The bZIP transcription factor Rca1p is a central regulator of a novel CO₂ sensing pathway in yeast.

Like many organisms the fungal pathogen Candida albicans senses changes in the environmental CO(2) concentration. This response involves two major proteins: adenylyl cyclase and carbonic anhydrase (CA). Here, we demonstrate that CA expression is tightly controlled by the availability of CO(2) and identify the bZIP transcription factor Rca1p as the first CO(2) regulator of CA expression in yeast. We show that Rca1p upregulates CA expression during contact with mammalian phagocytes and demonstrate that serine 124 is critical for Rca1p signaling, which occurs independently of adenylyl cyclase. ChIP-chip analysis and the identification of Rca1p orthologs in the model yeast Saccharomyces cerevisiae (Cst6p) point to the broad significance of this novel pathway in fungi. By using advanced microscopy we visualize for the first time the impact of CO(2) build-up on gene expression in entire fungal populations with an exceptional level of detail. Our results present the bZIP protein Rca1p as the first fungal regulator of carbonic anhydrase, and reveal the existence of an adenylyl cyclase independent CO(2) sensing pathway in yeast. Rca1p appears to regulate cellular metabolism in response to CO(2) availability in environments as diverse as the phagosome, yeast communities or liquid culture.

Diagnosis of invasive candidiasis in the ICU.

Invasive candidiasis ranges from 5 to 10 cases per 1,000 ICU admissions and represents 5% to 10% of all ICU-acquired infections, with an overall mortality comparable to that of severe sepsis/septic shock. A large majority of them are due to Candida albicans, but the proportion of strains with decreased sensitivity or resistance to fluconazole is increasingly reported. A high proportion of ICU patients become colonized, but only 5% to 30% of them develop an invasive infection. Progressive colonization and major abdominal surgery are common risk factors, but invasive candidiasis is difficult to predict and early diagnosis remains a major challenge. Indeed, blood cultures are positive in a minority of cases and often late in the course of infection. New nonculture-based laboratory techniques may contribute to early diagnosis and management of invasive candidiasis. Both serologic (mannan, antimannan, and betaglucan) and molecular (Candida-specific PCR in blood and serum) have been applied as serial screening procedures in high-risk patients. However, although reasonably sensitive and specific, these techniques are largely investigational and their clinical usefulness remains to be established. Identification of patients susceptible to benefit from empirical antifungal treatment remains challenging, but it is mandatory to avoid antifungal overuse in critically ill patients. Growing evidence suggests that monitoring the dynamic of Candida colonization in surgical patients and prediction rules based on combined risk factors may be used to identify ICU patients at high risk of invasive candidiasis susceptible to benefit from prophylaxis or preemptive antifungal treatment.

In Vitro Activity of Gentamicin-Loaded Bioabsorbable Beads against Different Microorganisms

Abstract: Osteomyelitis is responsible for high treatment costs, long hospital stays, and results in substantial morbidity. Treatment with surgical debridement and antibiotic-impregnated Polymethylmetacrylate (PMMA) beads is the standard of care, providing high local but low serum antibiotic concentrations, thereby avoiding systemic toxicity. However, for several reasons, the beads require surgical removal. Alternative antibiotic delivery systems should improve the treatment of bone infection, actively encourage bone healing and require no additional surgery for removal. We investigated the activity of gentamicin-loaded bioabsorbable beads against different microorganisms (Staphylococcus epidermidis, S. aureus, Escherichia coli, Enterococcus faecalis, Candida albicans) commonly causing surgical site bone infection, by microcalorimetry. Calcium sulphate beads containing gentamicin were incubated in microcalorimetry ampoules containing different concentrations of the corresponding microorganism. Growth medium with each germ and unloaded beads was used as positive control, growth medium with loaded beads alone as negative control. Bacterial growth-related heat production at 37 °C was measured for 24 h. Cultures without gentamicin-loaded beads produced heat-flow peaks corresponding to the exponential growth of the corresponding microorganisms in nutrient-rich medium. In contrast, cultures with gentamicin-loaded beads completely suppressed heat production during 24 h, demonstrating their antibiotic activity. Gentamicin-loaded beads effectively inhibited growth of susceptible microorganisms, under the described in vitro conditions.

