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

Energy Technology Data Exchange (ETDEWEB)

Wall, Andrew J. [National Energy Technology Lab. (NETL), Pittsburgh, PA, (United States); Capo, Rosemary C. [Univ. of Pittsburgh, PA (United States); Stewart, Brian W. [Univ. of Pittsburgh, PA (United States); Phan, Thai T. [Univ. of Pittsburgh, PA (United States); Jain, Jinesh C. [National Energy Technology Lab. (NETL), Pittsburgh, PA, (United States); Hakala, Alexandra [National Energy Technology Lab. (NETL), Pittsburgh, PA, (United States); Guthrie, George D. [National Energy Technology Lab. (NETL), Pittsburgh, PA, (United States)

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

Energy Technology Data Exchange (ETDEWEB)

Hakala, Jacqueline Alexandra [National Energy Technology Lab. (NETL), Morgantown, WV (United States)

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 high-throughput method for GMO multi-detection using a microfluidic dynamic array

NARCIS (Netherlands)

Brod, F.C.A.; Dijk, van J.P.; Voorhuijzen, M.M.; Dinon, A.Z.; Guimarães, L.H.S.; Scholtens, I.M.J.; Arisi, A.C.M.; Kok, E.J.

2014-01-01

The ever-increasing production of genetically modified crops generates a demand for high-throughput DNAbased methods for the enforcement of genetically modified organisms (GMO) labelling requirements. The application of standard real-time PCR will become increasingly costly with the growth of the

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

Science.gov (United States)

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

2016-01-01

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

DRABAL: novel method to mine large high-throughput screening assays using Bayesian active learning

KAUST Repository

Soufan, Othman; Ba Alawi, Wail; Afeef, Moataz A.; Essack, Magbubah; Kalnis, Panos; Bajic, Vladimir B.

2016-01-01

Mining high-throughput screening (HTS) assays is key for enhancing decisions in the area of drug repositioning and drug discovery. However, many challenges are encountered in the process of developing suitable and accurate methods

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

Science.gov (United States)

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.

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

Science.gov (United States)

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 sample adaptive offset hardware architecture for high-efficiency video coding

Science.gov (United States)

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.

High-throughput method for optimum solubility screening for homogeneity and crystallization of proteins

Science.gov (United States)

Kim, Sung-Hou [Moraga, CA; Kim, Rosalind [Moraga, CA; Jancarik, Jamila [Walnut Creek, CA

2012-01-31

An optimum solubility screen in which a panel of buffers and many additives are provided in order to obtain the most homogeneous and monodisperse protein condition for protein crystallization. The present methods are useful for proteins that aggregate and cannot be concentrated prior to setting up crystallization screens. A high-throughput method using the hanging-drop method and vapor diffusion equilibrium and a panel of twenty-four buffers is further provided. Using the present methods, 14 poorly behaving proteins have been screened, resulting in 11 of the proteins having highly improved dynamic light scattering results allowing concentration of the proteins, and 9 were crystallized.

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

Directory of Open Access Journals (Sweden)

Karson S Putt

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

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

Science.gov (United States)

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

2013-01-01

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

Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads.

Science.gov (United States)

Sasagawa, Yohei; Danno, Hiroki; Takada, Hitomi; Ebisawa, Masashi; Tanaka, Kaori; Hayashi, Tetsutaro; Kurisaki, Akira; Nikaido, Itoshi

2018-03-09

High-throughput single-cell RNA-seq methods assign limited unique molecular identifier (UMI) counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts. We thus developed a high-throughput single-cell RNA-seq method, Quartz-Seq2, to overcome these issues. Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts, at a rate of 30-50%, and detect more genes. To demonstrate the power of Quartz-Seq2, we analyzed approximately 10,000 transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads.

A high-throughput method for quantifying metabolically active yeast cells

DEFF Research Database (Denmark)

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

2015-01-01

By redesigning the established methylene blue reduction test for bacteria and yeast, we present a cheap and efficient methodology for quantitative physiology of eukaryotic cells applicable for high-throughput systems. Validation of themethod in fermenters and highthroughput systems proved....... The drop in metabolic activity associated with the diauxic shift in yeast proved more pronounced for the MBRT-derived curve compared with OD curves, consistent with a dramatic shift in the ratio between live and dead cells at this metabolic event. This method provides a tool with numerous applications, e.......g. characterizing the death phase of stationary phase cultures, or in drug screens with pathogenic yeasts....

A gas trapping method for high-throughput metabolic experiments.

Science.gov (United States)

Krycer, James R; Diskin, Ciana; Nelson, Marin E; Zeng, Xiao-Yi; Fazakerley, Daniel J; James, David E

2018-01-01

Research into cellular metabolism has become more high-throughput, with typical cell-culture experiments being performed in multiwell plates (microplates). This format presents a challenge when trying to collect gaseous products, such as carbon dioxide (CO2), which requires a sealed environment and a vessel separate from the biological sample. To address this limitation, we developed a gas trapping protocol using perforated plastic lids in sealed cell-culture multiwell plates. We used this trap design to measure CO2 production from glucose and fatty acid metabolism, as well as hydrogen sulfide production from cysteine-treated cells. Our data clearly show that this gas trap can be applied to liquid and solid gas-collection media and can be used to study gaseous product generation by both adherent cells and cells in suspension. Since our gas traps can be adapted to multiwell plates of various sizes, they present a convenient, cost-effective solution that can accommodate the trend toward high-throughput measurements in metabolic research.

A high-throughput method for GMO multi-detection using a microfluidic dynamic array.

Science.gov (United States)

Brod, Fábio Cristiano Angonesi; van Dijk, Jeroen P; Voorhuijzen, Marleen M; Dinon, Andréia Zilio; Guimarães, Luis Henrique S; Scholtens, Ingrid M J; Arisi, Ana Carolina Maisonnave; Kok, Esther J

2014-02-01

The ever-increasing production of genetically modified crops generates a demand for high-throughput DNA-based methods for the enforcement of genetically modified organisms (GMO) labelling requirements. The application of standard real-time PCR will become increasingly costly with the growth of the number of GMOs that is potentially present in an individual sample. The present work presents the results of an innovative approach in genetically modified crops analysis by DNA based methods, which is the use of a microfluidic dynamic array as a high throughput multi-detection system. In order to evaluate the system, six test samples with an increasing degree of complexity were prepared, preamplified and subsequently analysed in the Fluidigm system. Twenty-eight assays targeting different DNA elements, GM events and species-specific reference genes were used in the experiment. The large majority of the assays tested presented expected results. The power of low level detection was assessed and elements present at concentrations as low as 0.06 % were successfully detected. The approach proposed in this work presents the Fluidigm system as a suitable and promising platform for GMO multi-detection.

Cox-nnet: An artificial neural network method for prognosis prediction of high-throughput omics data.

Science.gov (United States)

Ching, Travers; Zhu, Xun; Garmire, Lana X

2018-04-01

Artificial neural networks (ANN) are computing architectures with many interconnections of simple neural-inspired computing elements, and have been applied to biomedical fields such as imaging analysis and diagnosis. We have developed a new ANN framework called Cox-nnet to predict patient prognosis from high throughput transcriptomics data. In 10 TCGA RNA-Seq data sets, Cox-nnet achieves the same or better predictive accuracy compared to other methods, including Cox-proportional hazards regression (with LASSO, ridge, and mimimax concave penalty), Random Forests Survival and CoxBoost. Cox-nnet also reveals richer biological information, at both the pathway and gene levels. The outputs from the hidden layer node provide an alternative approach for survival-sensitive dimension reduction. In summary, we have developed a new method for accurate and efficient prognosis prediction on high throughput data, with functional biological insights. The source code is freely available at https://github.com/lanagarmire/cox-nnet.

High Throughput Facility

Data.gov (United States)

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

High-throughput optical system for HDES hyperspectral imager

Science.gov (United States)

Václavík, Jan; Melich, Radek; Pintr, Pavel; Pleštil, Jan

2015-01-01

Affordable, long-wave infrared hyperspectral imaging calls for use of an uncooled FPA with high-throughput optics. This paper describes the design of the optical part of a stationary hyperspectral imager in a spectral range of 7-14 um with a field of view of 20°×10°. The imager employs a push-broom method made by a scanning mirror. High throughput and a demand for simplicity and rigidity led to a fully refractive design with highly aspheric surfaces and off-axis positioning of the detector array. The design was optimized to exploit the machinability of infrared materials by the SPDT method and a simple assemblage.

High-Throughput Block Optical DNA Sequence Identification.

Science.gov (United States)

Sagar, Dodderi Manjunatha; Korshoj, Lee Erik; Hanson, Katrina Bethany; Chowdhury, Partha Pratim; Otoupal, Peter Britton; Chatterjee, Anushree; Nagpal, Prashant

2018-01-01

Optical techniques for molecular diagnostics or DNA sequencing generally rely on small molecule fluorescent labels, which utilize light with a wavelength of several hundred nanometers for detection. Developing a label-free optical DNA sequencing technique will require nanoscale focusing of light, a high-throughput and multiplexed identification method, and a data compression technique to rapidly identify sequences and analyze genomic heterogeneity for big datasets. Such a method should identify characteristic molecular vibrations using optical spectroscopy, especially in the "fingerprinting region" from ≈400-1400 cm -1 . Here, surface-enhanced Raman spectroscopy is used to demonstrate label-free identification of DNA nucleobases with multiplexed 3D plasmonic nanofocusing. While nanometer-scale mode volumes prevent identification of single nucleobases within a DNA sequence, the block optical technique can identify A, T, G, and C content in DNA k-mers. The content of each nucleotide in a DNA block can be a unique and high-throughput method for identifying sequences, genes, and other biomarkers as an alternative to single-letter sequencing. Additionally, coupling two complementary vibrational spectroscopy techniques (infrared and Raman) can improve block characterization. These results pave the way for developing a novel, high-throughput block optical sequencing method with lossy genomic data compression using k-mer identification from multiplexed optical data acquisition. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microengineering methods for cell-based microarrays and high-throughput drug-screening applications

International Nuclear Information System (INIS)

Xu Feng; Wu Jinhui; Wang Shuqi; Gurkan, Umut Atakan; Demirci, Utkan; Durmus, Naside Gozde

2011-01-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time consuming, and often faces concerns due to the extensive use of animals. To improve cost effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems has facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell-based drug-screening models which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell-based drug-screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds great potential to provide repeatable 3D cell-based constructs with high temporal, spatial control and versatility.

Microengineering methods for cell-based microarrays and high-throughput drug-screening applications

Energy Technology Data Exchange (ETDEWEB)

Xu Feng; Wu Jinhui; Wang Shuqi; Gurkan, Umut Atakan; Demirci, Utkan [Department of Medicine, Demirci Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Center for Biomedical Engineering, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States); Durmus, Naside Gozde, E-mail: udemirci@rics.bwh.harvard.edu [School of Engineering and Division of Biology and Medicine, Brown University, Providence, RI (United States)

2011-09-15

Screening for effective therapeutic agents from millions of drug candidates is costly, time consuming, and often faces concerns due to the extensive use of animals. To improve cost effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems has facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell-based drug-screening models which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell-based drug-screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds great potential to provide repeatable 3D cell-based constructs with high temporal, spatial control and versatility.

High Throughput Analysis of Photocatalytic Water Purification

NARCIS (Netherlands)

Sobral Romao, J.I.; Baiao Barata, David; Habibovic, Pamela; Mul, Guido; Baltrusaitis, Jonas

2014-01-01

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

High-throughput open source computational methods for genetics and genomics

NARCIS (Netherlands)

Prins, J.C.P.

2015-01-01

Biology is increasingly data driven by virtue of the development of high-throughput technologies, such as DNA and RNA sequencing. Computational biology and bioinformatics are scientific disciplines that cross-over between the disciplines of biology, informatics and statistics; which is clearly

High throughput protein production screening

Science.gov (United States)

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

2009-09-08

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

HIGH THROUGHPUT SCREENING METHOD AND APPARATUS FOR ANALYSING INTERACTIONS BETWEEN SURFACES WITH DIFFERENT TOPOGRAPHY AND THE ENVIRONMENT

NARCIS (Netherlands)

de Boer, Jan; van Blitterswijk, Clemens; Unadkat, H.V.; Stamatialis, Dimitrios; Papenburg, B.J.; Wessling, Matthias

2009-01-01

The invention is directed to a high throughput screening method for analysing and interaction between a surface of a material and an environment. The screening method of the invention comprises: providing a micro-array comprising said material and having a multitude of units at least part of which

Lateral Temperature-Gradient Method for High-Throughput Characterization of Material Processing by Millisecond Laser Annealing.

Science.gov (United States)

Bell, Robert T; Jacobs, Alan G; Sorg, Victoria C; Jung, Byungki; Hill, Megan O; Treml, Benjamin E; Thompson, Michael O

2016-09-12

A high-throughput method for characterizing the temperature dependence of material properties following microsecond to millisecond thermal annealing, exploiting the temperature gradients created by a lateral gradient laser spike anneal (lgLSA), is presented. Laser scans generate spatial thermal gradients of up to 5 °C/μm with peak temperatures ranging from ambient to in excess of 1400 °C, limited only by laser power and materials thermal limits. Discrete spatial property measurements across the temperature gradient are then equivalent to independent measurements after varying temperature anneals. Accurate temperature calibrations, essential to quantitative analysis, are critical and methods for both peak temperature and spatial/temporal temperature profile characterization are presented. These include absolute temperature calibrations based on melting and thermal decomposition, and time-resolved profiles measured using platinum thermistors. A variety of spatially resolved measurement probes, ranging from point-like continuous profiling to large area sampling, are discussed. Examples from annealing of III-V semiconductors, CdSe quantum dots, low-κ dielectrics, and block copolymers are included to demonstrate the flexibility, high throughput, and precision of this technique.

A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

DEFF Research Database (Denmark)

Uren, Anthony G; Mikkers, Harald; Kool, Jaap

2009-01-01

sites has been a major limitation to performing screens on this scale. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA......Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion...

High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots.

Directory of Open Access Journals (Sweden)

Guangbo Liu

Full Text Available Saccharomyces cerevisiae (budding yeast is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids or genome mutation (e.g., gene mutation, deletion, epitope tagging is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.

High-Throughput Thermodynamic Modeling and Uncertainty Quantification for ICME

Science.gov (United States)

Otis, Richard A.; Liu, Zi-Kui

2017-05-01

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

High-throughput cloning and expression in recalcitrant bacteria

NARCIS (Netherlands)

Geertsma, Eric R.; Poolman, Bert

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

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

Science.gov (United States)

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.

High throughput integrated thermal characterization with non-contact optical calorimetry

Science.gov (United States)

Hou, Sichao; Huo, Ruiqing; Su, Ming

2017-10-01

Commonly used thermal analysis tools such as calorimeter and thermal conductivity meter are separated instruments and limited by low throughput, where only one sample is examined each time. This work reports an infrared based optical calorimetry with its theoretical foundation, which is able to provide an integrated solution to characterize thermal properties of materials with high throughput. By taking time domain temperature information of spatially distributed samples, this method allows a single device (infrared camera) to determine the thermal properties of both phase change systems (melting temperature and latent heat of fusion) and non-phase change systems (thermal conductivity and heat capacity). This method further allows these thermal properties of multiple samples to be determined rapidly, remotely, and simultaneously. In this proof-of-concept experiment, the thermal properties of a panel of 16 samples including melting temperatures, latent heats of fusion, heat capacities, and thermal conductivities have been determined in 2 min with high accuracy. Given the high thermal, spatial, and temporal resolutions of the advanced infrared camera, this method has the potential to revolutionize the thermal characterization of materials by providing an integrated solution with high throughput, high sensitivity, and short analysis time.

High efficient plastic solar cells fabricated with a high-throughput gravure printing method

Energy Technology Data Exchange (ETDEWEB)

Kopola, P.; Jin, H.; Tuomikoski, M.; Maaninen, A.; Hast, J. [VTT, Kaitovaeylae 1, FIN-90571 Oulu (Finland); Aernouts, T. [IMEC, Organic PhotoVoltaics, Polymer and Molecular Electronics, Kapeldreef 75, B-3001 Leuven (Belgium); Guillerez, S. [CEA-INES RDI, 50 Avenue Du Lac Leman, 73370 Le Bourget Du Lac (France)

2010-10-15

We report on polymer-based solar cells prepared by the high-throughput roll-to-roll gravure printing method. The engravings of the printing plate, along with process parameters like printing speed and ink properties, are studied to optimise the printability of the photoactive as well as the hole transport layer. For the hole transport layer, the focus is on testing different formulations to produce thorough wetting of the indium-tin-oxide (ITO) substrate. The challenge for the photoactive layer is to form a uniform layer with optimal nanomorphology in the poly-3-hexylthiophene (P3HT) and [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) blend. This results in a power conversion efficiency of 2.8% under simulated AM1.5G solar illumination for a solar cell device with gravure-printed hole transport and a photoactive layer. (author)

A High-Throughput UHPLC-QqQ-MS Method for Polyphenol Profiling in Rosé Wines

Directory of Open Access Journals (Sweden)

Marine Lambert

2015-04-01

Full Text Available A rapid, sensitive and selective analysis method using Ultra High Performance Liquid Chromatography coupled to triple-quadrupole Mass Spectrometry (UHPLC-QqQ-MS has been developed for the quantification of polyphenols in rosé wines. The compound detection being based on specific MS transitions in Multiple Reaction Monitoring (MRM mode, the present method allows the selective quantification of up to 152 phenolic and two additional non-phenolic wine compounds in 30 min without sample purification or pre-concentration, even at low concentration levels. This method was repeatably applied to a set of 12 rosé wines and thus proved to be suitable for high-throughput and large-scale metabolomics studies.

High-throughput theoretical design of lithium battery materials

International Nuclear Information System (INIS)

Ling Shi-Gang; Gao Jian; Xiao Rui-Juan; Chen Li-Quan

2016-01-01

The rapid evolution of high-throughput theoretical design schemes to discover new lithium battery materials is reviewed, including high-capacity cathodes, low-strain cathodes, anodes, solid state electrolytes, and electrolyte additives. With the development of efficient theoretical methods and inexpensive computers, high-throughput theoretical calculations have played an increasingly important role in the discovery of new materials. With the help of automatic simulation flow, many types of materials can be screened, optimized and designed from a structural database according to specific search criteria. In advanced cell technology, new materials for next generation lithium batteries are of great significance to achieve performance, and some representative criteria are: higher energy density, better safety, and faster charge/discharge speed. (topical review)

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

Science.gov (United States)

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.

Towards a high throughput droplet-based agglutination assay

KAUST Repository

Kodzius, Rimantas; Castro, David; Foulds, Ian G.

2013-01-01

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

Towards a high throughput droplet-based agglutination assay

KAUST Repository

Kodzius, Rimantas

2013-10-22

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

High throughput materials research and development for lithium ion batteries

Directory of Open Access Journals (Sweden)

Parker Liu

2017-09-01

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

Enzyme free cloning for high throughput gene cloning and expression

NARCIS (Netherlands)

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

2006-01-01

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

High-throughput screening of small molecule libraries using SAMDI mass spectrometry.

Science.gov (United States)

Gurard-Levin, Zachary A; Scholle, Michael D; Eisenberg, Adam H; Mrksich, Milan

2011-07-11

High-throughput screening is a common strategy used to identify compounds that modulate biochemical activities, but many approaches depend on cumbersome fluorescent reporters or antibodies and often produce false-positive hits. The development of "label-free" assays addresses many of these limitations, but current approaches still lack the throughput needed for applications in drug discovery. This paper describes a high-throughput, label-free assay that combines self-assembled monolayers with mass spectrometry, in a technique called SAMDI, as a tool for screening libraries of 100,000 compounds in one day. This method is fast, has high discrimination, and is amenable to a broad range of chemical and biological applications.

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds.

Science.gov (United States)

Lindsey, Benson E; Rivero, Luz; Calhoun, Chistopher S; Grotewold, Erich; Brkljacic, Jelena

2017-10-17

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and

A method for high throughput bioelectrochemical research based on small scale microbial electrolysis cells

KAUST Repository

Call, Douglas F.

2011-07-01

There is great interest in studying exoelectrogenic microorganisms, but existing methods can require expensive electrochemical equipment and specialized reactors. We developed a simple system for conducting high throughput bioelectrochemical research using multiple inexpensive microbial electrolysis cells (MECs) built with commercially available materials and operated using a single power source. MECs were small crimp top serum bottles (5mL) with a graphite plate anode (92m 2/m 3) and a cathode of stainless steel (SS) mesh (86m 2/m 3), graphite plate, SS wire, or platinum wire. The highest volumetric current density (240A/m 3, applied potential of 0.7V) was obtained using a SS mesh cathode and a wastewater inoculum (acetate electron donor). Parallel operated MECs (single power source) did not lead to differences in performance compared to non-parallel operated MECs, which can allow for high throughput reactor operation (>1000 reactors) using a single power supply. The utility of this method for cultivating exoelectrogenic microorganisms was demonstrated through comparison of buffer effects on pure (Geobacter sulfurreducens and Geobacter metallireducens) and mixed cultures. Mixed cultures produced current densities equal to or higher than pure cultures in the different media, and current densities for all cultures were higher using a 50mM phosphate buffer than a 30mM bicarbonate buffer. Only the mixed culture was capable of sustained current generation with a 200mM phosphate buffer. These results demonstrate the usefulness of this inexpensive method for conducting in-depth examinations of pure and mixed exoelectrogenic cultures. © 2011 Elsevier B.V.

Filtering high-throughput protein-protein interaction data using a combination of genomic features

Directory of Open Access Journals (Sweden)

Patil Ashwini

2005-04-01

Full Text Available Abstract Background Protein-protein interaction data used in the creation or prediction of molecular networks is usually obtained from large scale or high-throughput experiments. This experimental data is liable to contain a large number of spurious interactions. Hence, there is a need to validate the interactions and filter out the incorrect data before using them in prediction studies. Results In this study, we use a combination of 3 genomic features – structurally known interacting Pfam domains, Gene Ontology annotations and sequence homology – as a means to assign reliability to the protein-protein interactions in Saccharomyces cerevisiae determined by high-throughput experiments. Using Bayesian network approaches, we show that protein-protein interactions from high-throughput data supported by one or more genomic features have a higher likelihood ratio and hence are more likely to be real interactions. Our method has a high sensitivity (90% and good specificity (63%. We show that 56% of the interactions from high-throughput experiments in Saccharomyces cerevisiae have high reliability. We use the method to estimate the number of true interactions in the high-throughput protein-protein interaction data sets in Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens to be 27%, 18% and 68% respectively. Our results are available for searching and downloading at http://helix.protein.osaka-u.ac.jp/htp/. Conclusion A combination of genomic features that include sequence, structure and annotation information is a good predictor of true interactions in large and noisy high-throughput data sets. The method has a very high sensitivity and good specificity and can be used to assign a likelihood ratio, corresponding to the reliability, to each interaction.

InSourcerer: a high-throughput method to search for unknown metabolite modifications by mass spectrometry.

Science.gov (United States)

Mrzic, Aida; Lermyte, Frederik; Vu, Trung Nghia; Valkenborg, Dirk; Laukens, Kris

2017-09-15

Using mass spectrometry, the analysis of known metabolite structures has become feasible in a systematic high-throughput fashion. Nevertheless, the identification of previously unknown structures remains challenging, partially because many unidentified variants originate from known molecules that underwent unexpected modifications. Here, we present a method for the discovery of unknown metabolite modifications and conjugate metabolite isoforms in a high-throughput fashion. The method is based on user-controlled in-source fragmentation which is used to induce loss of weakly bound modifications. This is followed by the comparison of product ions from in-source fragmentation and collision-induced dissociation (CID). Diagonal MS 2 -MS 3 matching allows the detection of unknown metabolite modifications, as well as substructure similarities. As the method relies heavily on the advantages of in-source fragmentation and its ability to 'magically' elucidate unknown modification, we have named it inSourcerer as a portmanteau of in-source and sorcerer. The method was evaluated using a set of 15 different cytokinin standards. Product ions from in-source fragmentation and CID were compared. Hierarchical clustering revealed that good matches are due to the presence of common substructures. Plant leaf extract, spiked with a mix of all 15 standards, was used to demonstrate the method's ability to detect these standards in a complex mixture, as well as confidently identify compounds already present in the plant material. Here we present a method that incorporates a classic liquid chromatography/mass spectrometry (LC/MS) workflow with fragmentation models and computational algorithms. The assumptions upon which the concept of the method was built were shown to be valid and the method showed that in-source fragmentation can be used to pinpoint structural similarities and indicate the occurrence of a modification. Copyright © 2017 John Wiley & Sons, Ltd.

High throughput production of mouse monoclonal antibodies using antigen microarrays

DEFF Research Database (Denmark)

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

2005-01-01

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

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

Science.gov (United States)

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

2014-01-01

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

High-throughput screening to identify inhibitors of lysine demethylases.

Science.gov (United States)

Gale, Molly; Yan, Qin

2015-01-01

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

Ultra-high-throughput screening method for the directed evolution of glucose oxidase.

Science.gov (United States)

Ostafe, Raluca; Prodanovic, Radivoje; Nazor, Jovana; Fischer, Rainer

2014-03-20

Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

Solion ion source for high-efficiency, high-throughput solar cell manufacturing

Energy Technology Data Exchange (ETDEWEB)

Koo, John, E-mail: john-koo@amat.com; Binns, Brant; Miller, Timothy; Krause, Stephen; Skinner, Wesley; Mullin, James [Applied Materials, Inc., Varian Semiconductor Equipment Business Unit, 35 Dory Road, Gloucester, Massachusetts 01930 (United States)

2014-02-15

In this paper, we introduce the Solion ion source for high-throughput solar cell doping. As the source power is increased to enable higher throughput, negative effects degrade the lifetime of the plasma chamber and the extraction electrodes. In order to improve efficiency, we have explored a wide range of electron energies and determined the conditions which best suit production. To extend the lifetime of the source we have developed an in situ cleaning method using only existing hardware. With these combinations, source life-times of >200 h for phosphorous and >100 h for boron ion beams have been achieved while maintaining 1100 cell-per-hour production.

A high-throughput method for assessing chemical toxicity using a Caenorhabditis elegans reproduction assay

International Nuclear Information System (INIS)

Boyd, Windy A.; McBride, Sandra J.; Rice, Julie R.; Snyder, Daniel W.; Freedman, Jonathan H.

2010-01-01

The National Research Council has outlined the need for non-mammalian toxicological models to test the potential health effects of a large number of chemicals while also reducing the use of traditional animal models. The nematode Caenorhabditis elegans is an attractive alternative model because of its well-characterized and evolutionarily conserved biology, low cost, and ability to be used in high-throughput screening. A high-throughput method is described for quantifying the reproductive capacity of C. elegans exposed to chemicals for 48 h from the last larval stage (L4) to adulthood using a COPAS Biosort. Initially, the effects of exposure conditions that could influence reproduction were defined. Concentrations of DMSO vehicle ≤ 1% did not affect reproduction. Previous studies indicated that C. elegans may be influenced by exposure to low pH conditions. At pHs greater than 4.5, C. elegans reproduction was not affected; however below this pH there was a significant decrease in the number of offspring. Cadmium chloride was chosen as a model toxicant to verify that automated measurements were comparable to those of traditional observational studies. EC 50 values for cadmium for automated measurements (176-192 μM) were comparable to those previously reported for a 72-h exposure using manual counting (151 μM). The toxicity of seven test toxicants on C. elegans reproduction was highly correlative with rodent lethality suggesting that this assay may be useful in predicting the potential toxicity of chemicals in other organisms.

High Throughput Transcriptomics @ USEPA (Toxicology ...

Science.gov (United States)

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

High-throughput purification of recombinant proteins using self-cleaving intein tags.

Science.gov (United States)

Coolbaugh, M J; Shakalli Tang, M J; Wood, D W

2017-01-01

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format. Copyright © 2016 Elsevier Inc. All rights reserved.

Application of ToxCast High-Throughput Screening and ...

Science.gov (United States)

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

Quantitative in vitro-to-in vivo extrapolation in a high-throughput environment

International Nuclear Information System (INIS)

Wetmore, Barbara A.

2015-01-01

High-throughput in vitro toxicity screening provides an efficient way to identify potential biological targets for environmental and industrial chemicals while conserving limited testing resources. However, reliance on the nominal chemical concentrations in these in vitro assays as an indicator of bioactivity may misrepresent potential in vivo effects of these chemicals due to differences in clearance, protein binding, bioavailability, and other pharmacokinetic factors. Development of high-throughput in vitro hepatic clearance and protein binding assays and refinement of quantitative in vitro-to-in vivo extrapolation (QIVIVE) methods have provided key tools to predict xenobiotic steady state pharmacokinetics. Using a process known as reverse dosimetry, knowledge of the chemical steady state behavior can be incorporated with HTS data to determine the external in vivo oral exposure needed to achieve internal blood concentrations equivalent to those eliciting bioactivity in the assays. These daily oral doses, known as oral equivalents, can be compared to chronic human exposure estimates to assess whether in vitro bioactivity would be expected at the dose-equivalent level of human exposure. This review will describe the use of QIVIVE methods in a high-throughput environment and the promise they hold in shaping chemical testing priorities and, potentially, high-throughput risk assessment strategies

A rapid enzymatic assay for high-throughput screening of adenosine-producing strains

Science.gov (United States)

Dong, Huina; Zu, Xin; Zheng, Ping; Zhang, Dawei

2015-01-01

Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3, the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples. PMID:25580842

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

Science.gov (United States)

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

2012-05-01

Genomic selection (GS) and high-throughput phenotyping have recently been captivating the interest of the crop breeding community from both the public and private sectors world-wide. Both approaches promise to revolutionize the prediction of complex traits, including growth, yield and adaptation to stress. Whereas high-throughput phenotyping may help to improve understanding of crop physiology, most powerful techniques for high-throughput field phenotyping are empirical rather than analytical and comparable to genomic selection. Despite the fact that the two methodological approaches represent the extremes of what is understood as the breeding process (phenotype versus genome), they both consider the targeted traits (e.g. grain yield, growth, phenology, plant adaptation to stress) as a black box instead of dissecting them as a set of secondary traits (i.e. physiological) putatively related to the target trait. Both GS and high-throughput phenotyping have in common their empirical approach enabling breeders to use genome profile or phenotype without understanding the underlying biology. This short review discusses the main aspects of both approaches and focuses on the case of genomic selection of maize flowering traits and near-infrared spectroscopy (NIRS) and plant spectral reflectance as high-throughput field phenotyping methods for complex traits such as crop growth and yield. © 2012 Institute of Botany, Chinese Academy of Sciences.

High throughput experimentation for the discovery of new catalysts

International Nuclear Information System (INIS)

Thomson, S.; Hoffmann, C.; Johann, T.; Wolf, A.; Schmidt, H.-W.; Farrusseng, D.; Schueth, F.

2002-01-01

Full text: The use of combinatorial chemistry to obtain new materials has been developed extensively by the pharmaceutical and biochemical industries, but such approaches have been slow to impact on the field of heterogeneous catalysis. The reasons for this lie in with difficulties associated in the synthesis, characterisation and determination of catalytic properties of such materials. In many synthetic and catalytic reactions, the conditions used are difficult to emulate using High Throughput Experimentation (HTE). Furthermore, the ability to screen these catalysts simultaneously in real time, requires the development and/or modification of characterisation methods. Clearly, there is a need for both high throughput synthesis and screening of new and novel reactions, and we describe several new concepts that help to achieve these goals. Although such problems have impeded the development of combinatorial catalysis, the fact remains that many highly attractive processes still exist for which no suitable catalysts have been developed. The ability to decrease the tiFme needed to evaluate catalyst is therefore essential and this makes the use of high throughput techniques highly desirable. In this presentation we will describe the synthesis, catalytic testing, and novel screening methods developed at the Max Planck Institute. Automated synthesis procedures, performed by the use of a modified Gilson pipette robot, will be described, as will the development of two 16 and 49 sample fixed bed reactors and two 25 and 29 sample three phase reactors for catalytic testing. We will also present new techniques for the characterisation of catalysts and catalytic products using standard IR microscopy and infrared focal plane array detection, respectively

A conifer-friendly high-throughput α-cellulose extraction method for δ13C and δ18O stable isotope ratio analysis

Science.gov (United States)

Lin, W.; Noormets, A.; domec, J.; King, J. S.; Sun, G.; McNulty, S.

2012-12-01

Wood stable isotope ratios (δ13C and δ18O) offer insight to water source and plant water use efficiency (WUE), which in turn provide a glimpse to potential plant responses to changing climate, particularly rainfall patterns. The synthetic pathways of cell wall deposition in wood rings differ in their discrimination ratios between the light and heavy isotopes, and α-cellulose is broadly seen as the best indicator of plant water status due to its local and temporal fixation and to its high abundance within the wood. To use the effects of recent severe droughts on the WUE of loblolly pine (Pinus taeda) throughout Southeastern USA as a harbinger of future changes, an effort has been undertaken to sample the entire range of the species and to sample the isotopic composition in a consistent manner. To be able to accommodate the large number of samples required by this analysis, we have developed a new high-throughput method for α-cellulose extraction, which is the rate-limiting step in such an endeavor. Although an entire family of methods has been developed and perform well, their throughput in a typical research lab setting is limited to 16-75 samples per week with intensive labor input. The resin exclusion step in conifersis is particularly time-consuming. We have combined the recent advances of α-cellulose extraction in plant ecology and wood science, including a high-throughput extraction device developed in the Potsdam Dendro Lab and a simple chemical-based resin exclusion method. By transferring the entire extraction process to a multiport-based system allows throughputs of up to several hundred samples in two weeks, while minimizing labor requirements to 2-3 days per batch of samples.

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

Science.gov (United States)

Zaromb, Solomon

2000-01-01

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

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

International Nuclear Information System (INIS)

2000-01-01

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

High-throughput technology for novel SO2 oxidation catalysts

International Nuclear Information System (INIS)

Loskyll, Jonas; Stoewe, Klaus; Maier, Wilhelm F

2011-01-01

We review the state of the art and explain the need for better SO 2 oxidation catalysts for the production of sulfuric acid. A high-throughput technology has been developed for the study of potential catalysts in the oxidation of SO 2 to SO 3 . High-throughput methods are reviewed and the problems encountered with their adaptation to the corrosive conditions of SO 2 oxidation are described. We show that while emissivity-corrected infrared thermography (ecIRT) can be used for primary screening, it is prone to errors because of the large variations in the emissivity of the catalyst surface. UV-visible (UV-Vis) spectrometry was selected instead as a reliable analysis method of monitoring the SO 2 conversion. Installing plain sugar absorbents at reactor outlets proved valuable for the detection and quantitative removal of SO 3 from the product gas before the UV-Vis analysis. We also overview some elements used for prescreening and those remaining after the screening of the first catalyst generations. (topical review)

High throughput nonparametric probability density estimation.

Science.gov (United States)

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 Cloning and Expression Library Creation for Functional Proteomics

Science.gov (United States)

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 single nucleotide polymorphism genotyping using nanofluidic Dynamic Arrays

Directory of Open Access Journals (Sweden)

Crenshaw Andrew

2009-01-01

Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs have emerged as the genetic marker of choice for mapping disease loci and candidate gene association studies, because of their high density and relatively even distribution in the human genomes. There is a need for systems allowing medium multiplexing (ten to hundreds of SNPs with high throughput, which can efficiently and cost-effectively generate genotypes for a very large sample set (thousands of individuals. Methods that are flexible, fast, accurate and cost-effective are urgently needed. This is also important for those who work on high throughput genotyping in non-model systems where off-the-shelf assays are not available and a flexible platform is needed. Results We demonstrate the use of a nanofluidic Integrated Fluidic Circuit (IFC - based genotyping system for medium-throughput multiplexing known as the Dynamic Array, by genotyping 994 individual human DNA samples on 47 different SNP assays, using nanoliter volumes of reagents. Call rates of greater than 99.5% and call accuracies of greater than 99.8% were achieved from our study, which demonstrates that this is a formidable genotyping platform. The experimental set up is very simple, with a time-to-result for each sample of about 3 hours. Conclusion Our results demonstrate that the Dynamic Array is an excellent genotyping system for medium-throughput multiplexing (30-300 SNPs, which is simple to use and combines rapid throughput with excellent call rates, high concordance and low cost. The exceptional call rates and call accuracy obtained may be of particular interest to those working on validation and replication of genome- wide- association (GWA studies.

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

Directory of Open Access Journals (Sweden)

Mateusz G Adamski

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

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

Science.gov (United States)

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

High-throughput screening with micro-x-ray fluorescence

International Nuclear Information System (INIS)

Havrilla, George J.; Miller, Thomasin C.

2005-01-01

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

High-throughput fragment screening by affinity LC-MS.

Science.gov (United States)

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

2013-02-01

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

Novel method for high-throughput phenotyping of sleep in mice.

Science.gov (United States)

Pack, Allan I; Galante, Raymond J; Maislin, Greg; Cater, Jacqueline; Metaxas, Dimitris; Lu, Shan; Zhang, Lin; Von Smith, Randy; Kay, Timothy; Lian, Jie; Svenson, Karen; Peters, Luanne L

2007-01-17

Assessment of sleep in mice currently requires initial implantation of chronic electrodes for assessment of electroencephalogram (EEG) and electromyogram (EMG) followed by time to recover from surgery. Hence, it is not ideal for high-throughput screening. To address this deficiency, a method of assessment of sleep and wakefulness in mice has been developed based on assessment of activity/inactivity either by digital video analysis or by breaking infrared beams in the mouse cage. It is based on the algorithm that any episode of continuous inactivity of > or =40 s is predicted to be sleep. The method gives excellent agreement in C57BL/6J male mice with simultaneous assessment of sleep by EEG/EMG recording. The average agreement over 8,640 10-s epochs in 24 h is 92% (n = 7 mice) with agreement in individual mice being 88-94%. Average EEG/EMG determined sleep per 2-h interval across the day was 59.4 min. The estimated mean difference (bias) per 2-h interval between inactivity-defined sleep and EEG/EMG-defined sleep was only 1.0 min (95% confidence interval for mean bias -0.06 to +2.6 min). The standard deviation of differences (precision) was 7.5 min per 2-h interval with 95% limits of agreement ranging from -13.7 to +15.7 min. Although bias significantly varied by time of day (P = 0.0007), the magnitude of time-of-day differences was not large (average bias during lights on and lights off was +5.0 and -3.0 min per 2-h interval, respectively). This method has applications in chemical mutagenesis and for studies of molecular changes in brain with sleep/wakefulness.

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

Energy Technology Data Exchange (ETDEWEB)

NONE

2000-07-11

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

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

Science.gov (United States)

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.

