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PubMed2004

High-throughput expression of C. elegans proteins.

Proteome-scale studies of protein three-dimensional structures should provide valuable information for both investigating basic biology and developing therapeutics. Critical for these endeavors is the expression of recombinant proteins. We selected Caenorhabditis elegans as our model organism in a structural proteomics initiative because of the high quality of its genome sequence and the availability of its ORFeome, protein-encoding open reading frames (ORFs), in a flexible recombinational cloning format. We developed a robotic pipeline for recombinant protein expression, applying the Gateway cloning/expression technology and utilizing a stepwise automation strategy on an integrated robotic platform. Using the pipeline, we have carried out heterologous protein expression experiments on 10,167 ORFs of C. elegans. With one expression vector and one Escherichia coli strain, protein expression was observed for 4854 ORFs, and 1536 were soluble. Bioinformatics analysis of the data indicates that protein hydrophobicity is a key determining factor for an ORF to yield a soluble expression product. This protein expression effort has investigated the largest number of genes in any organism to date. The pipeline described here is applicable to high-throughput expression of recombinant proteins for other species, both prokaryotic and eukaryotic, provided that ORFeome resources become available.

Animals↗
PubMed2000

Development of a high-throughput process for detection and screening of genetic polymorphisms.

A method is described that uses the ABI PRISM 310 genetic analyzer in conjunction with custom-designed software to identify and classify RAPD products. This methodology will also work well with AFLPs and microsatellite analyses. The methodology uses the ABI PRISM 310's high-throughput (> 500 samples per week) capabilities and in-lane molecular weight standards to efficiently separate and size DNA products. Peak detection, locus classification and export of the data in a form accessible by several genetic analysis programs were accomplished through a custom-written software program (Peaks). Various criteria used by the program to identify and classify loci are described, and their effect on population analyses is examined. Criteria providing an effective, robust determination of population structure are presented.

Alleles↗
PubMed1996

High-throughput screening for drug discovery.

Recent progress in elucidating the mechanisms underlying human disease has dramatically increased the number of protein targets available for potential drug treatment. Concurrently, new approaches have increased the number of compounds that can be tested for activity against these targets. Together, these trends have stimulated the adoption of high-throughput screening as a primary tool for early-stage drug discovery.

Drug Evaluation, Preclinical↗
PubMed2002

Integration of NMR and high-throughput screening.

NMR-based screening has become a powerful method for the identification and analysis of low-molecular weight organic compounds that bind to protein targets and can be utilized in drug discovery programs. In particular, heteronuclear NMR-based screening can yield information about both the affinity and binding location of potential lead compounds. In addition, heteronuclear NMR-based screening has wide applications in complementing and facilitating conventional high-throughout screening programs. This article will describe several strategies for the integration of NMR-based screening and high-throughput screening. The marriage of these two techniques promises to be of tremendous benefit in the triage of hits that come from HTS, and can aid the medicinal chemist in the identification of quality leads that have high potential for further optimization.

Combinatorial Chemistry Techniques↗
PubMed2006

Identification of DNA sequence variation in Campylobacter jejuni strains associated with the Guillain-Barré syndrome by high-throughput AFLP analysis.

BACKGROUND: Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular. RESULTS: We compared 6 different isolates of the "genome strain" NCTC 11168 obtained from different laboratories. HtAFLP analysis generated approximately 3000 markers per stain, 19 of which were polymorphic. The DNA polymorphisms could not be confirmed by PCR-RFLP analysis, suggesting a baseline level of 0.6% AFLP artefacts. Comparison of NCTC 11168 with 4 GBS-associated strains revealed 23 potentially GBS-specific markers, 17 of which were identified by DNA sequencing. A collection of 27 GBS/MFS-associated and 17 enteritis control strains was analyzed with PCR-RFLP tests based on 11 of these markers. We identified 3 markers, located in the LOS biosynthesis genes cj1136, cj1138 and cj1139c, that were significantly associated with GBS (P = 0.024, P = 0.047 and P < 0.001, respectively). HtAFLP analysis of 13 highly clonal South African GBS/MFS-associated and enteritis control strains did not reveal GBS-specific markers. CONCLUSION: This study shows that bacterial GBS markers are limited in number and located in the LOS biosynthesis genes, which corroborates the current consensus that LOS mimicry may be the prime etiologic determinant of GBS. Furthermore, our results demonstrate that htAFLP, with its high reproducibility and resolution, is an effective technique for the detection and subsequent identification of putative bacterial disease markers.

