Abstract Cryptococcus neoformans and Cryptococcus gattii are the etiological agents of cryptococcosis, a high mortality disease. The development of such disease depends on the interaction of fungal cells with macrophages, in which they can reside and replicate. In order to dissect the molecular mechanisms by which cryptococcal cells modulate the activity of macrophages, a genome-scale comparative analysis of transcriptional changes in macrophages exposed to Cryptococcus spp. was conducted. Altered expression of nearly 40 genes was detected in macrophages exposed to cryptococcal cells. The major processes were associated with the mTOR pathway, whose associated genes exhibited decreased expression in macrophages incubated with cryptococcal cells. Phosphorylation of p70S6K and GSK-3β was also decreased in macrophages incubated with fungal cells. In this way, Cryptococci presence could drive the modulation of mTOR pathway in macrophages possibly to increase the survival of the pathogen.

Biodiesel purity expressed as fatty esters content is one of its most important quality parameters. Although several instrumental methods have been employed, gas chromatography with internal standard calibration has been the most used. Biodiesel is a very complex matrix, therefore finding a suitable internal standard (IS) is not straightforward. EN14103:2003 standard established methyl heptadecanoate (C17:0) as IS. Since this fatty ester is naturally present in some biodiesel feedstock as tallow, that standard was reviewed in 2011, and methyl nonadecanoate (C19:0) became the new IS. In turn, C19:0 produces a peak on the chromatogram that is difficult to resolve from methyl linoeate (C18:2) and metyl linolenate (C18:3) ones. In this paper, a GC-FID method for the determination of the esters content in methyl and ethyl biodiesel from canola and soy oils, using hexadecyl acetate as a new internal standard was validated. EN14103:2003 was used as reference method. Method selectivity, accuracy (t student parameter < 2.18 from both methods), repeatability (0.1 - 0.4%), intermediate precision (0.2 - 1.8%), and robustness (0.1 - 1.6%) were investigated and considered appropriate for the scope.

Cryptococcus neoformans and C. gattii are pathogenic yeasts that cause life-threatening diseases in humans and animals. Iron is an essential nutrient for virtually every organism as it functions as a cofactor in numerous essential enzymatic reactions. In the literature, the competition for iron between microbes and mammalian hosts during infection is well documented. In this study, we used representational difference analysis (RDA) in order to gain a better understanding of how C. gattii responds to iron starvation. A total of 15 and 29 genes were identified as having altered expression levels due to iron depletion after 3 h and 12 h, respectively. Of these, eight genes were identified in both libraries. The transcripts were related to many biological processes, such as cell cycle, ergosterol metabolism, cell wall organization, transportation, translation, cell respiration and the stress response. These data suggest a remodeling of C. gattii metabolism during conditions of iron deprivation.

The aim of this work was the partial purification and subsequent evaluation of chitinase expression during the various growth phases of Paracoccidioides brasiliensis. Initially, PbCTS1r was expressed as a recombinant protein and displayed enzymatic activity against 4-MU-[N-acetylglucosamine (GlcNAc)]3 and 4-MU-(GlcNAc)2. Two proteins, 45 kDa and 39 kDa in size, were partially purified from P. brasiliensis yeast crude extract using cation-exchange chromatography coupled with HPLC and were characterised as PbCTS1 and PbCTS2, respectively. Anti-PbCTS1r antibody recognised two proteins in the crude extracts of yeast and the transitional stage between mycelial and yeast phases. In crude extracts of mycelium, only the 45 kDa protein was detected. However, quantitative real-time polymerase chain reaction led to the detection of small quantities of Pbcts2 transcript in the mycelial phase. In the yeast cell wall extract, only the 39 kDa protein was detected. Moreover, both proteins were secreted by the yeast parasitic phase, suggesting that these proteins participate in the modulation of the fungal environment. Phylogenetic analysis of the predicted PbCTS1 and PbCTS2 proteins indicated that they code for distinct chitinases in P. brasiliensis. During evolution, P. brasiliensis could have acquired the paralogues Pbcts1 and Pbcts2 for growth and survival in diverse environments in both saprophytic and parasitic phases.

Neste trabalho foi investigado o índice de HMF (5-hidroximetilfurfural) em méis comercializados em Porto Alegre - RS, utilizando Cromatografia Capilar Eletrocinética Micelar. O HMF, produto da condensação da frutose, é um indicador da qualidade e conservação do mel. Foram analisadas 11 marcas de méis comercializados na cidade de Porto Alegre. O composto estudado esteve presente em todas as amostras, em um intervalo de concentração de 0,191 a 6,206 mg.kg-1. Para quantificar o HMF presente nos méis, utilizou-se a técnica de adição de padrão. A taxa de recuperação foi de 98% e o limite de detecção foi de 0,025 mg.kg-1. O limite permitido de HMF em méis, segundo a legislação brasileira, é de 60 mg.kg-1.

