bluuebunny/crossref_metadata_2025 huggingface dataset:
Phospholipase D2 stimulates integrin-mediated adhesion via phosphatidylinositol 4-phosphate 5-kinase Iγb
Authors: Powner Dale J. , Payne Roberta M. , Pettitt Trevor R. , Giudici M. Luisa , Irvine Robin F. , Wakelam Michael J. O.
Published: June 2005

Cellular adhesion can be regulated by, as yet, poorly defined intracellular signalling events. Phospholipase D enzymes generate the messenger lipid phosphatidate and here we demonstrate that suppression of this reaction inhibits cellular adhesion. This effect was reversed by the addition of cell-permeable analogues of either phosphatidate or phosphatidylinositol 4,5-bisphosphate. By contrast, neither diacylglycerol nor lysophosphatidic acid were able to reverse this effect suggesting that phosphatidate itself acts directly on a target protein(s) to regulate adhesion rather than as the result of its conversion to either of these metabolite lipids. Antibodies that block β1 and β2 integrin-substrate interactions inhibited adhesion stimulated by both phosphatidate and phosphatidylinositol 4,5-bisphosphate indicating that these lipids regulate β1 and β2 integrin-mediated adhesion. In vivo, these lipids can be generated by phospholipase D2 and phosphatidylinositol 4-phosphate 5-kinase Iγb, respectively, and over-expression of catalytically-functional forms of these enzymes dose-dependently stimulated adhesion while siRNA depletion of PLD2 levels inhibited adhesion. Furthermore the ability of over-expressed phospholipase D2 to stimulate adhesion was inhibited by a dominant-negative version of phosphatidylinositol 4-phosphate 5-kinase Iγb. Consistent with this, phosphatidylinositol 4-phosphate 5-kinase Iγb-mediated adhesion was dependent upon phospholipase D2's product, phosphatidate indicating that phosphatidylinositol 4-phosphate 5-kinase Iγb is downstream of, and necessary for, phospholipase D2's regulation of adhesion. It is likely that this phospholipase D2-generated phosphatidate directly stimulates phosphatidylinositol 4-phosphate 5-kinase Iγb to generate phosphatidylinositol 4,5-bisphosphate as this mechanism has previously been demonstrated in vitro. Thus, our data indicates that during the initial stages of adhesion, phospholipase D2-derived phosphatidate stimulates phosphatidylinositol 4-phosphate 5-kinase Iγb to generate phosphatidylinositol 4,5-bisphosphate and that consequently this inositol phospholipid promotes adhesion through its regulation of cell-surface integrins.
Characterization of the TGN exit signal of the human mannose 6-phosphate uncovering enzyme
Authors: Nair Prashant , Schaub Beat E. , Huang Kai , Chen Xiang , Murphy Robert F. , Griffith Janice M. , Geuze Hans J. , Rohrer Jack
Published: June 2005

The human mannose 6-phosphate uncovering enzyme participates in the uncovering of the mannose 6-phosphate recognition tag on lysosomal enzymes, a process that facilitates recognition of those enzymes by mannose 6-phosphate receptors to ensure delivery to lysosomes. Uncovering enzyme has been identified on the trans-Golgi network at steady state. It has been shown to traffic to the plasma membrane from where it is rapidly internalized via endosomal structures, the process being mediated by a tyrosine-based internalization motif, Y488HPL, in its cytoplasmic tail. Using immunogold electron microscopy a GFP-uncovering enzyme fusion construct was found to be colocalized with the cation-dependent mannose 6-phosophate receptor in regions of the trans-Golgi network, suggesting that uncovering enzyme might follow a similar pathway of exit from the trans-Golgi network as that of the cation-dependent mannose 6-phosohate receptor. In this study, we identified the signal sequence in the cytoplasmic tail of uncovering enzyme responsible for its exit from the trans-Golgi network. Using GFP fusion constructs of the transmembrane and cytoplasmic domains of uncovering enzyme, we could show, by automated analysis of confocal immunofluorescence images, that residues Q492EMN in the cytoplasmic tail of uncovering enzyme are involved in its exit from the trans-Golgi network. Detailed characterization of the exit signal revealed that residue Q492 is the most important to the exit function while M494 and N495 also contribute. The cytoplasmic tail of the uncovering enzyme does not possess any of the known canonical signal sequences for interaction with Golgi-associated gamma ear-containing adaptor proteins. The identification of a trans-Golgi network exit signal in its cytoplasmic tail elucidates the trafficking pathway of uncovering enzyme, a crucial player in the process of lysosomal biogenesis.
Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD133) from neural progenitors and other epithelial cells
Authors: Marzesco Anne-Marie , Janich Peggy , Wilsch-Bräuninger Michaela , Dubreuil Véronique , Langenfeld Katja , Corbeil Denis , Huttner Wieland B.
Published: June 2005

