Molecular variation among virulent and avirulent strains of the quarantine nematode Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is an emerging pathogenic nematode that is responsible for a devastating epidemic of pine wilt disease worldwide, causing severe ecological damage and economic losses to forestry. Two forms of this nematode have been reported, i.e., with strong and weak virulence, commonly referred as virulent and avirulent strains. However, the pathogenicity-related genes of B. xylophilus are not sufficiently characterized. In this study, to find pathogenesis related genes we re-sequenced and compared genomes of two virulent and two avirulent populations. We identified genes affected by genomic variation, and functional annotation of those genes indicated that some of them might play potential roles in pathogenesis. The performed analysis showed that both avirulent populations differed from the virulent ones by 1576 genes with high impact variants. Demonstration of genetic differences between virulent and avirulent strains will provide effective methods to distinguish these two nematode virulence forms at the molecular level. The reported results provide basic information that can facilitate development of a better diagnosis for B. xylophilus isolates/strains which present different levels of virulence and better understanding of the molecular mechanism involved in the development of the PWD.

First record and description of juvenile stages of Longidorus artemisiae Rubtsova, Chizhov & Subbotin, 1999 (Nematoda: Longidoridae) in Poland and new data on L. juglandicola Lisková, Robbins & Brown, 1997 based on topotype specimens from Slovakia.

This paper presents the first geographical record of the needle nematode Longidorus artemisiae Rubtsova, Chizhov & Subbotin, 1999 outside Russia. This species was found in Poland near the city of Skierniewice in association with nettle (Urtica dioica L.). Morphometric and morphological data are provided, including the first description of juveniles of this species. Nematodes of the Polish population differ from the type-population in Russia in possessing a thicker body (lower 'a' index) in both sexes; males having a longer body and longer spicules; different sex ratio (1:2 in Polish population vs 1:1 in the type-population) and somewhat less expanded lips. Molecularly, the Polish population was characterised by sequencing D2-D3 28S rDNA and ITS1 markers. Additionally, new data on these two markers are provided for another species, Longidorus juglandicola Lisková, Robbins & Brown, 1997, obtained from topotype specimens from Slovakia. Surprisingly, despite the high morphological similarity of these two species, analysis of their phylogenetic position did not show close phylogenetic relation and several other species (less similar in general morphology) appeared more closely related to both L. artemisiae and L. juglandicola.

A Compendium of Volatile Organic Compounds (VOCs) Released By Human Cell Lines.

Volatile organic compounds (VOCs) offer unique insights into ongoing biochemical processes in healthy and diseased humans. Yet, their diagnostic use is hampered by the limited understanding of their biochemical or cellular origin and their frequently unclear link to the underlying diseases. Major advancements are expected from the analyses of human primary cells, cell lines and cultures of microorganisms. In this review, a database of 125 reliably identified VOCs previously reported for human healthy and diseased cells was assembled and their potential origin is discussed. The majority of them have also been observed in studies with other human matrices (breath, urine, saliva, feces, blood, skin emanations). Moreover, continuing improvements of qualitative and quantitative analyses, based on the recommendations of the ISO-11843 guidelines, are suggested for the necessary standardization of analytical procedures and better comparability of results. The data provided contribute to arriving at a more complete human volatilome and suggest potential volatile biomarkers for future validation. Dedication:This review is dedicated to the memory of Prof. Dr. Anton Amann, who sadly passed away on January 6, 2015. He was motivator and motor for the field of breath research.

The use of real-time polymerase chain reaction with high resolution melting (real-time PCR-HRM) analysis for the detection and discrimination of nematodes Bursaphelenchus xylophilus and Bursaphelenchus mucronatus.

The real-time PCR-HRM analysis was developed for the detection and discrimination of the quarantine nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus. A set of primers was designed to target the ITS region of rDNA. The results have demonstrated that this analysis is a valuable tool for differentiation of these both species.

Breath analysis for in vivo detection of pathogens related to ventilator-associated pneumonia in intensive care patients: a prospective pilot study.

