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Results for “high throughput”

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PubMed1999

Improved cell line development by a high throughput affinity capture surface display technique to select for high secretors.

A novel process is described which permits rapid and objective selection of rare cells from a heterogeneous population based on quantity of secreted target protein. The process involves construction of an immobilised affinity surface display matrix that specifically binds secreted target product which is then detected using a fluorescent labelled ligand. Cells with the highest fluorescence can then be sorted using conventional flow cytometric technology. Overall, the whole process can be completed in less than 4 h during which time in the region of five million cells can be analysed. Cells are rapidly selected for in a quantitative manner compared to traditional methods which can take several months and have a reduced probability of finding low abundance high secretors due to practical limitations imposed on the number of cells which can be screened.

Affinity Labels↗
PubMed1999

Analyses of compound libraries obtained by high-throughput parallel synthesis: strategy of quality control by high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance techniques.

The growing interest in combinatorial chemistry has led to a new source of compounds from which a large number of leads has emerged over recent years. Parallel synthesis, in particular, allows a quick production of a wide number of individual compounds. A rapid analytical control is needed to determine their quality. A strategy using automated, fast reversed-phase C18 high-performance liquid chromatography with diode-array detection (LC-DAD-MS) followed by atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) and NMR has been developed for their characterisation and purity control. Complementary NMR analyses are done on selected compounds to provide a better structural characterisation of the expected compounds and their potential side-products. Validated libraries are then registered in ISIS databases using automated procedures.

Chemistry, Pharmaceutical↗
PubMed2001

Whole genome amplification and high-throughput allelotyping identified five distinct deletion regions on chromosomes 5 and 6 in microdissected early-stage ovarian tumors.

Investigation of genetic changes in tumors by loss of heterozygosity is a powerful technique for identifying chromosomal regions that may contain tumor suppressor genes. In this study, we determined allelic loss on chromosomes 5 and 6 in 29 primary early-stage epithelial ovarian carcinomas including 3 microscopically identified adenocarcinomas using a high-throughput PCR-based method combined with laser capture microdissection and whole genome amplification techniques. Twenty microsatellite markers spanning chromosomes 5 and 6 at an average distance of 20 cM were examined. High frequencies of loss on chromosome 5 were identified at loci D5S428 (48%), D5S424 (32%), and D5S630 (32%). Our study also showed that chromosome 6 exhibited high frequencies of loss of heterozygosity at loci D6S1574 (46%), D6S287 (42%), D6S441 (45%), D6S264 (60%), and D6S281 (35%). These results suggest that multiple tumor suppressor genes are located on five distinct regions on chromosomes 5 and 6, i.e., 5p15.2, 5q13-21, 6p24-25, 6q21-23, and 6q25.1-27, and may be involved in the early development of ovarian carcinomas.

Adenocarcinoma↗
PubMed2001

Detection of a decrease in green fluorescent protein fluorescence for the monitoring of cell death: an assay amenable to high-throughput screening technologies.

BACKGROUND: Reliable assessment of cell death is now pivotal to many research programs aiming at generating new anti-tumor compounds or at screening cDNA libraries. Such approaches need to rely on reproducible, easy-to-handle, and rapid microplate-based cytotoxicity assays that are amenable to high-throughput screening (HTS) technologies. We describe a method for the direct measurement of cell death, based on the detection of a decrease in fluorescence observed following death induction in cells expressing enhanced green fluorescent protein (EGFP). METHODS: Cell death was induced by a variety of apoptotic stimuli in various EGFP-expressing mammalian cell lines, including those routinely used in anti-cancer drug screening. Decrease in fluorescence was assessed either by flow cytometry (and compared with other apoptotic markers) or by a fluorescence microplate reader. RESULTS: Cells expressing EGFP exhibited a decrease in fluorescence when treated by various agents, such as chemotherapeutic drugs, UV irradiation, or caspase-independent cell death inducers. Kinetics and sensitivity of this EGFP-based assay were comparable to those of traditional apoptosis markers such as annexin-V binding, propidium iodide incorporation, or reactive oxygen species production. We also show that the decrease in EGFP fluorescence is directly quantifiable in a fluorescence-based microplate assay. Furthermore, analysis of EGFP protein content in cells undergoing cell death demonstrates that the decrease in fluorescence does not arise from degradation of the protein. CONCLUSIONS: This novel GFP-based microplate assay combines sensitivity and rapidity, is easily amenable to HTS setups, making it an assay of choice for cytotoxicity evaluation.

