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PubMed2001

Tissue microarrays (TMAs) for high-throughput molecular pathology research.

A rapidly increasing number of genes are being suspected to play a role in cancer biology. To evaluate the clinical significance of newly detected potential cancer genes, it is usually required to examine a high number of well-characterized primary tumors. Using traditional methods of molecular pathology, this is a time consuming endeavor rapidly exhausting precious tissue resources. To allow for a high throughput tissue analysis we have developed a "tissue chip" approach (Kononen et al., Nat. Med. 1998;4:844-7). Using this tissue microarray (TMA) technology, samples from up to 1,000 different tumors are arrayed in one recipient paraffin block, sections of which can be used for all kind of in situ analyses. Section from TMA blocks can then be utilized for the simultaneous analysis of up to 1,000 different tumors on the DNA, RNA or protein level. TMAs allow a high throughput molecular analysis of thousands of tumors within a few hours. All currently available data have suggested that minute arrayed tissue specimens are highly representative of their donor tissues. There are multiple different types of TMAs that can be utilized in cancer research including multi tumor arrays (containing different tumor types), tumor progression arrays (tumors of different stages) and prognostic arrays (tumors with clinical endpoints). The combination of multiple different TMAs allows a very quick but comprehensive characterization of biomarkers of interest. We anticipate that the use of TMAs will greatly accelerate the transition of basic research findings to clinical applications.

Humans↗
PubMed2004

Technology for high-throughput screens: the present and future using zebrafish.

The zebrafish is a popular vertebrate model organism with similar organ systems and gene sequences to humans. Zebrafish embryos are optically transparent enabling organ visualisation, which can be complemented with gene expression analysis at the transcript and protein levels. Furthermore, zebrafish can be treated with small molecules and drugs in a microtitre plate format for high-throughput analysis and for the identification and validation of drugs. High-throughput methodologies for use in zebrafish include phenotype-based visualisation, transcript studies using low-density DNA microarrays and proteomic analysis. These technologies offer significant whole-organism biological value in the drug discovery and drug development pipeline.

Animals↗
PubMed2006

High-throughput screen for poly-3-hydroxybutyrate in Escherichia coli and Synechocystis sp. strain PCC6803.

A novel, quantitative method for detecting poly-3-hydroxybutyrate (PHB) amounts in viable cells was developed to allow for high-throughput screening of mutant libraries. The staining technique was demonstrated and optimized for the cyanobacterium Synechocystis sp. strain PCC6803 and the eubacterium Escherichia coli to maximize the fluorescence difference between PHB-accumulating and control cells by flow cytometry. In Synechocystis, the level of nonspecific dye binding was reduced by using nonionic stain buffer that allowed quantitation of fluorescence levels. In E. coli, the use of a mild sucrose shock facilitated uptake of Nile red without significant loss of viability. The optimized staining protocols yielded a linear response for the mean fluorescence against (chemically measured) PHB. The staining protocols are novel methods useful in the high-throughput evaluation of combinatorial libraries of Synechocystis and E. coli using fluorescence-activated cell sorting to identify mutants with increased PHB-accumulating properties.

Escherichia coli↗
PubMed1995

High throughput assays of cloned adrenergic, muscarinic, neurokinin, and neurotrophin receptors in living mammalian cells.

Many receptors stimulate proliferation of NIH 3T3 cells in a ligand dependent fashion. Based on this observation, we developed a high throughput assay of cloned receptor pharmacology. In this assay, receptors are transiently co-expressed with the marker enzyme beta-galactosidase. Receptors that induce cellular proliferation select and amplify the cells that also express the marker, thus the ability of ligands to alter receptor activity are reported as changes in enzyme activity. In the present study, we used this assay to evaluate the ability of agonist ligands to stimulate four cloned receptors. The agonists phenylephrine, carbachol, substance P and nerve growth factor selectively stimulated cells transfected with the alpha-1b adrenergic, m4 muscarinic, NK1 neurokinin and trkA neurotrophin receptors, respectively. These data demonstrate that a high throughput colorimetric assay performed in 96 well plates can be used to evaluate the pharmacology of ligands for cloned receptors belonging to a wide range of functional and pharmacological classes.

