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PubMedDate not supplied

Use of statistical design of experiments in the optimization of amide synthesis utilizing polystyrene-supported N-hydroxybenzotriazole resin.

Two fields that routinely perform reaction optimization studies are chemical development (prior to scale-up) and medicinal or combinatorial chemistry (prior to analogue synthesis or library production). To date, the use of statistical design of experiments (DoE) in conjunction with automated synthesizers has been applied in process research to a greater extent than in the medicinal or combinatorial laboratories. We have applied DoE in conjunction with an automated synthesizer to optimize the synthesis of amides employing resin-bound N-hydroxybenzotriazole (PS-HOBt) active esters as intermediates. This methodology allowed the rapid development of an improved protocol for the parallel synthesis of amides by conversion of carboxylic acids to PS-HOBt esters followed by treatment with appropriate amines. Product isolation involved only simple filtration and evaporation.

Amides↗
PubMed1997

1H, 13C, and 15N NMR backbone assignments and chemical-shift-derived secondary structure of glutamine-binding protein of Escherichia coli.

1H, 13C, and 15N NMR assignments of the backbone atoms and beta-carbons have been made for liganded glutamine-binding protein (GlnBP) of Escherichia coli, a monomeric protein with 226 amino acid residues and a molecular weight of 24935 Da. GlnBP is a periplasmic binding protein which plays an essential role in the active transport of L-glutamine through the cytoplasmic membrane. The assignments have been obtained from three-dimensional triple-resonance NMR experiments on a 13C, 15N uniformly labeled sample as well as specifically labeled samples. Results from the 3D triple-resonance experiments, HNCO, HN(CO)CA, HN(COCA)HA, HNCA, HN(CA)HA, HN(CA)CO, and CBCA(CO)NH, are the main sources used to make the resonance assignments. Other 3D experiments, such as HNCACB, COCAH, HCACO, HCACON, and HOHAHA-HMQC, have been used to confirm the resonance assignments and to extend connections where resonance peaks are missing in some of the experiments mentioned above. We have assigned more than 95% of the polypeptide backbone resonances of GlnBP. The result of the standard manual assignment is in agreement with that predicted by an automated probailistic method developed in our laboratory. A solution secondary structure of the GlnBP-Gln complex has been proposed based on chemical shift deviations from random coil values. Eight alpha-helices and 10 beta-strands are derived using the Chemical Shift Index method.

Amino Acid Sequence↗
PubMed2001

Main findings and conclusions of the implementation of Directive 76/464/CEE concerning the monitoring of organic pollutants in surface waters (Portugal, April 1999-May 2000).

The purpose of this paper is to relate the implementation of Directive 76/464/CEE concerning the survey of priority dangerous substances and indicate the main priority pollutants encountered throughout a systematic monitoring program carried out in Portugal during a period of 14 months (April 1999-May 2000). Contrary to what it seems, the implementation of such a Directive is not straightforward owing to the large number of compounds and samples to be analyzed at trace levels. During this period of time, 115 priority semivolatile organic compounds (SVOCs), 41 volatile organic compounds (VOCs), six organotin compounds, 14 heavy metals, phosphate, nitrates, nitrites and ammonia were determined in 644 surface water samples (corresponding to 14 months and 46 sites). In addition, priority SVOCs were also determined in sediment, mussels and fish (muscle and liver). This paper reports the procedure and analytical tools used and necessary to determine the levels of 115 priority toxic and persistent semivolatile organic compounds selected from the EC Council Directive list (76/464/EEC). An advanced analytical protocol was developed involving enrichment of water samples in Oasis solid-phase extraction cartridges (SPE) in the Lisbon laboratory with a Gilson ASPEC XL automated extraction system and by sending the enriched cartridges to Barcelona by aeroplane at 4 degrees C, where elution and analysis were performed. Final determination was performed with gas chromatography-electron ionization mass spectrometry (GC-EI-MS). Acidic herbicides were determined by liquid chromatography with diode array detection. Owing to the great amount of data generated throughout the monitoring study, statistical analysis of the more representative contaminants was performed, allowing the identification of the more relevant contamination sources, percentages of findings and the description of their temporal and spatial (geographical) distributions. Recommendations and compounds that should be included in future monitoring programs are given.

