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At least 451 records · Page 25Linked to original sources

Preprocessing implementation for microarray (PRIM): an efficient method for processing cDNA microarray data.

cDNA microarray technology is useful for systematically analyzing the expression profiles of thousands of genes at once. Although many useful results inferred by using this technology and a hierarchical clustering method for statistical analysis have been confirmed using other methods, there are still questions about the reproducibility of the data. We have therefore developed a data processing method that very efficiently extracts reproducible data from the result of duplicate experiments. It is designed to automatically filter the raw results obtained from cDNA microarray image-analysis software. We optimize the threshold value for filtering the data by using the product of N and R, where N is the ratio of the number of spots that passed the filtering vs. the total number of spots, and R is the correlation coefficient for results obtained in the duplicate experiments. Using this method to process mouse tissue expression profile data that contain 1,881,600 points of analysis, we obtained clustered results more reasonable than those obtained using previously reported filtering methods.

Animals↗

Single-stranded linear amplification protocol results in reproducible and reliable microarray data from nanogram amounts of starting RNA.

The range of scientific questions utilizing DNA microarray techniques is limited by the fact that these methods require 5-40 microg of high-quality total RNA. Thus, methods that reliably amplify the starting RNA amount could expand the applicability of DNA microarray technology. We developed a single-stranded linear amplification protocol (SLAP) that combines the reproducibility of in vitro transcription and the amplification robustness of polymerase chain reactions. We compared SLAP to the NIH-IVT amplification protocol. SLAP displayed excellent conservation of the 5'/3' signal and demonstrated the most robust amplification, producing the recommended amounts of biotin-labeled RNA with as little as 0.002 microg of starting RNA. Both SLAP and NIH-IVT methods demonstrated good reproducibility, but SLAP maintained the highest level of reliability with RNA starting amounts of <0.05 microg. These results suggest that SLAP is an excellent alternative to IVT-based amplification protocols when RNA is limited by small sample size.

Biotin↗

Reproducibility of continuous 24 hour oesophageal pH monitoring in infants and children.

The reproducibility of 24 hour oesophageal pH monitoring was studied in 30 infants and children including the proportions of the investigation time with a pH less than 4 (reflux index), the number of episodes with a pH less than 4 or lasting longer than five minutes, and the duration of the longest episodes with a pH less than 4. Twenty four hour pH monitoring was performed using identical equipment on two consecutive days under similar conditions. Pearson correlation coefficients range from 0.88 to 0.98. The results obtained on two consecutive days were similar. The reflux index and the number of reflux episodes greater than five minutes were the most reproducible parameters. Despite the many patient related factors influencing pH data, the reproducibility of 24 hour pH monitoring data are satisfactory for routine clinical application.

Child↗

Comparative analysis of ancient ceramics by neutron activation analysis, inductively coupled plasma-optical-emission spectrometry, inductively coupled plasma-mass spectrometry, and X-ray fluorescence.

The accurate measurement of the maximum possible number of elements in ancient ceramic samples is the main requirement in provenance studies. For this reason neutron activation analysis (NAA) and X-ray fluorescence (XRF) have been successfully used for most of the studies. In this work the analytical performance of inductively coupled plasma-optical-emission spectrometry (ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS) has been compared with that of XRF and NAA for the chemical characterization of archaeological pottery. Correlation coefficients between ICP techniques and XRF or NAA data were generally better than 0.90. The reproducibility of data calculated on a sample prepared and analysed independently ten times was approximately 5% for most of the elements. Results from the ICP techniques were finally evaluated for their capacity to identify the same compositional pottery groups as results from XRF and NAA analysis, by use of multivariate statistics.

Journal Article↗

Repeatability and reproducibility of high concentration data in reversed-phase liquid chromatography. I. Overloaded band profiles on Kromasil-C18.