3T3 Cells↗
PubMed2006

Assessment of anti-metastatic drug efficacy via localization and quantification of ex vivo murine bone tumor load using high-throughput MRI T1 parametric analysis.

MRI methods show great utility for assessing the growth of tumors metastasized to bone in clinical studies. However, preclinical MRI methods in rodents do not translate well to high-throughput studies of bone tumors, especially for early-stage tumors typically examined in pharmaceutical discovery efforts. To overcome these limitations, an ex vivo MR T1 parametric mapping method has been developed to measure metastasized bone tumor load in murine long bones. This method has been used to assess the therapeutic efficacy of SU11248, a multi-targeted inhibitor with demonstrated anti-tumor activity and reduction of bone loss, in a murine model of metastasized breast tumor cells. The results show precise localizations of relative tumor loads within the bones and reveal significant differences between SU11248-treated and untreated animal groups. The procedures were optimized for simultaneous, high-throughput parallel image acquisition of MRI data for 30 samples and included an automated segmentation method for image processing. The merits of this T1 parametric mapping method are compared with clinical T1-weighted MRI methods, histopathology and bioluminescence imaging of the same murine bone tumor model.

Animals↗
PubMed2006

Analysis of high throughput protein expression in Escherichia coli.

The ability to efficiently produce hundreds of proteins in parallel is the most basic requirement of many aspects of proteomics. Overcoming the technical and financial barriers associated with high throughput protein production is essential for the development of an experimental platform to query and browse the protein content of a cell (e.g. protein and antibody arrays). Proteins are inherently different one from another in their physicochemical properties; therefore, no single protocol can be expected to successfully express most of the proteins. Instead of optimizing a protocol to express a specific protein, we used sequence analysis tools to estimate the probability of a specific protein to be expressed successfully using a given protocol, thereby avoiding a priori proteins with a low success probability. A set of 547 proteins, to be used for antibody production and selection, was expressed in Escherichia coli using a high throughput protein production pipeline. Protein properties derived from sequence alone were correlated to successful expression, and general guidelines are given to increase the efficiency of similar pipelines. A second set of 68 proteins was expressed to investigate the link between successful protein expression and inclusion body formation. More proteins were expressed in inclusion bodies; however, the formation of inclusion bodies was not a requirement for successful expression.

Cloning, Molecular↗
PubMed1998

Development of high-throughput flexible multichannel electron detectors at Daresbury Laboratory.

Continuing demands from Synchrotron Radiation Source (SRS) end-users for higher throughput and improved reliability in photoelectron spectroscopy experiments have driven an intensive development programme for new multichannel electron detectors. The development philosophy focuses on high throughput to match present and future source intensity, flexible structures to allow increased mobility of designs and modular design for easy maintenance and repair. Developments include parallel readout electronics and innovative detector heads for the hemispherical deflection analysers currently in use on the SRS. Novel anode arrays have been implemented in the detector heads and extensive microchannel plate (MCP) characterization has been undertaken to source the MCPs most suited to this application. The present multichannel detection systems provide a significant enhancement to single-channel detection systems. They have also surpassed previous multichannel detection systems due to their high throughput, flexible structure and modular design. Information on these developments and experimental results obtained at Daresbury Laboratory are presented.

Journal Article↗
PubMed2004

High throughput quantitative analysis of serum proteins using glycopeptide capture and liquid chromatography mass spectrometry.

It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins, generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. Here we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared with their control littermates.

Animals↗
PubMed2001

High throughput screening: a rapid way to recombinant allergens.

Complex allergenic sources such as moulds, foods and mites contain large panels of IgE-binding molecules which need to be cloned, produced and characterized in order to mimic the entire allergenicity of whole extracts reconstituted by mixing single standardized recombinant allergens. Phage display of cDNA libraries allows selective enrichment of allergen-expressing clones using IgE from allergic patients. For the characterization of all different clones present in enriched cDNA libraries in a fast and cost-effective way, however, high-throughput screening technology is required. We have used a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries in order to characterize whole allergenic repertoires from complex allergenic sources. The strategy, based on a combination of phage display and high-density arrays, allowed fast discovery of panels of related structures from different allergenic sources. They cover secreted, cytoplasmic and structural proteins with or without enzymatic activity and offer a rational explanation for the IgE-mediated cross-reactivity frequently encountered in clinical practice.

