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 1,189 records · Page 66Linked to original sources
PubMed2003

Semi-automatic high-throughput determination of plasma protein binding using a 96-well plate filtrate assembly and fast liquid chromatography-tandem mass spectrometry.

A semi-automatic, high-throughput method has been developed to rapidly assess plasma protein binding of new chemical entities in drug discovery phase. New chemical entities are mixed with plasma and the unbound fractions are separated from the bound fraction by ultrafiltration in a 96-well filtrate assembly. The unbound fractions are then analyzed by fast liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample handling is automated by a robotic system. Employing a cocktail approach where multiple new chemical entities are allowed to bind to plasma proteins in the same well has further increased the throughput. We have validated the method with 12 commercially available compounds. The plasma protein binding data obtained by this method are comparable with the literature values. This method enables the determination of protein binding for 32 compounds in one single experiment instead of 1-2 compounds using the conventional methods.

Blood Proteins↗
PubMed2005

High-throughput screening for enzyme inhibitors using frontal affinity chromatography with liquid chromatography and mass spectrometry.

This work presents new frontal affinity chromatography (FAC) methodologies for high-throughput screening of compound libraries, designed to increase screening rates and improve sensitivity and ruggedness in performance. A FAC column constructed around the enzyme N-acetylglucosaminyltransferase V (GnT-V) was implemented in the identification of potential enzyme inhibitors from two libraries of trisaccharides. Effluent from the FAC column was fractionated, sequentially processed via LC/MS, and referenced to a similar analysis through a control FAC column lacking the enzyme. The resulting multidimensional data sets were compared across corresponding sample and control fractions to identify binders, in a semiautomated approach. A strong binder in the protonated form at m/z 795 was identified from the first library of 81 compounds, exhibiting an estimated Kd value of 0.3 microM. Other binders yielded Kd values ranging from 0.35 to 3.35 microM. To demonstrate the improvement in performance of this FAC-LC/MS approach over the conventional online FAC/MS approach, 15 compounds from this library were blended with a second library of 1000 synthetic trisaccharides and screened against GnT-V. All ligands in the 15-compound set were identified in this larger screen, and no ligands of greater affinity than compound 1 were found. Our results show that FAC-LC/MS is a reliable method for screening large compound libraries directly and useful for large-scale ligand discovery initiatives.

Chromatography, Affinity↗
PubMed2005

Polymer-micelle incarcerated scandium as a polymer-supported catalyst for high-throughput organic synthesis.

A novel immobilization technique for Sc(OTf)3, a polymer-micelle incarcerated (PMI) method, has been developed. PMI Sc(OTf)3 is highly active in several fundamental carbon-carbon bond-forming reactions. The catalyst is recovered quantitatively by simple filtration and reused several times without loss of catalytic activity, and no Sc leaching was observed in all reactions (<0.1 ppm). In addition, several solvents are available, and these aspects are suitable for high-throughput organic synthesis (HTOS). To the best of our knowledge, this is the first example of immobilization of Lewis acids utilizing polymer micelles.

Catalysis↗
PubMed2002

Discovery of a new family of chromium ethylene polymerisation catalysts using high throughput screening methodology.

Following a discovery that salicylaldimines bearing bulky ortho-phenoxy substituents and small imine substituents give very active chromium catalysts for ethylene polymerisation, High Throughput Screening (HTS) methodology has been employed to facilitate a further discovery of exceptionally active catalysts based on tridentate salicylaldimine ligands with bulky triptycenyl groups.

Journal Article↗
PubMed2004

High-throughput screening with quantitation of ATP consumption: a universal non-radioisotope, homogeneous assay for protein kinase.

A number of assays have been developed for high-throughput screening (HTS) of potentially bioactive compounds. To screen millions of chemical compounds efficiently, the best detection technology prior to initiating HTS must be chosen. Ideally, a non-radioisotope (non-RI), homogeneous method, equivalent to the most reliable assay for a particular target, should be selected as an HTS method. Protein kinases are among the most important classes for drug discovery because they participate in various signaling pathways. Several HTS technologies are available for kinase activity: SPA (Amersham, Piscataway, NJ, U.S.A.), HTRF (CIS-US, Inc., Bedford, MA, U.S.A.), IMAP (Molecular Devices, Sunnyvale, CA, U.S.A.), and Z'-LYTE (Invitrogen, Carlsbad, CA, U.S.A.). The amount of phosphorylated product is detected by different methods in these assays. Recently, Kinase-Glo Luminescent Kinase Assay, a non-RI, homogeneous, adenosine triphosphate (ATP) quantitative kit useful for kinase activity detection, has become available from Promega (Madison, WI, U.S.A.). ATP is a universal substrate for kinases. Thus, the Kinase-Glo assay shows promise for becoming the primary method of determining kinase activity in HTS. We have developed a Kinase-Glo system for cyclin-dependent kinase 4 (Cdk4), and compare its results with those of the filtration method, the most reliable assay for in vitro Cdk4 activity. In addition, the reliability and sensitivity of the Kinase-Glo are discussed.

