Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “High Throughput Screening”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 487 records · Page 27Linked to original sources
PubMed2005

A systematic high-throughput screen of a yeast deletion collection for mutants defective in PHO5 regulation.

In response to phosphate limitation, Saccharomyces cerevisiae induces transcription of a set of genes important for survival. One of these genes is PHO5, which encodes a secreted acid phosphatase. A phosphate-responsive signal transduction pathway (the PHO pathway) mediates this response through three central components: a cyclin-dependent kinase (CDK), Pho85; a cyclin, Pho80; and a CDK inhibitor (CKI), Pho81. While signaling downstream of the Pho81/Pho80/Pho85 complex to PHO5 expression has been well characterized, little is known about factors acting upstream of these components. To identify missing factors involved in the PHO pathway, we carried out a high-throughput, quantitative enzymatic screen of a yeast deletion collection, searching for novel mutants defective in expression of PHO5. As a result of this study, we have identified at least nine genes that were previously not known to regulate PHO5 expression. The functional diversity of these genes suggests that the PHO pathway is networked with other important cellular signaling pathways. Among these genes, ADK1 and ADO1, encoding an adenylate kinase and an adenosine kinase, respectively, negatively regulate PHO5 expression and appear to function upstream of PHO81.

Acid Phosphatase↗
PubMedDate not supplied

High-throughput screening and optimization of photoembossed relief structures.

A methodology for the rapid design, screening, and optimization of coating systems with surface relief structures, using a combination of statistical experimental design, high-throughput experimentation, data mining, and graphical and mathematical optimization routines was developed. The methodology was applied to photopolymers used in photoembossing applications. A library of 72 films was prepared by dispensing a given amount of sample onto a chemically patterned substrate consisting of hydrophilic areas separated by fluorinated hydrophobic barriers. Film composition and film processing conditions were determined using statistical experimental design. The surface topology of the films was characterized by automated AFM. Subsequently, models explaining the dependence of surface topologies on sample composition and processing parameters were developed and used for screening a virtual 4000-membered in silico library of photopolymer lacquers. Simple graphical optimization or Pareto algorithms were subsequently used to find an ensemble of formulations, which were optimal with respect to a predefined set of properties, such as aspect ratio and shape of the relief structures.

Automation↗
PubMed2002

Asb4, Ata3, and Dcn are novel imprinted genes identified by high-throughput screening using RIKEN cDNA microarray.

Genes differentially expressed between parthenogenetic and androgenetic embryos are candidates for the identification of imprinted genes, which are expressed specifically from the maternal or paternal allele. To search for genes differentially expressed between parthenogenetic and androgenetic embryos, we used the RIKEN full-length enriched mouse cDNA microarray. The 25 candidates obtained included 8 known imprinted genes (such as IgfII, Snrpn, and Neuronatin) and 3 new ones--Asb4 (ankyrin repeat and SOCS box-containing protein 4), Ata3 (amino acid transport system A3), and Decorin--which were confirmed by using normal diploid embryos from the reciprocal F1 crosses of B6 and JF1 mice. The 25 candidates also included genes that showed no imprinting-associated expression in normal diploid embryos. We describe a feasible high-throughput method of screening for novel imprinted genes by using the RIKEN cDNA microarray.

Amino Acid Transport System A↗
PubMed2004

Characterization of CHO cells stably expressing a G alpha 16/z chimera for high throughput screening of GPCRs.

