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PubMedDate not supplied

Measurement of unidirectional transplacental flux: a simplified method.

A simple method for determining unidirectional transplacental flux in the in vitro perfusion (transfer factor analysis) has been derived from a general treatment of non-compartmental analysis describing transfer between two accessible pools. This method was validated by comparison of the unidirectional transfer fractions for Evans Blue in a two-pool hydraulic model with the true transfer fractions determined from the pump flow rates in the model. There was excellent agreement between calculated and true transfer fractions. Transfer fractions obtained using this method were also compared to the fractions determined by a previously described technique, deconvolution analysis, for a hydraulic model in which a third, inaccessible pool was interposed between the two accessible pools. Good agreement was found between the two methods. Similar agreement was found for the fractional transfer of [14C]L-lactate in the in vitro perfused human placenta, calculated using transfer factor and deconvolution analysis. The sample collection and data processing are much simpler using the former method but the quality of information obtained is reduced accordingly.

Female↗
PubMed2000

Fluorescence ratio intrinsic basis states analysis: a novel approach to monitor and analyze protein unfolding by fluorescence.

Fluorescence ratio intrinsic basis states analysis (FRIBSTA) is a novel method allowing quantitative estimation of the stability of proteins in aqueous solution as a function of temperature. In FRIBSTA emission fluorescence spectra are repeatedly recorded while ramping temperature from < or =-15 to > or =100 degrees C. Subsets of these are identified as reference spectra of the protein in either its folded or in its heat denatured configuration. Each reference spectrum of both sets is normalized by its own integrated fluorescence intensity to give a fractional area spectrum. Linear extrapolations of these normalized reference spectral shapes over the entire temperature range of measurement are then used to deconvolute each experimental emission spectrum to give a fraction of emission from native state and a fraction from denatured state. Additionally, the integrated emission fluorescence intensity for the native configuration is fitted and extrapolated over the temperature range of measurement. Division of the deconvoluted native integrated fluorescence intensity by the fitted-extrapolated integrated emission fluorescence intensity yields the fraction folded. The free energy functions derived from fraction unfolded are presented for beta-lactoglobulin and phosphoglycerate kinase. According to these results both proteins are considerably less stable than heretofore assumed at ambient temperatures and partially denatured at temperatures < or =0 degrees C. The method is employed to study the effect of denaturants on these proteins as well. The major usefulness of FRIBSTA is that one can directly measure the protein stability at ambient and subambient temperatures in the absence of denaturants rather than predicting it by extrapolation from heat denaturation data.

Animals↗
PubMed1999

Conformation, pH-induced conformational changes, and thermal unfolding of anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab and Fc fragments.

Conformation, acid-induced conformational changes and stability of the murine monoclonal antibody CB4-1 directed against the human immunodeficiency virus type 1 capsid protein p24, and its Fab and Fc fragments, were analysed by circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) measurements. CD spectra show the characteristics expected for beta-proteins. Lowering the pH to 3.5 reduces the stability, but does not change the conformation. Between pH 3.5 and 2.0 conformational changes and the formation of new structures are indicated. Deconvolution of the bimodal DSC curves of CB4-1 reveals five 'two-state' transitions at pH 7.5. At pH 5 and below, only four transitions are found. Half transition temperatures Tm and molar enthalpy changes DeltaHm gradually decrease at pH 4 and 3.4. At pH 2.1, two low-temperature (Tm=36.9 and 44.1 degrees C) and two high-temperature (Tm=74.6 and 76.8 degrees C) transitions are identified. The Fab and Fc fragments behave similarly. Deconvolution of their monophasic DSC curves yields two 'two-state' transitions for each fragment. Tm and DeltaHm values gradually decrease at pH 4.0 and 3.4; and at pH 2.1 and 2.8 for Fab and Fc, respectively, one of the transitions is found at high temperature (Tm=67.2 and 75.9 degrees C for Fab and Fc, respectively).

Antibodies, Monoclonal↗
PubMed2001

Calculation of the electronic structure of carbon films using electron energy loss spectroscopy.

