The role of bradykinin B(1) and B(2) receptors for secondary brain damage after traumatic brain injury in mice.

Inflammatory mechanisms are known to contribute to the pathophysiology of traumatic brain injury (TBI). Since bradykinin is one of the first mediators activated during inflammation, we investigated the role of bradykinin and its receptors in posttraumatic secondary brain damage. We subjected wild-type (WT), B(1)-, and B(2)-receptor-knockout mice to controlled cortical impact (CCI) and analyzed tissue bradykinin as well as kinin receptor mRNA and protein expression up to 48 h thereafter. Brain edema, contusion volume, and functional outcome were assessed 24 h and 7 days after CCI. Tissue bradykinin was maximally increased 2 h after trauma (P<0.01 versus sham). Kinin B(1) receptor mRNA was upregulated up to four-fold 24 h after CCI. Immunohistochemistry showed that B(1) and B(2) receptors were expressed in the brain and were significantly upregulated in the traumatic penumbra 1 to 24 h after CCI. B(2)R(-/-) mice had significantly less brain edema (-51% versus WT, 24 h; P<0.001), smaller contusion volumes ( approximately 50% versus WT 24 h and 7 d after CCI; P<0.05), and better functional outcome 7 days after TBI as compared with WT mice (P<0.05). The present results show that bradykinin and its B(2) receptors play a causal role for brain edema formation and cell death after TBI.

Use of transgenic mice for the characterization of human alpha 1-acid glycoprotein (orosomucoid) variants.

Sera from transgenic mice (TM) carrying human genes of alpha 1-acid glycoprotein (orosomucoid or ORM) have been analyzed by isoelectrofocusing and subsequent immunoblotting with antihuman ORM antibodies. With this technique it is possible to reveal selectively the human protein secreted in the TM sera. Orosomucoid bands present in TM sera have been compared with those of the most common human ORM phenotypes to correlate the products of specific genes to previously identified genetic variants. In this paper, we report the identification of the genes encoding for variants ORM1 F1 and ORM2 A, which are genes AGP-A and AGP-B/B' respectively. The nucleotide sequences of these genes are known; therefore a direct correlation between variants and specific amino acid sequences can be established.

Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum.

Localization of human MHC class I-restricted T cell epitopes in the circumsporozoite (CS) protein of the human parasite Plasmodium falciparum is an important objective in the development of antimalarial vaccines. To this purpose, we synthesized a series of overlapping synthetic 20-mer peptides, spanning the entire sequence of the 7G8 CS molecule except for the central repeat B cell domain. The P.f.CS peptides were first tested for their ability to bind to the human MHC class I HLA-A2.1 molecule on T2, a human cell line. Subsequently, the use of a series of shorter peptide analogues allowed us to determine the optimal A2.1 binding sequence present in several of the 20-mers. Binding P.f.CS peptides were further tested for their capacity to activate PBL from HLA-A2.1+ immune donors living in a malaria-endemic area. Specific IFN-gamma production was detected in the supernatant of cultures of PBL from exposed individuals. Cytotoxic T cell lines and clones were derived from the PBL of one responder, and their activity was shown to be HLA-A2.1-restricted and specific for the peptide 334-342 of the CS protein. In addition, double transgenic HLA-A2.1 x human beta 2-microglobulin mice were immunized with peptide 1-10 of the CS protein. T cells derived from immune lymph nodes displayed a peptide-specific HLA-A2.1-restricted cytolytic activity after one in vitro stimulation.

Rational design of 4-aryl-1,2,3-triazoles for indoleamine 2,3-dioxygenase 1 inhibition.

Indoleamine 2,3-dioxygenase 1 (IDO1) is an important therapeutic target for the treatment of diseases such as cancer that involve pathological immune escape. Starting from the scaffold of our previously discovered IDO1 inhibitor 4-phenyl-1,2,3-triazole, we used computational structure-based methods to design more potent ligands. This approach yielded highly efficient low molecular weight inhibitors, the most active being of nanomolar potency both in an enzymatic and in a cellular assay, while showing no cellular toxicity and a high selectivity for IDO1 over tryptophan 2,3-dioxygenase (TDO). A quantitative structure-activity relationship based on the electrostatic ligand-protein interactions in the docked binding modes and on the quantum chemically derived charges of the triazole ring demonstrated a good explanatory power for the observed activities.

