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At least 721 records · Page 40Linked to original sources
PubMed2001

Automated assessment of dipyridamole 201Tl myocardial SPECT perfusion scintigraphy by case-based reasoning.

UNLABELLED: This study evaluated the diagnostic accuracy of case-based reasoning (CBR) to automatically detect significant coronary artery disease from dipyridamole 201Tl myocardial SPECT perfusion scintigrams. METHODS: The study population included 240 patients (182 men, 58 women; mean age +/- SD, 61 +/- 12 y) on whom coronary angiography and perfusion scintigraphy were performed within 6 +/- 11 d of each other. The patients were divided into two groups according to the presence or absence of significant coronary disease in any major coronary vessel. Regional myocardial tracer uptake was observed in 84 segments by polar map analysis. For each scintigraphic image, a CBR algorithm based on a similarity metric was used to identify similar scintigraphic images within the case library. The angiographic results of these similar cases were used to obtain the CBR reading, which was compared with the true angiographic results. Myocardial scintigrams were also analyzed by a first-generation Cedars-Sinai (CS) method, including a comparison with a reference database, and by the visual analysis of an expert reader. RESULTS: By receiver-operating-characteristic analysis, the diagnostic accuracy of CBR was not different from the interpretation by the CS algorithm and from visual interpretation (P = not significant [NS]). For detection of significant coronary disease, the respective sensitivities at 50% and 80% specificity were 90% and 67% for CBR, 88% and 65% for CS polar map analysis, and 91% and 74% for visual interpretation. For the detection of coronary disease in the vascular territories assigned to the left anterior descending and the right coronary arteries, CBR and CS polar map analysis showed similar diagnostic accuracy (P = NS). However, for detection of disease in the circumflex artery, CS polar map analysis was slightly better than CBR (P = 0.03). CONCLUSION: Automated interpretation of dipyridamole 201Tl myocardial SPECT perfusion images by CBR has diagnostic accuracy similar to that of visual interpretation or CS analysis. Thus, use of a case library that includes a variety of normal and abnormal perfusion images does not appear to have greater diagnostic power than use of reference limits.

Adult↗
PubMed2002

Comparison of rapid, automated ribotyping and DNA macrorestriction analysis of Burkholderia pseudomallei.

An automated ribotyping device (RiboPrinter) was used to determine the ribotypes of a collection of Burkholderia pseudomallei isolates. In a preliminary evaluation with the restriction enzymes BamHI and EcoRI, the protocol with EcoRI was more discriminating. The reproducibilities of the ribotypes obtained with EcoRI (EcoRI ribotypes) were determined by testing three levels of bacterial loads. The performance of the manufacturer's software was assessed by comparing the machine-optimized ribotypes with the type determined from the original gel image analyzed with Bionumerics software. The library of B. pseudomallei EcoRI ribotypes was then compared with the ribotypes obtained by DNA macrorestriction analysis of XbaI digests by pulsed-field gel electrophoresis. The typeability of B. pseudomallei by EcoRI ribotyping was 100%, and the discrimination index was 0.94. The slightly greater discrimination provided by DNA macrorestriction analysis (0.96) was achieved at the expense of a significantly longer processing time of 6 days, although the method was only half the cost of automated ribotyping. Typeability by macrorestriction analysis was lower (97%) unless a thiourea step was added to neutralize the action of Tris-dependent endonucleases. The digital record of B. pseudomallei isolates analyzed thus far provides a useful resource for future epidemiological studies and will help shorten the response time in the event of a further melioidosis outbreak or the deliberate release of B. pseudomallei as a biohazard.

Automation↗
PubMed2000

Solid-phase synthesis and biological evaluation of a combinatorial library of philanthotoxin analogues.