The detailed understanding of the electronic properties of carbon-based materials requires the determination of their electronic structure and more precisely the calculation of their joint density of states (JDOS) and dielectric constant. Low electron energy loss spectroscopy (EELS) provides a continuous spectrum which represents all the excitations of the electrons within the material with energies ranging between zero and about 100 eV. Therefore, EELS is potentially more powerful than conventional optical spectroscopy which has an intrinsic upper information limit of about 6 eV due to absorption of light from the optical components of the system or the ambient. However, when analysing EELS data, the extraction of the single scattered data needed for Kramers Kronig calculations is subject to the deconvolution of the zero loss peak from the raw data. This procedure is particularly critical when attempting to study the near-bandgap region of materials with a bandgap below 1.5 eV. In this paper, we have calculated the electronic properties of three widely studied carbon materials; namely amorphous carbon (a-C), tetrahedral amorphous carbon (ta-C) and C60 fullerite crystal. The JDOS curve starts from zero for energy values below the bandgap and then starts to rise with a rate depending on whether the material has a direct or an indirect bandgap. Extrapolating a fit to the data immediately above the bandgap in the stronger energy loss region was used to get an accurate value for the bandgap energy and to determine whether the bandgap is direct or indirect in character. Particular problems relating to the extraction of the single scattered data for these materials are also addressed. The ta-C and C60 fullerite materials are found to be direct bandgap-like semiconductors having a bandgaps of 2.63 and 1.59eV, respectively. On the other hand, the electronic structure of a-C was unobtainable because it had such a small bandgap that most of the information is contained in the first 1.2 eV of the spectrum, which is a region removed during the zero loss deconvolution.

Journal Article↗
PubMed1999

Physico-chemical and biological analysis of true combinatorial libraries.

Combinatorial libraries offer new sources of compounds for the research of pharmacological agents such as receptor ligands, enzyme inhibitors or substrates and antibody-binding epitopes. The present review stresses the main roles played by both physico-chemical analysis, particularly when complex mixture of compounds are synthesized as libraries, and biological analysis from which active compounds are identified. After a brief discussion of semantic problems related to the designation of the product mixtures, the physico-chemical analysis of mixtures is reviewed with special emphasis on mass spectrometric techniques. These methods are able both to give a representative view of a library composition and to identify single critical compounds in large libraries. Then the biological screening of such combinatorial libraries is critically discussed with respect to the power and limitations of the methods used for the identification of the active components. Special attention is given to the complex process of library deconvolution. It is pointed out that while combinatorial techniques have evolved towards sophisticated high-tech methods, simple and robust biochemical tests should be used to deconvolute. From a large panel of published examples, a set of trends are identified which should help investigators to choose the most appropriate assay for the discovery of new entities.

Chemistry, Pharmaceutical↗
PubMed2000

Determination of possible impurities in piracetam using FTIR spectroscopy.

Piracetam (2-oxo-1-pyrrolidine acetamide (PAm)) may contain some structurally related impurities deriving from synthesis or degradation, mainly 2-oxo-1-pyrrolidine acetic acid (PAc). A rapid and sensitive Fourier transform infrared (FTIR) spectrophotometric method was developed for determination of possible impurities in piracetam. The results showed that the method was effective for the simultaneous determination of PAc in piracetam by FTIR spectrophotometry and may be a real alternative to HPLC. The apparent spectral resolution was first enhanced by using the Fourier self-deconvolution (FSD) method and the profiles were then fully deconvoluted by using a curve fitting procedure. FSD method followed by curve fitting allowed to evaluate quantitatively the areas under the pyrrolidine acetic acid peaks. Mixtures of known composition were used as standards to minimise errors due to the presence of both compounds in the same mixture. The detection limit of pyrrolidine acetic acid was estimated to be 0.1% with respect to piracetam.

Nootropic Agents↗
PubMedDate not supplied

The use of deblurring technique for improving the longitudinal resolution in helical CT of the head and neck region.

We evaluated the use of deblurring technique by digital deconvolution for improving the longitudinal resolution in helical CT in a phantom and CT images of head and neck patients. After performing the helical scanning, overlapped axial images were reconstructed at 1/10 of the actual collimation. Then, the deblurring technique was performed. Results revealed that the resolution of slice profiles was improved after deblurring. Further, the longitudinal resolution increased as the strength of the deconvolution increased, at the expense of an increase in helical artifacts. The sharpness and visualization of horizontally running structures were improved in the reconstructed MPR, MPVR, MIP and 3D images in both the phantom and the images of all patients. Also, visualization of the peripheral vessels in CTAs was improved.

Angiography↗
PubMed1998

Design of allele-specific inhibitors to probe protein kinase signaling.

