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ABSTRACT: Chaetomium globosum is one of the most common species of fungi found growing on damp building materials in North America and Europe. At doses that could be experienced in a building with some mould damage, exposure to metabolites from other fungi results in inflammatory changes in vivo and in vitro. This research requires knowledge of the dominant toxins produced by fungal strains from the built environment and characterization of pure compounds for toxicity testing. We examined 25 strains of C. globosum isolated from the built environment in Canada. In varying amounts, these strains primarily produced chaetoglobosin A, C and F, chaetomugilin D, and chaetoviridin A. Spectroscopic data of the major isolated compounds are provided. Previous studies reported a number of metabolites from this species that we did not find. However, this appears to be due to misidentifications of the fungi they examined as well as problems with the analytical methods used. In addition, our data support the use of metabolite profiles for resolving the taxonomy of some economically important Chaetomium species.
Mycotoxin Research 02/2013; 29(1):47-54.
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ABSTRACT: We are interested in identifying human fungal allergens and antigens from species common on water-damaged or damp building materials for use as marker proteins and diagnostic tests. The cellulolytic fungus Chaetomium globosum is common on damp materials in the building environment worldwide. ELISA and immunoblotting tests identified two related proteins of molecular weights 45 and 47 kDa which were identified as fungal antigens found on spore surfaces and in culture filtrate. The sequences were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS), which indicated that the two proteins were chitosanases, confirmed by enzyme assay. The 47 kDa protein was not glycosylated and had an acidic pI of 4.5. These proteins have not been reported from other fungi and similar antigens were not seen in other fungi common in buildings. The production of polyclonal antibodies in rabbits showed the antigenicity of the target proteins and confirmed they were not artifacts of the isolation process. The proteins isolated are useful biomarkers for the detection of C. globosum in the building environment.
Medical mycology: official publication of the International Society for Human and Animal Mycology 09/2012; · 2.13 Impact Factor
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ABSTRACT: Penicillium chrysogenum clade 4, is a common mold on damp building materials. A capture ELISA assay for the major allergen from P. chrysogenum Pch52 has been developed and tested in house dust samples and potential cross-reactivity examined. Minimal cross-reactivity with other relevant indoor fungi was observed for the assay following thorough purification of the monoclonal and polyclonal antibodies. The limit of quantification for ELISA analysis of Pch52 in sieved house dust is comparable to other assays of other fungi. The LOQ for Pch52 was 0.31 ng/mL in solution or 110 ng/g dust. The LOQ for Asp f1 and Alt a1 were 2.2 ng/g and 17 ng/g, respectively. These results indicate this assay is suitable for the quantification of Pch52 in sieved house dust.
Journal of Occupational and Environmental Hygiene 04/2012; 9(4):211-6. · 1.19 Impact Factor
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ABSTRACT: This study examined the response of various forms and sources of glucans toward two different Limulus amebocyte lysate (LAL) methods, the modified LAL, and Glucatell. The glucans studied were curdlan, laminarin, yeast glucan, barley glucan, paramylon, pullulan, pustulan, mannan, and pachyman (as part of the Glucatell kit). Both methods provided largely similar results for each of the glucans; however, the Glucatell method yielded slightly higher responses to certain structures that may not necessarily be of fungal origin, leading to falsely greater positive results. The performance of each method to measure fungal glucan concentration specifically was then assessed.
Journal of Occupational and Environmental Hygiene 09/2011; 8(9):540-3. · 1.19 Impact Factor
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ABSTRACT: Aspergillus versicolor, a fungus commonly found on damp building materials, produces the allergen, Asp v 13. Here we report a sensitive Asp v 13 capture ELISA for A. versicolor spores and spore- and mycelial fragments in house dust samples. The method is based on a double polyclonal capture ELSIA. The detection limits for Asp v 13 antigen and A. versicolor spores without dust were 2.44 pg and 12 ng (ca. 110 spores). Detection limits for Asp v 13 and A. versicolor spores in sieved house dust samples were 1.0 ng and 7.8 μg per gram dry weight house dust, respectively. This detection limit is lower than for other house dust allergen immunoassays including for Stachybotrys chartarum, Aspergillus fumigatus, but much lower than that from Alternaria alternata.
