In vitro characterization of Trichophyton rubrum and T. mentagrophytes biofilms

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

cDNA representational difference analysis used in the identification of genes expressed by Trichophyton rubrum during contact with keratin

Dermatophytes are adapted to infect skin, hair and nails by their ability to utilize keratin as a nutrient source. Trichophyton rubrum is an anthropophilic fungus, causing up to 90% of chronic cases of dermatophytosis. The understanding of the complex interactions between the fungus and its host should include the identification of genes expressed during infection. To identify the genes involved in the infection process, representational difference analysis (RDA) was applied to two cDNA populations from T. rubrum, one transcribed from the RNA of fungus cultured in the presence of keratin and the other from RNA generated during fungal growth in minimal medium. The analysis identified differentially expressed transcripts. Genes related to signal transduction, membrane protein, oxidative stress response, and some putative virulence factors were up-regulated during the contact of the fungus with keratin. The expression patterns of these genes were also verified by real-time PCR, in conidia of T. rubrum infecting primarily cultured human keratinocytes in vitro, revealing their potential role in the infective process. A better understanding of this interaction will contribute significantly to our knowledge of the process of dermatophyte infection.

Comparative Genome Analysis of Trichophyton rubrum and Related Dermatophytes Reveals Candidate Genes Involved in Infection

The major cause of athlete's foot is Trichophyton rubrum, a dermatophyte or fungal pathogen of human skin. To facilitate molecular analyses of the dermatophytes, we sequenced T. rubrum and four related species, Trichophyton tonsurans, Trichophyton equinum, Microsporum canis, and Microsporum gypseum. These species differ in host range, mating, and disease progression. The dermatophyte genomes are highly colinear yet contain gene family expansions not found in other human-associated fungi. Dermatophyte genomes are enriched for gene families containing the LysM domain, which binds chitin and potentially related carbohydrates. These LysM domains differ in sequence from those in other species in regions of the peptide that could affect substrate binding. The dermatophytes also encode novel sets of fungus-specific kinases with unknown specificity, including nonfunctional pseudokinases, which may inhibit phosphorylation by competing for kinase sites within substrates, acting as allosteric effectors, or acting as scaffolds for signaling. The dermatophytes are also enriched for a large number of enzymes that synthesize secondary metabolites, including dermatophyte-specific genes that could synthesize novel compounds. Finally, dermatophytes are enriched in several classes of proteases that are necessary for fungal growth and nutrient acquisition on keratinized tissues. Despite differences in mating ability, genes involved in mating and meiosis are conserved across species, suggesting the possibility of cryptic mating in species where it has not been previously detected. These genome analyses identify gene families that are important to our understanding of how dermatophytes cause chronic infections, how they interact with epithelial cells, and how they respond to the host immune response. IMPORTANCE Athlete's foot, jock itch, ringworm, and nail infections are common fungal infections, all caused by fungi known as dermatophytes (fungi that infect skin). This report presents the genome sequences of Trichophyton rubrum, the most frequent cause of athlete's foot, as well as four other common dermatophytes. Dermatophyte genomes are enriched for four gene classes that may contribute to the ability of these fungi to cause disease. These include (i) proteases secreted to degrade skin; (ii) kinases, including pseudokinases, that are involved in signaling necessary for adapting to skin; (iii) secondary metabolites, compounds that act as toxins or signals in the interactions between fungus and host; and (iv) a class of proteins (LysM) that appear to bind and mask cell wall components and carbohydrates, thus avoiding the host's immune response to the fungi. These genome sequences provide a strong foundation for future work in understanding how dermatophytes cause disease.

Susceptibilities of the dermatophytes Trichophyton mentagrophytes and T. rubrum microconidia to photodynamic antimicrobial chemotherapy with novel phenothiazinium photosensitizers and red light

Photodynamic antimicrobial chemotherapy (PACT) is a promising alternative to conventional chemotherapy that can be used to treat localized mycosis. The development of PACT depends on identifying effective and selective PS for the different pathogenic species. The in vitro susceptibilities of Trichophyton mentagrophytes and Trichophyton rubrum microconidia to PACT with methylene blue (MB), toluidine blue o (TBO), new methylene blue N (NMBN), and the novel pentacyclic phenothiazinium photosensitizer S137 were investigated. The efficacy of each PS was determined based on its minimal inhibitory concentration (MIC). Additionally, we evaluated the effect of PACT with NMBN and S137 on the survival of the microconidia of both species. 5137 showed the lowest MIC. MIC for S137 was 2.5 mu M both for T. mentagrophytes and T. rubrum, when a light dose of 5J cm(-2) was used. PACT with NMBN (10 mu M and 20J cm(-2)) resulted in a reduction of 4 logs in the survival of the T. rubrum and no survivor of T. mentagrophytes was observed. PACT with S137 at 1 mu M and 20J cm(-2) resulted in a reduction of approximately 3 logs in the survival of both species. When a S137 concentration of 10 mu M was used, no survivor was observed for both species at all light doses (5, 10 and 20J cm(-2)). (C) 2012 Elsevier B.V. All rights reserved.

rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum

The selection of reference genes used for data normalization to quantify gene expression by real-time PCR amplifications (qRT-PCR) is crucial for the accuracy of this technique. In spite of this, little information regarding such genes for qRT-PCR is available for gene expression analyses in pathogenic fungi. Thus, we investigated the suitability of eight candidate reference genes in isolates of the human dermatophyte Trichophyton rubrum subjected to several environmental challenges, such as drug exposure, interaction with human nail and skin, and heat stress. The stability of these genes was determined by geNorm, NormFinder and Best-Keeper programs. The gene with the most stable expression in the majority of the conditions tested was rpb2 (DNA-dependent RNA polymerase II), which was validated in three T. rubrum strains. Moreover, the combination of rpb2 and chs1 (chitin synthase) genes provided for the most reliable qRT-PCR data normalization in T. rubrum under a broad range of biological conditions. To the best of our knowledge this is the first report on the selection of reference genes for qRT-PCR data normalization in dermatophytes and the results of these studies should permit further analysis of gene expression under several experimental conditions, with improved accuracy and reliability.

Ri-abitare il margine urbano - Strategie di riqualificazione per il social housing: un edificio residenziale "ACER" come modello di intervento

La tesi individua alcune strategie di rigenerazione urbana e di riqualificazione edilizia, al fine di ottenere una serie di linee guida per l’intervento sul patrimonio di edilizia abitativa situata nelle periferie urbane. Tali principi sono stati poi applicati ad un edificio ACER collocato nella prima periferia di Forlì, per sperimentare l’efficacia delle strategie individuate. Dalla ricerca svolta sulle strategie di intervento volte alla riqualificazione sociale delle periferie, in particolare la teoria del “Defencible space” di Jacobs, si evidenzia l’importanza di accentuare nei residenti il sentimento di territorialità, ovvero la consapevolezza di far parte di una comunità specifica insediata in un particolare spazio, alimentata attraverso la frequentazione e l’appropriazione percettivo-funzionale degli spazi pubblici. Si è deciso quindi di allargare il campo di intervento alla rigenerazione dell’interno comparto, attraverso la riorganizzazione degli spazi verdi e la dotazione di attrezzature sportive e ricreative, in modo da offrire spazi specifici per le diverse utenze (anziani, giovani, bambini) e la definizione di un programma funzionale di servizi ricreativi e spazi destinati a piccolo commercio per integrare le dotazioni carenti dell’area. Dall’analisi approfondita dell’edificio sono emerse le criticità maggiori su cui intervenire: - l’intersezione dei percorsi di accesso all’edificio - la struttura portante rigida, non modificabile - la scarsa varietà tipologica degli alloggi - l’elevato fabbisogno energetico. La riqualificazione dell’edificio ha toccato quindi differenti campi: tecnologico, funzionale, energetico e sociale; il progetto è stato strutturato come una serie di fasi successive di intervento, eventualmente realizzabili in tempi diversi, in modo da consentire il raggiungimento di diversi obiettivi di qualità, in funzione della priorità data alle diverse esigenze. Secondo quest’ottica, il primo grado di intervento, la fase 1 - riqualificazione energetica, si limita all’adeguamento dello stato attuale alle prestazioni energetiche richieste dalla normativa vigente, in assenza di adeguamenti tipologici e spaziali. La fase 2 propone la sostituzione degli impianti di riscaldamento a caldaie autonome presenti attualmente con un impianto centralizzato con pompa di calore, un intervento invasivo che rende necessaria la realizzazione di un “involucro polifunzionale” che avvolge completamente l’edificio. Questo intervento nasce da tre necessità fondamentali : - architettonica: poter ampliare verso l’esterno le superfici degli alloggi, così da intervenire sulle unità abitative rendendole più rispondenti alle necessità odierne; - statica: non dover gravare in ciò sull’edificio esistente apportando ulteriori carichi, difficilmente sopportabili dalla struttura esistente, assicurando il rispetto della normativa antisismica in vigore; - impiantistica/tecnologica: alloggiare i condotti del nuovo impianto centralizzato per il riscaldamento, raffrescamento e acs; La fase 3 è invece incentrata sull’ampliamento dell’offerta abitativa, in modo da rispondere anche a necessità legate ad utenze speciali, come utenti disabili o anziani. L’addizione di nuovi volumi si sviluppa in tre direzioni: - un volume parassita, che aderisce all’edificio nel fronte sud/est, indipendente dal punto di vista strutturale, ruotato per sfruttare al meglio l’orientamento ottimale. - un volume satellite, indipendente, connesso all’edificio esistente tramite un elemento di raccordo, e nel quale sono collocati alcuni alloggi speciali. - un’addizione in copertura, che non appoggia direttamente sul solaio di copertura esistente, ma grava sull’elemento di chiusura del’involucro realizzato nella fase 2 Completano il progetto le addizioni volumetriche a piano terra, destinate a servizi quali un centro diurno, un micronido e un bar, i quali costituiscono la traduzione alla scala dell’edificio delle strategie applicate nel progetto di comparto. Questi interventi hanno consentito di trasformare un edificio costruito negli anni ’80 in un complesso residenziale moderno, dotato spazi accessori di grande qualità, tecnologie moderne che ne garantiscono il comfort abitativo, servizi alla persona disponibili in prossimità dell’edificio.

