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Results for “high throughput”

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At least 253 records · Page 14Linked to original sources
PubMed2005

[Establishment of high-throughput drug screening model targeting retinoic acid receptor in cells].

AIM: To screen new drug for the treatment of acute promyelocytic leukemia, psoriasis and acne, high-throughput drug screening cell models marked by green fluorescent protein (GFP) have been established. METHODS: Eight repeats of retinoic acid response element (RARE) were synthesized and cloned into a GFP expression vector. This construct was stably transfected into cells in vitro. Stable and sensitive cell clones with high copy numbers of RARE were selected by retinoic acid (RA) using fluorescence-activated cell sorting (FACS). RESULTS: A cell line has been chosen to be high-throughput drug screening cell model. This model was shown with low background, high sensitive and good reproducibility, and was convenient and inexpensive. CONCLUSION: This drug screening cell model can be used for retinoic acid receptor target high-throughput drug screening.

Antineoplastic Agents↗
PubMed2004

Chemoenzymatic synthesis and high-throughput screening of an aminoglycoside-polyamine library: identification of high-affinity displacers and DNA-binding ligands.

Chemoenzymatic parallel synthesis and high-throughput screening were employed to develop a multivalent aminoglycoside-polyamine library for use as high-affinity cation-exchange displacers and DNA-binding ligands. Regioselective lipase-catalyzed acylation, followed by chemical aminolysis, was used to generate vinyl carbonate and vinyl carbamate linkers, respectively, of the aminoglycosidic cores. These were further derivatized with polyamines, leading to library generation. A parallel batch-displacement assay was employed to identify the efficacy of the library candidates as potential displacers for protein purification. Using this approach, low-molecular-mass displacers with affinities higher than those previously observed have been identified. The aminoglycoside-polyamine library was also screened for DNA binding efficacy using an ethidium bromide displacement assay. These highly cationic molecules exhibited strong DNA-binding properties and may have potential for enhanced gene delivery.

Aminoglycosides↗
PubMed2004

Integrating high-throughput and computational data elucidates bacterial networks.

The flood of high-throughput biological data has led to the expectation that computational (or in silico) models can be used to direct biological discovery, enabling biologists to reconcile heterogeneous data types, find inconsistencies and systematically generate hypotheses. Such a process is fundamentally iterative, where each iteration involves making model predictions, obtaining experimental data, reconciling the predicted outcomes with experimental ones, and using discrepancies to update the in silico model. Here we have reconstructed, on the basis of information derived from literature and databases, the first integrated genome-scale computational model of a transcriptional regulatory and metabolic network. The model accounts for 1,010 genes in Escherichia coli, including 104 regulatory genes whose products together with other stimuli regulate the expression of 479 of the 906 genes in the reconstructed metabolic network. This model is able not only to predict the outcomes of high-throughput growth phenotyping and gene expression experiments, but also to indicate knowledge gaps and identify previously unknown components and interactions in the regulatory and metabolic networks. We find that a systems biology approach that combines genome-scale experimentation and computation can systematically generate hypotheses on the basis of disparate data sources.

Aerobiosis↗
PubMed2005

High-throughput MS-based protein phenotyping: application to haptoglobin.

A high-throughput affinity capture and reduction approach was developed for phenotype and post-translational modification analysis of a complexed globular protein, haptoglobin (Hp), directly from human plasma. Hp was selectively retrieved utilizing anti-Hp antibodies immobilized onto affinity pipette tips, eluted onto a formatted mass spectrometer target for reduction of Hp alpha-chains (Hpalpha1 and Hpalpha2) and subjected to subsequent MALDI-MS analysis. The affinity capture and reduction approach was originally developed from a pre-extraction reduction methodology that was optimized to an affinity capture post-reduction technique for intact Hp alpha-chain variant analysis, phenotype classification and ensuing post-translational variant detection. Three common Hp phenotypes (1-1, 2-1 and 2-2) were assigned according to detection of Hpalpha1 and/or Hpalpha2 reduced intact chain(s) average mass(es). The affinity capture post-reduction approach was scaled for high-throughput Hp alpha-chain phenotype analysis from a normal plasma cohort. The entire sample cohort was successfully analyzed and phenotyped using the developed approach. Additionally, Hp post-translational variants were detected and assigned via accurate MS analyses. The results of this study suggest use of the methodology in future analyses of other similarly complexed proteins and in normal versus disease cohort population proteomics studies.

Haptoglobins↗
PubMed2002

High throughput screening of transdermal formulations.

