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PubMed2006

High-throughput mutational analysis of the human cancer genome.

Cancer is essentially a genetic disease. Recent high-throughput mutational analyses of gene families in human colorectal, breast and lung cancer, myeloproliferative disorders, and other tumor types have identified a number of kinases and phosphatases that are mutated in the human cancer genome. This approach has been proven to be an efficient way to catalog tumor-specific mutations in human cancers. Although there are still some technical hurdles to overcome, it is not a far-reaching goal to perform genome-wide mutational analysis in all different tumor types. Systematic cataloging of tumor-specific mutations in the human cancer genome not only will lead to new insights into the mechanisms of tumorigenesis, but also provide unprecedented opportunities in the design of novel therapeutics for cancer patients. Personalized cancer therapy based on tumor-specific mutations will be a realistic goal in the near future.

Breast Neoplasms↗

PubMed2006

Slow-binding human serine racemase inhibitors from high-throughput screening of combinatorial libraries.

One-bead one-compound combinatorial chemistry together with a high-throughput screen based on fluorescently labeled enzyme allowed the identification of slow binding inhibitors of human serine racemase (hSR). A peptide library of topographically segregated encoded resin beads was synthesized, and several hSR-binding compounds were isolated, identified, and resynthesized for further kinetic study. Of these, several showed inhibitory effects with moderate potency (high micromolar K(I)s) toward hSR. A clear structural motif was identified consisting of 3-phenylpropionic acid and histidine moieties. Importantly, the inhibitors identified showed no structural similarities to the natural substrate, L-serine. Detailed kinetic analyses of the properties of selected inhibitors show that the screening protocol used here selectively identifies slow binding inhibitors. They provide a pharmacophore for the future isolation of more potent ligands that may prove useful in probing and understanding the biological role of hSR.

Combinatorial Chemistry Techniques↗

PubMed2006

Evaluation of the iCE280 Analyzer as a potential high-throughput tool for formulation development.

The iCE280 Analyzer (iCE280) was evaluated for its potential application as a high-throughput tool to determine pI and separate charge related species using glycosylated, non-glycosylated and pegylated protein therapeutics as models. Resolution was achieved for glycosylated and non-glycosylated molecules, but remained a challenge for pegylated proteins. The sources of charge variants were determined to be the presence of C-terminal lysine residues, sialic acid content, and deamidation. Limited assay performance evaluation demonstrated that the method was linear in the concentration range of 2-333 microg/ml of IgG with linear regression coefficients of 0.984, 0.998, and 0.990 for acidic, main and basic species, respectively. Limit of detection and limit of quantitation were determined to be 3 and 11 microg/ml. The R.S.D. for intra- and inter-day precision as well as reproducibility was determined to be 0.2% or less for all pI values and 1.4% or less for acidic and main peak area distribution; the R.S.D. for basic peak area distribution was 5.7% or less. Robustness testing was performed by deliberately deviating +/-50% of pharmalyte concentration away from the desired condition. This deviation revealed a pI shift of only 0.06 units and resulted in no significant impact on area percent distribution. Utilization of iCE280 Analyzer eliminated the mobilization step associated with traditional capillary isoelectric focusing analysis and increased analytical throughput at least 2-fold.

Carboxypeptidase B↗

PubMed2026

Identification and characterization of ectopic chromosomal amplifications in acute myeloid leukemia cell limes using high-throughput chromosome conformation capture screening.