Environmental Monitoring↗
PubMed1996

The performance of a variable-flow indirect calorimeter.

Indirect calorimetry estimates energy expenditure from measurement of respiratory gas exchange volumes. This paper considers the design and evaluation of an indirect calorimeter, the Europa GEM, suitable for use in nutritional research. The calorimeter is of the ventilated hood, flow-through type and is intended for use with spontaneously breathing patients. Our aim was to develop an accurate, flexible instrument with a high level of automation. Performance was assessed in a laboratory simulation using reference gas injections (n = 24) producing a mean error of 0.3 +/- 2% in oxygen consumption (VO2), 1.8 +/- 1% in carbon dioxide production (VCO2) and 1.4 +/- 1.5% in respiratory quotient (RQ). In order to investigate the effect of FeCO2 on error multiplication a further subdivision (n = 8) of tests at FeCO2 = 0.5%, 0.75% and 1% was made by modulating the air flow through the hood. However, the predicted increase in system accuracy with increasing FeCO2 was not apparent in practice.

Calorimetry, Indirect↗
PubMed1984

Intelligent computational assistance for experiment design.

We have developed an automated system for the design of laboratory experiments in molecular biology. The system uses a planning method known as skeletal plan refinement that attempts to emulate the human cognitive task of experiment design. This paper describes the theory, history, and implementation of the design system and illustrates its function in the domain of DNA cloning experiments.

Base Sequence↗
PubMed1988

Semi-automated analysis of manually reconstructed tracks of progressively motile human spermatozoa.

Several alternative algorithms for computer-assisted derivation of measurements of movement characteristics from manually reconstructed tracks of progressively motile human spermatozoa were compared. Fifty tracks were reconstructed at 30 Hz from video recordings and analysed using traditional manual methods and by four combinations of computer algorithms. The best algorithm set was identified ('Videomot.mdpt') and the values for the curvilinear, average path and linear velocities (VCVL, VAVE and VLIN respectively), the amplitude of lateral displacement of the sperm head about the axis of progression (AH) and the number of times the sperm head crossed the average path (the 'beat/cross frequency', BXF) obtained using it were compared to those obtained by manual analysis. There was a considerable time saving when the computer-assisted method was used and the values it gave for the various movement characteristics showed good correspondence with those obtained manually. In addition, repeated data entry and analysis was found to be highly reproducible. When the tracks were re-plotted at 6 Hz (as used by the multiple-exposure photomicrography method for sperm motility analysis) insufficient information remained in the tracks for reliable determination of anything other than VLIN. We conclude that the Videomot.mdpt program provides reliable values for the movement characteristics of progressively motile human spermatozoa, and believe it will be of great value in the validation of commercial systems providing automated sperm movement analysis and in laboratories which do not have access to such costly equipment.

Algorithms↗
PubMed1995

Two-centre evaluation of the Abbott CD3500 blood counter.

The CD3500 blood counter (Abbott Laboratories) is a 33 parameter fully automated blood counter that produces a five part differential count with flagging of leucocyte abnormalities. In this evaluation excellent correlation between CD3500 and Coulter STKR blood counter was found for all red cell and platelet parameters on the 221 samples tested. Studies of carryover, mixing efficiency and precision also gave excellent results. There was a good correlation with manual 400 cell differential counts for neutrophils, lymphocytes, monocytes and eosinophils for the 468 samples compared. Correlation of CD3500 and manual basophil counts was poor. Normal samples stored at 4 degrees C and analysed while cold showed satisfactory stability for WBC, RBC, Hb, MCV and platelets for 48 h and a stable differential for 24 h. Correlation with the differential count produced by the Coulter STKS showed good correlation for neutrophils, lymphocytes, monocytes and eosinophils; correlation with STKS basophils was poor. False positive flagging rate varied between 8.9% (Band and/or IG) and 0.9% (NRBC) depending on the nature of the flag; 5.8% of samples exhibited two or more false positive flags. No significant breakdowns were encountered during the period of the evaluation. The scatterplot displays of laser light scatter produced by the instrument provide an interesting adjunct to conventional morphology.

Blood Cell Count↗
PubMed2004

How to track and assess genotyping errors in population genetics studies.

