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© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved., Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans., Funding was provided by the Wellcome Trust (102705, 097377, 093378, 099197, 108430, 101873), the Medical Research Council Centre for Medical Mycology and the University of Aberdeen (MR/N006364/1). K.J.K.-C is supported by the intramural program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health; V.A. by an ANR-DST COMASPIN grant (ANR-13-ISV3-0004); B.H. by German Science Foundation (www.dfg.de) grant no. HE 7565/1-1; J.-P.L., I.V. and V.A. by the ANR and FRM DEQ2015-331722; A.C. by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013), and by the Fundação para a Ciência e Tecnologia (FCT) (IF/00735/2014 and SFRH/BPD/96176/2013)

5 pages; International audience; Behavioural laterality is a commonly observed phenomenon in many species suggesting there might be an advantage of using dominantly one side over the other for certain tasks. Indeed, lateralized individuals were often shown to be more successful in cognitive tasks compared to non-lateralized conspecifics. However, stressed individuals are also often, but not always, more strongly lateralized. Because barn owl (Tyto alba) females displaying larger black spots on the tip of their ventral feathers produce offspring that are more resistant to a variety of environmental stressful factors, we examined whether laterality is associated with melanin-based coloration. We recorded whether nestlings use more often the right or left foot to scratch their body and whether they preen more often one side of the body or the other using their bills. We found that the strength of lateralization of preening and scratching was less pronounced in individuals born from heavily spotted mothers. This result might be explained by plumage-related variation in the ability to resist stressful rearing conditions.

International audience; The master regulator of the melanocyte lineage Mitf is intimately involved in development as well as melanoma, controlling cell survival, differentiation, proliferation and metastasis/migration. Consistent with its central role, Mitf expression and Mitf post-translational modifications are tightly regulated. An additional potential level of regulation is afforded by differential splicing of Mitf exon-6 leading to the generation of two isoforms that differ by the presence of six amino-acids in the Mitf (+) isoform and which have differential effects on cell cycle progression. However, whether the ratio of the two isoforms is regulated and whether their expression correlates with melanoma progression is not known. Here, we show that the differential expression of the Mitf 6a/b isoforms is dependent on the MAPKinase signalling, being linked to the activation of MEK1-ERK2, but not to N-RAS/B-RAF mutation status. In addition, quantification of Mitf 6a/b splicing forms in 86 melanoma samples revealed substantially increased levels of the Mitf (-) form in a subset of metastatic melanomas. The results suggest that differential expression of the Mitf 6a/b isoforms may represent an additional mechanism for regulating Mitf function and melanoma biology.

We have morphologically characterized Candida tropicalis isolates resistant to amphotericin B (AmB). These isolates present an enlarged cell wall compared to isolates of regular susceptibility. This correlated with higher levels of β-1,3-glucan in the cell wall but not with detectable changes in chitin content. In line with this, AmB-resistant strains showed reduced susceptibility to Congo red. Moreover, mitogen-activated protein kinases (MAPKs) involved in cell integrity were already activated during regular growth in these strains. Finally, we investigated the response elicited by human blood cells and found that AmB-resistant strains induced a stronger proinflammatory response than susceptible strains. In agreement, AmB-resistant strains also induced stronger melanization of Galleria mellonella larvae, indicating that the effect of alterations of the cell wall on the immune response is conserved in different types of hosts. Our results suggest that resistance to AmB is associated with pleiotropic mechanisms that might have important consequences, not only for the efficacy of the treatment but also for the immune response elicited by the host.

