Francesca Boscaro

Cuticular hydrocarbons and reproductive status in the social wasp Polistes dominulus

Abstract. Most species of social insect are characterized by a reproductive division of labor among morphologically specialized individuals. In contrast, there exist many species where all individuals are morphologically identical and dominance relationships determine which individuals mate and/or reproduce. In newly founded multiple-foundress associations of the social wasp Polistes dominulus, foundresses establish dominance hierarchies where the top-ranked (alpha) female monopolizes egg laying. The possibility that chemical cues are used for recognition of egg-laying individuals has not been explored in this wasp. Using non-destructive techniques, we examined the relationship between ovarian activity and the proportions of cuticular hydrocarbons of three female types (dominant and subordinate foundresses and workers) in 11 colonies. Immediately after nest foundation, no differences were found between alpha and subordinate females. In contrast, at worker emergence, alpha females were statistically distinguishable from both subordinates and workers. We experimentally removed the alpha female in 5 of the original nests and reanalyzed hydrocarbon proportions of the new dominant individual. Replacement individuals were all found to acquire a cuticular signature characteristic of the alpha female. This suggests that cuticular hydrocarbons are used as cues of ovarian activity in P. dominulus, and we discuss our results in terms of a switch from behavioral dominance to chemical signaling in this wasp.

Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps

Background Histone deimination regulates gene function and contributes to antimicrobial response, allowing the formation of neutrophil extracellular traps (NETs). Deiminated proteins are target of anti-citrullinated peptides antibodies (ACPA) in rheumatoid arthritis (RA). Objective The objective of this paper is to test the hypothesis that RA sera react with deiminated histones contained in NETs. Methods Neutrophils from peripheral blood were stimulated with A23187 and acid treated; NETosis was induced by phorbol myristate acetate, and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs, and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were excised from gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) analysis, before and after derivatisation to detect citrullinated peptides. Results RA sera reacted with a deiminated antigen of 11 KDa from activated neutrophils, recognised also by anti-H4 and antideiminated H4 antibodies. A similar reactivity was observed with NET proteins. The antigen from neutrophils or NETs was identified as citrullinated H4 by MALDI-TOF analysis. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14–34 and 31–50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titre was correlated with anti-CCP2. Conclusions Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA, and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA.

Odour signals major histocompatibility complex genotype in an Old World monkey

The major histocompatibility complex (MHC) is an extraordinarily diverse cluster of genes that play a key role in the immune system. MHC gene products are also found in various body secretions, leading to the suggestion that MHC genotypes are linked to unique individual odourtypes that animals use to assess the suitability of other individuals as potential mates or social partners. We investigated the relationship between chemical odour profiles and genotype in a large, naturally reproducing population of mandrills, using gas chromatography–mass spectrometry and MHC genotyping. Odour profiles were not linked to the possession of particular MHC supertypes. Sex influenced some measures of odour diversity and dominance rank influenced some measures of odour diversity in males, but not in females. Odour similarity was strongly related to similarity at the MHC, and, in some cases, to pedigree relatedness. Our results suggest that odour provides both a cue of individual genetic quality and information against which the receiver can compare its own genotype to assess genetic similarity. These findings provide a potential mechanism underlying mate choice for genetic diversity and MHC similarity as well as kin selection.

Chemical Composition of Scent-Gland Secretions in an Old World Monkey (Mandrillus sphinx): Influence of Sex, Male Status, and Individual Identity

Primates are traditionally considered to be microsmatic, with decreased reliance on olfactory senses in comparison to other sensory modalities such as vision. This is particularly the case for Old World monkeys and apes (catarrhines). However, various lines of evidence suggest that chemical communication may be important in these species, including the presence of a sternal scent-gland in the mandrill. We investigated the volatile components of mandrill odor using gas chromatography-mass spectrometry. We identified a total of 97 volatile components in 88 swabs of the sternal gland secretion and 95 samples of sternal gland hair saturated with scent-gland secretion collected from 27 males and 18 females. We compared odor profiles with features of the signaler using principle components and discriminant function analyses and found that volatile profiles convey both variable (age, dominance rank in males) and fixed (sex, possibly individual identity) information about the signaler. The combination of an odor profile that signals sex, age, and rank with increased motivation to scent-mark and increased production of secretion in high-ranking males leads to a potent signal of the presence of a dominant, adult male with high testosterone levels. This may be particularly relevant in the dense Central African rain forest which mandrills inhabit. By contrast, we were unable to differentiate between either female cycle stage or female rank based on odor profiles, which accords with behavioral studies suggesting that odor signals are not as important in female mandrills as they are in males. The similarity of our findings to those for other mammals and in primates that are more distantly related to humans suggests a broader role for odor in primate communication than is currently recognized.