Single-component adsorption-isotherm data were acquired by frontal analysis (FA) for six low-molecular-mass compounds (phenol, aniline, caffeine, theophylline, ethylbenzene and propranolol) on one Kromasil-C18 column, using water-methanol solutions (between 70:30 and 20:80, v/v) as the mobile phase. Propranolol data were also acquired using an acetate buffer (0.2 M) instead of water. The data were modeled for best agreement between calculated and experimental overloaded band profiles. The adsorption energy distribution was also derived and used for the selection of the best isotherm model. Widely different isotherm models were found to model best the data obtained for these compounds, convex upward (i.e. Langmuirian), convex downward (i.e. anti-Langmuirian), and S-shaped isotherms. Using the same sample size for all columns (loading factor, Lf approximately 10%), overloaded band profiles were recorded on four different columns packed with the same batch of Kromasil-C18 and five other columns packed with different batches of Kromasil-C18. These experimental band profiles were compared to the profile calculated from the isotherm measured by FA on the first column. The repeatability as well as the column-to-column and the batch-to-batch reproducibilities of the band profiles are better than 4%.

Adsorption↗

Experimental validation of molecular dynamics simulations of lipid bilayers: a new approach.

A novel protocol has been developed for comparing the structural properties of lipid bilayers determined by simulation with those determined by diffraction experiments, which makes it possible to test critically the ability of molecular dynamics simulations to reproduce experimental data. This model-independent method consists of analyzing data from molecular dynamics bilayer simulations in the same way as experimental data by determining the structure factors of the system and, via Fourier reconstruction, the overall transbilayer scattering-density profiles. Multi-nanosecond molecular dynamics simulations of a dioleoylphosphatidylcholine bilayer at 66% RH (5.4 waters/lipid) were performed in the constant pressure and temperature ensemble using the united-atom GROMACS and the all-atom CHARMM22/27 force fields with the GROMACS and NAMD software packages, respectively. The quality of the simulated bilayer structures was evaluated by comparing simulation with experimental results for bilayer thickness, area/lipid, individual molecular-component distributions, continuous and discrete structure factors, and overall scattering-density profiles. Neither the GROMACS nor the CHARMM22/27 simulations reproduced experimental data within experimental error. The widths of the simulated terminal methyl distributions showed a particularly strong disagreement with the experimentally observed distributions. A comparison of the older CHARMM22 with the newer CHARMM27 force fields shows that significant progress is being made in the development of atomic force fields for describing lipid bilayer systems empirically.

Computer Simulation↗

Reproducibility of dietary and other data from a self-administered questionnaire.

This study examined the reproducibility of information obtained from a questionnaire covering dietary and other information by comparing the answers to the questionnaires with answers to the same questions one year later. Answers from 191 men and 220 women, aged 40 to 65, in Fukuoka prefecture, Japan were compared. The surveys were conducted in November 1989 and November 1990. In the second survey, 97.2% of the first respondents answered. The concordance between the two responses was high, and the differences between the mean intakes from the two surveys were within 5% for 7 food items and within 15% for 14 food items among the 20 items about which the respondents were asked. The intraclass correlation coefficients varied from 0.30 for eggs to 0.62 for milk, with 16 items greater than 0.4. The differences in reproducibility between the sexes and two age categories were not significant. Close values also were obtained for the estimated consumption of salt. A substantially high reproducibility was observed on items regarding drinking and smoking; most of the kappa statistics and the intraclass correlation coefficients were between 0.5 and 0.9. The above reproducibility on individual food items was comparable to or better than those reported from other studies. The results of the present study thus indicate that the self-administered semiquantitative food intake questionnaire used for our cross-sectional study is useful for epidemiologic studies to assess the association between diet and various diseases. In particular, the present questionnaire is highly dependable regarding the overall group intake of foods.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Retention of restorations placed in noncarious cervical lesions after centric and eccentric occlusal loading in a chewing simulator--A pilot study.