Apical plasma membrane constituents of mammalian neural stem/progenitor cells have recently been implicated in maintaining their stem/progenitor cell state. Here, we report that in the developing embryonic mouse brain, the fluid in the lumen of the neural tube contains membrane particles carrying the stem cell marker prominin-1 (CD133), a pentaspan membrane protein found on membrane protrusions of the apical surface of neuroepithelial cells. Two size classes of prominin-1-containing membrane particles were observed in the ventricular fluid: ≈600-nm particles, referred to as P2 particles, and 50-80-nm vesicles, referred to as P4 particles. The P2 and P4 particles appeared in the ventricular fluid at the very onset and during the early phase of neurogenesis, respectively. Concomitant with their appearance, the nature of the prominin-1-containing apical plasma membrane protrusions of neuroepithelial cells changed, in that microvilli were lost and large pleiomorphic protuberances appeared. P4 particles were found in various body fluids of adult humans, including saliva, seminal fluid and urine, and were released by the epithelial model cell line Caco-2 upon differentiation. Importantly, P4 particles were distinct from exosomes. Our results demonstrate the widespread occurrence of a novel class of extracellular membrane particles containing proteins characteristic of stem cells, and raise the possibility that the release of the corresponding membrane subdomains from the apical surface of neural progenitors and other epithelial cells may have a role in tissue development and maintenance. Moreover, the presence of prominin-1-containing membrane particles in human body fluids may provide the basis for a protein-based diagnosis of certain diseases.
Give lipids a START: the StAR-related lipid transfer (START) domain in mammals
Authors: Alpy Fabien , Tomasetto Catherine
Published: June 2005

The steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain is a protein module of ∼210 residues that binds lipids, including sterols. Fifteen mammalian proteins, STARD1-STARD15, possess a START domain and these can be grouped into six subfamilies. Cholesterol, 25-hydroxycholesterol, phosphatidylcholine, phosphatidylethanolamine and ceramides are ligands for STARD1/STARD3/STARD5, STARD5, STARD2/STARD10, STARD10 and STARD11, respectively. The lipids or sterols bound by the remaining 9 START proteins are unknown. Recent studies show that the C-terminal end of the domain plays a fundamental role, forming a lid over a deep lipid-binding pocket that shields the ligand from the external environment. The START domain can be regarded as a lipid-exchange and/or a lipid-sensing domain. Mammalian START proteins have diverse expression patterns and can be found free in the cytoplasm, attached to membranes or in the nucleus. They appear to function in a variety of distinct physiological processes, such as lipid transfer between intracellular compartments, lipid metabolism and modulation of signaling events. Mutation or misexpression of START proteins is linked to pathological processes, including genetic disorders, autoimmune disease and cancer.
Inhaled Iloprost in Pulmonary Arterial Hypertension
Authors: Baker Stacey E , Hockman Rebecca Haynes
Published: June 2005

OBJECTIVE

To review the pharmacology, pharmacodynamics, and clinical trials evaluating inhaled iloprost in pulmonary arterial hypertension (PAH).

DATA SOURCES

A MEDLINE search (1996–February 2005) was performed using the key words pulmonary hypertension, iloprost, and epoprostenol. Information regarding Food and Drug Administration approval was obtained via the Internet.

STUDY SELECTION AND DATA EXTRACTION

All clinical trials using inhaled iloprost in PAH published in English were identified. Additionally, references from the identified articles were reviewed.

DATA SYNTHESIS

A stable analog of prostacyclin, inhaled iloprost is thought to promote benefit in PAH through vasodilation, antiproliferative effects, and inhibition of platelet aggregation. In a placebo-controlled trial of 203 patients, inhaled iloprost significantly improved the combined endpoint of change in New York Heart Association functional class and 10% improvement in 6-minute walk distance (p = 0.007). Small, short-term clinical trials demonstrated hemodynamic benefits for inhaled iloprost alone and in combination with other pulmonary vasodilating agents. The aerosolized delivery route and low incidence of adverse events are positive attributes for inhaled iloprost, while the frequency of administration and lack of comparative data limit its role in PAH.

CONCLUSIONS

Currently, inhaled iloprost offers potential benefit for patients with contraindications to bosentan, preference for non-parenteral products, ineligibility for parenteral therapy, or as adjunctive therapy with other pulmonary vasodilators. Larger, long-term clinical trials are needed to solidify the role for inhaled iloprost in the management of PAH.
Analysis of Captan on Nitrile Glove Surfaces Using a Portable Attenuated Total Reflection Fourier Transform Infrared Spectrometer
Authors: Phalen R. N. , Hee Shane S. Que
Published: June 2005

This study developed a method to produce uniform captan surface films on a disposable nitrile glove for quantitation with a portable attenuated total reflection Fourier transform infrared (ATR-FTIR) spectrometer. A permeation test was performed using aqueous captan formulation. Uniform captan surface films were produced using solvent casting with 2-propanol and a 25 mm filter holder connected to a vacuum manifold to control solvent evaporation. The coefficient of variation of the reflectance at 1735 ± 5 cm−1 was minimized by selection of the optimum solvent volume, airflow rate, and evaporation time. At room temperature, the lower to upper quantifiable limits were 0.31–20.7 μg/cm2 ( r = 0.9967; p ≤ 0.05) for the outer glove surface and 0.55–17.5 μg/cm2 ( r = 0.9409; p ≤ 0.05) for the inner surface. Relative humidity and temperature did not affect the uncoated gloves at the wavelength of captan analysis. Glove screening using ATR-FTIR was necessary as a control for between-glove variation. Captan permeation, after 8 hours exposure to an aqueous concentration of 217 mg/mL of Captan 50-WP, was detected at 0.8 ± 0.3 μg/cm2 on the inner glove surface. ATR-FTIR can detect captan permeation and can determine the protectiveness of this glove in the field.
Vibrational Circular Dichroism in General Anisotropic Thin Solid Films: Measurement and Theoretical Approach
Authors: Buffeteau Thierry , Lagugné-Labarthet François , Sourisseau Claude
Published: June 2005

