Ib Søndergaard

Associations between Fungal Species and Water-Damaged Building Materials

ABSTRACT Fungal growth in damp or water-damaged buildings worldwide is an increasing problem, which has adverse effects on both the occupants and the buildings. Air sampling alone in moldy buildings does not reveal the full diversity of fungal species growing on building materials. One aim of this study was to estimate the qualitative and quantitative diversity of fungi growing on damp or water-damaged building materials. Another was to determine if associations exist between the most commonly found fungal species and different types of materials. More than 5,300 surface samples were taken by means of V8 contact plates from materials with visible fungal growth. Fungal identifications and information on building material components were analyzed using multivariate statistic methods to determine associations between fungi and material components. The results confirmed that Penicillium chrysogenum and Aspergillus versicolor are the most common fungal species in water-damaged buildings. The results also showed Chaetomium spp., Acremonium spp., and Ulocladium spp. to be very common on damp building materials. Analyses show that associated mycobiotas exist on different building materials. Associations were found between (i) Acremonium spp., Penicillium chrysogenum, Stachybotrys spp., Ulocladium spp., and gypsum and wallpaper, (ii) Arthrinium phaeospermum, Aureobasidium pullulans, Cladosporium herbarum, Trichoderma spp., yeasts, and different types of wood and plywood, and (iii) Aspergillus fumigatus, Aspergillus melleus, Aspergillus niger, Aspergillus ochraceus, Chaetomium spp., Mucor racemosus, Mucor spinosus, and concrete and other floor-related materials. These results can be used to develop new and resistant building materials and relevant allergen extracts and to help focus research on relevant mycotoxins, microbial volatile organic compounds (MVOCs), and microparticles released into the indoor environment.

Exploring the phenotypic expression of a regulatory proteome- altering gene by spectroscopy and chemometrics

Evaluating gene effects on proteomes and the resulting indirect pleiotropic effects through the cell machinery on the chemical phenotype constitutes a formidable challenge to the analytical chemist. This paper demonstrates that near-infrared (NIR) spectroscopy and chemometrics on the level of the barley seed phenotype is able to differentiate between genetic and environmental effects in a PCA model involving normal barley lines and the gene regulator lys3a in different genetic backgrounds. The gene drastically changes the proteome quantitatively and qualitatively, as displayed in two-dimensional electrophoresis, resulting in a radically changed amino acid and chemical composition. A synergy interval partial least squares regression model (si-PLSR) is tested to select combinations of spectral segments which have a high correlation to defined chemical components indicative of the lys3a gene, such as direct effects of the changed proteome, for example, the amide content, or indirect effects due to changes in carbohydrate and fat composition. It is concluded that the redundancy of biological information on the DNA sequence level is also represented at the phenotypic level in the dataset read by the NIR spectroscopic sensor from the chemical physical fingerprint. The PLS algorithm chooses spectral intervals which combine both direct and indirect proteome effects. This explains the robustness of NIR spectral predictions by PLSR for a wide range of chemical components. The new option of using spectroscopy, analytical chemistry and chemometrics in modeling the genetically based covariance of physical/chemical fingerprints of the intact phenotype in plant breeding and biotechnology is discussed.

Implications of high-temperature events and water deficits on protein profiles in wheat (Triticum aestivum L. cv. Vinjett) grain.

Increased climatic variability is resulting in an increase of both the frequency and the magnitude of extreme climate events. Therefore, cereals may be exposed to more than one stress event in the growing season, which may ultimately affect crop yield and quality. Here, effects are reported of interaction of water deficits and/or a high‐temperature event (32°C) during vegetative growth (terminal spikelet) with either of these stress events applied during generative growth (anthesis) in wheat. Influence of combinations of stress on protein fractions (albumins, globulins, gliadins and glutenins) in grains and stress‐induced changes on the albumin and gliadin proteomes were investigated by 2‐DE and MS. The synthesis of individual protein fractions was shown to be affected by both the type and time of the applied stresses. Identified drought or high‐temperature‐responsive proteins included proteins involved in primary metabolism, storage and stress response such as late embryogenesis abundant proteins, peroxiredoxins and α‐amylase/trypsin inhibitors. Several proteins, e.g. heat shock protein and 14‐3‐3 protein changed in abundance only under multiple high temperatures.

