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In pathogenic bacteria post-translationally modified proteins have been found to promote bacterial survival, replication and evasion from the host immune system. In the human pathogen Neisseria meningitidis, the protein PilE (15–18 kDa) is the major building block of type IV pili, extracellular filamentous organelles that play a major role in mediating pathogenesis. Previous reports have shown that PilE can be expressed as a number of different proteoforms, each harbouring its own set of post-translational modifications (PTMs) and that specific proteoforms are key in promoting bacterial virulence. Efficient tools that allow complete PTM mapping of proteins involved in bacterial infection are therefore strongly needed. As we show in this study, a simple combination of mass profiling and bottom-up proteomics is fundamentally unable to achieve this goal when more than two proteoforms are present simultaneously. In a N. meningitidis strain isolated from a patient with meningitis, mass profiling revealed the presence of four major proteoforms of PilE, in a 1:1:1:1 ratio. Due to the complexity of the sample, a top-down approach was required to achieve complete PTM mapping for all four proteoforms, highlighting an unprecedented extent of glycosylation. Top-down mass spectrometry therefore appears to be a promising tool for the analysis of highly post-translationally modified proteins involved in bacterial virulence.

Experimental investigations and kinetic interpretation indicate the presence of several peroxidic species during the oxidation of n-dodecane. The oxidation was performed either in the gaseous phase in a flow system, or with a liquid/gas interaction in a bulb, because in many combustion processes liquid and gas coexist during preignition. The different peroxides were identified by mass spectrometry. Simple hydroperoxides with two oxygen atoms per molecule were observed at relatively low temperatures above 370 K with liquid and gas present. Ketohydroperoxides with three oxygen atoms per molecules are formed at ∼500 K. Di-, and even tri-, ketohydroperoxides with four or five oxygen atoms per molecule are also present under these experimental conditions at 518 K. Their formation is explained by isomerization reactions of alkoxy radicals OR −2H O bearing a carbonyl group; these stem from the decomposition of carbonyl-hydroperoxides. Depending on the experimental conditions, the nature of the hydroperoxides is different and the respective reactions of these species should be introduced in low temperature oxidation mechanisms. These reaction schemes should include the isomerization of peroxy radicals RO 2 and of ketoalkoxy radicals OR −2H O, leading to the formation of peroxides with several carbonyl groups.

In the queenless ant, Diacamma ceylonense, the cuticular hydrocarbons (C25-C35) of nestmate workers vary in their proportions according to age and fertility. Newly eclosed adults ('callows') initially have the same cuticular profile, but with time this changes to that typical of foragers. In contrast, workers that begin to produce eggs develop a different cuticular profile. Several substances (n-C29 and some methyl C25 and C27) discriminate these different social categories (callows, foragers and egg-layers). In Diacamma ceylonense, inter-colony variation of the cuticular hydrocarbons was much lower than intra-colony variation. We also found qualitative differences between the sexes, with males having a clearly different profile with much more alkanes. We discuss these results in the context of physiological models of the relation between ovarian activity and the synthesis of cuticular hydrocarbons. Variations in cuticular profile are a reliable reflection of ovarian activity, and could be used by ants as a fertility signal.

Tetramorium bicarinatum(Myrmicinae) is an ant species frequently found in tropical and subtropical areas, particularly in Africa, Southeast Asia (Japan), and South America (Brazil). The species is polygynous, reproduces by budding, and has sterile workers. Since the nests are widely distributed in a given area, the problem arises of territorial defense against conspecifics. Because not all ants defend territories, we assessed the defensive behavior of T. bicarinatumworkers through intraspecific and interspecific aggressiveness tests. A detailed behavioral study of the interactions between workers from several different colonies of T. bicarinatum(originating from Japan and Brazil) showed that workers do not discriminate against conspecific nonnestmate individuals, but they are highly aggressive towards allospecifics (Myrmica rubra, Myrmicinae). The results suggest that each colony from this ant species possesses a similar colonial odor. Chemical analyses of the cuticular hydrocarbons of these species were made with gas chromatography coupled to mass spectrometry. Results showed that the different colonies of T. bicarinatumpossess a common chemical profile mainly composed of straight-chain alkanes and alkenes, while M. rubrapossess more methyl-branched alkanes. We suggest that methyl alkane cues play a determining role in colonial recognition and that these results could explain the underlaying basis of the lack of intraspecific aggressiveness in T. bicarinatum.

Solid-phase microextraction (SPME) samplings from live insects or natural secretion allowed one to identify the aggregation pheromones of the pest beetles Scapanes australis and Strategus aloeus by efficient and rapid isolation of their highly volatile (72

Microdochium nivale , a wheat pathogenic filamentous fungus, appeared to be very sensitive in in vivo laboratory assays to fenpropimorph and tridemorph and to a lesser extent to fenpropidin, piperalin, and spiroxamine. It accumulated Δ 8 -sterols when grown in the presence of one of these sterol biosynthesis inhibitors. Thus, in M. nivale , these fungicides seemed to be very good inhibitors of the sterol Δ 8 → Δ 7 -isomerase. This hypothesis was then confirmed by sterol Δ 8 → Δ 7 -isomerase inhibition assays in cell-free enzyme systems.