G protein-coupled receptors (GPCRs) are important therapeutic targets for drug discovery. The identification and characterization of new ligands ideally requires the use of high throughput assays that are applicable to all GPCR subtypes. To circumvent the problem of different GPCRs coupling to distinct intracellular second messenger pathways, we describe a new method that uses the chimeric Galpha protein 16z25 to facilitate this process. Stably expressed in Chinese hamster ovary cells, 16z25 allows G(i/o)- and G(s)-coupled receptors to mobilize intracellular Ca(2+) upon agonist stimulation. We have generated nine cell lines each stably expressing 16z25 and a GPCR. All cell lines respond to appropriate agonist stimulation in fluorometric imaging plate reader (FLIPR) assays with robust and potent Ca(2+) mobilization. Several of these lines have been pharmacologically characterized using agonists and antagonists. We also demonstrate that the coexpression of GPCR and 16z25 does not interfere with the receptors' ability to activate endogenous signaling pathways. The ability of 16z25 to functionally mediate the agonist stimulation of a broad spectrum of GPCRs indicates that the use of cell lines stably coexpressing this chimera and GPCRs will simplify the drug screening process and aid in the deorphanization of new receptors.

Amino Acid Sequence↗
PubMed2003

A cell-based high-throughput screening system for detecting ecdysteroid agonists and antagonists in plant extracts and libraries of synthetic compounds.

Screening systems for ecdysteroid mimetic or antiecdysteroid substances in plant extracts or libraries of synthetic compounds are commonly based on the observation of morphological and/or growth responses in insect cell lines. Because these responses are slow and require careful monitoring, existing screening systems are considered limited regarding their applicability to analysis in high-throughput (HT) formats. Here we describe the generation of transformed silkmoth (Bombyx mori) cell lines that respond to the addition of ecdysone-like substances through the expression of the green fluorescent protein (GFP) and the appearance of green fluorescence. Because tests consist of three simple steps, i.e., 1) distribution of transformed cells in microtiter plates; 2) addition of compounds/extracts at different concentrations; and 3) quantification of fluorescence intensity by a fluorescence plate reader, they can be performed quickly and be easily adapted to a HT format. The generated reporter cell lines are used for the screening of extracts from available plant collections for the presence of compounds with ecdysone mimetic or antagonistic activities as well as for monitoring subsequent activity during enrichment and purification steps. The same cell lines are also used here for the determination of structure-activity relationships among available synthetic dibenzoylhydrazine derivatives. Finally, for the identified agonists, we show that their activity as determined by the cell-based screening assays parallels their bioactivity in growth inhibition and toxicity assays carried out on live insects.

Animals↗
PubMed2004

Miniaturized, ultra-high throughput screening of tyrosine kinases using homogeneous, competitive fluorescence immunoassays.

Assay miniaturization and the implementation of high-density 1,536-microwell screening increase the speed and efficiency of screening and lead discovery. To serve this need, a platform of miniaturizable assay technologies has been assembled for specific biological targets. This platform will enable initiation and completion of uHTS screens in a straightforward and expeditious manner. Although faster primary screening does contribute to a reduction in timelines, the process of assay development can become a bottleneck. Assay technologies that do not require the use of target-specific reagents can reduce the time necessary for assay development. Assays that measure inhibition of tyrosine kinases can be configured in a competitive format where only the enzyme itself is specific to the assay. In this context, several technologies, including time-resolved fluorometry (also known as DELFIA), time-resolved fluorescence resonance energy transfer (also known as LANCE( trade mark )), fluorescence polarization, enzyme fragmentation complementation assay, and confocal laser scanning imaging, were examined. Quality parameters such as assay reproducibility, signal:background ratio, Z factor, and assay sensitivity were compared. Additionally, the relative merits of each of these technologies are assessed in terms of assay miniaturization, ease of development, ultimate screening capability, efficiency, and cost.

Amino Acid Sequence↗
PubMed2006

Use of early passage fetal intestinal epithelial cells in semi-high-throughput screening assays: an approach to identify new innate immune system adjuvants.