Existing methods for the early detection of infections in mechanically ventilated (MV) patients at intensive care units (ICUs) are unsatisfactory. Here we present an exploratory study assessing the feasibility of breath VOC analyses for the non-invasive detection of pathogens in the lower respiratory tract of ventilated patients. An open uncontrolled clinical pilot study was performed by enrolling 28 mechanically ventilated (MV) patients with severe intracranial disease, being at risk for the development of or already with confirmed ventilation-associated pneumonia (VAP). The recently developed sampling technique enabled the collection of breath gas with a maximized contribution of alveolar air directly from the respiratory circuit under continuous capnography control, adsorptive preconcentration and final analysis by means of gas chromatography-mass spectrometry (GC-MS).VAP was confirmed in 22/28 preselected patients (78%). The most common microorganisms were Staphylococcus aureus (5/22 VAP patients), Escherichia coli (5/22 VAP patients) and Candida spp. (5/22 VAP patients). 12/32 metabolites released by S. aureus in our previous in vitro studies were also detected in the end-tidal air of VAP patients infected with this pathogen. A similar overlap was seen in Candida albicans infections (8/29 VOCs). Moreover, the concentration profile of selected compounds correlated with the course of the infection.This prospective pilot study provides proof of the concept that the appearance and the concentration profile of pathogen-derived metabolites (elucidated from in vitro experiments) in the breath of ventilated patients during clinically confirmed VAP correlates with the presence of a particular pathogen.

Comparative analyses of volatile organic compounds (VOCs) from patients, tumors and transformed cell lines for the validation of lung cancer-derived breath markers.

Breath analysis for the purpose of non-invasive diagnosis of lung cancer has yielded numerous candidate compounds with still questionable clinical relevance. To arrive at suitable volatile organic compounds our approach combined the analysis of different sources: isolated tumor samples compared to healthy lung tissues, and exhaled breath from lung cancer patients and healthy controls. Candidate compounds were further compared to substances previously identified in the comparison of transformed and normal lung epithelial cell lines. For human studies, a breath sampling device was developed enabling automated and CO2-controlled collection of the end-tidal air. All samples were first preconcentrated on multibed sorption tubes and analyzed with gas chromatography mass spectrometry (GC-MS). Significantly (p < 0.05) higher concentrations in all three types of cancer samples studied were observed for ethanol and n-octane. Additional metabolites (inter alia 2-methylpentane, n-hexane) significantly released by lung cancer cells were observed at higher levels in cancer lung tissues and breath samples (compared to respective healthy controls) with statistical significance (p < 0.05) only in breath samples. The results obtained confirmed the cancer-related origin of volatile metabolites, e.g. ethanol and octane that were both detected at significantly (p < 0.05) elevated concentrations in all three kinds of cancer samples studied. This work is an important step towards identification of volatile breath markers of lung cancer through the demonstration of cancer-related origin of certain volatile metabolites.

Characterization of volatile metabolites taken up by or released from Streptococcus pneumoniae and Haemophilus influenzae by using GC-MS.

Volatile organic compounds (VOCs) released from or taken up by Streptococcus pneumoniae and Haemophilus influenzae cultures were analysed by means of GC-MS after adsorption of headspace samples on multi-bed sorption tubes. Sampling was performed at different time points during cultivation of bacteria to follow the dynamics of VOC metabolism. VOCs were identified not only by spectral library match but also based on retention times of native standards. As many as 34 volatile metabolites were released from S. pneumoniae and 28 from H. influenzae, comprising alcohols, aldehydes, esters, hydrocarbons, ketones and sulfur-containing compounds. For both species, acetic acid, acetaldehyde, methyl methacrylate, 2,3-butanedione and methanethiol were found in strongly elevated concentrations and 1-butanol and butanal in moderately elevated concentrations. In addition, characteristic volatile biomarkers were detected for both bacterial species and exclusively for S. pneumoniae, also catabolism of aldehydes (3-methylbutanal and hexanal) was found. The results obtained provide important input into the knowledge about volatile bacterial biomarkers, which may become particularly important for detection of pathogens in upper airways by breath-gas analysis in the future.

Molecular analysis of volatile metabolites released specifically by Staphylococcus aureus and Pseudomonas aeruginosa.