PURPOSE: To evaluate in a pilot study whether (1) loading of restored teeth in a chewing simulator is an adequate method to reproduce clinical data about the retention rate of restorations placed in noncarious cervical lesions by means of the one-step self-etching adhesive system Prompt-L-Pop, and (2) whether eccentric loading contributes to the loss of Class V fillings. MATERIALS AND METHODS: In 12 extracted mandibular premolars with noncarious buccal cervical lesions of similar dimensions, restorations were placed without preparation using Prompt-L-Pop and Tetric Ceram. The adhesive was applied in one layer and cured at 650 mW/cm2 for 10 s, while the composite was placed in two increments and cured at 1200 mW/cm2 for 10 s. After storage in water at 37 degrees C for 7 days, the teeth were mounted in a chewing simulator that uses pneumatic cylinders as force actuators and subjected to a centric load of 50 N and 1,200,000 load cycles at a frequency of 1.6 Hz and simultaneously to 3125 thermocycles (5 degrees C/55 degrees C). The antagonists were standardized and made of Empress ceramic material. Every 100,000 load cycles, the fillings were evaluated with regard to retention. In the second phase, the same teeth were loaded on the lingual cusps with new antagonists for another 1,200,000 cycles. RESULTS: Neither during centric nor eccentric loading was any restoration loss observed. CONCLUSION: The short-term loading of extracted teeth with Class V fillings in a chewing simulator was inadequate to reproduce the data of clinical studies on the retention rate of a self-etching adhesive system. Obviously, long-term degradation mechanisms (eg, hydrolysis) acting on the composite/dentin interface play a more crucial role when it comes to the loss of retention of Class V restorations. Eccentric loading alone did not contribute to the loss of fillings, so that the theory--which maintains that this type of occlusal stress is a major cause for abfractions or the retention loss of restorations placed in abfraction lesions--may be questioned.

Aluminum Silicates↗

The reproducibility of striatal uptake data obtained with positron emission tomography and fluorine-18-L-6-fluorodopa tracer in non-human primates.

Cynomolgus and rhesus monkeys have been studied via PET with [18F]-L-6 fluorodopa tracer. Striatal fluorodopa uptake rate constants have been derived by graphical analysis of transaxial slice images centered on the striata. The differences between pairs of values of the rate constant, obtained from two scans on the same monkey separated by two weeks or more, exhibited a relative standard deviation of 34.4%. If the two scans were conducted one immediately after the other, with the position of the monkey undisturbed, the standard deviation was reduced to 14.0%. The utility of this technique was demonstrated by comparing the effects on the scans of halothane and pentobarbital anesthesia and by the administration of NSD 1015, a peripheral and central inhibitor of L-aromatic amino-acid decarboxylase, between back-to-back scans. With NSD 1015, the fluorodopa uptake constant was reduced by an average of 76.0%.

Animals↗

Reproducibility of treadmill exercise data in patients with atrial fibrillation.

Serial submaximal treadmill tests are often used to evaluate the efficacy of therapy in patients with atrial fibrillation. Since the response to serial tests can be influenced by a 'learning phenomenon', we performed maximal exercise tests on 9 patients (mean age 63 +/- 4 years) with chronic atrial fibrillation. Points of analysis for the initial and follow-up treadmill exercise tests were 3 mph/0% grade, the gas exchange anaerobic threshold, and maximal exertion. Significant (p less than 0.05) reductions in ventilation (l/min) and oxygen uptake (ml/kg/min) were observed on follow-up at a standard submaximal work load of 3.0 mph/0% grade and at the gas exchange anaerobic threshold. There was no significant alteration in these variables at maximal exertion. A reduction in heart rate was observed throughout exercise during the follow-up test with the most marked reduction (21 beats/min) occurring at 3.0 mph/0% grade. There were no differences in respiratory exchange ratio or systolic blood pressure at any point. The reduction in submaximal heart rate and gas exchange variables without a significant change in these variables at maximal exertion is consistent with a learning effect. Therefore, studies comparing consecutive submaximal exercise test responses in patients with atrial fibrillation can be misleading.

Aged↗

An approximate model and empirical energy function for solute interactions with a water-phosphatidylcholine interface.

