georgia Institute of Technology research team. The team tracked changes in : o...

EMBRYONIC VASCULAR DISRUPTION ADVERSE OUTCOMES: LINKING HIGH THROUGHPUT SIGNALING SIGNATURES WITH FUNCTIONAL CONSEQUENCES

EPA Science Inventory

Embryonic vascular disruption is an important adverse outcome pathway (AOP) given the knowledge that chemical disruption of early cardiovascular system development leads to broad prenatal defects. High throughput screening (HTS) assays provide potential building blocks for AOP d...

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 ...

Advances in High-Throughput Speed, Low-Latency Communication for Embedded Instrumentation (7th Annual SFAF Meeting, 2012)

ScienceCinema

Jordan, Scott

2018-01-24

Scott Jordan on "Advances in high-throughput speed, low-latency communication for embedded instrumentation" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.

Inter-Individual Variability in High-Throughput Risk Prioritization of Environmental Chemicals (Sot)

EPA Science Inventory

We incorporate realistic human variability into an open-source high-throughput (HT) toxicokinetics (TK) modeling framework for use in a next-generation risk prioritization approach. Risk prioritization involves rapid triage of thousands of environmental chemicals, most which have...

Inter-individual variability in high-throughput risk prioritization of environmental chemicals (IVIVE)

EPA Science Inventory

We incorporate inter-individual variability into an open-source high-throughput (HT) toxicokinetics (TK) modeling framework for use in a next-generation risk prioritization approach. Risk prioritization involves rapid triage of thousands of environmental chemicals, most which hav...

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...

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...

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...

Evaluating and Refining High Throughput Tools for Toxicokinetics

EPA Science Inventory

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

Picking Cell Lines for High-Throughput Transcriptomic Toxicity Screening (SOT)

EPA Science Inventory

High throughput, whole genome transcriptomic profiling is a promising approach to comprehensively evaluate chemicals for potential biological effects. To be useful for in vitro toxicity screening, gene expression must be quantified in a set of representative cell types that captu...

20180312 - Uncertainty and Variability in High-Throughput Toxicokinetics for Risk Prioritization (SOT)

EPA Science Inventory

Streamlined approaches that use in vitro experimental data to predict chemical toxicokinetics (TK) are increasingly being used to perform risk-based prioritization based upon dosimetric adjustment of high-throughput screening (HTS) data across thousands of chemicals. However, ass...

Microfluidic Flow Injection Analysis with Thermal Lens Microscopic Detection for Determination of NGAL

NASA Astrophysics Data System (ADS)

Radovanović, Tatjana; Liu, Mingqiang; Likar, Polona; Klemenc, Matjaž; Franko, Mladen

2015-06-01

A combined microfluidic flow injection analysis-thermal lens microscopy (FIA-TLM) system was applied for determination of neutrophil gelatinase-associated lipocalin (NGAL)—a biomarker of acute kidney injury. NGAL was determined following a commercial ELISA assay and transfer of the resulting solution into the FIA-TLM system with a 100 m deep microchannel. At an excitation power of 100 mW, the FIA-TLM provided about seven times lower limits of detection (1.5 pg as compared to a conventional ELISA test, and a sample throughput of six samples per minute, which compares favorably with sample throughput of the microtiter plate reader, which reads 96 wells in about 30 min. Comparison of results for NGAL in plasma samples from healthy individuals and for NGAL dynamics in patients undergoing coronary angiography measured with transmission mode spectrometry on a microtiter plate reader and with FIA-TLM showed good agreement. In addition to improved LOD, the high sensitivity of FIA-TLM offers possibilities of a further reduction of the total reaction time of the NGAL ELISA test by sacrificing some of the sensitivity while reducing the duration of individual incubation steps.

High-Throughput Tabular Data Processor - Platform independent graphical tool for processing large data sets.

PubMed

Madanecki, Piotr; Bałut, Magdalena; Buckley, Patrick G; Ochocka, J Renata; Bartoszewski, Rafał; Crossman, David K; Messiaen, Ludwine M; Piotrowski, Arkadiusz

2018-01-01

High-throughput technologies generate considerable amount of data which often requires bioinformatic expertise to analyze. Here we present High-Throughput Tabular Data Processor (HTDP), a platform independent Java program. HTDP works on any character-delimited column data (e.g. BED, GFF, GTF, PSL, WIG, VCF) from multiple text files and supports merging, filtering and converting of data that is produced in the course of high-throughput experiments. HTDP can also utilize itemized sets of conditions from external files for complex or repetitive filtering/merging tasks. The program is intended to aid global, real-time processing of large data sets using a graphical user interface (GUI). Therefore, no prior expertise in programming, regular expression, or command line usage is required of the user. Additionally, no a priori assumptions are imposed on the internal file composition. We demonstrate the flexibility and potential of HTDP in real-life research tasks including microarray and massively parallel sequencing, i.e. identification of disease predisposing variants in the next generation sequencing data as well as comprehensive concurrent analysis of microarray and sequencing results. We also show the utility of HTDP in technical tasks including data merge, reduction and filtering with external criteria files. HTDP was developed to address functionality that is missing or rudimentary in other GUI software for processing character-delimited column data from high-throughput technologies. Flexibility, in terms of input file handling, provides long term potential functionality in high-throughput analysis pipelines, as the program is not limited by the currently existing applications and data formats. HTDP is available as the Open Source software (https://github.com/pmadanecki/htdp).

High-Throughput Tabular Data Processor – Platform independent graphical tool for processing large data sets

PubMed Central

Bałut, Magdalena; Buckley, Patrick G.; Ochocka, J. Renata; Bartoszewski, Rafał; Crossman, David K.; Messiaen, Ludwine M.; Piotrowski, Arkadiusz

2018-01-01

High-throughput technologies generate considerable amount of data which often requires bioinformatic expertise to analyze. Here we present High-Throughput Tabular Data Processor (HTDP), a platform independent Java program. HTDP works on any character-delimited column data (e.g. BED, GFF, GTF, PSL, WIG, VCF) from multiple text files and supports merging, filtering and converting of data that is produced in the course of high-throughput experiments. HTDP can also utilize itemized sets of conditions from external files for complex or repetitive filtering/merging tasks. The program is intended to aid global, real-time processing of large data sets using a graphical user interface (GUI). Therefore, no prior expertise in programming, regular expression, or command line usage is required of the user. Additionally, no a priori assumptions are imposed on the internal file composition. We demonstrate the flexibility and potential of HTDP in real-life research tasks including microarray and massively parallel sequencing, i.e. identification of disease predisposing variants in the next generation sequencing data as well as comprehensive concurrent analysis of microarray and sequencing results. We also show the utility of HTDP in technical tasks including data merge, reduction and filtering with external criteria files. HTDP was developed to address functionality that is missing or rudimentary in other GUI software for processing character-delimited column data from high-throughput technologies. Flexibility, in terms of input file handling, provides long term potential functionality in high-throughput analysis pipelines, as the program is not limited by the currently existing applications and data formats. HTDP is available as the Open Source software (https://github.com/pmadanecki/htdp). PMID:29432475

Validation of a spectrophotometer-based method for estimating daily sperm production and deferent duct transit.

