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PubMed2005

Inferential literacy for experimental high-throughput biology.

Many biologists believe that data analysis expertise lags behind the capacity for producing high-throughput data. One view within the bioinformatics community is that biological scientists need to develop algorithmic skills to meet the demands of the new technologies. In this article, we argue that the broader concept of inferential literacy, which includes understanding of data characteristics, experimental design and statistical analysis, in addition to computation, more adequately encompasses what is needed for efficient progress in high-throughput biology.

Animals↗
PubMed2004

High-throughput determination of the free fraction of drugs strongly bound to plasma proteins.

Quantification of protein binding of new chemical entities is an important early screening step during drug discovery and is of fundamental interest for the estimation of safety margins during drug development. In this publication, we describe the development of a new high-throughput assay for the determination of the free drug fraction in plasma (fu). The new technique is an enhancement of the previously published erythrocytes partition method. It is based on the distribution of drugs between plasma water, plasma proteins, and solid-supported lipid membranes (Transil). The execution of protein binding studies by partitioning is dramatically simplified by substituting erythrocytes with commercially available Transil beads, and makes the method particularly suitable for high-throughput studies. Eight Bayer compounds from different compound classes covering a wide range of lipophilicities (log P = 1.9-5.6) and fu values (0.018-35%) were selected for validation of the assay. The results obtained by the new method and by either the erythrocytes partitioning technique or more conventional methods (ultrafiltration and equilibrium dialysis) are identical, confirming that the new method produces valid results even for drugs that are strongly bound to plasma proteins. Precision and accuracy of the data in the cases of very low and high fu values are comparable, indicating that the method is especially suited for highly lipophilic drugs that tend to adsorb to surfaces compared with other methods, like ultrafiltration or equilibrium dialysis, that may produce biased data. The method is also useful for the determination of binding parameters and the pH dependence of fu. In summary, this assay is well suited for high-throughput determination of protein binding during drug discovery and for extended protein binding studies during drug development.

Animals↗
PubMed2003

Use of high-throughput DNA microarrays to identify biomarkers for bladder cancer.

BACKGROUND: Numerous markers have been described to correlate to some extent with tumor stage and prognosis of patients with bladder cancer. The power of many of these biomarkers in detecting superficial disease or predicting the clinical outcome of individual tumors is limited, and alternative markers are still in demand. High-throughput microarrays represent novel means for cancer research and tumor marker discovery. APPROACH: The aim of this report was to discuss the application of DNA technologies to provide novel biomarkers for bladder cancer. CONTENT: Specific bladder tumor subtypes have distinct gene expression profiles. The use of high-throughput DNA microarrays allows identification of the most prevalent and relevant alterations within bladder tumors. Clusters of differentially expressed genes will become biomarkers to discriminate subgroups of patients with different histopathology or clinical outcome. Additionally, the identified individual molecular targets might be further validated and developed into novel serum or urinary biomarkers for the diagnosis and/or as prognostic factors to be applied in clinical practice. The diagnosis and prognosis of bladder cancer would be enhanced by the use of such markers, and the marker itself may constitute a therapeutic target when studied in appropriate patients and control groups. SUMMARY: Expression profiling with high-throughput DNA microarrays has the potential of providing critical clues for the management of bladder cancer patients. As the quality, standardization, and ease of use of the technology increase and the costs decrease, DNA microarrays will move from being a technology restricted to research to clinical laboratories in the near future.

Biomarkers, Tumor↗
PubMed2002

A bilayer cell-free protein synthesis system for high-throughput screening of gene products.

A high-throughput cell-free protein synthesis method has been described. The methodology is based on a bilayer diffusion system that enables the continuous supply of substrates, together with the continuous removal of small byproducts, through a phase between the translation mixture and substrate mixture. With the use of a multititer plate the system was functional for a prolonged time, and as a consequence yielded more than 10 times that of the similar batch-mode reaction. Combining this method with a wheat germ cell-free translation system developed by us, the system could produce a large amount of protein sufficient for carrying out functional analyses. This novel bilayer-based cell-free protein synthesis system with its simplicity, minimum time and low cost may be useful practical methodology in the post-genome era.

Bacterial Proteins↗
PubMed2001

Determination of beta-2 microglobulin levels in plasma using a high-throughput mass spectrometric immunoassay system.