Peroxisome proliferator-activated receptor gamma activation is required for maintenance of innate antimicrobial immunity in the colon.

Crohn's disease (CD), a major form of human inflammatory bowel disease, is characterized by primary immunodeficiencies. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is essential for intestinal homeostasis in response to both dietary- and microbiota-derived signals. Its role in host defense remains unknown, however. We show that PPARgamma functions as an antimicrobial factor by maintaining constitutive epithelial expression of a subset of beta-defensin in the colon, which includes mDefB10 in mice and DEFB1 in humans. Colonic mucosa of Ppargamma mutant animals shows defective killing of several major components of the intestinal microbiota, including Candida albicans, Bacteroides fragilis, Enterococcus faecalis, and Escherichia coli. Neutralization of the colicidal activity using an anti-mDefB10 blocking antibody was effective in a PPARgamma-dependent manner. A functional promoter variant that is required for DEFB1 expression confers strong protection against Crohn's colitis and ileocolitis (odds ratio, 0.559; P = 0.018). Consistently, colonic involvement in CD is specifically linked to reduced expression of DEFB1 independent of inflammation. These findings support the development of PPARgamma-targeting therapeutic and/or nutritional approaches to prevent colonic inflammation by restoring antimicrobial immunity in CD.

In vitro activity of gentamicin-loaded bioabsorbable beads against different microorganisms

Grâce à la prophylaxie antibiotique per-opératoire, à l'amélioration des techniques chirurgicales et au flux laminaire dans les blocs opératoires le taux d'infections postopératoires a pu être diminué dans les interventions chirurgicales orthopédiques. Il stagne néanmoins à environ 0,5- 2% dans les fractures fermées ainsi que dans les interventions orthopédiques électives, et peut monter jusqu'à 30% dans les fractures ouvertes stade III. L'ostéomyelite et la pseudarthrose infectée, qui peuvent en découler, sont responsables de séjours hospitaliers prolongés, engendrent des coûts de traitement élevés, et conduisent souvent à une morbidité substantielle. Le traitement de l'ostéomyelite combine le débridement chirurgical et l'administration d'antibiotiques. Cependant l'administration systémique d'antibiotiques à hautes doses est problématique en raison de leur toxicité systémique et de leur faible pénétration dans les tissus ischémiques et nécrotiques, souvent présents dans l'ostéomyelite. Pour ces raisons, le traitement standard actuel consiste, après débridement chirurgical, en la mise en place de billes de Polyrnéthylmétacrylate (PMMA) imprégnées d'antibiotique, qui fournissent des concentrations locales élevées mais systémiques basses. Malheureusement, ces billes doivent être enlevées une fois l'infection guérie, ce qui nécessite une nouvelle intervention chirurgicale. Des systèmes de libération antibiotique alternatifs devraient non seulement guérir l'infection osseuse, mais également encourager activement la consolidation osseuse et ne pas nécessiter de nouvelle chirurgie pour leur ablation. Nous avons investigué l'activité de billes résorbables chargées en gentamicine contre différents microorganismes (Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Candida albicans), des germes communément responsables d'infections osseuses, par microcalorimétrie, une méthode novice basée sur la mesure de la chaleur produite par des microorganismes qui se répliquent dans une culture. Des billes composées essentiellement de sulfate de calcium et chargées en gentamicine ont été incubées dans des ampoules microcalorimétriques contenant différentes concentrations du germe correspondant. Les bouillons de culture avec chaque germe et billes non-chargées ont été utilisés comme contrôle positif, le bouillon de culture avec bille chargée mais sans germe comme contrôle négatif. La production de chaleur provenant de la croissance bactérienne à 37°C a été mesurée pendant 24 heures. Les cultures ne contenant pas de billes chargées en gentamicine ont. produit des pics de chaleur équivalents à la croissance exponentielle du microorganisme correspondant en milieu riche. Par contre, les germes susceptibles à la gentamicine incubés avec les billes chargées ont complètement supprimé leur production de chaleur pendant 24 heures, démontrant ainsi l'activité antibiotique des billes chargées en gentamicine contre ces germes. Les billes résorbables chargées en gentamicine inhibent donc de façon effective la croissance des germes susceptibles sous les conditions in vitro décrites. Des études sur animaux sont maintenant nécessaires pour déterminer la cinétique d'élution et l'effet antimicrobien de la gentamicine sous conditions in vivo. Finalement des études cliniques devront démontrer que l'utilisation de ces billes est effectivement une bonne option thérapeutique dans le traitement des infections osseuses.

