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PubMed2004

Microspotting streptavidin and double-stranded DNA arrays on gold for high-throughput studies of protein-DNA interactions by surface plasmon resonance microscopy.

We present two strategies for microspotting 10 x 12 arrays of double-stranded DNAs (dsDNAs) onto a gold-coated glass slide for high-throughput studies of protein-DNA interactions by surface plasmon resonance (SPR) microscopy. Both methods use streptavidin (SA) as a linker layer between a biotin-containing mixed self-assembled monolayer (SAM) and biotinylated dsDNAs to produce arrays with high packing density. The primary mixed SAM is produced from biotin- and oligo(ethylene glycol)-terminated thiols bonded as thiolates onto the gold surface. In the first method, a robotic microspotter is used to deliver nanoliter droplets of dsDNA solution onto a uniform layer of this SA ( approximately 2 x 10(12) SA/cm(2)). SPR microscopy shows a density of (5-6) x 10(11) dsDNA/cm(2) (0.2-0.3 dsDNA/SA) in the array elements. The second method uses instead a microspotted array of this SA linker layer, onto which the microspots of dsDNA are added with spatial registry. SPR microscopy before addition of the dsDNA shows a SA coverage of 2 x 10(12) SA/cm(2) within the spots and a dsDNA density of 8.5 +/- 3.5 x 10(11) dsDNA/cm(2) (0.3-0.7 dsDNA/SA, depending on the length of dsDNA) after dsDNA spotting. We demonstrate the ability to simultaneously monitor protein binding with the SPR microscope in many 200-microm spots with 1-s time resolution and sensitivity to <1 pg of protein.

Adsorption↗
PubMed2006

Identification of small-molecule inhibitors of protein kinase B (PKB/AKT) in an AlphaScreenTM high-throughput screen.

Protein kinase B (PKB/AKT) has been identified as a promising cancer drug target downstream of PI3 kinase. To find novel inhibitors of PKB/AKT kinase activity for progression as anticancer agents, the authors have used a high-throughput screen based on AlphaScreentrade mark technology. A known kinase inhibitor, the isoquinoline H8, was used as a positive control with mean inhibition in the screen of 43.4% +/- 13.1%. The performance of the screen was highly acceptable with Z' and Z factors of 0.83 +/- 0.07 and 0.75 +/- 0.04, respectively. A number of confirmed hits ( approximately 0.1% hit rate) were identified from 63,500 compounds screened. Five compounds have previously been described as PKB inhibitors, demonstrating the ability of the assay to find authentic inhibitors of the enzyme. Five hits had the potential to interfere with the assay signal and were deemed to be false positives. Two compounds were nonspecific inhibitors of PKB as enzyme inhibition in a filter-based assay was markedly reduced in the presence of 0.01% Triton X100. The authors now include an interference assay during hit confirmation procedures and check compound activity in the presence of Triton X100 in an attempt to eliminate nonspecific aggregators at an early stage.

Drug Evaluation, Preclinical↗
PubMed1999

High-Throughput Testing of Heterogeneous Catalyst Libraries Using Array Microreactors and Mass Spectrometry.

Eighty isolated channels are present in the array microreactor system that was coupled to a quadrupole mass spectrometer for high-throughput testing of heterogeneous catalyst libraries. The system was used to monitor the activities, selectivities, and reaction kinetics of 66 ternary Pt/Pd/In catalysts for the dehydrogenation of cyclohexane to benzene over 24 h. It was possible to screen the entire 80-channel library in less than 10 minutes (see picture).

Journal Article↗
PubMed2006

Streamlining lead discovery by aligning in silico and high-throughput screening.

Lead discovery in the pharmaceutical environment is largely an industrial-scale process in which it is typical to screen 1-5 million compounds in a matter of weeks using High Throughput Screening (HTS). This process is a very costly endeavor. Typically a HTS campaign of 1 million compounds will cost anywhere from $500000 to $1000000. There is consequently a great deal of pressure to maximize the return on investment by finding fast and more effective ways to screen. A panacea that has emerged over the past few years to help address this issue is in silico screening. In silico screening is now incorporated in all areas of lead discovery; from target identification and library design, to hit analysis and compound profiling. However, as lead discovery has evolved over the past few years, so has the role of in silico screening.