In this study, the measurement of the true vibrational circular dichroism (VCD) spectrum is considered from an experimental and theoretical approach for any general anisotropic thin solid sample exhibiting linear as well as circular birefringence (LB, CB) and dichroism (LD, CD) properties. For this purpose, we have made use of a simple model α-helix polypeptide, namely, the poly(γ-benzyl-L-glutamate) or PBLG, reference sample possessing a well-known VCD spectrum and giving rise to slightly oriented films by deposition onto a solid substrate. Also, we have used a different Fourier transform infrared modulation of polarization (PM-FTIR) optical setup with two-channel electronic processing in order to record the PM-VLD and PM-VCD spectra for various sample orientations in its film plane. All the corresponding general relations of the expected intensities in these experiments and the related properly designed calibration measurements were established using the Stokes–Mueller formalism; in addition, the residual birefringence of the optical setup and the transmittance anisotropy of the detector were estimated. From a comparative study of the results obtained in solution and in the solid state, we then propose a simple new experimental procedure to extract the true VCD spectrum of an oriented PBLG thin film: its consists of calculating the half-sum of two spectra recorded at θ and at θ ± 90° sample orientations. Moreover, the complete linear and circular birefringence and dichroism properties of the ordered PBLG thin film are estimated in the amide I and amide II vibrational regions. This allows us to establish for any sample orientation various theoretical simulations of the VCD spectra that agree nicely with the observed experimental results; this confirms that the measurement of LD and LB is in this case a prerequisite in simulating the true VCD spectrum of a partly oriented anisotropic sample. This validates our combined experimental and theoretical approach and opens the route to promising future vibrational CD studies on other macroscopic anisotropic thin film samples.
Low-Cost Approaches to Robust Temperature Compensation in Near-Infrared Calibration and Prediction Situations
Authors: Segtnan Vegard H. , Mevik Bjørn-Helge , Isaksson Tomas , Næs Tormod
Published: June 2005

The traditional way of handling temperature shifts and other perturbations in calibration situations is to incorporate the non-relevant spectral variation in the calibration set by measuring the samples at various conditions. The present paper proposes two low-cost approaches based on simulation and prior knowledge about the perturbations, and these are compared to traditional methods. The first approach is based on augmentation of the calibration matrix through adding simulated noise on the spectra. The second approach is a correction method that removes the non-relevant variation from new spectra. Neither method demands exact knowledge of the perturbation levels. Using the augmentation method it was found that a few, in this case four, selected samples run under different conditions gave approximately the same robustness as running all the calibration samples under different conditions. For the carbohydrate data set, all robustification methods investigated worked well, including the use of pure water spectra for temperature compensation. For the more complex meat data set, only the augmentation method gave comparable results to the full global model.
Assessing Hemlock Decline Using Visible and Near-Infrared Spectroscopy: Indices Comparison and Algorithm Development
Authors: Pontius Jennifer , Hallett Richard , Martin Mary
Published: June 2005

Near-infrared reflectance spectroscopy was evaluated for its effectiveness at predicting pre-visual decline in eastern hemlock trees. An ASD FieldSpec Pro FR field spectroradiometer measuring 2100 contiguous 1-nm-wide channels from 350 nm to 2500 nm was used to collect spectra from fresh hemlock foliage. Full spectrum partial least squares (PLS) regression equations and reduced stepwise linear regression equations were compared. The best decline predictive model was a 6-term linear regression equation ( R2 = 0.71, RMSE = 0.591) based on: Carter Miller Stress Index (R694/R760), Derivative Chlorophyll Index (FD705/FD723), Normalized Difference Vegetation Index ((R800 – R680)/(R800 + R680)), R950, R1922, and FD1388. Accuracy assessment showed that this equation predicted an 11-class decline rating with a 1-class tolerance accuracy of 96% and differentiated healthy trees from those in very early decline with 72% accuracy. These results indicate that narrow-band sensors could be developed to detect very early stages of hemlock decline, before visual symptoms are apparent. This capability would enable land managers to identify early hemlock woolly adelgid infestations and monitor forest health over large areas of the landscape.
Chemometric Determination of Blood Parameters Using Visible—Near-Infrared Spectra
Authors: Meinke M. , Gersonde I. , Friebel M. , Helfmann J. , Müller G.
Published: June 2005

Visible and near-infrared (NIR) integrating sphere spectroscopy and chemometric multivariate linear regression were applied to determine hematocrit (HCT) and oxygen saturation (SatO2) of circulating human blood. Diffuse transmission, total transmission, and diffuse reflectance were measured and the partial least squares method (PLS) was used for calibration considering different wavelength ranges and selected optical measurement parameters. HCT and SatO2 were changed independently. Each parameter was adjusted to different levels and four designs with blood from different donors were carried out for the calibration with PLS. The calibration included the changes in hemolysis as well as inter-individual differences in cell dimensions and hemoglobin content. At a sample thickness of 0.1 mm the HCT and SatO2 were predicted with a root mean square error (PRMSE) of 1.4% and 2.5%, respectively, using transmission and reflectance spectra and the full Vis-NIR range. Using only diffuse NIR reflectance spectroscopy and a sample thickness of 1 mm, HCT and SatO2 could be predicted with a PRMSE of 1.9% and 2.8%, respectively. Prediction of hemolysis was also possible for one blood sample with a PRMSE of 0.8% and keeping HCT and SatO2 stable with a PRMSE of 0.03%.
Standoff Detection of High Explosive Materials at 50 Meters in Ambient Light Conditions Using a Small Raman Instrument
Authors: Carter J. Chance , Angel S. Michael , Lawrence-Snyder Marion , Scaffidi Jon , Whipple Richard E. , Reynolds John G.
Published: June 2005