Item does not contain fulltext The Drosophila Toll-signaling pathway controls the systemic antifungal host response. Gram-negative binding protein 3 (GNBP3), a member of the beta-glucan recognition protein family senses fungal infections and activates this pathway. A second detection system perceives the activity of proteolytic fungal virulence factors and redundantly activates Toll. GNBP3(hades) mutant flies succumb more rapidly to Candida albicans and to entomopathogenic fungal infections than WT flies, despite normal triggering of the Toll pathway via the virulence detection system. These observations suggest that GNBP3 triggers antifungal defenses that are not dependent on activation of the Toll pathway. Here, we show that GNBP3 agglutinates fungal cells. Furthermore, it can activate melanization in a Toll-independent manner. Melanization is likely to be an essential defense against some fungal infections given that the entomopathogenic fungus Beauveria bassiana inhibits the activity of the main melanization enzymes, the phenol oxidases. Finally, we show that GNBP3 assembles "attack complexes", which comprise phenoloxidase and the necrotic serpin. We propose that Drosophila GNBP3 targets fungi immediately at the inception of the infection by bringing effector molecules in direct contact with the invading microorganisms. 01 mei 2010

International audience; Although a great deal is published on the MCH neurons, very few works were devoted to the study of their development. However, existing literature points out two important traits: first, these neurons differentiate a MCH phenotype very early in all species studied so far, which might suggest a role for the MCH peptide during development; second, in the rat, birth date greatly influence the phenotype of MCH neurons. At least two sub-populations were described on the basis of their chemical phenotype, projection pattern and birth date. The understanding of processes involved in the differentiation of these sub-populations may help understand the medio-lateral differentiation of the tuberal hypothalamus.

International audience; Prepro-RFRP-containing neurons have recently been described in the mammalian brain. These neurons are only found in the tuberal hypothalamus. In this work, we have provided a detailed analysis of the distribution of cells expressing the RFRP mRNA, and found them in seven anatomical structures of the tuberal hypothalamus. No co-expression with melanin-concentrating hormone (MCH) or hypocretin (Hcrt), that are also described in neurons of the tuberal hypothalamus, was observed. Using the BrdU method, we found that all RFRP cell bodies are generated between E13 and E14. Thus, RFRP neurons form a specific cell population with a complex distribution pattern in the tuberal hypothalamus. However, they are generated in one peak. These observations are discussed with data concerning the distribution and genesis of the MCH and Hcrt cell populations that are also distributed in the tuberal hypothalamus.

International audience; Background. Platelets were recently identified as a part of innate immunity. They are activated by contact with Aspergillus fumigatus; putative consequences include antifungal defense but also thrombosis, excessive inflammation, and thrombocytopenia. We aimed to identify those fungal surface structures that mediate interaction with platelets.Methods. Human platelets were incubated with Aspergillus conidia and hyphae, isolated wall components, or fungal surface mutants. Interaction was visualized microscopically; activation was quantified by flow cytometry of specific markers.Results. The capacity of A. fumigatus conidia to activate platelets is at least partly due to melanin, because this effect can be mimicked with “melanin ghosts”; a mutant lacking melanin showed reduced platelet stimulating potency. In contrast, conidial hydrophobin masks relevant structures, because an A. fumigatus mutant lacking the hydrophobin protein induced stronger platelet activation than wild-type conidia. A. fumigatus hyphae also contain surface structures that interact with platelets. Wall proteins, galactomannan, chitin, and β-glucan are not the relevant hyphal components; instead, the recently identified fungal polysaccharide galactosaminogalactan potently triggered platelet activation.Conclusions. Conidial melanin and hydrophobin as well as hyphal galactosaminogalactan represent important pathogenicity factors that modulate platelet activity and thus might influence immune responses, inflammation, and thrombosis in infected patients.