Allergens↗
PubMed2004

A general framework for development and data analysis of competitive high-throughput screens for small-molecule inhibitors of protein-protein interactions by fluorescence polarization.

Equilibrium binding experiments are widely used for the accurate characterization of binding and competitive binding behavior in biological systems. Modern high-throughput discovery efforts in chemical biology rely heavily upon this principle. Here, we derive exact analytical expressions for general competitive binding models which can also explain a commonly encountered phenomenon in these types of experiments, anticooperative incomplete displacement. We explore the effects of nonspecific binding behavior and parameter misestimation. All expressions are derived in terms of total concentrations determined a priori. We discuss a general framework for high-throughput screening assays based on fluorescence polarization and strategies for assay development, sensitivity regimes, data quality control, analysis, and ranking. Theoretical findings are visualized by simulations using realistic parameter sets. Our results are the basis for the discovery of small-molecule inhibitors of the protein-protein interaction between human calcineurin and NFAT transcription factors, as discussed in the subsequent paper (31).

Computer Simulation↗
PubMed2006

Development of a novel dicistronic reporter-selectable hepatitis C virus replicon suitable for high-throughput inhibitor screening.

Hepatitis C virus (HCV) research and drug discovery have been facilitated by the introduction of cell lines with self-replicating subgenomic HCV replicons. Early attempts to carry out robust, high-throughput screens (HTS) using HCV replicons have met with limited success. Specifically, selectable replicons have required laborious reverse transcription-PCR quantitation, and reporter replicons have generated low signal-to-noise ratios. In this study, we constructed a dicistronic single reporter (DSR)-selectable HCV replicon that contained a humanized Renilla luciferase (hRLuc) gene separated from the selectable Neo(r) marker by a short peptide cleavage site. The mutations E1202G, T1280I, and S2197P were introduced to enhance replicative capability. A dicistronic dual-reporter HCV replicon cell line (DDR) was subsequently created by transfection of Huh-7 cells with the DSR replicon to monitor antiviral activity and by the introduction of the firefly luciferase (FLuc) reporter gene into the host cell genome to monitor cytotoxicity. The DDR cell line demonstrated low signal variation within the HTS format, with a calculated Z' value of 0.8. A pilot HTS consisting of 20 96-well plates with a single concentration (10 microM) of 1,760 different compounds was executed. Hits were defined as compounds that reduced hRLuc and FLuc signals > or =50 and < or =40%, respectively, relative to those in a compound-free control. Good reproducibility was demonstrated, with a calculated confirmation rate of >75%. The development of a robust, high-throughput HCV replicon assay where the effects of inhibitors can be monitored for antiviral activity and cytotoxicity should greatly facilitate HCV drug discovery.

Antiviral Agents↗
PubMed2002

New generation pharmacogenomic tools: a SNP linkage disequilibrium Map, validated SNP assay resource, and high-throughput instrumentation system for large-scale genetic studies.

Since public and private efforts announced the first draft of the human genome last year, researchers have reported great numbers of single nucleotide polymorphisms (SNPs). We believe that the availability of well-mapped, quality SNP markers constitutes the gateway to a revolution in genetics and personalized medicine that will lead to better diagnosis and treatment of common complex disorders. A new generation of tools and public SNP resources for pharmacogenomic and genetic studies--specifically for candidate-gene, candidate-region, and whole-genome association studies--will form part of the new scientific landscape. This will only be possible through the greater accessibility of SNP resources and superior high-throughput instrumentation-assay systems that enable affordable, highly productive large-scale genetic studies. We are contributing to this effort by developing a high-quality linkage disequilibrium SNP marker map and an accompanying set of ready-to-use, validated SNP assays across every gene in the human genome. This effort incorporates both the public sequence and SNP data sources, and Celera Genomics' human genome assembly and enormous resource ofphysically mapped SNPs (approximately 4,000,000 unique records). This article discusses our approach and methodology for designing the map, choosing quality SNPs, designing and validating these assays, and obtaining population frequency ofthe polymorphisms. We also discuss an advanced, high-performance SNP assay chemisty--a new generation of the TaqMan probe-based, 5' nuclease assay-and high-throughput instrumentation-software system for large-scale genotyping. We provide the new SNP map and validation information, validated SNP assays and reagents, and instrumentation systems as a novel resource for genetic discoveries.

