Volatile profiles of human skin cell cultures in different degrees of senescence

It is known that skin releases volatile organic compounds to the environment, and also that its emission pattern changes with aging of the skin. It could be considered, that these compounds are intermediaries in cell metabolism, since many intermediaries of metabolic pathways have a volatile potential. In this work, a simple and non-destructive method consisting of SPME sampling and GC/MS analysis was developed to identify volatile organic emanations from cell cultures. This technique, applied to skin cells culture, indicates that the cells or cell metabolism produce several skin emissions. Chemometric analysis was performed in order to explore the relationship between a volatile profile and the senescence of cell cultures. Volatile profiles were different for cell cultures in different degrees of senescence, indicating that volatile compound patterns could be used to provide information about the age of skin cells.     

On-line monitoring of microbial volatile metabolites by proton transfer reaction-mass spectrometry

A method for analysis of volatile organic compounds (VOCs) from microbial cultures was established using proton transfer reaction-mass spectrometry (PTR-MS). A newly developed sampling system was coupled to a PTR-MS instrument to allow on-line monitoring of VOCs in the dynamic headspaces of microbial cultures. The novel PTR-MS method was evaluated for four reference organisms: Escherichia coli, Shigella flexneri, Salmonella enterica, and Candida tropicalis. Headspace VOCs in sampling bottles containing actively growing cultures and uninoculated culture medium controls were sequentially analyzed by PTR-MS. Characteristic marker ions were found for certain microbial cultures: C. tropicalis could be identified by several unique markers compared with the other three organisms, and E. coli and S. enterica were distinguishable from each other and from S. flexneri by specific marker ions, demonstrating the potential of this method to differentiate between even closely related microorganisms. Although the temporal profiles of some VOCs were similar to the growth dynamics of the microbial cultures, most VOCs showed a different temporal profile, characterized by constant or decreasing VOC levels or by single or multiple peaks over 24 h of incubation. These findings strongly indicate that the temporal evolution of VOC emissions during growth must be considered if characterization or differentiation based on microbial VOC emissions is attempted. Our study may help to establish the analysis of VOCs by on-line PTR-MS as a routine method in microbiology and as a tool for monitoring environmental and biotechnological processes.     

Investigation of volatile organic biomarkers derived from Plasmodium falciparum in vitro

ABSTRACT: BACKGROUND: There remains a need for techniques that improve the sensitive detection of viable Plasmodium falciparum as part of diagnosis and therapeutic monitoring in clinical studies and usual-care management of malaria infections. A non-invasive breath test based on P. falciparum-associated specific volatile organic compounds (VOCs) could fill this gap and provide insights into parasite metabolism and pathogenicity. The aim of this study was to determine whether VOCs are present in the headspace above in vitro P. falciparum cultures. METHODS: A novel, custom-designed apparatus was developed to enable efficient headspace sampling of infected and non-infected cultures. Conditions were optimized to support cultures of high parasitaemia (>20%) to improve the potential detection of parasite-specific VOCs. A number of techniques for VOC analysis were investigated including solid phase micro-extraction using two different polarity fibres, and purge and trap/thermal desorption, each coupled to gas chromatography-mass spectrometry. Each experiment and analysis method was performed at least on two occasions. VOCs were identified by comparing their mass spectra against commercial mass spectral libraries. RESULTS: No unique malarial-specific VOCs could be detected relative to those in the control red blood cell cultures. This could reflect sequestration of VOCs into cell membranes and/or culture media but solvent extractions of supernatants and cell lysates using hexane, dichloromethane and ethyl acetate also showed no obvious difference compared to control non-parasitized cultures. CONCLUSIONS: Future in vivo studies analysing the breath of patients with severe malaria who are harbouring a parasite biomass that is significantly greater than achievable in vitro may yet reveal specific clinically-useful volatile chemical biomarkers.     

Why plant volatile analysis needs bioinformatics--detecting signal from noise in increasingly complex profiles.

