The effect of chlorophyll on the enzyme-linked immunosorbent assay (ELISA) of procymidone in vegetables and the way to overcome the matrix interference.

BACKGROUND: There is now an increasing demand for the immunoassay of procymidone residue in foodstuffs. However, the matrix interference could significantly affect the analysis. Till now there is no detailed information on the source of the interference and the mechanism involved, which greatly limits the real application of these techniques. RESULTS: Significant matrix effect was observed in the enzyme-linked immunosorbent assay (ELISA) of procymidone in negative vegetable samples (leek, broccoli and cucumber). By the investigation with both vegetable extracts and standard solutions, the chlorophyll was confirmed as an important source of the matrix effect. Therefore, a new strategy was proposed for the pretreatment based on the exploitation of 5-sulfosalicylic acid. It was demonstrated to effectively eliminate chlorophyll and exhibited little effect on procymidone and the competitive indirect ELISA (ci-ELISA) performance. The established technique was validated with different vegetables. With the spiking concentration of procymidone investigated, the recovery rate of ci-ELISA was 71.52-120.37%, and the relative standard deviation was 4.05-17.61%. CONCLUSION: Chlorophyll was for the first time illuminated as an important source of matrix interference to the immunoassay of procymidone in vegetables. A new pretreatment based on 5-sulfosalicylic acid was established to remove chlorophyll and therefore eliminate the matrix effect. Validated with different vegetable samples, the new technique was demonstrated much better efficiency in comparison to conventional methods, which indicated its promising application for the development of immunoassays of herb-origin samples. © 2021 Society of Chemical Industry.

Schinus molle essential oil as a potential source of bioactive compounds: antifungal and antibacterial properties.

AIMS: The study was focused on the evaluation of antimicrobial activity in vitro of the essential oil (EO) of leaves from Schinus molle against bacteria and fungi of clinical importance in the search for the discovery of new active compounds. METHODS AND RESULTS: The chemical composition of the S. molle EO was determined by gas chromatography/mass spectrometry and its antimicrobial effect was verified by broth microdilution method. The major compounds found were ß-pinene (25·23%), epi-α-cadinol (21·29%), α-pinene (18·72%), myrcene (11·54%) and sabinene (5·02%). The EO showed significant antifungal activity against Paracoccidioides brasiliensis (39·06 µg ml-1 ), weak action against Cryptococcus neoformans (625 µg ml-1 ) and Trichophyton quinckeanum (625 µg ml-1 ) and was inactive against Candida sp. In the analysis of the antibacterial action, the micro-organisms tested did not show sensitivity. CONCLUSIONS: This study showed a promising result of S. molle volatiles against the fungus P. brasiliensis, which causes paracoccidioidomycosis (PCM), a systemic mycosis of great clinical importance in Latin America. SIGNIFICANCE AND IMPACT OF THE STUDY: The results found here are novel and encourage investigations of the compounds present in this EO, which represents a source of molecules with potential use in the treatment of PCM.

Piper nigrum Oil - Determination of Selected Terpenes for Quality Evaluation.

The growing demand and commercial value of black pepper (Piper nigrum) has resulted in considerable interest in developing suitable and cost-effective methods for chemical characterization and quality evaluation purposes. In the current study, an extensive set of oil samples (n = 23) that were extracted by steam distillation from black pepper seeds was investigated to compare the chemical profiles of samples originating from nine major producing countries, as well as to identify potential chemical markers for quality evaluation. The twenty-two most abundant volatile compounds, mainly terpenes, in these oils were determined by conventional GC/MS analysis. Principal component analysis with this set of data revealed distinct clusters for samples that originated from China and Malaysia. Relatively low concentrations of sabinene (< 0.2%) and high concentrations of 3-carene (10.9 - 21.1%) were observed in these samples, respectively, compared to oil samples from other countries. The enantiomeric distributions of key terpene markers, viz., ß-pinene, sabinene, limonene, and terpinen-4-ol, were determined by chiral GC/MS analysis. Interestingly, for these four monoterpenes, levo-isomers were found to be predominant, emphasizing the highly conserved enzymatic processes occurring in P. nigrum. Moreover, consistent enantiomeric ratios ((-) isomer/(+) isomer) of 92.2 ± 3.0% for ß-pinene, 94.8 ± 2.8% for sabinene, 60.7 ± 1.1% for limonene, and 78.3 ± 1.3% for terpinen-4-ol were observed, independent of geographical location. These results demonstrate the potential of using stereospecific compositions as chiral signatures for establishing the authenticity and quality of black pepper oil.