Adenosine Triphosphate↗
PubMed2002

High-throughput imaging of brain gene expression.

Voxelation is a new method for acquisition of three dimensional (3D) gene expression patterns in the brain. It employs high-throughput analysis of spatially registered voxels (cubes) to produce multiple volumetric maps of gene expression analogous to the images reconstructed in biomedical imaging systems. Using microarrays, 24 voxel images of coronal hemisections at the level of the hippocampus of both the normal human brain and Alzheimer's disease brain were acquired for 2000 genes. The analysis revealed a common network of coregulated genes, and allowed identification of putative control regions. In addition, singular value decomposition (SVD), a mathematical method used to provide economical explanations of complex data sets, produced images that distinguished between brain structures, including cortex, caudate, and hippocampus. The results suggest that voxelation will be a useful approach for understanding how the genome constructs the brain.

Adult↗
PubMed2000

Fluorescence polarization and anisotropy in high throughput screening: perspectives and primer.

Fluorescence polarization and anisotropy are two nearly equivalent techniques that have together, over the past 5 years, achieved wide use in high throughput screening in drug discovery. These are single-label methods that can be used to construct homogeneous assays that are fast, sensitive, and resistant to some significant interferences. Moreover, the assays are relatively inexpensive. This review surveys the peer-reviewed literature on the subject and explores some of the fundamental issues that bear on assay performance.

Binding, Competitive↗
PubMed2001

Development of a miniaturized 384-well high throughput screen for the detection of substrates of cytochrome P450 2D6 and 3A4 metabolism.

The identification of a large number of biologically active chemical entities during high throughput screening (HTS) necessitates the incorporation of new strategies to identify compounds with druglike properties early during the lead prioritization and development process. One of the major steps in lead prioritization is the assessment of drug metabolism mediated by the cytochrome P(450) (CYP) enzymes to evaluate the potential drug-drug interactions. CYP2D6 and CYP3A4 comprise the main human CYP enzymes involved in drug metabolism. The recent availability of specific CYP cDNA expression systems and the development of specific fluorescent probes have accelerated the ability to develop robust in vitro assays in HTS format. The aim of this study was to optimize conditions for the CYP2D6 and CYP3A4 HTS assays and subsequently adapt those assays to a miniaturized 384-well format. Assay conversion to a miniaturized format presents certain difficulties, such as robustness of the signal and of compound delivery. Thus the assay optimization involved the comparison of different substrates to identify those most suitable for use in a miniaturized format. Because of current technical limitations in liquid dispensing of nanoliter volumes, assay sensitivity to organic solvents also provides a main concern during assay miniaturization. Therefore, compound activity from redissolved dry films and from DMSO stocks directly delivered into assay buffer was compared. The data indicate that compound activity was comparable in both formats. The data support the conclusion that CYP2D6 and CYP3A4 in vitro metabolism assays can be successfully performed in 384-well plate format and the substrate potencies, as evaluated by the IC(50) values, determined.

Automation↗
PubMed2006

The application of fluorescence lifetime readouts in high-throughput screening.

Measurement of fluorescence lifetime is a well-established technique, which has recently been introduced into the portfolio of assay formats used in high-throughput screening (HTS). This investigation establishes appropriate conditions for using lifetime measurements to reduce the impact of compound interference effects during large-scale HTS of corporate screening files. Experimental data on mixtures of standard fluorophores and interfering compounds (from 5 HTS campaigns) have been combined with a theoretical model to identify the minimum data quality required, defined by the photon count in the peak channel, for discrimination of biological activity. Single-component fluorophore lifetimes can be recovered with an error of 1%, with a peak photon count of 10(2), but the same accuracy with a 2-component decay requires a peak photon count of 10(3). When a 3rd component is introduced, the minimum peak count increases to 10(4). The influence of scattered light on lifetime determination was investigated using an emulsion (diameters 25-675 nm). The measured decays of interfering compounds, identified as autofluorescent, show that the vast majority have a very short lifetime that can readily be resolved from the reporter fluorophore, using appropriate data-fitting methods.

Drug Evaluation, Preclinical↗
PubMed2005

Optimisation Issues of High Throughput Medical Data and Video Streaming Traffic in 3G Wireless Environments.

