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At least 235 records · Page 13Linked to original sources
PubMed2006

Cellular effect of high doses of silica-coated quantum dot profiled with high throughput gene expression analysis and high content cellomics measurements.

Quantum dots (Qdots) are now used extensively for labeling in biomedical research, and this use is predicted to grow because of their many advantages over alternative labeling methods. Uncoated Qdots made of core/shell CdSe/ZnS are toxic to cells because of the release of Cd2+ ions into the cellular environment. This problem has been partially overcome by coating Qdots with polymers, poly(ethylene glycol) (PEG), or other inert molecules. The most promising coating to date, for reducing toxicity, appears to be PEG. When PEG-coated silanized Qdots (PEG-silane-Qdots) are used to treat cells, toxicity is not observed, even at dosages above 10-20 nM, a concentration inducing death when cells are treated with polymer or mercaptoacid coated Qdots. Because of the importance of Qdots in current and future biomedical and clinical applications, we believe it is essential to more completely understand and verify this negative global response from cells treated with PEG-silane-Qdots. Consequently, we examined the molecular and cellular response of cells treated with two different dosages of PEG-silane-Qdots. Human fibroblasts were exposed to 8 and 80 nM of these Qdots, and both phenotypic as well as whole genome expression measurements were made. PEG-silane-Qdots did not induce any statistically significant cell cycle changes and minimal apoptosis/necrosis in lung fibroblasts (IMR-90) as measured by high content image analysis, regardless of the treatment dosage. A slight increase in apoptosis/necrosis was observed in treated human skin fibroblasts (HSF-42) at both the low and the high dosages. We performed genome-wide expression array analysis of HSF-42 exposed to doses 8 and 80 nM to link the global cell response to a molecular and genetic phenotype. We used a gene array containing approximately 22,000 total probe sets, containing 18,400 probe sets from known genes. Only approximately 50 genes (approximately 0.2% of all the genes tested) exhibited a statistically significant change in expression level of greater than 2-fold. Genes activated in treated cells included those involved in carbohydrate binding, intracellular vesicle formation, and cellular response to stress. Conversely, PEG-silane-Qdots induce a down-regulation of genes involved in controlling the M-phase progression of mitosis, spindle formation, and cytokinesis. Promoter analysis of these results reveals that expression changes may be attributed to the down-regulation of FOXM and BHLB2 transcription factors. Remarkably, PEG-silane-Qdots, unlike carbon nanotubes, do not activate genes indicative of a strong immune and inflammatory response or heavy-metal-related toxicity. The experimental evidence shows that CdSe/ZnS Qdots, if appropriately protected, induce negligible toxicity to the model cell system studied here, even when exposed to high dosages. This study indicates that PEG-coated silanized Qdots pose minimal impact to cells and are a very promising alternative to uncoated Qdots.

Cell Line↗
PubMed2006

First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value.

The EC 'Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, approximately 170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples.

Animals↗
PubMed2004

High throughput determination of gains and losses of genetic material using high resolution BAC arrays and comparative genomic hybridization.

Chromosome analysis has been a cornerstone both for the identification of genetic defects that predispose to a variety of genetic syndromes as well as for the analysis of cancer progression. The relatively low resolution of metaphase chromosomes, however, only allows characterization of major genetic events which are defined at the megabase level. The development of the human genome-wide bacterial artificial chromosome (BACs) libraries which were used as templates for the human genome project made it possible to design microarrays containing these BACs which can theoretically span the genome uninterrupted. Comparative genomic hybridization to these arrays using test and reference DNA samples reveals numerical chromosome abnormalities (deletions, gains and amplifications) which can be accurately defined with a resolution depending on the density of the arrays. Analysis of test DNA samples using these arrays reveals low level deletions and amplifications that cannot be detected by chromosome analysis and provides a global view of these genetic changes in a single overnight hybridization using a high throughput approach. The extent of the genetic changes can then be determined precisely and the gene content of the affected regions established. These BAC arrays have widespread application to the analysis of constitutional genetic abnormalities associated with human diseases as well as cancer patients and their tumors. The development of similar BAC arrays for the mouse genome means that it is now possible to extend the CGHa approach to the study of genetic disorders and cancer models in mice.

Animals↗
PubMed2003

TIP, a T-cell factor identified using high-throughput screening increases survival in a graft-versus-host disease model.

A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins.

Adjuvants, Immunologic↗
PubMed2006

Complex functionality of gene groups identified from high-throughput data.