Alleles↗
PubMed2003

High-throughput confirmation of differential display PCR results using reverse Northern blotting.

Nylon filter arrays spotted with differential display PCR (DD-PCR) clones and hybridized with radiolabeled cRNA generated from the source RNA pool (reverse Northern blot) provide a high-throughput means to screen clones for artifacts. Reverse Northern blots also confirm differential gene expression in parallel and require modest quantities of the source RNA pool. We describe a strategy to screen multiple candidates from DD-PCR by high-throughput ligation and transformation, followed by reverse Northern blotting. Purification of re-amplified DD-PCR clones and fabrication of nylon arrays was facilitated by a batch-processing protocol using the widely available Biomek laboratory robot and Bioworks scripts (available from the authors). A strategy to screen out DD-PCR product artifacts of an inappropriate size was also employed. Using these approaches, we identified several mRNAs that are differentially expressed in response to venlafaxine, fluoxetine or desipramine antidepressant treatment in rat C6 glioma cell lines and are candidates for full length clone isolation using 5'-RACE. Such an approach provides a rapid means to eliminate the high percentage of false positive clones from DD-PCR and enables independent confirmation of differential gene expression patterns generated by various experimental conditions.

Animals↗
PubMed2006

High-throughput pKa screening and prediction amenable for ADME profiling.

Recent technological advances have made it possible for several new pK(a) assays to be used in drug screening. In this review, a critical overview is provided of current new methodologies for high-throughput screening and prediction of pK(a). Typical applications of using pK(a )constants and charge state for absorption, distribution, metabolism and excretion (ADME) profiling and quantitative structure-activity relationship modelling complements the methodological comparisons and discussions. The experimental methods discussed include high-throughput screening of pK(a) by multiplexed capillary with ultraviolet absorbance detection on a 96-capillary format instrument, capillary electrophoresis and mass spectrometry (CEMS) based on sample pooling, determination of pK(a) by pH gradient high-performance liquid chromatography, and measurement of pK(a) by a mixed-buffer liner pH gradient system. Comparisons of the different experimental assays are made with emphasis on the newly developed CEMS method. The current status and recent progress in computational approaches to pK(a) prediction are also discussed. In particular, the accuracy limits of simple fragment-based approaches as well as quantum mechanical methods are addressed. Examples of pK(a) prediction from in-house drug candidates as well as commercially available drug molecules are shown and an outline is provided for how drug discovery companies can integrate experiments with computational approaches for increased applications for ADME profiling.

Animals↗
PubMed2001

High-throughput PCR in silicon based microchamber array.

Highly integrated hybridization assay and capillary electrophoresis have improved the throughput of DNA analysis. The shift to high throughput analysis requires a high speed DNA amplification system, and several rapid PCR systems have been developed. In these thermal cyclers, the temperature was controlled by effective methodology instead of a large heating/cooling block preventing rapid thermal cycling. In our research, high speed PCR was performed using a silicon-based microchamber array and three heat blocks. The highly integrated microchamber array was fabricated by semiconductor microfabrication techniques. The temperature of the PCR microchamber was controlled by alternating between three heat blocks of different temperature. In general, silicon has excellent thermal conductivity, and the heat capacity is small in the miniaturized sample volume. Hence, the heating/cooling rate was rapid, approximately 16 degrees C/s. The rapid PCR was therefore completed in 18 min for 40 cycles. The thermal cycle time was reduced to 1/10 of a commercial PCR instrument (Model 9600, PE Applied Biosystems-3 h).