PubMed

Froman, D P; Rhoads, D D

2012-10-01

The objectives of the present work were 3-fold. First, a new method for estimating daily sperm production was validated. This method, in turn, was used to evaluate testis output as well as deferent duct throughput. Next, this analytical approach was evaluated in 2 experiments. The first experiment compared left and right reproductive tracts within roosters. The second experiment compared reproductive tract throughput in roosters from low and high sperm mobility lines. Standard curves were constructed from which unknown concentrations of sperm cells and sperm nuclei could be predicted from observed absorbance. In each case, the independent variable was based upon hemacytometer counts, and absorbance was a linear function of concentration. Reproductive tracts were excised, semen recovered from each duct, and the extragonadal sperm reserve determined by multiplying volume by sperm cell concentration. Testicular sperm nuclei were procured by homogenization of a whole testis, overlaying a 20-mL volume of homogenate upon 15% (wt/vol) Accudenz (Accurate Chemical and Scientific Corporation, Westbury, NY), and then washing nuclei by centrifugation through the Accudenz layer. Daily sperm production was determined by dividing the predicted number of sperm nuclei within the homogenate by 4.5 d (i.e., the time sperm with elongated nuclei spend within the testis). Sperm transit through the deferent duct was estimated by dividing the extragonadal reserve by daily sperm production. Neither the efficiency of sperm production (sperm per gram of testicular parenchyma per day) nor deferent duct transit differed between left and right reproductive tracts (P > 0.05). Whereas efficiency of sperm production did not differ (P > 0.05) between low and high sperm mobility lines, deferent duct transit differed between lines (P < 0.001). On average, this process required 2.2 and 1.0 d for low and high lines, respectively. In summary, we developed and then tested a method for quantifying male reproductive tract throughput. This method makes the study of semen production amenable to systems biology.

A comparison of high-throughput techniques for assaying circadian rhythms in plants.

PubMed

Tindall, Andrew J; Waller, Jade; Greenwood, Mark; Gould, Peter D; Hartwell, James; Hall, Anthony

2015-01-01

Over the last two decades, the development of high-throughput techniques has enabled us to probe the plant circadian clock, a key coordinator of vital biological processes, in ways previously impossible. With the circadian clock increasingly implicated in key fitness and signalling pathways, this has opened up new avenues for understanding plant development and signalling. Our tool-kit has been constantly improving through continual development and novel techniques that increase throughput, reduce costs and allow higher resolution on the cellular and subcellular levels. With circadian assays becoming more accessible and relevant than ever to researchers, in this paper we offer a review of the techniques currently available before considering the horizons in circadian investigation at ever higher throughputs and resolutions.

Turning tumor-promoting copper into an anti-cancer weapon via high-throughput chemistry.

PubMed

Wang, F; Jiao, P; Qi, M; Frezza, M; Dou, Q P; Yan, B

2010-01-01

Copper is an essential element for multiple biological processes. Its concentration is elevated to a very high level in cancer tissues for promoting cancer development through processes such as angiogenesis. Organic chelators of copper can passively reduce cellular copper and serve the role as inhibitors of angiogenesis. However, they can also actively attack cellular targets such as proteasome, which plays a critical role in cancer development and survival. The discovery of such molecules initially relied on a step by step synthesis followed by biological assays. Today high-throughput chemistry and high-throughput screening have significantly expedited the copper-binding molecules discovery to turn "cancer-promoting" copper into anti-cancer agents.

HTP-OligoDesigner: An Online Primer Design Tool for High-Throughput Gene Cloning and Site-Directed Mutagenesis.

PubMed

Camilo, Cesar M; Lima, Gustavo M A; Maluf, Fernando V; Guido, Rafael V C; Polikarpov, Igor

2016-01-01

Following burgeoning genomic and transcriptomic sequencing data, biochemical and molecular biology groups worldwide are implementing high-throughput cloning and mutagenesis facilities in order to obtain a large number of soluble proteins for structural and functional characterization. Since manual primer design can be a time-consuming and error-generating step, particularly when working with hundreds of targets, the automation of primer design process becomes highly desirable. HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive online primer design tool for both laboratory-scale and high-throughput projects of sequence-independent gene cloning and site-directed mutagenesis and a Tm calculator for quick queries.

A high performance hardware implementation image encryption with AES algorithm

NASA Astrophysics Data System (ADS)

Farmani, Ali; Jafari, Mohamad; Miremadi, Seyed Sohrab

2011-06-01

This paper describes implementation of a high-speed encryption algorithm with high throughput for encrypting the image. Therefore, we select a highly secured symmetric key encryption algorithm AES(Advanced Encryption Standard), in order to increase the speed and throughput using pipeline technique in four stages, control unit based on logic gates, optimal design of multiplier blocks in mixcolumn phase and simultaneous production keys and rounds. Such procedure makes AES suitable for fast image encryption. Implementation of a 128-bit AES on FPGA of Altra company has been done and the results are as follow: throughput, 6 Gbps in 471MHz. The time of encrypting in tested image with 32*32 size is 1.15ms.

An optical method to determine the thermodynamics of hydrogen absorption and desorption in metals

NASA Astrophysics Data System (ADS)

Gremaud, R.; Slaman, M.; Schreuders, H.; Dam, B.; Griessen, R.

2007-12-01

Hydrogenography, an optical high-throughput combinatorial technique to find hydrogen storage materials, has so far been applied only to materials undergoing a metal-to-semiconductor transition during hydrogenation. We show here that this technique works equally well for metallic hydrides. Additionally, we find that the thermodynamic data obtained optically on thin Pd-H films agree very well with Pd-H bulk data. This confirms that hydrogenography is a valuable general method to determine the relevant parameters for hydrogen storage in metal hydrides.

State of the Art High-Throughput Approaches to Genotoxicity: Flow Micronucleus, Ames II, GreenScreen and Comet

EPA Pesticide Factsheets

State of the Art High-Throughput Approaches to Genotoxicity: Flow Micronucleus, Ames II, GreenScreen and Comet (Presented by Dr. Marilyn J. Aardema, Chief Scientific Advisor, Toxicology, Dr. Leon Stankowski, et. al. (6/28/2012)

Fun with High Throughput Toxicokinetics (CalEPA webinar)

EPA Science Inventory

Thousands of chemicals have been profiled by high-throughput screening (HTS) programs such as ToxCast and Tox21. These chemicals are tested in part because there are limited or no data on hazard, exposure, or toxicokinetics (TK). TK models aid in predicting tissue concentrations ...

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...

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...

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...

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...

