Transcriptional regulation of MDR1, a gene involved in antifungal drug resistance in Candida albicans

ABSTRACT Upregulation of the Major Facilitator transporter gene MDR1 (Multi_drug Resistance 1) is one of the mechanisms observed in Candida albicans clinical isolates developing resistance to azole antifungal agents. To better understand this phenomenon, the cis-acting regulatory elements present in a modulatable reporter system under the control of the MDR1 promoter were characterized. In an azole-susceptible strain, transcription of this reporter is transiently upregulated in response to either benomyl or H2O2, whereas its expression is constitutively high in an azole-resistant strain (FR2). Two cis-acting regulatory elements, that are necessary and sufficient to convey the same transcriptional responses to a heterologous promoter (CDR2), were identified within the MDR1promoter. The first element, called BRE (for Benomyl Response Element, -296 to -260 with respect to the ATG start codon), is required for benomyl-dependent MDR1 upregulation and for constitutive high expression of MDR1 in FR2. The second element, termed HRE (for H2O2 Response Element, -561 to -520), is required for H2O2-dependent MDR1 upregulation, but is dispensable for constitutive high expression. Two potential binding sites (TTAG/CTAA) for the blip transcription factor Cap1p lie within the HRE. Moreover, inactivation of CAP1 abolished the transient response to H2O2 and diminished significantly the transient response to benomyl. Cap1p, which has been previously implicated in cellular responses to oxidative stress, may thus play a transacting and positive regulatory role in benomyl- and H2O2-dependent transcription of MDR1. However, it is not the only transcription factor involved in the response of MDR1 to benomyl. A minimal BRE element (-290 to -273) that is sufficient to detect in vitro sequence-specific binding of protein complexes in crude extracts prepared from C. albicans was also delimited. Genome-wide transcript profiling analyses undertaken with a matched pair of clinical isolates, one of which being azole-resistant and upregulating MDR1, and with an azole-susceptible strain exposed to benomyl, revealed that genes specifically upregulated by benomyl harbour in their promoters Cap1p binding site(s). This strengthened the idea that Cap1p plays a role in benomyl-dependent upregulation of MDR1. BRE-like sequences were also identified in several genes co-regulated with MDR1 in both conditions, which was consistent with the involvement of the BRE in both processes. A set of 147 mutants lacking a single transcription factor gene was next screened for loss of MDR1response to benomyl. Unfortunately, none of the tested mutants showed a loss of benomyl-dependent MDR1 upregulation. Nevertheless, a significant diminution of the response was observed in the mutants in which the MADS-box transcription factor Mcm1p and the C2H2 zinc finger transcription factor orf19.13374p were inactivated, suggesting that Mcm1p and orf19.13374p are involved in MDR1response to benomyl. Interestingly, the BRE contains a perfect match to the binding consensus of Mcm1p, raising the possibility that MDR1may be a direct target of this transcriptional activator. In conclusion, while the identity of the trans-acting factors that bind to the BRE and HRE remains to be confirmed, the tools we have developed during characterization of the cis-acting elements of the MDR1promoter should now serve to elucidate the nature of the components that modulate its activity. RESUME La surexpression du gène MDR1 (pour Résistance Multidrogue 1), qui code pour un transporteur de la famille des Major Facilitators, est l'un des mécanismes observés dans les isolats cliniques de la levure Candida albicans développant une résistance aux agents antifongiques appelés azoles. Pour mieux comprendre ce phénomène, les éléments de régulation agissant en cis dans un système rapporteur modulable sous le contrôle du promoteur MDR1 ont été caractérisés. Dans une souche sensible aux azoles, la transcription de ce rapporteur est transitoirement surélevée en réponse soit au bénomyl soit à l'agent oxydant H2O2, alors que son expression est constitutivement élevée dans une souche résistante aux azoles (souche FR2). Deux éléments de régulation agissant en cis, nécessaires et suffisants pour transmettre les mêmes réponses transcriptionnelles à un promoteur hétérologue (CDR2), ont été identifiés dans le promoteur MDR1. Le premier élément, appelé BRE (pour Elément de Réponse au Bénomyl, de -296 à -260 par rapport au codon d'initiation ATG) est requis pour la surexpression de MDR1dépendante du bénomyl et pour l'expression constitutive de MDR1 dans FR2. Le deuxième élément, appelé HRE (pour Elément de Réponse à l'H2O2, de -561 à -520), est requis pour la surexpression de MDR1 dépendante de l'H2O2, mais n'est pas impliqué dans l'expression constitutive du gène MDR1. Deux sites de fixation potentiels (TTAG/CTAA) pour le facteur de transcription Cap1p ont été identifiés dans l'élément HRE. De plus, l'inactivation de CAP1 abolit la réponse transitoire à l'H2O2 et diminua significativement la réponse transitoire au bénomyl. Cap1p, qui est impliqué dans les réponses de la cellule au stress oxydatif, doit donc jouer un rôle positif en trans dans la surexpression de MDR1 dépendante du bénomyl et de l'H2O2. Cependant, ce n'est pas le seul facteur de transcription impliqué dans la réponse au bénomyl. Un élément BRE d'une longueur minimale (de -290 à -273) a également été défini et est suffisant pour détecter une interaction spécifique in vitro avec des protéines provenant d'extraits bruts de C. albicans. L'analyse du profil de transcription d'une paire d'isolats cliniques comprenant une souche résistante aux azoles surexprimant MDR1, et d'une souche sensible aux azoles exposée au bénomyl, a révélé que les gènes spécifiquement surexprimés par le bénomyl contiennent dans leurs promoteurs un ou plusieurs sites de fixation pour Cap1p. Ceci renforce l'idée que Cap1p joue un rôle dans la surexpression de MDR1dépendante du bénomyl. Une ou deux séquences ressemblant à l'élément BRE ont également été identifiées dans la plupart des gènes corégulés avec MDR1 dans ces deux conditions, ce qui était attendu compte-tenu du rôle joué par cet élément dans les deux processus. Une collection de 147 mutants dans lesquels un seul facteur de transcription est inactivé a été testée pour la perte de réponse au bénomyl de MDR1. Malheureusement, la surexpression de MDR1 dépendante du bénomyl n'a été perdue dans aucun des mutants testés. Néanmoins, une diminution significative de la réponse a été observée chez des mutants dans lesquels le facteur de transcription à MADS-box Mcm1p et le facteur de transcription à doigts de zinc de type C2H2 orf19.13374p ont été inactivés, suggérant que Mcm1p et orf19.13374p sont impliqués dans la réponse de MDR1au bénomyl. Il est intéressant de noter que la BRE contient une séquence qui s'aligne parfaitement avec la séquence consensus du site de fixation de Mcm1p, ce qui soulève la possibilité que MDR1 pourrait être une cible directe de cet activateur transcriptionnel. En conclusion, alors que l'identité des facteurs agissant en trans en se fixant à la BRE et à la HRE reste à être confirmée, les outils que nous avons développés au cours de la caractérisation des éléments agissant en cis sur le promoteur MDR1 peut maintenant servir à élucider la nature des composants modulant son activité.