We have designed and demonstrated a standoff Raman system for detecting high explosive materials at distances up to 50 meters in ambient light conditions. In the system, light is collected using an 8-in. Schmidt–Cassegrain telescope fiber-coupled to an f/1.8 spectrograph with a gated intensified charge-coupled device (ICCD) detector. A frequency-doubled Nd: YAG (532 nm) pulsed (10 Hz) laser is used as the excitation source for measuring remote spectra of samples containing up to 8% explosive materials. The explosives RDX, TNT, and PETN as well as nitrate- and chlorate-containing materials were used to evaluate the performance of the system with samples placed at distances of 27 and 50 meters. Laser power studies were performed to determine the effects of laser heating and photodegradation on the samples. Raman signal levels were found to increase linearly with increasing laser energy up to ∼3 × 106 W/cm2 for all samples except TNT, which showed some evidence of photo- or thermal degradation at higher laser power densities. Detector gate width studies showed that Raman spectra could be acquired in high levels of ambient light using a 10 microsecond gate width.
Framework for Multivariate Selectivity Analysis, Part I: Theoretical and Practical Merits
Authors: Brown Christopher D. , Ridder Trent D.
Published: June 2005

A number of definitions of multivariate selectivity have been proposed in the literature. Arguably, the one that enjoys the greatest chemometric attention has been the net analyte signal (NAS) based definitions of Lorber and Zinn. Recent works have suggested that similar inference can be made for inverse least-squares calibration methods (e.g., principal components regression). However, the properties of inverse calibration methods are markedly different than classical methods, so in many practical cases involving inverse models classically derived figures of merit cannot be transparently interpreted. In Part I of this work, we discuss a selectivity framework that is theoretically consistent regardless of the calibration method. Importantly, it is also experimentally measurable, either through controlled selectivity experiments, or through analysis on opportunistically acquired sample measurements. It is statistically advantageous to use the former if such control is achievable. Selectivity is defined to be a function of the change in predicted analyte concentration that will result from a change in the concentration of an interferant, an approach consistent with traditional definitions of analytical selectivity and National Committee for Clinical Laboratory Standards recommendations for interference testing. Unlike the NAS-based definition of selectivity, the definition discussed herein is relevant to only a particular analyte–interferant pair. The theoretical and experimental aspects of this approach are illustrated with simulated data herein and in Part II of this paper, which investigates several experimental near-infrared data sets.
Thermal Desorption Solid-Phase Microextraction Inlet for Differential Mobility Spectrometry
Authors: Rainsberg Matthew R. , de B. Harrington Peter
Published: June 2005

A splitless thermal desorber unit that interfaces a differential mobility spectrometry (DMS) sensor has been devised. This device was characterized by the detection of benzene, toluene, and xylene (BTX) in water. The detection of BTX in water is important for environmental monitoring, and ion mobility measurements are traditionally difficult for hydrocarbons in water because water competes for charge and quenches the hydrocarbon signals. This paper reports the use of a DMS with a photoionization source that is directly coupled to a solid-phase microextraction (SPME) desorber. The separation and detection capabilities of the DMS were demonstrated using BTX components. Detection limits for benzene, toluene, and m-xylene were 75, 50, and 5 μg mL−1, respectively.
Extended Multiplicative Signal Correction as a Tool for Separation and Characterization of Physical and Chemical Information in Fourier Transform Infrared Microscopy Images of Cryo-Sections of Beef Loin
Authors: Kohler A. , Kirschner C. , Oust A. , Martens H.
Published: June 2005

Extended multiplicative signal correction (EMSC) is used to separate and to characterize physical and chemical information in spectra from Fourier transform infrared (FT-IR) microscopy. This appears especially useful for applications in infrared spectroscopy where the scatter variance in spectra changes with the chemical variance in the sample set. In these cases the chemical information of specific bands that are assigned to functional groups is easier to interpret when the scatter information is removed from the spectra. We show that scatter (physical) information in FT-IR spectra of heat-treated beef loin is related to chemical changes due to heat treatment. This information is caused by textural changes induced by the heat treatment and expressed by physical effects as the optical path length. The chemical absorbance changes introduced in the FT-IR spectra due to heat treatment are shifts in the protein region of the infrared spectrum caused by changes in the secondary structure of the proteins. If the scatter and the chemical information is not separated properly, scatter information may erroneously be interpreted as chemical information.
Surface-Enhanced Raman Spectroscopy Using Silver-Coated Porous Glass-Ceramic Substrates
Authors: Pan Z. , Zavalin A. , Ueda A. , Guo M. , Groza M. , Burger A. , Mu R. , Morgan S. H.
Published: June 2005