Strategic Adaptation in Function of Chronicity of Schizophrenic Symptoms During A Fluency Task: An fMRI Study

Background: Language processing abnormalities and executive difficulties are hallmark features of schizophrenia. The objective of this study is to assess the blood oxygenation level-dependent (BOLD) response at two different stages of the illness (i.e. comparison between adolescents and adults with schizophrenic symptoms) during a fluency task.Methods: BOLD responses during a covert verbal fluency task were compared between 11 psychotic adolescents with schizophrenic symptoms (mean age 16,9 years) and 14 adults with schizophrenia (mean age 33,4 years). fMRI data were analyzed with standard routine of spm5.Results: First, expected activation's network was found for both groups, separately. Secondly, adolescents showed greater activation in left rolandic opercule (BA 48), left angular (BA 39) and right hippocampus compared to adults. Thirdly, adults demonstrated greater activation in presupplementary motor area (BA 6) and in precentral area (BA 4) compared to adolescents.Conclusions: The adolescents seemed to recruit a verbal network (Broca and Wernicke) and memory abilities to perform a fluency task. In contrast, adults seemed to recruit more executive function abilities to perform a similar task. Despite the evolution of schizophrenia, which is known to have a deleterious influence on the prefrontal cortex development, adult patients seemed to be able to recruit such areas to perform a verbal fluency / executive function task.

β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells.

β-Catenin signaling has recently been tied to the emergence of tolerogenic dendritic cells (DCs). In this article, we demonstrate a novel role for β-catenin in directing DC subset development through IFN regulatory factor 8 (IRF8) activation. We found that splenic DC precursors express β-catenin, and DCs from mice with CD11c-specific constitutive β-catenin activation upregulated IRF8 through targeting of the Irf8 promoter, leading to in vivo expansion of IRF8-dependent CD8α(+), plasmacytoid, and CD103(+)CD11b(-) DCs. β-Catenin-stabilized CD8α(+) DCs secreted elevated IL-12 upon in vitro microbial stimulation, and pharmacological β-catenin inhibition blocked this response in wild-type cells. Upon infections with Toxoplasma gondii and vaccinia virus, mice with stabilized DC β-catenin displayed abnormally high Th1 and CD8(+) T lymphocyte responses, respectively. Collectively, these results reveal a novel and unexpected function for β-catenin in programming DC differentiation toward subsets that orchestrate proinflammatory immunity to infection.

The adaptive function of melanin-based coloration in the tawny owl (Strix aluco) ; the proximate role of the melanocortin system