Implementation and evaluation of an internet health site for adolescents in Switzerland.

Ciao is a website specifically designed for young people and focuses mainly on health issues. This report presents the process of setting up the site and a first evaluation undertaken by using two self-administered questionnaires administered via the website itself. It suggests that it is possible to provide young people with authoritative health information and to facilitate their access to counseling and health care facilities by having young people use such a website.

Extracellular matrix formation after transplantation of human embryonic stem cell-derived cardiomyocytes.

Transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) for cardiac regeneration is hampered by the formation of fibrotic tissue around the grafts, preventing electrophysiological coupling. Investigating this process, we found that: (1) beating hESC-CM in vitro are embedded in collagens, laminin and fibronectin, which they bind via appropriate integrins; (2) after transplantation into the mouse heart, hESC-CM continue to secrete collagen IV, XVIII and fibronectin; (3) integrin expression on hESC-CM largely matches the matrix type they encounter or secrete in vivo; (4) co-transplantation of hESC-derived endothelial cells and/or cardiac progenitors with hESC-CM results in the formation of functional capillaries; and (5) transplanted hESC-CM survive and mature in vivo for at least 24 weeks. These results form the basis of future developments aiming to reduce the adverse fibrotic reaction that currently complicates cell-based therapies for cardiac disease, and to provide an additional clue towards successful engraftment of cardiomyocytes by co-transplanting endothelial cells.

Eager for fairness or revenge: psychological altruism in economics

To understand the human capacity for psychological altruism, one requires a proper understanding of how people actually think and feel. This paper addresses the possible relevance of recent findings in experimental economics and neuroeconomics to the philosophical controversy over altruism and egoism. After briefly sketching and contextualizing the controversy, we survey and discuss the results of various studies on behaviourally altruistic helping and punishing behaviour, which provide stimulating clues for the debate over psychological altruism. On closer analysis, these studies prove less relevant than originally expected because the data obtained admit competing interpretations such as people seeking fairness versus people seeking revenge. However, this mitigated conclusion does not preclude the possibility of more fruitful research in the area in the future. Throughout our analysis, we provide hints for the direction of future research on the question.

Atuação da enfermeira eleva o controle de hipertensos e diminui o efeito do avental branco

Realizou-se estudo comparativo randomizado para avaliar o controle de hipertensos, com uso da medida residencial da pressão arterial (MRPA) e medida casual, bem como para analisar o efeito do avental branco. Hipertensos atendidos em unidades básicas de saúde foram divididos aleatoriamente em: grupo I, participante das atividades educativas, e grupo II, que seguiu a rotina de atendimento. Os hipertensos do grupo I realizaram MRPA no início e final do estudo. Efeito do avental branco foi avaliado pela diferença entre a medida casual e MRPA. Foram incluídos 290 hipertensos, porém realizaram MRPA 82 hipertensos. Houve aumento no controle da pressão do início ao final do estudo nos hipertensos do grupo I (p < 0,05) avaliado pela MRPA (60% para 68,3%) e pela medida casual (62% para 71%); no grupo II o controle foi maior na MRPA do que na medida casual (63% vs 50%). O efeito do avental branco foi maior no grupo II.