Plant volatile analysis may be the oldest form of what now is called plant "metabolomic" analysis. A wide array of volatile organic compounds (VOCs), such as alkanes, alcohols, isoprenoids, and esters, can be collected simultaneously from the plant headspace, either within the laboratory or in the field. Increasingly faster and more sensitive analysis techniques allow detection of an ever-growing number of compounds in decreasing concentrations. However, the myriads of data becoming available from such experiments do not automatically increase our ecological and evolutionary understanding of the roles these VOCs play in plant-insect interactions. Herbivores and parasitoids responding to changes in VOC emissions are able to perceive minute changes within a complex VOC background. Plants modified in genes involved in VOC synthesis may be valuable for the evaluation of changes in plant-animal interactions compared to tests with synthetic compounds, as they allow changes to be made within the context of a more complex profile. We argue that bioinformatics is an essential tool to integrate statistical analysis of plant VOC profiles with insect behavioural data. The implementation of statistical techniques such as multivariate analysis (MVA) and meta-analysis is of the utmost importance to interpreting changes in plant VOC mixtures. MVA focuses on differences in volatile patterns rather than in single compounds. Therefore, it more closely resembles the information processing in insects that base their behavioural decisions on differences in VOC profiles between plants. Meta-analysis of different datasets will reveal general patterns pertaining to the ecological role of VOC in plant-insect interactions. Successful implementation of bioinformatics in VOC research also includes the development of MVA that integrate time-resolved chemical and behavioural analyses, as well as databases that link plant VOCs to their effects on insects.     

Volatile Emissions from Mycobacterium avium subsp. paratuberculosis Mirror Bacterial Growth and Enable Distinction of Different Strains

Control of paratuberculosis in livestock is hampered by the low sensitivity of established direct and indirect diagnostic methods. Like other bacteria, Mycobacterium avium subsp. paratuberculosis (MAP) emits volatile organic compounds (VOCs). Differences of VOC patterns in breath and feces of infected and not infected animals were described in first pilot experiments but detailed information on potential marker substances is missing. This study was intended to look for characteristic volatile substances in the headspace of cultures of different MAP strains and to find out how the emission of VOCs was affected by density of bacterial growth. One laboratory adapted and four field strains, three of MAP C-type and one MAP S-type were cultivated on Herrold’s egg yolk medium in dilutions of 10^-0, 10^-2, 10^-4 and 10^-6. Volatile substances were pre-concentrated from the headspace over the MAP cultures by means of Solid Phase Micro Extraction (SPME), thermally desorbed from the SPME fibers and separated and identified by means of GC-MS. Out of the large number of compounds found in the headspace over MAP cultures, 34 volatile marker substances could be identified as potential biomarkers for growth and metabolic activity. All five MAP strains could clearly be distinguished from blank culture media by means of emission patterns based on these 34 substances. In addition, patterns of volatiles emitted by the reference strain were significantly different from the field strains. Headspace concentrations of 2-ethylfuran, 2-methylfuran, 3-methylfuran, 2-pentylfuran, ethyl acetate, 1-methyl-1-H-pyrrole and dimethyldisulfide varied with density of bacterial growth. Analysis of VOCs emitted from mycobacterial cultures can be used to identify bacterial growth and, in addition, to differentiate between different bacterial strains. VOC emission patterns may be used to approximate bacterial growth density. In a perspective volatile marker substances could be used to diagnose MAP infections in animals and to identify different bacterial strains and origins.     

Production and effects of volatile organic compounds during interspecific interactions

Competition between mycelia of saprotrophic cord-forming basidiomycetes occurs both within dead woody resources and in the soil-litter interface, and involves a variety of antagonistic mechanisms including the production of volatile organic compounds (VOCs). The antagonistic potential of VOC profiles from interactions in wood blocks and in soil microcosms was assessed using shared headspace experiments, and the profile of VOCs emitted over the course of interactions elucidated using solid phase microextraction (SPME) with gas chromatography-mass spectrometry (GC–MS). Quantitative and qualitative changes in VOC production occurred in interactions compared to self-pairing controls, with different VOC profiles from fungi growing in wood blocks compared to soil trays. There were both stimulatory and inhibitory effects of VOCs on target mycelial extension rate, hyphal coverage and fractal dimension. VOC-mediated effects were greater in self-pairing controls compared to interactions, and differed depending on the substratum in which the VOC-producing fungi were growing.     

