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At least 163 records · Page 9Linked to original sources
PubMed2005

A new surface plasmon resonance sensor for high-throughput screening applications.

We report a new high-throughput surface plasmon resonance (SPR) sensor based on combination of SPR imaging with polarization contrast and a spatially patterned multilayer SPR structure. We demonstrate that this approach offers numerous advantageous features including high-contrast SPR images suitable for automated computer analysis, minimum crosstalk between neighboring sensing channels and inherent compensation for light level fluctuations. Applications of a laboratory prototype of the high-throughput SPR sensor with 108 sensing channels for refractometry and biosensing are described. In refractometric experiments, the noise-limited refractive index resolution of the system has been established to be 3 x 10(-6) refractive index unit (RIU). Experimental data on detection of human choriogonadotropin (hCG) suggest that in conjunction with monoclonal antibodies against hCG, the reported SPR imaging sensor is capable of detecting hCG at concentrations lower than 500 ng/ml.

Biosensing Techniques↗
PubMed2001

High-throughput screening for biocatalysts.

Recent progress in high-throughput enzyme assays includes new examples of fluorogenic and chromogenic substrates, fluorescence resonance energy transfer substrates, and applications of the pH and pM indicator methods. Recent developments of Horeau's pseudo-enantiomer derivatisation method to screen enantioselectivities in high-throughput have also been reported.

Automation↗
PubMed2005

Simple, robust methods for high-throughput nanoliter-scale DNA sequencing.

We have developed high-throughput DNA sequencing methods that generate high quality data from reactions as small as 400 nL, providing an approximate order of magnitude reduction in reagent use relative to standard protocols. Sequencing of clones from plasmid, fosmid, and BAC libraries yielded read lengths (PHRED20 bases) of 765 +/- 172 (n = 10,272), 621 +/- 201 (n = 1824), and 647 +/- 189 (n = 568), respectively. Implementation of these procedures at high-throughput genome centers could have a substantial impact on the amount of data that can be generated per unit cost.

Nanotechnology↗
PubMed2006

High-throughput physical organic chemistry--Hammett parameter evaluation.

High-throughput analysis techniques were developed to allow the rapid assessment of a range of Hammett parameters utilizing positive electrospray mass spectrometry (ESI+ -MS) as the sole quantitative tool, with the core of the approach being a so-called "analytical construct". Hammett substituent parameters were determined for a range of meta- and para-substituted anilines by high-throughput (HT) assessment of relative reaction rates for competitive amide bond formation reaction with up to five parameters determined in a single pot reaction. Sensitivity of the reaction to substituents' effects (materialized by Hammett's rho parameter) was determined in the first instance, with HT Hammett's sigma substituent parameter assessment then carried out successfully for over 30 anilines, with excellent correlation observed between the HT ESI+ -MS method of determination and literature values.

Aniline Compounds↗
PubMed2002

Finding the needle in the haystack: why high-throughput screening is good for your health.

High-throughput screening is an essential component of the toolbox of modern technologies that improve speed and efficiency in contemporary cancer drug development. This is particularly important as we seek to exploit, for maximum therapeutic benefit, the large number of new molecular targets emerging from the Human Genome Project and cancer genomics. Screening of diverse collections of low molecular weight compounds plays a key role in providing chemical starting points for iterative optimisation by medicinal chemistry. Examples of successful drug discovery programmes based on high-throughput screening are described, and these offer potential in the treatment of breast cancer and other malignancies.

Antineoplastic Agents↗
PubMed2002

A strategy for high-throughput assay development using leads derived from nuclear magnetic resonance-based screening.

A strategy is described for the development of high-throughput screening assays against targets of unknown function that involves the use of nuclear magnetic resonance (NMR) spectroscopy. Using this approach, molecules that bind to the protein target are identified from an NMR-based screen of a library of substrates, cofactors, and other compounds that are known to bind to many proteins and enzymes. Once a ligand has been discovered, a fluorescent or radiolabeled analog of the ligand is synthesized that can be used in a high-throughput screen. The approach is illustrated in the development of a high-throughput screening assay against HI-0033, a conserved protein from Haemophilus influenzae whose function is currently unknown. Adenosine was found to bind to HI-0033 by NMR, and fluorescent analogs were rapidly identified that bound to HI-0033 in the submicromolar range. Using these fluorescent compounds, a fluorescence polarization assay was developed that is suitable for high-throughput screening and obtaining detailed structure-activity relationships for lead optimization.