Genetic variability and structuring of deep Corallium rubrum (Linnaeus, 1758) populations

ABSTRACT Given the decline of shallow-water red coral populations resulting from over-exploitation and mass mortality events, deeper populations below 50 metres depth (mesophotic populations) are currently the most harvested; unfortunately, very little is known about their biology and ecology. The persistence of these populations is tightly linked to their adult density, reproductive success, larval dispersal and recruitment. Moreover, for their conservation, it is paramount understand processes such as connectivity within and among populations. Here, for the first time, genetic variability and structuring of Corallium rubrum populations collected in the Tyrrhenian Sea ranging from 58 to 118 metres were analyzed using ten microsatellite loci and two mitochondrial markers (mtMSH and MtC). The aims of the work were 1) to examine patterns of genetic diversity within each geographic area (Elba, Ischia and Praiano) and 2) to define population structuring at different spatial scales (from tens of metres to hundreds of kilometres). Based on microsatellite data set, significant deviations from Hardy-Weinberg equilibrium due to elevated heterozygote deficiencies were detected in all samples, probably related to the presence of null alleles and/or inbreeding, as was previously observed in shallow-water populations. Moreover, significant levels of genetic differentiation were observed at all spatial scale, suggesting a recent isolation of populations. Biological factors which act at small spatial scale and/or abiotic factors at larger scale (e.g. summer gyres or absence of suitable substrata for settlement) could determine this genetic isolation. Using mitochondrial markers, significant differences were found only at wider scale (between Tuscany and Campania regions). These results could be related to the different mutation rate of the molecular makers or to the occurrence of some historical links within regions. A significant isolation by distance pattern was then observed using both data sets, confirming the restricted larval dispersal capability of the species. Therefore, the hypothesis that deeper populations may act as a source of larvae helping recovery of threatened shallow-water populations is not proved. Conservation strategies have to take into account these results, and management plans of deep and currently harvested populations have to be defined at a regional or sub regional level, similarly to shallow-water populations. Nevertheless, further investigations should be needed to understand better the genetic structuring of this species in the mesophotic zone, e.g. extending studies to other Mediterranean deep-water populations.

Recuperare l'abitare sociale. Riqualificazione dell'edificio acer di via Wolfgang Goethe a Bologna.

Nel volume vengono proposte delle strategie di riqualificazione dell’edificio in via W. Goethe con numero civico 2-10 a Corticella zona Navile. L’edificio fa parte del quartiere PEEP realizzato a partire dagli anni ‘70 circa. L’intera area ricopre una superficie pari a 220.000 m2 di cui edificata 38.000 m2. Della superficie edificata ben 30.400 m2 è stata destinata al residenziale. A seguito delle evoluzioni economiche e sociali e alle restrizioni sempre più severe della normativa, il quartiere ha mostrato delle carenze e delle criticità: – Scarse prestazioni energetiche degli edifici con elevati costi di esercizio; – Inadeguata risposta alle azioni sismiche; – Tagli di alloggi non in grado di soddisfare l’odierna domanda; – Mancanza di efficaci spazi pubblici e di relazione; – Inefficace connessione urbana ai maggiori poli attrattivi e di circolazione. Come primo obbiettivo è stato affrontato il problema della mancanza di connessioni ciclopedonali adeguate al territorio di Corticella. Vi sono infatti alcune aree di nodale importanza che sono sprovviste di questi collegamenti rendendo difficoltoso il link tra i punti attrattivi fondamentali di Corticella e il nostro quartiere. Il progetto intende migliorare questo tipo di servizio in linea con il progetto “bike sharing & ride” promosso dalla Regione Emilia Romagna al fine di incentivare e promuovere la mobilità. Seguono poi gli interventi volti a risolvere le problematiche riscontrate nell’edificio. Interventi atti a riportare l’edificio in una condizione tale da renderlo adeguato alle necessità attuali e in grado di svolgere la sua funzione per gli anni a venire. Si tratta di strategie tecniche e parallelamente di strategie energetiche, con l’obbiettivo di definire spazi confortevoli all’interno degli alloggi. A tale fine sono state realizzate anche valutazioni sull’illuminazione naturale degli ambienti interni per valutare l’efficacia o meno del sistema utilizzato.