Despite advanced molecular diagnostics, improving outcomes for refractory acute myeloid leukemia (AML) remains challenging. Although many cancer-related genes are identified, their molecular mechanisms are not fully elucidated. Amplification is a mechanism of cancer-associated gene activation, and ectopic gene amplification may have particularly high pathological significance. However, research on ectopically amplified cancer-associated genes in leukemia remains limited. Here, we evaluated the usefulness of high-throughput chromosomal conformation capture (Hi-C) as a screening method for ectopic gene amplification and assessed whether ectopic amplification of cancer-associated genes may represent a general phenomenon in AML. We screened the U-937 and NB-4 cell lines using in situ Hi-C. Regions appearing as "high-intensity bands" in Hi-C contact maps were identified and validated using fluorescence in situ hybridization (FISH). Additionally, copy number variation analysis was performed using whole-genome sequencing (WGS) to extract cancer-associated genes with ectopic amplification. In the U-937, three genomic regions showing "high-intensity bands" were identified and confirmed as ectopic amplifications-including PDCD1LG2 (PD-L2), CD274 (PD-L1), and JAK2; that is, four copies were detected by WGS, and amplification signals were observed by FISH. In the NB-4, four such regions were detected, including MYC and KRAS, with expression level of 498 transcripts per million (TPM) and 34 TPM, respectively. Copy number variation analysis further identified multiple cancer-associated genes with ectopic amplification. Overall, these findings demonstrate the presence of ectopic amplification of cancer-associated genes in AML cell lines and support the usefulness of Hi-C as a screening method for detecting such genomic alterations.

Acute myeloid leukemia↗

PubMed1999

Multiple solenoidal microcoil probes for high-sensitivity, high-throughput nuclear magnetic resonance spectroscopy.

Two designs for incorporating multiple solenoidal microcoils into a single probe head are presented to increase the throughput of high-resolution NMR. Through a combination of radio frequency switches and low-noise amplifiers, multiple NMR spectra can be acquired in the same time as a single spectrum from a conventional probe consisting of one coil. Since this method does not compromise sensitivity with regard to the individual microcoils, throughput increases linearly with the number of coils. Only one receiver is needed, and data acquisition parameters can be optimized for each sample. Specifically, a four-coil system has been implemented for proton NMR at 250 MHz using a wide-bore magnet, with an observe volume of 28 nL for each microcoil. Signal cross-contamination was approximately 0.2% between individual coils, and simultaneous one- and two-dimensional spectra have been obtained from samples of fructose, galactose, adenosine triphosphate, and chloroquine (7 nmol of each compound). A more compact two-coil configuration has also been designed for operation at 500 MHz, with observe volumes of 5 and 31 nL for the two coils. One- and two-dimensional spectra were acquired from samples of 1-butanol (55 nmol) and ethylbenzene (250 nmol).

Image Processing, Computer-Assisted↗

PubMed2026

Development of a cell-based nanoluciferase reporter system for high-throughput screening of HBV cccDNA inhibitors.

Hepatitis B virus (HBV) persistence is sustained by the viral covalently closed circular DNA (cccDNA) minichromosome, which remains a major barrier to curative antiviral therapies. The lack of reliable quantitative cccDNA detection methods and surrogate markers has hindered efforts to target cccDNA in antiviral high-throughput screening (HTS). Here, we established a novel inducible cccDNA-dependent nanoluciferase (NLuc) reporter cell line, designated HepBLE12, by inserting an in-frame 11-amino acid split-NLuc HiBiT tag into the precore (pC) coding region of an HBV transgene. The resulting 1.3-kDa HiBiT tag on pC serves as the detection module of the split NLuc system, generating quantitative luminescence upon high-affinity complementation with the cognate 18-kDa LgBiT subunit in cell lysates. Notably, the HiBiT assay enables direct detection of intracellular HiBiT-pC protein rather than secreted HBeAg, providing a reporter signal more closely linked to cccDNA activity. HepBLE12 cells exhibited inducible and robust viral DNA replication, and the cccDNA-dependent HiBiT signal was validated under diverse experimental conditions that modulate cccDNA formation or transcription. We further miniaturized the assay to a 384-well format and optimized key parameters following standard HTS assay development practices. The assay was successfully automated and demonstrated excellent performance in a multi-day variability study and a pilot screen, with signal-to-background (S/B) ≈ 9, coefficient of variance (CV) < 10%, and average Z-factor value of 0.74, exceeding canonical HTS quality benchmarks. Together, the HepBLE12 cell-based HTS platform provides a robust and practical tool for identifying inhibitors targeting HBV cccDNA.

Hepatitis B virus↗

PubMed2005

Description and validation of a four-channel staggered LC-MS/MS system for high-throughput in vitro screens.