Innate immune system stimulants (innate adjuvants) offer complementary approaches to vaccines and antimicrobial compounds to increase host resistance to infection. The authors established fetal bovine intestinal epithelial cell (BIEC) cultures to screen natural product and synthetic compound libraries for novel mucosal adjuvants. They showed that BIECs from fetal intestine maintained an in vivo phenotype as reflected in cytokeratin expression, expression of antigens restricted to intestinal enterocytes, and induced interleukin-8 (IL-8) production. BIECs could be infected by and support replication of bovine rotavirus. A semi-high-throughput enzyme-linked immunosorbent assay-based assay that measured IL-8 production by BIECs was established and used to screen commercially available natural compounds for novel adjuvant activity. Five novel hits were identified, demonstrating the utility of the assay for selecting and screening new epithelial cell adjuvants. Although the identified compounds had not previously been shown to induce IL-8 production in epithelial cells, other known functions for 3 of the 5 were consistent with this activity. Statistical analysis of the throughput data demonstrated that the assay is adaptable to a high-throughput format for screening both synthetic and natural product derived compound libraries.

Adjuvants, Immunologic↗
PubMed2003

Enhanced crossover SCRATCHY: construction and high-throughput screening of a combinatorial library containing multiple non-homologous crossovers.

SCRATCHY is a methodology for the construction of libraries of chimeras between genes that display low sequence homology. We have developed a strategy for library creation termed enhanced crossover SCRATCHY, that significantly increases the number of clones containing multiple crossovers. Complementary chimeric gene libraries generated by incremental truncation (ITCHY) of two distinct parental sequences are created, and are then divided into arbitrarily defined sections. The respective sections are amplified by skewed sets of primers (i.e. a combination of gene A specific forward primer and gene B specific reverse primer, etc.) allowing DNA fragments containing non-homologous crossover points to be amplified. The amplified chimeric sections are then subjected to a DNA shuffling process generating an enhanced crossover SCRATCHY library. We have constructed such a library using the rat theta 2 glutathione transferase (rGSTT2) and the human theta 1 glutathione transferase (hGSTT1) genes (63% DNA sequence identity). DNA sequencing analysis of unselected library members revealed a greater diversity than that obtained by canonical family shuffling or with conventional SCRATCHY. Expression and high-throughput flow cytometric screening of the chimeric GST library identified several chimeric progeny that retained rat-like parental substrate specificity.

Animals↗
PubMed1999

High-throughput screening for ligand-induced c-fos mRNA expression by branched DNA assay in Chinese hamster ovary cells.

We have developed a generally useful screening assay for receptor agonists and antagonists in Chinese Hamster Ovary (CHO) cells. Three key features of the assay make it applicable to a broad range of receptors: (1) the use of CHO cells as host cells to overexpress receptors, (2) measurement of endogenous c-fos mRNA, which responds to a wide spectrum of stimuli, and (3) the use of branched chain DNA assay which is highly sensitive, quantifiable, amenable to high-throughput analysis, and easy to execute. The combination of these features provides a powerful means to screen rapidly for peptide and small molecule ligands for a variety of receptors. CHO cells overexpressing insulin receptor were used as a test system to compare conventional signaling assays with the high-throughput c-fos branched DNA assay.

Amino Acid Sequence↗
PubMed2004

Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena.

Comparative screening of gene expression libraries employing the potent industrial host Pichia pastoris for improving recombinant eukaryotic enzymes by protein engineering was an unsolved task. We simplified the protocol for protein expression by P. pastoris and scaled it down to 0.5-ml cultures. Optimising standard growth conditions and procedures, programmed cell death and necrosis of P. pastoris in microscale cultures were diminished. Uniform cell growth in 96-deep-well plates now allows for high-throughput protein expression and screening for improved enzyme variants. Furthermore, the change from one host for protein engineering to another host for enzyme production becomes dispensable, and this accelerates the protein breeding cycles and makes predictions for large-scale production more accurate.

Apoptosis↗
PubMed2003

High throughput screening of G-protein coupled receptors via flow cytometry.