The repellency and toxicity effects of essential oils from the Libyan plants Salvadora persica and Rosmarinus officinalis against nymphs of Ixodes ricinus.

Essential oils extracted from the leaves of Libyan Rosemary (Rosmarinus officinalis L.), and Miswak (Salvadora persica L.) were evaluated for their acaricidal and repellent effects on Ixodes ricinus L. nymphs (Acari: Ixodidae) using a bioassay based on an 'open filter paper method'. Rosmarinus officinalis leaf essential oil diluted to 0.5 and 1 µl/cm2 in acetone exhibited, respectively, 20 and 100% tick mortality after about 5 h of exposure. A total of 50 and 95% of I. ricinus nymphs were killed by direct contact with the oil when exposed to lethal concentrations (LC) of 0.7 µl/cm2 (LC50) and 0.95 µl/cm2 (LC95), respectively. The LC50 (0.5 µl/cm2) was reached before the end of the first 24 h of exposure time (ET), as tick mortality at 24 h was 60%. Salvadora persica leaf essential oil at 1 µl/cm2 showed a significant repellency effect against I. ricinus nymphs at 1.5 h ET. A 95% repellency was observed at a repellent concentration (RC95) of 1 µl/cm2 of S. persica, but no significant mortality was recorded at this dose of S. persica oil. Gas chromatography-mass spectrometry analyses showed that the main monoterpenes in both oils were 1,8-cineol, α-pinene, and ß-pinene, although in markedly different proportions. These results suggest that essential oils have substantial potential as alternative approaches for I. ricinus tick control.

Chemical Composition and Biological Activity of Five Essential Oils from the Ecuadorian Amazon Rain Forest.

The chemical composition and biological activity of essential oils isolated from the leaves of Siparuna aspera, Siparuna macrotepala, Piper leticianum, Piper augustum and the rhizome of Hedychium coronarium were evaluated. These species are used medicinally in different ways by the Amazonian communities that live near the Kutukú mountain range. Chemical studies revealed that the main components for the two Siparuna species were germacrene D, bicyclogermacrene, α-pinene, δ-cadinene, δ-elemene, α-copaene and ß-caryophyllene; for the two Piper species ß-caryophyllene, germacrene D, α-(E,E)-farnesene, ß-elemene, bicyclogermacrene, δ-cadinene and for H. coronarium 1,8-cineole, ß-pinene, α-pinene and α-terpineol. The antioxidant activity of all essential oils was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), photochemiluminescence (PCL) quantitative assays, and DPPH and ABTS bioautographic profiles, with different results for each of them. Antimicrobial activity studies were carried out on three yeasts, six Gram positive and four Gram negative bacteria, by means of the disc diffusion method. The essential oil of H. coronarium showed the most relevant results on L. grayi, K. oxytoca and S. mutans, P. augustum and P. leticianum on S. mutans. An antibacterial bioautographic test for H. coronarium was also carried out and highlighted the potential activity of terpinen-4-ol and 1,8-cineole.

Accurate and Sensitive Determination Method for Procymidone and Chlorflurenol in Municipal Wastewater, Medical Wastewater and Irrigation Canal Water by GC-MS After Vortex Assisted Switchable Solvent Liquid Phase Microextraction.