Bayes Theorem↗
PubMed2003

High-throughput miniature cylindrical ion trap array mass spectrometer.

A fully multiplexed cylindrical ion trap (CIT) array mass spectrometer with four parallel ion source/mass analyzer/detector channels has been built to allow simultaneous high-throughput analysis of multiple samples. A multielement external chemical ionization/electron ionization source was coupled to a parallel array of CITs each of equal size (internal radius 2.5 mm), and the signal was recorded using an array of four miniature (2-mm inner diameter) electron multipliers. Using external electron ionization, the spectra of four separate samples were recorded simultaneously in real time using a four-channel preamplifier system and a data acquisition program written using LabVIEW software. These experiments mark the first demonstration of externally generated ions being successfully trapped in a miniature CIT mass analyzer. The instrument currently provides mass/charge range of approximately m/z 50-500. Average peak width is m/z 0.3, corresponding to a resolution of 1000 at m/z 300. The four-channel mass spectrometer is housed in a single vacuum manifold and operated with a single set of control electronics. The modular design of this instrument allows scale-up to many more channels of analysis for future applications in the areas of industrial process monitoring and combinatorial analysis and in the fields of proteomics and metabolomics.

Journal Article↗
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↗
PubMed2004

Discovery of potent inhibitors of dihydroneopterin aldolase using CrystaLEAD high-throughput X-ray crystallographic screening and structure-directed lead optimization.

Potent inhibitors of 7,8-dihydroneopterin aldolase (DHNA; EC 4.1.2.25) have been discovered using CrystaLEAD X-ray crystallographic high-throughput screening followed by structure-directed optimization. Screening of a 10 000 compound random library provided several low affinity leads and their corresponding X-ray crystal structures bound to the enzyme. The presence of a common structural feature in each of the leads suggested a strategy for the construction of a directed library of approximately 1000 compounds that were screened for inhibitory activity in a traditional enzyme assay. Several lead compounds with IC(50) values of about 1 microM against DHNA were identified, and crystal structures of their enzyme-bound complexes were obtained by cocrystallization. Structure-directed optimization of one of the leads thus identified afforded potent inhibitors with submicromolar IC(50) values.

Aldehyde-Lyases↗
PubMed2005

Development of a cell-based high-throughput specificity screen using a hepatitis C virus-bovine viral diarrhea virus dual replicon assay.

The hepatitis C virus (HCV) replicon is a unique system for the development of a high-throughput screen (HTS), since the analysis of inhibitors requires the quantification of a decrease in a steady-state level of HCV RNA. HCV replicon replication is dependent on host cell factors, and any toxic effects may have a significant impact on HCV replicon replication. Therefore, determining the antiviral specificity of compounds presents a challenge for the identification of specific HCV inhibitors. Here we report the development of an HCV/bovine viral diarrhea virus (BVDV) dual replicon assay suitable for HTS to address these issues. The HCV reporter enzyme is the endogenous NS3 protease contained within the HCV genome, while the BVDV reporter enzyme is a luciferase enzyme engineered into the BVDV genome. The HTS uses a mixture of HCV and BVDV replicon cell lines placed in the same well of a 96-well plate and isolated in the same cell backgrounds (Huh-7). The format consists of three separate but compatible assays: the first quantitates the amount of cytotoxicity based upon the conversion of Alamar blue dye via cellular enzymes, while the second indirectly quantitates HCV replicon replication through measurement of the amount of NS3 protease activity present. The final assay measures the amount of luciferase activity present from the BVDV replicon cells, as an indicator of the specificity of the test compounds. This HCV/BVDV dual replicon assay provides a reliable format to determine the potency and specificity of HCV replicon inhibitors.

Animals↗
PubMed2006

High throughput modular chambers for rapid evaluation of anesthetic sensitivity.