Induction of systemic CTL responses in melanoma patients by dendritic cell vaccination: Cessation of CTL responses is associated with disease progression

Two HLA‐A2‐positive patients with advanced stage IV melanoma were treated with monocyte‐derived dendritic cells (DC) pulsed with either tumor peptide antigens from gp100, MART‐1 and MAGE‐3 alone or in combination with autologous oncolysates. Clinically, the rapid progression of disease was substantially stalled and both patients were alive for more than 15 months after initiation of therapy. Specific CTL reactivity against several tumor antigens was detectable in peripheral blood, which declined just before reactivation of disease progression. Furthermore, CD3 ζ‐chain expression detected by Western lotting was decreased in PBL at this time. In summary, our data confirm that DC‐based vaccinations induce peptide‐specific T cells in the peripheral blood of advanced‐stage melanoma patients. Although successful induction of systemic tumor antigen‐specific CTL may not lead to objective clinical tumor regression, their presence are indicative of a prolonged survival.

Hydrophobins from Aspergillus species cannot be clearly divided into two classes

BackgroundHydrophobins are a family of small secreted proteins with a characteristic pattern of eight cysteine residues found exclusively in filamentous fungi. They have originally been divided into two classes based on their physical properties and hydropathy patterns, and are involved in the attachment of hyphae to hydrophobic structures, the formation of aerial structures and appear to be involved in pathogenicity.FindingsAnalysis of nine genome sequences from seven Aspergilli revealed fifty hydrophobins, where each species displayed between two to eight hydrophobins. Twenty of the identified hydrophobins have not previously been described from these species. Apart from the cysteines, very little amino acid sequence homology was observed. Twenty-three of the identified hydrophobins could be classified as class I hydrophobins based on their conserved cysteine spacing pattern and hydropathy pattern. However twenty-six of the identified hydrophobins were intermediate forms. Notably, a single hydrophobin, ATEG_04730, from Aspergillus terreus displayed class II cysteine spacing and had a class II hydropathy pattern.ConclusionFifty hydrophobins were identified in Aspergillus, all containing the characteristic eight cysteine pattern. Aspergillus terreus exhibited both class I and class II hydrophobins. This is the first report of an Aspergillus species with the potential to express both class I and class II hydrophobins. Many of the identified hydrophobins could not directly be allocated to either class I or class II.

Combination of statistical approaches for analysis of 2-DE data gives complementary results

Five methods for finding significant changes in proteome data have been used to analyze a two-dimensional gel electrophoresis data set. We used both univariate (ANOVA) and multivariate (Partial Least Squares with jackknife, Cross Model Validation, Power-PLS and CovProc) methods. The gels were taken from a time-series experiment exploring the changes in metabolic enzymes in bovine muscle at five time-points after slaughter. The data set consisted of 1377 protein spots, and for each analysis, the data set were preprocessed to fit the requirements of the chosen method. The generated results were one list from each analysis method of proteins found to be significantly changed according to the experimental design. Although the number of selected variables varied between the methods, we found that this was dependent on the specific aim of each method. CovProc and P-PLS focused more on getting the minimum necessary subset of proteins to explain properties of the samples. These methods ended up with less selected proteins. There was also a correlation between level of significance and frequency of selection for the selected proteins.

Classification of wheat varieties: Use of two-dimensional gel electrophoresis for varieties that can not be classified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and an artificial neural network

Analyzing a gliadin extract by matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) combined with an artificial neural network (ANN) is a suitable method for identification of wheat varieties. However, the ANN can not distinguish between all different wheat varieties. Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was applied to three pairs of wheat varieties, which can not be classified correctly by ANN. By 2‐D PAGE the varieties in the three pairs can be discriminated and these six wheat varieties can be separated from each other, which could not be separated by MALDI‐TOF‐MS and NN.

Spectroscopic investigations on the highly purified lactoperoxidase Fe(III)-heme catalytic site

Purification of the lactoperoxidase (LPO) major cationic isoenzyme was significantly improved by the use of preparative chromatographic and electrophoretic methods combined with analytical electrophoretic techniques and image processing. A detailed report is given of the experimental procedure. Furthermore, electron paramagnetic resonance has played a fundamental role in evaluating the enzyme purity against lactoferrin and minor LPO isoenzyme components in setting the final steps of the purification. With the aim to completely clarify the Fe(III)-heme high-spin nature of the native LPO, two samples of lactoperoxidase, LPO1 and LPO2 (RZ = 0.95) from farm and commercial milk, respectively, were purified and characterized in particular by electron paramagnetic resonance (EPR) spectroscopy, in comparison with a commercial preparation (LPOs). The LPO1 EPR spectrum, at physiological pH, is clearly indictive of the presence of an iron(III)-heme high-spin catalytic site in the native enzyme. On the contrary, in the LPO2 spectrum a thermal equilibrium between high- and low-spin iron(III)-heme species is present. The low-spin component of the spectrum has been assigned to an LPO-NO2- adduct due to the presence of some nitrite impurities originating either from commercial unpasteurized milk or from external sources. The LPOs EPR spectrum shwos the presence of some spurious lines in the g approximately equal to 6 and 4 regions due to the minor LPO isoenzyme components and to lactoferrin, respectively. The LPO EPR spectra previously reported in the literature contain a variable number of spurious lines in the g approximately equal to 4 and 2 regions as a consequence of lactoferrin impurity and LPO low-spin adducts with endogenous or exogenous anions. Furthermore, the interaction of LPO with its native substrate (the thiocyanate anion), which previously was shown by NMR and EPR (at high substrate concentration) spectroscopies, has been confirmed by EPR at low temperature and low substrate concentration and by optical spectroscopy at room temperature and high substrate concentration as a function of pH. The LPO activity at optimum pH (approximately equal to 4-5) has been measured in phosphate and acetate buffer using as an oxidizable substrate the system dimethylamino benzoic acid 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (DMAB-MBTH), which was considered a good chromogen for other peroxidases such as HRP and zucchini peroxidases. The LPO vs SCN- activity at optimum pH (approximately equal to 5.5) has been measured in phosphate and acetate buffer.(ABSTRACT TRUNCATED AT 400 WORDS)