The molecular assemblies of signal transduction components, for example kinases and their target proteins or receptor-ligand complexes and intracellular signaling molecules, are critical for biological functions in cells. To better understand the interactions of these molecular assemblies and to screen for new pharmaceutics that could control and modulate these types of interactions, we have focused on developing high throughput approaches for the analysis of G-protein coupled receptors via flow cytometry. Flow cytometry offers a number of advantages including real-time collection of multicomponent data, and together with improvements in sample handling, the high throughput sampling rate is up to 100 samples per minute. For our targets, assemblies of solubilized GPCRs, a screening platform of a dextran bead has proven to be flexible, allowing different surface chemistries on the beads. The bead can be either ligand-labeled or have epitope-linked proteins attached to the bead surface, enabling several molecular assemblies to be constructed and analyzed. A major improvement with this system is that for screening ligands for GPCRs the underlying mechanism of action for these compounds can be investigated and incorporated into the definition of a 'hit'. Our current screening system is capable of simultaneously distinguishing GPCR agonists and antagonists.

Animals↗
PubMed2004

Seeing 'cool' and 'hot'--infrared thermography as a tool for non-invasive, high-throughput screening of Arabidopsis guard cell signalling mutants.

The use of Arabidopsis mutants defective in abscisic acid (ABA) perception has been instrumental in the understanding of stomatal function, in particular, ABA signalling in guard cells. The considerable attention devoted to ABA signalling in guard cells is due in part to (1) the fundamental role of ABA in drought stress and (2) the use of a screening protocol based on the sensitivity of seed germination to ABA. Such a screen has facilitated the isolation of ABA signalling mutants with genetic lesions that exert pleiotropic effects at the whole plant level. As such, there is a requirement for new approaches to complement the seed germination screen. The recent advances made in the use of infrared thermography as a non-invasive, high-throughput tool are reviewed here and the versatility of this technique for screening Arabidopsis defective in stomatal regulation is highlighted.

Abscisic Acid↗
PubMed2003

Hit and lead generation: beyond high-throughput screening.

The identification of small-molecule modulators of protein function, and the process of transforming these into high-content lead series, are key activities in modern drug discovery. The decisions taken during this process have far-reaching consequences for success later in lead optimization and even more crucially in clinical development. Recently, there has been an increased focus on these activities due to escalating downstream costs resulting from high clinical failure rates. In addition, the vast emerging opportunities from efforts in functional genomics and proteomics demands a departure from the linear process of identification, evaluation and refinement activities towards a more integrated parallel process. This calls for flexible, fast and cost-effective strategies to meet the demands of producing high-content lead series with improved prospects for clinical success.

Amino Acid Motifs↗
PubMed2000

Pattern recognition analysis of endogenous cell metabolites for high throughput mode of action identification: removing the postscreening dilemma associated with whole-organism high throughput screening.

Although whole-organism HTS can give clear indications of in vivo activity, typically few clues are given as to the mechanism of action (MOA), and determining the MOA for large numbers of active compounds can be costly and complex-an alternative approach is required. This report demonstrates that it is possible to conduct relatively high throughput MOA characterization of HTS hits utilizing a single sample preparation and analytical method. By monitoring a wide range of endogenous cellular metabolites via (1)H nuclear magnetic resonance spectroscopy, the MOA of herbicides can be predicted using computational methods to compare the metabolite perturbation patterns. Herbicides that induce a characteristic pattern of metabolic perturbation in maize include inhibitors of acetolactate synthase, acetyl co-enzyme A carboxylase, protoporphyrinogen oxidase, 5-enolpyruvylshikimate-3-phosphate synthase, and phytoene desaturase. In soya, photosystem II inhibitors can also be detected, further demonstrating that this method is not limited to inhibitors of enzymes that directly act upon endogenous metabolites, or a single species. The methods, including data analysis, can be readily automated, enabling relatively high throughput MOA elucidation of whole-organism screen hits. Additionally, for compounds with a novel MOA, this approach may lead to MOA identification faster than traditional methods. It is envisaged that application of these data analysis methods to other data types-for example, transcription (mRNA) or translation (protein) profiles-is likely to permit higher throughput with smaller sample requirements, along with ability to discriminate MOAs that are not adequately discriminated based upon endogenous metabolite profiles.