Human variability in high-throughput risk prioritization of environmental chemicals (Texas AM U. webinar)

EPA Science Inventory

We incorporate inter-individual variability into an open-source high-throughput (HT) toxicokinetics (TK) modeling framework for use in a next-generation risk prioritization approach. Risk prioritization involves rapid triage of thousands of environmental chemicals, most which hav...

HTTK: R Package for High-Throughput Toxicokinetics

EPA Science Inventory

Thousands of chemicals have been profiled by high-throughput screening programs such as ToxCast and Tox21; these chemicals are tested in part because most of them have limited or no data on hazard, exposure, or toxicokinetics. Toxicokinetic models aid in predicting tissue concent...

tcpl: The ToxCast Pipeline for High-Throughput Screening Data

EPA Science Inventory

Motivation: The large and diverse high-throughput chemical screening efforts carried out by the US EPAToxCast program requires an efficient, transparent, and reproducible data pipeline.Summary: The tcpl R package and its associated MySQL database provide a generalized platform fo...

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,...

In Vitro Toxicity Screening Technique for Volatile Substances Using Flow-Through System#

EPA Science Inventory

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However the cha...

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...

A review of the theory, methods and recent applications of high-throughput single-cell droplet microfluidics

NASA Astrophysics Data System (ADS)

Lagus, Todd P.; Edd, Jon F.

2013-03-01

Most cell biology experiments are performed in bulk cell suspensions where cell secretions become diluted and mixed in a contiguous sample. Confinement of single cells to small, picoliter-sized droplets within a continuous phase of oil provides chemical isolation of each cell, creating individual microreactors where rare cell qualities are highlighted and otherwise undetectable signals can be concentrated to measurable levels. Recent work in microfluidics has yielded methods for the encapsulation of cells in aqueous droplets and hydrogels at kilohertz rates, creating the potential for millions of parallel single-cell experiments. However, commercial applications of high-throughput microdroplet generation and downstream sensing and actuation methods are still emerging for cells. Using fluorescence-activated cell sorting (FACS) as a benchmark for commercially available high-throughput screening, this focused review discusses the fluid physics of droplet formation, methods for cell encapsulation in liquids and hydrogels, sensors and actuators and notable biological applications of high-throughput single-cell droplet microfluidics.

High-Throughput Cloning and Expression Library Creation for Functional Proteomics

PubMed Central

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-01-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

High-Throughput Lectin Microarray-Based Analysis of Live Cell Surface Glycosylation

PubMed Central

Li, Yu; Tao, Sheng-ce; Zhu, Heng; Schneck, Jonathan P.

2011-01-01

Lectins, plant-derived glycan-binding proteins, have long been used to detect glycans on cell surfaces. However, the techniques used to characterize serum or cells have largely been limited to mass spectrometry, blots, flow cytometry, and immunohistochemistry. While these lectin-based approaches are well established and they can discriminate a limited number of sugar isomers by concurrently using a limited number of lectins, they are not amenable for adaptation to a high-throughput platform. Fortunately, given the commercial availability of lectins with a variety of glycan specificities, lectins can be printed on a glass substrate in a microarray format to profile accessible cell-surface glycans. This method is an inviting alternative for analysis of a broad range of glycans in a high-throughput fashion and has been demonstrated to be a feasible method of identifying binding-accessible cell surface glycosylation on living cells. The current unit presents a lectin-based microarray approach for analyzing cell surface glycosylation in a high-throughput fashion. PMID:21400689

Next-generation sequencing coupled with a cell-free display technology for high-throughput production of reliable interactome data

PubMed Central

Fujimori, Shigeo; Hirai, Naoya; Ohashi, Hiroyuki; Masuoka, Kazuyo; Nishikimi, Akihiko; Fukui, Yoshinori; Washio, Takanori; Oshikubo, Tomohiro; Yamashita, Tatsuhiro; Miyamoto-Sato, Etsuko

2012-01-01

Next-generation sequencing (NGS) has been applied to various kinds of omics studies, resulting in many biological and medical discoveries. However, high-throughput protein-protein interactome datasets derived from detection by sequencing are scarce, because protein-protein interaction analysis requires many cell manipulations to examine the interactions. The low reliability of the high-throughput data is also a problem. Here, we describe a cell-free display technology combined with NGS that can improve both the coverage and reliability of interactome datasets. The completely cell-free method gives a high-throughput and a large detection space, testing the interactions without using clones. The quantitative information provided by NGS reduces the number of false positives. The method is suitable for the in vitro detection of proteins that interact not only with the bait protein, but also with DNA, RNA and chemical compounds. Thus, it could become a universal approach for exploring the large space of protein sequences and interactome networks. PMID:23056904

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

PubMed

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

2009-01-01

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

Advanced Virus Detection Technologies Interest Group (AVDTIG): Efforts on High Throughput Sequencing (HTS) for Virus Detection.

PubMed

Khan, Arifa S; Vacante, Dominick A; Cassart, Jean-Pol; Ng, Siemon H S; Lambert, Christophe; Charlebois, Robert L; King, Kathryn E

Several nucleic-acid based technologies have recently emerged with capabilities for broad virus detection. One of these, high throughput sequencing, has the potential for novel virus detection because this method does not depend upon prior viral sequence knowledge. However, the use of high throughput sequencing for testing biologicals poses greater challenges as compared to other newly introduced tests due to its technical complexities and big data bioinformatics. Thus, the Advanced Virus Detection Technologies Users Group was formed as a joint effort by regulatory and industry scientists to facilitate discussions and provide a forum for sharing data and experiences using advanced new virus detection technologies, with a focus on high throughput sequencing technologies. The group was initiated as a task force that was coordinated by the Parenteral Drug Association and subsequently became the Advanced Virus Detection Technologies Interest Group to continue efforts for using new technologies for detection of adventitious viruses with broader participation, including international government agencies, academia, and technology service providers. © PDA, Inc. 2016.

The application of the high throughput sequencing technology in the transposable elements.

PubMed

Liu, Zhen; Xu, Jian-hong

2015-09-01

High throughput sequencing technology has dramatically improved the efficiency of DNA sequencing, and decreased the costs to a great extent. Meanwhile, this technology usually has advantages of better specificity, higher sensitivity and accuracy. Therefore, it has been applied to the research on genetic variations, transcriptomics and epigenomics. Recently, this technology has been widely employed in the studies of transposable elements and has achieved fruitful results. In this review, we summarize the application of high throughput sequencing technology in the fields of transposable elements, including the estimation of transposon content, preference of target sites and distribution, insertion polymorphism and population frequency, identification of rare copies, transposon horizontal transfers as well as transposon tagging. We also briefly introduce the major common sequencing strategies and algorithms, their advantages and disadvantages, and the corresponding solutions. Finally, we envision the developing trends of high throughput sequencing technology, especially the third generation sequencing technology, and its application in transposon studies in the future, hopefully providing a comprehensive understanding and reference for related scientific researchers.