BACKGROUND: Anesthetic sensitivity is determined by the interaction of multiple genes. Hence, a dissection of genetic contributors would be aided by precise and high throughput behavioral screens. Traditionally, anesthetic phenotyping has addressed only induction of anesthesia, evaluated with dose-response curves, while ignoring potentially important data on emergence from anesthesia. METHODS: We designed and built a controlled environment apparatus to permit rapid phenotyping of twenty-four mice simultaneously. We used the loss of righting reflex to indicate anesthetic-induced unconsciousness. After fitting the data to a sigmoidal dose-response curve with variable slope, we calculated the MAC(LORR) (EC50), the Hill coefficient, and the 95% confidence intervals bracketing these values. Upon termination of the anesthetic, Emergence timeRR was determined and expressed as the mean +/- standard error for each inhaled anesthetic. RESULTS: In agreement with several previously published reports we find that the MAC(LORR) of halothane, isoflurane, and sevoflurane in 8-12 week old C57BL/6J mice is 0.79% (95% confidence interval = 0.78-0.79%), 0.91% (95% confidence interval = 0.90-0.93%), and 1.96% (95% confidence interval = 1.94-1.97%), respectively. Hill coefficients for halothane, isoflurane, and sevoflurane are 24.7 (95% confidence interval = 19.8-29.7%), 19.2 (95% confidence interval = 14.0-24.3%), and 33.1 (95% confidence interval = 27.3-38.8%), respectively. After roughly 2.5 MAC(LORR) x hr exposures, mice take 16.00 +/- 1.07, 6.19 +/- 0.32, and 2.15 +/- 0.12 minutes to emerge from halothane, isoflurane, and sevoflurane, respectively. CONCLUSION: This system enabled assessment of inhaled anesthetic responsiveness with a higher precision than that previously reported. It is broadly adaptable for delivering an inhaled therapeutic (or toxin) to a population while monitoring its vital signs, motor reflexes, and providing precise control over environmental conditions. This system is also amenable to full automation. Data presented in this manuscript prove the utility of the controlled environment chambers and should allow for subsequent phenotyping of mice with targeted mutations that are expected to alter sensitivity to induction or emergence from anesthesia.

Journal Article↗
PubMed1998

A high-throughput graphics library designed for a portable molecular structure viewer.

We have equipped our graphics library with efficient functions so that a molecular structure viewer program can provide both portability and high-throughput rendering without hardware acceleration. The library renders graphics primitives into off-screen image memory with novel functions such as a point list for the vertices of three-dimensional graphical primitives, scan conversions of sphere and cylinder primitives, and z-buffered bit-block transfer. A molecular structure viewer program was implemented with the graphics library, giving reasonable rendering performance on conventional UNIX workstations with the X-Window system. The use of dynamic linking also lends a flexible extension facility to this software system. An advanced polygon renderer can be provided as an plug-in style extension module as well as other functional modules that are specific to the application program. The design of our graphics library is not only effective in molecular graphics but is also applicable for general three-dimensional graphics software systems.

Computer Graphics↗
PubMed2003

A high-throughput method for the conversion of CO2 obtained from biochemical samples to graphite in septa-sealed vials for quantification of 14C via accelerator mass spectrometry.

The growth of accelerator mass spectrometry as a tool for quantitative isotope ratio analysis in the biosciences necessitates high-throughput sample preparation. A method has been developed to convert CO(2) obtained from carbonaceous samples to solid graphite for highly sensitive and precise (14)C quantification. Septa-sealed vials are used along with commercially available disposable materials, eliminating sample cross contamination, minimizing complex handling, and keeping per sample costs low. Samples containing between 0.25 and 10 mg of total carbon can be reduced to graphite in approximately 4 h in routine operation. Approximately 150 samples per 8-h day can be prepared by a single technician.

Biochemistry↗
PubMed2002

A homogeneous high throughput nonradioactive method for measurement of functional activity of Gs-coupled receptors in membranes.

