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PubMed2005

Fluoroprofile, a fluorescence-based assay for rapid and sensitive quantitation of proteins in solution.

The development of a sensitive fluorescence-based assay for the quantitative determination of protein concentration is described. The assay is based on the natural product epicocconone, which produces a large increase in fluorescence quantum yield upon binding to detergent-coated proteins in solution. There is a concomitant shift in the emission maximum from 520 to 605 nm after binding, which results in low background signal allowing a linear dynamic range of 40 ng/mL to 200 microg/mL for most proteins. There is little protein-to-protein variation except for iron-containing proteins and the assay can be used so that it is tolerant of chemicals commonly used in 2-D sample buffers. The assay is more sensitive than standard absorption assays such as the Bradford and Lowry assays, and has a greater dynamic range and sensitivity than other fluorescent assays.

Benzopyrans↗
PubMed1989

Solid-state imagers for microscopy.

Solid-state imagers offer a variety of options to the modern optical microscopist. Photodiode arrays, charge injection devices, and charge-coupled devices are the three basic types of solid-state imagers available for research imaging. The charge-coupled device (CCD) is the solid-state imager of choice, because of its superior characteristics and its widespread acceptance in the research environment. The performance characteristics of the CCD are well documented and understood, having been quantified by many experimenters, especially in the physical sciences. CCDs exhibit dynamic ranges up to 50,000:1, very high quantum efficiency, and ultralow noise. The camera system in which a device is used, however, dictates the overall performance which can be achieved. The CCD imaging system as it applies to cell biology is discussed in detail in Hiraoka et al. (1987). A video camera operating at 30 frames/second does not provide the resolution, low noise, dynamic range, and linearity of a slow scan, cooled camera operating at 1 frame/second. Conversely, a slow scan camera does not offer the user the facility to resolve rapidly changing events in real time. The analogy between an 8-mm movie camera (video camera) and a 35-mm snapshot camera (slow scan cooled camera) is a useful one to emphasize the different character of these two electronic imaging systems. These two basic camera systems both employ CCD imagers, and each has very different, but complementary, characteristics. No one camera system can address the wide variety of imaging problems which face the modern microscopist. The user of this new generation of instrumentation must decide which system best fits the problem at hand.

Calibration↗
PubMed1994

Grouping of somatosensory neurons in the spinal cord and the gracile nucleus of the rat by cluster analysis.

1. A set of 11 cutaneous stimuli defined previously to differentiate among different types of cutaneous sensory receptors in the rat hindpaw was also effective in differentially activating second-order sensory neurons in the dorsal horn and the gracile nucleus of rats. 2. All sampled units were responsive to more than 1 of the 11 stimuli. However, none responded to innocuous warming or cooling stimuli. Therefore further analysis was restricted to responses to nine of the selected stimuli. 3. Cluster analysis of the responses to nine selected innocuous and noxious mechanical stimuli and noxious thermal stimuli yielded seven classes that seemed functionally distinct from each other: a class of high-threshold neurons, three classes of convergent (wide dynamic range) neurons, a class of a mixture of poorly responsive neurons and neurons receiving Pacinian inputs, and two classes of low-threshold neurons. 4. High-threshold neurons responded predominantly to noxious mechanical and thermal stimuli and presumably received an input from both mechanically and thermally sensitive nociceptors. These cells were located in the dorsal horn, and some were spinothalamic tract cells. Wide dynamic range neurons were excited by innocuous and noxious stimuli, but better by noxious stimuli. These classes of cells were either in the dorsal horn (some were spinothalamic tract cells) or in the nucleus gracilis.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1986

Transducer action of isolated frog muscle spindle evoked by pseudorandom noise stimuli.