Colour polymorphism is common in wild population. One of the main questioning of evolutionary biologists is to understand how different colour variants could have evolved and be maintained in fluctuating environments, a selective process that forces individuals to constantly adapt their strategies in order to survive. This issue is particularly true for traits that are genetically inherited. Natural selection erodes genotypes with lowest fitness (less adapted), reducing in turn global genetic variation within population. In this context, the study of the evolution and maintenance of melanin- based coloration is relevant since inter-individual variation in the deposition of these pigments is common in animal and plant kingdoms and is under strong genetic control. In this thesis, I focus on the specific case of the tawny owl (Strix aluco), a species displaying continuous variation in reddish pheomelanin-based coloration. Interestingly, empirical studies highlighted covariations between melanin-based coloration and important behavioural, physiological and life history traits. Recently, a genetic model pointed out the melanocortin system and their pleiotropic effects as a potential regulator of these covariations. Accordingly, this PhD thesis further investigates colour-specific behavioural, physiological, or life history strategies, while examining the proximate mechanisms underlying these reaction norms. We found that differently coloured tawny owls differently resolve fundamental trade-off between offspring number and quality (Chapter 1), light melanic individuals producing many low- quality offspring and dark, melanic ones producing few high-quality offspring. These reproductive strategies are likely to induce alternative physiological constraints. Indeed, we demonstrated that light melanic individuals produced higher levels of reactive oxygen species (ROS, Chapter 2), but also expressed higher levels of antioxidant (GSH, Chapters 2 &amp; 3). Interestingly, we showed that light melanic breeding females could modulate their POMC prohormone levels according to the environmental conditions, while dark reddish ones produced constant levels of this prohormone {Chapter 4). Finally, we highlighted colour-specific patterns of prohormone convertase 1 (PCI) gene expression (Chapter 5), an enzyme responsible for POMC prohormone processing to ACTH and a- MSH, for instance. Altogether, these results provide strong evidence of colour-specific strategies, light and melanic tawny owls better coping with stressful and relaxed environments, respectively. Variation in melanin-based coloration is likely to be maintained by the heterogeneity of our study area and strong environmental stochasticity within and between years, these process favouring differently coloured tawny owls at different periods of time. From a proximate point of view, this PhD thesis supports the hypothesis that covariations between phenotypic traits and melanin-based coloration stems from the melanocortin system, especially the fundamental role of POMC gene expression and its processing to melanocortin peptides. - Le polymorphisme de couleur est une variation phénotypique très fréquente dans la nature. En biologie évolutive, une des problématiques clés est donc de comprendre comment différent morphes de couleur peuvent être apparus et maintenus au cours du temps dans des environnements aussi variables que les nôtres, surtout que ces fluctuations forcent ces morphes à s'adapter constamment pour assurer leur survie. Cette thématique est particulièrement réelle lorsque les variations phénotypiques sont héréditaires et donc sous forte influence génétique. La sélection naturelle a en effet le pouvoir d'éroder rapidement la variation génétique en éliminant les génotypes mal adaptés. Dans ce sens, l'étude de l'évolution, et de la maintenance de la coloration mélanique est donc tout à fait pertinente car la variation de coloration entre individus est très répandue à travers les règnes animal et végétal et sous forte influence génétique. Dans cette thèse, je me suis concentré sur le cas spécifique de la chouette hulotte (Strix aluco), une espèce présentant une variation continue dans la déposition de pigments pheomélaniques roux. De précédentes études ont déjà montré que cette variation de coloration était associée avec des variations de traits comportementaux, physiologiques ou d'histoire de vie. Récemment, une étude a souligné l'importance du système des mélanocortines et de leurs effets pléiotropes dans la régulation de ces covariations. En conséquence, cette thèse de doctoral a pour but d'étudier un peu plus les stratégies comportementales, physiologiques ou d'histoire de vie spécifiques à chaque morphe de couleur, tout en examinant un peu plus les mécanismes proximaux potentiellement à la base de ces normes de réactions. Nous constatons tout d'abord que les morphes de couleurs étaient associés à différentes stratégies dans la résolution de compromis telle que la production de beaucoup de jeunes ou des jeunes de qualité (Chapitre 1). Les morphes gris (dit peu mélaniques) ont tendance à produire beaucoup de jeunes mains de moindre qualité, alors que les morphes roux (dit fortement mélaniques) produisent moins de jeunes mais de meilleure qualité. Ces stratégies sont susceptibles alors d'induire certaines contraintes physiologiques. Par exemple, nous montrons que les morphes gris produisent plus de dérivés réactifs de l'oxygène (ROS, Chapitre 2), mais aussi plus d'antioxydants (GSH, Chapitres 2 &amp; 3). Nous montrons ensuite que les femelles grises ont une plus grande capacité à moduler leur niveau de POMC prohormone dans le sang en fonction des conditions environnementales, alors que les femelles rousses gardent un niveau constant (Chapitre 4). Finalement, nous démontrons que les patterns d'expression du gène codant pour la prohormone convertase 1 varient chez des jeunes issus de parents gris ou roux (Chapitre 5). Ceci est particulièrement intéressant car cette enzyme permet de scinder la POMC prohormone en plusieurs peptides importants tels que l'ACTH ou l'a-MSH. En conclusion, ces résultats démontrent qu'il y a bel et bien des stratégies évolutives différentes entre les morphes de couleurs, les chouettes hulottes grises et rousses étant respectivement plus adaptés à des environnements stressants ou favorables. L'hétérogénéité de notre zone d'étude et la stochasticité environnementale qui caractérise ses habitats pourraient donc agir comme une source de sélection temporelle, laquelle favoriserait les différents morphes de couleurs à diverses périodes. D'un point de vue plus proximale maintenant, cette thèse de doctorat soutient l'hypothèse que les covariations observées entre la coloration mélanique et des traits phénotypiques importants sont modulées par les effets pléiotropes du système des mélanocortines, et met en avant le rôle prépondérant que pourrait jouer l'expression du gène POMC et sa post traduction en mélanocortines.