Relating experimental data to biological knowledge is necessary to cope with the avalanches of new data emerging from recent developments in high-throughput technologies. Automatic functional profiling becomes the de facto standard approach for the secondary analysis of high-throughput data. A number of tools employing available gene functional annotations have been developed for this purpose. However, current annotations are derived mostly from traditional analysis of the individual gene function. The complex biological phenomena carried out by the concerted activity of many genes often requires the definition of new complex functionality (related to a group of genes), which is, in many cases, not available in current annotation vocabularies. Functional profiling with annotation terms related to the description of individual biological functions of a gene may fail to provide reasonable interpretation of biological relationships in a set of genes involved in complex biological phenomena. We introduce a novel procedure to profile a complex functionality of a gene set. Complex functionality is constructed as a combination of available annotation terms. By profiling ChIP-chip data from Saccharomyces cerevisiae we demonstrate that this technique produces deeper insights into the results of high-throughput experiments that are beyond the known facts described in the functional classifications.

Animals↗
PubMed2005

A novel HIV-1 antiviral high throughput screening approach for the discovery of HIV-1 inhibitors.

Antiviral high throughput screens remain a viable option for identifying novel target inhibitors. However, few antiviral screens have been reduced to practice on an industrial scale. In this study, we describe an HIV-1 dual reporter assay that allows for the simultaneous evaluation of the potential antiviral activities and cytotoxicities of compounds in a high throughput screen (HTS) format. We validate the assay with known HIV-1 inhibitors and show that the antiviral and cytotoxic activities of compounds are reproducibly measured under screening conditions. In addition, we show that the assay exhibits parameters (e.g., signal-to-background ratios and Z' coefficients) suitable for high throughout screening. In a pilot screen, we demonstrate that non-specific or cytotoxic compounds represent a significant fraction of the hits identified in an antiviral screen and that these false positives are identified and deprioritized by the HIV-1 dual reporter assay at the primary screening step. We propose that the HIV-1 dual reporter assay represents a novel approach to HIV-1 antiviral screening that allows for the effective execution of industrial scale HTS campaigns with significantly greater returns on resource investment when compared to previous methods.

Animals↗
PubMed2006

Development of a simple high-throughput screening protocol based on biosynthetic activity of Mycobacterium tuberculosis glutamine synthetase for the identification of novel Inhibitors.

A high-throughput screening protocol has been developed for Mycobacterium tuberculosis glutamine synthetase by quantitative estimation of inorganic phosphate. The K(m) values determined at pH 6.8 are 22 mM for L-glutamic acid, 0.75 mM for NH(4)Cl, 3.25 mM for MgCl(2), and 2.5 mM for adenosine triphosphate. The K(m) value for glutamine is affected significantly by the increase in pH of assay buffer. At the saturating level of the substrate, the enzyme activity at pH 6.8 and 25 degrees C is found to be linear up to 3 h. The reduction of enzyme activity is negligible even in presence of 10% DMSO. The Z' factor and signal-to-noise ratio are found to be 0.75 and 6.18, respectively, when the enzyme is used at 62.5 microg/ml concentration. The IC(50) values obtained at pH 6.8 for both L-methionine S-sulfoximine and DL-phosphothriacin are 500 microM and 30 microM, respectively, which is lowest compared to the values obtained at other pH levels. The Beckman Coulter high-throughput screening platform was found to take 5 h 9 min to complete the screening of 60 plates. For each assay plate, a replica plate is used to normalize the data. Screening of 1164 natural product fractions/extracts and synthetic molecules from an in-house library was able to identify 12 samples as confirmed hits. Altogether, the validation data from screening of a small set of an in-house library coupled with Z' and signal-to-noise values indicate that the protocol is robust for high-throughput screening of a diverse chemical library.

Combinatorial Chemistry Techniques↗
PubMed2006

Establishment and application of a high throughput model for Rho kinase inhibitors screening based on fluorescence polarization.

Rho kinase (ROCK) inhibitors are effective candidates for neural or cardiovascular disorders. High throughput model for screening ROCK inhibitors is a basic foundation to pick up ROCK inhibitors from thousands of compounds for drug developing. The high throughput model was established based on purified recombinant rat ROCK catalytic domain (rROCK-CD) from Escherichia coli (E. coli). There are two steps of reaction in the model: incubation of 5.0 microl recombinant rROCK-CD (2.0 microg/ml), 5.0 microl different compounds, 5.0 microl fluorescent S6-peptide (200 nM), and 5.0 microl ATP (10 microM) at 37 degrees C for 60 min was made the first reaction, and the second reaction was made by incubating them with additional 60 microl binding reagent at ambient temperature for 30 min. The phosphorylated S6 peptide can bind to a binding reagent, and the fluorescence varies from low polarization to high according to the amount of the phosphorylated peptide. IC50 was calculated based on polarization variation. Compound, which IC50 was less than 10 microM, was recognized as a lead compound which taken bioactivity evaluation in PC12 by observing neurite outgrowth. The Z'-factor of the model is 0.81 (above 0.5). The model screened five lead compounds from 3294, which promoted neurite outgrowth to different extent. The results suggested that the model is suitable for high throughput screening (HTS), and the five lead compounds are worth of further investigation.