Bacterial Proteins↗
PubMed2006

A new automated and high-throughput system for analysis of the forced swim test in mice based on magnetic field changes.

INTRODUCTION: MicroAct (Neuroscience, Tokyo, Japan) was originally developed as a new system for an automated analysis of scratching behaviors in mice. This system is based on the detection of electric current in the coils according to the movement of the magnets implanted in the hind paws. We applied and improved this system to establish an automated analysis system of forced swimming behaviors in mice, which is used as an animal model of depression. METHODS: One day before the test, male CD-1 mice were attached with a wire ring to their hind paws under inhalation anesthesia. After attaching a small magnet to both of the wire rings, each animal was placed for 6 min in a glass cylinder filled with water, which is surrounded by a coil. The swimming behaviors of the mouse were analyzed for the measurement of duration of immobility, a major marker of depression in rodents, by the detection system (MicroAct). The duration of immobility was also determined by manual measurement using the swimming behavior-recorded videotapes produced at the same time as the automated analysis. RESULTS: The difference of the duration of immobility between naïve mice and mice with the rings was not significant. The dose-response effect of imipramine (tricyclic antidepressant, 0, 7.5, 15, 30 and 60 mg/kg, p.o.) on the duration of immobility in the last 4-min of the 6-min testing period determined by MicroAct was similar to that assessed by the manual measurement. These data from the two different methods were significantly correlated (r=0.8805). Moreover, throughput of the automated analysis was 15 times more efficient than that of the manual analysis. DISCUSSION: These results suggest that the automated analysis system of forced swimming of mice using MicroAct can be used as a high-throughput method to examine antidepressive activity of a compound with objectivity and reliability.

Animals↗
PubMed2000

Neuroendocrine expression in metastatic prostate cancer: evaluation of high throughput tissue microarrays to detect heterogeneous protein expression.

The theory that poorly differentiated prostate carcinoma develops a neuroendocrine (NE) phenotype is controversial. Supportive data is variable with NE expression being observed in anywhere from 5% to 83% of prostate cancers. These percentages are derived from standard immunohistochemistry studies, which make no attempt to quantify the results. High-density tissue microarrays (TMAs), represent a novel method for evaluating up to 1000 tissue samples with a 0.6 mm diameter on a single glass slide. This high throughput technology for screening antibodies, however, requires validation to determine if TMAs are useful in evaluating heterogeneously expressed proteins such as the NE markers chromogranin A (CGA) and synaptophysin (SYN). This study compares results from standard slides to TMAs in 50 primary and metastatic prostate tumors taken from 12 rapid autopsies from men with hormone refractory prostate cancer. One hundred standard and 2 TMA slides were immunostained for CGA and SYN. Using standard slides, focal NE expression was seen in 1/12 primary prostate tumors. Overall, 13/100 (13%) standard slides showed focal NE expression for both primary and metastatic prostate tumors; NE expression was observed in 4/12 autopsy cases (33%) when all tumor sites per case were considered. 458 tissue elements (tumor and normal) were arrayed into one paraffin block. Seventy-three percent (332/458) of the elements placed into the TMA were confirmed histologically to represent tumor. Seventy-five percent (250/332) and 66% (218/332) could be evaluated for CGA and SYN expression, respectively. Six of the metastatic tumors expressed CGA and SYN or 2.4% (6/250; 95% CI = 0.9% to 5.2%) and 2.3% (6/218; 95% CI = 0.8% to 5.3%), respectively. In conclusion, only focal NE expression was observed by both methods (eg, standard and TMA slides). The focal expression in these advanced prostate tumors was unexpected given data from prostate tumor cell lines and animal models suggesting that progression to the NE phenotype parallels tumor progression. This study also supports the use of high density TMAs to screen for protein expression, even when expression is focal.

Adenocarcinoma↗
PubMed2002

Integration of Cot analysis, DNA cloning, and high-throughput sequencing facilitates genome characterization and gene discovery.