Effects of temperature and relative humidity on Aflatoxin B1 reduction in corn grains and antagonistic activities against Aflatoxin-producing Aspergillus flavus by a volatile organic compound-producing yeast,Kwoniella heveanensis DMKU-CE82

As shown in our previous study, Kwoniella heveanensis DMKU-CE82, a volatile organic compound (VOC)-producing yeast, demonstrated promising antagonistic activity against aflatoxin-producing strain of Aspergillus flavus. This yeast’s volatile organic compounds (VOCs) could reduce Aflatoxin B1 (AFB1) in corn grains. In the current study, we evaluated the effect of temperatures and relative humidity on AFB1 reduction during grain storage when co-incubated with this VOC-producing yeast. The VOCs produced by K. heveanensis DMKU-CE82 could promote reduction of AFB1 to less than 20 part per billion (ppb) in the fungal contaminated corn grains under most storage conditions at 35 °C. The major VOCs produced by 2-, 4-, and 6-day-old yeast cultures were closely matched to 3-methyl-1-butanol, 2-methyl-1-butanol, hydrazine-1-1-dimethyl, and butanoic acid-3-methyl. In addition, this yeast strain had the ability to produce β-1,3-glucanase, amylase, cellulase, chitinase, siderophores, and biofilms. Scanning electron microscopy also confirmed the antagonistic activity of K. heveanensis DMKU-CE82 as it caused structural damage to conidia and inhibited the development of mycelia and conidiophores in both direct fungal–yeast interaction and the VOC method in corn grains. These results demonstrated that this yeast strain could be a promising biocontrol agent against aflatoxin-producing fungi in agricultural products.

     

A study on volatile organic compounds (VOCs) produced by tropical ascomycetous yeasts

As a part of a program aiming at the selection of strains which might be of interest as sources of natural flavouring molecules, the production of volatile organic compounds (VOCs) by 98 ascomycetous yeast strains (representative of 40 species belonging to 12 genera) isolated from tropical environments was investigated. Volatiles produced were sampled by means of headspace solid-phase microextraction (SPME) and the compounds were analysed and identified by gas chromatography–mass spectroscopy (GC–MS). The VOCs produced were found to be alcohols (amyl alcohol and isoamyl alcohol), aldehydes (2-methyl-2-hexenal and 2-isopropyl-5-methyl-2-hexenal) and esters (ethyl isobutyrate, isobutyl acetate, isoamyl acetate, 2-methylbutyl acetate, ethyl isovalerate, isoamyl propionate and phenylmethyl acetate). Differences in VOC profiles were used to cluster the yeast strains into 25 VOC phenotypes. The different frequency of VOC phenotypes in three specific habitats was correlated to the divergent environmental conditions, possibly affecting the selection of specific yeasts. From a biotechnological viewpoint, this study reveals the potentiality of ascomycetous yeasts isolated from tropical environments as a promising source of VOCs relevant in food and fragrance industry. This revised version was published online in June 2006 with corrections to the Cover Date.     

Parasitoids select plants more heavily infested with their caterpillar hosts: a new approach to aid interpretation of plant headspace volatiles

Plants produce volatile organic compounds (VOCs) in response to herbivore attack, and these VOCs can be used by parasitoids of the herbivore as host location cues. We investigated the behavioural responses of the parasitoid Cotesia vestalis to VOCs from a plant-herbivore complex consisting of cabbage plants (Brassica oleracea) and the parasitoids host caterpillar, Plutella xylostella. A Y-tube olfactometer was used to compare the parasitoids' responses to VOCs produced as a result of different levels of attack by the caterpillar and equivalent levels of mechanical damage. Headspace VOC production by these plant treatments was examined using gas chromatography-mass spectrometry. Cotesia vestalis were able to exploit quantitative and qualitative differences in volatile emissions, from the plant-herbivore complex, produced as a result of different numbers of herbivores feeding. Cotesia vestalis showed a preference for plants with more herbivores and herbivore damage, but did not distinguish between different levels of mechanical damage. Volatile profiles of plants with different levels of herbivores/herbivore damage could also be separated by canonical discriminant analyses. Analyses revealed a number of compounds whose emission increased significantly with herbivore load, and these VOCs may be particularly good indicators of herbivore number, as the parasitoid processes cues from its external environment.     