An UPLC-MS/MS method for highly sensitive high-throughput analysis of phytohormones in plant tissues

Directory of Open Access Journals (Sweden)

Balcke Gerd Ulrich

2012-11-01

Full Text Available Abstract Background Phytohormones are the key metabolites participating in the regulation of multiple functions of plant organism. Among them, jasmonates, as well as abscisic and salicylic acids are responsible for triggering and modulating plant reactions targeted against pathogens and herbivores, as well as resistance to abiotic stress (drought, UV-irradiation and mechanical wounding. These factors induce dramatic changes in phytohormone biosynthesis and transport leading to rapid local and systemic stress responses. Understanding of underlying mechanisms is of principle interest for scientists working in various areas of plant biology. However, highly sensitive, precise and high-throughput methods for quantification of these phytohormones in small samples of plant tissues are still missing. Results Here we present an LC-MS/MS method for fast and highly sensitive determination of jasmonates, abscisic and salicylic acids. A single-step sample preparation procedure based on mixed-mode solid phase extraction was efficiently combined with essential improvements in mobile phase composition yielding higher efficiency of chromatographic separation and MS-sensitivity. This strategy resulted in dramatic increase in overall sensitivity, allowing successful determination of phytohormones in small (less than 50 mg of fresh weight tissue samples. The method was completely validated in terms of analyte recovery, sensitivity, linearity and precision. Additionally, it was cross-validated with a well-established GC-MS-based procedure and its applicability to a variety of plant species and organs was verified. Conclusion The method can be applied for the analyses of target phytohormones in small tissue samples obtained from any plant species and/or plant part relying on any commercially available (even less sensitive tandem mass spectrometry instrumentation.

Achieving high data throughput in research networks

International Nuclear Information System (INIS)

Matthews, W.; Cottrell, L.

2001-01-01

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

Achieving High Data Throughput in Research Networks

International Nuclear Information System (INIS)

Matthews, W

2004-01-01

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

High Throughput Determinations of Critical Dosing Parameters (IVIVE workshop)

Science.gov (United States)

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

High-Throughput Cancer Cell Sphere Formation for 3D Cell Culture.

Science.gov (United States)

Chen, Yu-Chih; Yoon, Euisik

2017-01-01

Three-dimensional (3D) cell culture is critical in studying cancer pathology and drug response. Though 3D cancer sphere culture can be performed in low-adherent dishes or well plates, the unregulated cell aggregation may skew the results. On contrary, microfluidic 3D culture can allow precise control of cell microenvironments, and provide higher throughput by orders of magnitude. In this chapter, we will look into engineering innovations in a microfluidic platform for high-throughput cancer cell sphere formation and review the implementation methods in detail.

A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers

Energy Technology Data Exchange (ETDEWEB)

Avonto, Cristina; Chittiboyina, Amar G. [National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677 (United States); Rua, Diego [The Center for Food Safety and Applied Nutrition, US Food and Drug Administration, College Park, MD 20740 (United States); Khan, Ikhlas A., E-mail: ikhan@olemiss.edu [National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677 (United States); Division of Pharmacognosy, Department of BioMolecular Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677 (United States)

2015-12-01

Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles after incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, ‘HTS-DCYA assay’, is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time. - Highlights: • A novel fluorescence-based method to detect electrophilic sensitizers is proposed. • A model fluorescent thiol was used to directly quantify the reaction products. • A discussion of the reaction workflow

A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers

International Nuclear Information System (INIS)

Avonto, Cristina; Chittiboyina, Amar G.; Rua, Diego; Khan, Ikhlas A.

2015-01-01

Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles after incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, ‘HTS-DCYA assay’, is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time. - Highlights: • A novel fluorescence-based method to detect electrophilic sensitizers is proposed. • A model fluorescent thiol was used to directly quantify the reaction products. • A discussion of the reaction workflow

High-throughput fractionation of human plasma for fast enrichment of low- and high-abundance proteins.

Science.gov (United States)

Breen, Lucas; Cao, Lulu; Eom, Kirsten; Srajer Gajdosik, Martina; Camara, Lila; Giacometti, Jasminka; Dupuy, Damian E; Josic, Djuro

2012-05-01

Fast, cost-effective and reproducible isolation of IgM from plasma is invaluable to the study of IgM and subsequent understanding of the human immune system. Additionally, vast amounts of information regarding human physiology and disease can be derived from analysis of the low abundance proteome of the plasma. In this study, methods were optimized for both the high-throughput isolation of IgM from human plasma, and the high-throughput isolation and fractionation of low abundance plasma proteins. To optimize the chromatographic isolation of IgM from human plasma, many variables were examined including chromatography resin, mobile phases, and order of chromatographic separations. Purification of IgM was achieved most successfully through isolation of immunoglobulin from human plasma using Protein A chromatography with a specific resin followed by subsequent fractionation using QA strong anion exchange chromatography. Through these optimization experiments, an additional method was established to prepare plasma for analysis of low abundance proteins. This method involved chromatographic depletion of high-abundance plasma proteins and reduction of plasma proteome complexity through further chromatographic fractionation. Purification of IgM was achieved with high purity as confirmed by SDS-PAGE and IgM-specific immunoblot. Isolation and fractionation of low abundance protein was also performed successfully, as confirmed by SDS-PAGE and mass spectrometry analysis followed by label-free quantitative spectral analysis. The level of purity of the isolated IgM allows for further IgM-specific analysis of plasma samples. The developed fractionation scheme can be used for high throughput screening of human plasma in order to identify low and high abundance proteins as potential prognostic and diagnostic disease biomarkers.

High Throughput Single-cell and Multiple-cell Micro-encapsulation

OpenAIRE

Lagus, Todd P.; Edd, Jon F.

2012-01-01

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of ...

High throughput LC-MS/MS method for the simultaneous analysis of multiple vitamin D analytes in serum.

Science.gov (United States)

Jenkinson, Carl; Taylor, Angela E; Hassan-Smith, Zaki K; Adams, John S; Stewart, Paul M; Hewison, Martin; Keevil, Brian G

2016-03-01

Recent studies suggest that vitamin D-deficiency is linked to increased risk of common human health problems. To define vitamin D 'status' most routine analytical methods quantify one particular vitamin D metabolite, 25-hydroxyvitamin D3 (25OHD3). However, vitamin D is characterized by complex metabolic pathways, and simultaneous measurement of multiple vitamin D metabolites may provide a more accurate interpretation of vitamin D status. To address this we developed a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyse multiple vitamin D analytes, with particular emphasis on the separation of epimer metabolites. A supportive liquid-liquid extraction (SLE) and LC-MS/MS method was developed to quantify 10 vitamin D metabolites as well as separation of an interfering 7α-hydroxy-4-cholesten-3-one (7αC4) isobar (precursor of bile acid), and validated by analysis of human serum samples. In a cohort of 116 healthy subjects, circulating concentrations of 25-hydroxyvitamin D3 (25OHD3), 3-epi-25-hydroxyvitamin D3 (3-epi-25OHD3), 24,25-dihydroxyvitamin D3 (24R,25(OH)2D3), 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3), and 25-hydroxyvitamin D2 (25OHD2) were quantifiable using 220μL of serum, with 25OHD3 and 24R,25(OH)2D3 showing significant seasonal variations. This high-throughput LC-MS/MS method provides a novel strategy for assessing the impact of vitamin D on human health and disease. Copyright © 2016 Elsevier B.V. All rights reserved.

High-throughput verification of transcriptional starting sites by Deep-RACE

DEFF Research Database (Denmark)

Olivarius, Signe; Plessy, Charles; Carninci, Piero

2009-01-01

We present a high-throughput method for investigating the transcriptional starting sites of genes of interest, which we named Deep-RACE (Deep–rapid amplification of cDNA ends). Taking advantage of the latest sequencing technology, it allows the parallel analysis of multiple genes and is free...

Applications of ambient mass spectrometry in high-throughput screening.

Science.gov (United States)

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

2013-06-07

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

Novel high-throughput cell-based hybridoma screening methodology using the Celigo Image Cytometer.

Science.gov (United States)

Zhang, Haohai; Chan, Leo Li-Ying; Rice, William; Kassam, Nasim; Longhi, Maria Serena; Zhao, Haitao; Robson, Simon C; Gao, Wenda; Wu, Yan

2017-08-01

Hybridoma screening is a critical step for antibody discovery, which necessitates prompt identification of potential clones from hundreds to thousands of hybridoma cultures against the desired immunogen. Technical issues associated with ELISA- and flow cytometry-based screening limit accuracy and diminish high-throughput capability, increasing time and cost. Conventional ELISA screening with coated antigen is also impractical for difficult-to-express hydrophobic membrane antigens or multi-chain protein complexes. Here, we demonstrate novel high-throughput screening methodology employing the Celigo Image Cytometer, which avoids nonspecific signals by contrasting antibody binding signals directly on living cells, with and without recombinant antigen expression. The image cytometry-based high-throughput screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on Chinese hamster ovary (CHO) cells. Next, the sensitivity of the image cytometer was demonstrated by serial dilution of purified CD39 antibody. Celigo was used to measure antibody affinities of commercial and in-house antibodies to membrane-bound CD39. This cell-based screening procedure can be completely accomplished within one day, significantly improving throughput and efficiency of hybridoma screening. Furthermore, measuring direct antibody binding to living cells eliminated both false positive and false negative hits. The image cytometry method was highly sensitive and versatile, and could detect positive antibody in supernatants at concentrations as low as ~5ng/mL, with concurrent K d binding affinity coefficient determination. We propose that this screening method will greatly facilitate antibody discovery and screening technologies. Copyright © 2017 Elsevier B.V. All rights reserved.

Controlling high-throughput manufacturing at the nano-scale

Science.gov (United States)

Cooper, Khershed P.

2013-09-01

Interest in nano-scale manufacturing research and development is growing. The reason is to accelerate the translation of discoveries and inventions of nanoscience and nanotechnology into products that would benefit industry, economy and society. Ongoing research in nanomanufacturing is focused primarily on developing novel nanofabrication techniques for a variety of applications—materials, energy, electronics, photonics, biomedical, etc. Our goal is to foster the development of high-throughput methods of fabricating nano-enabled products. Large-area parallel processing and highspeed continuous processing are high-throughput means for mass production. An example of large-area processing is step-and-repeat nanoimprinting, by which nanostructures are reproduced again and again over a large area, such as a 12 in wafer. Roll-to-roll processing is an example of continuous processing, by which it is possible to print and imprint multi-level nanostructures and nanodevices on a moving flexible substrate. The big pay-off is high-volume production and low unit cost. However, the anticipated cost benefits can only be realized if the increased production rate is accompanied by high yields of high quality products. To ensure product quality, we need to design and construct manufacturing systems such that the processes can be closely monitored and controlled. One approach is to bring cyber-physical systems (CPS) concepts to nanomanufacturing. CPS involves the control of a physical system such as manufacturing through modeling, computation, communication and control. Such a closely coupled system will involve in-situ metrology and closed-loop control of the physical processes guided by physics-based models and driven by appropriate instrumentation, sensing and actuation. This paper will discuss these ideas in the context of controlling high-throughput manufacturing at the nano-scale.

Comprehensive evaluation and optimization of amplicon library preparation methods for high-throughput antibody sequencing.

Science.gov (United States)

Menzel, Ulrike; Greiff, Victor; Khan, Tarik A; Haessler, Ulrike; Hellmann, Ina; Friedensohn, Simon; Cook, Skylar C; Pogson, Mark; Reddy, Sai T

2014-01-01

High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not been thoroughly investigated. Therefore, we compared three standard library preparation methods by performing Illumina HTS on antibody variable heavy genes from murine antibody-secreting cells. Clonal overlap and rank statistics demonstrated that the investigated methods produced equivalent HTS datasets. PCR-based methods were experimentally superior to ligation with respect to speed, efficiency, and practicality. Finally, using a two-step PCR based method we established a protocol for antibody repertoire library generation, beginning from inputs as low as 1 ng of total RNA. In summary, this study represents a major advance towards a standardized experimental framework for antibody HTS, thus opening up the potential for systems-based, cross-experiment meta-analyses of antibody repertoires.

Risk-based high-throughput chemical screening and prioritization using exposure models and in vitro bioactivity assays

DEFF Research Database (Denmark)

Shin, Hyeong-Moo; Ernstoff, Alexi; Arnot, Jon

2015-01-01

We present a risk-based high-throughput screening (HTS) method to identify chemicals for potential health concerns or for which additional information is needed. The method is applied to 180 organic chemicals as a case study. We first obtain information on how the chemical is used and identify....../oral contact, or dermal exposure. The method provides high-throughput estimates of exposure and important input for decision makers to identify chemicals of concern for further evaluation with additional information or more refined models....

Validation of a Microscale Extraction and High Throughput UHPLC-QTOF-MS Analysis Method for Huperzine A in Huperzia

Science.gov (United States)

Cuthbertson, Daniel; Piljac-Žegarac, Jasenka; Lange, Bernd Markus

2011-01-01

Herein we report on an improved method for the microscale extraction of huperzine A (HupA), an acetylcholinesterase-inhibiting alkaloid, from as little as 3 mg of tissue homogenate from the clubmoss Huperzia squarrosa (G. Forst.) Trevis with 99.95 % recovery. We also validated a novel UHPLC-QTOF-MS method for the high-throughput analysis of H. squarrosa extracts in only 6 min, which, in combination with the very low limit of detection (20 pg on column) and the wide linear range for quantification (20 to 10,000 pg on column), allow for a highly efficient screening of extracts containing varying amounts of HupA. Utilization of this methodology has the potential to conserve valuable plant resources. PMID:22275140

Toward reliable and repeatable automated STEM-EDS metrology with high throughput

Science.gov (United States)

Zhong, Zhenxin; Donald, Jason; Dutrow, Gavin; Roller, Justin; Ugurlu, Ozan; Verheijen, Martin; Bidiuk, Oleksii

2018-03-01

New materials and designs in complex 3D architectures in logic and memory devices have raised complexity in S/TEM metrology. In this paper, we report about a newly developed, automated, scanning transmission electron microscopy (STEM) based, energy dispersive X-ray spectroscopy (STEM-EDS) metrology method that addresses these challenges. Different methodologies toward repeatable and efficient, automated STEM-EDS metrology with high throughput are presented: we introduce the best known auto-EDS acquisition and quantification methods for robust and reliable metrology and present how electron exposure dose impacts the EDS metrology reproducibility, either due to poor signalto-noise ratio (SNR) at low dose or due to sample modifications at high dose conditions. Finally, we discuss the limitations of the STEM-EDS metrology technique and propose strategies to optimize the process both in terms of throughput and metrology reliability.

Spectrophotometric Enzyme Assays for High-Throughput Screening

Directory of Open Access Journals (Sweden)

Jean-Louis Reymond

2004-01-01

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

Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format

OpenAIRE

Salvo-Chirnside, Eliane; Kane, Steven; Kerr, Lorraine E

2011-01-01

Abstract The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated p...

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

Science.gov (United States)

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

Insights into Sonogashira cross-coupling by high-throughput kinetics and descriptor modeling

NARCIS (Netherlands)

an der Heiden, M.R.; Plenio, H.; Immel, S.; Burello, E.; Rothenberg, G.; Hoefsloot, H.C.J.

2008-01-01

A method is presented for the high-throughput monitoring of reaction kinetics in homogeneous catalysis, running up to 25 coupling reactions in a single reaction vessel. This method is demonstrated and validated on the Sonogashira reaction, analyzing the kinetics for almost 500 coupling reactions.

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

DEFF Research Database (Denmark)

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

2009-01-01

Technological advances in mass spectrometry and meticulous method development have produced several shotgun lipidomic approaches capable of characterizing lipid species by direct analysis of total lipid extracts. Shotgun lipidomics by hybrid quadrupole time-of-flight mass spectrometry allows...... the absolute quantification of hundreds of molecular glycerophospholipid species, glycerolipid species, sphingolipid species and sterol lipids. Future applications in clinical cohort studies demand detailed lipid molecule information and the application of high-throughput lipidomics platforms. In this review...... we describe a novel high-throughput shotgun lipidomic platform based on 96-well robot-assisted lipid extraction, automated sample infusion by mircofluidic-based nanoelectrospray ionization, and quantitative multiple precursor ion scanning analysis on a quadrupole time-of-flight mass spectrometer...

High-Throughput Flow Cytometric Method for the Simultaneous Measurement of CAR-T Cell Characterization and Cytotoxicity against Solid Tumor Cell Lines.

Science.gov (United States)

Martinez, Emily M; Klebanoff, Samuel D; Secrest, Stephanie; Romain, Gabrielle; Haile, Samuel T; Emtage, Peter C R; Gilbert, Amy E

2018-04-01

High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell-dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be challenging in these flow-based assays. Here, we present the development of a high-throughput flow-based assay to measure TDCC for a CAR-T construct co-cultured with multiple adherent tumor cell lines. We describe optimal assay conditions (such as adherent cell dissociation techniques to minimize impact on cell viability) that result in robust cytotoxicity assays. In addition, we report on the concurrent use of T cell transduction and activation antibody panels (CD25) that provide further dissection of engineered T cell function. In conclusion, we present the development of a high-throughput flow cytometry method allowing for in vitro interrogation of solid tumor, targeting CAR-T cell-mediated cytotoxicity, CAR transduction, and engineered T cell characterization in a single assay.

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

Science.gov (United States)

Moutsatsos, Ioannis K; Parker, Christian N

2016-01-01

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

High-throughput scoring of seed germination

NARCIS (Netherlands)

Ligterink, Wilco; Hilhorst, Henk W.M.

2017-01-01

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

ExSTA: External Standard Addition Method for Accurate High-Throughput Quantitation in Targeted Proteomics Experiments.

Science.gov (United States)

Mohammed, Yassene; Pan, Jingxi; Zhang, Suping; Han, Jun; Borchers, Christoph H

2018-03-01

Targeted proteomics using MRM with stable-isotope-labeled internal-standard (SIS) peptides is the current method of choice for protein quantitation in complex biological matrices. Better quantitation can be achieved with the internal standard-addition method, where successive increments of synthesized natural form (NAT) of the endogenous analyte are added to each sample, a response curve is generated, and the endogenous concentration is determined at the x-intercept. Internal NAT-addition, however, requires multiple analyses of each sample, resulting in increased sample consumption and analysis time. To compare the following three methods, an MRM assay for 34 high-to-moderate abundance human plasma proteins is used: classical internal SIS-addition, internal NAT-addition, and external NAT-addition-generated in buffer using NAT and SIS peptides. Using endogenous-free chicken plasma, the accuracy is also evaluated. The internal NAT-addition outperforms the other two in precision and accuracy. However, the curves derived by internal vs. external NAT-addition differ by only ≈3.8% in slope, providing comparable accuracies and precision with good CV values. While the internal NAT-addition method may be "ideal", this new external NAT-addition can be used to determine the concentration of high-to-moderate abundance endogenous plasma proteins, providing a robust and cost-effective alternative for clinical analyses or other high-throughput applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel mean-centering method for normalizing microRNA expression from high-throughput RT-qPCR data

Directory of Open Access Journals (Sweden)

Wylie Dennis

2011-12-01

Full Text Available Abstract Background Normalization is critical for accurate gene expression analysis. A significant challenge in the quantitation of gene expression from biofluids samples is the inability to quantify RNA concentration prior to analysis, underscoring the need for robust normalization tools for this sample type. In this investigation, we evaluated various methods of normalization to determine the optimal approach for quantifying microRNA (miRNA expression from biofluids and tissue samples when using the TaqMan® Megaplex™ high-throughput RT-qPCR platform with low RNA inputs. Findings We compared seven normalization methods in the analysis of variation of miRNA expression from biofluid and tissue samples. We developed a novel variant of the common mean-centering normalization strategy, herein referred to as mean-centering restricted (MCR normalization, which is adapted to the TaqMan Megaplex RT-qPCR platform, but is likely applicable to other high-throughput RT-qPCR-based platforms. Our results indicate that MCR normalization performs comparable to or better than both standard mean-centering and other normalization methods. We also propose an extension of this method to be used when migrating biomarker signatures from Megaplex to singleplex RT-qPCR platforms, based on the identification of a small number of normalizer miRNAs that closely track the mean of expressed miRNAs. Conclusions We developed the MCR method for normalizing miRNA expression from biofluids samples when using the TaqMan Megaplex RT-qPCR platform. Our results suggest that normalization based on the mean of all fully observed (fully detected miRNAs minimizes technical variance in normalized expression values, and that a small number of normalizer miRNAs can be selected when migrating from Megaplex to singleplex assays. In our study, we find that normalization methods that focus on a restricted set of miRNAs tend to perform better than methods that focus on all miRNAs, including

Applications of high-throughput clonogenic survival assays in high-LET particle microbeams

Directory of Open Access Journals (Sweden)

Antonios eGeorgantzoglou

2016-01-01

Full Text Available Charged particle therapy is increasingly becoming a valuable tool in cancer treatment, mainly due to the favorable interaction of particle radiation with matter. Its application is still limited due, in part, to lack of data regarding the radiosensitivity of certain cell lines to this radiation type, especially to high-LET particles. From the earliest days of radiation biology, the clonogenic survival assay has been used to provide radiation response data. This method produces reliable data but it is not optimized for high-throughput microbeam studies with high-LET radiation where high levels of cell killing lead to a very low probability of maintaining cells’ clonogenic potential. A new method, therefore, is proposed in this paper, which could potentially allow these experiments to be conducted in a high-throughput fashion. Cells are seeded in special polypropylene dishes and bright-field illumination provides cell visualization. Digital images are obtained and cell detection is applied based on corner detection, generating individual cell targets as x-y points. These points in the dish are then irradiated individually by a micron field size high-LET microbeam. Post-irradiation, time-lapse imaging follows cells’ response. All irradiated cells are tracked by linking trajectories in all time-frames, based on finding their nearest position. Cell divisions are detected based on cell appearance and individual cell temporary corner density. The number of divisions anticipated is low due to the high probability of cell killing from high-LET irradiation. Survival curves are produced based on cell’s capacity to divide at least 4-5 times. The process is repeated for a range of doses of radiation. Validation shows the efficiency of the proposed cell detection and tracking method in finding cell divisions.

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

Science.gov (United States)

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

2015-01-01

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

Blood group genotyping: from patient to high-throughput donor screening.

Science.gov (United States)

Veldhuisen, B; van der Schoot, C E; de Haas, M

2009-10-01

Blood group antigens, present on the cell membrane of red blood cells and platelets, can be defined either serologically or predicted based on the genotypes of genes encoding for blood group antigens. At present, the molecular basis of many antigens of the 30 blood group systems and 17 human platelet antigens is known. In many laboratories, blood group genotyping assays are routinely used for diagnostics in cases where patient red cells cannot be used for serological typing due to the presence of auto-antibodies or after recent transfusions. In addition, DNA genotyping is used to support (un)-expected serological findings. Fetal genotyping is routinely performed when there is a risk of alloimmune-mediated red cell or platelet destruction. In case of patient blood group antigen typing, it is important that a genotyping result is quickly available to support the selection of donor blood, and high-throughput of the genotyping method is not a prerequisite. In addition, genotyping of blood donors will be extremely useful to obtain donor blood with rare phenotypes, for example lacking a high-frequency antigen, and to obtain a fully typed donor database to be used for a better matching between recipient and donor to prevent adverse transfusion reactions. Serological typing of large cohorts of donors is a labour-intensive and expensive exercise and hampered by the lack of sufficient amounts of approved typing reagents for all blood group systems of interest. Currently, high-throughput genotyping based on DNA micro-arrays is a very feasible method to obtain a large pool of well-typed blood donors. Several systems for high-throughput blood group genotyping are developed and will be discussed in this review.

Optimization and high-throughput screening of antimicrobial peptides.

Science.gov (United States)

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.

Statistical methods for the analysis of high-throughput metabolomics data

Directory of Open Access Journals (Sweden)

Fabian J. Theis

2013-01-01

Full Text Available Metabolomics is a relatively new high-throughput technology that aims at measuring all endogenous metabolites within a biological sample in an unbiased fashion. The resulting metabolic profiles may be regarded as functional signatures of the physiological state, and have been shown to comprise effects of genetic regulation as well as environmental factors. This potential to connect genotypic to phenotypic information promises new insights and biomarkers for different research fields, including biomedical and pharmaceutical research. In the statistical analysis of metabolomics data, many techniques from other omics fields can be reused. However recently, a number of tools specific for metabolomics data have been developed as well. The focus of this mini review will be on recent advancements in the analysis of metabolomics data especially by utilizing Gaussian graphical models and independent component analysis.

Fluorescent foci quantitation for high-throughput analysis

Directory of Open Access Journals (Sweden)

Elena Ledesma-Fernández

2015-06-01

Full Text Available A number of cellular proteins localize to discrete foci within cells, for example DNA repair proteins, microtubule organizing centers, P bodies or kinetochores. It is often possible to measure the fluorescence emission from tagged proteins within these foci as a surrogate for the concentration of that specific protein. We wished to develop tools that would allow quantitation of fluorescence foci intensities in high-throughput studies. As proof of principle we have examined the kinetochore, a large multi-subunit complex that is critical for the accurate segregation of chromosomes during cell division. Kinetochore perturbations lead to aneuploidy, which is a hallmark of cancer cells. Hence, understanding kinetochore homeostasis and regulation are important for a global understanding of cell division and genome integrity. The 16 budding yeast kinetochores colocalize within the nucleus to form a single focus. Here we have created a set of freely-available tools to allow high-throughput quantitation of kinetochore foci fluorescence. We use this ‘FociQuant’ tool to compare methods of kinetochore quantitation and we show proof of principle that FociQuant can be used to identify changes in kinetochore protein levels in a mutant that affects kinetochore function. This analysis can be applied to any protein that forms discrete foci in cells.

High-Throughput Next-Generation Sequencing of Polioviruses

Science.gov (United States)

Montmayeur, Anna M.; Schmidt, Alexander; Zhao, Kun; Magaña, Laura; Iber, Jane; Castro, Christina J.; Chen, Qi; Henderson, Elizabeth; Ramos, Edward; Shaw, Jing; Tatusov, Roman L.; Dybdahl-Sissoko, Naomi; Endegue-Zanga, Marie Claire; Adeniji, Johnson A.; Oberste, M. Steven; Burns, Cara C.

2016-01-01

ABSTRACT The poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance. PMID:27927929

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

Science.gov (United States)

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...

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

DEFF Research Database (Denmark)

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

2011-01-01

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

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

Science.gov (United States)

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.

A high throughput array microscope for the mechanical characterization of biomaterials

Science.gov (United States)

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

2015-02-01

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

The introduction of high-throughput experimentation methods for Suzuki-Miyaura coupling reactions in University education

NARCIS (Netherlands)

Hoogenboom, R.; Meier, M.A.R.; Schubert, U.S.

2005-01-01

Use of high-throughput experimentation is becoming common in industry. To prepare students to work with those novel techniques in their future careers, the utilization of an automated synthesis robot was integrated into an undergraduate research project. The practical course included performing a

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

Science.gov (United States)

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

2015-01-01

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

High-throughput sequence alignment using Graphics Processing Units

Directory of Open Access Journals (Sweden)

Trapnell Cole

2007-12-01

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

Patterning cell using Si-stencil for high-throughput assay

KAUST Repository

Wu, Jinbo

2011-01-01

In this communication, we report a newly developed cell pattering methodology by a silicon-based stencil, which exhibited advantages such as easy handling, reusability, hydrophilic surface and mature fabrication technologies. Cell arrays obtained by this method were used to investigate cell growth under a temperature gradient, which demonstrated the possibility of studying cell behavior in a high-throughput assay. This journal is © The Royal Society of Chemistry 2011.

Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy

DEFF Research Database (Denmark)

Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H

2017-01-01

To target bacterial pathogens that invade and proliferate inside host cells, it is necessary to design intervention strategies directed against bacterial attachment, cellular invasion and intracellular proliferation. We present an automated microscopy-based, fast, high-throughput method for analy...

Modular high-throughput test stand for versatile screening of thin-film materials libraries

International Nuclear Information System (INIS)

Thienhaus, Sigurd; Hamann, Sven; Ludwig, Alfred

2011-01-01

Versatile high-throughput characterization tools are required for the development of new materials using combinatorial techniques. Here, we describe a modular, high-throughput test stand for the screening of thin-film materials libraries, which can carry out automated electrical, magnetic and magnetoresistance measurements in the temperature range of −40 to 300 °C. As a proof of concept, we measured the temperature-dependent resistance of Fe–Pd–Mn ferromagnetic shape-memory alloy materials libraries, revealing reversible martensitic transformations and the associated transformation temperatures. Magneto-optical screening measurements of a materials library identify ferromagnetic samples, whereas resistivity maps support the discovery of new phases. A distance sensor in the same setup allows stress measurements in materials libraries deposited on cantilever arrays. A combination of these methods offers a fast and reliable high-throughput characterization technology for searching for new materials. Using this approach, a composition region has been identified in the Fe–Pd–Mn system that combines ferromagnetism and martensitic transformation.

Polymerase chain reaction-hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation.

Science.gov (United States)

Xu, Chen; Zhang, Nan; Huo, Qianyu; Chen, Minghui; Wang, Rengfeng; Liu, Zhili; Li, Xue; Liu, Yunde; Bao, Huijing

2016-04-15

In this article, we discuss the polymerase chain reaction (PCR)-hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA-BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase-streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR-hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR-hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications. Copyright © 2016 Elsevier Inc. All rights reserved.

High throughput imaging cytometer with acoustic focussing.

Science.gov (United States)

Zmijan, Robert; Jonnalagadda, Umesh S; Carugo, Dario; Kochi, Yu; Lemm, Elizabeth; Packham, Graham; Hill, Martyn; Glynne-Jones, Peter

2015-10-31

We demonstrate an imaging flow cytometer that uses acoustic levitation to assemble cells and other particles into a sheet structure. This technique enables a high resolution, low noise CMOS camera to capture images of thousands of cells with each frame. While ultrasonic focussing has previously been demonstrated for 1D cytometry systems, extending the technology to a planar, much higher throughput format and integrating imaging is non-trivial, and represents a significant jump forward in capability, leading to diagnostic possibilities not achievable with current systems. A galvo mirror is used to track the images of the moving cells permitting exposure times of 10 ms at frame rates of 50 fps with motion blur of only a few pixels. At 80 fps, we demonstrate a throughput of 208 000 beads per second. We investigate the factors affecting motion blur and throughput, and demonstrate the system with fluorescent beads, leukaemia cells and a chondrocyte cell line. Cells require more time to reach the acoustic focus than beads, resulting in lower throughputs; however a longer device would remove this constraint.

High-throughput GPU-based LDPC decoding

Science.gov (United States)

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

2010-08-01

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

A Novel Multiparametric Drug-Scoring Method for High-Throughput Screening of 3D Multicellular Tumor Spheroids Using the Celigo Image Cytometer.

Science.gov (United States)

Cribbes, Scott; Kessel, Sarah; McMenemy, Scott; Qiu, Jean; Chan, Leo Li-Ying

2017-06-01

Three-dimensional (3D) tumor models have been increasingly used to investigate and characterize cancer drug compounds. The ability to perform high-throughput screening of 3D multicellular tumor spheroids (MCTS) can highly improve the efficiency and cost-effectiveness of discovering potential cancer drug candidates. Previously, the Celigo Image Cytometer has demonstrated a novel method for high-throughput screening of 3D multicellular tumor spheroids. In this work, we employed the Celigo Image Cytometer to examine the effects of 14 cancer drug compounds on 3D MCTS of the glioblastoma cell line U87MG in 384-well plates. Using parameters such as MCTS diameter and invasion area, growth and invasion were monitored for 9 and 3 d, respectively. Furthermore, fluorescent staining with calcein AM, propidium iodide, Hoechst 33342, and caspase 3/7 was performed at day 9 posttreatment to measure viability and apoptosis. Using the kinetic and endpoint data generated, we created a novel multiparametric drug-scoring system for 3D MCTS that can be used to identify and classify potential drug candidates earlier in the drug discovery process. Furthermore, the combination of quantitative and qualitative image data can be used to delineate differences between drugs that induce cytotoxic and cytostatic effects. The 3D MCTS-based multiparametric scoring method described here can provide an alternative screening method to better qualify tested drug compounds.

Evaluating High Throughput Toxicokinetics and Toxicodynamics for IVIVE (WC10)

Science.gov (United States)

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...

Study on a digital pulse processing algorithm based on template-matching for high-throughput spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Wen, Xianfei; Yang, Haori

2015-06-01

A major challenge in utilizing spectroscopy techniques for nuclear safeguards is to perform high-resolution measurements at an ultra-high throughput rate. Traditionally, piled-up pulses are rejected to ensure good energy resolution. To improve throughput rate, high-pass filters are normally implemented to shorten pulses. However, this reduces signal-to-noise ratio and causes degradation in energy resolution. In this work, a pulse pile-up recovery algorithm based on template-matching was proved to be an effective approach to achieve high-throughput gamma ray spectroscopy. First, a discussion of the algorithm was given in detail. Second, the algorithm was then successfully utilized to process simulated piled-up pulses from a scintillator detector. Third, the algorithm was implemented to analyze high rate data from a NaI detector, a silicon drift detector and a HPGe detector. The promising results demonstrated the capability of this algorithm to achieve high-throughput rate without significant sacrifice in energy resolution. The performance of the template-matching algorithm was also compared with traditional shaping methods. - Highlights: • A detailed discussion on the template-matching algorithm was given. • The algorithm was tested on data from a NaI and a Si detector. • The algorithm was successfully implemented on high rate data from a HPGe detector. • The performance of the algorithm was compared with traditional shaping methods. • The advantage of the algorithm in active interrogation was discussed.

Subnuclear foci quantification using high-throughput 3D image cytometry

Science.gov (United States)

Wadduwage, Dushan N.; Parrish, Marcus; Choi, Heejin; Engelward, Bevin P.; Matsudaira, Paul; So, Peter T. C.

2015-07-01

Ionising radiation causes various types of DNA damages including double strand breaks (DSBs). DSBs are often recognized by DNA repair protein ATM which forms gamma-H2AX foci at the site of the DSBs that can be visualized using immunohistochemistry. However most of such experiments are of low throughput in terms of imaging and image analysis techniques. Most of the studies still use manual counting or classification. Hence they are limited to counting a low number of foci per cell (5 foci per nucleus) as the quantification process is extremely labour intensive. Therefore we have developed a high throughput instrumentation and computational pipeline specialized for gamma-H2AX foci quantification. A population of cells with highly clustered foci inside nuclei were imaged, in 3D with submicron resolution, using an in-house developed high throughput image cytometer. Imaging speeds as high as 800 cells/second in 3D were achieved by using HiLo wide-field depth resolved imaging and a remote z-scanning technique. Then the number of foci per cell nucleus were quantified using a 3D extended maxima transform based algorithm. Our results suggests that while most of the other 2D imaging and manual quantification studies can count only up to about 5 foci per nucleus our method is capable of counting more than 100. Moreover we show that 3D analysis is significantly superior compared to the 2D techniques.

High-Throughput Dietary Exposure Predictions for Chemical Migrants from Food Packaging Materials

Science.gov (United States)

United States Environmental Protection Agency researchers have developed a Stochastic Human Exposure and Dose Simulation High -Throughput (SHEDS-HT) model for use in prioritization of chemicals under the ExpoCast program. In this research, new methods were implemented in SHEDS-HT...

High-throughput gene expression profiling of memory differentiation in primary human T cells

Directory of Open Access Journals (Sweden)

Russell Kate

2008-08-01

Full Text Available Abstract Background The differentiation of naive T and B cells into memory lymphocytes is essential for immunity to pathogens. Therapeutic manipulation of this cellular differentiation program could improve vaccine efficacy and the in vitro expansion of memory cells. However, chemical screens to identify compounds that induce memory differentiation have been limited by 1 the lack of reporter-gene or functional assays that can distinguish naive and memory-phenotype T cells at high throughput and 2 a suitable cell-line representative of naive T cells. Results Here, we describe a method for gene-expression based screening that allows primary naive and memory-phenotype lymphocytes to be discriminated based on complex genes signatures corresponding to these differentiation states. We used ligation-mediated amplification and a fluorescent, bead-based detection system to quantify simultaneously 55 transcripts representing naive and memory-phenotype signatures in purified populations of human T cells. The use of a multi-gene panel allowed better resolution than any constituent single gene. The method was precise, correlated well with Affymetrix microarray data, and could be easily scaled up for high-throughput. Conclusion This method provides a generic solution for high-throughput differentiation screens in primary human T cells where no single-gene or functional assay is available. This screening platform will allow the identification of small molecules, genes or soluble factors that direct memory differentiation in naive human lymphocytes.

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

Directory of Open Access Journals (Sweden)

Albert-Baskar Arul

2013-06-01

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

The French press: a repeatable and high-throughput approach to exercising zebrafish (Danio rerio).

Science.gov (United States)

Usui, Takuji; Noble, Daniel W A; O'Dea, Rose E; Fangmeier, Melissa L; Lagisz, Malgorzata; Hesselson, Daniel; Nakagawa, Shinichi

2018-01-01

Zebrafish are increasingly used as a vertebrate model organism for various traits including swimming performance, obesity and metabolism, necessitating high-throughput protocols to generate standardized phenotypic information. Here, we propose a novel and cost-effective method for exercising zebrafish, using a coffee plunger and magnetic stirrer. To demonstrate the use of this method, we conducted a pilot experiment to show that this simple system provides repeatable estimates of maximal swim performance (intra-class correlation [ICC] = 0.34-0.41) and observe that exercise training of zebrafish on this system significantly increases their maximum swimming speed. We propose this high-throughput and reproducible system as an alternative to traditional linear chamber systems for exercising zebrafish and similarly sized fishes.

Automated image alignment for 2D gel electrophoresis in a high-throughput proteomics pipeline.

Science.gov (United States)

Dowsey, Andrew W; Dunn, Michael J; Yang, Guang-Zhong

2008-04-01

The quest for high-throughput proteomics has revealed a number of challenges in recent years. Whilst substantial improvements in automated protein separation with liquid chromatography and mass spectrometry (LC/MS), aka 'shotgun' proteomics, have been achieved, large-scale open initiatives such as the Human Proteome Organization (HUPO) Brain Proteome Project have shown that maximal proteome coverage is only possible when LC/MS is complemented by 2D gel electrophoresis (2-DE) studies. Moreover, both separation methods require automated alignment and differential analysis to relieve the bioinformatics bottleneck and so make high-throughput protein biomarker discovery a reality. The purpose of this article is to describe a fully automatic image alignment framework for the integration of 2-DE into a high-throughput differential expression proteomics pipeline. The proposed method is based on robust automated image normalization (RAIN) to circumvent the drawbacks of traditional approaches. These use symbolic representation at the very early stages of the analysis, which introduces persistent errors due to inaccuracies in modelling and alignment. In RAIN, a third-order volume-invariant B-spline model is incorporated into a multi-resolution schema to correct for geometric and expression inhomogeneity at multiple scales. The normalized images can then be compared directly in the image domain for quantitative differential analysis. Through evaluation against an existing state-of-the-art method on real and synthetically warped 2D gels, the proposed analysis framework demonstrates substantial improvements in matching accuracy and differential sensitivity. High-throughput analysis is established through an accelerated GPGPU (general purpose computation on graphics cards) implementation. Supplementary material, software and images used in the validation are available at http://www.proteomegrid.org/rain/.