Animals↗
PubMed2004

SpeedScreen: label-free liquid chromatography-mass spectrometry-based high-throughput screening for the discovery of orphan protein ligands.

A high-throughput screening methodology tailored to the discovery of ligands for known and orphan proteins is presented. With this method, labeling of neither target protein nor screened compounds is required, as the ligands are affinity selected by incubation of the protein with mixtures of compounds in aqueous binding buffer. Unbound small-molecular-weight compounds are removed from the target protein:ligand complex by rapid size-exclusion chromatography in the 96-well format. The protein fraction is analyzed subsequently by liquid chromatography-mass spectrometry for detection and identification of the bound ligand. This screening method was validated with known protein:ligand model systems and optimized for selection of high-affinity binders in an industrial screening environment. All sample handling steps and the analytics are rapid, robust, and largely automated, adopting this technology to the needs of present high-throughput screening processes. This affinity-selection technology, termed SpeedScreen, is currently an integral part of our lead discovery process.

Chromatography, High Pressure Liquid↗
PubMed2000

High-throughput quantitative bioanalysis by LC/MS/MS.

This review article discusses the most recent significant advances in the sample preparation and mass spectrometry aspects of high-throughput bioanalysis by LC/MS/MS for the quantitation of drugs, metabolites and endogenous biomolecules in biological matrices. The introduction and implementation of automated 96-well extraction has brought about high-throughput approaches to the biological sample preparation techniques of solid-phase extraction, liquid-liquid extraction and protein precipitation. The fast-flow on-line extraction technique is a different high-throughput approach that has also significantly speeded up analysis by LC/MS/MS. The use of pierceable caps for biological tubes further enhances the analysis speed and improves the safety in handling biological samples. The need for adequate chromatographic separation in order to eliminate interferences due to metabolites and/or matrix effects in LC/MS/MS is discussed. To highlight our limited understanding of atmospheric pressure ionization mass spectrometry, results from recent investigations that appear to be counter-intuitive are presented. Looking ahead to the future, multiplexed LC/MS/MS systems and capillary LC are presented as areas that can bring about further improvements in analysis speed and sensitivity to quantitative bioanalysis by LC/MS/MS.

Acetonitriles↗
PubMed2006

An efficient and high-throughput approach for experimental validation of novel human gene predictions.

A highly automated RT-PCR-based approach has been established to validate novel human gene predictions with no prior experimental evidence of mRNA splicing (ab initio predictions). Ab initio gene predictions were selected for high-throughput validation using predicted protein classification, sequence similarity to other genomes, colocalization with an MPSS tag, or microarray expression. Initial microarray prioritization followed by RT-PCR validation was the most efficient combination, resulting in approximately 35% of the ab initio predictions being validated by RT-PCR. Of the 7252 novel genes that were prioritized and processed, 796 constituted real transcripts. In addition, high-throughput RACE successfully extended the 5' and/or 3' ends of >60% of RT-PCR-validated genes. Reevaluation of these transcripts produced 574 novel transcripts using RefSeq as a reference. RT-PCR sequencing in combination with RACE on ab initio gene predictions could be used to define the transcriptome across all species.

Algorithms↗
PubMed2005

A high-throughput, low-volume enzyme assay on solid support.

A high-throughput enzyme assay is described that uses 1 microL or less of enzyme solution for each test. Enzyme solutions are deposited by robotic handling in a throughput of over 1000 tests/h on the surface of silica gel plates that have been preimpregnated with fluorogenic substrates. The reaction is quantitated by fluorescence. The method is compatible with water-insoluble substrates (lipases), water-soluble substrates (glycosidases), whole-protein substrates (proteases), and enzyme inhibition measurements. Hydrolytically labile umbelliferyl esters can be used to assay lipases in this format without background hydrolysis. High throughput and reproducibility were tested by fingerprint analysis of lipases and esterases against 37 different fluorogenic ester substrates. A set of eight fluorogenic unbelliferyl esters was selected for optimal activity screening of lipases and esterases on silica gel plates.

Autoanalysis↗
PubMed2006

High throughput metabolic stability screen for lead optimization in drug discovery.

A high throughput approach for the determination of in vitro metabolic stability and metabolic profiles of drug candidates has been developed. This approach comprises the combination of a Biomek FX liquid handling system with 96-channel pipetting capability and a custom-designed 96-well format on-line incubator with efficient thermal conductivity. This combination facilitates automated reagent preparation, sample incubation, and sample purification for microsome stability studies. The overall process is both fast and accurate and meets the challenges of high throughput screening for drug discovery. A custom designed, user-friendly computer program has been incorporated for large-scale data processing and report generation. Several applications are discussed that implement this strategy for rapid selection of compounds in early drug discovery.