The function of a new regulator of epidermal differentiation, HOP, and pathomechanisms underlying epidermolytic hyperkeratosis

The process of epidermal differentiation involves proliferation, differentiation, migration and maturation of keratinocytes to form an impermeable barrier against water loss and outside environment. It is controlled by highly balanced regulatory machinery, involving many molecules that are still under investigation.Homeobox proteins are involved in body patterning and morphogenesis of organs and are studied as potentially good candidates to regulate this process. In the first project we investigated the role of a protein named HOP which belongs to a group of homeobox proteins. Even if HOP is a small protein almost completely composed of the homeodomain and without DNA binding capacity, it is considered as transcriptional regulator in different tissues. HOP interacts with serum response factor (SRF) and histone deacetylase type 2 (HDAC2). By microarray analysis we found that HOP expression increases in cultured human primary keratinocytes (NHK) which undergo calcium-induced differentiation. HOP protein was localized in granular layer of the epidermis of healthy individuals. Lack of HOP was demonstrated in psoriatic lesions, whereas a strong expression was demonstrated in the lesional skin of patients affected with lichen planus (LP). Since LP is characterized by hypergranulosis while psoriatic lesions by progressive lack of the granular layer, the obtained data indicated that HOP might have a potential function in granular layer of epidermis. To investigate HOP function, we inhibited its expression by using HOP specific StealthRNAi and we overexpressed HOP using lentiviral vectors in differentiating NHK. The conclusion of both experiments indicated that HOP positively regulates the expression of late differentiation markers, such as profilaggrin, loricrin and transglutaminase 1. The in vitro data were next confirmed in vivo using HOP knockout mouse model.The second part of my study involved analysis of mechanisms underlying the pathogenesis of epidermolytic hyperkeratosis (EHK). EHK is a genetic disorder characterized by erythema, skin blistering, keratinocyte hyperproliferation and hyperkeratosis. EHK is caused by mutations in keratin 1 or 10 (K1, K10) which are major structural proteins of differentiated keratinocytes and participate in the cellular scaffold formation. To investigate how the structural proteins carrying mutations alter cellular signaling, we established an in vitro model for EHK by overexpression of one of the most common K10 mutations reported so far (K10R156H), in primary human keratinocytes. In order to mimic the in vivo situation, mutated keratinocytes growing on silicone membranes were subjected to mechanical stretch. We observed strong collapse of KIF in K10R156H keratinocytes when subjected to stretch for 30 minutes. Our data demonstrated stronger activation of p38, a member of MAPK stress signaling pathways, in K10R156H when compared to control cells. We demonstrated also that K10R156H keratinocytes showed an induction of TNF-α and RANTES release in response to stretch.Taken together these studies characterize a novel regulator of epidermal differentiation - HOP and demonstrate new aspects implicated in the pathogenesis of EHK.

Determination of picogram levels of midazolam, and 1- and 4-hydroxymidazolam in human plasma by gas chromatography-negative chemical ionization-mass spectrometry.