Odour of King Penguin feathers analysed using direct thermal desorption discriminates between individuals but not sexes

The role and use of olfactory cues by penguins is largely under‐investigated, with only a few studies suggesting that odours are involved in prey detection, orientation and for interspecific communication. This also applies to King Penguins Aptenodytes patagonicus where little is known about their chemoreception abilities and, subsequently, the role of odours in their behavioural ecology. Here, we investigated the chemical composition of volatile organic compounds (VOCs) from feathers of King Penguins in the Kerguelen Archipelago and their potential to carry information on identity and sex. We analysed VOCs using direct thermal desorption, a novel approach for extracting volatile compounds directly from solid matrices. We were only able to test at desorption temperatures of 70 and 100 °C to optimize conditions for VOC analysis. We found a profile of 26 VOCs present in most individuals, which varied significantly between individuals but not between sexes. Results suggested that VOCs could possibly be used by King Penguins to locate the colony and recognize individuals, if similar VOCs are also present at ambient conditions. Further studies and behavioural experiments are encouraged to explore olfactory‐based communication in this species.     

Performance and Microbial Diversity of a Trickle‐Bed Air Biofilter under Interchanging Contaminants

A trickle‐bed air biofilter (TBAB) was evaluated under conditions of interchanging the feed volatile organic compounds (VOCs) in the sequence methyl ethyl ketone (MEK), toluene, methyl isobutyl ketone (MIBK), styrene, and then back to MEK. The obtained performance results revealed that the biofilter provided high removal efficiency within the critical loading of each VOC, which was previously defined in the non‐interchanging VOC fed biofilter. The biofilter easily acclimated to the oxygenated compounds (MEK and MIBK), but re‐acclimation was delayed for the aromatic compounds (toluene and styrene). Ratios of the molar mass of CO₂ produced per molar mass of VOC removed were investigated. It has been found that the ratios for the aromatic compounds closely resembled the theoretical complete chemical oxidation based ratios while larger differences were encountered with the oxygenated compounds. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes was used to assess the impact of interchanging VOCs on the bacterial community structure in the biofilter. The results from denaturing gradient gel electrophoresis (DGGE) showed that the structure of the microbial community in the biofilter was different after each interchange of VOCs.     

Development of accurate classification method based on the analysis of volatile organic compounds from human exhaled air

Analysis of exhaled air leads to the development of fast accurate and non-invasive diagnostics. A comprehensive analysis of the entire range of volatile organic compounds (VOCs) in exhaled air samples will enable the identification of VOCs unique for certain patient groups. This study demonstrates proof of principle of our developed method tested on a smoking/non-smoking study population. Thermal desorption and gas chromatography coupled to time-of-flight mass spectrometry were used to analyse exhaled air samples. The VOC profiles obtained from each individual were combined into one final database based on similarity of mass spectra and retention indexes (RI), which offers the possibility for a reliable selection of compounds of interest. As proof of principle we correctly classified all subjects from population of smoking (N=11) and non-smoking (N=11) based on the VOC profiles available in their exhaled air. Support vector machine (SVM) analysis identified 4 VOCs as biomarkers of recent exposure to cigarette smoke: 2,5-dimethyl hexane, dodecane, 2,5-dimethylfuran and 2-methylfuran. This approach contributes to future development of fast, accurate and non-invasive diagnostics of inflammatory diseases including pulmonary diseases.     

The rapid evaluation of bacterial growth in blood cultures by selected ion flow tube-mass spectrometry (SIFT-MS) and comparison with the BacT/ALERT automated blood culture system

We compared the performance of the BacT/ALERT automated blood culture system with real-time, quantitative volatile organic compound (VOC) detection by selected ion flow tube-mass spectrometry (SIFT-MS). Blood samples from healthy donors were artificially infected with 5 or 100 CFU of organisms commonly causing bacteremia. Positive results by SIFT-MS analysis of headspace gases were recorded for 53/60 (88.3%) bottles at 8h, and 58/60 (96.6%) bottles at 24 h. We conclude that SIFT-MS is a sensitive method for the detection of microbial VOCs. Furthermore, profiles of the VOCs detected may allow simultaneous identification of infecting organisms.     