The modular structure of philanthotoxins was exploited for construction of the first combinatorial library of these compounds using solid-phase parallel synthesis. (S)-Tyrosine and (S)-3-hydroxyphenylalanine were used as amino acid components, spermine, 1,12-dodecanediamine, and 4,9-dioxa-1,12-dodecanediamine as amine components, and butanoyl, phenylacetyl, and cyclohexylacetyl as N-acyl groups. Following automated preparative HPLC, the resulting 18 compounds were isolated as the S-forms in 40-70% yields. The purity of the products was determined by HPLC with evaporative light scattering detection and by (1)H and (13)C NMR. The thus obtained philanthotoxins were tested electrophysiologically for their antagonist properties on human muscle-type nicotinic acetylcholine receptors (nAChR) expressed in TE671 cells and on rat brain non-NMDA glutamate receptors (non-NMDAR) expressed in Xenopus oocytes. 4-Hydroxy analogues lacking the secondary amino groups (PhTX-12 and 4,9-dioxa-PhTX-12 and their analogues) were inactive on non-NMDAR, whereas the potency of the spermine derivatives (PhTX-343 and its analogues) increased with steric bulk of the N-acyl group. The analogue of PhTX-343 in which the N-butanoyl group was replaced by phenylacetyl group had IC(50) of 15 +/- 4 nM on non-NMDAR. Increasing the steric bulk of the N-acyl group was not advantageous for activity at nAChR, and a sharp decrease in potency with increased steric bulk was observed with the derivatives of PhTX-12. 3-Hydroxy analogues generally exhibited lower activity and different response to alterations of the N-acyl groups as compared to the 4-hydroxy analogues. Since the acyl group alterations in PhTX-343 and 4,9-dioxa-PhTX-12 have a similar effect on potency, which is distinctly different from that observed for PhTX-12, the two former compounds may bind to nAChR in a similar fashion but differently from that of PhTX-12. The combinatorial library approach described in this work represents a prototype methodology for future exploration of structure-activity relationships of philanthotoxins.

Animals↗
PubMed2005

Insights into TOR function and rapamycin response: chemical genomic profiling by using a high-density cell array method.

With the advent of complete genome sequences, large-scale functional analyses are generating new excitement in biology and medicine. To facilitate genomewide functional analyses, we developed a high-density cell array with quantitative and automated readout of cell fitness. Able to print at > x 10 higher density on a standard microtiter plate area than currently possible, our cell array allows single-plate screening of the complete set of Saccharomyces cerevisiae gene-deletion library and significantly reduces the amount of small molecules and other materials needed for the study. We used this method to map the relation between genes and cell fitness in response to rapamycin, a medically important natural product that targets the eukaryotic kinase Tor. We discuss the implications for pharmacogenomics and the uncharted complexity in genotype-dependent drug response in molecularly targeted therapies. Our analysis leads to several basic findings, including a class of gene deletions that confer better fitness in the presence of rapamycin. This result provides insights into possible therapeutic uses of rapamycin/CCI-779 in the treatment of neurodegenerative diseases (including Alzheimer's, Parkinson's, and Huntington's diseases), and cautions the possible existence of similar rapamycin-enhanceable mutations in cancer. It is well established in yeast that although TOR2 has a unique rapamycin-insensitive function, TOR1 and TOR2 are interchangeable in the rapamycin-sensitive functions. We show that even the rapamycin-sensitive functions are distinct between TOR1 and TOR2 and map the functional difference to a approximately 120-aa region at the N termini of the proteins. Finally, we discuss using cell-based genomic pattern recognition in designing electronic or optical biosensors.

Cell Cycle Proteins↗
PubMed1992

Automatable screening of yeast artificial-chromosome libraries based on the oligonucleotide-ligation assay.