Midazolam is a widely accepted probe for phenotyping cytochrome P4503A. A gas chromatography-mass spectrometry (GC-MS)-negative chemical ionization method is presented which allows measuring very low levels of midazolam (MID), 1-OH midazolam (1OHMID) and 4-OH midazolam (4OHMID), in plasma, after derivatization with the reagent N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. The standard curves were linear over a working range of 20 pg/ml to 5 ng/ml for the three compounds, with the mean coefficients of correlation of the calibration curves (n = 6) being 0.999 for MID and 1OHMID, and 1.0 for 4OHMID. The mean recoveries measured at 100 pg/ml, 500 pg/ml, and 2 ng/ml, ranged from 76 to 87% for MID, from 76 to 99% for 1OHMID, from 68 to 84% for 4OHMID, and from 82 to 109% for N-ethyloxazepam (internal standard). Intra- (n = 7) and inter-day (n = 8) coefficients of variation determined at three concentrations ranged from 1 to 8% for MID, from 2 to 13% for 1OHMID and from 1 to 14% for 4OHMID. The percent theoretical concentrations (accuracy) were within +/-8% for MID and 1OHMID, within +/-9% for 4OHMID at 500 pg/ml and 2 ng/ml, and within +/-28% for 4OHMID at 100 pg/ml. The limits of quantitation were found to be 10 pg/ml for the three compounds. This method can be used for phenotyping cytochrome P4503A in humans following the administration of a very low oral dose of midazolam (75 microg), without central nervous system side-effects.

Role of the epithelial sodium channel (ENaC) in the epidermal barrier function of the skin