Cot-based sequence discovery represents a powerful means by which both low-copy and repetitive sequences can be selectively and efficiently fractionated, cloned, and characterized. Based upon the results of a Cot analysis, hydroxyapatite chromatography was used to fractionate sorghum (Sorghum bicolor) genomic DNA into highly repetitive (HR), moderately repetitive (MR), and single/low-copy (SL) sequence components that were consequently cloned to produce HRCot, MRCot, and SLCot genomic libraries. Filter hybridization (blotting) and sequence analysis both show that the HRCot library is enriched in sequences traditionally found in high-copy number (e.g., retroelements, rDNA, centromeric repeats), the SLCot library is enriched in low-copy sequences (e.g., genes and "nonrepetitive ESTs"), and the MRCot library contains sequences of moderate redundancy. The Cot analysis suggests that the sorghum genome is approximately 700 Mb (in agreement with previous estimates) and that HR, MR, and SL components comprise 15%, 41%, and 24% of sorghum DNA, respectively. Unlike previously described techniques to sequence the low-copy components of genomes, sequencing of Cot components is independent of expression and methylation patterns that vary widely among DNA elements, developmental stages, and taxa. High-throughput sequencing of Cot clones may be a means of "capturing" the sequence complexity of eukaryotic genomes at unprecedented efficiency.

Base Composition↗
PubMed2003

Automated high-throughput probe production for DNA microarray analysis.

DNA microarrays have become an established tool for gene expression profiling. Construction of these microarrays using immobilized cDNAs is a common experimental strategy. However, this is extremely laborious, requiring the preparation of hundreds or thousands of cDNA probes. To minimize this initial bottleneck, we developed a comprehensive high-throughput robotic system to prepare DNA probes suitable for microarray analysis with minimal user intervention. We describe an automated system using the MultiPROBE Nucleic Acid Purification Workstation to provide the liquid handling and other functions needed to optimize this process. We were able to carry out fully automated plasmid cDNA isolation, PCR assay setup, and PCR purification and also to direct the characterization and tracking of DNA probes during processing. Protocols began with the initial preparation of a plasmid DNA archive of bacterial stocks in parallel 96-well plates (192 samples/run) and continued through to the dilution and reformatting of chip-ready DNA probes in 384-well format. These and other probe production procedures and additional instrument systems were used to process fully a set of mouse cDNA clones that were then validated by differential gene expression analysis.

Animals↗
PubMed1999

High-throughput DNA sequencing: a genomic data manufacturing process.

The progress trends in automated DNA sequencing operation are reviewed. Technological development in sequencing instruments, enzymatic chemistry and robotic stations has resulted in ever-increasing capacity of sequence data production. This progress leads to a higher demand on laboratory information management and data quality assessment. High-throughput laboratories face the challenge of organizational management, as well as technology management. Engineering principles of process control should be adopted in this biological data manufacturing procedure. While various systems attempt to provide solutions to automate different parts of, or even the entire process, new technical advances will continue to change the paradigm and provide new challenges.

Automation↗
PubMed2000

Chemoselective high-throughput purification mediated by solid-supported reagents: its application to the first 6, 9-disubstituted purine library synthesis.

A new application of solid-supported reagents was developed to separate the alkylated N7/N9 regioisomers derived from commercially available 2-amino-6-chloropurine. Simple filtration through an alumina/H+ pad or scavenging by AG/Dowex-50W-X8 resin provides diverse N9 regioisomers selectively in moderate yields with high purities (>90%). This purification method can be conveniently used in a high-throughput format and facilitates the synthesis of a purine library without laborious regioisomer separation and aqueous work-up. The first library synthesis of 6,9-disubstituted purines is reported using the combination of this novel separation method in conjunction with polymer-supported reagents.

2-Aminopurine↗
PubMed2003

High-throughput proteomics and protein biomarker discovery in an experimental model of inflammatory hyperalgesia: effects of nimesulide.