An empirical model of a liquid crystalline (L alpha phase) phosphatidylcholine (PC) bilayer interface is presented along with a function which calculates the position-dependent energy of associated solutes. The model approximates the interface as a gradual two-step transition, the first step being from an aqueous phase to a phase of reduced polarity, but which maintains a high enough concentration of water and/or polar head group moieties to satisfy the hydrogen bond-forming potential of the solute. The second transition is from the hydrogen bonding/low polarity region to an effectively anhydrous hydrocarbon phase. The "interfacial energies" of solutes within this variable medium are calculated based upon atomic positions and atomic parameters describing general polarity and hydrogen bond donor/acceptor propensities. This function was tested for its ability to reproduce experimental water-solvent partitioning energies and water-bilayer partitioning data. In both cases, the experimental data was reproduced fairly well. Energy minimizations carried out on beta-hexyl glucopyranoside led to identification of a global minimum for the interface-associated glycolipid which exhibited glycosidic torsion angles in agreement with prior results (Hare, B.J., K.P. Howard, and J.H. Prestegard. 1993. Biophys. J. 64:392-398). Molecular dynamics simulations carried out upon this same molecule within the simulated interface led to results which were consistent with a number of experimentally based conclusions from previous work, but failed to quantitatively reproduce an available NMR quadrupolar/dipolar coupling data set (Sanders, C.R., and J.H. Prestegard. 1991. J. Am. Chem. Soc. 113:1987-1996). The proposed model and functions are readily incorporated into computational energy modeling algorithms and may prove useful in future studies of membrane-associated molecules.

Biophysical Phenomena↗

The acquisition and validation of the surface electromyogram signal for evaluating muscle fatigue.

The advent of new USAF aircraft control concepts necessitates the investigation of muscle fatigue in complex dynamic environments. The Acceleration Effects Branch of the Aerospace Medical Research Laboratory at Wright-Patterson Air Force Base had undertaken such a study for evaluation of fatigue in lateral G environments. Investigation of the literature indicates that the Fast Fourier Transform (FFT) of the electromyogram (EMG) signal samples could be used to determine fatigue by analysis of the center frequencies and amplitudes of the sampled power spectra. Reproducibility of data, especially in a dynamic environment, proved to be a challenge. However, acquisition and processing of the EMG signal have been optimized to give consistently reproducible and useable data. The method of sampling data and its processing are discussed.

Acceleration↗

Contrast-reduced imaging of tissue concentration and arterial level (CRITICAL) for assessment of cerebral hemodynamics in acute stroke by magnetic resonance.

RATIONALE AND OBJECTIVES: To compare cerebral perfusion data obtained by using a low-dose, T1-weighted MRI technique with radionuclide (single positron emission computed tomography [SPECT]) brain imaging and to assess the reproducibility of parametric MRI data (cerebral blood flow [CBF], cerebral blood volume [CBV], and time to peak [TTP]) by applying a previously described nuclear medicine technique to derive quantitative perfusion data. METHODS: Single-slice brain and neck images were rapidly acquired during the passage of a small (1/10th of normal dose) bolus of contrast. Parametric images were constructed from the MR data by extracting the bolus transit curve for the brain and the peak arterial input curve from the carotid vessels in the neck. These were compared with SPECT perfusion imaging. Twenty-four patients with acute stroke were studied with both techniques; 13 underwent repeated scanning to assess data reproducibility. RESULTS: Relative CBF data were comparable to SPECT data (r = 0.584, P = 0.01). Results were reproducible for relative CBF, CBV, and TTP. The arterial input function was significantly different on the second injection with an average difference of 73.5, suggesting that the signal-concentration relationship had lost linearity with increased contrast load. Absolute quantitative MRI data produced values in the expected range (CBF = 42.6 mL x 100 g(-1) x min(-1)). CONCLUSIONS: This technique allows estimation of CBF in the setting of acute stroke with quantitative values in the expected range. Repeated measurements in the same patients showed that this technique provides a reproducible measure of relative CBF, CBV, and TTP.

Brain↗

Femoro-tibial cartilage metrics from coronal MR image data: Technique, test-retest reproducibility, and findings in osteoarthritis.

