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

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

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

2009-07-20

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

Saturated linkage map construction in Rubus idaeus using genotyping by sequencing and genome-independent imputation

USDA-ARS?s Scientific Manuscript database

Rapid development of highly saturated genetic maps aids molecular breeding, which can accelerate gain per breeding cycle in woody perennial plants such as Rubus idaeus (red raspberry). Recently, robust genotyping methods based on high-throughput sequencing were developed, which provide high marker d...

A Robust Framework for Microbial Archaeology

PubMed Central

Warinner, Christina; Herbig, Alexander; Mann, Allison; Yates, James A. Fellows; Weiβ, Clemens L.; Burbano, Hernán A.; Orlando, Ludovic; Krause, Johannes

2017-01-01

Microbial archaeology is flourishing in the era of high-throughput sequencing, revealing the agents behind devastating historical plagues, identifying the cryptic movements of pathogens in prehistory, and reconstructing the ancestral microbiota of humans. Here, we introduce the fundamental concepts and theoretical framework of the discipline, then discuss applied methodologies for pathogen identification and microbiome characterization from archaeological samples. We give special attention to the process of identifying, validating, and authenticating ancient microbes using high-throughput DNA sequencing data. Finally, we outline standards and precautions to guide future research in the field. PMID:28460196

A high-throughput microRNA expression profiling system.

PubMed

Guo, Yanwen; Mastriano, Stephen; Lu, Jun

2014-01-01

As small noncoding RNAs, microRNAs (miRNAs) regulate diverse biological functions, including physiological and pathological processes. The expression and deregulation of miRNA levels contain rich information with diagnostic and prognostic relevance and can reflect pharmacological responses. The increasing interest in miRNA-related research demands global miRNA expression profiling on large numbers of samples. We describe here a robust protocol that supports high-throughput sample labeling and detection on hundreds of samples simultaneously. This method employs 96-well-based miRNA capturing from total RNA samples and on-site biochemical reactions, coupled with bead-based detection in 96-well format for hundreds of miRNAs per sample. With low-cost, high-throughput, high detection specificity, and flexibility to profile both small and large numbers of samples, this protocol can be adapted in a wide range of laboratory settings.

tcpl: the ToxCast pipeline for high-throughput screening data.

PubMed

Filer, Dayne L; Kothiya, Parth; Setzer, R Woodrow; Judson, Richard S; Martin, Matthew T

2017-02-15

Large high-throughput screening (HTS) efforts are widely used in drug development and chemical toxicity screening. Wide use and integration of these data can benefit from an efficient, transparent and reproducible data pipeline. Summary: The tcpl R package and its associated MySQL database provide a generalized platform for efficiently storing, normalizing and dose-response modeling of large high-throughput and high-content chemical screening data. The novel dose-response modeling algorithm has been tested against millions of diverse dose-response series, and robustly fits data with outliers and cytotoxicity-related signal loss. tcpl is freely available on the Comprehensive R Archive Network under the GPL-2 license. martin.matt@epa.gov. Published by Oxford University Press 2016. This work is written by US Government employees and is in the public domain in the US.

A Fast and Robust UHPLC-MRM-MS Method to Characterize and Quantify Grape Skin Tannins after Chemical Depolymerization.

PubMed

Pinasseau, Lucie; Verbaere, Arnaud; Roques, Maryline; Meudec, Emmanuelle; Vallverdú-Queralt, Anna; Terrier, Nancy; Boulet, Jean-Claude; Cheynier, Véronique; Sommerer, Nicolas

2016-10-21

A rapid, sensitive, and selective analysis method using ultra high performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-QqQ-MS) has been developed for the characterization and quantification of grape skin flavan-3-ols after acid-catalysed depolymerization in the presence of phloroglucinol (phloroglucinolysis). The compound detection being based on specific MS transitions in Multiple Reaction Monitoring (MRM) mode, this fast gradient robust method allows analysis of constitutive units of grape skin proanthocyanidins, including some present in trace amounts, in a single injection, with a throughput of 6 samples per hour. This method was applied to a set of 214 grape skin samples from 107 different red and white grape cultivars grown under two conditions in the vineyard, irrigated or non-irrigated. The results of triplicate analyses confirmed the robustness of the method, which was thus proven to be suitable for high-throughput and large-scale metabolomics studies. Moreover, these preliminary results suggest that analysis of tannin composition is relevant to investigate the genetic bases of grape response to drought.

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

PubMed Central

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

2015-01-01

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. PMID:26501283

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

PubMed

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.

Robust ridge regression estimators for nonlinear models with applications to high throughput screening assay data.

PubMed

Lim, Changwon

2015-03-30

Nonlinear regression is often used to evaluate the toxicity of a chemical or a drug by fitting data from a dose-response study. Toxicologists and pharmacologists may draw a conclusion about whether a chemical is toxic by testing the significance of the estimated parameters. However, sometimes the null hypothesis cannot be rejected even though the fit is quite good. One possible reason for such cases is that the estimated standard errors of the parameter estimates are extremely large. In this paper, we propose robust ridge regression estimation procedures for nonlinear models to solve this problem. The asymptotic properties of the proposed estimators are investigated; in particular, their mean squared errors are derived. The performances of the proposed estimators are compared with several standard estimators using simulation studies. The proposed methodology is also illustrated using high throughput screening assay data obtained from the National Toxicology Program. Copyright © 2014 John Wiley & Sons, Ltd.

A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution.

PubMed

Jha, Ramesh K; Bingen, Jeremy M; Johnson, Christopher W; Kern, Theresa L; Khanna, Payal; Trettel, Daniel S; Strauss, Charlie E M; Beckham, Gregg T; Dale, Taraka

2018-06-01

Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. Here we demonstrate the optimization of an Escherichia coli- based biosensor in a robust microbial strain for the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators.

A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution

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

Jha, Ramesh K.; Bingen, Jeremy M.; Johnson, Christopher W.

Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. In this study, we demonstrate the optimization of an Escherichia coli-based biosensor in a robust microbial strain formore » the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators.« less

A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution

DOE PAGES

Jha, Ramesh K.; Bingen, Jeremy M.; Johnson, Christopher W.; ...

2018-06-01

Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. In this study, we demonstrate the optimization of an Escherichia coli-based biosensor in a robust microbial strain formore » the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators.« less

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

PubMed

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

2018-06-01

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

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

PubMed

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

2009-08-25

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

Microfluidic guillotine for single-cell wound repair studies

NASA Astrophysics Data System (ADS)

Blauch, Lucas R.; Gai, Ya; Khor, Jian Wei; Sood, Pranidhi; Marshall, Wallace F.; Tang, Sindy K. Y.

2017-07-01

Wound repair is a key feature distinguishing living from nonliving matter. Single cells are increasingly recognized to be capable of healing wounds. The lack of reproducible, high-throughput wounding methods has hindered single-cell wound repair studies. This work describes a microfluidic guillotine for bisecting single Stentor coeruleus cells in a continuous-flow manner. Stentor is used as a model due to its robust repair capacity and the ability to perform gene knockdown in a high-throughput manner. Local cutting dynamics reveals two regimes under which cells are bisected, one at low viscous stress where cells are cut with small membrane ruptures and high viability and one at high viscous stress where cells are cut with extended membrane ruptures and decreased viability. A cutting throughput up to 64 cells per minute—more than 200 times faster than current methods—is achieved. The method allows the generation of more than 100 cells in a synchronized stage of their repair process. This capacity, combined with high-throughput gene knockdown in Stentor, enables time-course mechanistic studies impossible with current wounding methods.

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

NASA Astrophysics Data System (ADS)

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.

Response to “Accurate Risk-Based Chemical Screening Relies on Robust Exposure Estimates”

EPA Science Inventory

This is a correspondence (letter to the editor) with reference to comments by Rudel and Perovich on the article "Integration of Dosimetry, Exposure, and High-Throughput Screening Data in Chemical Toxicity Assessment". Article Reference: SI # 238882

Development of a Rapid Fluorescence-Based High-Throughput Screening Assay to Identify Novel Kynurenine 3-Monooxygenase Inhibitor Scaffolds.

PubMed

Jacobs, K R; Guillemin, G J; Lovejoy, D B

2018-02-01

Kynurenine 3-monooxygenase (KMO) is a well-validated therapeutic target for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Huntington's disease (HD). This work reports a facile fluorescence-based KMO assay optimized for high-throughput screening (HTS) that achieves a throughput approximately 20-fold higher than the fastest KMO assay currently reported. The screen was run with excellent performance (average Z' value of 0.80) from 110,000 compounds across 341 plates and exceeded all statistical parameters used to describe a robust HTS assay. A subset of molecules was selected for validation by ultra-high-performance liquid chromatography, resulting in the confirmation of a novel hit with an IC 50 comparable to that of the well-described KMO inhibitor Ro-61-8048. A medicinal chemistry program is currently underway to further develop our novel KMO inhibitor scaffolds.

Spotsizer: High-throughput quantitative analysis of microbial growth.

PubMed

Bischof, Leanne; Převorovský, Martin; Rallis, Charalampos; Jeffares, Daniel C; Arzhaeva, Yulia; Bähler, Jürg

2016-10-01

Microbial colony growth can serve as a useful readout in assays for studying complex genetic interactions or the effects of chemical compounds. Although computational tools for acquiring quantitative measurements of microbial colonies have been developed, their utility can be compromised by inflexible input image requirements, non-trivial installation procedures, or complicated operation. Here, we present the Spotsizer software tool for automated colony size measurements in images of robotically arrayed microbial colonies. Spotsizer features a convenient graphical user interface (GUI), has both single-image and batch-processing capabilities, and works with multiple input image formats and different colony grid types. We demonstrate how Spotsizer can be used for high-throughput quantitative analysis of fission yeast growth. The user-friendly Spotsizer tool provides rapid, accurate, and robust quantitative analyses of microbial growth in a high-throughput format. Spotsizer is freely available at https://data.csiro.au/dap/landingpage?pid=csiro:15330 under a proprietary CSIRO license.

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

PubMed Central

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

2014-01-01

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

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

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

Wang, Yonggang, E-mail: wangyg@ustc.edu.cn; Hui, Cong; Liu, Chong

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,more » 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.« less

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

PubMed

Wang, Yonggang; Hui, Cong; Liu, Chong; Xu, Chao

2016-04-01

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.

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

PubMed

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.

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

PubMed

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

2018-01-01

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

Convenient, Sensitive and High-Throughput Method for Screening Botanic Origin

NASA Astrophysics Data System (ADS)

Yuan, Yuan; Jiang, Chao; Liu, Libing; Yu, Shulin; Cui, Zhanhu; Chen, Min; Lin, Shufang; Wang, Shu; Huang, Luqi

2014-06-01

In this work, a rapid (within 4-5 h), sensitive and visible new method for assessing botanic origin is developed by combining loop-mediated isothermal amplification with cationic conjugated polymers. The two Chinese medicinal materials (Jin-Yin-Hua and Shan-Yin-Hua) with similar morphology and chemical composition were clearly distinguished by gene SNP genotyping assays. The identification of plant species in Patented Chinese drugs containing Lonicera buds is successfully performed using this detection system. The method is also robust enough to be used in high-throughput screening. This new method is very helpful to identify herbal materials, and is beneficial for detecting safety and quality of botanic products.

RootGraph: a graphic optimization tool for automated image analysis of plant roots

PubMed Central

Cai, Jinhai; Zeng, Zhanghui; Connor, Jason N.; Huang, Chun Yuan; Melino, Vanessa; Kumar, Pankaj; Miklavcic, Stanley J.

2015-01-01

This paper outlines a numerical scheme for accurate, detailed, and high-throughput image analysis of plant roots. In contrast to existing root image analysis tools that focus on root system-average traits, a novel, fully automated and robust approach for the detailed characterization of root traits, based on a graph optimization process is presented. The scheme, firstly, distinguishes primary roots from lateral roots and, secondly, quantifies a broad spectrum of root traits for each identified primary and lateral root. Thirdly, it associates lateral roots and their properties with the specific primary root from which the laterals emerge. The performance of this approach was evaluated through comparisons with other automated and semi-automated software solutions as well as against results based on manual measurements. The comparisons and subsequent application of the algorithm to an array of experimental data demonstrate that this method outperforms existing methods in terms of accuracy, robustness, and the ability to process root images under high-throughput conditions. PMID:26224880

384 hanging drop arrays give excellent Z-factors and allow versatile formation of co-culture spheroids.

PubMed

Hsiao, Amy Y; Tung, Yi-Chung; Qu, Xianggui; Patel, Lalit R; Pienta, Kenneth J; Takayama, Shuichi

2012-05-01

We previously reported the development of a simple, user-friendly, and versatile 384 hanging drop array plate for 3D spheroid culture and the importance of utilizing 3D cellular models in anti-cancer drug sensitivity testing. The 384 hanging drop array plate allows for high-throughput capabilities and offers significant improvements over existing 3D spheroid culture methods. To allow for practical 3D cell-based high-throughput screening and enable broader use of the plate, we characterize the robustness of the 384 hanging drop array plate in terms of assay performance and demonstrate the versatility of the plate. We find that the 384 hanging drop array plate performance is robust in fluorescence- and colorimetric-based assays through Z-factor calculations. Finally, we demonstrate different plate capabilities and applications, including: spheroid transfer and retrieval for Janus spheroid formation, sequential addition of cells for concentric layer patterning of different cell types, and culture of a wide variety of cell types. Copyright © 2011 Wiley Periodicals, Inc.

384 Hanging Drop Arrays Give Excellent Z-factors and Allow Versatile Formation of Co-culture Spheroids

PubMed Central

Hsiao, Amy Y.; Tung, Yi-Chung; Qu, Xianggui; Patel, Lalit R.; Pienta, Kenneth J.; Takayama, Shuichi

2012-01-01

We previously reported the development of a simple, user-friendly, and versatile 384 hanging drop array plate for 3D spheroid culture and the importance of utilizing 3D cellular models in anti-cancer drug sensitivity testing. The 384 hanging drop array plate allows for high-throughput capabilities and offers significant improvements over existing 3D spheroid culture methods. To allow for practical 3D cell-based high-throughput screening and enable broader use of the plate, we characterize the robustness of the 384 hanging drop array plate in terms of assay performance and demonstrate the versatility of the plate. We find that the 384 hanging drop array plate performance is robust in fluorescence- and colorimetric-based assays through z-factor calculations. Finally, we demonstrate different plate capabilities and applications, including: spheroid transfer and retrieval for Janus spheroid formation, sequential addition of cells for concentric layer patterning of different cell types, and culture of a wide variety of cell types. PMID:22161651

Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates.

PubMed

Leung, Kaston; Klaus, Anders; Lin, Bill K; Laks, Emma; Biele, Justina; Lai, Daniel; Bashashati, Ali; Huang, Yi-Fei; Aniba, Radhouane; Moksa, Michelle; Steif, Adi; Mes-Masson, Anne-Marie; Hirst, Martin; Shah, Sohrab P; Aparicio, Samuel; Hansen, Carl L

2016-07-26

The genomes of large numbers of single cells must be sequenced to further understanding of the biological significance of genomic heterogeneity in complex systems. Whole genome amplification (WGA) of single cells is generally the first step in such studies, but is prone to nonuniformity that can compromise genomic measurement accuracy. Despite recent advances, robust performance in high-throughput single-cell WGA remains elusive. Here, we introduce droplet multiple displacement amplification (MDA), a method that uses commercially available liquid dispensing to perform high-throughput single-cell MDA in nanoliter volumes. The performance of droplet MDA is characterized using a large dataset of 129 normal diploid cells, and is shown to exceed previously reported single-cell WGA methods in amplification uniformity, genome coverage, and/or robustness. We achieve up to 80% coverage of a single-cell genome at 5× sequencing depth, and demonstrate excellent single-nucleotide variant (SNV) detection using targeted sequencing of droplet MDA product to achieve a median allelic dropout of 15%, and using whole genome sequencing to achieve false and true positive rates of 9.66 × 10(-6) and 68.8%, respectively, in a G1-phase cell. We further show that droplet MDA allows for the detection of copy number variants (CNVs) as small as 30 kb in single cells of an ovarian cancer cell line and as small as 9 Mb in two high-grade serous ovarian cancer samples using only 0.02× depth. Droplet MDA provides an accessible and scalable method for performing robust and accurate CNV and SNV measurements on large numbers of single cells.

Toward high throughput optical metamaterial assemblies.

PubMed

Fontana, Jake; Ratna, Banahalli R

2015-11-01

Optical metamaterials have unique engineered optical properties. These properties arise from the careful organization of plasmonic elements. Transitioning these properties from laboratory experiments to functional materials may lead to disruptive technologies for controlling light. A significant issue impeding the realization of optical metamaterial devices is the need for robust and efficient assembly strategies to govern the order of the nanometer-sized elements while enabling macroscopic throughput. This mini-review critically highlights recent approaches and challenges in creating these artificial materials. As the ability to assemble optical metamaterials improves, new unforeseen opportunities may arise for revolutionary optical devices.

High pressure inertial focusing for separating and concentrating bacteria at high throughput

NASA Astrophysics Data System (ADS)

Cruz, J.; Hooshmand Zadeh, S.; Graells, T.; Andersson, M.; Malmström, J.; Wu, Z. G.; Hjort, K.

2017-08-01

Inertial focusing is a promising microfluidic technology for concentration and separation of particles by size. However, there is a strong correlation of increased pressure with decreased particle size. Theory and experimental results for larger particles were used to scale down the phenomenon and find the conditions that focus 1 µm particles. High pressure experiments in robust glass chips were used to demonstrate the alignment. We show how the technique works for 1 µm spherical polystyrene particles and for Escherichia coli, not being harmful for the bacteria at 50 µl min-1. The potential to focus bacteria, simplicity of use and high throughput make this technology interesting for healthcare applications, where concentration and purification of a sample may be required as an initial step.

A simple and sensitive high-throughput GFP screening in woody and herbaceous plants.

PubMed

Hily, Jean-Michel; Liu, Zongrang

2009-03-01

Green fluorescent protein (GFP) has been used widely as a powerful bioluminescent reporter, but its visualization by existing methods in tissues or whole plants and its utilization for high-throughput screening remains challenging in many species. Here, we report a fluorescence image analyzer-based method for GFP detection and its utility for high-throughput screening of transformed plants. Of three detection methods tested, the Typhoon fluorescence scanner was able to detect GFP fluorescence in all Arabidopsis thaliana tissues and apple leaves, while regular fluorescence microscopy detected it only in Arabidopsis flowers and siliques but barely in the leaves of either Arabidopsis or apple. The hand-held UV illumination method failed in all tissues of both species. Additionally, the Typhoon imager was able to detect GFP fluorescence in both green and non-green tissues of Arabidopsis seedlings as well as in imbibed seeds, qualifying it as a high-throughput screening tool, which was further demonstrated by screening the seedlings of primary transformed T(0) seeds. Of the 30,000 germinating Arabidopsis seedlings screened, at least 69 GFP-positive lines were identified, accounting for an approximately 0.23% transformation efficiency. About 14,000 seedlings grown in 16 Petri plates could be screened within an hour, making the screening process significantly more efficient and robust than any other existing high-throughput screening method for transgenic plants.

Benchmarking Procedures for High-Throughput Context Specific Reconstruction Algorithms

PubMed Central

Pacheco, Maria P.; Pfau, Thomas; Sauter, Thomas

2016-01-01

Recent progress in high-throughput data acquisition has shifted the focus from data generation to processing and understanding of how to integrate collected information. Context specific reconstruction based on generic genome scale models like ReconX or HMR has the potential to become a diagnostic and treatment tool tailored to the analysis of specific individuals. The respective computational algorithms require a high level of predictive power, robustness and sensitivity. Although multiple context specific reconstruction algorithms were published in the last 10 years, only a fraction of them is suitable for model building based on human high-throughput data. Beside other reasons, this might be due to problems arising from the limitation to only one metabolic target function or arbitrary thresholding. This review describes and analyses common validation methods used for testing model building algorithms. Two major methods can be distinguished: consistency testing and comparison based testing. The first is concerned with robustness against noise, e.g., missing data due to the impossibility to distinguish between the signal and the background of non-specific binding of probes in a microarray experiment, and whether distinct sets of input expressed genes corresponding to i.e., different tissues yield distinct models. The latter covers methods comparing sets of functionalities, comparison with existing networks or additional databases. We test those methods on several available algorithms and deduce properties of these algorithms that can be compared with future developments. The set of tests performed, can therefore serve as a benchmarking procedure for future algorithms. PMID:26834640

Capturing Positive Transgressive Variation From Wild And Exotic Germplasm Resources

USDA-ARS?s Scientific Manuscript database

Only a small fraction of the naturally occurring genetic diversity available in rice germplasm repositories around the world has been explored to date. This is beginning to change with the advent of affordable, high throughput genotyping approaches coupled with robust statistical analysis methods th...

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

PubMed Central

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

Genome sequencing in microfabricated high-density picolitre reactors.

PubMed

Margulies, Marcel; Egholm, Michael; Altman, William E; Attiya, Said; Bader, Joel S; Bemben, Lisa A; Berka, Jan; Braverman, Michael S; Chen, Yi-Ju; Chen, Zhoutao; Dewell, Scott B; Du, Lei; Fierro, Joseph M; Gomes, Xavier V; Godwin, Brian C; He, Wen; Helgesen, Scott; Ho, Chun Heen; Ho, Chun He; Irzyk, Gerard P; Jando, Szilveszter C; Alenquer, Maria L I; Jarvie, Thomas P; Jirage, Kshama B; Kim, Jong-Bum; Knight, James R; Lanza, Janna R; Leamon, John H; Lefkowitz, Steven M; Lei, Ming; Li, Jing; Lohman, Kenton L; Lu, Hong; Makhijani, Vinod B; McDade, Keith E; McKenna, Michael P; Myers, Eugene W; Nickerson, Elizabeth; Nobile, John R; Plant, Ramona; Puc, Bernard P; Ronan, Michael T; Roth, George T; Sarkis, Gary J; Simons, Jan Fredrik; Simpson, John W; Srinivasan, Maithreyan; Tartaro, Karrie R; Tomasz, Alexander; Vogt, Kari A; Volkmer, Greg A; Wang, Shally H; Wang, Yong; Weiner, Michael P; Yu, Pengguang; Begley, Richard F; Rothberg, Jonathan M

2005-09-15

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation.

PubMed

D'Aiuto, Leonardo; Zhi, Yun; Kumar Das, Dhanjit; Wilcox, Madeleine R; Johnson, Jon W; McClain, Lora; MacDonald, Matthew L; Di Maio, Roberto; Schurdak, Mark E; Piazza, Paolo; Viggiano, Luigi; Sweet, Robert; Kinchington, Paul R; Bhattacharjee, Ayantika G; Yolken, Robert; Nimgaonka, Vishwajit L; Nimgaonkar, Vishwajit L

2014-01-01

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.

Convenient, sensitive and high-throughput method for screening botanic origin.

PubMed

Yuan, Yuan; Jiang, Chao; Liu, Libing; Yu, Shulin; Cui, Zhanhu; Chen, Min; Lin, Shufang; Wang, Shu; Huang, Luqi

2014-06-23

In this work, a rapid (within 4-5 h), sensitive and visible new method for assessing botanic origin is developed by combining loop-mediated isothermal amplification with cationic conjugated polymers. The two Chinese medicinal materials (Jin-Yin-Hua and Shan-Yin-Hua) with similar morphology and chemical composition were clearly distinguished by gene SNP genotyping assays. The identification of plant species in Patented Chinese drugs containing Lonicera buds is successfully performed using this detection system. The method is also robust enough to be used in high-throughput screening. This new method is very helpful to identify herbal materials, and is beneficial for detecting safety and quality of botanic products.

A high-throughput virus-induced gene silencing protocol identifies genes involved in multi-stress tolerance

PubMed Central

2013-01-01

Background Understanding the function of a particular gene under various stresses is important for engineering plants for broad-spectrum stress tolerance. Although virus-induced gene silencing (VIGS) has been used to characterize genes involved in abiotic stress tolerance, currently available gene silencing and stress imposition methodology at the whole plant level is not suitable for high-throughput functional analyses of genes. This demands a robust and reliable methodology for characterizing genes involved in abiotic and multi-stress tolerance. Results Our methodology employs VIGS-based gene silencing in leaf disks combined with simple stress imposition and effect quantification methodologies for easy and faster characterization of genes involved in abiotic and multi-stress tolerance. By subjecting leaf disks from gene-silenced plants to various abiotic stresses and inoculating silenced plants with various pathogens, we show the involvement of several genes for multi-stress tolerance. In addition, we demonstrate that VIGS can be used to characterize genes involved in thermotolerance. Our results also showed the functional relevance of NtEDS1 in abiotic stress, NbRBX1 and NbCTR1 in oxidative stress; NtRAR1 and NtNPR1 in salinity stress; NbSOS1 and NbHSP101 in biotic stress; and NtEDS1, NbETR1, NbWRKY2 and NbMYC2 in thermotolerance. Conclusions In addition to widening the application of VIGS, we developed a robust, easy and high-throughput methodology for functional characterization of genes involved in multi-stress tolerance. PMID:24289810

A Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery.

PubMed

Joslin, John; Gilligan, James; Anderson, Paul; Garcia, Catherine; Sharif, Orzala; Hampton, Janice; Cohen, Steven; King, Miranda; Zhou, Bin; Jiang, Shumei; Trussell, Christopher; Dunn, Robert; Fathman, John W; Snead, Jennifer L; Boitano, Anthony E; Nguyen, Tommy; Conner, Michael; Cooke, Mike; Harris, Jennifer; Ainscow, Ed; Zhou, Yingyao; Shaw, Chris; Sipes, Dan; Mainquist, James; Lesley, Scott

2018-05-01

The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.

The Robustness Analysis of Wireless Sensor Networks under Uncertain Interference

PubMed Central

Deng, Changjian

2013-01-01

Based on the complex network theory, robustness analysis of condition monitoring wireless sensor network under uncertain interference is present. In the evolution of the topology of sensor networks, the density weighted algebraic connectivity is taken into account, and the phenomenon of removing and repairing the link and node in the network is discussed. Numerical simulation is conducted to explore algebraic connectivity characteristics and network robustness performance. It is found that nodes density has the effect on algebraic connectivity distribution in the random graph model; high density nodes carry more connections, use more throughputs, and may be more unreliable. Moreover, the results show that, when network should be more error tolerant or robust by repairing nodes or adding new nodes, the network should be better clustered in median and high scale wireless sensor networks and be meshing topology in small scale networks. PMID:24363613

CA resist with high sensitivity and sub-100-nm resolution for advanced mask making

NASA Astrophysics Data System (ADS)

Huang, Wu-Song; Kwong, Ranee W.; Hartley, John G.; Moreau, Wayne M.; Angelopoulos, Marie; Magg, Christopher; Lawliss, Mark

2000-07-01

Recently, there is significant interest in using CA resist for electron beam (E-beam) applications including mask making, direct write, and projection printing. CA resists provide superior lithographic performance in comparison to traditional non-CA E-beam resist in particular high contrast, resolution, and sensitivity. However, most of the commercially available CA resist have the concern of airborne base contaminants and sensitivity to PAB and/or PEB temperatures. In this presentation, we will discuss a new improved ketal resists system referred to as KRS-XE which exhibits excellent lithography, is robust toward airborne base, compatible with 0.263N TMAH aqueous developer and exhibits excellent lithography, is robust toward airborne base, compatible with 0.263N TMAH aqueous developer and exhibits a large PAB/PEB latitude. With the combination of a high performance mask making E-beam exposure tool, high kV shaped beam system EL4+ and the KRS-XE resist, we have printed 75nm lines/space feature with excellent profile control at a dose of 13(mu) C/cm2 at 75kV. The shaped beam vector scan system used here provides a unique property in resolving small features in lithography and throughput. Overhead in EL4+$ limits the systems ability to fully exploit the sensitivity of the new resist for throughput. The EL5 system has sufficiently low overhead that it is projected to print a 4X, 16G DRAM mask with OPC in under 3 hours with the CA resist. We will discuss the throughput advantages of the next generation EL5 system over the existing EL4+.

Development of a High Throughput Assay for Rapid and Accurate 10-Plex Detection of Citrus Pathogens

USDA-ARS?s Scientific Manuscript database

The need to reliably detect and identify multiple plant pathogens simultaneously, especially in woody perennial hosts, has led to development of new molecular diagnostic approaches. In this study, a Luminex-based system was developed that provided a robust and sensitive test for simultaneous detect...

Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.

PubMed

Stanton, Richard J; McSharry, Brian P; Armstrong, Melanie; Tomasec, Peter; Wilkinson, Gavin W G

2008-12-01

With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

High Throughput, Polymeric Aqueous Two-Phase Printing of Tumor Spheroids

PubMed Central

Atefi, Ehsan; Lemmo, Stephanie; Fyffe, Darcy; Luker, Gary D.; Tavana, Hossein

2014-01-01

This paper presents a new 3D culture microtechnology for high throughput production of tumor spheroids and validates its utility for screening anti-cancer drugs. We use two immiscible polymeric aqueous solutions and microprint a submicroliter drop of the “patterning” phase containing cells into a bath of the “immersion” phase. Selecting proper formulations of biphasic systems using a panel of biocompatible polymers results in the formation of a round drop that confines cells to facilitate spontaneous formation of a spheroid without any external stimuli. Adapting this approach to robotic tools enables straightforward generation and maintenance of spheroids of well-defined size in standard microwell plates and biochemical analysis of spheroids in situ, which is not possible with existing techniques for spheroid culture. To enable high throughput screening, we establish a phase diagram to identify minimum cell densities within specific volumes of the patterning drop to result in a single spheroid. Spheroids show normal growth over long-term incubation and dose-dependent decrease in cellular viability when treated with drug compounds, but present significant resistance compared to monolayer cultures. The unprecedented ease of implementing this microtechnology and its robust performance will benefit high throughput studies of drug screening against cancer cells with physiologically-relevant 3D tumor models. PMID:25411577

A high-throughput screen of the GTPase activity of Escherichia coli EngA to find an inhibitor of bacterial ribosome biogenesis

PubMed Central

Bharat, Amrita; Blanchard, Jan E.; Brown, Eric D.

2014-01-01

The synthesis of ribosomes is an essential process, which is aided by a variety of transacting factors in bacteria. Among these is a group of GTPases essential for bacterial viability and emerging as promising targets for new antibacterial agents. Herein, we describe a robust high-throughput screening process for inhibitors of one such GTPase, the Escherichia coli EngA protein. The primary screen employed an assay of phosphate production in 384-well density. Reaction conditions were chosen to maximize sensitivity for the discovery of competitive inhibitors while maintaining a strong signal amplitude and low noise. In a pilot screen of 31,800 chemical compounds, 44 active compounds were identified. Further, we describe the elimination of non-specific inhibitors that were detergent-sensitive or reactive as well as those that interfered with the high-throughput phosphate assay. Four inhibitors survived these common counter-screens for non-specificity but these chemicals were also inhibitors of the unrelated enzyme dihydrofolate reductase, suggesting that they too were promiscuously active. The high-throughput screen of the EngA protein described here provides a meticulous pilot study in the search for specific inhibitors of GTPases involved in ribosome biogenesis. PMID:23606650

Optimization of a resazurin-based microplate assay for large-scale compound screenings against Klebsiella pneumoniae.

PubMed

Kim, Hyung Jun; Jang, Soojin

2018-01-01

A new resazurin-based assay was evaluated and optimized using a microplate (384-well) format for high-throughput screening of antibacterial molecules against Klebsiella pneumoniae . Growth of the bacteria in 384-well plates was more effectively measured and had a > sixfold higher signal-to-background ratio using the resazurin-based assay compared with absorbance measurements at 600 nm. Determination of minimum inhibitory concentrations of the antibiotics revealed that the optimized assay quantitatively measured antibacterial activity of various antibiotics. An edge effect observed in the initial assay was significantly reduced using a 1-h incubation of the bacteria-containing plates at room temperature. There was an approximately 10% decrease in signal variability between the edge and the middle wells along with improvement in the assay robustness ( Z ' = 0.99). This optimized resazurin-based assay is an efficient, inexpensive, and robust assay that can quantitatively measure antibacterial activity using a high-throughput screening system to assess a large number of compounds for discovery of new antibiotics against K. pneumoniae .

Valiant load-balanced robust routing under hose model for WDM mesh networks

NASA Astrophysics Data System (ADS)

Zhang, Xiaoning; Li, Lemin; Wang, Sheng

2006-09-01

In this paper, we propose Valiant Load-Balanced robust routing scheme for WDM mesh networks under the model of polyhedral uncertainty (i.e., hose model), and the proposed routing scheme is implemented with traffic grooming approach. Our Objective is to maximize the hose model throughput. A mathematic formulation of Valiant Load-Balanced robust routing is presented and three fast heuristic algorithms are also proposed. When implementing Valiant Load-Balanced robust routing scheme to WDM mesh networks, a novel traffic-grooming algorithm called MHF (minimizing hop first) is proposed. We compare the three heuristic algorithms with the VPN tree under the hose model. Finally we demonstrate in the simulation results that MHF with Valiant Load-Balanced robust routing scheme outperforms the traditional traffic-grooming algorithm in terms of the throughput for the uniform/non-uniform traffic matrix under the hose model.

A simple whole cell based high throughput screening protocol using Mycobacterium bovis BCG for inhibitors against dormant and active tubercle bacilli.

PubMed

Khan, Arshad; Sarkar, Dhiman

2008-04-01

This study aimed at developing a whole cell based high throughput screening protocol to identify inhibitors against both active and dormant tubercle bacilli. A respiratory type of nitrate reductase (NarGHJI), which was induced during dormancy, could reflect the viability of dormant bacilli of Mycobacterium bovis BCG in microplate adopted model of in vitro dormancy. Correlation between reduction in viability and nitrate reductase activity was seen clearly when dormant stage inhibitor metronidazole and itaconic anhydride were applied in this in vitro microplate model. Active replicating stage could also be monitored in the same assay by measuring the A(620) of the culture. MIC values of 0.08, 0.075, 0.3 and 3.0 microg/ml, determined through monitoring A(620) in this assay for rifampin, isoniazid, streptomycin and ethambutol respectively, were well in agreement with previously reported by BACTEC and Bio-Siv assays. S/N ratio and Z' factor for the assay were 8.5 and 0.81 respectively which indicated the robustness of the protocol. Altogether the assay provides an easy, inexpensive, rapid, robust and high content screening tool to search novel antitubercular molecules against both active and dormant bacilli.

Advanced scanning probe lithography.

PubMed

Garcia, Ricardo; Knoll, Armin W; Riedo, Elisa

2014-08-01

The nanoscale control afforded by scanning probe microscopes has prompted the development of a wide variety of scanning-probe-based patterning methods. Some of these methods have demonstrated a high degree of robustness and patterning capabilities that are unmatched by other lithographic techniques. However, the limited throughput of scanning probe lithography has prevented its exploitation in technological applications. Here, we review the fundamentals of scanning probe lithography and its use in materials science and nanotechnology. We focus on robust methods, such as those based on thermal effects, chemical reactions and voltage-induced processes, that demonstrate a potential for applications.

Potential of dynamically harmonized Fourier transform ion cyclotron resonance cell for high-throughput metabolomics fingerprinting: control of data quality.

PubMed

Habchi, Baninia; Alves, Sandra; Jouan-Rimbaud Bouveresse, Delphine; Appenzeller, Brice; Paris, Alain; Rutledge, Douglas N; Rathahao-Paris, Estelle

2018-01-01

Due to the presence of pollutants in the environment and food, the assessment of human exposure is required. This necessitates high-throughput approaches enabling large-scale analysis and, as a consequence, the use of high-performance analytical instruments to obtain highly informative metabolomic profiles. In this study, direct introduction mass spectrometry (DIMS) was performed using a Fourier transform ion cyclotron resonance (FT-ICR) instrument equipped with a dynamically harmonized cell. Data quality was evaluated based on mass resolving power (RP), mass measurement accuracy, and ion intensity drifts from the repeated injections of quality control sample (QC) along the analytical process. The large DIMS data size entails the use of bioinformatic tools for the automatic selection of common ions found in all QC injections and for robustness assessment and correction of eventual technical drifts. RP values greater than 10 6 and mass measurement accuracy of lower than 1 ppm were obtained using broadband mode resulting in the detection of isotopic fine structure. Hence, a very accurate relative isotopic mass defect (RΔm) value was calculated. This reduces significantly the number of elemental composition (EC) candidates and greatly improves compound annotation. A very satisfactory estimate of repeatability of both peak intensity and mass measurement was demonstrated. Although, a non negligible ion intensity drift was observed for negative ion mode data, a normalization procedure was easily applied to correct this phenomenon. This study illustrates the performance and robustness of the dynamically harmonized FT-ICR cell to perform large-scale high-throughput metabolomic analyses in routine conditions. Graphical abstract Analytical performance of FT-ICR instrument equipped with a dynamically harmonized cell.

Xi-CAM v1.2.3

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

PANDOLFI, RONALD; KUMAR, DINESH; VENKATAKRISHNAN, SINGANALLUR

Xi-CAM aims to provide a community driven platform for multimodal analysis in synchrotron science. The platform core provides a robust plugin infrastructure for extensibility, allowing continuing development to simply add further functionality. Current modules include tools for characterization with (GI)SAXS, Tomography, and XAS. This will continue to serve as a development base as algorithms for multimodal analysis develop. Seamless remote data access, visualization and analysis are key elements of Xi-CAM, and will become critical to synchrotron data infrastructure as expectations for future data volume and acquisition rates rise with continuously increasing throughputs. The highly interactive design elements of Xi-cam willmore » similarly support a generation of users which depend on immediate data quality feedback during high-throughput or burst acquisition modes.« less

Wisdom of crowds for robust gene network inference

PubMed Central

Marbach, Daniel; Costello, James C.; Küffner, Robert; Vega, Nicci; Prill, Robert J.; Camacho, Diogo M.; Allison, Kyle R.; Kellis, Manolis; Collins, James J.; Stolovitzky, Gustavo

2012-01-01

Reconstructing gene regulatory networks from high-throughput data is a long-standing problem. Through the DREAM project (Dialogue on Reverse Engineering Assessment and Methods), we performed a comprehensive blind assessment of over thirty network inference methods on Escherichia coli, Staphylococcus aureus, Saccharomyces cerevisiae, and in silico microarray data. We characterize performance, data requirements, and inherent biases of different inference approaches offering guidelines for both algorithm application and development. We observe that no single inference method performs optimally across all datasets. In contrast, integration of predictions from multiple inference methods shows robust and high performance across diverse datasets. Thereby, we construct high-confidence networks for E. coli and S. aureus, each comprising ~1700 transcriptional interactions at an estimated precision of 50%. We experimentally test 53 novel interactions in E. coli, of which 23 were supported (43%). Our results establish community-based methods as a powerful and robust tool for the inference of transcriptional gene regulatory networks. PMID:22796662

Adaptation of a Simple Microfluidic Platform for High-Dimensional Quantitative Morphological Analysis of Human Mesenchymal Stromal Cells on Polystyrene-Based Substrates.

PubMed

Lam, Johnny; Marklein, Ross A; Jimenez-Torres, Jose A; Beebe, David J; Bauer, Steven R; Sung, Kyung E

2017-12-01

Multipotent stromal cells (MSCs, often called mesenchymal stem cells) have garnered significant attention within the field of regenerative medicine because of their purported ability to differentiate down musculoskeletal lineages. Given the inherent heterogeneity of MSC populations, recent studies have suggested that cell morphology may be indicative of MSC differentiation potential. Toward improving current methods and developing simple yet effective approaches for the morphological evaluation of MSCs, we combined passive pumping microfluidic technology with high-dimensional morphological characterization to produce robust tools for standardized high-throughput analysis. Using ultraviolet (UV) light as a modality for reproducible polystyrene substrate modification, we show that MSCs seeded on microfluidic straight channel devices incorporating UV-exposed substrates exhibited morphological changes that responded accordingly to the degree of substrate modification. Substrate modification also effected greater morphological changes in MSCs seeded at a lower rather than higher density within microfluidic channels. Despite largely comparable trends in morphology, MSCs seeded in microscale as opposed to traditional macroscale platforms displayed much higher sensitivity to changes in substrate properties. In summary, we adapted and qualified microfluidic cell culture platforms comprising simple straight channel arrays as a viable and robust tool for high-throughput quantitative morphological analysis to study cell-material interactions.

High-throughput discovery of rare human nucleotide polymorphisms by Ecotilling

PubMed Central

Till, Bradley J.; Zerr, Troy; Bowers, Elisabeth; Greene, Elizabeth A.; Comai, Luca; Henikoff, Steven

2006-01-01

Human individuals differ from one another at only ∼0.1% of nucleotide positions, but these single nucleotide differences account for most heritable phenotypic variation. Large-scale efforts to discover and genotype human variation have been limited to common polymorphisms. However, these efforts overlook rare nucleotide changes that may contribute to phenotypic diversity and genetic disorders, including cancer. Thus, there is an increasing need for high-throughput methods to robustly detect rare nucleotide differences. Toward this end, we have adapted the mismatch discovery method known as Ecotilling for the discovery of human single nucleotide polymorphisms. To increase throughput and reduce costs, we developed a universal primer strategy and implemented algorithms for automated band detection. Ecotilling was validated by screening 90 human DNA samples for nucleotide changes in 5 gene targets and by comparing results to public resequencing data. To increase throughput for discovery of rare alleles, we pooled samples 8-fold and found Ecotilling to be efficient relative to resequencing, with a false negative rate of 5% and a false discovery rate of 4%. We identified 28 new rare alleles, including some that are predicted to damage protein function. The detection of rare damaging mutations has implications for models of human disease. PMID:16893952

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

PubMed

Kittelmann, Jörg; Ottens, Marcel; Hubbuch, Jürgen

2015-04-15

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

Essential attributes identified in the design of a Laboratory Information Management System for a high throughput siRNA screening laboratory.

PubMed

Grandjean, Geoffrey; Graham, Ryan; Bartholomeusz, Geoffrey

2011-11-01

In recent years high throughput screening operations have become a critical application in functional and translational research. Although a seemingly unmanageable amount of data is generated by these high-throughput, large-scale techniques, through careful planning, an effective Laboratory Information Management System (LIMS) can be developed and implemented in order to streamline all phases of a workflow. Just as important as data mining and analysis procedures at the end of complex processes is the tracking of individual steps of applications that generate such data. Ultimately, the use of a customized LIMS will enable users to extract meaningful results from large datasets while trusting the robustness of their assays. To illustrate the design of a custom LIMS, this practical example is provided to highlight the important aspects of the design of a LIMS to effectively modulate all aspects of an siRNA screening service. This system incorporates inventory management, control of workflow, data handling and interaction with investigators, statisticians and administrators. All these modules are regulated in a synchronous manner within the LIMS. © 2011 Bentham Science Publishers

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

PubMed

Massey, Andrew J

2018-01-01

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

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

PubMed

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

2016-10-01

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

Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells

PubMed Central

Cole, Russell H.; Tang, Shi-Yang; Siltanen, Christian A.; Shahi, Payam; Zhang, Jesse Q.; Poust, Sean; Gartner, Zev J.; Abate, Adam R.

2017-01-01

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay. PMID:28760972

Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells.

PubMed

Cole, Russell H; Tang, Shi-Yang; Siltanen, Christian A; Shahi, Payam; Zhang, Jesse Q; Poust, Sean; Gartner, Zev J; Abate, Adam R

2017-08-15

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.

Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells

NASA Astrophysics Data System (ADS)

Cole, Russell H.; Tang, Shi-Yang; Siltanen, Christian A.; Shahi, Payam; Zhang, Jesse Q.; Poust, Sean; Gartner, Zev J.; Abate, Adam R.

2017-08-01

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.

Automated Microfluidic Instrument for Label-Free and High-Throughput Cell Separation.

PubMed

Zhang, Xinjie; Zhu, Zhixian; Xiang, Nan; Long, Feifei; Ni, Zhonghua

2018-03-20

Microfluidic technologies for cell separation were reported frequently in recent years. However, a compact microfluidic instrument enabling thoroughly automated cell separation is still rarely reported until today due to the difficult hybrid between the macrosized fluidic control system and the microsized microfluidic device. In this work, we propose a novel and automated microfluidic instrument to realize size-based separation of cancer cells in a label-free and high-throughput manner. Briefly, the instrument is equipped with a fully integrated microfluidic device and a set of robust fluid-driven and control units, and the instrument functions of precise fluid infusion and high-throughput cell separation are guaranteed by a flow regulatory chip and two cell separation chips which are the key components of the microfluidic device. With optimized control programs, the instrument is successfully applied to automatically sort human breast adenocarcinoma cell line MCF-7 from 5 mL of diluted human blood with a high recovery ratio of ∼85% within a rapid processing time of ∼23 min. We envision that our microfluidic instrument will be potentially useful in many biomedical applications, especially cell separation, enrichment, and concentration for the purpose of cell culture and analysis.

Automated Purification of Recombinant Proteins: Combining High-throughput with High Yield

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

Lin, Chiann Tso; Moore, Priscilla A.; Auberry, Deanna L.

2006-05-01

Protein crystallography, mapping protein interactions and other approaches of current functional genomics require not only purifying large numbers of proteins but also obtaining sufficient yield and homogeneity for downstream high-throughput applications. There is a need for the development of robust automated high-throughput protein expression and purification processes to meet these requirements. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli: First - a filtration separation protocol based on expression of 800 ml E. coli cultures followed by filtration purification using Ni2+-NTATM Agarose (Qiagen). Second - a smallermore » scale magnetic separation method based on expression in 25 ml cultures of E.coli followed by 96-well purification on MagneHisTM Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins about 8 ug of purified protein per unit of OD at 600 nm of bacterial culture. We discuss advantages and limitations of the automated workflows that can provide proteins more than 90 % pure in the range of 100 ug – 45 mg per purification run as well as strategies for optimization of these protocols.« less

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

PubMed

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/.

Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET.

PubMed

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.

High-throughput olfactory conditioning and memory retention test show variation in Nasonia parasitic wasps

PubMed Central

Hoedjes, K M; Steidle, J L M; Werren, J H; Vet, L E M; Smid, H M

2012-01-01

Most of our knowledge on learning and memory formation results from extensive studies on a small number of animal species. Although features and cellular pathways of learning and memory are highly similar in this diverse group of species, there are also subtle differences. Closely related species of parasitic wasps display substantial variation in memory dynamics and can be instrumental to understanding both the adaptive benefit of and mechanisms underlying this variation. Parasitic wasps of the genus Nasonia offer excellent opportunities for multidisciplinary research on this topic. Genetic and genomic resources available for Nasonia are unrivaled among parasitic wasps, providing tools for genetic dissection of mechanisms that cause differences in learning. This study presents a robust, high-throughput method for olfactory conditioning of Nasonia using a host encounter as reward. A T-maze olfactometer facilitates high-throughput memory retention testing and employs standardized odors of equal detectability, as quantified by electroantennogram recordings. Using this setup, differences in memory retention between Nasonia species were shown. In both Nasonia vitripennis and Nasonia longicornis, memory was observed up to at least 5 days after a single conditioning trial, whereas Nasonia giraulti lost its memory after 2 days. This difference in learning may be an adaptation to species-specific differences in ecological factors, for example, host preference. The high-throughput methods for conditioning and memory retention testing are essential tools to study both ultimate and proximate factors that cause variation in learning and memory formation in Nasonia and other parasitic wasp species. PMID:22804968

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

PubMed

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 are used in the high-throughput community to determine assay robustness (Z'-value) demonstrate the suitability of this format for high-throughput screening applications for detection of inhibitors of enzyme activity. The QTL Lightspeed protein detection system provides a simple mix and measure "turn on" assay for the detection of kinase activity using natural protein substrates. The platform is robust and allows for identification of inhibitors of kinase activity.

Next-generation sequencing is a robust strategy for the high-throughput detection of zygosity in transgenic maize.

PubMed

Fritsch, Leonie; Fischer, Rainer; Wambach, Christoph; Dudek, Max; Schillberg, Stefan; Schröper, Florian

2015-08-01

Simple and reliable, high-throughput techniques to detect the zygosity of transgenic events in plants are valuable for biotechnology and plant breeding companies seeking robust genotyping data for the assessment of new lines and the monitoring of breeding programs. We show that next-generation sequencing (NGS) applied to short PCR products spanning the transgene integration site provides accurate zygosity data that are more robust and reliable than those generated by PCR-based methods. The NGS reads covered the 5' border of the transgenic events (incorporating part of the transgene and the flanking genomic DNA), or the genomic sequences flanking the unfilled transgene integration site at the wild-type locus. We compared the NGS method to competitive real-time PCR with transgene-specific and wild-type-specific primer/probe pairs, one pair matching the 5' genomic flanking sequence and 5' part of the transgene and the other matching the unfilled transgene integration site. Although both NGS and real-time PCR provided useful zygosity data, the NGS technique was favorable because it needed fewer optimization steps. It also provided statistically more-reliable evidence for the presence of each allele because each product was often covered by more than 100 reads. The NGS method is also more suitable for the genotyping of large panels of plants because up to 80 million reads can be produced in one sequencing run. Our novel method is therefore ideal for the rapid and accurate genotyping of large numbers of samples.

Numerical techniques for high-throughput reflectance interference biosensing

NASA Astrophysics Data System (ADS)

Sevenler, Derin; Ünlü, M. Selim

2016-06-01

We have developed a robust and rapid computational method for processing the raw spectral data collected from thin film optical interference biosensors. We have applied this method to Interference Reflectance Imaging Sensor (IRIS) measurements and observed a 10,000 fold improvement in processing time, unlocking a variety of clinical and scientific applications. Interference biosensors have advantages over similar technologies in certain applications, for example highly multiplexed measurements of molecular kinetics. However, processing raw IRIS data into useful measurements has been prohibitively time consuming for high-throughput studies. Here we describe the implementation of a lookup table (LUT) technique that provides accurate results in far less time than naive methods. We also discuss an additional benefit that the LUT method can be used with a wider range of interference layer thickness and experimental configurations that are incompatible with methods that require fitting the spectral response.

A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis.

PubMed

Collins, Tony J; Ylanko, Jarkko; Geng, Fei; Andrews, David W

2015-11-01

A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose-response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds.

A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis

PubMed Central

Collins, Tony J.; Ylanko, Jarkko; Geng, Fei

2015-01-01

Abstract A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose–response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds. PMID:26422066

High-throughput electrophysiological assays for voltage gated ion channels using SyncroPatch 768PE.

PubMed

Li, Tianbo; Lu, Gang; Chiang, Eugene Y; Chernov-Rogan, Tania; Grogan, Jane L; Chen, Jun

2017-01-01

Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel Nav1.7. By testing a group of Nav1.7 reference compounds' IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z' factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel Kv1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.

Experimental design and statistical methods for improved hit detection in high-throughput screening.

PubMed

Malo, Nathalie; Hanley, James A; Carlile, Graeme; Liu, Jing; Pelletier, Jerry; Thomas, David; Nadon, Robert

2010-09-01

Identification of active compounds in high-throughput screening (HTS) contexts can be substantially improved by applying classical experimental design and statistical inference principles to all phases of HTS studies. The authors present both experimental and simulated data to illustrate how true-positive rates can be maximized without increasing false-positive rates by the following analytical process. First, the use of robust data preprocessing methods reduces unwanted variation by removing row, column, and plate biases. Second, replicate measurements allow estimation of the magnitude of the remaining random error and the use of formal statistical models to benchmark putative hits relative to what is expected by chance. Receiver Operating Characteristic (ROC) analyses revealed superior power for data preprocessed by a trimmed-mean polish method combined with the RVM t-test, particularly for small- to moderate-sized biological hits.

Analytical Validation of a Portable Mass Spectrometer Featuring Interchangeable, Ambient Ionization Sources for High Throughput Forensic Evidence Screening

NASA Astrophysics Data System (ADS)

Lawton, Zachary E.; Traub, Angelica; Fatigante, William L.; Mancias, Jose; O'Leary, Adam E.; Hall, Seth E.; Wieland, Jamie R.; Oberacher, Herbert; Gizzi, Michael C.; Mulligan, Christopher C.

2017-06-01

Forensic evidentiary backlogs are indicative of the growing need for cost-effective, high-throughput instrumental methods. One such emerging technology that shows high promise in meeting this demand while also allowing on-site forensic investigation is portable mass spectrometric (MS) instrumentation, particularly that which enables the coupling to ambient ionization techniques. While the benefits of rapid, on-site screening of contraband can be anticipated, the inherent legal implications of field-collected data necessitates that the analytical performance of technology employed be commensurate with accepted techniques. To this end, comprehensive analytical validation studies are required before broad incorporation by forensic practitioners can be considered, and are the focus of this work. Pertinent performance characteristics such as throughput, selectivity, accuracy/precision, method robustness, and ruggedness have been investigated. Reliability in the form of false positive/negative response rates is also assessed, examining the effect of variables such as user training and experience level. To provide flexibility toward broad chemical evidence analysis, a suite of rapidly-interchangeable ion sources has been developed and characterized through the analysis of common illicit chemicals and emerging threats like substituted phenethylamines. [Figure not available: see fulltext.

Towards high-throughput automated targeted femtosecond laser-based transfection of adherent cells

NASA Astrophysics Data System (ADS)

Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Gunn-Moore, Frank; Dholakia, Kishan

2011-03-01

Femtosecond laser induced cell membrane poration has proven to be an attractive alternative to the classical methods of drug and gene delivery. It is a selective, sterile, non-contact technique that offers a highly localized operation, low toxicity and consistent performance. However, its broader application still requires the development of robust, high-throughput and user-friendly systems. We present a system capable of unassisted enhanced targeted optoinjection and phototransfection of adherent mammalian cells with a femtosecond laser. We demonstrate the advantages of a dynamic diffractive optical element, namely a spatial light modulator (SLM) for precise three dimensional positioning of the beam. It enables the implementation of a "point-and-shoot" system in which using the software interface a user simply points at the cell and a predefined sequence of precisely positioned doses can be applied. We show that irradiation in three axial positions alleviates the problem of exact beam positioning on the cell membrane and doubles the number of viably optoinjected cells when compared with a single dose. The presented system enables untargeted raster scan irradiation which provides transfection of adherent cells at the throughput of 1 cell per second.

Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli.

PubMed

Zerbini, Francesca; Zanella, Ilaria; Fraccascia, Davide; König, Enrico; Irene, Carmela; Frattini, Luca F; Tomasi, Michele; Fantappiè, Laura; Ganfini, Luisa; Caproni, Elena; Parri, Matteo; Grandi, Alberto; Grandi, Guido

2017-04-24

The exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a "brute force" validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions. We first set up our own version of the CRISPR/Cas9 protocol and then we evaluated the mutagenesis efficiency by changing different parameters including sequence of guide RNAs, length and concentration of donor DNAs, and use of single stranded and double stranded donor DNAs. We then validated the optimized conditions targeting 78 "dispensable" genes. This work led to the definition of a protocol, featuring the use of double stranded synthetic donor DNAs, which guarantees mutagenesis efficiencies consistently higher than 10% and a robustness of 100%. The procedure can be applied also for simultaneous gene deletions. This work defines for the first time the robustness of a CRISPR/Cas9-based protocol based on a large sample size. Since the technical solutions here proposed can be applied to other similar procedures, the data could be of general interest for the scientific community working on bacterial genome editing and, in particular, for those involved in synthetic biology projects requiring high throughput procedures.