Development of an aggregation assay to screen FimH antagonists.

Alpha-D-mannopyranosides are potent FimH antagonists, which inhibit the adhesion of Escherichia coli to highly mannosylated uroplakin Ia on the urothelium and therefore offer an efficient therapeutic opportunity for the treatment and prevention of urinary tract infection. For the evaluation of the therapeutic potential of FimH antagonists, their effect on the disaggregation of E. coli from Candida albicans and guinea pig erythrocytes (GPE) was studied. The mannose-specific binding of E. coli to yeast cells and erythrocytes is mediated by type 1 pili and can be monitored by aggregometry. Maximal aggregation of C. albicans or GPE to E. coli is reached after 600 s. Then the FimH antagonist was added and disaggregation determined by light transmission over a period of 1400 s. A FimH-deleted mutant of E. coli, which does not induce any aggregation, was used in a control experiment. The activities of FimH antagonists are expressed as IC(50)s, the half maximal inhibitory concentration of the disaggregation potential. n-Heptyl alpha-D-mannopyranoside (1) was used as a reference compound and exhibits an IC(50) of 77.14 microM , whereas methyl alpha-D-mannopyranoside (2) does not lead to any disaggregation at concentrations up to 800 microM. o-Chloro-p-[N-(2-ethoxy-3,4-dioxocyclobut-1-enyl)amino]phenyl alpha-D-mannopyranoside (3) shows a 90-fold and 2-chloro-4-nitrophenyl alpha-D-mannopyranoside (4) a 6-fold increased affinity compared to 1. Finally, 4-nitrophenyl alpha-D-mannopyranoside (5) exhibits an activity similar to 1. As negative control, D-galactose (6) was used. The standardized aggregation assay generates concentration-dependent, reproducible data allowing the evaluation of FimH antagonists according to their potency to inhibit E. coli adherence and can therefore be employed to select candidates for experimental and clinical studies for treatment and prevention of urinary tract infections.

Sensing of mammalian IL-17A regulates fungal adaptation and virulence.

Infections by opportunistic fungi have traditionally been viewed as the gross result of a pathogenic automatism, which makes a weakened host more vulnerable to microbial insults. However, fungal sensing of a host's immune environment might render this process more elaborate than previously appreciated. Here we show that interleukin (IL)-17A binds fungal cells, thus tackling both sides of the host-pathogen interaction in experimental settings of host colonization and/or chronic infection. Global transcriptional profiling reveals that IL-17A induces artificial nutrient starvation conditions in Candida albicans, resulting in a downregulation of the target of rapamycin signalling pathway and in an increase in autophagic responses and intracellular cAMP. The augmented adhesion and filamentous growth, also observed with Aspergillus fumigatus, eventually translates into enhanced biofilm formation and resistance to local antifungal defenses. This might exemplify a mechanism whereby fungi have evolved a means of sensing host immunity to ensure their own persistence in an immunologically dynamic environment.

Antifungal drug resistance mechanisms in fungal pathogens from the perspective of transcriptional gene regulation.

Fungi are primitive eukaryotes and have adapted to a variety of niches during evolution. Some fungal species may interact with other life forms (plants, insects, mammals), but are considered as pathogens when they cause mild to severe diseases. Chemical control strategies have emerged with the development of several drugs with antifungal activity against pathogenic fungi. Antifungal agents have demonstrated their efficacy by improving patient health in medicine. However, fungi have counteracted antifungal agents in several cases by developing resistance mechanisms. These mechanisms rely on drug resistance genes including multidrug transporters and drug targets. Their regulation is crucial for the development of antifungal drug resistance and therefore transcriptional factors critical for their regulation are being characterized. Recent genome-wide studies have revealed complex regulatory circuits involving these genetic and transcriptional regulators. Here, we review the current understanding of the transcriptional regulation of drug resistance genes from several fungal pathogens including Candida and Aspergillus species.