High-throughput tri-colour flow cytometry technique to assess Plasmodium falciparum parasitaemia in bioassays

DEFF Research Database (Denmark)

Tiendrebeogo, Regis W; Adu, Bright; Singh, Susheel K

2014-01-01

BACKGROUND: Unbiased flow cytometry-based methods have become the technique of choice in many laboratories for high-throughput, accurate assessments of malaria parasites in bioassays. A method to quantify live parasites based on mitotracker red CMXRos was recently described but consistent...... distinction of early ring stages of Plasmodium falciparum from uninfected red blood cells (uRBC) remains a challenge. METHODS: Here, a high-throughput, three-parameter (tri-colour) flow cytometry technique based on mitotracker red dye, the nucleic acid dye coriphosphine O (CPO) and the leucocyte marker CD45...... for enumerating live parasites in bioassays was developed. The technique was applied to estimate the specific growth inhibition index (SGI) in the antibody-dependent cellular inhibition (ADCI) assay and compared to parasite quantification by microscopy and mitotracker red staining. The Bland-Altman analysis...

CrossCheck: an open-source web tool for high-throughput screen data analysis.

Science.gov (United States)

Najafov, Jamil; Najafov, Ayaz

2017-07-19

Modern high-throughput screening methods allow researchers to generate large datasets that potentially contain important biological information. However, oftentimes, picking relevant hits from such screens and generating testable hypotheses requires training in bioinformatics and the skills to efficiently perform database mining. There are currently no tools available to general public that allow users to cross-reference their screen datasets with published screen datasets. To this end, we developed CrossCheck, an online platform for high-throughput screen data analysis. CrossCheck is a centralized database that allows effortless comparison of the user-entered list of gene symbols with 16,231 published datasets. These datasets include published data from genome-wide RNAi and CRISPR screens, interactome proteomics and phosphoproteomics screens, cancer mutation databases, low-throughput studies of major cell signaling mediators, such as kinases, E3 ubiquitin ligases and phosphatases, and gene ontological information. Moreover, CrossCheck includes a novel database of predicted protein kinase substrates, which was developed using proteome-wide consensus motif searches. CrossCheck dramatically simplifies high-throughput screen data analysis and enables researchers to dig deep into the published literature and streamline data-driven hypothesis generation. CrossCheck is freely accessible as a web-based application at http://proteinguru.com/crosscheck.

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

Science.gov (United States)

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

2016-01-29

Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3'-5' exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Infra-red thermography for high throughput field phenotyping in Solanum tuberosum.

Directory of Open Access Journals (Sweden)

Ankush Prashar

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

High Performance Computing Modernization Program Kerberos Throughput Test Report

Science.gov (United States)

2017-10-26

Naval Research Laboratory Washington, DC 20375-5320 NRL/MR/5524--17-9751 High Performance Computing Modernization Program Kerberos Throughput Test ...NUMBER 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 2. REPORT TYPE1. REPORT DATE (DD-MM-YYYY) 4. TITLE AND SUBTITLE 6. AUTHOR(S) 8. PERFORMING...PAGE 18. NUMBER OF PAGES 17. LIMITATION OF ABSTRACT High Performance Computing Modernization Program Kerberos Throughput Test Report Daniel G. Gdula* and

Label-free detection of cellular drug responses by high-throughput bright-field imaging and machine learning.

Science.gov (United States)

Kobayashi, Hirofumi; Lei, Cheng; Wu, Yi; Mao, Ailin; Jiang, Yiyue; Guo, Baoshan; Ozeki, Yasuyuki; Goda, Keisuke

2017-09-29

In the last decade, high-content screening based on multivariate single-cell imaging has been proven effective in drug discovery to evaluate drug-induced phenotypic variations. Unfortunately, this method inherently requires fluorescent labeling which has several drawbacks. Here we present a label-free method for evaluating cellular drug responses only by high-throughput bright-field imaging with the aid of machine learning algorithms. Specifically, we performed high-throughput bright-field imaging of numerous drug-treated and -untreated cells (N = ~240,000) by optofluidic time-stretch microscopy with high throughput up to 10,000 cells/s and applied machine learning to the cell images to identify their morphological variations which are too subtle for human eyes to detect. Consequently, we achieved a high accuracy of 92% in distinguishing drug-treated and -untreated cells without the need for labeling. Furthermore, we also demonstrated that dose-dependent, drug-induced morphological change from different experiments can be inferred from the classification accuracy of a single classification model. Our work lays the groundwork for label-free drug screening in pharmaceutical science and industry.

A high-throughput sample preparation method for cellular proteomics using 96-well filter plates.

Science.gov (United States)

Switzar, Linda; van Angeren, Jordy; Pinkse, Martijn; Kool, Jeroen; Niessen, Wilfried M A

2013-10-01

A high-throughput sample preparation protocol based on the use of 96-well molecular weight cutoff (MWCO) filter plates was developed for shotgun proteomics of cell lysates. All sample preparation steps, including cell lysis, buffer exchange, protein denaturation, reduction, alkylation and proteolytic digestion are performed in a 96-well plate format, making the platform extremely well suited for processing large numbers of samples and directly compatible with functional assays for cellular proteomics. In addition, the usage of a single plate for all sample preparation steps following cell lysis reduces potential samples losses and allows for automation. The MWCO filter also enables sample concentration, thereby increasing the overall sensitivity, and implementation of washing steps involving organic solvents, for example, to remove cell membranes constituents. The optimized protocol allowed for higher throughput with improved sensitivity in terms of the number of identified cellular proteins when compared to an established protocol employing gel-filtration columns. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-throughput microfluidics automated cytogenetic processing for effectively lowering biological process time and aid triage during radiation accidents

International Nuclear Information System (INIS)

Ramakumar, Adarsh

2016-01-01

Nuclear or radiation mass casualties require individual, rapid, and accurate dose-based triage of exposed subjects for cytokine therapy and supportive care, to save life. Radiation mass casualties will demand high-throughput individual diagnostic dose assessment for medical management of exposed subjects. Cytogenetic techniques are widely used for triage and definitive radiation biodosimetry. Prototype platform to demonstrate high-throughput microfluidic micro incubation to support the logistics of sample in miniaturized incubators from the site of accident to analytical labs has been developed. Efforts have been made, both at the level of developing concepts and advanced system for higher throughput in processing the samples and also implementing better and efficient methods of logistics leading to performance of lab-on-chip analyses. Automated high-throughput platform with automated feature extraction, storage, cross platform data linkage, cross platform validation and inclusion of multi-parametric biomarker approaches will provide the first generation high-throughput platform systems for effective medical management, particularly during radiation mass casualty events

Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematics of mushrooms.

Science.gov (United States)

Dentinger, Bryn T M; Margaritescu, Simona; Moncalvo, Jean-Marc

2010-07-01

We present two methods for DNA extraction from fresh and dried mushrooms that are adaptable to high-throughput sequencing initiatives, such as DNA barcoding. Our results show that these protocols yield ∼85% sequencing success from recently collected materials. Tests with both recent (100 years) specimens reveal that older collections have low success rates and may be an inefficient resource for populating a barcode database. However, our method of extracting DNA from herbarium samples using small amount of tissue is reliable and could be used for important historical specimens. The application of these protocols greatly reduces time, and therefore cost, of generating DNA sequences from mushrooms and other fungi vs. traditional extraction methods. The efficiency of these methods illustrates that standardization and streamlining of sample processing should be shifted from the laboratory to the field. © 2009 Blackwell Publishing Ltd.

Chromatographic Monoliths for High-Throughput Immunoaffinity Isolation of Transferrin from Human Plasma

Directory of Open Access Journals (Sweden)

Irena Trbojević-Akmačić

2016-06-01

Full Text Available Changes in protein glycosylation are related to different diseases and have a potential as diagnostic and prognostic disease biomarkers. Transferrin (Tf glycosylation changes are common marker for congenital disorders of glycosylation. However, biological interindividual variability of Tf N-glycosylation and genes involved in glycosylation regulation are not known. Therefore, high-throughput Tf isolation method and large scale glycosylation studies are needed in order to address these questions. Due to their unique chromatographic properties, the use of chromatographic monoliths enables very fast analysis cycle, thus significantly increasing sample preparation throughput. Here, we are describing characterization of novel immunoaffinity-based monolithic columns in a 96-well plate format for specific high-throughput purification of human Tf from blood plasma. We optimized the isolation and glycan preparation procedure for subsequent ultra performance liquid chromatography (UPLC analysis of Tf N-glycosylation and managed to increase the sensitivity for approximately three times compared to initial experimental conditions, with very good reproducibility. This work is licensed under a Creative Commons Attribution 4.0 International License.

Data for automated, high-throughput microscopy analysis of intracellular bacterial colonies using spot detection.

Science.gov (United States)

Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H; Nørregaard, Rikke; Møller-Jensen, Jakob; Nejsum, Lene N

2017-10-01

Quantification of intracellular bacterial colonies is useful in strategies directed against bacterial attachment, subsequent cellular invasion and intracellular proliferation. An automated, high-throughput microscopy-method was established to quantify the number and size of intracellular bacterial colonies in infected host cells (Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy, Ernstsen et al., 2017 [1]). The infected cells were imaged with a 10× objective and number of intracellular bacterial colonies, their size distribution and the number of cell nuclei were automatically quantified using a spot detection-tool. The spot detection-output was exported to Excel, where data analysis was performed. In this article, micrographs and spot detection data are made available to facilitate implementation of the method.

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

Directory of Open Access Journals (Sweden)

Sun Zhenyu

2001-08-01

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

A High-Throughput Antibody-Based Microarray Typing Platform

Directory of Open Access Journals (Sweden)

Ashan Perera

2013-05-01

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

Determining the optimal size of small molecule mixtures for high throughput NMR screening

International Nuclear Information System (INIS)

Mercier, Kelly A.; Powers, Robert

2005-01-01

High-throughput screening (HTS) using NMR spectroscopy has become a common component of the drug discovery effort and is widely used throughout the pharmaceutical industry. NMR provides additional information about the nature of small molecule-protein interactions compared to traditional HTS methods. In order to achieve comparable efficiency, small molecules are often screened as mixtures in NMR-based assays. Nevertheless, an analysis of the efficiency of mixtures and a corresponding determination of the optimum mixture size (OMS) that minimizes the amount of material and instrumentation time required for an NMR screen has been lacking. A model for calculating OMS based on the application of the hypergeometric distribution function to determine the probability of a 'hit' for various mixture sizes and hit rates is presented. An alternative method for the deconvolution of large screening mixtures is also discussed. These methods have been applied in a high-throughput NMR screening assay using a small, directed library

High Resolution Melting (HRM for High-Throughput Genotyping—Limitations and Caveats in Practical Case Studies

Directory of Open Access Journals (Sweden)

Marcin Słomka

2017-11-01

Full Text Available High resolution melting (HRM is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs. This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup.

High Resolution Melting (HRM) for High-Throughput Genotyping—Limitations and Caveats in Practical Case Studies

Science.gov (United States)

Słomka, Marcin; Sobalska-Kwapis, Marta; Wachulec, Monika; Bartosz, Grzegorz

2017-01-01

High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup. PMID:29099791

High Resolution Melting (HRM) for High-Throughput Genotyping-Limitations and Caveats in Practical Case Studies.

Science.gov (United States)

Słomka, Marcin; Sobalska-Kwapis, Marta; Wachulec, Monika; Bartosz, Grzegorz; Strapagiel, Dominik

2017-11-03

High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup.

High-throughput analysis of endogenous fruit glycosyl hydrolases using a novel chromogenic hydrogel substrate assay

DEFF Research Database (Denmark)

Schückel, Julia; Kracun, Stjepan Kresimir; Lausen, Thomas Frederik

2017-01-01

A broad range of enzyme activities can be found in a wide range of different fruits and fruiting bodies but there is a lack of methods where many samples can be handled in a high-throughput and efficient manner. In particular, plant polysaccharide degrading enzymes – glycosyl hydrolases (GHs) play...... led to a more profound understanding of the importance of GH activity and regulation, current methods for determining glycosyl hydrolase activity are lacking in throughput and fail to keep up with data output from transcriptome research. Here we present the use of a versatile, easy...

Combinatorial chemoenzymatic synthesis and high-throughput screening of sialosides.

Science.gov (United States)

Chokhawala, Harshal A; Huang, Shengshu; Lau, Kam; Yu, Hai; Cheng, Jiansong; Thon, Vireak; Hurtado-Ziola, Nancy; Guerrero, Juan A; Varki, Ajit; Chen, Xi

2008-09-19

Although the vital roles of structures containing sialic acid in biomolecular recognition are well documented, limited information is available on how sialic acid structural modifications, sialyl linkages, and the underlying glycan structures affect the binding or the activity of sialic acid-recognizing proteins and related downstream biological processes. A novel combinatorial chemoenzymatic method has been developed for the highly efficient synthesis of biotinylated sialosides containing different sialic acid structures and different underlying glycans in 96-well plates from biotinylated sialyltransferase acceptors and sialic acid precursors. By transferring the reaction mixtures to NeutrAvidin-coated plates and assaying for the yields of enzymatic reactions using lectins recognizing sialyltransferase acceptors but not the sialylated products, the biotinylated sialoside products can be directly used, without purification, for high-throughput screening to quickly identify the ligand specificity of sialic acid-binding proteins. For a proof-of-principle experiment, 72 biotinylated alpha2,6-linked sialosides were synthesized in 96-well plates from 4 biotinylated sialyltransferase acceptors and 18 sialic acid precursors using a one-pot three-enzyme system. High-throughput screening assays performed in NeutrAvidin-coated microtiter plates show that whereas Sambucus nigra Lectin binds to alpha2,6-linked sialosides with high promiscuity, human Siglec-2 (CD22) is highly selective for a number of sialic acid structures and the underlying glycans in its sialoside ligands.

A high-throughput pipeline for the design of real-time PCR signatures

Directory of Open Access Journals (Sweden)

Reifman Jaques

2010-06-01

Full Text Available Abstract Background Pathogen diagnostic assays based on polymerase chain reaction (PCR technology provide high sensitivity and specificity. However, the design of these diagnostic assays is computationally intensive, requiring high-throughput methods to identify unique PCR signatures in the presence of an ever increasing availability of sequenced genomes. Results We present the Tool for PCR Signature Identification (TOPSI, a high-performance computing pipeline for the design of PCR-based pathogen diagnostic assays. The TOPSI pipeline efficiently designs PCR signatures common to multiple bacterial genomes by obtaining the shared regions through pairwise alignments between the input genomes. TOPSI successfully designed PCR signatures common to 18 Staphylococcus aureus genomes in less than 14 hours using 98 cores on a high-performance computing system. Conclusions TOPSI is a computationally efficient, fully integrated tool for high-throughput design of PCR signatures common to multiple bacterial genomes. TOPSI is freely available for download at http://www.bhsai.org/downloads/topsi.tar.gz.

High-Throughput Scoring of Seed Germination.

Science.gov (United States)

Ligterink, Wilco; Hilhorst, Henk W M

2017-01-01

High-throughput analysis of seed germination for phenotyping large genetic populations or mutant collections is very labor intensive and would highly benefit from an automated setup. Although very often used, the total germination percentage after a nominated period of time is not very informative as it lacks information about start, rate, and uniformity of germination, which are highly indicative of such traits as dormancy, stress tolerance, and seed longevity. The calculation of cumulative germination curves requires information about germination percentage at various time points. We developed the GERMINATOR package: a simple, highly cost-efficient, and flexible procedure for high-throughput automatic scoring and evaluation of germination that can be implemented without the use of complex robotics. The GERMINATOR package contains three modules: (I) design of experimental setup with various options to replicate and randomize samples; (II) automatic scoring of germination based on the color contrast between the protruding radicle and seed coat on a single image; and (III) curve fitting of cumulative germination data and the extraction, recap, and visualization of the various germination parameters. GERMINATOR is a freely available package that allows the monitoring and analysis of several thousands of germination tests, several times a day by a single person.

High throughput salt separation from uranium deposits

Energy Technology Data Exchange (ETDEWEB)

Kwon, S.W.; Park, K.M.; Kim, J.G.; Kim, I.T.; Park, S.B., E-mail: swkwon@kaeri.re.kr [Korea Atomic Energy Research Inst. (Korea, Republic of)

2014-07-01

It is very important to increase the throughput of the salt separation system owing to the high uranium content of spent nuclear fuel and high salt fraction of uranium dendrites in pyroprocessing. Multilayer porous crucible system was proposed to increase a throughput of the salt distiller in this study. An integrated sieve-crucible assembly was also investigated for the practical use of the porous crucible system. The salt evaporation behaviors were compared between the conventional nonporous crucible and the porous crucible. Two step weight reductions took place in the porous crucible, whereas the salt weight reduced only at high temperature by distillation in a nonporous crucible. The first weight reduction in the porous crucible was caused by the liquid salt penetrated out through the perforated crucible during the temperature elevation until the distillation temperature. Multilayer porous crucibles have a benefit to expand the evaporation surface area. (author)

The JCSG high-throughput structural biology pipeline

International Nuclear Information System (INIS)

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

2010-01-01

The Joint Center for Structural Genomics high-throughput structural biology pipeline has delivered more than 1000 structures to the community over the past ten years and has made a significant contribution to the overall goal of the NIH Protein Structure Initiative (PSI) of expanding structural coverage of the protein universe. The Joint Center for Structural Genomics high-throughput structural biology pipeline has delivered more than 1000 structures to the community over the past ten years. The JCSG has made a significant contribution to the overall goal of the NIH Protein Structure Initiative (PSI) of expanding structural coverage of the protein universe, as well as making substantial inroads into structural coverage of an entire organism. Targets are processed through an extensive combination of bioinformatics and biophysical analyses to efficiently characterize and optimize each target prior to selection for structure determination. The pipeline uses parallel processing methods at almost every step in the process and can adapt to a wide range of protein targets from bacterial to human. The construction, expansion and optimization of the JCSG gene-to-structure pipeline over the years have resulted in many technological and methodological advances and developments. The vast number of targets and the enormous amounts of associated data processed through the multiple stages of the experimental pipeline required the development of variety of valuable resources that, wherever feasible, have been converted to free-access web-based tools and applications

COMPUTER APPROACHES TO WHEAT HIGH-THROUGHPUT PHENOTYPING

Directory of Open Access Journals (Sweden)

Afonnikov D.

2012-08-01

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

High throughput, low set-up time reconfigurable linear feedback shift registers

NARCIS (Netherlands)

Nas, R.J.M.; Berkel, van C.H.

2010-01-01

This paper presents a hardware design for a scalable, high throughput, configurable LFSR. High throughput is achieved by producing L consecutive outputs per clock cycle with a clock cycle period that, for practical cases, increases only logarithmically with the block size L and the length of the

High-throughput epitope identification for snakebite antivenom

DEFF Research Database (Denmark)

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

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

Bioassessment of a Drinking Water Reservoir Using Plankton: High Throughput Sequencing vs. Traditional Morphological Method

Directory of Open Access Journals (Sweden)

Wanli Gao

2018-01-01

Full Text Available Drinking water safety is increasingly perceived as one of the top global environmental issues. Plankton has been commonly used as a bioindicator for water quality in lakes and reservoirs. Recently, DNA sequencing technology has been applied to bioassessment. In this study, we compared the effectiveness of the 16S and 18S rRNA high throughput sequencing method (HTS and the traditional optical microscopy method (TOM in the bioassessment of drinking water quality. Five stations reflecting different habitats and hydrological conditions in Danjiangkou Reservoir, one of the largest drinking water reservoirs in Asia, were sampled May 2016. Non-metric multi-dimensional scaling (NMDS analysis showed that plankton assemblages varied among the stations and the spatial patterns revealed by the two methods were consistent. The correlation between TOM and HTS in a symmetric Procrustes analysis was 0.61, revealing overall good concordance between the two methods. Procrustes analysis also showed that site-specific differences between the two methods varied among the stations. Station Heijizui (H, a site heavily influenced by two tributaries, had the largest difference while station Qushou (Q, a confluence site close to the outlet dam, had the smallest difference between the two methods. Our results show that DNA sequencing has the potential to provide consistent identification of taxa, and reliable bioassessment in a long-term biomonitoring and assessment program for drinking water reservoirs.

Development of a high-throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design.

Science.gov (United States)

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.

Screening for methicillin-resistant Staphylococcus aureus in clinical swabs using a high-throughput real-time PCR-based method

DEFF Research Database (Denmark)

Ornskov, D; Kolmos, B; Bendix Horn, P

2008-01-01

2005, all patients and healthcare personnel have been screened for MRSA colonisation, involving analysis of 300-400 samples daily. To deal with this number of samples, a PCR-based method customised for high-throughput analysis and a system for fast reporting of MRSA carrier status were developed. Swab...... samples were incubated overnight in a selective tryptone soya broth and were analysed by PCR the following day. Using this strategy, non-colonised individuals were identified within 24 h, while MRSA-positive samples were analysed further by traditional microbiological methods to determine the resistance...... pattern. This is a cost-effective approach, as the greatest expense in hospitals involves the isolation of patients of unknown MRSA status. The method was evaluated by testing 2194 clinical samples, with a sensitivity and specificity of 100% and 94%, respectively. The analytical sensitivity was 97...

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

DEFF Research Database (Denmark)

Lundqvist, Magnus; Edfors, Fredrik; Sivertsson, Åsa

2015-01-01

We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We ...

Preliminary High-Throughput Metagenome Assembly

Energy Technology Data Exchange (ETDEWEB)

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

2007-03-26

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

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

Science.gov (United States)

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

2017-02-01

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

Computational and statistical methods for high-throughput analysis of post-translational modifications of proteins

DEFF Research Database (Denmark)

Schwämmle, Veit; Braga, Thiago Verano; Roepstorff, Peter

2015-01-01

The investigation of post-translational modifications (PTMs) represents one of the main research focuses for the study of protein function and cell signaling. Mass spectrometry instrumentation with increasing sensitivity improved protocols for PTM enrichment and recently established pipelines...... for high-throughput experiments allow large-scale identification and quantification of several PTM types. This review addresses the concurrently emerging challenges for the computational analysis of the resulting data and presents PTM-centered approaches for spectra identification, statistical analysis...

Liquid-phase sample preparation method for real-time monitoring of airborne asbestos fibers by dual-mode high-throughput microscopy.

Science.gov (United States)

Cho, Myoung-Ock; Kim, Jung Kyung; Han, Hwataik; Lee, Jeonghoon

2013-01-01

Asbestos that had been used widely as a construction material is a first-level carcinogen recognized by the World Health Organization. It can be accumulated in body by inhalation causing virulent respiratory diseases including lung cancer. In our previous study, we developed a high-throughput microscopy (HTM) system that can minimize human intervention accompanied by the conventional phase contrast microscopy (PCM) through automated counting of fibrous materials and thus significantly reduce analysis time and labor. Also, we attempted selective detection of chrysotile using DksA protein extracted from Escherichia coli through a recombinant protein production technique, and developed a dual-mode HTM (DM-HTM) by upgrading the HTM device. We demonstrated that fluorescently-labeled chrysotile asbestos fibers can be identified and enumerated automatically among other types of asbestos fibers or non-asbestos particles in a high-throughput manner through a newly modified HTM system for both reflection and fluorescence imaging. However there is a limitation to apply DM-HTM to airborne sample with current air collecting method due to the difficulty of applying the protein to dried asbestos sample. Here, we developed a technique for preparing liquid-phase asbestos sample using an impinger normally used to collect odor molecules in the air. It would be possible to improve the feasibility of the dual-mode HTM by integrating a sample preparation unit for making collected asbestos sample dispersed in a solution. The new technique developed for highly sensitive and automated asbestos detection can be a potential alternative to the conventional manual counting method, and it may be applied on site as a fast and reliable environmental monitoring tool.

Data for automated, high-throughput microscopy analysis of intracellular bacterial colonies using spot detection

DEFF Research Database (Denmark)

Ernstsen, Christina Lundgaard; Login, Frédéric H.; Jensen, Helene Halkjær

2017-01-01

Quantification of intracellular bacterial colonies is useful in strategies directed against bacterial attachment, subsequent cellular invasion and intracellular proliferation. An automated, high-throughput microscopy-method was established to quantify the number and size of intracellular bacteria...

High-Throughput LC-MS/MS Method for Direct Quantification of Glucuronidated, Sulfated and Free Enterolactone in Human Plasma

DEFF Research Database (Denmark)

Nørskov, Natalja; Kyrø, Cecilie; Olsen, Anja

2016-01-01

Sulfation and glucuronidation constitute a major pathway in humans and may play an important role in biological activity of metabolites including the enterolignan, enterolactone. Because the aromatic structure of enterolactone has similarities to steroid metabolites, it was hypothesized that ente......Sulfation and glucuronidation constitute a major pathway in humans and may play an important role in biological activity of metabolites including the enterolignan, enterolactone. Because the aromatic structure of enterolactone has similarities to steroid metabolites, it was hypothesized......−MS/MS and a fluoroimmunoassay; however, most of these methods measure the total concentration of enterolactone, without any specification of its conjugation pattern. Here for the first time we present a high-throughput LC−MS/MS method to quantify enterolactone in its intact form as glucuronide, sulfate, and free enterolactone....... The method has shown good accuracy and precision at low concentration and very high sensitivity, with LLOQ for enterolactone sulfate at 16 pM, enterolactone glucuronide at 26 pM, and free enterolactone at 86 pM. The short run time of 2.6 min combined with simple sample clean up and high sensitivity make...

A multilayer microdevice for cell-based high-throughput drug screening

International Nuclear Information System (INIS)

Liu, Chong; Wang, Lei; Li, Jingmin; Ding, Xiping; Chunyu, Li; Xu, Zheng; Wang, Qi

2012-01-01

A multilayer polydimethylsiloxane microdevice for cell-based high-throughput drug screening is described in this paper. This established microdevice was based on a modularization method and it integrated a drug/medium concentration gradient generator (CGG), pneumatic microvalves and a cell culture microchamber array. The CGG was able to generate five steps of linear concentrations with the same outlet flow rate. The medium/drug flowed through CGG and then into the pear-shaped cell culture microchambers vertically. This vertical perfusion mode was used to reduce the impact of the shear stress on the physiology of cells induced by the fluid flow in the microchambers. Pear-shaped microchambers with two arrays of miropillars at each outlet were adopted in this microdevice, which were beneficial to cell distribution. The chemotherapeutics Cisplatin (DDP)-induced Cisplatin-resistant cell line A549/DDP apoptotic experiments were performed well on this platform. The results showed that this novel microdevice could not only provide well-defined and stable conditions for cell culture, but was also useful for cell-based high-throughput drug screening with less reagents and time consumption. (paper)

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

International Nuclear Information System (INIS)

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

2011-01-01

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.

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

Science.gov (United States)

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.

In-situ nanoelectrospray for high-throughput screening of enzymes and real-time monitoring of reactions.

Science.gov (United States)

Yang, Yuhan; Han, Feifei; Ouyang, Jin; Zhao, Yunling; Han, Juan; Na, Na

2016-01-01

The in-situ and high-throughput evaluation of enzymes and real-time monitoring of enzyme catalyzed reactions in liquid phase is quite significant in the catalysis industry. In-situ nanoelectrospray, the direct sampling and ionization method for mass spectrometry, has been applied for high-throughput evaluation of enzymes, as well as the on-line monitoring of reactions. Simply inserting a capillary into a liquid system with high-voltage applied, analytes in liquid reaction system can be directly ionized at the capillary tip with small volume consumption. With no sample pre-treatment or injection procedure, different analytes such as saccharides, amino acids, alkaloids, peptides and proteins can be rapidly and directly extracted from liquid phase and ionized at the capillary tip. Taking irreversible transesterification reaction of vinyl acetate and ethanol as an example, this technique has been used for the high-throughput evaluation of enzymes, fast optimizations, as well as real-time monitoring of reaction catalyzed by different enzymes. In addition, it is even softer than traditional electrospray ionization. The present method can also be used for the monitoring of other homogenous and heterogeneous reactions in liquid phases, which will show potentials in the catalysis industry. Copyright © 2015 Elsevier B.V. All rights reserved.

Meta-Analysis of High-Throughput Datasets Reveals Cellular Responses Following Hemorrhagic Fever Virus Infection

Directory of Open Access Journals (Sweden)

Gavin C. Bowick

2011-05-01

Full Text Available The continuing use of high-throughput assays to investigate cellular responses to infection is providing a large repository of information. Due to the large number of differentially expressed transcripts, often running into the thousands, the majority of these data have not been thoroughly investigated. Advances in techniques for the downstream analysis of high-throughput datasets are providing additional methods for the generation of additional hypotheses for further investigation. The large number of experimental observations, combined with databases that correlate particular genes and proteins with canonical pathways, functions and diseases, allows for the bioinformatic exploration of functional networks that may be implicated in replication or pathogenesis. Herein, we provide an example of how analysis of published high-throughput datasets of cellular responses to hemorrhagic fever virus infection can generate additional functional data. We describe enrichment of genes involved in metabolism, post-translational modification and cardiac damage; potential roles for specific transcription factors and a conserved involvement of a pathway based around cyclooxygenase-2. We believe that these types of analyses can provide virologists with additional hypotheses for continued investigation.

High-throughput combinatorial chemical bath deposition: The case of doping Cu (In, Ga) Se film with antimony

Science.gov (United States)

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.

High Throughput Synthesis and Screening for Agents Inhibiting Androgen Receptor Mediated Gene Transcription

National Research Council Canada - National Science Library

Boger, Dale L

2005-01-01

.... This entails the high throughput synthesis of DNA binding agents related to distamycin, their screening for binding to androgen response elements using a new high throughput DNA binding screen...

High Throughput Synthesis and Screening for Agents Inhibiting Androgen Receptor Mediated Gene Transcription

National Research Council Canada - National Science Library

Boger, Dale

2004-01-01

.... This entails the high throughput synthesis of DNA binding agents related to distamycin, their screening for binding to androgen response elements using a new high throughput DNA binding screen...

A CRISPR CASe for High-Throughput Silencing

Directory of Open Access Journals (Sweden)

Jacob eHeintze

2013-10-01

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

High throughput route selection in multi-rate wireless mesh networks

Institute of Scientific and Technical Information of China (English)

WEI Yi-fei; GUO Xiang-li; SONG Mei; SONG Jun-de

2008-01-01

Most existing Ad-hoc routing protocols use the shortest path algorithm with a hop count metric to select paths. It is appropriate in single-rate wireless networks, but has a tendency to select paths containing long-distance links that have low data rates and reduced reliability in multi-rate networks. This article introduces a high throughput routing algorithm utilizing the multi-rate capability and some mesh characteristics in wireless fidelity (WiFi) mesh networks. It uses the medium access control (MAC) transmission time as the routing metric, which is estimated by the information passed up from the physical layer. When the proposed algorithm is adopted, the Ad-hoc on-demand distance vector (AODV) routing can be improved as high throughput AODV (HT-AODV). Simulation results show that HT-AODV is capable of establishing a route that has high data-rate, short end-to-end delay and great network throughput.

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

Directory of Open Access Journals (Sweden)

Mark D Mathew

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

High Throughput Screening Method for Systematic Surveillance of Drugs of Abuse by Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry.

Science.gov (United States)

DiBattista, Alicia; Rampersaud, Dianne; Lee, Howard; Kim, Marcus; Britz-McKibbin, Philip

2017-11-07

New technologies are urgently required for reliable drug screening given a worldwide epidemic of prescription drug abuse and its devastating socioeconomic impacts on public health. Primary screening of drugs of abuse (DoA) currently relies on immunoassays that are prone to bias and are not applicable to detect an alarming array of psychoactive stimulants, tranquilizers, and synthetic opioids. These limitations impact patient safety when monitoring for medication compliance, drug substitution, or misuse/abuse and require follow-up confirmatory testing by more specific yet lower throughput instrumental methods. Herein, we introduce a high throughput platform for nontargeted screening of a broad spectrum of DoA and their metabolites based on multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). We demonstrate that MSI-CE-MS enables serial injections of 10 samples within a single run (high resolution MS with full-scan data acquisition. Unambiguous drug identification was achieved by four or more independent parameters, including comigration with a deuterated internal standard or in silico prediction of electromigration behavior together with accurate mass, most likely molecular formula, as well as MS/MS as required for confirmation testing. Acceptable precision was demonstrated for over 50 DoA at 3 concentration levels over 4 days (median coefficient of variance = 13%, n = 117) with minimal ion suppression, isobaric interferences, and sample carry-over (screening cutoff levels in human urine while allowing for systematic surveillance, specimen verification, and retrospective testing of designer drugs that elude conventional drug tests.

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

Science.gov (United States)

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

Development of a Scintillation Proximity Assay (SPA) Based, High Throughput Screening Feasible Method for the Identification of PDE12 Activity Modulators.

Science.gov (United States)

Mang, Samuel; Bucher, Hannes; Nickolaus, Peter

2016-01-01

The scintillation proximity assay (SPA) technology has been widely used to establish high throughput screens (HTS) for a range of targets in the pharmaceutical industry. PDE12 (aka. 2'- phosphodiesterase) has been published to participate in the degradation of oligoadenylates that are involved in the establishment of an antiviral state via the activation of ribonuclease L (RNAse-L). Degradation of oligoadenylates by PDE12 terminates these antiviral activities, leading to decreased resistance of cells for a variety of viral pathogens. Therefore inhibitors of PDE12 are discussed as antiviral therapy. Here we describe the use of the yttrium silicate SPA bead technology to assess inhibitory activity of compounds against PDE12 in a homogeneous, robust HTS feasible assay using tritiated adenosine-P-adenylate ([3H]ApA) as substrate. We found that the used [3H]ApA educt, was not able to bind to SPA beads, whereas the product [3H]AMP, as known before, was able to bind to SPA beads. This enables the measurement of PDE12 activity on [3H]ApA as a substrate using a wallac microbeta counter. This method describes a robust and high throughput capable format in terms of specificity, commonly used compound solvents, ease of detection and assay matrices. The method could facilitate the search for PDE12 inhibitors as antiviral compounds.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)-A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes.

Science.gov (United States)

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare . However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

Directory of Open Access Journals (Sweden)

Karolina Chwialkowska

2017-11-01

Full Text Available Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq. We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation

Application of high-throughput DNA sequencing in phytopathology.

Science.gov (United States)

Studholme, David J; Glover, Rachel H; Boonham, Neil

2011-01-01

The new sequencing technologies are already making a big impact in academic research on medically important microbes and may soon revolutionize diagnostics, epidemiology, and infection control. Plant pathology also stands to gain from exploiting these opportunities. This manuscript reviews some applications of these high-throughput sequencing methods that are relevant to phytopathology, with emphasis on the associated computational and bioinformatics challenges and their solutions. Second-generation sequencing technologies have recently been exploited in genomics of both prokaryotic and eukaryotic plant pathogens. They are also proving to be useful in diagnostics, especially with respect to viruses. Copyright © 2011 by Annual Reviews. All rights reserved.

High-throughput ab-initio dilute solute diffusion database.

Science.gov (United States)

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.

Mechanical Conversion for High-Throughput TEM Sample Preparation

International Nuclear Information System (INIS)

Kendrick, Anthony B; Moore, Thomas M; Zaykova-Feldman, Lyudmila

2006-01-01

This paper presents a novel method of direct mechanical conversion from lift-out sample to TEM sample holder. The lift-out sample is prepared in the FIB using the in-situ liftout Total Release TM method. The mechanical conversion is conducted using a mechanical press and one of a variety of TEM coupons, including coupons for both top-side and back-side thinning. The press joins a probe tip point with attached TEM sample to the sample coupon and separates the complete assembly as a 3mm diameter TEM grid, compatible with commercially available TEM sample holder rods. This mechanical conversion process lends itself well to the high through-put requirements of in-line process control and to materials characterization labs where instrument utilization and sample security are critically important

HTTK: R Package for High-Throughput Toxicokinetics

Science.gov (United States)

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...

A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents

International Nuclear Information System (INIS)

Zhu, Xiaoming; Fu, Afu; Luo, Kathy Qian

2012-01-01

Highlights: ► An endothelial cell apoptosis assay using FRET-based biosensor was developed. ► The fluorescence of the cells changed from green to blue during apoptosis. ► This method was developed into a high-throughput assay in 96-well plates. ► This assay was applied to screen vascular disrupting agents. -- Abstract: In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z′ factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening.

High-Throughput Analysis and Automation for Glycomics Studies.

Science.gov (United States)

Shubhakar, Archana; Reiding, Karli R; Gardner, Richard A; Spencer, Daniel I R; Fernandes, Daryl L; Wuhrer, Manfred

This review covers advances in analytical technologies for high-throughput (HTP) glycomics. Our focus is on structural studies of glycoprotein glycosylation to support biopharmaceutical realization and the discovery of glycan biomarkers for human disease. For biopharmaceuticals, there is increasing use of glycomics in Quality by Design studies to help optimize glycan profiles of drugs with a view to improving their clinical performance. Glycomics is also used in comparability studies to ensure consistency of glycosylation both throughout product development and between biosimilars and innovator drugs. In clinical studies there is as well an expanding interest in the use of glycomics-for example in Genome Wide Association Studies-to follow changes in glycosylation patterns of biological tissues and fluids with the progress of certain diseases. These include cancers, neurodegenerative disorders and inflammatory conditions. Despite rising activity in this field, there are significant challenges in performing large scale glycomics studies. The requirement is accurate identification and quantitation of individual glycan structures. However, glycoconjugate samples are often very complex and heterogeneous and contain many diverse branched glycan structures. In this article we cover HTP sample preparation and derivatization methods, sample purification, robotization, optimized glycan profiling by UHPLC, MS and multiplexed CE, as well as hyphenated techniques and automated data analysis tools. Throughout, we summarize the advantages and challenges with each of these technologies. The issues considered include reliability of the methods for glycan identification and quantitation, sample throughput, labor intensity, and affordability for large sample numbers.

A high-throughput, multi-channel photon-counting detector with picosecond timing

CERN Document Server

Lapington, J S; Miller, G M; Ashton, T J R; Jarron, P; Despeisse, M; Powolny, F; Howorth, J; Milnes, J

2009-01-01

High-throughput photon counting with high time resolution is a niche application area where vacuum tubes can still outperform solid-state devices. Applications in the life sciences utilizing time-resolved spectroscopies, particularly in the growing field of proteomics, will benefit greatly from performance enhancements in event timing and detector throughput. The HiContent project is a collaboration between the University of Leicester Space Research Centre, the Microelectronics Group at CERN, Photek Ltd., and end-users at the Gray Cancer Institute and the University of Manchester. The goal is to develop a detector system specifically designed for optical proteomics, capable of high content (multi-parametric) analysis at high throughput. The HiContent detector system is being developed to exploit this niche market. It combines multi-channel, high time resolution photon counting in a single miniaturized detector system with integrated electronics. The combination of enabling technologies; small pore microchanne...