OBJECTIVE: The goal of this study was to derive a systematic approach for the identification of protein species profiles that selectively identify subgroups of similar but distinct pain models and correlate with the therapeutic efficacy (or lack thereof) of drug intervention. METHODS: Using high-throughput surface-enhanced laser desorption ionization (SELDI) mass spectrometry proteomic technology based on ProteinChip arrays, we conducted a comparative analysis of the profile of protein expression in cerebral spinal fluid (CSF) from rats exposed to either injection of complete Freund's adjuvant (CFA) [a model of inflammatory pain] or chronic constriction injury (CCI) [a model of neuropathic pain]. The CFA model was then further studied for the effects of treatment with the NSAID, nimesulide (a preferential cyclo-oxygenase [COX]-2 inhibitor). RESULTS: Among other observations, we found that the content of two metal (copper) binding protein species (2.9 and 3.2kDa) and three anionic protein species (4.0, 6.9 and 8.2kDa) were increased in the CSF of rats with inflammatory pain in a time-dependent fashion, at 7 and 14 days after CFA injection. These changes were highly selective for the CFA model, as no detectable increase in these protein biomarkers was found in the CCI neuropathic pain model. Further, we found that most of the changes in the biomarker protein species induced by the inflammatory pain were prevented by treatment with nimesulide and correlated with the antihyperalgesic effect of this drug. CONCLUSION: This study demonstrates that CSF biomarker profiles, as detected by SELDI technology, can consistently and reproducibly differentiate inflammatory from neuropathic pain, and reflect the analgesic action produced by the preferential COX-2 inhibitor, nimesulide. The characterisation and identification of these biomarkers will provide invaluable insight into the pathophysiology of pain mechanisms, in addition to further understanding of the value of nimesulide in the treatment of inflammatory pain.

Analysis of Variance↗
PubMed2001

Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi.

Genome-wide analysis of gene function is essential for the post-genome era, and development of efficient and economical technology suitable for it has been in demand. Here we report a large-scale inactivation of the expressed genes in the nematode Caenorhabditis elegans. For this purpose, we have established a high-throughput "RNAi-by-soaking" methodology by modifying the conventional RNAi method [1, 2]. A set of tag-sequenced, nonredundant cDNAs corresponding to approximately 10,000 genes [3] (representing half of the predicted genes [4]) was used for the systematic RNAi analysis. We have processed approximately 2500 genes to date. In development, 27% of them showed detectable phenotypes, such as embryonic lethality, post-embryonic lethality, sterility, and morphological abnormality. Of these, we analyzed the phenotypes of F1 sterility in detail, and we have identified 24 genes that might play important roles in germline development. Combined with the ongoing analysis of expression patterns of these cDNAs [3, 5], the functional information obtained in this work will provide a starting point for the further analysis of each gene. Another finding from this screening is that the incidence of essential genes is significantly lower in the X chromosome than in the autosomes.

Animals↗
PubMed2003

Testing hypotheses about determinants of protein structure with high-precision, high-throughput stability measurements and statistical modeling.

Statistical modeling provides the mathematics to use data from large numbers of mutant proteins to generate information about hypotheses concerning protein structure not easily obtained from anecdotal studies on small numbers of mutants. Here we use the unfolding free energies of 303 unique eglin c mutant proteins obtained from high-precision, high-throughput chemical denaturation measurements to assess models concerning helix stability. A model with helix propensity as the sole determinant of stability accounts for 83% of the mutant-to-mutant variation in stability for 99% of the mutant proteins (three outliers). When position effects and side chain-side chain interactions are added to the model, the fraction of variation explained increases to 92%. The propensity parameters in this model are identical to helix propensity values derived from other approaches. Measurement error accounts for another 1% of the mutant-to-mutant variation in stability. While the data support terms for several of the expected stabilizing/destabilizing effects, it does not support terms for several others, including i, i + 3 effects in the center of the helix and helix-dipole effects. In addition, the model does better with terms for several stabilizing/destabilizing effects for which we cannot identify the physical basis. The precision of our unfolding stability measurements (+/-0.087 kcal/mol) allows us to conclude that the 7% of variation in stabilities of the mutant proteins not accounted for by the model or by measurement variation is both real and large with respect to the nonpropensity terms in the model. The analysis also shows that the common practice of using C(m)m(av) instead of C(m)m(mut) to calculate DeltaG(HOH,N-D) values for each mutant protein results in a loss of information. We see no correlation between the residuals derived from the full model and m(mut) - m(wt), and hence it is unlikely our m(mut) values reflect mutant-to-mutant differences in the denatured state.

Cloning, Molecular↗
PubMed2004

Terminological mapping for high throughput comparative biology of phenotypes.