In this study, the detection power of a gas chromatography mass spectrometer (GC-MS) for procymidone and chlorflurenol was significantly enhanced using switchable solvent liquid phase microextraction (SS-LPME) as a preconcentration tool. This was achieved by a comprehensive optimization of significant parameters to the SS-LPME method such as switchable solvent amount, concentration and amount of sodium hydroxide, pH effect and mixing effect. The optimum experimental conditions obtained were used to determine analytical figures of merit for the analytes. The limits of detection obtained were 0.44 and 2.9 ng/mL for procymidone and chlorflurenol, respectively. The optimum method was applied to water sampled from an irrigation canal and two wastewater samples. The samples were spiked at two concentrations and the percent recovery results obtained ranged between 86 and 115% for both analytes. The recovery results together with the low standard deviations recorded validated the method as accurate and precise.

Morphophysiological and Phytochemical Variability in Some Wild Populations of Ducrosia anethifolia from Iran.

In order to determine the morphophysiological and phytochemical properties of various Ducrosia anethifolia populations, the plant samples were collected from 20 locations in native regions. Current study indicated significant differences in the morphophysiological and phytochemical characteristics of D. anethifolia populations collected from 20 locations in Sistan and Baluchestan Province, Iran. The highest value of plant height and the number of lateral stems, node per plant, umbellate per umbel, seeds per umbellate and the roots fresh and dry weight were related to the location with relatively high rainfall (130-161 mm) and low altitude (up to 1165 m) compared with others. Based on the essential oil components, D. anethifolia populations were divided into five different chemotypes. Chemotypes I, II and III were characterized by high amounts of methyl chavicol, chrysanthenyl acetate and decanal, respectively. Moreover, the populations with high amounts of decanal, anethole and dodecanal were placed in chemotype IV. Chemotype V was attributed to the Naserabad population with 1-decanol as the major compound.

Simultaneous determination of iprodione, procymidone, and chlorflurenol in lake water and wastewater matrices by GC-MS after multivariate optimization of binary dispersive liquid-liquid microextraction.

This study reports the optimization of a binary dispersive liquid-liquid microextraction method for the determination of iprodione, procymidone, and chlorflurenol by gas chromatography mass spectrometry. The study was aimed at using two extraction solvents to increase the extraction efficiency of all analytes. The binary solvents recorded results higher than the mono-solvents. After examining the effects of main experimental parameters and their interactions by analysis of variance, 200 µL of binary mixture (dichloromethane and 1,2-dichloroethane), 2.5 mL of ethanol, and 15 s vortex were obtained as optimum parameters. The detection and quantification limits calculated for the analytes were found to be between 0.30-1.6 and 1.0-5.3 ng/mL, respectively. Enhancement in detection power calculated as a ratio of the binary extraction detection limit to the detection limit of direct GC-MS analysis was 105-, 214-, and 233-fold for chlorflurenol, iprodione, and procymidone, respectively. In order to check the accuracy of the developed method, recovery study was performed. Water sampled from a lake and two wastewater samples from treatment facilities were spiked at two concentrations, and the percent recovery calculated for the samples ranged between 87 and 116%. These results confirmed the suitability of the method to real samples for accurate determination of the analytes at trace levels.

Abies Concolor Seeds and Cones as New Source of Essential Oils-Composition and Biological Activity.

The chemical composition, including the enantiomeric excess of the main terpenes, of essential oils from seeds and cones of Abies concolor was studied by chromatographic (GC) and spectroscopic methods (mass spectrometry, nuclear magnetic resonance), leading to the determination of 98 compounds. Essential oils were mainly composed of monoterpene hydrocarbons. The dominant volatiles of seed essential oil were: limonene (47 g/100 g, almost pure levorotary form) and α-pinene (40 g/100 g), while α-pinene (58 g/100 g), sabinene (11 g/100 g), and ß-pinene (4.5 g/100 g) were the predominant components of the cone oil. The seed and cone essential oils exhibited mild antibacterial activity, and the MIC ranged from 26 to 30 µL/mL against all of the tested bacterial standard strains: Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, and Klebsiella pneumoniae. The cytotoxic studies have demonstrated that tested essential oils were cytotoxic to human skin fibroblasts and human microvascular endothelial cells at concentrations much lower than the MIC. The essential oils from A. concolor seeds and cones had no toxic effect on human skin fibroblasts and human microvascular endothelial cells, when added to the cells at a low concentration (0-0.075 µL/mL) and (0-1.0 µL/mL), respectively, and cultured for 24 h.