Animals↗
PubMedDate not supplied

High-throughput DNA preparation system.

A system demonstrating the feasibility of high-throughput, centrifugation-based DNA separations and purifications has been constructed and tested. Samples are currently processed at a rate of 96 in approximately 2-3 h. The device implements an automation-optimized alkaline lysis protocol for the rapid extraction of plasmid or cosmid DNA from 1-ml bacteria cultures. The conditions for optimal culturing in deep-well (96 x 1 ml) microwell plates have been developed, and all sample manipulations are done within these plates. The use of microwell plates was essential to obtain high throughput and make manipulations following the DNA preparation (prep) easier because they can then be manipulated using a variety of commercially available robots. The entire prep system is constructed above a Beckman GPR centrifuge and operated under Macintosh IIcx control. This device has systems for fluid handling, microwell-plate manipulations, and centrifuge rotor alignment.

Automation↗
PubMedDate not supplied

High-throughput manual parallel synthesis using SynPhase crowns and lanterns.

The high-throughput manual solid-phase parallel synthesis of libraries comprising thousands of discrete samples using pellicular supports (i.e. SynPhase crowns and lanterns) and a suite of novel tools and techniques is described. Key aspects of this approach include the combination of a split-split-split synthesis strategy with spatial encoding to differentiate thousands of crowns, the rapid washing and filtration of up to 48 reaction vessels in parallel, the application of an inexpensive and environmentally friendly technique to remove trifluoroacetic acid from sixteen 96-well plates in parallel, and a high-throughput method for removing cleaved crowns from reusable pin racks. Tens of thousands of discrete samples have been produced in-house using this conceptually and operationally straightforward strategy.

Journal Article↗
PubMed2002

High-throughput receptor-binding methods for somatostatin receptor 2.

Three high-throughput screening methods for quantitating 125I-SS14 binding to human somatostatin receptor 2 (hSST2) have been developed. Microplate-based separation assays were performed in Packard Unifilter and Millipore Multiscreen plates. A homogeneous ligand-binding assay was developed by employing wheat germ agglutinin (WGA)-coated Flashplates. Apparent dissociation constants for 125I-SS14 binding to hSST2 were obtained with each method. IC(50) values were determined for 12 compounds using each of the methods. Similar IC(50) values were obtained for each compound with all of the methods. The WGA-Flashplate is suitable for fully automated high-throughput screening whereas the Unifilter and Multiscreen methods are more suitable for semiautomated and manual screening applications.

Animals↗
PubMed2006

A novel assay for monoacylglycerol hydrolysis suitable for high-throughput screening.

A simple assay for monoacylglycerol hydrolysis suitable for high-throughput screening is described. The assay uses [(3)H]2-oleoylglycerol as substrate, with the tritium label in the glycerol part of the molecule and the use of phenyl sepharose gel to separate the hydrolyzed product ([(3)H]glycerol) from substrate. Using cytosolic fractions derived from rat cerebella as a source of hydrolytic activity, the assay gives the appropriate pH profile and sensitivity to inhibition with compounds known to inhibit hydrolysis of this substrate. The assay could also be adapted to a 96-well plate format, using C6 cells as the source of hydrolytic activity. Thus the assay is simple and appropriate for high-throughput screening of inhibitors of monoacylglycerol hydrolysis.

Animals↗
PubMed2005

Desorption electrospray ionization mass spectrometry for high-throughput analysis of pharmaceutical samples in the ambient environment.

Desorption electrospray ionization (DESI) allows mass spectrometry to be used for on-line high-throughput monitoring of pharmaceutical samples in the ambient environment, without prior sample preparation. Positive and negative ion DESI are used to characterize the active ingredients in pharmaceutical samples formulated as tablets, ointments, and liquids. Compounds of a wide variety of chemical types are detected in these complex matrices. The effects on analytical performance of operating parameters, including the electrospray high voltage, heated capillary temperature, solvent infusion rate, and solvent composition, are evaluated and optimized. In addition to experiments in which a simple solvent is sprayed onto the solid analyte samples, reactive desorption is performed by adding reagents to the solvent spray to generate particularly stable or characteristic ions with the analytes of interest. A variable-speed moving belt was built for high-throughput sampling and used to provide rapid qualitative and semiquantitative information on drug constituents in tablets. Sampling rates as high as 3 samples/s are achieved in the ambient environment. Relative standard deviations of the relative ion abundances for major components in the mass spectra are in the range of 2-8%. Impurities and components present at levels as low as approximately 0.1% are identified and carryover effects are minimized in high-throughput on-line analysis of pharmaceutical samples.

Drug Combinations↗