International audience; Very little is known about the sub-cellular distribution of metal ions in cells. Some metals such as zinc, copper and iron are essential and play an important role in the cell metabolism. Dysfunctions in this delicate housekeeping may be at the origin of major diseases. There is also a prevalent use of metals in a wide range of diagnostic agents and drugs for the diagnosis or treatment of a variety of disorders. This is becoming more and more of a concern in the field of nanomedicine with the increasing development and use of nanoparticles, which are suspected of causing adverse effects on cells and organ tissues. Synchrotron-based X-ray and Fourier-transformed infrared microspectroscopies are developing into well-suited sub-micrometer analytical tools for addressing new problems when studying the role of metals in biology. As a complementary tool to optical and electron microscopes, developments and studies have demonstrated the unique capabilities of multi-keV microscopy: namely, an ultra-low detection limit, large penetration depth, chemical sensitivity and three-dimensional imaging capabilities. More recently, the capabilities have been extended towards sub-100nm lateral resolutions, thus enabling sub-cellular chemical imaging. Possibilities offered by these techniques in the biomedical field are described through examples of applications performed at the ESRF synchrotron-based microspectroscopy platform (ID21 and ID22 beamlines).

International audience; In rats and mice, ascending and descending axons from neurons producing melanin-concentrating hormone (MCH) reach the cerebral cortex and spinal cord. However, these ascending and descending projections originate from distinct sub-populations expressing or not "Cocaine-and-Amphetamine-Regulated-Transcript" (CART) peptide. Using a BrdU approach, MCH cell bodies are among the very first generated in the hypothalamus, within a longitudinal cell cord made of earliest delaminating neuroblasts in the diencephalon and extending from the chiasmatic region to the ventral midbrain. This region also specifically expresses the regulatory genes Sonic hedgehog (Shh) and Nkx2.2. First MCH axons run through the tractus postopticus (tpoc) which gathers pioneer axons from the cell cord and courses parallel to the Shh/Nkx2.2 expression domain. Subsequently generated MCH neurons and ascending MCH axons differentiate while neurogenesis and mantle layer differentiation are generalized in the prosencephalon, including telencephalon. Ascending MCH axons follow dopaminergic axons of the mesotelencephalic tract, both being an initial component of the medial forebrain bundle (mfb). Netrin1 and Slit2 proteins that are involved in the establishment of the tpoc and mfb, respectively attract or repulse MCH axons.We conclude that first generated MCH neurons develop in a diencephalic segment of a longitudinal Shh/Nkx2.2 domain. This region can be seen as a prosencephalic segment of a medial neurogenic column extending from the chiasmatic region through the ventral neural tube. However, as the telencephalon expends, it exerts a trophic action and the mfb expands, inducing a switch in the longitudinal axial organization of the prosencephalon.

International audience; Two neuronal populations of the lateral hypothalamus that, respectively, produce melanin-concentrating hormone (MCH) and orexin peptides are crucially involved in control of metabolism, feeding and related goal-oriented behaviors. In contrast to orexin neurons, mainly involved in short-term regulation of feeding, MCH neurons participate in long-term control of energy storage and body weight. Beyond its effect on feeding, MCH has also been shown to be involved in regulation of seeking behavior and addiction through modulation of dopamine (DA) metabolism. This regulation is essential for reinforcement-associated behaviors. Moreover, drugs of abuse, which increase extracellular DA levels, are known to decrease food intake. Consistent with this observation, DA has been shown to modulate orexin neurons of the lateral hypothalamus. However, no study is available concerning the effects of DA on MCH neurons. Whole-cell patch-clamp recordings were done in hypothalamic mouse brain slices. MCH neurons were identified by Tau-Cyan-GFP labeling using a transgenic mouse model (MCH-GFP). First, we show that DA (10-200 μM) induces an outward current in MCH neurons. However, this current is not due to activation of DA receptors, but mediated through activation of α2-noradrenergic receptors and subsequent opening of G-protein activated inward rectifier K+ (GIRK) channels. Current-clamp experiments revealed that this GIRK-activation leads to hyperpolarization, thus decreasing excitability of MCH neurons. Furthermore, we confirm that MCH neurons receive mainly GABAergic inputs rather than glutamatergic ones. We show that DA modulates these inputs in a complex manner: at low concentrations, DA activates D1-like receptors, promoting presynaptic activity, whereas, at higher concentrations (100 μM), D2-like receptor activation inhibits presynaptic activity. Overall, DA should lead to a decrease in MCH neuron excitability, likely resulting in down-regulation of MCH release and feeding behavior.