BACKGROUND: Deconvoluting protein kinase signaling pathways using conventional genetic and biochemical approaches has been difficult because of the overwhelming number of closely related kinases. If cell-permeable inhibitors of individual kinases could be designed, the role of each kinase could be systematically assessed. RESULTS: We have devised an approach combining chemistry and genetics to develop the first highly specific cell-permeable inhibitor of the oncogenic tyrosine kinase v-Src. A functionally silent active-site mutation was made in v-Src to distinguish it from all other cellular kinases. A tight-binding cell-permeable inhibitor of this mutant kinase that does not inhibit wild-type kinases was designed and synthesized. In vitro and whole-cell assays established the unique specificity of the mutant v-Src-inhibitor pair. The inhibitor reversed cell transformation by the engineered but not the 'wild type' v-Src, establishing that changes in cellular signaling can be attributed to specific inhibition of the engineered kinase. The generality of the method was tested by engineering another tyrosine kinase, Fyn, to contain the corresponding active-site mutation to the one in v-Src. The same compound that inhibited mutant v-Src could also potently inhibit the engineered Fyn kinase. CONCLUSIONS: Allele-specific cell-permeable inhibitors of individual Src family kinases can be rapidly developed in an approach that should be applicable to all kinases. This approach will be useful for the deconvolution of kinase-mediated cellular pathways and for validating novel kinases as good targets for drug discovery both in vitro and in vivo.

3T3 Cells↗
PubMedDate not supplied

Searching for active ribosomal genes in situ: light microscopy in light of the electron beam.

Light microscopy (LM) approaches are commonly used to attain a description of the cell structure. Even though LM, if compared to electron microscopy (EM), represents a very fast approach, its resolution is, in principle, much lower than in the case of EM. To improve the LM resolution, computational methods based on removal of the image blur are frequently implemented in cell biology studies. One of the standard deblurring approaches is image restoration through deconvolution algorithms. Even though this method of mathematical remodeling of microscopically observed objects represents an efficient tool of current cell biology, it is legitimate to ask what the limits of its use are. We demonstrate that, in the specific case of the fluorescence mapping of active ribosomal genes in HeLa cell nucleoli, restoration generates a biased result. On restoration of model images, we demonstrate the difficulties of one of the most effective deconvolution algorithms during the restoration of ring-shaped fluorescent objects of a diameter comparable to the microscope resolution limit. In the case involving the mapping of nucleolar transcription in HeLa cells, not the restored fluorescence images, but rather the EM images show the true distribution of active ribosomal genes.

Cell Nucleolus↗
PubMed2003

Region growing method for the analysis of functional MRI data.

Existing analytical techniques for functional magnetic resonance imaging (fMRI) data always need some specific assumptions on the time series. In this article, we present a new approach for fMRI activation detection, which can be implemented without any assumptions on the time series. Our method is based on a region growing method, which is very popular for image segmentation. A comparison of performance on fMRI activation detection is made between the proposed method and the deconvolution method and the fuzzy clustering method with receiver operating characteristic (ROC) methodology. In addition, we examine the effectiveness and usefulness of our method on real experimental data. Experimental results show that our method outperforms over the deconvolution method and the fuzzy clustering method on a number of aspects. These results suggest that our region growing method can serve as a reliable analysis of fMRI data.

Acoustic Stimulation↗
PubMed1998

Increased disorderliness and decreased mass and daily rate of endogenous growth hormone secretion in adult Turner syndrome: the impact of body composition, maximal oxygen uptake and treatment with sex hormones.

The objectives of this study were to (1) quantify pulsatility and orderliness of 24-h growth hormone (GH) secretion in adult Turner syndrome; (2) study the impact of sex steroid replacement therapy in adult Turner syndrome on these measures of GH secretion, and in addition examine the differential effect of oral vs transdermal estrogen administration on GH secretion patterns. To these ends, we used deconvolution analysis and approximate entropy (ApEn) to quantify GH release over 24 h in 21 patients with Turner syndrome before and during sex hormone substitution, compared to an age-matched control group. Deconvolution analysis revealed that the mass of GH secreted per burst and production rate was significantly lower in Turner patients compared to controls, resulting in a significantly lower integrated 24-h GH concentration. However, multiple stepwise regression revealed that lean body mass (LBM) and maximal oxygen uptake were significant discriminative variable, explaining a large part of the variation in mass secreted per burst (r = 0.72, P < 0.0005) and production rate (r = 0.73, P < 0.0005), while group (Turner or control) did not explain any of the difference. There was a significant difference in ApEn between Turner patients and controls, denoting more disorderly GH release in Turner syndrome. During administration of sex hormones, a significant increase was seen in basal secretion and GH secretory burst half-duration, as well as in integrated 24-h GH concentration. No change in ApEn was evident. We conclude that GH secretion in adult Turner syndrome is irregular, reduced in mass and production rate. The reduction in mass and production rate could be explained by differences in body composition and maximal oxygen uptake compared to relevant controls, while the irregularity of GH secretion was unexplained by the measured variables. We hypothesize that the increased irregularity could be attributable to low levels of circulating androgens or an increased biological age in the Turner patients.