Journal of immunological methods 07/2011; 372(1-2):89-94. · 2.35 Impact Factor
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ABSTRACT: The extracts of five foliar fungal endophytes isolated from Pinus strobus (eastern white pine) that showed antifungal activity in disc diffusion assays were selected for further study. From these strains, the aliphatic polyketide compound 1 and three related sesquiterpenes 2-4 were isolated and characterized. Compound 2 is reported for the first time as a natural product and the E/Z conformational isomers 3 and 4 were hitherto unknown. Additionally, the three known macrolides; pyrenophorol (5), dihydropyrenophorin (6), and pyrenophorin (7) were isolated and identified. Their structures were elucidated by spectroscopic analyses including 2D NMR, HRMS and by comparison to literature data where available. The isolated compounds 1, 2, and 5 were antifungal against both the rust Microbotryum violaceum and Saccharomyces cerevisae.
Phytochemistry 05/2011; 72(14-15):1833-7. · 3.35 Impact Factor
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Amanda J Wheeler,
Xiaohong Xu,
Ryan Kulka,
Hongyu You,
Lance Wallace,
Gary Mallach,
Keith Van Ryswyk,
Morgan MacNeill,
Jill Kearney,
Pat E Rasmussen,
Ewa Dabek-Zlotorzynska,
Daniel Wang,
Raymond Poon,
Ron Williams,
Corinne Stocco,
Angelos Anastassopoulos, J David Miller,
Robert Dales,
Jeffrey R Brook
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ABSTRACT: The Windsor, Ontario Exposure Assessment Study evaluated the contribution of ambient air pollutants to personal and indoor exposures of adults and asthmatic children living in Windsor, Ontario, Canada. In addition, the role of personal, indoor, and outdoor air pollution exposures upon asthmatic children's respiratory health was assessed. Several active and passive sampling methods were applied, or adapted, for personal, indoor, and outdoor residential monitoring of nitrogen dioxide, volatile organic compounds, particulate matter (PM; PM-2.5 pm [PM2.5] and < or =10 microm [PM10] in aerodynamic diameter), elemental carbon, ultrafine particles, ozone, air exchange rates, allergens in settled dust, and particulate-associated metals. Participants completed five consecutive days of monitoring during the winter and summer of 2005 and 2006. During 2006, in addition to undertaking the air pollution measurements, asthmatic children completed respiratory health measurements (including peak flow meter tests and exhaled breath condensate) and tracked respiratory symptoms in a diary. Extensive quality assurance and quality control steps were implemented, including the collocation of instruments at the National Air Pollution Surveillance site operated by Environment Canada and at the Michigan Department of Environmental Quality site in Allen Park, Detroit, MI. During field sampling, duplicate and blank samples were also completed and these data are reported. In total, 50 adults and 51 asthmatic children were recruited to participate, resulting in 922 participant days of data. When comparing the methods used in the study with standard reference methods, field blanks were low and bias was acceptable, with most methods being within 20% of reference methods. Duplicates were typically within less than 10% of each other, indicating that study results can be used with confidence. This paper covers study design, recruitment, methodology, time activity diary, surveys, and quality assurance and control results for the different methods used.
Journal of the Air & Waste Management Association (1995) 03/2011; 61(3):324-38. · 1.52 Impact Factor
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ABSTRACT: Extracts of fermentation cultures of a fungal endophyte (DAOM 221611) from spruce needles have afforded the known macrocyclic antibiotic vermiculin (1), 7α,8β,11-trihydroxydrimane (2), and eight novel 13-carbon γ-lactones, namely trans-3-methyldodec-cis-6-en-4-olide (3), trans-8-hydroxy-3-methyldodec-cis-6-en-4-olide (4), trans-8-acetoxy-3-methyldodec-cis-6-en-4-olide (5), trans-9-hydroxy-3-methyl-8-oxo-dodec-trans-6-en-4-olide (6), trans-8,9-dihydroxy-3-methyldodec-cis-6-en-4-olide (7), trans-9-hydroxy-8-oxo-3-methyldodecan-4-olide (8), trans-7,9-dihydroxy-3-methyl-8-oxo-dodecan-4-olide (9), and trans-6-hydroxymethyl-3-methyl-7-oxo-undecan-4-olide (10). A known JH III metabolism product, 10,11-dihydroxyfarnesenic acid (11), was also isolated and synthesized from farnesol. Other endophyte cultures from black spruce and white spruce afforded the novel 6,7-dihydroxy-2-propyl-2,4-octadien-4-olide (16), 5,6,8-trihydroxy-4-(1'-hydroxyethyl) isocoumarin (17) plus the known sescandelin (18), sescandelin B (19), and 4-hydroxy-2-methoxyacetanilide (20). Several of the γ-lactones showed toxicity to spruce budworm (Choristoneura fumiferana Clem.) larvae and vermiculin 1 and compound 16 were toxic to spruce budworm cells.Key words: toxigenic endophytes, insect toxins, γ-lactones, isocoumarins.
