Area under the maximum expiratory flow-volume curve--a sensitive parameter in the evaluation of airway patency.

BACKGROUND: The most frequently used parameters for assessing bronchoconstriction and bronchodilation are forced expiratory volume in 1 s (FEV(1)) and peak expiratory flow (PEF). OBJECTIVES: To assess the sensitivity of other parameters after induced bronchoconstriction and bronchodilation. METHODS: From maximum expiratory flow-volume (MEFV) curves, forced vital capacity, FEV(1), PEF, maximum expiratory flows (MEF) at 25, 50 and 75% of vital capacity and the area under the MEFV curve (A(ex)) were measured in two groups of asthmatic children after induced bronchoconstriction and bronchodilation, and in children with cystic fibrosis (CF) after bronchodilation. RESULTS: In 142 asthmatics without airway obstruction, bronchoconstriction was induced by inhalation of 1% histamine aerosol. The 20% fall in A(ex) compared to baseline was found in all asthmatics, while the 20 and 15% falls in FEV(1) were noted in 36 and 65% of the patients, respectively. Other parameters were less sensitive or interpretation was problematic. Another 110 asthmatics with mild-moderate airway obstruction were treated with various bronchodilators. The 20% increase in A(ex) was observed in all asthmatics, while the 20% increase in FEV(1) was found in only 33% of the patients and the 15% increase in FEV(1) in 51%. In 9 CF children, the pattern of changes in A(ex) and FEV(1) after bronchodilation was similar to that in asthmatics. CONCLUSIONS: A(ex) was a sensitive and less problematic parameter in the evaluation of airway patency in comparison with FEV(1) and other parameters measured from the MEFV curve in our study patients.

Identification of fungal microorganisms by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a reliable tool for fast identification and classification of microorganisms. In this regard, it represents a strong challenge to microscopic and molecular biology methods. Nowadays, commercial MALDI systems are accessible for biological research work as well as for diagnostic applications in clinical medicine, biotechnology and industry. They are employed namely in bacterial biotyping but numerous experimental strategies have also been developed for the analysis of fungi, which is the topic of the present review. Members of many fungal genera such as Aspergillus, Fusarium, Penicillium or Trichoderma and also various yeasts from clinical samples (e.g. Candida albicans) have been successfully identified by MALDI-TOF MS. However, there is no versatile method for fungi currently available even though the use of only a limited number of matrix compounds has been reported. Either intact cell/spore MALDI-TOF MS is chosen or an extraction of surface proteins is performed and then the resulting extract is measured. Biotrophic fungal phytopathogens can be identified via a direct acquisition of MALDI-TOF mass spectra e.g. from infected plant organs contaminated by fungal spores. Mass spectrometric peptide/protein profiles of fungi display peaks in the m/z region of 1000-20000, where a unique set of biomarker ions may appear facilitating a differentiation of samples at the level of genus, species or strain. This is done with the help of a processing software and spectral database of reference strains, which should preferably be constructed under the same standardized experimental conditions.

MALDI-based intact spore mass spectrometry of downy and powdery mildews.

Fast and easy identification of fungal phytopathogens is of great importance in agriculture. In this context, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a powerful tool for analyzing microorganisms. This study deals with a methodology for MALDI-TOF MS-based identification of downy and powdery mildews representing obligate biotrophic parasites of crop plants. Experimental approaches for the MS analyses were optimized using Bremia lactucae, cause of lettuce downy mildew, and Oidium neolycopersici, cause of tomato powdery mildew. This involved determining a suitable concentration of spores in the sample, selection of a proper MALDI matrix, looking for the optimal solvent composition, and evaluation of different sample preparation methods. Furthermore, using different MALDI target materials and surfaces (stainless steel vs polymer-based) and applying various conditions for sample exposure to the acidic MALDI matrix system were investigated. The dried droplet method involving solvent evaporation at room temperature was found to be the most suitable for the deposition of spores and MALDI matrix on the target and the subsequent crystallization. The concentration of spore suspension was optimal between 2 and 5 × 10(9) spores per ml. The best peptide/protein profiles (in terms of signal-to-noise ratio and number of peaks) were obtained by combining ferulic and sinapinic acids as a mixed MALDI matrix. A pretreatment of the spore cell wall with hydrolases was successfully introduced prior to MS measurements to obtain more pronounced signals. Finally, a novel procedure was developed for direct mass spectra acquisition from infected plant leaves.

A convenient purification and preconcentration of peptides with alpha-cyano-4-hydroxycinnamic acid matrix crystals in a pipette tip for matrix-assisted laser desorption/ionization mass spectrometry.

Peptide samples derived from enzymatic in-gel digestion of proteins resolved by gel electrophoresis often contain high amount of salts originating from reaction and separation buffers. Different methods are used for desalting prior to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), e.g. reversed-phase pipette tip purification, on-target washing, adding co-matrices, etc. As a suitable matrix for MALDI MS of peptides, alpha-cyano-4-hydroxycinnamic acid (CHCA) is frequently used. Crystalline CHCA shows the ability to bind peptides on its surface and because it is almost insoluble in acidic water solutions, the on-target washing of peptide samples can significantly improve MALDI MS signals. Although the common on-target washing represents a simple, cheap and fast procedure, only a small portion of the available peptide solution is efficiently used for the subsequent MS analysis. The present approach is a combination of the on-target washing principle carried out in a narrow-end pipette tip (e.g. GELoader tip) and preconcentration of peptides from acidified solution by passing it through small CHCA crystals captured inside the tip on a glass microfiber frit. The results of MALDI MS analysis using CHCA-tip peptide preconcentration are comparable with the use of homemade POROS R2 pipette tip microcolumns. Advantages and limitations of this approach are discussed.