Thirteen host-plant kairomone blends, including 28 compounds, were tested and showed moderate to high synergy with rhynchophorol. The blends plus rhynchophorol also attracted the related Dynamis borassi. Ethanol–ethyl acetate blends in various ratios showed moderate synergy. Two blends, including "characteristic coconut" odor molecules, were as efficient as sugarcane in synergizing rhynchophorol and field luring American Palm weevils (APWs). Preliminary olfactometer tests of natural host-plant volatiles demonstrated the role of fermentation in primary APW attraction. The synergists were chosen from a comparative study of the odors emitted by four plant materials attractive to the APW: sugarcane, coconut, Jacaratia digitata tree and Elaeis guineensis (Oil palm). The volatiles were isolated during 6 days of sequential trappings onto Supelpak-2 adsorbent. The highly volatile fraction of sugarcane volatiles was sampled by solid-phase microextraction (SPME). Odors were analyzed and identified by gas chromatography and mass spectrometry. Electroantennogram responses to the plant odors were recorded to help in screening for bioactivity. The odor compositions between plants prior to and during fermentation were compared using a principal component analysis (PCA) to determine common odor features of the plants and to design simplified blends for field activity screening. About 100 components were identified in the >4-carbon fraction of the odors, among which 65% were fermentation volatiles. Fermentation generated a strong increase in the amount and variety of the volatiles emitted. The palm materials emitted two- to threefold greater odor amounts than the other plants. The odors from each plant were distinct according to PCA, with few common abundant components: isopentanol, 2-methylbutanol, their acetates, acetoin, isobutyl acetate, 2,3-butanediol, and 2-phenylethanol. Ethanol and ethyl acetate accounted for 80–90% in the highly volatile fraction of sugarcane odors. Coconut odor was mainly characterized by phenol, guaiacol, 1,2-dimethoxybenzene, ethyl esters of tiglic and 3,3-dimethylacrylic acids, 2-hexanone, 2-nonanone; and, to a lesser extent, by 2-heptanone, menthone, β-phellandrene, ethyl octanoate and decanoate, which were also present in other plants.

The males of Nezara viridula (Heteroptera, Pentatomidae) produce a blend of four terpenoid compounds. The proportion of these four compounds varies among different populations. The terpenoid content of the volatile emission from individual males of a French population was monitored for several days with headspace solid-phase microextraction. The total amount of terpenoid compounds collected on an SPME fiber from one virgin male bug emitting over the course of 1 hr was very variable between samples. More than 50% of males released pheromone, at least during one part of the observation period. The relative percentages of bisabolene, nerolidol, trans-bisabolene epoxide, and cis-bisabolene epoxide were calculated and compared. The proportion of nerolidol in the blend showed a large dispersion between samples and a high coefficient of variation. The percentage values for bisabolene and its trans and cis epoxides were less dispersed, and the coefficients of variation for both epoxides were low. The proportion of trans epoxide relative to the two epoxides, which has been considered characteristic of pheromone strains of N. viridula, showed the smallest coefficient of variation. The repeatability index (RI) varied between 0.57 and 0.85 for the four compounds. Repeatability was particularly high for the two epoxide isomers. Stability in epoxide production was confirmed by statistical analysis (ANOVA), which revealed significant differences in the percentages of cis and trans epoxides between individuals. Correspondingly, the RI of the ratio of trans to cis + trans epoxides was over 0.8. Our study confirms the existence of an interindividual variation of the trans to cis epoxide ratio within populations of Nezara viridula but indicates that each individual reproducibly emits both isomers in the same proportion day after day.

Volatiles emitted by cones and foliage of the Swiss stone pine (Pinus cembra), as well as cone volatiles emitted by some other conifers (Picea abies, Abies alba, Pinus uncinata, P. sylvestris, P. koraiensis, P. pumila, P. peuce, P. strobus, P. parviflora, P. wallichiana and P. armandi) were collected using headspace techniques and analyzed by GC-mass spectrometry. The same major monoterpenes, i.e. α-pinene, β-pinene, and limonene+β-phellandrene were found in both the cone and foliage volatiles of Swiss stone pine. The monoterpene profile did not vary among cones from the same tree, or among different geographic origins. A few differences were noted between trees from the same stand, and over cone development. In other conifers, cone emissions consisted of the same monoterpenes, plus 3-carene, with specific proportions related to tree species.

Genetic control and mechanisms of resistance to tebuconazole, a sterol C14-demethylation inhibitor, were investigated in the phytopathogenic fungus Nectria haematococca . Resistant mutants have been selected from the laboratory, following UV irradiation. They have been characterized through genetic crosses and mutations in at least three genes were found to be responsible for resistance. The genes Teb1, Teb2 , and Teb3 were clearly identified, a fourth gene called Teb4 could be hypothesized. Mutations at loci Teb2 and Teb3 induced pleiotropic effects such as reduced sporulation and growth rate, mycelium pigmentation ( Teb2 ), or altered ascospore viability ( Teb3 ). The resistance levels determined by mutations in the different genes were relatively low (below 10). When associated in double mutants, the additive effect was recorded. Cross-resistance toward other sterol C14-demethylation inhibitors was observed in all the resistant strains, except in the Teb4 -carrying strain; moreover, for some C14-demethylation inhibitors hypersensitivity was expressed. A constitutive energy-dependent efflux seemed implicated in the mechanism of resistance for the Teb1 -carrying strain and probably also for the Teb2 and Teb3 -carrying strains. However, the Teb4 -carrying strain exhibited a kinetic of fungicide uptake similar to that of the wild-type strain. The sterol profile of the Teb4 -carrying strain was similar to that of all the other resistant mutants and wild-type strains. Thus the resistance mechanism induced by mutation at the Teb4 locus has not been found yet.