Surface-enhanced Raman scattering (SERS) has been studied using a silver-coated porous glass-ceramic material as a new type of substrate. The porous glass-ceramic is in the CaO–TiO2–P2O5 system prepared by controlled crystallization and subsequent chemical leaching of the dense glass-ceramic, leaving a solid skeleton with pores ranging in size from 50 nm to submicrometer. Silver was coated on the surface of the porous glass-ceramic by radio frequency (RF) sputtering or e-beam evaporation in vacuum. SERS spectra of excellent quality were obtained from several dyes and carboxylic acid molecules, including rhodamine 6G, crystal violet, isonicotinic acid, and benzoic acid, using this new substrate. This new substrate showed a good compatibility with these molecules. The porous glass-ceramic with a nanometer-structured surface accommodated both test molecules and silver film. The absorbed molecules were therefore better interfaced with silver for surface-enhanced Raman scattering.
Tunable kHz Deep Ultraviolet (193–210 nm) Laser for Raman Applications
Authors: Balakrishnan Gurusamy , Hu Ying , Nielsen Steen Brøndsted , Spiro Thomas G.
Published: June 2005

The performance characteristics of a kilohertz solid-state laser source for ultraviolet Raman spectroscopy are described. Deep ultraviolet (UV) excitation in the 193–210 nm region is provided by mixing of the fundamental and third harmonics of a Ti–sapphire laser, which is pumped by the second harmonic of a Q-Switched Nd–YLF laser. The combination of tunability, narrow linewidth, high average power, good stability, and kilohertz repetition rate makes this laser suitable for deep UV resonance Raman applications. The short pulse duration (∼20 ns) permits nanosecond time resolution in pump–probe applications. The low peak power and high data rate provide artifact-free spectra with a high signal-to-noise ratio. UV Raman cross-section and Raman excitation profiles are reported for gaseous O2 (relative to N2), aqueous ClO4−, tyrosine, phenylalanine, tryptophan, histidine, and hemoglobin excited between 193 nm and 210 nm to illustrate laser performance.
Using the Intrinsic Chirality of a Molecule as a Label-Free Probe to Detect Molecular Adsorption to a Surface by Second Harmonic Generation
Authors: Kriech Matthew A. , Conboy John C.
Published: June 2005

Chiral second harmonic generation (C-SHG) has been used for the label-free detection of (R)-(+)-1,1′-bi-2-naphthol (RBN) and (S)-(+)-1,1′-bi-2-naphthol (SBN) binding to planar-supported lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphotidylcholine (POPC) based on the intrinsic chirality of the molecules. C-SHG adsorption isotherms of RBN and SBN reveal Langmuir adsorption behavior with binding constants of 2.7 ± 0.2 × 105 M−1 and 3.0 ± 0.1 × 105 M−1, respectively. The kinetics of RBN binding to a POPC bilayer was also measured. It was determined that the adsorption rate for RBN was 5.7 ± 0.4 × 103 s−1M−1 and the desorption rate was 2.1 ± 0.8 × 10−2 s−1. From the kinetic data a binding constant of 2.7 ± 1.0 × 105 M−1 was calculated, which agrees well with the thermodynamic measurement. The C-SHG technique was correlated with surface tension measurements in order to determine the RBN surface excess within the POPC membrane. The maximum surface excess of RBN in a monolayer of POPC was 4.3 ± 0.5 × 10−11 mol cm2. Using the maximum surface excess in conjunction with the C-SHG binding data a lower limit of detection of 1.5 ±0.1 × 10−13 mols cm−2 was calculated. The results of these studies show that C-SHG is a powerful tool for the study of chiral molecular interactions at surfaces.
Visualization Strategies for Infrared Spectroscopic Images
Authors: Couts-Lendon Carrie , Koenig Jack L.
Published: June 2005

Although infrared imaging is becoming more commonplace and publications demonstrating its utility are on the rise, only a small portion of the literature involves visualization strategies and techniques. In order to fully realize the potential of imaging, visualization of the data in the form of images needs to be examined more thoroughly. Visualization techniques are discussed using the dynamic process of polymer dissolution. The dissolution of poly(ethylene oxide) (PEO) was captured as a series of images showing the changes of the polymer front brought about by the dissolving solvent. Using a unique infrared band for both the polymer and solvent, each species is tracked through the entire experiment. The ability to reconstruct the dissolution from the spatially resolved infrared data proves very valuable. However, the processing of the data is not trivial and steps must be taken to ensure that the images best display the specific attributes of the species. Scaling and color mapping are demonstrated to be two ways to achieve excellent imagery.
Framework for Multivariate Selectivity Analysis, Part II: Experimental Applications
Authors: Ridder Trent D. , Brown Christopher D. , Steeg Benjamin J. Ver
Published: June 2005

In Part I of this paper, a framework for multivariate selectivity was introduced that is both calculable from first principles and experimentally tractable. In this part, we employ the proposed selectivity framework for analyzing both in vitro and in vivo near-infrared experimental data. Two in vitro data sets are used to compare different methods for estimating selectivity and to demonstrate the benefits obtained from validation data with expanded interferant concentration ranges. The in vitro data also demonstrate that the experimentally estimated selectivities provide insights into the properties of the calibration models that are difficult or impossible to infer by other means. The merits of the proposed selectivity function are further demonstrated using a complex in vivo application: the noninvasive measurement of ethanol in humans. Results indicate that in vivo calibration model sensitivity, selectivity, and concentration correlations can be systematically interrogated using the proposed selectivity framework and judicious use of experimental measurements. These analyses not only provide selectivity and sensitivity information, but also the variance components of the total MSEP, which is invaluable information for both method development and analytical method characterization.
Strategies for Formaldehyde Detection in Flames and Engines Using a Single-Mode Nd:YAG/OPO Laser System
Authors: Brackmann Christian , Li Zhongshan , Rupinski Martin , Docquier Nicolas , Pengloan Gaelle , Aldén Marcus
Published: June 2005