3T3 Cells↗
PubMed2004

Eukaryotic arylamine N-acetyltransferase. Investigation of substrate specificity by high-throughput screening.

Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2.

Animals↗
PubMed2005

High throughput analysis of endogenous glutamate release using a fluorescence plate reader.

Recent discoveries of different modes of exocytosis and a plethora of molecules involved in neurotransmitter release has resulted in demand for more rapid and efficient methods for monitoring endogenous glutamate release from various tissue sources. In this article, we describe a high throughput microplate version of the enzyme-linked fluorescence detection method for the measurement of released glutamate, which utilises glutamate dehydrogenase, and the reduction of NADP to NADPH. Previous versions of this method rely upon cuvette-based fluorimeters for detection that are limited by large sample volumes and small numbers of samples that can be measured simultaneously. Comparison between the two methods shows that the microplate assay has comparable performance to the cuvette-based assay but has the capacity to analyse many times more samples in a given run. This increased capacity provides improved experimental design opportunities, higher experimental throughput and better comparison between experimental conditions.

Animals↗
PubMed2006

High-throughput, functional screening of the anti-HIV-1 humoral response by an enzymatic nanosensor.

The impact of antibodies on the target's epitope conformation is a major determinant of HIV-1 neutralization and a potential contributor to disease progression. We explore here a conformation-sensitive enzymatic nanosensor for the high-throughput functional screening of human anti-HIV-1 antibodies in sera. When displaying a model epitope from a gp41 immunodominant region (Env residues from 579 to 613), the sensing signal quantitatively distinguishes between adaptive and non-adaptive antibody binding. By using this tool, we have identified IgG4 as the immunoglobulin subpopulation most efficient in the structural modification of the target epitope.

Antibodies, Viral↗
PubMed2003

C. elegans, a model for aging with high-throughput capacity.

The 1 mm long nematode Caenorhabditis elegans is one of the prime animal models to study the genetics of aging. Wild type animals under laboratory conditions live only for an average of 18 days, whereas mutations in about 50 genes have been identified that extend longevity up to sixfold. High-throughput analyses have been devised that allow large-scale analysis to identify additional genes, as well as compounds that affect life-span.

Aging↗
PubMedDate not supplied

GFscore: a general nonlinear consensus scoring function for high-throughput docking.

Most of the recent published works in the field of docking and scoring protein/ligand complexes have focused on ranking true positives resulting from a Virtual Library Screening (VLS) through the use of a specified or consensus linear scoring function. In this work, we present a methodology to speed up the High Throughput Screening (HTS) process, by allowing focused screens or for hitlist triaging when a prohibitively large number of hits is identified in the primary screen, where we have extended the principle of consensus scoring in a nonlinear neural network manner. This led us to introduce a nonlinear Generalist scoring Function, GFscore, which was trained to discriminate true positives from false positives in a data set of diverse chemical compounds. This original Generalist scoring Function is a combination of the five scoring functions found in the CScore package from Tripos Inc. GFscore eliminates up to 75% of molecules, with a confidence rate of 90%. The final result is a Hit Enrichment in the list of molecules to investigate during a research campaign for biological active compounds where the remaining 25% of molecules would be sent to in vitro screening experiments. GFscore is therefore a powerful tool for the biologist, saving both time and money.

Molecular Structure↗
PubMed2006

ADP_EM: fast exhaustive multi-resolution docking for high-throughput coverage.