Administration, Cutaneous↗
PubMed1997

Fourier transform infrared spectral evidences for protein conformational changes in immature cataractous human lens capsules accelerated by myopia and/or systemic hypertension.

The possible changes in protein structures of the cataractous human lens capsules of the immature patients with myopia and/or systemic hypertension have been investigated using Fourier transform infrared (FT-IR) microspectroscopy. Second-derivative and deconvolution methods have been applied to obtain the position of the overlapping components of the amide I band and assign them to different secondary structures. Changes in the protein secondary structure and composition of amide I band were estimated quantitatively from Fourier self-deconvolution and curve fitting algorithms. The results indicate that myopia and/or systemic hypertension were found to significantly modify the protein secondary structure of the cataractous human lens capsules to increase the beta-type structure and random coil and decrease the alpha-helix structure. Myopia-induced conformational change in triple helix structure was more pronounced. In conclusion, myopia and/or systemic hypertension seem to modify the conformation of the protein structures in cataractous human lens capsule to change ionic permeation through lens capsule to accelerate the cataract formation of senile patients.

Aged↗
PubMed2000

High-performance liquid chromatography-electrospray ionization mass spectrometry of single- and double-stranded nucleic acids using monolithic capillary columns.

Monolithic capillary columns were prepared by copolymerization of styrene and divinylbenzene inside a 200-microm i.d. fused silica capillary using a mixture of tetrahydrofuran and decanol as porogen. With gradients of acetonitrile in 100 mM triethylammonium acetate, the synthesized columns allowed the rapid and highly efficient separation of single-stranded oligodeoxynucleotides and double-stranded DNA fragments by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). Compared with capillary columns packed with micropellicular, octadecylated poly-(styrene/divinylbenzene) particles, an improvement in column performance of approximately 40% was obtained, enabling the analysis of an 18-mer oligodeoxynucleotide with a column efficiency of more than 190000 plates per meter. The chromatographic separation system was on-line-coupled to electrospray ionization mass spectrometry (ESI-MS). To improve the mass spectrometric detectabilities, 25 mM triethylammonium bicarbonate was utilized as an ion-pair reagent at the cost of only little reduction in separation performance and acetonitrile was added postcolumn as the sheath liquid through the triaxial electrospray probe. High-quality mass spectra of femtomole amounts of 3-mer to 80-mer oligodeoxynucleotides were recorded showing very little cation adduction. Double-stranded DNA fragments ranging in size from 51 to 587 base pairs were separated and detected by IP-RP-HPLC-ESI-MS. Accurate mass determination by deconvolution of the mass spectra was feasible for DNA fragments up to the 267-mer with a molecular mass of 165 019, whereas the spectra of longer fragments were too complex for deconvolution because of incomplete separation due to overloading of the column. Finally, on-line IP-RP-HPLC tandem MS was applied to the sequencing of short oligodeoxynucleotides.

Chromatography, High Pressure Liquid↗
PubMed2001

Direct ultraviolet spectrophotometric determination of total sulfide and iodide in natural waters.

A technique is described that allows the determination of total dissolved sulfide in natural waters using direct ultraviolet detection of the HS- ion. The concentration of bisulfide is determined by measuring absorption from 214 to 300 nm and then deconvolution of the HS- spectra from the complex spectrum of natural fluids. A nonlinear least-squares fitting approach is used for the deconvolution. At a pH near 8, where >95% of total sulfide is present as HS-, the results are indistinguishable from total sulfide measured using the methylene blue method in a wide range of sample types and matrixes including freshwater from groundwater wells, marine hydrothermal vent fluids, and marine sediment porewaters. The method allows simultaneous determination of other UV-absorbing ions, including nitrate, bromide, and iodide, in samples with low total sulfide concentrations. Bisulfide concentrations can be determined in samples with low background absorption, such as well water and hydrothermal fluids, with a detection limit of < 1 microM. The detection limit for bisulfide in sediment porewaters that have a high organic loading, which produces background absorbances of approximately 0.5 A at 260 nm in a 1-cm cuvette, is 5 microM. The only chemical manipulation required is buffering acidic samples to pH > 7 and filtration of particulate-rich samples.