Phytochemical investigation of alpine plants : the cottonsedges Eriophorum scheuchzeri Hoppe, E. angustifolium Honck. and E. latifolium Hoppe (CYPERACEAE)

Résumé: Alpine plants living at high altitudes undergo a series of climatic stress factors (chilling, enhanced UV radiation, short growing season, low nutriment supply...) which may influence their secondary compounds composition. Many publications showed in these last years that plants under stress conditions do synthesize a range of specific defence compounds (terpenes, flavonoids, coumarines...). A careful phytochemical investigation of those plants could therefore lead to the discovery of active molecules. Thus, for the biological and chemical screening, about 30 alpine plants have been collected above 2000 metres, in the alpine grass-lands. Eriophorum scheuchzeri Hoppe (Cyperaceae), not yet investigated phytochemically, revealed in its lipophilic and polar extracts the presence of various radical scavengers in a TLC autography with the DPPH (2,2-dipheny1-1- picrylhydrazyl) radical as spray reagent, as well as several antifungal compounds acitve against Cladosporium cucumerinum and Candida albi cans. The first part of this study consisted in the detection, isolation and characterization of the bioactive natural compounds present in the lipophilic extract of Eriophorum scheuchzeri. Among the eight isolated compounds, six were isoflavones. No isoflavones have been reported in the Cyperaceae family yet, nor in related families such as Poaceae or Juncaceae. Besides, isoflavones are generally rare in the plant kingdom and and they occur only in some families, such as Fabaceae, Rosaceae or Myristicaceae. In addition, out of these six isoflavones, three were new isoflavones. The known compounds were parvisoflavone A and B and cajanin which are already known isoflavones in the Fabaceae family. Two of the new isoflavones were particular, as they were C-methylated on the B-ring at the C-3' position. Methylated flavonoids are particularly rare in the plant kingdom. At present, no C-methylated isoflavones with methyl groups on the B-ring have ever been reported. The fourth new compound was a prenylated flavanone. Flavanones are also rare in the Cyperaceae family since they were found only in two genera (Cyperus and Schoenus). Finally, the widespread flavone tricin, characteristic of the Cyperaceae and Poaceae family has also been isolated. The second part of this study consisted in the characterization of the polar components present in the Me0H extract. In order to obtain mass and UV information about the secondary compounds present in the Eriophorum scheuchzeri methanolic extract, a LC-UV/DAD-APCl/MSn analysis has been performed as a first dereplication step. The UV/DAD spectra showed the presence of polyphenol compounds (phenylpropanoids and flavonoids). The LC-APCI/MSn analysis allowed the determination of the molecular weight of these compounds. Moreover, the fragmentation pattern of the [M+H]+ ions indicated presence of mono-, di- and tri-glycosides. LC-UV in combination with UV shift reagents added post-column was used in a second phase for the structural elucidation of the flavonoids. It allowed the positioning of the sugars on the aglycones. Finally, LC-NMR was used for a more detailed structural investigation of the compounds present in the crude MEOH extract. Thus, 10 fiavonoids have been totally or partially characterized by LC-UV-MS and LC-1H-RMN and UV-shift reagents added post column. However, the information obtained on-line was not always sufficient to allow a complete identification of all the compounds. Some of these compounds especially those with more than two sugar units attached to them, have been isolated in order to proceed to their complete characterization. Moreover, the Eriophorum scheuchzeri species was compared