Chemical composition, toxicity and non-target effects of Pinus kesiya essential oil: An eco-friendly and novel larvicide against malaria, dengue and lymphatic filariasis mosquito vectors.

Mosquitoes (Diptera: Culicidae) are vectors of important parasites and pathogens causing death, poverty and social disability worldwide, with special reference to tropical and subtropical countries. The overuse of synthetic insecticides to control mosquito vectors lead to resistance, adverse environmental effects and high operational costs. Therefore, the development of eco-friendly control tools is an important public health challenge. In this study, the mosquito larvicidal activity of Pinus kesiya leaf essential oil (EO) was evaluated against the malaria vector Anopheles stephensi, the dengue vector Aedes aegypti and the lymphatic filariasis vector Culex quinquefasciatus. The chemical composition of the EO was analyzed by gas chromatography-mass spectroscopy. GC-MS revealed that the P. kesiya EO contained 18 compounds. Major constituents were α-pinene, ß-pinene, myrcene and germacrene D. In acute toxicity assays, the EO showed significant toxicity against early third-stage larvae of An. stephensi, Ae. aegypti and Cx. quinquefasciatus, with LC50 values of 52, 57, and 62µg/ml, respectively. Notably, the EO was safer towards several aquatic non-target organisms Anisops bouvieri, Diplonychus indicus and Gambusia affinis, with LC50 values ranging from 4135 to 8390µg/ml. Overall, this research adds basic knowledge to develop newer and safer natural larvicides from Pinaceae plants against malaria, dengue and filariasis mosquito vectors.

Essential-Oil Variability in Natural Populations of Pinus mugo Turra from the Julian Alps.

The composition and variability of the terpenes and their derivatives isolated from the needles of a representative pool of 114 adult trees originating from four natural populations of dwarf mountain pine (Pinus mugo Turra) from the Julian Alps were investigated by GC-FID and GC/MS analyses. In total, 54 of the 57 detected essential-oil components were identified. Among the different compound classes present in the essential oils, the chief constituents belonged to the monoterpenes, comprising an average content of 79.67% of the total oil composition (74.80% of monoterpene hydrocarbons and 4.87% of oxygenated monoterpenes). Sesquiterpenes were present in smaller amounts (average content of 19.02%), out of which 16.39% were sesquiterpene hydrocarbons and 2.62% oxygenated sesquiterpenes. The most abundant components in the needle essential oils were the monoterpenes δ-car-3-ene, ß-phellandrene, α-pinene, ß-myrcene, and ß-pinene and the sesquiterpene ß-caryophyllene. From the total data set of 57 detected compounds, 40 were selected for principal-component analysis (PCA), discriminant analysis (DA), and cluster analysis (CA). The overlap tendency of the four populations suggested by PCA, was as well observed by DA. CA also demonstrated similarity among the populations, which was the highest between Populations I and II.

Organic aerosol molecular composition and gas-particle partitioning coefficients at a Mediterranean site (Corsica).

Molecular speciation of atmospheric organic matter was investigated during a short summer field campaign performed in a citrus fruit field in northern Corsica (June 2011). Aimed at assessing the performance on the field of newly developed analytical protocols, this work focuses on the molecular composition of both gas and particulate phases and provides an insight into partitioning behavior of the semi-volatile oxygenated fraction. Limonene ozonolysis tracers were specifically searched for, according to gas chromatography-mass spectrometry (GC-MS) data previously recorded for smog chamber experiments. A screening of other oxygenated species present in the field atmosphere was also performed. About sixty polar molecules were positively or tentatively identified in gas and/or particle phases. These molecules comprise a wide range of branched and linear, mono and di-carbonyls (C3-C7), mono and di-carboxylic acids (C3-C18), and compounds bearing up to three functionalities. Among these compounds, some can be specifically attributed to limonene oxidation and others can be related to α- or ß-pinene oxidation. This provides an original snapshot of the organic matter composition at a Mediterranean site in summer. Furthermore, for compounds identified and quantified in both gaseous and particulate phases, an experimental gas/particle partitioning coefficient was determined. Several volatile products, which are not expected in the particulate phase assuming thermodynamic equilibrium, were nonetheless present in significant concentrations. Hypotheses are proposed to explain these observations, such as the possible aerosol viscosity that could hinder the theoretical equilibrium to be rapidly reached.