MRI-based measures of cartilage morphology are being increasingly used as surrogate markers in osteoarthritis. In contrast to other knee joint surfaces, quantitative analysis of the femoral condyles from sagittal MRI suffers from limited precision. The objective, therefore, was to develop a technique for reproducible assessment of femoral cartilage morphology from coronal image data. Coronal MR images (3D T(1)-w FLASHwe) of the knee were obtained in 16 healthy volunteers and in 7 patients with severe osteoarthritis (OA, prior to knee arthroplasty), with repositioning between repeated scans. After segmentation the cartilage volume, thickness, and joint surface areas were quantified in the tibia and in an anatomically defined region of the femoral condyle. Immediate test-retest interscan precision errors (CV%) for femoral cartilage volume were 3.0% (SD = 26 microl) and 3.2% (29 microl) medially and laterally in volunteers, and 3.0% (34 microl) and 7.0% (37 microl) in OA patients. The estimated loss, from cross sectional data, in the patients in the medial femoral condyle (-61%/-4.4 SD) was higher than that in the medial tibia (-45%/-3.1 SD) and compared favorably with precision errors (ratio > 16:1). The technique proposed overcomes some of the problems associated with sagittal scans and thus shows high promise for reliable assessment of femoro-tibial cartilage loss in OA.

Adult↗

The reproducible acquisition of comparative liquid chromatography/tandem mass spectrometry data from complex biological samples.

An in-depth study of the reproducibility of data acquired for comparative proteomics analysis using a prototype two-stage heated laminar flow chamber fitted to a commercial high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) instrument was undertaken. The study is based on 24 replicate samples from four independent membrane preparations derived from two matched breast cancer cell lines. Variation and reproducibility in the data were evaluated at several levels highlighting the relative efficiency and variability of the acquisition routines used. Specifically, variation in the number and relative intensities of chromatographic peaks eluted from the LC column, precursor ion selection and sequence identification were evaluated. On average, approximately 6500 chromatographic peaks were generated for each acquisition with a corresponding coefficient of variance (CV) of less than 20%. Precursor ion selection and sequence identification averaged 1380 and 780 events per acquisition sample, respectively, with corresponding CVs of less than 10% for each. The reproducibility in the precursor ion selection was typically better than 60% between similar replicates. Using protein and peptide internal standards, it was found that the CV in retention time across the gradient between two acquisition pairs was typically less than 5%, whereas the average intensity ratio was 1.0 (expected) with a CV approaching 20%. An evaluation of the intensity ratios calculated from endogenous peptide sequences, identified across the acquisition set, indicated a CV of approximately 30%. Similarly, the CV associated with the top 1000 peptides indicated a mean and median of 28.4 and 26.95%. For a given acquisition pair it was also found that approximately 11% of the chromatographic peaks eluting from the column were linked to a sequence or identified. For these experiments, less than 10% of the peak pairs had absolute ratios greater than 2.0 and of those only approximately 10% had sequences linked to them. For each matched acquisition set on average 406 proteins were identified with a CV of less than 10%. Of the proteins that were identified approximately 30% had at least one predicted trans-membrane domain, indicating a four-fold increase over a crude homogenate sample with only minor enrichment. During these experiments it was found that the interface did not significantly alter the relative charge state distribution of ions, nor did it introduce significant interference from background ions. The interface was capable of 24-hour acquisition cycles.

Amino Acid Sequence↗

A mathematical model for the description of the Coomassie brilliant blue protein assay.

The Coomassie brilliant blue dye-binding method for protein assay has become important relatively recently. The basis of the assay method is the binding of dye to protein, with production of a dye-protein complex which absorbs light intensely at 620 nm, but the mechanism of the binding process is not well understood. In this paper, two mathematical models for the binding process are developed, one involving the binding of both protonated (green) and deprotonated (blue) forms of the dye. The second model allows only binding of the blue species to proteins. These models are tested for their ability to estimate number of dye-binding sites (n) and binding constants (Kd) from protein assay data. The models are also tested for their ability to reproduce the experimental assay curve using either known values or reasonable estimates of the equation parameters. The models are shown to be approximately equal in ability to reproduce experimental data related to the protein assay, which somewhat favors the simpler of the two models. In this paper, a method for estimating n and Kd from standard curve-fitting procedures is established. Hitherto, binding constants were only available from assay data taken under conditions of very large molar protein/dye ratios. The possibility of protonated forms of the dye binding to proteins was not ruled out by this study, but for many purposes the use of the simple dye-binding model, in which only the deprotonated dye species binds, is sufficient.

Indicators and Reagents↗