Effects of camptothecin derivatives and topoisomerase dual inhibitors on Trypanosoma cruzi growth and ultrastructure

BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas’ disease that is an endemic disease in Latin America and affects about 8 million people. This parasite belongs to the Trypanosomatidae family which contains a single mitochondrion with an enlarged region, named kinetoplast that harbors the mitochondrial DNA (kDNA). The kinetoplast and the nucleus present a great variety of essential enzymes involved in DNA replication and topology, including DNA topoisomerases. Such enzymes are considered to be promising molecular targets for cancer treatment and for antiparasitic chemotherapy. In this work, the proliferation and ultrastructure of T. cruzi epimastigotes were evaluated after treatment with eukaryotic topoisomerase I inhibitors, such as topotecan and irinotecan, as well as with dual inhibitors (compounds that block eukaryotic topoisomerase I and topoisomerase II activities), such as baicalein, luteolin and evodiamine. Previous studies have shown that such inhibitors were able to block the growth of tumor cells, however most of them have never been tested on trypanosomatids. RESULTS: Considering the effects of topoisomerase I inhibitors, our results showed that topotecan decreased cell proliferation and caused unpacking of nuclear heterochromatin, however none of these alterations were observed after treatment with irinotecan. The dual inhibitors baicalein and evodiamine decreased cell growth; however the nuclear and kinetoplast ultrastructures were not affected. CONCLUSIONS: Taken together, our data showed that camptothecin is more efficient than its derivatives in decreasing T. cruzi proliferation. Furthermore, we conclude that drugs pertaining to a certain class of topoisomerase inhibitors may present different efficiencies as chemotherapeutical agents

Polyphasic identification of Aspergillus isolates belonging to section Nigri with clinical relevance

Aspergillosis is the name of a group of diseases in humans and animals caused by opportunist moulds of the genus Aspergillus. The vast majority of infections are caused by A. fumigatus, followed by other species such as A. flavus, A. terreus and A. niger. Among the pulmonary infection, aspergillosis is gaining prominent position not only in immunocompromised patients, but also in immunosuppressed. The absence of a reliable fungal identification system affects the control of systemic fungal infections. Matrix-Assisted Laser Desorption⁄Ionisation Time-Of- Flight Mass Spectrometry (MALDI-TOF MS) is a spectral technique that analysis the chemical molecular mass of the microbial cellular composition providing rapid and discriminatory fingerprints for identification. This technique starts to have application in clinical laboratories.This work aimed to get a reliable identification of Aspergillus isolates from section Nigri deposited at University of Recife Mycology (URM) culture collection. These materials were used as clinical reference strains to attend the great variability (morphology, biochemical, genomics and proteomics patterns) of the Brazilian fungal population. A polyphasic approach based on morphological, biochemical and spectral analysis by MALDI-TOF MS was applied for the characterisation and identification of 74 Aspergillus isolates from section Nigri deposited at URM. In addition, 12 Aspergillus type strains belonging to section Nigri deposited at Micoteca da Universidade do Minho (MUM) culture collection were used as reference for MALDI-TOF MS studies. The data obtained from the polyphasic approach indicates that MALDI-TOF MS results corroborate with those data obtained using classical taxonomy, biochemical and molecular biology analyses. Finally, MALDI-TOF MS technique is rapid, reliable and inexpensive in terms of labour and consumables when compared with other biological techniques. At present, it adds an additional step for polyphasic identification which is essential for the clinical diagnostic.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Brazil and Erasmus Mundus External Cooperation Window (EMECW

Antifungal susceptibility of the endophytic fungus Rhinocladiella similis (URM 7800) isolated from the Caatinga dry forest in Brazil

The present study reports a new occurrence of Rhinocladiella similis isolated as an endophytic fungus in the Caatinga dry tropical forest in Brazil and describes its antifungal susceptibility. The isolate R. similis URM 7800 was obtained from leaves of the medicinal plant Myracrodruon urundeuva. Its morphological characterization was performed on potato dextrose agar medium and molecular analysis using the ITS rDNA sequence. The antifungal susceptibility profile was defined using the Clinical and Laboratory Standards Institute (CLSI) protocol M38-A2. The colony of isolate URM 7800 showed slow growth, with an olivaceous-gray color and powdery mycelium; in microculture, it showed the typical features of R. similis. In the antifungal susceptibility test, isolate URM 7800 showed high minimal inhibitory concentration (MIC) values for amphotericin B (>16 μg/mL), voriconazole (16 μg/mL), terbinafine (>0.5 μg/mL), and caspofungin (>8 μg/mL), among other antifungal drugs. Pathogenic melanized fungi are frequently isolated in environments where humans may be exposed, and these data show that it is essential to know if these isolates possess antifungal resistance