The vinyl methyl ether (VME) reactivity towards alkenes R 1 –CHCH–R 2 in the gas phase giving rise to substituted VME radical cations [R 1 –CHCH–OCH 3 ] +° and/or [R 2 –CHCH–OCH 3 ] +° is a well known and elegant approach for the localization of the carbon–carbon double bonds. However, and despite the improvements proposed in the past to increase its specificity, real applications have rarely (if ever) been reported. In this paper, we describe the applicability of GC–VME chemical ionization as a method directly performed on crude cuticular extracts of social insects (three ant species namely Myrmica alaskensis , Formicoxenus quebecensis and Formica selysi and the honey bee). Assignment of the double bond position is done without ambiguity for a number of Δ 8 , Δ 9 and Δ 10 long chain alkenes (C 23 to C 33 ) on the basis of the obtained substituted VME radical cations and corresponding ions after loss of methanol.

The composition of volatile chemicals released by fresh coffee berries at different stages of ripeness for Coffea canephora var. Robusta and Coffea arabica was investigated by dynamic headspace collection and GC and GC/MS analyses. Green berries produced few compounds whereas red berries were characterized by high levels of terpenes for C. arabica and by both terpenes and sesquiterpenes for C. canephora var. Robusta. As berries dried out, these compounds decreased and oxygenated compounds became more abundant. The results showed changes in volatile blends released by the coffee berries during ripening. Red berry effluvia are already known to attract colonizing females of the coffee berry borer; thus, specific compounds identified will be considered as candidate chemicals involved in the attraction.

We extracted the cuticular hydrocarbons from live Dinoponera quadriceps ants (10 colonies collected from Brazil) with the solventless solid-phase microextraction (SPME) technique. Gas chromatography of the SPME samples (N = 233 measurements) compared with pentane extracts (N = 10) resulted in similar profiles. Eighty-one compounds belonging to the main long-chain hydrocarbon families were identified by GC-MS. There is no morphologically specialized queen in D. quadriceps and only one aggressively dominant worker (alpha) mates and reproduces in each colony. The alpha ant (N = 26 individuals) always yielded higher amounts and percentages of 9-hentriacontene (9-C31 : 1) than her sterile nestmates (N = 47). Since SPME is not destructive, it allowed for the repeated extraction of the same individuals, demonstrating that the alpha ant (virgin or mated) always had higher levels of 9-hentriacontene. This difference appears related to ovarian activity and may function as a signal of the alpha's dominance status.

The biotransformation of benzo[a]pyrene by purified extracellular laccase of Pycnoporus cinnabarinus was investigated in bench scale reactors. The reaction required the presence of exogenous mediator ABTS. Most of 95% of the substrate was converted within 24 hours. The enzyme preparation oxidised the substrate mainly to benzo[a]pyrene 1,6- 3,6- and 6,12-quinones in a 2/1/1 ratio after 24 h incubation.

This study is an attempt to identity the active chemicals (signal) of the odor of dog feces that suppress feeding in domestic sheep. The repellent effects of the odors of dog, wolf, pig, and sheep feces (potential predator and nonpredator species) were assessed on sheep, using a food-choice situation. The odors of wolf and dog feces had the highest repellent effect. A total pentane extract of dog feces was split by micropreparative gas chromatography and the fractions obtained were analyzed and presented to sheep in a food-choice situation. The quantitatively major constituents of the pentane extract, identified by gas chromatography–mass spectrometry, are indole and fatty acids. In food repellency tests, indole had no repellent effect. The active odorous signal contained in dog feces appears to consist of fatty acids mixed with neutral compounds acting synergically. These experiments underline the complexity of this biological signal and constitute a first step in the development of a practical repellent for ungulates.

Attraction of the large pine weevil, Hylobius abietis (L.) (Coleoptera: Curculionidae) to pine logs was enhanced by the presence of weevils already on the logs. We investigated the volatiles emitted by insects feeding on host trees to determine the mechanism of the attraction. Chemical analyses of the collected volatiles showed large amounts of monoterpenes. There were no qualitative differences in the collections, between Scots pine alone and pine plus feeding weevils, nor between males and females, mated or virgin. Strong quantitative differences in monoterpenes existed that were correlated to the surface of cut bark. Electroantennograms (EAGs) were recorded on male and female antennae stimulated by increasing doses of collected volatiles. EAGs for pine alone and pine plus weevils at the same stimulus loading in (a + P)-pinene gave superimposable curves. EAGs of male and female volatiles were also identical. These results suggest an attraction mediated by host plant allelochemicals rather than by an aggregation pheromone.

Ants of the genus Formicoxenus are called xenobiotic ants because they live in close association with their Myrmica host; they cannot live independently of the host but they keep their brood apart. They forage in the host's galleries and obtain food from it through trophallaxies. They also very frequently lick the Myrmica workers and for that reason are called “shampoo ants”. The chemical basis of the integration of the parasite into the host society is studied. In the two host-parasite pairs F. provancheri/M. incompleta and F. quebecensis/M. alaskensis , chemical mimicry in the cuticular hydrocarbons was found. With adoption experiments it is shown that this mimicry was not innate but is acquired during the first days of adult life.

Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material., Author Summary Natural genetic transformation, first discovered in Streptococcus pneumoniae by Griffith in 1928, is observed in many Gram-negative and Gram-positive bacteria. This process promotes genome plasticity and adaptability. In particular, it enables many human pathogens such as Streptococcus pneumoniae, Staphylococcus aureus or Neisseria gonorrhoeae to acquire resistance to antibiotics and/or to escape vaccines through the binding and incorporation of new genetic material. While it is well established that this process requires the binding and internalization of external DNA, the molecular details of these steps are unknown. In this study, we discovered a new appendage at the surface of S. pneumoniae cells. We show that this appendage is similar in morphology and composition to appendages called Type IV pili commonly found in Gram-negative bacteria. We demonstrate that this new pneumococcal pilus is essential for transformation and that it directly binds DNA. We propose that the transformation pilus is an essential piece of the transformation apparatus by capturing exogenous DNA at the bacterial cell surface.

International audience; Nicotinic acetylcholine receptors (nAChRs) are one of the most important families in the ligand-gated ion channel superfamily due to their involvement in primordial brain functions and in several neurodegenerative pathologies. The discovery of new ligands which can bind with high affinity and selectivity to nAChR subtypes is of prime interest in order to study these receptors and to potentially discover new drugs for treating various pathologies. Predatory cone snails of the genus Conus hunt their prey using venoms containing a large number of small, highly structured peptides called conotoxins. Conotoxins are classified in different structural families and target a large panel of receptors and ion channels. Interestingly, nAChRs represent the only subgroup for which Conus has developed seven distinct families of conotoxins. Conus venoms have thus received much attention as they could represent a potential source of selective ligands of nAChR subtypes. We describe the mass spectrometric-based approaches which led to the discovery of a novel α-conotoxin targeting muscular nAChR from the venom of Conus ermineus. The presence of several posttranslational modifications complicated the N-terminal sequencing. To discriminate between the different possible sequences, analogs with variable N-terminus were synthesized and fragmented by MS/MS. Understanding the fragmentation pathways in the low m/z range appeared crucial to determine the right sequence. The biological activity of this novel α-conotoxin (α-EIIA) that belongs to the unusual α4/4 subfamily was determined by binding experiments. The results revealed not only its selectivity for the muscular nAChR, but also a clear discrimination between the two binding sites described for this receptor.

Biotransformation of the insecticide lindane by the white rot basidiomycete Phanerochaete chrysosporium has been investigated in liquid cultures. Some polar metabolites and carbon dioxide were produced from the pesticide. Among the metabolites identified were tetrachlorocyclohexene, tetrachlorocyclohexene epoxide and tetrachlorocyclohexenol. When used as a substrate, tetrachlorocyclohexene was also converted by the fungus to tetrachlorocyclohexenol, polar metabolites and carbon dioxide. Three incubation conditions leading to low and high peroxidase production were assayed. Data from these experiments, as well as in-vitro incubations with purified enzymes, ruled out any involvement of the peroxidases in lindane biotransformation and mineralization. Moreover, 1-aminobenzotriazole (a P450 inactivator) drastically reduced pesticide metabolism. Conversely, phenobarbital (a P450 inducer) did not significantly increase lindane breakdown.

Five hydroxylated aliphatic molecules were identified as the pheromone produced by male West Indian Sugarcane Borer (WISB): 4-methyl-5-nonanol (1), 2-methyl-4-heptanol (2), 2-methyl-4-octanol (3), 5-nonanol (4) and 3-hydroxy-4-methyl-5-nonanone (5). Electroantennographic recordings revealed antennal responses to compounds 1, 2, 3 and 4. Significant EAGs were also recorded in response to pheromone compounds of weevils belonging to the same subfamily and structurally related to the WISB pheromone. The natural pheromone elicited aggregation behavior on WISB adults in laboratory bioassays.

The diastereoselective synthesis of (1S∗, 3R∗, 5R∗, 7S∗) 2,8-dioxa 1-ethyl 3,5,7-trimethyl bicylo [3,2,1] octane 1d has been achieved using as key-step the regioselective Baeyer-Villiger reaction of 2,6-disubstituted cyclohexanone. It confirms the identification of a new male pheromone emitted by the banana weevil, Cosmopolites sordidus. The trivial name sordidin is proposed.

International audience; The Gram-negative bacterium Neisseria meningitidis asymptomatically colonizes the throat of 10 to 30% of the human population, but throat colonization can also act as the port of entry to the blood (septicemia) and then the brain (meningitis). Colonization is mediated by filamentous organelles referred to as type IV pili, which allow the formation of bacterial aggregates associated with host cells. We found that proliferation of N. meningitidis in contact with host cells increased the transcription of a bacterial gene encoding a transferase that adds phosphoglycerol onto type IV pili. This unusual posttranslational modification specifically released type IV pili-dependent contacts between bacteria. In turn, this regulated detachment process allowed propagation of the bacterium to new colonization sites and also migration across the epithelium, a prerequisite for dissemination and invasive disease.

The enantioselective synthesis of both (4R,5R)- and (4S,5S)-4-methyl-5-nonanol (1) was achieved by starting from an optically active epoxide 5. A GC comparison on a chiral stationary phase of the synthetic enantiomers of 1 with the naturally occurring 1 was executed, and (4S,5S)-1 was shown to be identical with the natural 1 isolated as the male-produced pheromone compound of the weevils Rhynchophorus vulneratus and Metamasius hemipterus.

