Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “high throughput”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 847 records · Page 47Linked to original sources
PubMed2000

High-throughput screening: new technology for the 21st century.

New technologies in high-throughput screening have significantly increased throughput and reduced assay volumes. Key advances over the past few years include new fluorescence methods, detection platforms and liquid-handling technologies. Screening 100,000 samples per day in miniaturized assay volumes will soon become routine. Furthermore, new technologies are now being applied to information-rich cell-based assays, and this is beginning to remove one of the key bottlenecks downstream from primary screening.

Drug Evaluation, Preclinical↗
PubMed2000

High-throughput sequencing in the population analysis of bacterial pathogens of humans.

High-throughput nucleotide sequence determination technologies present new opportunities for studies of bacterial pathogens by enabling the accumulation of large volumes of biodiversity information from isolate collections. Population studies, which combine these data with epidemiological, phylogenetic, and evolutionary concepts, provide insights into the behaviour of pathogens that are unavailable from other approaches as they address questions of relevance to pathogenesis from the perspective of the infectious organism rather from that of the host. Hypothesis-driven analyses applied to these data permit the determination of microbial population diversity and structure, the identification of the mechanisms of genetic change in bacterial populations, and the generation of models of pathogen evolution. The nucleotide sequence-based population studies performed to date demonstrate a spectrum of nucleotide sequence diversity, population structure, and evolutionary mechanisms among pathogenic bacteria. The rapid development of nucleotide sequence determination and analysis techniques provides the tools necessary for the prosecution of population studies on an increasing number of bacterial pathogens.

Bacteria↗
PubMedDate not supplied

High-throughput prediction of blood-brain partitioning: a thermodynamic approach.

A high-throughput in silico screening tool for potentially CNS active compounds was developed on the basis of the correlation of solvation free energies and blood-brain partitioning (log(cbrain/cblood) = log BB) data available from experimental sources. Utilizing a thermodynamic approach, solvation free energies were calculated by the fast and efficient generalized Born/surface area continuum solvation model, which enabled us to evaluate more than 10 compounds/min. Our training set involved a structurally diverse set of 55 compounds and yielded a function of log BB = 0.035Gsolv + 0.2592 (r = 0.85, standard error 0.37). Calculation of solvation free energies for 8700 CNS active compounds (CIPSLINE database) revealed that Gsolv is higher than -50 kJ/mol for the 96% of these compounds which can be used as suitable criteria for the identification of compounds preferable for CNS penetration.

Blood-Brain Barrier↗
PubMed2002

A high-throughput screening protocol for fast evaluation of enantioselective catalysts.

A new high-throughput screening protocol that allows fast evaluation of enantioselective catalysts has been developed. The usefulness of norephedrine-derived beta-amino alcohols as catalysts for the enantioselective alkylation of prochiral aldehydes has been determined by simultaneous screening of three representative substrates. GC analysis of the crude product mixture using a selectively modified cyclodextrin as the chiral stationary phase avoids time-consuming workup procedures. The chemical yield, enantioselectivity, substrate specificity, and catalytic activity of the chiral catalysts as well as the induced absolute configuration have been determined in a single screening experiment and two short GC runs.

Journal Article↗
PubMed2002

Applications of CpG island microarrays for high-throughput analysis of DNA methylation.

Differential methylation hybridization (DMH) is a high-throughput microarray technique designed to identify changes in DNA methylation patterns commonly observed in cancer and other disease states. The DMH methodology comprises three fundamental components: the arraying of CpG island clones on glass slides, the preparation of the sample amplicons under investigation, and the hybridization of amplicon targets onto the CpG island microarray. Herein, we outline the DMH protocol and illustrate its utility and the validation approaches used in analyzing the hypermethylation profile of breast tumor and normal samples.

CpG Islands↗
PubMed1998

Green fluorescent protein reporter microplate assay for high-throughput screening of compounds against Mycobacterium tuberculosis.

An optimal assay for high-throughput screening for new antituberculosis agents would combine the microplate format and low cost of firefly luciferase reporter assays and redox dyes with the ease of kinetic monitoring inherent in the BACTEC system. The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is a useful reporter molecule which requires neither substrates nor cofactors due to the intrinsically fluorescent nature of the protein. The gene encoding a red-shifted, higher-intensity GFP variant was introduced by electroporation into Mycobacterium tuberculosis H37Ra and M. tuberculosis H37Rv on expression vector pFPV2. A microplate-based fluorescence assay (GFP microplate assay [GFPMA]) was developed and evaluated by determining the MICs of existing antimycobacterial agents. The MICs of isoniazid, rifampin, ethambutol, streptomycin, amikacin, ofloxacin, ethionamide, thiacetazone, and capreomycin, but not cycloserine, determined by GFPMA were within 1 log2 dilution of those determined with the BACTEC 460 system and were available in 7 days. Equivalent MICs of antituberculosis agents in the BACTEC 460 system for both the reporter and parent strains suggested that introduction of pFPV2 did not influence drug susceptibility, in general. GFPMA provides a unique tool with which the dynamic response of M. tuberculosis to the existing and potential antituberculosis agents can easily, rapidly, and inexpensively be monitored.

