Surfactant-aided, matrix-assisted laser desorption/ionization mass spectrometry of hydrophobic and hydrophilic peptides

The analysis of hydrophobic and hydrophilic peptides in an aqueous medium using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported. The key development allowing for simultaneous analysis of both hydrophobic and hydrophilic components of the sample mixture is the use of surfactants to solubilize the hydrophobic components in the MALDI matrix solution. A wide variety of anionic, cationic, zwitterionic, and nonionic surfactants were evaluated for their ability to assist in the generation of an abundant pseudomolecular ion from a model hydrophobic peptide ([tert-butoxycarbonyl]Glu[gamma-O-benzyl]-Ala-Leu-Ala[O-phenacyl ester]). The results indicate that the most successful surfactant among those studied for analyzing the model hydrophobic peptide is sodium dodecyl sulfate (SDS). SDS exhibited no interfering surfactant background ions, little to no loss of the acid-labile protecting groups from the model hydrophobic peptide, and an abundant pseudomolecular ion of the analyte. In addition, the use of surfactants is shown to be compatible with hydrophilic peptides as well. Mixtures of hydrophobic and hydrophilic peptides were characterized using surfactant-aided (SA) MALDI-MS, and it is demonstrated that all components are detectable once the surfactant is included in the sample solution. We conclude that the key benefit of using SA-MALDI-MS is its ability to simultaneously analyze hydrophobic and hydrophilic peptides from a single sample mixture, including synthetic peptides containing acid- and base-labile protecting groups.     

Testing the significance of microorganism identification by mass spectrometry and proteome database search

We derive and validate a simple statistical model that predicts the distribution of false matches between peaks in matrix-assisted laser desorption/ionization mass spectrometry data and proteins in proteome databases. The model allows us to calculate the significance of previously reported microorganism identification results. In particular, for deltam = +/-1.5 Da, we find that the computed significance levels are sufficient to demonstrate the ability to identify microorganisms, provided the number of candidate microorganisms is limited to roughly three Escherichia coli-like or roughly 10 Bacillus subtilis-like microorganisms (in the sense of having roughly the same number of proteins per unit-mass interval). We conclude that, given the cluttered and incomplete nature of the data, it is likely that neither simple ranking nor simple hypothesis testing will be sufficient for truly robust microorganism identification over a large number of candidate microorganisms.     

Implementing evidence-based recommendations for health care: a roundtable comparing European and American experiences

BACKGROUND: A roundtable held October 5-6 1999, in Maidstone, Kent, United Kingdom, was convened to identify current strategies and ongoing challenges in implementing evidence-based practice guidelines in health care. Despite numerous new medical research findings for improving health care and despite the dissemination of many practice guidelines, the recommendations from these efforts are not being uniformly adopted. Overuse, underuse, and misuse plague the practice of medicine today. IMPLEMENTING GUIDELINES: Multiple implementation strategies are more likely to succeed that a single implementation method; local selection and adaptation of guidelines are critical; and reminders, educational outreach (for prescribing), and interactive educational workshops are generally effective. EXPERIENCE IN EUROPE: In most countries, guideline development has progressed from consensus conference, to evidence-based statements, and finally to evidence-based guidelines that also consider cost-effectiveness. Guideline development is the most advanced in The Netherlands, where physicians have coordinated their efforts with the government to achieve more uniformity than is found elsewhere. EXPERIENCE IN THE UNITED STATES: Designing systems that will facilitate change--not changing physician behavior--should be the focus. The concern for effecting improvement in health care is now more acute because of the increased attention being given to medical errors and patient safety. SUMMARY STATEMENT: Multifaceted approaches are clearly the most important method for improving care. Such approaches may include many improvement methods, none of which work well alone most of the time or any of the time.     

In-chair movement: validity, reliability and implications for measuring sitting discomfort

Sitting discomfort is traditionally evaluated with subjective rating scales which are referenced to an objective correlate (e.g. sitting posture) measured on a static (i.e. non-continuous) basis. Since sitting discomfort is dynamic in nature, it requires continuous, objective measurement. We therefore adapted an interface pressure mat to continuously record in-chair movement (ICM) as an indirect measurement of sitting discomfort by tracking the center of pressure (COP) at the buttock-chair interface. Here we report on two phases in the development of the COP system: laboratory validity and field reliability. In the laboratory study we confirmed system validity by simultaneously tracking ICM with the mat and a force platform (r2 > 0.80) and by comparing subjects' COP movement with their gross trunk movements (r2 > 0.86). In our field study we used the intraclass correlation coefficient to establish a data sampling (i.e. selection) protocol that was reliable. We collected ICM data on seated telecommunications Directory Assistance operators during 2 h field tests. Results showed that using a minimum sampling time of 5 min and then averaging a series of 5 min samples of ICM was more reliable than single discrete samples. Using the averaging protocol, we also showed that ICM increased significantly over 2 h and that ICM did not differ between trials.     

