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At least 433 records · Page 24Linked to original sources
PubMed2006

Metabolomic analysis using optimized NMR and statistical methods.

NMR-based metabolomics requires robust automated methodologies, and the accuracy of NMR-based metabolomics data is greatly influenced by the reproducibility of data acquisition and processing methods. Effective water resonance signal suppression and reproducible spectral phasing and baseline traces across series of related samples are crucial for statistical analysis. We assess robustness, repeatability, sensitivity, selectivity, and practicality of commonly used solvent peak suppression methods in the NMR analysis of biofluids with respect to the automated processing of the NMR spectra and the impact of pulse sequence and data processing methods on the sensitivity of pattern recognition and statistical analysis of the metabolite profiles. We introduce two modifications to the excitation sculpting pulse sequence whereby the excitation solvent suppression pulse cascade is preceded by low-power water resonance presaturation pulses during the relaxation delay. Our analysis indicates that combining water presaturation with excitation sculpting water suppression delivers the most reproducible and information-rich NMR spectra of biofluids.

Animals↗
PubMed1995

Reversed-phase high-performance liquid chromatography of salmon calcitonin and its degradation products in biological samples using column switching and flow-through radioisotope detection.

For the determination of salmon calcitonin and its degradation products in biological samples, a reversed-phase HPLC method with column switching and flow-through radioisotope detection has been developed using high specific activity [125I]salmon calcitonin. Effects of the precolumn packing material and washing solvent were examined in terms of [125I]salmon calcitonin recovery. Spiked samples of [125I]salmon calcitonin in plasma and kidney homogenate were injected onto a LiChroprep RP-8 precolumn after dilution with 0.1% trifluoroacetic acid. After washing the polar interfering compounds with 0.1% trifluoroacetic acid, the concentrated [125I]salmon calcitonin and its degradation products were eluted and separated on a W-Porex C18 column with a gradient of 0.1% trifluoroacetic acid in acetonitrile-water. Detection and calibration of [125I]salmon calcitonin were possible down to picogram levels. Reproducible kinetic data for the degradation of intact [125I]salmon calcitonin were possible down to picogram levels. Reproducible kinetic data for the degradation of intact [125I]salmon calcitonin by rat kidney homogenate could be traced.

Animals↗
PubMed2001

Identification of the factors that influence the reproducibility of chromatographic retention data.

Principal component analysis was used to identify the parameters that influence the column-to-column and batch-to-batch reproducibility of retention times and retention factors measured on Symmetry C18, Kromasil C18, Luna C18 (2) and Vydac RP C18, all reversed-phase silica columns. We devised a procedure that allows the determination of the differences in column volume and packing density between two columns, provided that these columns are packed with identical stationary phases (i.e., phases that originate from the same batch). Principal component analysis of the retention times confirmed that the column-to-column variations of the column volume and the total porosity of the bed are the factors that influence the reproducibility of the retention times, the column volume being the major factor. For the fluctuations of the retention factors, the column phase ratios (or the bed porosities) and some specific, secondary retention mechanisms are responsible. All the C18 columns investigated proved to behave in a very similar fashion. Two principal components were always sufficient to characterize the variations of either the retention times or the retention factors.

Chromatography, Liquid↗
PubMed2001

A multiple source model for 6 MV photon beam dose calculations using Monte Carlo.

A multiple source model (MSM) for the 6 MV beam of a Varian Clinac 2300 C/D was developed by simulating radiation transport through the accelerator head for a set of square fields using the GEANT Monte Carlo (MC) code. The corresponding phase space (PS) data enabled the characterization of 12 sources representing the main components of the beam defining system. By parametrizing the source characteristics and by evaluating the dependence of the parameters on field size, it was possible to extend the validity of the model to arbitrary rectangular fields which include the central 3 x 3 cm2 field without additional precalculated PS data. Finally, a sampling procedure was developed in order to reproduce the PS data. To validate the MSM, the fluence, energy fluence and mean energy distributions determined from the original and the reproduced PS data were compared and showed very good agreement. In addition, the MC calculated primary energy spectrum was verified by an energy spectrum derived from transmission measurements. Comparisons of MC calculated depth dose curves and profiles, using original and PS data reproduced by the MSM, agree within 1% and 1 mm. Deviations from measured dose distributions are within 1.5% and 1 mm. However, the real beam leads to some larger deviations outside the geometrical beam area for large fields. Calculated output factors in 10 cm water depth agree within 1.5% with experimentally determined data. In conclusion, the MSM produces accurate PS data for MC photon dose calculations for the rectangular fields specified.