Fresh Water↗
PubMed2004

Broadband phase correction of FT-ICR mass spectra via simultaneous excitation and detection.

In typical Fourier transform ion cyclotron resonance (FT-ICR) mass spectra, temporally dispersed excitation and the delay between excitation and detection result in continuous variation of signal phase with frequency in the detected time-domain ion signal. The complex frequency-domain spectrum of such a signal is a linear combination of absorption- and dispersion-mode spectral components with corresponding asymmetric peaks. For this reason, magnitude-mode spectral display is usually employed to yield a phase-independent uniform and symmetrical peak shape at the expense of spectral resolution. In this work, we implement simultaneous excitation and detection to enable Fourier deconvolution to recover absorption-mode spectra for both low- and high-field FT-ICR instruments. These spectra yield resolving power improvement factors approaching the maximum theoretical limit of 2.0, as well as reduction in frequency assignment errors relative to conventional magnitude-mode spectra. The Fourier deconvolution procedure has the additional benefit of correcting for spectral variation resulting from nonuniform power distribution over the excitation bandwidth and the potential benefit of providing useful diagnostic information for interpretation of experimental performance.

Journal Article↗
PubMed2005

High-throughput data analysis for detecting and identifying differences between samples in GC/MS-based metabolomic analyses.

In metabolomics, the objective is to identify differences in metabolite profiles between samples. A widely used tool in metabolomics investigations is gas chromatography-mass spectrometry (GC/MS). More than 400 compounds can be detected in a single analysis, if overlapping GC/MS peaks are deconvoluted. However, the deconvolution process is time-consuming and difficult to automate, and additional processing is needed in order to compare samples. Therefore, there is a need to improve and automate the data processing strategy for data generated in GC/MS-based metabolomics; if not, the processing step will be a major bottleneck for high-throughput analyses. Here we describe a new semiautomated strategy using a hierarchical multivariate curve resolution approach that processes all samples simultaneously. The presented strategy generates (after appropriate treatment, e.g., multivariate analysis) tables of all the detected metabolites that differ in relative concentrations between samples. The processing of 70 samples took similar time to that of the GC/TOFMS analyses of the samples. The strategy has been validated using two different sets of samples: a complex mixture of standard compounds and Arabidopsis samples.

Arabidopsis↗
PubMed2006

Multiple and simultaneous fluorophore detection using fluorescence spectrometry and partial least-squares regression with sample-specific confidence intervals.

Fluorescent labeling is widely used in biological and chemical analysis, and the drive for increased throughput is stretching multiplexing capabilities to the limit. The limiting factor in multiplexed analyses is the ability to subsequently deconvolute the signals. Consequently, alternative approaches for interpreting complex data sets are required to allow individual components to be identified. Here we have investigated the application of a novel approach to multiplexed analysis that does not rely on multivariate curve resolution to achieve signal deconvolution. The approach calculates a sample-specific confidence interval for a multivariate (partial least-squares regression (PLSR)) prediction, thereby enabling the estimation of the presence or absence of each fluorophore based on the total spectral signal. This approach could potentially be applied to any multiplexed measurement system and has the advantage over the current algorithm-based methods that the requirement for resolution of spectral peaks is not central to the method. Here, PLSR was used to obtain the concentrations for up to eight dye-labeled oligonucleotides at levels of (0.6-5.3) x 10(-6) M. The sample-specific prediction intervals show good discrimination for the presence/absence of seven of the eight labeled oligonucleotides with efficiencies ranging from approximately 91 to 100%.

Confidence Intervals↗
PubMed2006

Estimation of the (n,m) concentration distribution of single-walled carbon nanotubes from photoabsorption spectra.

Deconvolution of the absorption spectrum of single-walled carbon nanotubes (SWNTs) into distinct (n,m) contributions is complicated because transition energies are closely spaced. The algorithm presented in this work attempts to simplify the problem by grouping nanotubes with similar transition energies and assigning weights to their spectral contributions. Voigt line shapes were used to fit absorption spectra of sodium dodecyl sulfate suspended HiPco SWNT and CoMoCat SWNT. Line widths for the metallic (93.42 meV) and two semiconducting regions (57.96 and 29.86 meV) were obtained from the absorption spectra of DNA-wrapped SWNT fractionated by ion-exchange chromatography. The method is used to describe the reaction kinetics of certain HiPco SWNTs upon reaction with 4-chlorobenzene diazonium and 4-hydroxybenzene diazonium salts. The code for deconvolution has been provided as open source in the Supporting Information for future modifications.

Algorithms↗