Reduction of Tubulin Expression in Angomonas deanei by RNAi Modifies the Ultrastructure of the Trypanosomatid Protozoan and Impairs Division of Its Endosymbiotic Bacterium

In the last two decades, RNA interference pathways have been employed as a useful tool for reverse genetics in trypanosomatids. Angomonas deanei is a non-pathogenic trypanosomatid that maintains an obligatory endosymbiosis with a bacterium related to the Alcaligenaceae family. Studies of this symbiosis can help us to understand the origin of eukaryotic organelles. The recent elucidation of both the A. deanei and the bacterium symbiont genomes revealed that the host protozoan codes for the enzymes necessary for RNAi activity in trypanosomatids. Here we tested the functionality of the RNAi machinery by transfecting cells with dsRNA to a reporter gene (green fluorescent protein), which had been previously expressed in the parasite and to α-tubulin, an endogenous gene. In both cases, protein expression was reduced by the presence of specific dsRNA, inducing, respectively, a decreased GFP fluorescence and the formation of enlarged cells with modified arrangement of subpellicular microtubules. Furthermore, symbiont division was impaired. These results indicate that the RNAi system is active in A. deanei and can be used to further explore gene function in symbiont-containing trypanosoma tids and to clarify important aspects of symbiosis and cell evolution. This article is protected by copyright. All rights reserved

Cellulolytic ability of Penicillium strains isolated from soil of the Brazilian Atlantic forest

Penicillium spp. are capable of degrading plant wastes by producing large amounts of enzymes such as cellulases. These form a complex capable of acting on cellulosic materials and producing sugars with industrial interest (e.g., ethanol production). Cellulases are also used for (a) pulp and paper industry (b) in the textile industry. The aim of this study was to evaluate the cellulolytic capability of 17 strains of Penicillium isolated from soil of the Brazilian Atlantic Forest and conserved under mineral oil at the URM Culture Collection. All strains were re-grown from mineral oil and re-identifiied. Each strain was grown in synthetic medium with carboxymethylcellulose as the carbon source and incubated for 5 days at 28°C. Strains were subjected to heat shock for 16h at 50°C. Thereafter, onto each colony was added 5 ml of Congo red stain solution in Tris-HCl. After 30 min this solution was removed and the cultures were washed and submerged under 0.1 M NaCl aqueous solution for 5 min. Finally, an enzymatic index was calculated from the ratio of the diameter of the halo around each colony to the diameter of the colony. All of the 17 strains tested showed a halo of cellulose degradation, indicating enzyme production. The enzymatic ratios varied between 0.2 (Penicillium brevicompactum URM5994) and 3.3 (Penicillium glabrum URM6009). Thus, Penicillium glabrum URM6009 is evaluated as a high producer of cellulase. It was selected for quantitative production of this enzyme and additional studies are taking place in order to verify potential industrial application for clarification of fruit juices

The importance of a quick identification of Aspergillus niger for a proper mycotoxin related diagnosis

Food safety has become an important food quality attribute within the last decade. Moreover, consumers have a better perception about the food contamination with mycotoxins. These secondary metabolites produced by filamentous fungi can cause acute toxic, mutagenic, teratogenic and carcinogenic effects on animals and humans. Mycotoxins can appear in the food chain and are greatly resistant to decomposition or being broken down in digestion. They remain intact in the food chain in livestock and dairy products and even after temperature treatments, such as cooking and freezing. Aspergillus niger aggregate species are commonly found on soil and are pathogenic to several crops. This group of filamentous fungi is formed by a series of morphologically indistinguishable species. Aspergillus niger is one of the species in the aggregate and, apart from its economic value (it is used for industrial purposes), it is also an important mycotoxin producer, such as ochratoxin A (OTA) and, more recently, was described as fumonisin B2 (FB2) producer. Both mycotoxins were evaluated by the International Agency for Research on Cancer (IARC) as “Group 2B carcinogens”, i.e., probably carcinogenic to humans. The continued exposure to these mycotoxins can cause chronic toxicity which is characterised by low-dose exposure over a long time period, resulting in cancers and other generally irreversible effects. Hence, a proper diagnosis is important, which will allow correct treatment. Fast identification of fungi is, therefore, a must needed necessity. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) allows rapid and reliable identification of microorganisms, being sensitive and accurate for the discrimination between species and strains of Aspergillus. This work consisted in the identification of fungal isolates belonging to the Aspergillus niger aggregate. Fungal identification was based on a polyphasic approach consisting of macro- and micro-morphology, mycotoxigenic profiles (OTA and FB2) and MALDI-TOF MS. About 250 isolates belonging to Aspergillus niger aggregate were analysed and results obtained were compared with type strains. Final results showed that all isolates were identified as Aspergillus niger, A. lacticoffeatus and A. vadensis.Fundação para a Ciência e Tecnologia, Portugal (reference SFRH/BD/37264/2007), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Brazil and Erasmus Mundus External Cooperation Window (EMECW)