Near-common-path interferometer for imaging Fourier-transform spectroscopy in wide-field microscopy

PubMed Central

Wadduwage, Dushan N.; Singh, Vijay Raj; Choi, Heejin; Yaqoob, Zahid; Heemskerk, Hans; Matsudaira, Paul; So, Peter T. C.

2017-01-01

Imaging Fourier-transform spectroscopy (IFTS) is a powerful method for biological hyperspectral analysis based on various imaging modalities, such as fluorescence or Raman. Since the measurements are taken in the Fourier space of the spectrum, it can also take advantage of compressed sensing strategies. IFTS has been readily implemented in high-throughput, high-content microscope systems based on wide-field imaging modalities. However, there are limitations in existing wide-field IFTS designs. Non-common-path approaches are less phase-stable. Alternatively, designs based on the common-path Sagnac interferometer are stable, but incompatible with high-throughput imaging. They require exhaustive sequential scanning over large interferometric path delays, making compressive strategic data acquisition impossible. In this paper, we present a novel phase-stable, near-common-path interferometer enabling high-throughput hyperspectral imaging based on strategic data acquisition. Our results suggest that this approach can improve throughput over those of many other wide-field spectral techniques by more than an order of magnitude without compromising phase stability. PMID:29392168

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

PubMed

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

2013-03-01

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

Graph-based signal integration for high-throughput phenotyping

PubMed Central

2012-01-01

Background Electronic Health Records aggregated in Clinical Data Warehouses (CDWs) promise to revolutionize Comparative Effectiveness Research and suggest new avenues of research. However, the effectiveness of CDWs is diminished by the lack of properly labeled data. We present a novel approach that integrates knowledge from the CDW, the biomedical literature, and the Unified Medical Language System (UMLS) to perform high-throughput phenotyping. In this paper, we automatically construct a graphical knowledge model and then use it to phenotype breast cancer patients. We compare the performance of this approach to using MetaMap when labeling records. Results MetaMap's overall accuracy at identifying breast cancer patients was 51.1% (n=428); recall=85.4%, precision=26.2%, and F1=40.1%. Our unsupervised graph-based high-throughput phenotyping had accuracy of 84.1%; recall=46.3%, precision=61.2%, and F1=52.8%. Conclusions We conclude that our approach is a promising alternative for unsupervised high-throughput phenotyping. PMID:23320851

High-throughput cloning and expression library creation for functional proteomics.

PubMed

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-05-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single-gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator(TM) DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR library

PubMed Central

Zhu, Shiyou; Li, Wei; Liu, Jingze; Chen, Chen-Hao; Liao, Qi; Xu, Ping; Xu, Han; Xiao, Tengfei; Cao, Zhongzheng; Peng, Jingyu; Yuan, Pengfei; Brown, Myles; Liu, Xiaole Shirley; Wei, Wensheng

2017-01-01

CRISPR/Cas9 screens have been widely adopted to analyse coding gene functions, but high throughput screening of non-coding elements using this method is more challenging, because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. Herein, we report a high throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We individually validated 9 lncRNAs using CRISPR/Cas9-mediated genomic deletion and functional rescue, CRISPR activation or inhibition, and gene expression profiling. Our high-throughput pgRNA genome deletion method should enable rapid identification of functional mammalian non-coding elements. PMID:27798563

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

PubMed Central

Choudhry, Priya

2016-01-01

Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays. PMID:26848849

High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics

NASA Astrophysics Data System (ADS)

Beneyton, Thomas; Wijaya, I. Putu Mahendra; Postros, Prexilia; Najah, Majdi; Leblond, Pascal; Couvent, Angélique; Mayot, Estelle; Griffiths, Andrew D.; Drevelle, Antoine

2016-06-01

Filamentous fungi are an extremely important source of industrial enzymes because of their capacity to secrete large quantities of proteins. Currently, functional screening of fungi is associated with low throughput and high costs, which severely limits the discovery of novel enzymatic activities and better production strains. Here, we describe a nanoliter-range droplet-based microfluidic system specially adapted for the high-throughput sceening (HTS) of large filamentous fungi libraries for secreted enzyme activities. The platform allowed (i) compartmentalization of single spores in ~10 nl droplets, (ii) germination and mycelium growth and (iii) high-throughput sorting of fungi based on enzymatic activity. A 104 clone UV-mutated library of Aspergillus niger was screened based on α-amylase activity in just 90 minutes. Active clones were enriched 196-fold after a single round of microfluidic HTS. The platform is a powerful tool for the development of new production strains with low cost, space and time footprint and should bring enormous benefit for improving the viability of biotechnological processes.

A simple dual online ultra-high pressure liquid chromatography system (sDO-UHPLC) for high throughput proteome analysis.

PubMed

Lee, Hangyeore; Mun, Dong-Gi; Bae, Jingi; Kim, Hokeun; Oh, Se Yeon; Park, Young Soo; Lee, Jae-Hyuk; Lee, Sang-Won

2015-08-21

We report a new and simple design of a fully automated dual-online ultra-high pressure liquid chromatography system. The system employs only two nano-volume switching valves (a two-position four port valve and a two-position ten port valve) that direct solvent flows from two binary nano-pumps for parallel operation of two analytical columns and two solid phase extraction (SPE) columns. Despite the simple design, the sDO-UHPLC offers many advantageous features that include high duty cycle, back flushing sample injection for fast and narrow zone sample injection, online desalting, high separation resolution and high intra/inter-column reproducibility. This system was applied to analyze proteome samples not only in high throughput deep proteome profiling experiments but also in high throughput MRM experiments.

Optimization and high-throughput screening of antimicrobial peptides.

PubMed

Blondelle, Sylvie E; Lohner, Karl

2010-01-01

While a well-established process for lead compound discovery in for-profit companies, high-throughput screening is becoming more popular in basic and applied research settings in academia. The development of combinatorial libraries combined with easy and less expensive access to new technologies have greatly contributed to the implementation of high-throughput screening in academic laboratories. While such techniques were earlier applied to simple assays involving single targets or based on binding affinity, they have now been extended to more complex systems such as whole cell-based assays. In particular, the urgent need for new antimicrobial compounds that would overcome the rapid rise of drug-resistant microorganisms, where multiple target assays or cell-based assays are often required, has forced scientists to focus onto high-throughput technologies. Based on their existence in natural host defense systems and their different mode of action relative to commercial antibiotics, antimicrobial peptides represent a new hope in discovering novel antibiotics against multi-resistant bacteria. The ease of generating peptide libraries in different formats has allowed a rapid adaptation of high-throughput assays to the search for novel antimicrobial peptides. Similarly, the availability nowadays of high-quantity and high-quality antimicrobial peptide data has permitted the development of predictive algorithms to facilitate the optimization process. This review summarizes the various library formats that lead to de novo antimicrobial peptide sequences as well as the latest structural knowledge and optimization processes aimed at improving the peptides selectivity.