PURPOSE: Applications of transdermal drug delivery are limited by low skin permeability. Many chemicals have been used to enhance skin permeability, however, only a handful are actually used in practice. Combinations of chemicals are likely to be more efficient in enhancing skin permeability compared to individual enhancers. However, identification of efficient enhancer combinations is quite challenging because many chemical enhancers interact with each other and with the skin in a complex manner. In the absence of a fundamental knowledge of such interactions, we need to rely on rapid methods to screen various enhancer combinations for their effectiveness. In this paper, we report a novel high throughput (HTP) method that is at least 50-fold more efficient in terms of skin utilization and up to 30-fold more efficient in terms of holdup times than the current methods for formulation screening (Franz diffusion cells). METHODS: A high throughput method was developed based on skin conductivity and mannitol penetration into the skin. This method was used to perform at least 100 simultaneous tests per day. Detailed studies were performed using two model enhancers, sodium lauryl sulfate (SLS) and dodecyl pyridinium chloride (DPC). The predictions of the high throughput method were validated using Franz diffusion cells. RESULT: High throughput screening revealed that mixtures of SLS and DPC are significantly more effective in enhancing transdermal transport compared to each of them alone. Maximum efficiency was observed with near-equimolar mixtures of SLS: DPC. The predictions of the HTP method compared well against those made using Franz diffusion cells. Specifically, the effect of surfactant mixtures on skin conductivity and mannitol permeability measured using Franz cells also showed a maximum at near-equimolar mixtures of SLS: DPC. CONCLUSIONS: The novel HTP method allows rapid screening of enhancer formulations for transdermal applications. This method can be used to discover new and effective enhancer mixtures. At the same time, these data may also broaden our understanding of the effect of enhancers on skin permeability.

Adjuvants, Pharmaceutic↗
PubMed2003

Evaluation of a high throughput toxicity biosensor and comparison with a Daphnia magna bioassay.

A high throughput toxicity biosensor has been designed and constructed using recombinant Escherichia coli cells, containing stress specific promoters (recA, fabA, or katG) or constitutive promoters (lac) fused to luciferase genes originating from Vibrio fisheri. These genetically engineered cells were immobilized in 96 well plates. By optimizing cell immobilization conditions and the strains' response specificity to toxic chemicals, bioluminescent outputs decreased or increased dose-dependently upon adding test chemicals. However, to date the toxicity data obtained using this biosensor have not been compared with the results of other toxicity tests. Phenolics were chosen to evaluate the correlation between the LD50 and the EC50 (GC2) or EC120 (DPD2540) of Daphnia magna and E. coli, respectively. Toxicity data obtained from constitutive strains by bioluminescent level decrements were compared with the results from D. magna as a standard. LD50 values were used as parameters of D. magna toxicity and EC50 of EC120 values were used for the immobilized biosensor. In the DPD2540 test, phenolics, membrane damaging toxic chemicals, for testing immobilized stress specific bacterial strains trigger dose-dependant bioluminescence increase within specific concentration. Although the stress specific responsiveness from the strains could not be compared with D. magna's LD50 values, these responses offer additional information, such as upon the mode of toxic action in the sample, in addition to the cellular toxicity results as indicated by the EC50. This novel high throughput toxicity biosensor can be implemented to investigate the toxicity of any other soluble materials, and can be used as a standardization tool for the evaluation of toxicity.

Animals↗
PubMed2005

High-throughput assays for promiscuous inhibitors.

High-throughput screening (HTS) searches large libraries of chemical compounds for those that can modulate the activity of a particular biological target; it is the dominant technique used in early-stage drug discovery. A key problem in HTS is the prevalence of nonspecific or 'promiscuous' inhibitors. These molecules have peculiar properties, act on unrelated targets and can dominate the results from screening campaigns. Several explanations have been proposed to account for promiscuous inhibitors, including chemical reactivity, interference in assay read-out, high molecular flexibility and hydrophobicity. The diversity of these models reflects the apparently unrelated molecules whose behaviors they seek to explain. However, a single mechanism may explain the effects of many promiscuous inhibitors: some organic molecules form large colloid-like aggregates that sequester and thereby inhibit enzymes. Hits from HTS, leads for drug discovery and even several drugs appear to act through this mechanism at micromolar concentrations. Here, we report two rapid assays for detecting promiscuous aggregates that we tested against 1,030 'drug-like' molecules. The results from these assays were used to test two preliminary computational models of this phenomenon and as benchmarks to develop new models.

Biological Assay↗
PubMed1999

New technique for high-throughput synthesis.