Anti-Infective Agents↗
PubMed2001

Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists.

Drug discovery by high-throughput screening is a promising approach to develop new therapies for the most common lethal genetic disease, cystic fibrosis. Because disease-causing mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein produce epithelial cells with reduced or absent Cl(-) permeability, the goal of screening is to identify compounds that restore cell Cl(-) transport. We have developed a rapid, quantitative screening procedure for analysis of CFTR-mediated halide transport in cells with the use of a conventional fluorescence plate reader. Doubly transfected cell lines were generated that express wild-type or mutant CFTR together with a yellow fluorescent protein (YFP)-based halide sensor. CFTR function was assayed from the time course of cell fluorescence in response to extracellular addition of 100 mM I(-) followed by forskolin, resulting in decreased YFP fluorescence due to CFTR-mediated I(-) entry. Cell lines were chosen, and conditions were optimized to minimize basal halide transport to maximize assay sensitivity. In cells cultured on 96-well plastic dishes, the assay gave reproducible halide permeabilities from well to well and could reliably detect a 2% activation of CFTR-dependent halide transport produced by low concentrations of forskolin. Applications of the assay are shown, including comparative dose-dependent CFTR activation by genistein, apigenin, 8-cyclopentyl-1,3-dipropylxanthine, IBMX, 8-methoxypsoralen, and milrinone as well as activation of alternative Cl(-) channels. The fluorescence assay and cell lines should facilitate the screening of novel CFTR activators and the characterization of alternative Cl(-) channels and transporters.

Animals↗
PubMed2005

Paraffin-embedded cell line microarray (PECLIMA): development and validation of a high-throughput method for antigen profiling of cell lines.

The introduction of high-throughput techniques is increasingly providing abundant information on molecular alterations requiring validation at the posttranscriptional level. Protein expression is now efficiently evaluated in large series of tumors included in tissue microarrays. We propose, describe and validate a technique to elaborate paraffin-embedded cell line microarrays (PECLIMA) from fixed cell cultures, which can be processed like standard surgical pathology biopsies prior to immunophenotyping. Our results show a reliable protein immunoexpression profiling in six widely used cell lines under different fixation conditions. This technique permits the simultaneous analysis of multiple antigens in multiple cell lines under different experimental conditions. Additional features of these arrays are long-term storage, their suitability for a variety of techniques including immunocytochemistry and in situ hybridization and their low cost.

Antigens, Neoplasm↗
PubMed2004

Validating bioluminescence imaging as a high-throughput, quantitative modality for assessing tumor burden.

Bioluminescence imaging (BLI) is a highly sensitive tool for visualizing tumors, neoplastic development, metastatic spread, and response to therapy. Although BLI has engendered much excitement due to its apparent simplicity and ease of implementation, few rigorous studies have been presented to validate the measurements. Here, we characterize the nature of bioluminescence output from mice bearing subcutaneous luciferase-expressing tumors over a 4-week period. Following intraperitoneal or direct intratumoral administration of luciferin substrate, there was a highly dynamic kinetic profile of light emission. Although bioluminescence was subject to variability, strong correlations (r >.8, p <.001) between caliper measured tumor volumes and peak light signal, area under light signal curve and light emission at specific time points were determined. Moreover, the profile of tumor growth, as monitored with bioluminescence, closely resembled that for caliper measurements. The study shows that despite the dynamic and variable nature of bioluminescence, where appropriate experimental precautions are taken, single time point BLI may be useful for noninvasive, high-throughput, quantitative assessment of tumor burden.

Animals↗
PubMed2005

Evaluation of the InteraX system technology in a high-throughput screening environment.