Probabilistic Assessment of High-Throughput Wireless Sensor Networks

PubMed Central

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

2016-01-01

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

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

PubMed

Yu, Xiaobo; LaBaer, Joshua

2015-05-01

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

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

PubMed

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

2006-01-01

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

Performance of Identifiler Direct and PowerPlex 16 HS on the Applied Biosystems 3730 DNA Analyzer for processing biological samples archived on FTA cards.

PubMed

Laurin, Nancy; DeMoors, Anick; Frégeau, Chantal

2012-09-01

Direct amplification of STR loci from biological samples collected on FTA cards without prior DNA purification was evaluated using Identifiler Direct and PowerPlex 16 HS in conjunction with the use of a high throughput Applied Biosystems 3730 DNA Analyzer. In order to reduce the overall sample processing cost, reduced PCR volumes combined with various FTA disk sizes were tested. Optimized STR profiles were obtained using a 0.53 mm disk size in 10 μL PCR volume for both STR systems. These protocols proved effective in generating high quality profiles on the 3730 DNA Analyzer from both blood and buccal FTA samples. Reproducibility, concordance, robustness, sample stability and profile quality were assessed using a collection of blood and buccal samples on FTA cards from volunteer donors as well as from convicted offenders. The new developed protocols offer enhanced throughput capability and cost effectiveness without compromising the robustness and quality of the STR profiles obtained. These results support the use of these protocols for processing convicted offender samples submitted to the National DNA Data Bank of Canada. Similar protocols could be applied to the processing of casework reference samples or in paternity or family relationship testing. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

High-Throughput RNA Interference Screening: Tricks of the Trade

PubMed Central

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

2016-01-01

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

Development and Optimization of a High-Throughput Assay To Measure Neutralizing Antibodies against Clostridium difficile Binary Toxin

PubMed Central

Xie, Jinfu; Horton, Melanie; Zorman, Julie; Antonello, Joseph M.; Zhang, Yuhua; Arnold, Beth A.; Secore, Susan; Xoconostle, Rachel; Miezeiewski, Matthew; Wang, Su; Price, Colleen E.; Thiriot, David; Goerke, Aaron; Gentile, Marie-Pierre; Skinner, Julie M.

2014-01-01

Clostridium difficile strains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence of C. difficile infection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized with C. difficile binary toxin-containing vaccines. The assay will be useful for C. difficile diagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates. PMID:24623624

High-Throughput Assessment of Cellular Mechanical Properties.

PubMed

Darling, Eric M; Di Carlo, Dino

2015-01-01

Traditionally, cell analysis has focused on using molecular biomarkers for basic research, cell preparation, and clinical diagnostics; however, new microtechnologies are enabling evaluation of the mechanical properties of cells at throughputs that make them amenable to widespread use. We review the current understanding of how the mechanical characteristics of cells relate to underlying molecular and architectural changes, describe how these changes evolve with cell-state and disease processes, and propose promising biomedical applications that will be facilitated by the increased throughput of mechanical testing: from diagnosing cancer and monitoring immune states to preparing cells for regenerative medicine. We provide background about techniques that laid the groundwork for the quantitative understanding of cell mechanics and discuss current efforts to develop robust techniques for rapid analysis that aim to implement mechanophenotyping as a routine tool in biomedicine. Looking forward, we describe additional milestones that will facilitate broad adoption, as well as new directions not only in mechanically assessing cells but also in perturbing them to passively engineer cell state.

High-Performance Flexible Perovskite Solar Cells by Using a Combination of Ultrasonic Spray-Coating and Low Thermal Budget Photonic Curing

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

Sanjib, Das; Yang, Bin; Gu, Gong

Realizing the commercialization of high-performance and robust perovskite solar cells urgently requires the development of economically scalable processing techniques. Here we report a high-throughput ultrasonic spray-coating (USC) process capable of fabricating perovskite film-based solar cells on glass substrates with power conversion efficiency (PCE) as high as 13.04%. Perovskite films with high uniformity, crystallinity, and surface coverage are obtained in a single step. Moreover, we report USC processing on TiOx/ITO-coated polyethylene terephthalate (PET) substrates to realize flexible perovskite solar cells with PCE as high as 8.02% that are robust under mechanical stress. In this case, an optical curing technique was usedmore » to achieve a highly-conductive TiOx layer on flexible PET substrates for the first time. The high device performance and reliability obtained by this combination of USC processing with optical curing appears very promising for roll-to-roll manufacturing of high-efficiency, flexible perovskite solar cells.« less

All solid state mid-infrared dual-comb spectroscopy platform based on QCL technology

NASA Astrophysics Data System (ADS)

Hugi, Andreas; Geiser, Markus; Villares, Gustavo; Cappelli, Francesco; Blaser, Stephane; Faist, Jérôme

2015-01-01

We develop a spectroscopy platform for industrial applications based on semiconductor quantum cascade laser (QCL) frequency combs. The platform's key features will be an unmatched combination of bandwidth of 100 cm-1, resolution of 100 kHz, speed of ten to hundreds of μs as well as size and robustness, opening doors to beforehand unreachable markets. The sensor can be built extremely compact and robust since the laser source is an all-electrically pumped semiconductor optical frequency comb and no mechanical elements are required. However, the parallel acquisition of dual-comb spectrometers comes at the price of enormous data-rates. For system scalability, robustness and optical simplicity we use free-running QCL combs. Therefore no complicated optical locking mechanisms are required. To reach high signal-to-noise ratios, we develop an algorithm, which is based on combination of coherent and non-coherent averaging. This algorithm is specifically optimized for free-running and small footprint, therefore high-repetition rate, comb sources. As a consequence, our system generates data-rates of up to 3.2 GB/sec. These data-rates need to be reduced by several orders of magnitude in real-time in order to be useful for spectral fitting algorithms. We present the development of a data-treatment solution, which reaches a single-channel throughput of 22% using a standard laptop-computer. Using a state-of-the art desktop computer, the throughput is increased to 43%. This is combined with a data-acquisition board to a stand-alone data processing unit, allowing real-time industrial process observation and continuous averaging to achieve highest signal fidelity.

Identification of Novel "Inks" for 3D Printing Using High-Throughput Screening: Bioresorbable Photocurable Polymers for Controlled Drug Delivery.

PubMed

Louzao, Iria; Koch, Britta; Taresco, Vincenzo; Ruiz-Cantu, Laura; Irvine, Derek J; Roberts, Clive J; Tuck, Christopher; Alexander, Cameron; Hague, Richard; Wildman, Ricky; Alexander, Morgan R

2018-02-28

A robust methodology is presented to identify novel biomaterials suitable for three-dimensional (3D) printing. Currently, the application of additive manufacturing is limited by the availability of functional inks, especially in the area of biomaterials; this is the first time when this method is used to tackle this problem, allowing hundreds of formulations to be readily assessed. Several functional properties, including the release of an antidepressive drug (paroxetine), cytotoxicity, and printability, are screened for 253 new ink formulations in a high-throughput format as well as mechanical properties. The selected candidates with the desirable properties are successfully scaled up using 3D printing into a range of object architectures. A full drug release study and degradability and tensile modulus experiments are presented on a simple architecture to validating the suitability of this methodology to identify printable inks for 3D printing devices with bespoke properties.

Quantitative profiling of immune repertoires for minor lymphocyte counts using unique molecular identifiers.

PubMed

Egorov, Evgeny S; Merzlyak, Ekaterina M; Shelenkov, Andrew A; Britanova, Olga V; Sharonov, George V; Staroverov, Dmitriy B; Bolotin, Dmitriy A; Davydov, Alexey N; Barsova, Ekaterina; Lebedev, Yuriy B; Shugay, Mikhail; Chudakov, Dmitriy M

2015-06-15

Emerging high-throughput sequencing methods for the analyses of complex structure of TCR and BCR repertoires give a powerful impulse to adaptive immunity studies. However, there are still essential technical obstacles for performing a truly quantitative analysis. Specifically, it remains challenging to obtain comprehensive information on the clonal composition of small lymphocyte populations, such as Ag-specific, functional, or tissue-resident cell subsets isolated by sorting, microdissection, or fine needle aspirates. In this study, we report a robust approach based on unique molecular identifiers that allows profiling Ag receptors for several hundred to thousand lymphocytes while preserving qualitative and quantitative information on clonal composition of the sample. We also describe several general features regarding the data analysis with unique molecular identifiers that are critical for accurate counting of starting molecules in high-throughput sequencing applications. Copyright © 2015 by The American Association of Immunologists, Inc.

Continuous cell introduction and rapid dynamic lysis for high-throughput single-cell analysis on microfludic chips with hydrodynamic focusing.

PubMed

Xu, Chun-Xiu; Yin, Xue-Feng

2011-02-04

A chip-based microfluidic system for high-throughput single-cell analysis is described. The system was integrated with continuous introduction of individual cells, rapid dynamic lysis, capillary electrophoretic (CE) separation and laser induced fluorescence (LIF) detection. A cross microfluidic chip with one sheath-flow channel located on each side of the sampling channel was designed. The labeled cells were hydrodynamically focused by sheath-flow streams and sequentially introduced into the cross section of the microchip under hydrostatic pressure generated by adjusting liquid levels in the reservoirs. Combined with the electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 33ms at the entry of the separation channel by Triton X-100 added in the sheath-flow solution. The maximum rate for introducing individual cells into the separation channel was about 150cells/min. The introduction of sheath-flow streams also significantly reduced the concentration of phosphate-buffered saline (PBS) injected into the separation channel along with single cells, thus reducing Joule heating during electrophoretic separation. The performance of this microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single erythrocytes. A throughput of 38cells/min was obtained. The proposed method is simple and robust for high-throughput single-cell analysis, allowing for analysis of cell population with considerable size to generate results with statistical significance. Copyright © 2010 Elsevier B.V. All rights reserved.

A Customizable Flow Injection System for Automated, High Throughput, and Time Sensitive Ion Mobility Spectrometry and Mass Spectrometry Measurements

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

Orton, Daniel J.; Tfaily, Malak M.; Moore, Ronald J.

To better understand disease conditions and environmental perturbations, multi-omic studies (i.e. proteomic, lipidomic, metabolomic, etc. analyses) are vastly increasing in popularity. In a multi-omic study, a single sample is typically extracted in multiple ways and numerous analyses are performed using different instruments. Thus, one sample becomes many analyses, making high throughput and reproducible evaluations a necessity. One way to address the numerous samples and varying instrumental conditions is to utilize a flow injection analysis (FIA) system for rapid sample injection. While some FIA systems have been created to address these challenges, many have limitations such as high consumable costs, lowmore » pressure capabilities, limited pressure monitoring and fixed flow rates. To address these limitations, we created an automated, customizable FIA system capable of operating at diverse flow rates (~50 nL/min to 500 µL/min) to accommodate low- and high-flow instrument sources. This system can also operate at varying analytical throughputs from 24 to 1200 samples per day to enable different MS analysis approaches. Applications ranging from native protein analyses to molecular library construction were performed using the FIA system. The results from these studies showed a highly robust platform, providing consistent performance over many days without carryover as long as washing buffers specific to each molecular analysis were utilized.« less

High-throughput tandem mass spectrometry multiplex analysis for newborn urinary screening of creatine synthesis and transport disorders, Triple H syndrome and OTC deficiency.

PubMed

Auray-Blais, Christiane; Maranda, Bruno; Lavoie, Pamela

2014-09-25

Creatine synthesis and transport disorders, Triple H syndrome and ornithine transcarbamylase deficiency are treatable inborn errors of metabolism. Early screening of patients was found to be beneficial. Mass spectrometry analysis of specific urinary biomarkers might lead to early detection and treatment in the neonatal period. We developed a high-throughput mass spectrometry methodology applicable to newborn screening using dried urine on filter paper for these aforementioned diseases. A high-throughput methodology was devised for the simultaneous analysis of creatine, guanidineacetic acid, orotic acid, uracil, creatinine and respective internal standards, using both positive and negative electrospray ionization modes, depending on the compound. The precision and accuracy varied by <15%. Stability during storage at different temperatures was confirmed for three weeks. The limits of detection and quantification for each biomarker varied from 0.3 to 6.3 μmol/l and from 1.0 to 20.9 μmol/l, respectively. Analyses of urine specimens from affected patients revealed abnormal results. Targeted biomarkers in urine were detected in the first weeks of life. This rapid, simple and robust liquid chromatography/tandem mass spectrometry methodology is an efficient tool applicable to urine screening for inherited disorders by biochemical laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

Validation of a high-throughput real-time polymerase chain reaction assay for the detection of capripoxviral DNA.

PubMed

Stubbs, Samuel; Oura, Chris A L; Henstock, Mark; Bowden, Timothy R; King, Donald P; Tuppurainen, Eeva S M

2012-02-01

Capripoxviruses, which are endemic in much of Africa and Asia, are the aetiological agents of economically devastating poxviral diseases in cattle, sheep and goats. The aim of this study was to validate a high-throughput real-time PCR assay for routine diagnostic use in a capripoxvirus reference laboratory. The performance of two previously published real-time PCR methods were compared using commercially available reagents including the amplification kits recommended in the original publication. Furthermore, both manual and robotic extraction methods used to prepare template nucleic acid were evaluated using samples collected from experimentally infected animals. The optimised assay had an analytical sensitivity of at least 63 target DNA copies per reaction, displayed a greater diagnostic sensitivity compared to conventional gel-based PCR, detected capripoxviruses isolated from outbreaks around the world and did not amplify DNA from related viruses in the genera Orthopoxvirus or Parapoxvirus. The high-throughput robotic DNA extraction procedure did not adversely affect the sensitivity of the assay compared to manual preparation of PCR templates. This laboratory-based assay provides a rapid and robust method to detect capripoxviruses following suspicion of disease in endemic or disease-free countries. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells.

PubMed

Spidel, Jared L; Vaessen, Benjamin; Chan, Yin Yin; Grasso, Luigi; Kline, J Bradford

2016-12-01

Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential. A protocol was developed to decrease the time, cost, and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol and from the stable cell line process by utilizing bicistronic plasmids to generate stable semi-clonal cell pools facilitated an increased throughput of the entire process from plasmid assembly through transient transfections and selection of stable semi-clonal cell pools. Furthermore, the time required by a single individual to clone, express, and select stable cell pools in a high-throughput format was reduced from 4 to 6months to only 4 to 6weeks. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Development of a high-throughput screen to detect inhibitors of TRPS1 sumoylation.

PubMed

Brandt, Martin; Szewczuk, Lawrence M; Zhang, Hong; Hong, Xuan; McCormick, Patricia M; Lewis, Tia S; Graham, Taylor I; Hung, Sunny T; Harper-Jones, Amber D; Kerrigan, John J; Wang, Da-Yuan; Dul, Edward; Hou, Wangfang; Ho, Thau F; Meek, Thomas D; Cheung, Mui H; Johanson, Kyung O; Jones, Christopher S; Schwartz, Benjamin; Kumar, Sanjay; Oliff, Allen I; Kirkpatrick, Robert B

2013-06-01

Small ubiquitin-like modifier (SUMO) belongs to the family of ubiquitin-like proteins (Ubls) that can be reversibly conjugated to target-specific lysines on substrate proteins. Although covalently sumoylated products are readily detectible in gel-based assays, there has been little progress toward the development of robust quantitative sumoylation assay formats for the evaluation of large compound libraries. In an effort to identify inhibitors of ubiquitin carrier protein 9 (Ubc9)-dependent sumoylation, a high-throughput fluorescence polarization assay was developed, which allows detection of Lys-1201 sumoylation, corresponding to the major site of functional sumoylation within the transcriptional repressor trichorhino-phalangeal syndrome type I protein (TRPS1). A minimal hexapeptide substrate peptide, TMR-VVK₁₂₀₁TEK, was used in this assay format to afford high-throughput screening of the GlaxoSmithKline diversity compound collection. A total of 728 hits were confirmed but no specific noncovalent inhibitors of Ubc9 dependent trans-sumoylation were found. However, several diaminopyrimidine compounds were identified as inhibitors in the assay with IC₅₀ values of 12.5 μM. These were further characterized to be competent substrates which were subject to sumoylation by SUMO-Ubc9 and which were competitive with the sumoylation of the TRPS1 peptide substrates.

Screening Chemicals for Estrogen Receptor Bioactivity Using a Computational Model.

PubMed

Browne, Patience; Judson, Richard S; Casey, Warren M; Kleinstreuer, Nicole C; Thomas, Russell S

2015-07-21

The U.S. Environmental Protection Agency (EPA) is considering high-throughput and computational methods to evaluate the endocrine bioactivity of environmental chemicals. Here we describe a multistep, performance-based validation of new methods and demonstrate that these new tools are sufficiently robust to be used in the Endocrine Disruptor Screening Program (EDSP). Results from 18 estrogen receptor (ER) ToxCast high-throughput screening assays were integrated into a computational model that can discriminate bioactivity from assay-specific interference and cytotoxicity. Model scores range from 0 (no activity) to 1 (bioactivity of 17β-estradiol). ToxCast ER model performance was evaluated for reference chemicals, as well as results of EDSP Tier 1 screening assays in current practice. The ToxCast ER model accuracy was 86% to 93% when compared to reference chemicals and predicted results of EDSP Tier 1 guideline and other uterotrophic studies with 84% to 100% accuracy. The performance of high-throughput assays and ToxCast ER model predictions demonstrates that these methods correctly identify active and inactive reference chemicals, provide a measure of relative ER bioactivity, and rapidly identify chemicals with potential endocrine bioactivities for additional screening and testing. EPA is accepting ToxCast ER model data for 1812 chemicals as alternatives for EDSP Tier 1 ER binding, ER transactivation, and uterotrophic assays.

Dynamic Environmental Photosynthetic Imaging Reveals Emergent Phenotypes

DOE PAGES

Cruz, Jeffrey A.; Savage, Linda J.; Zegarac, Robert; ...

2016-06-22

Understanding and improving the productivity and robustness of plant photosynthesis requires high-throughput phenotyping under environmental conditions that are relevant to the field. Here we demonstrate the dynamic environmental photosynthesis imager (DEPI), an experimental platform for integrated, continuous, and high-throughput measurements of photosynthetic parameters during plant growth under reproducible yet dynamic environmental conditions. Using parallel imagers obviates the need to move plants or sensors, reducing artifacts and allowing simultaneous measurement on large numbers of plants. As a result, DEPI can reveal phenotypes that are not evident under standard laboratory conditions but emerge under progressively more dynamic illumination. We show examples inmore » mutants of Arabidopsis of such “emergent phenotypes” that are highly transient and heterogeneous, appearing in different leaves under different conditions and depending in complex ways on both environmental conditions and plant developmental age. Finally, these emergent phenotypes appear to be caused by a range of phenomena, suggesting that such previously unseen processes are critical for plant responses to dynamic environments.« less

Design and engineering of intracellular-metabolite-sensing/regulation gene circuits in Saccharomyces cerevisiae.

PubMed

Wang, Meng; Li, Sijin; Zhao, Huimin

2016-01-01

The development of high-throughput phenotyping tools is lagging far behind the rapid advances of genotype generation methods. To bridge this gap, we report a new strategy for design, construction, and fine-tuning of intracellular-metabolite-sensing/regulation gene circuits by repurposing bacterial transcription factors and eukaryotic promoters. As proof of concept, we systematically investigated the design and engineering of bacterial repressor-based xylose-sensing/regulation gene circuits in Saccharomyces cerevisiae. We demonstrated that numerous properties, such as induction ratio and dose-response curve, can be fine-tuned at three different nodes, including repressor expression level, operator position, and operator sequence. By applying these gene circuits, we developed a cell sorting based, rapid and robust high-throughput screening method for xylose transporter engineering and obtained a sugar transporter HXT14 mutant with 6.5-fold improvement in xylose transportation capacity. This strategy should be generally applicable and highly useful for evolutionary engineering of proteins, pathways, and genomes in S. cerevisiae. © 2015 Wiley Periodicals, Inc.

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

PubMed Central

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

2015-01-01

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

Saccharomyces cerevisiae as a platform for assessing sphingolipid lipid kinase inhibitors

PubMed Central

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

2018-01-01

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

iPSC-derived neurons as a higher-throughput readout for autism: Promises and pitfalls

PubMed Central

Prilutsky, Daria; Palmer, Nathan P.; Smedemark-Margulies, Niklas; Schlaeger, Thorsten M.; Margulies, David M.; Kohane, Isaac S.

2014-01-01

The elucidation of disease etiologies and establishment of robust, scalable, high-throughput screening assays for autism spectrum disorders (ASDs) have been impeded by both inaccessibility of disease-relevant neuronal tissue and the genetic heterogeneity of the disorder. Neuronal cells derived from induced pluripotent stem cells (iPSCs) from autism patients may circumvent these obstacles and serve as relevant cell models. To date, derived cells are characterized and screened by assessing their neuronal phenotypes. These characterizations are often etiology-specific or lack reproducibility and stability. In this manuscript, we present an overview of efforts to study iPSC-derived neurons as a model for autism, and we explore the plausibility of gene expression profiling as a reproducible and stable disease marker. PMID:24374161

Gas pressure assisted microliquid-liquid extraction coupled online to direct infusion mass spectrometry: a new automated screening platform for bioanalysis.

PubMed

Raterink, Robert-Jan; Witkam, Yoeri; Vreeken, Rob J; Ramautar, Rawi; Hankemeier, Thomas

2014-10-21

In the field of bioanalysis, there is an increasing demand for miniaturized, automated, robust sample pretreatment procedures that can be easily connected to direct-infusion mass spectrometry (DI-MS) in order to allow the high-throughput screening of drugs and/or their metabolites in complex body fluids like plasma. Liquid-Liquid extraction (LLE) is a common sample pretreatment technique often used for complex aqueous samples in bioanalysis. Despite significant developments that have been made in automated and miniaturized LLE procedures, fully automated LLE techniques allowing high-throughput bioanalytical studies on small-volume samples using direct infusion mass spectrometry, have not been matured yet. Here, we introduce a new fully automated micro-LLE technique based on gas-pressure assisted mixing followed by passive phase separation, coupled online to nanoelectrospray-DI-MS. Our method was characterized by varying the gas flow and its duration through the solvent mixture. For evaluation of the analytical performance, four drugs were spiked to human plasma, resulting in highly acceptable precision (RSD down to 9%) and linearity (R(2) ranging from 0.990 to 0.998). We demonstrate that our new method does not only allow the reliable extraction of analytes from small sample volumes of a few microliters in an automated and high-throughput manner, but also performs comparable or better than conventional offline LLE, in which the handling of small volumes remains challenging. Finally, we demonstrate the applicability of our method for drug screening on dried blood spots showing excellent linearity (R(2) of 0.998) and precision (RSD of 9%). In conclusion, we present the proof of principe of a new high-throughput screening platform for bioanalysis based on a new automated microLLE method, coupled online to a commercially available nano-ESI-DI-MS.

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

PubMed

Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

2003-01-01

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing

PubMed Central

Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

2003-01-01

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing. PMID:14519201

High-throughput prediction of eucalypt lignin syringyl/guaiacyl content using multivariate analysis: a comparison between mid-infrared, near-infrared, and Raman spectroscopies for model development

PubMed Central

2014-01-01

Background In order to rapidly and efficiently screen potential biofuel feedstock candidates for quintessential traits, robust high-throughput analytical techniques must be developed and honed. The traditional methods of measuring lignin syringyl/guaiacyl (S/G) ratio can be laborious, involve hazardous reagents, and/or be destructive. Vibrational spectroscopy can furnish high-throughput instrumentation without the limitations of the traditional techniques. Spectral data from mid-infrared, near-infrared, and Raman spectroscopies was combined with S/G ratios, obtained using pyrolysis molecular beam mass spectrometry, from 245 different eucalypt and Acacia trees across 17 species. Iterations of spectral processing allowed the assembly of robust predictive models using partial least squares (PLS). Results The PLS models were rigorously evaluated using three different randomly generated calibration and validation sets for each spectral processing approach. Root mean standard errors of prediction for validation sets were lowest for models comprised of Raman (0.13 to 0.16) and mid-infrared (0.13 to 0.15) spectral data, while near-infrared spectroscopy led to more erroneous predictions (0.18 to 0.21). Correlation coefficients (r) for the validation sets followed a similar pattern: Raman (0.89 to 0.91), mid-infrared (0.87 to 0.91), and near-infrared (0.79 to 0.82). These statistics signify that Raman and mid-infrared spectroscopy led to the most accurate predictions of S/G ratio in a diverse consortium of feedstocks. Conclusion Eucalypts present an attractive option for biofuel and biochemical production. Given the assortment of over 900 different species of Eucalyptus and Corymbia, in addition to various species of Acacia, it is necessary to isolate those possessing ideal biofuel traits. This research has demonstrated the validity of vibrational spectroscopy to efficiently partition different potential biofuel feedstocks according to lignin S/G ratio, significantly reducing experiment and analysis time and expense while providing non-destructive, accurate, global, predictive models encompassing a diverse array of feedstocks. PMID:24955114

High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection.

PubMed

Islam, Md Koushikul; Baudin, Maria; Eriksson, Jonas; Öberg, Christopher; Habjan, Matthias; Weber, Friedemann; Överby, Anna K; Ahlm, Clas; Evander, Magnus

2016-04-01

Rift Valley fever virus (RVFV) is an emerging virus that causes serious illness in humans and livestock. There are no approved vaccines or treatments for humans. The purpose of the study was to identify inhibitory compounds of RVFV infection without any preconceived idea of the mechanism of action. A whole-cell-based high-throughput drug screening assay was developed to screen 28,437 small chemical compounds targeting RVFV infection. To accomplish both speed and robustness, a replication-competent NSs-deleted RVFV expressing a fluorescent reporter gene was developed. Inhibition of fluorescence intensity was quantified by spectrophotometry and related to virus infection in human lung epithelial cells (A549). Cell toxicity was assessed by the Resazurin cell viability assay. After primary screening, 641 compounds were identified that inhibited RVFV infection by ≥80%, with ≥50% cell viability at 50 µM concentration. These compounds were subjected to a second screening regarding dose-response profiles, and 63 compounds with ≥60% inhibition of RVFV infection at 3.12 µM compound concentration and ≥50% cell viability at 25 µM were considered hits. Of these, six compounds with high inhibitory activity were identified. In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection. © 2016 Society for Laboratory Automation and Screening.

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

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

Shin, Jong; Phelan, Paul J.; Chhum, Panharith

2014-11-15

Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCVmore » DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.« less

MetaUniDec: High-Throughput Deconvolution of Native Mass Spectra

NASA Astrophysics Data System (ADS)

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.

A High-Throughput Automated Microfluidic Platform for Calcium Imaging of Taste Sensing.

PubMed

Hsiao, Yi-Hsing; Hsu, Chia-Hsien; Chen, Chihchen

2016-07-08

The human enteroendocrine L cell line NCI-H716, expressing taste receptors and taste signaling elements, constitutes a unique model for the studies of cellular responses to glucose, appetite regulation, gastrointestinal motility, and insulin secretion. Targeting these gut taste receptors may provide novel treatments for diabetes and obesity. However, NCI-H716 cells are cultured in suspension and tend to form multicellular aggregates, preventing high-throughput calcium imaging due to interferences caused by laborious immobilization and stimulus delivery procedures. Here, we have developed an automated microfluidic platform that is capable of trapping more than 500 single cells into microwells with a loading efficiency of 77% within two minutes, delivering multiple chemical stimuli and performing calcium imaging with enhanced spatial and temporal resolutions when compared to bath perfusion systems. Results revealed the presence of heterogeneity in cellular responses to the type, concentration, and order of applied sweet and bitter stimuli. Sucralose and denatonium benzoate elicited robust increases in the intracellular Ca(2+) concentration. However, glucose evoked a rapid elevation of intracellular Ca(2+) followed by reduced responses to subsequent glucose stimulation. Using Gymnema sylvestre as a blocking agent for the sweet taste receptor confirmed that different taste receptors were utilized for sweet and bitter tastes. This automated microfluidic platform is cost-effective, easy to fabricate and operate, and may be generally applicable for high-throughput and high-content single-cell analysis and drug screening.

Optically enhanced acoustophoresis

NASA Astrophysics Data System (ADS)

McDougall, Craig; O'Mahoney, Paul; McGuinn, Alan; Willoughby, Nicholas A.; Qiu, Yongqiang; Demore, Christine E. M.; MacDonald, Michael P.

2017-08-01

Regenerative medicine has the capability to revolutionise many aspects of medical care, but for it to make the step from small scale autologous treatments to larger scale allogeneic approaches, robust and scalable label free cell sorting technologies are needed as part of a cell therapy bioprocessing pipeline. In this proceedings we describe several strategies for addressing the requirements for high throughput without labeling via: dimensional scaling, rare species targeting and sorting from a stable state. These three approaches are demonstrated through a combination of optical and ultrasonic forces. By combining mostly conservative and non-conservative forces from two different modalities it is possible to reduce the influence of flow velocity on sorting efficiency, hence increasing robustness and scalability. One such approach can be termed "optically enhanced acoustophoresis" which combines the ability of acoustics to handle large volumes of analyte with the high specificity of optical sorting.

A Bootstrap Based Measure Robust to the Choice of Normalization Methods for Detecting Rhythmic Features in High Dimensional Data.

PubMed

Larriba, Yolanda; Rueda, Cristina; Fernández, Miguel A; Peddada, Shyamal D

2018-01-01

Motivation: Gene-expression data obtained from high throughput technologies are subject to various sources of noise and accordingly the raw data are pre-processed before formally analyzed. Normalization of the data is a key pre-processing step, since it removes systematic variations across arrays. There are numerous normalization methods available in the literature. Based on our experience, in the context of oscillatory systems, such as cell-cycle, circadian clock, etc., the choice of the normalization method may substantially impact the determination of a gene to be rhythmic. Thus rhythmicity of a gene can purely be an artifact of how the data were normalized. Since the determination of rhythmic genes is an important component of modern toxicological and pharmacological studies, it is important to determine truly rhythmic genes that are robust to the choice of a normalization method. Results: In this paper we introduce a rhythmicity measure and a bootstrap methodology to detect rhythmic genes in an oscillatory system. Although the proposed methodology can be used for any high-throughput gene expression data, in this paper we illustrate the proposed methodology using several publicly available circadian clock microarray gene-expression datasets. We demonstrate that the choice of normalization method has very little effect on the proposed methodology. Specifically, for any pair of normalization methods considered in this paper, the resulting values of the rhythmicity measure are highly correlated. Thus it suggests that the proposed measure is robust to the choice of a normalization method. Consequently, the rhythmicity of a gene is potentially not a mere artifact of the normalization method used. Lastly, as demonstrated in the paper, the proposed bootstrap methodology can also be used for simulating data for genes participating in an oscillatory system using a reference dataset. Availability: A user friendly code implemented in R language can be downloaded from http://www.eio.uva.es/~miguel/robustdetectionprocedure.html.

A Bootstrap Based Measure Robust to the Choice of Normalization Methods for Detecting Rhythmic Features in High Dimensional Data

PubMed Central

Larriba, Yolanda; Rueda, Cristina; Fernández, Miguel A.; Peddada, Shyamal D.

2018-01-01

Motivation: Gene-expression data obtained from high throughput technologies are subject to various sources of noise and accordingly the raw data are pre-processed before formally analyzed. Normalization of the data is a key pre-processing step, since it removes systematic variations across arrays. There are numerous normalization methods available in the literature. Based on our experience, in the context of oscillatory systems, such as cell-cycle, circadian clock, etc., the choice of the normalization method may substantially impact the determination of a gene to be rhythmic. Thus rhythmicity of a gene can purely be an artifact of how the data were normalized. Since the determination of rhythmic genes is an important component of modern toxicological and pharmacological studies, it is important to determine truly rhythmic genes that are robust to the choice of a normalization method. Results: In this paper we introduce a rhythmicity measure and a bootstrap methodology to detect rhythmic genes in an oscillatory system. Although the proposed methodology can be used for any high-throughput gene expression data, in this paper we illustrate the proposed methodology using several publicly available circadian clock microarray gene-expression datasets. We demonstrate that the choice of normalization method has very little effect on the proposed methodology. Specifically, for any pair of normalization methods considered in this paper, the resulting values of the rhythmicity measure are highly correlated. Thus it suggests that the proposed measure is robust to the choice of a normalization method. Consequently, the rhythmicity of a gene is potentially not a mere artifact of the normalization method used. Lastly, as demonstrated in the paper, the proposed bootstrap methodology can also be used for simulating data for genes participating in an oscillatory system using a reference dataset. Availability: A user friendly code implemented in R language can be downloaded from http://www.eio.uva.es/~miguel/robustdetectionprocedure.html PMID:29456555

High-Precision Pinpointing of Luminescent Targets in Encoder-Assisted Scanning Microscopy Allowing High-Speed Quantitative Analysis.

PubMed

Zheng, Xianlin; Lu, Yiqing; Zhao, Jiangbo; Zhang, Yuhai; Ren, Wei; Liu, Deming; Lu, Jie; Piper, James A; Leif, Robert C; Liu, Xiaogang; Jin, Dayong

2016-01-19

Compared with routine microscopy imaging of a few analytes at a time, rapid scanning through the whole sample area of a microscope slide to locate every single target object offers many advantages in terms of simplicity, speed, throughput, and potential for robust quantitative analysis. Existing techniques that accommodate solid-phase samples incorporating individual micrometer-sized targets generally rely on digital microscopy and image analysis, with intrinsically low throughput and reliability. Here, we report an advanced on-the-fly stage scanning method to achieve high-precision target location across the whole slide. By integrating X- and Y-axis linear encoders to a motorized stage as the virtual "grids" that provide real-time positional references, we demonstrate an orthogonal scanning automated microscopy (OSAM) technique which can search a coverslip area of 50 × 24 mm(2) in just 5.3 min and locate individual 15 μm lanthanide luminescent microspheres with standard deviations of 1.38 and 1.75 μm in X and Y directions. Alongside implementation of an autofocus unit that compensates the tilt of a slide in the Z-axis in real time, we increase the luminescence detection efficiency by 35% with an improved coefficient of variation. We demonstrate the capability of advanced OSAM for robust quantification of luminescence intensities and lifetimes for a variety of micrometer-scale luminescent targets, specifically single down-shifting and upconversion microspheres, crystalline microplates, and color-barcoded microrods, as well as quantitative suspension array assays of biotinylated-DNA functionalized upconversion nanoparticles.

OPTIMA: sensitive and accurate whole-genome alignment of error-prone genomic maps by combinatorial indexing and technology-agnostic statistical analysis.

PubMed

Verzotto, Davide; M Teo, Audrey S; Hillmer, Axel M; Nagarajan, Niranjan

2016-01-01

Resolution of complex repeat structures and rearrangements in the assembly and analysis of large eukaryotic genomes is often aided by a combination of high-throughput sequencing and genome-mapping technologies (for example, optical restriction mapping). In particular, mapping technologies can generate sparse maps of large DNA fragments (150 kilo base pairs (kbp) to 2 Mbp) and thus provide a unique source of information for disambiguating complex rearrangements in cancer genomes. Despite their utility, combining high-throughput sequencing and mapping technologies has been challenging because of the lack of efficient and sensitive map-alignment algorithms for robustly aligning error-prone maps to sequences. We introduce a novel seed-and-extend glocal (short for global-local) alignment method, OPTIMA (and a sliding-window extension for overlap alignment, OPTIMA-Overlap), which is the first to create indexes for continuous-valued mapping data while accounting for mapping errors. We also present a novel statistical model, agnostic with respect to technology-dependent error rates, for conservatively evaluating the significance of alignments without relying on expensive permutation-based tests. We show that OPTIMA and OPTIMA-Overlap outperform other state-of-the-art approaches (1.6-2 times more sensitive) and are more efficient (170-200 %) and precise in their alignments (nearly 99 % precision). These advantages are independent of the quality of the data, suggesting that our indexing approach and statistical evaluation are robust, provide improved sensitivity and guarantee high precision.

Development of Multiwell-Plate Methods Using Pure Cultures of Methanogens To Identify New Inhibitors for Suppressing Ruminant Methane Emissions.

PubMed

Weimar, M R; Cheung, J; Dey, D; McSweeney, C; Morrison, M; Kobayashi, Y; Whitman, W B; Carbone, V; Schofield, L R; Ronimus, R S; Cook, G M

2017-08-01

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H 2 and CO 2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions. IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a high-throughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions. Copyright © 2017 American Society for Microbiology.

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

PubMed

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

2015-03-01

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

High-Throughput Screening of Myometrial Calcium-Mobilization to Identify Modulators of Uterine Contractility

PubMed Central

Herington, Jennifer L.; Swale, Daniel R.; Brown, Naoko; Shelton, Elaine L.; Choi, Hyehun; Williams, Charles H.; Hong, Charles C.; Paria, Bibhash C.; Denton, Jerod S.; Reese, Jeff

2015-01-01

The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10μM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility. PMID:26600013

High throughput selection of antibiotic-resistant transgenic Arabidopsis plants.

PubMed

Nagashima, Yukihiro; Koiwa, Hisashi

2017-05-15

Kanamycin resistance is the most frequently used antibiotic-resistance marker for Arabidopsis transformations, however, this method frequently causes escape of untransformed plants, particularly at the high seedling density during the selection. Here we developed a robust high-density selection method using top agar for Arabidopsis thaliana. Top agar effectively suppressed growth of untransformed wild-type plants on selection media at high density. Survival of the transformed plants during the selection were confirmed by production of green true leaves and expression of a firefly luciferase reporter gene. Top agar method allowed selection using a large amount of seeds in Arabidopsis transformation. Copyright © 2017 Elsevier Inc. All rights reserved.

A robust high-throughput fungal biosensor assay for the detection of estrogen activity.

PubMed

Zutz, Christoph; Wagener, Karen; Yankova, Desislava; Eder, Stefanie; Möstl, Erich; Drillich, Marc; Rychli, Kathrin; Wagner, Martin; Strauss, Joseph

2017-10-01

Estrogenic active compounds are present in a variety of sources and may alter biological functions in vertebrates. Therefore, it is crucial to develop innovative analytical systems that allow us to screen a broad spectrum of matrices and deliver fast and reliable results. We present the adaptation and validation of a fungal biosensor for the detection of estrogen activity in cow derived samples and tested the clinical applicability for pregnancy diagnosis in 140 mares and 120 cows. As biosensor we used a previously engineered genetically modified strain of the filamentous fungus Aspergillus nidulans, which contains the human estrogen receptor alpha and a reporter construct, in which β-galactosidase gene expression is controlled by an estrogen-responsive-element. The estrogen response of the fungal biosensor was validated with blood, urine, feces, milk and saliva. All matrices were screened for estrogenic activity prior to and after chemical extraction and the results were compared to an enzyme immunoassay (EIA). The biosensor showed consistent results in milk, urine and feces, which were comparable to those of the EIA. In contrast to the EIA, no sample pre-treatment by chemical extraction was needed. For 17β-estradiol, the biosensor showed a limit of detection of 1ng/L. The validation of the biosensor for pregnancy diagnosis revealed a specificity of 100% and a sensitivity of more than 97%. In conclusion, we developed and validated a highly robust fungal biosensor for detection of estrogen activity, which is highly sensitive and economic as it allows analyzing in high-throughput formats without the necessity for organic solvents. Copyright © 2017 Elsevier Inc. All rights reserved.

Robust transceiver design for reciprocal M × N interference channel based on statistical linearization approximation

NASA Astrophysics Data System (ADS)

Mayvan, Ali D.; Aghaeinia, Hassan; Kazemi, Mohammad

2017-12-01

This paper focuses on robust transceiver design for throughput enhancement on the interference channel (IC), under imperfect channel state information (CSI). In this paper, two algorithms are proposed to improve the throughput of the multi-input multi-output (MIMO) IC. Each transmitter and receiver has, respectively, M and N antennas and IC operates in a time division duplex mode. In the first proposed algorithm, each transceiver adjusts its filter to maximize the expected value of signal-to-interference-plus-noise ratio (SINR). On the other hand, the second algorithm tries to minimize the variances of the SINRs to hedge against the variability due to CSI error. Taylor expansion is exploited to approximate the effect of CSI imperfection on mean and variance. The proposed robust algorithms utilize the reciprocity of wireless networks to optimize the estimated statistical properties in two different working modes. Monte Carlo simulations are employed to investigate sum rate performance of the proposed algorithms and the advantage of incorporating variation minimization into the transceiver design.

An Energy-Efficient and Robust Multipath Routing Protocol for Cognitive Radio Ad Hoc Networks.

PubMed

Singh, Kishor; Moh, Sangman

2017-09-04

Routing in cognitive radio ad hoc networks (CRAHNs) is a daunting task owing to dynamic topology, intermittent connectivity, spectrum heterogeneity, and energy constraints. Other prominent aspects such as channel stability, path reliability, and route discovery frequency should also be exploited. Several routing protocols have been proposed for CRAHNs in the literature. By stressing on one of the aspects more than any other, however, they do not satisfy all requirements of throughput, energy efficiency, and robustness. In this paper, we propose an energy-efficient and robust multipath routing (ERMR) protocol for CRAHNs by considering all prominent aspects including residual energy and channel stability in design. Even when the current routing path fails, the alternative routing path is immediately utilized. In establishing primary and alternative routing paths, both residual energy and channel stability are exploited simultaneously. Our simulation study shows that the proposed ERMR outperforms the conventional protocol in terms of network throughput, packet delivery ratio, energy consumption, and end-to-end delay.

An Energy-Efficient and Robust Multipath Routing Protocol for Cognitive Radio Ad Hoc Networks

PubMed Central

Singh, Kishor

2017-01-01

Routing in cognitive radio ad hoc networks (CRAHNs) is a daunting task owing to dynamic topology, intermittent connectivity, spectrum heterogeneity, and energy constraints. Other prominent aspects such as channel stability, path reliability, and route discovery frequency should also be exploited. Several routing protocols have been proposed for CRAHNs in the literature. By stressing on one of the aspects more than any other, however, they do not satisfy all requirements of throughput, energy efficiency, and robustness. In this paper, we propose an energy-efficient and robust multipath routing (ERMR) protocol for CRAHNs by considering all prominent aspects including residual energy and channel stability in design. Even when the current routing path fails, the alternative routing path is immediately utilized. In establishing primary and alternative routing paths, both residual energy and channel stability are exploited simultaneously. Our simulation study shows that the proposed ERMR outperforms the conventional protocol in terms of network throughput, packet delivery ratio, energy consumption, and end-to-end delay. PMID:28869551

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

DOE PAGES

Rames, Matthew; Yu, Yadong; Ren, Gang

2014-08-15

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

Asymmetrical Deterministic Lateral Displacement Gaps for Dual Functions of Enhanced Separation and Throughput of Red Blood Cells

PubMed Central

Zeming, Kerwin Kwek; Salafi, Thoriq; Chen, Chia-Hung; Zhang, Yong

2016-01-01

Deterministic lateral displacement (DLD) method for particle separation in microfluidic devices has been extensively used for particle separation in recent years due to its high resolution and robust separation. DLD has shown versatility for a wide spectrum of applications for sorting of micro particles such as parasites, blood cells to bacteria and DNA. DLD model is designed for spherical particles and efficient separation of blood cells is challenging due to non-uniform shape and size. Moreover, separation in sub-micron regime requires the gap size of DLD systems to be reduced which exponentially increases the device resistance, resulting in greatly reduced throughput. This paper shows how simple application of asymmetrical DLD gap-size by changing the ratio of lateral-gap (GL) to downstream-gap (GD) enables efficient separation of RBCs without greatly restricting throughput. This method reduces the need for challenging fabrication of DLD pillars and provides new insight to the current DLD model. The separation shows an increase in DLD critical diameter resolution (separate smaller particles) and increase selectivity for non-spherical RBCs. The RBCs separate better as compared to standard DLD model with symmetrical gap sizes. This method can be applied to separate non-spherical bacteria or sub-micron particles to enhance throughput and DLD resolution. PMID:26961061

Asymmetrical Deterministic Lateral Displacement Gaps for Dual Functions of Enhanced Separation and Throughput of Red Blood Cells.

PubMed

Zeming, Kerwin Kwek; Salafi, Thoriq; Chen, Chia-Hung; Zhang, Yong

2016-03-10

Deterministic lateral displacement (DLD) method for particle separation in microfluidic devices has been extensively used for particle separation in recent years due to its high resolution and robust separation. DLD has shown versatility for a wide spectrum of applications for sorting of micro particles such as parasites, blood cells to bacteria and DNA. DLD model is designed for spherical particles and efficient separation of blood cells is challenging due to non-uniform shape and size. Moreover, separation in sub-micron regime requires the gap size of DLD systems to be reduced which exponentially increases the device resistance, resulting in greatly reduced throughput. This paper shows how simple application of asymmetrical DLD gap-size by changing the ratio of lateral-gap (GL) to downstream-gap (GD) enables efficient separation of RBCs without greatly restricting throughput. This method reduces the need for challenging fabrication of DLD pillars and provides new insight to the current DLD model. The separation shows an increase in DLD critical diameter resolution (separate smaller particles) and increase selectivity for non-spherical RBCs. The RBCs separate better as compared to standard DLD model with symmetrical gap sizes. This method can be applied to separate non-spherical bacteria or sub-micron particles to enhance throughput and DLD resolution.

High-throughput Crystallography for Structural Genomics

PubMed Central

Joachimiak, Andrzej

2009-01-01

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

Human Leukocyte Antigen Typing Using a Knowledge Base Coupled with a High-Throughput Oligonucleotide Probe Array Analysis

PubMed Central

Zhang, Guang Lan; Keskin, Derin B.; Lin, Hsin-Nan; Lin, Hong Huang; DeLuca, David S.; Leppanen, Scott; Milford, Edgar L.; Reinherz, Ellis L.; Brusic, Vladimir

2014-01-01

Human leukocyte antigens (HLA) are important biomarkers because multiple diseases, drug toxicity, and vaccine responses reveal strong HLA associations. Current clinical HLA typing is an elimination process requiring serial testing. We present an alternative in situ synthesized DNA-based microarray method that contains hundreds of thousands of probes representing a complete overlapping set covering 1,610 clinically relevant HLA class I alleles accompanied by computational tools for assigning HLA type to 4-digit resolution. Our proof-of-concept experiment included 21 blood samples, 18 cell lines, and multiple controls. The method is accurate, robust, and amenable to automation. Typing errors were restricted to homozygous samples or those with very closely related alleles from the same locus, but readily resolved by targeted DNA sequencing validation of flagged samples. High-throughput HLA typing technologies that are effective, yet inexpensive, can be used to analyze the world’s populations, benefiting both global public health and personalized health care. PMID:25505899

Nuclear Magnetic Resonance Spectroscopy-Based Identification of Yeast.

PubMed

Himmelreich, Uwe; Sorrell, Tania C; Daniel, Heide-Marie

2017-01-01

Rapid and robust high-throughput identification of environmental, industrial, or clinical yeast isolates is important whenever relatively large numbers of samples need to be processed in a cost-efficient way. Nuclear magnetic resonance (NMR) spectroscopy generates complex data based on metabolite profiles, chemical composition and possibly on medium consumption, which can not only be used for the assessment of metabolic pathways but also for accurate identification of yeast down to the subspecies level. Initial results on NMR based yeast identification where comparable with conventional and DNA-based identification. Potential advantages of NMR spectroscopy in mycological laboratories include not only accurate identification but also the potential of automated sample delivery, automated analysis using computer-based methods, rapid turnaround time, high throughput, and low running costs.We describe here the sample preparation, data acquisition and analysis for NMR-based yeast identification. In addition, a roadmap for the development of classification strategies is given that will result in the acquisition of a database and analysis algorithms for yeast identification in different environments.

High throughput exploration of process-property linkages in Al-6061 using instrumented spherical microindentation and microstructurally graded samples

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

Weaver, Jordan S.; Khosravani, Ali; Castillo, Andrew

Recent spherical nanoindentation protocols have proven robust at capturing the local elastic-plastic response of polycrystalline metal samples at length scales much smaller than the grain size. In this work, we extend these protocols to length scales that include multiple grains to recover microindentation stress-strain curves. These new protocols are first established in this paper and then demonstrated for Al-6061 by comparing the measured indentation stress-strain curves with the corresponding measurements from uniaxial tension tests. More specifically, the scaling factors between the uniaxial yield strength and the indentation yield strength was determined to be about 1.9, which is significantly lower thanmore » the value of 2.8 used commonly in literature. Furthermore, the reasons for this difference are discussed. Second, the benefits of these new protocols in facilitating high throughput exploration of process-property relationships are demonstrated through a simple case study.« less

High throughput exploration of process-property linkages in Al-6061 using instrumented spherical microindentation and microstructurally graded samples

DOE PAGES

Weaver, Jordan S.; Khosravani, Ali; Castillo, Andrew; ...

2016-06-14

Recent spherical nanoindentation protocols have proven robust at capturing the local elastic-plastic response of polycrystalline metal samples at length scales much smaller than the grain size. In this work, we extend these protocols to length scales that include multiple grains to recover microindentation stress-strain curves. These new protocols are first established in this paper and then demonstrated for Al-6061 by comparing the measured indentation stress-strain curves with the corresponding measurements from uniaxial tension tests. More specifically, the scaling factors between the uniaxial yield strength and the indentation yield strength was determined to be about 1.9, which is significantly lower thanmore » the value of 2.8 used commonly in literature. Furthermore, the reasons for this difference are discussed. Second, the benefits of these new protocols in facilitating high throughput exploration of process-property relationships are demonstrated through a simple case study.« less

Building biochips: a protein production pipeline

NASA Astrophysics Data System (ADS)

de Carvalho-Kavanagh, Marianne G. S.; Albala, Joanna S.

2004-06-01

Protein arrays are emerging as a practical format in which to study proteins in high-throughput using many of the same techniques as that of the DNA microarray. The key advantage to array-based methods for protein study is the potential for parallel analysis of thousands of samples in an automated, high-throughput fashion. Building protein arrays capable of this analysis capacity requires a robust expression and purification system capable of generating hundreds to thousands of purified recombinant proteins. We have developed a method to utilize LLNL-I.M.A.G.E. cDNAs to generate recombinant protein libraries using a baculovirus-insect cell expression system. We have used this strategy to produce proteins for analysis of protein/DNA and protein/protein interactions using protein microarrays in order to understand the complex interactions of proteins involved in homologous recombination and DNA repair. Using protein array techniques, a novel interaction between the DNA repair protein, Rad51B, and histones has been identified.

An automated compound screening for anti-aging effects on the function of C. elegans sensory neurons.

PubMed

Bazopoulou, Daphne; Chaudhury, Amrita R; Pantazis, Alexandros; Chronis, Nikos

2017-08-24

Discovery of molecular targets or compounds that alter neuronal function can lead to therapeutic advances that ameliorate age-related neurodegenerative pathologies. Currently, there is a lack of in vivo screening technologies for the discovery of compounds that affect the age-dependent neuronal physiology. Here, we present a high-throughput, microfluidic-based assay for automated manipulation and on-chip monitoring and analysis of stimulus-evoked calcium responses of intact C. elegans at various life stages. First, we successfully applied our technology to quantify the effects of aging and age-related genetic and chemical factors in the calcium transients of the ASH sensory neuron. We then performed a large-scale screen of a library of 107 FDA-approved compounds to identify hits that prevented the age-dependent functional deterioration of ASH. The robust performance of our assay makes it a valuable tool for future high-throughput applications based on in vivo functional imaging.

Two-dimensional materials from high-throughput computational exfoliation of experimentally known compounds.

PubMed

Mounet, Nicolas; Gibertini, Marco; Schwaller, Philippe; Campi, Davide; Merkys, Andrius; Marrazzo, Antimo; Sohier, Thibault; Castelli, Ivano Eligio; Cepellotti, Andrea; Pizzi, Giovanni; Marzari, Nicola

2018-03-01

Two-dimensional (2D) materials have emerged as promising candidates for next-generation electronic and optoelectronic applications. Yet, only a few dozen 2D materials have been successfully synthesized or exfoliated. Here, we search for 2D materials that can be easily exfoliated from their parent compounds. Starting from 108,423 unique, experimentally known 3D compounds, we identify a subset of 5,619 compounds that appear layered according to robust geometric and bonding criteria. High-throughput calculations using van der Waals density functional theory, validated against experimental structural data and calculated random phase approximation binding energies, further allowed the identification of 1,825 compounds that are either easily or potentially exfoliable. In particular, the subset of 1,036 easily exfoliable cases provides novel structural prototypes and simple ternary compounds as well as a large portfolio of materials to search from for optimal properties. For a subset of 258 compounds, we explore vibrational, electronic, magnetic and topological properties, identifying 56 ferromagnetic and antiferromagnetic systems, including half-metals and half-semiconductors.

Characterizing and controlling the inflammatory network during influenza A virus infection

NASA Astrophysics Data System (ADS)

Jin, Suoqin; Li, Yuanyuan; Pan, Ruangang; Zou, Xiufen

2014-01-01

To gain insights into the pathogenesis of influenza A virus (IAV) infections, this study focused on characterizing the inflammatory network and identifying key proteins by combining high-throughput data and computational techniques. We constructed the cell-specific normal and inflammatory networks for H5N1 and H1N1 infections through integrating high-throughput data. We demonstrated that better discrimination between normal and inflammatory networks by network entropy than by other topological metrics. Moreover, we identified different dynamical interactions among TLR2, IL-1β, IL10 and NFκB between normal and inflammatory networks using optimization algorithm. In particular, good robustness and multistability of inflammatory sub-networks were discovered. Furthermore, we identified a complex, TNFSF10/HDAC4/HDAC5, which may play important roles in controlling inflammation, and demonstrated that changes in network entropy of this complex negatively correlated to those of three proteins: TNFα, NFκB and COX-2. These findings provide significant hypotheses for further exploring the molecular mechanisms of infectious diseases and developing control strategies.

Two-dimensional materials from high-throughput computational exfoliation of experimentally known compounds

NASA Astrophysics Data System (ADS)

Mounet, Nicolas; Gibertini, Marco; Schwaller, Philippe; Campi, Davide; Merkys, Andrius; Marrazzo, Antimo; Sohier, Thibault; Castelli, Ivano Eligio; Cepellotti, Andrea; Pizzi, Giovanni; Marzari, Nicola

2018-02-01

Two-dimensional (2D) materials have emerged as promising candidates for next-generation electronic and optoelectronic applications. Yet, only a few dozen 2D materials have been successfully synthesized or exfoliated. Here, we search for 2D materials that can be easily exfoliated from their parent compounds. Starting from 108,423 unique, experimentally known 3D compounds, we identify a subset of 5,619 compounds that appear layered according to robust geometric and bonding criteria. High-throughput calculations using van der Waals density functional theory, validated against experimental structural data and calculated random phase approximation binding energies, further allowed the identification of 1,825 compounds that are either easily or potentially exfoliable. In particular, the subset of 1,036 easily exfoliable cases provides novel structural prototypes and simple ternary compounds as well as a large portfolio of materials to search from for optimal properties. For a subset of 258 compounds, we explore vibrational, electronic, magnetic and topological properties, identifying 56 ferromagnetic and antiferromagnetic systems, including half-metals and half-semiconductors.