A parallel four-channel LC-MS/MS system for quantitative high-throughput in vitro screens is described. The system comprises four pumps, a four-valve autosampler equipped with a nine-port stream selector valve, and a triple quadrupole mass spectrometer. Staggered injections across the sample list are performed by four injector valves onto separate columns in such a manner that the chromatographic window of interest is always eluting from one of the columns into the mass spectrometer. To demonstrate the applicability of the system, three different compounds (verapamil, propanolol, dextromethorphan) were analyzed. The results showed that the sample throughput could be increased more than three times in comparison with a conventional single-column LC-MS/MS system. Validation results for the three compounds showed an accuracy of 85.0-108%, a precision (given as relative standard deviation) of 2.60-10.7%, and a carryover of less than 0.1%.

Caco-2 Cells↗

PubMed2004

Generic three-column parallel LC-MS/MS system for high-throughput in vitro screens.

A parallel three-column LC-MS/MS system for quantitative high-throughput in vitro screens is described. The robust novel system is composed of three LC pumps, an autosampler and a triple quadrupole mass spectrometer used in combination with three valves and three analytical LC columns in parallel configuration. Two of the three valves work in unison to select which column receives the injection, and the third valve selects which column is to be in line with the mass spectrometer. Improved sample throughput is achieved without sacrificing chromatographic separation quality or sensitivity. To demonstrate the applicability of the system, pools of five compounds (phenacetin, S-mephenytoin, bufuralol, midazolam and clomethiazole) were analyzed, together with an internal standard. The results show that the sample throughput can be increased significantly by reducing analysis time to 3 min per sample as compared to 8 min with a general gradient single-column system. Analysis of the five compounds shows an accuracy of 81-108% and a precision (given as relative standard deviation) of 1.5-14%. The system was further applied to samples from a metabolic stability assay in liver microsomes.

Chromatography, High Pressure Liquid↗

PubMed2000

Printing proteins as microarrays for high-throughput function determination.

Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

Biochemistry↗

PubMed2026

Comparison of endothelin-1 levels in plasma from human coronary arteries measured by enzyme linked immunosorbent assay and Olink high-throughput proteomics platform.

Endothelin-1 (ET-1) antagonists are increasingly being approved for new treatments for cardiovascular disease, where elevated ET-1 levels contribute to increased vasoconstriction. Further therapeutic targets, including coronary artery disease, are under investigation. The Olink Explore 3072 Proximity Extension Assay platform enables multiplexed high-throughput measurement of ~3000 plasma proteins, from minimal (≤6 µL) sample volumes. However, it is not known if the two oligonucleotide-tagged antibodies raised against preproET-11-212, used in this Olink assay, specifically measure biologically active ET-1 or the other inactive EDN1-encoded peptides, also secreted by human endothelial cells. Paired plasma samples from 29 patients with coronary artery disease were obtained, using a specialised intra-coronary sampling catheter, designed to obtain site specific biochemical information from within coronary arteries. We compared ET-1 concentrations measured with an ET-1 specific ELISA, demonstrated to have no cross-reactivity with other EDN1-encoded peptides versus values obtained using Olink Explore platform. Olink-measured ET-1 correlated significantly with ELISA-derived ET-1 levels (r = 0.53, p = 0.003), and Olink values predicted ELISA results. Olink ET-1 concentrations also correlated with ETB receptor levels (r = 0.40, p < 0.05). These findings indicate that the Olink Explore platform can detect relative changes in biologically active ET-1, supporting its use as a biomarker tool in clinical and translational studies.

Humans↗

PubMed2006

Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists.

Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources. Moreover, significant variability invariably occurs in cell yield, viability, expression levels, and target activities. In particular, stable expression of targets such as transient receptor potential A1 (TRPA1) causes toxicity, cell line degeneration, and loss of functional activity. Therefore, in an effort to identify TRPA1 antagonists, the authors used large-scale transiently transfected (LSTT) cells, enabling rapid establishment of assays suitable for HTS. LSTT cells, which could- be stored frozen for a long period of time (e.g., at least 42 weeks), retained TRPA1 protein expression and could be easily revived to produce robust and consistent signals in calcium influx and electrophysiological assays. Using cells from a single transfection, a chemical library of 700,000 compounds was screened, and TRPA1 antagonists were identified. The use of LSTT circumvented issues associated with stable TRPA1 expression, increased flexibility and consistency, and greatly reduced labor and cost. This approach will also be applicable to other pharmaceutical targets.

Calcium↗

PubMed2007

Microbial composition in bioaerosols of a high-throughput chicken-slaughtering facility.

The microbial composition of the air in various areas of a high-throughput chicken-slaughtering facility was investigated. Over a 4-mo period, 6 processing areas were sampled, and the influence of environmental factors was monitored. The highest counts of microorganisms were recorded in the initial stages of processing, comprising the receiving-killing and defeathering areas, whereas counts decreased toward the evisceration, air-chilling, packaging, and dispatch areas. Maximum microbial counts were as follows: coliforms, 4.9 x 10(3) cfu/m(3); Escherichia coli 3.4 x 10(3) cfu/m(3); Bacillus cereus, 5.0 x 10(4) cfu/m(3); Staphylococcus aureus, 1.6 x 10(4) cfu/m(3); Pseudomonas aeruginosa, 7.0 x 10(4) cfu/m(3); presumptive Salmonella spp., 1.5 x 10(4) cfu/m(3); Listeria monocytogenes, 1.6 x 10(4) cfu/m(3); and fungi, 1.4 x 10(4) cfu/m(3). Higher counts of airborne microorganisms found in the receiving-killing and defeathering areas indicate the importance of controlling microbial levels before processing to prevent the spread of organisms downstream. This should limit the risk of carrying over contaminants from areas known to generate high counts to areas where the final food product is exposed to air and surface contamination.

Abattoirs↗

PubMed2006

Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries.

High-throughput screening (HTS) of chemical compounds to identify modulators of molecular targets is a mainstay of pharmaceutical development. Increasingly, HTS is being used to identify chemical probes of gene, pathway, and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal will require methodologies that efficiently generate pharmacological data from the primary screen and reliably profile the range of biological activities associated with large chemical libraries. Traditional HTS, which tests compounds at a single concentration, is not suited to this task, because HTS is burdened by frequent false positives and false negatives and requires extensive follow-up testing. We have developed a paradigm, quantitative HTS (qHTS), tested with the enzyme pyruvate kinase, to generate concentration-response curves for >60,000 compounds in a single experiment. We show that this method is precise, refractory to variations in sample preparation, and identifies compounds with a wide range of activities. Concentration-response curves were classified to rapidly identify pyruvate kinase activators and inhibitors with a variety of potencies and efficacies and elucidate structure-activity relationships directly from the primary screen. Comparison of qHTS with traditional single-concentration HTS revealed a high prevalence of false negatives in the single-point screen. This study demonstrates the feasibility of qHTS for accurately profiling every compound in large chemical libraries (>10(5) compounds). qHTS produces rich data sets that can be immediately mined for reliable biological activities, thereby providing a platform for chemical genomics and accelerating the identification of leads for drug discovery.

Combinatorial Chemistry Techniques↗

PubMed2001

Simple absorbance-based assays for ultra-high throughput screening.

In order to accommodate the predicted increase in screening required of successful pharmaceutical companies, miniaturized, high-speed HTS formats are necessary. Much emphasis has been placed on sensitive fluorescence techniques, but some systems, particularly enzymes interconverting small substrates, are likely to be refractory to such approaches. We show here that simple absorbance-based assays can be miniaturized to 10-microl volumes in 1536-well microplates compatible with the requirements for ultra-high throughput screening. We demonstrate that, with low-cost hardware, assay performance is wholly predictable from the 2-fold decrease in pathlength for fully filled 1536-well plates compared to 96- and 384-well microplates. A number of enzyme systems are shown to work in this high-density format, and the inhibition parameters determined are comparable with those in standard assay formats. We also demonstrate the utility of kinetics measurements in miniaturized format with improvements in assay quality and the ability to extract detailed mechanistic information about inhibitors.