to two other species from the same genus. A LC-UV-ESI/MS analysis enabled a survey of the chemical composition of the DCM extracts of two related species E. angustifolium (Honck) and E. latifolium (Hoppe). The chromatograms of the three species showed some similarities in their flavonoid contents, especially by the recurrent presence of three compounds. The MEOH extracts of all three species have been compared by means of LC-UV-APCl/MS analyses. The chromatographic profile of all the three species showed even closer similarities than those found in the DCM extracts. E. angustifolium Honck. and E. latifolium species showed 7 compounds in common. Finally, the pure compounds obtained from the DCM (CH2Cl2) fraction were tested at different concentration, in order to evaluate their chemical and biological activities. All eight compounds showed an anti-scavenger activity against the DPPH radical, and four compounds showed antifungal activities against Cladosporium cucumerinum and Candida albicans. The pure compounds isolated from the MeOH extract were tested only for their biological activities as their antioxidant activity is already well documented in the literature. No compound showed a biological activity against Cladosporium cucumerinum and Candida albicans. Résumé: De nombreux travaux ont démontré ces dernières décennies que les plantes soumises à différents types de stress (basse température, UV, stress hydrique) synthétisent des composés secondaires (fiavonoides, coumarines, terpènes...) de protection et de défense. Les plantes d'altitude par exemple qui sont exposées à des conditions climatiques et environnementales difficiles, ont tendance à synthétiser des substances antioxydantes et antiradicalaires. Une investigation phytochirnique de ces plantes a conduit à la découverte de nouvelles molécules actives. Ainsi plusieurs plantes alpines ont été sélectionnées en fonction de leur habitat en vue de les soumettre aux tests biologiques (antifongiques) et chimiques (antiradicalaires) menés en routine dans notre laboratoire. Dans ce criblage biologique préliminaire, les extraits d'Eriophorum scheuchzeri Hoppe (Cyperaceae) ont réagi positivement aux différents tests. Il a donc été décidé d'entreprendre l'isolement des composés actifs. La première partie de ce travail a consisté à détecter, isoler et caractériser les composés naturels actifs présents dans l'extrait apolaire d' Eriophorum scheuchzeri. Parmi les huit composés isolés, quatre d'entre eux sont nouveaux. Un de ces produits est une flavanone et trois sont de nouvelles isoflavones, particulièrement intéressantes car elles possèdent des groupements C-méthylés au niveau du cycle B. Les flavonoides C-méthylés sont peu répandus dans le règne végétal et les rares exemples connus sont généralement C-méthylés sur le cycle A. Les quatre autres composés isolés n'ont jamais été décrits dans cette famille. Il s'agit d' isoflavones, les parvisoflavones A et B et la cajanine. Enfin, la flavone tricine, flavonoide caractéristique des Cyperaceae et des Poaceae a également été isolée. La deuxième partie de ce travail a consisté à caractériser les constituants polaires présents dans l'extrait methanolique. L'extrait a été analysé par chromatographie analytique couplée à différentes méthodes spectroscopiques (LC-UV-MS et LC-UV-1H RMN). De cette façon, douze flavonoides et un dérivé du phénylpropane, l'acide chlorogénique ont été identifiés. Les flavonoides tri-glycosylés ont dû être isolés afin de déterminer la nature et l'enchaînement des sucres. Finalement, l'espèce Eriophorum scheuchzeri a été comparée à deux autres espèces d' Eriophorum, soit E. angustifolium et E. latifolium. En conclusion, cette étude phytochimique a abouti à l'isolement de plusieurs nouvelles isoflavones aux activités antioxydantes et antifongiques ainsi qu'oestrogéniques.