Space Link Extension Protocol Emulation for High-Throughput, High-Latency Network Connections

Science.gov (United States)

Tchorowski, Nicole; Murawski, Robert

2014-01-01

New space missions require higher data rates and new protocols to meet these requirements. These high data rate space communication links push the limitations of not only the space communication links, but of the ground communication networks and protocols which forward user data to remote ground stations (GS) for transmission. The Consultative Committee for Space Data Systems, (CCSDS) Space Link Extension (SLE) standard protocol is one protocol that has been proposed for use by the NASA Space Network (SN) Ground Segment Sustainment (SGSS) program. New protocol implementations must be carefully tested to ensure that they provide the required functionality, especially because of the remote nature of spacecraft. The SLE protocol standard has been tested in the NASA Glenn Research Center's SCENIC Emulation Lab in order to observe its operation under realistic network delay conditions. More specifically, the delay between then NASA Integrated Services Network (NISN) and spacecraft has been emulated. The round trip time (RTT) delay for the continental NISN network has been shown to be up to 120ms; as such the SLE protocol was tested with network delays ranging from 0ms to 200ms. Both a base network condition and an SLE connection were tested with these RTT delays, and the reaction of both network tests to the delay conditions were recorded. Throughput for both of these links was set at 1.2Gbps. The results will show that, in the presence of realistic network delay, the SLE link throughput is significantly reduced while the base network throughput however remained at the 1.2Gbps specification. The decrease in SLE throughput has been attributed to the implementation's use of blocking calls. The decrease in throughput is not acceptable for high data rate links, as the link requires constant data a flow in order for spacecraft and ground radios to stay synchronized, unless significant data is queued a the ground station. In cases where queuing the data is not an option

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

Directory of Open Access Journals (Sweden)

Velasco Riccardo

2008-01-01

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

A robust robotic high-throughput antibody purification platform.

Science.gov (United States)

Schmidt, Peter M; Abdo, Michael; Butcher, Rebecca E; Yap, Min-Yin; Scotney, Pierre D; Ramunno, Melanie L; Martin-Roussety, Genevieve; Owczarek, Catherine; Hardy, Matthew P; Chen, Chao-Guang; Fabri, Louis J

2016-07-15

Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant. Copyright © 2016 Elsevier B.V. All rights reserved.

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

DEFF Research Database (Denmark)

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

2014-01-01

A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its α......-amylase production, close to the theoretical maximum enrichment. Furthermore, a 105 member yeast cell library is screened yielding a clone with a more than 2-fold increase in α-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host...

Theory and implementation of a very high throughput true random number generator in field programmable gate array

Energy Technology Data Exchange (ETDEWEB)

Wang, Yonggang, E-mail: wangyg@ustc.edu.cn; Hui, Cong; Liu, Chong; Xu, Chao [Department of Modern Physics, University of Science and Technology of China, Hefei 230026 (China)

2016-04-15

The contribution of this paper is proposing a new entropy extraction mechanism based on sampling phase jitter in ring oscillators to make a high throughput true random number generator in a field programmable gate array (FPGA) practical. Starting from experimental observation and analysis of the entropy source in FPGA, a multi-phase sampling method is exploited to harvest the clock jitter with a maximum entropy and fast sampling speed. This parametrized design is implemented in a Xilinx Artix-7 FPGA, where the carry chains in the FPGA are explored to realize the precise phase shifting. The generator circuit is simple and resource-saving, so that multiple generation channels can run in parallel to scale the output throughput for specific applications. The prototype integrates 64 circuit units in the FPGA to provide a total output throughput of 7.68 Gbps, which meets the requirement of current high-speed quantum key distribution systems. The randomness evaluation, as well as its robustness to ambient temperature, confirms that the new method in a purely digital fashion can provide high-speed high-quality random bit sequences for a variety of embedded applications.

High throughput electrospinning of high-quality nanofibers via an aluminum disk spinneret

Science.gov (United States)

Zheng, Guokuo

In this work, a simple and efficient needleless high throughput electrospinning process using an aluminum disk spinneret with 24 holes is described. Electrospun mats produced by this setup consisted of fine fibers (nano-sized) of the highest quality while the productivity (yield) was many times that obtained from conventional single-needle electrospinning. The goal was to produce scaled-up amounts of the same or better quality nanofibers under variable concentration, voltage, and the working distance than those produced with the single needle lab setting. The fiber mats produced were either polymer or ceramic (such as molybdenum trioxide nanofibers). Through experimentation the optimum process conditions were defined to be: 24 kilovolt, a distance to collector of 15cm. More diluted solutions resulted in smaller diameter fibers. Comparing the morphologies of the nanofibers of MoO3 produced by both the traditional and the high throughput set up it was found that they were very similar. Moreover, the nanofibers production rate is nearly 10 times than that of traditional needle electrospinning. Thus, the high throughput process has the potential to become an industrial nanomanufacturing process and the materials processed by it may be used as filtration devices, in tissue engineering, and as sensors.

High-throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass.

Science.gov (United States)

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.

An Unsupervised kNN Method to Systematically Detect Changes in Protein Localization in High-Throughput Microscopy Images.

Directory of Open Access Journals (Sweden)

Alex Xijie Lu

Full Text Available Despite the importance of characterizing genes that exhibit subcellular localization changes between conditions in proteome-wide imaging experiments, many recent studies still rely upon manual evaluation to assess the results of high-throughput imaging experiments. We describe and demonstrate an unsupervised k-nearest neighbours method for the detection of localization changes. Compared to previous classification-based supervised change detection methods, our method is much simpler and faster, and operates directly on the feature space to overcome limitations in needing to manually curate training sets that may not generalize well between screens. In addition, the output of our method is flexible in its utility, generating both a quantitatively ranked list of localization changes that permit user-defined cut-offs, and a vector for each gene describing feature-wise direction and magnitude of localization changes. We demonstrate that our method is effective at the detection of localization changes using the Δrpd3 perturbation in Saccharomyces cerevisiae, where we capture 71.4% of previously known changes within the top 10% of ranked genes, and find at least four new localization changes within the top 1% of ranked genes. The results of our analysis indicate that simple unsupervised methods may be able to identify localization changes in images without laborious manual image labelling steps.

An Unsupervised kNN Method to Systematically Detect Changes in Protein Localization in High-Throughput Microscopy Images.

Science.gov (United States)

Lu, Alex Xijie; Moses, Alan M

2016-01-01

Despite the importance of characterizing genes that exhibit subcellular localization changes between conditions in proteome-wide imaging experiments, many recent studies still rely upon manual evaluation to assess the results of high-throughput imaging experiments. We describe and demonstrate an unsupervised k-nearest neighbours method for the detection of localization changes. Compared to previous classification-based supervised change detection methods, our method is much simpler and faster, and operates directly on the feature space to overcome limitations in needing to manually curate training sets that may not generalize well between screens. In addition, the output of our method is flexible in its utility, generating both a quantitatively ranked list of localization changes that permit user-defined cut-offs, and a vector for each gene describing feature-wise direction and magnitude of localization changes. We demonstrate that our method is effective at the detection of localization changes using the Δrpd3 perturbation in Saccharomyces cerevisiae, where we capture 71.4% of previously known changes within the top 10% of ranked genes, and find at least four new localization changes within the top 1% of ranked genes. The results of our analysis indicate that simple unsupervised methods may be able to identify localization changes in images without laborious manual image labelling steps.

High-throughput screening for bioactive components from traditional Chinese medicine.

Science.gov (United States)

Zhu, Yanhui; Zhang, Zhiyun; Zhang, Meng; Mais, Dale E; Wang, Ming-Wei

2010-12-01

Throughout the centuries, traditional Chinese medicine has been a rich resource in the development of new drugs. Modern drug discovery, which relies increasingly on automated high throughput screening and quick hit-to-lead development, however, is confronted with the challenges of the chemical complexity associated with natural products. New technologies for biological screening as well as library building are in great demand in order to meet the requirements. Here we review the developments in these techniques under the perspective of their applicability in natural product drug discovery. Methods in library building, component characterizing, biological evaluation, and other screening methods including NMR and X-ray diffraction are discussed.

Meeting Report: High-Throughput Technologies for In Vivo Imaging Agents

Directory of Open Access Journals (Sweden)

Robert J. Gillies

2005-04-01

Full Text Available Combinatorial chemistry and high-throughput screening have become standard tools for discovering new drug candidates with suitable pharmacological properties. Now, those same technologies are starting to be applied to the problem of discovering novel in vivo imaging agents. Important differences in the biological and pharmacological properties needed for imaging agents, compared to those for a therapeutic agent, require new screening methods that emphasize those characteristics, such as optimized residence time and tissue specificity, that make for a good imaging agent candidate.

High-throughput Methods Redefine the Rumen Microbiome and Its Relationship with Nutrition and Metabolism

Science.gov (United States)

McCann, Joshua C.; Wickersham, Tryon A.; Loor, Juan J.

2014-01-01

Diversity in the forestomach microbiome is one of the key features of ruminant animals. The diverse microbial community adapts to a wide array of dietary feedstuffs and management strategies. Understanding rumen microbiome composition, adaptation, and function has global implications ranging from climatology to applied animal production. Classical knowledge of rumen microbiology was based on anaerobic, culture-dependent methods. Next-generation sequencing and other molecular techniques have uncovered novel features of the rumen microbiome. For instance, pyrosequencing of the 16S ribosomal RNA gene has revealed the taxonomic identity of bacteria and archaea to the genus level, and when complemented with barcoding adds multiple samples to a single run. Whole genome shotgun sequencing generates true metagenomic sequences to predict the functional capability of a microbiome, and can also be used to construct genomes of isolated organisms. Integration of high-throughput data describing the rumen microbiome with classic fermentation and animal performance parameters has produced meaningful advances and opened additional areas for study. In this review, we highlight recent studies of the rumen microbiome in the context of cattle production focusing on nutrition, rumen development, animal efficiency, and microbial function. PMID:24940050

Analysis of high-throughput biological data using their rank values.

Science.gov (United States)

Dembélé, Doulaye

2018-01-01

High-throughput biological technologies are routinely used to generate gene expression profiling or cytogenetics data. To achieve high performance, methods available in the literature become more specialized and often require high computational resources. Here, we propose a new versatile method based on the data-ordering rank values. We use linear algebra, the Perron-Frobenius theorem and also extend a method presented earlier for searching differentially expressed genes for the detection of recurrent copy number aberration. A result derived from the proposed method is a one-sample Student's t-test based on rank values. The proposed method is to our knowledge the only that applies to gene expression profiling and to cytogenetics data sets. This new method is fast, deterministic, and requires a low computational load. Probabilities are associated with genes to allow a statistically significant subset selection in the data set. Stability scores are also introduced as quality parameters. The performance and comparative analyses were carried out using real data sets. The proposed method can be accessed through an R package available from the CRAN (Comprehensive R Archive Network) website: https://cran.r-project.org/web/packages/fcros .

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

Science.gov (United States)

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

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

DEFF Research Database (Denmark)

Knudsen, Peter Boldsen

producing the heterologous model polyketide, 6-methylsalicylic acid (6-MSA). An automated methodology for high throughput screening focusing on growth rates, together with a fully automated method for quantitative physiological characterisation in microtiter plates, was established for yeast. Full...

MIPHENO: Data normalization for high throughput metabolic analysis.

Science.gov (United States)

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

A high-throughput liquid bead array-based screening technology for Bt presence in GMO manipulation.

Science.gov (United States)

Fu, Wei; Wang, Huiyu; Wang, Chenguang; Mei, Lin; Lin, Xiangmei; Han, Xueqing; Zhu, Shuifang

2016-03-15

The number of species and planting areas of genetically modified organisms (GMOs) has been rapidly developed during the past ten years. For the purpose of GMO inspection, quarantine and manipulation, we have now devised a high-throughput Bt-based GMOs screening method based on the liquid bead array. This novel method is based on the direct competitive recognition between biotinylated antibodies and beads-coupled antigens, searching for Bt presence in samples if it contains Bt Cry1 Aa, Bt Cry1 Ab, Bt Cry1 Ac, Bt Cry1 Ah, Bt Cry1 B, Bt Cry1 C, Bt Cry1 F, Bt Cry2 A, Bt Cry3 or Bt Cry9 C. Our method has a wide GMO species coverage so that more than 90% of the whole commercialized GMO species can be identified throughout the world. Under our optimization, specificity, sensitivity, repeatability and availability validation, the method shows a high specificity and 10-50 ng/mL sensitivity of quantification. We then assessed more than 1800 samples in the field and food market to prove capacity of our method in performing a high throughput screening work for GMO manipulation. Our method offers an applicant platform for further inspection and research on GMO plants. Copyright © 2015 Elsevier B.V. All rights reserved.

Optimised method to estimate octanol water distribution coefficient (logD) in a high throughput format.

Science.gov (United States)

Low, Ying Wei Ivan; Blasco, Francesca; Vachaspati, Prakash

2016-09-20

Lipophilicity is one of the molecular properties assessed in early drug discovery. Direct measurement of the octanol-water distribution coefficient (logD) requires an analytical method with a large dynamic range or multistep dilutions, as the analyte's concentrations span across several orders of magnitude. In addition, water/buffer and octanol phases which have very different polarity could lead to matrix effects and affect the LC-MS response, leading to erroneous logD values. Most compound libraries use DMSO stocks as it greatly reduces the sample requirement but the presence of DMSO has been shown to underestimate the lipophilicity of the analyte. The present work describes the development of an optimised shake flask logD method using deepwell 96 well plate that addresses the issues related to matrix effects, DMSO concentration and incubation conditions and is also amenable to high throughput. Our results indicate that the equilibrium can be achieved within 30min by flipping the plate on its side while even 0.5% of DMSO is not tolerated in the assay. This study uses the matched matrix concept to minimise the errors in analysing the two phases namely buffer and octanol in LC-MS. Copyright © 2016 Elsevier B.V. All rights reserved.

A cell-based high-throughput screening assay for radiation susceptibility using automated cell counting

International Nuclear Information System (INIS)

Hodzic, Jasmina; Dingjan, Ilse; Maas, Mariëlle JP; Meulen-Muileman, Ida H van der; Menezes, Renee X de; Heukelom, Stan; Verheij, Marcel; Gerritsen, Winald R; Geldof, Albert A; Triest, Baukelien van; Beusechem, Victor W van

2015-01-01

Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening. We developed a new high-throughput screening method for determining radiation susceptibility. Fast and uniform irradiation of batches up to 30 microplates was achieved using a Perspex container and a clinically employed linear accelerator. The readout was done by automated counting of fluorescently stained nuclei using the Acumen eX3 laser scanning cytometer. Assay performance was compared to that of the CFA and the CellTiter-Blue homogeneous uniform-well cell viability assay. The assay was validated in a whole-genome siRNA library screening setting using PC-3 prostate cancer cells. On 4 different cancer cell lines, the automated cell counting assay produced radiation dose response curves that followed a linear-quadratic equation and that exhibited a better correlation to the results of the CFA than did the cell viability assay. Moreover, the cell counting assay could be used to detect radiosensitization by silencing DNA-PKcs or by adding caffeine. In a high-throughput screening setting, using 4 Gy irradiated and control PC-3 cells, the effects of DNA-PKcs siRNA and non-targeting control siRNA could be clearly discriminated. We developed a simple assay for radiation susceptibility that can be used for high-throughput screening. This will aid

High-Throughput Analysis and Automation for Glycomics Studies

NARCIS (Netherlands)

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

2015-01-01

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

Association Study of Gut Flora in Coronary Heart Disease through High-Throughput Sequencing

OpenAIRE

Cui, Li; Zhao, Tingting; Hu, Haibing; Zhang, Wen; Hua, Xiuguo

2017-01-01

Objectives. We aimed to explore the impact of gut microbiota in coronary heart disease (CHD) patients through high-throughput sequencing. Methods. A total of 29 CHD in-hospital patients and 35 healthy volunteers as controls were included. Nucleic acids were extracted from fecal samples, followed by ? diversity and principal coordinate analysis (PCoA). Based on unweighted UniFrac distance matrices, unweighted-pair group method with arithmetic mean (UPGMA) trees were created. Results. After dat...

High-throughput bioinformatics with the Cyrille2 pipeline system

Directory of Open Access Journals (Sweden)

de Groot Joost CW

2008-02-01

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

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

Science.gov (United States)

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.

High-throughput screening assay used in pharmacognosy: Selection, optimization and validation of methods of enzymatic inhibition by UV-visible spectrophotometry

Directory of Open Access Journals (Sweden)

Graciela Granados-Guzmán

2014-02-01

Full Text Available In research laboratories of both organic synthesis and extraction of natural products, every day a lot of products that can potentially introduce some biological activity are obtained. Therefore it is necessary to have in vitro assays, which provide reliable information for further evaluation in in vivo systems. From this point of view, in recent years has intensified the use of high-throughput screening assays. Such trials should be optimized and validated for accurate and precise results, i.e. reliable. The present review addresses the steps needed to develop and validate bioanalytical methods, emphasizing UV-Visible spectrophotometry as detection system. Particularly focuses on the selection of the method, the optimization to determine the best experimental conditions, validation, implementation of optimized and validated method to real samples, and finally maintenance and possible transfer it to a new laboratory.

Uplink SDMA with Limited Feedback: Throughput Scaling

Directory of Open Access Journals (Sweden)

Jeffrey G. Andrews

2008-01-01

Full Text Available Combined space division multiple access (SDMA and scheduling exploit both spatial multiplexing and multiuser diversity, increasing throughput significantly. Both SDMA and scheduling require feedback of multiuser channel sate information (CSI. This paper focuses on uplink SDMA with limited feedback, which refers to efficient techniques for CSI quantization and feedback. To quantify the throughput of uplink SDMA and derive design guidelines, the throughput scaling with system parameters is analyzed. The specific parameters considered include the numbers of users, antennas, and feedback bits. Furthermore, different SNR regimes and beamforming methods are considered. The derived throughput scaling laws are observed to change for different SNR regimes. For instance, the throughput scales logarithmically with the number of users in the high SNR regime but double logarithmically in the low SNR regime. The analysis of throughput scaling suggests guidelines for scheduling in uplink SDMA. For example, to maximize throughput scaling, scheduling should use the criterion of minimum quantization errors for the high SNR regime and maximum channel power for the low SNR regime.

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

Science.gov (United States)

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

2017-09-01

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

Using high-throughput barcode sequencing to efficiently map connectomes.

Science.gov (United States)

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.

A high-throughput, multi-channel photon-counting detector with picosecond timing

Science.gov (United States)

Lapington, J. S.; Fraser, G. W.; Miller, G. M.; Ashton, T. J. R.; Jarron, P.; Despeisse, M.; Powolny, F.; Howorth, J.; Milnes, J.

2009-06-01

High-throughput photon counting with high time resolution is a niche application area where vacuum tubes can still outperform solid-state devices. Applications in the life sciences utilizing time-resolved spectroscopies, particularly in the growing field of proteomics, will benefit greatly from performance enhancements in event timing and detector throughput. The HiContent project is a collaboration between the University of Leicester Space Research Centre, the Microelectronics Group at CERN, Photek Ltd., and end-users at the Gray Cancer Institute and the University of Manchester. The goal is to develop a detector system specifically designed for optical proteomics, capable of high content (multi-parametric) analysis at high throughput. The HiContent detector system is being developed to exploit this niche market. It combines multi-channel, high time resolution photon counting in a single miniaturized detector system with integrated electronics. The combination of enabling technologies; small pore microchannel plate devices with very high time resolution, and high-speed multi-channel ASIC electronics developed for the LHC at CERN, provides the necessary building blocks for a high-throughput detector system with up to 1024 parallel counting channels and 20 ps time resolution. We describe the detector and electronic design, discuss the current status of the HiContent project and present the results from a 64-channel prototype system. In the absence of an operational detector, we present measurements of the electronics performance using a pulse generator to simulate detector events. Event timing results from the NINO high-speed front-end ASIC captured using a fast digital oscilloscope are compared with data taken with the proposed electronic configuration which uses the multi-channel HPTDC timing ASIC.

A high-throughput, multi-channel photon-counting detector with picosecond timing

International Nuclear Information System (INIS)

Lapington, J.S.; Fraser, G.W.; Miller, G.M.; Ashton, T.J.R.; Jarron, P.; Despeisse, M.; Powolny, F.; Howorth, J.; Milnes, J.

2009-01-01

High-throughput photon counting with high time resolution is a niche application area where vacuum tubes can still outperform solid-state devices. Applications in the life sciences utilizing time-resolved spectroscopies, particularly in the growing field of proteomics, will benefit greatly from performance enhancements in event timing and detector throughput. The HiContent project is a collaboration between the University of Leicester Space Research Centre, the Microelectronics Group at CERN, Photek Ltd., and end-users at the Gray Cancer Institute and the University of Manchester. The goal is to develop a detector system specifically designed for optical proteomics, capable of high content (multi-parametric) analysis at high throughput. The HiContent detector system is being developed to exploit this niche market. It combines multi-channel, high time resolution photon counting in a single miniaturized detector system with integrated electronics. The combination of enabling technologies; small pore microchannel plate devices with very high time resolution, and high-speed multi-channel ASIC electronics developed for the LHC at CERN, provides the necessary building blocks for a high-throughput detector system with up to 1024 parallel counting channels and 20 ps time resolution. We describe the detector and electronic design, discuss the current status of the HiContent project and present the results from a 64-channel prototype system. In the absence of an operational detector, we present measurements of the electronics performance using a pulse generator to simulate detector events. Event timing results from the NINO high-speed front-end ASIC captured using a fast digital oscilloscope are compared with data taken with the proposed electronic configuration which uses the multi-channel HPTDC timing ASIC.

Development on the High-throughput Vol-oxidizer for Decladding and Voloxidation of Spent Fuel Rod-cuts

International Nuclear Information System (INIS)

Kim, Young Hwang; Jung, Jae Hoo; Kim, Ki Ho; Park, Byung Buk; Lee, Hyo Jik; Kim, Sung Hyun; Park, Hee Sung; Lee, Jong Kwang; Kim, Ho Dong

2009-12-01

A high-throughput vol-oxidizer which can handle a several ten kg HM/batch is being developed to supply U 3 O 8 powders to an electrolytic reduction reactor in pyro-processing. At the first year step(2007), for enhancement of oxidation and recovery rate, we analyzed the mechanical and chemical methods, and devised the main mechanism with ball drop methods and rotary kiln type. Also, the main devices for oxidation and recovery of rod-cuts were designed by using the Solid Works and COSMOS program tools, and manufactured after thermal/mechanical analysis. In order to verify the main devices, simulation fuels(W 90%+SiO 2 10%) were manufactured and the main devices were tested for the oxidation and recovery rate of its. Here the expansion ratio of simulation fuel is similar to U 3 O 8 (2.7). At the second year step(2008), with the constant ration of rod-cuts volume and expansion ratio of U 3 O 8 (2.7), we produced a theoretical equation that can estimate the volume of rod-cuts according to a variation of their weight and lengths. We considered various materials such as ceramics and Ni-Cr, finally, the APM material which can constantly maintain against high temperature(1,200 .deg. C) and vacuum(1 torr) was selected and a vol-oxidizer was designed. At the third year step(2009), in order to manufacture a high-throughput vol-oxidizer, we have analyzed the vol-oxidizer for remote operability and maintainability, also the remote assembling and disassembling possibilities of the selected modules have been analyzed in terms of visibility, interference, approach, weight, and so on. We have presented final modular design and manufactured a high-throughput vol-oxidizer. Also, we have conducted the blank, heating(over 500 .deg. C) and hull separation test(capacity : 50 kg HM/batch, hull length 50mm) on the high-throughput vol-oxidizer. Also, these design technologies for the high-throughput vol-oxidizer will be utilized in the development of a more efficient vol-oxidizer with higher

A high-throughput method to detect RNA profiling by integration of RT-MLPA with next generation sequencing technology.

Science.gov (United States)

Wang, Jing; Yang, Xue; Chen, Haofeng; Wang, Xuewei; Wang, Xiangyu; Fang, Yi; Jia, Zhenyu; Gao, Jidong

2017-07-11

RNA in formalin-fixed and paraffin-embedded (FFPE) tissues provides large amount of information indicating disease stages, histological tumor types and grades, as well as clinical outcomes. However, Detection of RNA expression levels in formalin-fixed and paraffin-embedded samples is extremely difficult due to poor RNA quality. Here we developed a high-throughput method, Reverse Transcription-Multiple Ligation-dependent Probe Sequencing (RT-MLPSeq), to determine expression levels of multiple transcripts in FFPE samples. By combining Reverse Transcription-Multiple Ligation-dependent Amplification method and next generation sequencing technology, RT-MLPSeq overcomes the limit of probe length in multiplex ligation-dependent probe amplification assay and thus could detect expression levels of transcripts without quantitative limitations. We proved that different RT-MLPSeq probes targeting on the same transcripts have highly consistent results and the starting RNA/cDNA input could be as little as 1 ng. RT-MLPSeq also presented consistent relative RNA levels of selected 13 genes with reverse transcription quantitative PCR. Finally, we demonstrated the application of the new RT-MLPSeq method by measuring the mRNA expression levels of 21 genes which can be used for accurate calculation of the breast cancer recurrence score - an index that has been widely used for managing breast cancer patients.

Digital imaging of root traits (DIRT): a high-throughput computing and collaboration platform for field-based root phenomics.

Science.gov (United States)

Das, Abhiram; Schneider, Hannah; Burridge, James; Ascanio, Ana Karine Martinez; Wojciechowski, Tobias; Topp, Christopher N; Lynch, Jonathan P; Weitz, Joshua S; Bucksch, Alexander

2015-01-01

Plant root systems are key drivers of plant function and yield. They are also under-explored targets to meet global food and energy demands. Many new technologies have been developed to characterize crop root system architecture (CRSA). These technologies have the potential to accelerate the progress in understanding the genetic control and environmental response of CRSA. Putting this potential into practice requires new methods and algorithms to analyze CRSA in digital images. Most prior approaches have solely focused on the estimation of root traits from images, yet no integrated platform exists that allows easy and intuitive access to trait extraction and analysis methods from images combined with storage solutions linked to metadata. Automated high-throughput phenotyping methods are increasingly used in laboratory-based efforts to link plant genotype with phenotype, whereas similar field-based studies remain predominantly manual low-throughput. Here, we present an open-source phenomics platform "DIRT", as a means to integrate scalable supercomputing architectures into field experiments and analysis pipelines. DIRT is an online platform that enables researchers to store images of plant roots, measure dicot and monocot root traits under field conditions, and share data and results within collaborative teams and the broader community. The DIRT platform seamlessly connects end-users with large-scale compute "commons" enabling the estimation and analysis of root phenotypes from field experiments of unprecedented size. DIRT is an automated high-throughput computing and collaboration platform for field based crop root phenomics. The platform is accessible at http://www.dirt.iplantcollaborative.org/ and hosted on the iPlant cyber-infrastructure using high-throughput grid computing resources of the Texas Advanced Computing Center (TACC). DIRT is a high volume central depository and high-throughput RSA trait computation platform for plant scientists working on crop roots

Fluorescence-based high-throughput screening of dicer cleavage activity.

Science.gov (United States)

Podolska, Katerina; Sedlak, David; Bartunek, Petr; Svoboda, Petr

2014-03-01

Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

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

Science.gov (United States)

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 Screening Using Fourier-Transform Infrared Imaging

Directory of Open Access Journals (Sweden)

Erdem Sasmaz

2015-06-01

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

Evaluation of Capacity on a High Throughput Vol-oxidizer for Operability

International Nuclear Information System (INIS)

Kim, Young Hwan; Park, Geun Il; Lee, Jung Won; Jung, Jae Hoo; Kim, Ki Ho; Lee, Yong Soon; Lee, Do Youn; Kim, Su Sung

2010-01-01

KAERI is developing a pyro-process. As a piece of process equipment, a high throughput vol-oxidizer which can handle a several tens kg HM/batch was developed to supply U 3 O 8 powders to an electrolytic reduction(ER) reactor. To increase the reduction yield, UO 2 pellets should be converted into uniform powders. In this paper, we aim at the evaluation of a high throughput vol-oxidizer for operability. The evaluation consisted of 3 targets, a mechanical motion test, a heating test and hull separation test. In order to test a high throughput vol-oxidizer, By using a control system, mechanical motion tests of the vol-oxidizer were conducted, and heating rates were analyzed. Also the separation tests of hulls for recovery rate were conducted. The test results of the vol-oxidizer are going to be applied for operability. A study on the characteristics of the volatile gas produced during a vol-oxidation process is not included in this study

Fun with High Throughput Toxicokinetics (CalEPA webinar)

Science.gov (United States)

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 ...

A high throughput architecture for a low complexity soft-output demapping algorithm

Science.gov (United States)

Ali, I.; Wasenmüller, U.; Wehn, N.

2015-11-01

Iterative channel decoders such as Turbo-Code and LDPC decoders show exceptional performance and therefore they are a part of many wireless communication receivers nowadays. These decoders require a soft input, i.e., the logarithmic likelihood ratio (LLR) of the received bits with a typical quantization of 4 to 6 bits. For computing the LLR values from a received complex symbol, a soft demapper is employed in the receiver. The implementation cost of traditional soft-output demapping methods is relatively large in high order modulation systems, and therefore low complexity demapping algorithms are indispensable in low power receivers. In the presence of multiple wireless communication standards where each standard defines multiple modulation schemes, there is a need to have an efficient demapper architecture covering all the flexibility requirements of these standards. Another challenge associated with hardware implementation of the demapper is to achieve a very high throughput in double iterative systems, for instance, MIMO and Code-Aided Synchronization. In this paper, we present a comprehensive communication and hardware performance evaluation of low complexity soft-output demapping algorithms to select the best algorithm for implementation. The main goal of this work is to design a high throughput, flexible, and area efficient architecture. We describe architectures to execute the investigated algorithms. We implement these architectures on a FPGA device to evaluate their hardware performance. The work has resulted in a hardware architecture based on the figured out best low complexity algorithm delivering a high throughput of 166 Msymbols/second for Gray mapped 16-QAM modulation on Virtex-5. This efficient architecture occupies only 127 slice registers, 248 slice LUTs and 2 DSP48Es.

Development of High-Throughput Method for Measurement of Vascular Nitric Oxide Generation in Microplate Reader

Directory of Open Access Journals (Sweden)

Soad S. Abd El-Hay

2017-01-01

Full Text Available Background: Despite the importance of nitric oxide (NO in vascular physiology and pathology, a high-throughput method for the quantification of its vascular generation is lacking. Objective: By using the fluorescent probe 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM, we have optimized a simple method for the determination of the generation of endothelial nitric oxide in a microplate format. Methods: A nitric oxide donor was used (3-morpholinosydnonimine hydrochloride, SIN-1. Different factors affecting the method were studied, such as the effects of dye concentration, different buffers, time of reaction, gain, and number of flashes. Results: Beer’s law was linear over a nanomolar range (1–10 nM of SIN-1 with wavelengths of maximum excitation and emission at 495 and 525 nm; the limit of detection reached 0.897 nM. Under the optimized conditions, the generation of rat aortic endothelial NO was measured by incubating DAF-FM with serial concentrations (10–1000 µM of acetylcholine (ACh for 3 min. To confirm specificity, Nω-Nitro-l-arginine methyl ester (l-NAME—the standard inhibitor of endothelial NO synthase—was found to inhibit the ACh-stimulated generation of NO. In addition, vessels pre-exposed for 1 h to 400 µM of the endothelial damaging agent methyl glyoxal showed inhibited NO generation when compared to the control stimulated by ACh. Conclusions: The capability of the method to measure micro-volume samples makes it convenient for the simultaneous handling of a very large number of samples. Additionally, it allows samples to be run simultaneously with their replicates to ensure identical experimental conditions, thus minimizing the effect of biological variability.

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

Science.gov (United States)

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

2017-12-01

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.

Comparing the normalization methods for the differential analysis of Illumina high-throughput RNA-Seq data.

Science.gov (United States)

Li, Peipei; Piao, Yongjun; Shon, Ho Sun; Ryu, Keun Ho

2015-10-28

Recently, rapid improvements in technology and decrease in sequencing costs have made RNA-Seq a widely used technique to quantify gene expression levels. Various normalization approaches have been proposed, owing to the importance of normalization in the analysis of RNA-Seq data. A comparison of recently proposed normalization methods is required to generate suitable guidelines for the selection of the most appropriate approach for future experiments. In this paper, we compared eight non-abundance (RC, UQ, Med, TMM, DESeq, Q, RPKM, and ERPKM) and two abundance estimation normalization methods (RSEM and Sailfish). The experiments were based on real Illumina high-throughput RNA-Seq of 35- and 76-nucleotide sequences produced in the MAQC project and simulation reads. Reads were mapped with human genome obtained from UCSC Genome Browser Database. For precise evaluation, we investigated Spearman correlation between the normalization results from RNA-Seq and MAQC qRT-PCR values for 996 genes. Based on this work, we showed that out of the eight non-abundance estimation normalization methods, RC, UQ, Med, TMM, DESeq, and Q gave similar normalization results for all data sets. For RNA-Seq of a 35-nucleotide sequence, RPKM showed the highest correlation results, but for RNA-Seq of a 76-nucleotide sequence, least correlation was observed than the other methods. ERPKM did not improve results than RPKM. Between two abundance estimation normalization methods, for RNA-Seq of a 35-nucleotide sequence, higher correlation was obtained with Sailfish than that with RSEM, which was better than without using abundance estimation methods. However, for RNA-Seq of a 76-nucleotide sequence, the results achieved by RSEM were similar to without applying abundance estimation methods, and were much better than with Sailfish. Furthermore, we found that adding a poly-A tail increased alignment numbers, but did not improve normalization results. Spearman correlation analysis revealed that RC, UQ

Modeling Steroidogenesis Disruption Using High-Throughput ...

Science.gov (United States)

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

Towards low-delay and high-throughput cognitive radio vehicular networks

Directory of Open Access Journals (Sweden)

Nada Elgaml

2017-12-01

Full Text Available Cognitive Radio Vehicular Ad-hoc Networks (CR-VANETs exploit cognitive radios to allow vehicles to access the unused channels in their radio environment. Thus, CR-VANETs do not only suffer the traditional CR problems, especially spectrum sensing, but also suffer new challenges due to the highly dynamic nature of VANETs. In this paper, we present a low-delay and high-throughput radio environment assessment scheme for CR-VANETs that can be easily incorporated with the IEEE 802.11p standard developed for VANETs. Simulation results show that the proposed scheme significantly reduces the time to get the radio environment map and increases the CR-VANET throughput.

Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

Science.gov (United States)

Lam, Kathy N; Hall, Michael W; Engel, Katja; Vey, Gregory; Cheng, Jiujun; Neufeld, Josh D; Charles, Trevor C

2014-01-01

High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.

High throughput 16S rRNA gene amplicon sequencing

DEFF Research Database (Denmark)

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

S rRNA gene amplicon sequencing has been developed over the past few years and is now ready to use for more comprehensive studies related to plant operation and optimization thanks to short analysis time, low cost, high throughput, and high taxonomic resolution. In this study we show how 16S r......RNA gene amplicon sequencing can be used to reveal factors of importance for the operation of full-scale nutrient removal plants related to settling problems and floc properties. Using optimized DNA extraction protocols, indexed primers and our in-house Illumina platform, we prepared multiple samples...... be correlated to the presence of the species that are regarded as “strong” and “weak” floc formers. In conclusion, 16S rRNA gene amplicon sequencing provides a high throughput approach for a rapid and cheap community profiling of activated sludge that in combination with multivariate statistics can be used...

A high-throughput colorimetric assay for glucose detection based on glucose oxidase-catalyzed enlargement of gold nanoparticles

Science.gov (United States)

Xiong, Yanmei; Zhang, Yuyan; Rong, Pengfei; Yang, Jie; Wang, Wei; Liu, Dingbin

2015-09-01

We developed a simple high-throughput colorimetric assay to detect glucose based on the glucose oxidase (GOx)-catalysed enlargement of gold nanoparticles (AuNPs). Compared with the currently available glucose kit method, the AuNP-based assay provides higher clinical sensitivity at lower cost, indicating its great potential to be a powerful tool for clinical screening of glucose.We developed a simple high-throughput colorimetric assay to detect glucose based on the glucose oxidase (GOx)-catalysed enlargement of gold nanoparticles (AuNPs). Compared with the currently available glucose kit method, the AuNP-based assay provides higher clinical sensitivity at lower cost, indicating its great potential to be a powerful tool for clinical screening of glucose. Electronic supplementary information (ESI) available: Experimental section and additional figures. See DOI: 10.1039/c5nr03758a

Development of combinatorial chemistry methods for coatings: high-throughput adhesion evaluation and scale-up of combinatorial leads.

Science.gov (United States)

Potyrailo, Radislav A; Chisholm, Bret J; Morris, William G; Cawse, James N; Flanagan, William P; Hassib, Lamyaa; Molaison, Chris A; Ezbiansky, Karin; Medford, George; Reitz, Hariklia

2003-01-01

Coupling of combinatorial chemistry methods with high-throughput (HT) performance testing and measurements of resulting properties has provided a powerful set of tools for the 10-fold accelerated discovery of new high-performance coating materials for automotive applications. Our approach replaces labor-intensive steps with automated systems for evaluation of adhesion of 8 x 6 arrays of coating elements that are discretely deposited on a single 9 x 12 cm plastic substrate. Performance of coatings is evaluated with respect to their resistance to adhesion loss, because this parameter is one of the primary considerations in end-use automotive applications. Our HT adhesion evaluation provides previously unavailable capabilities of high speed and reproducibility of testing by using a robotic automation, an expanded range of types of tested coatings by using the coating tagging strategy, and an improved quantitation by using high signal-to-noise automatic imaging. Upon testing, the coatings undergo changes that are impossible to quantitatively predict using existing knowledge. Using our HT methodology, we have developed several coatings leads. These HT screening results for the best coating compositions have been validated on the traditional scales of coating formulation and adhesion loss testing. These validation results have confirmed the superb performance of combinatorially developed coatings over conventional coatings on the traditional scale.

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

Directory of Open Access Journals (Sweden)

Soichi Inagaki

Full Text Available 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.

Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis.

Science.gov (United States)

Mei, Feng; Fancy, Stephen P J; Shen, Yun-An A; Niu, Jianqin; Zhao, Chao; Presley, Bryan; Miao, Edna; Lee, Seonok; Mayoral, Sonia R; Redmond, Stephanie A; Etxeberria, Ainhoa; Xiao, Lan; Franklin, Robin J M; Green, Ari; Hauser, Stephen L; Chan, Jonah R

2014-08-01

Functional screening for compounds that promote remyelination represents a major hurdle in the development of rational therapeutics for multiple sclerosis. Screening for remyelination is problematic, as myelination requires the presence of axons. Standard methods do not resolve cell-autonomous effects and are not suited for high-throughput formats. Here we describe a binary indicant for myelination using micropillar arrays (BIMA). Engineered with conical dimensions, micropillars permit resolution of the extent and length of membrane wrapping from a single two-dimensional image. Confocal imaging acquired from the base to the tip of the pillars allows for detection of concentric wrapping observed as 'rings' of myelin. The platform is formatted in 96-well plates, amenable to semiautomated random acquisition and automated detection and quantification. Upon screening 1,000 bioactive molecules, we identified a cluster of antimuscarinic compounds that enhance oligodendrocyte differentiation and remyelination. Our findings demonstrate a new high-throughput screening platform for potential regenerative therapeutics in multiple sclerosis.