Proteome profiling-pitfalls and progress

In this review we examine the current state of analytical methods in proteomics. The conventional methodology using two-dimensional electrophoresis gels and mass spectrometry is discussed, with particular reference to the advantages and shortcomings thereof. Two recently published methods which offer an alternative approach are presented and discussed, with emphasis on how they can provide information not available via two-dimensional gel electrophoresis. These two methods are the isotope-coded affinity tags approach of Gygi et al. and the two-dimensional liquid chromatography-tandem mass spectrometry approach as presented by Link et al. We conclude that both of these new techniques represent significant advances in analytical methodology for proteome analysis. Furthermore, we believe that in the future biological research will continue to be enhanced by the continuation of such developments in proteomic analytical technology.     

Experimental and theoretical evaluation of microbubble behavior: effect of transmitted phase and bubble size

Ultrasound contrast agents provide new opportunities to image vascular volume and flow rate directly. To accomplish this goal, new pulse sequences can be developed to detect specifically the presence of a microbubble or group of microbubbles. We consider a new scheme to detect the presence of contrast agents in the body by examining the effect of transmitted phase on the received echoes from single bubbles. In this study, three tools are uniquely combined to aid in the understanding of the effects of transmission parameters and bubble radius on the received echo. These tools allow for optical measurement of radial oscillations of single bubbles during insonation, acoustical study of echoes from single contrast agent bubbles, and the comparison of these experimental observations with theoretical predictions. A modified Herring equation with shell terms is solved for the time-dependent bubble radius and wall velocity, and these outputs are used to formulate the predicted echo from a single encapsulated bubble. The model is validated by direct comparison of the predicted radial oscillations with those measured optically. The transient bubble response is evaluated with a transducer excitation consisting of one-cycle pulses with a center frequency of 2.4-MHz. The experimental and theoretical results are in good agreement and predict that the transmission of two pulses with opposite polarity will yield similar time domain echoes with the first significant portion of the echo generated when the rarefactional half-cycle reaches the bubble.     

A novel method for determining calibration and behavior of PVDF ultrasonic hydrophone probes in the frequency range up to 100 MHz

A new calibration technique for PVDF ultrasonic hydrophone probes is described. Current implementation of the technique allows determination of hydrophone frequency response between 2 and 100 MHz and is based on the comparison of theoretically predicted and experimentally determined pressure-time waveforms produced by a focused, circular source. The simulation model was derived from the time domain algorithm that solves the non linear KZK (Khokhlov-Zabolotskaya-Kuznetsov) equation describing acoustic wave propagation. The calibration technique data were experimentally verified using independent calibration procedures in the frequency range from 2 to 40 MHz using a combined time delay spectrometry and reciprocity approach or calibration data provided by the National Physical Laboratory (NPL), UK. The results of verification indicated good agreement between the results obtained using KZK and the above-mentioned independent calibration techniques from 2 to 40 MHz, with the maximum discrepancy of 18% at 30 MHz. The frequency responses obtained using different hydrophone designs, including several membrane and needle probes, are presented, and it is shown that the technique developed provides a desirable tool for independent verification of primary calibration techniques such as those based on optical interferometry. Fundamental limitations of the presented calibration method are also examined.     

Growth of human endothelial cells on plasma-treated polyethyleneterephthalate surfaces

Different methods have been proposed to reduce the surface thrombogenicity of small caliber vascular grafts, using plasma treatments of polymer surfaces in order to improve the adhesion and the proliferation of human endothelial cells (HEC). Plasma modified polyethyleneterephthalate (PET) substrates were employed to grow HEC, isolated from the umbilical vein. A combination of X-ray photoelectron spectroscopy (XPS) and Sessile contact angle (SCA) measurements allowed the study of the surface modifications produced soon after nitrogen and hydrogen plasma treatments with respect to an untreated PET substrate, used as reference. It was possible to select a number of PET substrates while actually performing the HEC seeding experiments. The HEC proliferation was evaluated by light microscope image analyzes. ©2000 Kluwer Academic Publishers     

Performance and design of InGaAs /InP photodiodes for single-photon counting at 1.55 microm

The performance of selected, commercially available InGaAs/InP avalanche photodiodes operating in a photon-counting mode at an incident wavelength of 1.55 mum is described. A discussion on the optimum operating conditions and their relationship to the electric field distribution within the device is presented.     

Calibration-free method to determine the size and hemoglobin concentration of individual red blood cells from light scattering

At present, hemoglobin concentration and the volume of an erythrocyte can be determined from the intensities of light scattered by an individual cell at fixed angular intervals. This method is used in modern hemoglobin analyzers, but it requires calibration of optical and electronic units by certified particles of known size and refractive index. We describe a method that is based on the parametric solution of an inverse light-scattering problem and does not require a calibration procedure. The method is based on the use of parameters of the entire angular light-scattering pattern, called an indicatrix here. These parameters do not depend on the absolute intensity of light scattering. The indicatrix parameters form approximating equations that relate these parameters to the size and the phase-shift parameters of the particle. The applicability of the method is demonstrated by measurement of the indicatrices of individual sphered erythrocytes. The indicatrices of the individual erythrocytes were measured with a scanning flow cytometer at an angular range of from 15 to 55 deg. The volume and the hemoglobin concentration have been calculated by use of the developed method and by fitting of the experimental indicatrices to the indicatrices calculated from the Mie theory.