Algorithms↗
PubMed1986

Follicular center cell lymphoma. Morphologic data relating to observer reproducibility.

Subjectivity and observer variation in non-Hodgkin's lymphoma continues to plague current classification schemes. It was thought important to assess objectively derived morphometric data to see if three categories of follicular center cell (FCC) lymphomas, follicular small cleaved cell, follicular mixed small cleaved and large cell, and diffuse and nodular large cell, actually fall into distinctly separate classes based on nuclear parameters. Mean nuclear area, contour index and invagination depth of neoplastic lymphocytes, and the percentage of invaginated and clefted nuclear profiles in each example of FCC lymphoma were evaluated by three different approaches. Results obtained from distribution of the morphometric data in scatter diagrams, calculation of the overlap index, and linear regression values, all revealed considerable (but variable) degrees of overlap between the three FCC subtypes regardless of the nuclear parameter employed. Where separation between FCC lymphomas was maximal, although still incomplete, there was no consistent correlation between the nuclear parameter and the pair of FCC lymphomas being compared. At least in terms of nuclear morphological features, non-Hodgkin's lymphomas of FCC type seem to represent a continuum of one disease process. The information provides a basis for understanding some reasons underlying the problem of observer variation in non-Hodgkin's lymphoma.

Cell Nucleus↗
PubMed2001

Validation of the Tracmor triaxial accelerometer system for walking.

PURPOSE: Walking is likely to contribute substantially to nonexercise activity thermogenesis. The Tracmor triaxial accelerometer system (Maastricht, The Netherlands) is the most widely validated system for detecting body movement in free-living subjects. The aim of this study was to validate the Tracmor triaxial accelerometer system for estimating the energy expenditure of walking. METHODS: Experiments were conducted in healthy subjects. First, baseline variability for Tracmor output was determined for subjects standing still. Second, Tracmor output was compared for walking on a treadmill and on level ground. Third, both Tracmor output and energy expenditure were compared for walking on a treadmill and walking on level ground. Finally, the effect of gradient on Tracmor output and energy expenditure was compared for subjects walking on a treadmill. RESULTS: The data demonstrated excellent reproducibility for comparing Tracmor output for standing (CV < 2%). There were excellent log-linear relationships between velocity and Tracmor output walking on a treadmill (r = 0.998) and on level ground (r = 0.999). Tracmor output and the energy expenditure of walking were inseparable for the two modalities of walking. However, the variance in response was such that to reliably derive the relationship between Tracmor output and energy expenditure, separate regression equations are needed for each subject. Finally, the Tracmor accelerometer did not detect the increased energy expenditure of walking that occurs as gradient increases. CONCLUSION: The Tracmor triaxial accelerometer provides reproducible and reliable data on the body motion associated with walking regardless of whether a subject walks on a treadmill or level ground. Tracmor units can be used to predict the energetic cost of walking provided that separate regression equations are derived for each subject to convert Tracmor output to energy expenditure.

Adult↗
PubMed1982

Reproducibility of electron diffraction intensity data obtained from hydrated microcrystals of rat hemoglobin.

Analysis of electron diffraction patterns from rat hemoglobin taken at 200 kV on a wet stage yields intensity data to a resolution of 2-3 A which are as reproducible as those from typical X-ray diffraction. Some crystals were so similar that the differences in measured intensities were insignificant (R = 0.056), but in other cases real differences between crystals were observed (R = 0.33). Dynamic scattering was insignificant under our diffraction conditions; however, exposures to electron doses as low as 10(-2) e/A2 produced detectable changes in measured intensities. Limits to the reproducibility of the data are set by radiation damage and errors in microdensitometry.

Animals↗
PubMed1977

Quantitative osmotic fragility and disease states: a preliminary study.