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

PubMed

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

2012-12-01

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

Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells

PubMed Central

Leister, Kristin P; Huang, Ruili; Goodwin, Bonnie L; Chen, Andrew; Austin, Christopher P; Xia, Menghang

2011-01-01

Tumor Necrosis Factor-α (TNF-α), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders, and is a potential target for drug development. The traditional assays for detecting TNF-α, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay, are not suitable for the large size compound screens. Both assays suffer from a complicated protocol, multiple plate wash steps and/or excessive radioactive waste. A simple and quick measurement of TNF-α production in a cell based assay is needed for high throughput screening to identify the lead compounds from the compound library. We have developed and optimized two homogeneous TNF-α assays using the HTRF (homogeneous time resolved fluorescence) and AlphaLISA assay formats. We have validated the HTRF based TNF-α assay in a 1536-well plate format by screening a library of 1280 pharmacologically active compounds. The active compounds identified from the screen were confirmed in the AlphaLISA TNF-α assay using a bead-based technology. These compounds were also confirmed in a traditional ELISA assay. From this study, several beta adrenergic agonists have been identified as TNF-α inhibitors. We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316. The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening. PMID:21643507

Development of a high-throughput microfluidic integrated microarray for the detection of chimeric bioweapons.

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

Sheppod, Timothy; Satterfield, Brent; Hukari, Kyle W.

2006-10-01

The advancement of DNA cloning has significantly augmented the potential threat of a focused bioweapon assault, such as a terrorist attack. With current DNA cloning techniques, toxin genes from the most dangerous (but environmentally labile) bacterial or viral organism can now be selected and inserted into robust organism to produce an infinite number of deadly chimeric bioweapons. In order to neutralize such a threat, accurate detection of the expressed toxin genes, rather than classification on strain or genealogical decent of these organisms, is critical. The development of a high-throughput microarray approach will enable the detection of unknowns chimeric bioweapons. Themore » development of a high-throughput microarray approach will enable the detection of unknown bioweapons. We have developed a unique microfluidic approach to capture and concentrate these threat genes (mRNA's) upto a 30 fold concentration. These captured oligonucleotides can then be used to synthesize in situ oligonucleotide copies (cDNA probes) of the captured genes. An integrated microfluidic architecture will enable us to control flows of reagents, perform clean-up steps and finally elute nanoliter volumes of synthesized oligonucleotides probes. The integrated approach has enabled a process where chimeric or conventional bioweapons can rapidly be identified based on their toxic function, rather than being restricted to information that may not identify the critical nature of the threat.« less

Hydrogel-Based Fluorescent Dual pH and Oxygen Sensors Loaded in 96-Well Plates for High-Throughput Cell Metabolism Studies.

PubMed

Wu, Shanshan; Wu, Siying; Yi, Zheyuan; Zeng, Fei; Wu, Weizhen; Qiao, Yuan; Zhao, Xingzhong; Cheng, Xing; Tian, Yanqing

2018-02-13

In this study, we developed fluorescent dual pH and oxygen sensors loaded in multi-well plates for in-situ and high-throughput monitoring of oxygen respiration and extracellular acidification during microbial cell growth for understanding metabolism. Biocompatible PHEMA-co-PAM materials were used as the hydrogel matrix. A polymerizable oxygen probe (OS2) derived from PtTFPP and a polymerizable pH probe (S2) derived from fluorescein were chemically conjugated into the matrix to solve the problem of the probe leaching from the matrix. Gels were allowed to cure directly on the bottom of 96-well plates at room-temperature via redox polymerization. The influence of matrix's composition on the sensing behaviors was investigated to optimize hydrogels with enough robustness for repeatable use with good sensitivity. Responses of the dual sensing hydrogels to dissolved oxygen (DO) and pH were studied. These dual oxygen-pH sensing plates were successfully used for microbial cell-based screening assays, which are based on the measurement of fluorescence intensity changes induced by cellular oxygen consumption and pH changes during microbial growth. This method may provide a real-time monitoring of cellular respiration, acidification, and a rapid kinetic assessment of multiple samples for cell viability as well as high-throughput drug screening. All of these assays can be carried out by a conventional plate reader.

Flexible Plasmonic Sensors

PubMed Central

Shir, Daniel; Ballard, Zachary S.; Ozcan, Aydogan

2016-01-01

Mechanical flexibility and the advent of scalable, low-cost, and high-throughput fabrication techniques have enabled numerous potential applications for plasmonic sensors. Sensitive and sophisticated biochemical measurements can now be performed through the use of flexible plasmonic sensors integrated into existing medical and industrial devices or sample collection units. More robust sensing schemes and practical techniques must be further investigated to fully realize the potentials of flexible plasmonics as a framework for designing low-cost, embedded and integrated sensors for medical, environmental, and industrial applications. PMID:27547023

Roll-to-Roll Gravure Printed Electrochemical Sensors for Wearable and Medical Devices.

PubMed

Bariya, Mallika; Shahpar, Ziba; Park, Hyejin; Sun, Junfeng; Jung, Younsu; Gao, Wei; Nyein, Hnin Yin Yin; Liaw, Tiffany Sun; Tai, Li-Chia; Ngo, Quynh P; Chao, Minghan; Zhao, Yingbo; Hettick, Mark; Cho, Gyoujin; Javey, Ali

2018-06-25

As recent developments in noninvasive biosensors spearhead the thrust toward personalized health and fitness monitoring, there is a need for high throughput, cost-effective fabrication of flexible sensing components. Toward this goal, we present roll-to-roll (R2R) gravure printed electrodes that are robust under a range of electrochemical sensing applications. We use inks and electrode morphologies designed for electrochemical and mechanical stability, achieving devices with uniform redox kinetics printed on 150 m flexible substrate rolls. We show that these electrodes can be functionalized into consistently high performing sensors for detecting ions, metabolites, heavy metals, and other small molecules in noninvasively accessed biofluids, including sensors for real-time, in situ perspiration monitoring during exercise. This development of robust and versatile R2R gravure printed electrodes represents a key translational step in enabling large-scale, low-cost fabrication of disposable wearable sensors for personalized health monitoring applications.

Experimental demonstration of topologically protected efficient sound propagation in an acoustic waveguide network

NASA Astrophysics Data System (ADS)

Wei, Qi; Tian, Ye; Zuo, Shu-Yu; Cheng, Ying; Liu, Xiao-Jun

2017-03-01

Acoustic topological states support sound propagation along the boundary in a one-way direction with inherent robustness against defects and disorders, leading to the revolution of the manipulation on acoustic waves. A variety of acoustic topological states relying on circulating fluid, chiral coupling, or temporal modulation have been proposed theoretically. However, experimental demonstration has so far remained a significant challenge, due to the critical limitations such as structural complexity and high losses. Here, we experimentally demonstrate an acoustic anomalous Floquet topological insulator in a waveguide network. The acoustic gapless edge states can be found in the band gap when the waveguides are strongly coupled. The scheme features simple structure and high-energy throughput, leading to the experimental demonstration of efficient and robust topologically protected sound propagation along the boundary. The proposal may offer a unique, promising application for design of acoustic devices in acoustic guiding, switching, isolating, filtering, etc.

QoS-aware integrated fiber-wireless standard compliant architecture based on XGPON and EDCA

NASA Astrophysics Data System (ADS)

Kaur, Ravneet; Srivastava, Anand

2018-01-01

Converged Fiber-Wireless (FiWi) broadband access network proves to be a promising candidate that is reliable, robust, cost efficient, ubiquitous and capable of providing huge amount of bandwidth. To meet the ever-increasing bandwidth requirements, it has become very crucial to investigate the performance issues that arise with the deployment of next-generation Passive Optical Network (PON) and its integration with various wireless technologies. Apart from providing high speed internet access for mass use, this combined architecture aims to enable delivery of high quality and effective e-services in different categories including health, education, finance, banking, agriculture and e-government. In this work, we present an integrated architecture of 10-Gigabit-capable PON (XG-PON) and Enhanced Distributed Channel Access (EDCA) that combines the benefits of both technologies to meet the QoS demands of subscribers. Performance evaluation of the standards-compliant hybrid network is done using discrete-event Network Simulator-3 (NS-3) and results are reported in terms of throughput, average delay, average packet loss rate and fairness index. Per-class throughput signifies effectiveness of QoS distribution whereas aggregate throughput indicates effective utilization of wireless channel. This work has not been reported so far to the best of our knowledge.

Agarose droplet microfluidics for highly parallel and efficient single molecule emulsion PCR.

PubMed

Leng, Xuefei; Zhang, Wenhua; Wang, Chunming; Cui, Liang; Yang, Chaoyong James

2010-11-07

An agarose droplet method was developed for highly parallel and efficient single molecule emulsion PCR. The method capitalizes on the unique thermoresponsive sol-gel switching property of agarose for highly efficient DNA amplification and amplicon trapping. Uniform agarose solution droplets generated via a microfluidic chip serve as robust and inert nanolitre PCR reactors for single copy DNA molecule amplification. After PCR, agarose droplets are gelated to form agarose beads, trapping all amplicons in each reactor to maintain the monoclonality of each droplet. This method does not require cocapsulation of primer labeled microbeads, allows high throughput generation of uniform droplets and enables high PCR efficiency, making it a promising platform for many single copy genetic studies.

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

PubMed

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.

Microfluidic based high throughput synthesis of lipid-polymer hybrid nanoparticles with tunable diameters

PubMed Central

Feng, Qiang; Zhang, Lu; Liu, Chao; Li, Xuanyu; Hu, Guoqing; Sun, Jiashu; Jiang, Xingyu

2015-01-01

Core-shell hybrid nanoparticles (NPs) for drug delivery have attracted numerous attentions due to their enhanced therapeutic efficacy and good biocompatibility. In this work, we fabricate a two-stage microfluidic chip to implement a high-throughput, one-step, and size-tunable synthesis of mono-disperse lipid-poly (lactic-co-glycolic acid) NPs. The size of hybrid NPs is tunable by varying the flow rates inside the two-stage microfluidic chip. To elucidate the mechanism of size-controllable generation of hybrid NPs, we observe the flow field in the microchannel with confocal microscope and perform the simulation by a numerical model. Both the experimental and numerical results indicate an enhanced mixing effect at high flow rate, thus resulting in the assembly of small and mono-disperse hybrid NPs. In vitro experiments show that the large hybrid NPs are more likely to be aggregated in serum and exhibit a lower cellular uptake efficacy than the small ones. This microfluidic chip shows great promise as a robust platform for optimization of nano drug delivery system. PMID:26180574

Imaging flow cytometry for the screening of compounds that disrupt the Plasmodium falciparum digestive vacuole.

PubMed

Chia, Wan Ni; Lee, Yan Quan; Tan, Kevin Shyong-Wei

2017-01-01

Malaria, despite being one of the world's oldest infectious diseases, remains difficult to eradicate because the parasite is rapidly developing resistance to frontline chemotherapies. Previous studies have shown that the parasite exhibits features resembling programmed cell death upon treatment with drugs that disrupt its digestive vacuole (DV), providing a phenotypic readout that can be detected using the imaging flow cytometer. Large compound collections can thus be screened to identify drugs that are able to disrupt the DV of the malaria parasite using this high-content high-throughput screening platform. As a proof-of-concept, 4440 compounds were screened using this platform in 4months and 254 hits (5.7% hit rate) were obtained. Additionally, 25 compounds (0.6% top hit rate) were observed to retain potent DV disruption activity that was comparable to the canonical DV disruptive drug chloroquine when tested at a ten-fold lower concentration from the original screen. This pilot study demonstrates the robustness and high-throughput capability of the imaging flow cytometer and we report herein the methodology of this screening assay. Copyright © 2016 Elsevier Inc. All rights reserved.

FusionAnalyser: a new graphical, event-driven tool for fusion rearrangements discovery

PubMed Central

Piazza, Rocco; Pirola, Alessandra; Spinelli, Roberta; Valletta, Simona; Redaelli, Sara; Magistroni, Vera; Gambacorti-Passerini, Carlo

2012-01-01

Gene fusions are common driver events in leukaemias and solid tumours; here we present FusionAnalyser, a tool dedicated to the identification of driver fusion rearrangements in human cancer through the analysis of paired-end high-throughput transcriptome sequencing data. We initially tested FusionAnalyser by using a set of in silico randomly generated sequencing data from 20 known human translocations occurring in cancer and subsequently using transcriptome data from three chronic and three acute myeloid leukaemia samples. in all the cases our tool was invariably able to detect the presence of the correct driver fusion event(s) with high specificity. In one of the acute myeloid leukaemia samples, FusionAnalyser identified a novel, cryptic, in-frame ETS2–ERG fusion. A fully event-driven graphical interface and a flexible filtering system allow complex analyses to be run in the absence of any a priori programming or scripting knowledge. Therefore, we propose FusionAnalyser as an efficient and robust graphical tool for the identification of functional rearrangements in the context of high-throughput transcriptome sequencing data. PMID:22570408

FusionAnalyser: a new graphical, event-driven tool for fusion rearrangements discovery.

PubMed

Piazza, Rocco; Pirola, Alessandra; Spinelli, Roberta; Valletta, Simona; Redaelli, Sara; Magistroni, Vera; Gambacorti-Passerini, Carlo

2012-09-01

Gene fusions are common driver events in leukaemias and solid tumours; here we present FusionAnalyser, a tool dedicated to the identification of driver fusion rearrangements in human cancer through the analysis of paired-end high-throughput transcriptome sequencing data. We initially tested FusionAnalyser by using a set of in silico randomly generated sequencing data from 20 known human translocations occurring in cancer and subsequently using transcriptome data from three chronic and three acute myeloid leukaemia samples. in all the cases our tool was invariably able to detect the presence of the correct driver fusion event(s) with high specificity. In one of the acute myeloid leukaemia samples, FusionAnalyser identified a novel, cryptic, in-frame ETS2-ERG fusion. A fully event-driven graphical interface and a flexible filtering system allow complex analyses to be run in the absence of any a priori programming or scripting knowledge. Therefore, we propose FusionAnalyser as an efficient and robust graphical tool for the identification of functional rearrangements in the context of high-throughput transcriptome sequencing data.

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

PubMed

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

2007-03-01

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

Application of Computational and High-Throughput in vitro ...

EPA Pesticide Factsheets

Abstract: There are tens of thousands of man-made chemicals to which humans are exposed, but only a fraction of these have the extensive in vivo toxicity data used in most traditional risk assessments. This lack of data, coupled with concerns about testing costs and animal use, are driving the development of new methods for assessing the risk of toxicity. These methods include the use of in vitro high-throughput screening assays and computational models. This talk will review a variety of high-throughput, non-animal methods being used at the U.S. EPA to screen chemicals for a variety of toxicity endpoints, with a focus on their potential to be endocrine disruptors as part of the Endocrine Disruptor Screening Program (EDSP). These methods all start with the use of in vitro assays, e.g. for activity against the estrogen and androgen receptors (ER and AR) and targets in the steroidogenesis and thyroid signaling pathways. Because all individual assays are subject to a variety of noise processes and technology-specific assay artefacts, we have developed methods to create consensus predictions from multiple assays against the same target. The goal of these models is to both robustly predict in vivo activity, and also to provide quantitative estimates of uncertainty. This talk will describe these models, and how they are validated against both in vitro and in vivo reference chemicals. The U.S. EPA has deemed the in vitro ER model results to be of high enough accuracy t

Application of computational and high-throughput in vitro ...

EPA Pesticide Factsheets

Abstract: There are tens of thousands of man-made chemicals to which humans are exposed, but only a fraction of these have the extensive in vivo toxicity data used in most traditional risk assessments. This lack of data, coupled with concerns about testing costs and animal use, are driving the development of new methods for assessing the risk of toxicity. These methods include the use of in vitro high-throughput screening assays and computational models. This talk will review a variety of high-throughput, non-animal methods being used at the U.S. EPA to screen chemicals for their potential to be endocrine disruptors as part of the Endocrine Disruptor Screening Program (EDSP). These methods all start with the use of in vitro assays, e.g. for activity against the estrogen and androgen receptors (ER and AR) and targets in the steroidogenesis and thyroid signaling pathways. Because all individual assays are subject to a variety of noise processes and technology-specific assay artefacts, we have developed methods to create consensus predictions from multiple assays against the same target. The goal of these models is to both robustly predict in vivo activity, and also to provide quantitative estimates of uncertainty. This talk will describe these models, and how they are validated against both in vitro and in vivo reference chemicals. The U.S. EPA has deemed the in vitro ER model results to be of high enough accuracy to be used as a substitute for the current EDSP Ti

Differential Expression and Functional Analysis of High-Throughput -Omics Data Using Open Source Tools.

PubMed

Kebschull, Moritz; Fittler, Melanie Julia; Demmer, Ryan T; Papapanou, Panos N

2017-01-01

Today, -omics analyses, including the systematic cataloging of messenger RNA and microRNA sequences or DNA methylation patterns in a cell population, organ, or tissue sample, allow for an unbiased, comprehensive genome-level analysis of complex diseases, offering a large advantage over earlier "candidate" gene or pathway analyses. A primary goal in the analysis of these high-throughput assays is the detection of those features among several thousand that differ between different groups of samples. In the context of oral biology, our group has successfully utilized -omics technology to identify key molecules and pathways in different diagnostic entities of periodontal disease.A major issue when inferring biological information from high-throughput -omics studies is the fact that the sheer volume of high-dimensional data generated by contemporary technology is not appropriately analyzed using common statistical methods employed in the biomedical sciences.In this chapter, we outline a robust and well-accepted bioinformatics workflow for the initial analysis of -omics data generated using microarrays or next-generation sequencing technology using open-source tools. Starting with quality control measures and necessary preprocessing steps for data originating from different -omics technologies, we next outline a differential expression analysis pipeline that can be used for data from both microarray and sequencing experiments, and offers the possibility to account for random or fixed effects. Finally, we present an overview of the possibilities for a functional analysis of the obtained data.

Optimization and quality control of genome-wide Hi-C library preparation.

PubMed

Zhang, Xiang-Yuan; He, Chao; Ye, Bing-Yu; Xie, De-Jian; Shi, Ming-Lei; Zhang, Yan; Shen, Wen-Long; Li, Ping; Zhao, Zhi-Hu

2017-09-20

Highest-throughput chromosome conformation capture (Hi-C) is one of the key assays for genome- wide chromatin interaction studies. It is a time-consuming process that involves many steps and many different kinds of reagents, consumables, and equipments. At present, the reproducibility is unsatisfactory. By optimizing the key steps of the Hi-C experiment, such as crosslinking, pretreatment of digestion, inactivation of restriction enzyme, and in situ ligation etc., we established a robust Hi-C procedure and prepared two biological replicates of Hi-C libraries from the GM12878 cells. After preliminary quality control by Sanger sequencing, the two replicates were high-throughput sequenced. The bioinformatics analysis of the raw sequencing data revealed the mapping-ability and pair-mate rate of the raw data were around 90% and 72%, respectively. Additionally, after removal of self-circular ligations and dangling-end products, more than 96% of the valid pairs were reached. Genome-wide interactome profiling shows clear topological associated domains (TADs), which is consistent with previous reports. Further correlation analysis showed that the two biological replicates strongly correlate with each other in terms of both bin coverage and all bin pairs. All these results indicated that the optimized Hi-C procedure is robust and stable, which will be very helpful for the wide applications of the Hi-C assay.

Polymer coating on a micropillar chip for robust attachment of PuraMatrix peptide hydrogel for 3D hepatic cell culture.

PubMed

Roth, Alexander David; Lama, Pratap; Dunn, Stephen; Hong, Stephen; Lee, Moo-Yeal

2018-09-01

For better mimicking tissues in vivo and developing predictive cell models for high-throughput screening (HTS) of potential drug candidates, three-dimensional (3D) cell cultures have been performed in various hydrogels. In this study, we have investigated several polymer coating materials to robustly attach PuraMatrix peptide hydrogel on a micropillar chip for 3D culture of Hep3B human hepatic cells, which can be used as a tool for high-throughput assessment of compound hepatotoxicity. Among several amphiphilic polymers with maleic anhydride groups tested, 0.01% (w/v) poly(maleic anhydride-alt-1-octadecene) (PMA-OD) provided superior coating properties with no PuraMatrix spot detachment from the micropillar chip and no air bubble entrapment in a complementary microwell chip. To maintain Hep3B cell viability in PuraMatrix gel on the chip, gelation conditions were optimized in the presence of additional salts, at different seeding densities, and for growth medium washes. As a result, salts in growth media were sufficient for gelation, and relatively high cell seeding at 6 million cells/mL and two media washes for pH neutralization were required. With optimized 3D cell culture conditions, controlled gene expression and compound toxicity assessment were successfully demonstrated by using recombinant adenoviruses carrying genes for green and red fluorescent proteins as well as six model compounds. Overall, PuraMatrix hydrogel on the chip was suitable for 3D cell encapsulation, gene expression, and rapid toxicity assessment. Published by Elsevier B.V.

Robust and Sensitive Analysis of Mouse Knockout Phenotypes

PubMed Central

Karp, Natasha A.; Melvin, David; Mott, Richard F.

2012-01-01

A significant challenge of in-vivo studies is the identification of phenotypes with a method that is robust and reliable. The challenge arises from practical issues that lead to experimental designs which are not ideal. Breeding issues, particularly in the presence of fertility or fecundity problems, frequently lead to data being collected in multiple batches. This problem is acute in high throughput phenotyping programs. In addition, in a high throughput environment operational issues lead to controls not being measured on the same day as knockouts. We highlight how application of traditional methods, such as a Student’s t-Test or a 2-way ANOVA, in these situations give flawed results and should not be used. We explore the use of mixed models using worked examples from Sanger Mouse Genome Project focusing on Dual-Energy X-Ray Absorptiometry data for the analysis of mouse knockout data and compare to a reference range approach. We show that mixed model analysis is more sensitive and less prone to artefacts allowing the discovery of subtle quantitative phenotypes essential for correlating a gene’s function to human disease. We demonstrate how a mixed model approach has the additional advantage of being able to include covariates, such as body weight, to separate effect of genotype from these covariates. This is a particular issue in knockout studies, where body weight is a common phenotype and will enhance the precision of assigning phenotypes and the subsequent selection of lines for secondary phenotyping. The use of mixed models with in-vivo studies has value not only in improving the quality and sensitivity of the data analysis but also ethically as a method suitable for small batches which reduces the breeding burden of a colony. This will reduce the use of animals, increase throughput, and decrease cost whilst improving the quality and depth of knowledge gained. PMID:23300663

Demonstration of analyzers for multimode photonic time-bin qubits

NASA Astrophysics Data System (ADS)

Jin, Jeongwan; Agne, Sascha; Bourgoin, Jean-Philippe; Zhang, Yanbao; Lütkenhaus, Norbert; Jennewein, Thomas

2018-04-01

We demonstrate two approaches for unbalanced interferometers as time-bin qubit analyzers for quantum communication, robust against mode distortions and polarization effects as expected from free-space quantum communication systems including wavefront deformations, path fluctuations, pointing errors, and optical elements. Despite strong spatial and temporal distortions of the optical mode of a time-bin qubit, entangled with a separate polarization qubit, we verify entanglement using the Negative Partial Transpose, with the measured visibility of up to 0.85 ±0.01 . The robustness of the analyzers is further demonstrated for various angles of incidence up to 0 .2∘ . The output of the interferometers is coupled into multimode fiber yielding a high system throughput of 0.74. Therefore, these analyzers are suitable and efficient for quantum communication over multimode optical channels.

High-throughput screening identifies artesunate as selective inhibitor of cancer stemness: Involvement of mitochondrial metabolism.

PubMed

Subedi, Amit; Futamura, Yushi; Nishi, Mayuko; Ryo, Akihide; Watanabe, Nobumoto; Osada, Hiroyuki

2016-09-02

Cancer stem cells (CSCs) have robust systems to maintain cancer stemness and drug resistance. Thus, targeting such robust systems instead of focusing on individual signaling pathways should be the approach allowing the identification of selective CSC inhibitors. Here, we used the alkaline phosphatase (ALP) assay to identify inhibitors for cancer stemness in induced cancer stem-like (iCSCL) cells. We screened several compounds from natural product chemical library and evaluated hit compounds for their efficacy on cancer stemness in iCSCL tumorspheres. We identified artesunate, an antimalarial drug, as a selective inhibitor of cancer stemness. Artesunate induced mitochondrial dysfunction that selectively inhibited cancer stemness of iCSCL cells, indicating an essential role of mitochondrial metabolism in cancer stemness. Copyright © 2016 Elsevier Inc. All rights reserved.

Design and Validation of High Date Rate Ka-Band Software Defined Radio for Small Satellite

NASA Technical Reports Server (NTRS)

Xia, Tian

2016-01-01

The Design and Validation of High Date Rate Ka- Band Software Defined Radio for Small Satellite project will develop a novel Ka-band software defined radio (SDR) that is capable of establishing high data rate inter-satellite links with a throughput of 500 megabits per second (Mb/s) and providing millimeter ranging precision. The system will be designed to operate with high performance and reliability that is robust against various interference effects and network anomalies. The Ka-band radio resulting from this work will improve upon state of the art Ka-band radios in terms of dimensional size, mass and power dissipation, which limit their use in small satellites.

Development of resazurin-based assay in 384-well format for high throughput whole cell screening of Trypanosoma brucei rhodesiense strain STIB 900 for the identification of potential anti-trypanosomal agents.

PubMed

Lim, Kah Tee; Zahari, Zuriati; Amanah, Azimah; Zainuddin, Zafarina; Adenan, Mohd Ilham

2016-03-01

To accelerate the discovery of novel leads for the treatment of Human African Trypanosomiasis (HAT), it is necessary to have a simple, robust and cost-effective assay to identify positive hits by high throughput whole cell screening. Most of the fluorescence assay was made in black plate however in this study the HTS assay developed in 384-well format using clear plate and black plate, for comparison. The HTS assay developed is simple, sensitive, reliable and reproducible in both types of plates. Assay robustness and reproducibility were determined under the optimized conditions in 384-well plate was well tolerated in the HTS assay, including percentage of coefficient of variation (% CV) of 4.68% and 4.74% in clear and black 384-well plate, signal-to-background ratio (S/B) of 12.75 in clear 384-well plate and 12.07 in black 384-well plate, Z' factor of 0.79 and 0.82 in clear 384-well plate and black 384-well plate, respectively and final concentration of 0.30% dimethylsulfoxide (DMSO) in both types of plate. Drug sensitivity was found to be comparable to the reported anti-trypanosomal assay in 96-well format. The reproducibility and sensitivity of this assay make it compliant to automated liquid handler use in HTS applications. Copyright © 2016 Elsevier Inc. All rights reserved.

Illumina GA IIx& HiSeq 2000 Production Sequenccing and QC Analysis Pipelines at the DOE Joint Genome Institute

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

Daum, Christopher; Zane, Matthew; Han, James

2011-01-31

The U.S. Department of Energy (DOE) Joint Genome Institute's (JGI) Production Sequencing group is committed to the generation of high-quality genomic DNA sequence to support the mission areas of renewable energy generation, global carbon management, and environmental characterization and clean-up. Within the JGI's Production Sequencing group, a robust Illumina Genome Analyzer and HiSeq pipeline has been established. Optimization of the sesequencer pipelines has been ongoing with the aim of continual process improvement of the laboratory workflow, reducing operational costs and project cycle times to increases ample throughput, and improving the overall quality of the sequence generated. A sequence QC analysismore » pipeline has been implemented to automatically generate read and assembly level quality metrics. The foremost of these optimization projects, along with sequencing and operational strategies, throughput numbers, and sequencing quality results will be presented.« less

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

PubMed

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

2014-12-30

Microfluidics has recently emerged as a new method of manufacturing liposomes, which allows for reproducible mixing in miliseconds on the nanoliter scale. Here we investigate microfluidics-based manufacturing of liposomes. The aim of these studies was to assess the parameters in a microfluidic process by varying the total flow rate (TFR) and the flow rate ratio (FRR) of the solvent and aqueous phases. Design of experiment and multivariate data analysis were used for increased process understanding and development of predictive and correlative models. High FRR lead to the bottom-up synthesis of liposomes, with a strong correlation with vesicle size, demonstrating the ability to in-process control liposomes size; the resulting liposome size correlated with the FRR in the microfluidics process, with liposomes of 50 nm being reproducibly manufactured. Furthermore, we demonstrate the potential of a high throughput manufacturing of liposomes using microfluidics with a four-fold increase in the volumetric flow rate, maintaining liposome characteristics. The efficacy of these liposomes was demonstrated in transfection studies and was modelled using predictive modeling. Mathematical modelling identified FRR as the key variable in the microfluidic process, with the highest impact on liposome size, polydispersity and transfection efficiency. This study demonstrates microfluidics as a robust and high-throughput method for the scalable and highly reproducible manufacture of size-controlled liposomes. Furthermore, the application of statistically based process control increases understanding and allows for the generation of a design-space for controlled particle characteristics. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

High-Throughput Histopathological Image Analysis via Robust Cell Segmentation and Hashing

PubMed Central

Zhang, Xiaofan; Xing, Fuyong; Su, Hai; Yang, Lin; Zhang, Shaoting

2015-01-01

Computer-aided diagnosis of histopathological images usually requires to examine all cells for accurate diagnosis. Traditional computational methods may have efficiency issues when performing cell-level analysis. In this paper, we propose a robust and scalable solution to enable such analysis in a real-time fashion. Specifically, a robust segmentation method is developed to delineate cells accurately using Gaussian-based hierarchical voting and repulsive balloon model. A large-scale image retrieval approach is also designed to examine and classify each cell of a testing image by comparing it with a massive database, e.g., half-million cells extracted from the training dataset. We evaluate this proposed framework on a challenging and important clinical use case, i.e., differentiation of two types of lung cancers (the adenocarcinoma and squamous carcinoma), using thousands of lung microscopic tissue images extracted from hundreds of patients. Our method has achieved promising accuracy and running time by searching among half-million cells. PMID:26599156

High throughput automated microbial bioreactor system used for clone selection and rapid scale‐down process optimization

PubMed Central

Velez‐Suberbie, M. Lourdes; Betts, John P. J.; Walker, Kelly L.; Robinson, Colin; Zoro, Barney

2017-01-01

High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed‐batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled‐up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale‐up. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:58–68, 2018 PMID:28748655

A Novel High-Throughput 1536-well Notch1 γ-Secretase AlphaLISA Assay

PubMed Central

Chau, De-ming; Shum, David; Radu, Constantin; Bhinder, Bhavneet; Gin, David; Gilchrist, M. Lane; Djaballah, Hakim; Li, Yue-Ming

2013-01-01

The Notch pathway plays a crucial role in cell fate decisions through controlling various cellular processes. Overactive Notch signal contributes to cancer development from leukemias to solid tumors. γ-Secretase is an intramembrane protease responsible for the final proteolytic step of Notch that releases the membrane-tethered Notch fragment for signaling. Therefore, γ-secretase is an attractive drug target in treating Notch-mediated cancers. However, the absence of high-throughput γ-secretase assay using Notch substrate has limited the identification and development of γ-secretase inhibitors that specifically target the Notch signaling pathway. Here, we report on the development of a 1536-well γ-secretase assay using a biotinylated recombinant Notch1 substrate. We effectively assimilated and miniaturized this newly developed Notch1 substrate with the AlphaLISA detection technology and demonstrated its robustness with a calculated Z’ score of 0.66. We further validated this optimized assay by performing a pilot screening against a chemical library consisting of ~5,600 chemicals and identified known γ-secretase inhibitors e.g. DAPT, and Calpeptin; as well as a novel γ-secretase inhibitor referred to as KD-I-085. This assay is the first reported 1536-well AlphaLISA format and represents a novel high-throughput Notch1-γ-secretase assay, which provides an unprecedented opportunity to discover Notch-selective γ-secretase inhibitors that can be potentially used for the treatment of cancer and other human disorders. PMID:23448293

High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells

PubMed Central

Gerby, Bastien; Veiga, Diogo F.T.; Krosl, Jana; Nourreddine, Sami; Ouellette, Julianne; Haman, André; Lavoie, Geneviève; Fares, Iman; Tremblay, Mathieu; Litalien, Véronique; Ottoni, Elizabeth; Geoffrion, Dominique; Maddox, Paul S.; Chagraoui, Jalila; Hébert, Josée; Sauvageau, Guy; Kwok, Benjamin H.; Roux, Philippe P.

2016-01-01

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non–cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy. PMID:27797342

Aminopyrazinamides: novel and specific GyrB inhibitors that kill replicating and nonreplicating Mycobacterium tuberculosis.

PubMed

Shirude, Pravin S; Madhavapeddi, Prashanti; Tucker, Julie A; Murugan, Kannan; Patil, Vikas; Basavarajappa, Halesha; Raichurkar, Anandkumar V; Humnabadkar, Vaishali; Hussein, Syeed; Sharma, Sreevalli; Ramya, V K; Narayan, Chandan B; Balganesh, Tanjore S; Sambandamurthy, Vasan K

2013-03-15

Aminopyrazinamides originated from a high throughput screen targeting the Mycobacterium smegmatis (Msm) GyrB ATPase. This series displays chemical tractability, robust structure-activity relationship, and potent antitubercular activity. The crystal structure of Msm GyrB in complex with one of the aminopyrazinamides revealed promising attributes of specificity against other broad spectrum pathogens and selectivity against eukaryotic kinases due to novel interactions at hydrophobic pocket, unlike other known GyrB inhibitors. The aminopyrazinamides display excellent mycobacterial kill under in vitro, intracellular, and hypoxic conditions.

The Road to Information Dominance: "System of Systems" Concept for the United States Armed Forces.

DTIC Science & Technology

1998-04-06

information dominance through the U.S. Armed Forces "System of Systems" concept. It addresses and analyzes current and future strategic implications and requirements for U.S. warfighting communications and information systems. It proposes a more flexible, reliable, responsive, robust and survivable high capacity throughput communications and "bitways" system to support future force projection operations for the Force and/or Army After Next. Lastly, it concludes with a suggested methodology to implement the "System of Systems" concept to enable

A Simple and Resource-efficient Setup for the Computer-aided Drug Design Laboratory.

PubMed

Moretti, Loris; Sartori, Luca

2016-10-01

Undertaking modelling investigations for Computer-Aided Drug Design (CADD) requires a proper environment. In principle, this could be done on a single computer, but the reality of a drug discovery program requires robustness and high-throughput computing (HTC) to efficiently support the research. Therefore, a more capable alternative is needed but its implementation has no widespread solution. Here, the realization of such a computing facility is discussed, from general layout to technical details all aspects are covered. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Personalized In Vitro and In Vivo Cancer Models to Guide Precision Medicine | Office of Cancer Genomics

Cancer.gov

Precision medicine is an approach that takes into account the influence of individuals' genes, environment, and lifestyle exposures to tailor interventions. Here, we describe the development of a robust precision cancer care platform that integrates whole-exome sequencing with a living biobank that enables high-throughput drug screens on patient-derived tumor organoids. To date, 56 tumor-derived organoid cultures and 19 patient-derived xenograft (PDX) models have been established from the 769 patients enrolled in an Institutional Review Board-approved clinical trial.

Supercritical fluid chromatography: a promising alternative to current bioanalytical techniques.

PubMed

Dispas, Amandine; Jambo, Hugues; André, Sébastien; Tyteca, Eva; Hubert, Philippe

2018-01-01

During the last years, chemistry was involved in the worldwide effort toward environmental problems leading to the birth of green chemistry. In this context, green analytical tools were developed as modern Supercritical Fluid Chromatography in the field of separative techniques. This chromatographic technique knew resurgence a few years ago, thanks to its high efficiency, fastness and robustness of new generation equipment. These advantages and its easy hyphenation to MS fulfill the requirements of bioanalysis regarding separation capacity and high throughput. In the present paper, the technical aspects focused on bioanalysis specifications will be detailed followed by a critical review of bioanalytical supercritical fluid chromatography methods published in the literature.

Shrink-induced sorting using integrated nanoscale magnetic traps.

PubMed

Nawarathna, Dharmakeerthi; Norouzi, Nazila; McLane, Jolie; Sharma, Himanshu; Sharac, Nicholas; Grant, Ted; Chen, Aaron; Strayer, Scott; Ragan, Regina; Khine, Michelle

2013-02-11

We present a plastic microfluidic device with integrated nanoscale magnetic traps (NSMTs) that separates magnetic from non-magnetic beads with high purity and throughput, and unprecedented enrichments. Numerical simulations indicate significantly higher localized magnetic field gradients than previously reported. We demonstrated >20 000-fold enrichment for 0.001% magnetic bead mixtures. Since we achieve high purity at all flow-rates tested, this is a robust, rapid, portable, and simple solution to sort target species from small volumes amenable for point-of-care applications. We used the NSMT in a 96 well format to extract DNA from small sample volumes for quantitative polymerase chain reaction (qPCR).

MBTH: A novel approach to rapid, spectrophotometric quantitation of total algal carbohydrates

DOE PAGES

Van Wychen, Stefanie; Long, William; Black, Stuart K.; ...

2016-11-24

A high-throughput and robust application of the 3-methyl-2-benzothiazolinone hydrazone (MBTH) method was developed for carbohydrate determination in microalgae. The traditional phenol-sulfuric acid method to quantify carbohydrates is strongly affected by algal biochemical components and exhibits a highly variable response to microalgal monosaccharides. We present a novel use of the MBTH method to accurately quantify carbohydrates in hydrolyzate after acid hydrolysis of algal biomass, without a need for neutralization. As a result, the MBTH method demonstrated consistent and sensitive quantitation of algae-specific monosaccharides down to 5 ug mL -1 without interference from other algae acidic hydrolyzate components.

MBTH: A novel approach to rapid, spectrophotometric quantitation of total algal carbohydrates

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

Van Wychen, Stefanie; Long, William; Black, Stuart K.

A high-throughput and robust application of the 3-methyl-2-benzothiazolinone hydrazone (MBTH) method was developed for carbohydrate determination in microalgae. The traditional phenol-sulfuric acid method to quantify carbohydrates is strongly affected by algal biochemical components and exhibits a highly variable response to microalgal monosaccharides. We present a novel use of the MBTH method to accurately quantify carbohydrates in hydrolyzate after acid hydrolysis of algal biomass, without a need for neutralization. As a result, the MBTH method demonstrated consistent and sensitive quantitation of algae-specific monosaccharides down to 5 ug mL -1 without interference from other algae acidic hydrolyzate components.

Detection of Botulinum Neurotoxin Serotype A, B, and F Proteolytic Activity in Complex Matrices with Picomolar to Femtomolar Sensitivity

PubMed Central

Dunning, F. Mark; Ruge, Daniel R.; Piazza, Timothy M.; Stanker, Larry H.; Zeytin, Füsûn N.

2012-01-01

Rapid, high-throughput assays that detect and quantify botulinum neurotoxin (BoNT) activity in diverse matrices are required for environmental, clinical, pharmaceutical, and food testing. The current standard, the mouse bioassay, is sensitive but is low in throughput and precision. In this study, we present three biochemical assays for the detection and quantification of BoNT serotype A, B, and F proteolytic activities in complex matrices that offer picomolar to femtomolar sensitivity with small assay volumes and total assay times of less than 24 h. These assays consist of magnetic beads conjugated with BoNT serotype-specific antibodies that are used to purify BoNT from complex matrices before the quantification of bound BoNT proteolytic activity using the previously described BoTest reporter substrates. The matrices tested include human serum, whole milk, carrot juice, and baby food, as well as buffers containing common pharmaceutical excipients. The limits of detection were below 1 pM for BoNT/A and BoNT/F and below 10 pM for BoNT/B in most tested matrices using 200-μl samples and as low as 10 fM for BoNT/A with an increased sample volume. Together, these data describe rapid, robust, and high-throughput assays for BoNT detection that are compatible with a wide range of matrices. PMID:22923410

Moving Liquids with Sound: The Physics of Acoustic Droplet Ejection for Robust Laboratory Automation in Life Sciences.

PubMed

Hadimioglu, Babur; Stearns, Richard; Ellson, Richard

2016-02-01

Liquid handling instruments for life science applications based on droplet formation with focused acoustic energy or acoustic droplet ejection (ADE) were introduced commercially more than a decade ago. While the idea of "moving liquids with sound" was known in the 20th century, the development of precise methods for acoustic dispensing to aliquot life science materials in the laboratory began in earnest in the 21st century with the adaptation of the controlled "drop on demand" acoustic transfer of droplets from high-density microplates for high-throughput screening (HTS) applications. Robust ADE implementations for life science applications achieve excellent accuracy and precision by using acoustics first to sense the liquid characteristics relevant for its transfer, and then to actuate transfer of the liquid with customized application of sound energy to the given well and well fluid in the microplate. This article provides an overview of the physics behind ADE and its central role in both acoustical and rheological aspects of robust implementation of ADE in the life science laboratory and its broad range of ejectable materials. © 2015 Society for Laboratory Automation and Screening.

Topology based data analysis identifies a subgroup of breast cancers with a unique mutational profile and excellent survival.

PubMed

Nicolau, Monica; Levine, Arnold J; Carlsson, Gunnar

2011-04-26

High-throughput biological data, whether generated as sequencing, transcriptional microarrays, proteomic, or other means, continues to require analytic methods that address its high dimensional aspects. Because the computational part of data analysis ultimately identifies shape characteristics in the organization of data sets, the mathematics of shape recognition in high dimensions continues to be a crucial part of data analysis. This article introduces a method that extracts information from high-throughput microarray data and, by using topology, provides greater depth of information than current analytic techniques. The method, termed Progression Analysis of Disease (PAD), first identifies robust aspects of cluster analysis, then goes deeper to find a multitude of biologically meaningful shape characteristics in these data. Additionally, because PAD incorporates a visualization tool, it provides a simple picture or graph that can be used to further explore these data. Although PAD can be applied to a wide range of high-throughput data types, it is used here as an example to analyze breast cancer transcriptional data. This identified a unique subgroup of Estrogen Receptor-positive (ER(+)) breast cancers that express high levels of c-MYB and low levels of innate inflammatory genes. These patients exhibit 100% survival and no metastasis. No supervised step beyond distinction between tumor and healthy patients was used to identify this subtype. The group has a clear and distinct, statistically significant molecular signature, it highlights coherent biology but is invisible to cluster methods, and does not fit into the accepted classification of Luminal A/B, Normal-like subtypes of ER(+) breast cancers. We denote the group as c-MYB(+) breast cancer.

A robust robotic high-throughput antibody purification platform.

PubMed

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

2016-07-15

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

Repliscan: a tool for classifying replication timing regions.

PubMed

Zynda, Gregory J; Song, Jawon; Concia, Lorenzo; Wear, Emily E; Hanley-Bowdoin, Linda; Thompson, William F; Vaughn, Matthew W

2017-08-07

Replication timing experiments that use label incorporation and high throughput sequencing produce peaked data similar to ChIP-Seq experiments. However, the differences in experimental design, coverage density, and possible results make traditional ChIP-Seq analysis methods inappropriate for use with replication timing. To accurately detect and classify regions of replication across the genome, we present Repliscan. Repliscan robustly normalizes, automatically removes outlying and uninformative data points, and classifies Repli-seq signals into discrete combinations of replication signatures. The quality control steps and self-fitting methods make Repliscan generally applicable and more robust than previous methods that classify regions based on thresholds. Repliscan is simple and effective to use on organisms with different genome sizes. Even with analysis window sizes as small as 1 kilobase, reliable profiles can be generated with as little as 2.4x coverage.

The essential roles of chemistry in high-throughput screening triage

PubMed Central

Dahlin, Jayme L; Walters, Michael A

2015-01-01

It is increasingly clear that academic high-throughput screening (HTS) and virtual HTS triage suffers from a lack of scientists trained in the art and science of early drug discovery chemistry. Many recent publications report the discovery of compounds by screening that are most likely artifacts or promiscuous bioactive compounds, and these results are not placed into the context of previous studies. For HTS to be most successful, it is our contention that there must exist an early partnership between biologists and medicinal chemists. Their combined skill sets are necessary to design robust assays and efficient workflows that will weed out assay artifacts, false positives, promiscuous bioactive compounds and intractable screening hits, efforts that ultimately give projects a better chance at identifying truly useful chemical matter. Expertise in medicinal chemistry, cheminformatics and purification sciences (analytical chemistry) can enhance the post-HTS triage process by quickly removing these problematic chemotypes from consideration, while simultaneously prioritizing the more promising chemical matter for follow-up testing. It is only when biologists and chemists collaborate effectively that HTS can manifest its full promise. PMID:25163000

High Throughput Multispectral Image Processing with Applications in Food Science.

PubMed

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

2015-01-01

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

A high-throughput, simultaneous analysis of carotenoids, chlorophylls and tocopherol using sub two micron core shell technology columns.

PubMed

Chebrolu, Kranthi K; Yousef, Gad G; Park, Ryan; Tanimura, Yoshinori; Brown, Allan F

2015-09-15

A high-throughput, robust and reliable method for simultaneous analysis of five carotenoids, four chlorophylls and one tocopherol was developed for rapid screening large sample populations to facilitate molecular biology and plant breeding. Separation was achieved for 10 known analytes and four unknown carotenoids in a significantly reduced run time of 10min. Identity of the 10 analytes was confirmed by their UV-Vis absorption spectras. Quantification of tocopherol, carotenoids and chlorophylls was performed at 290nm, 460nm and 650nm respectively. In this report, two sub two micron particle core-shell columns, Kinetex from Phenomenex (1.7μm particle size, 12% carbon load) and Cortecs from Waters (1.6μm particle size, 6.6% carbon load) were investigated and their separation efficiencies were evaluated. The peak resolutions were >1.5 for all analytes except for chlorophyll-a' with Cortecs column. The ruggedness of this method was evaluated in two identical but separate instruments that produced CV<2 in peak retentions for nine out of 10 analytes separated. Copyright © 2015 Elsevier B.V. All rights reserved.

Pathway analysis of high-throughput biological data within a Bayesian network framework.

PubMed

Isci, Senol; Ozturk, Cengizhan; Jones, Jon; Otu, Hasan H

2011-06-15

Most current approaches to high-throughput biological data (HTBD) analysis either perform individual gene/protein analysis or, gene/protein set enrichment analysis for a list of biologically relevant molecules. Bayesian Networks (BNs) capture linear and non-linear interactions, handle stochastic events accounting for noise, and focus on local interactions, which can be related to causal inference. Here, we describe for the first time an algorithm that models biological pathways as BNs and identifies pathways that best explain given HTBD by scoring fitness of each network. Proposed method takes into account the connectivity and relatedness between nodes of the pathway through factoring pathway topology in its model. Our simulations using synthetic data demonstrated robustness of our approach. We tested proposed method, Bayesian Pathway Analysis (BPA), on human microarray data regarding renal cell carcinoma (RCC) and compared our results with gene set enrichment analysis. BPA was able to find broader and more specific pathways related to RCC. Accompanying BPA software (BPAS) package is freely available for academic use at http://bumil.boun.edu.tr/bpa.

Towards ambient temperature-stable vaccines: the identification of thermally stabilizing liquid formulations for measles virus using an innovative high-throughput infectivity assay.

PubMed

Schlehuber, Lisa D; McFadyen, Iain J; Shu, Yu; Carignan, James; Duprex, W Paul; Forsyth, William R; Ho, Jason H; Kitsos, Christine M; Lee, George Y; Levinson, Douglas A; Lucier, Sarah C; Moore, Christopher B; Nguyen, Niem T; Ramos, Josephine; Weinstock, B André; Zhang, Junhong; Monagle, Julie A; Gardner, Colin R; Alvarez, Juan C

2011-07-12

As a result of thermal instability, some live attenuated viral (LAV) vaccines lose substantial potency from the time of manufacture to the point of administration. Developing regions lacking extensive, reliable refrigeration ("cold-chain") infrastructure are particularly vulnerable to vaccine failure, which in turn increases the burden of disease. Development of a robust, infectivity-based high throughput screening process for identifying thermostable vaccine formulations offers significant promise for vaccine development across a wide variety of LAV products. Here we describe a system that incorporates thermal stability screening into formulation design using heat labile measles virus as a prototype. The screening of >11,000 unique formulations resulted in the identification of liquid formulations with marked improvement over those used in commercial monovalent measles vaccines, with <1.0 log loss of activity after incubation for 8h at 40°C. The approach was shown to be transferable to a second unrelated virus, and therefore offers significant promise towards the optimization of formulation for LAV vaccine products. Copyright © 2011 Elsevier Ltd. All rights reserved.

Targeting α-synuclein oligomers by protein-fragment complementation for drug discovery in synucleinopathies.

PubMed

Moussaud, Simon; Malany, Siobhan; Mehta, Alka; Vasile, Stefan; Smith, Layton H; McLean, Pamela J

2015-05-01

Reducing the burden of α-synuclein oligomeric species represents a promising approach for disease-modifying therapies against synucleinopathies such as Parkinson's disease and dementia with Lewy bodies. However, the lack of efficient drug discovery strategies that specifically target α-synuclein oligomers has been a limitation to drug discovery programs. Here we describe an innovative strategy that harnesses the power of bimolecular protein-fragment complementation to monitor synuclein-synuclein interactions. We have developed two robust models to monitor α-synuclein oligomerization by generating novel stable cell lines expressing α-synuclein fusion proteins for either fluorescent or bioluminescent protein-fragment complementation under the tetracycline-controlled transcriptional activation system. A pilot screen was performed resulting in the identification of two potential hits, a p38 MAPK inhibitor and a casein kinase 2 inhibitor, thereby demonstrating the suitability of our protein-fragment complementation assay for the measurement of α-synuclein oligomerization in living cells at high throughput. The application of the strategy described herein to monitor α-synuclein oligomer formation in living cells with high throughput will facilitate drug discovery efforts for disease-modifying therapies against synucleinopathies and other proteinopathies.

Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS)

PubMed Central

Sexton, Jonathan Z; Danshina, Polina V; Lamson, David R; Hughes, Mark; House, Alan J; Yeh, Li-An; O’Brien, Deborah A; Williams, Kevin P

2011-01-01

Glycolytic isozymes that are restricted to the male germline are potential targets for the development of reversible, non-hormonal male contraceptives. GAPDHS, the sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase, is an essential enzyme for glycolysis making it an attractive target for rational drug design. Toward this goal, we have optimized and validated a high-throughput spectrophotometric assay for GAPDHS in 384-well format. The assay was stable over time and tolerant to DMSO. Whole plate validation experiments yielded Z’ values >0.8 indicating a robust assay for HTS. Two compounds were identified and confirmed from a test screen of the Prestwick collection. This assay was used to screen a diverse chemical library and identified fourteen small molecules that modulated the activity of recombinant purified GAPDHS with confirmed IC50 values ranging from 1.8 to 42 µM. These compounds may provide useful scaffolds as molecular tools to probe the role of GAPDHS in sperm motility and long term to develop potent and selective GAPDHS inhibitors leading to novel contraceptive agents. PMID:21760877

Personalized In Vitro and In Vivo Cancer Models to Guide Precision Medicine

PubMed Central

Pauli, Chantal; Hopkins, Benjamin D.; Prandi, Davide; Shaw, Reid; Fedrizzi, Tarcisio; Sboner, Andrea; Sailer, Verena; Augello, Michael; Puca, Loredana; Rosati, Rachele; McNary, Terra J.; Churakova, Yelena; Cheung, Cynthia; Triscott, Joanna; Pisapia, David; Rao, Rema; Mosquera, Juan Miguel; Robinson, Brian; Faltas, Bishoy M.; Emerling, Brooke E.; Gadi, Vijayakrishna K.; Bernard, Brady; Elemento, Olivier; Beltran, Himisha; Dimichelis, Francesca; Kemp, Christopher J.; Grandori, Carla; Cantley, Lewis C.; Rubin, Mark A.

2017-01-01

Precision Medicine is an approach that takes into account the influence of individuals' genes, environment and lifestyle exposures to tailor interventions. Here, we describe the development of a robust precision cancer care platform, which integrates whole exome sequencing (WES) with a living biobank that enables high throughput drug screens on patient-derived tumor organoids. To date, 56 tumor-derived organoid cultures, and 19 patient-derived xenograft (PDX) models have been established from the 769 patients enrolled in an IRB approved clinical trial. Because genomics alone was insufficient to identify therapeutic options for the majority of patients with advanced disease, we used high throughput drug screening effective strategies. Analysis of tumor derived cells from four cases, two uterine malignancies and two colon cancers, identified effective drugs and drug combinations that were subsequently validated using 3D cultures and PDX models. This platform thereby promotes the discovery of novel therapeutic approaches that can be assessed in clinical trials and provides personalized therapeutic options for individual patients where standard clinical options have been exhausted. PMID:28331002

An Engineered Virus Library as a Resource for the Spectrum-wide Exploration of Virus and Vector Diversity.