In this paper we describe some of the optimisation issues relevant to the requirements of high throughput of medical data and video streaming traffic in 3G wireless environments. In particular we present a challenging 3G mobile health care application that requires a demanding 3G medical data throughput. We also describe the 3G QoS requirement of mObile Tele-Echography ultra-Light rObot system (OTELO that is designed to provide seamless 3G connectivity for real-time ultrasound medical video streams and diagnosis from a remote site (robotic and patient station) manipulated by an expert side (specialists) that is controlling the robotic scanning operation and presenting a real-time feedback diagnosis using 3G wireless communication links.

Humans↗
PubMed2003

Beta galactosidase enzyme fragment complementation as a novel technology for high throughput screening.

In this review, the applications of beta galactosidase complementation are described. alpha Complementation is a naturally occurring process in bacteria and in engineered cells, and can also occur in eukaryotic cells. Two forms of alpha complementation have been used in high throughput screening (HTS), in which interacting fragments complement with either low or high affinity. Low affinity complementation is used to monitor protein protein interactions, such as those occurring in homodimerization of the epidermal growth factor receptor (EGFR), and provides a robust screen for detection of EGFR inhibitors. High affinity complementation provides the basis for several HTS assays, in which analytes, such as cAMP or IP(3), are detected in crude cell lysates. A development of the latter approach is protein labeling, providing for measurement of cell protein expression and trafficking. Collectively, the use of beta galactosidase complementation provides a novel and flexible technology for highly sensitive, homogeneous HTS assay development.

Animals↗
PubMed1994

High throughput and economical mutation detection and RFLP analysis using a minimethod for DNA preparation from whole blood and acrylamide gel electrophoresis.

We report a simple, rapid, and high throughput method which allows the simultaneous processing of multiple whole blood samples for routine DNA purification and analysis. The method is based on a microscale DNA preparation and digestion using minimal amounts of reagents and handling. The amount of material necessary for a Southern blot analysis (5-7 micrograms) is obtained from 200 microliters of whole blood. All steps involved in DNA preparation and restriction digestion are processed in a single 1.5-ml Eppendorf tube. DNA preparation is performed using a salting out procedure with a proteinase K digestion step but no phenol/chloroform extraction. Restricted fragments are separated by electrophoresis through polyacrylamide slab gels followed by electrotransfer to nylon membranes. By varying the electrophoresis parameters (V/cm or duration), fragments of interest up to 12 kb length can be separated with high resolution. At least 80 samples can be processed at once per DNA preparation, and multiples of this number depend on the available equipment. This economical and rapid method allows routine DNA analysis for mutation or RFLP detection to be performed on a large scale which is a mandatory feature in any DNA-based population screening program. In addition, the DNA purified by the minimethod can be used as an economical source for PCR analysis.

Blotting, Southern↗
PubMedDate not supplied

High-throughput modeling of human G-protein coupled receptors: amino acid sequence alignment, three-dimensional model building, and receptor library screening.

The current study describes the development of a computer package (GPCRmod) aimed at the high-throughput modeling of the therapeutically important family of human G-protein coupled receptors (GPCRs). GPCRmod first proposes a reliable alignment of the seven transmembrane domains (7 TMs) of most druggable human GPCRs based on pattern/motif recognition for each of the 7 TMs that are considered independently. It then converts the alignment into knowledge-based three-dimensional (3-D) models starting from a set of 3-D backbone templates and two separate rotamer libraries for side chain positioning. The 7 TMs of 277 human GPCRs have been accurately aligned, unambiguously clustered in three different classes (rhodopsin-like, secretin-like, metabotropic glutamate-like), and converted into high-quality 3-D models at a remarkable throughput (ca. 3s/model). A 3-D GPCR target library of 277 receptors has consequently been setup. Its utility for "in silico" inverse screening purpose has been demonstrated by recovering among top scorers the receptor of a selective GPCR antagonist as well as the receptors of a promiscuous antagonist. The current GPCR target library thus constitutes a 3-D database of choice to address as soon as possible the "virtual selectivity" profile of any GPCR antagonist or inverse agonist in an early hit optimization process.

Amino Acid Sequence↗
PubMed2004

Application of high-throughput Fourier-transform infrared spectroscopy in toxicology studies: contribution to a study on the development of an animal model for idiosyncratic toxicity.