Reverse Phase Protein Arrays for High-throughput Toxicity Screening

DEFF Research Database (Denmark)

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

High-throughput screening is extensively applied for identification of drug targets and drug discovery and recently it found entry into toxicity testing. Reverse phase protein arrays (RPPAs) are used widespread for quantification of protein markers. We reasoned that RPPAs also can be utilized...... beneficially in automated high-throughput toxicity testing. An advantage of using RPPAs is that, in addition to the baseline toxicity readout, they allow testing of multiple markers of toxicity, such as inflammatory responses, which do not necessarily cumulate in cell death. We used transfection of si......RNAs with known killing effects as a model system to demonstrate that RPPA-based protein quantification can serve as substitute readout of cell viability, hereby reliably reflecting toxicity. In terms of automation, cell exposure, protein harvest, serial dilution and sample reformatting were performed using...

Laterally orienting C. elegans using geometry at microscale for high-throughput visual screens in neurodegeneration and neuronal development studies.

Directory of Open Access Journals (Sweden)

Ivan de Carlos Cáceres

Full Text Available C. elegans is an excellent model system for studying neuroscience using genetics because of its relatively simple nervous system, sequenced genome, and the availability of a large number of transgenic and mutant strains. Recently, microfluidic devices have been used for high-throughput genetic screens, replacing traditional methods of manually handling C. elegans. However, the orientation of nematodes within microfluidic devices is random and often not conducive to inspection, hindering visual analysis and overall throughput. In addition, while previous studies have utilized methods to bias head and tail orientation, none of the existing techniques allow for orientation along the dorso-ventral body axis. Here, we present the design of a simple and robust method for passively orienting worms into lateral body positions in microfluidic devices to facilitate inspection of morphological features with specific dorso-ventral alignments. Using this technique, we can position animals into lateral orientations with up to 84% efficiency, compared to 21% using existing methods. We isolated six mutants with neuronal development or neurodegenerative defects, showing that our technology can be used for on-chip analysis and high-throughput visual screens.

DAG expression: high-throughput gene expression analysis of real-time PCR data using standard curves for relative quantification.

Directory of Open Access Journals (Sweden)

María Ballester

Full Text Available BACKGROUND: Real-time quantitative PCR (qPCR is still the gold-standard technique for gene-expression quantification. Recent technological advances of this method allow for the high-throughput gene-expression analysis, without the limitations of sample space and reagent used. However, non-commercial and user-friendly software for the management and analysis of these data is not available. RESULTS: The recently developed commercial microarrays allow for the drawing of standard curves of multiple assays using the same n-fold diluted samples. Data Analysis Gene (DAG Expression software has been developed to perform high-throughput gene-expression data analysis using standard curves for relative quantification and one or multiple reference genes for sample normalization. We discuss the application of DAG Expression in the analysis of data from an experiment performed with Fluidigm technology, in which 48 genes and 115 samples were measured. Furthermore, the quality of our analysis was tested and compared with other available methods. CONCLUSIONS: DAG Expression is a freely available software that permits the automated analysis and visualization of high-throughput qPCR. A detailed manual and a demo-experiment are provided within the DAG Expression software at http://www.dagexpression.com/dage.zip.

High-throughput search for caloric materials: the CaloriCool approach

Science.gov (United States)

Zarkevich, N. A.; Johnson, D. D.; Pecharsky, V. K.

2018-01-01

The high-throughput search paradigm adopted by the newly established caloric materials consortium—CaloriCool®—with the goal to substantially accelerate discovery and design of novel caloric materials is briefly discussed. We begin with describing material selection criteria based on known properties, which are then followed by heuristic fast estimates, ab initio calculations, all of which has been implemented in a set of automated computational tools and measurements. We also demonstrate how theoretical and computational methods serve as a guide for experimental efforts by considering a representative example from the field of magnetocaloric materials.

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

DEFF Research Database (Denmark)

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

2017-01-01

and machine learning identifies protein properties that hinder the HPA high-throughput antibody production pipeline. We predict protein expression and solubility with accuracies of 70% and 80%, respectively, based on a subset of key properties (aromaticity, hydropathy and isoelectric point). We guide...... the selection of protein fragments based on these characteristics to optimize high-throughput experimentation. Availability and implementation: We present the machine learning workflow as a series of IPython notebooks hosted on GitHub (https://github.com/SBRG/Protein_ML). The workflow can be used as a template...

Quality control methodology for high-throughput protein-protein interaction screening.

Science.gov (United States)

Vazquez, Alexei; Rual, Jean-François; Venkatesan, Kavitha

2011-01-01

Protein-protein interactions are key to many aspects of the cell, including its cytoskeletal structure, the signaling processes in which it is involved, or its metabolism. Failure to form protein complexes or signaling cascades may sometimes translate into pathologic conditions such as cancer or neurodegenerative diseases. The set of all protein interactions between the proteins encoded by an organism constitutes its protein interaction network, representing a scaffold for biological function. Knowing the protein interaction network of an organism, combined with other sources of biological information, can unravel fundamental biological circuits and may help better understand the molecular basics of human diseases. The protein interaction network of an organism can be mapped by combining data obtained from both low-throughput screens, i.e., "one gene at a time" experiments and high-throughput screens, i.e., screens designed to interrogate large sets of proteins at once. In either case, quality controls are required to deal with the inherent imperfect nature of experimental assays. In this chapter, we discuss experimental and statistical methodologies to quantify error rates in high-throughput protein-protein interactions screens.

High-throughput screening to enhance oncolytic virus immunotherapy

Directory of Open Access Journals (Sweden)

Allan KJ

2016-04-01

Full Text Available KJ Allan,1,2 David F Stojdl,1–3 SL Swift1 1Children’s Hospital of Eastern Ontario (CHEO Research Institute, 2Department of Biology, Microbiology and Immunology, 3Department of Pediatrics, University of Ottawa, Ottawa, ON, Canada Abstract: High-throughput screens can rapidly scan and capture large amounts of information across multiple biological parameters. Although many screens have been designed to uncover potential new therapeutic targets capable of crippling viruses that cause disease, there have been relatively few directed at improving the efficacy of viruses that are used to treat disease. Oncolytic viruses (OVs are biotherapeutic agents with an inherent specificity for treating malignant disease. Certain OV platforms – including those based on herpes simplex virus, reovirus, and vaccinia virus – have shown success against solid tumors in advanced clinical trials. Yet, many of these OVs have only undergone minimal engineering to solidify tumor specificity, with few extra modifications to manipulate additional factors. Several aspects of the interaction between an OV and a tumor-bearing host have clear value as targets to improve therapeutic outcomes. At the virus level, these include delivery to the tumor, infectivity, productivity, oncolysis, bystander killing, spread, and persistence. At the host level, these include engaging the immune system and manipulating the tumor microenvironment. Here, we review the chemical- and genome-based high-throughput screens that have been performed to manipulate such parameters during OV infection and analyze their impact on therapeutic efficacy. We further explore emerging themes that represent key areas of focus for future research. Keywords: oncolytic, virus, screen, high-throughput, cancer, chemical, genomic, immunotherapy

Adaptation to high throughput batch chromatography enhances multivariate screening.

Science.gov (United States)

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

2015-09-01

High throughput process development offers unique approaches to explore complex process design spaces with relatively low material consumption. Batch chromatography is one technique that can be used to screen chromatographic conditions in a 96-well plate. Typical batch chromatography workflows examine variations in buffer conditions or comparison of multiple resins in a given process, as opposed to the assessment of protein loading conditions in combination with other factors. A modification to the batch chromatography paradigm is described here where experimental planning, programming, and a staggered loading approach increase the multivariate space that can be explored with a liquid handling system. The iterative batch chromatography (IBC) approach is described, which treats every well in a 96-well plate as an individual experiment, wherein protein loading conditions can be varied alongside other factors such as wash and elution buffer conditions. As all of these factors are explored in the same experiment, the interactions between them are characterized and the number of follow-up confirmatory experiments is reduced. This in turn improves statistical power and throughput. Two examples of the IBC method are shown and the impact of the load conditions are assessed in combination with the other factors explored. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Leveraging the Power of High Performance Computing for Next Generation Sequencing Data Analysis: Tricks and Twists from a High Throughput Exome Workflow

Science.gov (United States)

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

2015-01-01

Next generation sequencing (NGS) has been a great success and is now a standard method of research in the life sciences. With this technology, dozens of whole genomes or hundreds of exomes can be sequenced in rather short time, producing huge amounts of data. Complex bioinformatics analyses are required to turn these data into scientific findings. In order to run these analyses fast, automated workflows implemented on high performance computers are state of the art. While providing sufficient compute power and storage to meet the NGS data challenge, high performance computing (HPC) systems require special care when utilized for high throughput processing. This is especially true if the HPC system is shared by different users. Here, stability, robustness and maintainability are as important for automated workflows as speed and throughput. To achieve all of these aims, dedicated solutions have to be developed. In this paper, we present the tricks and twists that we utilized in the implementation of our exome data processing workflow. It may serve as a guideline for other high throughput data analysis projects using a similar infrastructure. The code implementing our solutions is provided in the supporting information files. PMID:25942438

MetaUniDec: High-Throughput Deconvolution of Native Mass Spectra

Science.gov (United States)

Reid, Deseree J.; Diesing, Jessica M.; Miller, Matthew A.; Perry, Scott M.; Wales, Jessica A.; Montfort, William R.; Marty, Michael T.

2018-04-01

The expansion of native mass spectrometry (MS) methods for both academic and industrial applications has created a substantial need for analysis of large native MS datasets. Existing software tools are poorly suited for high-throughput deconvolution of native electrospray mass spectra from intact proteins and protein complexes. The UniDec Bayesian deconvolution algorithm is uniquely well suited for high-throughput analysis due to its speed and robustness but was previously tailored towards individual spectra. Here, we optimized UniDec for deconvolution, analysis, and visualization of large data sets. This new module, MetaUniDec, centers around a hierarchical data format 5 (HDF5) format for storing datasets that significantly improves speed, portability, and file size. It also includes code optimizations to improve speed and a new graphical user interface for visualization, interaction, and analysis of data. To demonstrate the utility of MetaUniDec, we applied the software to analyze automated collision voltage ramps with a small bacterial heme protein and large lipoprotein nanodiscs. Upon increasing collisional activation, bacterial heme-nitric oxide/oxygen binding (H-NOX) protein shows a discrete loss of bound heme, and nanodiscs show a continuous loss of lipids and charge. By using MetaUniDec to track changes in peak area or mass as a function of collision voltage, we explore the energetic profile of collisional activation in an ultra-high mass range Orbitrap mass spectrometer. [Figure not available: see fulltext.

DRABAL: novel method to mine large high-throughput screening assays using Bayesian active learning

KAUST Repository

Soufan, Othman

2016-11-10

Background Mining high-throughput screening (HTS) assays is key for enhancing decisions in the area of drug repositioning and drug discovery. However, many challenges are encountered in the process of developing suitable and accurate methods for extracting useful information from these assays. Virtual screening and a wide variety of databases, methods and solutions proposed to-date, did not completely overcome these challenges. This study is based on a multi-label classification (MLC) technique for modeling correlations between several HTS assays, meaning that a single prediction represents a subset of assigned correlated labels instead of one label. Thus, the devised method provides an increased probability for more accurate predictions of compounds that were not tested in particular assays. Results Here we present DRABAL, a novel MLC solution that incorporates structure learning of a Bayesian network as a step to model dependency between the HTS assays. In this study, DRABAL was used to process more than 1.4 million interactions of over 400,000 compounds and analyze the existing relationships between five large HTS assays from the PubChem BioAssay Database. Compared to different MLC methods, DRABAL significantly improves the F1Score by about 22%, on average. We further illustrated usefulness and utility of DRABAL through screening FDA approved drugs and reported ones that have a high probability to interact with several targets, thus enabling drug-multi-target repositioning. Specifically DRABAL suggests the Thiabendazole drug as a common activator of the NCP1 and Rab-9A proteins, both of which are designed to identify treatment modalities for the Niemann–Pick type C disease. Conclusion We developed a novel MLC solution based on a Bayesian active learning framework to overcome the challenge of lacking fully labeled training data and exploit actual dependencies between the HTS assays. The solution is motivated by the need to model dependencies between existing

High-Throughput Tools for Characterization of Antibody Epitopes

DEFF Research Database (Denmark)

Christiansen, Anders

mapping. In Chapter 1, it was examined whether combining phage display, a traditional epitope mapping approach, with HTS would improve the method. The developed approach was successfully used to map Ara h 1 epitopes in sera from patients with peanut allergy. Notably, the sera represented difficult...... proliferation advantages. Finally, in Chapter 4, a different emerging technology, next-generation peptide microarrays, was applied for epitope mapping of major peanut allergens using sera from allergic patients. New developments in the peptide microarray have enabled a greatly increased throughput....... In this study, these improvements were utilized to characterize epitopes at high resolution, i.e. determine the importance of each residue for antibody binding, for all major peanut allergens. Epitope reactivity among patients often converged on known epitope hotspots, however the binding patterns were somewhat...

MGI-oriented High-throughput Measurement of Interdiffusion Coefficient Matrices in Ni-based Superalloys

Directory of Open Access Journals (Sweden)

TANG Ying

2017-01-01

Full Text Available One of the research hotspots in the field of high-temperature alloys was to search the substitutional elements for Re in order to prepare the single-crystal Ni-based superalloys with less or even no Re addition. To find the elements with similar or even lower diffusion coefficients in comparison with that of Re was one of the effective strategies. In multicomponent alloys, the interdiffusivity matrix were used to comprehensively characterize the diffusion ability of any alloying elements. Therefore, accurate determination of the composition-dependant and temperature-dependent interdiffusivities matrices of different elements in γ and γ' phases of Ni-based superalloys was high priority. The paper briefly introduces of the status of the interdiffusivity matrices determination in Ni-based superalloys, and the methods for determining the interdiffusivities in multicomponent alloys, including the traditional Matano-Kirkaldy method and recently proposed numerical inverse method. Because the traditional Matano-Kirkaldy method is of low efficiency, the experimental reports on interdiffusivity matrices in ternary and higher order sub-systems of the Ni-based superalloys were very scarce in the literature. While the numerical inverse method newly proposed in our research group based on Fick's second law can be utilized for high-throughput measurement of accurate interdiffusivity matrices in alloys with any number of components. After that, the successful application of the numerical inverse method in the high-throughput measurement of interdiffusivity matrices in alloys is demonstrated in fcc (γ phase of the ternary Ni-Al-Ta system. Moreover, the validation of the resulting composition-dependant and temperature-dependent interdiffusivity matrices is also comprehensively made. Then, this paper summarizes the recent progress in the measurement of interdiffusivity matrices in γ and γ' phases of a series of core ternary Ni-based superalloys achieved in

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

Science.gov (United States)

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

High throughput electrophysiology: new perspectives for ion channel drug discovery

DEFF Research Database (Denmark)

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

2003-01-01

Proper function of ion channels is crucial for all living cells. Ion channel dysfunction may lead to a number of diseases, so-called channelopathies, and a number of common diseases, including epilepsy, arrhythmia, and type II diabetes, are primarily treated by drugs that modulate ion channels....... A cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct...... characterization of ion channel properties. However, patch clamp is a slow, labor-intensive, and thus expensive, technique. New techniques combining the reliability and high information content of patch clamping with the virtues of high throughput philosophy are emerging and predicted to make a number of ion...

A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC libraries using High Resolution Melt analysis

Directory of Open Access Journals (Sweden)

Caligari Peter DS

2010-05-01

Full Text Available Abstract Background The high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. Multidimentional BAC pooling strategies for PCR-based screening of large insert libraries is a widely used alternative to high density filter hybridisation of bacterial colonies. To date, concerns over reliability have led most if not all groups engaged in high throughput physical mapping projects to favour BAC DNA isolation prior to amplification by conventional PCR. Results Here, we report the first combined use of Multiplex Tandem PCR (MT-PCR and High Resolution Melt (HRM analysis on bacterial stocks of BAC library superpools as a means of rapidly anchoring markers to BAC colonies and thereby to integrate genetic and physical maps. We exemplify the approach using a BAC library of the model plant Arabidopsis thaliana. Super pools of twenty five 384-well plates and two-dimension matrix pools of the BAC library were prepared for marker screening. The entire procedure only requires around 3 h to anchor one marker. Conclusions A pre-amplification step during MT-PCR allows high multiplexing and increases the sensitivity and reliability of subsequent HRM discrimination. This simple gel-free protocol is more reliable, faster and far less costly than conventional PCR screening. The option to screen in parallel 3 genetic markers in one MT-PCR-HRM reaction using templates from directly pooled bacterial stocks of BAC-containing bacteria further reduces time for anchoring markers in physical maps of species with large genomes.

Statistical removal of background signals from high-throughput 1H NMR line-broadening ligand-affinity screens

International Nuclear Information System (INIS)

Worley, Bradley; Sisco, Nicholas J.; Powers, Robert

2015-01-01

NMR ligand-affinity screens are vital to drug discovery, are routinely used to screen fragment-based libraries, and used to verify chemical leads from high-throughput assays and virtual screens. NMR ligand-affinity screens are also a highly informative first step towards identifying functional epitopes of unknown proteins, as well as elucidating the biochemical functions of protein–ligand interaction at their binding interfaces. While simple one-dimensional 1 H NMR experiments are capable of indicating binding through a change in ligand line shape, they are plagued by broad, ill-defined background signals from protein 1 H resonances. We present an uncomplicated method for subtraction of protein background in high-throughput ligand-based affinity screens, and show that its performance is maximized when phase-scatter correction is applied prior to subtraction

High throughput screening of starch structures using carbohydrate microarrays

DEFF Research Database (Denmark)

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

2016-01-01

In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated...

A simpler sampling interface of venturi easy ambient sonic-spray ionization mass spectrometry for high-throughput screening enzyme inhibitors.

Science.gov (United States)

Liu, Ning; Liu, Yang; Yang, YuHan; He, Lan; Ouyang, Jin

2016-03-24

High-throughput screening (HTS) is often required in enzyme inhibitor drugs screening. Mass spectrometry (MS) provides a powerful method for high-throughput screening enzyme inhibitors because its high speed, sensitivity and property of lable free. However, most of the MS methods need complicated sampling interface system. Overall throughput was limited by sample loading in these cases. In this study, we develop a simple interface which coupled droplet segmented system to a venturi easy ambient sonic-spray ionization mass spectrometer. It is fabricated by using a single capillary to act as both sampling probe and the emitter, which simplifies the construction, reduces the cost and shorten the sampling time. Samples sucked by venturi effect are segmented to nanoliter plugs by air, then the plugs can be detected by MS directly. This system eliminated the need for flow injection which was popular used in classic scheme. The new system is applied to screen angiotensin converting enzyme inhibitors. High-throughput was achieved in analyzing 96 samples at 1.6 s per sample. The plugs formation was at 0.5s per sample. Carry-over between samples was less than 5%, the peak height RSD was 2.92% (n = 15). Dose-response curves of 3 known inhibitors were also measured to validate its potential in drug discovery. The calculated IC50 agreed well with reported values. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of High-Throughput Method for Measurement of Vascular Nitric Oxide Generation in Microplate Reader.

Science.gov (United States)

Abd El-Hay, Soad S; Colyer, Christa L

2017-01-13

Despite the importance of nitric oxide (NO) in vascular physiology and pathology, a high-throughput method for the quantification of its vascular generation is lacking. By using the fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), we have optimized a simple method for the determination of the generation of endothelial nitric oxide in a microplate format. A nitric oxide donor was used (3-morpholinosydnonimine hydrochloride, SIN-1). Different factors affecting the method were studied, such as the effects of dye concentration, different buffers, time of reaction, gain, and number of flashes. Beer's law was linear over a nanomolar range (1-10 nM) of SIN-1 with wavelengths of maximum excitation and emission at 495 and 525 nm; the limit of detection reached 0.897 nM. Under the optimized conditions, the generation of rat aortic endothelial NO was measured by incubating DAF-FM with serial concentrations (10-1000 µM) of acetylcholine (ACh) for 3 min. To confirm specificity, N ω -Nitro-l-arginine methyl ester (l-NAME)-the standard inhibitor of endothelial NO synthase-was found to inhibit the ACh-stimulated generation of NO. In addition, vessels pre-exposed for 1 h to 400 µM of the endothelial damaging agent methyl glyoxal showed inhibited NO generation when compared to the control stimulated by ACh. The capability of the method to measure micro-volume samples makes it convenient for the simultaneous handling of a very large number of samples. Additionally, it allows samples to be run simultaneously with their replicates to ensure identical experimental conditions, thus minimizing the effect of biological variability.

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

Science.gov (United States)

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

2016-02-01

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

A priori Considerations When Conducting High-Throughput Amplicon-Based Sequence Analysis

Directory of Open Access Journals (Sweden)

Aditi Sengupta

2016-03-01

Full Text Available Amplicon-based sequencing strategies that include 16S rRNA and functional genes, alongside “meta-omics” analyses of communities of microorganisms, have allowed researchers to pose questions and find answers to “who” is present in the environment and “what” they are doing. Next-generation sequencing approaches that aid microbial ecology studies of agricultural systems are fast gaining popularity among agronomy, crop, soil, and environmental science researchers. Given the rapid development of these high-throughput sequencing techniques, researchers with no prior experience will desire information about the best practices that can be used before actually starting high-throughput amplicon-based sequence analyses. We have outlined items that need to be carefully considered in experimental design, sampling, basic bioinformatics, sequencing of mock communities and negative controls, acquisition of metadata, and in standardization of reaction conditions as per experimental requirements. Not all considerations mentioned here may pertain to a particular study. The overall goal is to inform researchers about considerations that must be taken into account when conducting high-throughput microbial DNA sequencing and sequences analysis.

High-throughput micro-scale cultivations and chromatography modeling: Powerful tools for integrated process development.

Science.gov (United States)

Baumann, Pascal; Hahn, Tobias; Hubbuch, Jürgen

2015-10-01

Upstream processes are rather complex to design and the productivity of cells under suitable cultivation conditions is hard to predict. The method of choice for examining the design space is to execute high-throughput cultivation screenings in micro-scale format. Various predictive in silico models have been developed for many downstream processes, leading to a reduction of time and material costs. This paper presents a combined optimization approach based on high-throughput micro-scale cultivation experiments and chromatography modeling. The overall optimized system must not necessarily be the one with highest product titers, but the one resulting in an overall superior process performance in up- and downstream. The methodology is presented in a case study for the Cherry-tagged enzyme Glutathione-S-Transferase from Escherichia coli SE1. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for direct product analytics by simple VIS absorption measurements. High-throughput cultivations were carried out in a 48-well format in a BioLector micro-scale cultivation system (m2p-Labs, Germany). The downstream process optimization for a set of randomly picked upstream conditions producing high yields was performed in silico using a chromatography modeling software developed in-house (ChromX). The suggested in silico-optimized operational modes for product capturing were validated subsequently. The overall best system was chosen based on a combination of excellent up- and downstream performance. © 2015 Wiley Periodicals, Inc.

Neural progenitor cells as models for high-throughput screens of developmental neurotoxicity: State of the science

NARCIS (Netherlands)

Breier, J.M.; Gassmann, K.; Kayser, R.; Stegeman, H.; Groot, D.de; Fritsche, E.; Shafer, T.J.

2010-01-01

In vitro, high-throughput methods have been widely recommended as an approach to screen chemicals for the potential to cause developmental neurotoxicity and prioritize them for additional testing. The choice of cellular models for such an approach will have important ramifications for the accuracy,

High-throughput creation of micropatterned PDMS surfaces using microscale dual roller casting

International Nuclear Information System (INIS)

DiBartolomeo, Franklin J; Ge, Ning; Trinkle, Christine A

2012-01-01

This work introduces microscale dual roller casting (MDRC), a novel high-throughput fabrication method for creating continuous micropatterned surfaces using thermosetting polymers. MDRC utilizes a pair of rotating, heated cylindrical molds with microscale surface patterns to cure a continuous microstructured film. Using unmodified polydimethylsiloxane as the thermosetting polymer, we were able to create optically transparent, biocompatible surfaces with submicron patterning fidelity. Compared to other roll-to-roll fabrication processes, this method offers increased flexibility in the types of materials and topography that can be generated, including dual-sided patterning, embedded materials and tunable film thickness. (paper)

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

Science.gov (United States)

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

2002-02-01

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

High-Throughput Analysis of Enzyme Activities

Energy Technology Data Exchange (ETDEWEB)

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

2007-01-01

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

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

Science.gov (United States)

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.

Multi-channel counter-current chromatography for high-throughput fractionation of natural products for drug discovery.

Science.gov (United States)

Wu, Shihua; Yang, Lu; Gao, Yuan; Liu, Xiaoyue; Liu, Feiyan

2008-02-08

A multi-channel counter-current chromatography (CCC) method has been designed and fabricated for the high-throughput fractionation of natural products without complications sometimes encountered with other conventional chromatographic systems, such as irreversible adsorptive constituent losses and deactivation, tailing of solute peaks and contamination. It has multiple independent CCC channels and each channel connects independent separation column(s) by parallel flow tubes, and thus the multi-channel CCC apparatus can achieve simultaneously two or more independent chromatographic processes. Furthermore, a high-throughput CCC fractionation method for natural products has been developed by a combination of a new three-channel CCC apparatus and conventional parallel chromatographic devices including pumps, sample injectors, effluent detectors and collectors, and its performance has been displayed on the fractionation of ethyl acetate extracts of three natural materials Solidago canadensis, Suillus placidus, and Trichosanthes kirilowii, which are found to be potent cytotoxic to tumor cell lines in the course of screening the antitumor candidates. By combination of biological screening programs and preparative high-performance liquid chromatography (HPLC) purification, 22.8 mg 6 beta-angeloyloxykolavenic acid and 29.4 mg 6 beta-tigloyloxykolavenic acid for S. canadensis, 25.3mg suillin for S. placidus, and 6.8 mg 23,24-dihydrocucurbitacin B for T. Kirilowii as their major cytotoxic principles were isolated from each 1000 mg crude ethyl acetate extract. Their chemical structures were characterized by electrospray ionization mass spectrometry, one- and two-dimensional nuclear magnetic resonance. The overall results indicate the multi-channel CCC is very useful for high-throughput fractionation of natural products for drug discovery in spite of the solvent balancing requirement and the lower resolution of the shorter CCC columns.

High-throughput full-automatic synchrotron-based tomographic microscopy

International Nuclear Information System (INIS)

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

2011-01-01

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

Investigation of Human Cancers for Retrovirus by Low-Stringency Target Enrichment and High-Throughput Sequencing

DEFF Research Database (Denmark)

Vinner, Lasse; Mourier, Tobias; Friis-Nielsen, Jens

2015-01-01

-stringency in-solution hybridization method enables detection of discovery of hitherto unknown viral sequences by high-throughput sequencing. The sensitivity was sufficient to detect retroviral...... sequences in clinical samples. We used this method to conduct an investigation for novel retrovirus in samples from three cancer types. In accordance with recent studies our investigation revealed no retroviral infections in human B-cell lymphoma cells, cutaneous T-cell lymphoma or colorectal cancer...

High-throughput characterization for solar fuels materials discovery

Science.gov (United States)

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

2014-03-01

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

The Protein Maker: an automated system for high-throughput parallel purification

International Nuclear Information System (INIS)

Smith, Eric R.; Begley, Darren W.; Anderson, Vanessa; Raymond, Amy C.; Haffner, Taryn E.; Robinson, John I.; Edwards, Thomas E.; Duncan, Natalie; Gerdts, Cory J.; Mixon, Mark B.; Nollert, Peter; Staker, Bart L.; Stewart, Lance J.

2011-01-01

The Protein Maker instrument addresses a critical bottleneck in structural genomics by allowing automated purification and buffer testing of multiple protein targets in parallel with a single instrument. Here, the use of this instrument to (i) purify multiple influenza-virus proteins in parallel for crystallization trials and (ii) identify optimal lysis-buffer conditions prior to large-scale protein purification is described. The Protein Maker is an automated purification system developed by Emerald BioSystems for high-throughput parallel purification of proteins and antibodies. This instrument allows multiple load, wash and elution buffers to be used in parallel along independent lines for up to 24 individual samples. To demonstrate its utility, its use in the purification of five recombinant PB2 C-terminal domains from various subtypes of the influenza A virus is described. Three of these constructs crystallized and one diffracted X-rays to sufficient resolution for structure determination and deposition in the Protein Data Bank. Methods for screening lysis buffers for a cytochrome P450 from a pathogenic fungus prior to upscaling expression and purification are also described. The Protein Maker has become a valuable asset within the Seattle Structural Genomics Center for Infectious Disease (SSGCID) and hence is a potentially valuable tool for a variety of high-throughput protein-purification applications

Sensitivity of neuroprogenitor cells to chemical-induced apoptosis using a multiplexed assay suitable for high-throughput screening*

Science.gov (United States)

AbstractHigh-throughput methods are useful for rapidly screening large numbers of chemicals for biological activity, including the perturbation of pathways that may lead to adverse cellular effects. In vitro assays for the key events of neurodevelopment, including apoptosis, may ...

HDAT: web-based high-throughput screening data analysis tools

International Nuclear Information System (INIS)

Liu, Rong; Hassan, Taimur; Rallo, Robert; Cohen, Yoram

2013-01-01

The increasing utilization of high-throughput screening (HTS) in toxicity studies of engineered nano-materials (ENMs) requires tools for rapid and reliable processing and analyses of large HTS datasets. In order to meet this need, a web-based platform for HTS data analyses tools (HDAT) was developed that provides statistical methods suitable for ENM toxicity data. As a publicly available computational nanoinformatics infrastructure, HDAT provides different plate normalization methods, various HTS summarization statistics, self-organizing map (SOM)-based clustering analysis, and visualization of raw and processed data using both heat map and SOM. HDAT has been successfully used in a number of HTS studies of ENM toxicity, thereby enabling analysis of toxicity mechanisms and development of structure–activity relationships for ENM toxicity. The online approach afforded by HDAT should encourage standardization of and future advances in HTS as well as facilitate convenient inter-laboratory comparisons of HTS datasets. (paper)

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

Directory of Open Access Journals (Sweden)

Martin Florian

2012-05-01

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

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

Directory of Open Access Journals (Sweden)

Rok Gaber

2013-11-01

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

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

Science.gov (United States)

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

2013-01-01

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

A high-throughput screening strategy for nitrile-hydrolyzing enzymes based on ferric hydroxamate spectrophotometry.

Science.gov (United States)

He, Yu-Cai; Ma, Cui-Luan; Xu, Jian-He; Zhou, Li

2011-02-01

Nitrile-hydrolyzing enzymes (nitrilase or nitrile hydratase/amidase) have been widely used in the pharmaceutical industry for the production of carboxylic acids and their derivatives, and it is important to build a method for screening for nitrile-hydrolyzing enzymes. In this paper, a simple, rapid, and high-throughput screening method based on the ferric hydroxamate spectrophotometry has been proposed. To validate the accuracy of this screening strategy, the nitrilases from Rhodococcus erythropolis CGMCC 1.2362 and Alcaligenes sp. ECU0401 were used for evaluating the method. As a result, the accuracy for assaying aliphatic and aromatic carboxylic acids was as high as the HPLC-based method. Therefore, the method may be potentially used in the selection of microorganisms or engineered proteins with nitrile-hydrolyzing enzymes.

A versatile and efficient high-throughput cloning tool for structural biology.

Science.gov (United States)

Geertsma, Eric R; Dutzler, Raimund

2011-04-19

Methods for the cloning of large numbers of open reading frames into expression vectors are of critical importance for challenging structural biology projects. Here we describe a system termed fragment exchange (FX) cloning that facilitates the high-throughput generation of expression constructs. The method is based on a class IIS restriction enzyme and negative selection markers. FX cloning combines attractive features of established recombination- and ligation-independent cloning methods: It allows the straightforward transfer of an open reading frame into a variety of expression vectors and is highly efficient and very economic in its use. In addition, FX cloning avoids the common but undesirable feature of significantly extending target open reading frames with cloning related sequences, as it leaves a minimal seam of only a single extra amino acid to either side of the protein. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems. It considerably speeds up the generation of expression constructs compared to traditional methods and thus facilitates a broader expression screening.

High-throughput electrical characterization for robust overlay lithography control

Science.gov (United States)

Devender, Devender; Shen, Xumin; Duggan, Mark; Singh, Sunil; Rullan, Jonathan; Choo, Jae; Mehta, Sohan; Tang, Teck Jung; Reidy, Sean; Holt, Jonathan; Kim, Hyung Woo; Fox, Robert; Sohn, D. K.

2017-03-01

Realizing sensitive, high throughput and robust overlay measurement is a challenge in current 14nm and advanced upcoming nodes with transition to 300mm and upcoming 450mm semiconductor manufacturing, where slight deviation in overlay has significant impact on reliability and yield1). Exponentially increasing number of critical masks in multi-patterning lithoetch, litho-etch (LELE) and subsequent LELELE semiconductor processes require even tighter overlay specification2). Here, we discuss limitations of current image- and diffraction- based overlay measurement techniques to meet these stringent processing requirements due to sensitivity, throughput and low contrast3). We demonstrate a new electrical measurement based technique where resistance is measured for a macro with intentional misalignment between two layers. Overlay is quantified by a parabolic fitting model to resistance where minima and inflection points are extracted to characterize overlay control and process window, respectively. Analyses using transmission electron microscopy show good correlation between actual overlay performance and overlay obtained from fitting. Additionally, excellent correlation of overlay from electrical measurements to existing image- and diffraction- based techniques is found. We also discuss challenges of integrating electrical measurement based approach in semiconductor manufacturing from Back End of Line (BEOL) perspective. Our findings open up a new pathway for accessing simultaneous overlay as well as process window and margins from a robust, high throughput and electrical measurement approach.

High-throughput Sequencing Based Immune Repertoire Study during Infectious Disease

Directory of Open Access Journals (Sweden)

Dongni Hou

2016-08-01

Full Text Available The selectivity of the adaptive immune response is based on the enormous diversity of T and B cell antigen-specific receptors. The immune repertoire, the collection of T and B cells with functional diversity in the circulatory system at any given time, is dynamic and reflects the essence of immune selectivity. In this article, we review the recent advances in immune repertoire study of infectious diseases that achieved by traditional techniques and high-throughput sequencing techniques. High-throughput sequencing techniques enable the determination of complementary regions of lymphocyte receptors with unprecedented efficiency and scale. This progress in methodology enhances the understanding of immunologic changes during pathogen challenge, and also provides a basis for further development of novel diagnostic markers, immunotherapies and vaccines.

A high-throughput screening method of bisphenols, bisphenols digycidyl ethers and their derivatives in dairy products by ultra-high performance liquid chromatography-tandem mass spectrometry.

Science.gov (United States)

Cheng, Yan; Nie, Xue-Mei; Wu, Han-Qiu; Hong, Yun-He; Yang, Bing-Cheng; Liu, Tong; Zhao, Dan; Wang, Jian-Feng; Yao, Gui-Hong; Zhang, Feng

2017-01-15

A simple and universal analytical method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for high throughput screening of 21 bisphenols, bisphenols digycidyl ethers and their derivatives in dairy products was developed. Response Surface Methodology (RSM) was used to optimize sample preparation conditions based on a quick, easy, cheap, effective, rugged and safe (QuEChERS) method. The analytes were extracted by using 15 mL acetonitrile with 1% acetic acid, and the extracts were further purified by using 190 mg of C18 and 390 mg of PSA. The extracts were analyzed by UHPLC-MS/MS with electrospray ionization (ESI) source. Linearity was assessed by using matrix-matched standard calibration and good correlation coefficients (r 2  > 0.99) were obtained. The limits of quantitation (LOQs) for the analytes ranged from 0.02 to 5 μg kg -1 . The extraction recoveries were in a range of 88.2%-108.2%. Good method reproducibility in terms of intra- and inter-day precision was observed, yielding relative standard deviations (RSDs) less than 8.9% and 9.9%, respectively. The validation method results revealed that the proposed method was sensitive and reliable. Finally, this method was successfully applied to dairy product analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Science.gov (United States)

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.

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

Science.gov (United States)

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

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

Science.gov (United States)

Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

2009-08-01

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

Targeted DNA Methylation Analysis by High Throughput Sequencing in Porcine Peri-attachment Embryos

OpenAIRE

MORRILL, Benson H.; COX, Lindsay; WARD, Anika; HEYWOOD, Sierra; PRATHER, Randall S.; ISOM, S. Clay

2013-01-01

Abstract The purpose of this experiment was to implement and evaluate the effectiveness of a next-generation sequencing-based method for DNA methylation analysis in porcine embryonic samples. Fourteen discrete genomic regions were amplified by PCR using bisulfite-converted genomic DNA derived from day 14 in vivo-derived (IVV) and parthenogenetic (PA) porcine embryos as template DNA. Resulting PCR products were subjected to high-throughput sequencing using the Illumina Genome Analyzer IIx plat...

High-throughput bioinformatics with the Cyrille2 pipeline system.

NARCIS (Netherlands)

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

2008-01-01

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

Mosquitoes meet microfluidics: High-throughput microfluidic tools for insect-parasite ecology in field conditions

Science.gov (United States)

Prakash, Manu; Mukundarajan, Haripriya

2013-11-01

A simple bite from an insect is the transmission mechanism for many deadly diseases worldwide--including malaria, yellow fever, west nile and dengue. Very little is known about how populations of numerous insect species and disease-causing parasites interact in their natural habitats due to a lack of measurement techniques. At present, vector surveillance techniques involve manual capture by using humans as live bait, which is hard to justify on ethical grounds. Individual mosquitoes are manually dissected to isolate salivary glands to detect sporozites. With typical vector infection rates being very low even in endemic areas, it is almost impossible to get an accurate picture of disease distribution, in both space and time. Here we present novel high-throughput microfluidic tools for vector surveillance, specifically mosquitoes. A two-dimensional high density array with baits provide an integrated platform for multiplex PCR for detection of both vector and parasite species. Combining techniques from engineering and field ecology, methods and tools developed here will enable high-throughput measurement of infection rates for a number of diseases in mosquito populations in field conditions. Pew Foundation.

GxGrare: gene-gene interaction analysis method for rare variants from high-throughput sequencing data.

Science.gov (United States)

Kwon, Minseok; Leem, Sangseob; Yoon, Joon; Park, Taesung

2018-03-19

With the rapid advancement of array-based genotyping techniques, genome-wide association studies (GWAS) have successfully identified common genetic variants associated with common complex diseases. However, it has been shown that only a small proportion of the genetic etiology of complex diseases could be explained by the genetic factors identified from GWAS. This missing heritability could possibly be explained by gene-gene interaction (epistasis) and rare variants. There has been an exponential growth of gene-gene interaction analysis for common variants in terms of methodological developments and practical applications. Also, the recent advancement of high-throughput sequencing technologies makes it possible to conduct rare variant analysis. However, little progress has been made in gene-gene interaction analysis for rare variants. Here, we propose GxGrare which is a new gene-gene interaction method for the rare variants in the framework of the multifactor dimensionality reduction (MDR) analysis. The proposed method consists of three steps; 1) collapsing the rare variants, 2) MDR analysis for the collapsed rare variants, and 3) detect top candidate interaction pairs. GxGrare can be used for the detection of not only gene-gene interactions, but also interactions within a single gene. The proposed method is illustrated with 1080 whole exome sequencing data of the Korean population in order to identify causal gene-gene interaction for rare variants for type 2 diabetes. The proposed GxGrare performs well for gene-gene interaction detection with collapsing of rare variants. GxGrare is available at http://bibs.snu.ac.kr/software/gxgrare which contains simulation data and documentation. Supported operating systems include Linux and OS X.