Hot Temperature↗
PubMed2000

High-throughput cytochrome P450 inhibition screening via cassette probe-dosing strategy. IV. Validation of a direct injection on-line guard cartridge extraction/tandem mass spectrometry method for simultaneous CYP3A4, 2D6 and 2E1 inhibition assessment.

A highly efficient direct injection on-line guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS) method has been validated for high-throughput evaluation of cytochrome P450 (CYP) 3A4, 2D6 and 2E1 inhibition potential via cassette dosing of midazolam, dextromethorphan and chlorzoxazone using human hepatic microsomes and 96-well microtiter plates. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for analysis by DI-GCE/MS/MS. Due to the novel use of an extremely short C(18) guard cartridge (4 mm in length), this method exhibits several advantages such as no sample preparation, excellent on-line extraction, short run time (2.5 min), and minimized source contamination and performance deterioration. The DI-GCE/MS/MS method demonstrates acceptable accuracy and precision for the simultaneous quantification of 1'-hydroxymidazolam, dextrorphan and 6-hydroxychlorzoxazone in microsomal incubations. The inhibition potential of CYP3A4, 2D6 and 2E1 has been evaluated using their known selective inhibitors. The IC(50) values measured by the cassette dosing approach (high-throughput) are consistent with those observed by an individual dosing regimen (conventional) and are all in good agreement with the literature values. The results suggest that the cassette probe-dosing strategy may provide an in vitro approach to minimize cost while maximizing throughput of CYP inhibition evaluation of new chemical entities in support of drug discovery and development.

Calibration↗
PubMed2000

Evaluation of a novel high-throughput assay for cytochrome P450 2D6 using 7-methoxy-4-(aminomethyl)-coumarin.

We recently reported on the design, synthesis and characterisation of a novel and selective substrate of human cytochrome P450 2D6 (CYP2D6), 7-methoxy-4-(aminomethyl)-coumarin (MAMC). Here, we describe a high-throughput microplate reader assay, which makes use of MAMC as a fluorescent probe for determining the inhibition and activity of CYP2D6 in heterologously expressed systems and human liver microsomes. The high-throughput screening (HTS) assay can be used both in an end-point and real-time configuration, and is easy to use, rapid and sensitive. In addition, end-point measurements by means of flow injection analysis have also successfully been performed. The HTS-assay was validated by performing inhibition experiments for several low- and high-affinity ligands (n=6) of CYP2D6, and comparing the findings to those obtained with the standard O-demethylation assay of dextromethorphan. The results indicate that all compounds tested display competitive inhibition in both the MAMC and dextromethorphan assay, and the K(i) values reveal a very good correlation (R(2)=0.984) between the two assays. To further demonstrate the usefulness of the HTS-assay, IC(50) values of a series of five N-substituted analogs of 3, 4-methylenedioxyamphetamine for CYP2D6 have been determined. The results obtained demonstrate that the current HTS-assay represents a significant improvement over previous assays, with a higher turnover of MAMC and a higher selectivity for CYP2D6.

Calibration↗
PubMed2004

High-throughput S-SAP by fluorescent multiplex PCR and capillary electrophoresis in plants.

The inherent replicative mode of transposition endows retrotransposons with considerable advantages as genetic tools in plant genome analysis. Here we present a high-throughput sequence-specific amplification polymorphism (S-SAP) method based on copia-like retrotransposons to fulfill the increasing desire of screening large numbers of samples in plants. Classic approach for digestion, ligation and pre-amplification was combined with optimized fluorescent multiplex PCR for simultaneously selective amplifying S-SAP fragments, and multiple S-SAPs were subsequently detected by capillary electrophoresis using ABI PRISM 3700 capillary instruments. Comparisons of results from multiplex PCR with simplex PCR, and from capillary electrophoresis with slab-gel electrophoresis demonstrated that this method is an efficient, economical, and accurate means for high-throughput and large-scale genotyping retrotransposon variation in plants.

Chromosome Mapping↗