A new technique for high throughput solid phase synthesis using the centrifuge based liquid removal from readily available standard microtiterplates is described. This technique eliminates the filtration step and is therefore applicable to simultaneous processing of an unlimited number of reaction compartments. Its application is illustrated on the synthesis of an array of 380 tetrahydroisoquinolinones.

Centrifugation↗
PubMed2003

A fluorescence-based high-throughput assay for antimicrotubule drugs.

With the advent of combinatorial chemistry and the extensive libraries of potential drugs produced from it, there is a growing need for rapid sensitive, high-throughput screening for drug potency. Microtubules are important targets for anticancer agents, and new antimicrotubule compounds are of continued interest in drug development. The in vitro potency of antimicrotubule drugs may be evaluated by measuring the extent of tubulin assembly. The extent of polymerization is proportional to the turbidity of the solution, which usually has been measured as apparent absorption. The turbidity method has inherent problems that hinder its adaptation to a high-throughput format, such as a requirement for high protein concentrations and a high coefficient of variation. We present here a high-throughput assay for antimicrotubule activity in which fluorescence is used to monitor microtubule assembly. Both assembly-inhibiting and assembly-promoting compounds can be evaluated. The assay is rapid and easy to perform, and the data are reliable, with good accuracy and reproducibility.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Column switching in high-performance liquid chromatography with tandem mass spectrometric detection for high-throughput preclinical pharmacokinetic studies.

A high-throughput liquid chromatography-tandem mass spectrometry method is described for the determination of multiple compounds in dog and rat plasma. After acetonitrile precipitation of plasma proteins, the analytes are pre-concentrated and back-flushed on a reversed-phase column for separation using a switching valve. The analytes are ionized using TurboIon Spray in a positive mode, and detected by multiple reaction monitoring. Automatic tuning software is used for fast method development. The data processing is greatly speeded up by using a powerful quantitation software package. Chromatography of multiple compounds takes only 4 min. The linear calibration curve ranges from 0.5 to 1000 ng/ml. This method was successfully used in the analysis of multi-compounds for preclinical pharmacokinetic studies.

Animals↗
PubMed2002

Comparison of assay technologies for a tyrosine kinase assay generates different results in high throughput screening.

In today's high-throughput screening (HTS) environment, an increasing number of assay detection technologies are routinely utilized in lead finding programs. Because of the relatively broad applicability of several of these technologies, one is often faced with a choice of which technology to utilize for a specific assay. The aim of this study was to address the question of whether the same compounds would be identified from screening a set of samples in three different versions of an HTS assay. Here, three different versions of a tyrosine kinase assay were established using scintillation proximity assay (SPA), homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET), and fluorescence polarization (FP) technologies. In this study, 30,000 compounds were evaluated in each version of the kinase assay in primary screening, deconvolution, and dose-response experiments. From this effort, there was only a small degree of overlap of active compounds identified subsequent to the deconvolution experiment. When all active compounds were then profiled in all three assays, 100 and 101 active compounds were identified in the HTR-FRET and FP assays, respectively. In contrast, 40 compounds were identified in the SPA version of the kinase assay, whereas all of these compounds were detected in the HTR-FRET assay only 35 were active in the FP assay. Although there was good correlation between the IC(50) values obtained in the HTR-FRET and FP assays, poor correlations were obtained with the IC(50) values obtained in the SPA assay. These findings suggest that significant differences can be observed from HTS depending on the assay technology that is utilized, particularly in assays with high hit rates.

Biological Assay↗
PubMed2006

SPINE bioinformatics and data-management aspects of high-throughput structural biology.

SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high-throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high-throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high-throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited.

Computational Biology↗
PubMed2003

Tracking chemical kinetics in high-throughput systems.

Combinatorial chemistry and high-throughput experimentation (HTE) have revolutionized the pharmaceutical industry-but can chemists truly repeat this success in the fields of catalysis and materials science? We propose to bridge the traditional "discovery" and "optimization" stages in HTE by enabling parallel kinetic analysis of an array of chemical reactions. We present here the theoretical basis to extract concentration profiles from reaction arrays and derive the optimal criteria to follow (pseudo)first-order reactions in time in parallel systems. We use the information vector f and introduce in this context the information gain ratio, chi(r), to quantify the amount of useful information that can be obtained by measuring the extent of a specified reaction r in the array at any given time. Our method is general and independent of the analysis technique, but it is more effective if the analysis is performed on-line. The feasibility of this new approach is demonstrated in the fast kinetic analysis of the carbon-sulfur coupling between 3-chlorophenylhydrazonopropane dinitrile and beta-mercaptoethanol. The theory agrees well with the results obtained from 31 repeated C-S coupling experiments.