High-throughput preparation and testing of ion-exchanged zeolites

International Nuclear Information System (INIS)

Janssen, K.P.F.; Paul, J.S.; Sels, B.F.; Jacobs, P.A.

2007-01-01

A high-throughput research platform was developed for the preparation and subsequent catalytic liquid-phase screening of ion-exchanged zeolites, for instance with regard to their use as heterogeneous catalysts. In this system aqueous solutions and other liquid as well as solid reagents are employed as starting materials and 24 samples are prepared on a library plate with a 4 x 6 layout. Volumetric dispensing of metal precursor solutions, weighing of zeolite and subsequent mixing/washing cycles of the starting materials and distributing reaction mixtures to the library plate are automatically performed by liquid and solid handlers controlled by a single common and easy-to-use programming software interface. The thus prepared materials are automatically contacted with reagent solutions, heated, stirred and sampled continuously using a modified liquid handling. The high-throughput platform is highly promising in enhancing synthesis of catalysts and their screening. In this paper the preparation of lanthanum-exchanged NaY zeolites (LaNaY) on the platform is reported, along with their use as catalyst for the conversion of renewables

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

Science.gov (United States)

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.

Quantitative high throughput analytics to support polysaccharide production process development.

Science.gov (United States)

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

2014-05-19

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2015-04-13

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

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

Science.gov (United States)

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.

'High throughput': new technique to evaluation of biocides for biofouling control in oil fields; 'High throughput': nova tecnologia para avaliacao da eficacia de biocidas no controle de biofilme na industria do petroleo

Energy Technology Data Exchange (ETDEWEB)

Yin, Bei [DOW, IL(United States); Yang, Jeff [DOW, Shangai (China); Bertheas, Ute [DOW, Horgen (Switzerland); Takahashi, Debora F. [DOW, Rio de Janeiro, RJ (Brazil)

2008-07-01

The wide metabolism diversifications and versatile surviving mechanisms lead to the broad existence of microorganisms in oil fields. Water flooding in secondary production can encourage microbial growth and biofilm build-up. Microbial contamination in oil field can cause many problems including microbiologically induced corrosion, oil and gas souring, deposition of iron sulfide, degradation of polymer additives, and plugging oil and gas pipelines and water purification systems. In general, biocides are needed both topside and down hole to control problematic microorganisms. In this study, a high throughput test method was developed that enables a more realistic determination of biocides efficacy against anaerobic microorganisms commonly found in oil field environments. Using this method, a thorough comparison of several commonly used biocides products in oil field for their efficacy against oil field anaerobic sulfate-reducing bacteria isolates was executed. This study showed that for each individual application, it is important to screen biocides and their combinations against microorganisms cultured from the field. Since biocides vary in their mode of action, this study also demonstrated the critical importance of utilizing the high throughput method for determining the best and most customized solution for each application. (author)

High throughput nanoimprint lithography for semiconductor memory applications

Science.gov (United States)

Ye, Zhengmao; Zhang, Wei; Khusnatdinov, Niyaz; Stachowiak, Tim; Irving, J. W.; Longsine, Whitney; Traub, Matthew; Fletcher, Brian; Liu, Weijun

2017-03-01

Imprint lithography is a promising technology for replication of nano-scale features. For semiconductor device applications, Canon deposits a low viscosity resist on a field by field basis using jetting technology. A patterned mask is lowered into the resist fluid which then quickly flows into the relief patterns in the mask by capillary action. Following this filling step, the resist is crosslinked under UV radiation, and then the mask is removed, leaving a patterned resist on the substrate. There are two critical components to meeting throughput requirements for imprint lithography. Using a similar approach to what is already done for many deposition and etch processes, imprint stations can be clustered to enhance throughput. The FPA-1200NZ2C is a four station cluster system designed for high volume manufacturing. For a single station, throughput includes overhead, resist dispense, resist fill time (or spread time), exposure and separation. Resist exposure time and mask/wafer separation are well understood processing steps with typical durations on the order of 0.10 to 0.20 seconds. To achieve a total process throughput of 17 wafers per hour (wph) for a single station, it is necessary to complete the fluid fill step in 1.2 seconds. For a throughput of 20 wph, fill time must be reduced to only one 1.1 seconds. There are several parameters that can impact resist filling. Key parameters include resist drop volume (smaller is better), system controls (which address drop spreading after jetting), Design for Imprint or DFI (to accelerate drop spreading) and material engineering (to promote wetting between the resist and underlying adhesion layer). In addition, it is mandatory to maintain fast filling, even for edge field imprinting. In this paper, we address the improvements made in all of these parameters to first enable a 1.20 second filling process for a device like pattern and have demonstrated this capability for both full fields and edge fields. Non

Cell surface profiling using high-throughput flow cytometry: a platform for biomarker discovery and analysis of cellular heterogeneity.

Directory of Open Access Journals (Sweden)

Craig A Gedye

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

Cell surface profiling using high-throughput flow cytometry: a platform for biomarker discovery and analysis of cellular heterogeneity.

Science.gov (United States)

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

2014-01-01

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

SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes.

Science.gov (United States)

Pruesse, Elmar; Peplies, Jörg; Glöckner, Frank Oliver

2012-07-15

In the analysis of homologous sequences, computation of multiple sequence alignments (MSAs) has become a bottleneck. This is especially troublesome for marker genes like the ribosomal RNA (rRNA) where already millions of sequences are publicly available and individual studies can easily produce hundreds of thousands of new sequences. Methods have been developed to cope with such numbers, but further improvements are needed to meet accuracy requirements. In this study, we present the SILVA Incremental Aligner (SINA) used to align the rRNA gene databases provided by the SILVA ribosomal RNA project. SINA uses a combination of k-mer searching and partial order alignment (POA) to maintain very high alignment accuracy while satisfying high throughput performance demands. SINA was evaluated in comparison with the commonly used high throughput MSA programs PyNAST and mothur. The three BRAliBase III benchmark MSAs could be reproduced with 99.3, 97.6 and 96.1 accuracy. A larger benchmark MSA comprising 38 772 sequences could be reproduced with 98.9 and 99.3% accuracy using reference MSAs comprising 1000 and 5000 sequences. SINA was able to achieve higher accuracy than PyNAST and mothur in all performed benchmarks. Alignment of up to 500 sequences using the latest SILVA SSU/LSU Ref datasets as reference MSA is offered at http://www.arb-silva.de/aligner. This page also links to Linux binaries, user manual and tutorial. SINA is made available under a personal use license.

High-Throughput Screening for a Moderately Halophilic Phenol-Degrading Strain and Its Salt Tolerance Response

Science.gov (United States)

Lu, Zhi-Yan; Guo, Xiao-Jue; Li, Hui; Huang, Zhong-Zi; Lin, Kuang-Fei; Liu, Yong-Di

2015-01-01

A high-throughput screening system for moderately halophilic phenol-degrading bacteria from various habitats was developed to replace the conventional strain screening owing to its high efficiency. Bacterial enrichments were cultivated in 48 deep well microplates instead of shake flasks or tubes. Measurement of phenol concentrations was performed in 96-well microplates instead of using the conventional spectrophotometric method or high-performance liquid chromatography (HPLC). The high-throughput screening system was used to cultivate forty-three bacterial enrichments and gained a halophilic bacterial community E3 with the best phenol-degrading capability. Halomonas sp. strain 4-5 was isolated from the E3 community. Strain 4-5 was able to degrade more than 94% of the phenol (500 mg·L−1 starting concentration) over a range of 3%–10% NaCl. Additionally, the strain accumulated the compatible solute, ectoine, with increasing salt concentrations. PCR detection of the functional genes suggested that the largest subunit of multicomponent phenol hydroxylase (LmPH) and catechol 1,2-dioxygenase (C12O) were active in the phenol degradation process. PMID:26020478

High-throughput screening of carbohydrate-degrading enzymes using novel insoluble chromogenic substrate assay kits

DEFF Research Database (Denmark)

Schückel, Julia; Kracun, Stjepan Kresimir; Willats, William George Tycho

2016-01-01

for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay...... CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used...... for screening the activities of enzymes, enzyme cocktails, and broths....

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

DEFF Research Database (Denmark)

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

into automated robotic high-throughput screens, which allows subsequent protein quantification. In this integrated solution, samples are directly forwarded to automated cell lysate preparation and preparation of dilution series, including reformatting to a protein spotter-compatible format after the high......-throughput screening. Tracking of huge sample numbers and data analysis from a high-content screen to RPPAs is accomplished via MIRACLE, a custom made software suite developed by us. To this end, we demonstrate that the RPPAs generated in this manner deliver reliable protein readouts and that GAPDH and TFR levels can...

High-throughput measurement of recombination rates and genetic interference in Saccharomyces cerevisiae.

Science.gov (United States)

Raffoux, Xavier; Bourge, Mickael; Dumas, Fabrice; Martin, Olivier C; Falque, Matthieu

2018-06-01

Allelic recombination owing to meiotic crossovers is a major driver of genome evolution, as well as a key player for the selection of high-performing genotypes in economically important species. Therefore, we developed a high-throughput and low-cost method to measure recombination rates and crossover patterning (including interference) in large populations of the budding yeast Saccharomyces cerevisiae. Recombination and interference were analysed by flow cytometry, which allows time-consuming steps such as tetrad microdissection or spore growth to be avoided. Moreover, our method can also be used to compare recombination in wild-type vs. mutant individuals or in different environmental conditions, even if the changes in recombination rates are small. Furthermore, meiotic mutants often present recombination and/or pairing defects affecting spore viability but our method does not involve growth steps and thus avoids filtering out non-viable spores. Copyright © 2018 John Wiley & Sons, Ltd.

High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics

Science.gov (United States)

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.

The application of the high throughput sequencing technology in the transposable elements.

Science.gov (United States)

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.

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

Directory of Open Access Journals (Sweden)

Jacques Yaacoub Bou Khalil

2016-01-01

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

A novel, simple, high-throughput method for isolation of genome-wide transposon insertion mutants of Escherichia coli K-12.

Science.gov (United States)

Miki, Takeyoshi; Yamamoto, Yoshihiro; Matsuda, Hideo

2008-01-01

We developed a novel, simple, high-throughput method for isolation of genome-wide transposon insertion mutants of Escherichia coli K-12. The basic idea of the method is to randomly disrupt the genes on the DNA fragments cloned on the Kohara library by inserting a mini-transposon first, and then transfer the disrupted genes from the lambda vector to the E. coli chromosome by homologous recombination. Using this method, we constructed a set of 8402 Km(r) cis-diploid mutants harboring a mini-Tn10 insertion mutation and the corresponding wild-type gene on a chromosome, as well as a set of 6954 haploid mutants derived from the cis-diploid mutants. The major advantage of the strategy used is that the indispensable genes or sites for growth can be identified. Preliminary results suggest that 415 open reading frames are indispensable for growth in E. coli cells. A total of 6404 haploid mutants were deposited to Genetic Strains Research Center, National Institute of Genetics, Japan (Chapter 26) and are available for public distribution upon request (http://shigen.lab.nig.ac.jp/ecoli/strain/nbrp/resource.jsp).

High-throughput differentiation of heparin from other glycosaminoglycans by pyrolysis mass spectrometry.

Science.gov (United States)

Nemes, Peter; Hoover, William J; Keire, David A

2013-08-06

Sensors with high chemical specificity and enhanced sample throughput are vital to screening food products and medical devices for chemical or biochemical contaminants that may pose a threat to public health. For example, the rapid detection of oversulfated chondroitin sulfate (OSCS) in heparin could prevent reoccurrence of heparin adulteration that caused hundreds of severe adverse events including deaths worldwide in 2007-2008. Here, rapid pyrolysis is integrated with direct analysis in real time (DART) mass spectrometry to rapidly screen major glycosaminoglycans, including heparin, chondroitin sulfate A, dermatan sulfate, and OSCS. The results demonstrate that, compared to traditional liquid chromatography-based analyses, pyrolysis mass spectrometry achieved at least 250-fold higher sample throughput and was compatible with samples volume-limited to about 300 nL. Pyrolysis yielded an abundance of fragment ions (e.g., 150 different m/z species), many of which were specific to the parent compound. Using multivariate and statistical data analysis models, these data enabled facile differentiation of the glycosaminoglycans with high throughput. After method development was completed, authentically contaminated samples obtained during the heparin crisis by the FDA were analyzed in a blinded manner for OSCS contamination. The lower limit of differentiation and detection were 0.1% (w/w) OSCS in heparin and 100 ng/μL (20 ng) OSCS in water, respectively. For quantitative purposes the linear dynamic range spanned approximately 3 orders of magnitude. Moreover, this chemical readout was successfully employed to find clues in the manufacturing history of the heparin samples that can be used for surveillance purposes. The presented technology and data analysis protocols are anticipated to be readily adaptable to other chemical and biochemical agents and volume-limited samples.

An Automated High Performance Capillary Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometer for High-Throughput Proteomics

International Nuclear Information System (INIS)

Belov, Mikhail E.; Anderson, Gordon A.; Wingerd, Mark A.; Udseth, Harold R.; Tang, Keqi; Prior, David C.; Swanson, Kenneth R.; Buschbach, Michael A.; Strittmatter, Eric F.; Moore, Ronald J.; Smith, Richard D.

2004-01-01

We report on a fully automated 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer coupled to reverse-phase chromatography for high-throughput proteomic studies. Modifications made to the front-end of a commercial FTICR instrument--a dual-ESI-emitter ion source; dual-channel electrodynamic ion funnel; and collisional-cooling, selection and accumulation quadrupoles--significantly improved the sensitivity, dynamic range and mass measurement accuracy of the mass spectrometer. A high-pressure capillary liquid chromatography (LC) system was incorporated with an autosampler that enabled 24 h/day operation. A novel method for accumulating ions in the ICR cell was also developed. Unattended operation of the instrument revealed the exceptional reproducibility (1-5% deviation in elution times for peptides from a bacterial proteome), repeatability (10-20% deviation in detected abundances for peptides from the same aliquot analyzed a few weeks apart) and robustness (high-throughput operation for 5 months without downtime) of the LC/FTICR system. When combined with modulated-ion-energy gated trapping, the internal calibration of FTICR mass spectra decreased dispersion of mass measurement errors for peptide identifications in conjunction with high resolution capillary LC separations to < 5 ppm over a dynamic range for each spectrum of 10 3

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

OpenAIRE

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

2015-01-01

A high throughput method for species identification and classification through chemometric processing of direct analysis in real time (DART) mass spectrometry-derived fingerprint signatures has been developed. The method entails introduction of samples to the open air space between the DART ion source and the mass spectrometer inlet, with the entire observed mass spectral fingerprint subjected to unsupervised hierarchical clustering processing. A range of both polar and non-polar chemotypes a...

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

Science.gov (United States)

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

2016-01-01

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

A high-throughput assay for quantifying appetite and digestive dynamics

Science.gov (United States)

Guggiana-Nilo, Drago; Soucy, Edward; Song, Erin Yue; Lei Wee, Caroline; Engert, Florian

2015-01-01

Food intake and digestion are vital functions, and their dysregulation is fundamental for many human diseases. Current methods do not support their dynamic quantification on large scales in unrestrained vertebrates. Here, we combine an infrared macroscope with fluorescently labeled food to quantify feeding behavior and intestinal nutrient metabolism with high temporal resolution, sensitivity, and throughput in naturally behaving zebrafish larvae. Using this method and rate-based modeling, we demonstrate that zebrafish larvae match nutrient intake to their bodily demand and that larvae adjust their digestion rate, according to the ingested meal size. Such adaptive feedback mechanisms make this model system amenable to identify potential chemical modulators. As proof of concept, we demonstrate that nicotine, l-lysine, ghrelin, and insulin have analogous impact on food intake as in mammals. Consequently, the method presented here will promote large-scale translational research of food intake and digestive function in a naturally behaving vertebrate. PMID:26108871

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

International Nuclear Information System (INIS)

Takamiya, Mari; Sakurai, Masaaki; Teranishi, Fumie; Ikeda, Tomoko; Kamiyama, Tsutomu; Asai, Akira

2016-01-01

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 a 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 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

High-throughput determination of RNA structure by proximity ligation.

Science.gov (United States)

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.

High-throughput volumetric reconstruction for 3D wheat plant architecture studies

Directory of Open Access Journals (Sweden)

Wei Fang

2016-09-01

Full Text Available For many tiller crops, the plant architecture (PA, including the plant fresh weight, plant height, number of tillers, tiller angle and stem diameter, significantly affects the grain yield. In this study, we propose a method based on volumetric reconstruction for high-throughput three-dimensional (3D wheat PA studies. The proposed methodology involves plant volumetric reconstruction from multiple images, plant model processing and phenotypic parameter estimation and analysis. This study was performed on 80 Triticum aestivum plants, and the results were analyzed. Comparing the automated measurements with manual measurements, the mean absolute percentage error (MAPE in the plant height and the plant fresh weight was 2.71% (1.08cm with an average plant height of 40.07cm and 10.06% (1.41g with an average plant fresh weight of 14.06g, respectively. The root mean square error (RMSE was 1.37cm and 1.79g for the plant height and plant fresh weight, respectively. The correlation coefficients were 0.95 and 0.96 for the plant height and plant fresh weight, respectively. Additionally, the proposed methodology, including plant reconstruction, model processing and trait extraction, required only approximately 20s on average per plant using parallel computing on a graphics processing unit (GPU, demonstrating that the methodology would be valuable for a high-throughput phenotyping platform.

Screening and Crystallization Plates for Manual and High-throughput Protein Crystal Growth

Science.gov (United States)

Thorne, Robert E. (Inventor); Berejnov, Viatcheslav (Inventor); Kalinin, Yevgeniy (Inventor)

2010-01-01

In one embodiment, a crystallization and screening plate comprises a plurality of cells open at a top and a bottom, a frame that defines the cells in the plate, and at least two films. The first film seals a top of the plate and the second film seals a bottom of the plate. At least one of the films is patterned to strongly pin the contact lines of drops dispensed onto it, fixing their position and shape. The present invention also includes methods and other devices for manual and high-throughput protein crystal growth.

High-Throughput Screening Using Mass Spectrometry within Drug Discovery.

Science.gov (United States)

Rohman, Mattias; Wingfield, Jonathan

2016-01-01

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

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

Science.gov (United States)

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

2017-08-15

The Human Protein Atlas (HPA) enables the simultaneous characterization of thousands of proteins across various tissues to pinpoint their spatial location in the human body. This has been achieved through transcriptomics and high-throughput immunohistochemistry-based approaches, where over 40 000 unique human protein fragments have been expressed in E. coli. These datasets enable quantitative tracking of entire cellular proteomes and present new avenues for understanding molecular-level properties influencing expression and solubility. Combining computational biology and machine learning identifies protein properties that hinder the HPA high-throughput antibody production pipeline. We predict protein expression and solubility with accuracies of 70% and 80%, respectively, based on a subset of key properties (aromaticity, hydropathy and isoelectric point). We guide the selection of protein fragments based on these characteristics to optimize high-throughput experimentation. We present the machine learning workflow as a series of IPython notebooks hosted on GitHub (https://github.com/SBRG/Protein_ML). The workflow can be used as a template for analysis of further expression and solubility datasets. ebrunk@ucsd.edu or johanr@biotech.kth.se. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

New high-throughput material-exploration system based on combinatorial chemistry and electrostatic atomization

International Nuclear Information System (INIS)

Fujimoto, K.; Takahashi, H.; Ito, S.; Inoue, S.; Watanabe, M.

2006-01-01

As a tool to facilitate future material explorations, our group has developed a new combinatorial system for the high-throughput preparation of compounds made up of more than three components. The system works in two steps: the atomization of a liquid by a high electric field followed by deposition to a grounded substrate. The combinatorial system based on this method has plural syringe pumps. The each starting materials are fed through the syringe pumps into a manifold, thoroughly mixed as they pass through the manifold, and atomized from the tip of a stainless steel nozzle onto a grounded substrate

Optimizing transformations for automated, high throughput analysis of flow cytometry data.

Science.gov (United States)

Finak, Greg; Perez, Juan-Manuel; Weng, Andrew; Gottardo, Raphael

2010-11-04

In a high throughput setting, effective flow cytometry data analysis depends heavily on proper data preprocessing. While usual preprocessing steps of quality assessment, outlier removal, normalization, and gating have received considerable scrutiny from the community, the influence of data transformation on the output of high throughput analysis has been largely overlooked. Flow cytometry measurements can vary over several orders of magnitude, cell populations can have variances that depend on their mean fluorescence intensities, and may exhibit heavily-skewed distributions. Consequently, the choice of data transformation can influence the output of automated gating. An appropriate data transformation aids in data visualization and gating of cell populations across the range of data. Experience shows that the choice of transformation is data specific. Our goal here is to compare the performance of different transformations applied to flow cytometry data in the context of automated gating in a high throughput, fully automated setting. We examine the most common transformations used in flow cytometry, including the generalized hyperbolic arcsine, biexponential, linlog, and generalized Box-Cox, all within the BioConductor flowCore framework that is widely used in high throughput, automated flow cytometry data analysis. All of these transformations have adjustable parameters whose effects upon the data are non-intuitive for most users. By making some modelling assumptions about the transformed data, we develop maximum likelihood criteria to optimize parameter choice for these different transformations. We compare the performance of parameter-optimized and default-parameter (in flowCore) data transformations on real and simulated data by measuring the variation in the locations of cell populations across samples, discovered via automated gating in both the scatter and fluorescence channels. We find that parameter-optimized transformations improve visualization, reduce

Optimizing transformations for automated, high throughput analysis of flow cytometry data

Directory of Open Access Journals (Sweden)

Weng Andrew

2010-11-01

Full Text Available Abstract Background In a high throughput setting, effective flow cytometry data analysis depends heavily on proper data preprocessing. While usual preprocessing steps of quality assessment, outlier removal, normalization, and gating have received considerable scrutiny from the community, the influence of data transformation on the output of high throughput analysis has been largely overlooked. Flow cytometry measurements can vary over several orders of magnitude, cell populations can have variances that depend on their mean fluorescence intensities, and may exhibit heavily-skewed distributions. Consequently, the choice of data transformation can influence the output of automated gating. An appropriate data transformation aids in data visualization and gating of cell populations across the range of data. Experience shows that the choice of transformation is data specific. Our goal here is to compare the performance of different transformations applied to flow cytometry data in the context of automated gating in a high throughput, fully automated setting. We examine the most common transformations used in flow cytometry, including the generalized hyperbolic arcsine, biexponential, linlog, and generalized Box-Cox, all within the BioConductor flowCore framework that is widely used in high throughput, automated flow cytometry data analysis. All of these transformations have adjustable parameters whose effects upon the data are non-intuitive for most users. By making some modelling assumptions about the transformed data, we develop maximum likelihood criteria to optimize parameter choice for these different transformations. Results We compare the performance of parameter-optimized and default-parameter (in flowCore data transformations on real and simulated data by measuring the variation in the locations of cell populations across samples, discovered via automated gating in both the scatter and fluorescence channels. We find that parameter

High-throughput analysis of amino acids in plant materials by single quadrupole mass spectrometry

DEFF Research Database (Denmark)

Dahl-Lassen, Rasmus; van Hecke, Jan Julien Josef; Jørgensen, Henning

2018-01-01

that it is very time consuming with typical chromatographic run times of 70 min or more. Results: We have here developed a high-throughput method for analysis of amino acid profiles in plant materials. The method combines classical protein hydrolysis and derivatization with fast separation by UHPLC and detection...... reducing the overall analytical costs compared to methods based on more advanced mass spectrometers....... by a single quadrupole (QDa) mass spectrometer. The chromatographic run time is reduced to 10 min and the precision, accuracy and sensitivity of the method are in line with other recent methods utilizing advanced and more expensive mass spectrometers. The sensitivity of the method is at least a factor 10...

Simultaneous measurements of auto-immune and infectious disease specific antibodies using a high throughput multiplexing tool.

Directory of Open Access Journals (Sweden)

Atul Asati

Full Text Available 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.

Application Of Empirical Phase Diagrams For Multidimensional Data Visualization Of High Throughput Microbatch Crystallization Experiments.

Science.gov (United States)

Klijn, Marieke E; Hubbuch, Jürgen

2018-04-27

Protein phase diagrams are a tool to investigate cause and consequence of solution conditions on protein phase behavior. The effects are scored according to aggregation morphologies such as crystals or amorphous precipitates. Solution conditions affect morphological features, such as crystal size, as well as kinetic features, such as crystal growth time. Common used data visualization techniques include individual line graphs or symbols-based phase diagrams. These techniques have limitations in terms of handling large datasets, comprehensiveness or completeness. To eliminate these limitations, morphological and kinetic features obtained from crystallization images generated with high throughput microbatch experiments have been visualized with radar charts in combination with the empirical phase diagram (EPD) method. Morphological features (crystal size, shape, and number, as well as precipitate size) and kinetic features (crystal and precipitate onset and growth time) are extracted for 768 solutions with varying chicken egg white lysozyme concentration, salt type, ionic strength and pH. Image-based aggregation morphology and kinetic features were compiled into a single and easily interpretable figure, thereby showing that the EPD method can support high throughput crystallization experiments in its data amount as well as its data complexity. Copyright © 2018. Published by Elsevier Inc.

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

Science.gov (United States)

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

2015-01-01

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

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

Science.gov (United States)

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

A high-throughput method for the simultaneous determination of multiple mycotoxins in human and laboratory animal biological fluids and tissues by PLE and HPLC-MS/MS.

Science.gov (United States)

Cao, Xiaoqin; Wu, Shuangchan; Yue, Yuan; Wang, Shi; Wang, Yuting; Tao, Li; Tian, Hui; Xie, Jianmei; Ding, Hong

2013-12-30

A high-throughput method for the determination of 28 mycotoxins involving pressurised liquid extraction (PLE) coupled with liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has been optimised and validated for determination in various biological fluids and tissues of human and laboratory animals. High-throughput analysis was achieved using PLE pre-treatment and without the need for any cleanup. The extraction solvent was acetonitrile/water/acetic acid (80/19/1, v/v/v). The static extraction time was 5min. The extraction pressure and temperature were 1500psi and 140°C, respectively. The flush volume was 60%. The limits of detection, which were defined as CCα, varied from 0.01μg/kg (μg/L) to 0.69μg/kg (μg/L). The recoveries of spiked samples from 0.20μg/kg (μg/L) to 2μg/kg (μg/L) ranged from 71% to 100.5% with relative standard deviations of less than 17.5%, except FB1 and FB2 recoveries, which were lower than 60%. The method was successfully applied in real samples, and the data indicate that this technique is a useful analytical method for the determination of mycotoxins from humans and animals. To the best of our knowledge, this method is the first for the large-scale testing of multi-class mycotoxins in all types of biological fluids and tissues that uses PLE and HPLC-MS/MS. Copyright © 2013 Elsevier B.V. All rights reserved.

High-throughput screening of effective siRNAs using luciferase-linked chimeric mRNA.

Directory of Open Access Journals (Sweden)

Shen Pang

Full Text Available The use of siRNAs to knock down gene expression can potentially be an approach to treat various diseases. To avoid siRNA toxicity the less transcriptionally active H1 pol III promoter, rather than the U6 promoter, was proposed for siRNA expression. To identify highly efficacious siRNA sequences, extensive screening is required, since current computer programs may not render ideal results. Here, we used CCR5 gene silencing as a model to investigate a rapid and efficient screening approach. We constructed a chimeric luciferase-CCR5 gene for high-throughput screening of siRNA libraries. After screening approximately 900 shRNA clones, 12 siRNA sequences were identified. Sequence analysis demonstrated that most (11 of the 12 sequences of these siRNAs did not match those identified by available siRNA prediction algorithms. Significant inhibition of CCR5 in a T-lymphocyte cell line and primary T cells by these identified siRNAs was confirmed using the siRNA lentiviral vectors to infect these cells. The inhibition of CCR5 expression significantly protected cells from R5 HIV-1JRCSF infection. These results indicated that the high-throughput screening method allows efficient identification of siRNA sequences to inhibit the target genes at low levels of expression.

High-resolution and high-throughput multichannel Fourier transform spectrometer with two-dimensional interferogram warping compensation

Science.gov (United States)

Watanabe, A.; Furukawa, H.

2018-04-01

The resolution of multichannel Fourier transform (McFT) spectroscopy is insufficient for many applications despite its extreme advantage of high throughput. We propose an improved configuration to realise both performance using a two-dimensional area sensor. For the spectral resolution, we obtained the interferogram of a larger optical path difference by shifting the area sensor without altering any optical components. The non-linear phase error of the interferometer was successfully corrected using a phase-compensation calculation. Warping compensation was also applied to realise a higher throughput to accumulate the signal between vertical pixels. Our approach significantly improved the resolution and signal-to-noise ratio by factors of 1.7 and 34, respectively. This high-resolution and high-sensitivity McFT spectrometer will be useful for detecting weak light signals such as those in non-invasive diagnosis.

Computational tools for high-throughput discovery in biology

OpenAIRE

Jones, Neil Christopher

2007-01-01

High throughput data acquisition technology has inarguably transformed the landscape of the life sciences, in part by making possible---and necessary---the computational disciplines of bioinformatics and biomedical informatics. These fields focus primarily on developing tools for analyzing data and generating hypotheses about objects in nature, and it is in this context that we address three pressing problems in the fields of the computational life sciences which each require computing capaci...

High throughput single-cell and multiple-cell micro-encapsulation.

Science.gov (United States)

Lagus, Todd P; Edd, Jon F

2012-06-15

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 μm polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency P(k=1) of 83.7% and a double-particle encapsulation efficiency P(k=2) of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify

A pocket device for high-throughput optofluidic holographic microscopy

Science.gov (United States)

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

2017-06-01

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

A comparison of high-throughput techniques for assaying circadian rhythms in plants.

Science.gov (United States)

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.

Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti.

Science.gov (United States)

Cai, Yingying; Xia, Miaomiao; Dong, Huina; Qian, Yuan; Zhang, Tongcun; Zhu, Beiwei; Wu, Jinchuan; Zhang, Dawei

2018-05-11

As a very important coenzyme in the cell metabolism, Vitamin B 12 (cobalamin, VB 12 ) has been widely used in food and medicine fields. The complete biosynthesis of VB 12 requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB 12 production. High-yield VB 12 -producing strains are usually obtained by mutagenesis treatments, thus developing an efficient screening approach is urgently needed. By the help of engineered strains with varied capacities of VB 12 production, a riboswitch library was constructed and screened, and the btuB element from Salmonella typhimurium was identified as the best regulatory device. A flow cytometry high-throughput screening system was developed based on the btuB riboswitch with high efficiency to identify positive mutants. Mutation of Sinorhizobium meliloti (S. meliloti) was optimized using the novel mutation technique of atmospheric and room temperature plasma (ARTP). Finally, the mutant S. meliloti MC5-2 was obtained and considered as a candidate for industrial applications. After 7 d's cultivation on a rotary shaker at 30 °C, the VB 12 titer of S. meliloti MC5-2 reached 156 ± 4.2 mg/L, which was 21.9% higher than that of the wild type strain S. meliloti 320 (128 ± 3.2 mg/L). The genome of S. meliloti MC5-2 was sequenced, and gene mutations were identified and analyzed. To our knowledge, it is the first time that a riboswitch element was used in S. meliloti. The flow cytometry high-throughput screening system was successfully developed and a high-yield VB 12 producing strain was obtained. The identified and analyzed gene mutations gave useful information for developing high-yield strains by metabolic engineering. Overall, this work provides a useful high-throughput screening method for developing high VB 12 -yield strains.

Noise and non-linearities in high-throughput data

International Nuclear Information System (INIS)

Nguyen, Viet-Anh; Lió, Pietro; Koukolíková-Nicola, Zdena; Bagnoli, Franco

2009-01-01

High-throughput data analyses are becoming common in biology, communications, economics and sociology. The vast amounts of data are usually represented in the form of matrices and can be considered as knowledge networks. Spectra-based approaches have proved useful in extracting hidden information within such networks and for estimating missing data, but these methods are based essentially on linear assumptions. The physical models of matching, when applicable, often suggest non-linear mechanisms, that may sometimes be identified as noise. The use of non-linear models in data analysis, however, may require the introduction of many parameters, which lowers the statistical weight of the model. According to the quality of data, a simpler linear analysis may be more convenient than more complex approaches. In this paper, we show how a simple non-parametric Bayesian model may be used to explore the role of non-linearities and noise in synthetic and experimental data sets

A high throughput capillary electrophoresis method to obtain pharmacokinetics and quality attributes of a therapeutic molecule in circulation

Science.gov (United States)

Piparia, Reema; Ouellette, David; Stine, W. Blaine; Grinnell, Christine; Tarcsa, Edit; Radziejewski, Czeslaw; Correia, Ivan

2012-01-01

Therapeutic proteins circulating in blood are in a highly crowded, redox environment at high temperatures of ~37°C. These molecules circulate in the presence of enzymes and other serum proteins making it difficult to predict from in vitro studies the stability, aggregation or pharmacokinetics of a therapeutic protein in vivo. Here, we describe use of a high throughput capillary electrophoresis based microfluidic device (LabChip GXII) to obtain pharmacokinetics (PK) of a fluorescently labeled human mAb directly from serum. The non-labeled and labeled mAbs were evaluated in single dose rat PK studies using a traditional ELISA method or LabChip GXII, respectively. The fluorescent dye did not significantly alter clearance of this particular mAb, and PK parameters were comparable for labeled and unlabeled molecules. Further, from the CE profile we concluded that the mAb was resistant to fragmentation or aggregation during circulation. In a follow-up experiment, dimers were generated from the mAb using photo-induced cross-linking of unmodified proteins (PICUP) and labeled with the same fluorophore. The extent of dimerization was incomplete and some monomer and higher molecular weight species were found in the preparation. In rat PK studies, the serum concentration-time profile of the three entities present in the dimer preparation could be followed simultaneously with the GXII technology. While further studies are warranted, we believe this method could be adapted to obtain PK of different forms of antibodies (oxidized, deamidated or various glycosylated species) and other proteins. PMID:22647389

High-Throughput Tabular Data Processor - Platform independent graphical tool for processing large data sets.

Science.gov (United States)

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 selection for cellulase catalysts using chemical complementation.

Science.gov (United States)

Peralta-Yahya, Pamela; Carter, Brian T; Lin, Hening; Tao, Haiyan; Cornish, Virginia W

2008-12-24

Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases, however, is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Because of the large number of enzyme variants that selections can now test as compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity.

High-throughput assessment of context-dependent effects of chromatin proteins

NARCIS (Netherlands)

Brueckner, L. (Laura); Van Arensbergen, J. (Joris); Akhtar, W. (Waseem); L. Pagie (Ludo); B. van Steensel (Bas)

2016-01-01

textabstractBackground: Chromatin proteins control gene activity in a concerted manner. We developed a high-throughput assay to study the effects of the local chromatin environment on the regulatory activity of a protein of interest. The assay combines a previously reported multiplexing strategy

tcpl: The ToxCast Pipeline for High-Throughput Screening Data

Science.gov (United States)

Motivation: The large and diverse high-throughput chemical screening efforts carried out by the US EPAToxCast program requires an efficient, transparent, and reproducible data pipeline.Summary: The tcpl R package and its associated MySQL database provide a generalized platform fo...

A high-throughput surface plasmon resonance biosensor based on differential interferometric imaging

International Nuclear Information System (INIS)

Wang, Daqian; Ding, Lili; Zhang, Wei; Zhang, Enyao; Yu, Xinglong; Luo, Zhaofeng; Ou, Huichao

2012-01-01

A new high-throughput surface plasmon resonance (SPR) biosensor based on differential interferometric imaging is reported. The two SPR interferograms of the sensing surface are imaged on two CCD cameras. The phase difference between the two interferograms is 180°. The refractive index related factor (RIRF) of the sensing surface is calculated from the two simultaneously acquired interferograms. The simulation results indicate that the RIRF exhibits a linear relationship with the refractive index of the sensing surface and is unaffected by the noise, drift and intensity distribution of the light source. The affinity and kinetic information can be extracted in real time from continuously acquired RIRF distributions. The results of refractometry experiments show that the dynamic detection range of SPR differential interferometric imaging system can be over 0.015 refractive index unit (RIU). High refractive index resolution is down to 0.45 RU (1 RU = 1 × 10 −6 RIU). Imaging and protein microarray experiments demonstrate the ability of high-throughput detection. The aptamer experiments demonstrate that the SPR sensor based on differential interferometric imaging has a great capability to be implemented for high-throughput aptamer kinetic evaluation. These results suggest that this biosensor has the potential to be utilized in proteomics and drug discovery after further improvement. (paper)

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

Science.gov (United States)

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...

Development of glycoprotein capture-based label-free method for the high-throughput screening of differential glycoproteins in hepatocellular carcinoma.

Science.gov (United States)

Chen, Rui; Tan, Yexiong; Wang, Min; Wang, Fangjun; Yao, Zhenzhen; Dong, Liwei; Ye, Mingliang; Wang, Hongyang; Zou, Hanfa

2011-07-01

A robust, reproducible, and high throughput method was developed for the relative quantitative analysis of glycoprotein abundances in human serum. Instead of quantifying glycoproteins by glycopeptides in conventional quantitative glycoproteomics, glycoproteins were quantified by nonglycosylated peptides derived from the glycoprotein digest, which consists of the capture of glycoproteins in serum samples and the release of nonglycopeptides by trypsin digestion of captured glycoproteins followed by two-dimensional liquid chromatography-tandem MS analysis of released peptides. Protein quantification was achieved by comparing the spectrum counts of identified nonglycosylated peptides of glycoproteins between different samples. This method was demonstrated to have almost the same specificity and sensitivity in glycoproteins quantification as capture at glycopeptides level. The differential abundance of proteins present at as low as nanogram per milliliter levels was quantified with high confidence. The established method was applied to the analysis of human serum samples from healthy people and patients with hepatocellular carcinoma (HCC) to screen differential glycoproteins in HCC. Thirty eight glycoproteins were found with substantial concentration changes between normal and HCC serum samples, including α-fetoprotein, the only clinically used marker for HCC diagnosis. The abundance changes of three glycoproteins, i.e. galectin-3 binding protein, insulin-like growth factor binding protein 3, and thrombospondin 1, which were associated with the development of HCC, were further confirmed by enzyme-linked immunosorbent assay. In conclusion, the developed method was an effective approach to quantitatively analyze glycoproteins in human serum and could be further applied in the biomarker discovery for HCC and other cancers.

Management of High-Throughput DNA Sequencing Projects: Alpheus.