This paper presents technical developments for the detection of formaldehyde (CH2O) using laser-induced fluorescence. The easily accessible third harmonic of the Nd:YAG laser at 355 nm was used for excitation of formaldehyde. In order to investigate potential background fluorescence, e.g., from large molecules such as polyaromatic hydrocarbons, special attention was paid to investigating the possibility of scanning the wavelength of a single-mode Nd:YAG laser under the gain profile, ∼3 cm−1, on and off resonance. Furthermore, a technique for simultaneous detection of formaldehyde and OH using one laser system is presented. The single-mode Nd: YAG laser at 355 nm in combination with an optical parametric oscillator (OPO) laser tuned to 283 nm was used for simultaneous two-dimensional imaging of both species using one charge-coupled device (CCD) detector equipped with a dual filter image separator. The techniques are demonstrated with measurements in laboratory flames and the combined measurements are also demonstrated in an engine.
Calibration Curves for Real-Time PCR
Authors: Lai K Kay-Yin , Cook Linda , Krantz Elizabeth M , Corey Lawrence , Jerome Keith R
Published: June 2005

Background: Despite the increasing use of real-time PCR in the diagnosis and management of viral infections, there are no published studies adequately addressing the optimum number of calibrators, the number of replicates of each calibrator, and the frequency with which calibration needs to be repeated. This study was designed to address these issues.

Methods: Cycle threshold data (ABI 7700) was collected from >50 consecutive real-time PCR runs for hepatitis B and Epstein–Barr viruses. Our routine calibration curve made from serial 10-fold dilutions run in duplicate was compared with alternative options, including duplicate 100-fold dilutions, inclusion of a low-copy calibrator, and omission of the duplicate determination. Control data were used to examine the use of an average calibration curve made from multiple runs.

Results: Use of duplicate serial 10-fold dilutions led to the least imprecision, duplicate 100-fold dilutions had slightly higher imprecision, and calibration curves obtained with singlet measurements showed the greatest imprecision. For patient data, the duplicate 100-fold dilution calibration curve produced results that best matched those from the routine calibration curve. Use of singlet dilutions or inclusion of a low-copy calibrator produced poorer agreement. Variability in controls was lower with a daily calibration curve than with an average calibration curve.

Conclusions: Duplicate 100-fold dilution calibration curves produced equivalent results and the same imprecision as curves with more calibrators, and thus are a valid alternative. Laboratories should carefully evaluate the variability resulting from the use of average calibration curves before adopting this approach.
Evaluation of the Quantitative Analytical Methods Real-Time PCR for HER-2 Gene Quantification and ELISA of Serum HER-2 Protein and Comparison with Fluorescence in Situ Hybridization and Immunohistochemistry for Determining HER-2 Status in Breast Cancer Patients
Authors: Tse Chantal , Brault Didier , Gligorov Joseph , Antoine Martine , Neumann Rainer , Lotz Jean-Pierre , Capeau Jacqueline
Published: June 2005

Background: HER-2 status is generally determined by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). Both methods are only semiquantitative, require a tumor sample, and can be difficult to reproduce. We compared these methods with 2 quantitative approaches, one measuring HER-2 gene copy number in tissue by real-time quantitative PCR (qPCR), and the other measuring shed HER-2 protein in serum by ELISA in patients with metastatic disease.

Methods: We analyzed 52 cases of metastatic breast cancer for which both serum collected at the diagnosis of metastasis and stored primary breast tumor specimens were available. The within- and between-run imprecision of real-time qPCR and ELISA were evaluated according to Clinical and Laboratory Standards Institute (formerly known as NCCLS) recommendations. Concordance among the 4 methods was assessed by calculating the κ statistic and its 95% confidence interval (95% CI).

Results: The CVs for within- and between-run imprecision were both <10% with qPCR and ELISA. There was good agreement of results between qPCR and IHC (κ = 0.81; 95% CI, 0.64–0.99), qPCR and FISH (κ = 0.77; 95% CI, 0.58–0.96), ELISA and IHC (κ = 0.65; 95% CI, 0.41–0.89); and ELISA and FISH (κ = 0.69; 95% CI, 0.46–0.92).

Conclusions: Measurements of HER-2 gene expression by qPCR and of serum HER-2 protein by ELISA are highly reproducible approaches for determining HER-2 status in metastatic breast cancer. In addition, ELISA eliminates the need for biopsy.
Iron Stores, Blood Donation, and Insulin Sensitivity and Secretion
Authors: Fernández-Real José Manuel , López-Bermejo Abel , Ricart Wifredo
Published: June 2005

Background: Epidemiologists have observed that blood donation is associated with decreased risk of type 2 diabetes and cardiovascular disease.

Methods: We investigated the relationship between iron stores and insulin sensitivity, after controlling for known confounding factors, and compared insulin sensitivity between blood donors and individuals who had never donated blood (nondonors). In 181 men, insulin sensitivity and insulin secretion were evaluated through frequently sampled intravenous glucose tolerance tests with minimal model analysis. Men who donated blood between 6 months and 5 years before inclusion (n = 21) were carefully matched with nondonors (n = 66) for age, body mass index, waist-to-hip ratio, and cardiovascular risk profile, including blood lipids, blood pressure, and smoking status.