Canadian Journal of Chemistry 02/2011; 81(4):284-292. · 1.24 Impact Factor
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ABSTRACT: Liquid culture fermentations of Fusariumculmorum yield a wide variety of secondary metabolites. In addition to known isoprenoids, such as the trichothecenes and modified trichothecenes, four new derivatives of culmorin have been isolated and characterized by spectral analysis. These compounds are 15-hydroxyculmorone (7), and 5-hydroxy- (8), 12-hydroxy- (10), and 15-hydroxyculmorin (9). The structure of culmorone is confirmed. The biosynthesis of culmorin from farnesyl pyrophosphate is substantiated through the use of 13C-acetate and the oxidation of these derivatives is discussed.
Canadian Journal of Chemistry 02/2011; 70(5):1308-1316. · 1.24 Impact Factor
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ABSTRACT: The formation of 3-acetyldeoxynivalenol (ADON) and other secondary metabolites of Fusarium culmorum in a stirred jar fermentor is described in relation to changes in concentrations of sugars, N, P, and O2, and in pH in the medium as well as changes in cellular parameters. The addition of sodium [1-13C]acetate to the fermentation demonstrated that the conditions used resulted in the biosynthesis of ADON instead of other primary and secondary metabolites such as culmorin, dihydroxycalonectrin, and sambucinol for all but the early stages of the fermentation. Enrichment studies also revealed that dihydroxycalonectrin was not an important intermediate of ADON. Biosynthesis of ADON was apparently induced by nitrogen limitation. At the end of the fermentation, ca. 710 mg L−1 ADON was obtained.
Canadian Journal of Botany 01/2011; 64(1):1-5. · 1.40 Impact Factor
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ABSTRACT: SchS21 and SchS34 are proteins from Stachybotrys chartarum sensu latto that are antigenic in goats, mice and humans. Monoclonal antibodies to these proteins react with spores of S. chartarum and S. chlorohalonata but do not cross-react with a diverse taxonomic and ecological array of other fungi.
Based on partial sequences of the 21- and 34-kDa proteins, obtained from tandem mass spectra and Edman degradation, degenerate primers were designed for touchdown PCR and the resulting amplicons were sequenced. Subsequently, inverse-PCR was used to obtain genomic DNA sequences encoding SchS21 and SchS34. RT-PCR products were sequenced to predict the mature protein sequences of SchS21 and SchS34. Based on the speculation that SchS21 protein was a DNase, the enzymatic properties were investigated.
Sequences of 435 and 666 bp in length were obtained from SchS21 and SchS34 cDNAs. The SchS21 open reading frame encodes a mature protein of 144 amino acids, while that of SchS34 is 221 amino acids in length. SchS21 is a secretory, alkaline, Mg-dependent exodeoxyribonuclease, while SchS34 is a secretory protein of unknown function. His-tagged forms of the mature SchS21 and SchS34 proteins were separately overexpressed in Escherichia coli and purified using Ni-NTA columns (0.5 mg/l yield).
Based on Western blots, the expressed proteins were similar in molecular weight and bound to the respective monoclonal antibodies to SchS21 and SchS34 proteins from S. chartarum. Interactions with human sera IgE confirmed the expressed forms of SchS21 and SchS34 as naturally occurring allergens.
International Archives of Allergy and Immunology 11/2010; 155(1):74-85. · 2.40 Impact Factor
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ABSTRACT: Whether or not indoor mold growth causes acute childhood respiratory illness is controversial.
To determine the influence of indoor fungus on the incidence of acute respiratory illness episodes during the first two years of life.
Fungal indicators were measured in homes of children followed by daily symptom diaries and twice monthly telephone contact up to two years.
357 children born in Prince Edward Island, Canada.
Generally, fungal contamination was not excessive with a geometric mean mold surface area (MSA) of 1012cm(2) (geometric standard deviation (GSD) 24.2). The annual mean illness episodes per child were 6.85 (Standard Deviation (SD) 2.80). The incidence of respiratory illness episodes was not significantly related to any of the mold indicators: Analysis of variance (ANOVA) derived F-statistic (p values) was 0.14 (0.7090) for mold surface area.