Monoterpene separation by coupling proton transfer reaction time-of-flight mass spectrometry with fastGC.

Proton transfer reaction mass spectrometry (PTR-MS) is a well-established technique for real-time analysis of volatile organic compounds (VOCs). Although it is extremely sensitive (with sensitivities of up to 4500 cps/ppbv, limits of detection <1 pptv and the response times of approximately 100 ms), the selectivity of PTR-MS is still somewhat limited, as isomers cannot be separated. Recently, selectivity-enhancing measures, such as manipulation of drift tube parameters (reduced electric field strength) and using primary ions other than H3O(+), such as NO(+) and O2 (+), have been introduced. However, monoterpenes, which belong to the most important plant VOCs, still cannot be distinguished so more traditional technologies, such as gas chromatography mass spectrometry (GC-MS), have to be utilised. GC-MS is very time consuming (up to 1 h) and cannot be used for real-time analysis. Here, we introduce a sensitive, near-to-real-time method for plant monoterpene research-PTR-MS coupled with fastGC. We successfully separated and identified six of the most abundant monoterpenes in plant studies (α- and ß-pinenes, limonene, 3-carene, camphene and myrcene) in less than 80 s, using both standards and conifer branch enclosures (Norway spruce, Scots pine and black pine). Five monoterpenes usually present in Norway spruce samples with a high abundance were separated even when the compound concentrations were diluted to 20 ppbv. Thus, fastGC-PTR-ToF-MS was shown to be an adequate one-instrument solution for plant monoterpene research.

Impact of gas chromatography and mass spectrometry combined with gas chromatography and olfactometry for the sex differentiation of Baccharis articulata by the analysis of volatile compounds.

The Baccharis genus has more than 400 species of aromatic plants. However, only approximately 50 species have been studied in oil composition to date. From these studies, very few take into consideration differences between male and female plants, which is a significant and distinctive factor in Baccharis in the Asteraceae family. Baccharis articulata is a common shrub that grows wild in south Brazil, northern and central Argentina, Bolivia, Paraguay and Uruguay. It is considered to be a medicinal plant and is employed in traditional medicine. We report B. articulata male and female volatile composition obtained by simultaneous distillation-extraction technique and analyzed by gas chromatography with mass spectrometry. Also, an assessment of aromatic differences between volatile extracts was evaluated by gas chromatography with olfactometry. The results show a very similar chemical composition between male and female extracts, with a high proportion of terpene compounds of which ß-pinene, limonene and germacrene D are the main components. Despite the chemical similarity, great differences in aromatic profile were found: male plant samples exhibited the strongest odorants in number and intensity of aromatic attributes. These differences explain field observations which indicate differences between male and female flower aroma, and might be of ecological significance in the attraction of pollinating insects.

Volatiles as Chemosystematic Markers for Distinguishing Closely Related Species within the Pinus mugo Complex.

Headspace solid-phase microextraction (HS-SPME) coupled to GC/MS analysis was used to identify the constituents of pine-needle volatiles differentiating three closely-related pine species within the Pinus mugo complex, i.e., P. uncinata Ramond ex DC., P. uliginosa G.E.Neumann ex Wimm., and P. mugo Turra. Moreover, chemosystematic markers were proposed for the three analyzed pine species. The major constituents of the pine-needle volatiles were α-pinene (28.4%) and bornyl acetate (10.8%) for P. uncinata, δ-car-3-ene (21.5%) and α-pinene (16.1%) for P. uliginosa, and α-pinene (20%) and δ-car-3-ene (18.1%) for P. mugo. This study is the first report on the application of the composition of pine-needle volatiles for the reliable identification of closely-related pine species within the Pinus mugo complex.