The dynamic response properties of the isolated frog muscle spindle receptor were investigated by recording the receptor potential evoked by pseudorandom noise (PRN) stimuli. The entire dynamic range of the receptor was determined by measuring the sensory response either at different intensities of the PRN stimulus (sigma = 8-30 microns) around a constant mean length or at the same intensity while varying the mean length from resting length L0 up to L0 + 150 microns. The 3-dB bandwidth of the test signal was 130 Hz. Random stimuli often evoked brief receptor potentials with variable size but characteristic shape. This shape contained a fast depolarization transient of the receptor potential during the stretching phase of the stimulus and a slowly decaying repolarization transient during release of stretch. The depolarization transient rose faster in proportion to the increasing amplitude of the receptor potential, so that larger receptor potentials were more phasic in character than smaller ones. The repolarization transient exhibited two segments of different exponential decay: The first brief repolarization phase lasted for 5 ms; its decline (tau = 2-5 ms) was faster for larger receptor potentials. The second slowly decaying repolarization transient was the same for different receptor potential amplitudes (tau = 47 ms). Consequently, the slow repolarization transients of succeeding receptor potentials displayed temporal summation. Since the amplitude and shape of the receptor potential remained constant during repeated sequences of PRN stimuli, this test stimulus was the most appropriate for the investigation of dynamic response properties under stationary conditions. Long-term stimulation caused a small shift of the mean membrane voltage towards hyperpolarizing values. This finding together with the marked "off effect" after termination of the stimulus indicate the action of an electrogenic pumping mechanism. The dynamic range of the muscle spindle receptor extended from resting length L0 up to L0 + 100 microns. Within this range static prestretches placed a bias upon the transducing site and effectively enhanced the amplitude of the receptor potential. Further prestretch beyond the dynamic region kept the receptor potential constant at its maximum amplitude. The receptor potential amplitude distribution was not symmetrical about the mean but was skewed in favor of depolarization values responding to the stretch trajectories of the PRN stimulus. Variation of the operating point by increasing the static prestretch also shifted the mode of the response distribution towards depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Acoustic Stimulation↗
PubMed2002

Continuous sample deposition from reversed-phase liquid chromatography to tracks on a matrix-assisted laser desorption/ionization precoated target for the analysis of protein digests.

Peptide mass fingerprinting by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) is one of the standard high-throughput methods for protein identification today. Traditionally this method has been based on spotting peptide mixtures onto MALDI targets. While this method works well for more abundant proteins, low-abundance proteins mixed with high-abundance proteins tend to go undetected due to ion suppression effects, instrumental dynamic range limitations and chemical noise interference. We present an alternative approach where liquid chromatography (LC) effluent is continuously collected as linear tracks on a MALDI target. In this manner the chromatographic separation is spatially preserved on the target, which enables generation of off-line LC-MS and LC-MS/MS data by MALDI. LC-MALDI sample collection provides improved sensitivity and dynamic range, spatial resolution of peptides along the sample track, and permits peptide mass mapping of low-abundance proteins in mixtures containing high-abundance proteins. In this work, standard and ribosomal protein digests are resolved and captured using LC-MALDI sample collection and analyzed by MALDI-TOF-MS.

Amino Acid Sequence↗
PubMed1997

Processing of natural time series of intensities by the visual system of the blowfly.

A major problem that a visual system faces is how to fit the large intensity variation of natural image streams into the limited dynamic range of its neurons. One of the means to accomplish this is through the use of gain control. In order to investigate this, natural time series of intensities were measured, as well as the responses of blowfly photoreceptors and Large Monopolar Cells (LMCs) to these time series. Time series representative of what each photoreceptor of a real visual system would normally receive were measured with an optical system measuring the light intensity of a spot comparable with the field of view of single human foveal cones. This system was worn on a headband by a freely walking person. Resulting time series have rms-contrasts ranging from an average of 0.45 for 1-sec segments to 1.39 for 100-sec segments (both when limited to frequencies up to 100 Hz). Power spectra behave approximately as 1/f (f: temporal frequency). Measured time series were subsequently presented to fly photoreceptors and LMCs by playing them back on an LED. The results show that fast gain controls indeed keep the response within the dynamic range of the cells and that a large part of this range is actually used for packing the information in natural time series.

Animals↗
PubMed2006

A model of technical variation of microarray signals.