A method is described for measuring the activity of G(s)-coupled receptors in a nonradioactive homogeneous membrane-based assay. This method has several major advantages over currently used methods for measuring functional activity of G(s)-coupled receptors. The assay is high throughput (>150,000 data points/day using a single reader). Dimethyl sulfoxide tolerance is high ( approximately 10%). Compared to complex cell-based assays, there is limited potential for nonspecific compound action. This resulted in low compound hit rates in robustness screening, where hit rates from a simulated screen were 1.0% (antagonist screen) and 0.1% (agonist screen). No continuous cell culture is required for the assay, reducing cell culture overheads and allowing the screen to run every day. Automation is simple and requires no temperature- or humidity-controlled incubation. No radioactivity is required. The method relies on measurement of cyclic AMP (cAMP) generation by fluorescence polarization assay using commercially available reagents. Membranes (1-2 microg protein per well, containing anti-cAMP antibody) are transferred to 384-well plates containing 1 microl test compound. For antagonist screens, agonist is added 15 min later. After 30 min incubation at room temperature, one further assay reagent (fluorescein-cAMP in a buffer containing detergent) is added. The signal may be read after 1 h and is stable for greater than 12 h. Typical Z' for the assay is approximately 0.5.

Adrenergic beta-Agonists↗
PubMed2001

High-throughput single molecule screening of DNA and proteins.

We report a novel imaging technology for real time comprehensive analysis of molecular alterations in cells and tissues appropriate for automation and adaptation to high-throughput applications. With these techniques it should eventually be possible to perform simultaneous analysis of the entire contents of individual biological cells with a sensitivity and selectivity sufficient to determine the presence or absence of a single copy of a targeted analyte (e.g., DNA region, RNA region, protein), and to do so at a relatively low cost. The technology is suitable for DNA and RNA through sizing or through fluorescent hybridization probes, and for proteins and small molecules through fluorescence immunoassays. This combination of the lowest possible detection limit and the broadest applicability to biomolecules represents the final frontier in bioanalysis. The general scheme is based on novel concepts for single molecule detection (SMD) and characterization recently demonstrated in our laboratory. Since minimal manipulation is involved, it should be possible to screen large numbers of cells in a short time to facilitate practical applications. This opens up the possibility of finding single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification.

Animals↗
PubMed2000

High-throughput SNP genotyping with the Masscode system.

QIAGEN Genomics, Inc, has developed the Masscode tagging system for DNA labeling and detection. In this application, the Masscode system is described as applied to high-throughput single-nucleotide polymorphism (SNP) genotyping. The labeling system is based on a small-molecular-weight tag that is covalently attached through a photocleavable linker to a DNA oligonucleotide. The tagged oligonucleotide is used as a primer in an allele-specific PCR SNP discrimination assay. The allele-specific amplicons are differentiated through their Masscode tag assignments. After a photolysis step to cleave the tags from the amplicon, the samples are introduced into a single quadrupole mass spectrometry detection system for analysis. Genotyping determinations are based on the relative proportions of the paired allele tags. The system has a lower limit of detection in the femtomolar range (10(-15) M). At present, 30 different Masscode tags may be used simultaneously in a multiplex fashion to routinely provide more than 40,000 SNP genotyping measurements daily. Further developments will allow for the simultaneous detection of several hundred tags.

Alleles↗
PubMed2006

High-throughput human metabolism and toxicity analysis.

Poor drug candidate safety profiles are often identified late in the drug development process, manifesting themselves in the preclinical and clinical phases and significantly contributing to the high cost and low yield of drug discovery. As a result, new tools are needed to accelerate the assessment of drug candidate toxicity and human metabolism earlier in the drug development process, from primary drug candidate screening to lead optimization. Although high-throughput screens exist for much of the discovery phase of drug development, translating such screening techniques into platforms that can accurately mimic the human in vivo response and predict the impact of drug candidates on human toxicology has proven difficult. Nevertheless, some success has been achieved in recent years, which may ultimately yield widespread acceptance in the pharmaceutical industry.