High-throughput combinatorial chemical bath deposition: The case of doping Cu (In, Ga) Se film with antimony

NASA Astrophysics Data System (ADS)

Yan, Zongkai; Zhang, Xiaokun; Li, Guang; Cui, Yuxing; Jiang, Zhaolian; Liu, Wen; Peng, Zhi; Xiang, Yong

2018-01-01

The conventional methods for designing and preparing thin film based on wet process remain a challenge due to disadvantages such as time-consuming and ineffective, which hinders the development of novel materials. Herein, we present a high-throughput combinatorial technique for continuous thin film preparation relied on chemical bath deposition (CBD). The method is ideally used to prepare high-throughput combinatorial material library with low decomposition temperatures and high water- or oxygen-sensitivity at relatively high-temperature. To check this system, a Cu(In, Ga)Se (CIGS) thin films library doped with 0-19.04 at.% of antimony (Sb) was taken as an example to evaluate the regulation of varying Sb doping concentration on the grain growth, structure, morphology and electrical properties of CIGS thin film systemically. Combined with the Energy Dispersive Spectrometer (EDS), X-ray Photoelectron Spectroscopy (XPS), automated X-ray Diffraction (XRD) for rapid screening and Localized Electrochemical Impedance Spectroscopy (LEIS), it was confirmed that this combinatorial high-throughput system could be used to identify the composition with the optimal grain orientation growth, microstructure and electrical properties systematically, through accurately monitoring the doping content and material composition. According to the characterization results, a Sb2Se3 quasi-liquid phase promoted CIGS film-growth model has been put forward. In addition to CIGS thin film reported here, the combinatorial CBD also could be applied to the high-throughput screening of other sulfide thin film material systems.

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

PubMed

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

2017-04-01

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

High Throughput Transcriptomics @ USEPA (Toxicology ...

EPA Pesticide Factsheets

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

Mobile element biology – new possibilities with high-throughput sequencing

PubMed Central

Xing, Jinchuan; Witherspoon, David J.; Jorde, Lynn B.

2014-01-01

Mobile elements compose more than half of the human genome, but until recently their large-scale detection was time-consuming and challenging. With the development of new high-throughput sequencing technologies, the complete spectrum of mobile element variation in humans can now be identified and analyzed. Thousands of new mobile element insertions have been discovered, yielding new insights into mobile element biology, evolution, and genomic variation. We review several high-throughput methods, with an emphasis on techniques that specifically target mobile element insertions in humans, and we highlight recent applications of these methods in evolutionary studies and in the analysis of somatic alterations in human cancers. PMID:23312846

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.

Accelerating Virtual High-Throughput Ligand Docking: current technology and case study on a petascale supercomputer.

PubMed

Ellingson, Sally R; Dakshanamurthy, Sivanesan; Brown, Milton; Smith, Jeremy C; Baudry, Jerome

2014-04-25

In this paper we give the current state of high-throughput virtual screening. We describe a case study of using a task-parallel MPI (Message Passing Interface) version of Autodock4 [1], [2] to run a virtual high-throughput screen of one-million compounds on the Jaguar Cray XK6 Supercomputer at Oak Ridge National Laboratory. We include a description of scripts developed to increase the efficiency of the predocking file preparation and postdocking analysis. A detailed tutorial, scripts, and source code for this MPI version of Autodock4 are available online at http://www.bio.utk.edu/baudrylab/autodockmpi.htm.

An economical and effective high-throughput DNA extraction protocol for molecular marker analysis in honey bees

USDA-ARS?s Scientific Manuscript database

Extraction of DNA from tissue samples can be expensive both in time and monetary resources and can often require handling and disposal of hazardous chemicals. We have developed a high throughput protocol for extracting DNA from honey bees that is of a high enough quality and quantity to enable hundr...

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

NASA Astrophysics Data System (ADS)

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

2013-06-01

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

20171015 - Capabilities and Evaluation of the US EPA’s HTTK (High Throughput Toxicokinetics) R package (ISES)

EPA Science Inventory

Toxicokinetics (TK) provides a bridge between toxicity and exposure assessment by predicting tissue concentrations due to exposure, however traditional TK methods are resource intensive. Relatively high throughput TK (HTTK) methods have been used by the pharmaceutical industry to...

High-throughput dietary exposure predictions for chemical migrants from food contact substances for use in chemical prioritization

EPA Science Inventory

Under the ExpoCast program, United States Environmental Protection Agency (EPA) researchers have developed a high-throughput (HT) framework for estimating aggregate exposures to chemicals from multiple pathways to support rapid prioritization of chemicals. Here, we present method...

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...

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...

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...

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...

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...

New Challenges of the Computation of Multiple Sequence Alignments in the High-Throughput Era (2010 JGI/ANL HPC Workshop)

ScienceCinema

Notredame, Cedric

2018-05-02

Cedric Notredame from the Centre for Genomic Regulation gives a presentation on New Challenges of the Computation of Multiple Sequence Alignments in the High-Throughput Era at the JGI/Argonne HPC Workshop on January 26, 2010.

Molecular characterization of a novel Nucleorhabdovirus from black currant identified by high-throughput sequencing

USDA-ARS?s Scientific Manuscript database

Contigs with sequence similarities to several nucleorhabdoviruses were identified by high-throughput sequencing analysis from a black currant (Ribes nigrum L.) cultivar. The complete genomic sequence of this new nucleorhabdovirus is 14,432 nucleotides. Its genomic organization is typical of nucleorh...

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...

Incorporating Population Variability and Susceptible Subpopulations into Dosimetry for High-Throughput Toxicity Testing

EPA Science Inventory

Momentum is growing worldwide to use in vitro high-throughput screening (HTS) to evaluate human health effects of chemicals. However, the integration of dosimetry into HTS assays and incorporation of population variability will be essential before its application in a risk assess...

Retrofit Strategies for Incorporating Xenobiotic Metabolism into High Throughput Screening Assays (EMGS)

EPA Science Inventory

The US EPA’s ToxCast program is designed to assess chemical perturbations of molecular and cellular endpoints using a variety of high-throughput screening (HTS) assays. However, existing HTS assays have limited or no xenobiotic metabolism which could lead to a mischaracterization...

Defining the taxonomic domain of applicability for mammalian-based high-throughput screening assays

EPA Science Inventory

Cell-based high throughput screening (HTS) technologies are becoming mainstream in chemical safety evaluations. The US Environmental Protection Agency (EPA) Toxicity Forecaster (ToxCastTM) and the multi-agency Tox21 Programs have been at the forefront in advancing this science, m...

High Throughput Plasmid Sequencing with Illumina and CLC Bio (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

ScienceCinema

Athavale, Ajay

2018-01-04

Ajay Athavale (Monsanto) presents "High Throughput Plasmid Sequencing with Illumina and CLC Bio" at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

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…

High-Throughput Cancer Cell Sphere Formation for 3D Cell Culture.