Anamorsin Is a [2Fe-2S] Cluster-Containing Substrate of the Mia40-Dependent Mitochondrial Protein Trapping Machinery.

Human anamorsin was implicated in cytosolic iron-sulfur (Fe/S) protein biogenesis. Here, the structural and metal-binding properties of anamorsin and its interaction with Mia40, a well-known oxidoreductase involved in protein trapping in the mitochondrial intermembrane space (IMS), were characterized. We show that (1), anamorsin contains two structurally independent domains connected by an unfolded linker; (2), the C-terminal domain binds a [2Fe-2S] cluster through a previously unknown cysteine binding motif in Fe/S proteins; (3), Mia40 specifically introduces two disulfide bonds in a twin CX(2)C motif of the C-terminal domain; (4), anamorsin and Mia40 interact through an intermolecular disulfide-bonded intermediate; and (5), anamorsin is imported into mitochondria. Hence, anamorsin is the first identified Fe/S protein imported into the IMS, raising the possibility that it plays a role in cytosolic Fe/S cluster biogenesis also once trapped in the IMS.

A proteomic investigation of soluble olfactory proteins in Anopheles gambiae

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector.

Electrospray MS and MALDI imaging show that non‐specific lipid‐transfer proteins (LTPs) in tomato are present as several isoforms and are concentrated in seeds

Non-specific lipid-transfer proteins (nsLTPs) are major human allergens in many plant species, albeit their role in plant biochemistry is still undefined. They are found in many plant species, either as one or several isoforms according to the species, and usually they are found to concentrate in the outer part of the fruits. In this work, the characterization of tomato nsLTP isoforms was performed on the three main fractions of Piccadilly tomato fruit (peel, pulp and seeds) by using ultracentrifuge devices with molecular cut-off able to retain proteins with molecular weight typical of plant LTPs. The isolated proteins were further analysed by LC-MS, in order to investigate the occurrence and the localization of tomato LTP isoforms. The chromatographic retention times, the molecular masses, the presence of eight cysteine residues in their tertiary structures and the sequence information obtained by MS, although not complete yet, allowed us to identify four different LTP isoforms, not yet reported in the literature, which were found to be concentrated in the seed fractions. None of the molecular masses of these potential LTPs was already present in the UniProtKB/SwissProt database. MALDI imaging experiments confirmed their presence and main localization in seeds, although the actual data hinted at their presence around seeds, rather than exactly in them. These data hint to a complicated scenario concerning LTP proteins in tomato.

Hippocampal long term memory: Effect of the cholinergic system on local protein synthesis

The present study was aimed at establishing a link between the cholinergic system and the pathway of mTOR and its downstream effector p70S6K, likely actors in long term memory encoding. We performed in vivo behavioral experiments using the step down inhibitory avoidance test (IA) in adult Wistar rats to evaluate memory formation under different conditions, and immunohistochemistry on hippocampal slices to evaluate the level and the time-course of mTOR and p70S6K activation. We also examined the effect of RAPA, inhibitor of mTORC1 formation, and of the acetylcholine (ACh) muscarinic receptor antagonist scopolamine (SCOP) or ACh nicotinic receptor antagonist mecamylamine (MECA) on short and long term memory formation and on the functionality of the mTOR pathway. Acquisition test was performed 30 min after i.c.v. injection of RAPA, a time sufficient for the drug to diffuse to CA1 pyramidal neurons, as demonstrated by MALDI-TOF-TOF imaging. Recall test was performed 1 h, 4 h or 24 h after acquisition. To confirm our results we performed in vitro experiments on live hippocampal slices: we evaluated whether stimulation of the cholinergic system with the cholinergic receptor agonist carbachol (CCh) activated the mTOR pathway and whether the administration of the above-mentioned antagonists together with CCh could revert this activation. We found that (1) mTOR and p70S6K activation in the hippocampus were involved in long term memory formation; (2) RAPA administration caused inhibition of mTOR activation at 1 h and 4 h and of p70S6K activation at 4 h, and long term memory impairment at 24 h after acquisition; (3) scopolamine treatment caused short but not long term memory impairment with an early increase of mTOR/p70S6K activation at 1 h followed by stabilization at longer times; (4) mecamylamine plus scopolamine treatment caused short term memory impairment at 1 h and 4 h and reduced the scopolamine-induced increase of mTOR/p70S6K activation at 1 h and 4 h; (5) mecamylamine plus scopolamine treatment did not impair long term memory formation; (6) in vitro treatment with carbachol activated mTOR and p70S6K and this effect was blocked by scopolamine and mecamylamine. Taken together our data reinforce the idea that distinct molecular mechanisms are at the basis of the two different forms of memory and are in accordance with data presented by other groups that there exist molecular mechanisms that underlie short term memory, others that underlie long term memories, but some mechanisms are involved in both.