The Kalmedic D-3 Fragiligraph was used to obtain both cumulative and derivative osmotic fragility data. Several instrumental modifications and several procedural changes combined to make the data more reproducible and more easily obtained. An analytical expression employing but two parameters was found to give excellent fits for the cumulative data, and when differentiated, reproduced the experimental derivative data equally well. The curve fitting procedure used is given in detail. Data obtained on a limited number of subjects with beta-thalassemia or multiple sclerosis are presented. The interpretation of these data by techniques common to the literature is compared with the procedure developed in this paper.

Hemolysis↗
PubMed2005

Utility of lab-on-a-chip technology for high-throughput nucleic acid and protein analysis.

On-chip electrophoresis can provide size separations of nucleic acids and proteins similar to more traditional slab gel electrophoresis. Lab-on-a-chip (LoaC) systems utilize on-chip electrophoresis in conjunction with sizing calibration, sensitive detection schemes, and sophisticated data analysis to achieve rapid analysis times (<120 s). This work describes the utility of LoaC systems to enable and augment systems biology investigations. RNA quality, as assessed by an RNA integrity number score, is compared to existing quality control (QC) measurements. High-throughput DNA analysis of multiplex PCR samples is used to stratify gene sets for disease discovery. Finally, the applicability of a high-throughput LoaC system for assessing protein purification is demonstrated. The improvements in workflow processes, speed of analysis, data accuracy and reproducibility, and automated data analysis are illustrated.

DNA↗
PubMed2006

A mathematical model of the treatment and survival of patients with high-grade brain tumours.

More years of life per patient are lost as the result of primary brain tumours than any other form of cancer. The most aggressive of these is known as glioblastoma (GBM). The median survival time of patients with GBM is under 10 months and the outlook has hardly improved over the past 20 years. Generally, these tumours are remarkably resistant to radiotherapy and yet about 2-3% of all GBMs appear to be cured. The objectives of this study were to formulate a mathematical and phenomenological model of tumour growth in a population of patients with GBM to predict survival, and to use the model to extract biological information from clinical data. The model describes the growth of the tumour and the resulting damage to the normal brain using simple concepts borrowed from chemical reaction engineering. Death is assumed to result when the amount of surviving normal brain falls to a critical level. Radiotherapy is assumed to destroy tumour but not healthy brain. Simple rules are included to represent approximately the clinician's decisions about what type of treatment to offer each patient. A population of patients is constructed by assuming that key parameters can be sampled from statistical distributions. Following Monte Carlo simulation, the model can be fitted to data from clinical trials. The model reproduces clinical data extremely accurately. This suggests that the long-term survivors are not a separate sub-population but are the 'lucky tail' of a unimodal distribution. The estimated values of radiation sensitivity (represented as SF2, the survival fraction after 2Gy) suggest the presence of severe hypoxia, which renders cells less sensitive to radiation. The model can predict the probable age distribution of tumours at presentation. The model shows the complicated effects of waiting times for treatment on the survival outcomes, and is used to predict the effects of escalation of radiotherapy dose. The model may aid the design of clinical trials using radiotherapy for patients with GBM, especially in helping to estimate the size of trial required. It is also designed in a generic form, and might be applicable to other tumour types.

Age Distribution↗
PubMed1985

A high-precision automatic closed-circuit respirometer for small animals.

An automatic apparatus for the continuous measurement of O2 consumption of small laboratory animals is described. By use of a high-sensitivity pressure transducer with associated circuitry together with a peristaltic O2 delivery system, the closed respirometer chamber is maintained at atmospheric pressure +/- 0.5 mmH2O. O2 delivery is measured to within 0.25 ml by recording rotations of the peristaltic pump, following calibration by the withdrawal of a preset volume of air from the chamber. Static trials (with the chamber empty) indicate a high degree of reproducibility of data with the chamber pressure remaining at atmospheric pressure +/- 0.5 mmH2O as a result of the proportional, as opposed to fixed-volume, delivery of O2. Trials with mice and rats have likewise produced data with a high degree of reproducibility.

Animals↗
PubMed1996

Rapid determination of amphetamine stereoisomer ratios in urine by gas chromatography-mass spectroscopy.