An evaluation of high-throughput Fourier-transform infrared spectroscopy (FT-IR) as a technology that could support a "metabonomics" component in toxicological studies of drug candidates is presented. The hypothesis tested in this study was that FT-IR had sufficient resolving power to discriminate between urine collected from control rat populations and rats subjected to treatment with a potent inflammatory agent, bacterial lipopolysaccharide (LPS). It was also hypothesized that co-administration of LPS with ranitidine, a drug associated with reports of idiosyncratic susceptibility, would induce hepatotoxicity in rats and that this could be detected non-invasively by an FT-IR-based metabonomics approach. The co-administration of LPS with "idiosyncratic" drugs represents an attempt to develop a predictive model of idiosyncratic toxicity and FT-IR is used herein to support characterization of this model. FT-IR spectra are high dimensional and the use of genetic programming to identify spectral sub-regions that most contribute to discrimination is demonstrated. FT-IR is rapid, reagentless, highly reproducible and inexpensive. Results from this pilot study indicate it could be extended to routine applications in toxicology and to supporting characterization of a new animal model for idiosyncratic susceptibility.

Animals↗
PubMed2002

High throughput genotyping technologies.

A comprehensive genetic map containing several hundred microsatellite markers resulted from a large microsatellite mapping project. This was the first real study that introduced high throughput methods to the genetic community. This map and the concurrent technological advances, which will briefly be reviewed, led to further numerous mapping investigations of simple and complex diseases. The annotated draft sequence of approximately three billion base pairs (bp) of the human genome has been completed much sooner than many imagined, due to considerable technological advancements and the international enterprise that resulted. This was a major development for the genetics community, but is only the precursor to the next phase of studying and understanding the variation within the human genome. The awareness of the differences may help us understand the effects on the genetics of the variation between individuals and disease. It is these variations at the nucleotide level that determine the physiological differences, or phenotypes of each individual, including all biological functions at the cellular and body level. Single nucleotide polymorphisms (SNPs) will provide the next high density map, and be the genetic tool to study these genetic variations. There are many sources of SNPs and exhaustive numbers of methods of SNP detection to be considered. The focus in this paper will be on the merits of selected, varied SNP typing methodologies that are emerging to genotype many individuals with the required huge number of SNPs to make the study of complex diseases and pharmacogenomics a practical and economically viable option.

Genotype↗
PubMed2000

High-throughput radioassays for autoantibodies to recombinant autoantigens.

Advances in immunology, biochemistry, and molecular biology have combined to allow the development of a large series of autoantibody assays utilizing recombinantly produced autoantigens. Labeled target proteins can be readily produced by in vitro transcription and translation of relevant cloned cDNA. The assays are carried out in the fluid phase and for most assays are more specific and sensitive than ELISA based assays. For some antigens (e.g. insulin) though ELISA assays detect antibodies following immunization, workshops indicate they are almost worthless for the diagnosis and prediction of type 1A diabetes. This new generation of radioassays is usually carried out in 96-well microtiter filtration plates that allow high throughput. Given such assays, individuals at high risk for type 1A diabetes, celiac disease, and Addison's disease can now be readily identified.

Addison Disease↗
PubMed2006

A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening.

Hydroxynitrile lyases (Hnls) are important biocatalysts for the synthesis of optically pure cyanohydrins, which are used as precursors and building blocks for a wide range of high price fine chemicals. Although two Hnl enzymes, from the tropical rubber tree Hevea brasiliensis and from the almond tree Prunus amygdalus, are already used for large scale industrial applications, the enzymes still need to be improved and adapted to the special demands of industrial processes. In many cases directed evolution has been the method of choice to improve enzymes, which are applied as industrial biocatalysts. The screening procedure is the most crucial point in every directed evolution experiment. Herein, we describe the successful development of a novel screening assay for Hnls and its application in high-throughput screening of Escherichia coli mutant libraries. The new assay allows rapid screening of mutant libraries and facilitates the discovery of improved enzyme variants. Hnls catalyze the cleavage of cyanohydrins to hydrocyanic acid and the corresponding aldehyde or ketone. The enzyme assay is based on the detection of hydrocyanic acid produced, making it an all-purpose screening assay, without restriction to any kind of substrate. The gaseous HCN liberated within the Hnl reaction is detected by a visible colorimetric reaction. The facile, highly sensitive and reproducible screening method was validated by identifying new enzyme variants with novel substrate specificities.

Aldehyde-Lyases↗
PubMed2002

Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions.

We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS). By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, a single fluorophore can be efficiently incorporated into a protein at the carboxyl terminus during in vitro translation. We confirmed that the resulting fluorescently labeled proteins are useful for probing protein-protein and protein-DNA interactions by means of pulldown assay, DNA microarrays, and FCCS in model experiments. These fluorescence assay systems can be easily extended to highly parallel analysis of protein interactions in studies of functional genomics.

DNA-Binding Proteins↗