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See detailSniffing out the hypoxia volatile metabolic signature of Aspergillus fumigatus
Rees, Christiaan A; Stefanuto, Pierre-Hugues ULg; Beattie, Sarah R et al

in Journal of Breath Research (2017), 036003

Invasive aspergillosis (IA) is a life-threatening infectious disease caused by fungi from the genus Aspergillus, with an associated mortality as high as 90% in certain populations. IA-associated pulmonary ... [more ▼]

Invasive aspergillosis (IA) is a life-threatening infectious disease caused by fungi from the genus Aspergillus, with an associated mortality as high as 90% in certain populations. IA-associated pulmonary lesions are characteristically depleted in oxygen relative to normal lung tissue, and it has been shown that the most common causal agent of IA, Aspergillus fumigatus, must respond to lowoxygen environments for pathogenesis and disease progression. Previous studies have demonstrated marked alterations to the Aspergillus fumigatus transcriptome in response to low-oxygen environments that induce a ‘hypoxia response’. Consequently, we hypothesized that these transcriptomic changes would alter the volatile metabolome and generate a volatile hypoxia signature. In the present study, we analyzed the volatile molecules produced by A. fumigatus in both oxygen replete (normoxia) and depleted (hypoxia) environments via headspace solid-phase micro-extraction coupled to twodimensional gas chromatography-time-of-flight mass spectrometry. Using the machine learning algorithm random forest, we identified 19 volatile molecules that were discriminatory between the four growth conditions assessed in this study (i.e., early hypoxia (1 h), late hypoxia (8 h), early normoxia (1 h), and late normoxia (8 h)), as well as a set of 19 that were discriminatory between late hypoxia cultures and all other growth conditions in aggregate. Nine molecules were common to both comparisons, while the remaining 20 were specific to only one of two.Weassigned putative identifications to 13 molecules, of which six were most highly abundant in late hypoxia cultures. Previously acquired transcriptomic data identified putative biochemical pathways induced in hypoxia conditions that plausibly account for the production of a subset of these molecules, including 2,3-butanedione and 3-hydroxy-2-butanone. These two molecules may represent a novel hypoxia fitness pathway in A. fumigatus, and could be useful in the detection of hypoxia-associated A. fumigatus lesions that develop in established IA infections. [less ▲]

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See detailIdentifying infectious agents from lung transplant patients using GC×GC-TOFMS
Stefanuto, Pierre-Hugues ULg; Rees, Christiaan A.; Romamno, Rosalba et al

Poster (2017, May)

During the first year post-surgery, bacterial and fungal infections are the main causes of death in lung transplant recipients. Contributing substantially to the high mortality rate (35%) is the lengthy ... [more ▼]

During the first year post-surgery, bacterial and fungal infections are the main causes of death in lung transplant recipients. Contributing substantially to the high mortality rate (35%) is the lengthy time-to-diagnosis, which includes both pathogen identification and antibiotic resistance profiling. A novel diagnostic strategy based on the detection of volatile compounds in breath seems a promising option to improve these patients’ outcomes. In order to demonstrate the feasibility of this approach, we characterized the volatile fraction of different clinical matrices (i.e., bronchoalveolar lavage (BAL) and blind bronchial aspirate (BBA)). Due to the complexity of the mixture and high dynamic range of the concentrations, we applied headspace solid phase microextraction (HS-SPME) combined with comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS) to obtain a comprehensive overview of the sample headspace composition. Following the analytical step, we applied various univariate and multivariate statistical methods (e.g. random forest, principal component analysis, and hierarchical cluster analysis) in order to extract required information from the data. Based on the composition of the volatile fraction, we were able to differentiate between BAL and BBA with a classification accuracy of 88.4 %. Furthermore, we could correctly distinguish between individuals with active respiratory infections and those without infection with a 72.2 % classification accuracy. In some cases, it was also possible to differentiate between pathogens at the species level. These results are preliminary evidence to suggest the possibility of detecting infections in these patient without the use of time-consuming culture techniques. [less ▲]

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See detailAdvanced method optimization for volatile aroma profiling of beer using two-dimensional gas chromatography time-of-flight mass spectrometry
Stefanuto, Pierre-Hugues ULg; Perrault, Katelynn ULg; Dubois, Lena ULg et al

in Journal of Chromatography. A (2017)

The complex mixture of volatile organic compounds (VOCs) present in the headspace of Trappist and craft beers was studied to illustrate the efficiency of thermal desorption (TD) comprehensive two ... [more ▼]

The complex mixture of volatile organic compounds (VOCs) present in the headspace of Trappist and craft beers was studied to illustrate the efficiency of thermal desorption (TD) comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS) for highlighting subtle differences between highly complex mixtures of VOCs. Headspace solid-phase microextraction (HS-SPME), multiple (and classical) stir bar sorptive extraction (mSBSE), static headspace (SHS), and dynamic headspace (DHS) were compared for the extraction of a set of 21 representative flavor compounds of beer aroma. A Box-Behnken surface response methodology experimental design optimization (DOE) was used for convex hull calculation (Delaunay’s triangulation algorithms) of peak dispersion in the chromatographic space. The predicted value of 0.5 for the ratio between the convex hull and the available space was 10% higher than the experimental value, demonstrating the usefulness of the approach to improve optimization of the GC × GC separation. Chemical variations amongst aligned chromatograms were studied by means of Fisher Ratio (FR) determination and F‐distribution threshold filtration at different significance levels (α = 0.05 and 0.01) and based on z‐score normalized area for data reduction. Statistically significant compounds were highlighted following principal component analysis (PCA) and hierarchical cluster analysis (HCA). The dendrogram structure not only provided clear visual information about similarities between products but also permitted direct identification of the chemicals and their relative weight in clustering. The effective coupling of DHS-TD-GC × GC-TOFMS with PCA and HCA was able to highlight the differences and common typical VOC patterns among 24 samples of different Trappist and selected Canadian craft beers. [less ▲]