We describe a method for the determination of amphetamine optical isomer ratios. A simple basic extraction of amphetamine from urine is followed by on-column derivatization with (S)-(-)-N-(trifluoroacetyl)-L-prolyl chloride and analysis by gas chromatography-mass spectroscopy. This approach has been shown to be satisfactory for the separation of d- and l-amphetamine isomers and precision data shows the method to be reproducible. Patient data is presented to demonstrate the ability to differentiate between patients taking d-amphetamine and those using illicit (racemic) amphetamine by their l/d amphetamine ratio.

Amphetamine↗
PubMed2007

Reproducibility of postprocessing of quantitative CT perfusion maps.

OBJECTIVE: The purpose of this study was to assess interobserver and intraobserver variability in evaluation of the reproducibility of quantitative data obtained in semiautomated postprocessing of CT perfusion data sets by observers of different levels of skill and experience and in fully automated postprocessing. MATERIALS AND METHODS: Twenty CT perfusion data sets were postprocessed by a neuroradiologist using an automated postprocessing program and by five observers (neuroradiology attending, neurology attending, radiology resident, senior and junior CT technologists) who received a brief training session in use of software for semiautomated postprocessing. For assessment of intraobserver variability, each observer repeated postprocessing of 10 CT perfusion data sets. Standard regions of interest were placed on identical locations for each observer's cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) maps of three brain regions: an ischemia-infarct region, normal cortical gray matter, and white matter. RESULTS: The variability in mean quantitative values of CBF, CBV, and MTT was 2.5-9.5% among all observers. Greater variability (20.4%) was introduced with the automated program. High correlation was found among all possible pairings of observers (r = 0.87-0.99). Low correlation was observed between automated postprocessing and postprocessing by all observers. Intraobserver variability in quantitative CT perfusion data ranged from 0.29% to 10.8%. High intraobserver correlation (r = 0.91-0.99) was found for the observers. CONCLUSION: Quantitative CBF, CBV, and MTT data obtained from postprocessing of CT perfusion data sets are reproducible among observers with varying levels of skill and experience. Observer interaction with the software is an important component for correct identification of user-defined parameters. Establishing a uniform and standard postprocessing technique is essential for maintaining good reproducibility.

Algorithms↗
PubMed2003

Whole-genome microarrays of fission yeast: characteristics, accuracy, reproducibility, and processing of array data.

BACKGROUND: The genome of the fission yeast Schizosaccharomyces pombe has recently been sequenced, setting the stage for the post-genomic era of this increasingly popular model organism. We have built fission yeast microarrays, optimised protocols to improve array performance, and carried out experiments to assess various characteristics of microarrays. RESULTS: We designed PCR primers to amplify specific probes (180-500 bp) for all known and predicted fission yeast genes, which are printed in duplicate onto separate regions of glass slides together with control elements (approximately 13,000 spots/slide). Fluorescence signal intensities depended on the size and intragenic position of the array elements, whereas the signal ratios were largely independent of element properties. Only the coding strand is covalently linked to the slides, and our array elements can discriminate transcriptional direction. The microarrays can distinguish sequences with up to 70% identity, above which cross-hybridisation contributes to the signal intensity. We tested the accuracy of signal ratios and measured the reproducibility of array data caused by biological and technical factors. Because the technical variability is lower, it is best to use samples prepared from independent biological experiments to obtain repeated measurements with swapping of fluorochromes to prevent dye bias. We also developed a script that discards unreliable data and performs a normalization to correct spatial artefacts. CONCLUSIONS: This paper provides data for several microarray properties that are rarely measured. The results define critical parameters for microarray design and experiments and provide a framework to optimise and interpret array data. Our arrays give reproducible and accurate expression ratios with high sensitivity. The scripts for primer design and initial data processing as well as primer sequences and detailed protocols are available from our website.

DNA Primers↗
PubMed1994

A scheme to account for the effects of Rb+ and K+ on inward rectifier K channels of bovine artery endothelial cells.