PubMed

Zhang, Wenli; Fu, Jun; Liu, Jing; Wang, Hailong; Schiwon, Maren; Janz, Sebastian; Schaffarczyk, Lukas; von der Goltz, Lukas; Ehrke-Schulz, Eric; Dörner, Johannes; Solanki, Manish; Boehme, Philip; Bergmann, Thorsten; Lieber, Andre; Lauber, Chris; Dahl, Andreas; Petzold, Andreas; Zhang, Youming; Stewart, A Francis; Ehrhardt, Anja

2017-05-23

Adenoviruses (Ads) are large human-pathogenic double-stranded DNA (dsDNA) viruses presenting an enormous natural diversity associated with a broad variety of diseases. However, only a small fraction of adenoviruses has been explored in basic virology and biomedical research, highlighting the need to develop robust and adaptable methodologies and resources. We developed a method for high-throughput direct cloning and engineering of adenoviral genomes from different sources utilizing advanced linear-linear homologous recombination (LLHR) and linear-circular homologous recombination (LCHR). We describe 34 cloned adenoviral genomes originating from clinical samples, which were characterized by next-generation sequencing (NGS). We anticipate that this recombineering strategy and the engineered adenovirus library will provide an approach to study basic and clinical virology. High-throughput screening (HTS) of the reporter-tagged Ad library in a panel of cell lines including osteosarcoma disease-specific cell lines revealed alternative virus types with enhanced transduction and oncolysis efficiencies. This highlights the usefulness of this resource. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

A new pooling strategy for high-throughput screening: the Shifted Transversal Design

PubMed Central

Thierry-Mieg, Nicolas

2006-01-01

Background In binary high-throughput screening projects where the goal is the identification of low-frequency events, beyond the obvious issue of efficiency, false positives and false negatives are a major concern. Pooling constitutes a natural solution: it reduces the number of tests, while providing critical duplication of the individual experiments, thereby correcting for experimental noise. The main difficulty consists in designing the pools in a manner that is both efficient and robust: few pools should be necessary to correct the errors and identify the positives, yet the experiment should not be too vulnerable to biological shakiness. For example, some information should still be obtained even if there are slightly more positives or errors than expected. This is known as the group testing problem, or pooling problem. Results In this paper, we present a new non-adaptive combinatorial pooling design: the "shifted transversal design" (STD). It relies on arithmetics, and rests on two intuitive ideas: minimizing the co-occurrence of objects, and constructing pools of constant-sized intersections. We prove that it allows unambiguous decoding of noisy experimental observations. This design is highly flexible, and can be tailored to function robustly in a wide range of experimental settings (i.e., numbers of objects, fractions of positives, and expected error-rates). Furthermore, we show that our design compares favorably, in terms of efficiency, to the previously described non-adaptive combinatorial pooling designs. Conclusion This method is currently being validated by field-testing in the context of yeast-two-hybrid interactome mapping, in collaboration with Marc Vidal's lab at the Dana Farber Cancer Institute. Many similar projects could benefit from using the Shifted Transversal Design. PMID:16423300

Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells

PubMed Central

Shalek, Alex K.; Robinson, Jacob T.; Karp, Ethan S.; Lee, Jin Seok; Ahn, Dae-Ro; Yoon, Myung-Han; Sutton, Amy; Jorgolli, Marsela; Gertner, Rona S.; Gujral, Taranjit S.; MacBeath, Gavin; Yang, Eun Gyeong; Park, Hongkun

2010-01-01

A generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed. Here, we demonstrate spatially localized, efficient, and universal delivery of biomolecules into immortalized and primary mammalian cells using surface-modified vertical silicon nanowires. The method relies on the ability of the silicon nanowires to penetrate a cell’s membrane and subsequently release surface-bound molecules directly into the cell’s cytosol, thus allowing highly efficient delivery of biomolecules without chemical modification or viral packaging. This modality enables one to assess the phenotypic consequences of introducing a broad range of biological effectors (DNAs, RNAs, peptides, proteins, and small molecules) into almost any cell type. We show that this platform can be used to guide neuronal progenitor growth with small molecules, knock down transcript levels by delivering siRNAs, inhibit apoptosis using peptides, and introduce targeted proteins to specific organelles. We further demonstrate codelivery of siRNAs and proteins on a single substrate in a microarray format, highlighting this technology’s potential as a robust, monolithic platform for high-throughput, miniaturized bioassays. PMID:20080678

Adapting AlphaLISA high throughput screen to discover a novel small-molecule inhibitor targeting protein arginine methyltransferase 5 in pancreatic and colorectal cancers

PubMed Central

Prabhu, Lakshmi; Wei, Han; Chen, Lan; Demir, Özlem; Sandusky, George; Sun, Emily; Wang, John; Mo, Jessica; Zeng, Lifan; Fishel, Melissa; Safa, Ahmad; Amaro, Rommie; Korc, Murray; Zhang, Zhong-Yin; Lu, Tao

2017-01-01

Pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) are notoriously challenging for treatment. Hyperactive nuclear factor κB (NF-κB) is a common culprit in both cancers. Previously, we discovered that protein arginine methyltransferase 5 (PRMT5) methylated and activated NF-κB. Here, we show that PRMT5 is highly expressed in PDAC and CRC. Overexpression of PRMT5 promoted cancer progression, while shRNA knockdown showed an opposite effect. Using an innovative AlphaLISA high throughput screen, we discovered a lead compound, PR5-LL-CM01, which exhibited robust tumor inhibition effects in both cancers. An in silico structure prediction suggested that PR5-LL-CM01 inhibits PRMT5 by binding with its active pocket. Importantly, PR5-LL-CM01 showed higher anti-tumor efficacy than the commercial PRMT5 inhibitor, EPZ015666, in both PDAC and CRC. This study clearly highlights the significant potential of PRMT5 as a therapeutic target in PDAC and CRC, and establishes PR5-LL-CM01 as a promising basis for new drug development in the future. PMID:28591716

A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo

PubMed Central

Coles, Andrew H.; Osborn, Maire F.; Alterman, Julia F.; Turanov, Anton A.; Godinho, Bruno M.D.C.; Kennington, Lori; Chase, Kathryn; Aronin, Neil

2016-01-01

Preclinical development of RNA interference (RNAi)-based therapeutics requires a rapid, accurate, and robust method of simultaneously quantifying mRNA knockdown in hundreds of samples. The most well-established method to achieve this is quantitative real-time polymerase chain reaction (qRT-PCR), a labor-intensive methodology that requires sample purification, which increases the potential to introduce additional bias. Here, we describe that the QuantiGene® branched DNA (bDNA) assay linked to a 96-well Qiagen TissueLyser II is a quick and reproducible alternative to qRT-PCR for quantitative analysis of mRNA expression in vivo directly from tissue biopsies. The bDNA assay is a high-throughput, plate-based, luminescence technique, capable of directly measuring mRNA levels from tissue lysates derived from various biological samples. We have performed a systematic evaluation of this technique for in vivo detection of RNAi-based silencing. We show that similar quality data is obtained from purified RNA and tissue lysates. In general, we observe low intra- and inter-animal variability (around 10% for control samples), and high intermediate precision. This allows minimization of sample size for evaluation of oligonucleotide efficacy in vivo. PMID:26595721

Impedance feedback control of microfluidic valves for reliable post processing combinatorial droplet injection.

PubMed

Axt, Brant; Hsieh, Yi-Fan; Nalayanda, Divya; Wang, Tza-Huei

2017-09-01

Droplet microfluidics has found use in many biological assay applications as a means of high-throughput sample processing. One of the challenges of the technology, however, is the ability to control and merge droplets on-demand as they flow through the microdevices. It is in the interest of developing lab-on-chip devices to be able to combinatorically program additive mixing steps for more complex multistep and multiplex assays. Existing technologies to merge droplets are either passive in nature or require highly predictable droplet movement for feedforward control, making them vulnerable to errors during high throughput operation. In this paper, we describe and demonstrate a microfluidic valve-based device for the purpose of combinatorial droplet injection at any stage in a multistep assay. Microfluidic valves are used to robustly control fluid flow, droplet generation, and droplet mixing in the device on-demand, while on-chip impedance measurements taken in real time are used as feedback to accurately time the droplet injections. The presented system is contrasted to attempts without feedback, and is shown to be 100% reliable over long durations. Additionally, content detection and discretionary injections are explored and successfully executed.

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens.

PubMed

Saeidi, Saman; McKain, Michael R; Kellogg, Elizabeth A

2018-03-08

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

Parallel Workflow for High-Throughput (>1,000 Samples/Day) Quantitative Analysis of Human Insulin-Like Growth Factor 1 Using Mass Spectrometric Immunoassay

PubMed Central

Oran, Paul E.; Trenchevska, Olgica; Nedelkov, Dobrin; Borges, Chad R.; Schaab, Matthew R.; Rehder, Douglas S.; Jarvis, Jason W.; Sherma, Nisha D.; Shen, Luhui; Krastins, Bryan; Lopez, Mary F.; Schwenke, Dawn C.; Reaven, Peter D.; Nelson, Randall W.

2014-01-01

Insulin-like growth factor 1 (IGF1) is an important biomarker for the management of growth hormone disorders. Recently there has been rising interest in deploying mass spectrometric (MS) methods of detection for measuring IGF1. However, widespread clinical adoption of any MS-based IGF1 assay will require increased throughput and speed to justify the costs of analyses, and robust industrial platforms that are reproducible across laboratories. Presented here is an MS-based quantitative IGF1 assay with performance rating of >1,000 samples/day, and a capability of quantifying IGF1 point mutations and posttranslational modifications. The throughput of the IGF1 mass spectrometric immunoassay (MSIA) benefited from a simplified sample preparation step, IGF1 immunocapture in a tip format, and high-throughput MALDI-TOF MS analysis. The Limit of Detection and Limit of Quantification of the resulting assay were 1.5 μg/L and 5 μg/L, respectively, with intra- and inter-assay precision CVs of less than 10%, and good linearity and recovery characteristics. The IGF1 MSIA was benchmarked against commercially available IGF1 ELISA via Bland-Altman method comparison test, resulting in a slight positive bias of 16%. The IGF1 MSIA was employed in an optimized parallel workflow utilizing two pipetting robots and MALDI-TOF-MS instruments synced into one-hour phases of sample preparation, extraction and MSIA pipette tip elution, MS data collection, and data processing. Using this workflow, high-throughput IGF1 quantification of 1,054 human samples was achieved in approximately 9 hours. This rate of assaying is a significant improvement over existing MS-based IGF1 assays, and is on par with that of the enzyme-based immunoassays. Furthermore, a mutation was detected in ∼1% of the samples (SNP: rs17884626, creating an A→T substitution at position 67 of the IGF1), demonstrating the capability of IGF1 MSIA to detect point mutations and posttranslational modifications. PMID:24664114

Rapid analysis of colipase gene variants by multicapillary electrophoresis.

PubMed

Jaczó, Zsuzsanna; Pál, Eszter; Dénes, Réka; Somogyi, Anikó; Sasvári-Székely, Mária; Guttman, András; Rónai, Zsolt

2015-06-01

Despite of the fact that the Human Genome Project was completed more than a decade ago, identification of the genetic background of polygenic diseases is still challenging. Several somewhat different approaches are available to investigate inheritable factors of complex phenotypes, all require, however efficient, high-throughput techniques for SNP genotyping. In this paper, we report a robust and reliable multiplex PCR-RFLP for genotype and haplotype analysis of six SNPs (rs41270082, rs3748051, rs142027015, rs3748048, rs73404011, and rs72925892) of the colipase (CLPS) gene. A multicapillary (12 capillaries) electrophoresis unit was used for high throughput and sensitive analysis of the digestion fragments. A Microsoft Excel-based spreadsheet was designed for the flexible visualization and evaluation of the electrophoretic separations, which is readily adaptable for any kind of electrophoresis application. Haplotype analysis of the two loci localized in close proximity of each other was carried out by molecular method, extended haplotypes including all five SNPs in the 5' upstream region were calculated. The techniques were applied in a case-control association study of type 2 diabetes mellitus. Although, single marker analysis did not reveal any significant association, it was observed that the rare GGCCG haplotype of the five 5' upstream region SNPs was about three times more frequent among patients compared to healthy control population. Our results demonstrated the applicability of multicapillary CGE in large-scale, high-throughput SNP analysis, and suggested that the CLPS gene polymorphisms might be considered as genetic risk factor for type 2 diabetes mellitus. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High throughput automated microbial bioreactor system used for clone selection and rapid scale-down process optimization.

PubMed

Velez-Suberbie, M Lourdes; Betts, John P J; Walker, Kelly L; Robinson, Colin; Zoro, Barney; Keshavarz-Moore, Eli

2018-01-01

High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed-batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled-up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale-up. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:58-68, 2018. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.

High-Throughput Quantification of GFP-LC3+ Dots by Automated Fluorescence Microscopy.

PubMed

Bravo-San Pedro, J M; Pietrocola, F; Sica, V; Izzo, V; Sauvat, A; Kepp, O; Maiuri, M C; Kroemer, G; Galluzzi, L

2017-01-01

Macroautophagy is a specific variant of autophagy that involves a dedicated double-membraned organelle commonly known as autophagosome. Various methods have been developed to quantify the size of the autophagosomal compartment, which is an indirect indicator of macroautophagic responses, based on the peculiar ability of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; best known as LC3) to accumulate in forming autophagosomes upon maturation. One particularly convenient method to monitor the accumulation of mature LC3 within autophagosomes relies on a green fluorescent protein (GFP)-tagged variant of this protein and fluorescence microscopy. In physiological conditions, cells transfected temporarily or stably with a GFP-LC3-encoding construct exhibit a diffuse green fluorescence over the cytoplasm and nucleus. Conversely, in response to macroautophagy-promoting stimuli, the GFP-LC3 signal becomes punctate and often (but not always) predominantly cytoplasmic. The accumulation of GFP-LC3 in cytoplasmic dots, however, also ensues the blockage of any of the steps that ensure the degradation of mature autophagosomes, calling for the implementation of strategies that accurately discriminate between an increase in autophagic flux and an arrest in autophagic degradation. Various cell lines have been engineered to stably express GFP-LC3, which-combined with the appropriate controls of flux, high-throughput imaging stations, and automated image analysis-offer a relatively straightforward tool to screen large chemical or biological libraries for inducers or inhibitors of autophagy. Here, we describe a simple and robust method for the high-throughput quantification of GFP-LC3 + dots by automated fluorescence microscopy. © 2017 Elsevier Inc. All rights reserved.

Identification of Small Molecule Inhibitors of Clostridium perfringens ε-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen.

PubMed

Lewis, Michelle; Weaver, Charles David; McClain, Mark S

2010-07-01

The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ε-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics.

Identification of Small Molecule Inhibitors of Clostridium perfringens ε-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen

PubMed Central

Lewis, Michelle; Weaver, Charles David; McClain, Mark S.

2010-01-01

The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ε-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics. PMID:20721308

Evaluation of Impermeant, DNA-Binding Dye Fluorescence as a Real-Time Readout of Eukaryotic Cell Toxicity in a High Throughput Screening Format

PubMed Central

Chiaraviglio, Lucius

2014-01-01

Abstract Interpretation of high throughput screening (HTS) data in cell-based assays may be confounded by cytotoxic properties of screening compounds. Therefore, assessing cell toxicity in real time during the HTS process itself would be highly advantageous. Here, we investigate the potential of putatively impermeant, fluorescent, DNA-binding dyes to give cell toxicity readout during HTS. Amongst 19 DNA-binding dyes examined, three classes were identified that were (1) permeant, (2) cytotoxic, or (3) neither permeant nor cytotoxic during 3-day incubation with a macrophage cell line. In the last class, four dyes (SYTOX Green, CellTox Green, GelGreen, and EvaGreen) gave highly robust cytotoxicity data in 384-well screening plates. As proof of principle, successful combination with a luminescence-based assay in HTS format was demonstrated. Here, both intracellular growth of Legionella pneumophila (luminescence) and host cell viability (SYTOX Green exclusion) were assayed in the same screening well. Incorporation of membrane-impermeant, DNA-binding, fluorescent dyes in HTS assays should prove useful by allowing evaluation of cytotoxicity in real time, eliminating reagent addition steps and effort associated with endpoint cell viability analysis, and reducing the need for follow-up cytotoxicity screening. PMID:24831788

G-Protein Coupled Receptors: Surface Display and Biosensor Technology

NASA Astrophysics Data System (ADS)

McMurchie, Edward; Leifert, Wayne

Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

MBTH: A novel approach to rapid, spectrophotometric quantitation of total algal carbohydrates.

PubMed

Van Wychen, Stefanie; Long, William; Black, Stuart K; Laurens, Lieve M L

2017-02-01

A high-throughput and robust application of the 3-methyl-2-benzothiazolinone hydrazone (MBTH) method was developed for carbohydrate determination in microalgae. The traditional phenol-sulfuric acid method to quantify carbohydrates is strongly affected by algal biochemical components and exhibits a highly variable response to microalgal monosaccharides. We present a novel use of the MBTH method to accurately quantify carbohydrates in hydrolyzate after acid hydrolysis of algal biomass, without a need for neutralization. The MBTH method demonstrated consistent and sensitive quantitation of algae-specific monosaccharides down to 5 μg mL -1 without interference from other algae acidic hydrolyzate components. Copyright © 2016 Elsevier Inc. All rights reserved.

Micropore-free surface-activated carbon for the analysis of polychlorinated dibenzo-p-dioxins-dibenzofurans and non-ortho-substituted polychlorinated biphenyls in environmental samples.

PubMed

Kemmochi, Yukio; Tsutsumi, Kaori; Arikawa, Akihiro; Nakazawa, Hiroyuki

2002-11-22

2,3,7,8-Substituted polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans (PCDD/Fs) and non-ortho-substituted polychlorinated biphenyls (PCBs) account for almost all of the total toxic equivalents (TEQ) in environmental samples. Activated carbon columns are used to fractionate the samples for GC-MS analysis or bioassay. Micropore-free surface-activated carbon is highly selective for PCDD/Fs and non-ortho-PCBs and can improve the conventional activated carbon column clean-up. Along with sulfuric acid-coated diatomaceous earth columns, micropore-free surface-activated carbon provides a rapid, robust, and high-throughput sample preparation method for PCDD/Fs and non-ortho-PCBs analysis.

Non-clinical studies required for new drug development - Part I: early in silico and in vitro studies, new target discovery and validation, proof of principles and robustness of animal studies.

PubMed

Andrade, E L; Bento, A F; Cavalli, J; Oliveira, S K; Freitas, C S; Marcon, R; Schwanke, R C; Siqueira, J M; Calixto, J B

2016-10-24

This review presents a historical overview of drug discovery and the non-clinical stages of the drug development process, from initial target identification and validation, through in silico assays and high throughput screening (HTS), identification of leader molecules and their optimization, the selection of a candidate substance for clinical development, and the use of animal models during the early studies of proof-of-concept (or principle). This report also discusses the relevance of validated and predictive animal models selection, as well as the correct use of animal tests concerning the experimental design, execution and interpretation, which affect the reproducibility, quality and reliability of non-clinical studies necessary to translate to and support clinical studies. Collectively, improving these aspects will certainly contribute to the robustness of both scientific publications and the translation of new substances to clinical development.

Development of an Intracellular Screen for New Compounds Able To Inhibit Mycobacterium tuberculosis Growth in Human Macrophages.

PubMed

Sorrentino, Flavia; Gonzalez del Rio, Ruben; Zheng, Xingji; Presa Matilla, Jesus; Torres Gomez, Pedro; Martinez Hoyos, Maria; Perez Herran, Maria Esther; Mendoza Losana, Alfonso; Av-Gay, Yossef

2016-01-01

Here we describe the development and validation of an intracellular high-throughput screening assay for finding new antituberculosis compounds active in human macrophages. The assay consists of a luciferase-based primary identification assay, followed by a green fluorescent protein-based secondary profiling assay. Standard tuberculosis drugs and 158 previously recognized active antimycobacterial compounds were used to evaluate assay robustness. Data show that the assay developed is a short and valuable tool for the discovery of new antimycobacterial compounds. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Assay Guidance Manual: Quantitative Biology and Pharmacology in Preclinical Drug Discovery.

PubMed

Coussens, Nathan P; Sittampalam, G Sitta; Guha, Rajarshi; Brimacombe, Kyle; Grossman, Abigail; Chung, Thomas D Y; Weidner, Jeffrey R; Riss, Terry; Trask, O Joseph; Auld, Douglas; Dahlin, Jayme L; Devanaryan, Viswanath; Foley, Timothy L; McGee, James; Kahl, Steven D; Kales, Stephen C; Arkin, Michelle; Baell, Jonathan; Bejcek, Bruce; Gal-Edd, Neely; Glicksman, Marcie; Haas, Joseph V; Iversen, Philip W; Hoeppner, Marilu; Lathrop, Stacy; Sayers, Eric; Liu, Hanguan; Trawick, Bart; McVey, Julie; Lemmon, Vance P; Li, Zhuyin; McManus, Owen; Minor, Lisa; Napper, Andrew; Wildey, Mary Jo; Pacifici, Robert; Chin, William W; Xia, Menghang; Xu, Xin; Lal-Nag, Madhu; Hall, Matthew D; Michael, Sam; Inglese, James; Simeonov, Anton; Austin, Christopher P

2018-06-07

The Assay Guidance Manual (AGM) is an eBook of best-practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a "testing funnel" of assays to identify genuine hits using high-throughput screening (HTS) and advancing them through pre-clinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Periodic nanostructural materials for nanoplasmonics

NASA Astrophysics Data System (ADS)

Choi, Dukhyun

2017-02-01

Nanoscale periodic material design and fabrication are essentially fundamental requirement for basic scientific researches and industrial applications of nanoscience and engineering. Innovative, effective, reproducible, large-area uniform, tunable and robust nanostructure/material syntheses are still challenging. Here, I would like to introduce the novel periodic nanostructural materials particularly with uniformly ordered nanoporous or nanoflower structures, which are fabricated by simple, cost-effective, and high-throughput wet chemical methods. I also report large-area periodic plasmonic nanostructures based on template-based nanolithography. The surface morphology and optical properties are characterized by SEM and UV-vis. spectroscopy. Furthermore, their enhancement factor is evaluated by using SERS signals.

Staged anticonvulsant screening for chronic epilepsy.

PubMed

Berdichevsky, Yevgeny; Saponjian, Yero; Park, Kyung-Il; Roach, Bonnie; Pouliot, Wendy; Lu, Kimberly; Swiercz, Waldemar; Dudek, F Edward; Staley, Kevin J

2016-12-01

Current anticonvulsant screening programs are based on seizures evoked in normal animals. One-third of epileptic patients do not respond to the anticonvulsants discovered with these models. We evaluated a tiered program based on chronic epilepsy and spontaneous seizures, with compounds advancing from high-throughput in vitro models to low-throughput in vivo models. Epileptogenesis in organotypic hippocampal slice cultures was quantified by lactate production and lactate dehydrogenase release into culture media as rapid assays for seizure-like activity and cell death, respectively. Compounds that reduced these biochemical measures were retested with in vitro electrophysiological confirmation (i.e., second stage). The third stage involved crossover testing in the kainate model of chronic epilepsy, with blinded analysis of spontaneous seizures after continuous electrographic recordings. We screened 407 compound-concentration combinations. The cyclooxygenase inhibitor, celecoxib, had no effect on seizures evoked in normal brain tissue but demonstrated robust antiseizure activity in all tested models of chronic epilepsy. The use of organotypic hippocampal cultures, where epileptogenesis occurs on a compressed time scale, and where seizure-like activity and seizure-induced cell death can be easily quantified with biomarker assays, allowed us to circumvent the throughput limitations of in vivo chronic epilepsy models. Ability to rapidly screen compounds in a chronic model of epilepsy allowed us to find an anticonvulsant that would be missed by screening in acute models.

High-Throughput Screening of Chemical Effects on Steroidogenesis Using H295R Human Adrenocortical Carcinoma Cells.

PubMed

Karmaus, Agnes L; Toole, Colleen M; Filer, Dayne L; Lewis, Kenneth C; Martin, Matthew T

2016-04-01

Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2060 chemical samples on steroidogenesis via high-performance liquid chromatography followed by tandem mass spectrometry quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a 3 stage screening strategy. The first stage established the maximum tolerated concentration (MTC; ≥ 70% viability) per sample. The second stage quantified changes in hormone levels at the MTC whereas the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were prestimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2060 chemical samples evaluated, 524 samples were selected for 6-point CR screening, based in part on significantly altering at least 4 hormones at the MTC. CR screening identified 232 chemical samples with concentration-dependent effects on 17β-estradiol and/or testosterone, with 411 chemical samples showing an effect on at least one hormone across the steroidogenesis pathway. Clustering of the concentration-dependent chemical-mediated steroid hormone effects grouped chemical samples into 5 distinct profiles generally representing putative mechanisms of action, including CYP17A1 and HSD3B inhibition. A distinct pattern was observed between imidazole and triazole fungicides suggesting potentially distinct mechanisms of action. From a chemical testing and prioritization perspective, this assay platform provides a robust model for high-throughput screening of chemicals for effects on steroidogenesis. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology.

CA resist with high sensitivity and sub-100-nm resolution for advanced mask and device making

NASA Astrophysics Data System (ADS)

Kwong, Ranee W.; Huang, Wu-Song; Hartley, John G.; Moreau, Wayne M.; Robinson, Christopher F.; Angelopoulos, Marie; Magg, Christopher; Lawliss, Mark

2000-07-01

Recently, there is significant interest in using CA resists for electron beam (E-Beam) applications including mask making, direct write, and projection printing. CA resists provide superior lithographic performance in comparison to traditional non CA E-beam resists in particular high contrast, resolution, and sensitivity. However, most of the commercially available CA resists have the concern of airborne base contaminants and sensitivity to PAB and/or PEB temperatures. In this presentation, we will discuss a new improved ketal resist system referred to as KRS-XE which exhibits excellent lithography, is robust toward airborne base, compatible with 0.263 N TMAH aqueous developer and exhibits a large PAB/PEB latitude. With the combination of a high performance mask making E-beam exposure tool, high kV (75 kV) shaped beam system EL4+ and the KRS-XE resist, we have printed 75 nm lines/space features with excellent profile control at a dose of 13 (mu) C/cm2 at 75 kV. The shaped beam vector scan system used here provides an unique property in resolving small features in lithography and throughput. Overhead in EL4+ limits the systems ability to fully exploit the sensitivity of the new resist for throughput. The EL5 system, currently in the build phase, has sufficiently low overhead that it is projected to print a 4X, 16G, DRAM mask with OPC in under 3 hours with the CA resist. We will discuss the throughput advantages of the next generation EL5 system over the existing EL4+. In addition we will show the resolution of KRS-XE down to 70 nm using the PREVAIL projection printing system.

High-Throughput Screening of Chemical Effects on Steroidogenesis Using H295R Human Adrenocortical Carcinoma Cells

PubMed Central

Toole, Colleen M.; Filer, Dayne L.; Lewis, Kenneth C.; Martin, Matthew T.

2016-01-01

Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2060 chemical samples on steroidogenesis via high-performance liquid chromatography followed by tandem mass spectrometry quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a 3 stage screening strategy. The first stage established the maximum tolerated concentration (MTC; ≥ 70% viability) per sample. The second stage quantified changes in hormone levels at the MTC whereas the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were prestimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2060 chemical samples evaluated, 524 samples were selected for 6-point CR screening, based in part on significantly altering at least 4 hormones at the MTC. CR screening identified 232 chemical samples with concentration-dependent effects on 17β-estradiol and/or testosterone, with 411 chemical samples showing an effect on at least one hormone across the steroidogenesis pathway. Clustering of the concentration-dependent chemical-mediated steroid hormone effects grouped chemical samples into 5 distinct profiles generally representing putative mechanisms of action, including CYP17A1 and HSD3B inhibition. A distinct pattern was observed between imidazole and triazole fungicides suggesting potentially distinct mechanisms of action. From a chemical testing and prioritization perspective, this assay platform provides a robust model for high-throughput screening of chemicals for effects on steroidogenesis. PMID:26781511

High-Throughput Screening of Na(V)1.7 Modulators Using a Giga-Seal Automated Patch Clamp Instrument.

PubMed

Chambers, Chris; Witton, Ian; Adams, Cathryn; Marrington, Luke; Kammonen, Juha

2016-03-01

Voltage-gated sodium (Na(V)) channels have an essential role in the initiation and propagation of action potentials in excitable cells, such as neurons. Of these channels, Na(V)1.7 has been indicated as a key channel for pain sensation. While extensive efforts have gone into discovering novel Na(V)1.7 modulating compounds for the treatment of pain, none has reached the market yet. In the last two years, new compound screening technologies have been introduced, which may speed up the discovery of such compounds. The Sophion Qube(®) is a next-generation 384-well giga-seal automated patch clamp (APC) screening instrument, capable of testing thousands of compounds per day. By combining high-throughput screening and follow-up compound testing on the same APC platform, it should be possible to accelerate the hit-to-lead stage of ion channel drug discovery and help identify the most interesting compounds faster. Following a period of instrument beta-testing, a Na(V)1.7 high-throughput screen was run with two Pfizer plate-based compound subsets. In total, data were generated for 158,000 compounds at a median success rate of 83%, which can be considered high in APC screening. In parallel, IC50 assay validation and protocol optimization was completed with a set of reference compounds to understand how the IC50 potencies generated on the Qube correlate with data generated on the more established Sophion QPatch(®) APC platform. In summary, the results presented here demonstrate that the Qube provides a comparable but much faster approach to study Na(V)1.7 in a robust and reliable APC assay for compound screening.

A device for high-throughput monitoring of degradation in soft tissue samples.

PubMed

Tzeranis, D S; Panagiotopoulos, I; Gkouma, S; Kanakaris, G; Georgiou, N; Vaindirlis, N; Vasileiou, G; Neidlin, M; Gkousioudi, A; Spitas, V; Macheras, G A; Alexopoulos, L G

2018-06-06

This work describes the design and validation of a novel device, the High-Throughput Degradation Monitoring Device (HDD), for monitoring the degradation of 24 soft tissue samples over incubation periods of several days inside a cell culture incubator. The device quantifies sample degradation by monitoring its deformation induced by a static gravity load. Initial instrument design and experimental protocol development focused on quantifying cartilage degeneration. Characterization of measurement errors, caused mainly by thermal transients and by translating the instrument sensor, demonstrated that HDD can quantify sample degradation with <6 μm precision and <10 μm temperature-induced errors. HDD capabilities were evaluated in a pilot study that monitored the degradation of fresh ex vivo human cartilage samples by collagenase solutions over three days. HDD could robustly resolve the effects of collagenase concentration as small as 0.5 mg/ml. Careful sample preparation resulted in measurements that did not suffer from donor-to-donor variation (coefficient of variance <70%). Due to its unique combination of sample throughput, measurement precision, temporal sampling and experimental versality, HDD provides a novel biomechanics-based experimental platform for quantifying the effects of proteins (cytokines, growth factors, enzymes, antibodies) or small molecules on the degradation of soft tissues or tissue engineering constructs. Thereby, HDD can complement established tools and in vitro models in important applications including drug screening and biomaterial development. Copyright © 2018 Elsevier Ltd. All rights reserved.

Interference-Robust Transmission in Wireless Sensor Networks

PubMed Central

Han, Jin-Seok; Lee, Yong-Hwan

2016-01-01

Low-power wireless sensor networks (WSNs) operating in unlicensed spectrum bands may seriously suffer from interference from other coexisting radio systems, such as IEEE 802.11 wireless local area networks. In this paper, we consider the improvement of the transmission performance of low-power WSNs by adjusting the transmission rate and the payload size in response to the change of co-channel interference. We estimate the probability of transmission failure and the data throughput and then determine the payload size to maximize the throughput performance. We investigate that the transmission time maximizing the normalized throughput is not much affected by the transmission rate, but rather by the interference condition. We adjust the transmission rate and the transmission time in response to the change of the channel and interference condition, respectively. Finally, we verify the performance of the proposed scheme by computer simulation. The simulation results show that the proposed scheme significantly improves data throughput compared with conventional schemes while preserving energy efficiency even in the presence of interference. PMID:27854249

Interference-Robust Transmission in Wireless Sensor Networks.

PubMed

Han, Jin-Seok; Lee, Yong-Hwan

2016-11-14

Low-power wireless sensor networks (WSNs) operating in unlicensed spectrum bands may seriously suffer from interference from other coexisting radio systems, such as IEEE 802.11 wireless local area networks. In this paper, we consider the improvement of the transmission performance of low-power WSNs by adjusting the transmission rate and the payload size in response to the change of co-channel interference. We estimate the probability of transmission failure and the data throughput and then determine the payload size to maximize the throughput performance. We investigate that the transmission time maximizing the normalized throughput is not much affected by the transmission rate, but rather by the interference condition. We adjust the transmission rate and the transmission time in response to the change of the channel and interference condition, respectively. Finally, we verify the performance of the proposed scheme by computer simulation. The simulation results show that the proposed scheme significantly improves data throughput compared with conventional schemes while preserving energy efficiency even in the presence of interference.

The developmental genetics of biological robustness

PubMed Central

Mestek Boukhibar, Lamia; Barkoulas, Michalis

2016-01-01

Background Living organisms are continuously confronted with perturbations, such as environmental changes that include fluctuations in temperature and nutrient availability, or genetic changes such as mutations. While some developmental systems are affected by such challenges and display variation in phenotypic traits, others continue consistently to produce invariable phenotypes despite perturbation. This ability of a living system to maintain an invariable phenotype in the face of perturbations is termed developmental robustness. Biological robustness is a phenomenon observed across phyla, and studying its mechanisms is central to deciphering the genotype–phenotype relationship. Recent work in yeast, animals and plants has shown that robustness is genetically controlled and has started to reveal the underlying mechinisms behind it. Scope and Conclusions Studying biological robustness involves focusing on an important property of developmental traits, which is the phenotypic distribution within a population. This is often neglected because the vast majority of developmental biology studies instead focus on population aggregates, such as trait averages. By drawing on findings in animals and yeast, this Viewpoint considers how studies on plant developmental robustness may benefit from strict definitions of what is the developmental system of choice and what is the relevant perturbation, and also from clear distinctions between gene effects on the trait mean and the trait variance. Recent advances in quantitative developmental biology and high-throughput phenotyping now allow the design of targeted genetic screens to identify genes that amplify or restrict developmental trait variance and to study how variation propagates across different phenotypic levels in biological systems. The molecular characterization of more quantitative trait loci affecting trait variance will provide further insights into the evolution of genes modulating developmental robustness. The study of robustness mechanisms in closely related species will address whether mechanisms of robustness are evolutionarily conserved. PMID:26292993

Robust reflective pupil slicing technology

NASA Astrophysics Data System (ADS)

Meade, Jeffrey T.; Behr, Bradford B.; Cenko, Andrew T.; Hajian, Arsen R.

2014-07-01

Tornado Spectral Systems (TSS) has developed the High Throughput Virtual Slit (HTVSTM), robust all-reflective pupil slicing technology capable of replacing the slit in research-, commercial- and MIL-SPEC-grade spectrometer systems. In the simplest configuration, the HTVS allows optical designers to remove the lossy slit from pointsource spectrometers and widen the input slit of long-slit spectrometers, greatly increasing throughput without loss of spectral resolution or cross-dispersion information. The HTVS works by transferring etendue between image plane axes but operating in the pupil domain rather than at a focal plane. While useful for other technologies, this is especially relevant for spectroscopic applications by performing the same spectral narrowing as a slit without throwing away light on the slit aperture. HTVS can be implemented in all-reflective designs and only requires a small number of reflections for significant spectral resolution enhancement-HTVS systems can be efficiently implemented in most wavelength regions. The etendueshifting operation also provides smooth scaling with input spot/image size without requiring reconfiguration for different targets (such as different seeing disk diameters or different fiber core sizes). Like most slicing technologies, HTVS provides throughput increases of several times without resolution loss over equivalent slitbased designs. HTVS technology enables robust slit replacement in point-source spectrometer systems. By virtue of pupilspace operation this technology has several advantages over comparable image-space slicer technology, including the ability to adapt gracefully and linearly to changing source size and better vertical packing of the flux distribution. Additionally, this technology can be implemented with large slicing factors in both fast and slow beams and can easily scale from large, room-sized spectrometers through to small, telescope-mounted devices. Finally, this same technology is directly applicable to multi-fiber spectrometers to achieve similar enhancement. HTVS also provides the ability to anamorphically "stretch" the slit image in long-slit spectrometers, allowing the instrument designer to optimize the plate scale in the dispersion axis and cross-dispersion axes independently without sacrificing spatial information. This allows users to widen the input slit, with the associated gain of throughput and loss of spatial selectivity, while maintaining the spectral resolution of the spectrometer system. This "stretching" places increased requirements on detector focal plane height, as with image slicing techniques, but provides additional degrees of freedom to instrument designers to build the best possible spectrometer systems. We discuss the details of this technology for an astronomical context, covering the applicability from small telescope mounted spectrometers through long-slit imagers and radial-velocity engines. This powerful tool provides additional degrees of freedom when designing a spectrometer, enabling instrument designers to further optimize systems for the required scientific goals.

Developing High-Throughput HIV Incidence Assay with Pyrosequencing Platform

PubMed Central

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

2014-01-01

ABSTRACT Human immunodeficiency virus (HIV) incidence is an important measure for monitoring the epidemic and evaluating the efficacy of intervention and prevention trials. This study developed a high-throughput, single-measure incidence assay by implementing a pyrosequencing platform. We devised a signal-masking bioinformatics pipeline, which yielded a process error rate of 5.8 × 10−4 per base. The pipeline was then applied to analyze 18,434 envelope gene segments (HXB2 7212 to 7601) obtained from 12 incident and 24 chronic patients who had documented HIV-negative and/or -positive tests. The pyrosequencing data were cross-checked by using the single-genome-amplification (SGA) method to independently obtain 302 sequences from 13 patients. Using two genomic biomarkers that probe for the presence of similar sequences, the pyrosequencing platform correctly classified all 12 incident subjects (100% sensitivity) and 23 of 24 chronic subjects (96% specificity). One misclassified subject's chronic infection was correctly classified by conducting the same analysis with SGA data. The biomarkers were statistically associated across the two platforms, suggesting the assay's reproducibility and robustness. Sampling simulations showed that the biomarkers were tolerant of sequencing errors and template resampling, two factors most likely to affect the accuracy of pyrosequencing results. We observed comparable biomarker scores between AIDS and non-AIDS chronic patients (multivariate analysis of variance [MANOVA], P = 0.12), indicating that the stage of HIV disease itself does not affect the classification scheme. The high-throughput genomic HIV incidence marks a significant step toward determining incidence from a single measure in cross-sectional surveys. IMPORTANCE Annual HIV incidence, the number of newly infected individuals within a year, is the key measure of monitoring the epidemic's rise and decline. Developing reliable assays differentiating recent from chronic infections has been a long-standing quest in the HIV community. Over the past 15 years, these assays have traditionally measured various HIV-specific antibodies, but recent technological advancements have expanded the diversity of proposed accurate, user-friendly, and financially viable tools. Here we designed a high-throughput genomic HIV incidence assay based on the signature imprinted in the HIV gene sequence population. By combining next-generation sequencing techniques with bioinformatics analysis, we demonstrated that genomic fingerprints are capable of distinguishing recently infected patients from chronically infected patients with high precision. Our high-throughput platform is expected to allow us to process many patients' samples from a single experiment, permitting the assay to be cost-effective for routine surveillance. PMID:24371062

A sensitive assay using a native protein substrate for screening HIV-1 maturation inhibitors targeting the protease cleavage site between the matrix and capsid.

PubMed

Lee, Sook-Kyung; Cheng, Nancy; Hull-Ryde, Emily; Potempa, Marc; Schiffer, Celia A; Janzen, William; Swanstrom, Ronald

2013-07-23

The matrix/capsid processing site in the HIV-1 Gag precursor is likely the most sensitive target to inhibit HIV-1 replication. We have previously shown that modest incomplete processing at the site leads to a complete loss of virion infectivity. In the study presented here, a sensitive assay based on fluorescence polarization that can monitor cleavage at the MA/CA site in the context of the folded protein substrate is described. The substrate, an MA/CA fusion protein, was labeled with the fluorescein-based FlAsH (fluorescein arsenical hairpin) reagent that binds to a tetracysteine motif (CCGPCC) that was introduced within the N-terminal domain of CA. By limiting the size of CA and increasing the size of MA (with an N-terminal GST fusion), we were able to measure significant differences in polarization values as a function of HIV-1 protease cleavage. The sensitivity of the assay was tested in the presence of increasing amounts of an HIV-1 protease inhibitor, which resulted in a gradual decrease in the fluorescence polarization values demonstrating that the assay is sensitive in discerning changes in protease processing. The high-throughput screening assay validation in 384-well plates showed that the assay is reproducible and robust with an average Z' value of 0.79 and average coefficient of variation values of <3%. The robustness and reproducibility of the assay were further validated using the LOPAC(1280) compound library, demonstrating that the assay provides a sensitive high-throughput screening platform that can be used with large compound libraries for identifying novel maturation inhibitors targeting the MA/CA site of the HIV-1 Gag polyprotein.

WormSizer: high-throughput analysis of nematode size and shape.

PubMed

Moore, Brad T; Jordan, James M; Baugh, L Ryan

2013-01-01

The fundamental phenotypes of growth rate, size and morphology are the result of complex interactions between genotype and environment. We developed a high-throughput software application, WormSizer, which computes size and shape of nematodes from brightfield images. Existing methods for estimating volume either coarsely model the nematode as a cylinder or assume the worm shape or opacity is invariant. Our estimate is more robust to changes in morphology or optical density as it only assumes radial symmetry. This open source software is written as a plugin for the well-known image-processing framework Fiji/ImageJ. It may therefore be extended easily. We evaluated the technical performance of this framework, and we used it to analyze growth and shape of several canonical Caenorhabditis elegans mutants in a developmental time series. We confirm quantitatively that a Dumpy (Dpy) mutant is short and fat and that a Long (Lon) mutant is long and thin. We show that daf-2 insulin-like receptor mutants are larger than wild-type upon hatching but grow slow, and WormSizer can distinguish dauer larvae from normal larvae. We also show that a Small (Sma) mutant is actually smaller than wild-type at all stages of larval development. WormSizer works with Uncoordinated (Unc) and Roller (Rol) mutants as well, indicating that it can be used with mutants despite behavioral phenotypes. We used our complete data set to perform a power analysis, giving users a sense of how many images are needed to detect different effect sizes. Our analysis confirms and extends on existing phenotypic characterization of well-characterized mutants, demonstrating the utility and robustness of WormSizer.

What time is it? Deep learning approaches for circadian rhythms.

PubMed

Agostinelli, Forest; Ceglia, Nicholas; Shahbaba, Babak; Sassone-Corsi, Paolo; Baldi, Pierre

2016-06-15

Circadian rhythms date back to the origins of life, are found in virtually every species and every cell, and play fundamental roles in functions ranging from metabolism to cognition. Modern high-throughput technologies allow the measurement of concentrations of transcripts, metabolites and other species along the circadian cycle creating novel computational challenges and opportunities, including the problems of inferring whether a given species oscillate in circadian fashion or not, and inferring the time at which a set of measurements was taken. We first curate several large synthetic and biological time series datasets containing labels for both periodic and aperiodic signals. We then use deep learning methods to develop and train BIO_CYCLE, a system to robustly estimate which signals are periodic in high-throughput circadian experiments, producing estimates of amplitudes, periods, phases, as well as several statistical significance measures. Using the curated data, BIO_CYCLE is compared to other approaches and shown to achieve state-of-the-art performance across multiple metrics. We then use deep learning methods to develop and train BIO_CLOCK to robustly estimate the time at which a particular single-time-point transcriptomic experiment was carried. In most cases, BIO_CLOCK can reliably predict time, within approximately 1 h, using the expression levels of only a small number of core clock genes. BIO_CLOCK is shown to work reasonably well across tissue types, and often with only small degradation across conditions. BIO_CLOCK is used to annotate most mouse experiments found in the GEO database with an inferred time stamp. All data and software are publicly available on the CircadiOmics web portal: circadiomics.igb.uci.edu/ fagostin@uci.edu or pfbaldi@uci.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

3-Dimensional culture systems for anti-cancer compound profiling and high-throughput screening reveal increases in EGFR inhibitor-mediated cytotoxicity compared to monolayer culture systems.

PubMed

Howes, Amy L; Richardson, Robyn D; Finlay, Darren; Vuori, Kristiina

2014-01-01

3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery.

What time is it? Deep learning approaches for circadian rhythms

PubMed Central

Agostinelli, Forest; Ceglia, Nicholas; Shahbaba, Babak; Sassone-Corsi, Paolo; Baldi, Pierre

2016-01-01

Motivation: Circadian rhythms date back to the origins of life, are found in virtually every species and every cell, and play fundamental roles in functions ranging from metabolism to cognition. Modern high-throughput technologies allow the measurement of concentrations of transcripts, metabolites and other species along the circadian cycle creating novel computational challenges and opportunities, including the problems of inferring whether a given species oscillate in circadian fashion or not, and inferring the time at which a set of measurements was taken. Results: We first curate several large synthetic and biological time series datasets containing labels for both periodic and aperiodic signals. We then use deep learning methods to develop and train BIO_CYCLE, a system to robustly estimate which signals are periodic in high-throughput circadian experiments, producing estimates of amplitudes, periods, phases, as well as several statistical significance measures. Using the curated data, BIO_CYCLE is compared to other approaches and shown to achieve state-of-the-art performance across multiple metrics. We then use deep learning methods to develop and train BIO_CLOCK to robustly estimate the time at which a particular single-time-point transcriptomic experiment was carried. In most cases, BIO_CLOCK can reliably predict time, within approximately 1 h, using the expression levels of only a small number of core clock genes. BIO_CLOCK is shown to work reasonably well across tissue types, and often with only small degradation across conditions. BIO_CLOCK is used to annotate most mouse experiments found in the GEO database with an inferred time stamp. Availability and Implementation: All data and software are publicly available on the CircadiOmics web portal: circadiomics.igb.uci.edu/. Contacts: fagostin@uci.edu or pfbaldi@uci.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307647

3-Dimensional Culture Systems for Anti-Cancer Compound Profiling and High-Throughput Screening Reveal Increases in EGFR Inhibitor-Mediated Cytotoxicity Compared to Monolayer Culture Systems

PubMed Central

Howes, Amy L.; Richardson, Robyn D.; Finlay, Darren; Vuori, Kristiina

2014-01-01

3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery. PMID:25247711

Next generation of decision making software for nanopatterns characterization: application to semiconductor industry

NASA Astrophysics Data System (ADS)

Dervilllé, A.; Labrosse, A.; Zimmermann, Y.; Foucher, J.; Gronheid, R.; Boeckx, C.; Singh, A.; Leray, P.; Halder, S.

2016-03-01

The dimensional scaling in IC manufacturing strongly drives the demands on CD and defect metrology techniques and their measurement uncertainties. Defect review has become as important as CD metrology and both of them create a new metrology paradigm because it creates a completely new need for flexible, robust and scalable metrology software. Current, software architectures and metrology algorithms are performant but it must be pushed to another higher level in order to follow roadmap speed and requirements. For example: manage defect and CD in one step algorithm, customize algorithms and outputs features for each R&D team environment, provide software update every day or every week for R&D teams in order to explore easily various development strategies. The final goal is to avoid spending hours and days to manually tune algorithm to analyze metrology data and to allow R&D teams to stay focus on their expertise. The benefits are drastic costs reduction, more efficient R&D team and better process quality. In this paper, we propose a new generation of software platform and development infrastructure which can integrate specific metrology business modules. For example, we will show the integration of a chemistry module dedicated to electronics materials like Direct Self Assembly features. We will show a new generation of image analysis algorithms which are able to manage at the same time defect rates, images classifications, CD and roughness measurements with high throughput performances in order to be compatible with HVM. In a second part, we will assess the reliability, the customization of algorithm and the software platform capabilities to follow new specific semiconductor metrology software requirements: flexibility, robustness, high throughput and scalability. Finally, we will demonstrate how such environment has allowed a drastic reduction of data analysis cycle time.

Evaluation of High Throughput Screening Methods in Picking up Differences between Cultivars of Lignocellulosic Biomass for Ethanol Production

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

Lindedam, Jane; Bruun, Sander; Jorgensen, Henning

2014-07-01

Here, we present a unique evaluation of three advanced high throughput pretreatment and enzymatic hydrolysis systems (HTPH-systems) for screening of lignocellulosic biomass for enzymatic saccharification. Straw from 20 cultivars of winter wheat from two sites in Denmark was hydrothermally pretreated and enzymatically processed in each of the separately engineered HTPH-systems at 1) University of California, Riverside, 2) National Renewable Energy Laboratory (NREL), Colorado, and 3) University of Copenhagen (CPH). All three systems were able to detect significant differences between the cultivars in the release of fermentable sugars, with average cellulose conversions of 57%, 64%, and 71% from Riverside, NREL andmore » CPH, respectively. We found the best correlation of glucose yields between the Riverside and NREL systems (R2 = 0.2139), and the best correlation for xylose yields was found between Riverside and CPH (R2 = 0.4269). The three systems identified Flair as the highest yielding cultivar and Dinosor, Glasgow, and Robigus as low yielding cultivars. Despite different conditions in the three HTPH-systems, the approach of microscale screening for phenotypically less recalcitrant feedstock seems sufficiently robust to be used as a generic analytical platform.« less

Development and validation of a high throughput assay for the quantification of multiple green tea-derived catechins in human plasma.

PubMed

Mawson, Deborah H; Jeffrey, Keon L; Teale, Philip; Grace, Philip B

2018-06-19

A rapid, accurate and robust method for the determination of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) concentrations in human plasma has been developed. The method utilises protein precipitation following enzyme hydrolysis, with chromatographic separation and detection using reversed-phase liquid chromatography - tandem mass spectrometry (LC-MS/MS). Traditional issues such as lengthy chromatographic run times, sample and extract stability, and lack of suitable internal standards have been addressed. The method has been evaluated using a comprehensive validation procedure, confirming linearity over appropriate concentration ranges, and inter/intra batch precision and accuracies within suitable thresholds (precisions within 13.8% and accuracies within 12.4%). Recoveries of analytes were found to be consistent between different matrix samples, compensated for using suitable internal markers and within the performance of the instrumentation used. Similarly, chromatographic interferences have been corrected using the internal markers selected. Stability of all analytes in matrix is demonstrated over 32 days and throughout extraction conditions. This method is suitable for high throughput sample analysis studies. This article is protected by copyright. All rights reserved.

In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen

PubMed Central

Plouffe, David; Brinker, Achim; McNamara, Case; Henson, Kerstin; Kato, Nobutaka; Kuhen, Kelli; Nagle, Advait; Adrián, Francisco; Matzen, Jason T.; Anderson, Paul; Nam, Tae-gyu; Gray, Nathanael S.; Chatterjee, Arnab; Janes, Jeff; Yan, S. Frank; Trager, Richard; Caldwell, Jeremy S.; Schultz, Peter G.; Zhou, Yingyao; Winzeler, Elizabeth A.

2008-01-01

The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of ≈1.7 million compounds, we identified a diverse collection of ≈6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 μM). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities. PMID:18579783

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

PubMed

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.

High-Content, High-Throughput Screening for the Identification of Cytotoxic Compounds Based on Cell Morphology and Cell Proliferation Markers

PubMed Central

Martin, Heather L.; Adams, Matthew; Higgins, Julie; Bond, Jacquelyn; Morrison, Ewan E.; Bell, Sandra M.; Warriner, Stuart; Nelson, Adam; Tomlinson, Darren C.

2014-01-01

Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays. PMID:24505478

Applying high-throughput methods to develop a purification process for a highly glycosylated protein.

PubMed

Sanaie, Nooshafarin; Cecchini, Douglas; Pieracci, John

2012-10-01

Micro-scale chromatography formats are becoming more routinely used in purification process development because of their ability to rapidly screen large number of process conditions at a time with minimal material. Given the usual constraints that exist on development timelines and resources, these systems can provide a means to maximize process knowledge and process robustness compared to traditional packed column formats. In this work, a high-throughput, 96-well filter plate format was used in the development of the cation exchange and hydrophobic interaction chromatography steps of a purification process designed to alter the glycoform distribution of a small protein. The significant input parameters affecting process performance were rapidly identified for both steps and preliminary operating conditions were identified. These ranges were verified in a packed chromatography column in order to assess the ability of the 96-well plate to predict packed column performance. In both steps, the 96-well plate format consistently led to underestimated glycoform-enrichment levels and to overestimated product recovery rates compared to the column-based approach. These studies demonstrate that the plate format can be used as a screening tool to narrow the operating ranges prior to further optimization on packed chromatography columns. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multiplexed SNP genotyping using the Qbead™ system: a quantum dot-encoded microsphere-based assay

PubMed Central

Xu, Hongxia; Sha, Michael Y.; Wong, Edith Y.; Uphoff, Janet; Xu, Yanzhang; Treadway, Joseph A.; Truong, Anh; O’Brien, Eamonn; Asquith, Steven; Stubbins, Michael; Spurr, Nigel K.; Lai, Eric H.; Mahoney, Walt

2003-01-01

We have developed a new method using the Qbead™ system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot™ semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral ‘barcodes’ are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein–protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications. PMID:12682378

An infrared optical pacing system for high-throughput screening of cardiac electrophysiology in human cardiomyocytes (Conference Presentation)

NASA Astrophysics Data System (ADS)

McPheeters, Matt T.; Wang, Yves T.; Laurita, Kenneth R.; Jenkins, Michael W.

2017-02-01

Cardiomyocytes derived from human induced pluripotent stem cells (hiPS-HCM) have the potential to provide individualized therapies for patients and to test drug candidates for cardiac toxicity. In order for hiPS-CM to be useful for such applications, there is a need for high-throughput technology to rapidly assess cardiac electrophysiology parameters. Here, we designed and tested a fully contactless optical mapping (OM) and optical pacing (OP) system capable of imaging and point stimulation of hiPS-CM in small wells. OM allowed us to characterize cardiac electrophysiological parameters (conduction velocity, action potential duration, etc.) using voltage-sensitive dyes with high temporal and spatial resolution over the entire well. To improve OM signal-to-noise ratio, we tested a new voltage-sensitive dye (Fluovolt) for accuracy and phototoxicity. Stimulation is essential because most electrophysiological parameters are rate dependent; however, traditional methods utilizing electrical stimulation is difficult in small wells. To overcome this limitation, we utilized OP (λ = 1464 nm) to precisely control heart rate with spatial precision without the addition of exogenous agents. We optimized OP parameters (e.g., well size, pulse width, spot size) to achieve robust pacing and minimize the threshold radiant exposure. Finally, we tested system sensitivity using Flecainide, a drug with well described action on multiple electrophysiological properties.

High throughput-screening of animal urine samples: It is fast but is it also reliable?

PubMed

Kaufmann, Anton

2016-05-01

Advanced analytical technologies like ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry can be used for veterinary drug screening of animal urine. The technique is sufficiently robust and reliable to detect veterinary drugs in urine samples of animals where the maximum residue limit of these compounds in organs like muscle, kidney, or liver has been exceeded. The limitations and possibilities of the technique are discussed. The most critical point is the variability of the drug concentration ratio between the tissue and urine. Ways to manage the false positive and false negatives are discussed. The capability to confirm findings and the possibility of semi-targeted analysis are also addressed. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

High throughput photo-oxidations in a packed bed reactor system.

PubMed

Kong, Caleb J; Fisher, Daniel; Desai, Bimbisar K; Yang, Yuan; Ahmad, Saeed; Belecki, Katherine; Gupton, B Frank

2017-12-01

The efficiency gains produced by continuous-flow systems in conducting photochemical transformations have been extensively demonstrated. Recently, these systems have been used in developing safe and efficient methods for photo-oxidations using singlet oxygen generated by photosensitizers. Much of the previous work has focused on the use of homogeneous photocatalysts. The development of a unique, packed-bed photoreactor system using immobilized rose bengal expands these capabilities as this robust photocatalyst allows access to and elaboration from these highly useful building blocks without the need for further purification. With this platform we were able to demonstrate a wide scope of singlet oxygen ene, [4+2] cycloadditions and heteroatom oxidations. Furthermore, we applied this method as a strategic element in the synthesis of the high-volume antimalarial artemisinin. Copyright © 2017. Published by Elsevier Ltd.

From Lab to Fab: Developing a Nanoscale Delivery Tool for Scalable Nanomanufacturing

NASA Astrophysics Data System (ADS)

Safi, Asmahan A.

The emergence of nanomaterials with unique properties at the nanoscale over the past two decades carries a capacity to impact society and transform or create new industries ranging from nanoelectronics to nanomedicine. However, a gap in nanomanufacturing technologies has prevented the translation of nanomaterial into real-world commercialized products. Bridging this gap requires a paradigm shift in methods for fabricating structured devices with a nanoscale resolution in a repeatable fashion. This thesis explores the new paradigms for fabricating nanoscale structures devices and systems for high throughput high registration applications. We present a robust and scalable nanoscale delivery platform, the Nanofountain Probe (NFP), for parallel direct-write of functional materials. The design and microfabrication of NFP is presented. The new generation addresses the challenges of throughput, resolution and ink replenishment characterizing tip-based nanomanufacturing. To achieve these goals, optimized probe geometry is integrated to the process along with channel sealing and cantilever bending. The capabilities of the newly fabricated probes are demonstrated through two type of delivery: protein nanopatterning and single cell nanoinjection. The broad applications of the NFP for single cell delivery are investigated. An external microfluidic packaging is developed to enable delivery in liquid environment. The system is integrated to a combined atomic force microscope and inverted fluorescence microscope. Intracellular delivery is demonstrated by injecting a fluorescent dextran into Hela cells in vitro while monitoring the injection forces. Such developments enable in vitro cellular delivery for single cell studies and high throughput gene expression. The nanomanufacturing capabilities of NFPs are explored. Nanofabrication of carbon nanotube-based electronics presents all the manufacturing challenges characterizing of assembling nanomaterials precisely onto devices. The presented study combines top-down and bottom-approaches by integrating the catalyst patterning and carbon nanotube growth directly on structures. Large array of iron-rich catalyst are patterned on an substrate for subsequent carbon nanotubes synthesis. The dependence of probe geometry and substrate wetting is assessed by modeling and experimental studies. Finally preliminary results on synthesis of carbon nanotube by catalyst assisted chemical vapor deposition suggest increasing the catalyst yield is critical. Such work will enable high throughput nanomanufacturing of carbon nanotube based devices.

Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies

PubMed Central

Asati, Atul; Kachurina, Olga; Karol, Alex; Dhir, Vipra; Nguyen, Michael; Parkhill, Robert; Kouiavskaia, Diana; Chumakov, Konstantin; Warren, William; Kachurin, Anatoly

2016-01-01

Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013–2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of Biologics Evaluation and Research, with plaque reduction neutralization test performed by Focus Diagnostics, and with hemaglutination inhibition assay performed in-house at Sanofi Pasteur. Taken together, fADI assay appears to be a useful high throughput functional immunoassay for assessment of antibody-related neutralization of the viral infections for which pre-attachment neutralization pathway is predominant, such as polio, influenza, yellow fever and dengue. PMID:26863313

Multi-shot PROPELLER for high-field preclinical MRI

PubMed Central

Pandit, Prachi; Qi, Yi; Story, Jennifer; King, Kevin F.; Johnson, G. Allan

2012-01-01

With the development of numerous mouse models of cancer, there is a tremendous need for an appropriate imaging technique to study the disease evolution. High-field T2-weighted imaging using PROPELLER MRI meets this need. The 2-shot PROPELLER technique presented here, provides (a) high spatial resolution, (b) high contrast resolution, and (c) rapid and non-invasive imaging, which enables high-throughput, longitudinal studies in free-breathing mice. Unique data collection and reconstruction makes this method robust against motion artifacts. The 2-shot modification introduced here, retains more high-frequency information and provides higher SNR than conventional single-shot PROPELLER, making this sequence feasible at high-fields, where signal loss is rapid. Results are shown in a liver metastases model to demonstrate the utility of this technique in one of the more challenging regions of the mouse, which is the abdomen. PMID:20572138

Multishot PROPELLER for high-field preclinical MRI.

PubMed

Pandit, Prachi; Qi, Yi; Story, Jennifer; King, Kevin F; Johnson, G Allan

2010-07-01

With the development of numerous mouse models of cancer, there is a tremendous need for an appropriate imaging technique to study the disease evolution. High-field T(2)-weighted imaging using PROPELLER (Periodically Rotated Overlapping ParallEL Lines with Enhanced Reconstruction) MRI meets this need. The two-shot PROPELLER technique presented here provides (a) high spatial resolution, (b) high contrast resolution, and (c) rapid and noninvasive imaging, which enables high-throughput, longitudinal studies in free-breathing mice. Unique data collection and reconstruction makes this method robust against motion artifacts. The two-shot modification introduced here retains more high-frequency information and provides higher signal-to-noise ratio than conventional single-shot PROPELLER, making this sequence feasible at high fields, where signal loss is rapid. Results are shown in a liver metastases model to demonstrate the utility of this technique in one of the more challenging regions of the mouse, which is the abdomen. (c) 2010 Wiley-Liss, Inc.

A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.

PubMed

Xie, Wensheng; Silvers, Robert; Ouellette, Michael; Wu, Zining; Lu, Quinn; Li, Hu; Gallagher, Kathleen; Johnson, Kathy; Montoute, Monica

2016-01-01

Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic β-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase.

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

PubMed Central

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

2015-01-01

The use of high-throughput phenotyping systems and non-destructive imaging is widely regarded as a key technology allowing scientists and breeders to develop crops with the ability to perform well under diverse environmental conditions. However, many of these phenotyping studies have been optimized using the model plant Arabidopsis thaliana. In this study, The Plant Accelerator® at The University of Adelaide, Australia, was used to investigate the growth and phenotypic response of the important cereal crop, Sorghum bicolor L. Moench and related hybrids to water-limited conditions and different levels of fertilizer. Imaging in different spectral ranges was used to monitor plant composition, chlorophyll, and moisture content. Phenotypic image analysis accurately measured plant biomass. The data set obtained enabled the responses of the different sorghum varieties to the experimental treatments to be differentiated and modelled. Plant architectural instead of architecture elements were determined using imaging and found to correlate with an improved tolerance to stress, for example diurnal leaf curling and leaf area index. Analysis of colour images revealed that leaf ‘greenness’ correlated with foliar nitrogen and chlorophyll, while near infrared reflectance (NIR) analysis was a good predictor of water content and leaf thickness, and correlated with plant moisture content. It is shown that imaging sorghum using a high-throughput system can accurately identify and differentiate between growth and specific phenotypic traits. R scripts for robust, parsimonious models are provided to allow other users of phenomic imaging systems to extract useful data readily, and thus relieve a bottleneck in phenotypic screening of multiple genotypes of key crop plants. PMID:25697789

Direct RNA detection without nucleic acid purification and PCR: Combining sandwich hybridization with signal amplification based on branched hybridization chain reaction.

PubMed

Xu, Yao; Zheng, Zhi

2016-05-15

We have developed a convenient, robust and low-cost RNA detection system suitable for high-throughput applications. This system uses a highly specific sandwich hybridization to capture target RNA directly onto solid support, followed by on-site signal amplification via 2-dimensional, branched hybridizing chain polymerization through toehold-mediated strand displacement reaction. The assay uses SYBR Green to detect targets at concentrations as low as 1 pM, without involving nucleic acid purification or any enzymatic reaction, using ordinary oligonucleotides without modification or labeling. The system was demonstrated in the detection of malaria RNA in blood and GAPDH gene expression in cell lysate. Copyright © 2015 Elsevier B.V. All rights reserved.

Tape transfer printing of a liquid metal alloy for stretchable RF electronics.

PubMed

Jeong, Seung Hee; Hjort, Klas; Wu, Zhigang

2014-09-03

In order to make conductors with large cross sections for low impedance radio frequency (RF) electronics, while still retaining high stretchability, liquid-alloy-based microfluidic stretchable electronics offers stretchable electronic systems the unique opportunity to combine various sensors on our bodies or organs with high-quality wireless communication with the external world (devices/systems), without sacrificing enhanced user comfort. This microfluidic approach, based on printed circuit board technology, allows large area processing of large cross section conductors and robust contacts, which can handle a lot of stretching between the embedded rigid active components and the surrounding system. Although it provides such benefits, further development is needed to realize its potential as a high throughput, cost-effective process technology. In this paper, tape transfer printing is proposed to supply a rapid prototyping batch process at low cost, albeit at a low resolution of 150 μm. In particular, isolated patterns can be obtained in a simple one-step process. Finally, a stretchable radio frequency identification (RFID) tag is demonstrated. The measured results show the robustness of the hybrid integrated system when the tag is stretched at 50% for 3000 cycles.

A robust multi-shot scan strategy for high-resolution diffusion weighted MRI enabled by multiplexed sensitivity-encoding (MUSE)

PubMed Central

Chen, Nan-kuei; Guidon, Arnaud; Chang, Hing-Chiu; Song, Allen W.

2013-01-01

Diffusion weighted magnetic resonance imaging (DWI) data have been mostly acquired with single-shot echo-planar imaging (EPI) to minimize motion induced artifacts. The spatial resolution, however, is inherently limited in single-shot EPI, even when the parallel imaging (usually at an acceleration factor of 2) is incorporated. Multi-shot acquisition strategies could potentially achieve higher spatial resolution and fidelity, but they are generally susceptible to motion-induced phase errors among excitations that are exacerbated by diffusion sensitizing gradients, rendering the reconstructed images unusable. It has been shown that shot-to-shot phase variations may be corrected using navigator echoes, but at the cost of imaging throughput. To address these challenges, a novel and robust multi-shot DWI technique, termed multiplexed sensitivity-encoding (MUSE), is developed here to reliably and inherently correct nonlinear shot-to-shot phase variations without the use of navigator echoes. The performance of the MUSE technique is confirmed experimentally in healthy adult volunteers on 3 Tesla MRI systems. This newly developed technique should prove highly valuable for mapping brain structures and connectivities at high spatial resolution for neuroscience studies. PMID:23370063

An open-source computational and data resource to analyze digital maps of immunopeptidomes

DOE PAGES

Caron, Etienne; Espona, Lucia; Kowalewski, Daniel J.; ...

2015-07-08

We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.

A computational image analysis glossary for biologists.

PubMed

Roeder, Adrienne H K; Cunha, Alexandre; Burl, Michael C; Meyerowitz, Elliot M

2012-09-01

Recent advances in biological imaging have resulted in an explosion in the quality and quantity of images obtained in a digital format. Developmental biologists are increasingly acquiring beautiful and complex images, thus creating vast image datasets. In the past, patterns in image data have been detected by the human eye. Larger datasets, however, necessitate high-throughput objective analysis tools to computationally extract quantitative information from the images. These tools have been developed in collaborations between biologists, computer scientists, mathematicians and physicists. In this Primer we present a glossary of image analysis terms to aid biologists and briefly discuss the importance of robust image analysis in developmental studies.

Commentary: The Materials Project: A materials genome approach to accelerating materials innovation

NASA Astrophysics Data System (ADS)

Jain, Anubhav; Ong, Shyue Ping; Hautier, Geoffroy; Chen, Wei; Richards, William Davidson; Dacek, Stephen; Cholia, Shreyas; Gunter, Dan; Skinner, David; Ceder, Gerbrand; Persson, Kristin A.

2013-07-01

Accelerating the discovery of advanced materials is essential for human welfare and sustainable, clean energy. In this paper, we introduce the Materials Project (www.materialsproject.org), a core program of the Materials Genome Initiative that uses high-throughput computing to uncover the properties of all known inorganic materials. This open dataset can be accessed through multiple channels for both interactive exploration and data mining. The Materials Project also seeks to create open-source platforms for developing robust, sophisticated materials analyses. Future efforts will enable users to perform ``rapid-prototyping'' of new materials in silico, and provide researchers with new avenues for cost-effective, data-driven materials design.

Matrix preconditioning: a robust operation for optical linear algebra processors.

PubMed

Ghosh, A; Paparao, P

1987-07-15

Analog electrooptical processors are best suited for applications demanding high computational throughput with tolerance for inaccuracies. Matrix preconditioning is one such application. Matrix preconditioning is a preprocessing step for reducing the condition number of a matrix and is used extensively with gradient algorithms for increasing the rate of convergence and improving the accuracy of the solution. In this paper, we describe a simple parallel algorithm for matrix preconditioning, which can be implemented efficiently on a pipelined optical linear algebra processor. From the results of our numerical experiments we show that the efficacy of the preconditioning algorithm is affected very little by the errors of the optical system.

High-throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA

PubMed Central

Schaaf, Tory M.; Peterson, Kurt C.; Grant, Benjamin D.; Bawaskar, Prachi; Yuen, Samantha; Li, Ji; Muretta, Joseph M.; Gillispie, Gregory D.; Thomas, David D.

2017-01-01

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green (GFP, donor) and red (RFP, acceptor) fluorescent proteins fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS. PMID:27899691

MultiGeMS: detection of SNVs from multiple samples using model selection on high-throughput sequencing data.

PubMed

Murillo, Gabriel H; You, Na; Su, Xiaoquan; Cui, Wei; Reilly, Muredach P; Li, Mingyao; Ning, Kang; Cui, Xinping

2016-05-15

Single nucleotide variant (SNV) detection procedures are being utilized as never before to analyze the recent abundance of high-throughput DNA sequencing data, both on single and multiple sample datasets. Building on previously published work with the single sample SNV caller genotype model selection (GeMS), a multiple sample version of GeMS (MultiGeMS) is introduced. Unlike other popular multiple sample SNV callers, the MultiGeMS statistical model accounts for enzymatic substitution sequencing errors. It also addresses the multiple testing problem endemic to multiple sample SNV calling and utilizes high performance computing (HPC) techniques. A simulation study demonstrates that MultiGeMS ranks highest in precision among a selection of popular multiple sample SNV callers, while showing exceptional recall in calling common SNVs. Further, both simulation studies and real data analyses indicate that MultiGeMS is robust to low-quality data. We also demonstrate that accounting for enzymatic substitution sequencing errors not only improves SNV call precision at low mapping quality regions, but also improves recall at reference allele-dominated sites with high mapping quality. The MultiGeMS package can be downloaded from https://github.com/cui-lab/multigems xinping.cui@ucr.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Single Nucleobase Identification Using Biophysical Signatures from Nanoelectronic Quantum Tunneling.

PubMed

Korshoj, Lee E; Afsari, Sepideh; Khan, Sajida; Chatterjee, Anushree; Nagpal, Prashant

2017-03-01

Nanoelectronic DNA sequencing can provide an important alternative to sequencing-by-synthesis by reducing sample preparation time, cost, and complexity as a high-throughput next-generation technique with accurate single-molecule identification. However, sample noise and signature overlap continue to prevent high-resolution and accurate sequencing results. Probing the molecular orbitals of chemically distinct DNA nucleobases offers a path for facile sequence identification, but molecular entropy (from nucleotide conformations) makes such identification difficult when relying only on the energies of lowest-unoccupied and highest-occupied molecular orbitals (LUMO and HOMO). Here, nine biophysical parameters are developed to better characterize molecular orbitals of individual nucleobases, intended for single-molecule DNA sequencing using quantum tunneling of charges. For this analysis, theoretical models for quantum tunneling are combined with transition voltage spectroscopy to obtain measurable parameters unique to the molecule within an electronic junction. Scanning tunneling spectroscopy is then used to measure these nine biophysical parameters for DNA nucleotides, and a modified machine learning algorithm identified nucleobases. The new parameters significantly improve base calling over merely using LUMO and HOMO frontier orbital energies. Furthermore, high accuracies for identifying DNA nucleobases were observed at different pH conditions. These results have significant implications for developing a robust and accurate high-throughput nanoelectronic DNA sequencing technique. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Strategic and Operational Plan for Integrating Transcriptomics ...

EPA Pesticide Factsheets

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

High-Throughput Experimental Approach Capabilities | Materials Science |

Science.gov Websites

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

A Novel, Poly-Etalon, Fabry-Perot for Planetary Research

NASA Technical Reports Server (NTRS)

Kerr, Robert B.; Doe, Richard; Noto, John

1997-01-01

In an effort to develop a mechanically robust, high throughput and solid state spectrometer several liquid crystal Fabry-Perot etalons were constructed. The etalons were tested for spectral response, radiation resistance and optical transmission. The first year of this project was spent developing and understanding the properties of the liquid crystal etalons; in the second year an intensified all-sky imaging system was developed around a pair of LC etalons. The imaging system, developed jointly with SRI International represents a unique brassboard to demonstrate the use of LC etalons as tunable filters. The first set of etalons constructed in year one of this project were tested for spectral response and throughput while etalon surrogates were exposed to proton radiation simulating the exposure of an object in Low Earth Orbit (LEO). The 2" diameter etalons had a measure finesse of approximately 10 and were tunable over five orders. Liquid crystals exposed to proton irradiation showed no signs of damage. In year two two larger diameter (3") etalons were constructed with gaps of 3 and 5 microns. This pair of etalons is for use in a high resolution, all-sky spectral imager. The WATUMI imager system follows the heritage of all sky, narrow band, intensified imagers however it includes two LC Fabry-Perot etalons to provide tunability and the ability to switch wavelengths rapidly, an import consideration in auroral airglow imaging. This work also resulted in two publications and one poster presentation. The instrument will be uniquely capable, with superior throughput and speed, to measure optical airglow of multiple emission lines in harsh conditions.

Development of a high-throughput brain slice method for studying drug distribution in the central nervous system.

PubMed

Fridén, Markus; Ducrozet, Frederic; Middleton, Brian; Antonsson, Madeleine; Bredberg, Ulf; Hammarlund-Udenaes, Margareta

2009-06-01

New, more efficient methods of estimating unbound drug concentrations in the central nervous system (CNS) combine the amount of drug in whole brain tissue samples measured by conventional methods with in vitro estimates of the unbound brain volume of distribution (V(u,brain)). Although the brain slice method is the most reliable in vitro method for measuring V(u,brain), it has not previously been adapted for the needs of drug discovery research. The aim of this study was to increase the throughput and optimize the experimental conditions of this method. Equilibrium of drug between the buffer and the brain slice within the 4 to 5 h of incubation is a fundamental requirement. However, it is difficult to meet this requirement for many of the extensively binding, lipophilic compounds in drug discovery programs. In this study, the dimensions of the incubation vessel and mode of stirring influenced the equilibration time, as did the amount of brain tissue per unit of buffer volume. The use of cassette experiments for investigating V(u,brain) in a linear drug concentration range increased the throughput of the method. The V(u,brain) for the model compounds ranged from 4 to 3000 ml . g brain(-1), and the sources of variability are discussed. The optimized setup of the brain slice method allows precise, robust estimation of V(u,brain) for drugs with diverse properties, including highly lipophilic compounds. This is a critical step forward for the implementation of relevant measurements of CNS exposure in the drug discovery setting.

Vortex coronagraphs for the Habitable Exoplanet Imaging Mission concept: theoretical performance and telescope requirements

NASA Astrophysics Data System (ADS)

Ruane, Garreth; Mawet, Dimitri; Mennesson, Bertrand; Jewell, Jeffrey; Shaklan, Stuart

2018-01-01

The Habitable Exoplanet Imaging Mission concept requires an optical coronagraph that provides deep starlight suppression over a broad spectral bandwidth, high throughput for point sources at small angular separation, and insensitivity to temporally varying, low-order aberrations. Vortex coronagraphs are a promising solution that performs optimally on off-axis, monolithic telescopes and may also be designed for segmented telescopes with minor losses in performance. We describe the key advantages of vortex coronagraphs on off-axis telescopes such as (1) unwanted diffraction due to aberrations is passively rejected in several low-order Zernike modes relaxing the wavefront stability requirements for imaging Earth-like planets from <10 to >100 pm rms, (2) stars with angular diameters >0.1 λ / D may be sufficiently suppressed, (3) the absolute planet throughput is >10 % , even for unfavorable telescope architectures, and (4) broadband solutions (Δλ / λ > 0.1) are readily available for both monolithic and segmented apertures. The latter make use of grayscale apodizers in an upstream pupil plane to provide suppression of diffracted light from amplitude discontinuities in the telescope pupil without inducing additional stroke on the deformable mirrors. We set wavefront stability requirements on the telescope, based on a stellar irradiance threshold set at an angular separation of 3 ± 0.5λ / D from the star, and discuss how some requirements may be relaxed by trading robustness to aberrations for planet throughput.

Decreasing-Rate Pruning Optimizes the Construction of Efficient and Robust Distributed Networks.

PubMed

Navlakha, Saket; Barth, Alison L; Bar-Joseph, Ziv

2015-07-01

Robust, efficient, and low-cost networks are advantageous in both biological and engineered systems. During neural network development in the brain, synapses are massively over-produced and then pruned-back over time. This strategy is not commonly used when designing engineered networks, since adding connections that will soon be removed is considered wasteful. Here, we show that for large distributed routing networks, network function is markedly enhanced by hyper-connectivity followed by aggressive pruning and that the global rate of pruning, a developmental parameter not previously studied by experimentalists, plays a critical role in optimizing network structure. We first used high-throughput image analysis techniques to quantify the rate of pruning in the mammalian neocortex across a broad developmental time window and found that the rate is decreasing over time. Based on these results, we analyzed a model of computational routing networks and show using both theoretical analysis and simulations that decreasing rates lead to more robust and efficient networks compared to other rates. We also present an application of this strategy to improve the distributed design of airline networks. Thus, inspiration from neural network formation suggests effective ways to design distributed networks across several domains.

Decreasing-Rate Pruning Optimizes the Construction of Efficient and Robust Distributed Networks

PubMed Central

Navlakha, Saket; Barth, Alison L.; Bar-Joseph, Ziv

2015-01-01

Robust, efficient, and low-cost networks are advantageous in both biological and engineered systems. During neural network development in the brain, synapses are massively over-produced and then pruned-back over time. This strategy is not commonly used when designing engineered networks, since adding connections that will soon be removed is considered wasteful. Here, we show that for large distributed routing networks, network function is markedly enhanced by hyper-connectivity followed by aggressive pruning and that the global rate of pruning, a developmental parameter not previously studied by experimentalists, plays a critical role in optimizing network structure. We first used high-throughput image analysis techniques to quantify the rate of pruning in the mammalian neocortex across a broad developmental time window and found that the rate is decreasing over time. Based on these results, we analyzed a model of computational routing networks and show using both theoretical analysis and simulations that decreasing rates lead to more robust and efficient networks compared to other rates. We also present an application of this strategy to improve the distributed design of airline networks. Thus, inspiration from neural network formation suggests effective ways to design distributed networks across several domains. PMID:26217933

A 181 GOPS AKAZE Accelerator Employing Discrete-Time Cellular Neural Networks for Real-Time Feature Extraction.

PubMed

Jiang, Guangli; Liu, Leibo; Zhu, Wenping; Yin, Shouyi; Wei, Shaojun

2015-09-04

This paper proposes a real-time feature extraction VLSI architecture for high-resolution images based on the accelerated KAZE algorithm. Firstly, a new system architecture is proposed. It increases the system throughput, provides flexibility in image resolution, and offers trade-offs between speed and scaling robustness. The architecture consists of a two-dimensional pipeline array that fully utilizes computational similarities in octaves. Secondly, a substructure (block-serial discrete-time cellular neural network) that can realize a nonlinear filter is proposed. This structure decreases the memory demand through the removal of data dependency. Thirdly, a hardware-friendly descriptor is introduced in order to overcome the hardware design bottleneck through the polar sample pattern; a simplified method to realize rotation invariance is also presented. Finally, the proposed architecture is designed in TSMC 65 nm CMOS technology. The experimental results show a performance of 127 fps in full HD resolution at 200 MHz frequency. The peak performance reaches 181 GOPS and the throughput is double the speed of other state-of-the-art architectures.

In Vivo High-Content Evaluation of Three-Dimensional Scaffolds Biocompatibility

PubMed Central

Oliveira, Mariana B.; Ribeiro, Maximiano P.; Miguel, Sónia P.; Neto, Ana I.; Coutinho, Paula; Correia, Ilídio J.

2014-01-01

While developing tissue engineering strategies, inflammatory response caused by biomaterials is an unavoidable aspect to be taken into consideration, as it may be an early limiting step of tissue regeneration approaches. We demonstrate the application of flat and flexible films exhibiting patterned high-contrast wettability regions as implantable platforms for the high-content in vivo study of inflammatory response caused by biomaterials. Screening biomaterials by using high-throughput platforms is a powerful method to detect hit spots with promising properties and to exclude uninteresting conditions for targeted applications. High-content analysis of biomaterials has been mostly restricted to in vitro tests where crucial information is lost, as in vivo environment is highly complex. Conventional biomaterials implantation requires the use of high numbers of animals, leading to ethical questions and costly experimentation. Inflammatory response of biomaterials has also been highly neglected in high-throughput studies. We designed an array of 36 combinations of biomaterials based on an initial library of four polysaccharides. Biomaterials were dispensed onto biomimetic superhydrophobic platforms with wettable regions and processed as freeze-dried three-dimensional scaffolds with a high control of the array configuration. These chips were afterward implanted subcutaneously in Wistar rats. Lymphocyte recruitment and activated macrophages were studied on-chip, by performing immunocytochemistry in the miniaturized biomaterials after 24 h and 7 days of implantation. Histological cuts of the surrounding tissue of the implants were also analyzed. Localized and independent inflammatory responses were detected. The integration of these data with control data proved that these chips are robust platforms for the rapid screening of early-stage in vivo biomaterials' response. PMID:24568682

Computational toxicology as implemented by the U.S. EPA: providing high throughput decision support tools for screening and assessing chemical exposure, hazard and risk.

PubMed

Kavlock, Robert; Dix, David

2010-02-01

Computational toxicology is the application of mathematical and computer models to help assess chemical hazards and risks to human health and the environment. Supported by advances in informatics, high-throughput screening (HTS) technologies, and systems biology, the U.S. Environmental Protection Agency EPA is developing robust and flexible computational tools that can be applied to the thousands of chemicals in commerce, and contaminant mixtures found in air, water, and hazardous-waste sites. The Office of Research and Development (ORD) Computational Toxicology Research Program (CTRP) is composed of three main elements. The largest component is the National Center for Computational Toxicology (NCCT), which was established in 2005 to coordinate research on chemical screening and prioritization, informatics, and systems modeling. The second element consists of related activities in the National Health and Environmental Effects Research Laboratory (NHEERL) and the National Exposure Research Laboratory (NERL). The third and final component consists of academic centers working on various aspects of computational toxicology and funded by the U.S. EPA Science to Achieve Results (STAR) program. Together these elements form the key components in the implementation of both the initial strategy, A Framework for a Computational Toxicology Research Program (U.S. EPA, 2003), and the newly released The U.S. Environmental Protection Agency's Strategic Plan for Evaluating the Toxicity of Chemicals (U.S. EPA, 2009a). Key intramural projects of the CTRP include digitizing legacy toxicity testing information toxicity reference database (ToxRefDB), predicting toxicity (ToxCast) and exposure (ExpoCast), and creating virtual liver (v-Liver) and virtual embryo (v-Embryo) systems models. U.S. EPA-funded STAR centers are also providing bioinformatics, computational toxicology data and models, and developmental toxicity data and models. The models and underlying data are being made publicly available through the Aggregated Computational Toxicology Resource (ACToR), the Distributed Structure-Searchable Toxicity (DSSTox) Database Network, and other U.S. EPA websites. While initially focused on improving the hazard identification process, the CTRP is placing increasing emphasis on using high-throughput bioactivity profiling data in systems modeling to support quantitative risk assessments, and in developing complementary higher throughput exposure models. This integrated approach will enable analysis of life-stage susceptibility, and understanding of the exposures, pathways, and key events by which chemicals exert their toxicity in developing systems (e.g., endocrine-related pathways). The CTRP will be a critical component in next-generation risk assessments utilizing quantitative high-throughput data and providing a much higher capacity for assessing chemical toxicity than is currently available.

DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors

NASA Astrophysics Data System (ADS)

Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana

2017-03-01

Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.

MEMS-based beam-steerable free-space optical communication link for reconfigurable wireless data center

NASA Astrophysics Data System (ADS)

Deng, Peng; Kavehrad, Mohsen; Lou, Yan

2017-01-01

Flexible wireless datacenter networks based on free space optical communication (FSO) links are being considered as promising solutions to meet the future datacenter demands of high throughput, robustness to dynamic traffic patterns, cabling complexity and energy efficiency. Robust and precise steerable FSO links over dynamic traffic play a key role in the reconfigurable optical wireless datacenter inter-rack network. In this work, we propose and demonstrate a reconfigurable 10Gbps FSO system incorporated with smart beam acquisition and tracking mechanism based on gimballess two-axis MEMS micro-mirror and retro-reflective film marked aperture. The fast MEMS-based beam acquisition switches laser beam of FSO terminal from one rack to the next for reconfigurable networks, and the precise beam tracking makes FSO device auto-correct the misalignment in real-time. We evaluate the optical power loss and bit error rate performance of steerable FSO links at various directions. Experimental results suggest that the MEMS based beam steerable FSO links hold considerable promise for the future reconfigurable wireless datacenter networks.

Discovery of highly selective brain-penetrant vasopressin 1a antagonists for the potential treatment of autism via a chemogenomic and scaffold hopping approach.

PubMed

Ratni, Hasane; Rogers-Evans, Mark; Bissantz, Caterina; Grundschober, Christophe; Moreau, Jean-Luc; Schuler, Franz; Fischer, Holger; Alvarez Sanchez, Ruben; Schnider, Patrick

2015-03-12

From a micromolar high throughput screening hit 7, the successful complementary application of a chemogenomic approach and of a scaffold hopping exercise rapidly led to a low single digit nanomolar human vasopressin 1a (hV1a) receptor antagonist 38. Initial optimization of the mouse V1a activities delivered suitable tool compounds which demonstrated a V1a mediated central in vivo effect. This novel series was further optimized through parallel synthesis with a focus on balancing lipophilicity to achieve robust aqueous solubility while avoiding P-gp mediated efflux. These efforts led to the discovery of the highly potent and selective brain-penetrant hV1a antagonist RO5028442 (8) suitable for human clinical studies in people with autism.

Criteria for predicting the formation of single-phase high-entropy alloys

DOE PAGES

Troparevsky, M Claudia; Morris, James R..; Kent, Paul R.; ...

2015-03-15

High entropy alloys constitute a new class of materials whose very existence poses fundamental questions. Originally thought to be stabilized by the large entropy of mixing, these alloys have attracted attention due to their potential applications, yet no model capable of robustly predicting which combinations of elements will form a single-phase currently exists. Here we propose a model that, through the use of high-throughput computation of the enthalpies of formation of binary compounds, is able to confirm all known high-entropy alloys while rejecting similar alloys that are known to form multiple phases. Despite the increasing entropy, our model predicts thatmore » the number of potential single-phase multicomponent alloys decreases with an increasing number of components: out of more than two million possible 7-component alloys considered, fewer than twenty single-phase alloys are likely.« less

Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography

PubMed Central

Mueller, C.; Marx, A.; Epp, S. W.; Zhong, Y.; Kuo, A.; Balo, A. R.; Soman, J.; Schotte, F.; Lemke, H. T.; Owen, R. L.; Pai, E. F.; Pearson, A. R.; Olson, J. S.; Anfinrud, P. A.; Ernst, O. P.; Dwayne Miller, R. J.

2015-01-01

We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). The chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs. PMID:26798825

Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography.

PubMed

Mueller, C; Marx, A; Epp, S W; Zhong, Y; Kuo, A; Balo, A R; Soman, J; Schotte, F; Lemke, H T; Owen, R L; Pai, E F; Pearson, A R; Olson, J S; Anfinrud, P A; Ernst, O P; Dwayne Miller, R J

2015-09-01

We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). The chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.

Machine learning bandgaps of double perovskites

PubMed Central

Pilania, G.; Mannodi-Kanakkithodi, A.; Uberuaga, B. P.; Ramprasad, R.; Gubernatis, J. E.; Lookman, T.

2016-01-01

The ability to make rapid and accurate predictions on bandgaps of double perovskites is of much practical interest for a range of applications. While quantum mechanical computations for high-fidelity bandgaps are enormously computation-time intensive and thus impractical in high throughput studies, informatics-based statistical learning approaches can be a promising alternative. Here we demonstrate a systematic feature-engineering approach and a robust learning framework for efficient and accurate predictions of electronic bandgaps of double perovskites. After evaluating a set of more than 1.2 million features, we identify lowest occupied Kohn-Sham levels and elemental electronegativities of the constituent atomic species as the most crucial and relevant predictors. The developed models are validated and tested using the best practices of data science and further analyzed to rationalize their prediction performance. PMID:26783247

Managing evaporation for more robust microscale assays. Part 2. Characterization of convection and diffusion for cell biology.

PubMed

Berthier, Erwin; Warrick, Jay; Yu, Hongmeiy; Beebe, David J

2008-06-01

Cell based microassays allow the screening of a multitude of culture conditions in parallel, which can be used for various applications from drug screening to fundamental cell biology research. Tubeless microfluidic devices based on passive pumping are a step towards accessible high throughput microassays, however they are vulnerable to evaporation. In addition to volume loss, evaporation can lead to the generation of small flows. Here, we focus on issues of convection and diffusion for cell culture in microchannels and particularly the transport of soluble factors secreted by cells. We find that even for humidity levels as high as 95%, convection in a passive pumping channel can significantly alter distributions of these factors and that appropriate system design can prevent convection.

Receptor binding assay for paralytic shellfish poisoning toxins: optimization and interlaboratory comparison.

PubMed

Ruberu, Shryamalie R; Liu, Yun-Gang; Wong, Carolyn T; Perera, S Kusum; Langlois, Gregg W; Doucette, Gregory J; Powell, Christine L

2003-01-01

A receptor binding assay (RBA) for detection of paralytic shellfish poisoning (PSP) toxins was formatted for use in a high throughput detection system using microplate scintillation counting. The RBA technology was transferred from the National Ocean Service, which uses a Wallac TriLux 1450 MicroBeta microplate scintillation counter, to the California Department of Health Services, which uses a Packard TopCount scintillation counter. Due to differences in the detector arrangement between these 2 counters, markedly different counting efficiencies were exhibited, requiring optimization of the RBA protocol for the TopCount instrument. Precision, accuracy, and sensitivity [limit of detection = 0.2 microg saxitoxin (STX) equiv/100 g shellfish tissue] of the modified protocol were equivalent to those of the original protocol. The RBA robustness and adaptability were demonstrated by an interlaboratory study, in which STX concentrations in shellfish generated by the TopCount were consistent with MicroBeta-derived values. Comparison of STX reference standards obtained from the U.S. Food and Drug Administration and the National Research Council, Canada, showed no observable differences. This study confirms the RBA's value as a rapid, high throughput screen prior to testing by the conventional mouse bioassay (MBA) and its suitability for providing an early warning of increasing PSP toxicity when toxin levels are below the MBA limit of detection.

Uncertainty Quantification in High Throughput Screening ...

EPA Pesticide Factsheets

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

Coprolites as a source of information on the genome and diet of the cave hyena

PubMed Central

Bon, Céline; Berthonaud, Véronique; Maksud, Frédéric; Labadie, Karine; Poulain, Julie; Artiguenave, François; Wincker, Patrick; Aury, Jean-Marc; Elalouf, Jean-Marc

2012-01-01

We performed high-throughput sequencing of DNA from fossilized faeces to evaluate this material as a source of information on the genome and diet of Pleistocene carnivores. We analysed coprolites derived from the extinct cave hyena (Crocuta crocuta spelaea), and sequenced 90 million DNA fragments from two specimens. The DNA reads enabled a reconstruction of the cave hyena mitochondrial genome with up to a 158-fold coverage. This genome, and those sequenced from extant spotted (Crocuta crocuta) and striped (Hyaena hyaena) hyena specimens, allows for the establishment of a robust phylogeny that supports a close relationship between the cave and the spotted hyena. We also demonstrate that high-throughput sequencing yields data for cave hyena multi-copy and single-copy nuclear genes, and that about 50 per cent of the coprolite DNA can be ascribed to this species. Analysing the data for additional species to indicate the cave hyena diet, we retrieved abundant sequences for the red deer (Cervus elaphus), and characterized its mitochondrial genome with up to a 3.8-fold coverage. In conclusion, we have demonstrated the presence of abundant ancient DNA in the coprolites surveyed. Shotgun sequencing of this material yielded a wealth of DNA sequences for a Pleistocene carnivore and allowed unbiased identification of diet. PMID:22456883

Investigating the viral ecology of global bee communities with high-throughput metagenomics.

PubMed

Galbraith, David A; Fuller, Zachary L; Ray, Allyson M; Brockmann, Axel; Frazier, Maryann; Gikungu, Mary W; Martinez, J Francisco Iturralde; Kapheim, Karen M; Kerby, Jeffrey T; Kocher, Sarah D; Losyev, Oleksiy; Muli, Elliud; Patch, Harland M; Rosa, Cristina; Sakamoto, Joyce M; Stanley, Scott; Vaudo, Anthony D; Grozinger, Christina M

2018-06-11

Bee viral ecology is a fascinating emerging area of research: viruses exert a range of effects on their hosts, exacerbate impacts of other environmental stressors, and, importantly, are readily shared across multiple bee species in a community. However, our understanding of bee viral communities is limited, as it is primarily derived from studies of North American and European Apis mellifera populations. Here, we examined viruses in populations of A. mellifera and 11 other bee species from 9 countries, across 4 continents and Oceania. We developed a novel pipeline to rapidly and inexpensively screen for bee viruses. This pipeline includes purification of encapsulated RNA/DNA viruses, sequence-independent amplification, high throughput sequencing, integrated assembly of contigs, and filtering to identify contigs specifically corresponding to viral sequences. We identified sequences for (+)ssRNA, (-)ssRNA, dsRNA, and ssDNA viruses. Overall, we found 127 contigs corresponding to novel viruses (i.e. previously not observed in bees), with 27 represented by >0.1% of the reads in a given sample, and 7 contained an RdRp or replicase sequence which could be used for robust phylogenetic analysis. This study provides a sequence-independent pipeline for viral metagenomics analysis, and greatly expands our understanding of the diversity of viruses found in bee communities.

From astronomy and telecommunications to biomedicine

NASA Astrophysics Data System (ADS)

Behr, Bradford B.; Baker, Scott A.; Bismilla, Yusuf; Cenko, Andrew T.; DesRoches, Brandon; Hajian, Arsen R.; Meade, Jeffrey T.; Nitkowski, Arthur; Preston, Kyle J.; Schmidt, Bradley S.; Sherwood-Droz, Nicolás.; Slaa, Jared

2015-03-01

Photonics is an inherently interdisciplinary endeavor, as technologies and techniques invented or developed in one scientific field are often found to be applicable to other fields or disciplines. We present two case studies in which optical spectroscopy technologies originating from stellar astrophysics and optical telecommunications multiplexing have been successfully adapted for biomedical applications. The first case involves a design concept called the High Throughput Virtual Slit, or HTVS, which provides high spectral resolution without the throughput inefficiency typically associated with a narrow spectrometer slit. HTVS-enhanced spectrometers have been found to significantly improve the sensitivity and speed of fiber-fed Raman analysis systems, and the method is now being adapted for hyperspectral imaging for medical and biological sensing. The second example of technology transfer into biomedicine centers on integrated optics, in which optical waveguides are fabricated on to silicon substrates in a substantially similar fashion as integrated circuits in computer chips. We describe an architecture referred to as OCTANE which implements a small and robust "spectrometer-on-a-chip" which is optimized for optical coherence tomography (OCT). OCTANE-based OCT systems deliver three-dimensional imaging resolution at the micron scale with greater stability and lower cost than equivalent conventional OCT approaches. Both HTVS and OCTANE enable higher precision and improved reliability under environmental conditions that are typically found in a clinical or laboratory setting.

Gentle, fast and effective crystal soaking by acoustic dispensing

PubMed Central

Ng, Jia Tsing; Talon, Romain; Nekrosiute, Karolina; Krojer, Tobias; Douangamath, Alice; Brandao-Neto, Jose; Pearce, Nicholas M.; von Delft, Frank

2017-01-01

The steady expansion in the capacity of modern beamlines for high-throughput data collection, enabled by increasing X-ray brightness, capacity of robotics and detector speeds, has pushed the bottleneck upstream towards sample preparation. Even in ligand-binding studies using crystal soaking, the experiment best able to exploit beamline capacity, a primary limitation is the need for gentle and nontrivial soaking regimens such as stepwise concentration increases, even for robust and well characterized crystals. Here, the use of acoustic droplet ejection for the soaking of protein crystals with small molecules is described, and it is shown that it is both gentle on crystals and allows very high throughput, with 1000 unique soaks easily performed in under 10 min. In addition to having very low compound consumption (tens of nanolitres per sample), the positional precision of acoustic droplet ejection enables the targeted placement of the compound/solvent away from crystals and towards drop edges, allowing gradual diffusion of solvent across the drop. This ensures both an improvement in the reproducibility of X-ray diffraction and increased solvent tolerance of the crystals, thus enabling higher effective compound-soaking concentrations. The technique is detailed here with examples from the protein target JMJD2D, a histone lysine demethylase with roles in cancer and the focus of active structure-based drug-design efforts. PMID:28291760

A DNA fingerprinting procedure for ultra high-throughput genetic analysis of insects.

PubMed

Schlipalius, D I; Waldron, J; Carroll, B J; Collins, P J; Ebert, P R

2001-12-01

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular information is often non-existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability in three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced approximately 50 scoreable polymorphic DNA markers, between individuals of three independent field isolates of R. dominica. Multivariate cluster analysis using forty-nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 +/- 6.0% between clades and of 21.0 +/- 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from individual DNA samples that had been combined to create the bulked samples.

Analysis of Protein Expression in Cell Microarrays: A Tool for Antibody-based Proteomics

PubMed Central

Andersson, Ann-Catrin; Strömberg, Sara; Bäckvall, Helena; Kampf, Caroline; Uhlen, Mathias; Wester, Kenneth; Pontén, Fredrik

2006-01-01

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome. PMID:16957166

The FIFE Project at Fermilab

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

Box, D.; Boyd, J.; Di Benedetto, V.

2016-01-01

The FabrIc for Frontier Experiments (FIFE) project is an initiative within the Fermilab Scientific Computing Division designed to steer the computing model for non-LHC Fermilab experiments across multiple physics areas. FIFE is a collaborative effort between experimenters and computing professionals to design and develop integrated computing models for experiments of varying size, needs, and infrastructure. The major focus of the FIFE project is the development, deployment, and integration of solutions for high throughput computing, data management, database access and collaboration management within an experiment. To accomplish this goal, FIFE has developed workflows that utilize Open Science Grid compute sites alongmore » with dedicated and commercial cloud resources. The FIFE project has made significant progress integrating into experiment computing operations several services including a common job submission service, software and reference data distribution through CVMFS repositories, flexible and robust data transfer clients, and access to opportunistic resources on the Open Science Grid. The progress with current experiments and plans for expansion with additional projects will be discussed. FIFE has taken the leading role in defining the computing model for Fermilab experiments, aided in the design of experiments beyond those hosted at Fermilab, and will continue to define the future direction of high throughput computing for future physics experiments worldwide.« less

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

PubMed

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

2014-06-01

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

Novel LC/MS/MS and High-Throughput Mass Spectrometric Assays for Monoacylglycerol Acyltransferase Inhibitors.

PubMed

Qi, Jenson; Masucci, John A; Lang, Wensheng; Connelly, Margery A; Caldwell, Gary W; Petrounia, Ioanna; Kirkpatrick, Jennifer; Barnakov, Alexander N; Struble, Geoffrey; Miller, Robyn; Dzordzorine, Keli; Kuo, Gee-Hong; Gaul, Michael; Pocai, Alessandro; Lee, Seunghun

2017-04-01

Monoacylglycerol acyltransferase enzymes (MGAT1, MGAT2, and MGAT3) convert monoacylglycerol to diacylglycerol (DAG). MGAT1 and MGAT2 are both implicated in obesity-related metabolic diseases. Conventional MGAT enzyme assays use radioactive substrates, wherein the product of the MGAT-catalyzed reaction is usually resolved by time-consuming thin layer chromatography (TLC) analysis. Furthermore, microsomal membrane preparations typically contain endogenous diacylglycerol acyltransferase (DGAT) from the host cells, and these DGAT activities can further acylate DAG to form triglyceride (TG). Our mass spectrometry (liquid chromatography-tandem mass spectrometry, or LC/MS/MS) MGAT2 assay measures human recombinant MGAT2-catalyzed formation of didecanoyl-glycerol from 1-decanoyl-rac-glycerol and decanoyl-CoA, to produce predominantly 1,3-didecanoyl-glycerol. Unlike 1,2-DAG, 1,3-didecanoyl-glycerol is proved to be not susceptible to further acylation to TG. 1,3-Didecanoyl-glycerol product can be readily solubilized and directly subjected to high-throughput mass spectrometry (HTMS) without further extraction in a 384-well format. We also have established the LC/MS/MS MGAT activity assay in the intestinal microsomes from various species. Our assay is proved to be highly sensitive, and thus it allows measurement of endogenous MGAT activity in cell lysates and tissue preparations. The implementation of the HTMS MGAT activity assay has facilitated the robust screening and evaluation of MGAT inhibitors for the treatment of metabolic diseases.

Identification of Microbial Communities in Open and Closed Circuit Bioelectrochemical MBRs by High-Throughput 454 Pyrosequencing

PubMed Central

Huang, Jian; Wang, Zhiwei; Zhu, Chaowei; Ma, Jinxing; Zhang, Xingran; Wu, Zhichao

2014-01-01

Two bioelectrochemical membrane bioreactors (MBRs) developed by integrating microbial fuel cell and MBR technology were operated under closed-circuit and open-circuit modes, and high-throughput 454 pyrosequencing was used to investigate the effects of the power generation on the microbial community of bio-anode and bio-cathode. Microbes on the anode under open-circuit operation (AO) were enriched and highly diverse when compared to those on the anode under closed-circuit operation (AC). However, among the cathodes the closed-circuit mode (CC) had richer and more diverse microbial community compared to the cathode under open-circuit mode (CO). On the anodes AO and AC, Proteobacteria and Bacteroidetes were the dominant phyla, while Firmicutes was enriched only on AC. Deltaproteobacteria affiliated to Proteobacteria were also more abundant on AC than AO. Furthermore, the relative abundance of Desulfuromonas, which are well-known electrogenic bacteria, were much higher on AC (10.2%) when compared to AO (0.11%), indicating that closed-circuit operation was more conducive for the growth of electrogenic bacteria on the anodes. On the cathodes, Protebacteria was robust on CC while Bacteroidetes was more abundant on CO. Rhodobacter and Hydrogenophaga were also enriched on CC than CO, suggesting that these genera play a role in electron transfer from the cathode surface to the terminal electron acceptors in the bioelectrochemical MBR under closed-circuit operation. PMID:24705450

Genomic-assisted haplotype analysis and the development of high-throughput SNP markers for salinity tolerance in soybean

PubMed Central

Patil, Gunvant; Do, Tuyen; Vuong, Tri D.; Valliyodan, Babu; Lee, Jeong-Dong; Chaudhary, Juhi; Shannon, J. Grover; Nguyen, Henry T.

2016-01-01

Soil salinity is a limiting factor of crop yield. The soybean is sensitive to soil salinity, and a dominant gene, Glyma03g32900 is primarily responsible for salt-tolerance. The identification of high throughput and robust markers as well as the deployment of salt-tolerant cultivars are effective approaches to minimize yield loss under saline conditions. We utilized high quality (15x) whole-genome resequencing (WGRS) on 106 diverse soybean lines and identified three major structural variants and allelic variation in the promoter and genic regions of the GmCHX1 gene. The discovery of single nucleotide polymorphisms (SNPs) associated with structural variants facilitated the design of six KASPar assays. Additionally, haplotype analysis and pedigree tracking of 93 U.S. ancestral lines were performed using publically available WGRS datasets. Identified SNP markers were validated, and a strong correlation was observed between the genotype and salt treatment phenotype (leaf scorch, chlorophyll content and Na+ accumulation) using a panel of 104 soybean lines and, an interspecific bi-parental population (F8) from PI483463 x Hutcheson. These markers precisely identified salt-tolerant/sensitive genotypes (>91%), and different structural-variants (>98%). These SNP assays, supported by accurate phenotyping, haplotype analyses and pedigree tracking information, will accelerate marker-assisted selection programs to enhance the development of salt-tolerant soybean cultivars. PMID:26781337

PCR Primers to Study the Diversity of Expressed Fungal Genes Encoding Lignocellulolytic Enzymes in Soils Using High-Throughput Sequencing

PubMed Central

Barbi, Florian; Bragalini, Claudia; Vallon, Laurent; Prudent, Elsa; Dubost, Audrey; Fraissinet-Tachet, Laurence; Marmeisse, Roland; Luis, Patricia

2014-01-01

Plant biomass degradation in soil is one of the key steps of carbon cycling in terrestrial ecosystems. Fungal saprotrophic communities play an essential role in this process by producing hydrolytic enzymes active on the main components of plant organic matter. Open questions in this field regard the diversity of the species involved, the major biochemical pathways implicated and how these are affected by external factors such as litter quality or climate changes. This can be tackled by environmental genomic approaches involving the systematic sequencing of key enzyme-coding gene families using soil-extracted RNA as material. Such an approach necessitates the design and evaluation of gene family-specific PCR primers producing sequence fragments compatible with high-throughput sequencing approaches. In the present study, we developed and evaluated PCR primers for the specific amplification of fungal CAZy Glycoside Hydrolase gene families GH5 (subfamily 5) and GH11 encoding endo-β-1,4-glucanases and endo-β-1,4-xylanases respectively as well as Basidiomycota class II peroxidases, corresponding to the CAZy Auxiliary Activity family 2 (AA2), active on lignin. These primers were experimentally validated using DNA extracted from a wide range of Ascomycota and Basidiomycota species including 27 with sequenced genomes. Along with the published primers for Glycoside Hydrolase GH7 encoding enzymes active on cellulose, the newly design primers were shown to be compatible with the Illumina MiSeq sequencing technology. Sequences obtained from RNA extracted from beech or spruce forest soils showed a high diversity and were uniformly distributed in gene trees featuring the global diversity of these gene families. This high-throughput sequencing approach using several degenerate primers constitutes a robust method, which allows the simultaneous characterization of the diversity of different fungal transcripts involved in plant organic matter degradation and may lead to the discovery of complex patterns in gene expression of soil fungal communities. PMID:25545363

High Throughput UPLC®-MSMS Method for the Analysis of Phosphatidylethanol (PEth) 16:0/18:1, a Specific Biomarker for Alcohol Consumption, in Whole Blood.

PubMed

Andreassen, Trine Naalsund; Havnen, Hilde; Spigset, Olav; Falch, Berit Margrethe Hasle; Skråstad, Ragnhild Bergene

2018-01-01

Phosphatidylethanol (PEth) is an alcohol biomarker formed in the presence of ethanol in the body. Both due to its specificity and because it has a detection window of up to several weeks after alcohol intake, its application potential is broader than for other ethanol biomarkers. The aim of this study was to develop and validate a robust method for PEth in whole blood with fast and efficient sample extraction and a short analytical runtime, suitable for high throughput routine purposes. A validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC®-MSMS) method for quantification of PEth 16:0/18:1 in the range 0.05-4.00 μM (R2 ≥ 0.999) is presented. PEth 16:0/18:1 and the internal standard (IS) PEth-d5 (0.55 μM), were extracted from whole blood (150 μL) by simple protein precipitation with 2-propanol (450 μL). Chromatography was achieved using a BEH-phenyl (2.1 × 30 mm, 1.7 μm) column and a gradient elution combining ammonium formate (5 mM, pH 10.1) and acetonitrile at a flow rate of 0.5 mL/min. Runtime was 2.3 min. The mass spectrometer was monitored in negative mode with multiple reaction monitoring (MRM). The m/z 701.7 > 255.2 and 701.7 > 281.3 transitions were monitored for PEth 16:0/18:1 and the m/z 706.7 > 255.3 for PEth-d5. Limit of quantification was 0.03 μM (coefficient of variation, CV = 6.7%, accuracy = 99.3%). Within-assay and between-assay imprecision were 0.4-3.3% (CV ≤ 7.1%). Recoveries were 95-102% (CV ≤ 4.9%). Matrix effects after IS correction ranged from 107% to 112%. PEth 16:0/18:1 in patient samples were stable for several days at 30°C. Repeated freezing (-80°C) and thawing did not affect the concentration. After thawing and analysis patient samples were stable at 4-8°C for at least 4 weeks. Results from a proficiency test program, showing |Z| values ≤1.2, confirm the validity of the method. Analysis of the first 3,169 samples sent to our laboratory for routine use has demonstrated its properties as a robust method suitable for high throughput purposes.

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

EPA Pesticide Factsheets

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

Towards Real Time Diagnostics of Hybrid Welding Laser/GMAW

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

Timothy Mcjunkin; Dennis C. Kunerth; Corrie Nichol

2013-07-01

Methods are currently being developed towards a more robust system real time feedback in the high throughput process combining laser welding with gas metal arc welding. A combination of ultrasonic, eddy current, electronic monitoring, and visual techniques are being applied to the welding process. Initial simulation and bench top evaluation of proposed real time techniques on weld samples are presented along with the concepts to apply the techniques concurrently to the weld process. Consideration for the eventual code acceptance of the methods and system are also being researched as a component of this project. The goal is to detect defectsmore » or precursors to defects and correct when possible during the weld process.« less

Towards real time diagnostics of Hybrid Welding Laser/GMAW

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

McJunkin, T. R.; Kunerth, D. C.; Nichol, C. I.

2014-02-18

Methods are currently being developed towards a more robust system real time feedback in the high throughput process combining laser welding with gas metal arc welding. A combination of ultrasonic, eddy current, electronic monitoring, and visual techniques are being applied to the welding process. Initial simulation and bench top evaluation of proposed real time techniques on weld samples are presented along with the concepts to apply the techniques concurrently to the weld process. Consideration for the eventual code acceptance of the methods and system are also being researched as a component of this project. The goal is to detect defectsmore » or precursors to defects and correct when possible during the weld process.« less

Towards real time diagnostics of Hybrid Welding Laser/GMAW

NASA Astrophysics Data System (ADS)

McJunkin, T. R.; Kunerth, D. C.; Nichol, C. I.; Todorov, E.; Levesque, S.

2014-02-01

Methods are currently being developed towards a more robust system real time feedback in the high throughput process combining laser welding with gas metal arc welding. A combination of ultrasonic, eddy current, electronic monitoring, and visual techniques are being applied to the welding process. Initial simulation and bench top evaluation of proposed real time techniques on weld samples are presented along with the concepts to apply the techniques concurrently to the weld process. Consideration for the eventual code acceptance of the methods and system are also being researched as a component of this project. The goal is to detect defects or precursors to defects and correct when possible during the weld process.

Network coding multiuser scheme for indoor visible light communications

NASA Astrophysics Data System (ADS)

Zhang, Jiankun; Dang, Anhong

2017-12-01

Visible light communication (VLC) is a unique alternative for indoor data transfer and developing beyond point-to-point. However, for realizing high-capacity networks, VLC is facing challenges including the constrained bandwidth of the optical access point and random occlusion. A network coding scheme for VLC (NC-VLC) is proposed, with increased throughput and system robustness. Based on the Lambertian illumination model, theoretical decoding failure probability of the multiuser NC-VLC system is derived, and the impact of the system parameters on the performance is analyzed. Experiments demonstrate the proposed scheme successfully in the indoor multiuser scenario. These results indicate that the NC-VLC system shows a good performance under the link loss and random occlusion.

NMR in the SPINE Structural Proteomics project.

PubMed

Ab, E; Atkinson, A R; Banci, L; Bertini, I; Ciofi-Baffoni, S; Brunner, K; Diercks, T; Dötsch, V; Engelke, F; Folkers, G E; Griesinger, C; Gronwald, W; Günther, U; Habeck, M; de Jong, R N; Kalbitzer, H R; Kieffer, B; Leeflang, B R; Loss, S; Luchinat, C; Marquardsen, T; Moskau, D; Neidig, K P; Nilges, M; Piccioli, M; Pierattelli, R; Rieping, W; Schippmann, T; Schwalbe, H; Travé, G; Trenner, J; Wöhnert, J; Zweckstetter, M; Kaptein, R

2006-10-01

This paper describes the developments, role and contributions of the NMR spectroscopy groups in the Structural Proteomics In Europe (SPINE) consortium. Focusing on the development of high-throughput (HTP) pipelines for NMR structure determinations of proteins, all aspects from sample preparation, data acquisition, data processing, data analysis to structure determination have been improved with respect to sensitivity, automation, speed, robustness and validation. Specific highlights are protonless (13)C-direct detection methods and inferential structure determinations (ISD). In addition to technological improvements, these methods have been applied to deliver over 60 NMR structures of proteins, among which are five that failed to crystallize. The inclusion of NMR spectroscopy in structural proteomics pipelines improves the success rate for protein structure determinations.

Open Port Probe Sampling Interface for the Direct Coupling of Biocompatible Solid-Phase Microextraction to Atmospheric Pressure Ionization Mass Spectrometry.

PubMed

Gómez-Ríos, Germán Augusto; Liu, Chang; Tascon, Marcos; Reyes-Garcés, Nathaly; Arnold, Don W; Covey, Thomas R; Pawliszyn, Janusz

2017-04-04

In recent years, the direct coupling of solid phase microextraction (SPME) and mass spectrometry (MS) has shown its great potential to improve limits of quantitation, accelerate analysis throughput, and diminish potential matrix effects when compared to direct injection to MS. In this study, we introduce the open port probe (OPP) as a robust interface to couple biocompatible SPME (Bio-SPME) fibers to MS systems for direct electrospray ionization. The presented design consisted of minimal alterations to the front-end of the instrument and provided better sensitivity, simplicity, speed, wider compound coverage, and high-throughput in comparison to the LC-MS based approach. Quantitative determination of clenbuterol, fentanyl, and buprenorphine was successfully achieved in human urine. Despite the use of short extraction/desorption times (5 min/5 s), limits of quantitation below the minimum required performance levels (MRPL) set by the world antidoping agency (WADA) were obtained with good accuracy (≥90%) and linearity (R 2 > 0.99) over the range evaluated for all analytes using sample volumes of 300 μL. In-line technologies such as multiple reaction monitoring with multistage fragmentation (MRM 3 ) and differential mobility spectrometry (DMS) were used to enhance the selectivity of the method without compromising analysis speed. On the basis of calculations, once coupled to high throughput, this method can potentially yield preparation times as low as 15 s per sample based on the 96-well plate format. Our results demonstrated that Bio-SPME-OPP-MS efficiently integrates sampling/sample cleanup and atmospheric pressure ionization, making it an advantageous configuration for several bioanalytical applications, including doping in sports, in vivo tissue sampling, and therapeutic drug monitoring.