Science.gov (United States)

Miller, Neil A; Kingsmore, Stephen F; Farmer, Andrew; Langley, Raymond J; Mudge, Joann; Crow, John A; Gonzalez, Alvaro J; Schilkey, Faye D; Kim, Ryan J; van Velkinburgh, Jennifer; May, Gregory D; Black, C Forrest; Myers, M Kathy; Utsey, John P; Frost, Nicholas S; Sugarbaker, David J; Bueno, Raphael; Gullans, Stephen R; Baxter, Susan M; Day, Steve W; Retzel, Ernest F

2008-12-26

High-throughput DNA sequencing has enabled systems biology to begin to address areas in health, agricultural and basic biological research. Concomitant with the opportunities is an absolute necessity to manage significant volumes of high-dimensional and inter-related data and analysis. Alpheus is an analysis pipeline, database and visualization software for use with massively parallel DNA sequencing technologies that feature multi-gigabase throughput characterized by relatively short reads, such as Illumina-Solexa (sequencing-by-synthesis), Roche-454 (pyrosequencing) and Applied Biosystem's SOLiD (sequencing-by-ligation). Alpheus enables alignment to reference sequence(s), detection of variants and enumeration of sequence abundance, including expression levels in transcriptome sequence. Alpheus is able to detect several types of variants, including non-synonymous and synonymous single nucleotide polymorphisms (SNPs), insertions/deletions (indels), premature stop codons, and splice isoforms. Variant detection is aided by the ability to filter variant calls based on consistency, expected allele frequency, sequence quality, coverage, and variant type in order to minimize false positives while maximizing the identification of true positives. Alpheus also enables comparisons of genes with variants between cases and controls or bulk segregant pools. Sequence-based differential expression comparisons can be developed, with data export to SAS JMP Genomics for statistical analysis.

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

Science.gov (United States)

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.

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

Science.gov (United States)

Wiesler, Simone C; Weinzierl, Robert O J

2015-01-01

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

High Throughput Plasma Water Treatment

Science.gov (United States)

Mujovic, Selman; Foster, John

2016-10-01

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

High throughput nanostructure-initiator mass spectrometry screening of microbial growth conditions for maximal β-glucosidase production.

Science.gov (United States)

Cheng, Xiaoliang; Hiras, Jennifer; Deng, Kai; Bowen, Benjamin; Simmons, Blake A; Adams, Paul D; Singer, Steven W; Northen, Trent R

2013-01-01

Production of biofuels via enzymatic hydrolysis of complex plant polysaccharides is a subject of intense global interest. Microbial communities are known to express a wide range of enzymes necessary for the saccharification of lignocellulosic feedstocks and serve as a powerful reservoir for enzyme discovery. However, the growth temperature and conditions that yield high cellulase activity vary widely, and the throughput to identify optimal conditions has been limited by the slow handling and conventional analysis. A rapid method that uses small volumes of isolate culture to resolve specific enzyme activity is needed. In this work, a high throughput nanostructure-initiator mass spectrometry (NIMS)-based approach was developed for screening a thermophilic cellulolytic actinomycete, Thermobispora bispora, for β-glucosidase production under various growth conditions. Media that produced high β-glucosidase activity were found to be I/S + glucose or microcrystalline cellulose (MCC), Medium 84 + rolled oats, and M9TE + MCC at 45°C. Supernatants of cell cultures grown in M9TE + 1% MCC cleaved 2.5 times more substrate at 45°C than at all other temperatures. While T. bispora is reported to grow optimally at 60°C in Medium 84 + rolled oats and M9TE + 1% MCC, approximately 40% more conversion was observed at 45°C. This high throughput NIMS approach may provide an important tool in discovery and characterization of enzymes from environmental microbes for industrial and biofuel applications.

High throughput nanostructure-initiator mass spectrometry screening of microbial growth conditions for maximal β-glucosidase production

Directory of Open Access Journals (Sweden)

Xiaoliang eCheng

2013-12-01

Full Text Available Production of biofuels via enzymatic hydrolysis of complex plant polysaccharides is a subject of intense global interest. Microbial communities are known to express a wide range of enzymes necessary for the saccharification of lignocellulosic feedstocks and serve as a powerful reservoir for enzyme discovery. However, the growth temperature and conditions that yield high cellulase activity vary widely, and the throughput to identify optimal conditions has been limited by the slow handling and conventional analysis. A rapid method that uses small volumes of isolate culture to resolve specific enzyme activity is needed. In this work, a high throughput nanostructure-initiator mass spectrometry (NIMS based approach was developed for screening a thermophilic cellulolytic actinomycete, Thermobispora bispora, for β-glucosidase production under various growth conditions. Media that produced high β-glucosidase activity were found to be I/S + glucose or microcrystalline cellulose (MCC, Medium 84 + rolled oats, and M9TE + MCC at 45 °C. Supernatants of cell cultures grown in M9TE + 1% MCC cleaved 2.5 times more substrate at 45 °C than at all other temperatures. While T. bispora is reported to grow optimally at 60 °C in Medium 84 + rolled oats and M9TE + 1% MCC, approximately 40% more conversion was observed at 45 °C. This high throughput NIMS approach may provide an important tool in discovery and characterization of enzymes from environmental microbes for industrial and biofuel applications.

Newborn screening for X-linked adrenoleukodystrophy: further evidence high throughput screening is feasible.

Science.gov (United States)

Theda, Christiane; Gibbons, Katy; Defor, Todd E; Donohue, Pamela K; Golden, W Christopher; Kline, Antonie D; Gulamali-Majid, Fizza; Panny, Susan R; Hubbard, Walter C; Jones, Richard O; Liu, Anita K; Moser, Ann B; Raymond, Gerald V

2014-01-01

X-linked adrenoleukodystrophy (ALD) is characterized by adrenal insufficiency and neurologic involvement with onset at variable ages. Plasma very long chain fatty acids are elevated in ALD; even in asymptomatic patients. We demonstrated previously that liquid chromatography tandem mass spectrometry measuring C26:0 lysophosphatidylcholine reliably identifies affected males. We prospectively applied this method to 4689 newborn blood spot samples; no false positives were observed. We show that high throughput neonatal screening for ALD is methodologically feasible. Copyright © 2013 Elsevier Inc. All rights reserved.

A simplified high-throughput method for pyrethroid knock-down resistance (kdr) detection in Anopheles gambiae

Science.gov (United States)

Lynd, Amy; Ranson, Hilary; McCall, P J; Randle, Nadine P; Black, William C; Walker, Edward D; Donnelly, Martin J

2005-01-01

Background A single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR) which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation. Methods A Hot Ligation Oligonucleotide Assay (HOLA) was developed to detect both the East and West African kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique. Results and Discussion The HOLA assay, developed for detection of the kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA) technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous. Conclusion The method is capable of detecting both the East and West African kdr alleles in the homozygous and

A simplified high-throughput method for pyrethroid knock-down resistance (kdr detection in Anopheles gambiae

Directory of Open Access Journals (Sweden)

Walker Edward D

2005-03-01

Full Text Available Abstract Background A single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation. Methods A Hot Ligation Oligonucleotide Assay (HOLA was developed to detect both the East and West African kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique. Results and Discussion The HOLA assay, developed for detection of the kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous. Conclusion The method is capable of detecting both the East and West African kdr alleles

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

Science.gov (United States)

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 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.

Virtual high screening throughput and design of 14α-lanosterol ...

African Journals Online (AJOL)

STORAGESEVER

2009-07-06

Jul 6, 2009 ... Virtual high screening throughput and design of. 14α-lanosterol demethylase inhibitors against. Mycobacterium tuberculosis. Hildebert B. Maurice1*, Esther Tuarira1 and Kennedy Mwambete2. 1School of Pharmaceutical Sciences, Institute of Allied Health Sciences, Muhimbili University of Health and.

PUFKEY: A High-Security and High-Throughput Hardware True Random Number Generator for Sensor Networks

Directory of Open Access Journals (Sweden)

Dongfang Li

2015-10-01

Full Text Available Random number generators (RNG play an important role in many sensor network systems and applications, such as those requiring secure and robust communications. In this paper, we develop a high-security and high-throughput hardware true random number generator, called PUFKEY, which consists of two kinds of physical unclonable function (PUF elements. Combined with a conditioning algorithm, true random seeds are extracted from the noise on the start-up pattern of SRAM memories. These true random seeds contain full entropy. Then, the true random seeds are used as the input for a non-deterministic hardware RNG to generate a stream of true random bits with a throughput as high as 803 Mbps. The experimental results show that the bitstream generated by the proposed PUFKEY can pass all standard national institute of standards and technology (NIST randomness tests and is resilient to a wide range of security attacks.

PUFKEY: a high-security and high-throughput hardware true random number generator for sensor networks.

Science.gov (United States)

Li, Dongfang; Lu, Zhaojun; Zou, Xuecheng; Liu, Zhenglin

2015-10-16

Random number generators (RNG) play an important role in many sensor network systems and applications, such as those requiring secure and robust communications. In this paper, we develop a high-security and high-throughput hardware true random number generator, called PUFKEY, which consists of two kinds of physical unclonable function (PUF) elements. Combined with a conditioning algorithm, true random seeds are extracted from the noise on the start-up pattern of SRAM memories. These true random seeds contain full entropy. Then, the true random seeds are used as the input for a non-deterministic hardware RNG to generate a stream of true random bits with a throughput as high as 803 Mbps. The experimental results show that the bitstream generated by the proposed PUFKEY can pass all standard national institute of standards and technology (NIST) randomness tests and is resilient to a wide range of security attacks.

High Throughput Measurement of Locomotor Sensitization to Volatilized Cocaine in Drosophila melanogaster.

Science.gov (United States)

Filošević, Ana; Al-Samarai, Sabina; Andretić Waldowski, Rozi

2018-01-01

Drosophila melanogaster can be used to identify genes with novel functional roles in neuronal plasticity induced by repeated consumption of addictive drugs. Behavioral sensitization is a relatively simple behavioral output of plastic changes that occur in the brain after repeated exposures to drugs of abuse. The development of screening procedures for genes that control behavioral sensitization has stalled due to a lack of high-throughput behavioral tests that can be used in genetically tractable organism, such as Drosophila . We have developed a new behavioral test, FlyBong, which combines delivery of volatilized cocaine (vCOC) to individually housed flies with objective quantification of their locomotor activity. There are two main advantages of FlyBong: it is high-throughput and it allows for comparisons of locomotor activity of individual flies before and after single or multiple exposures. At the population level, exposure to vCOC leads to transient and concentration-dependent increase in locomotor activity, representing sensitivity to an acute dose. A second exposure leads to further increase in locomotion, representing locomotor sensitization. We validate FlyBong by showing that locomotor sensitization at either the population or individual level is absent in the mutants for circadian genes period (per) , Clock (Clk) , and cycle (cyc) . The locomotor sensitization that is present in timeless (tim) and pigment dispersing factor (pdf) mutant flies is in large part not cocaine specific, but derived from increased sensitivity to warm air. Circadian genes are not only integral part of the neural mechanism that is required for development of locomotor sensitization, but in addition, they modulate the intensity of locomotor sensitization as a function of the time of day. Motor-activating effects of cocaine are sexually dimorphic and require a functional dopaminergic transporter. FlyBong is a new and improved method for inducing and measuring locomotor sensitization

High Throughput Measurement of Locomotor Sensitization to Volatilized Cocaine in Drosophila melanogaster

Directory of Open Access Journals (Sweden)

Ana Filošević

2018-02-01

Full Text Available Drosophila melanogaster can be used to identify genes with novel functional roles in neuronal plasticity induced by repeated consumption of addictive drugs. Behavioral sensitization is a relatively simple behavioral output of plastic changes that occur in the brain after repeated exposures to drugs of abuse. The development of screening procedures for genes that control behavioral sensitization has stalled due to a lack of high-throughput behavioral tests that can be used in genetically tractable organism, such as Drosophila. We have developed a new behavioral test, FlyBong, which combines delivery of volatilized cocaine (vCOC to individually housed flies with objective quantification of their locomotor activity. There are two main advantages of FlyBong: it is high-throughput and it allows for comparisons of locomotor activity of individual flies before and after single or multiple exposures. At the population level, exposure to vCOC leads to transient and concentration-dependent increase in locomotor activity, representing sensitivity to an acute dose. A second exposure leads to further increase in locomotion, representing locomotor sensitization. We validate FlyBong by showing that locomotor sensitization at either the population or individual level is absent in the mutants for circadian genes period (per, Clock (Clk, and cycle (cyc. The locomotor sensitization that is present in timeless (tim and pigment dispersing factor (pdf mutant flies is in large part not cocaine specific, but derived from increased sensitivity to warm air. Circadian genes are not only integral part of the neural mechanism that is required for development of locomotor sensitization, but in addition, they modulate the intensity of locomotor sensitization as a function of the time of day. Motor-activating effects of cocaine are sexually dimorphic and require a functional dopaminergic transporter. FlyBong is a new and improved method for inducing and measuring locomotor

An automatic on-line 2,2-diphenyl-1-picrylhydrazyl-high performance liquid chromatography method for high-throughput screening of antioxidants from natural products.

Science.gov (United States)

Lu, Yanzhen; Wu, Nan; Fang, Yingtong; Shaheen, Nusrat; Wei, Yun

2017-10-27

Many natural products are rich in antioxidants which play an important role in preventing or postponing a variety of diseases, such as cardiovascular and inflammatory disease, diabetes as well as breast cancer. In this paper, an automatic on-line 2,2-diphenyl-1-picrylhydrazyl-high performance liquid chromatography (DPPH-HPLC) method was established for antioxidants screening with nine standards including organic acids (4-hydroxyphenylacetic acid, p-coumaric acid, ferulic acid, and benzoic acid), alkaloids (coptisine and berberine), and flavonoids (quercitrin, astragalin, and quercetin). The optimal concentration of DPPH was determined, and six potential antioxidants including 4-hydroxyphenylacetic acid, p-coumaric acid, ferulic acid, quercitrin, astragalin, and quercetin, and three non-antioxidants including benzoic acid, coptisine, and berberine, were successfully screened out and validated by conventional DPPH radical scavenging activity assay. The established method has been applied to the crude samples of Saccharum officinarum rinds, Coptis chinensis powders, and Malus pumila leaves, consecutively. Two potential antioxidant compounds from Saccharum officinarum rinds and five potential antioxidant compounds from Malus pumila eaves were rapidly screened out. Then these seven potential antioxidants were purified and identified as p-coumaric acid, ferulic acid, phloridzin, isoquercitrin, quercetin-3-xyloside, quercetin-3-arabinoside, and quercetin-3-rhamnoside using countercurrent chromatography combined with mass spectrometry and their antioxidant activities were further evaluated by conventional DPPH radical scavenging assay. The activity result was in accordance with that of the established method. This established method is cheap and automatic, and could be used as an efficient tool for high-throughput antioxidant screening from various complex natural products. Copyright © 2017 Elsevier B.V. All rights reserved.

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

Directory of Open Access Journals (Sweden)

Weatherford Wendy

2005-05-01

Full Text Available Abstract Background High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Results Using a modified QTL Lightspeed™ assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP, Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1. Phosphorylation of the proteins was detected by Protein Kinase Cα (PKCα and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4. Enzyme inhibition yielded IC50 values that were comparable to those obtained using

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

Science.gov (United States)

Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan

2005-05-31

High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that

[Morphometry of pulmonary tissue: From manual to high throughput automation].

Science.gov (United States)

Sallon, C; Soulet, D; Tremblay, Y

2017-12-01

Weibel's research has shown that any alteration of the pulmonary structure has effects on function. This demonstration required a quantitative analysis of lung structures called morphometry. This is possible thanks to stereology, a set of methods based on principles of geometry and statistics. His work has helped to better understand the morphological harmony of the lung, which is essential for its proper functioning. An imbalance leads to pathophysiology such as chronic obstructive pulmonary disease in adults and bronchopulmonary dysplasia in neonates. It is by studying this imbalance that new therapeutic approaches can be developed. These advances are achievable only through morphometric analytical methods, which are increasingly precise and focused, in particular thanks to the high-throughput automation of these methods. This review makes a comparison between an automated method that we developed in the laboratory and semi-manual methods of morphometric analyzes. The automation of morphometric measurements is a fundamental asset in the study of pulmonary pathophysiology because it is an assurance of robustness, reproducibility and speed. This tool will thus contribute significantly to the acceleration of the race for the development of new drugs. Copyright © 2017 SPLF. Published by Elsevier Masson SAS. All rights reserved.

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

Science.gov (United States)

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

2016-01-01

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

Crystal Symmetry Algorithms in a High-Throughput Framework for Materials

Science.gov (United States)

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.

Development of Control Applications for High-Throughput Protein Crystallography Experiments

International Nuclear Information System (INIS)

Gaponov, Yurii A.; Matsugaki, Naohiro; Honda, Nobuo; Sasajima, Kumiko; Igarashi, Noriyuki; Hiraki, Masahiko; Yamada, Yusuke; Wakatsuki, Soichi

2007-01-01

An integrated client-server control system (PCCS) with a unified relational database (PCDB) has been developed for high-throughput protein crystallography experiments on synchrotron beamlines. The major steps in protein crystallographic experiments (purification, crystallization, crystal harvesting, data collection, and data processing) are integrated into the software. All information necessary for performing protein crystallography experiments is stored in the PCDB database (except raw X-ray diffraction data, which is stored in the Network File Server). To allow all members of a protein crystallography group to participate in experiments, the system was developed as a multi-user system with secure network access based on TCP/IP secure UNIX sockets. Secure remote access to the system is possible from any operating system with X-terminal and SSH/X11 (Secure Shell with graphical user interface) support. Currently, the system covers the high-throughput X-ray data collection stages and is being commissioned at BL5A and NW12A (PF, PF-AR, KEK, Tsukuba, Japan)

Fluorescence-based high-throughput screening of dicer cleavage activity

Czech Academy of Sciences Publication Activity Database

Podolská, Kateřina; Sedlák, David; Bartůněk, Petr; Svoboda, Petr

2014-01-01

Roč. 19, č. 3 (2014), s. 417-426 ISSN 1087-0571 R&D Projects: GA ČR GA13-29531S; GA MŠk(CZ) LC06077; GA MŠk LM2011022 Grant - others:EMBO(DE) 1483 Institutional support: RVO:68378050 Keywords : Dicer * siRNA * high-throughput screening Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.423, year: 2014

High throughput electrophysiology: new perspectives for ion channel drug discovery

DEFF Research Database (Denmark)

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

2003-01-01

. A cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct....... The introduction of new powerful HTS electrophysiological techniques is predicted to cause a revolution in ion channel drug discovery....

A modified FASP protocol for high-throughput preparation of protein samples for mass spectrometry.

Directory of Open Access Journals (Sweden)

Jeremy Potriquet

Full Text Available To facilitate high-throughput proteomic analyses we have developed a modified FASP protocol which improves the rate at which protein samples can be processed prior to mass spectrometry. Adapting the original FASP protocol to a 96-well format necessitates extended spin times for buffer exchange due to the low centrifugation speeds tolerated by these devices. However, by using 96-well plates with a more robust polyethersulfone molecular weight cutoff membrane, instead of the cellulose membranes typically used in these devices, we could use isopropanol as a wetting agent, decreasing spin times required for buffer exchange from an hour to 30 minutes. In a typical work flow used in our laboratory this equates to a reduction of 3 hours per plate, providing processing times similar to FASP for the processing of up to 96 samples per plate. To test whether our modified protocol produced similar results to FASP and other FASP-like protocols we compared the performance of our modified protocol to the original FASP and the more recently described eFASP and MStern-blot. We show that all FASP-like methods, including our modified protocol, display similar performance in terms of proteins identified and reproducibility. Our results show that our modified FASP protocol is an efficient method for the high-throughput processing of protein samples for mass spectral analysis.

High Throughput Screening of Valganciclovir in Acidic Microenvironments of Polyester Thin Films

Directory of Open Access Journals (Sweden)

Teilo Schaller

2015-04-01

Full Text Available Ganciclovir and valganciclor are antiviral agents used for the treatment of cytomegalovirus retinitis. The conventional method for administering ganciclovir in cytomegalovirus retinitis patients is repeated intravitreal injections. In order to obviate the possible detrimental effects of repeated intraocular injections, to improve compliance and to eliminate systemic side-effects, we investigated the tuning of the ganciclovir pro-drug valganciclovir and the release from thin films of poly(lactic-co-glycolic acid (PLGA, polycaprolactone (PCL, or mixtures of both, as a step towards prototyping periocular valganciclovir implants. To investigate the drug release, we established and evaluated a high throughput fluorescence-based quantification screening assay for the detection of valganciclovir. Our protocol allows quantifying as little as 20 ng of valganciclovir in 96-well polypropylene plates and a 50× faster analysis compared to traditional HPLC measurements. This improvement can hence be extrapolated to other polyester matrix thin film formulations using a high-throughput approach. The acidic microenvironment within the polyester matrix was found to protect valganciclovir from degradation with resultant increases in the half-life of the drug in the periocular implant to 100 days. Linear release profiles were obtained using the pure polyester polymers for 10 days and 60 days formulations; however, gross phase separations of PCL and acid-terminated PLGA prevented tuning within these timeframes due to the phase separation of the polymer, valganciclovir, or both.

web cellHTS2: A web-application for the analysis of high-throughput screening data

Directory of Open Access Journals (Sweden)

Boutros Michael

2010-04-01

Full Text Available Abstract Background The analysis of high-throughput screening data sets is an expanding field in bioinformatics. High-throughput screens by RNAi generate large primary data sets which need to be analyzed and annotated to identify relevant phenotypic hits. Large-scale RNAi screens are frequently used to identify novel factors that influence a broad range of cellular processes, including signaling pathway activity, cell proliferation, and host cell infection. Here, we present a web-based application utility for the end-to-end analysis of large cell-based screening experiments by cellHTS2. Results The software guides the user through the configuration steps that are required for the analysis of single or multi-channel experiments. The web-application provides options for various standardization and normalization methods, annotation of data sets and a comprehensive HTML report of the screening data analysis, including a ranked hit list. Sessions can be saved and restored for later re-analysis. The web frontend for the cellHTS2 R/Bioconductor package interacts with it through an R-server implementation that enables highly parallel analysis of screening data sets. web cellHTS2 further provides a file import and configuration module for common file formats. Conclusions The implemented web-application facilitates the analysis of high-throughput data sets and provides a user-friendly interface. web cellHTS2 is accessible online at http://web-cellHTS2.dkfz.de. A standalone version as a virtual appliance and source code for platforms supporting Java 1.5.0 can be downloaded from the web cellHTS2 page. web cellHTS2 is freely distributed under GPL.

High throughput diffractive multi-beam femtosecond laser processing using a spatial light modulator

Energy Technology Data Exchange (ETDEWEB)

Kuang Zheng [Laser Group, Department of Engineering, University of Liverpool Brownlow Street, Liverpool L69 3GQ (United Kingdom)], E-mail: z.kuang@liv.ac.uk; Perrie, Walter [Laser Group, Department of Engineering, University of Liverpool Brownlow Street, Liverpool L69 3GQ (United Kingdom); Leach, Jonathan [Department of Physics and Astronomy, University of Glasgow, Glasgow G12 8QQ (United Kingdom); Sharp, Martin; Edwardson, Stuart P. [Laser Group, Department of Engineering, University of Liverpool Brownlow Street, Liverpool L69 3GQ (United Kingdom); Padgett, Miles [Department of Physics and Astronomy, University of Glasgow, Glasgow G12 8QQ (United Kingdom); Dearden, Geoff; Watkins, Ken G. [Laser Group, Department of Engineering, University of Liverpool Brownlow Street, Liverpool L69 3GQ (United Kingdom)

2008-12-30

High throughput femtosecond laser processing is demonstrated by creating multiple beams using a spatial light modulator (SLM). The diffractive multi-beam patterns are modulated in real time by computer generated holograms (CGHs), which can be calculated by appropriate algorithms. An interactive LabVIEW program is adopted to generate the relevant CGHs. Optical efficiency at this stage is shown to be {approx}50% into first order beams and real time processing has been carried out at 50 Hz refresh rate. Results obtained demonstrate high precision surface micro-structuring on silicon and Ti6Al4V with throughput gain >1 order of magnitude.

HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications.

Science.gov (United States)

Imaduwage, Kasun P; Go, Eden P; Zhu, Zhikai; Desaire, Heather

2016-11-01

A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have K i values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods. Graphical Abstract ᅟ.

An Improved Methodology for Multidimensional High-Throughput Preformulation Characterization of Protein Conformational Stability

Science.gov (United States)

Maddux, Nathaniel R.; Rosen, Ilan T.; Hu, Lei; Olsen, Christopher M.; Volkin, David B.; Middaugh, C. Russell

2013-01-01

The Empirical Phase Diagram (EPD) technique is a vector-based multidimensional analysis method for summarizing large data sets from a variety of biophysical techniques. It can be used to provide comprehensive preformulation characterization of a macromolecule’s higher-order structural integrity and conformational stability. In its most common mode, it represents a type of stimulus-response diagram using environmental variables such as temperature, pH, and ionic strength as the stimulus, with alterations in macromolecular structure being the response. Until now EPD analysis has not been available in a high throughput mode because of the large number of experimental techniques and environmental stressor/stabilizer variables typically employed. A new instrument has been developed that combines circular dichroism, UV-absorbance, fluorescence spectroscopy and light scattering in a single unit with a 6-position temperature controlled cuvette turret. Using this multifunctional instrument and a new software system we have generated EPDs for four model proteins. Results confirm the reproducibility of the apparent phase boundaries and protein behavior within the boundaries. This new approach permits two EPDs to be generated per day using only 0.5 mg of protein per EPD. Thus, the new methodology generates reproducible EPDs in high-throughput mode, and represents the next step in making such determinations more routine. PMID:22447621

Uncertainty Quantification in High Throughput Screening ...

Science.gov (United States)

Using uncertainty quantification, we aim to improve the quality of modeling data from high throughput screening assays for use in risk assessment. ToxCast is a large-scale screening program that analyzes thousands of chemicals using over 800 assays representing hundreds of biochemical and cellular processes, including endocrine disruption, cytotoxicity, and zebrafish development. Over 2.6 million concentration response curves are fit to models to extract parameters related to potency and efficacy. Models built on ToxCast results are being used to rank and prioritize the toxicological risk of tested chemicals and to predict the toxicity of tens of thousands of chemicals not yet tested in vivo. However, the data size also presents challenges. When fitting the data, the choice of models, model selection strategy, and hit call criteria must reflect the need for computational efficiency and robustness, requiring hard and somewhat arbitrary cutoffs. When coupled with unavoidable noise in the experimental concentration response data, these hard cutoffs cause uncertainty in model parameters and the hit call itself. The uncertainty will then propagate through all of the models built on the data. Left unquantified, this uncertainty makes it difficult to fully interpret the data for risk assessment. We used bootstrap resampling methods to quantify the uncertainty in fitting models to the concentration response data. Bootstrap resampling determines confidence intervals for

Modeling Disordered Materials with a High Throughput ab-initio Approach

Science.gov (United States)

2015-11-13

Modeling Disordered Materials with a High Throughput ab - initio Approach Kesong Yang,1 Corey Oses,2 and Stefano Curtarolo3, 4 1Department of...J. Furthmüller, Efficient iterative schemes for ab initio total-energy calculations using a plane-wave basis set, Phys. Rev. B 54, 11169–11186 (1996

A nanofluidic bioarray chip for fast and high-throughput detection of antibodies in biological fluids

Science.gov (United States)

Lee, Jonathan; Gulzar, Naveed; Scott, Jamie K.; Li, Paul C. H.

2012-10-01

Immunoassays have become a standard in secretome analysis in clinical and research analysis. In this field there is a need for a high throughput method that uses low sample volumes. Microfluidics and nanofluidics have been developed for this purpose. Our lab has developed a nanofluidic bioarray (NBA) chip with the goal being a high throughput system that assays low sample volumes against multiple probes. A combination of horizontal and vertical channels are produced to create an array antigens on the surface of the NBA chip in one dimension that is probed by flowing in the other dimension antibodies from biological fluids. We have tested the NBA chip by immobilizing streptavidin and then biotinylated peptide to detect the presence of a mouse monoclonal antibody (MAb) that is specific for the peptide. Bound antibody is detected by an AlexaFluor 647 labeled goat (anti-mouse IgG) polyclonal antibody. Using the NBA chip, we have successfully detected peptide binding by small-volume (0.5 μl) samples containing 50 attomoles (100 pM) MAb.

High-throughput, temperature-controlled microchannel acoustophoresis device made with rapid prototyping

DEFF Research Database (Denmark)

Adams, Jonathan D; Ebbesen, Christian L.; Barnkob, Rune

2012-01-01

-slide format using low-cost, rapid-prototyping techniques. This high-throughput acoustophoresis chip (HTAC) utilizes a temperature-stabilized, standing ultrasonic wave, which imposes differential acoustic radiation forces that can separate particles according to size, density and compressibility. The device...

Mining Chemical Activity Status from High-Throughput Screening Assays

KAUST Repository

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

2015-01-01

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

Mining Chemical Activity Status from High-Throughput Screening Assays

KAUST Repository

Soufan, Othman

2015-12-14

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

High-throughput heterodyne thermoreflectance: Application to thermal conductivity measurements of a Fe-Si-Ge thin film alloy library

Science.gov (United States)

d'Acremont, Quentin; Pernot, Gilles; Rampnoux, Jean-Michel; Furlan, Andrej; Lacroix, David; Ludwig, Alfred; Dilhaire, Stefan

2017-07-01

A High-Throughput Time-Domain ThermoReflectance (HT-TDTR) technique was developed to perform fast thermal conductivity measurements with minimum user actions required. This new setup is based on a heterodyne picosecond thermoreflectance system. The use of two different laser oscillators has been proven to reduce the acquisition time by two orders of magnitude and avoid the experimental artefacts usually induced by moving the elements present in TDTR systems. An amplitude modulation associated to a lock-in detection scheme is included to maintain a high sensitivity to thermal properties. We demonstrate the capabilities of the HT-TDTR setup to perform high-throughput thermal analysis by mapping thermal conductivity and interface resistances of a ternary thin film silicide library FexSiyGe100-x-y (20 deposited by wedge-type multi-layer method on a 100 mm diameter sapphire wafer offering more than 300 analysis areas of different ternary alloy compositions.

A Functional High-Throughput Assay of Myelination in Vitro

Science.gov (United States)

2014-07-01

Human induced pluripotent stem cells, hydrogels, 3D culture, electrophysiology, high-throughput assay 16. SECURITY CLASSIFICATION OF: 17...image the 3D rat dorsal root ganglion ( DRG ) cultures with sufficiently low background as to detect electrically-evoked depolarization events, as...of voltage-sensitive dyes. 8    We have made substantial progress in Task 4.1. We have fabricated neural fiber tracts from DRG explants and

High-throughput anisotropic plasma etching of polyimide for MEMS

International Nuclear Information System (INIS)

Bliznetsov, Vladimir; Manickam, Anbumalar; Ranganathan, Nagarajan; Chen, Junwei

2011-01-01

This note describes a new high-throughput process of polyimide etching for the fabrication of MEMS devices with an organic sacrificial layer approach. Using dual frequency superimposed capacitively coupled plasma we achieved a vertical profile of polyimide with an etching rate as high as 3.5 µm min −1 . After the fabrication of vertical structures in a polyimide material, additional steps were performed to fabricate structural elements of MEMS by deposition of a SiO 2 layer and performing release etching of polyimide. (technical note)

REDItools: high-throughput RNA editing detection made easy.

Science.gov (United States)

Picardi, Ernesto; Pesole, Graziano

2013-07-15

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

Caveats and limitations of plate reader-based high-throughput kinetic measurements of intracellular calcium levels

International Nuclear Information System (INIS)

Heusinkveld, Harm J.; Westerink, Remco H.S.

2011-01-01

Calcium plays a crucial role in virtually all cellular processes, including neurotransmission. The intracellular Ca 2+ concentration ([Ca 2+ ] i ) is therefore an important readout in neurotoxicological and neuropharmacological studies. Consequently, there is an increasing demand for high-throughput measurements of [Ca 2+ ] i , e.g. using multi-well microplate readers, in hazard characterization, human risk assessment and drug development. However, changes in [Ca 2+ ] i are highly dynamic, thereby creating challenges for high-throughput measurements. Nonetheless, several protocols are now available for real-time kinetic measurement of [Ca 2+ ] i in plate reader systems, though the results of such plate reader-based measurements have been questioned. In view of the increasing use of plate reader systems for measurements of [Ca 2+ ] i a careful evaluation of current technologies is warranted. We therefore performed an extensive set of experiments, using two cell lines (PC12 and B35) and two fluorescent calcium-sensitive dyes (Fluo-4 and Fura-2), for comparison of a linear plate reader system with single cell fluorescence microscopy. Our data demonstrate that the use of plate reader systems for high-throughput real-time kinetic measurements of [Ca 2+ ] i is associated with many pitfalls and limitations, including erroneous sustained increases in fluorescence, limited sensitivity and lack of single cell resolution. Additionally, our data demonstrate that probenecid, which is often used to prevent dye leakage, effectively inhibits the depolarization-evoked increase in [Ca 2+ ] i . Overall, the data indicate that the use of current plate reader-based strategies for high-throughput real-time kinetic measurements of [Ca 2+ ] i is associated with caveats and limitations that require further investigation. - Research highlights: → The use of plate readers for high-throughput screening of intracellular Ca 2+ is associated with many pitfalls and limitations. → Single cell

High-Throughput Accurate Single-Cell Screening of Euglena gracilis with Fluorescence-Assisted Optofluidic Time-Stretch Microscopy.

Directory of Open Access Journals (Sweden)

Baoshan Guo

Full Text Available The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a method that combines the strengths of microfluidic cell focusing, optical time-stretch microscopy, and fluorescence detection used in conventional flow cytometry. Specifically, our fluorescence-assisted optofluidic time-stretch microscope consists of an optical time-stretch microscope and a fluorescence analyzer on top of a hydrodynamically focusing microfluidic device and can detect fluorescence from every E. gracilis cell in a population and simultaneously obtain its image with a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary and nitrogen-deficient (lipid-accumulated E. gracilis cells with a low false positive rate of 1.0%. This method holds promise for evaluating cultivation techniques and selective breeding for microalgae-based biofuel production.

Reverse Phase Protein Arrays for High-Throughput Protein Measurements in Mammospheres

DEFF Research Database (Denmark)

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

Protein Array (RPPA)-based readout format integrated into robotic siRNA screening. This technique would allow post-screening high-throughput quantification of protein changes. Recently, breast cancer stem cells (BCSCs) have attracted much attention, as a tumor- and metastasis-driving subpopulation...

High Throughput WAN Data Transfer with Hadoop-based Storage

Science.gov (United States)

Amin, A.; Bockelman, B.; Letts, J.; Levshina, T.; Martin, T.; Pi, H.; Sfiligoi, I.; Thomas, M.; Wüerthwein, F.

2011-12-01

Hadoop distributed file system (HDFS) is becoming more popular in recent years as a key building block of integrated grid storage solution in the field of scientific computing. Wide Area Network (WAN) data transfer is one of the important data operations for large high energy physics experiments to manage, share and process datasets of PetaBytes scale in a highly distributed grid computing environment. In this paper, we present the experience of high throughput WAN data transfer with HDFS-based Storage Element. Two protocols, GridFTP and fast data transfer (FDT), are used to characterize the network performance of WAN data transfer.

High Throughput WAN Data Transfer with Hadoop-based Storage

International Nuclear Information System (INIS)

Amin, A; Thomas, M; Bockelman, B; Letts, J; Martin, T; Pi, H; Sfiligoi, I; Wüerthwein, F; Levshina, T

2011-01-01

Hadoop distributed file system (HDFS) is becoming more popular in recent years as a key building block of integrated grid storage solution in the field of scientific computing. Wide Area Network (WAN) data transfer is one of the important data operations for large high energy physics experiments to manage, share and process datasets of PetaBytes scale in a highly distributed grid computing environment. In this paper, we present the experience of high throughput WAN data transfer with HDFS-based Storage Element. Two protocols, GridFTP and fast data transfer (FDT), are used to characterize the network performance of WAN data transfer.

Fluorographene as a Mass Spectrometry Probe for High-Throughput Identification and Screening of Emerging Chemical Contaminants in Complex Samples.

Science.gov (United States)

Huang, Xiu; Liu, Qian; Huang, Xiaoyu; Nie, Zhou; Ruan, Ting; Du, Yuguo; Jiang, Guibin

2017-01-17

Mass spectrometry techniques for high-throughput analysis of complex samples are of profound importance in many areas such as food safety, omics studies, and environmental health science. Here we report the use of fluorographene (FG) as a new mass spectrometry probe for high-throughput identification and screening of emerging chemical contaminants in complex samples. FG was facilely synthesized by one-step exfoliation of fluorographite. With FG as a matrix or probe in matrix-assisted or surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (MALDI- or SELDI-TOF MS), higher sensitivity (detection limits at ppt or subppt levels), and better reproducibility were achieved than with other graphene-based materials due to the unique chemical structure and self-assembly properties of FG. The method was validated with different types of real complex samples. By using FG as a SELDI probe, we could easily detect trace amount of bisphenol S in paper products and high-fat canned food samples. Furthermore, we have successfully identified and screened as many as 28 quaternary ammonium halides in sewage sludge samples collected from municipal wastewater treatment plants. These results demonstrate that FG probe is a powerful tool for high-throughput analysis of complex samples by MS.

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

Energy Technology Data Exchange (ETDEWEB)

Guo, Xuejiang; Tang, Keqi

2017-06-14

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

Cancer panomics: computational methods and infrastructure for integrative analysis of cancer high-throughput "omics" data

DEFF Research Database (Denmark)

Brunak, Søren; De La Vega, Francisco M.; Rätsch, Gunnar

2014-01-01

Targeted cancer treatment is becoming the goal of newly developed oncology medicines and has already shown promise in some spectacular cases such as the case of BRAF kinase inhibitors in BRAF-mutant (e.g. V600E) melanoma. These developments are driven by the advent of high-throughput sequencing......, which continues to drop in cost, and that has enabled the sequencing of the genome, transcriptome, and epigenome of the tumors of a large number of cancer patients in order to discover the molecular aberrations that drive the oncogenesis of several types of cancer. Applying these technologies...... in the clinic promises to transform cancer treatment by identifying therapeutic vulnerabilities of each patient's tumor. These approaches will need to address the panomics of cancer--the integration of the complex combination of patient-specific characteristics that drive the development of each person's tumor...

A Primer on High-Throughput Computing for Genomic Selection

Directory of Open Access Journals (Sweden)

Xiao-Lin eWu

2011-02-01

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

IS-seq: a novel high throughput survey of in vivo IS6110 transposition in multiple Mycobacterium tuberculosis genomes

Directory of Open Access Journals (Sweden)

Reyes Alejandro

2012-06-01

Full Text Available Abstract Background The insertion element IS6110 is one of the main sources of genomic variability in Mycobacterium tuberculosis, the etiological agent of human tuberculosis. Although IS 6110 has been used extensively as an epidemiological marker, the identification of the precise chromosomal insertion sites has been limited by technical challenges. Here, we present IS-seq, a novel method that combines high-throughput sequencing using Illumina technology with efficient combinatorial sample multiplexing to simultaneously probe 519 clinical isolates, identifying almost all the flanking regions of the element in a single experiment. Results We identified a total of 6,976 IS6110 flanking regions on the different isolates. When validated using reference strains, the method had 100% specificity and 98% positive predictive value. The insertions mapped to both coding and non-coding regions, and in some cases interrupted genes thought to be essential for virulence or in vitro growth. Strains were classified into families using insertion sites, and high agreement with previous studies was observed. Conclusions This high-throughput IS-seq method, which can also be used to map insertions in other organisms, extends previous surveys of in vivo interrupted loci and provides a baseline for probing the consequences of disruptions in M. tuberculosis strains.