We describe a mathematical model of signal from single-channel direct hybridization microarray platforms. The model establishes a linear relationship between microarray signals and their standard deviations from a minimum set of assumptions. We use the model to precisely define important microarray quality characteristics: resolved fold change and dynamic range. The definitions lead to closed form expressions relating these characteristics to physical parameters of the microarray experiment in the case when both specific and nonspecific binding of target to probe are governed by the Langmuir hybridization isotherm. The predictions of the model are in close agreement to data obtained from spike-in experiments. Given the generality of the model, the introduced definitions of dynamic range and resolved concentration fold-change can be used to conduct cross-platform comparisons and to guide improvement of the microarray platform.

Computational Biology↗
PubMed1992

Factors predicting postoperative sentence scores in postlinguistically deaf adult cochlear implant patients.

A sample of 64 postlinguistically profoundly to totally deaf adult cochlear implant patients were tested without lipreading by means of the Central Institute for the Deaf (CID) sentence test 3 months postoperatively. Preoperative promontory stimulation results (thresholds, gap detection, and frequency discrimination), age, duration of profound deafness, cause of deafness, lipreading ability, postoperative intracochlear thresholds and dynamic ranges for electrical stimulation, depth of insertion of the electrode array into the scala tympani, and number of electrodes in use were considered as possible factors that might be related to the postoperative sentence scores. A multiple regression analysis with stepwise inclusion of independent variables indicated that good gap detection and frequency discrimination during preoperative promontory testing, larger numbers of electrodes in use, and greater dynamic ranges for intracochlear electrical stimulation were associated with better CID scores. The CID scores tended to decrease with longer periods of profound deafness.

Adolescent↗
PubMed2001

Demonstration of correlations between clinical and physical image quality measures in chest and lumbar spine screen-film radiography.

The ability to predict clinical image quality from physical measures is useful for optimization in diagnostic radiology. In this work, clinical and physical assessments of image quality are compared and correlations between the two are derived. Clinical assessment has been made by a group of expert radiologists who evaluated fulfillment of the European image criteria for chest and lumbar spine radiography using two scoring methods: image criteria score (ICS) and visual grading analysis score (VGAS). Physical image quality measures were calculated using a Monte Carlo simulation model of the complete imaging system. This model includes a voxelized male anatomy and was used to calculate contrast and signal-to-noise ratio of various important anatomical details and measures of dynamic range. Correlations between the physical image quality measures on the one hand and the ICS and VGAS on the other were sought. 16 chest and 4 lumbar spine imaging system configurations were compared in frontal projection. A statistically significant correlation with clinical image quality was found in chest posteroanterior radiography for the contrast of blood vessels in the retrocardiac area and a measure of useful dynamic range. In lumbar spine anteroposterior radiography, a similar significant correlation with clinical image quality was found between the contrast and signal-to-noise ratio of the trabecular structures in the L1-L5 vertebrae. The significant correlation shows that clinical image quality can, at least in some cases, be predicted from appropriate measures of physical image quality.

Computer Simulation↗
PubMed1983

Effects of muscle model parameter dispersion and multi-loop segmental interaction on the neuromuscular system performance.

The effects of parameter dispersion among motor units on the neuromuscular system performance as well as interaction between muscle segments and spinal cord mechanisms are investigated. Elementary components of the system are modeled to simulate with simple models their input-output characteristics. A leaky SS-IPFM encoder with a time-dependent threshold simulates the motor-neuron encoding characteristics. An amplitude and time dependent nonlinear model represent the motor unit mechanical output to neuronal input relationship. The dispersion of parameters in the components of the whole muscle control model is investigated in the open loop mode. It is shown that the dispersion of parameters in the multi-efferent channels converging on a common tendon provides a spatial filtration generating a smoother muscle force in addition to extending the linear dynamic range compared to a similar system having identical motor units. Muscle segmental interaction is investigated in this distributed model by closing the loop through a coupling matrix, representing afferent-motorneuron interaction on the spinal cord level. A diagonal matrix represents no segmental interaction and a uniform matrix represents a uniform interaction between segments through the muscle spindles and Golgi tendon feedback elements. The close loop simulation studied shows that (a). The type of segmental interaction has little effect on the overall system performance, i.e., range of linerity and stability, which is the result of having a muscle system with a large number of motor units. (b) There are only minor differences in results between the uniform and normal parameter distributions tested. (c) A loop gain of 4 divided by 8 in the distributed model can provide linearity through the full physiological force range. (d) Type of segmental interaction has significant effects on the individual segment. A uniform matrix provides a more stable segment due to the spatial filtration resulting from the segmental interaction, while the diagonal noninteracting matrix shows instabilities on the local segmental level despite global stability. The more realistic exponentially decaying spatial interaction matrix yields both global neuromuscular and local segmental stability with the same linear dynamic range generated with the uniform or diagonal matrices.