Practical considerations for improving the productivity of mass spectrometry-based proteomics.

Mass spectrometry (MS) is currently the most sensitive and selective analytical technique for routine peptide and protein structure analysis. Top-down proteomics is based on tandem mass spectrometry (MS/ MS) of intact proteins, where multiply charged precursor ions are fragmented in the gas phase, typically by electron transfer or electron capture dissociation, to yield sequence-specific fragment ions. This approach is primarily used for the study of protein isoforms, including localization of post-translational modifications and identification of splice variants. Bottom-up proteomics is utilized for routine high-throughput protein identification and quantitation from complex biological samples. The proteins are first enzymatically digested into small (usually less than ca. 3 kDa) peptides, these are identified by MS or MS/MS, usually employing collisional activation techniques. To overcome the limitations of these approaches while combining their benefits, middle-down proteomics has recently emerged. Here, the proteins are digested into long (3-15 kDa) peptides via restricted proteolysis followed by the MS/MS analysis of the obtained digest. With advancements of high-resolution MS and allied techniques, routine implementation of the middle-down approach has been made possible. Herein, we present the liquid chromatography (LC)-MS/MS-based experimental design of our middle-down proteomic workflow coupled with post-LC supercharging.

Investigation phytochimique d'une plante médicinale malgache Hypoestes serpens (Vahl) R. Br. (Acanthaceae) variété glabre et étude comparative avec la variété poilue

Résumé A Madagascar, comme dans plusieurs pays en développement, une grande partie de la population utilise les plantes pour se soigner. Plusieurs espèces des plantes sont ainsi utilisées en médecine traditionnelle malgache. Par ailleurs, la plupart de ces plantes ne font l'objet que de très peu d'étude scientifique. En effet, dans le cadre de l'investigation phytochimique de plantes utilisées en médecine traditionnelle malgache et dans la recherche des nouvelles substances biologiquement actives, Hypoestes serpens (Vahl) R. Br. (Acanthaceae) a été étudiée. Elle se présente sous deux variétés (glabre et poilue) qui sont tous utilisées dans la région sud-centre de Madagascar pour traiter la blennorragie. De l'extrait dichlorométhanique des feuilles de H. serpens (Vahl) R. Br. variété glabre, 12 diterpénoides dont 8 nouveaux ont été isolés. Ils ont tous montré une activité antifongique contre un champignon pathogène des plantes, Cladosporium cucumerinum, dans la bioautographie directe sur CCM. Quelques-uns ont également présenté une activité contre une levure saprophyte chez l'homme, Candida albicans et une activité inhibitrice de l'enzyme acétylcholinesterase. Les diterpènoïdes sont déjà considérés comme les principaux métabolites secondaires du genre Hypoestes. Le fractionnement de l'extrait méthanolique a conduit à l'isolement de 5 glycosides des flavonoïdes dont 4 sous formes C-g,lycosides qui n'ont jamais été identifiés dans la famille Acanthaceae. Ces flavonoïdes ont présenté une activité antiradicalaire contre le DPPH. Le fractionnement et la purification des extraits ont été effectués à l'aide des différentes techniques chromatographiques telles que la chromatographie sur colonne ouverte, la filtration sur gel, la chromatographie liquide à haute pression, la chromatographie liquide à moyenne pression et la chromatographie liquide à basse pression. Par ailleurs, les structures des composés isolés ont été élucidées par des techniques spectroscopiques (UV, MS, RMN) et de méthode chimique (hydrolyse acide). En plus de ces techniques, certaines méthodes physiques (cristallographie par rayons-X, mesure de rotation optique) ont été réalisées pour confirmer certaines structures. Comme l'espèce Hypoestes serpens (Vahl) R. Br. se présente en deux variétés, une étude comparative a été effectuée. Cette étude avait montré que ces deux variétés ont une activité biologique similaire. Finalement, une technique analytique couplée, HPLC-UV-APC1-MS a permis de montrer la présence de toutes les substances isolées de la variété glabre dans la variété poilue. Second résumé Depuis des milliers d'almées, l'homme utilise les plantes pour se soigner. De nos jours, même avec le développement de la médecine moderne, la phytothérapie reste toujours la forme des soins de santé abordable et accessible