BACKGROUND: The routinely used microbiological diagnosis of ventilator associated pneumonia (VAP) is time consuming and often requires invasive methods for collection of human specimens (e.g. bronchoscopy). Therefore, it is of utmost interest to develop a non-invasive method for the early detection of bacterial infection in ventilated patients, preferably allowing the identification of the specific pathogens. The present work is an attempt to identify pathogen-derived volatile biomarkers in breath that can be used for early and non- invasive diagnosis of ventilator associated pneumonia (VAP). For this purpose, in vitro experiments with bacteria most frequently found in VAP patients, i.e. Staphylococcus aureus and Pseudomonas aeruginosa, were performed to investigate the release or consumption of volatile organic compounds (VOCs). RESULTS: Headspace samples were collected and preconcentrated on multibed sorption tubes at different time points and subsequently analyzed with gas chromatography mass spectrometry (GC-MS). As many as 32 and 37 volatile metabolites were released by S. aureus and P. aeruginosa, respectively. Distinct differences in the bacteria-specific VOC profiles were found, especially with regard to aldehydes (e.g. acetaldehyde, 3-methylbutanal), which were taken up only by P. aeruginosa but released by S. aureus. Differences in concentration profiles were also found for acids (e.g. isovaleric acid), ketones (e.g. acetoin, 2-nonanone), hydrocarbons (e.g. 2-butene, 1,10-undecadiene), alcohols (e.g. 2-methyl-1-propanol, 2-butanol), esters (e.g. ethyl formate, methyl 2-methylbutyrate), volatile sulfur compounds (VSCs, e.g. dimethylsulfide) and volatile nitrogen compounds (VNCs, e.g. 3-methylpyrrole).Importantly, a significant VOC release was found already 1.5 hours after culture start, corresponding to cell numbers of ~8*106 [CFUs/ml]. CONCLUSIONS: The results obtained provide strong evidence that the detection and perhaps even identification of bacteria could be achieved by determination of characteristic volatile metabolites, supporting the clinical use of breath-gas analysis as non-invasive method for early detection of bacterial lung infections.

Optimization of sampling parameters for collection and preconcentration of alveolar air by needle traps.

The approach for breath-VOCs' collection and preconcentration by applying needle traps was developed and optimized. The alveolar air was collected from only a few exhalations under visual control of expired CO(2) into a large gas-tight glass syringe and then warmed up to 45 °C for a short time to avoid condensation. Subsequently, a specially constructed sampling device equipped with Bronkhorst® electronic flow controllers was used for automated adsorption. This sampling device allows time-saving collection of expired/inspired air in parallel onto three different needle traps as well as improvement of sensitivity and reproducibility of NT-GC-MS analysis by collection of relatively large (up to 150 ml) volume of exhaled breath. It was shown that the collection of alveolar air derived from only a few exhalations into a large syringe followed by automated adsorption on needle traps yields better results than manual sorption by up/down cycles with a 1 ml syringe, mostly due to avoided condensation and electronically controlled stable sample flow rate. The optimal profile and composition of needle traps consists of 2 cm Carbopack X and 1 cm Carboxen 1000, allowing highly efficient VOCs' enrichment, while injection by a fast expansive flow technique requires no modifications in instrumentation and fully automated GC-MS analysis can be performed with a commercially available autosampler. This optimized analytical procedure considerably facilitates the collection and enrichment of alveolar air, and is therefore suitable for application at the bedside of critically ill patients in an intensive care unit. Due to its simplicity it can replace the time-consuming sampling of sufficient breath volume by numerous up/down cycles with a 1 ml syringe.

Link between defoliation and light treatments on root vitality of five understory shrubs with different resistance to insect herbivory.