An electrochemical gating model is presented to account for the effects described in the companion paper by M. R. Silver, M. S. Shapiro, and T. E. DeCoursey (1994. Journal of General Physiology, 103:519-548) of Rb+ and Rb+/K+ mixtures on the kinetics and voltage dependence of an inwardly rectifying (IR) K+ channel. The model proposes that both Rb+ and K+ act as allosteric modulators of an intrinsically voltage dependent isomerization between open and closed states. Occupancy of binding sites on the outside of the channel promotes channel opening and stabilizes the open state. Rb+ binds to separate sites within the pore and plugs IR channels. Occupancy of the pore by Rb+ can modify the rates of isomerization and the affinity of the allosteric sites for activator ions. The model also incorporates the proposed triple-barreled nature of the IR channel (Matsuda, H., 1988. Journal of Physiology. 397:237-258.) by proposing that plugging of the channel is a cooperative process involving a single site in each of the three bores, 80% of the way through the membrane field. Interaction between bores during plugging and permeation is consistent with correlated flux models of the properties of the IR channel. Parallel bores multiply the number allosteric sites associated with the macromolecular channel and allow for steep voltage dependence without compromising the parallel shift of the half-activation potential with reversal potential. Our model proposes at least six and possibly 12 such allosteric binding sites for activator ions. We derive algebraic relations that permit derivation of parameters that define simple versions of our model from the data of Silver et al. (1994). Numerical simulations based on those parameters closely reproduce that data. The model reproduces the RS+ induced slowing of IR kinetics and the negative shift of the relation between the half-activation voltage (V1/2) and reversal potential when channel plugging is associated with (a) a slowing of the isomerization rates; (b) an increase in the affinity of allosteric sites on closed channels that promote opening; and (c) a decrease in the affinity of sites on open channels that slow closing. Rb+ also slows closing at positive potentials where open channel blockade is unlikely. Allowing Rb+ to be 1.5 times more potent than K+ as an activator in the model can account for this effect and improves the match between the predicted and observed relation between the Rb+ to K+ mole fraction and the opening rate at V1/2.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2002

Gene expression during acute allograft rejection: novel statistical analysis of microarray data.

High-throughput microarrays promise a comprehensive analysis of complex biological processes, yet their applicability is hampered by problems of reproducibility and data management. The current study examines some of the major questions of microarray use in a well-described model of allograft rejection. Using the Brown Norway to Lewis heterotopic heart transplant model, highly purified RNA was isolated from cardiac tissue at postoperative days (POD) 3, 5 and 7 and hybridized onto Affymetrix U34A microarrays. Using the log average ratio (LAR), changes in gene expression were monitored at each timepoint and p-values generated through statistical analysis. Microarray data were verified for 13 significant transcripts using RT-PCR. Of the 8800 transcripts studied, 2864 were increased on POD 3, 1418 on POD 5 and 2745 on POD 7. Verifying previous studies, many up-regulated genes appeared to be associated with the inflammatory process and graft infiltrating cells. Down-regulated transcripts included many novel molecules such as SC1 and decorin. LAR analysis provides a useful approach to analyze microarray data. Results were reproducible and correlated well with both RT-PCR and prior studies. Most importantly, these results provide new insights into the pathogenesis of acute rejection and suggest new molecules for future studies.

Animals↗
PubMedDate not supplied

Comparison of automated cellular imaging system and manual microscopy for immunohistochemically stained cryostat sections of lung cancer specimens applying p53, ki-67 and p120.

Seventy-one frozen lung tissue specimens from 52 patients suffering from non-small cell lung cancer (NSCLC) were analysed in this study. Cryostat sections were stained with monoclonal antibodies against p53, ki-67 and p120, all of which are of prognostic value in NSCLC. Slides were evaluated by standard manual microscopy (MM) and using a new Automated Cellular Imaging System (ACIS). The results obtained with ACIS correlated significantly with MM examination (p<0.001). However, ACIS showed a higher sensitivity, especially for specimens with a low infiltration volume. In 15 (6.8%) MM negative cases singular positive cells were identified with ACIS. In cases with a high infiltration volume subjective (MM) quantitation tended to overestimate the number of infiltrating cells. ACIS guaranteed a high reproducibility of data. We conclude that ACIS-assisted analysis is a valid means of investigating the p53, ki-67 and p120 antibody expression in cryostat sections of lung cancer specimens. ACIS can complement conventional manual microscopy due to its higher accuracy, sensitivity and better reproducibility of data.

Adenocarcinoma↗