In this work, the occurrence of HMF (5-hydroxymethylfurfural) in honey marketed in Porto Alegre - RS was investigated using Micellar Electrokinetic Capillary Chromatography. The HMF, which is the product of the fructose condensation, is an indicator of honey quality and conservation. Eleven types of honey commercialized in Porto Alegre were analyzed, and all of them contained HMF in a range from 0.191 to 6.206 mg.kg-1. In order to quantify the HMF present in the samples, the technique of standard addition was employed. The recovery was 98% and the detection limit was 0.025 mg.kg-1. The allowed limit of HMF in honey, according to the Brazilian Legislation, is 60 mg.kg-1.

O presente estudo teve por objetivo determinar os padrões moleculares e de resistência aos antimicrobianos de isolados de E. coli provenientes do trato urinário de suínos no Sul do Brasil. Os fatores estudados dividiram os patotipos ETEC, STEC e UPEC. Trinta e quatro (38,63%) isolados avaliados apresentavam um ou mais dos fatores de virulência pesquisados. A freqüência dos genes de virulência detectados foram: pap (10,97%), hlyA (10,97%), iha (9,75%), lt (8,53%), sta (7,31%) sfa (6,09%), f4 (4,87%), f5 (4,87%), stb (4,87%), f6 (1,21%) e f41 (1,21%). Os isolados foram resistentes à penicilina (95,12%), lincomicina (93,9%), eritromicina (92,68%), tetraciclina (90,24%), amoxacilina (82,92%), ampicilina (74,39%), josamicina (79,26%), norfloxacina (58,53%), enrofloxacina (57,31%), gentamicina (39,02%), neomicina (37,8%), apramicina (30,48%), colistina (30,48%) e cefalexina (6,09%). Trinta e dois (39,02%) isolados de E. coli continham plasmídeos.

The present study determined the molecular and resistance patterns of E. coli isolates from urinary tract of swine in Southern of Brazil. Molecular characterization of urinary vesicle samples was performed by PCR detection of virulence factors from ETEC, STEC and UPEC. From a total of 82 E. coli isolates, 34 (38.63%) harbored one or more virulence factors. The frequency of virulence factors genes detected by PCR were: pap (10.97%), hlyA (10.97%), iha (9.75%), lt (8.53%), sta (7.31%) sfa (6.09%), f4 (4.87%), f5 (4.87%), stb (4.87%), f6 (1.21%) and f41 (1.21%). Isolates were resistant to penicillin (95.12%), lincomycin (93.9%), erythromycin (92.68%), tetracycline (90.24%), amoxicillin (82.92%), ampicillin (74.39%), josamycin (79.26%), norfloxacin (58.53%), enrofloxacin (57.31%), gentamicin (39.02%), neomycin (37.8%), apramycin (30.48%), colistine (30.48%) and cefalexin (6.09%). A number of 32 (39.02%) E. coli isolates harbored plasmids.

A patulina é uma micotoxina produzida por diversas espécies de fungos dos gêneros Penicillium e Aspergillus, encontrada em diversas espécies frutíferas e, notadamente, em maçãs e produtos derivados da maçã. A patulina tem um amplo espectro de toxicidade, incluindo carcinogenicidade e teratogenicidade em animais. Devido a sua estabilidade, níveis consideráveis de patulina permanecem nos produtos derivados da maçã, mesmo após as etapas do processamento. Este artigo revisa os diferentes métodos analíticos usados para a determinação de patulina e métodos empregados para reduzir os níveis de patulina nos produtos alimentícios.

Patulin is a mycotoxin produced by several fungal species of the genera Penicillium and Aspergillus, found on several fruit species and, remarkably, in apples and apple products. Patulin has a broad spectrum of toxicity, including carcinogenicity and teratogenicity in animals. Due to the stability of the molecule, considerable amounts of patulin still remain in apple products after processing. This paper reviews different analytical methods for patulin determination and methods to reduce levels of patulin in apple products as well.