MOTIVATION: Efficient fitting tools are needed to take advantage of a fast growth of atomic models of protein domains from crystallography or comparative modeling, and low-resolution density maps of larger molecular assemblies. Here, we report a novel fitting algorithm for the exhaustive and fast overlay of partial high-resolution models into a low-resolution density map. The method incorporates a fast rotational search based on spherical harmonics (SH) combined with a simple translational scanning. RESULTS: This novel combination makes it possible to accurately dock atomic structures into low-resolution electron-density maps in times ranging from seconds to a few minutes. The high-efficiency achieved with simulated and experimental test cases preserves the exhaustiveness needed in these heterogeneous-resolution merging tools. The results demonstrate its efficiency, robustness and high-throughput coverage. AVAILABILITY: http://sbg.cib.csic.es/Software/ADP_EM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Algorithms↗
PubMed2002

Robotic high-throughput assay for isolating apoptosis-inducing genes.

The need to assign functions to genes will increase in upcoming years because of the accumulation of sequence information from various organisms. Here we present a genetic screen for the systematic functional analysis of genes in mammalian cells. The screen is based on high-throughput, transient transfections of individual cDNAs by a robotic system. This construction uses a moveable pipetting head with a novel 96-channel parallel tube-pump array and an integrated de-lidding device. Because of the robot design, the processing speed can reach 2,000 transfections/h. We demonstrate the suitability of the screen to isolate apoptosis-inducing genes as a first read-out. This is achieved in combination with co-transfected reporter plasmids to produce a novel and convenient assay for apoptosis induction.

Alkaline Phosphatase↗
PubMed2003

High-throughput Pyrosequencing of a phage display library for the identification of enriched target-specific peptides.

Gene therapy clinical trials have highlighted the importance of specific cellular/tissue targeting of gene delivery vectors. Phage display libraries are powerful tools for the selection of novel peptide ligands as targeting moieties because of their high-throughput screening potential. However, a severe rate-limiting step in this procedure in terms of time, numbers, and cost is the sequence identification of selected phages. Here we describe the application of Pyrosequencing technology for sequencing phage isolates after panning a random 7-mer peptide expressing phage library against the A549 bronchial epithelial cell line to search for enrichment of possible targeting peptides. Pyrosequencing allows sequencing of 96 phages at one time in approximately 45 min at only a sixth of the cost of conventional sequencing methods. Using this technology, we have identified four sequences of interest. A phage binding assay revealed that three of the four sequences show a significant increase in binding abilities and specificity for A549 cells when compared to an unrelated cell line.

Amino Acid Sequence↗
PubMed2000

A quantitative, high-throughput screen for protein stability.

In proteomic research, it is often necessary to screen a large number of polypeptides for the presence of stable structure. Described here is a technique (referred to as SUPREX, stability of unpurified proteins from rates of H/D exchange) for measuring the stability of proteins in a rapid, high-throughput fashion. The method uses hydrogen exchange to estimate the stability of microgram quantities of unpurified protein extracts by using matrix-assisted laser desorption/ionization MS. The stabilities of maltose binding protein and monomeric lambda repressor variants determined by SUPREX agree well with stability data obtained from conventional CD denaturation of purified protein. The method also can detect the change in stability caused by the binding of maltose to maltose binding protein. The results demonstrate the precision of the method over a wide range of stabilities.

Bacterial Proteins↗
PubMed2004

High-throughput metabolic fingerprinting of legume silage fermentations via Fourier transform infrared spectroscopy and chemometrics.

Silage quality is typically assessed by the measurement of several individual parameters, including pH, lactic acid, acetic acid, bacterial numbers, and protein content. The objective of this study was to use a holistic metabolic fingerprinting approach, combining a high-throughput microtiter plate-based fermentation system with Fourier transform infrared (FT-IR) spectroscopy, to obtain a snapshot of the sample metabolome (typically low-molecular-weight compounds) at a given time. The aim was to study the dynamics of red clover or grass silage fermentations in response to various inoculants incorporating lactic acid bacteria (LAB). The hyperspectral multivariate datasets generated by FT-IR spectroscopy are difficult to interpret visually, so chemometrics methods were used to deconvolute the data. Two-phase principal component-discriminant function analysis allowed discrimination between herbage types and different LAB inoculants and modeling of fermentation dynamics over time. Further analysis of FT-IR spectra by the use of genetic algorithms to identify the underlying biochemical differences between treatments revealed that the amide I and amide II regions (wavenumbers of 1,550 to 1,750 cm(-1)) of the spectra were most frequently selected (reflecting changes in proteins and free amino acids) in comparisons between control and inoculant-treated fermentations. This corresponds to the known importance of rapid fermentation for the efficient conservation of forage proteins.