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Background Aspergillus fumigatus is the most common agent of invasive aspergillosis, a feared complication in severely immunocompromised patients. Despite the recent commercialisation of new antifungal drugs, the prognosis for this infection remains uncertain. Thus, there is a real need to discover new targets for therapy. Particular attention has been paid to the biochemical composition and organisation of the fungal cell wall, because it mediates the host-fungus interplay. Conidia, which are responsible for infections, have melanin as one of the cell wall components. Melanin has been established as an important virulence factor, protecting the fungus against the host's immune defences. We suggested that it might also have an indirect role in virulence, because it is required for correct assembly of the cell wall layers of the conidia. Results We used three A. fumigatus isolates which grew as white or brown powdery colonies, to demonstrate the role of melanin. Firstly, sequencing the genes responsible for biosynthesis of melanin (ALB1, AYG1, ARP1, ARP2, ABR1 and ABR2) showed point mutations (missense mutation, deletion or insertion) in the ALB1 gene for pigmentless isolates or in ARP2 for the brownish isolate. The isolates were then shown by scanning electron microscopy to produce numerous, typical conidial heads, except that the conidia were smooth-walled, as previously observed for laboratory mutants with mutations in the PKSP/ALB1 gene. Flow cytometry showed an increase in the fibronectin binding capacity of conidia from mutant isolates, together with a marked decrease in the binding of laminin to the conidial surface. A marked decrease in the electronegative charge of the conidia and cell surface hydrophobicity was also seen by microelectrophoresis and two-phase partitioning, respectively. Ultrastructural studies of mutant isolates detected considerable changes in the organisation of the conidial wall, with the loss of the outermost electron dense layer responsible for the ornamentations seen on the conidial surface in wild-type strains. Finally, analysis of the conidial surface of mutant isolates by atomic force microscopy demonstrated the absence of the outer cell wall rodlet layer which is composed of hydrophobins. Conclusion These results suggest that, in addition to a protective role against the host's immune defences, melanin is also a structural component of the conidial wall that is required for correct assembly of the cell wall layers and the expression at the conidial surface of adhesins and other virulence factors.

International audience; Numerous works associate the MCH peptide, and the hypothalamic neurons that produce it, to the feeding behavior and energy homeostasis. It is commonly admitted that MCH is an orexigenic peptide, and MCH neurons could be under the control of arcuate NPY and POMC neurons. However, the literature data is not always concordant. In particular questions about the intrahypothalamic circuit involving other neuropeptides and about the mechanisms through which MCH could act are not yet clearly answered. For example, which receptors mediate a MCH response to NPY or alpha-MSH, does MCH act alone, is there any local anatomical organization within the tuberal LHA? A review of the current literature is then needed to help focus attention on these unresolved and often neglected issues.

Pls1 tetraspanins were shown for some pathogenic fungi to be essential for appressorium-mediated penetration into their host plants. We show here that Podospora anserina , a saprobic fungus lacking appressorium, contains PaPls1 , a gene orthologous to known PLS1 genes. Inactivation of PaPls1 demonstrates that this gene is specifically required for the germination of ascospores in P. anserina . These ascospores are heavily melanized cells that germinate under inducing conditions through a specific pore. On the contrary, MgPLS1 , which fully complements a Δ PaPls1 ascospore germination defect, has no role in the germination of Magnaporthe grisea nonmelanized ascospores but is required for the formation of the penetration peg at the pore of its melanized appressorium. P. anserina mutants with mutation of PaNox2 , which encodes the NADPH oxidase of the NOX2 family, display the same ascospore-specific germination defect as the Δ PaPls1 mutant. Both mutant phenotypes are suppressed by the inhibition of melanin biosynthesis, suggesting that they are involved in the same cellular process required for the germination of P. anserina melanized ascospores. The analysis of the distribution of PLS1 and NOX2 genes in fungal genomes shows that they are either both present or both absent. These results indicate that the germination of P. anserina ascospores and the formation of the M. grisea appressorium penetration peg use the same molecular machinery that includes Pls1 and Nox2. This machinery is specifically required for the emergence of polarized hyphae from reinforced structures such as appressoria and ascospores. Its recurrent recruitment during fungal evolution may account for some of the morphogenetic convergence observed in fungi.