Recent findings in the chemistry of odorants from four Baccharis species and their impact as chemical markers.

Baccharis is a widespread genus belonging to the Asteraceae family that includes almost 400 species exclusively from the Americas. Even when studied in detail, the taxonomic classification among species from this genus is not yet fully defined. Within the framework of our study of the volatile composition of the Baccharis genus, four species (B. trimera, B. milleflora, B. tridentata, and B. uncinella) were collected from the 'Campos de Cima da Serra' highlands of the Brazilian state of Rio Grande do Sul. The aerial parts were dried and extracted by the simultaneous distillation extraction (SDE) procedure. This is the first time that SDE has been applied to obtain and compare the volatile-extract composition in the Baccharis genus. Characterization of the volatile extracts allowed the identification of 180 peaks with many coeluting components; these latter being detailed for the first time for this genus. The multivariate statistical analyses allowed separating the volatile extracts of the four populations of Baccharis into two separate groups. The first one included the B. milleflora, B. trimera, and B. uncinella volatile extracts. The three species showed a high degree of similarity in their volatile composition, which was characterized by the presence of high contents of sesquiterpene compounds, in particular of spathulenol. The second group comprised the extract of B. tridentata, which contained α-pinene, ß-pinene, limonene, and (E)-ß-ocimene in high amounts.

The scope for using the volatile profiles of Pinus caribaea var. bahamensis as indicators of susceptibility to pine tortoise scale and as predictors of environmental stresses.

Climate change, unseasonal fire and urbanization are contributing to the decline of Pinus caribaea var. bahamensis populations in the Turks and Caicos Islands (TCI). Infestation of pines with the invasive pine tortoise scale (PTS, Toumeyella parvicornis) is accelerating this decline. Pine trees in the Bahamas are larger and healthier and are not infested with PTS although they are subject to some of the same environmental pressures as the trees in TCI. Volatile compounds were collected from wild and nursery-reared P. caribaea var. bahamensis from TCI and the Bahamas and characterized using GC/MS analysis, to look for differences between the compounds detected in insect-infested pines of TCI and the healthy pines of the Bahamas. Ten compounds contributing at least 1% of the total detected peak areas in any one of the samples were selected for further study. Eight of these compounds were identified using authentic standards and mass spectral libraries. The main constituents in the samples were α- and ß-pinene as well as ß-phellandrene, and, together with ß-myrcene, their contents varied the most between samples collected at different locations. Principal-component analysis showed that the two structural isomers of pinene, together with ß-myrcene and ß-phellandrene, contributed 98.4% of the variance between samples. There was a positive relationship between the concentrations of the two structural isomers of pinene and between levels of ß-myrcene and ß-phellandrene. The results are discussed in relation to the biology and adaptations of invasive scale insects, the importance of monoterpenes in pine as a defense against insect predation, whether these compounds can be used as indicators of tree health, and future directions for research into conserving the Caicos pine.

Pesticide residues in stone fruits from the south-eastern region of Poland in 2012-2104.