A high-throughput mass spectrometric immunoassay (MSIA) system for the analysis of proteins directly from biological fluids is reported. A 96-well-format robotic workstation equipped with antibody-derivatized affinity pipet tips was used for the parallel extraction of specific proteins from samples and subsequent deposition onto 96-well arrayed matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) targets. Interferences from nonspecifically bound proteins were minimized through choice of appropriate affinity pipet tip derivatization chemistries. Sample preparation for MALDI-TOFMS was enhanced through the use of hydrophobic/hydrophilic contrasting targets, which also presented functionalities found to promote matrix/analyte crystal growth. Automated mass spectrometry was used in the unattended acquisition of data, resulting in an analysis rate of approximately 100 samples/h (biological fluid-->data). The quantitative MSIA of beta2m levels present in human plasma samples is given as illustration.

Adult↗
PubMedDate not supplied

Prediction of biological activity for high-throughput screening using binary kernel discrimination.

High-throughput screening has made a significant impact on drug discovery, but there is an acknowledged need for quantitative methods to analyze screening results and predict the activity of further compounds. In this paper we introduce one such method, binary kernel discrimination, and investigate its performance on two datasets; the first is a set of 1650 monoamine oxidase inhibitors, and the second a set of 101 437 compounds from an in-house enzyme assay. We compare the performance of binary kernel discrimination with a simple procedure which we call "merged similarity search", and also with a feedforward neural network. Binary kernel discrimination is shown to perform robustly with varying quantities of training data and also in the presence of noisy data. We conclude by highlighting the importance of the judicious use of general pattern recognition techniques for compound selection.

Algorithms↗
PubMed2004

Finding more needles in the haystack: A simple and efficient method for improving high-throughput docking results.

The technology underpinning high-throughput docking (HTD) has developed over the past few years to where it has become a vital tool in modern drug discovery. Although the performance of various docking algorithms is adequate, the ability to accurately and consistently rank compounds using a scoring function remains problematic. We show that by employing a simple machine learning method (naïve Bayes) it is possible to significantly overcome this deficiency. Compounds from the Available Chemical Directory (ACD), along with known active compounds, were docked into two protein targets using three software packages. In cases where HTD alone was able to show some enrichment, the application of naïve Bayes was able to improve upon the enrichment. The application of this methodology to enrich HTD results can be carried out without a priori knowledge of the activity of compounds and results in superior enrichment of known actives compared to the use of scoring methods alone.

Bayes Theorem↗
PubMed2005

Suaveolindole, a new mass-limited antibacterial indolosesquiterpene from Greenwayodendron suaveolens obtained via high-throughput natural products chemistry methods.

Utilizing high-throughput isolation, purification, and analysis methods applied to a natural products library, a new mass-limited antibacterial indolosesquiterpene, suaveolindole (1), was obtained from Greenwayodendron suaveolens. The miniaturization of the structure elucidation of 1 was performed primarily using the CapNMR probe. Compound 1 was found to possess significant in vitro antibacterial activity against the Gram-positive bacteria Bacillus subtilis (ATCC 43223), Staphylococcus aureus (ATTC 6538P), and methicillin-resistant Staphylococcus aureus (ATTC 33591), with MIC values of 4, 8, and 8 microg/mL, respectively.

Anti-Bacterial Agents↗
PubMed1998

Kaleidaseq: a Web-based tool to monitor data flow in a high throughput sequencing facility.

Tracking data flow in high throughput sequencing is important in maintaining a consistent number of successfully sequenced samples, making decisions on scheduling the flow of sequencing steps, resolving problems at various steps and tracking the status of different projects. This is especially critical when the laboratory is handling a multitude of projects. We have built a Web-based data flow tracking package, called Kaleidaseq, which allows us to monitor the flow and quality of sequencing samples through the steps of preparation of library plates, plaque-picking, preparation of templates, conducting sequencing reactions, loading of samples on gels, base-calling the traces, and calculating the quality of the sequenced samples. Kaleidaseq's suite of displays allows for outstanding monitoring of the production sequencing process. The online display of current information that Kaleidaseq provides on both project status and process queues sorted by project enables accurate real-time assessment of the necessary samples that must be processed to complete the project. This information allows the process manager to allocate future resources optimally and schedule tasks according to scientific priorities. Quality of the sequenced samples can be tracked on a daily basis, which allows the sequencing laboratory to maintain a steady performance level and quickly resolve dips in quality. Kaleidaseq has a simple easy-to-use interface that allows access to all major functions and process queues from one Web page. This software package is modular and designed to allow additional processing steps and new monitoring variables to be added and tracked with ease. Access to the underlying relational database is through the Perl DBI interface, which allows for the use of different relational databases. Kaleidaseq is available for free use by the academic community from http://www.cshl.org/kaleidaseq.

Arabidopsis↗
PubMed2004

Probabilistic cross-link analysis and experiment planning for high-throughput elucidation of protein structure.