pour la majorité des populations rurales des pays en développement. En outre, les plantes médicinales constituent une source potentielle de molécules biologiquement actives pour les industries pharmaceutiques et actuellement, on estime que 25% des médicaments commercialisés dans le monde sont à base de plantes Dans le cadre de la recherche des nouvelles molécules à intérêt thérapeutique qui pourraient devenir un médicament ou un modèle de structure ("lead compound") pour le développement de nouveaux médicaments, nous avons fait une étude sur l'espèce, Hypoestes serpens (Vahl) R. Br, plante utilisée en médecine traditionnelle malgache. Cette espèce existe en deux variétés, une glabre et une autre poilue qui sont tous utilisées dans la région sud-centre de Madagascar pour traiter la blennorragie. Par ailleurs, les tradipraticiens utilisent de préférence la variété poilue. Dans la première partie de ce présent travail, une investigation phytochimique de H serpens, variété glabre (variété moins utilisée) a d'abord été effectuée afm d'isoler et d'identifier le maximum des molécules biologiquement actives qu'elle contient. De ce fait, 17 composés dont 8 nouveaux ont été isolés. Les potentiels d'activités thérapeutiques des substances isolées ont ensuite été dépistés sur les différents cibles suivants.: deux souches de champignons (Cladosporium cucumerinum et Candida albicans), l'enzyme acétylcholinesterase et le radical DPPH. La deuxième partie de ce travail a été consacrée sur l'étude comparative des deux variétés (glabre et poilue) de H. serpens à la fois sur le plan biologique et sur le plan phytochimique. A l'issue de cette comparaison, nous avons constaté que l'utilisation de ces deux variétés en médecine traditionnelle malgache n'est pas un hasard ; les deux variétés avaient présenté une activité biologique très remarquable et contiennent les mêmes substances actives. Ces résultats démontrent les potentiels thérapeutiques de H serpens en médecine traditionnelle malgache et pourraient également encourager les tradipraticiens à utiliser la variété glabre tout en protégeant la variété poilue qui est en voie de disparition actuellement. En bref, l'investigation phytochimique de H. serpens justifiée par l'isolement et l'identification de certains de ses principes actifs ouvre la voie aux recherches des médicaments d'origine naturelle. Abstract In Madagascar, as in many developing countries, most people use plants to cure. A large number of plant species are employed in Malagasy traditional medicine. Moreover, most of these plants have been subject only very little scientific study. As part of a phytochemical investigation of plants used in Malagasy traditional medicine and in the search for new biologically active substances, Hypoestes serpens (Vah1) R.Br. (Acanthaceae) was investigated. This species exists in two varieties (glabrous and hairy) which are used in the south-center part of Madagascar to treat gonorrhoea. From the dichloromethane extract of the leaves of H. serpens (Vah1) R. Br. glabrous variety, 12 diterpenoids 8 of which were new, were isolated. They showed antifungal activity against the plant pathogen Cladosporium cucumerinum, in the direct TLC bioautography. Some of them also had activity against the yeast Candida athicans and inhibited acetylcholinesterase. The diterpenes are considered as the principal secondary metabolites of the genus Hypoestes. Fractionation of the methanol extract led to the isolation of 5 flavonoid glycosides, 4 of which were C-glycosides, never before identified in the Acanthaceae family. These flavonoids showed radical scavenging activity against DPPH. The fractionation and the purification of the extracts were achieved by different chromatographic techniques such as open-column chromatography, gel filtration, high- pressure liquid chromatography, medium-pressure liquid chromatography and low-pressure liquid chromatography. Moreover, the structures of the isolated compounds were elucidated by spectroscopic techniques (UV, MS, NMR) and chemical technique (acid hydrolysis). In addition, some physical methods (X-ray crystallography, measurement of optical rotation) were performed to confirm some structures. As the species Hypoestes serpens (Vah1) R. Br. is present in two varieties, a comparative study was carried out. This study showed that these two varieties had similar biological activity. Finally, a coupled analytical technique HPLC-UV-APCI-MS showed the presence of the same compounds in both the glabrous and hairy varieties.