The diastereoselective synthesis of (1S∗, 3R∗, 5R∗, 7S∗) 2,8-dioxa 1-ethyl 3,5,7-trimethyl bicylo [3,2,1] octane 1d has been achieved using as key-step the regioselective Baeyer-Villiger reaction of 2,6-disubstituted cyclohexanone. It confirms the identification of a new male pheromone emitted by the banana weevil, Cosmopolites sordidus. The trivial name sordidin is proposed.

International audience; PURPOSE: To determine the human centrin 2 (Hscen 2) protein response to oxidising radicals in vitro and to evaluate the consequences on its biological functions. MATERIALS AND METHODS: Hscen 2 was submitted to hydroxyl and azide radicals produced by radiolysis in the absence of oxygen. The resulting products were characterised by biochemical, spectroscopic and mass spectrometry techniques. Their thermodynamics parameters of complexation with C-terminal fragment of Xeroderma pigmentosum C protein (C-XPC), one of the Hscen 2 cellular partners, were quantified by isothermal titration calorimetry (ITC). RESULTS: Both hydroxyl and azide radicals induce centrin 2 polymerisation as we characterised several intermolecular cross-links generating dimers, trimers, tetramers and higher molecular mass species. These cross-links result from the formation of a covalent bond between the only tyrosine residue (Tyr 172) located in the C-terminal region of each monomer. Remarkably, dimerisation occurs for doses as low as a few grays. Moreover, this Hscen2 dimer has a lower affinity and stoechiometry binding to C-XPC. CONCLUSIONS: These results show that as oxidative radicals induce high proportions of irreversible damages (polymerisation) centrin 2 is highly sensitive to ionising radiation. This could have important consequences on its biological functions.

In the mycelium of all the Fusarium species studied, the major sterol was ergosterol in the absence of fenpropimorph. In the presence of this fungicide fenpropimorph-sensitive strains, with the exception of F. nivale , accumulated mainly Δ 8,14 -sterols and tolerant ones accumulated either both Δ 8,14 - and Δ 8 -sterols or only Δ 8 -sterols. This seemed to indicate that fenpropimorph toxicity was related to sterol Δ 14 -reductase sensitivity. In F. nivale , which was highly sensitive to fenpropimorph, the accumulation of only Δ 8 -sterols indicated that the main target enzyme was sterol Δ 8 →Δ 7 -isomerase. In addition to Δ 8,14 -and Δ 8 -sterols, squalene was accumulated in very high amounts in several strains; the level of squalene accumulation did not seem to be correlated with fenpropimorph sensitivity.

International audience; The relationship between pheromone composition and mitochondrial haplotype clades was investigated by coupling DNA analyses with pheromone identification and male mate searching behavior among different geographic populations of Busseola fusca. The within-population variations in pheromone blend were as great as those observed between geographic populations, suggesting that the female sex pheromone blend was not the basis of reproductive isolation between the geographic clades. Furthermore, while data from wind tunnel experiments demonstrated that most of the tested males were sensitive to small variations in pheromone mixture, there was considerable within-population variability in the observed response. The study identified a new pheromone component, (Z)-11-hexadecen-1-yl acetate, which when added to the currently used three-component synthetic blend resulted in significantly higher traps catches. The new recommended blend for monitoring flight phenology and for timing control measures for optimal efficacy of B. fusca is (Z)-11-tetradecen-1-yl acetate (62%), (E)-11-tetradecen-1-yl acetate (15%), (Z)-9-tetradecen-1-yl acetate (13%), and (Z)-11-hexadecen-1-yl acetate (10%).

1 - Article; 1-Hydroxymethylene-1,1-bisphosphonic acids (HMBPs) are important drugs for the treatment of a variety of bone diseases. Since these compounds have no chromophore, their detection is challenging and mass spectrometry (MS) appears to be an appropriate sensitive tool. Our work deals with the analysis by electrospray ionization tandem mass spectrometry (ESI-MSn) of the well-known nitrogen-containing HMBP alendronate and of three analogues, considered as potential prodrugs. These four molecules share a common structure with different protecting groups on the phosphonic acid and on the amine functions. We describe the dissociation mechanisms of nitrogen-containing HMBPs in positive ion mode and we compare, in negative ion mode, our results with literature data. In both modes, the dissociations are essentially losses of ROH, and of phosphorus-containing species (HPO2, ROP(OH)(2) and ROPO(OH)(2)), where R = H, C6H5, or CH3OC6H5. These fingerprints will be of great value for differentiating alendronate from its potential prodrugs in complex biological mixtures. Copyright (c) 2008 John Wiley & Sons, Ltd.

Pheromonal secretions produced by females and males of the noctuid moth,Mocis megas (Guenee) have been analyzed by gas chromatography and mass spectrometry (EI (electron impact) and CI (chemical ionization)). The female sex pheromone was a blend of (Z,Z,Z) 3,6,9 heneicosatriene (55%) and (Z,Z) 3,6-cis-9S, 10R-epoxyheneicosadiene (45%). Male secretion produced at the level of a prothoracic organ was a blend of two unsaturated major hydrocarbons: (Z,Z) 6,9 heneicosadiene, (64%) and (Z,Z,Z) 3,6,9 heneicosatriene (24%) and C19, C20 and C22 homologues (total ratio 12%), as minor components. The trienic hydrocarbon was present in both sexes. The behavioral role of this male secretion has not yet been elucidated.