The systematic screening of yeast artificial-chromosome (YAC) libraries is the limiting step in many physical mapping projects. To improve the screening throughput for a human YAC library, we designed an automatable strategy to identify YAC clones containing a specific segment of DNA. Our approach combines amplification of the target sequence from pooled YAC DNA by the polymerase chain reaction (PCR) with detection of the sequence by an ELISA-based oligonucleotide-ligation assay (OLA). The PCR-OLA approach eliminates the use of radioactive isotopes and gel electrophoresis, two of the major obstacles to automated YAC screening. Furthermore, the use of the OLA to test for the presence of sequences internal to PCR primers provides an additional level of sensitivity and specificity in comparison to methods that rely solely on the PCR.

Base Sequence↗
PubMed2004

Top-down characterization of nucleic acids modified by structural probes using high-resolution tandem mass spectrometry and automated data interpretation.

A top-down approach based on sustained off-resonance irradiation collision-induced dissociation (SORI-CID) has been implemented on an electrospray ionization (ESI) Fourier transform mass spectrometer (FTMS) to characterize nucleic acid substrates modified by structural probes. Solvent accessibility reagents, such as dimethyl sulfate (DMS), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT), and beta-ethoxy-alpha-ketobutyraldehyde (kethoxal, KT) are widely employed to reveal the position of single- vs double-stranded regions and obtain the footprint of bound proteins onto nucleic acids structures. Established methods require end-labeling of the nucleic acid constructs, probe-specific chemistry to produce strand cleavage at the modified nucleotides, and analysis by polyacrylamide gel electrophoresis to determine the position of the susceptible sites. However, these labor-intensive procedures can be avoided when mass spectrometry is used to identify the probe-induced modifications from their characteristic mass signatures. In particular, ESI-FTMS can be directly employed to monitor the conditions of probe application to avoid excessive alkylation, which could induce unwanted distortion or defolding of the substrate of interest. The sequence position of the covalent modifications can be subsequently obtained from classic tandem techniques, which allow for the analysis of individual target adducts present in complex reaction mixtures with no need for separation techniques. Selection and activation by SORI-CID has been employed to reveal the position of adducts in nucleic acid substrates in excess of 6 kDa. The stability of the different covalent modifications under SORI-CID conditions was investigated. Multiple stages of isolation and activation were employed in MS(n)() experiments to obtain the desired sequence information whenever the adduct stability was not particularly favorable, and SORI-CID induced the facile loss of the modified base. A new program called MS2Links was developed for the automated reduction and interpretation of fragmentation data obtained from modified nucleic acids. Based on an algorithm that searches for plausible isotopic patterns, the data reduction module is capable of discriminating legitimate signals from noise spikes of comparable intensity. The fragment identification module calculates the monoisotopic mass of ion products expected from a certain sequence and user-defined covalent modifications, which are finally matched with the signals selected by the data reduction program. Considering that MS2Links can generate similar fragment libraries for peptides and their covalent conjugates with other peptides or nucleic acids, this program provides an integrated platform for the structural investigation of protein-nucleic acid complexes based on cross-linking strategies and top-down ESI-FTMS.

Aldehydes↗
PubMedDate not supplied

Optimizing photo-embossed gratings: a gradient library approach.

Methodologies for the rapid screening of coating systems were developed and applied to photopolymer lacquers for photoembossing applications. Continuous and discrete gradient libraries were prepared with a gradient in grating period along the short axis and along the long axis, a gradient in exposure energy, development temperature, film thickness, photoinitiator concentration, or monomer to polymer mass ratio. Discrete gradient libraries consisted of arrays of rectangular films made by pipetting a certain amount of sample onto a chemically patterned substrate consisting of hydrophilic patches surrounded by hydrophobic, fluorinated barriers. The shape and height of the photoembossed gratings were measured using an automated AFM. Optimum grating height was obtained for a 20-microm period at intermediate exposure energies, photoinitiator concentrations, or both. Height improves with development temperature (max 110 degrees C), monomer-to-polymer ratio (max 55 wt % monomer), and film thickness. Surface topography can also be optimized, depending on any specific application.

Calorimetry, Differential Scanning↗
PubMed1992

Computer-assisted pattern recognition model for the identification of slowly growing mycobacteria including Mycobacterium tuberculosis.