In homes not selected by degree of fungal contamination, fungal burden was generally not excessive and was not found to be a risk factor for acute respiratory illness episodes during the first two years of life.
Environmental Research 10/2010; 110(7):692-8. · 3.40 Impact Factor
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ABSTRACT: This study examines decontamination processes that were developed on an emergency basis to eliminate Bacillus anthracis spores from deliberately contaminated buildings. The recommended steps include a survey with sampling, the removal of sensitive items, and HEPA vacuuming of all readily available surfaces, followed by biocide treatment and subsequent analyses for viable cells. There are several analytical challenges posed by this approach. These include the ability to discriminate the added strain from naturally occurring resident microbes, determining detection limits for anthrax spores in settled dusts, and detecting viable but nonculturable spores. There are also logistical issues relating to the various skill sets required from investigation to reconstruction. In the present study, a model office was constructed, and a strain of Bacillus pumilus was isolated from the carpet and reintroduced to the office in excess. The abundance of the B. pumilus strain was monitored in settled dust using a strain-specific, quantitative polymerase chain reaction (QPCR)-based detection method following repeated HEPA vacuum cleanings. The QPCR method had a limit of detection corresponding to < or = 10(2) colony forming units per gram of settled dust. QPCR results were compared with measures of dust recoveries and fungal glucan and endotoxin levels in the dust samples. The largest fraction (ca. 81%) of added spores was recovered during the first HEPA cleaning. Subsequent cleanings resulted in incrementally lower recoveries, with removal of 93% of the initial inoculum by the third HEPA vacuuming. HEPA vacuuming prior to removal of items such as office contents and furnishings would result in much less resuspension of dust and limiting the extent of contamination. This approach also ensures that residual contaminants are as low as can be reasonably achieved.
Journal of Occupational and Environmental Hygiene 10/2010; 7(10):585-92. · 1.19 Impact Factor
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ABSTRACT: The extracts of a selection of 150 foliar fungal endophytes isolated from Picea rubens (red spruce) needles were screened by LC-MS and assayed for toxicity. Three of these strains that were toxic to the forest pest Choristoneura fumiferana (eastern spruce budworm) in dietary bioassays were selected for further study. Their culture extracts were analyzed by LC-NMR spectroscopy, and the major metabolites were isolated by LC-MS-SPE or PTLC/column chromatography and characterized. The structures were elucidated by spectroscopic analyses including 2D NMR, HRMS and by comparison to literature data. Compounds 1 and 5-7 are hitherto unknown whereas compounds 2 and 3 are natural products described for the first time. Compound 4 is reported for the first time as a fungal metabolite and 8-9 were identified as known fungal metabolites in genera.
Phytochemistry 02/2010; 71(7):760-5. · 3.35 Impact Factor
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ABSTRACT: Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize). Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC). The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX) for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B1. Six unique sequences were obtained, each showing improved binding to fumonisin B1 compared to controls. Sequence FB1 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.
International Journal of Molecular Sciences. 01/2010;
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ABSTRACT: Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize). Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC). The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX) for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B(1). Six unique sequences were obtained, each showing improved binding to fumonisin B(1) compared to controls. Sequence FB(1) 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.
International Journal of Molecular Sciences 01/2010; 11(12):4864-81. · 2.60 Impact Factor
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ABSTRACT: Choristoneura fumiferana is the most economically-important insect pest in eastern North America. Historically, strategies to control epidemics have relied on chemical pesticides that are no longer approved for use. The presence of fungal endophytes in cool area grass species and their role in reducing the impact of herbivorous insects is well understood. Recent work has demonstrated that foliar endophytes of conifers also produce anti-insect toxins. Field and nursery studies testing trees infected with the rugulosin producing endophyte Phialocephala scopiformis reduced the growth and development of C. fumiferana. The study of foliar endophytes from a variety of conifers including: Picea mariana, P. rubens and P. glauca as well as Abies balsamea and Larix laricina for the discovery of other anti-insect toxins are discussed. These endophytes are horizontally transmitted thus they are not present in nursery seedlings. Inoculating seedlings with toxigenic endophyte strains has been demonstrated to be effective in providing the tree with tolerance to herbivorous insects.