Bacterial Proteins↗
PubMed2006

A novel tracing algorithm for high throughput imaging Screening of neuron-based assays.

High throughput neuron image processing is an important method for drug screening and quantitative neurobiological studies. The method usually includes detection of neurite structures, feature extraction, quantification, and statistical analysis. In this paper, we present a new algorithm for fast and automatic extraction of neurite structures in microscopy neuron images. The algorithm is based on novel methods for soma segmentation, seed point detection, recursive center-line detection, and 2D curve smoothing. The algorithm is fully automatic without any human interaction, and robust enough for usage on images with poor quality, such as those with low contrast or low signal-to-noise ratio. It is able to completely and accurately extract neurite segments in neuron images with highly complicated neurite structures. Robustness comes from the use of 2D smoothening techniques and the idea of center-line extraction by estimating the surrounding edges. Efficiency is achieved by processing only pixels that are close enough to the line structures, and by carefully chosen stopping conditions. These make the proposed approach suitable for demanding image processing tasks in high throughput screening of neuron-based assays. Detailed results on experimental validation of the proposed method and on its comparative performance with other proposed schemes are included.

Algorithms↗
PubMed2006

Statistical practice in high-throughput screening data analysis.

High-throughput screening is an early critical step in drug discovery. Its aim is to screen a large number of diverse chemical compounds to identify candidate 'hits' rapidly and accurately. Few statistical tools are currently available, however, to detect quality hits with a high degree of confidence. We examine statistical aspects of data preprocessing and hit identification for primary screens. We focus on concerns related to positional effects of wells within plates, choice of hit threshold and the importance of minimizing false-positive and false-negative rates. We argue that replicate measurements are needed to verify assumptions of current methods and to suggest data analysis strategies when assumptions are not met. The integration of replicates with robust statistical methods in primary screens will facilitate the discovery of reliable hits, ultimately improving the sensitivity and specificity of the screening process.

Biological Assay↗
PubMed2006

High-throughput screening of biocatalytic activity: applications in drug discovery.

Enzymes catalyze a diverse set of reactions that propel life's processes and hence serve as valuable therapeutic targets. High-throughput screening methods have become essential for sifting through large chemical libraries in search of drug candidates, and several sensitive and reliable analytical techniques have been specifically adapted to high-throughput measurements of biocatalytic activity. High-throughput biocatalytic assay platforms thus enable rapid screening against enzymatic targets, and have vast potential to impact various stages of the drug discovery process, including lead identification and optimization, and ADME/Tox assessment. These advances are paving the way for the adoption of high-throughput biocatalytic assays as an indispensable tool for the pharmaceutical industry.

Catalysis↗
PubMed2005

Nanoliter homogenous ultra-high throughput screening microarray for lead discoveries and IC50 profiling.

Microfluidic technologies offer the potential for highly productive and low-cost ultra-high throughput screening and high throughput selectivity profiling. Such technologies need to provide the flexibility of plate-based assays as well as be less expensive to operate. Presented here is a unique microarray system (the Reaction Biology [Malvern, PA] DiscoveryDot), which runs over 6,000 homogeneous reactions per 1" x 3" microarray using chemical libraries or compound dilutions printed in 1-nl volumes. A simple and rapid piezo-activation method delivers from 30 to 300 pl of biochemical targets and detector chemistries to each reaction. The fluorescent signals are detected and analyzed with conventional microarray scanners and software. The DiscoveryDot platform is highly customizable, and reduces consumption of targets and reaction chemistries by >40-fold and the consumption of compounds by >10,000-fold, compared to 384-well plate assay. We demonstrate here that the DiscoveryDot platform is compatible with conventional large-volume well-based reactions, with a Z' factor of >0.6 for many enzymes, such as the caspase family enzymes, matrix metalloproteinase, serine proteases, kinases, and histone deacetylases. The platform is well equipped for 50% inhibitory concentration (IC50) profiling studies of enzyme inhibitors, with up to 10 dilution conditions of each test compound printed in duplicate, and each microarray chip can generate over 300 IC50 measurements against a given target.

