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We describe here the integration of image analysis and robotics to produce a fully automated colony picking/plate handling system. Biological tests were performed to verify its performance in terms of sterilisation and accuracy of picking. The machine was then used by a single operative to pick a 36,000 clone cDNA library in approximately 42 hrs over 5 days.
A computer system (MSSMET), using methylene unit retention indices for an off-line reverse library search analysis of selected ion chromatograms from gas-liquid chromatographic mass spectrometric data, has been applied for the qualitative and quantitative determination of daily variations in the excreted levels of urinary steroids of two individuals, using capillary column gas-liquid chromatography. Aliquots of 24 h urine collections and morning spot urine samples were examined. The daily excretion pattern of most of the major steroid metabolites was fairly consistent from day to day (i.e. 3 alpha-hydroxy-5 alpha-androstane-17-one, androsterone; 3 alpha-hydroxy-5 beta-androstane-17-one, etiocheolanolone; 3 alpha, 17 alpha, 21-trihydroxy-5 beta-pregnane-11,20-dione, THE; 3 alpha, 11 beta, 17 alpha, 21-tetrahydroxy-5 beta-pregnane-20-one, THF; 3 alpha, 11 beta, 17 alpha, 21-tetrahydroxy-5 alpha-pregnane-20-one; allo-THF; 3 alpha, 17 alpha, 20 alpha, 21-tetrahydroxy-5 beta-pregnane-11-one, cortolone; 3 alpha, 17 alpha, 20 beta, 21-tetrahydroxy-5 beta-pregnane-11-one, beta-cortolone; 5 beta-pregnane-3 alpha, 11 beta,17 alpha,20 alpha,21-pentol, cortol; 5 beta-pregnane-3 alpha, 11 beta, 17 alpha, 20 beta, 21-pentol, beta-cortol), while certain other steroid metabolites had a less consistent excretion pattern (3 beta-hydroxy-5-androstene-17-one, for example). Advantages and disadvantages of using capillary columns for the automated metabolic profile analysis of urinary steroids by reverse library search of selected mass chromatograms.
Combinatorial chemistry has emerged as a set of novel strategies for the synthesis of large sets of compounds (combinatorial libraries) for biological evaluation. Within a few years combinatorial chemistry has undergone a series of changes in trends, which are closely related to two important factors in libraries: numbers and quality. While the number of compounds in a library may be easily expressed, it is a lot more difficult to indicate the degree of quality of a library. This degree of quality can be split into two aspects: purity and diversity. The changing trends in combinatorial chemistry with respect to the strategies, the technologies, the libraries themselves (numbers and purity aspects) and the molecular diversity are outlined in this paper.
A new tool for analyzing compound libraries by NMR has been developed. Aliquots of solution-state samples (between 120 and 350 microL) are directly injected, using a standard liquids handler, into an NMR (LC-NMR) flow probe. Automated NMR software tracks--and suppresses--intense signals arising from the nondeuterated solvents used (if any) and acquires high-sensitivity one-dimensional 1H NMR spectra. An 88-member combinatorial library, dissolved in DMSO and stored in a 96-well microtiter plate, has been analyzed a number of ways using this technique. This nondestructive technique, which we call direct-injection NMR (DI-NMR) and which is embodied in our versatile automated sample changer (VAST) hardware, has proven to be both routine and robust. Our success in automatically acquiring the NMR data for entire plates of library compounds (within 4-8 h) has caused us to develop new ways to display and analyze the resulting NMR data, as will be shown here.
The increasing size of the collections used for drug-discovery purposes has demanded both hardware and software automation of compound management in order to cope with the increasing demands of HTS. Splitting the collection into a number of copies in different formats is a desirable approach to keep a balance between rapid response to the demands and best storage conditions. Flexibility to different assay configurations can be provided with the appropriate selection of liquid handlers, and the informatic management systems should be accessible to keep track of the samples and link them to a variety of information that can help interpret HTS data. In this respect, QC data on the compounds and quality checks of the equipment used are highly desirable. It is also prudent for a large organization with different research sites to have a unified database and compatible plate format and concentration in order to be able to exchange samples and share screening results. The accomplishment of all the previous requirements is the only way to ensure an efficient and effective compound library management.
An automated capillary microinjection system with computer-controlled positioning of the cells and of the capillary, and its applications and advantages, is described. The system is easy in handling and manipulation. About 1500 injections are possible in 1 h, with high reproducibility. In cytoplasmic and nuclear injections more than 90 and 85% of the cells are successfully injected. Using FITC-dextran at a concentration of 0.5% as a fluorescently labeled coinjection marker, 99% of the cells can be retrieved in culture medium even 48 h after injection. The coordinates of the cells are stored in the computer and accuracy in statistical evaluation of experiments is improved in comparison to the manual techniques. Methods for preparation and handling of glass capillaries were developed resulting in reproducible form and significantly reduced clogging rate. The improved characteristics offered by this system are demonstrated in studies leading to the confirmation of existence of an mRNA inhibiting DNA synthesis in cells. Functional screening by cell injections of cDNA libraries and of size-fractionated mRNA molecules can be performed efficiently with the automated microinjection system.