(Z)-9-dodecenyl acetate (85%) and (Z)-9-dodecenol (15%) were identified from female gland extract of Lobesia bicinctana (Dup.) by capillary gas chromatography linked either to mass spectrometry or to electroantennography. Attractivity of this blend used at a dose of 1000 μg as bait for field trapping was inferior to the attractivity of (Z)-9-dodecenyl acetate applied alone at the same dose. This chemical can be used now to survey flights and populations of this pest in onion crops. Resume Un attractif sexuel pour Lobesia bicinctana (Dup.) (Lep., Tortricidae) La chromatographie en phase gazeuse, seule ou couplee a la spectrometrie de masse ou a l'electroantennographie met en evidence 2 composes de nature pheromonale dans les extraits hexaniques de glandes a pheromone. Il s'agit de l'acetoxy-1 dodecene-9Z (85%) et du dodecenol-9Z (15%). Le melange de ces 2 constituants, appliques dans les memes proportions a la dose totale de 1000 μg est moins attractif que l' acetoxy-1 dodecene-9Z utilises seul a la meme dose. Cette derniere formulation doit permettre de surveiller les vols et les populations de ce ravageur dans les cultures d'oignons a graines. Zusammenfassung Ein Sexuallockstoff fur Lobesia bicinctana (Dup.) (Lep., Tortricidae) (Z)-9-dodecenyl Acetat (85%) und (Z)-9-dodecenol (15%) Konnten gaschromatographisch aus dem Drusenextrakt von Lobesia bicinctana Weibchen identifiziert werden; die Ergebnisse wurden entweder mit einem Massenspektrometer oder einem Elektroantennogramm nachgewiesen. Die Attraktivitat dieser beiden Substanzen in Mischung war bei einer Dosis von insgesamt 100 μg geringer als die Attraktivitat von (Z)-9-dodecenyl Acetat bei gleicher Dosis. Diese Substanz kann zur Populations-uberwachung von L. bicinctana in Zwiebelfeldern eingesetzt werden.

(2E,4E)-4,6,10-trimethyl-2,4-dodecadien-7-one and (2E,4Z)-4,6,10-trimethyl-2,4-dodecadien-7-one have been identified mostly through 2D NMR and MS/MS techniques as primary and secondary components of the sex pheromone of Matsucoccus feytaudi.

The importance of protein glycosylation in the interaction of pathogenic bacteria with their host is becoming increasingly clear. Neisseria meningitidis , the etiological agent of cerebrospinal meningitis, crosses cellular barriers after adhering to host cells through type IV pili. Pilin glycosylation genes ( pgl ) are responsible for the glycosylation of PilE, the major subunit of type IV pili, with the 2,4-diacetamido-2,4,6-trideoxyhexose residue. Nearly half of the clinical isolates, however, display an insertion in the pglBCD operon, which is anticipated to lead to a different, unidentified glycosylation. Here the structure of pilin glycosylation was determined in such a strain by “top-down” MS approaches. MALDI-TOF, nanoelectrospray ionization Fourier transform ion cyclotron resonance, and nanoelectrospray ionization quadrupole TOF MS analysis of purified pili preparations originating from N. meningitidis strains, either wild type or deficient for pilin glycosylation, revealed a glycan mass inconsistent with 2,4-diacetamido-2,4,6-trideoxyhexose or any sugar in the databases. This unusual modification was determined by in-source dissociation of the sugar from the protein followed by tandem MS analysis with collision-induced fragmentation to be a hexose modified with a glyceramido and an acetamido group. We further show genetically that the nature of the sugar present on the pilin is determined by the carboxyl-terminal region of the pglB gene modified by the insertion in the pglBCD locus. We thus report a previously undiscovered monosaccharide involved in posttranslational modification of type IV pilin subunits by a MS-based approach and determine the molecular basis of its biosynthesis.

Background Allopolyploidy is a preeminent process in plant evolution that results from the merger of distinct genomes in a common nucleus via inter-specific hybridization. Allopolyploid formation is usually related to genome-wide structural and functional changes though the underlying mechanisms operating during this "genomic shock" still remain poorly known. The aim of the present study was to investigate the modifications occurring at the proteomic level following an allopolyploidization event and to determine whether these changes are related to functional properties of the proteins. In a previous report, we applied comparative proteomics to synthetic amphiploids of Brassica napus and to its diploid progenitors B. rapa and B. oleracea. Although several hundred polypeptides displayed additivity (i.e. mid-parent values) in the amphiploids, many of them showed non-additivity. Here, we report the in silico functional characterization of the "non-additive" proteins (the ones with a non-additive pattern of regulation) in synthetic B. napus. Results The complete set of non-additive proteins (335 in the stem and 205 in the root), as well as a subset of additive polypeptides (200 per organ), was identified by mass spectrometry. Several protein isoforms were found, and most of them (~55%) displayed "different" or "opposite" patterns of regulation in the amphiploids, i.e. isoforms of the same protein showing both up-regulation and down-regulation in the synthetic B. napus compared to the mid-parent value. Components of protein complexes were identified of which ~50% also displayed "different" or "opposite" patterns of regulation in the allotetraploids. In silico functional categorization of the identified proteins was carried out, and showed that neither functional category nor metabolic pathway were systematically affected by non-additivity in the synthetic amphiploids. In addition, no subcellular compartment was found to be over- or under-represented among the proteins displaying non-additive values in the allopolyploids. Conclusion Protein identification showed that functionally related polypeptides (isoforms and complex subunits) could be differentially regulated in synthetic B. napus in comparison to its diploid progenitors while such proteins are usually expected to display co-regulation. The genetic redundancy within an allopolyploid could explain why functionally related proteins could display imbalanced levels of expression. No functional category, no metabolic pathway and no subcellular localization was found to be over- or under-represented within non-additive polypeptides, suggesting that the differential regulation of gene products was not related to functional properties of the proteins. Thus, at the protein level, there is no evidence for the "genomic shock" expected in neo-polyploids and the overall topology of protein networks and metabolic pathways is conserved in synthetic allotetraploids of B. napus in comparison to its diploid progenitors B. rapa and B. oleracea.