Mycoplasmas are very fastidious in their nutritional requirements for in vitro growth and have limited biosynthetic capacity, a reflection of their reduced genomes. As a result, these bacteria depend upon external metabolites for nutrition and growth and have developed dependence on their hosts for survival and maintenance. Protein degradation and peptide importation play an important role in Mycoplasma spp. nutrition, and proteases can play a role in host adaptation and pathogenicity. Here, we present a general survey on the genes involved in protein degradation, secretion and importation, comparing all available Mollicute genomes.

Dezenove culturas de C. dubliniensis isoladas no Brasil, previamente identificadas através de métodos genotípicos, foram avaliadas pelo kit comercial ID 32C (bioMerieux). Treze culturas foram identificadas como C. dubliniensis, mas as demais (seis) evidenciaram perfil duvidoso, embora o software do sistema sugerisse 83,6% de chances das mesmas pertencerem à espécie C. dubliniensis. A literatura tem registrado grande variabilidade fenotípica com esta espécie e, por isto, as identificações obtidas com este sistema deverão ser consideradas como presuntivas.

The purpose of the present study was to evaluate the identification of 19 Brazilian C. dubliniensis based on the biochemical profile exhibited when tested by the commercial identification kit ID 32C (bioMerieux). Thirteen of the isolates were rigorously identified as C. dubliniensis and the remaining isolates (six) were considered as having a doubtful profile but the software also suggested that there was 83.6% of chances for them to be C. dubliniensis. As well as pointed by the literature the identification obtained by phenotypic tests should be considered presumptive for C. dubliniensis due to variability of this new species.

Este estudo foi realizado com os primeiros isolados ambientais de C. neoformans sorotipo D, obtidos no Rio Grande do Sul. Objetivando-se avaliar a susceptibilidade a agentes antifúngicos de forma mais rigorosa, utilizou-se a técnica de referência proposta pelo NCCLS, Caldo Yeast Nitrogen Base (YNB) proposto por Ghannoum et al., Antibiotic medium 3, caldo YNB adequado à metodologia do NCCLS e o E-test. Os resultados indicaram que todos os isolados foram sensíveis à anfotericina B (0,0625-0,5 µg/mL), fluconazol (0,125-4,0 µg/mL), itraconazol (0,031-0,25 µg/ml) e fluorocitosina (0,125-4,0 µg/mL) através das técnicas empregadas. Nos testes de termotolerância (47ºC/30 min), observou-se que as culturas de C. neoformans sorotipo D são mais sensíveis do que as de C. neoformans sorotipo A.

In this work we studied the susceptibility to heat and antifungal agents of the first strains of environmental Cryptococcus neoformans var neoformans (serotype D) isolated in the state of Rio Grande do Sul, Brazil. In order to achieve a rigorous analysis, we employed the methodology recommended by NCCLS, Yeast Nitrogen Base (YNB) proposed by Ghannoum et al (YNB-1), Antibiotic medium 3 (AM3) indicated by others, YNB adjusted to the NCCLS methodology (YNB-2) and Etest. Our results indicate that all strains were susceptible to amphotericin B (0.0625-0.5 µg/mL), fluconazole (0.125-8.0 µg/mL), itraconazole (0.031-0.25 µg/mL) and flucytosine (0.125-4.0 µg/mL). The C. neoformans serotype D strains were more susceptible to heat (47ºC/30 min) than C. neoformans serotype A.

Comparou-se a suscetibilidade de 69 amostras de C. neoformans isoladas de pacientes com SIDA com 13 amostras de C. neoformans isoladas do meio ambiente, frente a quatro agentes antifúngicos. Com base na metodologia preconizada pelo NCCLS (M27-A) as concentrações inibitórias mínimas (CIMs) obtidas para a anfotericina B, itraconazol e cetoconazol foram muito semelhantes nos dois grupos estudados. Todavia, frente ao fluconazol, os isolados clínicos evidenciaram menor sensibilidade do que os provenientes do meio ambiente. Alguns aspectos envolvendo a importância e dificuldades dos testes de suscetibilidade com Cryptococcus neoformans são também discutidos pelos autores.

The purpose of the present study was to compare the susceptibility to four antifungal agents of 69 Cryptococcus neoformans strains isolated from AIDS patients with that of 13 C. neoformans strains isolated from the environment. Based on the NCCLS M27-A methodology the Minimal Inhibitory Concentrations (MICs) obtained for amphotericin B, itraconazole and ketoconazole were very similar for clinical and environmental isolates. Clinical isolates were less susceptible to fluconazole than environmental isolates. The significance of these findings and aspects concerning the importance, role and difficulties of C. neoformans susceptibility testing are also discussed.