An automated cellular fatty acid (CFA) bacterial identification system, Microbial Identification System (MIS; Microbial ID, Newark, Del.), was compared with a conventional system for the identification of 573 strains of gram-negative nonfermentative bacteria. MIS identifications were based exclusively on the CFA composition following 22 to 26 h of growth at 28 degrees C on Trypticase soy agar. MIS identifications were listed with a confidence measurement (similarity index [SI]) on a scale of 0 to 1.0. A value of greater than or equal to 0.5 was considered a good match. The MIS correctly listed as the first choice 478 of 532 (90%) strains contained in the data base. However, only 314 (59%) had SI values of greater than or equal to 0.5. Of the 54 strains in which there was not agreement, 37 belonged to the genera Acinetobacter, Moraxella, or Alcaligenes or were Pseudomonas pickettii. Reproducibility studies suggest that SI variation is most likely a function of a difference in culture age at the time of analysis, which is due to the relatively low temperature and time of incubation. Other discrepancies were attributable to insufficiently characterized library entries or an inability to differentiate chemotaxonomically closely related species. The MIS, as the first automated CFA identification system, is an accurate, efficient, and relatively rapid method for the identification of gram-negative nonfermentative bacteria. The development of a CFA library with the media and incubation conditions routinely used for the isolation of clinical pathogens could further decrease the identification time and provide an increase in accuracy.
We have previously reported the characterization of a DNA-binding protein isolated from Xenopus laevis mitochondria (mtDBP-C). The amino terminal sequence of this protein (26 residues) has been determined by automated Edman degradation and used to search for sequence similarity with the NBRF library. A segment of 17 amino acids displays 47.1% of identity with proteins HMG-1 and 2 of various vertebrate species.
PURPOSE: Pigment epithelium-derived factor (PEDF), a neurotrophic protein, is a member of the serine protease inhibitor supergene family. PEDF promotes both neuronal phenotype in cultured Y79 cells and neuronal survival in cultured cerebellar granulocytes. The purpose of this study was to clone the mouse PEDF gene and to determine its structure and levels of expression in different tissues. METHODS: The mouse PEDF cDNA was cloned from a mouse liver cDNA library using human PEDF cDNA as a probe. The mouse PEDF gene was cloned from a mouse ES genomic P1 library. DNA sequencing was performed using a PE-Applied Biosystems model 373 automated fluorescent sequencer. RESULTS: The mouse PEDF cDNA is 1461 bp in length and contains an open reading frame of 417 amino acids. The mouse PEDF gene spans approximately 13 kb and, like the human, it is fragmented into 8 exons. The splice sites follow the AG/GT consensus rule. Southern blot analysis indicates that the mouse genome contains only one gene for PEDF. Northern blot analysis shows the presence of the PEDF transcript in a broad range of adult mouse tissues with liver showing the highest level of expression. CONCLUSIONS: The mouse and human PEDF promoters share overall 27% similarity but are nearly identical between mouse +86 to -166 and human +102 to -96. The present study will allow us to move from in vitro experiments to in vivo studies through the development of a "knock-out" mouse model.
MOTIVATION: Computer-based selection of entries from sequence databases with respect to a related functional description, e.g. with respect to a common cellular localization or contributing to the same phenotypic function, is a difficult task. Automatic semantic analysis of annotations is not only hampered by incomplete functional assignments. A major problem is that annotations are written in a rich, non-formalized language and are meant for reading by a human expert. This person can extract from the text considerably more information than is immediately apparent due to his extended biological background knowledge and logical reasoning. APPROACH: A technique of automated annotation evaluation based on a combination of lexical analysis and the usage of biological rule libraries has been developed. The proposed algorithm generates new functional descriptors from the annotation of a given entry using the semantic units of the annotation as prepositions for implications executed in accordance with the rule library. RESULTS: The prototype of a software system, the Meta_A(nnotator) program, is described and the results of its application to sequence attribute assignment and sequence selection problems, such as cellular localization and sequence domain annotation of SWISS-PROT entries, are presented. The current software version assigns useful subcellular localization qualifiers to approximately 88% of all SWISS-PROT entries. As shown by demonstrative examples, the combination of sequence and annotation analysis is a powerful approach for the detection of mutual annotation/sequence inconsistencies. AVAILABILITY: Results for the cellular localization assignment can be viewed at the URL http://www.bork. embl-heidelberg.de/CELL_LOC/CELL_LOC.html.
A library of spreadsheets has been developed to facilitate the practice of diagnostic physics quality assurance. Each spreadsheet follows a standard template and uses the highest ranking controlling authority (within the United States) for pass/fail criteria and testing procedures. Sheets are now available for CT (computed tomography), MR (magnetic resonance), US (ultrasound), screen-film mammography, stereotactic breast, radiographic, fluoroscopic, computed radiography, film digitizer, and display quality control. Use is made of spreadsheet "workbooks" so that each testing event is a single sheet in the workbook. Thus, results over the lifetime of the device are gathered in a single file, and historical control charts are gathered on the first sheet. The spreadsheets are available at http://radweb.mcis.washington.edu/~sglanger, and are released under the Gnu (a recursive acronym. Gnu's Not Unix) public license. It is expected that others will add improvements, and they are expected and requested to submit them back to the author to be shared with the diagnostic physicist community at large.