Animals↗
PubMed2006

Validation of an optical sensor-based high-throughput bioreactor system for mammalian cell culture.

Cell culture optimization is a labor-intensive process requiring a large number of experiments to be conducted under varying conditions. Here we describe a high-throughput bioreactor system that allows 12 mini stirred-tank bioreactors to be operated simultaneously. All bioreactors are monitored by low-cost minimally invasive optical sensors for pH and dissolved oxygen. The sensors consist of single-use patches affixed inside the bioreactors and monitored optically from the outside. Experimental results show that different sensing patches with the same composition respond consistently. The discrepancy between different pH sensors is less than 0.1 pH units over most of their responsive range. The discrepancy between different dissolved oxygen sensors is less than 10% over the whole range from 0% to 100% dissolved oxygen. The consistency of the sensing system ensures that only an initial one-time calibration is required for the sensing patches. After that, a calibration code is generated and sensing patches of the same composition can be used directly. This greatly reduces the time and cost required for monitored multi-bioreactor operations. We used SP2/0 myeloma/mouse hybridoma cell cultures to demonstrate reactor performance consistency. Transcriptional profiling, HPLC analysis, viable cell count, and viability inspection show that the presence of sensing patches and the use of optical monitoring have no apparent effect on the metabolism of the cells.

Animals↗
PubMed2006

A bifunctional monolithic column for combined protein preconcentration and digestion for high throughput proteomics research.

Enzymatic digestion of proteins is a key step in protein identification by mass spectrometry (MS). Traditional solution-based protein digestion methods require long incubation times and are limitations for high throughput proteomics research. Recently, solid phase digestion (e.g. trypsin immobilization on solid supports) has become a useful strategy to accelerate the speed of protein digestion and eliminate autodigestion by immobilizing and isolating the enzyme moieties on solid supports. Monolithic media is an attractive support for immobilization of enzymes due to its unique properties that include fast mass transfer, stability in most solvents, and versatility of functional groups on the surfaces of monoliths. We prepared immobilized trypsin monolithic capillaries for on-column protein digestion, analyzed the digested peptides through LC/FTICR tandem MS, and compared peptide mass fingerprinting by MALDI-TOF-MS. To further improve the digestion efficiency for low abundance proteins, we introduced C4 functional groups onto the monolith surfaces to combine on-column protein enrichment and digestion. Compared with immobilized trypsin monolithic capillaries without C4, the immobilized trypsin-C4 monolith showed improved digestion efficiency. A mechanism for increased efficiency from the combination of sample enrichment and on-column digestion is also proposed in this paper. Moreover, we investigated the effects of organic solvent on digestion and detection by comparing the observed digested peptide sequences. Our data demonstrated that all columns showed good tolerance to organic solvents and maintained reproducible enzymatic activity for at least 30 days.

Animals↗
PubMed2006

An improved, high-throughput method for detection of bluetongue virus RNA in Culicoides midges utilizing infrared-dye-labeled primers for reverse transcriptase PCR.

A new rapid (less than 6h from insect-to-results) high-throughput assay that is sensitive and specific for detecting BTV RNA in Culicoides biting midges is reported. Homogenization and extraction of nucleic acids from individual Culicoides specimens were performed in a 96-well plate format using specialized beads in a homogenization buffer compatible with cell culture and RNA extraction. A portion of homogenate (10%) from each specimen was retained for confirmatory infectious virus isolation, while the remaining 90% was used for RNA extraction. The RNA was used in a single step reverse transcriptase PCR (RT-PCR) reaction with infrared (IR)-dye-labeled primers. The RT-PCR products were visualized in agarose gels with an infrared scanner. The adaptation of IR-dye-labeled primers in combination with a one step RT-PCR resulted in a detection limit of 0.5 pfu of purified BTV RNA. All 24 serotypes of BTV prototype strains and none of the 8 serotypes of the closely related epizootic hemorrhagic disease virus (EHDV) prototype strains were detected.

Animals↗