BACKGROUND: Peaches, sour cherries, nectarines, apricots, plums and cherries are fruit commonly known as "stone fruit". Their nutritional properties namely, vitamins, minerals, fiber and numerous microelements, make them a very important component of human diet. As fruit trees can be attacked by numerous diseases and pests, chemical protection of these crops is used. Therefore, it is important that the relevant governmental agencies or institutions ensure correct application of pesticides. OBJECTIVE: The aim of the study was to evaluate the occurrence of pesticide residues in stone fruits south-eastern region of Poland in 2012-2014 in order to provide data to estimate health risk to consumers. MATERIAL AND METHODS: Validated analytical methods based on liquid / liquid extraction coupled with gas chromatography with electron capture and nitrogen phosphorus detection (GC-ECD/NPD) and spectrophotometry (dithiocarbamates residues) were used for the analysis. 92 samples of stone fruits were tested for the presence of pesticide residues. RESULTS: 13 of all samples (14%) contained pesticide residues. 7 active substances were detected, including 5 fungicides: boscalide, bupirimate, difenoconazole, dithiocarbamates and captan, and 2 insecticides: cypermethrin and pirimicarb. In the analysed samples, the use of not recommended plant protection products in orchard crops were found. However, neither maximum residue levels (MRLs) recommended by the Regulation (EC) No 396/2005 were exceeded nor pesticides being unapproved by the Regulation (EC) No 1107/2009 detected in the analysed samples. CONCLUSIONS: Lack of plant protection products for control specific diseases or pests in crops results in the use of formulations not recommended for use in certain orchard crops. On a basis of results reported in previous years it can be concluded that occurrence of pesticide residues in stone fruit samples dropped significantly.

A breath fungal secondary metabolite signature to diagnose invasive aspergillosis.

BACKGROUND: Invasive aspergillosis (IA) remains a leading cause of mortality in immunocompromised patients, in part due to the difficulty of diagnosing this infection. METHODS: Using thermal desorption-gas chromatography/mass spectrometry, we characterized the in vitro volatile metabolite profile of Aspergillus fumigatus, the most common cause of IA, and other pathogenic aspergilli. We prospectively collected breath samples from patients with suspected invasive fungal pneumonia from 2011 to 2013, and assessed whether we could discriminate patients with proven or probable IA from patients without aspergillosis, as determined by European Organization for Research and Treatment of Cancer/Mycoses Study Group consensus definitions, by direct detection of fungal volatile metabolites in these breath samples. RESULTS: The monoterpenes camphene, α- and ß-pinene, and limonene, and the sesquiterpene compounds α- and ß-trans-bergamotene were distinctive volatile metabolites of A. fumigatus in vitro, distinguishing it from other pathogenic aspergilli. Of 64 patients with suspected invasive fungal pneumonia based on host risk factors, clinical symptoms, and radiologic findings, 34 were diagnosed with IA, whereas 30 were ultimately diagnosed with other causes of pneumonia, including other invasive mycoses. Detection of α-trans-bergamotene, ß-trans-bergamotene, a ß-vatirenene-like sesquiterpene, or trans-geranylacetone identified IA patients with 94% sensitivity (95% confidence interval [CI], 81%-98%) and 93% specificity (95% CI, 79%-98%). CONCLUSIONS: In patients with suspected fungal pneumonia, an Aspergillus secondary metabolite signature in breath can identify individuals with IA. These results provide proof-of-concept that direct detection of exogenous fungal metabolites in breath can be used as a novel, noninvasive, pathogen-specific approach to identifying the precise microbial cause of pneumonia.

Copper-free click reactions with polar bicyclononyne derivatives for modulation of cellular imaging.

The ability of cells to incorporate azidosugars metabolically is a useful tool for extracellular glycan labelling. The exposed azide moiety can covalently react with alkynes, such as bicyclo[6.1.0]nonyne (BCN), by strain-promoted alkyne-azide cycloaddition (SPAAC). However, the use of SPAAC can be hampered by low specificity of the cycloalkyne. In this article we describe the synthesis of more polar BCN derivatives and their properties for selective cellular glycan labelling. The new polar derivatives [amino-BCN, glutarylamino-BCN and bis(hydroxymethyl)-BCN] display reaction rates similar to those of BCN and are less cell-permeable. The labelling specificity in HEK293 cells is greater than that of BCN, as determined by confocal microscopy and flow cytometry. Interestingly, amino-BCN appears to be highly specific for the Golgi apparatus. In addition, the polar BCN derivatives label the N-glycan of the membrane calcium channel TRPV5 in HEK293 cells with significantly enhanced signal-to-noise ratios.