Natural product communications 11/2009; 4(11):1497-504. · 1.24 Impact Factor
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ABSTRACT: We are interested in isolating and identifying antigenic fungal proteins from species that grow on damp building materials. The indoor clade of Penicillium chrysogenum, the so-called Fleming clade, is the most common species of Penicillium on moldy building materials. We have identified a 52-kDa marker protein for the indoor clade of P. chrysogenum not present in a taxonomically diverse selection of fungi. It is found in high concentrations in protein extracted from the fungus grown on paper-faced gypsum wallboard. During this process, we illuminated the variability in response to patient sera and of strains of the fungus collected over a wide geographic area. From a collection of sera from all over the USA, 25 of the 48 patients reacted to the 52-kDa protein from this prescreened collection of sera. Most strain/antibody combinations had proportionate ELISA response associated with the presence of the target. However, approximately 25% of the strain/patient serum combinations included people who responded to many common allergens from the Penicillia. All the P. chrysogenum strains tested produced the target protein. However, there was considerable variability in patient IgG response to 32-, 30-, and 18-kDa antigens and in their production by the various clade 4 strains. The target protein was not found in spores or culture extracts of a wide selection of relevant fungi. It appears that the previous studies have been conducted on strains of the fungus from the three clades not those associated with the built environment.
Mycopathologia 08/2009; 168(5):213-26. · 1.65 Impact Factor
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ABSTRACT: BACKGROUND: Three matrices—corn (maize) meal, distiller's dried grains with solubles (DDGS) and condensed distiller's solubles (CDS)—were sampled in sequence from a continuous dry-milling process plant for the determination of mass balance of deoxynivalenol (DON). Four commercially available enzyme-linked immunosorbent assay (ELISA) kits were evaluated for their ability to measure the presence of DON. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) was used as standard method to detect DON and other Fusarium toxins.RESULTS: The concentrations of DON in DDGS and especially CDS were overestimated or underestimated by ELISA. However, for both matrices, all ELISA methods were not significantly different in their mean results from the LC/MS/MS standard, although the variability in results was much higher. DON concentrations in the CDS and the final DDGS co-product were significantly higher (P ≤ 0.01) than in the starting material (corn grain). Toxin concentration increased by a factor of 3 on a dry weight basis in DDGS compared with the starting corn and by a factor of 4 in CDS. Mean concentration of DON in CDS was four times higher (7.11 mg kg−1) than in corn grains (1.80 mg kg−1) and 1.4 times higher than in DDGS (5.24 mg kg−1). Mass balance calculations showed that CDS was the main source of contamination of DON, comprising ca 70% of the toxin found in the final product (DDGS). Most DON (87%) was accounted for by this analysis.CONCLUSION: Concentrations in the grain corn entering ethanol plants should be close to the dietary values recommended for swine in Canada and the USA for DON (1 mg kg−1). Small amounts of acetyldeoxynivalenol and DON glucoside were also found in the three matrices along with a small amount of zearalenone. Unlike the situation for DON, the DON glucoside was not concentrated into DDGS and CDS, indicating that it was hydrolysed during the fermentation process. Copyright © 2009 Society of Chemical Industry
Journal of the Science of Food and Agriculture 05/2009; 89(9):1574 - 1580. · 1.44 Impact Factor
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ABSTRACT: As part of studies of metabolites from fungi common in the built environment in Canadian homes, we investigated metabolites from strains of three Eurotium species, namely E. herbariorum, E. amstelodami, and E. rubrum as well as a number of isolates provisionally identified as Aspergillus ustus. The latter have been recently assigned as the new species A. insuetus and A. calidoustus. E. amstelodami produced neoechinulin A and neoechinulin B, epiheveadride, flavoglaucin, auroglaucin, and isotetrahydroauroglaucin as major metabolites. Minor metabolites included echinulin, preechinulin and neoechinulin E. E. rubrum produced all of these metabolites, but epiheveadride was detected as a minor metabolite. E. herbariorum produced cladosporin as a major metabolite, in addition to those found in E. amstelodami. This species also produced questin and neoechinulin E as minor metabolites. This is the first report of epiheveadride occurring as a natural product, and the first nonadride isolated from Eurotium species. Unlike strains from mainly infection-related samples, largely from Europe, neither ophiobolins G and H nor austins were detected in the Canadian strains of A. insuetus and A. calidoustus tested, all of which had been reported from the latter species. TMC-120 A, B, C and a sesquiterpene drimane are reported with certainty for the first time from indoor isolates, as well as two novel related methyl isoquinoline alkaloids.
Mycological Research 05/2009; 113(Pt 4):480-90. · 2.81 Impact Factor