Results: Frequent blood donors (2–10 donations) had increased insulin sensitivity [3.42 (1.03) vs 2.45 (1.2) × 10−4 · min−1 · mIU/L; P = 0.04], decreased insulin secretion [186 (82) vs 401.7 (254) mIU/L · min; P <0.0001], and significantly lower iron stores [serum ferritin, 101.5 (74) vs 162 (100) μg/L; P = 0.017] than nondonors, but the 2 groups had similar blood hematocrits and blood hemoglobin concentrations.

Conclusions: Blood donation is simultaneously associated with increased insulin sensitivity and decreased iron stores. Stored iron seems to impact negatively on insulin action even in healthy people, and not just in classic pathologic conditions associated with iron overload (hemochromatosis and hemosiderosis). According to these observations, it is imperative that a definition of excessive iron stores in healthy people be formulated.
Multiplexed Analysis of Biomarkers Related to Obesity and the Metabolic Syndrome in Human Plasma, Using the Luminex-100 System
Authors: Liu Mine Y , Xydakis Antonios M , Hoogeveen Ron C , Jones Peter H , Smith E O’Brian , Nelson Kathleen W , Ballantyne Christie M
Published: June 2005

Background: The complex pathology of disease has sparked the development of novel protein expression profiling techniques that require validation in clinical settings. This study focuses on multiplexed analyses of adipocytokines and biomarkers linked to the metabolic syndrome, diabetes, and cardiovascular disease.

Methods: Multiplexed immunoassays using fluorescent microspheres and the Luminex-100 system were performed on plasma from 80 obese patients (40 with the metabolic syndrome) before and after 6–8 weeks of diet-induced weight loss. Leptin, insulin, C-peptide, monocyte chemoattractant protein-1 (MCP-1), eotaxin, interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and IL-6 concentrations measured with multiplex panels from 3 different manufacturers were compared with results from commercial ELISAs. Detection limits and between- and within-run imprecision were determined for each analyte. Bland–Altman analysis was used to determine agreement between multiplexed immunoassays and ELISAs.

Results: Correlation between the Luminex multiplexed assays and ELISAs was good for leptin (Linco), insulin (Linco), MCP-1 (Biosource and Upstate), and eotaxin (Biosource) with correlation coefficients of 0.711–0.895; fair for eotaxin (Upstate) and C-peptide (Linco) with correlation coefficients of 0.496–0.582; and poor for TNF-α, IL-8, and IL-6 (Linco, Biosource, Upstate, and R&D) with correlation coefficients of −0.107 to 0.318. Within- and between-run imprecision values for the multiplex method were generally <15%. Relative changes in plasma leptin and insulin concentrations after diet-induced weight loss were similar whether assessed by multiplex assay or ELISA.

Conclusion: Although this technology appears useful in clinical research studies, low assay sensitivity and poor correlations with conventional ELISA methods for some analytes with very low plasma concentrations should be considered when using the Luminex platform in clinical studies.
Postanalytical External Quality Assessment of Blood Glucose and Hemoglobin A1c: An International Survey
Authors: Skeie Svein , Perich Carmen , Ricos Carmen , Araczki Agnes , Horvath Andrea R , Oosterhuis Wytze P , Bubner Tanya , Nordin Gunnar , Delport Rhena , Thue Geir , Sandberg Sverre
Published: June 2005

Background: Diabetes mellitus (DM) is diagnosed and monitored worldwide by blood glucose (BG) and glycohemoglobin A1c (HbA1c) testing, respectively. Methods for quality assessment of clinician interpretations of changes in these laboratory results have been developed. This study uses survey responses from general practitioners (GPs) in different countries to investigate possible differences in interpretation of results, as well as the feasibility of performing international postanalytical external quality assessment surveys (P-EQAS).

Methods: GPs recruited from 7 countries received questionnaires requesting interpretation of changes in a potentially diagnostic capillary BG result and an HbA1c value obtained during monitoring of a patient with type 2 DM. GPs were asked to estimate clinically significant differences between 2 consecutive laboratory results [critical difference (CD)/reference change value] for both BG and HbA1c. The CDs reported by GPs were used to calculate the analytical variation (CVa), which was taken as the quality specification for analytical imprecision. Participants received national benchmarking feedback reports after the survey.

Results: The study included responses from 2538 GPs. CDs in BG results showed the same pattern and were comparable among countries. Calculated median CVa values would be possible to attain at 80% confidence but not at the conventional 95% confidence. For HbA1c, the same pattern was shown across countries, but with lower changes considered true when HbA1c increased than when it decreased. Despite the consistent pattern, variations among GPs were considerable in all countries.

Conclusions: Assessments of CDs for BG and HbA1c were similar internationally, and quality specifications for these analytes based on clinicians’ opinions are therefore interchangeable among countries. International P-EQAS may contribute to a more rational use of laboratory services and clinical guidelines.
High-Throughput Purification of Viral RNA Based on Novel Aqueous Chemistry for Nucleic Acid Isolation
Authors: Hourfar Michael K , Michelsen Uwe , Schmidt Michael , Berger Annemarie , Seifried Erhard , Roth W Kurt
Published: June 2005

Background: Extraction protocols using magnetic solid phases offer a high potential for automation. However, commercially available magnetic-bead–based assays either lack the sensitivity required for viral diagnostics or are disproportionately expensive.