Emerging metrology for high-throughput nanomaterial genotoxicology.

PubMed

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

2017-01-01

The rapid development of the engineered nanomaterial (ENM) manufacturing industry has accelerated the incorporation of ENMs into a wide variety of consumer products across the globe. Unintentionally or not, some of these ENMs may be introduced into the environment or come into contact with humans or other organisms resulting in unexpected biological effects. It is thus prudent to have rapid and robust analytical metrology in place that can be used to critically assess and/or predict the cytotoxicity, as well as the potential genotoxicity of these ENMs. Many of the traditional genotoxicity test methods [e.g. unscheduled DNA synthesis assay, bacterial reverse mutation (Ames) test, etc.,] for determining the DNA damaging potential of chemical and biological compounds are not suitable for the evaluation of ENMs, due to a variety of methodological issues ranging from potential assay interferences to problems centered on low sample throughput. Recently, a number of sensitive, high-throughput genotoxicity assays/platforms (CometChip assay, flow cytometry/micronucleus assay, flow cytometry/γ-H2AX assay, automated 'Fluorimetric Detection of Alkaline DNA Unwinding' (FADU) assay, ToxTracker reporter assay) have been developed, based on substantial modifications and enhancements of traditional genotoxicity assays. These new assays have been used for the rapid measurement of DNA damage (strand breaks), chromosomal damage (micronuclei) and for detecting upregulated DNA damage signalling pathways resulting from ENM exposures. In this critical review, we describe and discuss the fundamental measurement principles and measurement endpoints of these new assays, as well as the modes of operation, analytical metrics and potential interferences, as applicable to ENM exposures. An unbiased discussion of the major technical advantages and limitations of each assay for evaluating and predicting the genotoxic potential of ENMs is also provided. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society 2016.

Analytical challenges for conducting rapid metabolism characterization for QIVIVE.

PubMed

Tolonen, Ari; Pelkonen, Olavi

2015-06-05

For quantitative in vitro-in vivo extrapolation (QIVIVE) of metabolism for the purposes of toxicokinetics prediction, a precise and robust analytical technique for identifying and measuring a chemical and its metabolites is an absolute prerequisite. Currently, high-resolution mass spectrometry (HR-MS) is a tool of choice for a majority of organic relatively lipophilic molecules, linked with a LC separation tool and simultaneous UV-detection. However, additional techniques such as gas chromatography, radiometric measurements and NMR, are required to cover the whole spectrum of chemical structures. To accumulate enough reliable and robust data for the validation of QIVIVE, there are some partially opposing needs: Detailed delineation of the in vitro test system to produce a reliable toxicokinetic measure for a studied chemical, and a throughput capacity of the in vitro set-up and the analytical tool as high as possible. We discuss current analytical challenges for the identification and quantification of chemicals and their metabolites, both stable and reactive, focusing especially on LC-MS techniques, but simultaneously attempting to pinpoint factors associated with sample preparation, testing conditions and strengths and weaknesses of a particular technique available for a particular task. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

A high-throughput Sanger strategy for human mitochondrial genome sequencing

PubMed Central

2013-01-01

Background A population reference database of complete human mitochondrial genome (mtGenome) sequences is needed to enable the use of mitochondrial DNA (mtDNA) coding region data in forensic casework applications. However, the development of entire mtGenome haplotypes to forensic data quality standards is difficult and laborious. A Sanger-based amplification and sequencing strategy that is designed for automated processing, yet routinely produces high quality sequences, is needed to facilitate high-volume production of these mtGenome data sets. Results We developed a robust 8-amplicon Sanger sequencing strategy that regularly produces complete, forensic-quality mtGenome haplotypes in the first pass of data generation. The protocol works equally well on samples representing diverse mtDNA haplogroups and DNA input quantities ranging from 50 pg to 1 ng, and can be applied to specimens of varying DNA quality. The complete workflow was specifically designed for implementation on robotic instrumentation, which increases throughput and reduces both the opportunities for error inherent to manual processing and the cost of generating full mtGenome sequences. Conclusions The described strategy will assist efforts to generate complete mtGenome haplotypes which meet the highest data quality expectations for forensic genetic and other applications. Additionally, high-quality data produced using this protocol can be used to assess mtDNA data developed using newer technologies and chemistries. Further, the amplification strategy can be used to enrich for mtDNA as a first step in sample preparation for targeted next-generation sequencing. PMID:24341507

Multiplexed transcriptome analysis to detect ALK, ROS1 and RET rearrangements in lung cancer

PubMed Central

Rogers, Toni-Maree; Arnau, Gisela Mir; Ryland, Georgina L.; Huang, Stephen; Lira, Maruja E.; Emmanuel, Yvette; Perez, Omar D.; Irwin, Darryl; Fellowes, Andrew P.; Wong, Stephen Q.; Fox, Stephen B.

2017-01-01

ALK, ROS1 and RET gene fusions are important predictive biomarkers for tyrosine kinase inhibitors in lung cancer. Currently, the gold standard method for gene fusion detection is Fluorescence In Situ Hybridization (FISH) and while highly sensitive and specific, it is also labour intensive, subjective in analysis, and unable to screen a large numbers of gene fusions. Recent developments in high-throughput transcriptome-based methods may provide a suitable alternative to FISH as they are compatible with multiplexing and diagnostic workflows. However, the concordance between these different methods compared with FISH has not been evaluated. In this study we compared the results from three transcriptome-based platforms (Nanostring Elements, Agena LungFusion panel and ThermoFisher NGS fusion panel) to those obtained from ALK, ROS1 and RET FISH on 51 clinical specimens. Overall agreement of results ranged from 86–96% depending on the platform used. While all platforms were highly sensitive, both the Agena panel and Thermo Fisher NGS fusion panel reported minor fusions that were not detectable by FISH. Our proof–of–principle study illustrates that transcriptome-based analyses are sensitive and robust methods for detecting actionable gene fusions in lung cancer and could provide a robust alternative to FISH testing in the diagnostic setting. PMID:28181564

Application of ToxCast High-Throughput Screening and ...

EPA Pesticide Factsheets

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

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

PubMed

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

2014-05-01

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

Development of Genome Engineering Tools from Plant-Specific PPR Proteins Using Animal Cultured Cells.

PubMed

Kobayashi, Takehito; Yagi, Yusuke; Nakamura, Takahiro

2016-01-01

The pentatricopeptide repeat (PPR) motif is a sequence-specific RNA/DNA-binding module. Elucidation of the RNA/DNA recognition mechanism has enabled engineering of PPR motifs as new RNA/DNA manipulation tools in living cells, including for genome editing. However, the biochemical characteristics of PPR proteins remain unknown, mostly due to the instability and/or unfolding propensities of PPR proteins in heterologous expression systems such as bacteria and yeast. To overcome this issue, we constructed reporter systems using animal cultured cells. The cell-based system has highly attractive features for PPR engineering: robust eukaryotic gene expression; availability of various vectors, reagents, and antibodies; highly efficient DNA delivery ratio (>80 %); and rapid, high-throughput data production. In this chapter, we introduce an example of such reporter systems: a PPR-based sequence-specific translational activation system. The cell-based reporter system can be applied to characterize plant genes of interested and to PPR engineering.

Mass spectrometric-based stable isotopic 2-aminobenzoic acid glycan mapping for rapid glycan screening of biotherapeutics.

PubMed

Prien, Justin M; Prater, Bradley D; Qin, Qiang; Cockrill, Steven L

2010-02-15

Fast, sensitive, robust methods for "high-level" glycan screening are necessary during various stages of a biotherapeutic product's lifecycle, including clone selection, process changes, and quality control for lot release testing. Traditional glycan screening involves chromatographic or electrophoretic separation-based methods, and, although reproducible, these methods can be time-consuming. Even ultrahigh-performance chromatographic and microfluidic integrated LC/MS systems, which work on the tens of minute time scale, become lengthy when hundreds of samples are to be analyzed. Comparatively, a direct infusion mass spectrometry (MS)-based glycan screening method acquires data on a millisecond time scale, exhibits exquisite sensitivity and reproducibility, and is amenable to automated peak annotation. In addition, characterization of glycan species via sequential mass spectrometry can be performed simultaneously. Here, we demonstrate a quantitative high-throughput MS-based mapping approach using stable isotope 2-aminobenzoic acid (2-AA) for rapid "high-level" glycan screening.

High Throughput Screening For Hazard and Risk of Environmental Contaminants

EPA Science Inventory

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

Use of planar array electrophysiology for the development of robust ion channel cell lines.

PubMed

Clare, Jeffrey J; Chen, Mao Xiang; Downie, David L; Trezise, Derek J; Powell, Andrew J

2009-01-01

The tractability of ion channels as drug targets has been significantly improved by the advent of planar array electrophysiology platforms which have dramatically increased the capacity for electrophysiological profiling of lead series compounds. However, the data quality and through-put obtained with these platforms is critically dependent on the robustness of the expression reagent being used. The generation of high quality, recombinant cell lines is therefore a key step in the early phase of ion channel drug discovery and this can present significant challenges due to the diversity and organisational complexity of many channel types. This article focuses on several complex and difficult to express ion channels and illustrates how improved stable cell lines can be obtained by integration of planar array electrophysiology systems into the cell line generation process per se. By embedding this approach at multiple stages (e.g., during development of the expression strategy, during screening and validation of clonal lines, and during characterisation of the final cell line), the cycle time and success rate in obtaining robust expression of complex multi-subunit channels can be significantly improved. We also review how recent advances in this technology (e.g., population patch clamp) have further widened the versatility and applicability of this approach.

High Throughput Transcriptomics: From screening to pathways

EPA Science Inventory

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

A novel unbalanced multiple description coder for robust video transmission over ad hoc wireless networks

NASA Astrophysics Data System (ADS)

Huang, Feng; Sun, Lifeng; Zhong, Yuzhuo

2006-01-01

Robust transmission of live video over ad hoc wireless networks presents new challenges: high bandwidth requirements are coupled with delay constraints; even a single packet loss causes error propagation until a complete video frame is coded in the intra-mode; ad hoc wireless networks suffer from bursty packet losses that drastically degrade the viewing experience. Accordingly, we propose a novel UMD coder capable of quickly recovering from losses and ensuring continuous playout. It uses 'peg' frames to prevent error propagation in the High-Resolution (HR) description and improve the robustness of key frames. The Low-Resolution (LR) coder works independent of the HR one, but they can also help each other recover from losses. Like many UMD coders, our UMD coder is drift-free, disruption-tolerant and able to make good use of the asymmetric available bandwidths of multiple paths. The simulation results under different conditions show that the proposed UMD coder has the highest decoded quality and lowest probability of pause when compared with concurrent UMDC techniques. The coder also has a comparable decoded quality, lower startup delay and lower probability of pause than a state-of-the-art FEC-based scheme. To provide robustness for video multicast applications, we propose non-end-to-end UMDC-based video distribution over a multi-tree multicast network. The multiplicity of parents decorrelates losses and the non-end-to-end feature increases the throughput of UMDC video data. We deploy an application-level service of LR description reconstruction in some intermediate nodes of the LR multicast tree. The principle behind this is to reconstruct the disrupted LR frames by the correctly received HR frames. As a result, the viewing experience at the downstream nodes benefits from the protection reconstruction at the upstream nodes.

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

NASA Astrophysics Data System (ADS)

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

2017-12-01

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

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

PubMed

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

2017-01-01

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

High Throughput Experimental Materials Database

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

Zakutayev, Andriy; Perkins, John; Schwarting, Marcus

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

Characterization of Differentiated SH-SY5Y as Neuronal Screening Model Reveals Increased Oxidative Vulnerability

PubMed Central

Forster, J. I.; Köglsberger, S.; Trefois, C.; Boyd, O.; Baumuratov, A. S.; Buck, L.; Balling, R.; Antony, P. M. A.

2016-01-01

The immortalized and proliferative cell line SH-SY5Y is one of the most commonly used cell lines in neuroscience and neuroblastoma research. However, undifferentiated SH-SY5Y cells share few properties with mature neurons. In this study, we present an optimized neuronal differentiation protocol for SH-SY5Y that requires only two work steps and 6 days. After differentiation, the cells present increased levels of ATP and plasma membrane activity but reduced expression of energetic stress response genes. Differentiation results in reduced mitochondrial membrane potential and decreased robustness toward perturbations with 6-hydroxydopamine. We are convinced that the presented differentiation method will leverage genetic and chemical high-throughput screening projects targeting pathways that are involved in the selective vulnerability of neurons with high energetic stress levels. PMID:26738520

A microfluidic tubing method and its application for controlled synthesis of polymeric nanoparticles.

PubMed

Wang, Jidong; Chen, Wenwen; Sun, Jiashu; Liu, Chao; Yin, Qifang; Zhang, Lu; Xianyu, Yunlei; Shi, Xinghua; Hu, Guoqing; Jiang, Xingyu

2014-05-21

This report describes a straightforward but robust tubing method for connecting polydimethylsiloxane (PDMS) microfluidic devices to external equipment. The interconnection is irreversible and can sustain a pressure of up to 4.5 MPa that is characterized experimentally and theoretically. To demonstrate applications of this high-pressure tubing technique, we fabricate a semicircular microfluidic channel to implement a high-throughput, size-controlled synthesis of poly(lactic-co-glycolic acid) (PLGA) nanoparticles ranging from 55 to 135 nm in diameter. This microfluidic device allows for a total flow rate of 410 mL h(-1), resulting in enhanced convective mixing which can be utilized to precipitate small size nanoparticles with a good dispersion. We expect that this tubing technique would be widely used in microfluidic chips for nanoparticle synthesis, cell manipulation, and potentially nanofluidic applications.

Machine learning bandgaps of double perovskites

DOE PAGES

Pilania, G.; Mannodi-Kanakkithodi, A.; Uberuaga, B. P.; ...

2016-01-19

The ability to make rapid and accurate predictions on bandgaps of double perovskites is of much practical interest for a range of applications. While quantum mechanical computations for high-fidelity bandgaps are enormously computation-time intensive and thus impractical in high throughput studies, informatics-based statistical learning approaches can be a promising alternative. Here we demonstrate a systematic feature-engineering approach and a robust learning framework for efficient and accurate predictions of electronic bandgaps of double perovskites. After evaluating a set of more than 1.2 million features, we identify lowest occupied Kohn-Sham levels and elemental electronegativities of the constituent atomic species as the mostmore » crucial and relevant predictors. As a result, the developed models are validated and tested using the best practices of data science and further analyzed to rationalize their prediction performance.« less

Validation and perspectives of a femtosecond laser fabricated monolithic optical stretcher

PubMed Central

Bellini, Nicola; Bragheri, Francesca; Cristiani, Ilaria; Guck, Jochen; Osellame, Roberto; Whyte, Graeme

2012-01-01

The combination of high power laser beams with microfluidic delivery of cells is at the heart of high-throughput, single-cell analysis and disease diagnosis with an optical stretcher. So far, the challenges arising from this combination have been addressed by externally aligning optical fibres with microfluidic glass capillaries, which has a limited potential for integration into lab-on-a-chip environments. Here we demonstrate the successful production and use of a monolithic glass chip for optical stretching of white blood cells, featuring microfluidic channels and optical waveguides directly written into bulk glass by femtosecond laser pulses. The performance of this novel chip is compared to the standard capillary configuration. The robustness, durability and potential for intricate flow patterns provided by this monolithic optical stretcher chip suggest its use for future diagnostic and biotechnological applications. PMID:23082304

Biologically inspired robots elicit a robust fear response in zebrafish

NASA Astrophysics Data System (ADS)

Ladu, Fabrizio; Bartolini, Tiziana; Panitz, Sarah G.; Butail, Sachit; Macrı, Simone; Porfiri, Maurizio

2015-03-01

We investigate the behavioral response of zebrafish to three fear-evoking stimuli. In a binary choice test, zebrafish are exposed to a live allopatric predator, a biologically-inspired robot, and a computer-animated image of the live predator. A target tracking algorithm is developed to score zebrafish behavior. Unlike computer-animated images, the robotic and live predator elicit a robust avoidance response. Importantly, the robotic stimulus elicits more consistent inter-individual responses than the live predator. Results from this effort are expected to aid in hypothesis-driven studies on zebrafish fear response, by offering a valuable approach to maximize data-throughput and minimize animal subjects.

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

EPA Science Inventory

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

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

EPA Science Inventory

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

Designing Robust and Resilient Tactical MANETs

DTIC Science & Technology

2014-09-25

Bounds on the Throughput Efficiency of Greedy Maximal Scheduling in Wireless Networks , IEEE/ACM Transactions on Networking , (06 2011): 0. doi: N... Wireless Sensor Networks and Effects of Long Range Dependant Data, Special IWSM Issue of Sequential Analysis, (11 2012): 0. doi: A. D. Dominguez...Bushnell, R. Poovendran. A Convex Optimization Approach for Clone Detection in Wireless Sensor Networks , Pervasive and Mobile Computing, (01 2012

Automatic and integrated micro-enzyme assay (AIμEA) platform for highly sensitive thrombin analysis via an engineered fluorescence protein-functionalized monolithic capillary column.

PubMed

Lin, Lihua; Liu, Shengquan; Nie, Zhou; Chen, Yingzhuang; Lei, Chunyang; Wang, Zhen; Yin, Chao; Hu, Huiping; Huang, Yan; Yao, Shouzhuo

2015-04-21

Nowadays, large-scale screening for enzyme discovery, engineering, and drug discovery processes require simple, fast, and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel automatic and integrated micro-enzyme assay (AIμEA) platform was proposed based on a unique microreaction system fabricated by a engineered green fluorescence protein (GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and to be released after thrombin digestion. Combined with capillary electrophoresis-laser-induced fluorescence (CE-LIF), all the procedures, including thrombin injection, online enzymatic digestion in the microreaction system, and label-free detection of the released GFP, were integrated in a single electrophoretic process. By taking advantage of the ultrahigh loading capacity of the AIμEA platform and the CE automatic programming setup, one microreaction column was sufficient for many times digestion without replacement. The novel microreaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly, the AIμEA platform was highly sensitive with a limit of detection down to 1 pM of thrombin. Moreover, the AIμEA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AIμEA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics, and drug screening.

Normalization and standardization of electronic health records for high-throughput phenotyping: the SHARPn consortium

PubMed Central

Pathak, Jyotishman; Bailey, Kent R; Beebe, Calvin E; Bethard, Steven; Carrell, David S; Chen, Pei J; Dligach, Dmitriy; Endle, Cory M; Hart, Lacey A; Haug, Peter J; Huff, Stanley M; Kaggal, Vinod C; Li, Dingcheng; Liu, Hongfang; Marchant, Kyle; Masanz, James; Miller, Timothy; Oniki, Thomas A; Palmer, Martha; Peterson, Kevin J; Rea, Susan; Savova, Guergana K; Stancl, Craig R; Sohn, Sunghwan; Solbrig, Harold R; Suesse, Dale B; Tao, Cui; Taylor, David P; Westberg, Les; Wu, Stephen; Zhuo, Ning; Chute, Christopher G

2013-01-01

Research objective To develop scalable informatics infrastructure for normalization of both structured and unstructured electronic health record (EHR) data into a unified, concept-based model for high-throughput phenotype extraction. Materials and methods Software tools and applications were developed to extract information from EHRs. Representative and convenience samples of both structured and unstructured data from two EHR systems—Mayo Clinic and Intermountain Healthcare—were used for development and validation. Extracted information was standardized and normalized to meaningful use (MU) conformant terminology and value set standards using Clinical Element Models (CEMs). These resources were used to demonstrate semi-automatic execution of MU clinical-quality measures modeled using the Quality Data Model (QDM) and an open-source rules engine. Results Using CEMs and open-source natural language processing and terminology services engines—namely, Apache clinical Text Analysis and Knowledge Extraction System (cTAKES) and Common Terminology Services (CTS2)—we developed a data-normalization platform that ensures data security, end-to-end connectivity, and reliable data flow within and across institutions. We demonstrated the applicability of this platform by executing a QDM-based MU quality measure that determines the percentage of patients between 18 and 75 years with diabetes whose most recent low-density lipoprotein cholesterol test result during the measurement year was <100 mg/dL on a randomly selected cohort of 273 Mayo Clinic patients. The platform identified 21 and 18 patients for the denominator and numerator of the quality measure, respectively. Validation results indicate that all identified patients meet the QDM-based criteria. Conclusions End-to-end automated systems for extracting clinical information from diverse EHR systems require extensive use of standardized vocabularies and terminologies, as well as robust information models for storing, discovering, and processing that information. This study demonstrates the application of modular and open-source resources for enabling secondary use of EHR data through normalization into standards-based, comparable, and consistent format for high-throughput phenotyping to identify patient cohorts. PMID:24190931

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR).

PubMed

Crosby, Priya; Hoyle, Nathaniel P; O'Neill, John S

2017-12-13

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

High Throughput Determination of Critical Human Dosing Parameters (SOT)

EPA Science Inventory

High throughput toxicokinetics (HTTK) is a rapid approach that uses in vitro data to estimate TK for hundreds of environmental chemicals. Reverse dosimetry (i.e., reverse toxicokinetics or RTK) based on HTTK data converts high throughput in vitro toxicity screening (HTS) data int...

High Throughput Determinations of Critical Dosing Parameters (IVIVE workshop)

EPA Science Inventory

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...

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

EPA Science Inventory

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

High-throughput screening of triplex DNA binders from complicated samples by 96-well pate format in conjunction with peak area-fading UHPLC-Orbitrap MS.

PubMed

Yang, Hongmei; Yao, Wenbin; Wang, Yihan; Shi, Lei; Su, Rui; Wan, Debin; Xu, Niusheng; Lian, Wenhui; Chen, Changbao; Liu, Shuying

2017-02-14

Conventional strategies for the screening of DNA triplex binders cannot be used for complicated samples, such as ligand libraries created by combinatorial chemistry or from natural product extracts. In the current study, an ultra-high-performance liquid chromatography coupled with an Orbitrap mass spectrometry (UHPLC-Orbitrap-MS)-based approach, which we call peak area-fading (PAF) UHPLC-Orbitrap-MS and was designed for just such a purpose, is reported. The triplex DNA modified 96-well plate and the single stranded oligonucleotide modified 96-well plate (as control) were incubated with ligand libraries, and the unbound ligands were directly determined via UHPLC-ESI-MS. The binders were detected through the decrease (fading) in the peak areas compared to those of the control group. Several factors, such as incubation time, incubation temperature, and buffer, which might affect the binding affinity and reproducibility, were optimized. The potential of the approach was examined using the extracts of Rhizoma Coptidis and Phellodendron chinense Schneid cortexe. The triplex DNA-binding capabilities of the five components (epiberberine, coptisine, jatrorrhizine, berberrubine, and columbamine) were found for the first time, indicating their efficiency for the analysis of complicated samples. In contrast to our previous study, which suffered from a serious drawback of poor reproducibility, this method is more robust and more suitable for high-throughput measurements, opening a new experimental strategy in assessing large libraries of potential drug candidates that work by forming a drug/DNA complex.

High-throughput, 384-well, LC-MS/MS CYP inhibition assay using automation, cassette-analysis technique, and streamlined data analysis.

PubMed

Halladay, Jason S; Delarosa, Erlie Marie; Tran, Daniel; Wang, Leslie; Wong, Susan; Khojasteh, S Cyrus

2011-08-01

Here we describe a high capacity and high-throughput, automated, 384-well CYP inhibition assay using well-known HLM-based MS probes. We provide consistently robust IC(50) values at the lead optimization stage of the drug discovery process. Our method uses the Agilent Technologies/Velocity11 BioCel 1200 system, timesaving techniques for sample analysis, and streamlined data processing steps. For each experiment, we generate IC(50) values for up to 344 compounds and positive controls for five major CYP isoforms (probe substrate): CYP1A2 (phenacetin), CYP2C9 ((S)-warfarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4/5 (testosterone and midazolam). Each compound is incubated separately at four concentrations with each CYP probe substrate under the optimized incubation condition. Each incubation is quenched with acetonitrile containing the deuterated internal standard of the respective metabolite for each probe substrate. To minimize the number of samples to be analyzed by LC-MS/MS and reduce the amount of valuable MS runtime, we utilize timesaving techniques of cassette analysis (pooling the incubation samples at the end of each CYP probe incubation into one) and column switching (reducing the amount of MS runtime). Here we also report on the comparison of IC(50) results for five major CYP isoforms using our method compared to values reported in the literature.

High Throughput Discovery of Solar Fuels Photoanodes in the CuO-V 2 O 5 System

DOE PAGES

Zhou, Lan; Yan, Qimin; Shinde, Aniketa; ...

2015-08-26

Solar photoelectrochemical generation of fuel is a promising energy technology yet the lack of an efficient, robust photoanode remains a primary materials challenge in the development and deployment of solar fuels generators. Metal oxides comprise the most promising class of photoanode materials, but no known material meets the demanding requirements of low band gap energy, photoelectrocatalysis of the oxygen evolution reaction, and stability under highly oxidizing conditions. Here, we report the identification of new photoelectroactive materials through a strategic combination of combinatorial materials synthesis, high-throughput photoelectrochemistry, optical spectroscopy, and detailed electronic structure calculations. We identify 4 photoelectrocatalyst phases - α-Cumore » 2V 2O 7, β-Cu 2V 2O 7, γ-Cu 3V 2O 8, and Cu 11V 6O 26 - with band gap energy at or below 2 eV. The photoelectrochemical properties and 30-minute stability of these copper vanadate phases are demonstrated in 3 different aqueous electrolytes (pH 7, pH 9, and pH 13), with select combinations of phase and electrolyte exhibiting unprecedented photoelectrocatalytic stability for metal oxides with sub-2 eV band gap. Through integration of experimental and theoretical techniques, we determine new structure-property relationships and establish CuO-V 2O 5 as the most prominent composition system for OER photoelectrocatalysts, providing crucial information for materials genomes initiatives and paving the way for continued development of solar fuels photoanodes.« less

A hydrogel-based versatile screening platform for specific biomolecular recognition in a well plate format.

PubMed

Beer, Meike V; Rech, Claudia; Diederichs, Sylvia; Hahn, Kathrin; Bruellhoff, Kristina; Möller, Martin; Elling, Lothar; Groll, Jürgen

2012-04-01

Precise determination of biomolecular interactions in high throughput crucially depends on a surface coating technique that allows immobilization of a variety of interaction partners in a non-interacting environment. We present a one-step hydrogel coating system based on isocyanate functional six-arm poly(ethylene oxide)-based star polymers for commercially available 96-well microtiter plates that combines a straightforward and robust coating application with versatile bio-functionalization. This system generates resistance to unspecific protein adsorption and cell adhesion, as demonstrated with fluorescently labeled bovine serum albumin and primary human dermal fibroblasts (HDF), and high specificity for the assessment of biomolecular recognition processes when ligands are immobilized on this surface. One particular advantage is the wide range of biomolecules that can be immobilized and convert the per se inert coating into a specifically interacting surface. We here demonstrate the immobilization and quantification of a broad range of biochemically important ligands, such as peptide sequences GRGDS and GRGDSK-biotin, the broadly applicable coupler molecule biocytin, the protein fibronectin, and the carbohydrates N-acetylglucosamine and N-acetyllactosamine. A simplified protocol for an enzyme-linked immunosorbent assay was established for the detection and quantification of ligands on the coating surface. Cell adhesion on the peptide and protein-modified surfaces was assessed using HDF. All coatings were applied using a one-step preparation technique, including bioactivation, which makes the system suitable for high-throughput screening in a format that is compatible with the most routinely used testing systems.

NiftyPET: a High-throughput Software Platform for High Quantitative Accuracy and Precision PET Imaging and Analysis.

PubMed

Markiewicz, Pawel J; Ehrhardt, Matthias J; Erlandsson, Kjell; Noonan, Philip J; Barnes, Anna; Schott, Jonathan M; Atkinson, David; Arridge, Simon R; Hutton, Brian F; Ourselin, Sebastien

2018-01-01

We present a standalone, scalable and high-throughput software platform for PET image reconstruction and analysis. We focus on high fidelity modelling of the acquisition processes to provide high accuracy and precision quantitative imaging, especially for large axial field of view scanners. All the core routines are implemented using parallel computing available from within the Python package NiftyPET, enabling easy access, manipulation and visualisation of data at any processing stage. The pipeline of the platform starts from MR and raw PET input data and is divided into the following processing stages: (1) list-mode data processing; (2) accurate attenuation coefficient map generation; (3) detector normalisation; (4) exact forward and back projection between sinogram and image space; (5) estimation of reduced-variance random events; (6) high accuracy fully 3D estimation of scatter events; (7) voxel-based partial volume correction; (8) region- and voxel-level image analysis. We demonstrate the advantages of this platform using an amyloid brain scan where all the processing is executed from a single and uniform computational environment in Python. The high accuracy acquisition modelling is achieved through span-1 (no axial compression) ray tracing for true, random and scatter events. Furthermore, the platform offers uncertainty estimation of any image derived statistic to facilitate robust tracking of subtle physiological changes in longitudinal studies. The platform also supports the development of new reconstruction and analysis algorithms through restricting the axial field of view to any set of rings covering a region of interest and thus performing fully 3D reconstruction and corrections using real data significantly faster. All the software is available as open source with the accompanying wiki-page and test data.

A robust clustering algorithm for identifying problematic samples in genome-wide association studies.

PubMed

Bellenguez, Céline; Strange, Amy; Freeman, Colin; Donnelly, Peter; Spencer, Chris C A

2012-01-01

High-throughput genotyping arrays provide an efficient way to survey single nucleotide polymorphisms (SNPs) across the genome in large numbers of individuals. Downstream analysis of the data, for example in genome-wide association studies (GWAS), often involves statistical models of genotype frequencies across individuals. The complexities of the sample collection process and the potential for errors in the experimental assay can lead to biases and artefacts in an individual's inferred genotypes. Rather than attempting to model these complications, it has become a standard practice to remove individuals whose genome-wide data differ from the sample at large. Here we describe a simple, but robust, statistical algorithm to identify samples with atypical summaries of genome-wide variation. Its use as a semi-automated quality control tool is demonstrated using several summary statistics, selected to identify different potential problems, and it is applied to two different genotyping platforms and sample collections. The algorithm is written in R and is freely available at www.well.ox.ac.uk/chris-spencer chris.spencer@well.ox.ac.uk Supplementary data are available at Bioinformatics online.

Microfluidic model of the platelet-generating organ: beyond bone marrow biomimetics

NASA Astrophysics Data System (ADS)

Blin, Antoine; Le Goff, Anne; Magniez, Aurélie; Poirault-Chassac, Sonia; Teste, Bruno; Sicot, Géraldine; Nguyen, Kim Anh; Hamdi, Feriel S.; Reyssat, Mathilde; Baruch, Dominique

2016-02-01

We present a new, rapid method for producing blood platelets in vitro from cultured megakaryocytes based on a microfluidic device. This device consists in a wide array of VWF-coated micropillars. Such pillars act as anchors on megakaryocytes, allowing them to remain trapped in the device and subjected to hydrodynamic shear. The combined effect of anchoring and shear induces the elongation of megakaryocytes and finally their rupture into platelets and proplatelets. This process was observed with megakaryocytes from different origins and found to be robust. This original bioreactor design allows to process megakaryocytes at high throughput (millions per hour). Since platelets are produced in such a large amount, their extensive biological characterisation is possible and shows that platelets produced in this bioreactor are functional.

Microfluidic model of the platelet-generating organ: beyond bone marrow biomimetics.

PubMed

Blin, Antoine; Le Goff, Anne; Magniez, Aurélie; Poirault-Chassac, Sonia; Teste, Bruno; Sicot, Géraldine; Nguyen, Kim Anh; Hamdi, Feriel S; Reyssat, Mathilde; Baruch, Dominique

2016-02-22

We present a new, rapid method for producing blood platelets in vitro from cultured megakaryocytes based on a microfluidic device. This device consists in a wide array of VWF-coated micropillars. Such pillars act as anchors on megakaryocytes, allowing them to remain trapped in the device and subjected to hydrodynamic shear. The combined effect of anchoring and shear induces the elongation of megakaryocytes and finally their rupture into platelets and proplatelets. This process was observed with megakaryocytes from different origins and found to be robust. This original bioreactor design allows to process megakaryocytes at high throughput (millions per hour). Since platelets are produced in such a large amount, their extensive biological characterisation is possible and shows that platelets produced in this bioreactor are functional.

Meeting review: Bioinformatics and Medicine - from molecules to humans, virtual and real.

PubMed

Russell, Roslin

2002-01-01

The Industrialization Workshop Series aims to promote and discuss integration, automation, simulation, quality, availability and standards in the high-throughput life sciences. The main issues addressed being the transformation of bioinformatics and bioinformaticsbased drug design into a robust discipline in industry, the government, research institutes and academia. The latest workshop emphasized the influence of the post-genomic era on medicine and healthcare with reference to advanced biological systems modeling and simulation, protein structure research, protein-protein interactions, metabolism and physiology. Speakers included Michael Ashburner, Kenneth Buetow, Francois Cambien, Cyrus Chothia, Jean Garnier, Francois Iris, Matthias Mann, Maya Natarajan, Peter Murray-Rust, Richard Mushlin, Barry Robson, David Rubin, Kosta Steliou, John Todd, Janet Thornton, Pim van der Eijk, Michael Vieth and Richard Ward.

Case study: impact of technology investment on lead discovery at Bristol-Myers Squibb, 1998-2006.

PubMed

Houston, John G; Banks, Martyn N; Binnie, Alastair; Brenner, Stephen; O'Connell, Jonathan; Petrillo, Edward W

2008-01-01

We review strategic approaches taken over an eight-year period at BMS to implement new high-throughput approaches to lead discovery. Investments in compound management infrastructure and chemistry library production capability allowed significant growth in the size, diversity and quality of the BMS compound collection. Screening platforms were upgraded with robust automated technology to support miniaturized assay formats, while workflows and information handling technologies were streamlined for improved performance. These technology changes drove the need for a supporting organization in which critical engineering, informatics and scientific skills were more strongly represented. Taken together, these investments led to significant improvements in speed and productivity as well a greater impact of screening campaigns on the initiation of new drug discovery programs.

Selected reaction monitoring mass spectrometry: a methodology overview.

PubMed

Ebhardt, H Alexander

2014-01-01

Moving past the discovery phase of proteomics, the term targeted proteomics combines multiple approaches investigating a certain set of proteins in more detail. One such targeted proteomics approach is the combination of liquid chromatography and selected or multiple reaction monitoring mass spectrometry (SRM, MRM). SRM-MS requires prior knowledge of the fragmentation pattern of peptides, as the presence of the analyte in a sample is determined by measuring the m/z values of predefined precursor and fragment ions. Using scheduled SRM-MS, many analytes can robustly be monitored allowing for high-throughput sample analysis of the same set of proteins over many conditions. In this chapter, fundaments of SRM-MS are explained as well as an optimized SRM pipeline from assay generation to data analyzed.

AMPA RECEPTOR POTENTIATORS: FROM DRUG DESIGN TO COGNITIVE ENHANCEMENT

PubMed Central

PARTIN, KATHRYN M.

2014-01-01

Positive allosteric modulators of ionotropic glutamate receptors have emerged as a target for treating cognitive impairment and neurodegeneration, but also mental illnesses such as major depressive disorder. The possibility of creating a new class of pharmaceutical agent to treat refractive mental health issues has compelled researchers to redouble their efforts to develop a safe, effective treatment for memory and cognition impairments. Coupled with the more robust research methodologies that have emerged, including more sophisticated high-throughput-screens, higher resolution structural biology techniques, and more focused assessment on pharmacokinetics, the development of positive modulators of AMPA receptors holds great promise. We describe recent approaches that improve our understanding of the basic physiology underlying memory and cognition, and their application towards promoting human health. PMID:25462292

Multiplex engineering of industrial yeast genomes using CRISPRm.

PubMed

Ryan, Owen W; Cate, Jamie H D

2014-01-01

Global demand has driven the use of industrial strains of the yeast Saccharomyces cerevisiae for large-scale production of biofuels and renewable chemicals. However, the genetic basis of desired domestication traits is poorly understood because robust genetic tools do not exist for industrial hosts. We present an efficient, marker-free, high-throughput, and multiplexed genome editing platform for industrial strains of S. cerevisiae that uses plasmid-based expression of the CRISPR/Cas9 endonuclease and multiple ribozyme-protected single guide RNAs. With this multiplex CRISPR (CRISPRm) system, it is possible to integrate DNA libraries into the chromosome for evolution experiments, and to engineer multiple loci simultaneously. The CRISPRm tools should therefore find use in many higher-order synthetic biology applications to accelerate improvements in industrial microorganisms.

Resolving Off-Nominal Situations in Schedule-Based Terminal Area Operations: Results from a Human-in-the-Loop Simulation

NASA Technical Reports Server (NTRS)

Mercer, Joey; Callantine, Todd; Martin, Lynne

2012-01-01

A recent human-in-the-loop simulation in the Airspace Operations Laboratory (AOL) at NASA's Ames Research Center investigated the robustness of Controller-Managed Spacing (CMS) operations. CMS refers to AOL-developed controller tools and procedures for enabling arrivals to conduct efficient Optimized Profile Descents with sustained high throughput. The simulation provided a rich data set for examining how a traffic management supervisor and terminal-area controller participants used the CMS tools and coordinated to respond to off-nominal events. This paper proposes quantitative measures for characterizing the participants responses. Case studies of go-around events, replicated during the simulation, provide insights into the strategies employed and the role the CMS tools played in supporting them.

Automated imaging system for single molecules

DOEpatents

Schwartz, David Charles; Runnheim, Rodney; Forrest, Daniel

2012-09-18

There is provided a high throughput automated single molecule image collection and processing system that requires minimal initial user input. The unique features embodied in the present disclosure allow automated collection and initial processing of optical images of single molecules and their assemblies. Correct focus may be automatically maintained while images are collected. Uneven illumination in fluorescence microscopy is accounted for, and an overall robust imaging operation is provided yielding individual images prepared for further processing in external systems. Embodiments described herein are useful in studies of any macromolecules such as DNA, RNA, peptides and proteins. The automated image collection and processing system and method of same may be implemented and deployed over a computer network, and may be ergonomically optimized to facilitate user interaction.

An open-source computational and data resource to analyze digital maps of immunopeptidomes

PubMed Central

Caron, Etienne; Espona, Lucia; Kowalewski, Daniel J; Schuster, Heiko; Ternette, Nicola; Alpízar, Adán; Schittenhelm, Ralf B; Ramarathinam, Sri H; Lindestam Arlehamn, Cecilia S; Chiek Koh, Ching; Gillet, Ludovic C; Rabsteyn, Armin; Navarro, Pedro; Kim, Sangtae; Lam, Henry; Sturm, Theo; Marcilla, Miguel; Sette, Alessandro; Campbell, David S; Deutsch, Eric W; Moritz, Robert L; Purcell, Anthony W; Rammensee, Hans-Georg; Stevanovic, Stefan; Aebersold, Ruedi

2015-01-01

We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies. DOI: http://dx.doi.org/10.7554/eLife.07661.001 PMID:26154972

Functional genomic analysis of drug sensitivity pathways to guide adjuvant strategies in breast cancer

PubMed Central

Swanton, Charles; Szallasi, Zoltan; Brenton, James D; Downward, Julian

2008-01-01

The widespread introduction of high throughput RNA interference screening technology has revealed tumour drug sensitivity pathways to common cytotoxics such as paclitaxel, doxorubicin and 5-fluorouracil, targeted agents such as trastuzumab and inhibitors of AKT and Poly(ADP-ribose) polymerase (PARP) as well as endocrine therapies such as tamoxifen. Given the limited power of microarray signatures to predict therapeutic response in associative studies of small clinical trial cohorts, the use of functional genomic data combined with expression or sequence analysis of genes and microRNAs implicated in drug response in human tumours may provide a more robust method to guide adjuvant treatment strategies in breast cancer that are transferable across different expression platforms and patient cohorts. PMID:18986507

False biochemical diagnosis of hyperthyroidism in streptavidin-biotin-based immunoassays: the problem of biotin intake and related interferences.

PubMed

Piketty, Marie-Liesse; Polak, Michel; Flechtner, Isabelle; Gonzales-Briceño, Laura; Souberbielle, Jean-Claude

2017-05-01

Immunoassays are now commonly used for hormone measurement, in high throughput analytical platforms. Immunoassays are generally robust to interference. However, endogenous analytical error may occur in some patients; this may be encountered in biotin supplementation or in the presence of anti-streptavidin antibody, in immunoassays involving streptavidin-biotin interaction. In these cases, the interference may induce both false positive and false negative results, and simulate a seemingly coherent hormonal profile. It is to be feared that this type of errors will be more frequently observed. This review underlines the importance of keeping close interactions between biologists and clinicians to be able to correlate the hormonal assay results with the clinical picture.

High-Throughput Method of Whole-Brain Sectioning, Using the Tape-Transfer Technique.

PubMed

Pinskiy, Vadim; Jones, Jamie; Tolpygo, Alexander S; Franciotti, Neil; Weber, Kevin; Mitra, Partha P

2015-01-01

Cryostat sectioning is a popular but labor-intensive method for preparing histological brain sections. We have developed a modification of the commercially available CryoJane tape collection method that significantly improves the ease of collection and the final quality of the tissue sections. The key modification involves an array of UVLEDs to achieve uniform polymerization of the glass slide and robust adhesion between the section and slide. This report presents system components and detailed procedural steps, and provides examples of end results; that is, 20 μm mouse brain sections that have been successfully processed for routine Nissl, myelin staining, DAB histochemistry, and fluorescence. The method is also suitable for larger brains, such as rat and monkey.

High-Throughput Method of Whole-Brain Sectioning, Using the Tape-Transfer Technique

PubMed Central

Pinskiy, Vadim; Jones, Jamie; Tolpygo, Alexander S.; Franciotti, Neil; Weber, Kevin; Mitra, Partha P.

2015-01-01

Cryostat sectioning is a popular but labor-intensive method for preparing histological brain sections. We have developed a modification of the commercially available CryoJane tape collection method that significantly improves the ease of collection and the final quality of the tissue sections. The key modification involves an array of UVLEDs to achieve uniform polymerization of the glass slide and robust adhesion between the section and slide. This report presents system components and detailed procedural steps, and provides examples of end results; that is, 20μm mouse brain sections that have been successfully processed for routine Nissl, myelin staining, DAB histochemistry, and fluorescence. The method is also suitable for larger brains, such as rat and monkey. PMID:26181725

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

DOE PAGES

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

2015-01-07

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

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

PubMed Central

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

2017-01-01

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

Systematic interpolation method predicts protein chromatographic elution with salt gradients, pH gradients and combined salt/pH gradients.

PubMed

Creasy, Arch; Barker, Gregory; Carta, Giorgio

2017-03-01

A methodology is presented to predict protein elution behavior from an ion exchange column using both individual or combined pH and salt gradients based on high-throughput batch isotherm data. The buffer compositions are first optimized to generate linear pH gradients from pH 5.5 to 7 with defined concentrations of sodium chloride. Next, high-throughput batch isotherm data are collected for a monoclonal antibody on the cation exchange resin POROS XS over a range of protein concentrations, salt concentrations, and solution pH. Finally, a previously developed empirical interpolation (EI) method is extended to describe protein binding as a function of the protein and salt concentration and solution pH without using an explicit isotherm model. The interpolated isotherm data are then used with a lumped kinetic model to predict the protein elution behavior. Experimental results obtained for laboratory scale columns show excellent agreement with the predicted elution curves for both individual or combined pH and salt gradients at protein loads up to 45 mg/mL of column. Numerical studies show that the model predictions are robust as long as the isotherm data cover the range of mobile phase compositions where the protein actually elutes from the column. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The use of high-throughput screening techniques to evaluate mitochondrial toxicity.

PubMed

Wills, Lauren P

2017-11-01

Toxicologists and chemical regulators depend on accurate and effective methods to evaluate and predict the toxicity of thousands of current and future compounds. Robust high-throughput screening (HTS) experiments have the potential to efficiently test large numbers of chemical compounds for effects on biological pathways. HTS assays can be utilized to examine chemical toxicity across multiple mechanisms of action, experimental models, concentrations, and lengths of exposure. Many agricultural, industrial, and pharmaceutical chemicals classified as harmful to human and environmental health exert their effects through the mechanism of mitochondrial toxicity. Mitochondrial toxicants are compounds that cause a decrease in the number of mitochondria within a cell, and/or decrease the ability of mitochondria to perform normal functions including producing adenosine triphosphate (ATP) and maintaining cellular homeostasis. Mitochondrial dysfunction can lead to apoptosis, necrosis, altered metabolism, muscle weakness, neurodegeneration, decreased organ function, and eventually disease or death of the whole organism. The development of HTS techniques to identify mitochondrial toxicants will provide extensive databases with essential connections between mechanistic mitochondrial toxicity and chemical structure. Computational and bioinformatics approaches can be used to evaluate compound databases for specific chemical structures associated with toxicity, with the goal of developing quantitative structure-activity relationship (QSAR) models and mitochondrial toxicophores. Ultimately these predictive models will facilitate the identification of mitochondrial liabilities in consumer products, industrial compounds, pharmaceuticals and environmental hazards. Copyright © 2017 Elsevier B.V. All rights reserved.

High- and low-throughput scoring of fat mass and body fat distribution in C. elegans

PubMed Central

Wählby, Carolina; Lee-Conery, Annie; Bray, Mark-Anthony; Kamentsky, Lee; Larkins-Ford, Jonah; Sokolnicki, Katherine L.; Veneskey, Matthew; Michaels, Kerry; Carpenter, Anne E.; O’Rourke, Eyleen J.

2014-01-01

Fat accumulation is a complex phenotype affected by factors such as neuroendocrine signaling, feeding, activity, and reproductive output. Accordingly, the most informative screens for genes and compounds affecting fat accumulation would be those carried out in whole living animals. Caenorhabditis elegans is a well-established and effective model organism, especially for biological processes that involve organ systems and multicellular interactions, such as metabolism. Every cell in the transparent body of C. elegans is visible under a light microscope. Consequently, an accessible and reliable method to visualize worm lipid-droplet fat depots would make C. elegans the only metazoan in which genes affecting not only fat mass but also body fat distribution could be assessed at a genome-wide scale. Here we present a radical improvement in oil red O worm staining together with high-throughput image-based phenotyping. The three-step sample preparation method is robust, formaldehyde-free, and inexpensive, and requires only 15 minutes of hands-on time to process a 96-well plate. Together with our free and user-friendly automated image analysis package, this method enables C. elegans sample preparation and phenotype scoring at a scale that is compatible with genome-wide screens. Thus we present a feasible approach to small-scale phenotyping and large-scale screening for genetic and/or chemical perturbations that lead to alterations in fat quantity and distribution in whole animals. PMID:24784529

Application of a high-throughput relative chemical stability assay to screen therapeutic protein formulations by assessment of conformational stability and correlation to aggregation propensity.

PubMed

Rizzo, Joseph M; Shi, Shuai; Li, Yunsong; Semple, Andrew; Esposito, Jessica J; Yu, Shenjiang; Richardson, Daisy; Antochshuk, Valentyn; Shameem, Mohammed

2015-05-01

In this study, an automated high-throughput relative chemical stability (RCS) assay was developed in which various therapeutic proteins were assessed to determine stability based on the resistance to denaturation post introduction to a chaotrope titration. Detection mechanisms of both intrinsic fluorescence and near UV circular dichroism (near-UV CD) are demonstrated. Assay robustness was investigated by comparing multiple independent assays and achieving r(2) values >0.95 for curve overlays. The complete reversibility of the assay was demonstrated by intrinsic fluorescence, near-UV CD, and biologic potency. To highlight the method utility, we compared the RCS assay with differential scanning calorimetry and dynamic scanning fluorimetry methodologies. Utilizing C1/2 values obtained from the RCS assay, formulation rank-ordering of 12 different mAb formulations was performed. The prediction of long-term stability on protein aggregation is obtained by demonstrating a good correlation with an r(2) of 0.83 between RCS and empirical aggregation propensity data. RCS promises to be an extremely useful tool to aid in candidate formulation development efforts based on the complete reversibility of the method to allow for multiple assessments without protein loss and the strong correlation between the C1/2 data obtained and accelerated stability under stressed conditions. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Time to "go large" on biofilm research: advantages of an omics approach.

PubMed

Azevedo, Nuno F; Lopes, Susana P; Keevil, Charles W; Pereira, Maria O; Vieira, Maria J

2009-04-01

In nature, the biofilm mode of life is of great importance in the cell cycle for many microorganisms. Perhaps because of biofilm complexity and variability, the characterization of a given microbial system, in terms of biofilm formation potential, structure and associated physiological activity, in a large-scale, standardized and systematic manner has been hindered by the absence of high-throughput methods. This outlook is now starting to change as new methods involving the utilization of microtiter-plates and automated spectrophotometry and microscopy systems are being developed to perform large-scale testing of microbial biofilms. Here, we evaluate if the time is ripe to start an integrated omics approach, i.e., the generation and interrogation of large datasets, to biofilms--"biofomics". This omics approach would bring much needed insight into how biofilm formation ability is affected by a number of environmental, physiological and mutational factors and how these factors interplay between themselves in a standardized manner. This could then lead to the creation of a database where biofilm signatures are identified and interrogated. Nevertheless, and before embarking on such an enterprise, the selection of a versatile, robust, high-throughput biofilm growing device and of appropriate methods for biofilm analysis will have to be performed. Whether such device and analytical methods are already available, particularly for complex heterotrophic biofilms is, however, very debatable.

Neural network control of focal position during time-lapse microscopy of cells.

PubMed

Wei, Ling; Roberts, Elijah

2018-05-09

Live-cell microscopy is quickly becoming an indispensable technique for studying the dynamics of cellular processes. Maintaining the specimen in focus during image acquisition is crucial for high-throughput applications, especially for long experiments or when a large sample is being continuously scanned. Automated focus control methods are often expensive, imperfect, or ill-adapted to a specific application and are a bottleneck for widespread adoption of high-throughput, live-cell imaging. Here, we demonstrate a neural network approach for automatically maintaining focus during bright-field microscopy. Z-stacks of yeast cells growing in a microfluidic device were collected and used to train a convolutional neural network to classify images according to their z-position. We studied the effect on prediction accuracy of the various hyperparameters of the neural network, including downsampling, batch size, and z-bin resolution. The network was able to predict the z-position of an image with ±1 μm accuracy, outperforming human annotators. Finally, we used our neural network to control microscope focus in real-time during a 24 hour growth experiment. The method robustly maintained the correct focal position compensating for 40 μm of focal drift and was insensitive to changes in the field of view. About ~100 annotated z-stacks were required to train the network making our method quite practical for custom autofocus applications.

High-Throughput Screening for Human Galactokinase Inhibitors

PubMed Central

WIERENGA, KLAAS J.; LAI, KENT; BUCHWALD, PETER; TANG, MANSHU

2009-01-01

Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) can result in a potentially lethal disorder called classic galactosemia. Although the neonatal lethality associated with this disease can be prevented through early diagnosis and a galactose-restricted diet, the lack of effective therapy continues to have consequences: developmental delay, neurological disorders, and premature ovarian failure are common sequelae in childhood and adulthood. Several lines of evidence indicate that an elevated level of galactose-1-phosphate (gal-1-p), the product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in patients with classic galactosemia. The authors hypothesize that elimination of gal-1-p production by inhibiting GALK will relieve GALT-deficient cells from galactose toxicity. To test this hypothesis, they obtained human GALK using a bacterial expression system. They developed a robust, miniaturized, high-throughput GALK assay (Z′ factor =0.91) and used this assay to screen against libraries composed of 50,000 chemical compounds with diverse structural scaffolds. They selected 150 compounds that, at an average concentration of 33.3 μM, inhibited GALK activity in vitro more than 86.5% and with a reproducibility score of at least 0.7 for a confirmatory screen under identical experimental conditions. Of these 150 compounds, 34 were chosen for further characterization. Preliminary results indicated that these 34 compounds have potential to serve as leads to the development of more effective therapy of classic galactosemia. PMID:18490662

High-throughput screening for human galactokinase inhibitors.

PubMed

Wierenga, Klaas J; Lai, Kent; Buchwald, Peter; Tang, Manshu

2008-06-01

Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) can result in a potentially lethal disorder called classic galactosemia. Although the neonatal lethality associated with this disease can be prevented through early diagnosis and a galactose-restricted diet, the lack of effective therapy continues to have consequences: developmental delay, neurological disorders, and premature ovarian failure are common sequelae in childhood and adulthood. Several lines of evidence indicate that an elevated level of galactose-1-phosphate (gal-1-p), the product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in patients with classic galactosemia. The authors hypothesize that elimination of gal-1-p production by inhibiting GALK will relieve GALT-deficient cells from galactose toxicity. To test this hypothesis, they obtained human GALK using a bacterial expression system. They developed a robust, miniaturized, high-throughput GALK assay (Z' factor = 0.91) and used this assay to screen against libraries composed of 50,000 chemical compounds with diverse structural scaffolds. They selected 150 compounds that, at an average concentration of 33.3 microM, inhibited GALK activity in vitro more than 86.5% and with a reproducibility score of at least 0.7 for a confirmatory screen under identical experimental conditions. Of these 150 compounds, 34 were chosen for further characterization. Preliminary results indicated that these 34 compounds have potential to serve as leads to the development of more effective therapy of classic galactosemia.

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

EPA Pesticide Factsheets

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

Evaluating High Throughput Toxicokinetics and Toxicodynamics for IVIVE (WC10)

EPA Science Inventory

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

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

USDA-ARS?s Scientific Manuscript database

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

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

EPA Science Inventory

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

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

PubMed Central

2011-01-01

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

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

PubMed

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

2016-09-12

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

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

PubMed Central

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

2011-01-01

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

Realizing the promise of reverse phase protein arrays for clinical, translational, and basic research: a workshop report: the RPPA (Reverse Phase Protein Array) society.