Headspace measurements of irradiated in vitro cultured cells using PTR-MS

A pilot study was performed to evaluate a new concept for a radiation biodosimetry method. Proton transfer reaction-mass spectrometry (PTR-MS) was used to find out whether radiation induces changes in the composition of volatile organic compounds (VOCs) in the headspace of in vitro cultured cells. Two different cell lines, retinal pigment epithelium cells hTERT-RPE1 and lung epithelium cells A-549, were irradiated with gamma radiation at doses of 4 Gy and 8 Gy. For measuring the cell-specific effects, the VOC concentrations in the headspace of flasks containing cells plus medium, as well as of flasks containing pure medium were analyzed for changes before and after irradiation. No significant radiation-induced alterations in VOC concentrations in the headspace could be observed after irradiation.     

Volatile organic compounds cytotoxicity and expression of HSP72, HSP90 and GRP78 stress proteins in cultured human cells

The aim of this study was to determine whether overexpression of stress proteins (SPs) could be a sensitive biomarker for cell injury due to exposure to low doses of volatile organic compounds (VOCs) such as benzene, ethylbenzene, toluene, xylene, and chlorinated derivatives (ClB). Sublethal and cytotoxic threshold concentrations of the VOCs were determined by studying the growth rate of normal (fibroblasts) or tumor-derived human cell lines (A549, HepG2) exposed for 4 days to VOCs. Changes in SP expression as a function of concentrations were investigated by Western blotting.VOC toxicity was found to be correlated with their degree of chlorination and their hydrophobicity. Cytotoxic threshold concentrations (no-observed effect concentration, NOEC) were found to be similar for the three cell lines. It was observed that using a mixture of VOCs, each of them at concentration below the NOEC, resulted in an actual toxicity to the cells. This finding reveals a synergistic effect and should be taken into account when assessing threshold risk and exposure limit values in the worker's environment when several pollutants may be present. HSP72 and HSP90 expression levels were not affected whereas GRP78 expression was increased by all the VOCs. Taking into account the specific molecular function of GRP78, it suggests that VOC exposure results in misfolded or underglycosylated protein accumulation in the endoplasmic reticulum. GRP78 overexpression was closely related to the magnitude of growth inhibition due to increasing concentrations of each VOC. The overexpression was found to be significant for concentrations 5 to 30 times higher than NOEC, indicating that, under our experimental conditions, GRP78 expression cannot be considered as a sensitive biomarker of exposure to environmental VOCs.     

Arthrobacter agilis UMCV2 induces iron acquisition in Medicago truncatula (strategy I plant) in vitro via dimethylhexadecylamine emission

Background and aims

Iron is an essential nutrient for plant growth. Although abundant in soil, iron is poorly available. Therefore, plants have evolved mechanisms for iron mobilization and uptake from the rhizospheric environment. In this study, we examined the physiological responses to iron deficiency in Medicago truncatula plants exposed to volatile organic compounds (VOCs) produced by Arthrobacter agilis UMCV2.

Methods

The VOC profiles of the plant and bacterium were determined separately and during interaction assays using gas chromatography. M. truncatula plants exposed to A. agilis VOCs and pure dimethylhexadecylamine were transferred to conditions of iron deficiency, and parameters associated with iron nutritional status were measured.

Results

The relative abundance of the bacterial VOC dimethylhexadecylamine increased 12-fold when in co-cultures of A. agilis and M. truncatula, compared to axenic cultures. Plants exposed to bacterial VOCs or dimethylhexadecylamine exhibited a higher rhizosphere acidification capacity, enhanced ferric reductase activity, higher biomass generation, and elevated chlorophyll and iron content relative to controls.