Investigating within-canopy variation of functional traits and cellular structure of sugar maple (Acer saccharum) leaves

Patterns of increasing leaf mass per area (LMA), area-based leaf nitrogen (Narea), and carbon isotope composition (δ13C) with increasing height in the canopy have been attributed to light gradients or hydraulic limitation in tall trees. Theoretical optimal distributions of LMA and Narea that scale with light maximize canopy photosynthesis; however, sub-optimal distributions are often observed due to hydraulic constraints on leaf development. Using observational, experimental, and modeling approaches, we investigated the response of leaf functional traits (LMA, density, thickness, and leaf nitrogen), leaf carbon isotope composition (δ13C), and cellular structure to light availability, height, and leaf water potential (Ψl) in an Acer saccharum forest to tease apart the influence of light and hydraulic limitations. LMA, leaf and palisade layer thickness, and leaf density were greater at greater light availability but similar heights, highlighting the strong control of light on leaf morphology and cellular structure. Experimental shading decreased both LMA and area-based leaf nitrogen (Narea) and revealed that LMA and Narea were more strongly correlated with height earlier in the growing season and with light later in the growing season. The supply of CO2 to leaves at higher heights appeared to be constrained by stomatal sensitivity to vapor pressure deficit (VPD) or midday leaf water potential, as indicated by increasing δ13C and VPD and decreasing midday Ψl with height. Model simulations showed that daily canopy photosynthesis was biased during the early growing season when seasonality was not accounted for, and was biased throughout the growing season when vertical gradients in LMA and Narea were not accounted for. Overall, our results suggest that leaves acclimate to light soon after leaf expansion, through an accumulation of leaf carbon, thickening of palisade layers and increased LMA, and reduction in stomatal sensitivity to Ψl or VPD. This period of light acclimation in leaves appears to optimize leaf function over time, despite height-related constraints early in the growing season. Our results imply that vertical gradients in leaf functional traits and leaf acclimation to light should be incorporated in canopy function models in order to refine estimates of canopy photosynthesis.

Hypoglycin A concentrations in seeds of Acer pseudoplatanus trees growing on atypical myopathy-affected and control pastures.

BACKGROUND Hypoglycin A, found in seeds of Acer negundo, appears to cause seasonal pasture myopathy (SPM) in North America and is implicated in atypical myopathy (AM) in Europe. Acer negundo is uncommon in Europe. Thus, the potential source of hypoglycin A in Europe is unknown. HYPOTHESIS AND OBJECTIVES We hypothesized that seeds of Acer pseudoplatanus were the source of hypoglycin A in Europe. Our objective was to determine the concentration of hypoglycin A in seeds of A. pseudoplatanus trees located in pastures where previous cases of AM had occurred. ANIMALS None. METHODS University of Berne records were searched to retrospectively identify 6 farms with 10 AM cases and 11 suspected AM deaths between 2007 and 2011. During October 2012, A. pseudoplatanus seeds were collected from 2 to 6 trees per pasture on 6 AM farms (7 pastures) from trees in or close to 2 pastures on 2 control farms where AM had not been previously reported. Hypoglycin A in seeds was analyzed by GC-MS. RESULTS Acer pseudoplatanus trees were identified on all AM pastures. Hypoglycin A was detected in all A. pseudoplatanus seeds in highly variable concentrations ranging from 0.04 to 2.81 μg/mg (mean 0.69) on AM farms and 0.10 to 9.12 μg/mg (mean 1.59) on control farms. CONCLUSION AND CLINICAL IMPORTANCE Preventing horses from grazing pastures containing A. pseudoplatanus seeds during late fall and early spring might be the best means to prevent AM.

Acer californicum T. et Gr.

Dr. G. Dieck

Ueber Acer Ginale, Ceanothus und Clematis und deren Verwendung bei Gruppenpflanzungen in Gärten

Von August Schütz

Blumenmißbildung an Ribes rubrum

Von Prof. v. Schlechtendal