PubMed

Akbani, Rehan; Becker, Karl-Friedrich; Carragher, Neil; Goldstein, Ted; de Koning, Leanne; Korf, Ulrike; Liotta, Lance; Mills, Gordon B; Nishizuka, Satoshi S; Pawlak, Michael; Petricoin, Emanuel F; Pollard, Harvey B; Serrels, Bryan; Zhu, Jingchun

2014-07-01

Reverse phase protein array (RPPA) technology introduced a miniaturized "antigen-down" or "dot-blot" immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories. Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following: preservation and optimization of pre-analytical sample quality, application of validated high-affinity and specific antibody (or other protein affinity) detection reagents, dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, and quality-assured procedures and data analysis workflows compatible with application within regulated clinical environments. In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and opportunities. The outcomes of the workshops included a number of key opportunities to advance the RPPA field and provide added benefit to existing and future participants in the RPPA research community. The purpose of this report is to share and disseminate, as a community, current knowledge and future directions of the RPPA technology. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Using iterative cluster merging with improved gap statistics to perform online phenotype discovery in the context of high-throughput RNAi screens

PubMed Central

Yin, Zheng; Zhou, Xiaobo; Bakal, Chris; Li, Fuhai; Sun, Youxian; Perrimon, Norbert; Wong, Stephen TC

2008-01-01

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 image-based datasets derived from a wide spectrum of experimental conditions and is suitable to adaptively process new images which are continuously added to existing datasets. Validations were carried out on different dataset, including published RNAi screening using Drosophila embryos [Additional files 1, 2], dataset for cell cycle phase identification using HeLa cells [Additional files 1, 3, 4] and synthetic dataset using polygons, our methods tackled three aforementioned tasks effectively with an accuracy range of 85%–90%. When our method is implemented in the context of a Drosophila genome-scale RNAi image-based screening of cultured cells aimed to identifying the contribution of individual genes towards the regulation of cell-shape, it efficiently discovers meaningful new phenotypes and provides novel biological insight. We also propose a two-step procedure to modify the novelty detection method based on one-class SVM, so that it can be used to online phenotype discovery. In different conditions, we compared the SVM based method with our method using various datasets and our methods consistently outperformed SVM based method in at least two of three tasks by 2% to 5%. These results demonstrate that our methods can be used to better identify novel phenotypes in image-based datasets from a wide range of conditions and organisms. Conclusion We demonstrate that our method can detect various novel phenotypes effectively in complex datasets. Experiment results also validate that our method performs consistently under different order of image input, variation of starting conditions including the number and composition of existing phenotypes, and dataset from different screens. In our findings, the proposed method is suitable for online phenotype discovery in diverse high-throughput image-based genetic and chemical screens. PMID:18534020

Application of targeted quantitative proteomics analysis in human cerebrospinal fluid using a liquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (LC MALDI TOF/TOF) platform.

PubMed

Pan, Sheng; Rush, John; Peskind, Elaine R; Galasko, Douglas; Chung, Kathryn; Quinn, Joseph; Jankovic, Joseph; Leverenz, James B; Zabetian, Cyrus; Pan, Catherine; Wang, Yan; Oh, Jung Hun; Gao, Jean; Zhang, Jianpeng; Montine, Thomas; Zhang, Jing

2008-02-01

Targeted quantitative proteomics by mass spectrometry aims to selectively detect one or a panel of peptides/proteins in a complex sample and is particularly appealing for novel biomarker verification/validation because it does not require specific antibodies. Here, we demonstrated the application of targeted quantitative proteomics in searching, identifying, and quantifying selected peptides in human cerebrospinal spinal fluid (CSF) using a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF)-based platform. The approach involved two major components: the use of isotopic-labeled synthetic peptides as references for targeted identification and quantification and a highly selective mass spectrometric analysis based on the unique characteristics of the MALDI instrument. The platform provides high confidence for targeted peptide detection in a complex system and can potentially be developed into a high-throughput system. Using the liquid chromatography (LC) MALDI TOF/TOF platform and the complementary identification strategy, we were able to selectively identify and quantify a panel of targeted peptides in the whole proteome of CSF without prior depletion of abundant proteins. The effectiveness and robustness of the approach associated with different sample complexity, sample preparation strategies, as well as mass spectrometric quantification were evaluated. Other issues related to chromatography separation and the feasibility for high-throughput analysis were also discussed. Finally, we applied targeted quantitative proteomics to analyze a subset of previously identified candidate markers in CSF samples of patients with Parkinson's disease (PD) at different stages and Alzheimer's disease (AD) along with normal controls.

Bringing the light to high throughput screening: use of optogenetic tools for the development of recombinant cellular assays

NASA Astrophysics Data System (ADS)

Agus, Viviana; Di Silvio, Alberto; Rolland, Jean Francois; Mondini, Anna; Tremolada, Sara; Montag, Katharina; Scarabottolo, Lia; Redaelli, Loredana; Lohmer, Stefan

2015-03-01

The use of light-activated proteins represents a powerful tool to control biological processes with high spatial and temporal precision. These so called "optogenetic" technologies have been successfully validated in many recombinant systems, and have been widely applied to the study of cellular mechanisms in intact tissues or behaving animals; to do that, complex, high-intensity, often home-made instrumentations were developed to achieve the optimal power and precision of light stimulation. In our study we sought to determine if this optical modulation can be obtained also in a miniaturized format, such as a 384-well plate, using the instrumentations normally dedicated to fluorescence analysis in High Throughput Screening (HTS) activities, such as for example the FLIPR (Fluorometric Imaging Plate Reader) instrument. We successfully generated optogenetic assays for the study of different ion channel targets: the CaV1.3 calcium channel was modulated by the light-activated Channelrhodopsin-2, the HCN2 cyclic nucleotide gated (CNG) channel was modulated by the light activated bPAC adenylyl cyclase, and finally the genetically encoded voltage indicator ArcLight was efficiently used to measure potassium, sodium or chloride channel activity. Our results showed that stable, robust and miniaturized cellular assays can be developed using different optogenetic tools, and efficiently modulated by the FLIPR instrument LEDs in a 384-well format. The spatial and temporal resolution delivered by this technology might enormously advantage the early stages of drug discovery, leading to the identification of more physiological and effective drug molecules.

A High Content Screening (HCS) Assay for the Identification of Chemical Inducers of PML Oncogenic Domains (PODs)

PubMed Central

Yip, Kenneth W.; Cuddy, Michael; Pinilla, Clemencia; Giulanotti, Marc; Heynen-Genel, Susanne; Matsuzawa, Shu-ichi; Reed, John C.

2014-01-01

PML is a tumor suppressor that promotes apoptosis through both p53-dependent and - independent mechanisms, participates in Rb-mediated cell cycle arrest, inhibits neoangiogenesis, and contributes to maintenance of genomic stability. PML also plays a role in host defense against viruses, conferring antiviral activity. When active, PML localizes to subnuclear structures named PML oncogenic domains (PODs) or PML nuclear bodies (PML-NBs), whereas inactive PML is located diffusely throughout the nucleus of cells, thus providing a morphological indicator. Known activators of PML include arsenicals and interferons, however, these agents induce a plethora of toxic effects, limiting their effectiveness. The objective of the current study was to develop a high content screening (HCS) assay for the identification of chemical activators of PML. We describe methods for automated analysis of POD formation using high throughput microscopy (HTM) to localize PML immunofluorescence in conjunction with image analysis software for POD quantification. Using this HCS assay in 384 well format, we performed pilot screens of a small synthetic chemical library and mixture-based combinatorial libraries, demonstrating the robust performance of the assay. HCS counter-screening assays were also developed for hit characterization, based on immunofluorescence analyses of the subcellular location of phosphorylated H2AX or phosphorylated CHK1, which increase in a punctate nuclear pattern in response to DNA damage. Thus, the HCS assay devised here represents a high throughput screen that can be utilized to discover POD-inducing compounds that may restore the tumor suppressor activity of PML in cancers or possibly promote anti-viral states. PMID:21233309

TCP Throughput Profiles Using Measurements over Dedicated Connections

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

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

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

Mitigated FPGA design of multi-gigabit transceivers for application in high radiation environments of High Energy Physics experiments

DOE PAGES

Brusati, M.; Camplani, A.; Cannon, M.; ...

2017-02-20

SRAM-ba8ed Field Programmable Gate Array (FPGA) logic devices arc very attractive in applications where high data throughput is needed, such as the latest generation of High Energy Physics (HEP) experiments. FPGAs have been rarely used in such experiments because of their sensitivity to radiation. The present paper proposes a mitigation approach applied to commercial FPGA devices to meet the reliability requirements for the front-end electronics of the Liquid Argon (LAr) electromagnetic calorimeter of the ATLAS experiment, located at CERN. Particular attention will be devoted to define a proper mitigation scheme of the multi-gigabit transceivers embedded in the FPGA, which ismore » a critical part of the LAr data acquisition chain. A demonstrator board is being developed to validate the proposed methodology. :!\\litigation techniques such as Triple Modular Redundancy (T:t\\IR) and scrubbing will be used to increase the robustness of the design and to maximize the fault tolerance from Single-Event Upsets (SEUs).« less

Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography

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

Mueller, C.; Marx, A.; Epp, S. W.

We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linacmore » Coherent Light Source (LCLS, Menlo Park, California, USA). As a result, the chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.« less

Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography

DOE PAGES

Mueller, C.; Marx, A.; Epp, S. W.; ...

2015-08-18

We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linacmore » Coherent Light Source (LCLS, Menlo Park, California, USA). As a result, the chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.« less

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

EPA Science Inventory

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

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

EPA Science Inventory

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

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

NASA Astrophysics Data System (ADS)

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

2015-01-01

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

Micropatterned comet assay enables high throughput and sensitive DNA damage quantification

PubMed Central

Ge, Jing; Chow, Danielle N.; Fessler, Jessica L.; Weingeist, David M.; Wood, David K.; Engelward, Bevin P.

2015-01-01

The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H2O2-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies. PMID:25527723

Micropatterned comet assay enables high throughput and sensitive DNA damage quantification.

PubMed

Ge, Jing; Chow, Danielle N; Fessler, Jessica L; Weingeist, David M; Wood, David K; Engelward, Bevin P

2015-01-01

The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H2O2-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies. © The Author 2014. Published by Oxford University Press on behalf of the Mutagenesis Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

A Robust Scalable Transportation System Concept

NASA Technical Reports Server (NTRS)

Hahn, Andrew; DeLaurentis, Daniel

2006-01-01

This report documents the 2005 Revolutionary System Concept for Aeronautics (RSCA) study entitled "A Robust, Scalable Transportation System Concept". The objective of the study was to generate, at a high-level of abstraction, characteristics of a new concept for the National Airspace System, or the new NAS, under which transportation goals such as increased throughput, delay reduction, and improved robustness could be realized. Since such an objective can be overwhelmingly complex if pursued at the lowest levels of detail, instead a System-of-Systems (SoS) approach was adopted to model alternative air transportation architectures at a high level. The SoS approach allows the consideration of not only the technical aspects of the NAS", but also incorporates policy, socio-economic, and alternative transportation system considerations into one architecture. While the representations of the individual systems are basic, the higher level approach allows for ways to optimize the SoS at the network level, determining the best topology (i.e. configuration of nodes and links). The final product (concept) is a set of rules of behavior and network structure that not only satisfies national transportation goals, but represents the high impact rules that accomplish those goals by getting the agents to "do the right thing" naturally. The novel combination of Agent Based Modeling and Network Theory provides the core analysis methodology in the System-of-Systems approach. Our method of approach is non-deterministic which means, fundamentally, it asks and answers different questions than deterministic models. The nondeterministic method is necessary primarily due to our marriage of human systems with technological ones in a partially unknown set of future worlds. Our goal is to understand and simulate how the SoS, human and technological components combined, evolve.

Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish.

PubMed

Egan, Rupert J; Bergner, Carisa L; Hart, Peter C; Cachat, Jonathan M; Canavello, Peter R; Elegante, Marco F; Elkhayat, Salem I; Bartels, Brett K; Tien, Anna K; Tien, David H; Mohnot, Sopan; Beeson, Esther; Glasgow, Eric; Amri, Hakima; Zukowska, Zofia; Kalueff, Allan V

2009-12-14

The zebrafish (Danio rerio) is emerging as a promising model organism for experimental studies of stress and anxiety. Here we further validate zebrafish models of stress by analyzing how environmental and pharmacological manipulations affect their behavioral and physiological phenotypes. Experimental manipulations included exposure to alarm pheromone, chronic exposure to fluoxetine, acute exposure to caffeine, as well as acute and chronic exposure to ethanol. Acute (but not chronic) alarm pheromone and acute caffeine produced robust anxiogenic effects, including reduced exploration, increased erratic movements and freezing behavior in zebrafish tested in the novel tank diving test. In contrast, ethanol and fluoxetine had robust anxiolytic effects, including increased exploration and reduced erratic movements. The behavior of several zebrafish strains was also quantified to ascertain differences in their behavioral profiles, revealing high-anxiety (leopard, albino) and low-anxiety (wild type) strains. We also used LocoScan (CleverSys Inc.) video-tracking tool to quantify anxiety-related behaviors in zebrafish, and dissect anxiety-related phenotypes from locomotor activity. Finally, we developed a simple and effective method of measuring zebrafish physiological stress responses (based on a human salivary cortisol assay), and showed that alterations in whole-body cortisol levels in zebrafish parallel behavioral indices of anxiety. Collectively, our results confirm zebrafish as a valid, reliable, and high-throughput model of stress and affective disorders.

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

PubMed Central

JR, Luft; EH, Snell; GT, DeTitta

2011-01-01

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

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

NASA Astrophysics Data System (ADS)

Zhu, Chenggang; Fei, Yiyan; Zhu, Xiangdong

2017-02-01

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

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

Brusati, M.; Camplani, A.; Cannon, M.

SRAM-ba8ed Field Programmable Gate Array (FPGA) logic devices arc very attractive in applications where high data throughput is needed, such as the latest generation of High Energy Physics (HEP) experiments. FPGAs have been rarely used in such experiments because of their sensitivity to radiation. The present paper proposes a mitigation approach applied to commercial FPGA devices to meet the reliability requirements for the front-end electronics of the Liquid Argon (LAr) electromagnetic calorimeter of the ATLAS experiment, located at CERN. Particular attention will be devoted to define a proper mitigation scheme of the multi-gigabit transceivers embedded in the FPGA, which ismore » a critical part of the LAr data acquisition chain. A demonstrator board is being developed to validate the proposed methodology. :!\\litigation techniques such as Triple Modular Redundancy (T:t\\IR) and scrubbing will be used to increase the robustness of the design and to maximize the fault tolerance from Single-Event Upsets (SEUs).« less

Fluorescent Cell Barcoding for Multiplex Flow Cytometry

PubMed Central

Krutzik, Peter O.; Clutter, Matthew R.; Trejo, Angelica; Nolan, Garry P.

2011-01-01

Fluorescent Cell Barcoding (FCB) enables high throughput, i.e. high content flow cytometry by multiplexing samples prior to staining and acquisition on the cytometer. Individual cell samples are barcoded, or labeled, with unique signatures of fluorescent dyes so that they can be mixed together, stained, and analyzed as a single sample. By mixing samples prior to staining, antibody consumption is typically reduced 10 to 100-fold. In addition, data robustness is increased through the combination of control and treated samples, which minimizes pipetting error, staining variation, and the need for normalization. Finally, speed of acquisition is enhanced, enabling large profiling experiments to be run with standard cytometer hardware. In this unit, we outline the steps necessary to apply the FCB method to cell lines as well as primary peripheral blood samples. Important technical considerations such as choice of barcoding dyes, concentrations, labeling buffers, compensation, and software analysis are discussed. PMID:21207359

Multiplex Reverse Transcription-PCR for Simultaneous Surveillance of Influenza A and B Viruses

PubMed Central

Zhou, Bin; Barnes, John R.; Sessions, October M.; Chou, Tsui-Wen; Wilson, Malania; Stark, Thomas J.; Volk, Michelle; Spirason, Natalie; Halpin, Rebecca A.; Kamaraj, Uma Sangumathi; Ding, Tao; Stockwell, Timothy B.; Ghedin, Elodie; Barr, Ian G.

2017-01-01

ABSTRACT Influenza A and B viruses are the causative agents of annual influenza epidemics that can be severe, and influenza A viruses intermittently cause pandemics. Sequence information from influenza virus genomes is instrumental in determining mechanisms underpinning antigenic evolution and antiviral resistance. However, due to sequence diversity and the dynamics of influenza virus evolution, rapid and high-throughput sequencing of influenza viruses remains a challenge. We developed a single-reaction influenza A/B virus (FluA/B) multiplex reverse transcription-PCR (RT-PCR) method that amplifies the most critical genomic segments (hemagglutinin [HA], neuraminidase [NA], and matrix [M]) of seasonal influenza A and B viruses for next-generation sequencing, regardless of viral type, subtype, or lineage. Herein, we demonstrate that the strategy is highly sensitive and robust. The strategy was validated on thousands of seasonal influenza A and B virus-positive specimens using multiple next-generation sequencing platforms. PMID:28978683

Decentralized Grid Scheduling with Evolutionary Fuzzy Systems

NASA Astrophysics Data System (ADS)

Fölling, Alexander; Grimme, Christian; Lepping, Joachim; Papaspyrou, Alexander

In this paper, we address the problem of finding workload exchange policies for decentralized Computational Grids using an Evolutionary Fuzzy System. To this end, we establish a non-invasive collaboration model on the Grid layer which requires minimal information about the participating High Performance and High Throughput Computing (HPC/HTC) centers and which leaves the local resource managers completely untouched. In this environment of fully autonomous sites, independent users are assumed to submit their jobs to the Grid middleware layer of their local site, which in turn decides on the delegation and execution either on the local system or on remote sites in a situation-dependent, adaptive way. We find for different scenarios that the exchange policies show good performance characteristics not only with respect to traditional metrics such as average weighted response time and utilization, but also in terms of robustness and stability in changing environments.

Text Stream Trend Analysis using Multiscale Visual Analytics with Applications to Social Media Systems

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

Steed, Chad A; Beaver, Justin M; BogenII, Paul L.

In this paper, we introduce a new visual analytics system, called Matisse, that allows exploration of global trends in textual information streams with specific application to social media platforms. Despite the potential for real-time situational awareness using these services, interactive analysis of such semi-structured textual information is a challenge due to the high-throughput and high-velocity properties. Matisse addresses these challenges through the following contributions: (1) robust stream data management, (2) automated sen- timent/emotion analytics, (3) inferential temporal, geospatial, and term-frequency visualizations, and (4) a flexible drill-down interaction scheme that progresses from macroscale to microscale views. In addition to describing thesemore » contributions, our work-in-progress paper concludes with a practical case study focused on the analysis of Twitter 1% sample stream information captured during the week of the Boston Marathon bombings.« less

OpenMS: a flexible open-source software platform for mass spectrometry data analysis.

PubMed

Röst, Hannes L; Sachsenberg, Timo; Aiche, Stephan; Bielow, Chris; Weisser, Hendrik; Aicheler, Fabian; Andreotti, Sandro; Ehrlich, Hans-Christian; Gutenbrunner, Petra; Kenar, Erhan; Liang, Xiao; Nahnsen, Sven; Nilse, Lars; Pfeuffer, Julianus; Rosenberger, George; Rurik, Marc; Schmitt, Uwe; Veit, Johannes; Walzer, Mathias; Wojnar, David; Wolski, Witold E; Schilling, Oliver; Choudhary, Jyoti S; Malmström, Lars; Aebersold, Ruedi; Reinert, Knut; Kohlbacher, Oliver

2016-08-30

High-resolution mass spectrometry (MS) has become an important tool in the life sciences, contributing to the diagnosis and understanding of human diseases, elucidating biomolecular structural information and characterizing cellular signaling networks. However, the rapid growth in the volume and complexity of MS data makes transparent, accurate and reproducible analysis difficult. We present OpenMS 2.0 (http://www.openms.de), a robust, open-source, cross-platform software specifically designed for the flexible and reproducible analysis of high-throughput MS data. The extensible OpenMS software implements common mass spectrometric data processing tasks through a well-defined application programming interface in C++ and Python and through standardized open data formats. OpenMS additionally provides a set of 185 tools and ready-made workflows for common mass spectrometric data processing tasks, which enable users to perform complex quantitative mass spectrometric analyses with ease.

Robust and versatile ionic liquid microarrays achieved by microcontact printing

NASA Astrophysics Data System (ADS)

Gunawan, Christian A.; Ge, Mengchen; Zhao, Chuan

2014-04-01

Lab-on-a-chip and miniaturized systems have gained significant popularity motivated by marked differences in material performance at the micro-to-nano-scale realm. However, to fully exploit micro-to-nano-scale chemistry, solvent volatility and lack of reproducibility need to be overcome. Here, we combine the non-volatile and versatile nature of ionic liquids with microcontact printing in an attempt to establish a facile protocol for high throughput fabrication of open microreactors and microfluidics. The micropatterned ionic liquid droplets have been demonstrated as electrochemical cells and reactors for microfabrication of metals and charge transfer complexes, substrates for immobilization of proteins and as membrane-free high-performance amperometric gas sensor arrays. The results suggest that miniaturized ionic liquid systems can be used to solve the problems of solvent volatility and slow mass transport in viscous ionic liquids in lab-on-a-chip devices, thus providing a versatile platform for a diverse number of applications.

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

PubMed Central

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

2015-01-01

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

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

PubMed

Erickson, Heidi S

2012-09-28

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

Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers*

PubMed Central

Chen, Hsiao-Wei; Wu, Chun-Feng; Chu, Lichieh Julie; Chiang, Wei-Fang; Wu, Chih-Ching; Yu, Jau-Song; Tsai, Cheng-Han; Liang, Kung-Hao; Chang, Yu-Sun; Wu, Maureen; Ou Yang, Wei-Ting

2017-01-01

Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research. PMID:28235782

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

Code of Federal Regulations, 2010 CFR

2010-07-01

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

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

PubMed

Liu, Yun; Grossman, Jeffrey C

2014-12-10

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

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

PubMed

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

2017-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2018-03-01

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

Multi-Band Multi-Tone Tunable Millimeter-Wave Frequency Synthesizer For Satellite Beacon Transmitter

NASA Technical Reports Server (NTRS)

Simons, Rainee N.; Wintucky, Edwin G.

2016-01-01

This paper presents the design and test results of a multi-band multi-tone tunable millimeter-wave frequency synthesizer, based on a solid-state frequency comb generator. The intended application of the synthesizer is in a satellite beacon transmitter for radio wave propagation studies at K-band (18 to 26.5 GHz), Q-band (37 to 42 GHz), and E-band (71 to 76 GHz). In addition, the architecture for a compact beacon transmitter, which includes the multi-tone synthesizer, polarizer, horn antenna, and power/control electronics, has been investigated for a notional space-to-ground radio wave propagation experiment payload on a small satellite. The above studies would enable the design of robust high throughput multi-Gbps data rate future space-to-ground satellite communication links.

Crystal structures of ASK1-inhibtor complexes provide a platform for structure-based drug design

PubMed Central

Singh, Onkar; Shillings, Anthony; Craggs, Peter; Wall, Ian; Rowland, Paul; Skarzynski, Tadeusz; Hobbs, Clare I; Hardwick, Phil; Tanner, Rob; Blunt, Michelle; Witty, David R; Smith, Kathrine J

2013-01-01

ASK1, a member of the MAPK Kinase Kinase family of proteins has been shown to play a key role in cancer, neurodegeneration and cardiovascular diseases and is emerging as a possible drug target. Here we describe a ‘replacement-soaking’ method that has enabled the high-throughput X-ray structure determination of ASK1/ligand complexes. Comparison of the X-ray structures of five ASK1/ligand complexes from 3 different chemotypes illustrates that the ASK1 ATP binding site is able to accommodate a range of chemical diversity and different binding modes. The replacement-soaking system is also able to tolerate some protein flexibility. This crystal system provides a robust platform for ASK1/ligand structure determination and future structure based drug design. PMID:23776076

Eliminating barriers to personalized medicine

PubMed Central

2014-01-01

With the emergence of high-throughput discovery platforms, robust preclinical small-animal models, and efficient clinical trial pipelines, it is becoming possible to envision a time when the treatment of human neurologic diseases will become personalized. The emergence of precision medicine will require the identification of subgroups of patients most likely to respond to specific biologically based therapies. This stratification only becomes possible when the determinants that contribute to disease heterogeneity become more fully elucidated. This review discusses the defining factors that underlie disease heterogeneity relevant to the potential for individualized brain tumor (optic pathway glioma) treatments arising in the common single-gene cancer predisposition syndrome, neurofibromatosis type 1 (NF1). In this regard, NF1 is posited as a model genetic condition to establish a workable paradigm for actualizing precision therapeutics for other neurologic disorders. PMID:24975854

Identification of Modules in Protein-Protein Interaction Networks

NASA Astrophysics Data System (ADS)

Erten, Sinan; Koyutürk, Mehmet

In biological systems, most processes are carried out through orchestration of multiple interacting molecules. These interactions are often abstracted using network models. A key feature of cellular networks is their modularity, which contributes significantly to the robustness, as well as adaptability of biological systems. Therefore, modularization of cellular networks is likely to be useful in obtaining insights into the working principles of cellular systems, as well as building tractable models of cellular organization and dynamics. A common, high-throughput source of data on molecular interactions is in the form of physical interactions between proteins, which are organized into protein-protein interaction (PPI) networks. This chapter provides an overview on identification and analysis of functional modules in PPI networks, which has been an active area of research in the last decade.

Microbial Ecology: Where are we now?

PubMed

Boughner, Lisa A; Singh, Pallavi

2016-11-01

Conventional microbiological methods have been readily taken over by newer molecular techniques due to the ease of use, reproducibility, sensitivity and speed of working with nucleic acids. These tools allow high throughput analysis of complex and diverse microbial communities, such as those in soil, freshwater, saltwater, or the microbiota living in collaboration with a host organism (plant, mouse, human, etc). For instance, these methods have been robustly used for characterizing the plant (rhizosphere), animal and human microbiome specifically the complex intestinal microbiota. The human body has been referred to as the Superorganism since microbial genes are more numerous than the number of human genes and are essential to the health of the host. In this review we provide an overview of the Next Generation tools currently available to study microbial ecology, along with their limitations and advantages.

Impact of synthetic biology and metabolic engineering on industrial production of fine chemicals.

PubMed

Jullesson, David; David, Florian; Pfleger, Brian; Nielsen, Jens

2015-11-15

Industrial bio-processes for fine chemical production are increasingly relying on cell factories developed through metabolic engineering and synthetic biology. The use of high throughput techniques and automation for the design of cell factories, and especially platform strains, has played an important role in the transition from laboratory research to industrial production. Model organisms such as Saccharomyces cerevisiae and Escherichia coli remain widely used host strains for industrial production due to their robust and desirable traits. This review describes some of the bio-based fine chemicals that have reached the market, key metabolic engineering tools that have allowed this to happen and some of the companies that are currently utilizing these technologies for developing industrial production processes. Copyright © 2015 Elsevier Inc. All rights reserved.

Microfluidic model of the platelet-generating organ: beyond bone marrow biomimetics

PubMed Central

Blin, Antoine; Le Goff, Anne; Magniez, Aurélie; Poirault-Chassac, Sonia; Teste, Bruno; Sicot, Géraldine; Nguyen, Kim Anh; Hamdi, Feriel S.; Reyssat, Mathilde; Baruch, Dominique

2016-01-01

We present a new, rapid method for producing blood platelets in vitro from cultured megakaryocytes based on a microfluidic device. This device consists in a wide array of VWF-coated micropillars. Such pillars act as anchors on megakaryocytes, allowing them to remain trapped in the device and subjected to hydrodynamic shear. The combined effect of anchoring and shear induces the elongation of megakaryocytes and finally their rupture into platelets and proplatelets. This process was observed with megakaryocytes from different origins and found to be robust. This original bioreactor design allows to process megakaryocytes at high throughput (millions per hour). Since platelets are produced in such a large amount, their extensive biological characterisation is possible and shows that platelets produced in this bioreactor are functional. PMID:26898346

Alternative Affinity Ligands for Immunoglobulins.

PubMed

Kruljec, Nika; Bratkovič, Tomaž

2017-08-16

The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond.

Material-Independent Nanotransfer onto a Flexible Substrate Using Mechanical-Interlocking Structure.

PubMed

Seo, Min-Ho; Choi, Seon-Jin; Park, Sang Hyun; Yoo, Jae-Young; Lim, Sung Kyu; Lee, Jae-Shin; Choi, Kwang-Wook; Jo, Min-Seung; Kim, Il-Doo; Yoon, Jun-Bo

2018-05-22

Nanowire-transfer technology has received much attention thanks to its capability to fabricate high-performance flexible nanodevices with high simplicity and throughput. However, it is still challenging to extend the conventional nanowire-transfer method to the fabrication of a wide range of devices since a chemical-adhesion-based nanowire-transfer mechanism is complex and time-consuming, hindering successful transfer of diverse nanowires made of various materials. Here, we introduce a material-independent mechanical-interlocking-based nanowire-transfer (MINT) method, fabricating ultralong and fully aligned nanowires on a large flexible substrate (2.5 × 2 cm 2 ) in a highly robust manner. For the material-independent nanotransfer, we developed a mechanics-based nanotransfer method, which employs a dry-removable amorphous carbon (a-C) sacrificial layer between a vacuum-deposited nanowire and the underlying master mold. The controlled etching of the sacrificial layer enables the formation of a mechanical-interlocking structure under the nanowire, facilitating peeling off of the nanowire from the master mold robustly and reliably. Using the developed MINT method, we successfully fabricated various metallic and semiconductor nanowire arrays on flexible substrates. We further demonstrated that the developed method is well suited to the reliable fabrication of highly flexible and high-performance nanoelectronic devices. As examples, a fully aligned gold (Au) microheater array exhibited high bending stability (10 6 cycling) and ultrafast (∼220 ms) heating operation up to ∼100 °C. An ultralong Au heater-embedded cuprous-oxide (Cu 2 O) nanowire chemical gas sensor showed significantly improved reversible reaction kinetics toward NO 2 with 10-fold enhancement in sensitivity at 100 °C.

Rapid, low dose X-ray diffractive imaging of the malaria parasite Plasmodium falciparum.

PubMed

Jones, Michael W M; Dearnley, Megan K; van Riessen, Grant A; Abbey, Brian; Putkunz, Corey T; Junker, Mark D; Vine, David J; McNulty, Ian; Nugent, Keith A; Peele, Andrew G; Tilley, Leann

2014-08-01

Phase-diverse X-ray coherent diffractive imaging (CDI) provides a route to high sensitivity and spatial resolution with moderate radiation dose. It also provides a robust solution to the well-known phase-problem, making on-line image reconstruction feasible. Here we apply phase-diverse CDI to a cellular sample, obtaining images of an erythrocyte infected by the sexual stage of the malaria parasite, Plasmodium falciparum, with a radiation dose significantly lower than the lowest dose previously reported for cellular imaging using CDI. The high sensitivity and resolution allow key biological features to be identified within intact cells, providing complementary information to optical and electron microscopy. This high throughput method could be used for fast tomographic imaging, or to generate multiple replicates in two-dimensions of hydrated biological systems without freezing or fixing. This work demonstrates that phase-diverse CDI is a valuable complementary imaging method for the biological sciences and ready for immediate application. © 2013 Elsevier B.V. All rights reserved.

Application of high performance asynchronous socket communication in power distribution automation

NASA Astrophysics Data System (ADS)

Wang, Ziyu

2017-05-01

With the development of information technology and Internet technology, and the growing demand for electricity, the stability and the reliable operation of power system have been the goal of power grid workers. With the advent of the era of big data, the power data will gradually become an important breakthrough to guarantee the safe and reliable operation of the power grid. So, in the electric power industry, how to efficiently and robustly receive the data transmitted by the data acquisition device, make the power distribution automation system be able to execute scientific decision quickly, which is the pursuit direction in power grid. In this paper, some existing problems in the power system communication are analysed, and with the help of the network technology, a set of solutions called Asynchronous Socket Technology to the problem in network communication which meets the high concurrency and the high throughput is proposed. Besides, the paper also looks forward to the development direction of power distribution automation in the era of big data and artificial intelligence.

A Method for Label-Free, Differential Top-Down Proteomics.

PubMed

Ntai, Ioanna; Toby, Timothy K; LeDuc, Richard D; Kelleher, Neil L

2016-01-01

Biomarker discovery in the translational research has heavily relied on labeled and label-free quantitative bottom-up proteomics. Here, we describe a new approach to biomarker studies that utilizes high-throughput top-down proteomics and is the first to offer whole protein characterization and relative quantitation within the same experiment. Using yeast as a model, we report procedures for a label-free approach to quantify the relative abundance of intact proteins ranging from 0 to 30 kDa in two different states. In this chapter, we describe the integrated methodology for the large-scale profiling and quantitation of the intact proteome by liquid chromatography-mass spectrometry (LC-MS) without the need for metabolic or chemical labeling. This recent advance for quantitative top-down proteomics is best implemented with a robust and highly controlled sample preparation workflow before data acquisition on a high-resolution mass spectrometer, and the application of a hierarchical linear statistical model to account for the multiple levels of variance contained in quantitative proteomic comparisons of samples for basic and clinical research.

Miniaturized high throughput detection system for capillary array electrophoresis on chip with integrated light emitting diode array as addressed ring-shaped light source.

PubMed

Ren, Kangning; Liang, Qionglin; Mu, Xuan; Luo, Guoan; Wang, Yiming

2009-03-07

A novel miniaturized, portable fluorescence detection system for capillary array electrophoresis (CAE) on a microfluidic chip was developed, consisting of a scanning light-emitting diode (LED) light source and a single point photoelectric sensor. Without charge coupled detector (CCD), lens, fibers and moving parts, the system was extremely simplified. Pulsed driving of the LED significantly increased the sensitivity, and greatly reduced the power consumption and photobleaching effect. The highly integrated system was robust and easy to use. All the advantages realized the concept of a portable micro-total analysis system (micro-TAS), which could work on a single universal serial bus (USB) port. Compared with traditional CAE detecting systems, the current system could scan the radial capillary array with high scanning rate. An 8-channel CAE of fluorescein isothiocyanate (FITC) labeled arginine (Arg) on chip was demonstrated with this system, resulting in a limit of detection (LOD) of 640 amol.

High-throughput and site-specific identification of 2'-O-methylation sites using ribose oxidation sequencing (RibOxi-seq).

PubMed

Zhu, Yinzhou; Pirnie, Stephan P; Carmichael, Gordon G

2017-08-01

Ribose methylation (2'- O -methylation, 2'- O Me) occurs at high frequencies in rRNAs and other small RNAs and is carried out using a shared mechanism across eukaryotes and archaea. As RNA modifications are important for ribosome maturation, and alterations in these modifications are associated with cellular defects and diseases, it is important to characterize the landscape of 2'- O -methylation. Here we report the development of a highly sensitive and accurate method for ribose methylation detection using next-generation sequencing. A key feature of this method is the generation of RNA fragments with random 3'-ends, followed by periodate oxidation of all molecules terminating in 2',3'-OH groups. This allows only RNAs harboring 2'-OMe groups at their 3'-ends to be sequenced. Although currently requiring microgram amounts of starting material, this method is robust for the analysis of rRNAs even at low sequencing depth. © 2017 Zhu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Transparent Wood Smart Windows: Polymer Electrochromic Devices Based on Poly(3,4-Ethylenedioxythiophene):Poly(Styrene Sulfonate) Electrodes.

PubMed

Lang, Augustus W; Li, Yuanyuan; De Keersmaecker, Michel; Shen, D Eric; Österholm, Anna M; Berglund, Lars; Reynolds, John R

2018-03-09

Transparent wood composites, with their high strength and toughness, thermal insulation, and excellent transmissivity, offer a route to replace glass for diffusely transmitting windows. Here, conjugated-polymer-based electrochromic devices (ECDs) that switch on-demand are demonstrated using transparent wood coated with poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) as a transparent conducting electrode. These ECDs exhibit a vibrant magenta-to-clear color change that results from a remarkably colorless bleached state. Furthermore, they require low energy and power inputs of 3 mWh m -2 at 2 W m -2 to switch due to a high coloration efficiency (590 cm 2  C -1 ) and low driving voltage (0.8 V). Each device component is processed with high-throughput methods, which highlights the opportunity to apply this approach to fabricate mechanically robust, energy-efficient smart windows on a large scale. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

3D Structure Determination of Native Mammalian Cells using Cryo-FIB and Cryo-electron Tomography

PubMed Central

Wang, Ke; Strunk, Korrinn; Zhao, Gongpu; Gray, Jennifer L.; Zhang, Peijun

2012-01-01

Cryo-electron tomography (cryo-ET) has enabled high resolution three-dimensional (3D) structural analysis of virus and host cell interactions and many cell signaling events; these studies, however, have largely been limited to very thin, peripheral regions of eukaryotic cells or to small prokaryotic cells. Recent efforts to make thin, vitreous sections using cryo-ultramicrotomy have been successful, however, this method is technically very challenging and with many artifacts. Here, we report a simple and robust method for creating in situ, frozen-hydrated cell lamellas using a focused ion beam at cryogenic temperature (cryo-FIB), allowing access to any interior cellular regions of interest. We demonstrate the utility of cryo-FIB with high resolution 3D cellular structures from both bacterial cells and large mammalian cells. The method will not only facilitate high-throughput 3D structural analysis of biological specimens, but is also broadly applicable to sample preparation of thin films and surface materials without the need for FIB “lift-out”. PMID:22796867

Rotary-Atomizer Electric Power Generator

NASA Astrophysics Data System (ADS)

Nguyen, Trieu; Tran, Tuan; de Boer, Hans; van den Berg, Albert; Eijkel, Jan C. T.

2015-03-01

We report experimental and theoretical results on a ballistic energy-conversion method based on a rotary atomizer working with a droplet acceleration-deceleration cycle. In a rotary atomizer, liquid is fed onto the center of a rotating flat surface, where it spreads out under the action of the centrifugal force and creates "atomized" droplets at its edge. The advantage of using a rotary atomizer is that the centrifugal force exerted on the fluid on a smooth, large surface is not only a robust form of acceleration, as it avoids clogging, but also easily allows high throughput, and produces high electrical power. We successfully demonstrate an output power of 4.9 mW and a high voltage up to 3120 V. At present, the efficiency of the system is still low (0.14%). However, the conversion mechanism of the system is fully interpreted in this paper, permitting a conceptual understanding of system operation and providing a roadmap for system optimization. This observation will open up a road for building power-generation systems in the near future.

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

Hornemann, Andrea, E-mail: andrea.hornemann@ptb.de; Hoehl, Arne, E-mail: arne.hoehl@ptb.de; Ulm, Gerhard, E-mail: gerhard.ulm@ptb.de

Bio-diagnostic assays of high complexity rely on nanoscaled assay recognition elements that can provide unique selectivity and design-enhanced sensitivity features. High throughput performance requires the simultaneous detection of various analytes combined with appropriate bioassay components. Nanoparticle induced sensitivity enhancement, and subsequent multiplexed capability Surface-Enhanced InfraRed Absorption (SEIRA) assay formats are fitting well these purposes. SEIRA constitutes an ideal platform to isolate the vibrational signatures of targeted bioassay and active molecules. The potential of several targeted biolabels, here fluorophore-labeled antibody conjugates, chemisorbed onto low-cost biocompatible gold nano-aggregates substrates have been explored for their use in assay platforms. Dried films were analyzedmore » by synchrotron radiation based FTIR/SEIRA spectro-microscopy and the resulting complex hyperspectral datasets were submitted to automated statistical analysis, namely Principal Components Analysis (PCA). The relationships between molecular fingerprints were put in evidence to highlight their spectral discrimination capabilities. We demonstrate that robust spectral encoding via SEIRA fingerprints opens up new opportunities for fast, reliable and multiplexed high-end screening not only in biodiagnostics but also in vitro biochemical imaging.« less

High density lipoproteins: Measurement techniques and potential biomarkers of cardiovascular risk

PubMed Central

Hafiane, Anouar; Genest, Jacques

2015-01-01

Plasma high density lipoprotein cholesterol (HDL) comprises a heterogeneous family of lipoprotein species, differing in surface charge, size and lipid and protein compositions. While HDL cholesterol (C) mass is a strong, graded and coherent biomarker of cardiovascular risk, genetic and clinical trial data suggest that the simple measurement of HDL-C may not be causal in preventing atherosclerosis nor reflect HDL functionality. Indeed, the measurement of HDL-C may be a biomarker of cardiovascular health. To assess the issue of HDL function as a potential therapeutic target, robust and simple analytical methods are required. The complex pleiotropic effects of HDL make the development of a single measurement challenging. Development of laboratory assays that accurately HDL function must be developed validated and brought to high-throughput for clinical purposes. This review discusses the limitations of current laboratory technologies for methods that separate and quantify HDL and potential application to predict CVD, with an emphasis on emergent approaches as potential biomarkers in clinical practice. PMID:26674734

Capturing the Alternative Cleavage and Polyadenylation Sites of 14 NAC Genes in Populus Using a Combination of 3'-RACE and High-Throughput Sequencing.

PubMed

Wang, Haoran; Wang, Mingxiu; Cheng, Qiang

2018-03-08

Detection of complex splice sites (SSs) and polyadenylation sites (PASs) of eukaryotic genes is essential for the elucidation of gene regulatory mechanisms. Transcriptome-wide studies using high-throughput sequencing (HTS) have revealed prevalent alternative splicing (AS) and alternative polyadenylation (APA) in plants. However, small-scale and high-depth HTS aimed at detecting genes or gene families are very few and limited. We explored a convenient and flexible method for profiling SSs and PASs, which combines rapid amplification of 3'-cDNA ends (3'-RACE) and HTS. Fourteen NAC (NAM, ATAF1/2, CUC2) transcription factor genes of Populus trichocarpa were analyzed by 3'-RACE-seq. Based on experimental reproducibility, boundary sequence analysis and reverse transcription PCR (RT-PCR) verification, only canonical SSs were considered to be authentic. Based on stringent criteria, candidate PASs without any internal priming features were chosen as authentic PASs and assumed to be PAS-rich markers. Thirty-four novel canonical SSs, six intronic/internal exons and thirty 3'-UTR PAS-rich markers were revealed by 3'-RACE-seq. Using 3'-RACE and real-time PCR, we confirmed that three APA transcripts ending in/around PAS-rich markers were differentially regulated in response to plant hormones. Our results indicate that 3'-RACE-seq is a robust and cost-effective method to discover SSs and label active regions subjected to APA for genes or gene families. The method is suitable for small-scale AS and APA research in the initial stage.

Technological Innovations for High-Throughput Approaches to In Vitro Allergy Diagnosis.

PubMed

Chapman, Martin D; Wuenschmann, Sabina; King, Eva; Pomés, Anna

2015-07-01

Allergy diagnostics is being transformed by the advent of in vitro IgE testing using purified allergen molecules, combined with multiplex technology and biosensors, to deliver discriminating, sensitive, and high-throughput molecular diagnostics at the point of care. Essential elements of IgE molecular diagnostics are purified natural or recombinant allergens with defined purity and IgE reactivity, planar or bead-based multiplex systems to enable IgE to multiple allergens to be measured simultaneously, and, most recently, nanotechnology-based biosensors that facilitate rapid reaction rates and delivery of test results via mobile devices. Molecular diagnostics relies on measurement of IgE to purified allergens, the "active ingredients" of allergenic extracts. Typically, this involves measuring IgE to multiple allergens which is facilitated by multiplex technology and biosensors. The technology differentiates between clinically significant cross-reactive allergens (which could not be deduced by conventional IgE assays using allergenic extracts) and provides better diagnostic outcomes. Purified allergens are manufactured under good laboratory practice and validated using protein chemistry, mass spectrometry, and IgE antibody binding. Recently, multiple allergens (from dog) were expressed as a single molecule with high diagnostic efficacy. Challenges faced by molecular allergy diagnostic companies include generation of large panels of purified allergens with known diagnostic efficacy, access to flexible and robust array or sensor technology, and, importantly, access to well-defined serum panels form allergic patients for product development and validation. Innovations in IgE molecular diagnostics are rapidly being brought to market and will strengthen allergy testing at the point of care.

Development of Glycoprotein Capture-Based Label-Free Method for the High-throughput Screening of Differential Glycoproteins in Hepatocellular Carcinoma*

PubMed Central

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

2011-01-01

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. PMID:21474793

An automated maze task for assessing hippocampus-sensitive memory in mice☆

PubMed Central

Pioli, Elsa Y.; Gaskill, Brianna N.; Gilmour, Gary; Tricklebank, Mark D.; Dix, Sophie L.; Bannerman, David; Garner, Joseph P.

2014-01-01

Memory deficits associated with hippocampal dysfunction are a key feature of a number of neurodegenerative and psychiatric disorders. The discrete-trial rewarded alternation T-maze task is highly sensitive to hippocampal dysfunction. Normal mice have spontaneously high levels of alternation, whereas hippocampal-lesioned mice are dramatically impaired. However, this is a hand-run task and handling has been shown to impact crucially on behavioural responses, as well as being labour-intensive and therefore unsuitable for high-throughput studies. To overcome this, a fully automated maze was designed. The maze was attached to the mouse's home cage and the subject earned all of its food by running through the maze. In this study the hippocampal dependence of rewarded alternation in the automated maze was assessed. Bilateral hippocampal-lesioned mice were assessed in the standard, hand-run, discrete-trial rewarded alternation paradigm and in the automated paradigm, according to a cross-over design. A similarly robust lesion effect on alternation performance was found in both mazes, confirming the sensitivity of the automated maze to hippocampal lesions. Moreover, the performance of the animals in the automated maze was not affected by their handling history whereas performance in the hand-run maze was affected by prior testing history. By having more stable performance and by decreasing human contact the automated maze may offer opportunities to reduce extraneous experimental variation and therefore increase the reproducibility within and/or between laboratories. Furthermore, automation potentially allows for greater experimental throughput and hence suitability for use in assessment of cognitive function in drug discovery. PMID:24333574

Determination of red blood cell fatty acid profiles: Rapid and high-confident analysis by chemical ionization-gas chromatography-tandem mass spectrometry.

PubMed

Schober, Yvonne; Wahl, Hans Günther; Renz, Harald; Nockher, Wolfgang Andreas

2017-01-01

Cellular fatty acid (FA) profiles have been acknowledged as biomarkers in various human diseases. Nevertheless, common FA analysis by gas chromatography mass spectrometry (GC-MS) requires long analysis time. Hence, there is a need for feasible methods for high throughput analysis in clinical studies. FA was extracted from red blood cells (RBC) and derivatized to fatty acid methyl esters (FAME). A method using gas chromatography tandem mass spectrometry (GC-MS/MS) with ammonia-induced chemical ionization (CI) was developed for the analysis of FA profiles in human RBC. We compared this method with classical single GC-MS using electron impact ionization (EI). The FA profiles of 703 RBC samples were determined by GC-MS/MS. In contrast to EI ammonia-induced CI resulted in adequate amounts of molecular ions for further fragmentation of FAME. Specific fragments for confident quantification and fragmentation were determined for 45 FA. The GC-MS/MS method has a total run time of 9min compared to typical analysis times of up to 60min in conventional GC-MS. Intra and inter assay variations were <10% for all FA analyzed. Analysis of RBC FA composition revealed an age-dependent increase of the omega-3 eicosapentaenoic and docosahexaenoic acid, and a decline of the omega-6 linoleic acid with a corresponding rise of the omega-3 index. The combination of ammonia-induced CI and tandem mass spectrometry after GC separation allows for high-throughput, robust and confident analysis of FA profiles in the clinical laboratory. Copyright © 2016. Published by Elsevier B.V.

High-throughput screening of chromatographic separations: IV. Ion-exchange.

PubMed

Kelley, Brian D; Switzer, Mary; Bastek, Patrick; Kramarczyk, Jack F; Molnar, Kathleen; Yu, Tianning; Coffman, Jon

2008-08-01

Ion-exchange (IEX) chromatography steps are widely applied in protein purification processes because of their high capacity, selectivity, robust operation, and well-understood principles. Optimization of IEX steps typically involves resin screening and selection of the pH and counterion concentrations of the load, wash, and elution steps. Time and material constraints associated with operating laboratory columns often preclude evaluating more than 20-50 conditions during early stages of process development. To overcome this limitation, a high-throughput screening (HTS) system employing a robotic liquid handling system and 96-well filterplates was used to evaluate various operating conditions for IEX steps for monoclonal antibody (mAb) purification. A screening study for an adsorptive cation-exchange step evaluated eight different resins. Sodium chloride concentrations defining the operating boundaries of product binding and elution were established at four different pH levels for each resin. Adsorption isotherms were measured for 24 different pH and salt combinations for a single resin. An anion-exchange flowthrough step was then examined, generating data on mAb adsorption for 48 different combinations of pH and counterion concentration for three different resins. The mAb partition coefficients were calculated and used to estimate the characteristic charge of the resin-protein interaction. Host cell protein and residual Protein A impurity levels were also measured, providing information on selectivity within this operating window. The HTS system shows promise for accelerating process development of IEX steps, enabling rapid acquisition of large datasets addressing the performance of the chromatography step under many different operating conditions. (c) 2008 Wiley Periodicals, Inc.

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

DOE PAGES

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

2016-09-23

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

Dual-Pulse Pulse Position Modulation (DPPM) for Deep-Space Optical Communications: Performance and Practicality Analysis

NASA Technical Reports Server (NTRS)

Li, Jing; Hylton, Alan; Budinger, James; Nappier, Jennifer; Downey, Joseph; Raible, Daniel

2012-01-01

Due to its simplicity and robustness against wavefront distortion, pulse position modulation (PPM) with photon counting detector has been seriously considered for long-haul optical wireless systems. This paper evaluates the dual-pulse case and compares it with the conventional single-pulse case. Analytical expressions for symbol error rate and bit error rate are first derived and numerically evaluated, for the strong, negative-exponential turbulent atmosphere; and bandwidth efficiency and throughput are subsequently assessed. It is shown that, under a set of practical constraints including pulse width and pulse repetition frequency (PRF), dual-pulse PPM enables a better channel utilization and hence a higher throughput than it single-pulse counterpart. This result is new and different from the previous idealistic studies that showed multi-pulse PPM provided no essential information-theoretic gains than single-pulse PPM.

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

PubMed

Li, Fumin; Wang, Jun; Jenkins, Rand

2016-05-01

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

DEEM, a versatile platform of FRD measurement for highly multiplexed fibre systems in astronomy

NASA Astrophysics Data System (ADS)

Yan, Yunxiang; Yan, Qi; Wang, Gang; Sun, Weimin; Luo, A.-Li; Ma, Zhenyu; Zhang, Qiong; Li, Jian; Wang, Shuqing

2018-06-01

We present a new method of DEEM, the direct energy encircling method, for characterizing the performance of fibres in most astronomical spectroscopic applications. It is a versatile platform to measure focal ratio degradation (FRD), throughput, and point spread function. The principle of DEEM and the relation between the encircled energy and the spot size were derived and simulated based on the power distribution model (PDM). We analysed the errors of DEEM and pointed out the major error source for better understanding and optimization. The validation of DEEM has been confirmed by comparing the results with conventional method which shows that DEEM has good robustness with high accuracy in both stable and complex experiment environments. Applications on the integral field unit (IFU) show that the FRD of 50 μm core fibre is substandard for the requirement which requires the output focal ratio to be slower than 4.5. The homogeneity of throughput is acceptable and higher than 85 per cent. The prototype IFU of the first generation helps to find out the imperfections to optimize the new design of the next generation based on the staggered structure with 35 μm core fibres of N.A. = 0.12, which can improve the FRD performance. The FRD dependence on wavelength and core size is revealed that higher output focal ratio occurs at shorter wavelengths for large core fibres, which is in agreement with the prediction of PDM. But the dependence of the observed data is weaker than the prediction.

Designing optimal nanofocusing with a gradient hyperlens

NASA Astrophysics Data System (ADS)

Shen, Lian; Prokopeva, Ludmila J.; Chen, Hongsheng; Kildishev, Alexander V.

2017-11-01

We report the design of a high-throughput gradient hyperbolic lenslet built with real-life materials and capable of focusing a beam into a deep sub-wavelength spot of λ/23. This efficient design is achieved through high-order transformation optics and circular effective-medium theory (CEMT), which are used to engineer the radially varying anisotropic artificial material based on the thin alternating cylindrical metal and dielectric layers. The radial gradient of the effective anisotropic optical constants allows for matching the impedances at the input and output interfaces, drastically improving the throughput of the lenslet. However, it is the use of the zeroth-order CEMT that enables the practical realization of a gradient hyperlens with realistic materials. To illustrate the importance of using the CEMT versus the conventional planar effective-medium theory (PEMT) for cylindrical anisotropic systems, such as our hyperlens, both the CEMT and PEMT are adopted to design gradient hyperlenses with the same materials and order of elemental layers. The CEMT- and PEMT-based designs show similar performance if the number of metal-dielectric binary layers is sufficiently large (9+ pairs) and if the layers are sufficiently thin. However, for the manufacturable lenses with realistic numbers of layers (e.g. five pairs) and thicknesses, the performance of the CEMT design continues to be practical, whereas the PEMT-based design stops working altogether. The accurate design of transformation optics-based layered cylindrical devices enabled by CEMT allow for a new class of robustly manufacturable nanophotonic systems, even with relatively thick layers of real-life materials.

speaq 2.0: A complete workflow for high-throughput 1D NMR spectra processing and quantification.

PubMed

Beirnaert, Charlie; Meysman, Pieter; Vu, Trung Nghia; Hermans, Nina; Apers, Sandra; Pieters, Luc; Covaci, Adrian; Laukens, Kris

2018-03-01

Nuclear Magnetic Resonance (NMR) spectroscopy is, together with liquid chromatography-mass spectrometry (LC-MS), the most established platform to perform metabolomics. In contrast to LC-MS however, NMR data is predominantly being processed with commercial software. Meanwhile its data processing remains tedious and dependent on user interventions. As a follow-up to speaq, a previously released workflow for NMR spectral alignment and quantitation, we present speaq 2.0. This completely revised framework to automatically analyze 1D NMR spectra uses wavelets to efficiently summarize the raw spectra with minimal information loss or user interaction. The tool offers a fast and easy workflow that starts with the common approach of peak-picking, followed by grouping, thus avoiding the binning step. This yields a matrix consisting of features, samples and peak values that can be conveniently processed either by using included multivariate statistical functions or by using many other recently developed methods for NMR data analysis. speaq 2.0 facilitates robust and high-throughput metabolomics based on 1D NMR but is also compatible with other NMR frameworks or complementary LC-MS workflows. The methods are benchmarked using a simulated dataset and two publicly available datasets. speaq 2.0 is distributed through the existing speaq R package to provide a complete solution for NMR data processing. The package and the code for the presented case studies are freely available on CRAN (https://cran.r-project.org/package=speaq) and GitHub (https://github.com/beirnaert/speaq).

speaq 2.0: A complete workflow for high-throughput 1D NMR spectra processing and quantification

PubMed Central

Pieters, Luc; Covaci, Adrian

2018-01-01

Nuclear Magnetic Resonance (NMR) spectroscopy is, together with liquid chromatography-mass spectrometry (LC-MS), the most established platform to perform metabolomics. In contrast to LC-MS however, NMR data is predominantly being processed with commercial software. Meanwhile its data processing remains tedious and dependent on user interventions. As a follow-up to speaq, a previously released workflow for NMR spectral alignment and quantitation, we present speaq 2.0. This completely revised framework to automatically analyze 1D NMR spectra uses wavelets to efficiently summarize the raw spectra with minimal information loss or user interaction. The tool offers a fast and easy workflow that starts with the common approach of peak-picking, followed by grouping, thus avoiding the binning step. This yields a matrix consisting of features, samples and peak values that can be conveniently processed either by using included multivariate statistical functions or by using many other recently developed methods for NMR data analysis. speaq 2.0 facilitates robust and high-throughput metabolomics based on 1D NMR but is also compatible with other NMR frameworks or complementary LC-MS workflows. The methods are benchmarked using a simulated dataset and two publicly available datasets. speaq 2.0 is distributed through the existing speaq R package to provide a complete solution for NMR data processing. The package and the code for the presented case studies are freely available on CRAN (https://cran.r-project.org/package=speaq) and GitHub (https://github.com/beirnaert/speaq). PMID:29494588

Enhanced HTS hit selection via a local hit rate analysis.