Emerging high-throughput techniques for the characterization of protein and protein-complex structures yield noisy data with sparse information content, placing a significant burden on computation to properly interpret the experimental data. One such technique uses cross-linking (chemical or by cysteine oxidation) to confirm or select among proposed structural models (e.g., from fold recognition, ab initio prediction, or docking) by testing the consistency between cross-linking data and model geometry. This paper develops a probabilistic framework for analyzing the information content in cross-linking experiments, accounting for anticipated experimental error. This framework supports a mechanism for planning experiments to optimize the information gained. We evaluate potential experiment plans using explicit trade-offs among key properties of practical importance: discriminability, coverage, balance, ambiguity, and cost. We devise a greedy algorithm that considers those properties and, from a large number of combinatorial possibilities, rapidly selects sets of experiments expected to discriminate pairs of models efficiently. In an application to residue-specific chemical cross-linking, we demonstrate the ability of our approach to plan experiments effectively involving combinations of cross-linkers and introduced mutations. We also describe an experiment plan for the bacteriophage lambda Tfa chaperone protein in which we plan dicysteine mutants for discriminating threading models by disulfide formation. Preliminary results from a subset of the planned experiments are consistent and demonstrate the practicality of planning. Our methods provide the experimenter with a valuable tool (available from the authors) for understanding and optimizing cross-linking experiments.

Algorithms↗
PubMed2006

Identification of novel keratinocyte differentiation modulating compounds by high-throughput screening.

The authors have designed high-throughput screens to identify compounds that promote or inhibit terminal differentiation of primary human epidermal keratinocytes. Eleven known inhibitors of signaling pathways and approximately 4000 compounds of diverse structure were screened using an In-Cell Western system based on immunofluorescent staining of the terminal differentiation marker, involucrin. Staurosporine, a nonspecific protein kinase C inhibitor, and H89, a protein kinase A inhibitor, promoted expression of involucrin. Conversely, U0126, a MEK inhibitor, and SAHA or SBHA, 2 histone deacetylase inhibitors, reduced the expression of involucrin during calcium-induced stratification. In addition, the authors found 1 novel compound that induced keratinocyte differentiation and 2 novel compounds that were inhibitory to calcium-induced differentiation. The differentiation-inducing compound also inhibited growth of a human squamous cell carcinoma line by stimulating both differentiation and apoptosis. Because the compound affected the tumor cells at a lower concentration than primary keratinocytes, it may have potential as an antitumor therapy.

Apoptosis↗
PubMed1999

Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology.

High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm(2) digitized image of a 100-µmm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twistertrade mark (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software.

Journal Article↗
PubMed2005

High-throughput peptide synthesis.

The methodologies of high-throughput peptide synthesis are overviewed and discussed. Particular focus is given to the techniques applicable to laboratories with a limited budget. Automated solutions for synthetic problems are also discussed.

Automation↗
PubMed2006

Heat-inducible autolytic vector for high-throughput screening.

In directed evolution, a high-throughput screening system is often a prerequisite for sampling the enzyme variants. When the target enzyme is expressed intracellularly, for example when Escherichia coli is used as the host, chemical or enzymatic disruption of cell membrane is often required in many cases, which can be tedious, time-consuming, and costly. In this study, a set of heat-inducible autolytic vectors were constructed to solve this problem, in which the SRRz lysis gene cassette from bacteriophage lambda was placed downstream of heat-inducible promoters, lambda cI857/pR promoter and its mutant, c1857/pR(M). The artificial autolytic units were inserted into the backbone of pUC18 (away from the multiple cloning sites). For the wild promoter; cI857/pR, the SRRz lysis cassette was expressed by temperature up-shift from 28 degrees to 38 degrees C, and the lysis efficiency of transformed bacterial cells was found to be consistent and could reach 96.3% as measured by the reporter beta3-galactosidase assay. In order to obtain a higher cell growth rate, the mutant promoter cI857/pR(M) was utilized to allow bacteria growth at 35 degrees C and lysis at 42 degrees C. However; this heat-inducible system showed significant inconsistency in terms of lysis efficiency. Bacillus subtilis 168 lipase A gene was further inserted into the multiple cloning sites of the autolytic vector containing cI857/pR, and 93.7% of the expressed lipase activity was found in the culture medium upon heat induction, demonstrating the utility of the vector for expression and rapid extracellular assay of heterologous enzymes.

Bacillus subtilis↗
PubMed2004

Multiplexed RT- PCR for high throughput screening applications.