Application of high-throughput sequencing in understanding human oral microbiome related with health and disease

OpenAIRE

Chen, Hui; Jiang, Wen

2014-01-01

The oral microbiome is one of most diversity habitat in the human body and they are closely related with oral health and disease. As the technique developing,, high throughput sequencing has become a popular approach applied for oral microbial analysis. Oral bacterial profiles have been studied to explore the relationship between microbial diversity and oral diseases such as caries and periodontal disease. This review describes the application of high-throughput sequencing for characterizati...

Engineering design on main mechanism of a high throughput vol-oxidizer for decladding and vol-oxidation of rod-cuts

International Nuclear Information System (INIS)

Kim, Y. H.; Park, B. S.; Jung, J. H.; Yoon, J. S.; Kim, H. D.; Hwang, J. S.; Yoon, K. H.

2008-01-01

In this paper, we designed the main mechanisms for a high throughput device for the rod-cuts of a spent fuel. To design the main mechanisms, we evaluated the current mechanical (slitting, ball mill, roller straightening) and chemical methods (muffle furnace, rotary kiln). As a result, the methods for a ball drop and a rotary drum as concepts were selected at the analysis step. For an enhancement of the oxidation rate, we devised blades for the reactor as a mesh type. Also, for an enhancement of the decladding rate, we designed the ball size and the rotation of the reactor as a mesh type and devised a vacuum system for the fission products. We also designed the main mechanisms devices and tested the capacity of these devices. Mechanisms for the oxidation and recovery can simultaneously handle the rod-cuts of a spent fuel and provide an independent recovery. The results of the mechanisms designs can be used for a scale-up of a high throughput device

High-Throughput Assay for Enantiomeric Excess Determination in 1,2- and 1,3-Diols and Direct Asymmetric Reaction Screening.

Science.gov (United States)

Shcherbakova, Elena G; Brega, Valentina; Lynch, Vincent M; James, Tony D; Anzenbacher, Pavel

2017-07-26

A simple and efficient method for determination of the yield, enantiomeric/diasteriomeric excess (ee/de), and absolute configuration of crude chiral diols without the need of work-up and product isolation in a high throughput setting is described. This approach utilizes a self-assembled iminoboronate ester formed as a product by dynamic covalent self-assembly of a chiral diol with an enantiopure fluorescent amine such as tryptophan methyl ester or tryptophanol and 2-formylphenylboronic acid. The resulting diastereomeric boronates display different photophysical properties and allow for fluorescence-based ee determination of molecules containing a 1,2- or 1,3-diol moiety. This method has been utilized for the screening of ee in a number of chiral diols including atorvastatin, a statin used for the treatment of hypercholesterolemia. Noyori asymmetric hydrogenation of benzil was performed in a highly parallel fashion with errors products from the parallel asymmetric synthesis in real time and in a high-throughput screening (HTS) fashion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Early diagnosis of adenylosuccinate lyase deficiency using a high-throughput screening method and a trial of oral S-adenosyl-l-methionine as a treatment method.

Science.gov (United States)

van Werkhoven, Michiel A; Duley, John A; McGown, Ivan; Munce, Teresa; Freeman, Jeremy L; Pitt, James J

2013-11-01

The aim of this study was to develop a high-throughput urine screening technique for adenylosuccinate lyase (ADSL) deficiency and to evaluate S-adenosyl-l-methionine (SAMe) as a potential treatment for this disorder. Testing for succinyladenosine (S-Ado), a marker of ADSL deficiency, was incorporated into a screening panel for urine biomarkers for inborn errors of metabolism using electrospray tandem mass spectrometry. Liquid chromatography-mass spectrometry and high-performance liquid chromatography were used to confirm and monitor the response of metabolites to oral SAMe treatment. Increased levels of S-Ado were detected in a 3-month-old male infant with hypotonia and seizures. ADSL gene sequencing revealed a previously described c.-49T>C mutation and a novel c.889_891dupAAT mutation, which was likely to disrupt enzyme function. After 9 months of SAMe treatment, there was no clear response evidenced in urine metabolite levels or clinical parameters. These results demonstrate proof of the principle for the high-throughput urine screening technique, allowing earlier diagnosis of patients with ADSL deficiency. However, early treatment with SAMe does not appear to be effective in ADSL deficiency. It is suggested that although SAMe treatment may ameliorate purine nucleotide deficiency, it cannot correct metabolic syndromes in which a toxic nucleotide is present, in this case presumed to be succinylaminoimidazole carboxamide ribotide. © 2013 Mac Keith Press.

PCR cycles above routine numbers do not compromise high-throughput DNA barcoding results.

Science.gov (United States)

Vierna, J; Doña, J; Vizcaíno, A; Serrano, D; Jovani, R

2017-10-01

High-throughput DNA barcoding has become essential in ecology and evolution, but some technical questions still remain. Increasing the number of PCR cycles above the routine 20-30 cycles is a common practice when working with old-type specimens, which provide little amounts of DNA, or when facing annealing issues with the primers. However, increasing the number of cycles can raise the number of artificial mutations due to polymerase errors. In this work, we sequenced 20 COI libraries in the Illumina MiSeq platform. Libraries were prepared with 40, 45, 50, 55, and 60 PCR cycles from four individuals belonging to four species of four genera of cephalopods. We found no relationship between the number of PCR cycles and the number of mutations despite using a nonproofreading polymerase. Moreover, even when using a high number of PCR cycles, the resulting number of mutations was low enough not to be an issue in the context of high-throughput DNA barcoding (but may still remain an issue in DNA metabarcoding due to chimera formation). We conclude that the common practice of increasing the number of PCR cycles should not negatively impact the outcome of a high-throughput DNA barcoding study in terms of the occurrence of point mutations.

Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET.

Science.gov (United States)

Beeman, Katrin; Baumgärtner, Jens; Laubenheimer, Manuel; Hergesell, Karlheinz; Hoffmann, Martin; Pehl, Ulrich; Fischer, Frank; Pieck, Jan-Carsten

2017-12-01

Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated "in-line reader" for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for drug discovery.

A high throughput mechanical screening device for cartilage tissue engineering.

Science.gov (United States)

Mohanraj, Bhavana; Hou, Chieh; Meloni, Gregory R; Cosgrove, Brian D; Dodge, George R; Mauck, Robert L

2014-06-27

Articular cartilage enables efficient and near-frictionless load transmission, but suffers from poor inherent healing capacity. As such, cartilage tissue engineering strategies have focused on mimicking both compositional and mechanical properties of native tissue in order to provide effective repair materials for the treatment of damaged or degenerated joint surfaces. However, given the large number design parameters available (e.g. cell sources, scaffold designs, and growth factors), it is difficult to conduct combinatorial experiments of engineered cartilage. This is particularly exacerbated when mechanical properties are a primary outcome, given the long time required for testing of individual samples. High throughput screening is utilized widely in the pharmaceutical industry to rapidly and cost-effectively assess the effects of thousands of compounds for therapeutic discovery. Here we adapted this approach to develop a high throughput mechanical screening (HTMS) system capable of measuring the mechanical properties of up to 48 materials simultaneously. The HTMS device was validated by testing various biomaterials and engineered cartilage constructs and by comparing the HTMS results to those derived from conventional single sample compression tests. Further evaluation showed that the HTMS system was capable of distinguishing and identifying 'hits', or factors that influence the degree of tissue maturation. Future iterations of this device will focus on reducing data variability, increasing force sensitivity and range, as well as scaling-up to even larger (96-well) formats. This HTMS device provides a novel tool for cartilage tissue engineering, freeing experimental design from the limitations of mechanical testing throughput. © 2013 Published by Elsevier Ltd.

A continuous high-throughput bioparticle sorter based on 3D traveling-wave dielectrophoresis.

Science.gov (United States)

Cheng, I-Fang; Froude, Victoria E; Zhu, Yingxi; Chang, Hsueh-Chia; Chang, Hsien-Chang

2009-11-21

We present a high throughput (maximum flow rate approximately 10 microl/min or linear velocity approximately 3 mm/s) continuous bio-particle sorter based on 3D traveling-wave dielectrophoresis (twDEP) at an optimum AC frequency of 500 kHz. The high throughput sorting is achieved with a sustained twDEP particle force normal to the continuous through-flow, which is applied over the entire chip by a single 3D electrode array. The design allows continuous fractionation of micron-sized particles into different downstream sub-channels based on differences in their twDEP mobility on both sides of the cross-over. Conventional DEP is integrated upstream to focus the particles into a single levitated queue to allow twDEP sorting by mobility difference and to minimize sedimentation and field-induced lysis. The 3D electrode array design minimizes the offsetting effect of nDEP (negative DEP with particle force towards regions with weak fields) on twDEP such that both forces increase monotonically with voltage to further increase the throughput. Effective focusing and separation of red blood cells from debris-filled heterogeneous samples are demonstrated, as well as size-based separation of poly-dispersed liposome suspensions into two distinct bands at 2.3 to 4.6 microm and 1.5 to 2.7 microm, at the highest throughput recorded in hand-held chips of 6 microl/min.

Structural, dielectric and ferroelectric properties of (Bi,Na)TiO3–BaTiO3 system studied by high throughput screening

International Nuclear Information System (INIS)

Hayden, Brian E.; Yakovlev, Sergey

2016-01-01

Thin-film materials libraries of the Bi 2 O 3 –Na 2 O–TiO 2 –BaO system in a broad composition range have been deposited in ultra-high vacuum from elemental evaporation sources and an oxygen plasma source. A high throughput approach was used for systematic compositional and structural characterization and the screening of the dielectric and ferroelectric properties. The perovskite (Bi,Na)TiO 3 –BaTiO 3 phase with a Ba concentration near the morphotropic phase boundary (ca. 6 at.%) exhibited a relative dielectric permittivity of 180, a loss tangent of 0.04 and remnant polarization of 19 μC/cm 2 . Compared to published data, observed remnant polarization is close to that known for epitaxially grown films but higher than the values reported for polycrystalline films. The high throughput methodology and systematic nature of the study allowed us to establish the composition boundaries of the phase with optimal dielectric and ferroelectric characteristics. - Highlights: • Bi 2 O 3 –Na 2 O–TiO 2 –BaO high throughput materials library was deposited using PVD method. • Materials were processed from individual molecular beam epitaxy sources of elements. • High throughput approach was used for structural, dielectric and ferroelectric study. • Composition boundaries of perovskite compounds with optimum properties are reported.

High-Throughput Fabrication Method for Producing a Silver-Nanoparticles-Doped Nanoclay Polymer Composite with Novel Synergistic Antibacterial Effects at the Material Interface.

Science.gov (United States)

Cai, Shaobo; Pourdeyhimi, Behnam; Loboa, Elizabeth G

2017-06-28

In this study, we report a high-throughput fabrication method at industrial pilot scale to produce a silver-nanoparticles-doped nanoclay-polylactic acid composite with a novel synergistic antibacterial effect. The obtained nanocomposite has a significantly lower affinity for bacterial adhesion, allowing the loading amount of silver nanoparticles to be tremendously reduced while maintaining satisfactory antibacterial efficacy at the material interface. This is a great advantage for many antibacterial applications in which cost is a consideration. Furthermore, unlike previously reported methods that require additional chemical reduction processes to produce the silver-nanoparticles-doped nanoclay, an in situ preparation method was developed in which silver nanoparticles were created simultaneously during the composite fabrication process by thermal reduction. This is the first report to show that altered material surface submicron structures created with the loading of nanoclay enables the creation of a nanocomposite with significantly lower affinity for bacterial adhesion. This study provides a promising scalable approach to produce antibacterial polymeric products with minimal changes to industry standard equipment, fabrication processes, or raw material input cost.

Risk-based high-throughput chemical screening and prioritization using exposure models and in vitro bioactivity assays

International Nuclear Information System (INIS)

Shin, Hyeong-Moo; Ernstoff, Alexi; Csiszar, Susan A.

2015-01-01

We present a risk-based high-throughput screening (HTS) method to identify chemicals for potential health concerns or for which additional information is needed. The method is applied to 180 organic chemicals as a case study. We first obtain information on how the chemical is used and identify relevant use scenarios (e.g., dermal application, indoor emissions). For each chemical and use scenario, exposure models are then used to calculate a chemical intake fraction, or a product intake fraction, accounting for chemical properties and the exposed population. We then combine these intake fractions with use scenario-specific estimates of chemical quantity to calculate daily intake rates (iR; mg/kg/day). These intake rates are compared to oral equivalent doses (OED; mg/kg/day), calculated from a suite of ToxCast in vitro bioactivity assays using in vitro-to-in vivo extrapolation and reverse dosimetry. Bioactivity quotients (BQs) are calculated as iR/OED to obtain estimates of potential impact associated with each relevant use scenario. Of the 180 chemicals considered, 38 had maximum iRs exceeding minimum OEDs (i.e., BQs > 1). For most of these compounds, exposures are associated with direct intake, food/oral contact, or dermal exposure. The method provides high-throughput estimates of exposure and important input for decision makers to identify chemicals of concern for further evaluation with additional information or more refined models

High-throughput sockets over RDMA for the Intel Xeon Phi coprocessor

CERN Document Server

Santogidis, Aram

2017-01-01

In this paper we describe the design, implementation and performance of Trans4SCIF, a user-level socket-like transport library for the Intel Xeon Phi coprocessor. Trans4SCIF library is primarily intended for high-throughput applications. It uses RDMA transfers over the native SCIF support, in a way that is transparent for the application, which has the illusion of using conventional stream sockets. We also discuss the integration of Trans4SCIF with the ZeroMQ messaging library, used extensively by several applications running at CERN. We show that this can lead to a substantial, up to 3x, increase of application throughput compared to the default TCP/IP transport option.

A High-throughput Selection for Cellulase Catalysts Using Chemical Complementation

Science.gov (United States)

Peralta-Yahya, Pamela; Carter, Brian T.; Lin, Hening; Tao, Haiyan; Cornish, Virginia W.

2010-01-01

Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases however is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Due to the large number of enzyme variants selections can test compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity. PMID:19053460

SNP calling using genotype model selection on high-throughput sequencing data

KAUST Repository

You, Na

2012-01-16

Motivation: A review of the available single nucleotide polymorphism (SNP) calling procedures for Illumina high-throughput sequencing (HTS) platform data reveals that most rely mainly on base-calling and mapping qualities as sources of error when calling SNPs. Thus, errors not involved in base-calling or alignment, such as those in genomic sample preparation, are not accounted for.Results: A novel method of consensus and SNP calling, Genotype Model Selection (GeMS), is given which accounts for the errors that occur during the preparation of the genomic sample. Simulations and real data analyses indicate that GeMS has the best performance balance of sensitivity and positive predictive value among the tested SNP callers. © The Author 2012. Published by Oxford University Press. All rights reserved.

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

Science.gov (United States)

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

Development of a high-throughput method for the systematic identification of human proteins nuclear translocation potential

Directory of Open Access Journals (Sweden)

Kawai Jun

2009-09-01

Full Text Available Abstract Background Important clues to the function of novel and uncharacterized proteins can be obtained by identifying their ability to translocate in the nucleus. In addition, a comprehensive definition of the nuclear proteome undoubtedly represents a key step toward a better understanding of the biology of this organelle. Although several high-throughput experimental methods have been developed to explore the sub-cellular localization of proteins, these methods tend to focus on the predominant localizations of gene products and may fail to provide a complete catalog of proteins that are able to transiently locate into the nucleus. Results We have developed a method for examining the nuclear localization potential of human gene products at the proteome scale by adapting a mammalian two-hybrid system we have previously developed. Our system is composed of three constructs co-transfected into a mammalian cell line. First, it contains a PCR construct encoding a fusion protein composed of a tested protein, the PDZ-protein TIP-1, and the transactivation domain of TNNC2 (referred to as ACT construct. Second, our system contains a PCR construct encoding a fusion protein composed of the DNA binding domain of GAL4 and the PDZ binding domain of rhotekin (referred to as the BIND construct. Third, a GAL4-responsive luciferase reporter is used to detect the reconstitution of a transcriptionally active BIND-ACT complex through the interaction of TIP-1 and rhotekin, which indicates the ability of the tested protein to translocate into the nucleus. We validated our method in a small-scale feasibility study by comparing it to green fluorescent protein (GFP fusion-based sub-cellular localization assays, sequence-based computational prediction of protein sub-cellular localization, and current sub-cellular localization data available from the literature for 22 gene products. Conclusion Our reporter-based system can rapidly screen gene products for their ability

A high throughput platform for understanding the influence of excipients on physical and chemical stability

DEFF Research Database (Denmark)

Raijada, Dhara; Cornett, Claus; Rantanen, Jukka

2013-01-01

The present study puts forward a miniaturized high-throughput platform to understand influence of excipient selection and processing on the stability of a given drug compound. Four model drugs (sodium naproxen, theophylline, amlodipine besylate and nitrofurantoin) and ten different excipients were...... for chemical degradation. The proposed high-throughput platform can be used during early drug development to simulate typical processing induced stress in a small scale and to understand possible phase transformation behaviour and influence of excipients on this....

Fabrication of combinatorial nm-planar electrode array for high throughput evaluation of organic semiconductors

International Nuclear Information System (INIS)

Haemori, M.; Edura, T.; Tsutsui, K.; Itaka, K.; Wada, Y.; Koinuma, H.

2006-01-01

We have fabricated a combinatorial nm-planar electrode array by using photolithography and chemical mechanical polishing processes for high throughput electrical evaluation of organic devices. Sub-nm precision was achieved with respect to the average level difference between each pair of electrodes and a dielectric layer. The insulating property between the electrodes is high enough to measure I-V characteristics of organic semiconductors. Bottom-contact field-effect-transistors (FETs) of pentacene were fabricated on this electrode array by use of molecular beam epitaxy. It was demonstrated that the array could be used as a pre-patterned device substrate for high throughput screening of the electrical properties of organic semiconductors

High-Throughput Quantification of Nanoparticle Degradation Using Computational Microscopy and Its Application to Drug Delivery Nanocapsules

KAUST Repository

Ray, Aniruddha

2017-04-25

Design and synthesis of degradable nanoparticles are very important in drug delivery and biosensing fields. Although accurate assessment of nanoparticle degradation rate would improve the characterization and optimization of drug delivery vehicles, current methods rely on estimating the size of the particles at discrete points over time using, for example, electron microscopy or dynamic light scattering (DLS), among other techniques, all of which have drawbacks and practical limitations. There is a significant need for a high-throughput and cost-effective technology to accurately monitor nanoparticle degradation as a function of time and using small amounts of sample. To address this need, here we present two different computational imaging-based methods for monitoring and quantification of nanoparticle degradation. The first method is suitable for discrete testing, where a computational holographic microscope is designed to track the size changes of protease-sensitive protein-core nanoparticles following degradation, by periodically sampling a subset of particles mixed with proteases. In the second method, a sandwich structure was utilized to observe, in real-time, the change in the properties of liquid nanolenses that were self-assembled around degrading nanoparticles, permitting continuous monitoring and quantification of the degradation process. These cost-effective holographic imaging based techniques enable high-throughput monitoring of the degradation of any type of nanoparticle, using an extremely small amount of sample volume that is at least 3 orders of magnitude smaller than what is required by, for example, DLS-based techniques.

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

Science.gov (United States)

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...

Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres.

Directory of Open Access Journals (Sweden)

Delyan P Ivanov

Full Text Available Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

GUItars: a GUI tool for analysis of high-throughput RNA interference screening data.

Directory of Open Access Journals (Sweden)

Asli N Goktug

Full Text Available High-throughput RNA interference (RNAi screening has become a widely used approach to elucidating gene functions. However, analysis and annotation of large data sets generated from these screens has been a challenge for researchers without a programming background. Over the years, numerous data analysis methods were produced for plate quality control and hit selection and implemented by a few open-access software packages. Recently, strictly standardized mean difference (SSMD has become a widely used method for RNAi screening analysis mainly due to its better control of false negative and false positive rates and its ability to quantify RNAi effects with a statistical basis. We have developed GUItars to enable researchers without a programming background to use SSMD as both a plate quality and a hit selection metric to analyze large data sets.The software is accompanied by an intuitive graphical user interface for easy and rapid analysis workflow. SSMD analysis methods have been provided to the users along with traditionally-used z-score, normalized percent activity, and t-test methods for hit selection. GUItars is capable of analyzing large-scale data sets from screens with or without replicates. The software is designed to automatically generate and save numerous graphical outputs known to be among the most informative high-throughput data visualization tools capturing plate-wise and screen-wise performances. Graphical outputs are also written in HTML format for easy access, and a comprehensive summary of screening results is written into tab-delimited output files.With GUItars, we demonstrated robust SSMD-based analysis workflow on a 3840-gene small interfering RNA (siRNA library and identified 200 siRNAs that increased and 150 siRNAs that decreased the assay activities with moderate to stronger effects. GUItars enables rapid analysis and illustration of data from large- or small-scale RNAi screens using SSMD and other traditional analysis

Using iterative cluster merging with improved gap statistics to perform online phenotype discovery in the context of high-throughput RNAi screens

Directory of Open Access Journals (Sweden)

Sun Youxian

2008-06-01

Full Text Available Abstract Background The recent emergence of high-throughput automated image acquisition technologies has forever changed how cell biologists collect and analyze data. Historically, the interpretation of cellular phenotypes in different experimental conditions has been dependent upon the expert opinions of well-trained biologists. Such qualitative analysis is particularly effective in detecting subtle, but important, deviations in phenotypes. However, while the rapid and continuing development of automated microscope-based technologies now facilitates the acquisition of trillions of cells in thousands of diverse experimental conditions, such as in the context of RNA interference (RNAi or small-molecule screens, the massive size of these datasets precludes human analysis. Thus, the development of automated methods which aim to identify novel and biological relevant phenotypes online is one of the major challenges in high-throughput image-based screening. Ideally, phenotype discovery methods should be designed to utilize prior/existing information and tackle three challenging tasks, i.e. restoring pre-defined biological meaningful phenotypes, differentiating novel phenotypes from known ones and clarifying novel phenotypes from each other. Arbitrarily extracted information causes biased analysis, while combining the complete existing datasets with each new image is intractable in high-throughput screens. Results Here we present the design and implementation of a novel and robust online phenotype discovery method with broad applicability that can be used in diverse experimental contexts, especially high-throughput RNAi screens. This method features phenotype modelling and iterative cluster merging using improved gap statistics. A Gaussian Mixture Model (GMM is employed to estimate the distribution of each existing phenotype, and then used as reference distribution in gap statistics. This method is broadly applicable to a number of different types of

The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond.

Science.gov (United States)

Haslam, Carl; Hellicar, John; Dunn, Adrian; Fuetterer, Arne; Hardy, Neil; Marshall, Peter; Paape, Rainer; Pemberton, Michelle; Resemannand, Anja; Leveridge, Melanie

2016-02-01

Mass spectrometry (MS) offers a label-free, direct-detection method, in contrast to fluorescent or colorimetric methodologies. Over recent years, solid-phase extraction-based techniques, such as the Agilent RapidFire system, have emerged that are capable of analyzing samples in high-throughput screening (HTS). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) offers an alternative for high-throughput MS detection. However, sample preparation and deposition onto the MALDI target, as well as interference from matrix ions, have been considered limitations for the use of MALDI for screening assays. Here we describe the development and validation of assays for both small-molecule and peptide analytes using MALDI-TOF coupled with nanoliter liquid handling. Using the JMJD2c histone demethylase and acetylcholinesterase as model systems, we have generated robust data in a 1536 format and also increased sample deposition to 6144 samples per target. Using these methods, we demonstrate that this technology can deliver fast sample analysis time with low sample volume, and data comparable to that of current RapidFire assays. © 2015 Society for Laboratory Automation and Screening.

Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

Science.gov (United States)

Sun, Changhong; Fan, Yu; Li, Juan; Wang, Gancheng; Zhang, Hanshuo; Xi, Jianzhong Jeff

2015-02-01

Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line. © 2014 Society for Laboratory Automation and Screening.

A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

Science.gov (United States)

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

2017-08-01

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

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

Science.gov (United States)

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

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

Science.gov (United States)

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.

Ethoscopes: An open platform for high-throughput ethomics.

Directory of Open Access Journals (Sweden)

Quentin Geissmann

2017-10-01

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

Correction of Microplate Data from High-Throughput Screening.

Science.gov (United States)

Wang, Yuhong; Huang, Ruili

2016-01-01

High-throughput screening (HTS) makes it possible to collect cellular response data from a large number of cell lines and small molecules in a timely and cost-effective manner. The errors and noises in the microplate-formatted data from HTS have unique characteristics, and they can be generally grouped into three categories: run-wise (temporal, multiple plates), plate-wise (background pattern, single plate), and well-wise (single well). In this chapter, we describe a systematic solution for identifying and correcting such errors and noises, mainly basing on pattern recognition and digital signal processing technologies.

High Throughput Multispectral Image Processing with Applications in Food Science.

Directory of Open Access Journals (Sweden)

Panagiotis Tsakanikas

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

Digital Biomass Accumulation Using High-Throughput Plant Phenotype Data Analysis.

Science.gov (United States)

Rahaman, Md Matiur; Ahsan, Md Asif; Gillani, Zeeshan; Chen, Ming

2017-09-01

Biomass is an important phenotypic trait in functional ecology and growth analysis. The typical methods for measuring biomass are destructive, and they require numerous individuals to be cultivated for repeated measurements. With the advent of image-based high-throughput plant phenotyping facilities, non-destructive biomass measuring methods have attempted to overcome this problem. Thus, the estimation of plant biomass of individual plants from their digital images is becoming more important. In this paper, we propose an approach to biomass estimation based on image derived phenotypic traits. Several image-based biomass studies state that the estimation of plant biomass is only a linear function of the projected plant area in images. However, we modeled the plant volume as a function of plant area, plant compactness, and plant age to generalize the linear biomass model. The obtained results confirm the proposed model and can explain most of the observed variance during image-derived biomass estimation. Moreover, a small difference was observed between actual and estimated digital biomass, which indicates that our proposed approach can be used to estimate digital biomass accurately.

High Throughput Multispectral Image Processing with Applications in Food Science.

Science.gov (United States)

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

2015-01-01

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

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

Science.gov (United States)

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.

High-Throughput Network Communication with NetIO

CERN Document Server

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

2016-01-01

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

High-Throughput Printing Process for Flexible Electronics

Science.gov (United States)

Hyun, Woo Jin

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

High-throughput screening of tick-borne pathogens in Europe

DEFF Research Database (Denmark)

Michelet, Lorraine; Delannoy, Sabine; Devillers, Elodie

2014-01-01

was conducted on 7050 Ixodes ricinus nymphs collected from France, Denmark, and the Netherlands using a powerful new high-throughput approach. This advanced methodology permitted the simultaneous detection of 25 bacterial, and 12 parasitic species (including; Borrelia, Anaplasma, Ehrlichia, Rickettsia......, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia, and Theileria genus) across 94 samples. We successfully determined the prevalence of expected (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia helvetica, Candidatus Neoehrlichia mikurensis, Babesia divergens, Babesia...

HiHiMap: single-cell quantitation of histones and histone posttranslational modifications across the cell cycle by high-throughput imaging.

Science.gov (United States)

Zane, Linda; Chapus, Fleur; Pegoraro, Gianluca; Misteli, Tom

2017-08-15

We describe Hi gh-throughput Hi stone Map ping (HiHiMap), a high-throughput imaging method to measure histones and histone posttranslational modifications (PTMs) in single cells. HiHiMap uses imaging-based quantification of DNA and cyclin A to stage individual cells in the cell cycle to determine the levels of histones or histone PTMs in each stage of the cell cycle. As proof of principle, we apply HiHiMap to measure the level of 21 core histones, histone variants, and PTMs in primary, immortalized, and transformed cells. We identify several histone modifications associated with oncogenic transformation. HiHiMap allows the rapid, high-throughput study of histones and histone PTMs across the cell cycle and the study of subpopulations of cells. © 2017 Zane et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Low Cost, High-Throughput 3-D Pulmonary Imager Using Hyperpolarized Contrast Agents and Low-Field MRI

Science.gov (United States)

2017-10-01

greater gas polarizations and production amounts/ throughputs- benefiting in particular from the advent of com- pact, high-power, relatively low- cost ...Award Number: W81XWH-15-1-0271 TITLE: Low- Cost , High-Throughput 3-D Pulmonary Imager Using Hyperpolarized Contrast Agents and Low-Field MRI...DISTRIBUTION STATEMENT: Approved for Public Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the

High throughput octal alpha/gamma spectrometer for low level bioassay estimations

International Nuclear Information System (INIS)

Bhasin, B.D.; Shirke, S.H.; Suri, M.M.; Vaidya, P.P.; Ghodgaonkar, M.D.

1995-01-01

The present paper describes the development of a high throughput octal alpha spectrometry system specially developed for the estimation of low levels of actinides in bioassay and environmental samples. The system processes simultaneously the outputs coming from eight independent detectors. It can be configured to simultaneously record low level alpha and gamma spectra. The high throughput is achieved by using a prioritised multiplexer router. The prioritised multiplexing and routing coupled with fast 8K ADC (conversion time 20 μsec) allow simultaneous acquisition of multiple spectra without any significant loss in counts. The dual (8K, 24bit) port memory facilitates easy online viewing of spectrum buildup. A menu driven user friendly software makes the operating system convenient to use. A specially developed software provides built-in routines for processing the spectra and estimating the isotopic activity. The interactive mode of software provides easy identification of isotopes compatible with the separation chemistry of different actinides. (author). 6 refs., 2 figs

Design of a High-Throughput Biological Crystallography Beamline for Superconducting Wiggler

International Nuclear Information System (INIS)

Tseng, P.C.; Chang, C.H.; Fung, H.S.; Ma, C.I.; Huang, L.J.; Jean, Y.C.; Song, Y.F.; Huang, Y.S.; Tsang, K.L.; Chen, C.T.

2004-01-01

We are constructing a high-throughput biological crystallography beamline BL13B, which utilizes the radiation generated from a 3.2 Tesla, 32-pole superconducting multipole wiggler, for multi-wavelength anomalous diffraction (MAD), single-wavelength anomalous diffraction (SAD), and other related experiments. This beamline is a standard double crystal monochromator (DCM) x-ray beamline equipped with a collimating mirror (CM) and a focusing mirror (FM). Both the CM and FM are one meter long and made of Si substrate, and the CM is side-cooled by water. Based on detailed thermal analysis, liquid nitrogen (LN2) cooling for both crystals of the DCM has been adopted to optimize the energy resolution and photon beam throughput. This beamline will deliver, through a 100 μm diameter pinhole, photon flux of greater than 1011 photons/sec in the energy range from 6.5 keV to 19 keV, which is comparable to existing protein crystallography beamlines from bending magnet source at high energy storage rings

Affinity selection-mass spectrometry and its emerging application to the high throughput screening of G protein-coupled receptors.

Science.gov (United States)

Whitehurst, Charles E; Annis, D Allen

2008-07-01

Advances in combinatorial chemistry and genomics have inspired the development of novel affinity selection-based screening techniques that rely on mass spectrometry to identify compounds that preferentially bind to a protein target. Of the many affinity selection-mass spectrometry techniques so far documented, only a few solution-based implementations that separate target-ligand complexes away from unbound ligands persist today as routine high throughput screening platforms. Because affinity selection-mass spectrometry techniques do not rely on radioactive or fluorescent reporters or enzyme activities, they can complement traditional biochemical and cell-based screening assays and enable scientists to screen targets that may not be easily amenable to other methods. In addition, by employing mass spectrometry for ligand detection, these techniques enable high throughput screening of massive library collections of pooled compound mixtures, vastly increasing the chemical space that a target can encounter during screening. Of all drug targets, G protein coupled receptors yield the highest percentage of therapeutically effective drugs. In this manuscript, we present the emerging application of affinity selection-mass spectrometry to the high throughput screening of G protein coupled receptors. We also review how affinity selection-mass spectrometry can be used as an analytical tool to guide receptor purification, and further used after screening to characterize target-ligand binding interactions, enabling the classification of orthosteric and allosteric binders.

Integrative Analysis of High-throughput Cancer Studies with Contrasted Penalization

Science.gov (United States)

Shi, Xingjie; Liu, Jin; Huang, Jian; Zhou, Yong; Shia, BenChang; Ma, Shuangge

2015-01-01

In cancer studies with high-throughput genetic and genomic measurements, integrative analysis provides a way to effectively pool and analyze heterogeneous raw data from multiple independent studies and outperforms “classic” meta-analysis and single-dataset analysis. When marker selection is of interest, the genetic basis of multiple datasets can be described using the homogeneity model or the heterogeneity model. In this study, we consider marker selection under the heterogeneity model, which includes the homogeneity model as a special case and can be more flexible. Penalization methods have been developed in the literature for marker selection. This study advances from the published ones by introducing the contrast penalties, which can accommodate the within- and across-dataset structures of covariates/regression coefficients and, by doing so, further improve marker selection performance. Specifically, we develop a penalization method that accommodates the across-dataset structures by smoothing over regression coefficients. An effective iterative algorithm, which calls an inner coordinate descent iteration, is developed. Simulation shows that the proposed method outperforms the benchmark with more accurate marker identification. The analysis of breast cancer and lung cancer prognosis studies with gene expression measurements shows that the proposed method identifies genes different from those using the benchmark and has better prediction performance. PMID:24395534

Turbocharged molecular discovery of OLED emitters: from high-throughput quantum simulation to highly efficient TADF devices

Science.gov (United States)

Gómez-Bombarelli, Rafael; Aguilera-Iparraguirre, Jorge; Hirzel, Timothy D.; Ha, Dong-Gwang; Einzinger, Markus; Wu, Tony; Baldo, Marc A.; Aspuru-Guzik, Alán.

2016-09-01

Discovering new OLED emitters requires many experiments to synthesize candidates and test performance in devices. Large scale computer simulation can greatly speed this search process but the problem remains challenging enough that brute force application of massive computing power is not enough to successfully identify novel structures. We report a successful High Throughput Virtual Screening study that leveraged a range of methods to optimize the search process. The generation of candidate structures was constrained to contain combinatorial explosion. Simulations were tuned to the specific problem and calibrated with experimental results. Experimentalists and theorists actively collaborated such that experimental feedback was regularly utilized to update and shape the computational search. Supervised machine learning methods prioritized candidate structures prior to quantum chemistry simulation to prevent wasting compute on likely poor performers. With this combination of techniques, each multiplying the strength of the search, this effort managed to navigate an area of molecular space and identify hundreds of promising OLED candidate structures. An experimentally validated selection of this set shows emitters with external quantum efficiencies as high as 22%.

Supplementary Material for: DRABAL: novel method to mine large high-throughput screening assays using Bayesian active learning

KAUST Repository

Soufan, Othman

2016-01-01

Abstract Background Mining high-throughput screening (HTS) assays is key for enhancing decisions in the area of drug repositioning and drug discovery. However, many challenges are encountered in the process of developing suitable and accurate methods for extracting useful information from these assays. Virtual screening and a wide variety of databases, methods and solutions proposed to-date, did not completely overcome these challenges. This study is based on a multi-label classification (MLC) technique for modeling correlations between several HTS assays, meaning that a single prediction represents a subset of assigned correlated labels instead of one label. Thus, the devised method provides an increased probability for more accurate predictions of compounds that were not tested in particular assays. Results Here we present DRABAL, a novel MLC solution that incorporates structure learning of a Bayesian network as a step to model dependency between the HTS assays. In this study, DRABAL was used to process more than 1.4 million interactions of over 400,000 compounds and analyze the existing relationships between five large HTS assays from the PubChem BioAssay Database. Compared to different MLC methods, DRABAL significantly improves the F1Score by about 22%, on average. We further illustrated usefulness and utility of DRABAL through screening FDA approved drugs and reported ones that have a high probability to interact with several targets, thus enabling drug-multi-target repositioning. Specifically DRABAL suggests the Thiabendazole drug as a common activator of the NCP1 and Rab-9A proteins, both of which are designed to identify treatment modalities for the Niemannâ Pick type C disease. Conclusion We developed a novel MLC solution based on a Bayesian active learning framework to overcome the challenge of lacking fully labeled training data and exploit actual dependencies between the HTS assays. The solution is motivated by the need to model dependencies between

Nanoliter Centrifugal Liquid Dispenser Coupled with Superhydrophobic Microwell Array Chips for High-Throughput Cell Assays

Directory of Open Access Journals (Sweden)

Yuyi Wang

2018-06-01

Full Text Available Microfluidic systems have been regarded as a potential platform for high-throughput screening technology in drug discovery due to their low sample consumption, high integration, and easy operation. The handling of small-volume liquid is an essential operation in microfluidic systems, especially in investigating large-scale combination conditions. Here, we develop a nanoliter centrifugal liquid dispenser (NanoCLD coupled with superhydrophobic microwell array chips for high-throughput cell-based assays in the nanoliter scale. The NanoCLD consists of a plastic stock block with an array of drilled through holes, a reagent microwell array chip (reagent chip, and an alignment bottom assembled together in a fixture. A simple centrifugation at 800 rpm can dispense ~160 nL reagents into microwells in 5 min. The dispensed reagents are then delivered to cells by sandwiching the reagent chip upside down with another microwell array chip (cell chip on which cells are cultured. A gradient of doxorubicin is then dispensed to the cell chip using the NanoCLD for validating the feasibility of performing drug tests on our microchip platform. This novel nanoliter-volume liquid dispensing method is simple, easy to operate, and especially suitable for repeatedly dispensing many different reagents simultaneously to microwells.

Large-scale DNA Barcode Library Generation for Biomolecule Identification in High-throughput Screens.

Science.gov (United States)

Lyons, Eli; Sheridan, Paul; Tremmel, Georg; Miyano, Satoru; Sugano, Sumio

2017-10-24

High-throughput screens allow for the identification of specific biomolecules with characteristics of interest. In barcoded screens, DNA barcodes are linked to target biomolecules in a manner allowing for the target molecules making up a library to be identified by sequencing the DNA barcodes using Next Generation Sequencing. To be useful in experimental settings, the DNA barcodes in a library must satisfy certain constraints related to GC content, homopolymer length, Hamming distance, and blacklisted subsequences. Here we report a novel framework to quickly generate large-scale libraries of DNA barcodes for use in high-throughput screens. We show that our framework dramatically reduces the computation time required to generate large-scale DNA barcode libraries, compared with a naїve approach to DNA barcode library generation. As a proof of concept, we demonstrate that our framework is able to generate a library consisting of one million DNA barcodes for use in a fragment antibody phage display screening experiment. We also report generating a general purpose one billion DNA barcode library, the largest such library yet reported in literature. Our results demonstrate the value of our novel large-scale DNA barcode library generation framework for use in high-throughput screening applications.

High-throughput analysis for preparation, processing and analysis of TiO2 coatings on steel by chemical solution deposition

International Nuclear Information System (INIS)

Cuadrado Gil, Marcos; Van Driessche, Isabel; Van Gils, Sake; Lommens, Petra; Castelein, Pieter; De Buysser, Klaartje

2012-01-01