Homeodomain-only protein HOP is a novel modulator of late differentiation in keratinocytes.

The homeodomain-only protein (HOP) contains an atypical homeodomain which is unable to bind to DNA due to mutations in residues important for DNA binding. Recently, HOP was reported to regulate proliferation/differentiation homeostasis in different cell types. In the present study, we performed transcriptional profiling of cultured primary human keratinocytes and noted a robust induction of HOP upon calcium-induced cell differentiation. Immunohistochemistry of human skin localized HOP to the granular layer in the epidermis. Overexpression of HOP using a lentiviral vector up-regulated FLG and LOR expression during keratinocyte differentiation. Conversely, decreasing HOP expression using small interfering RNA markedly reduced the calcium-induced expression of late markers of differentiation in vitro, with the most prominent effect on profilaggrin (FLG) mRNA. Moreover, mRNA levels of profilaggrin and loricrin were downregulated in the epidermis of HOP knockout mice. Analysis of skin disorders revealed altered HOP expression in lichen planus, psoriasis and squamous cell carcinoma (SCC). Our data indicate that HOP is a novel modulator of late terminal differentiation in keratinocytes.

Julius Firmicus Maternus, Mathesis, libri IV.

Les ff. 2-7v, 12-15v, 19-27v et 30-42v ont été copiés au XIe s., les autres ont été rajoutés au XVe s.

U-Pb zircon dating of granitoids from the Dora-Maira massif (western Italian Alps)

Among the large number of granitic intrusions within the Dora-Maira massif, several main types can be distinguished. In this study we report field, petrographic and geochemical investigations as well as zircon typology and conventional U-Pb zircon dating of plutons representing these types. The main results are as follows: the Punta Muret augengneiss is a polymetamorphosed peraluminous granite of anatectic origin. It is 457 +/- 2 Ma old and represents one of the numerous Caledonian orthogneisses of the Alpine basement. All other dated granites are of Late Variscan age. The Cavour leucogranite is an evolved granite of probably calc-alkaline affiliation, dated at 304 +/- 2 Ma. The dioritic and granodioritic facies of the Malanaggio diorite (auct.) are typical calc-alkaline rocks, whose respective age of 290 +/- 2 and 288 +/- 2 Ma overlap within errors. The Sangone and Freidour granite types have very similar alkali-calcic characteristics; their ages are poorly constrained between 267-279 and 268-283 Ma, respectively. The new data for the Dora-Maira granites are in keeping with models of the overall evolution of the Late- to Post-Variscan magmatism in the Alpine area in terms of age distribution and progressive geochemical evolution towards alkaline melts. In a first approximation, granitic rocks across the Variscan belt seem to be increasingly younger towards the internal (southern) parts of the orogen. A Carboniferous, distensive Basin and Range situation is thought to be responsible for the magmatic activity. This tectonic context is comparable to the back-are opening of an active continental margin. The observed southward migration of the magmatism could be linked to the roll-back of the subducting Paleotethyan oceanic plate along the Variscan cordillera.

VOLUMES RELIES du Cabinet des titres : recherches de noblesse, armoriaux, preuves, histoires généalogiques. « Les arbres de généalogie de la noblesse des provinces de Bourgogne et de Bresse, et des quatre comtéz de son gouvernement, avec le blason de leurs armes ».

Contient : « Procès-verbaux de production de preuves de noblesse, faites par-devant M. Bouchu, intendant en Bourgogne »

VOLUMES RELIES du Cabinet des titres : recherches de noblesse, armoriaux, preuves, histoires généalogiques. Inventaire des titres de propriété et autres de l'amiral Gaspard de Coligny.

L'inventaire débute par le détail d'une « liasse en laquelle sont les principaulx tiltres comme la chastellenie de Chastillon a esté vendue à la maison des Bragues, et comme de ladicte maison elle est venue ès maisons de Lourdin, de Salligny et de Colligny. »

Desigualdade no Acesso ao Mercado de Trabalho dos Recem Formados

A natureza do mercado de trabalho em Cabo Verde nos últimos anos, tem influenciado constantemente a área de formação profissional. Exigindo assim, um numero cada vez maior de jovens quadros melhor qualificado que consegue dar resposta positivamente as necessidades do mercado. Entretanto, não podemos falar do mercado de trabalho sem realçar as instituições de formação, entidades responsáveis por aquilo que estratégias de formação para a manutenção do equilíbrio do mercado, dando assim prioridades áreas prioritárias para o desenvolvimento económico e social do país. Cabo Verde é um pequeno país arquipelágico, desprovido de uma base de recursos naturais economicamente exploráveis e fortemente dependente do exterior (importa cerca de 65% dos bens de consumo, 15% do seu PIB provem sob forma de remessas de emigrantes e 15%-da ajuda pública ao desenvolvimento)1.