Adenosine Triphosphatases↗

PubMed1998

High-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates.

Capillary array electrophoresis (CAE) microplates that can analyze 96 samples in less than 8 min have been produced by bonding 10-cm-diameter micromachined glass wafers to form a glass sandwich structure. The microplate has 96 sample wells and 48 separation channels with an injection unit that permits the serial analysis of two different samples on each capillary. An elastomer sheet with an 8 by 12 array of holes is placed on top of the glass sandwich structure to define the sample wells. Samples are addressed with an electrode array that makes up the third layer of the assembly. Detection of all lanes with high temporal resolution was achieved by using a laser-excited confocal fluorescence scanner. To demonstrate the functionality of these microplates, electrophoretic separation and fluorescence detection of a restriction fragment marker for the diagnosis of hereditary hemochromatosis were performed. CAE microplates will facilitate all types of high-throughput genetic analysis because their high assay speed provides a throughput that is 50 to 100 times greater than that of conventional slab gels.

Capillary Action↗

PubMedDate not supplied

High throughput encapsulation of murine fibroblasts in alginate using the JetCutter technology.

The JetCutter technology originally developed for high-throughput encapsulation of particles and substances into small beads was applied in this study for the entrapment of mammalian cells in alginate beads. In contrast to other established techniques such as the air jet droplet generation or laminar jet break-up, the JetCutter is capable of working with highly viscous fluids necessary for the production of stable beads based on hydrogels. A 1.5% (w/v) sodium alginate solution containing 2.0 x 106 murine fibroblasts/ml was processed under good manufacturing practice (GMP) conditions to beads with a mean diameter of 320 microm. The production capacity of the JetCutter technology was 5200 beads/s or to approximately 330 ml bead suspension per h. Beads were coated with poly-L-lysine and with an additional alginate layer to produce hollow microcapsules containing living cells. The influence of this method of encapsulation on the cell viability and morphology was investigated by light microscopic techniques. Encapsulated cells showed unchanged rates of proliferation and preserved morphology. They were able to survive in culture for extended periods of time. In conclusion, the JetCutter technology seems to be well suitable for alginate bead encapsulation of living mammalian cells.

Alginates↗

PubMed2004

High-throughput assay of rotenone in olive oil using atmospheric pressure chemical ionization tandem mass spectrometry.

A high-throughput method is presented for the determination of rotenone in contaminated matrices. The procedure involves atmospheric pressure chemical ionization tandem mass spectrometry with a triple-quadrupole instrument and the use as internal standard of a cycloadduct derivative of rotenone itself. The method was applied to the evaluation of the decay of rotenone in olives and olive oil at various times after exposure to the contaminant.

Indicators and Reagents↗

PubMed2003

Virtual screening to enrich hit lists from high-throughput screening: a case study on small-molecule inhibitors of angiogenin.

"Hit lists" generated by high-throughput screening (HTS) typically contain a large percentage of false positives, making follow-up assays necessary to distinguish active from inactive substances. Here we present a method for improving the accuracy of HTS hit lists by computationally based virtual screening (VS) of the corresponding chemical libraries and selecting hits by HTS/VS consensus. This approach was applied in a case study on the target-enzyme angiogenin, a potent inducer of angiogenesis. In conjunction with HTS of the National Cancer Institute Diversity Set and ChemBridge DIVERSet E (approximately 18,000 compounds total), VS was performed with two flexible library docking/scoring methods, DockVision/Ludi and GOLD. Analysis of the results reveals that dramatic enrichment of the HTS hit rate can be achieved by selecting compounds in consensus with one or both of the VS functions. For example, HTS hits ranked in the top 2% by GOLD included 42% of the true hits, but only 8% of the false positives; this represents a sixfold enrichment over the HTS hit rate. Notably, the HTS/VS method was effective in selecting out inhibitors with midmicromolar dissociation constants typical of leads commonly obtained in primary screens.

Binding Sites↗