Methods: We developed an aqueous chemistry for extraction of viral nucleic acids from plasma samples by use of common magnetic silica beads. Nucleic acids were bound to the beads at acidic conditions in the presence of a kosmotropic salt and were eluted at a slightly alkaline pH. The method was implemented on a standard pipetting workstation for fully automated extraction of up to 48 samples of 240 μL plasma in 1 batch.

Results: The detection limit of the method was comparable to the spin-column–based QIAamp Viral RNA Mini Kit, which relies on chaotropic salts and binding to a silica membrane, as the comparison method. The 95% detection limit was 23.1 IU per PCR for HIV-1 and 10.7 IU per PCR for hepatitis C virus (HCV). Suitability for clinical routine testing was confirmed in a total of 178 HIV-1- or HCV-positive plasma samples. The method linearity (R2) was >0.99 for the viruses evaluated.

Conclusions: Use of reagents without organic solvents allows simple and cost-effective automation of this method on common pipetting robots with low risk of contamination. Performance characteristics of the novel extraction method make it suitable for use in diagnosis of infectious diseases and viral load determinations.
Multianalyte Quantification of Vitamin B6 and B2 Species in the Nanomolar Range in Human Plasma by Liquid Chromatography–Tandem Mass Spectrometry
Authors: Midttun Øivind , Hustad Steinar , Solheim Einar , Schneede Jørn , Ueland Per M
Published: June 2005

Background: Homocysteine, a risk factor of cardiovascular disease, cognitive disorders, and pregnancy complications, exists at a point of metabolic convergence of several B vitamins, including vitamins B6 and B2 (riboflavin). Measurement of the various forms of these vitamins may be useful for the study of hyperhomocysteinemia as well as for the assessment of vitamin status.

Methods: Plasma (60 μL) was deproteinized by mixing with an equal volume of 50 g/L trichloroacetic acid that contained d2-pyridoxal 5′-phosphate, d3-pyridoxal, and d8-riboflavin as internal standards. Pyridoxal (PL), pyridoxal 5′-phosphate (PLP), pyridoxine (PN), pyridoxine 5′-phosphate, pyridoxamine (PM), pyridoxamine 5′-phosphate, 4-pyridoxic acid (PA), riboflavin, flavin mononucleotide (FMN), and FAD were separated on a C8 reversed-phase column, which was developed with an acetonitrile gradient in a buffer containing acetic acid and heptafluorobutyric acid. The analytes were detected by tandem mass spectrometry in the positive-ion mode.

Results: The chromatographic run lasted 8 min. Within- and between-day CVs were 3%–20% and 6%–22%, respectively, and recoveries were 78%–163%. Limits of detection (signal-to-noise ratio = 5) were in the range 0.1–4.0 nmol/L, and the response was linear over several orders of magnitude. In samples from 94 healthy persons, we obtained median concentrations (nmol/L) of 35.4 for PLP, 16.9 for PL, 22.4 for PA, 10.3 for riboflavin, 7.5 for FMN, and 63.1 for FAD. PN and PM were also detected in some cardiovascular patients taking B6 supplements.

Conclusions: This method based on liquid chromatography–tandem mass spectrometry measures all known plasma forms of vitamins B6 and B2, which span a wide range of polarity. The assay is characterized by simple sample processing with no derivatization, low sample volume requirement, and a short run time.
Fourth Biannual Festival Ondes de Liberté (Waves of Freedom Festival). Bamako, Mali, 27–29 November 2003
Authors: Tower Craig
Published: June 2005

Held at the modern Palais des Congres convention centre near the Niger River in central Bamako, Festival Ondes de Liberté was primarily intended for media professionals and representatives of governmental and non-governmental organizations that use radio for publicity. The majority
of the participants were directors of francophone FM radio stations in West Africa, although a handful of lusophones, mainly from Guinea-Bissau, also participated. The conference organizers are trying to expand the appeal of the conference to radio professionals from all of Africa, and attracted
participants from as far away as Algeria, the Democratic Republic of the Congo and Burundi. The importance of the event within Mali was highlighted by an opening performance by the country's renowned Ensemble Instrumentale Nationale
Vocal Pathways Modulate Efferent Neurons to the Inner Ear and Lateral Line
Authors: Weeg Matthew S. , Land Bruce R. , Bass Andrew H.
Published: June 2005

All sonic vertebrates face the problem of sound production interfering with their ability to detect and process external acoustic signals, including conspecific vocalizations. Direct efferent inputs to the inner ear of all vertebrates, and the lateral line system of some aquatic vertebrates, represent a potential mechanism to adjust peripheral sensitivity during sound production. We recorded from single efferent neurons that innervate the inner ear and lateral line in a sound-producing teleost fish while evoking fictive vocalizations predictive of the temporal features of natural vocalizations. The majority of efferent neurons showed an increase in activity that occurred in-phase with modulations in the fine temporal structure of the fictive vocalizations. Many of these neurons also showed a decrease in activity at fictive vocal offset. Efferents to the sacculus, the main auditory end organ, showed features especially well adapted for maintaining sensitivity to external acoustic signals during sound production. These included robust phase locking of efferent activity to each cycle of a fictive vocalization and a long-duration rebound suppression after each fictive vocalization that could provide a rapid, long-lasting period of sensitization to external acoustic stimuli such as the call of a conspecific. These results suggest that efferent activation by the vocal motor system can directly modulate auditory sensitivity to self-generated sounds and maintain sensitivity to ongoing external sounds. Given the conserved organization of the auditory efferent system across vertebrates, such mechanisms may be operative among all sonic vertebrates.