A brief overview of the Bibliographic Access and Control System developed by the Washington University School of Medicine Library is presented. Because the system has been described in two previous reports, this paper focuses on its relationship to other automated programs (i.e., PHILSOM and OCTANET), education of users, evaluation of the system, and outreach to the medical center. In operation for more than two years, BACS represents the computerization of much of the managerial and operational functions of the library, and marks the completion of stage 1 of the three stages of library evolution described in the AAMC report Academic Information in the Academic Health Sciences Center: Roles for the Library in Information Management.
High-throughput automated sequencing has enabled researchers to examine large numbers of clones from a cDNA library as a measure of the steady-state levels of mRNA species. The past year has witnessed many new applications of this technique to allow the qualitative and quantitative comparison of the changes in transcript levels from multiple genes.
MOTIVATION: Mining the biomedical literature for references to genes and proteins always involves a tradeoff between high precision with false negatives, and high recall with false positives. Having a reliable method for assessing the relevance of literature mining results is crucial to finding ways to balance precision and recall, and for subsequently building automated systems to analyze these results. We hypothesize that abstracts and titles that discuss the same gene or protein use similar words. To validate this hypothesis, we built a dictionary- and rule-based system to mine Medline for references to genes and proteins, and used a Bayesian metric for scoring the relevance of each reference assignment. RESULTS: We analyzed the entire set of Medline records from 1966 to late 2001, and scored each gene and protein reference using a Bayesian estimated probability (EP) based on word frequency in a training set of 137837 known assignments from 30594 articles to 36197 gene and protein symbols. Two test sets of 148 and 150 randomly chosen assignments, respectively, were hand-validated and categorized as either good or bad. The distributions of EP values, when plotted on a log-scale histogram, are shown to markedly differ between good and bad assignments. Using EP values, recall was 100% at 61% precision (EP=2 x 10(-5)), 63% at 88% precision (EP=0.008), and 10% at 100% precision (EP=0.1). These results show that Medline entries discussing the same gene or protein have similar word usage, and that our method of assessing this similarity using EP values is valid, and enables an EP cutoff value to be determined that accurately and reproducibly balances precision and recall, allowing automated analysis of literature mining results. .
Efficient procedures for managing a large number of M13 or plasmid clones have been developed. In addition to picking, clones are directly arrayed in multiwell plates by dispensing diluted transformation mixtures. Metal pin arrays are used for fast inoculations of preparative plates filled by medium or by PCR mixture. Growth of M13 clones in multiwell plates is optimized to obtain a consistently high yield, and a PCR protocol is defined for reliable amplification of several thousand M13 or plasmid inserts per day in BioOvens. Over 80,000 cDNA inserts have been amplified. The phages or amplified inserts are spotted on nylon filters using an array of pins having a flat bottom, 0.3 mm in diameter. The procedures are suitable for an automated processing of hundreds of thousands of short clones from representative cDNA and genomic libraries. Hybridization of arrayed clones with oligonucleotide and complex probes can simplify the search for new genes and accelerate large-scale sequencing.
In the use of combinatorial chemistry as a novel strategy for drug discovery, the chirality assessment of building blocks used for library construction is particularly important in the evaluation of biological actions of generated libraries. The procedure for chiral analysis of lead compounds in screening may be of a high priority, particularly in the case of the protection of intellectual rights. Previously, an automated amino acid analysis system using enantiomer labeling was developed. The system incorporates a reactor, which allows automated esterification and acylation of amino acids, and is connected to an on-line gas chromatographic system. A capillary column coated with a chiral phase is employed for the separation of the enantiomers. This particular system is improved with a newly constructed "high-throughput auto-derivatizer" in combination with a new GC-system. The resulting data can be processed by newly constructed software. The analyses of amino acid derivatives or hydrolysates of proteins and peptides are carried out routinely within ca. 45 min, including derivatization. Using this system several non-natural amino acids were tested with respect to the stereoisomeric configuration. In addition, acid hydrolysates of food proteins and tissues obtained by autopsy were analyzed as an application to proteome research.
We consider the application of high-throughput experimentation (HTE), including combinatorial methods, to catalyst discovery and early-phase optimization. While combinatorial- and parallel-testing methods have an already substantial history in catalysis, recent work by several groups promises significant efficiency gains. In molecular catalysis, progress is noted in library design, library synthesis by pooled, parallel, and discrete formats, catalyst testing and reaction optimization; the prime constraint for organometallic catalysts is the limited scope of synthesis procedures for non-peptide-based ligand libraries. Routes described for the synthesis of heterogeneous catalysts include hydrothermal synthesis, arraying of solution precursors, automated impregnation and precipitation, and arraying of solid precursors. The key challenge in applying HTE to heterogeneous catalysis is testing; we distinguish here between Stage 1, or "discovery" testing and Stage 2, optimization testing, and describe techniques with potential in each case. Recent examples from the literature and our own work are used to illustrate these principles and the prospects for HTE applied to catalysis.