POLYPLOIDY is a major evolutionary process of speciation in eukaryotes and particularly in plants where several economically important crop species including oilseed rape, wheat, and sugarcane are polyploid (Grant 1981; Wendel 2000). Moreover, an increasing number of diploid-considered species are shown to actually be paleopolyploids, such as maize (Gaut and Doebley 1997), Arabidopsis (Arabidopsis Genome Initiative 2000), and rice (Zhang et al. 2005). The high occurrence of polyploid plants has been the source of considerable interest, notably to determine why polyploidy represents such an indisputable evolutionary success. Several authors have noted that polyploidy must confer an immediate selective advantage (Osborn et al. 2003; Comai 2005) and have underlined the necessity to analyze the very first stages of polyploid formation. Accordingly, the study of newly synthesized polyploids has led to the description of structural changes such as chromosomal rearrangements (Pontes et al. 2004) and gains or losses of DNA sequences (Song et al. 1995; Liu et al. 1998; Madlung et al. 2005). Additionally, investigations have demonstrated the occurrence of modifications at the level of gene expression. Several studies on resynthesized Arabidopsis suecica (Comai et al. 2000; Wang et al. 2004, 2006), Gossypium allotetraploids (Adams et al. 2003, 2004), and allotetraploids and allohexaploids of wheat (Ni et al. 2000; Kashkush et al. 2002, 2003; He et al. 2003; Wu et al. 2003) have shown that some genes are silenced after polyploidization, while others are derepressed. Nearly all of these approaches were undertaken at the transcript level and most of them used qualitative or semiqualitative techniques like cDNA–AFLP display that allowed the detection of gene silencing or novel expression patterns, but lacked precision for more subtle variations (up- or downregulation). Other innovating techniques like cDNA–SSCP (Adams et al. 2003) give quantitative information on homeologous gene expression, but are restricted to a small number of genes. Genomewide analyses can now be undertaken using the promising cDNA microarray technology. This approach is planned for Brassica polyploids (Chen et al. 2004; Lee et al. 2004; Wang et al. 2005) and has been recently applied to the synthetic allopolyploids Senecio cambrensis (Hegarty et al. 2005) and A. suecica (Wang et al. 2006), revealing several modifications of gene expression. Proteomic tools complement transcriptomic approaches: cellular biochemistry is essentially modulated by proteins rather than by transcripts, and the correlation between mRNA abundance and protein amounts is erratic due to post-transcriptional regulations (Gygi et al. 1999). Additionally, post-translational modifications play a key role in the cell as they modulate protein activity, turnover, subcellular location, or interactions with proteins or nucleic acids and can be investigated only at the protein level. Comparative proteomics can be achieved using the two-dimensional electrophoresis (2-DE) technique that allows the resolution of hundreds of polypeptides in a single gel. Accurate and reproducible quantification of the corresponding spots can be performed using dedicated software and the relevant proteins identified by mass spectrometry. Comparative proteomics provides pertinent and reliable quantitative results on genomewide expression and it is becoming more and more popular in plant research (Thiellement et al. 1999, 2002). In this study, we used the Brassica napus model system to investigate the early consequences of allopolyploid formation on gene expression. Four independent neosynthesized B. napus amphidiploids, their diploid progenitors B. oleracea and B. rapa, as well as the amphihaploid hybrids were submitted to comparative proteomics. The additivity hypothesis (predicting a midparent proteome for the amphiploids) was tested in two distinct organs, the stem and root, and polypeptides displaying nonadditive patterns were identified using mass spectrometry.

International audience; Recognition of the mother is of major importance for the survival of mammalian neonates. This recognition is based, immediately after birth, on the detection of odours that have been learned by the fetus in utero. If the ethological basis of a transnatal olfactory continuity is well established, little is known on the nature of its olfactory cues, and nothing about the presence of potential carrier proteins in the maternal fluids such as amniotic fluid, colostrum and milk. We have identified the components of the pig putative maternal pheromone in these fluids of the sow. We also used a ligand-oriented approach to functionally characterize carrier proteins for these compounds in the maternal fluids. Six proteins were identified, using binding assay, immunodetection and peptide mapping by mass spectrometry. These proteins are known to transport hydrophobic ligands in biological fluids. Among them, alpha-1 acid glycoprotein (AGP) and odorant-binding protein (OBP) have been described in the oral sphere of piglets as being involved in the detection of pig putative maternal pheromone components. These are the first chemical and biochemical data supporting a transnatal olfactory continuity between the fetal and the postnatal environments.