Drug Design↗
PubMed2005

High-throughput alternative splicing quantification by primer extension and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Alternative splicing is a significant contributor to transcriptome diversity, and a high-throughput experimental method to quantitatively assess predictions from expressed sequence tag and microarray analyses may help to answer questions about the extent and functional significance of these variants. Here, we describe a method for high-throughput analysis of known or suspected alternative splicing variants (ASVs) using PCR, primer extension and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Reverse-transcribed mRNA is PCR amplified with primers surrounding the site of alternative splicing, followed by a primer extension reaction designed to target sequence disparities between two or more variants. These primer extension products are assayed on a MALDI-TOF mass spectrometer and analyzed automatically. This method is high-throughput, highly accurate and reproducible, allowing for the verification of the existence of splicing variants in a variety of samples. An example given also demonstrates how this method can eliminate potential pitfalls from ordinary gel electrophoretic analysis of splicing variants where heteroduplexes formed from different variants can produce erroneous results. The new method can be used to create alternative variant profiles for cancer markers, to study complex splicing regulation, or to screen potential splicing therapies.

Actinin↗
PubMed2003

Facilities and methods for the high-throughput crystal structural analysis of human proteins.

Facilities and methods for the high-throughput crystal structure analysis of human proteins are described as recently established in the Protein Structure Factory, a Berlin-area structural genomics project. Genes encoding human proteins are expressed in either recombinant Escherichia coli or yeast (Saccharomyces cerevisiae or Pichia pastoris). To facilitate and standardize protein purification, the target proteins are produced with various tags for affinity chromatography. For high-throughput crystallization, a robotic station is being set up that has the capacity to handle 960 000 experiments simultaneously. The resulting protein crystals will be subjected to X-ray diffraction experiments at the third-generation synchrotron storage ring BESSY where protein crystallography beamlines are currently under construction. The Protein Structure Factory's strategy for high-throughput production and structure analysis of human proteins is evaluated based on first results.

Cloning, Molecular↗
PubMed2005

High-throughput measurement of protein stability in microtiter plates.

The direct determination of protein stability at high throughput has applications in proteomics, directed evolution, and formulation. Each application places different requirements on the accuracy of stability or transition midpoint determination. The measurement of protein stability by chemical denaturation has been previously performed at medium throughput and high accuracy using autotitrating fluorometers, after removal of proteins from the 96-well plate format in which they were expressed and purified. Herein we present a higher-throughput method for measuring and indexing the stability of proteins maintained within the 96-well format using a fluorescence microplate reader. Protein unfolding transitions were monitored by tryptophan fluorescence at 340 nm and assessed using bovine and equine cytochrome c (cyt c), as well as bovine serum albumin (BSA) stabilized with various amounts of palmitic acid. Two different approaches for generating unfolding curves in microtiter plates have been evaluated for their accuracy and applicability. Unfolding curves generated by the serial addition of denaturant into single wells allowed high-throughput stability screens capable of identifying protein variants with unfolding midpoint differences of 0.15 M denaturant concentration or larger. Such a method would be suitable for screening large numbers of proteins, as typically generated for directed evolution. Unfolding curves generated using one well per denaturant concentration allowed for medium-throughput stability screening and generated more accurate and precise stability values (C(1/2) +/- 0.05 M, m(G), and DeltaG(H2O)) for cyt c that are similar to values reported in literature. This method is suitable for screening the smaller numbers of proteins generated in proteomic research programmes. By using BSA stabilized with various palmitate concentrations and simple numerical indexing, it was shown that both experimental methods can successfully rank the order of protein stability.

Animals↗
PubMed2004

High-throughput comprehensive analysis of human plasma proteins: a step toward population proteomics.