Exploring abiotic stress on asynchronous protein metabolism in single kernels of wheat studied by NMR spectroscopy and chemometrics

Extreme climate events are being recognized as important factors in the effects on crop growth and yield. Increased climatic variability leads to more frequent extreme conditions which may result in crops being exposed to more than one extreme event within a growing season. The aim of this study was to examine the implications of different drought treatments on the protein fractions in grains of winter wheat using 1H nuclear magnetic resonance spectroscopy followed by chemometric analysis. Triticum aestivum L. cv. Vinjett was studied in a semi-field experiment and subjected to drought episodes either at terminal spikelet, during grain-filling or at both stages. Principal component trajectories of the total protein content and the protein fractions of flour as well as the 1H NMR spectra of single wheat kernels, wheat flour, and wheat methanol extracts were analysed to elucidate the metabolic development during grain-filling. The results from both the 1H NMR spectra of methanol extracts and the 1H HR-MAS NMR of single kernels showed that a single drought event during the generative stage had as strong an influence on protein metabolism as two consecutive events of drought. By contrast, a drought event at the vegetative growth stage had little effect on the parameters investigated. For the first time, 1H HR-MAS NMR spectra of grains taken during grain-filling were analysed by an advanced multiway model. In addition to the results from the chemical protein analysis and the 1H HR-MAS NMR spectra of single kernels indicating that protein metabolism is influenced by multiple drought events, the 1H NMR spectra of the methanol extracts of flour from mature grains revealed that the amount of fumaric acid is particularly sensitive to water deficits.

Spontaneous T-cell responses against peptides derived from the Taxol resistance–associated gene-3 (TRAG-3) protein in cancer patients

Expression of the cancer-testis antigen Taxol resistance–associated gene-3 (TRAG-3) protein is associated with acquired paclitaxel (Taxol) resistance, and is expressed in various cancer types; e.g., breast cancer, leukemia, and melanoma. Thus, TRAG-3 represents an attractive target for immunotherapy of cancer. To identify HLA-A*02.01–restricted epitopes from TRAG-3, we screened cancer patients for spontaneous cytotoxic T-cell responses against TRAG-3–derived peptides. The TRAG-3 protein sequence was screened for 9mer and 10mer peptides possessing HLA-A*02.01–binding motifs. Of 12 potential binders, 9 peptides were indeed capable of binding to the HLA-A*02.01 molecule, with binding affinities ranging from strong to weak binders. Subsequently, lymphocytes from cancer patients (9 breast cancer patients, 12 melanoma patients, and 13 patients with hematopoietic malignancies) were analyzed for spontaneous reactivity against the panel of peptides by ELISpot assay. Spontaneous immune responses were detected against 8 epitope candidates in 7 of 9 breast cancer patients, 7 of 12 melanoma patients, and 5 of 13 patients with hematopoietic malignancies. In several cases, TRAG-3–specific CTL responses were scattered over several epitopes. Hence, no immunodominance of any single peptide was observed. Furthermore, single-peptide responses were detected in 2 of 12 healthy HLA-A2+ donors, but no responses were detectable in 9 HLA-A2− healthy donors or 4 HLA-A2− melanoma patients. The identified HLA-A*02.01–restricted TRAG-3–derived epitopes are targets for spontaneous immune responses in breast cancer, hematopoietic cancer, and melanoma patients. Hence, these epitopes represent potential target structures for future therapeutic vaccinations against cancer, possibly appropriate for strategies that combine vaccination and chemotherapy; i.e., paclitaxel treatment.