Automation↗
PubMed2006

High-throughput screening for the discovery of inhibitors of fatty acid amide hydrolase using a microsome-based fluorescent assay.

Fatty acid amide hydrolase (FAAH) is a membrane-associated enzyme that catalyzes the hydrolysis of several endogenous bioactive lipids, including anandamide (AEA), N-palmitoylethanolamine (PEA), oleamide, and N-oleoylethanolamine (OEA). These fatty acid amides participate in many physiological activities such as analgesia, anxiety, sleep modulation, anti inflammatory responses, and appetite suppression. Because FAAH plays an essential role in controlling the tone and activity of these endogenous bioactive lipids, this enzyme has been implicated to be a drug target for the therapeutic management of pain, anxiety, and other disorders. In an effort to discover FAAH inhibitors, the authors have previously reported the development of a novel fluorescent assay using purified FAAH microsomes as an enzyme source and a fluorogenic substrate, arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA). Herein, the authors have adapted this assay to a high-throughput format and have screened a large library of small organic compounds, identifying a number of novel FAAH inhibitors. These data further verify that this fluorescent assay is sufficiently robust, efficient, and low-cost for the identification of FAAH inhibitory molecules and open this class of enzymes for therapeutic exploration.

Amidohydrolases↗
PubMed2004

Identification of selective inhibitors for the glycosyltransferase MurG via high-throughput screening.

Nucleotide-glycosyltransferases (NDP-Gtfs) play key roles in a wide range of biological processes. It is difficult to probe the roles of individual glycosyltransferases or their products because, with few exceptions, selective glycosyltransferase inhibitors do not exist. Here, we investigate a high-throughput approach to identify glycosyltransferase inhibitors based on a fluorescent donor displacement assay. We have applied the screen to E. coli MurG, an enzyme that is both a potential antibiotic target and a paradigm for a large family of glycosyltransferases. We show that the compounds identified in the donor-displacement screen of MurG are selective for MurG over other enzymes that use similar or identical substrates, including structurally related enzymes. The donor displacement assay described here should be adaptable to many other NDP-Gtfs and represents a new strategy to identify selective NDP-Gtf inhibitors.

Animals↗
PubMed2005

High throughput screening of meningioma biomarkers using a tissue microarray.

Meningiomas are histologically and clinically diverse CNS neoplasms with few available immunohistochemical markers of differentiation and progression. Therefore, we investigated a panel of potentially useful meningioma-associated biomarkers using high throughput tissue microarray immunohistochemistry (TMA-IHC) with a TMA that includes 9 hemangiopericytomas (HPCs) and 41 meningiomas spanning all grades, as well as two subsets of atypical meningiomas, stratified according to clinical behavior. Antibodies utilized were progesterone receptor (PR), epithelial membrane antigen (EMA), cathepsin D, E-cadherin, platelet derived growth factor (PDGF) receptor beta, PDGF BB ligand, survivin, epithelial growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF). In most cases, frequencies of tumor positivity were similar to those previously reported using whole section IHC. EMA, E-cadherin, and PDGFR-beta staining patterns distinguished the anaplastic meningiomas from the HPCs (P < 0.001, P = 0.02, P = 0.015, respectively). As in prior studies, PR and cathepsin D expression were inversely proportional to tumor grade. However, PR and EGFR were also differentially expressed between symptomatic, surgically resected benign meningiomas and incidental meningiomas found at autopsy. We conclude that (1) TMA-IHC is an accurate and efficient way to rapidly assess biomarkers in meningeal tumors, (2) EMA, E-cadherin, and PDGFR-beta are useful in distinguishing anaplastic meningiomas from HPCs, and (3) the expression patterns for incidental meningiomas differ slightly from their surgically resected symptomatic counterparts.

Biomarkers, Tumor↗