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See detailSniffing Out the Hypoxia Volatile Metabolic Signature of Aspergillus fumigatus
Stefanuto, Pierre-Hugues ULg; Rees, Christiaan A.; Beattie, Sarah R. et al

Poster (2017, May)

Background: Aspergillus fumigatus is responsible for the life-threatening infection invasive aspergillosis (IA) that is associated with high mortality in immunocompromised individuals. Current diagnostics ... [more ▼]

Background: Aspergillus fumigatus is responsible for the life-threatening infection invasive aspergillosis (IA) that is associated with high mortality in immunocompromised individuals. Current diagnostics for IA are either highly invasive or suffer from inadequate sensitivity and/or specificity. Improvement of IA diagnostic assays requires a better understanding of fungal metabolism inside the host. The infection microenvironment becomes progressively depleted of oxygen during IA. Thus, metabolites associated with hypoxia metabolism may yield to novel diagnostic biomarkers. Here, we report on the volatile metabolites emitted from A. fumigatus batch cultures under normoxia and hypoxia conditions. Methods: A. fumigatus strain CEA10 was incubated in glucose minimal medium at 37 °C under either normoxia (21% O2, 0.04% CO2) or hypoxia (0.2% O2, 5% CO2) growth conditions. Volatile molecules from the supernatants of early (1 h) and late (8 h) cultures were collected using headspace solid-phase microextraction (HS-SPME) and analyzed using two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-TOFMS). Results: We identified 10 volatile compounds that are significantly more abundant in late hypoxia cultures compared with all other experimental conditions. Among these compounds, we identified ethanol and acetaldehyde, which are known to result from hypoxia metabolism of glucose by A. fumigatus. In addition, we detected 2-propanol, which can be generated by the reduction of acetone via alcohol dehydrogenase, as well as 2,3-butanedione (diacetyl) and 3-hydroxy-2-butanone (acetoin), which results from the catabolism of acetolactate. Transcriptomic data collected from normoxia and hypoxia cultures validates our volatilomic findings through the identification of putative catabolic pathways that are upregulated during hypoxia growth. Diacetyl is known to be produced by bacteria as a pH-neutral fermentative byproduct to prevent lethal acidification, and we speculate that A. fumigatus may use the same pathway. Conclusion: The composition of A. fumigatus’ volatile metabolome changes according to the oxygen tension in the environment. We measured hypoxia-associated compounds, ethanol and acetaldehyde which have been previously identified as byproducts of hypoxia metabolism in this organism. 2-propanol, 2, 3-butanedione and 3-hydroxy-2-butanone represent novel metabolites that suggests the existence of other catabolic pathways activated in A. fumigatus under hypoxia growth conditions. [less ▲]

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See detailIdentifying novel biomarkers for the rapid diagnosis of invasive aspergillosis using GC×GC-TOFMS
Stefanuto, Pierre-Hugues ULg; Rees, Christiaan A.; Beattie, Sarah R. et al

Conference (2017, May)

Aspergillus fumigatus is a filamentous fungus responsible for the life-threatening infection, invasive aspergillosis (IA), in immunocompromised individuals. Currently, IA diagnostic methods take days to ... [more ▼]

Aspergillus fumigatus is a filamentous fungus responsible for the life-threatening infection, invasive aspergillosis (IA), in immunocompromised individuals. Currently, IA diagnostic methods take days to produce a result and methods to obtain samples are invasive. One approach to improve the time-to-diagnosis is based on utilizing our knowledge of fungal metabolism during pathogenesis. Recent studies have demonstrated the importance of oxygen availability during IA, which presents a potential diagnostic opportunity. In this study, we analyzed the early (1 hr) and late (8 hr) volatile profile of A. fumigatus strain CEA1o under oxygen replete (~20% O2) and deplete (0.2% O2) conditions. In order to comprehensively screen the headspace of these cultures, we applied headspace solid phase microextraction (HS-SPME) associated with comprehensive two-dimensional gas chromatography coupled to time of flight mass spectrometry (GC×GC-TOFMS). Comparing the headspace of the four culture conditions, we were able to identify specific volatile biomarkers of low oxygen fungal growth: ethanol, acetaldehyde, 2-propanol, 2,3-butanedione, and 3-hydroxy-2-butanone. Based on the putative identifications of these compounds, we identified potential metabolic pathways that are responsible for their production. The two first compounds are known to be produced during hypoxia metabolism of glucose, however, the three others suggest that previously uncharacterized metabolic pathways may be utilized by A. fumigatus in low oxygen conditions. Validation of these biomarker identities and translation into patients may generate metabolic insight into these organisms during IA pathogenesis. [less ▲]

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See detailThe Case of the Decaying Cadaver
Stefanuto, Pierre-Hugues ULg; Focant, Jean-François ULg

in The Analytical Scientist (2017), 51

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See detailEnhanced Chemical Profiling of Human Decomposition in a Case Study
Dubois, Lena ULg; Perrault, Katelynn ULg; Stefanuto, Pierre-Hugues ULg et al

in Forensic Science International (2017), 277

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See detailMonitoring Changes in the Volatile Profile of Aging Human Blood
Dubois, Lena ULg; Perrault, Katelynn ULg; Stefanuto, Pierre-Hugues ULg et al

in Forensic Science International (2017), 277

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