Animals↗
PubMed2004

Quantitative assessment of a novel flow-through porous microarray for the rapid analysis of gene expression profiles.

A novel microarray system that utilizes a porous aluminum-oxide substrate and flow-through incubation has been developed for rapid molecular biological testing. To assess its utility in gene expression analysis, we determined hybridization kinetics, variability, sensitivity and dynamic range of the system using amplified RNA. To show the feasibility with complex biological RNA, we subjected Jurkat cells to heat-shock treatment and analyzed the transcriptional regulation of 23 genes. We found that trends (regulation or no change) acquired on this platform are in good agreement with data obtained from real-time quantitative PCR and Affymetrix GeneChips. Additionally, the system demonstrates a linear dynamic range of 3 orders of magnitude and at least 10-fold decreased hybridization time compared to conventional microarrays. The minimum amount of transcript that could be detected in 20 microl volume is 2-5 amol, which enables the detection of 1 in 300,000 copies of a transcript in 1 microg of amplified RNA. Hybridization and subsequent analysis are completed within 2 h. Replicate hybridizations on 24 identical arrays with two complex biological samples revealed a mean coefficient of variation of 11.6%. This study shows the potential of flow-through porous microarrays for the rapid analysis of gene expression profiles in clinical applications.

Aluminum Oxide↗
PubMed2004

Performance characteristics of a quantitative TaqMan hepatitis C virus RNA analyte-specific reagent.

We determined the dynamic range, reproducibility, accuracy, genotype bias, and sensitivity of the TaqMan hepatitis C virus (HCV) analyte-specific reagent (ASR). Serum samples were processed using the MagNA Pure LC instrument and run on the COBAS TaqMan 48 analyzer. The performance characteristics of the ASR were also compared with those of the qualitative AMPLICOR and quantitative AMPLICOR MONITOR HCV tests. The ASR exhibited a >/=6-log(10) linear dynamic range and excellent reproducibility, with a mean coefficient of variation of 14%. HCV RNA concentration measured with the ASR agreed within an average of 0.42 log(10) (2.6-fold) of the labeled concentration with members of a standard reference panel. HCV genotypes 1 to 4 were amplified with similar efficiencies with the ASR. The ASR and AMPLICOR MONITOR viral load results were significantly correlated (r = 0.8898; P < 0.01), but the agreement was poor (mean difference, 0.45 +/- 0.35 log(10)) for 72 HCV RNA-positive clinical samples. However, 98.9% agreement between the ASR and qualitative AMPLICOR test results was found with 60 positive and 29 negative samples. Limiting-dilution experiments demonstrated that the limits of detection for ASR and AMPLICOR tests were 84 and 26 IU/ml, respectively. The performance characteristics of the TaqMan HCV ASR are appropriate for all clinical applications of HCV RNA testing.

Gene Amplification↗
PubMed2003

Modeling and predicting hearing aid outcome.