PubMed

Posner, Bruce A; Xi, Hualin; Mills, James E J

2009-10-01

The postprocessing of high-throughput screening (HTS) results is complicated by the occurrence of false positives (inactive compounds misidentified as active by the primary screen) and false negatives (active compounds misidentified as inactive by the primary screen). An activity cutoff is frequently used to select "active" compounds from HTS data; however, this approach is insensitive to both false positives and false negatives. An alternative method that can minimize the occurrence of these artifacts will increase the efficiency of hit selection and therefore lead discovery. In this work, rather than merely using the activity of a given compound, we look at the presence and absence of activity among all compounds in its "chemical space neighborhood" to give a degree of confidence in its activity. We demonstrate that this local hit rate (LHR) analysis method outperforms hit selection based on ranking by primary screen activity values across ten diverse high throughput screens, spanning both cell-based and biochemical assay formats of varying biology and robustness. On average, the local hit rate analysis method was approximately 2.3-fold and approximately 1.3-fold more effective in identifying active compounds and active chemical series, respectively, than selection based on primary activity alone. Moreover, when applied to finding false negatives, this method was 2.3-fold better than ranking by primary activity alone. In most cases, novel hit series were identified that would have otherwise been missed. Additional uses of and observations regarding this HTS analysis approach are also discussed.

Identifying initiation and elongation inhibitors of dengue virus RNA polymerase in a high-throughput lead-finding campaign.

PubMed

Smith, Thomas M; Lim, Siew Pheng; Yue, Kimberley; Busby, Scott A; Arora, Rishi; Seh, Cheah Chen; Wright, S Kirk; Nutiu, Razvan; Niyomrattanakit, Pornwaratt; Wan, Kah Fei; Beer, David; Shi, Pei-Yong; Benson, Timothy E

2015-01-01

Dengue virus (DENV) is the most significant mosquito-borne viral pathogen in the world and is the cause of dengue fever. The DENV RNA-dependent RNA polymerase (RdRp) is conserved among the four viral serotypes and is an attractive target for antiviral drug development. During initiation of viral RNA synthesis, the polymerase switches from a "closed" to "open" conformation to accommodate the viral RNA template. Inhibitors that lock the "closed" or block the "open" conformation would prevent viral RNA synthesis. Herein, we describe a screening campaign that employed two biochemical assays to identify inhibitors of RdRp initiation and elongation. Using a DENV subgenomic RNA template that promotes RdRp de novo initiation, the first assay measures cytosine nucleotide analogue (Atto-CTP) incorporation. Liberated Atto fluorophore allows for quantification of RdRp activity via fluorescence. The second assay uses the same RNA template but is label free and directly detects RdRp-mediated liberation of pyrophosphates of native ribonucleotides via liquid chromatography-mass spectrometry. The ability of inhibitors to bind and stabilize a "closed" conformation of the DENV RdRp was further assessed in a differential scanning fluorimetry assay. Last, active compounds were evaluated in a renilla luciferase-based DENV replicon cell-based assay to monitor cellular efficacy. All assays described herein are medium to high throughput, are robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase. © 2014 Society for Laboratory Automation and Screening.

The origin, global distribution, and functional impact of the human 8p23 inversion polymorphism.

PubMed

Salm, Maximilian P A; Horswell, Stuart D; Hutchison, Claire E; Speedy, Helen E; Yang, Xia; Liang, Liming; Schadt, Eric E; Cookson, William O; Wierzbicki, Anthony S; Naoumova, Rossi P; Shoulders, Carol C

2012-06-01

Genomic inversions are an increasingly recognized source of genetic variation. However, a lack of reliable high-throughput genotyping assays for these structures has precluded a full understanding of an inversion's phylogenetic, phenotypic, and population genetic properties. We characterize these properties for one of the largest polymorphic inversions in man (the ∼4.5-Mb 8p23.1 inversion), a structure that encompasses numerous signals of natural selection and disease association. We developed and validated a flexible bioinformatics tool that utilizes SNP data to enable accurate, high-throughput genotyping of the 8p23.1 inversion. This tool was applied retrospectively to diverse genome-wide data sets, revealing significant population stratification that largely follows a clinal "serial founder effect" distribution model. Phylogenetic analyses establish the inversion's ancestral origin within the Homo lineage, indicating that 8p23.1 inversion has occurred independently in the Pan lineage. The human inversion breakpoint was localized to an inverted pair of human endogenous retrovirus elements within the large, flanking low-copy repeats; experimental validation of this breakpoint confirmed these elements as the likely intermediary substrates that sponsored inversion formation. In five data sets, mRNA levels of disease-associated genes were robustly associated with inversion genotype. Moreover, a haplotype associated with systemic lupus erythematosus was restricted to the derived inversion state. We conclude that the 8p23.1 inversion is an evolutionarily dynamic structure that can now be accommodated into the understanding of human genetic and phenotypic diversity.

The origin, global distribution, and functional impact of the human 8p23 inversion polymorphism

PubMed Central

Salm, Maximilian P.A.; Horswell, Stuart D.; Hutchison, Claire E.; Speedy, Helen E.; Yang, Xia; Liang, Liming; Schadt, Eric E.; Cookson, William O.; Wierzbicki, Anthony S.; Naoumova, Rossi P.; Shoulders, Carol C.

2012-01-01

Genomic inversions are an increasingly recognized source of genetic variation. However, a lack of reliable high-throughput genotyping assays for these structures has precluded a full understanding of an inversion's phylogenetic, phenotypic, and population genetic properties. We characterize these properties for one of the largest polymorphic inversions in man (the ∼4.5-Mb 8p23.1 inversion), a structure that encompasses numerous signals of natural selection and disease association. We developed and validated a flexible bioinformatics tool that utilizes SNP data to enable accurate, high-throughput genotyping of the 8p23.1 inversion. This tool was applied retrospectively to diverse genome-wide data sets, revealing significant population stratification that largely follows a clinal “serial founder effect” distribution model. Phylogenetic analyses establish the inversion's ancestral origin within the Homo lineage, indicating that 8p23.1 inversion has occurred independently in the Pan lineage. The human inversion breakpoint was localized to an inverted pair of human endogenous retrovirus elements within the large, flanking low-copy repeats; experimental validation of this breakpoint confirmed these elements as the likely intermediary substrates that sponsored inversion formation. In five data sets, mRNA levels of disease-associated genes were robustly associated with inversion genotype. Moreover, a haplotype associated with systemic lupus erythematosus was restricted to the derived inversion state. We conclude that the 8p23.1 inversion is an evolutionarily dynamic structure that can now be accommodated into the understanding of human genetic and phenotypic diversity. PMID:22399572

Fast quantitation of opioid isomers in human plasma by differential mobility spectrometry/mass spectrometry via SPME/open-port probe sampling interface.

PubMed

Liu, Chang; Gómez-Ríos, Germán Augusto; Schneider, Bradley B; Le Blanc, J C Yves; Reyes-Garcés, Nathaly; Arnold, Don W; Covey, Thomas R; Pawliszyn, Janusz

2017-10-23

Mass spectrometry (MS) based quantitative approaches typically require a thorough sample clean-up and a decent chromatographic step in order to achieve needed figures of merit. However, in most cases, such processes are not optimal for urgent assessments and high-throughput determinations. The direct coupling of solid phase microextraction (SPME) to MS has shown great potential to shorten the total sample analysis time of complex matrices, as well as to diminish potential matrix effects and instrument contamination. In this study, we demonstrate the use of the open-port probe (OPP) as a direct and robust sampling interface to couple biocompatible-SPME (Bio-SPME) fibres to MS for the rapid quantitation of opioid isomers (i.e. codeine and hydrocodone) in human plasma. In place of chromatography, a differential mobility spectrometry (DMS) device was implemented to provide the essential selectivity required to quantify these constitutional isomers. Taking advantage of the simplified sample preparation process based on Bio-SPME and the fast separation with DMS-MS coupling via OPP, a high-throughput assay (10-15 s per sample) with limits of detection in the sub-ng/mL range was developed. Succinctly, we demonstrated that by tuning adequate ion mobility separation conditions, SPME-OPP-MS can be employed to quantify non-resolved compounds or those otherwise hindered by co-extracted isobaric interferences without further need of coupling to other separation platforms. Copyright © 2017 Elsevier B.V. All rights reserved.

High throughput system for magnetic manipulation of cells, polymers, and biomaterials

PubMed Central

Spero, Richard Chasen; Vicci, Leandra; Cribb, Jeremy; Bober, David; Swaminathan, Vinay; O’Brien, E. Timothy; Rogers, Stephen L.; Superfine, R.

2008-01-01

In the past decade, high throughput screening (HTS) has changed the way biochemical assays are performed, but manipulation and mechanical measurement of micro- and nanoscale systems have not benefited from this trend. Techniques using microbeads (particles ∼0.1–10 μm) show promise for enabling high throughput mechanical measurements of microscopic systems. We demonstrate instrumentation to magnetically drive microbeads in a biocompatible, multiwell magnetic force system. It is based on commercial HTS standards and is scalable to 96 wells. Cells can be cultured in this magnetic high throughput system (MHTS). The MHTS can apply independently controlled forces to 16 specimen wells. Force calibrations demonstrate forces in excess of 1 nN, predicted force saturation as a function of pole material, and powerlaw dependence of F∼r−2.7±0.1. We employ this system to measure the stiffness of SR2+ Drosophila cells. MHTS technology is a key step toward a high throughput screening system for micro- and nanoscale biophysical experiments. PMID:19044357

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

PubMed

Ulitsky, Igor; Shamir, Ron

2007-01-26

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

High density diffusion-free nanowell arrays.

PubMed

Takulapalli, Bharath R; Qiu, Ji; Magee, D Mitchell; Kahn, Peter; Brunner, Al; Barker, Kristi; Means, Steven; Miersch, Shane; Bian, Xiaofang; Mendoza, Alex; Festa, Fernanda; Syal, Karan; Park, Jin G; LaBaer, Joshua; Wiktor, Peter

2012-08-03

Proteomics aspires to elucidate the functions of all proteins. Protein microarrays provide an important step by enabling high-throughput studies of displayed proteins. However, many functional assays of proteins include untethered intermediates or products, which could frustrate the use of planar arrays at very high densities because of diffusion to neighboring features. The nucleic acid programmable protein array (NAPPA) is a robust in situ synthesis method for producing functional proteins just-in-time, which includes steps with diffusible intermediates. We determined that diffusion of expressed proteins led to cross-binding at neighboring spots at very high densities with reduced interspot spacing. To address this limitation, we have developed an innovative platform using photolithographically etched discrete silicon nanowells and used NAPPA as a test case. This arrested protein diffusion and cross-binding. We present confined high density protein expression and display, as well as functional protein-protein interactions, in 8000 nanowell arrays. This is the highest density of individual proteins in nanovessels demonstrated on a single slide. We further present proof of principle results on ultrahigh density protein arrays capable of up to 24000 nanowells on a single slide.

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

PubMed

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

2016-04-01

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

A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry.

PubMed

Chan, Leo Li-Ying; Smith, Tim; Kumph, Kendra A; Kuksin, Dmitry; Kessel, Sarah; Déry, Olivier; Cribbes, Scott; Lai, Ning; Qiu, Jean

2016-10-01

To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture.

Further development of a robust workup process for solution-phase high-throughput library synthesis to address environmental and sample tracking issues.

PubMed

Kuroda, Noritaka; Hird, Nick; Cork, David G

2006-01-01

During further improvement of a high-throughput, solution-phase synthesis system, new workup tools and apparatus for parallel liquid-liquid extraction and evaporation have been developed. A combination of in-house design and collaboration with external manufacturers has been used to address (1) environmental issues concerning solvent emissions and (2) sample tracking errors arising from manual intervention. A parallel liquid-liquid extraction unit, containing miniature high-speed magnetic stirrers for efficient mixing of organic and aqueous phases, has been developed for use on a multichannel liquid handler. Separation of the phases is achieved by dispensing them into a newly patented filter tube containing a vertical hydrophobic porous membrane, which allows only the organic phase to pass into collection vials positioned below. The vertical positioning of the membrane overcomes the hitherto dependence on the use of heavier-than-water, bottom-phase, organic solvents such as dichloromethane, which are restricted due to environmental concerns. Both small (6-mL) and large (60-mL) filter tubes were developed for parallel phase separation in library and template synthesis, respectively. In addition, an apparatus for parallel solvent evaporation was developed to (1) remove solvent from the above samples with highly efficient recovery and (2) avoid the movement of individual samples between their collection on a liquid handler and registration to prevent sample identification errors. The apparatus uses a diaphragm pump to achieve a dynamic circulating closed system with a heating block for the rack of 96 sample vials and an efficient condenser to trap the solvents. Solvent recovery is typically >98%, and convenient operation and monitoring has made the apparatus the first choice for removal of volatile solvents.

Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples

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

Clair, Geremy; Piehowski, Paul D.; Nicola, Teodora

Global proteomics approaches allow characterization of whole tissue lysates to an impressive depth. However, it is now increasingly recognized that to better understand the complexity of multicellular organisms, global protein profiling of specific spatially defined regions/substructures of tissues (i.e. spatially-resolved proteomics) is essential. Laser capture microdissection (LCM) enables microscopic isolation of defined regions of tissues preserving crucial spatial information. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, and that impact measurement robustness, quantification, and throughput. Here, we coupled LCM with a fully automated sample preparation workflow thatmore » with a single manual step allows: protein extraction, tryptic digestion, peptide cleanup and LC-MS/MS analysis of proteomes from microdissected tissues. Benchmarking against the current state of the art in ultrasensitive global proteomic analysis, our approach demonstrated significant improvements in quantification and throughput. Using our LCM-SNaPP proteomics approach, we characterized to a depth of more than 3,400 proteins, the ontogeny of protein changes during normal lung development in laser capture microdissected alveolar tissue containing ~4,000 cells per sample. Importantly, the data revealed quantitative changes for 350 low abundance transcription factors and signaling molecules, confirming earlier transcript-level observations and defining seven modules of coordinated transcription factor/signaling molecule expression patterns, suggesting that a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes. Our LCM-proteomics approach facilitates efficient, spatially-resolved, ultrasensitive global proteomics analyses in high-throughput that will be enabling for several clinical and biological applications.« less

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

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

Liu, Y; Grossman, JC

2014-12-01

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

MICCA: a complete and accurate software for taxonomic profiling of metagenomic data.

PubMed

Albanese, Davide; Fontana, Paolo; De Filippo, Carlotta; Cavalieri, Duccio; Donati, Claudio

2015-05-19

The introduction of high throughput sequencing technologies has triggered an increase of the number of studies in which the microbiota of environmental and human samples is characterized through the sequencing of selected marker genes. While experimental protocols have undergone a process of standardization that makes them accessible to a large community of scientist, standard and robust data analysis pipelines are still lacking. Here we introduce MICCA, a software pipeline for the processing of amplicon metagenomic datasets that efficiently combines quality filtering, clustering of Operational Taxonomic Units (OTUs), taxonomy assignment and phylogenetic tree inference. MICCA provides accurate results reaching a good compromise among modularity and usability. Moreover, we introduce a de-novo clustering algorithm specifically designed for the inference of Operational Taxonomic Units (OTUs). Tests on real and synthetic datasets shows that thanks to the optimized reads filtering process and to the new clustering algorithm, MICCA provides estimates of the number of OTUs and of other common ecological indices that are more accurate and robust than currently available pipelines. Analysis of public metagenomic datasets shows that the higher consistency of results improves our understanding of the structure of environmental and human associated microbial communities. MICCA is an open source project.

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

Xu, Zhe; Wu, Chaochao; Xie, Fang

Aberrant degradation of proteins is associated with many pathological states, including cancers. Mass spectrometric analysis of tumor peptidomes, the intracellular and intercellular products of protein degradation, has the potential to provide biological insights on proteolytic processing in cancer. However, attempts to use the information on these smaller protein degradation products from tumors for biomarker discovery and cancer biology studies have been fairly limited to date, largely due to the lack of effective approaches for robust peptidomics identification and quantification, and the prevalence of confounding factors and biases associated with sample handling and processing. Herein, we have developed an effective andmore » robust analytical platform for comprehensive analyses of tissue peptidomes, which is suitable for high throughput quantitative studies. The reproducibility and coverage of the platform, as well as the suitability of clinical ovarian tumor and patient-derived breast tumor xenograft samples with post-excision delay of up to 60 min before freezing for peptidomics analysis, have been demonstrated. Moreover, our data also show that the peptidomics profiles can effectively separate breast cancer subtypes, reflecting tumor-associated protease activities. Peptidomics complements results obtainable from conventional bottom-up proteomics, and provides insights not readily obtainable from such approaches.« less

Comprehensive Quantitative Analysis of Ovarian and Breast Cancer Tumor Peptidomes

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

Xu, Zhe; Wu, Chaochao; Xie, Fang

Aberrant degradation of proteins is associated with many pathological states, including cancers. Mass spectrometric analysis of tumor peptidomes, the intracellular and intercellular products of protein degradation, has the potential to provide biological insights on proteolytic processing in cancer. However, attempts to use the information on these smaller protein degradation products from tumors for biomarker discovery and cancer biology studies have been fairly limited to date, largely due to the lack of effective approaches for robust peptidomics identification and quantification, and the prevalence of confounding factors and biases associated with sample handling and processing. Herein, we have developed an effective andmore » robust analytical platform for comprehensive analyses of tissue peptidomes, and which is suitable for high throughput quantitative studies. The reproducibility and coverage of the platform, as well as the suitability of clinical ovarian tumor and patient-derived breast tumor xenograft samples with post-excision delay of up to 60 min before freezing for peptidomics analysis, have been demonstrated. Additionally, our data also show that the peptidomics profiles can effectively separate breast cancer subtypes, reflecting tumor-associated protease activities. In conclusion, peptidomics complements results obtainable from conventional bottom-up proteomics, and provides insights not readily obtainable from such approaches.« less

A quality-by-design approach to risk reduction and optimization for human embryonic stem cell cryopreservation processes.

PubMed

Mitchell, Peter D; Ratcliffe, Elizabeth; Hourd, Paul; Williams, David J; Thomas, Robert J

2014-12-01

It is well documented that cryopreservation and resuscitation of human embryonic stem cells (hESCs) is complex and ill-defined, and often suffers poor cell recovery and increased levels of undesirable cell differentiation. In this study we have applied Quality-by-Design (QbD) concepts to the critical processes of slow-freeze cryopreservation and resuscitation of hESC colony cultures. Optimized subprocesses were linked together to deliver a controlled complete process. We have demonstrated a rapid, high-throughput, and stable system for measurement of cell adherence and viability as robust markers of in-process and postrecovery cell state. We observed that measurement of adherence and viability of adhered cells at 1 h postseeding was predictive of cell proliferative ability up to 96 h in this system. Application of factorial design defined the operating spaces for cryopreservation and resuscitation, critically linking the performance of these two processes. Optimization of both processes resulted in enhanced reattachment and post-thaw viability, resulting in substantially greater recovery of cryopreserved, pluripotent cell colonies. This study demonstrates the importance of QbD concepts and tools for rapid, robust, and low-risk process design that can inform manufacturing controls and logistics.

An Automated Pipeline for Engineering Many-Enzyme Pathways: Computational Sequence Design, Pathway Expression-Flux Mapping, and Scalable Pathway Optimization.

PubMed

Halper, Sean M; Cetnar, Daniel P; Salis, Howard M

2018-01-01

Engineering many-enzyme metabolic pathways suffers from the design curse of dimensionality. There are an astronomical number of synonymous DNA sequence choices, though relatively few will express an evolutionary robust, maximally productive pathway without metabolic bottlenecks. To solve this challenge, we have developed an integrated, automated computational-experimental pipeline that identifies a pathway's optimal DNA sequence without high-throughput screening or many cycles of design-build-test. The first step applies our Operon Calculator algorithm to design a host-specific evolutionary robust bacterial operon sequence with maximally tunable enzyme expression levels. The second step applies our RBS Library Calculator algorithm to systematically vary enzyme expression levels with the smallest-sized library. After characterizing a small number of constructed pathway variants, measurements are supplied to our Pathway Map Calculator algorithm, which then parameterizes a kinetic metabolic model that ultimately predicts the pathway's optimal enzyme expression levels and DNA sequences. Altogether, our algorithms provide the ability to efficiently map the pathway's sequence-expression-activity space and predict DNA sequences with desired metabolic fluxes. Here, we provide a step-by-step guide to applying the Pathway Optimization Pipeline on a desired multi-enzyme pathway in a bacterial host.

MICCA: a complete and accurate software for taxonomic profiling of metagenomic data

PubMed Central

Albanese, Davide; Fontana, Paolo; De Filippo, Carlotta; Cavalieri, Duccio; Donati, Claudio

2015-01-01

The introduction of high throughput sequencing technologies has triggered an increase of the number of studies in which the microbiota of environmental and human samples is characterized through the sequencing of selected marker genes. While experimental protocols have undergone a process of standardization that makes them accessible to a large community of scientist, standard and robust data analysis pipelines are still lacking. Here we introduce MICCA, a software pipeline for the processing of amplicon metagenomic datasets that efficiently combines quality filtering, clustering of Operational Taxonomic Units (OTUs), taxonomy assignment and phylogenetic tree inference. MICCA provides accurate results reaching a good compromise among modularity and usability. Moreover, we introduce a de-novo clustering algorithm specifically designed for the inference of Operational Taxonomic Units (OTUs). Tests on real and synthetic datasets shows that thanks to the optimized reads filtering process and to the new clustering algorithm, MICCA provides estimates of the number of OTUs and of other common ecological indices that are more accurate and robust than currently available pipelines. Analysis of public metagenomic datasets shows that the higher consistency of results improves our understanding of the structure of environmental and human associated microbial communities. MICCA is an open source project. PMID:25988396

Robustness of Massively Parallel Sequencing Platforms

PubMed Central

Kavak, Pınar; Yüksel, Bayram; Aksu, Soner; Kulekci, M. Oguzhan; Güngör, Tunga; Hach, Faraz; Şahinalp, S. Cenk; Alkan, Can; Sağıroğlu, Mahmut Şamil

2015-01-01

The improvements in high throughput sequencing technologies (HTS) made clinical sequencing projects such as ClinSeq and Genomics England feasible. Although there are significant improvements in accuracy and reproducibility of HTS based analyses, the usability of these types of data for diagnostic and prognostic applications necessitates a near perfect data generation. To assess the usability of a widely used HTS platform for accurate and reproducible clinical applications in terms of robustness, we generated whole genome shotgun (WGS) sequence data from the genomes of two human individuals in two different genome sequencing centers. After analyzing the data to characterize SNPs and indels using the same tools (BWA, SAMtools, and GATK), we observed significant number of discrepancies in the call sets. As expected, the most of the disagreements between the call sets were found within genomic regions containing common repeats and segmental duplications, albeit only a small fraction of the discordant variants were within the exons and other functionally relevant regions such as promoters. We conclude that although HTS platforms are sufficiently powerful for providing data for first-pass clinical tests, the variant predictions still need to be confirmed using orthogonal methods before using in clinical applications. PMID:26382624

Comprehensive Quantitative Analysis of Ovarian and Breast Cancer Tumor Peptidomes

DOE PAGES

Xu, Zhe; Wu, Chaochao; Xie, Fang; ...

2014-10-28

Aberrant degradation of proteins is associated with many pathological states, including cancers. Mass spectrometric analysis of tumor peptidomes, the intracellular and intercellular products of protein degradation, has the potential to provide biological insights on proteolytic processing in cancer. However, attempts to use the information on these smaller protein degradation products from tumors for biomarker discovery and cancer biology studies have been fairly limited to date, largely due to the lack of effective approaches for robust peptidomics identification and quantification, and the prevalence of confounding factors and biases associated with sample handling and processing. Herein, we have developed an effective andmore » robust analytical platform for comprehensive analyses of tissue peptidomes, and which is suitable for high throughput quantitative studies. The reproducibility and coverage of the platform, as well as the suitability of clinical ovarian tumor and patient-derived breast tumor xenograft samples with post-excision delay of up to 60 min before freezing for peptidomics analysis, have been demonstrated. Additionally, our data also show that the peptidomics profiles can effectively separate breast cancer subtypes, reflecting tumor-associated protease activities. In conclusion, peptidomics complements results obtainable from conventional bottom-up proteomics, and provides insights not readily obtainable from such approaches.« less

Robust statistical methods for hit selection in RNA interference high-throughput screening experiments.

PubMed

Zhang, Xiaohua Douglas; Yang, Xiting Cindy; Chung, Namjin; Gates, Adam; Stec, Erica; Kunapuli, Priya; Holder, Dan J; Ferrer, Marc; Espeseth, Amy S

2006-04-01

RNA interference (RNAi) high-throughput screening (HTS) experiments carried out using large (>5000 short interfering [si]RNA) libraries generate a huge amount of data. In order to use these data to identify the most effective siRNAs tested, it is critical to adopt and develop appropriate statistical methods. To address the questions in hit selection of RNAi HTS, we proposed a quartile-based method which is robust to outliers, true hits and nonsymmetrical data. We compared it with the more traditional tests, mean +/- k standard deviation (SD) and median +/- 3 median of absolute deviation (MAD). The results suggested that the quartile-based method selected more hits than mean +/- k SD under the same preset error rate. The number of hits selected by median +/- k MAD was close to that by the quartile-based method. Further analysis suggested that the quartile-based method had the greatest power in detecting true hits, especially weak or moderate true hits. Our investigation also suggested that platewise analysis (determining effective siRNAs on a plate-by-plate basis) can adjust for systematic errors in different plates, while an experimentwise analysis, in which effective siRNAs are identified in an analysis of the entire experiment, cannot. However, experimentwise analysis may detect a cluster of true positive hits placed together in one or several plates, while platewise analysis may not. To display hit selection results, we designed a specific figure called a plate-well series plot. We thus suggest the following strategy for hit selection in RNAi HTS experiments. First, choose the quartile-based method, or median +/- k MAD, for identifying effective siRNAs. Second, perform the chosen method experimentwise on transformed/normalized data, such as percentage inhibition, to check the possibility of hit clusters. If a cluster of selected hits are observed, repeat the analysis based on untransformed data to determine whether the cluster is due to an artifact in the data. If no clusters of hits are observed, select hits by performing platewise analysis on transformed data. Third, adopt the plate-well series plot to visualize both the data and the hit selection results, as well as to check for artifacts.

Comparison of mapping algorithms used in high-throughput sequencing: application to Ion Torrent data

PubMed Central

2014-01-01

Background The rapid evolution in high-throughput sequencing (HTS) technologies has opened up new perspectives in several research fields and led to the production of large volumes of sequence data. A fundamental step in HTS data analysis is the mapping of reads onto reference sequences. Choosing a suitable mapper for a given technology and a given application is a subtle task because of the difficulty of evaluating mapping algorithms. Results In this paper, we present a benchmark procedure to compare mapping algorithms used in HTS using both real and simulated datasets and considering four evaluation criteria: computational resource and time requirements, robustness of mapping, ability to report positions for reads in repetitive regions, and ability to retrieve true genetic variation positions. To measure robustness, we introduced a new definition for a correctly mapped read taking into account not only the expected start position of the read but also the end position and the number of indels and substitutions. We developed CuReSim, a new read simulator, that is able to generate customized benchmark data for any kind of HTS technology by adjusting parameters to the error types. CuReSim and CuReSimEval, a tool to evaluate the mapping quality of the CuReSim simulated reads, are freely available. We applied our benchmark procedure to evaluate 14 mappers in the context of whole genome sequencing of small genomes with Ion Torrent data for which such a comparison has not yet been established. Conclusions A benchmark procedure to compare HTS data mappers is introduced with a new definition for the mapping correctness as well as tools to generate simulated reads and evaluate mapping quality. The application of this procedure to Ion Torrent data from the whole genome sequencing of small genomes has allowed us to validate our benchmark procedure and demonstrate that it is helpful for selecting a mapper based on the intended application, questions to be addressed, and the technology used. This benchmark procedure can be used to evaluate existing or in-development mappers as well as to optimize parameters of a chosen mapper for any application and any sequencing platform. PMID:24708189

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

Code of Federal Regulations, 2010 CFR

2010-07-01

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

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

PubMed

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

2016-02-01

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

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

PubMed Central

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

2010-01-01

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

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

PubMed

Cannon, Taylor M; Shah, Amy T; Walsh, Alex J; Skala, Melissa C

2015-01-01

There is a need for accurate, high-throughput, functional measures to gauge the efficacy of potential drugs in living cells. As an early marker of drug response in cells, cellular metabolism provides an attractive platform for high-throughput drug testing. Optical techniques can noninvasively monitor NADH and FAD, two autofluorescent metabolic coenzymes. The autofluorescent redox ratio, defined as the autofluorescence intensity of NADH divided by that of FAD, quantifies relative rates of cellular glycolysis and oxidative phosphorylation. However, current microscopy methods for redox ratio quantification are time-intensive and low-throughput, limiting their practicality in drug screening. Alternatively, high-throughput commercial microplate readers quickly measure fluorescence intensities for hundreds of wells. This study found that a commercial microplate reader can differentiate the receptor status of breast cancer cell lines (p < 0.05) based on redox ratio measurements without extrinsic contrast agents. Furthermore, microplate reader redox ratio measurements resolve response (p < 0.05) and lack of response (p > 0.05) in cell lines that are responsive and nonresponsive, respectively, to the breast cancer drug trastuzumab. These studies indicate that the microplate readers can be used to measure the redox ratio in a high-throughput manner and are sensitive enough to detect differences in cellular metabolism that are consistent with microscopy results.

Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers.

PubMed

Chen, Yi-Ting; Chen, Hsiao-Wei; Wu, Chun-Feng; Chu, Lichieh Julie; Chiang, Wei-Fang; Wu, Chih-Ching; Yu, Jau-Song; Tsai, Cheng-Han; Liang, Kung-Hao; Chang, Yu-Sun; Wu, Maureen; Ou Yang, Wei-Ting

2017-05-01

Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed Central

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

2012-01-01

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

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

PubMed Central

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

2018-01-01

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

Experimental Design for Combinatorial and High Throughput Materials Development

NASA Astrophysics Data System (ADS)

Cawse, James N.

2002-12-01

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

Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing

PubMed Central

Chandran, Anandhakumar; Syed, Junetha; Taylor, Rhys D.; Kashiwazaki, Gengo; Sato, Shinsuke; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

2016-01-01

Chemically engineered small molecules targeting specific genomic sequences play an important role in drug development research. Pyrrole-imidazole polyamides (PIPs) are a group of molecules that can bind to the DNA minor-groove and can be engineered to target specific sequences. Their biological effects rely primarily on their selective DNA binding. However, the binding mechanism of PIPs at the chromatinized genome level is poorly understood. Herein, we report a method using high-throughput sequencing to identify the DNA-alkylating sites of PIP-indole-seco-CBI conjugates. High-throughput sequencing analysis of conjugate 2 showed highly similar DNA-alkylating sites on synthetic oligos (histone-free DNA) and on human genomes (chromatinized DNA context). To our knowledge, this is the first report identifying alkylation sites across genomic DNA by alkylating PIP conjugates using high-throughput sequencing. PMID:27098039

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

PubMed

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

1994-01-01

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

Quantum-Sequencing: Fast electronic single DNA molecule sequencing

NASA Astrophysics Data System (ADS)

Casamada Ribot, Josep; Chatterjee, Anushree; Nagpal, Prashant

2014-03-01

A major goal of third-generation sequencing technologies is to develop a fast, reliable, enzyme-free, high-throughput and cost-effective, single-molecule sequencing method. Here, we present the first demonstration of unique ``electronic fingerprint'' of all nucleotides (A, G, T, C), with single-molecule DNA sequencing, using Quantum-tunneling Sequencing (Q-Seq) at room temperature. We show that the electronic state of the nucleobases shift depending on the pH, with most distinct states identified at acidic pH. We also demonstrate identification of single nucleotide modifications (methylation here). Using these unique electronic fingerprints (or tunneling data), we report a partial sequence of beta lactamase (bla) gene, which encodes resistance to beta-lactam antibiotics, with over 95% success rate. These results highlight the potential of Q-Seq as a robust technique for next-generation sequencing.

Microbial Ecology: Where are we now?

PubMed Central

2016-01-01

Conventional microbiological methods have been readily taken over by newer molecular techniques due to the ease of use, reproducibility, sensitivity and speed of working with nucleic acids. These tools allow high throughput analysis of complex and diverse microbial communities, such as those in soil, freshwater, saltwater, or the microbiota living in collaboration with a host organism (plant, mouse, human, etc). For instance, these methods have been robustly used for characterizing the plant (rhizosphere), animal and human microbiome specifically the complex intestinal microbiota. The human body has been referred to as the Superorganism since microbial genes are more numerous than the number of human genes and are essential to the health of the host. In this review we provide an overview of the Next Generation tools currently available to study microbial ecology, along with their limitations and advantages. PMID:27975077

Second International Conference on Accelerating Biopharmaceutical Development

PubMed Central

2009-01-01

The Second International Conference on Accelerating Biopharmaceutical Development was held in Coronado, California. The meeting was organized by the Society for Biological Engineering (SBE) and the American Institute of Chemical Engineers (AIChE); SBE is a technological community of the AIChE. Bob Adamson (Wyeth) and Chuck Goochee (Centocor) were co-chairs of the event, which had the theme “Delivering cost-effective, robust processes and methods quickly and efficiently.” The first day focused on emerging disruptive technologies and cutting-edge analytical techniques. Day two featured presentations on accelerated cell culture process development, critical quality attributes, specifications and comparability, and high throughput protein formulation development. The final day was dedicated to discussion of technology options and new analysis methods provided by emerging disruptive technologies; functional interaction, integration and synergy in platform development; and rapid and economic purification process development. PMID:20065637

Evolutionary Origins of Cancer Driver Genes and Implications for Cancer Prognosis

PubMed Central

Chu, Xin-Yi; Zhou, Xiong-Hui; Cui, Ze-Jia; Zhang, Hong-Yu

2017-01-01

The cancer atavistic theory suggests that carcinogenesis is a reverse evolution process. It is thus of great interest to explore the evolutionary origins of cancer driver genes and the relevant mechanisms underlying the carcinogenesis. Moreover, the evolutionary features of cancer driver genes could be helpful in selecting cancer biomarkers from high-throughput data. In this study, through analyzing the cancer endogenous molecular networks, we revealed that the subnetwork originating from eukaryota could control the unlimited proliferation of cancer cells, and the subnetwork originating from eumetazoa could recapitulate the other hallmarks of cancer. In addition, investigations based on multiple datasets revealed that cancer driver genes were enriched in genes originating from eukaryota, opisthokonta, and eumetazoa. These results have important implications for enhancing the robustness of cancer prognosis models through selecting the gene signatures by the gene age information. PMID:28708071

Evolutionary Origins of Cancer Driver Genes and Implications for Cancer Prognosis.

PubMed

Chu, Xin-Yi; Jiang, Ling-Han; Zhou, Xiong-Hui; Cui, Ze-Jia; Zhang, Hong-Yu

2017-07-14

The cancer atavistic theory suggests that carcinogenesis is a reverse evolution process. It is thus of great interest to explore the evolutionary origins of cancer driver genes and the relevant mechanisms underlying the carcinogenesis. Moreover, the evolutionary features of cancer driver genes could be helpful in selecting cancer biomarkers from high-throughput data. In this study, through analyzing the cancer endogenous molecular networks, we revealed that the subnetwork originating from eukaryota could control the unlimited proliferation of cancer cells, and the subnetwork originating from eumetazoa could recapitulate the other hallmarks of cancer. In addition, investigations based on multiple datasets revealed that cancer driver genes were enriched in genes originating from eukaryota, opisthokonta, and eumetazoa. These results have important implications for enhancing the robustness of cancer prognosis models through selecting the gene signatures by the gene age information.

RAFT polymerization and some of its applications.

PubMed

Moad, Graeme; Rizzardo, Ezio; Thang, San H

2013-08-01

Reversible addition-fragmentation chain transfer (RAFT) is one of the most robust and versatile methods for controlling radical polymerization. With appropriate selection of the RAFT agent for the monomers and reaction conditions, it is applicable to the majority of monomers subject to radical polymerization. The process can be used in the synthesis of well-defined homo-, gradient, diblock, triblock, and star polymers and more complex architectures, which include microgels and polymer brushes. In this Focus Review we describe how the development of RAFT and RAFT application has been facilitated by the adoption of continuous flow techniques using tubular reactors and through the use of high-throughput methodology. Applications described include the use of RAFT in the preparation of polymers for optoelectronics, block copolymer therapeutics, and star polymer rheology control agents. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Theory, Design, and Algorithms for Optimal Control of wireless Networks

DTIC Science & Technology

2010-06-09

The implementation of network-centric warfare technologies is an abiding, critical interest of Air Force Science and Technology efforts for the Warfighter. Wireless communications, strategic signaling are areas of critical Air Force Mission need. Autonomous networks of multiple, heterogeneous Throughput enhancement and robust connectivity in communications and sensor networks are critical factors in net-centric USAF operations. This research directly supports the Air Force vision of information dominance and the development of anywhere, anytime operational readiness.

High-Throughput/High-Content Screening Assays with Engineered Nanomaterials in ToxCast

EPA Science Inventory

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

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

PubMed

Moore, Priscilla A; Kery, Vladimir

2009-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

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

PubMed

Yang, Darren; Wong, Wesley P

2018-01-01

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

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

DTIC Science & Technology

2016-12-01

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

Differentiating Pathway-Specific From Nonspecific Effects in High-Throughput Toxicity Data: A Foundation for Prioritizing Adverse Outcome Pathway Development.

PubMed

Fay, Kellie A; Villeneuve, Daniel L; Swintek, Joe; Edwards, Stephen W; Nelms, Mark D; Blackwell, Brett R; Ankley, Gerald T

2018-06-01

The U.S. Environmental Protection Agency's ToxCast program has screened thousands of chemicals for biological activity, primarily using high-throughput in vitro bioassays. Adverse outcome pathways (AOPs) offer a means to link pathway-specific biological activities with potential apical effects relevant to risk assessors. Thus, efforts are underway to develop AOPs relevant to pathway-specific perturbations detected in ToxCast assays. Previous work identified a "cytotoxic burst" (CTB) phenomenon wherein large numbers of the ToxCast assays begin to respond at or near test chemical concentrations that elicit cytotoxicity, and a statistical approach to defining the bounds of the CTB was developed. To focus AOP development on the molecular targets corresponding to ToxCast assays indicating pathway-specific effects, we conducted a meta-analysis to identify which assays most frequently respond at concentrations below the CTB. A preliminary list of potentially important, target-specific assays was determined by ranking assays by the fraction of chemical hits below the CTB compared with the number of chemicals tested. Additional priority assays were identified using a diagnostic-odds-ratio approach which gives greater ranking to assays with high specificity but low responsivity. Combined, the two prioritization methods identified several novel targets (e.g., peripheral benzodiazepine and progesterone receptors) to prioritize for AOP development, and affirmed the importance of a number of existing AOPs aligned with ToxCast targets (e.g., thyroperoxidase, estrogen receptor, aromatase). The prioritization approaches did not appear to be influenced by inter-assay differences in chemical bioavailability. Furthermore, the outcomes were robust based on a variety of different parameters used to define the CTB.

A robust, high-throughput method for computing maize ear, cob, and kernel attributes automatically from images.

PubMed

Miller, Nathan D; Haase, Nicholas J; Lee, Jonghyun; Kaeppler, Shawn M; de Leon, Natalia; Spalding, Edgar P

2017-01-01

Grain yield of the maize plant depends on the sizes, shapes, and numbers of ears and the kernels they bear. An automated pipeline that can measure these components of yield from easily-obtained digital images is needed to advance our understanding of this globally important crop. Here we present three custom algorithms designed to compute such yield components automatically from digital images acquired by a low-cost platform. One algorithm determines the average space each kernel occupies along the cob axis using a sliding-window Fourier transform analysis of image intensity features. A second counts individual kernels removed from ears, including those in clusters. A third measures each kernel's major and minor axis after a Bayesian analysis of contour points identifies the kernel tip. Dimensionless ear and kernel shape traits that may interrelate yield components are measured by principal components analysis of contour point sets. Increased objectivity and speed compared to typical manual methods are achieved without loss of accuracy as evidenced by high correlations with ground truth measurements and simulated data. Millimeter-scale differences among ear, cob, and kernel traits that ranged more than 2.5-fold across a diverse group of inbred maize lines were resolved. This system for measuring maize ear, cob, and kernel attributes is being used by multiple research groups as an automated Web service running on community high-throughput computing and distributed data storage infrastructure. Users may create their own workflow using the source code that is staged for download on a public repository. © 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

Quantifying Stochastic Noise in Cultured Circadian Reporter Cells

DOE PAGES

John, Peter C.; Doyle, III, Francis J.

2015-11-20

We report that stochastic noise at the cellular level has been shown to play a fundamental role in circadian oscillations, influencing how groups of cells entrain to external cues and likely serving as the mechanism by which cell-autonomous rhythms are generated. Despite this importance, few studies have investigated how clock perturbations affect stochastic noise—even as increasing numbers of high-throughput screens categorize how gene knockdowns or small molecules can change clock period and amplitude. This absence is likely due to the difficulty associated with measuring cell-autonomous stochastic noise directly, which currently requires the careful collection and processing of single-cell data. Inmore » this study, we show that the damping rate of population-level bioluminescence recordings can serve as an accurate measure of overall stochastic noise, and one that can be applied to future and existing high-throughput circadian screens. Using cell-autonomous fibroblast data, we first show directly that higher noise at the single-cell results in faster damping at the population level. Next, we show that the damping rate of cultured cells can be changed in a dose-dependent fashion by small molecule modulators, and confirm that such a change can be explained by single-cell noise using a mathematical model. We further demonstrate the insights that can be gained by applying our method to a genome-wide siRNA screen, revealing that stochastic noise is altered independently from period, amplitude, and phase. Finally, we hypothesize that the unperturbed clock is highly optimized for robust rhythms, as very few gene perturbations are capable of simultaneously increasing amplitude and lowering stochastic noise. Ultimately, this study demonstrates the importance of considering the effect of circadian perturbations on stochastic noise, particularly with regard to the development of small-molecule circadian therapeutics.« less

Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol

PubMed Central

2017-01-01

Tight and tunable control of gene expression is a highly desirable goal in synthetic biology for constructing predictable gene circuits and achieving preferred phenotypes. Elucidating the sequence–function relationship of promoters is crucial for manipulating gene expression at the transcriptional level, particularly for inducible systems dependent on transcriptional regulators. Sort-seq methods employing fluorescence-activated cell sorting (FACS) and high-throughput sequencing allow for the quantitative analysis of sequence–function relationships in a robust and rapid way. Here we utilized a massively parallel sort-seq approach to analyze the formaldehyde-inducible Escherichia coli promoter (Pfrm) with single-nucleotide resolution. A library of mutated formaldehyde-inducible promoters was cloned upstream of gfp on a plasmid. The library was partitioned into bins via FACS on the basis of green fluorescent protein (GFP) expression level, and mutated promoters falling into each expression bin were identified with high-throughput sequencing. The resulting analysis identified two 19 base pair repressor binding sites, one upstream of the −35 RNA polymerase (RNAP) binding site and one overlapping with the −10 site, and assessed the relative importance of each position and base therein. Key mutations were identified for tuning expression levels and were used to engineer formaldehyde-inducible promoters with predictable activities. Engineered variants demonstrated up to 14-fold lower basal expression, 13-fold higher induced expression, and a 3.6-fold stronger response as indicated by relative dynamic range. Finally, an engineered formaldehyde-inducible promoter was employed to drive the expression of heterologous methanol assimilation genes and achieved increased biomass levels on methanol, a non-native substrate of E. coli. PMID:28463494

Spectrophotometric Analysis of Pigments: A Critical Assessment of a High-Throughput Method for Analysis of Algal Pigment Mixtures by Spectral Deconvolution

PubMed Central

Thrane, Jan-Erik; Kyle, Marcia; Striebel, Maren; Haande, Sigrid; Grung, Merete; Rohrlack, Thomas; Andersen, Tom

2015-01-01

The Gauss-peak spectra (GPS) method represents individual pigment spectra as weighted sums of Gaussian functions, and uses these to model absorbance spectra of phytoplankton pigment mixtures. We here present several improvements for this type of methodology, including adaptation to plate reader technology and efficient model fitting by open source software. We use a one-step modeling of both pigment absorption and background attenuation with non-negative least squares, following a one-time instrument-specific calibration. The fitted background is shown to be higher than a solvent blank, with features reflecting contributions from both scatter and non-pigment absorption. We assessed pigment aliasing due to absorption spectra similarity by Monte Carlo simulation, and used this information to select a robust set of identifiable pigments that are also expected to be common in natural samples. To test the method’s performance, we analyzed absorbance spectra of pigment extracts from sediment cores, 75 natural lake samples, and four phytoplankton cultures, and compared the estimated pigment concentrations with concentrations obtained using high performance liquid chromatography (HPLC). The deviance between observed and fitted spectra was generally very low, indicating that measured spectra could successfully be reconstructed as weighted sums of pigment and background components. Concentrations of total chlorophylls and total carotenoids could accurately be estimated for both sediment and lake samples, but individual pigment concentrations (especially carotenoids) proved difficult to resolve due to similarity between their absorbance spectra. In general, our modified-GPS method provides an improvement of the GPS method that is a fast, inexpensive, and high-throughput alternative for screening of pigment composition in samples of phytoplankton material. PMID:26359659

Perspectives on zebrafish models of hallucinogenic drugs and related psychotropic compounds.

PubMed

Neelkantan, Nikhil; Mikhaylova, Alina; Stewart, Adam Michael; Arnold, Raymond; Gjeloshi, Visar; Kondaveeti, Divya; Poudel, Manoj K; Kalueff, Allan V

2013-08-21

Among different classes of psychotropic drugs, hallucinogenic agents exert one of the most prominent effects on human and animal behaviors, markedly altering sensory, motor, affective, and cognitive responses. The growing clinical and preclinical interest in psychedelic, dissociative, and deliriant hallucinogens necessitates novel translational, sensitive, and high-throughput in vivo models and screens. Primate and rodent models have been traditionally used to study cellular mechanisms and neural circuits of hallucinogenic drugs' action. The utility of zebrafish ( Danio rerio ) in neuroscience research is rapidly growing due to their high physiological and genetic homology to humans, ease of genetic manipulation, robust behaviors, and cost effectiveness. Possessing a fully characterized genome, both adult and larval zebrafish are currently widely used for in vivo screening of various psychotropic compounds, including hallucinogens and related drugs. Recognizing the growing importance of hallucinogens in biological psychiatry, here we discuss hallucinogenic-induced phenotypes in zebrafish and evaluate their potential as efficient preclinical models of drug-induced states in humans.

NMR approaches in structure-based lead discovery: Recent developments and new frontiers for targeting multi-protein complexes

PubMed Central

Dias, David M.; Ciulli, Alessio

2014-01-01

Nuclear magnetic resonance (NMR) spectroscopy is a pivotal method for structure-based and fragment-based lead discovery because it is one of the most robust techniques to provide information on protein structure, dynamics and interaction at an atomic level in solution. Nowadays, in most ligand screening cascades, NMR-based methods are applied to identify and structurally validate small molecule binding. These can be high-throughput and are often used synergistically with other biophysical assays. Here, we describe current state-of-the-art in the portfolio of available NMR-based experiments that are used to aid early-stage lead discovery. We then focus on multi-protein complexes as targets and how NMR spectroscopy allows studying of interactions within the high molecular weight assemblies that make up a vast fraction of the yet untargeted proteome. Finally, we give our perspective on how currently available methods could build an improved strategy for drug discovery against such challenging targets. PMID:25175337

Perspectives on Zebrafish Models of Hallucinogenic Drugs and Related Psychotropic Compounds

PubMed Central

2013-01-01

Among different classes of psychotropic drugs, hallucinogenic agents exert one of the most prominent effects on human and animal behaviors, markedly altering sensory, motor, affective, and cognitive responses. The growing clinical and preclinical interest in psychedelic, dissociative, and deliriant hallucinogens necessitates novel translational, sensitive, and high-throughput in vivo models and screens. Primate and rodent models have been traditionally used to study cellular mechanisms and neural circuits of hallucinogenic drugs’ action. The utility of zebrafish (Danio rerio) in neuroscience research is rapidly growing due to their high physiological and genetic homology to humans, ease of genetic manipulation, robust behaviors, and cost effectiveness. Possessing a fully characterized genome, both adult and larval zebrafish are currently widely used for in vivo screening of various psychotropic compounds, including hallucinogens and related drugs. Recognizing the growing importance of hallucinogens in biological psychiatry, here we discuss hallucinogenic-induced phenotypes in zebrafish and evaluate their potential as efficient preclinical models of drug-induced states in humans. PMID:23883191

Compartmentalized microchannel array for high-throughput analysis of single cell polarized growth and dynamics

DOE PAGES

Geng, Tao; Bredeweg, Erin L.; Szymanski, Craig J.; ...

2015-11-04

Here, interrogating polarized growth is technologically challenging due to extensive cellular branching and uncontrollable environmental conditions in conventional assays. Here we present a robust and high-performance microfluidic system that enables observations of polarized growth with enhanced temporal and spatial control over prolonged periods. The system has built-in tunability and versatility to accommodate a variety of science applications requiring precisely controlled environments. Using the model filamentous fungus, Neurospora crassa, this microfluidic system enabled direct visualization and analysis of cellular heterogeneity in a clonal fungal cell population, nuclear distribution and dynamics at the subhyphal level, and quantitative dynamics of gene expression withmore » single hyphal compartment resolution in response to carbon source starvation and exchange experiments. Although the microfluidic device is demonstrated on filamentous fungi, our technology is immediately extensible to a wide array of other biosystems that exhibit similar polarized cell growth with applications ranging from bioenergy production to human health.« less

Pitfalls to avoid when using phage display for snake toxins.

PubMed

Laustsen, Andreas Hougaard; Lauridsen, Line Præst; Lomonte, Bruno; Andersen, Mikael Rørdam; Lohse, Brian

2017-02-01

Antivenoms against bites and stings from snakes, spiders, and scorpions are associated with immunological side effects and high cost of production, since these therapies are still derived from the serum of hyper-immunized production animals. Biotechnological innovations within envenoming therapies are thus warranted, and phage display technology may be a promising avenue for bringing antivenoms into the modern era of biologics. Although phage display technology represents a robust and high-throughput approach for the discovery of antibody-based antitoxins, several pitfalls may present themselves when animal toxins are used as targets for phage display selection. Here, we report selected critical challenges from our own phage display experiments associated with biotinylation of antigens, clone picking, and the presence of amber codons within antibody fragment structures in some phage display libraries. These challenges may be detrimental to the outcome of phage display experiments, and we aim to help other researchers avoiding these pitfalls by presenting their solutions. Copyright © 2016 Elsevier Ltd. All rights reserved.

The discriminatory power of ribotyping as automatable technique for differentiation of bacteria.

PubMed

Schumann, Peter; Pukall, Rüdiger

2013-09-01

Since the introduction of ribonucleic acid gene restriction patterns as taxonomic tools in 1986, ribotyping has become an established method for systematics, epidemiological, ecological and population studies of microorganisms. In the last 25 years, several modifications have improved the convenience, reproducibility and turn-around time of this technique. The technological development culminated in the automation of ribotyping which allowed for high-throughput applications e.g. in the quality control of food production, pharmaceutical industry and culture collections. The capability of the fully automated RiboPrinter(®) System for the differentiation of bacteria below the species level is compared with the discriminatory power of traditional ribotyping, of molecular fingerprint techniques like PFGE, MLST and MLVA as well as of MALDI-TOF mass spectrometry. While automated RiboPrinting is advantageous with respect to standardization, ease and speed, PCR ribotyping has proved being a highly discriminatory, flexible, robust and cost-efficient routine technique which makes inter-laboratory comparison and build of ribotype databases possible, too. Copyright © 2013 Elsevier GmbH. All rights reserved.

Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection

PubMed Central

Kang, Dong-Ku; Ali, M. Monsur; Zhang, Kaixiang; Huang, Susan S.; Peterson, Ellena; Digman, Michelle A.; Gratton, Enrico; Zhao, Weian

2014-01-01

Blood stream infection or sepsis is a major health problem worldwide, with extremely high mortality, which is partly due to the inability to rapidly detect and identify bacteria in the early stages of infection. Here we present a new technology termed ‘Integrated Comprehensive Droplet Digital Detection’ (IC 3D) that can selectively detect bacteria directly from milliliters of diluted blood at single-cell sensitivity in a one-step, culture- and amplification-free process within 1.5–4 h. The IC 3D integrates real-time, DNAzyme-based sensors, droplet microencapsulation and a high-throughput 3D particle counter system. Using Escherichia coli as a target, we demonstrate that the IC 3D can provide absolute quantification of both stock and clinical isolates of E. coli in spiked blood within a broad range of extremely low concentration from 1 to 10,000 bacteria per ml with exceptional robustness and limit of detection in the single digit regime. PMID:25391809

Fast and Efficient Fragment-Based Lead Generation by Fully Automated Processing and Analysis of Ligand-Observed NMR Binding Data.

PubMed

Peng, Chen; Frommlet, Alexandra; Perez, Manuel; Cobas, Carlos; Blechschmidt, Anke; Dominguez, Santiago; Lingel, Andreas

2016-04-14

NMR binding assays are routinely applied in hit finding and validation during early stages of drug discovery, particularly for fragment-based lead generation. To this end, compound libraries are screened by ligand-observed NMR experiments such as STD, T1ρ, and CPMG to identify molecules interacting with a target. The analysis of a high number of complex spectra is performed largely manually and therefore represents a limiting step in hit generation campaigns. Here we report a novel integrated computational procedure that processes and analyzes ligand-observed proton and fluorine NMR binding data in a fully automated fashion. A performance evaluation comparing automated and manual analysis results on (19)F- and (1)H-detected data sets shows that the program delivers robust, high-confidence hit lists in a fraction of the time needed for manual analysis and greatly facilitates visual inspection of the associated NMR spectra. These features enable considerably higher throughput, the assessment of larger libraries, and shorter turn-around times.

A versatile pipeline for the multi-scale digital reconstruction and quantitative analysis of 3D tissue architecture

PubMed Central

Morales-Navarrete, Hernán; Segovia-Miranda, Fabián; Klukowski, Piotr; Meyer, Kirstin; Nonaka, Hidenori; Marsico, Giovanni; Chernykh, Mikhail; Kalaidzidis, Alexander; Zerial, Marino; Kalaidzidis, Yannis

2015-01-01

A prerequisite for the systems biology analysis of tissues is an accurate digital three-dimensional reconstruction of tissue structure based on images of markers covering multiple scales. Here, we designed a flexible pipeline for the multi-scale reconstruction and quantitative morphological analysis of tissue architecture from microscopy images. Our pipeline includes newly developed algorithms that address specific challenges of thick dense tissue reconstruction. Our implementation allows for a flexible workflow, scalable to high-throughput analysis and applicable to various mammalian tissues. We applied it to the analysis of liver tissue and extracted quantitative parameters of sinusoids, bile canaliculi and cell shapes, recognizing different liver cell types with high accuracy. Using our platform, we uncovered an unexpected zonation pattern of hepatocytes with different size, nuclei and DNA content, thus revealing new features of liver tissue organization. The pipeline also proved effective to analyse lung and kidney tissue, demonstrating its generality and robustness. DOI: http://dx.doi.org/10.7554/eLife.11214.001 PMID:26673893