PubMed

Chen, Yu-Chih; Yoon, Euisik

2017-01-01

Three-dimensional (3D) cell culture is critical in studying cancer pathology and drug response. Though 3D cancer sphere culture can be performed in low-adherent dishes or well plates, the unregulated cell aggregation may skew the results. On contrary, microfluidic 3D culture can allow precise control of cell microenvironments, and provide higher throughput by orders of magnitude. In this chapter, we will look into engineering innovations in a microfluidic platform for high-throughput cancer cell sphere formation and review the implementation methods in detail.

Determination of red blood cell fatty acid profiles: Rapid and high-confident analysis by chemical ionization-gas chromatography-tandem mass spectrometry.

PubMed

Schober, Yvonne; Wahl, Hans Günther; Renz, Harald; Nockher, Wolfgang Andreas

2017-01-01

Cellular fatty acid (FA) profiles have been acknowledged as biomarkers in various human diseases. Nevertheless, common FA analysis by gas chromatography mass spectrometry (GC-MS) requires long analysis time. Hence, there is a need for feasible methods for high throughput analysis in clinical studies. FA was extracted from red blood cells (RBC) and derivatized to fatty acid methyl esters (FAME). A method using gas chromatography tandem mass spectrometry (GC-MS/MS) with ammonia-induced chemical ionization (CI) was developed for the analysis of FA profiles in human RBC. We compared this method with classical single GC-MS using electron impact ionization (EI). The FA profiles of 703 RBC samples were determined by GC-MS/MS. In contrast to EI ammonia-induced CI resulted in adequate amounts of molecular ions for further fragmentation of FAME. Specific fragments for confident quantification and fragmentation were determined for 45 FA. The GC-MS/MS method has a total run time of 9min compared to typical analysis times of up to 60min in conventional GC-MS. Intra and inter assay variations were <10% for all FA analyzed. Analysis of RBC FA composition revealed an age-dependent increase of the omega-3 eicosapentaenoic and docosahexaenoic acid, and a decline of the omega-6 linoleic acid with a corresponding rise of the omega-3 index. The combination of ammonia-induced CI and tandem mass spectrometry after GC separation allows for high-throughput, robust and confident analysis of FA profiles in the clinical laboratory. Copyright © 2016. Published by Elsevier B.V.

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

PubMed

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

2009-08-25

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

Microfluidic guillotine for single-cell wound repair studies

NASA Astrophysics Data System (ADS)

Blauch, Lucas R.; Gai, Ya; Khor, Jian Wei; Sood, Pranidhi; Marshall, Wallace F.; Tang, Sindy K. Y.

2017-07-01

Wound repair is a key feature distinguishing living from nonliving matter. Single cells are increasingly recognized to be capable of healing wounds. The lack of reproducible, high-throughput wounding methods has hindered single-cell wound repair studies. This work describes a microfluidic guillotine for bisecting single Stentor coeruleus cells in a continuous-flow manner. Stentor is used as a model due to its robust repair capacity and the ability to perform gene knockdown in a high-throughput manner. Local cutting dynamics reveals two regimes under which cells are bisected, one at low viscous stress where cells are cut with small membrane ruptures and high viability and one at high viscous stress where cells are cut with extended membrane ruptures and decreased viability. A cutting throughput up to 64 cells per minute—more than 200 times faster than current methods—is achieved. The method allows the generation of more than 100 cells in a synchronized stage of their repair process. This capacity, combined with high-throughput gene knockdown in Stentor, enables time-course mechanistic studies impossible with current wounding methods.

Computational tool for the early screening of monoclonal antibodies for their viscosities

PubMed Central

Agrawal, Neeraj J; Helk, Bernhard; Kumar, Sandeep; Mody, Neil; Sathish, Hasige A.; Samra, Hardeep S.; Buck, Patrick M; Li, Li; Trout, Bernhardt L

2016-01-01

Highly concentrated antibody solutions often exhibit high viscosities, which present a number of challenges for antibody-drug development, manufacturing and administration. The antibody sequence is a key determinant for high viscosity of highly concentrated solutions; therefore, a sequence- or structure-based tool that can identify highly viscous antibodies from their sequence would be effective in ensuring that only antibodies with low viscosity progress to the development phase. Here, we present a spatial charge map (SCM) tool that can accurately identify highly viscous antibodies from their sequence alone (using homology modeling to determine the 3-dimensional structures). The SCM tool has been extensively validated at 3 different organizations, and has proved successful in correctly identifying highly viscous antibodies. As a quantitative tool, SCM is amenable to high-throughput automated analysis, and can be effectively implemented during the antibody screening or engineering phase for the selection of low-viscosity antibodies. PMID:26399600

Present and future of membrane protein structure determination by electron crystallography.

PubMed

Ubarretxena-Belandia, Iban; Stokes, David L

2010-01-01

Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This chapter describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

Present and future of membrane protein structure determination by electron crystallography

PubMed Central

Ubarretxena-Belandia, Iban; Stokes, David L.

2011-01-01

Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This review describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins. PMID:21115172

CrossCheck: an open-source web tool for high-throughput screen data analysis.

PubMed

Najafov, Jamil; Najafov, Ayaz

2017-07-19

Modern high-throughput screening methods allow researchers to generate large datasets that potentially contain important biological information. However, oftentimes, picking relevant hits from such screens and generating testable hypotheses requires training in bioinformatics and the skills to efficiently perform database mining. There are currently no tools available to general public that allow users to cross-reference their screen datasets with published screen datasets. To this end, we developed CrossCheck, an online platform for high-throughput screen data analysis. CrossCheck is a centralized database that allows effortless comparison of the user-entered list of gene symbols with 16,231 published datasets. These datasets include published data from genome-wide RNAi and CRISPR screens, interactome proteomics and phosphoproteomics screens, cancer mutation databases, low-throughput studies of major cell signaling mediators, such as kinases, E3 ubiquitin ligases and phosphatases, and gene ontological information. Moreover, CrossCheck includes a novel database of predicted protein kinase substrates, which was developed using proteome-wide consensus motif searches. CrossCheck dramatically simplifies high-throughput screen data analysis and enables researchers to dig deep into the published literature and streamline data-driven hypothesis generation. CrossCheck is freely accessible as a web-based application at http://proteinguru.com/crosscheck.

Ion channel drug discovery and research: the automated Nano-Patch-Clamp technology.