International audience; This study targets to express the piglet odorant-binding protein (p1OBP) and compare the engineered product to the corresponding native protein forms, i.e. plOBP and adult porcine OBP (pOBP). Using the natural signal peptide from the cDNA sequence, up to 40 mg 1-1 of secreted recombinant piglet OBP (rOBP) has been produced in a minimal culture medium. No significant difference in molecular mass between rOBP and native plOBP could be observed by mass spectrometry following or not trypsin digestion. rOBP and pOBP shared similar immunoreactivity towards polyclonal anti-pOBP antibodies, suggesting a proper processing and folding of the recombinant product. Both plOBP and rOBP displayed comparable binding properties towards fatty acids present in the putative maternal pheromone and a steroid, component of the boar sex pheromone. Furthermore, the rOBP product was found to bind to an olfactory receptor, for which pOBP binding was previously characterized. Taken together, these findings suggest that rOBP, produced in Pichia pastoris, exhibits structural and functional properties comparable to those of the native lipocalins from both young or adult animal.

International audience

Social insects often show an extreme reproductive skew. In queenless ants, colonies consist of morphologically identical workers that can all potentially reproduce sexually. Similarly to that in social vertebrates, aggression in these ants functions to select the reproductive(s). We investigated the mechanisms underlying reproductive skew in the monogynous queenless ant Streblognathus peetersi. Behavioural observations of disturbed hierarchies were integrated with physiological measures of fertility (vitellogenin titre in the haemolymph) and chemical analysis of cuticular hydrocarbons, which are putative fertility pheromones. This multifaceted approach revealed that the colony reproductive is determined as a result of aggression between high-ranking workers, but once an alpha is established, chemical signalling is enough to maintain reproductive skew. As already reported in several species of ants and also in a social wasp, egg layers have distinct profiles of cuticular hydrocarbons compared with infertile workers. Importantly, ‘high rankers’ who are unable to lay eggs also have a specific cuticular profile; this is consistent with their intermediate state of fertility indicated by vitellogenin levels. Rather than just ovarian activity, the cuticular hydrocarbon profile thus reveals the individual hormonal state that underlies reproductive activity. We compare the fertility signal in queenless ants with the ‘badge of status’ reported in various birds. We discuss the evolutionary stability of this communication system and give special emphasis to ecological constraints and the high degree of intracolonial relatedness typical of social insects.

International audience; Improvement of the catalytic properties of fungal laccases is a current challenge for the efficient bioremediation of natural media polluted by xenobiotics. We developed the heterologous expression of a laccase from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica as a first step for enzyme evolution. The full-length cDNA consisted of a 1,561-bp open reading frame encoding lacIIIb, a 499-amino-acid protein and a 21-amino-acid signal peptide. Native and yeast secretion signals were used to direct the secretion of the enzyme, with the native signal yielding higher enzyme activity in the culture medium. The level of laccase activity secreted by the transformed yeast was similar to that observed for the non-induced wild-type strain of T. versicolor. The identity of the recombinant enzyme was checked by Western blot and matrix-assisted laser desorption/ionization time-of-flight analysis. Electrophoresis separation in native conditions indicated a molecular mass of the recombinant protein slightly higher (5 kDa) than that of the mature T. versicolor laccase IIIb, suggesting a limited excess of glycosylation. The laccase production level reached 2.5 mg/l (0.23 units/ml), which is suitable for engineering purpose.

International audience; Laboratory and field investigations were carried out to characterize the chemical communication system of the date palm fruit stalk borer, Oryctes elegans, and to develop pheromone-based trapping in Eastern Iran. Adults of both sexes feeding on date palm pieces attracted conspecifics, whereas date palm alone was minimally attractive. Males were twice as attractive as females. More beetles were captured at the palm crown than at ground level. Odors from adults feeding on sugarcane were sampled and analyzed by gas chromatography and mass spectrometry. Whereas females did not emit sex specific volatiles, males emitted a blend of 4-methyloctanoic acid (1: major component) and ethyl 4–methyloctanoate (2), occasionally mixed with minor components: 4-methyloctanyl acetate (3), methyl 4-methyloctanoate (4), 4-methyloctanol (5), and nonanyl acetate (6). Electroantennography and field trapping experiments demonstrated that compound 1 is an essential component of the male aggregation pheromone of O. elegans. It was barely attractive by itself but synergistic with fresh date palm odor. It attracted many more beetles than any of compounds 2–6. The addition of one or several of compounds 2–6 to 1 did not improve trap captures. During the course of 2 years, we captured 4000 beetles, with a weekly average of 6.3 beetles/trap, and were able to monitor the seasonal flight of O. elegans. Our results provide the basis for developing mass trapping for control of this pest.

The interaction between the aphid Rhopalosiphum padi (L.) and the leafhopper Psammotettix alienus was investigated in the laboratory. The leafhoppers, when physically separated from aphid but with a common air supply, caused a reduction in aphid offspring production. One of the hypotheses to explain this result is that volatile compounds might be responsible for the effect on the aphid reproduction. To verify this hypothesis, leafhopper static headspace was subjected to GC-MS (gas chromatography-mass spectrometry) analyses. An original compound was identified in the leafhopper headspace as mevalonolactone (Mev) and was found to be produced only by adults. This is the first report of Mev released in the headspace of either an insect or living organisms in general. Two other new compounds in insects were also identified in both leafhopper nymphs and adults: 2-(2-hydroxypropoxy)-1-propanol and 3,3'-oxybis-2-butanol.