To increase efficiency of high throughput gene expression profiling, we established a new TaqMan RT-PCR (real-time reverse transcriptase-polymerase chain reaction with internal probes for the quantification of PCR products) method for quantitative mRNA expression analysis. In this procedure, we utilized poly-A mRNA capture plates and validated a multiplexed single tube RT-PCR assay for cell culture applications, including compound testing via gene induction measurement. In the described procedure, all steps including RNA extraction, RT and PCR are performed in the same tube, thus significantly enhancing throughput of this method. Optimization of conditions, including the number of cells necessary for detection of mRNA signal was performed. With a relatively abundant message such as GAPDH, we saw a linear response for all of the concentrations tested, from 10,000 cells to 10 cells. We have also demonstrated multiplexing of different targets within the PCR reactions. In these experiments, we combined VIC-labeled probes for GAPDH with several FAM-labeled probes obtained from Assays On Demand (Applied Biosystems). In the reported experiments, multiplexing did not affect the efficiency of RT-PCR. We also demonstrated the utility of this technology for compound screening applications. The described technology also has the potential to accelerate studies on target and biomarker identification and toxicity assessment in ADMET (absorption, distribution, metabolism, elimination, and toxicity) testing.

Cell Count↗
PubMed2006

Robust statistical methods for hit selection in RNA interference high-throughput screening experiments.

RNA interference (RNAi) high-throughput screening (HTS) experiments carried out using large (>5000 short interfering [si]RNA) libraries generate a huge amount of data. In order to use these data to identify the most effective siRNAs tested, it is critical to adopt and develop appropriate statistical methods. To address the questions in hit selection of RNAi HTS, we proposed a quartile-based method which is robust to outliers, true hits and nonsymmetrical data. We compared it with the more traditional tests, mean +/- k standard deviation (SD) and median +/- 3 median of absolute deviation (MAD). The results suggested that the quartile-based method selected more hits than mean +/- k SD under the same preset error rate. The number of hits selected by median +/- k MAD was close to that by the quartile-based method. Further analysis suggested that the quartile-based method had the greatest power in detecting true hits, especially weak or moderate true hits. Our investigation also suggested that platewise analysis (determining effective siRNAs on a plate-by-plate basis) can adjust for systematic errors in different plates, while an experimentwise analysis, in which effective siRNAs are identified in an analysis of the entire experiment, cannot. However, experimentwise analysis may detect a cluster of true positive hits placed together in one or several plates, while platewise analysis may not. To display hit selection results, we designed a specific figure called a plate-well series plot. We thus suggest the following strategy for hit selection in RNAi HTS experiments. First, choose the quartile-based method, or median +/- k MAD, for identifying effective siRNAs. Second, perform the chosen method experimentwise on transformed/normalized data, such as percentage inhibition, to check the possibility of hit clusters. If a cluster of selected hits are observed, repeat the analysis based on untransformed data to determine whether the cluster is due to an artifact in the data. If no clusters of hits are observed, select hits by performing platewise analysis on transformed data. Third, adopt the plate-well series plot to visualize both the data and the hit selection results, as well as to check for artifacts.

Cluster Analysis↗
PubMed2002

Primer design for PCR and sequencing in high-throughput analysis of SNPs.

To achieve high-throughput analysis of allele frequencies in human SNPs, we have developed automated methodsfor designing PCR and DNA sequencing primers. We found we could run the PCR assays at quite stringent, uniform conditions. The design process used freely available databases, including dbSNP, SNPper, and TSC, and publicly available software including RepeatMasker and Primer3. We describe parameters for the software and other considerations that increase experimental success. As anticipated. some assays filed at the design stage due primarily to the genomic locations of repetitive sequences, extreme GC content regions, or lack of sufficient sequence. However, over 23,000 assays, including 96% of those recently analyzed, have been experimentally successfuL Similar design methods could be usedfor PCR assays in any organism with substantial available sequence.

DNA Mutational Analysis↗
PubMed2003

High-throughput crystallography at an affordable cost: the TB Structural Genomics Consortium Crystallization Facility.

The crystallization facility of the TB Structural Genomics Consortium, one of nine P50 structural genomics centers sponsored by the National Institutes of Health, provides TB consortium members with automated crystallization, data collection, and basic molecular replacement structure solution up to bias-minimized maps. In contrast to venture capital -funded commercial enterprises, the TB consortium facilities are decentralized and aim to develop high-throughput crystallography methods and automation on a comparatively small budget. In addition to financial constraints, the logistics and organization of a production environment differ considerably from academic settings. The TB Structural Genomics Consortium crystallization facility may thus provide a model for cost-effective, efficient high-throughput crystallography. Processes and methods presented in this review should assist academic institutions planning to invest in high-throughput structural biology to assess both the rewards and risks of establishing structural genomics programs.

Bacterial Proteins↗