PubMed

Brueggemann, A; George, M; Klau, M; Beckler, M; Steindl, J; Behrends, J C; Fertig, N

2004-01-01

Unlike the genomics revolution, which was largely enabled by a single technological advance (high throughput sequencing), rapid advancement in proteomics will require a broader effort to increase the throughput of a number of key tools for functional analysis of different types of proteins. In the case of ion channels -a class of (membrane) proteins of great physiological importance and potential as drug targets- the lack of adequate assay technologies is felt particularly strongly. The available, indirect, high throughput screening methods for ion channels clearly generate insufficient information. The best technology to study ion channel function and screen for compound interaction is the patch clamp technique, but patch clamping suffers from low throughput, which is not acceptable for drug screening. A first step towards a solution is presented here. The nano patch clamp technology, which is based on a planar, microstructured glass chip, enables automatic whole cell patch clamp measurements. The Port-a-Patch is an automated electrophysiology workstation, which uses planar patch clamp chips. This approach enables high quality and high content ion channel and compound evaluation on a one-cell-at-a-time basis. The presented automation of the patch process and its scalability to an array format are the prerequisites for any higher throughput electrophysiology instruments.

High-Reflectivity Coatings for a Vacuum Ultraviolet Spectropolarimeter

NASA Astrophysics Data System (ADS)

Narukage, Noriyuki; Kubo, Masahito; Ishikawa, Ryohko; Ishikawa, Shin-nosuke; Katsukawa, Yukio; Kobiki, Toshihiko; Giono, Gabriel; Kano, Ryouhei; Bando, Takamasa; Tsuneta, Saku; Auchère, Frédéric; Kobayashi, Ken; Winebarger, Amy; McCandless, Jim; Chen, Jianrong; Choi, Joanne

2017-03-01

Precise polarization measurements in the vacuum ultraviolet (VUV) region are expected to be a new tool for inferring the magnetic fields in the upper atmosphere of the Sun. High-reflectivity coatings are key elements to achieving high-throughput optics for precise polarization measurements. We fabricated three types of high-reflectivity coatings for a solar spectropolarimeter in the hydrogen Lyman-α (Lyα; 121.567 nm) region and evaluated their performance. The first high-reflectivity mirror coating offers a reflectivity of more than 80 % in Lyα optics. The second is a reflective narrow-band filter coating that has a peak reflectivity of 57 % in Lyα, whereas its reflectivity in the visible light range is lower than 1/10 of the peak reflectivity (˜ 5 % on average). This coating can be used to easily realize a visible light rejection system, which is indispensable for a solar telescope, while maintaining high throughput in the Lyα line. The third is a high-efficiency reflective polarizing coating that almost exclusively reflects an s-polarized beam at its Brewster angle of 68° with a reflectivity of 55 %. This coating achieves both high polarizing power and high throughput. These coatings contributed to the high-throughput solar VUV spectropolarimeter called the Chromospheric Lyman-Alpha SpectroPolarimeter (CLASP), which was launched on 3 September, 2015.

Nonlinear Optical Characterization of Membrane Protein Microcrystals and Nanocrystals.

PubMed

Newman, Justin A; Simpson, Garth J

2016-01-01

Nonlinear optical methods such as second harmonic generation (SHG) and two-photon excited UV fluorescence (TPE-UVF) imaging are promising approaches to address bottlenecks in the membrane protein structure determination pipeline. The general principles of SHG and TPE-UVF are discussed here along with instrument design considerations. Comparisons to conventional methods in high throughput crystallization condition screening and crystal quality assessment prior to X-ray diffraction are also discussed.

Development of Personalized Cancer Therapy for Men with Advanced Prostate Cancer

DTIC Science & Technology

2017-10-01

cascade is significantly associated with the beta isoform. Based on these studies , we hypothesize that FGFR1 alpha and beta confers different phenotypes...Goals: This is an open label study to determine the effect of Alpharadin on the bone marrow microenvironment in patients with castrate resistant...and analysis of studies using high-throughput technologies. Effort added. Specific Aims: Same as above. Role: Co-Investigator Prostate Moon Shot

Development of Antibacterials Targeting the MEP Pathway of Select Agents

DTIC Science & Technology

2015-03-01

inhibitor discovery, evaluation of lead inhibitors in microbial growth assays, determining X- ray crystal structures of the MEP pathway enzymes MEP...recombinant proteins to WRAIR for X- ray crystallography. Reportable Outcomes Haymond A, Johny C, Dowdy T, Schweibenz B, Villarroel K, Young R, Mantooth...journal.pone.0020884. 9 3. Zhang, Chung, Oldenburg (1999) A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening

Chirality sensing with stereodynamic copper(I) complexes.

PubMed

De Los Santos, Zeus A; Legaux, Nicholas M; Wolf, Christian

2017-11-01

Three Cu(I) complexes derived from stereodynamic diphosphine ligands were synthesized and used for chirality sensing. The coordination of diamines and amino acids to these complexes generates distinct circular dichroism signals. The chiroptical sensor response allows determination of the absolute configuration and the enantiomeric excess of the analyte at low concentrations. This method is operationally simple, fast, and attractive for high-throughput sensing applications. © 2017 Wiley Periodicals, Inc.

A simple and sensitive high-throughput GFP screening in woody and herbaceous plants.

PubMed

Hily, Jean-Michel; Liu, Zongrang

2009-03-01

Green fluorescent protein (GFP) has been used widely as a powerful bioluminescent reporter, but its visualization by existing methods in tissues or whole plants and its utilization for high-throughput screening remains challenging in many species. Here, we report a fluorescence image analyzer-based method for GFP detection and its utility for high-throughput screening of transformed plants. Of three detection methods tested, the Typhoon fluorescence scanner was able to detect GFP fluorescence in all Arabidopsis thaliana tissues and apple leaves, while regular fluorescence microscopy detected it only in Arabidopsis flowers and siliques but barely in the leaves of either Arabidopsis or apple. The hand-held UV illumination method failed in all tissues of both species. Additionally, the Typhoon imager was able to detect GFP fluorescence in both green and non-green tissues of Arabidopsis seedlings as well as in imbibed seeds, qualifying it as a high-throughput screening tool, which was further demonstrated by screening the seedlings of primary transformed T(0) seeds. Of the 30,000 germinating Arabidopsis seedlings screened, at least 69 GFP-positive lines were identified, accounting for an approximately 0.23% transformation efficiency. About 14,000 seedlings grown in 16 Petri plates could be screened within an hour, making the screening process significantly more efficient and robust than any other existing high-throughput screening method for transgenic plants.

Development of a high-throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design.

PubMed

Bláha, Benjamin A F; Morris, Stephen A; Ogonah, Olotu W; Maucourant, Sophie; Crescente, Vincenzo; Rosenberg, William; Mukhopadhyay, Tarit K

2018-01-01

The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high-throughput disruption methods exist. The development of an automated, miniaturized, high-throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high-pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130-140, 2018. © 2017 American Institute of Chemical Engineers.

A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics.