We present a computerized pattern recognition model used to speciate mycobacteria based on their restriction fragment length polymorphism (RFLP) banding patterns. DNA fragment migration distances were normalized to minimize lane-to-lane variability of band location both within and among gels through the inclusion of two internal size standards in each sample. The computer model used a library of normalized RFLP patterns derived from samples of known origin to create a probability matrix which was then used to classify the RFLP patterns from samples of unknown origin. The probability matrix contained the proportion of bands that fell within defined migration distance windows for each species in the library of reference samples. These proportions were then used to compute the likelihood that the banding pattern of an unknown sample corresponded to that of each species represented in the probability matrix. As a test of this process, we developed an automated, computer-assisted model for the identification of Mycobacterium species based on their normalized RFLP banding patterns. The probability matrix contained values for the M. tuberculosis complex, M. avium, M. intracellulare, M. kansasii and M. gordonae species. Thirty-nine independent strains of known origin, not included in the probability matrix, were used to test the accuracy of the method in classifying unknowns: 37 of 39 (94.9%) were classified correctly. An additional set of 16 strains of known origin representing species not included in the model were tested to gauge the robustness of the probability matrix. Every sample was correctly identified as an outlier, i.e. a member of a species not included in the original matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Bacterial↗
PubMed2025

Automated chromatin profiling with spa-ChIP-seq uncovers the impacts of condition variations.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is widely used to study the genomic localization of DNA-associated proteins. However, conventional protocols include multiple manual steps that can introduce inconsistency and limit scalability, thereby restricting the inclusion of appropriate replicates and controls. Although the introduction of liquid handling platforms has improved reproducibility, most existing efforts have automated only a subset of the workflow, and extending automation to efficiently map non-histone proteins, such as chromatin regulators, remains challenging. Here, we present a fully automated implementation of our previously developed single-pot ChIP-seq protocol (Texari et al. 2021), named spa-ChIP-seq, which enables scalable processing of 8 to 96 ChIP-seq samples from crosslinked cells to sequencing-ready library in approximately three days with an estimated cost of $70 per sample. Benchmarking spa-ChIP-seq against manual ChIP-seq performed in parallel demonstrates comparable signal-to-noise ratio between the two workflows. Using spa-ChIP-seq, we systematically evaluate multiple parameters including shearing and crosslinking conditions, buffer compositions, and the ratio of antibody to cell-number. We find, for the first time to our knowledge, that weaker genomic localization signals are sensitive to changing the antibody to cell-number ratio, whereas the stronger signals remain unaffected. This finding underscores the importance of maintaining consistent antibody-to-cell-number ratio for comparative studies, such as treatment responses or chromatin-QTL mapping. The spa-ChIP-seq protocol is publicly available, including deck setups, operational parameters, and scripts. We envision that this robust, cost-efficient protocol will facilitate high-throughput, reproducible ChIP-seq analyses, supporting large-scale studies of antibody validation, compound screening, population genomics, and diagnostic frameworks.

Journal Article↗
PubMed2003

An automated method to position prosthetic components within multiple anatomical spaces.

The level of fit and fill of a stem in the host femur is the most critical factor for the mechanical stability and success of the prosthesis. It would be useful to have a simulation tool able to investigate the anatomical compatibility of a new implant in a large library of femoral anatomies in the early phases of the design process. In order to realise this tool, it is necessary to develop an automatic method for the positioning of the stem in a database of anatomies. The aim of this study was to develop and evaluate a method for the automatic positioning of the stem geometry in the anatomical CT dataset. Two different strategies were considered: a completely automatic registration technique and a semi-automatic method based on an anatomical referencing. The two procedures were compared to the manual positioning obtained by an expert surgeon in a set of nine CT datasets. For both methods in each femur the positioning and the orientation of the stem were good. The results showed a better level of fitting for the automatic method, while the shift of the hip joint centre was lower for the anatomical referencing technique. However, the anatomical referencing method requires a higher computational effort without being significantly better than the automatic method. For this reason, the automatic method should be chosen to develop the automatic positioning of a stem in a database of anatomies.