Journal Article↗
PubMed2006

Strategies for high-throughput gene cloning and expression.

High-throughput approaches for gene cloning and expression require the development of new, nonstandard tools for use by molecular biologists and biochemists. We have developed and implemented a series of methods that enable the production of expression constructs in 96-well plate format. A screening process is described that facilitates the identification of bacterial clones expressing soluble protein. Application of the solubility screen then provides a plate map that identifies the location of wells containing clones producing soluble proteins. A series of semi-automated methods can then be applied for validation of solubility and production of freezer stocks for the protein production group. This process provides an 80% success rate for the identification of clones producing soluble protein and results in a significant decrease in the level of effort required for the labor-intensive components of validation and preparation of freezer stocks. This process is customized for large-scale structural genomics programs that rely on the production of large amounts of soluble proteins for crystallization trials.

Animals↗
PubMed2006

Robust regression for high throughput drug screening.

Effective analysis of high throughput screening (HTS) data requires automation of dose-response curve fitting for large numbers of datasets. Datasets with outliers are not handled well by standard non-linear least squares methods, and manual outlier removal after visual inspection is tedious and potentially biased. We propose robust non-linear regression via M-estimation as a statistical technique for automated implementation. The approach of finding M-estimates by Iteratively Reweighted Least Squares (IRLS) and the resulting optimization problem are described. Initial parameter estimates for iterative methods are important, so self-starting methods for our model are presented. We outline the software implementation, done in Matlab and deployed as an Excel application via the Matlab Excel Builder Toolkit. Results of M-estimation are compared with least squares estimates before and after manual editing.

Algorithms↗
PubMed2006

High-throughput screening for enhanced protein stability.

High thermostability of proteins is a prerequisite for their implementation in biocatalytic processes and in the evolution of new functions. Various protein engineering methods have been applied to the evolution of increased thermostability, including the use of combinatorial design where a diverse library of proteins is generated and screened for variants with increased stability. Current trends are toward the use of data-driven methods that reduce the library size by using available data to choose areas of the protein to target, without specifying the precise changes. For example, the half-lives of subtilisin and a Bacillus subtilis lipase were increased 1500-fold and 300-fold, respectively, using a crystal structure to guide mutagenesis choices. Sequence homology based methods have also produced libraries where 50% of the variants have improved thermostability. Moreover, advances in the high-throughput measurement of denaturation curves and the application of selection methods to thermostability evolution have enabled the screening of larger libraries. The combination of these methods will lead to the rapid improvement of protein stability for biotechnological purposes.

Computational Biology↗
PubMedDate not supplied

MS-Express: data-extracting and -processing software for high-throughput experimentation with mass spectrometry.

High-throughput experiments (HTE) result in large amounts of raw data that have to be evaluated for sample classification. Especially mass spectrometry, a widely used detection method in catalytic HTE applications, produces enormous amounts of data. In the past few years, in catalysts research, several test rigs based on mass spectrometric detection have been independently reported by different groups. In a typical HTE, the catalysts are tested sequentially; the recording of the scans, however, occurs continuously. For this reason, the scans of interest have to be extracted from the raw data, and scans belonging to the same sample have to be averaged in a tedious procedure before further processing. In this publication, we present our custom-designed software MS-Express (mass spectrometry data-extracting and -processing software), an efficient tool for HTE MS data evaluation. MS-Express not only sorts the data, it also establishes statistical significance with the help of reference and blank data and provides concise information about abundance and intensity distributions of expected peaks. A special feature is that the program also reports unexpected MS signals, which potentially lead to unexpected discoveries.

Mass Spectrometry↗
PubMed2006

GenoProfiler: batch processing of high-throughput capillary fingerprinting data.

UNLABELLED: High-throughput content fingerprinting techniques employing capillary electrophoresis place new demands on the editing of fingerprint files for the downstream contig assembly program, FPC. A cross-platform software application, GenoProfiler, was developed for automated editing of sized fingerprinting profiles generated by the ABI Genetic Analyzers. The batch-processing module extracts the sized fragment information directly from the ABI raw trace files, or from data files exported from GeneMapper or other size calling software, removes the background noise and undesired fragments, and generates fragment size files compatible with the FPC software. AVAILABILITY: http://wheat.pw.usda.gov/PhysicalMapping/

Algorithms↗