PubMed

Alterman, Julia F; Coles, Andrew H; Hall, Lauren M; Aronin, Neil; Khvorova, Anastasia; Didiot, Marie-Cécile

2017-08-20

Primary neurons represent an ideal cellular system for the identification of therapeutic oligonucleotides for the treatment of neurodegenerative diseases. However, due to the sensitive nature of primary cells, the transfection of small interfering RNAs (siRNA) using classical methods is laborious and often shows low efficiency. Recent progress in oligonucleotide chemistry has enabled the development of stabilized and hydrophobically modified small interfering RNAs (hsiRNAs). This new class of oligonucleotide therapeutics shows extremely efficient self-delivery properties and supports potent and durable effects in vitro and in vivo . We have developed a high-throughput in vitro assay to identify and test hsiRNAs in primary neuronal cultures. To simply, rapidly, and accurately quantify the mRNA silencing of hundreds of hsiRNAs, we use the QuantiGene 2.0 quantitative gene expression assay. This high-throughput, 96-well plate-based assay can quantify mRNA levels directly from sample lysate. Here, we describe a method to prepare short-term cultures of mouse primary cortical neurons in a 96-well plate format for high-throughput testing of oligonucleotide therapeutics. This method supports the testing of hsiRNA libraries and the identification of potential therapeutics within just two weeks. We detail methodologies of our high throughput assay workflow from primary neuron preparation to data analysis. This method can help identify oligonucleotide therapeutics for treatment of various neurological diseases.

High-throughput screening with nanoimprinting 3D culture for efficient drug development by mimicking the tumor environment.

PubMed

Yoshii, Yukie; Furukawa, Takako; Waki, Atsuo; Okuyama, Hiroaki; Inoue, Masahiro; Itoh, Manabu; Zhang, Ming-Rong; Wakizaka, Hidekatsu; Sogawa, Chizuru; Kiyono, Yasushi; Yoshii, Hiroshi; Fujibayashi, Yasuhisa; Saga, Tsuneo

2015-05-01

Anti-cancer drug development typically utilizes high-throughput screening with two-dimensional (2D) cell culture. However, 2D culture induces cellular characteristics different from tumors in vivo, resulting in inefficient drug development. Here, we report an innovative high-throughput screening system using nanoimprinting 3D culture to simulate in vivo conditions, thereby facilitating efficient drug development. We demonstrated that cell line-based nanoimprinting 3D screening can more efficiently select drugs that effectively inhibit cancer growth in vivo as compared to 2D culture. Metabolic responses after treatment were assessed using positron emission tomography (PET) probes, and revealed similar characteristics between the 3D spheroids and in vivo tumors. Further, we developed an advanced method to adopt cancer cells from patient tumor tissues for high-throughput drug screening with nanoimprinting 3D culture, which we termed Cancer tissue-Originated Uniformed Spheroid Assay (COUSA). This system identified drugs that were effective in xenografts of the original patient tumors. Nanoimprinting 3D spheroids showed low permeability and formation of hypoxic regions inside, similar to in vivo tumors. Collectively, the nanoimprinting 3D culture provides easy-handling high-throughput drug screening system, which allows for efficient drug development by mimicking the tumor environment. The COUSA system could be a useful platform for drug development with patient cancer cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Quantification of locomotor activity in larval zebrafish: considerations for the design of high-throughput behavioral studies

PubMed Central

Ingebretson, Justin J.; Masino, Mark A.

2013-01-01

High-throughput behavioral studies using larval zebrafish often assess locomotor activity to determine the effects of experimental perturbations. However, the results reported by different groups are difficult to compare because there is not a standardized experimental paradigm or measure of locomotor activity. To address this, we investigated the effects that several factors, including the stage of larval development and the physical dimensions (depth and diameter) of the behavioral arena, have on the locomotor activity produced by larval zebrafish. We provide evidence for differences in locomotor activity between larvae at different stages and when recorded in wells of different depths, but not in wells of different diameters. We also show that the variability for most properties of locomotor activity is less for older than younger larvae, which is consistent with previous reports. Finally, we show that conflicting interpretations of activity level can occur when activity is assessed with a single measure of locomotor activity. Thus, we conclude that although a combination of factors should be considered when designing behavioral experiments, the use of older larvae in deep wells will reduce the variability of locomotor activity, and that multiple properties of locomotor activity should be measured to determine activity level. PMID:23772207

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

PubMed Central

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

2012-01-01

High-throughput screening (HTS) is one of the newest techniques used in drug design and may be applied in biological and chemical sciences. This method, due to utilization of robots, detectors and software that regulate the whole process, enables a series of analyses of chemical compounds to be conducted in a short time and the affinity of biological structures which is often related to toxicity to be defined. Since 2008 we have implemented the automation of this technique and as a consequence, the possibility to examine 100,000 compounds per day. The HTS method is more frequently utilized in conjunction with analytical techniques such as NMR or coupled methods e.g., LC-MS/MS. Series of studies enable the establishment of the rate of affinity for targets or the level of toxicity. Moreover, researches are conducted concerning conjugation of nanoparticles with drugs and the determination of the toxicity of such structures. For these purposes there are frequently used cell lines. Due to the miniaturization of all systems, it is possible to examine the compound’s toxicity having only 1–3 mg of this compound. Determination of cytotoxicity in this way leads to a significant decrease in the expenditure and to a reduction in the length of the study. PMID:22312262

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

RSCA genotyping of MHC for high-throughput evolutionary studies in the model organism three-spined stickleback Gasterosteus aculeatus

PubMed Central

Lenz, Tobias L; Eizaguirre, Christophe; Becker, Sven; Reusch, Thorsten BH

2009-01-01

Background In all jawed vertebrates, highly polymorphic genes of the major histocompatibility complex (MHC) encode antigen presenting molecules that play a key role in the adaptive immune response. Their polymorphism is composed of multiple copies of recently duplicated genes, each possessing many alleles within populations, as well as high nucleotide divergence between alleles of the same species. Experimental evidence is accumulating that MHC polymorphism is a result of balancing selection by parasites and pathogens. In order to describe MHC diversity and analyse the underlying mechanisms that maintain it, a reliable genotyping technique is required that is suitable for such highly variable genes. Results We present a genotyping protocol that uses Reference Strand-mediated Conformation Analysis (RSCA), optimised for recently duplicated MHC class IIB genes that are typical for many fish and bird species, including the three-spined stickleback, Gasterosteus aculeatus. In addition we use a comprehensive plasmid library of MHC class IIB alleles to determine the nucleotide sequence of alleles represented by RSCA allele peaks. Verification of the RSCA typing by cloning and sequencing demonstrates high congruency between both methods and provides new insight into the polymorphism of classical stickleback MHC genes. Analysis of the plasmid library additionally reveals the high resolution and reproducibility of the RSCA technique. Conclusion This new RSCA genotyping protocol offers a fast, but sensitive and reliable way to determine the MHC allele repertoire of three-spined sticklebacks. It therefore provides a valuable tool to employ this highly polymorphic and adaptive marker in future high-throughput studies of host-parasite co-evolution and ecological speciation in this emerging model organism. PMID:19291291