Arthroplasty, Replacement, Hip↗
PubMed1998

Desktop document delivery using portable document format (PDF) files and the Web.

Desktop access to electronic full-text literature was rated one of the most desirable services in a client survey conducted by the University of Washington Libraries. The University of Washington Health Sciences Libraries (UW HSL) conducted a ten-month pilot test from August 1996 to May 1997 to determine the feasibility of delivering electronic journal articles via the Internet to remote faculty. Articles were scanned into Adobe Acrobat Portable Document Format (PDF) files and delivered to individuals using Multipurpose Internet Mail Extensions (MIME) standard e-mail attachments and the Web. Participants retrieved scanned articles and used the Adobe Acrobat Reader software to view and print files. The pilot test required a special programming effort to automate the client notification and file deletion processes. Test participants were satisfied with the pilot test despite some technical difficulties. Desktop delivery is now offered as a routine delivery method from the UW HSL.

Computer Systems↗
PubMed2006

A fully automated correction method of EOG artifacts in EEG recordings.

OBJECTIVE: A fully automated method for reducing EOG artifacts is presented and validated. METHODS: The correction method is based on regression analysis and was applied to 18 recordings with 22 channels and approx. 6 min each. Two independent experts scored the original and corrected EEG in a blinded evaluation. RESULTS: The expert scorers identified in 5.9% of the raw data some EOG artifacts; 4.7% were corrected. After applying the EOG correction, the expert scorers identified in another 1.9% of the data some EOG artifacts, which were not recognized in the uncorrected data. CONCLUSIONS: The advantage of a fully automated reduction of EOG artifacts justifies the small additional effort of the proposed method and is a viable option for reducing EOG artifacts. The method has been implemented for offline and online analysis and is available through BioSig, an open source software library for biomedical signal processing. SIGNIFICANCE: Visual identification and rejection of EOG-contaminated EEG segments can miss many EOG artifacts, and is therefore not sufficient for removing EOG artifacts. The proposed method was able to reduce EOG artifacts by 80%.

Adolescent↗
PubMed1997

New technologies for high-throughput screening.

To screen efficiently the millions of compounds that are synthesized using combinatorial and automated methods, dramatically improved assay technologies are currently needed. In 96-well microtiter plates, nonradioactive techniques (primarily fluorimetric) and cell-based functional methods have moved to the cutting edge, while clever assays that extract information from large bead-based combinatorial libraries have begun to show considerable promise. In the future, miniaturized assays that break out of the 96-well format will be enabled by innovative technologies for high-throughput screening.

Chemistry, Organic↗
PubMed1998

Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells.

The dramatic increase in sequence information in the form of expressed sequence tags (ESTs) and genomic sequence has created a 'gene function gap' with the identification of new genes far outpacing the rate at which their function can be identified. The ability to create mutations in embryonic stem (ES) cells on a large scale by tagged random mutagenesis provides a powerful approach for determining gene function in a mammalian system; this approach is well established in lower organisms. Here we describe a high-throughput mutagenesis method based on gene trapping that allows the automated identification of sequence tags from the mutated genes. This method traps and mutates genes regardless of their expression status in ES cells. To facilitate the study of gene function on a large scale, we are using these techniques to create a library of ES cells called Omnibank, from which sequence-tagged mutations in 2,000 genes are described.

Animals↗
PubMed2005

NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins.