Abstract In humans, the skin is the largest organ of the body, covering up to 2m2 and weighing up to 4kg in an average adult. Its function is to preserve the body from external insults and also to retain water inside. This barrier function termed epidermal permeability barrier (EPB) is localized in the functional part of the skin: the epidermis. For this, evolution has built a complex structure of cells and lipids sealing the surface, the stratum corneum. The formation of this structure is finely tuned since it is not only formed once at birth, but renewed all life long. This active process gives a high plasticity and reactivity to skin, but also leads to various pathologies. ENaC is a sodium channel extensively studied in organs like kidney and lung due to its importance in regulating sodium homeostasis and fluid volume. It is composed of three subunits α, ß and r which are forming sodium selective channel through the cell membrane. Its presence in the skin has been demonstrated, but little is known about its physiological role. Previous work has shown that αENaC knockout mice displayed an abnormal epidermis, suggesting a role in differentiation processes that might be implicated in the EPB. The principal aim of this thesis has been to study the consequences for EPB function in mice deficient for αENaC by molecular and physiological means and to investigate the underlying molecular mechanisms. Here, the barrier function of αENaC knockout pups is impaired. Apparently not immediately after birth (permeability test) but 24h later, when evident water loss differences appeared compared to wildtypes. Neither the structural proteins of the epithelium nor the tights junctions showed any obvious alterations. In contrary, stratum corneum lipid disorders are most likely responsible for the barrier defect, accompanied by an impairment of skin surface acidification. To analyze in details this EPB defect, several hypotheses have been proposed: reduced sensibility to calcium which is the key activator far epidermal formation, or modification of ENaC-mediated ion fluxes/currents inside the epidermis. The cellular localization of ENaC and the action in the skin of CAPl, a positive regulator of ENaC, have been also studied in details. In summary, this study clearly demonstrates that ENaC is a key player in the EPB maintenance, because αENaC knockout pups are not able to adapt to the new environment (ex utero) as efficiently as the wildtypes, most likely due to impaired of sodium handling inside the epidermis. Résumé Chez l'homme, la peau est le plus grand organe, couvrant presque 2m2 et pesant près de 4kg chez l'adulte. Sa fonction principale est de protéger l'organisme des agressions extérieures mais également de conserver l'eau à l'intérieur du corps. Cette fonction nommée barrière épithéliale est localisée dans la partie fonctionnelle de la peau : l'épiderme. A cette fin, l'évolution s'est dotée d'une structure complexe composée de cellules et de lipides recouvrant la surface, la couche cornée. Sa formation est finement régulée, car elle n'est pas seulement produite à la naissance mais constamment renouvelée tout au long de la vie, ce qui lui confère une grande plasticité mais ce qui est également la cause de nombreuses pathologies. ENaC est un canal sodique très étudié dans le rein et le poumon pour son importance dans la régulation de l'homéostasie sodique et la régulation du volume du milieu intérieur. Il est composé de 3 sous unités, α, ß et y qui forment un pore sélectif pour le sodium dans les membranes. Ce canal est présent dans la peau mais sa fonction n'y est pas connue. Des travaux précédents ont pu montrer que les souris dont le gène codant pour αENaC a été invalidé présentent un épiderme pathologique, suggérant un rôle dans la différentiation et pourrait même être impliqué dans la barrière épithéliale. Le but de cette thèse fut l'étude de la barrière dans ces souris knockouts avec des méthodes moléculaires et physiologiques et la caractérisation des mécanismes moléculaire impliqués. Dans ce travail, il a été montré que les souris mutantes présentaient un défaut de la barrière. Ce défaut n'est pas visible immédiatement à la naissance (test de perméabilité), mais 24h plus tard, lorsque les tests de perte d'eau transépithéliale montrent une différence évidente avec les animaux contrôles. Ni les protéines de structures ni les jonctions serrées de l'épiderme ne présentaient d'imperfections majeures. A l'inverse, les lipides de la couche cornée présentaient un problème de maturation (expliquant le phénotype de la barrière), certainement consécutif au défaut d'acidification à la surface de la peau que nous avons observé. D'autres mécanismes ont été explorées afin d'investiguer cette anomalie de la barrière, comme la réduction de sensibilité au calcium qui est le principal activateur de la formation de l'épiderme, ou la modification des flux d'ions entre les couches de l'épiderme. La localisation cellulaire d'ENaC, et l'action de son activateur CAPl ont également été étudiés en détails. En résumé, cette étude démontre clairement qu'ENaC est un acteur important dans la formation de la barrière épithéliale, car la peau des knockouts ne s'adapte pas aussi bien que celle des sauvages au nouvel environnement ex utero à cause de la fonction d'ENaC dans les mouvements de sodium au sein même de l'épiderme. Résumé tout public Chez l'homme, la peau est le plus grand organe, couvrant presque 2m2 et pesant près de 4kg chez l'adulte. Sa fonction principale est de protéger l'organisme des agressions extérieures mais également de conserver l'eau à l'intérieur du corps. Cette fonction nommée barrière épithéliale est localisée dans la partie fonctionnelle de la peau : l'épiderme. A cette fin, l'évolution s'est dotée d'une structure complexe composée de cellules et de lipides recouvrant la surface, la couche cornée. Sa formation est finement régulée, car elle n'est pas seulement produite à la naissance mais constamment renouvelée tout au long de la vie, ce qui lui confère une grande plasticité mais ce qui est également la cause de nombreuses maladies. ENaC est une protéine formant un canal qui permet le passage sélectif de l'ion sodium à travers la paroi des cellules. Il est très étudié dans le rein pour son importance dans la récupération du sel lors de la concentration de l'urine. Ce canal est présent dans la peau mais sa fonction n'y est pas connue. Des travaux précédents ont pu montrer que les souris où le gène codant pour αENaC a été invalidé présentent un épiderme pathologique, suggérant un rôle dans la peau et plus particulièrement la fonction de barrière de l'épiderme. Le but de cette thèse fut l'étude de la fonction de barrière dans ces souris mutantes, au niveau tissulaire et cellulaire. Dans ce travail, il a été montré que les souris mutantes présentaient une peau plus perméable que celle des animaux contrôles, grâce à une machine mesurant la perte d'eau à travers la peau. Ce défaut n'est visible que 24h après la naissance, mais nous avons pu montrer que les animaux mutants perdaient quasiment 2 fois plus d'eau que les contrôles. Au niveau moléculaire, nous avons pu montrer que ce défaut provenait d'un problème de maturation des lipides qui composent la barrière de la peau. Cette maturation est incomplète vraisemblablement à cause d'un défaut de mouvement des ions dans les couches les plus superficielles de l'épiderme, et cela à cause de l'absence du canal ENaC. En résumé, cette étude démontre clairement qu'ENaC est un acteur important dans la formation de la barrière épithéliale, car la peau des mutants ne s'adapte pas aussi bien que celle des sauvages au nouvel environnement ex utero à cause de la fonction d'ENaC dans les mouvements de sodium au sein même de l'épiderme.