Aspergillus protein degradation pathways with different secreted protease sets at neutral and acidic pH.

Aspergillus fumigatus grows well at neutral and acidic pH in a medium containing protein as the sole nitrogen source by secreting two different sets of proteases. Neutral pH favors the secretion of neutral and alkaline endoproteases, leucine aminopeptidases (Laps) which are nonspecific monoaminopeptidases, and an X-prolyl dipeptidase (DppIV). Acidic pH environment promotes the secretion of an aspartic endoprotease of pepsin family (Pep1) and tripeptidyl-peptidases of the sedolisin family (SedB and SedD). A novel prolyl peptidase, AfuS28, was found to be secreted in both alkaline and acidic conditions. In previous studies, Laps were shown to degrade peptides from their N-terminus until an X-Pro sequence acts as a stop signal. X-Pro sequences can be then removed by DppIV, which allows Laps access to the following residues. We have shown that at acidic pH Seds degrade large peptides from their N-terminus into tripeptides until Pro in P1 or P'1 position acts as a stop for these exopeptidases. However, X-X-Pro and X-X-X-Pro sequences can be removed by AfuS28 thus allowing Seds further sequential proteolysis. In conclusion, both alkaline and acidic sets of proteases contain exoprotease activity capable of cleaving after proline residues that cannot be removed during sequential digestion by nonspecific exopeptidases.

Malassezia yeasts activate the NLRP3 inflammasome in antigen-presenting cells via Syk-kinase signalling.

Although being a normal part of the skin flora, yeasts of the genus Malassezia are associated with several common dermatologic conditions including pityriasis versicolour, seborrhoeic dermatitis (SD), folliculitis, atopic eczema/dermatitis (AE/AD) and dandruff. While Malassezia spp. are aetiological agents of pityriasis versicolour, a causal role of Malassezia spp. in AE/AD and SD remains to be established. Previous reports have shown that fungi such as Candida albicans and Aspergillus fumigatus are able to efficiently activate the NLRP3 inflammasome leading to robust secretion of the pro-inflammatory cytokine IL-1β. To date, innate immune responses to Malassezia spp. are not well characterized. Here, we show that different Malassezia species could induce NLRP3 inflammasome activation and subsequent IL-1β secretion in human antigen-presenting cells. In contrast, keratinocytes were not able to secrete IL-1β when exposed to Malassezia spp. Moreover, we demonstrate that IL-1β secretion in antigen-presenting cells was dependent on Syk-kinase signalling. Our results identify Malassezia spp. as potential strong inducers of pro-inflammatory responses when taken up by antigen-presenting cells and identify C-type lectin receptors and the NLRP3 inflammasome as crucial actors in this process.

Ultra-performance liquid chromatography mass spectrometry and sensitive bioassay methods for quantification of posaconazole plasma concentrations after oral dosing.

Posaconazole (POS) is a new antifungal agent for prevention and therapy of mycoses in immunocompromised patients. Variable POS pharmacokinetics after oral dosing may influence efficacy: a trough threshold of 0.5 ?g/ml has been recently proposed. Measurement of POS plasma concentrations by complex chromatographic techniques may thus contribute to optimize prevention and management of life-threatening infections. No microbiological analytical method is available. The objective of this study was to develop and validate a new simplified ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method and a sensitive bioassay for quantification of POS over the clinical plasma concentration range. The UPLC-MS/MS equipment consisted of a triple quadrupole mass spectrometer, an electrospray ionization (ESI) source, and a C(18) analytical column. The Candida albicans POS-hypersusceptible mutant (MIC of 0.002 ?g/ml) ?cdr1 ?cdr2 ?flu ?mdr1 ?can constructed by targeted deletion of multidrug efflux transporters and calcineurin genes was used for the bioassay. POS was extracted from plasma by protein precipitation with acetonitrile-methanol (75%/25%, vol/vol). Reproducible standard curves were obtained over the range 0.014 to 12 (UPLC-MS/MS) and 0.028 to 12 ?g/ml (bioassay). Intra- and interrun accuracy levels were 106% ± 2% and 103% ± 4% for UPLC-MS/MS and 102% ± 8% and 104% ± 1% for bioassay, respectively. The intra- and interrun coefficients of variation were 7% ± 4% and 7% ± 3% for UPLC-MS/MS and 5% ± 3% and 4% ± 2% for bioassay, respectively. An excellent correlation between POS plasma concentrations measured by UPLC-MS/MS and bioassay was found (concordance, 0.96). In 26 hemato-oncological patients receiving oral POS, 27/69 (39%) trough plasma concentrations were lower than 0.5 ?g/ml. The UPLC-MS/MS method and sensitive bioassay offer alternative tools for accurate and precise quantification of the plasma concentrations in patients receiving oral posaconazole.