Genotyping errors occur when the genotype determined after molecular analysis does not correspond to the real genotype of the individual under consideration. Virtually every genetic data set includes some erroneous genotypes, but genotyping errors remain a taboo subject in population genetics, even though they might greatly bias the final conclusions, especially for studies based on individual identification. Here, we consider four case studies representing a large variety of population genetics investigations differing in their sampling strategies (noninvasive or traditional), in the type of organism studied (plant or animal) and the molecular markers used [microsatellites or amplified fragment length polymorphisms (AFLPs)]. In these data sets, the estimated genotyping error rate ranges from 0.8% for microsatellite loci from bear tissues to 2.6% for AFLP loci from dwarf birch leaves. Main sources of errors were allelic dropouts for microsatellites and differences in peak intensities for AFLPs, but in both cases human factors were non-negligible error generators. Therefore, tracking genotyping errors and identifying their causes are necessary to clean up the data sets and validate the final results according to the precision required. In addition, we propose the outline of a protocol designed to limit and quantify genotyping errors at each step of the genotyping process. In particular, we recommend (i) several efficient precautions to prevent contaminations and technical artefacts; (ii) systematic use of blind samples and automation; (iii) experience and rigor for laboratory work and scoring; and (iv) systematic reporting of the error rate in population genetics studies.

Animals↗
PubMed1986

Comparative evaluation of the new Titertek Enterobac Rapid Automated System (TTE-RAS) for identification of members of the family Enterobacteriaceae.

The Titertek Enterobac Rapid Automated System (TTE-RAS; Flow Laboratories, SpA, Milan, Italy), a new semiautomated system for the identification of members of the family Enterobacteriaceae, was compared with the API 20E system (API System P.A., Montalieu Vercieu, France) by using 284 clinically isolated strains that were previously identified by conventional methods. Six strains from the American Type Culture Collection (Rockville, Md.) were included to evaluate the reproducibility of identification by both systems. Correct identifications at the species level were 93.7% with TTE-RAS and 96.1% with API 20E. Although some of the features of the TTE-RAS data base were not satisfactory, we consider this new miniaturized system to be a very valuable tool for the rapid identification of the most frequently isolated opportunistic bacteria.

Enterobacteriaceae↗
PubMed1994

Influence of serum cholesterol and cholesterol subfractions on restenosis after successful coronary angioplasty. A quantitative angiographic analysis of 3336 lesions.

BACKGROUND: Previous reports have suggested that hyperlipidemia may be associated with increased restenosis after successful coronary angioplasty. These studies have been compromised, however, by their retrospective nature, the small numbers involved, differences in the definition of restenosis, and inadequate quantitative angiographic follow-up at a prespecified time interval. The objective of the study was to examine the relation between serum cholesterol and long-term restenosis after coronary angioplasty, using quantitative angiography, at a predetermined time interval. METHODS AND RESULTS: The study population comprised 2753 patients (3336 lesions) prospectively enrolled and successfully completing four major restenosis trials. Cineangiographic films were processed and analyzed at a central angiographic core laboratory with the use of an automated interpolated edge-detection technique. Serum total cholesterol was measured at trial entry and at 6 months. Hypercholesterolemia was defined as total cholesterol > 7.8 mmol.L-1 at trial entry. Two approaches were used to assess restenosis: first, a categorical approach using the cutoff point of > 50% diameter stenosis at follow-up and second, a continuous approach examining changes in minimal luminal dimensions, the absolute loss (change in minimum luminal diameter after PTCA to follow-up, in mm) and relative loss (absolute loss corrected for vessel size), which may give a better understanding of the underlying pathological process involved. One hundred sixty patients with 191 lesions (5.73%) had hypercholesterolemia (total cholesterol, > 7.8 mmol.L-1; mean +/- SD, 8.46 +/- 0.75 mmol.L-1) and 2593 patients with 3145 lesions (94.27%) normal cholesterol (5.67 +/- 1.06 mmol.L-1). The restenosis rate was similar in patients with and without hypercholesterolemia (31.9% versus 33.7%, respectively; relative risk, 0.975; 95% CI, 0.882 to 1.077; P = .68). Similarly, there was no difference in either the absolute or relative loss between patients with and without hypercholesterolemia (0.31 +/- 0.53 versus 0.32 +/- 0.53 mm and 0.12 +/- 0.20 versus 0.13 +/- 0.21, respectively, P = NS for both). Conversely, the total serum cholesterol in patients with restenosis (using the categorical definition) was similar to those without restenosis (5.84 +/- 1.24 versus 5.81 +/- 1.22 mmol/L, respectively, P = NS). Dividing the population into deciles according to total cholesterol and examining the categorical restenosis rate (by chi 2) as well as the absolute and relative loss by ANOVA again revealed no significant differences between deciles. Subgroup analysis of 579 patients (667 lesions) with HDL and LDL cholesterol levels available again revealed no differences in the categorical restenosis rate (by chi 2) or the absolute or relative loss between deciles according to LDL, HDL, or LDL:HDL ratio, suggesting no influence of these cholesterol subfractions on restenosis. CONCLUSIONS: Our results indicate that there is no association between cholesterol and restenosis by either a categorical or continuous approach, suggesting that measures aimed at reducing total cholesterol are unlikely to significantly influence postangioplasty restenosis.