Sternal Gland Scent-Marking Signals Sex, Age, Rank, and Group Identity in Captive Mandrills.

Mandrills are one of the few Old World primates to show scent-marking. We combined ethological and chemical approaches to improve our understanding of this behavior in 3 zoo-managed groups. We observed the olfactory behavior performed by adults and adolescents (N = 39) for 775h. We investigated the volatile components of sternal scent-marks using gas chromatography-mass spectrometry and compared volatile profiles with traits of the signaler. Males marked more than females and within each sex the frequency of scent-marking was related to age and dominance status, but alpha males scent-marked most frequently and particularly in specific areas at the enclosure boundaries. We identified a total of 77 volatile components of sternal gland secretion, including compounds functioning as male sex pheromones in other mammals, in scent-marks spontaneously released on filter paper by 27 male and 18 female mandrills. We confirmed our previous findings that chemical profiles contain information including sex, male age and rank, and we also found that odor may encode information about group membership in mandrills. Our results support the hypotheses that scent-marking signals the status of the dominant male as well as playing territorial functions but also suggest that it is part of sociosexual communication.

A novel allergen-adjuvant conjugate suitable for specific immunotherapy of respiratory allergy.

BACKGROUND Several approaches to find a better adjuvant, focus immunomodulation, and reduce allergenicity are under investigation to improve the efficacy and safety of specific immunotherapy. OBJECTIVE We performed an investigation of the in vitro and in vivo effects of a purified allergen chemically conjugated to a novel 8-OH modified adenine as an adjuvant. METHODS Purified group 2 major allergen from house dust mite chemically conjugated to 4-(6-amino-9-benzyl-8-hydroxy-9H-purin-2-ylsulfanyl)-butyric acid succinimidyl ester was analyzed by using mass spectrometry. The adduct (nDer p 2-Conj) was assayed for Toll-like receptor activation on transfected HEK293 cells, stimulation of innate cells, and effects on the functional phenotype of specific T-cell lines and clones by means of flow cytometry, real-time PCR, and expression of TH-related transcription factors. Lung cells and sera of nDer p 2-Conj-sensitized C57Bl/6 mice were studied by means of cytology, histology, real-time PCR, and ELISA. RESULTS nDer p 2-Conj stimulated IL-12 and IFN-α production from monocytes and plasmacytoid dendritic cells, respectively, retaining the ability to trigger Toll-like receptor 7 exclusively, and expanded human allergen-specific lymphocytes with reduced ability to produce T(H)2-related cytokines and increased IFN-γ levels, as based on GATA-3/T-bet expression. In vivo adduct-sensitized mice exhibited reduced eosinophil infiltration and IL-13 expression in the airways, IFN-γ upregulation together with IgE downregulation, and an increase in allergen-specific IgG(2a) levels in sera. The conjugate exhibited reduced ability to activate human FcεRI(+) cells without inducing T(H)17 cells or autoantibodies. CONCLUSIONS The codelivery of an allergen with a modified adenine as a conjugate inducing modulatory cytokines from innate cells redirects in vitro and in vivo pathogenic TH2 responses without eliciting harmful effects.