International audience; This study aims to assess type of melanin as a risk indicator for skin tumors, in a sample of melanoma cases and controls within a larger multicenter study (Helios 2), held in Europe and South America in 2001-2002. In each case and control, the melanin content in hair was assessed by three methods: 1) the amount of 2,3,5-pyrroletricarboxylic acid (PTCA); 2) the absorbance ratio with ultraviolet spectroscopy; and 3) the spectra of near-infrared spectroscopy. Statistical analysis was performed in a Bayesian setting, defining priors for confounders and effect modifiers from the larger study data set. Subjects with values of PTCA of less than 85 ng/mg carried an increased risk (26 vs. seven discordant pairs: odds ratio = 4.4, 95% confidence interval: 1.52, 14.54), adjusted by hair color, eye color, and number of nevi (n = > or =40). The absorbance ratio showed a weaker and nonsignificant odds ratio of 1.5. After correction by misclassification, near-infrared spectroscopy was associated with an odds ratio of 2.3 (95% confidence interval: 1.36, 4.22). The amount of PTCA is thus a strong and independent risk indicator for melanoma. Incorporating PTCA determination into epidemiologic studies is therefore recommended.

A number of different neuropeptides exert powerful concerted controls on feeding behavior and energy balance, most of them being produced in hypothalamic neuronal networks under stimulation by anabolic and catabolic peripheral hormones such as ghrelin and leptin, respectively. These peptide-expressing neurons interconnect extensively to integrate the multiple opposing signals that mediate changes in energy expenditure. In the present review I have summarized our current knowledge about two key peptidic systems involved in regulating appetite and energy homeostasis, the melanocortin system (alpha-MSH, agouti and Agouti-related peptides, MC receptors and mahogany protein) and the melanin-concentrating hormone system (proMCH-derived peptides and MCH receptors) that contribute to satiety and feeding-initiation, respectively, with concurrent effects on energy expenditure. I have focused particularly on recent data concerning transgenic mice and the ongoing development of MC/MCH receptor antagonists/agonists that may represent promising drugs to treat human eating disorders on both sides of the energy balance (anorexia, obesity).

The melanization reaction is used as an immune mechanism in arthropods to encapsulate and kill microbial pathogens. In Drosophila, the serpin Spn27A regulates melanization apparently by inhibiting the protease that activates phenoloxidase, the key enzyme in melanin synthesis. Here, we have described the genetic characterization of two immune inducible serine proteases, MP1 and MP2, which act in a melanization cascade regulated by Spn27A. MP1 is required to activate melanization in response to both bacterial and fungal infection, whereas MP2 is mainly involved during fungal infection. Pathogenic bacteria and fungi may therefore trigger two different melanization cascades that use MP1 as a common downstream protease to activate phenoloxidase. We have also shown that the melanization reaction activated by MP1 and MP2 plays an important role in augmenting the effectiveness of other immune reactions, thereby promoting resistance of Drosophila to microbial infection.

International audience; The perifornical-lateral hypothalamic area is implicated in regulating waking and paradoxical sleep. The blockade of GABAA receptors by iontophoretic applications of bicuculline (or gabazine) into the perifornical-lateral hypothalamic area induced a continuous quiet waking state associated to a robust muscle tone in head-restrained rats. During the effects, sleep was totally suppressed. In rats killed at the end of a 90 min ejection of bicuculline, Fos expression was induced in approximately 28% of the neurons immunoreactive for hypocretin and in approximately 3% of the neurons immunostained for melanin-concentrating hormone within the ejection site. These results suggest that neurons containing melanin-concentrating hormone are not active during waking and that the lack of a potent GABAergic influence during waking is consistent with their role in sleep regulation.