Aged↗
PubMed1995

Long-term luminal renarrowing after successful elective coronary angioplasty of total occlusions. A quantitative angiographic analysis.

BACKGROUND: The long-term angiographic outcome after successful dilatation of coronary occlusions remains unclear. The objective of this study was to examine long-term restenosis after successful balloon dilatation of coronary occlusions at a predetermined time interval with quantitative angiography and compare this with a control population of stenoses. METHODS AND RESULTS: The study population comprised 2950 patients (3583 lesions) prospectively enrolled in and successfully completing four major restenosis trials (86% quantitative angiographic follow-up). Cineangiographic films were processed and analyzed at a central core laboratory with the use of an automated interpolated edge detection technique. The study population comprised 266 occlusions (7%) defined as total when there was absent anterograde filling beyond the lesion (109 lesions) and functional (157 lesions) when faint, late anterograde opacification of the distal segment was seen in the absence of a discernible luminal continuity; 3317 lesions were defined as stenoses (93%). Restenosis was significantly higher after successful dilatation of occlusions than of stenoses. With the categorical (> 50% diameter stenosis at follow-up) approach, the restenosis rate was 44.7% in occlusions compared with 34.0% in stenoses (P < .001; relative risk, 1.575; CI, 1.224 to 2.027). Similarly, the absolute loss (defined as the change in minimal lumen diameter between post coronary angioplasty and follow-up; in millimeters, mean +/- SD) (0.43 +/- 0.68) in occlusions was significantly higher than in stenoses (0.31 +/- 0.51, P < .001), as was the relative loss, defined as the change in minimal lumen diameter between postangioplasty and follow-up, adjusted for the vessel size (0.17 +/- 0.28 versus 0.12 +/- 0.20, P < .001). The higher restenosis rate in the occlusions group was due predominantly to an increased number of occlusions at follow-up angiography in this group (19.2% compared with 5.0% for stenoses, P < .001). Within the occlusions group, there were no significant differences in long-term outcome between total and functional occlusions (restenosis rate, 45.0% versus 44.6%; reocclusion rate, 23.9% versus 15.9%; absolute loss, 0.53 +/- 0.69 versus 0.36 +/- 0.67; relative loss, 0.21 +/- 0.28 versus 0.15 +/- 0.28; P = NS). CONCLUSIONS: These results indicate that successfully dilated coronary occlusions, both total and functional, have a higher rate of angiographic restenosis at 6 months than stenoses. This is due chiefly to a higher rate of occlusion at follow-up angiography in this group of lesions. Measures aimed at reducing restenosis after successful dilatation of coronary occlusion should be focused in this direction.

Angioplasty, Balloon, Coronary↗
PubMed2000

Development of a facile method for high throughput screening with reporter gene assays.

This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.

Animals↗
PubMed2003

Typing single-nucleotide polymorphisms using a gel-based sequencer: a new data analysis tool and suggestions for improved efficiency.