The authors have developed a cell-based high-throughput screening (HTS)-compatible assay to measure EGFR dimerization using the InteraX enzyme complementation technology of Applied Biosystems. The cells contain 2 chimeric proteins with complementing deletion mutants of the beta galactosidase enzyme, each fused to the extracellular and transmembrane part of EGFR. On binding of EGF, EGF receptor dimerizes and an active beta galactosidase is built. The authors used this homogeneous 384-well assay to screen about 20,000 diverse compounds. From 2 independent primary screen runs 239 hits were identified. For run 1, a mean S/B ratio of 4.26 and a mean Z' factor of 0.74 were obtained, for run 2 a mean S/B ratio of 3.88 and a mean Z' factor of 0.71 were obtained. After hit confirmation, repeated 4 times, 112 hits remained with a confirmation rate of 48.9%. Thirty of the 112 could be identified as cytotoxic. Fifty-one of the remaining 82 compounds could be shown to be inhibitors of the beta galactosidase enzyme itself. In summary, 31 compounds remained as potential EGFR dimerization or EGF stimulation inhibitors. The authors conclude that the InteraX system technology is HTS capable and can detect small molecule inhibitors capable of inhibiting protein-protein interactions.

Animals↗
PubMed2003

Enabling high-throughput discovery.

During the past few years, the introduction of ultra-high-throughput screening and new assay design and detection technologies has exponentially increased the amount and complexity of screening data. Effective use of this data implies a process that begins with assay design. An effective data management system should control a range of processes, from the initial selection of compounds and storage and mining of the assay result to more complex tasks, such as extracting patterns from these data. Remarkable advances have been made during the last year to increase efficiency at different phases of the screening, shifting the bottleneck of this process to data analysis. The challenge facing drug discovery today is to extract knowledge from these data. Knowledge discovery is defined as 'the non-trivial extraction of implicit, unknown, and potentially useful information from data'. A large amount of research is being devoted to optimize the extraction of knowledge from screening data. In this review, we discuss the screening process and its progress during the last year. Some of the challenges for the future, such as optimization of the knowledge discovery process and the sharing of data across an organization, will also be presented.

Databases, Factual↗
PubMed2000

[Introduction of the high throughput screening system].

In this article, we introduce the system of HTS (high throughput screening). Its role in new drug study and current development are described. The relationship between research achievements of genome study and new type screening model of new drugs is emphasized. The personal opinions of current problems about HTS study in China are raised.

Autoanalysis↗
PubMed2001

Radioligand binding assays in the drug discovery process: potential pitfalls of high throughput screenings.

Radioligand binding assays evaluating directly the ability of a drug to interact with a defined molecular target is part of the drug discovery process. The need for a high throughput rate in screening drugs is actually leading to simplified experimental schemes that increase the probability of false negative results. Special concern involves voltage-gated ion channel drug discovery where a great care is required in designing assays because of frequent multiplicity of (interacting) binding sites. To clearly illustrate this situation, three different assays used in the academic drug discovery program of the authors were selected because they are rich of intrinsic artifacts: (I) (20 mmol/l caffeine almost duplicated [3H]ryanodine binding (89% higher than control) to rat heart microsomes at 0.3 mumol/l free calcium but did not exert any effect when using a high (107 mumol/l) free calcium, as mostly used in ryanodine binding assays; (II) An agonist for the ionotropic glutamate receptor of the kainate type can distinctly affect [3H]kainate binding to chicken cerebellum membranes depending on its concentration: unlabelled kainic acid per se either stimulated about 30% (at 50-100 nmol/l), had no effect (at 200 nmol/l) or even progressively decreased (at 0.3-2 mumol/l) the binding of 5 nmol/l [3H]kainate, emphasizing the risk of using a single concentration for screening a drug; (III) in a classical [3H]flunitrazepam binding assay, the stimulatory effect of a GABA (gamma-aminobutyric acid) agonist was only observed when using extensively washed rat brain synaptosomes (10 mumol/l GABA increased flunitrazepam binding by 90%). On the other hand, the inhibitory effect of a GABA antagonist was only observed when using crude synaptosomes (10 mumol/l bicuculine reduced [3H]flunitrazepam binding by 40%). It can be concluded that carefully designed radioligand assays which can be performed in an academic laboratory are appropriate for screening a small number of drugs, especially if these are potential hits because of their rational design. Therefore, the low throughput rate could be partially balanced by a higher performance when compared to what is done in a robotic high throughput screening where simplification of assay conditions can lead to false negative results.

Animals↗
PubMed2000

Parallel ultra-high flow rate liquid chromatography with mass spectrometric detection using a multiplex electrospray source for direct, sensitive determination of pharmaceuticals in plasma at extremely high throughput.

Recent years have seen increasing usage of large particle size stationary phases and ultra-high flow rate liquid chromatography/mass spectrometry (LC/MS) for rapid determination of pharmaceuticals in plasma without prior sample preparation. This lack of sample preparation prior to analysis, together with the extremely high throughput of the chromatography, makes the technique extremely attractive to the bioanalyst. Further, the introduction of multiple sprayer interfaces to mass spectrometers provides the potential for even higher throughput. In this paper, we present parallel ultra-high flow rate liquid chromatography using four columns in parallel and a four-way multiple sprayer interface to the mass spectrometer. We have applied this on both the narrow-bore and capillary scale. This technique enables the quantification of drugs from four plasma samples simultaneously, at nanogram per millilitre concentrations, from small aliquots of plasma without sample preparation and with throughputs of up to 120 samples per hour.