Understory shrubs are frequently attacked by insect herbivores. However, very little is known regarding possible interactions between light condition, defoliation (D) and fine root vitality (% live roots) and metabolic activity, and whether different plant strategies (compensation, trade-off and equilibrium) to defoliation depend on individual species light requirements. To explore the response of roots to such conditions, an experiment was established in which we experimentally removed 50% of leaves in 1-year-old seedlings of Sambucus nigra, Cornus sanguinea, Prunus serotina, Frangula alnus and Corylus avellana grown in 15% and full sunlight. On average, defoliation leads to a 15% reduction in fine root (< 2 mm) vitality (% live roots). However, a statistically significant reduction in root vitality after defoliation was detected only in those species that are less herbivorized in nature (48% in S. nigra and 5% in C. sanguinea). On average, shade conditions (L) resulted in 18% decline in root vitality, and the effects of defoliation were also 22% higher than for plants grown in full light. Root vitality in both treatments (D and L) was significantly correlated with their dry mass, concentration of total phenol (TPh) and carbon to nitrogen ratio, and negatively correlated with nitrogen, soluble carbohydrates, starch and total non-structural carbohydrates (TNC). To a large extent, root vitality and chemistry varied by species. Higher root vitality was related to higher concentrations of phenolics, more than to N and TNC concentrations. Concentrations of phenolics also differed significantly between defoliated plants and controls. However, in defoliated plants, an increase in TPh was observed only in two species, which belong to two different groups in light requirements and susceptibility to insect grazing (C. sanguinea and P. serotina). This study indicated that higher vitality of roots occurred in species that are characterized by higher insect defoliation under natural conditions. It is likely that higher root vitality of these species was related to their high level of TPh and tannins. This was especially noticeable for the reduced light treatment, which represents natural conditions under which insect defoliation is highest. Our results suggest that varied strategies of resource allocation were used by the different species in response to variations in light and defoliation.

TD-GC-MS analysis of volatile metabolites of human lung cancer and normal cells in vitro.

The aim of this study was to confirm the existence of volatile organic compounds (VOC) specifically released or consumed by the lung cancer cell line A549, which could be used in future screens as biomarkers for the early detection of lung cancer. For comparison, primary human bronchial epithelial cells (HBEpC) and human fibroblasts (hFB) were included. VOCs were detected in the headspace of cell cultures or medium controls following adsorption on solid sorbents, thermodesorption, and analysis by gas chromatography mass spectrometry. Using this approach, we identified VOCs that behaved similarly in normal and transformed cells. Thus, concentrations of 2-pentanone and 2,4-dimethyl-1-heptene were found to increase in the headspace of A549, HBEpC, and hFB cell cultures. In addition, the ethers methyl tert-butyl ether and ethyl tert-butyl ether could be detected at elevated levels in the case of A549 cells and one of the untransformed cell lines. However, especially branched hydrocarbons and alcohols were seen increased more frequently in untransformed than A549 cells. A big variety of predominantly aldehydes and the ester n-butyl acetate were found at decreased concentrations in the headspace of all cell lines tested compared with medium controls. Again, more different aldehydes were found to be decreased in hFB and HBEpC cells compared with A549 cells and 2-butenal was metabolized exclusively by both control cell lines. These data suggest that certain groups of VOCs may be preferentially associated with the transformed phenotype.

Isolation, purification and determination of 4-n-nonylphenol and 4-tert-octylphenol in aqueous and biological samples.

The aim of the present study was to attempt to describe the procedure of isolation, purification, enrichment and determination of 4-n-nonylphenol (4-n-NP) and 4-tert-octylphenol (4-t-OP) in water and biological samples (fish tissue). There were five procedures of solid phase extraction (SPE) tested using different sorbents for the isolation of analytes from water samples. Moreover, we isolated these chemicals from biological matrices with the aid of various extraction methods. The purpose of it was to perform an optimisation of ultrasonic bath, accelerated solvent extraction (ASE) and solid phase extraction process of alkylphenols from biological samples, through the choice of selective sorbents (octadecyl, octadecyl end-capped and amine) and search solvents (methylene chloride, methanol, hexane). Reversed-phase HPLC with diode array detection was used for the determination of 4-n-NP and 4-t-OP in water and fish tissue samples. Sensitivity was evaluated by determining the limit of detection (LOD=0.06 and 0.04ng microL(-1)) and limit of quantification (LOQ=0.18 and 0.16ng microL(-1)) of 4-NP and 4-t-OP, respectively. A series of standard solutions for 4-n-NP and 4-t-OP provided the basis for plotting an analytical curve and obtaining a linear dependence in the range of approximately 1-25ng microL(-1). The best efficiencies obtained for 4-n-NP and 4-t-OP in water samples were 76.65% (+/-1.49) and 83.08% (+/-3.73), respectively. In the case of fish tissue, different situation was observed because the obtained values were considerably lower, being 68.32% for 4-t-OP using hexane (program 1) as solvent and 72.35% (program 2) for 4-n-NP using acetonitrile.