Following a brief tutorial on the application of factor analysis to hearing aid outcome measures, three studies of hearing aid outcome measures in elderly adults are presented and analyzed. Two of the studies were completed at Indiana University (IU-1 and IU-2), and one was a collaborative multisite study by the Veterans Administration and the National Institute of Deafness and other Communication Disorders (NIDCD/VA). IU-1 measured hearing aid outcome in 173 elderly wearers of single-channel, linear, in-the-ear hearing aids with output-limiting compression, whereas IU-2 obtained the same extensive set of outcome measures from 53 elderly wearers of two-channel, wide-dynamic-range compression, in-the-canal hearing aids. In the NIDCD/VA study, 333 to 338 participants wore three single-channel circuits in succession, with each circuit housed within an in-the-ear shell. The three circuits included in that study and in this analysis were: (1) linear with peak clipping, (2) linear with output-limiting compression, and (3) single-channel, wide-dynamic-range compression. Evaluation of the many outcome measures completed in each study using principal components factor analysis revealed that from three (both IU studies) to five (NIDCD/VA study) principal components captured the individual differences in hearing aid outcome. This was independent of hearing aid type (in-the-ear or in-the-canal) and circuitry. Subsequent multiple regression analyses of individual differences in performance along each dimension of hearing aid outcome revealed that these individual differences could be accounted for reasonably well by various prefit variables for some dimensions of outcome, but not others. In general, measures of speech recognition performance were well accounted for by prefit measures, with the best predictors being hearing loss, cognitive performance, and age. Measures of hearing aid usage were less well accounted for by prefit measures, with the most accurate predictor of current hearing aid use being prior hearing aid use. The outcome dimension accounted for most poorly was that associated with hearing aid satisfaction, with subjective measures of aided sound quality being the best predictor of performance along this dimension of hearing aid outcome. Additional multicenter, large-scale studies are needed to develop more complete models of hearing aid outcome and to identify the variables that influence various aspects of hearing aid outcome. It is only through this additional research that it will be possible to optimize outcome for hearing aid wearers.

Journal Article↗
PubMed2004

FTICR mass spectrometry for qualitative and quantitative bioanalyses.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is playing an increasing role in the characterization of cellular systems owing to its capabilities for providing higher confidence of identification, increased dynamic range and sensitivity unmatched by other MS platforms. Particularly in proteomics, where global and quantitative approaches are essential, the attributes of FTICR-MS are poised to make significant contributions. Recent advances in the field that have particular importance for proteomic applications include the use of high-performance micro-capillary column separation techniques coupled to FTICR, as well as methods that improve protein identification, sensitivity, dynamic range and throughput.

Animals↗
PubMed2004

Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement.

BACKGROUND: The antiviral effect of anti-influenza drugs such as zanamivir may be demonstrated in patients as an increased rate of decline in viral load over a time course of treatment as compared with placebo. Historically this was measured using plaque assays, or Culture Enhanced Enzyme Linked Immunosorbent Assay (CE-ELISA). OBJECTIVES: to develop and characterise real time quantitative PCR (qPCR) assays to measure influenza A and B viral load in clinical samples, that offer improvements over existing methods, in particular virus infectivity assays. STUDY DESIGN: The dynamic range and robustness were established for the real time qPCR assays along with stability of the assay components. Cross validation of the real time PCR assays with CE-ELISA was performed by parallel testing of both serial dilutions of three different subtypes of cultured virus and a panel of influenza positive throat swab specimens. RESULTS: the assays were specific for influenza A and B and the dynamic ranges were at least seven logs. The assay variability was within acceptable limits but increased towards the lower limit of quantification, which was 3.33 log(10) viral cDNA copies/ml of virus transport medium (ten viral RNA copies/PCR). The components of the assay were robust enough to withstand extended storage and several freeze-thaw cycles. For the real time PCR assays the limit of quantification was equivalent to the virus infectivity cut off, which equates to a 93-fold increase in sensitivity. CONCLUSION: Well characterised real time PCR assays offer significant improvements over the existing methods for measuring the viral load of strains of influenza A and B in clinical specimens.

DNA, Complementary↗
PubMed1991

Rapid and sensitive chemiluminescent enzyme immunoassay for measuring tumor markers.

We developed a chemiluminescent enzyme immunoassay (CLEIA) to quantify such tumor markers as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CA19-9, and CA125. We used a novel chemiluminescent substrate, a derivative of 1,2-dioxetane phosphate (AMPPD), to measure alkaline phosphatase as a labeling enzyme to Fab' fragments of antibody. Regardless of the solid phase, i.e., polystyrene beads (6 mm diameter) or ferrite-coated particles (0.3 microns diameter), the standard curves within the dynamic ranges of the conventional RIA or enzyme immunoassay (EIA) were linear in all cases except for those with AFP. Use of the ferrite particles further shortens the immunoreaction, so the assay can be performed in 30 min. In addition, the relationships between concentrations of the marker and chemiluminescent signals for CA19-9, CA125, and CEA were linear up to concentrations about 10-fold greater than the ordinary dynamic ranges. Intra- and interassay CVs (averages for individual analyte) were 2.2%-4.9% and 2.0%-5.8%, respectively. In an analysis of serum samples, results of the CLEIA correlated reasonably well with those of RIA or EIA. The lower limit of detection by CLEIA with ferrite particles was 390 arb. units/L for CA19-9, 990 arb. units/L for CA125, 0.06 micrograms/L for CEA, and 0.03 micrograms/L for AFP. Thus, the sensitivity increased to between two- and 10-fold that of RIA or colorimetric EIA, depending on the analytes.