Fabaceae↗
PubMed2000

Microbiological high throughput screening: an opportunity for the lead discovery process.

Microbial HTS has been implemented at Rhône-Poulenc Rorer through the development of a dedicated robotic platform. This robot (Turbo) has been designed with the aim of fully integrating microbial HTS into the lead discovery processes. Innovative solutions have been found to reach high throughput as well as flexibility. This opens up new prospects for solid-phase microbial screening, taking advantage of the easy implementation and the very low costs of such screens. The different types of microbial screens done in our laboratory, as well as the throughputs and outputs obtained, are described. Some of the specific aspects of microbial HTS, as compared to biochemical and cell-based assays, are also discussed.

Bacteria↗
PubMed2002

High-throughput quantification of drugs and their metabolites in biosamples by LC-MS/MS and CE-MS/MS: possibilities and limitations.

Off-line solid phase extraction with C18 disk plates and turbulent flow chromatography were evaluated versus on-line solid phase extraction using column-switching HPLC as sample preparation techniques for high-throughput analysis of pharmaceutical compounds and their metabolites by LC-MS/MS. Turbulent flow chromatography was found to be very straightforward in its applicaton, but the LOQs were more than fivefold higher compared with off-line or other on-line solid phase extraction methods. Solid phase extraction (SPE) on disk was found to be fast and sufficient efficient to minimize matrix effects and therefore an apprach to provide sensitive and reliable LC-MS/MS methods. Column-switching HPLC with microbore columns (0.5 mm i.d.) were used for fast analysis of a parent drug and four of its metabolites utilizing steep gradients in 1 minute. The application of CZE-MS/MS for bionalysis of pharamaceutical compounds is also discussed.

Chromatography, High Pressure Liquid↗
PubMed2004

Identification of novel small-molecule inhibitors for human transketolase by high-throughput screening with fluorescent intensity (FLINT) assay.

The metabolic enzyme transketolase (TK) plays a crucial role in tumor cell nucleic acid synthesis, using glucose through the elevated nonoxidative pentose phosphate pathway (PPP). Identification of inhibitors specifically targeting TK and preventing the nonoxidative PPP from generating the RNA ribose precursor, ribose-5-phosphate, provides a novel approach for developing effective anticancer therapeutic agents. The full-length human transketolase gene was cloned and expressed in Escherichia coli and the recombinant human transketolase protein purified to homogeneity. A fluorescent intensity (FLINT) assay was developed and optimized. Library compounds were screened in a high-throughput screening (HTS) campaign using the FLINT assay. Fifty-four initial hits were identified. Among them, 2 scaffolds with high selectivity, ideal physiochemical properties, and low molecular weight were selected for lead optimization studies. These compounds specifically inhibited in vitro TK enzyme activity and suppressed tumor cell proliferation in at least 3 cancer cell lines: SW620, LS174T, and MIA PaCa-2. Identification of these active scaffolds represents a good starting point for development of drugs specifically targeting TK and the nonoxidative PPP for cancer therapy.

Cell Line, Tumor↗
PubMed2005

From drug to protein: using yeast genetics for high-throughput target discovery.

The budding yeast Saccharomyces cerevisiae has long been an effective eukaryotic model system for understanding basic cellular processes. The genetic tractability and ease of manipulation in the laboratory make yeast well suited for large-scale chemical and genetic screens. Several recent studies describing the use of yeast genetics for high-throughput drug target identification are discussed in this review.

Drug Design↗