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Background Analytical imaging by secondary ion mass spectrometry (SIMS) provides images representative of the distribution of a specific ion within a sample surface. For the last fifteen years, concerted collaborative research to design a new ion microprobe with high technical standards in both mass and lateral resolution as well as in sensitivity has led to the CAMECA NanoSims 50, recently introduced onto the market. This instrument has decisive capabilities, which allow biological applications of SIMS microscopy at a level previously inaccessible. Its potential is illustrated here by the demonstration of the specific affinity of a melanoma marker for melanin. This finding is of great importance for the diagnosis and/or treatment of malignant melanoma, a tumour whose worldwide incidence is continuously growing. Methods The characteristics of the instrument are briefly described and an example of application is given. This example deals with the intracellular localization of an iodo-benzamide used as a diagnostic tool for the scintigraphic detection of melanic cells (e.g. metastasis of malignant melanoma). B16 melanoma cells were injected intravenously to C57BL6/J1/co mice. Multiple B16 melanoma colonies developed in the lungs of treated animals within three weeks. Iodobenzamide was injected intravenously in tumour bearing mice six hours before sacrifice. Small pieces of lung were prepared for SIMS analysis. Results Mouse lung B16 melanoma colonies were observed with high lateral resolution. Cyanide ions gave "histological" images of the cell, representative of the distribution of C and N containing molecules (e.g. proteins, nucleic acids, melanin, etc.) while phosphorus ions are mainly produced by nucleic acids. Iodine was detected only in melanosomes, confirming the specific affinity of the drug for melanin. No drug was found in normal lung tissue. Conclusion This study demonstrates the potential of SIMS microscopy, which allows the study of ultra structural distribution of a drug within a cell. On the basis of our observations, drug internalization via membrane sigma receptors can be excluded.

Background Peptidergic neurons containing the melanin-concentrating hormone (MCH) and the hypocretins (or orexins) are intermingled in the zona incerta, perifornical nucleus and lateral hypothalamic area. Both types of neurons have been implicated in the integrated regulation of energy homeostasis and body weight. Hypocretin neurons have also been involved in sleep-wake regulation and narcolepsy. We therefore sought to determine whether hypocretin and MCH neurons express Fos in association with enhanced paradoxical sleep (PS or REM sleep) during the rebound following PS deprivation. Next, we compared the effect of MCH and NaCl intracerebroventricular (ICV) administrations on sleep stage quantities to further determine whether MCH neurons play an active role in PS regulation. Results Here we show that the MCH but not the hypocretin neurons are strongly active during PS, evidenced through combined hypocretin, MCH, and Fos immunostainings in three groups of rats (PS Control, PS Deprived and PS Recovery rats). Further, we show that ICV administration of MCH induces a dose-dependant increase in PS (up to 200%) and slow wave sleep (up to 70%) quantities. Conclusion These results indicate that MCH is a powerful hypnogenic factor. MCH neurons might play a key role in the state of PS via their widespread projections in the central nervous system.

International audience; Obesity is a complex disease involving genetic components and environmental factors and probably associated with the dysregulation of central homeostasis normally maintained by the hypothalamic neuroendocrine/neurotransmitter network. We previously reported that canine distemper virus (CDV), which is closely related to human measles virus, can target hypothalamic nuclei, and lead to obesity syndrome in the late stages of infection. Here, using differential display PCR, we demonstrate specific down-regulation of melanin-concentrating hormone precursor mRNA (ppMCH) in infected-obese mice. Although ppMCH was down-regulated in all infected mice during the acute stage of infection, this was only seen during the late stage of infection in infected-obese mice. In addition, ppMCH mRNA and protein expression in the lateral hypothalamus was decreased in the absence of neuronal death. These results show the importance of ppMCH in the establishment and maintenance of obesity and the involvement of a virus as an environmental factor.