The National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) database (http://www.ncbi.nlm.nih.gov/RefSeq/) provides a non-redundant collection of sequences representing genomic data, transcripts and proteins. Although the goal is to provide a comprehensive dataset representing the complete sequence information for any given species, the database pragmatically includes sequence data that are currently publicly available in the archival databases. The database incorporates data from over 2400 organisms and includes over one million proteins representing significant taxonomic diversity spanning prokaryotes, eukaryotes and viruses. Nucleotide and protein sequences are explicitly linked, and the sequences are linked to other resources including the NCBI Map Viewer and Gene. Sequences are annotated to include coding regions, conserved domains, variation, references, names, database cross-references, and other features using a combined approach of collaboration and other input from the scientific community, automated annotation, propagation from GenBank and curation by NCBI staff.

Animals↗
PubMed2005

DNA sequencing by indexer walking.

BACKGROUND: There is a need for DNA sequencing methods that are faster, more accurate, and less expensive than existing techniques. Here we present a new method for DNA analysis by means of indexer walking. METHODS: For DNA sequencing by indexer walking, we ligated double-stranded synthetic oligonucleotides (indexers) to DNA fragments that were produced by type IIS restriction endonucleases, which generate nonidentical 4-nucleotide 5' overhangs. The subsequent amplification (30 thermal cycles) of indexed DNA provided a template for automated DNA sequencing with fluorescent dideoxy terminators. The data gathered in the first sequencing reaction permitted further movement into the unknown nucleotide sequence by digestion of analyzed DNA with selected type IIS restriction endonuclease followed by ligation of the next indexer. A library of presynthesized indexers consisting of 256 oligonucleotides was used for bidirectional analysis of DNA molecules and provided universal primers for sequencing. RESULTS: The proposed protocol was successfully applied to sequencing of cryptic plasmids isolated from pathogenic strains of Escherichia coli. The overall error rate for base-calling was 0.5%, with a mean read length of 550 nucleotides. Approximately 1000 nucleotides of high-quality sequence could be obtained per day from a single clone. CONCLUSIONS: Indexer walking can be used as a low-cost procedure for nucleotide sequence determination of DNA molecules, such as natural plasmids, cDNA clones, and longer DNA fragments. It can also serve as an alternative method for gap filling at the final stage of genome sequencing projects.

DNA Primers↗
PubMed2004

Protein expression arrays for proteomics.

As biology approaches the 50th year of deciphering the DNA code, the next frontier toward understanding cell function has protein biochemistry in the form of structural and functional proteomics. To accomplish the needs of proteomics, novel strategies must be devised to examine the gene products or proteins, emerged as en masse. The authors have developed a high-throughput system for the expression and purification of eukaryotic proteins to provide the resources for structural studies and protein functional analysis. The long-term objective is to overexpress and purify thousands of proteins encoded by the human genome. This library of proteins--the human proteome--can be arrayed in addressable format in quantities and purities suitable for high-throughput studies. Critical technology involved in efficiently moving from genome to proteome includes parallel sample handling, robust expression, and rapid purification procedures. Automation of these processes is essential for the production of thousands of recombinant proteins and the reduction of human error.

Animals↗
PubMed2006

Linking cDNA-AFLP-based gene expression patterns and ESTs.

Massive amounts of DNA sequence data, generated from expressed sequence tag (EST) and genome sequencing projects, require efficient methods to link sequence databases with temporal and spatial expression profiles. To meet this need, we have developed a powerful computer program (GenEST), which links cDNA sequence data (including EST sequences) with transcript profiles revealed by cDNA-amplified fragment length polymorphism (AFLP). cDNA-AFLP is a highly reproducible differential display method based on restriction enzyme digests and selective amplification under high stringency conditions. GenEST predicts the sizes of virtual transcript derived fragments (TDFs) from cDNA sequences digested in silico. The resulting virtual TDFs could be traced back among the thousands of TDFs displayed on cDNA-AFLP gels. As a consequence, cDNA sequence databases can be screened very efficiently to identify genes with relevant expression profiles. Vice versa, using the restriction enzyme recognition sites, the primer extensions and the estimated TDF size as identifiers, the DNA sequence(s) corresponding to a TDF with an interesting expression pattern can be identified.

Automation↗