A high-throughput (HT) comprehensive analysis approach was developed for assaying proteins directly from human plasma. Proteins were selectively retrieved, by utilizing antibodies immobilized within affinity pipet tips, and eluted onto enzymatically active mass spectrometer targets for subsequent digestion and structural characterization. Several parameters, including uniform parallel protein elution from 96 affinity pipet tips, proper buffering for on-target digestion, termination of the digestion, and MALDI matrix (re)introduction, were evaluated and optimized. The approach was validated via parallel, high-throughput analysis of transthyretin (TTR) and transferrin (TRFE) from 96 identical plasma samples. The 96 parallel analyses for each protein were completed in less than 90 min, measured from protein extraction to insertion in the mass spectrometer. Virtually identical mass spectra were obtained from the 96 TTR analyses, characterized by the presence of 14 tryptic fragments that allowed TTR sequence mapping with 100% coverage. Database search returned TTR as the best match for all 96 data sets. In regard to the TRFE analyses, database searching using data from the 96 spectra returned TRFE as the best match for all but 1 of the spectra. TRFE was mapped with 47-69% sequence coverage, with gaps in the sequence coverage corresponding to the carbohydrate-containing peptide fragments and large and small trypsin fragments that fell outside the window of mass analysis. Overall, the combined high-throughput affinity capture-protein digestion approach showed high reproducibility and speed and yielded an exceptional level of protein characterization, suggesting its use in future population proteomics endeavors.

Blood Proteins↗
PubMed2006

HTS-Corrector: software for the statistical analysis and correction of experimental high-throughput screening data.

MOTIVATION: High-throughput screening (HTS) plays a central role in modern drug discovery, allowing for testing of >100,000 compounds per screen. The aim of our work was to develop and implement methods for minimizing the impact of systematic error in the analysis of HTS data. To the best of our knowledge, two new data correction methods included in HTS-Corrector are not available in any existing commercial software or freeware. RESULTS: This paper describes HTS-Corrector, a software application for the analysis of HTS data, detection and visualization of systematic error, and corresponding correction of HTS signals. Three new methods for the statistical analysis and correction of raw HTS data are included in HTS-Corrector: background evaluation, well correction and hit-sigma distribution procedures intended to minimize the impact of systematic errors. We discuss the main features of HTS-Corrector and demonstrate the benefits of the algorithms.

Algorithms↗
PubMed2005

An empirical comparison of case-control and trio based study designs in high throughput association mapping.

Motivated by high throughput genotyping technology, our aim in this study was to experimentally compare the power and accuracy of case-control and family trio based approaches for haplotype based, large scale, association gene mapping. We compared trio based and case-control study designs in different disease models, and partitioned the performance differences into separate components: those from the sample ascertainment, the effective sample size, and the haplotyping approaches. For systematic and controlled tests, we simulated a rapidly expanding and relatively young isolated population. The experiments were also replicated with real asthma data. We used computationally efficient methods that scale up to large amounts of both markers and individuals. Mapping is based on a haplotype association test for haplotypes of 1-10 markers. For population based haplotype reconstruction, we use HaploRec, and compare it to both a simple trio based inference and true haplotypes. Firstly and surprisingly, statistically inferred population based haplotypes can be equally powerful as true haplotypes. Secondly, as expected, the effective sample size has a clear effect on both gene detection power and mapping accuracy. Thirdly, the sample ascertainment method does not have much effect on mapping accuracy. Finally, an interesting side result is that the simple haplotype association test clearly outperformed exhaustive allelic transmission disequilibrium tests. The results suggest that the case-control design is a powerful alternative to the more laborious family based ascertainment approach, especially for large datasets, and wherever population stratification can be controlled.

Algorithms↗
PubMed2006

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

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

Computer Graphics↗
PubMed2001

[High throughput pharmacology for drug discovery].

High Throughput Screening (HTS) now plays an important role in the discovery of new lead compounds for novel therapeutic targets. The advantage of HTS over the conventional method, now termed as Low Throughput Screening (LTS), is that valuable compounds can be selected rapidly from a large number of samples with minimal human involvement. In spite of the growing awareness of HTS, the importance of the LTS in the drug discovery and development is still not changed. Advances in pharmacogenomics will also provide us many pharmacological targets, and thus increase the number of compounds that should be assayed by HTS and LTS. In this review, we will first describe the outline of HTS. We will next describe new approaches to develop and brush up the LTS: 1) screening method of drugs acting on ion channels by voltage-sensitive fluorescent dye, 2) functional assay method using reconstituted smooth muscle fiber, and 3) organ culture method as a useful model of vascular proliferative disease. These approaches, which work cooperatively with HTS, will contribute greatly to the development of new drugs.

Animals↗