Calibration↗
PubMed2025

Characterization of Novel Luteoviruses in Canadian Highbush Blueberries Using High-Throughput Sequencing.

The Fraser Valley of British Columbia, Canada is among the top ten blueberry producing regions globally. Viral diseases are established in the region and significantly reduce average yields. While testing for two viruses is routine, characterization of all the viruses present in the region is incomplete. We used high-throughput sequencing to obtain an unbiased overview of RNA viruses present in 97 plants collected across the region. In addition to known viruses, we identified four luteoviruses previously unidentified in the region. Two of them matched the blueberry virus L (BlVL) and blueberry virus M (BlVM). recently found in the USA, while the third constitutes a new major variant of BlVM (BlVM-2), and the fourth a new luteovirus, which we named blueberry virus N (BlVN). The genome sequences were ~5 kbp long and contained four open-reading frames similar to other luteoviruses. PCR screening revealed that these luteoviruses are widespread in the region, and that plants typically harbour more than one of these luteoviruses. While luteoviruses are typically vectored by aphids, they were also present in nursery stock, indicating that spread also occurs via vegetative propagation.

High-Throughput Nucleotide Sequencing↗
PubMed2005

High-throughput SNP genotyping on universal bead arrays.

We have developed a flexible, accurate and highly multiplexed SNP genotyping assay for high-throughput genetic analysis of large populations on a bead array platform. The novel genotyping system combines high assay conversion rate and data quality with >1500 multiplexing, and Array of Arrays formats. Genotyping assay oligos corresponding to specific SNP sequences are each linked to a unique sequence (address) that can hybridize to its complementary strand on universal arrays. The arrays are made of beads located in microwells of optical fiber bundles (Sentrix Array Matrix) or silicon slides (Sentrix BeadChip). The optical fiber bundles are further organized into a matrix that matches a 96-well microtiter plate. The arrays on the silicon slides are multi-channel pipette compatible for loading multiple samples onto a single silicon slide. These formats allow many samples to be processed in parallel. This genotyping system enables investigators to generate approximately 300,000 genotypes per day with minimal equipment requirements and greater than 1.6 million genotypes per day in a robotics-assisted process. With a streamlined and comprehensive assay, this system brings a new level of flexibility, throughput, and affordability to genetic research.

DNA Methylation↗
PubMed2004

Phagemid encoded small molecules for high throughput screening of chemical libraries.

A new strategy for monovalently displaying small molecules on phage surfaces was developed and applied to high throughput screening for molecules with high binding affinity to the target protein. Peptidyl carrier protein (PCP) excised from nonribosomal peptide synthetase was monovalently displayed on the surface of M13 phage as pIII fusion proteins. Small molecules of diverse structures were conjugated to coenzyme A (CoA) and then covalently attached to the phage displayed PCP by Sfp phosphopantetheinyl transferase. Because Sfp is broadly promiscuous for the transfer of small molecule linked phosphopantetheinyl moieties to apo PCP domains, this approach will enable displaying libraries of small molecules on phage surfaces. Unique 20-base-pair (bp) DNA sequences were also incorporated into the phagemid DNA so that each compound displayed on the phage surface was encoded by a DNA bar code encapsulated inside the phage coat protein. Single round selection of phage displayed small molecules achieved more than 2000-fold enrichment of small molecules with nM binding affinity to the target protein. The selection process is further accelerated by the use of DNA decoding arrays for identifying the selected small molecules.

Bacterial Proteins↗
PubMed2006

High-throughput analysis of catechins and theaflavins by high performance liquid chromatography with diode array detection.

Increased interest in potential health-protective activities of flavonoid-rich tea has created the need to take advantage of HPLC column and system advances in order to optimize methodologies for flavonoid analysis. Two new RP-C18 methods for HPLC-DAD analysis of tea flavonoids were developed to facilitate separation of catechins within 5 min and separation of catechins and theaflavins within 10 min total analysis time. Calibration results indicate that these methods have on-column limits of detection on the order of 1-10 pmol for most tea catechins, and method replication generally resulted in intraday and interday peak area variation of <5% for catechins and <9% for theaflavins in green and black tea infusions. These new methods are therefore sensitive, reproducible, and represent a 2-4-fold reduction in HPLC analysis time from existing analytical methods. These improvements are readily achievable with commonly used HPLC equipment, thus facilitating increased sample throughput and efficiency across a broad range of experimental applications.

Biflavonoids↗