Adamantane↗
PubMed2004

Performance assessment of eight high-throughput PCR assays for viral load quantitation of oncogenic HPV types.

Infection with mucosotropic human papillomavirus (HPV) is the necessary cause of cervical intraepithelial neoplasia. Several epidemiological studies suggest that HPV viral load can be a risk factor of cervical dysplasia. The purpose of the present study was to evaluate a methodology to determine HPV viral load of eight oncogenic HPV types (16, 18, 31, 39, 45, 51, 52, and 58). The quantitation assay is based on a high-throughput real-time PCR. The E6-E7 region of HPV types 16, 18, 45, and 51 were used to amplify specific DNA sequences and cloned into a plasmid vector. The constructs for HPV types 16, 18, 45, and 51, and the whole genome for HPV types 31, 39, 52, and 58 were quantitated using a limiting dilution analysis and used to create standard curves. Type-specific HPV clones were used to determine specificity, linearity, and intra- and inter-assay variation. The sensitivity of our assay covered a dynamic range of up to seven orders of magnitude with a coefficient of intra-assay variation less than 6% and the inter-assay variation less than 20%. No cross-reactivity was observed on any of the type-specific standard curves when phylogenetically close HPV types were used as templates. Our real-time PCR methodologies are highly reproducible, sensitive, and specific over a sevenfold magnitude dynamic range.

DNA Probes, HPV↗
PubMed2003

Effects of neutralizing antibodies to tumor necrosis factor-alpha on nucleus pulposus-induced abnormal nociresponses in rat dorsal horn neurons.

STUDY DESIGN: The effect of an anti-tumor necrosis factor alpha (anti-TNFalpha) antibody on abnormal discharges caused by application of nucleus pulposus to the nerve root was investigated in an electrophysiologic study. OBJECTIVES: To assess whether inhibition of TNFalpha can reduce nucleus pulposus-induced abnormal discharges. SUMMARY OF BACKGROUND DATA: It has been shown that TNFalpha, a proinflammatory cytokine, is a key pathogenic factor in the development of nucleus pulposus-induced abnormal discharges as a pain sensation. However, the electrophysiologic mechanisms involved in sciatica after disc herniation still have not been elucidated. METHODS: Extracellular activities of wide-dynamic-range neurons were assessed in 21 rats. Autologous nucleus pulposus harvested from the tail was applied to the L5 nerve root. The animals were simultaneously treated with antibodies to TNFalpha (anti-TNF + nucleus pulposus group) and with phosphate-buffered saline (nucleus pulposus group). As a control (control group), a similar volume of muscle was applied to the nerve root with phosphate-buffered saline. Responses of wide-dynamic-range neurons to noxious and innocuous stimuli were examined for 2 hours. RESULTS: Discharges evoked during noxious stimulation and discharges after withdrawal of stimulation in the nucleus pulposus group were significantly higher than those in the control group (P < 0.05). In the anti-TNF + nucleus pulposus group, discharges after withdrawal of stimulation were remarkably inhibited, as compared with those of the nucleus pulposus group (P < 0.05). However, evoked discharges during stimulation apparently were not inhibited. Responses to innocuous stimulation did not change throughout the measurements. CONCLUSIONS: These data indicate that application of TNFalpha antibodies to the nerve root partially prevents the nucleus pulposus-induced abnormal nociresponses. Therefore, anti-TNFalpha treatment may have a therapeutic effect on sciatica after lumbar disc herniation.

Animals↗