Maladaptive exploratory behavior and neuropathology of the PS-1 P117L Alzheimer transgenic mice.

Patients with the early-onset Alzheimer's disease P117L mutation in the presenilin-1 gene (PS-1) present pathological hallmarks in the hippocampus, the frontal cortex and the basal ganglia. In the present work we determined by immunohistochemistry which brain regions were injured in the transgenic PS-1 P117L mice, in comparison to their littermates, the B6D2 mice. Furthermore, as these regions are involved in novelty detection, we investigated the behavior of these mice in tests for object and place novelty recognition. Limited numbers of senile plaques and neurofibrillary tangles were detected in aged PS-1 P117L mice in the CA1 only, indicating that the disease is restrained to an initial neuropathological stage. Western blots showed a change in PSD-95 expression (p=0.03), not in NR2A subunit, NR2B subunit and synaptophysin expressions in the frontal cortex, suggesting specific synaptic alterations. The behavioral tests repeatedly revealed, despite a non-significant preference for object or place novelty, maladaptive exploratory behavior of the PS-1 P117L mice in novel environmental conditions, not due to locomotor problems. These mice, unlike the B6D2 mice, were less inhibited to visit the center of the cages (p=0.01) and they continued to move excessively in the presence of a displaced object (p=0.021). Overall, the PS-1 P117L mice appear to be in an initial Alzheimer's disease-like neuropathological stage, and they showed a lack of reaction toward novel environmental conditions.

Azole resistance by loss of function of the sterol 16;⁵,S10;-desaturase gene (ERG3) in Candida albicans does not necessarily decrease virulence.

The inactivation of ERG3, a gene encoding sterol 16;⁵,S10;-desaturase (essential for ergosterol biosynthesis), is a known mechanism of in vitro resistance to azole antifungal drugs in the human pathogen Candida albicans. ERG3 inactivation typically results in loss of filamentation and attenuated virulence in animal models of disseminated candidiasis. In this work, we identified a C. albicans clinical isolate (VSY2) with high-level resistance to azole drugs in vitro and an absence of ergosterol but normal filamentation. Sequencing of ERG3 in VSY2 revealed a double base deletion leading to a premature stop codon and thus a nonfunctional enzyme. The reversion of the double base deletion in the mutant allele (erg3-1) restored ergosterol biosynthesis and full fluconazole susceptibility in VSY2, confirming that ERG3 inactivation was the mechanism of azole resistance. Additionally, the replacement of both ERG3 alleles by erg3-1 in the wild-type strain SC5314 led to the absence of ergosterol and to fluconazole resistance without affecting filamentation. In a mouse model of disseminated candidiasis, the clinical ERG3 mutant VSY2 produced kidney fungal burdens and mouse survival comparable to those obtained with the wild-type control. Interestingly, while VSY2 was resistant to fluconazole both in vitro and in vivo, the ERG3-derived mutant of SC5314 was resistant only in vitro and was less virulent than the wild type. This suggests that VSY2 compensated for the in vivo fitness defect of ERG3 inactivation by a still unknown mechanism(s). Taken together, our results provide evidence that contrary to previous reports inactivation of ERG3 does not necessarily affect filamentation and virulence.