Single-nucleotide polymorphisms (SNPs) are increasingly used as genetic markers. Although a high number of SNP-genotyping techniques have been described, most techniques still have low throughput or require major investments. For laboratories that have access to an automated sequencer, a single-base extension (SBE) assay can be implemented using the ABI SNaPshot trade mark kit. Here we present a modified protocol comprising multiplex template generation, multiplex SBE reaction, and multiplex sample analysis on a gel-based sequencer such as the ABI 377. These sequencers run on a Macintosh platform, but on this platform the software available for analysis of data from the ABI 377 has limitations. First, analysis of the size standard included with the kit is not facilitated. Therefore a new size standard was designed. Second, using Genotyper (ABI), the analysis of the data is very tedious and time consuming. To enable automated batch analysis of 96 samples, with 10 SNPs each, we developed SNPtyper. This is a spreadsheet-based tool that uses the data from Genotyper and offers the user a convenient interface to set parameters required for correct allele calling. In conclusion, the method described will enable any lab having access to an ABI sequencer to genotype up to 1000 SNPs per day for a single experimenter, without investing in new equipment.

Computational Biology↗
PubMed2003

A guide to building image-centric databases.

There is a paucity of image-centric neuroinformatics infrastructure within the individual investigator's laboratory despite the obvious need for automation and integration of experimental results. Yet, solutions can often be readily built using off-the-shelf databases and associated tools. Doing so simplifies day-to-day research operation and increases throughput. Proper construction of in-house solutions may also expedite community-wide integration of private and public data repositories. Here we describe neuroinformatics approaches at different levels of functionality, required expertise, and size of image datasets. The simplest approach offers ease of image browsing and rudimentary searching. More sophisticated systems provide powerful search capabilities, a means of tracking analysis, and even automated serial processing pipelines. In this practicum, we provide guidance in selecting among the different options.

Animals↗
PubMed1988

An alternative approach to the prevention of succinyldicholine-induced apnoea.

Succinyldithiocholine was utilized as a substrate analogue of succinyldicholine to study normal and atypical serum pseudocholinesterase (EC 3.1.1.8). In the method, the enzyme acts on succinyldithiocholine to release thiocholine, which reacts with 5,5'-dithio-bis-(2-nitrobenzoic acid) to produce a coloured compound with maximal absorbance at 410 nm. The procedure appears to be precise (between-day analysis gives a coefficient of variation between 1.1 and 3.7%) and amenable to automation, permitting routine use in any laboratory. The reference interval for 300 healthy adults with "usual" cholinesterase genotype was estimated to be 34-77 U/l, with a significant difference between males and females (40-78 U/l for men and 33-76 U/l for women, p less than 0.01). The median activity in 105 individuals with "heterozygous" cholinesterase genotype was 22 U/l (range 5-35 U/l), and for 14 "atypical" homozygotes 1.5 U/l (range 1-4 U/l). The assay with succinyldithiocholine may offer a direct procedure for preoperative screening of individuals with an abnormal response to the muscle relaxant succinyldicholine, thus avoiding the determination of genotype by measurement of inhibitor numbers.

Apnea↗
PubMed1991

Evaluation of the automated haematology analyser Sysmex NE-8000.

The automated haematology analyser Sysmex NE-8000 was compared with the Technicon H-1, the automated analyser routinely used in our laboratory, and with manual cell differentiation results. One hundred and seventy samples from the daily routine workload, comprising specimens from healthy adults and patients with various ailments, were analysed on the Sysmex NE-8000 and the Technicon H-1. A manual-400 leukocyte differential count was performed on each specimen. Comparison of the results from the two blood cell counters showed good correlation (r greater than 0.9) for the white blood cell count, haemoglobin, haematocrit and platelet count. For the red blood cell count and mean cellular volume, the correlation coefficients were greater than 0.8. In the leukocyte differential count, Sysmex NE-8000 and Technicon H-1 showed good correlations for the neutrophil (r = 0.953), lymphocyte (r = 0.763), and eosinophil counts (r = 0.904). Correlation coefficients were very low for monocyte (r = 0.130) and basophil counts (r = 0.006). Correlation between the manual-400 method and the electronic leukocyte differential count showed similar results. Two hundred and twenty six normal and abnormal samples were compared with respect to morphology flagging with the two analysers, using the manual differentiation as the reference method. The abnormal specimens were representative of the range of leukocyte abnormalities seen in our laboratory. Sensitivity for detecting blasts was equal for both analysers. Sysmex NE-8000 was much more sensitive for detecting immature granulocytes than Technicon H-1. Low ranges of atypical lymphocytes were missed by Sysmex NE-8000. Left shift was also frequently missed.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Cell Count↗