Conclusions

The VOCs emitted by A. agilis UMCV2 induce iron acquisition mechanisms in vitro in the Strategy I plant M. truncatula. Dimethylhexadecylamine is the signal molecule responsible for producing the beneficial effects.     

Induced defenses of Veronica spicata: Variability in herbivore-induced volatile organic compounds

Plants release volatile organic compounds (VOCs) that have many eco-physiological functions. Induction of plant VOCs is known to occur upon herbivory. Herbivore-induced VOCs are involved in the attraction of predators and parasitoids, a phenomenon known as an indirect defense of plants. We measured the VOC profiles of the wild species Veronica spicata with and without larval feeding and oviposition by the specialist butterfly Melitaea cinxia. V. spicata showed great plasticity when deploying indirect defences. The induction of several ubiquitous terpenoids and green leaf volatiles (GLVs) was associated with larval feeding, whereas the increase of two ketones, 6-methyl-5-hepten-2-one and t-geranylacetone and the suppression of GLVs were associated with oviposition by the butterfly.     

Petrol exhaust pollution impairs honey bee learning and memory

Volatile organic compounds (VOCs) serve as important infochemicals, mediating several ecological interactions including herbivory and pollination. Atmospheric pollutants including traffic‐related air pollution may impair the detection of VOCs used by insects in insect–plant interactions. We investigated the indirect effect of petrol exhaust pollution on olfactory learning and memory (short and long term) in honey bees. Using appetitive olfactory conditioning, we trained bees to learn one of four floral VOC profiles; linalool, dipentene, myrcene and geranium. VOCs were unpolluted or polluted with exhaust collected from a petrol generator. Exhaust emissions included concentrations of CO (246.07 + 17 ppm), NO (20.50 + 6.90 ppb) and NO2 (20.93 + 0.05 ppb) consistent with those typically encountered in urban areas and near roads. Once bees had learnt the training VOC, we tested whether they could recognise that VOC 1 h, 24 h and 48 h post‐training. Bees took significantly longer to learn polluted VOCs and forgot them faster than unpolluted ones. We also tested the ‘masking’ potential of pollution on floral VOCs. Using gas chromatography mass spectroscopy we noted differences in the chemical profile of polluted versus unpolluted VOCs and tested whether bees could recognise polluted VOCs if trained using unpolluted ones. For several VOCs tested, bees could distinguish between polluted and unpolluted VOCs. Ultimately, our results show that air pollution changes the recognition and retention of floral VOCs by bees which may consequently impact foraging efficiency.     

Defining adult asthma endotypes by clinical features and patterns of volatile organic compounds in exhaled air

Background

Several classifications of adult asthma patients using cluster analyses based on clinical and demographic information has resulted in clinical phenotypic clusters that do not address molecular mechanisms. Volatile organic compounds (VOC) in exhaled air are released during inflammation in response to oxidative stress as a result of activated leukocytes. VOC profiles in exhaled air could distinguish between asthma patients and healthy subjects. In this study, we aimed to classify new asthma endotypes by combining inflammatory mechanisms investigated by VOC profiles in exhaled air and clinical information of asthma patients.

Methods

Breath samples were analyzed for VOC profiles by gas chromatography–mass spectrometry from asthma patients (n = 195) and healthy controls (n = 40). A total of 945 determined compounds were subjected to discriminant analysis to find those that could discriminate healthy from asthmatic subjects. 2-step cluster analysis based on clinical information and VOCs in exhaled air were used to form asthma endotypes.

Results

We identified 16 VOCs, which could distinguish between healthy and asthma subjects with a sensitivity of 100% and a specificity of 91.1%. Cluster analysis based on VOCs in exhaled air and the clinical parameters FEV1, FEV1 change after 3 weeks of hospitalization, allergic sensitization, Junipers symptoms score and asthma medications resulted in the formation of 7 different asthma endotype clusters. We identified asthma clusters with different VOC profiles but similar clinical characteristics and endotypes with similar VOC profiles, but distinct clinical characteristics.

Conclusion

This study demonstrates that both, clinical presentation of asthma and inflammatory mechanisms in the airways should be considered for classification of asthma subtypes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0136-8) contains supplementary material, which is available to authorized users.