Comparative biological studies have led to remarkable biomedical discoveries. While genomic science and technologies are advancing rapidly, our ability to precisely specify a phenotype and compare it to related phenotypes of other organisms remains challenging. This study has examined the systematic use of terminology and knowledge based technologies to enable high-throughput comparative phenomics. More specifically, we measured the accuracy of a multi-strategy automated classification method to bridge the phenotype gap between a phenotypic terminology (MGD: Phenoslim) and a broad-coverage clinical terminology (SNOMED CT). Furthermore, we qualitatively evaluate the additional emerging properties of the combined terminological network for comparative biology and discovery science. According to the gold standard (n = 100), the accuracies (precision / recall) of the composite automated methods were 67% / 97% (mapping for identical concepts) and 85% / 98% (classification). Quantitatively, only 2% of the phenotypic concepts were missing from the clinical terminology, however, qualitatively the gap was larger: conceptual scope, granularity and subtle yet significant, homonymy problems were observed. These results suggest that, as observed in other domains, additional strategies are required for combining terminologies.

Animals↗
PubMed2001

High throughput genotyping technologies for pharmacogenomics.

Genetic differences between individuals play a role in determining susceptibility to diseases as well as in drug response. The challenge today is first to discover the range of genetic variability in the human population and then to define the particular gene variants, or alleles, that contribute to clinically important outcomes. Consequently, high throughput, automated methods are being developed that allow rapid scoring of microsatellite alleles and single nucleotide polymorphisms (SNPs). Many detection technologies are being used to accomplish this goal, including electrophoresis, standard fluorescence, fluorescence polarization, fluorescence resonance energy transfer, and mass spectrometry. SNP alleles may be distinguished by any one of several methods, including single nucleotide primer extension, allele-specific hybridization, allele-specific primer extension, oligonucleotide ligation assay, and invasive signal amplification. Newer methods require less sample manipulation, increase sensitivity, allow more flexibility, and decrease reagent costs. Recent developments show promise for continuing these trends by combining amplification and detection steps and providing flexible, miniaturized platforms for genotyping.

Automation↗
PubMed2005

Mass spectrometry for high throughput quantitative proteomics in plant research: lessons from thylakoid membranes.

Proteomics seeks to monitor the flux of protein through cells under variable developmental and environmental influences as programmed by the genome. Consequently, it is necessary to measure changes in protein abundance and turnover rate as faithfully as possible. In the absence of non-invasive technologies, the majority of proteomics approaches involve destructive sampling at various time points to obtain 'snapshots' that periodically report the genomes's product. The work has fallen to separations technologies coupled to mass spectrometry, for high throughput protein identification. Quantitation has become the major challenge facing proteomics as the field matures. Because of the variability of day-to-day measurements of protein quantities by mass spectrometry, a common feature of quantitative proteomics is the use of stable isotope coding to distinguish control and experimental samples in a mixture that can be profiled in a single experiment. To address limitations with separation technologies such as 2D-gel electrophoresis, alternative systems are being introduced including multi-dimensional chromatography. Strategies that accelerate throughput for mass spectrometry are also emerging and the benefits of these 'shotgun' protocols will be considered in the context of the thylakoid membrane and photosynthesis. High resolution Fourier-transform mass spectrometry is bringing increasingly accurate mass measurements to peptides and a variety of gas-phase dissociation mechanisms are permitting 'top-down' sequencing of intact proteins. Finally, a versatile workflow for sub-cellular compartments including membranes is presented that allows for intact protein mass measurements, localization of post-translational modifications and relative quantitation or turnover measurement.

Mass Spectrometry↗
PubMed2002

Automated sample preparation and LC-MS for high-throughput ADME quantification.

Bioanalytical groups in the pharmaceutical industry provide quantitative data to support all stages of drug discovery. The increased use of 96-well plates and robotic liquid handling systems, the availability of robust triple quadruple mass spectrometers, and developments in chromatographic and samples preparation techniques, have all increased the rate at which this data can be generated. This review describes currently used methods and emerging technologies for automation of high-throughput quantitative bioanalysis. The focus is on recent applications of sample preparation and chromatography techniques compatible with detection by triple quadruple mass spectrometers.

Absorption↗