The microbial culture collections of the Federal University of Pernambuco (UFPE) and the new consortium towards the establishment of BRC-UFPE

The UFPE from Recife in Brazil hosts a bacterial (UFPEDA) and a fungal (URM) collections since 1951 and 1954, respectively. The UFPEDA was established by Prof. Oswaldo Gonçalves de Lima and is register in WDCM as 114. It is hosted at Antibiotic Department (DA) of UFPE and started out with 200 species mainly of the genus Streptomyces. Nowadays this collection holds 4000 strains of actinomycetes isolated from all the Brazilian places and from the International Streptomyces Project (ISP). The URM – University of Recife Mycology was established by Prof. Augusto Chaves Batista and is register in WDCM as 604. Actual it holds 9000 identified species including 1400 yeasts and 7600 filamentous fungi. All major fungal taxonomic groups are cover by this collection. The collections preserve each strain at least by two different techniques. Water and mineral oil storage were used for long operation time while freeze-drying and freezing at -80 ºC become the main techniques used at this stage. Special care is taken to test whether cultures recovered from preserved material conform to the original deposit. These collections have a range of services which are acceptance of free and confidential deposits, supply strains for academia, industry and services, support research and education (graduate and post-graduate students, as well as advanced training courses), identification services and confidential contracts (e.g. fungal medical diagnosis, starters for agro-industry companies, etc.). The OECD initiative related to guidance for the operation of Biological Resource Centres (BRC) is now a key reference for these collections. The right management of biological resources and their associate information including quality control are perused by these collections. The recent national projects, with reasonable budgets to support their activities, either on networking activities or requalification and management create a new breath and responsibilities to these collections. Taking advantage of good and well equipped premises of LIKA these collections are now open new avenues working in consortium to improve the quality control of their holdings using new tools from molecular biology and spectral analysis (MALDI-TOF) to achieve in the future a certified BRC for the UFPE microbial culture collections

Oficinas multiprofissionais:: educação em saúde para idosos de uma comunidade

Objetivos: Descrever o processo de elaboração de oficinas de promoção da saúde voltadas a um grupo de convivência para idosos e compartilhar a experiência de uma equipe de residentes multiprofissionais na construção de metodologias para se discutir saúde dentro de grupos. Métodos: Entre 2007 e 2009, a equipe de residentes multiprofissionais acompanhou um grupo de convivência, constituído de aproximadamente quarenta idosos. Foram observadas as principais demandas em saúde a serem trabalhadas junto ao grupo e desenvolvidas oficinas para promoção da saúde. Resultados: Os temas trabalhados nas oficinas foram osteoporose, diabetes mellitus, dislipidemia, planejando o futuro e relações de cuidado. Os residentes construíram materiais didáticos, como cartazes, folders, bolsas coloridas, cartões ilustrativos, que ilustraram os temas abordados de forma lúdica. As oficinas possibilitaram que os participantes fossem agentes ativos no processo de aprendizagem e de fazer saúde, o que pressupõe benefícios à saúde física, mental e social desse grupo

A quick identification of Aspergillus niger strains using MALDI-TOF MS

Publicado em "Biological resource centres : closing the gap between science and society : abstracts book...". ISBN 978-972-97916-5-9Food safety has become an important food quality attribute within the last decade; moreover, consumers have a better perception about the food contamination with mycotoxins. These secondary metabolites produced by filamentous fungi can cause acute toxic, mutagenic, teratogenic and carcinogenic effects on animals and humans. Mycotoxins can appear in the food chain either by being eaten directly by humans, or by being used as livestock feed. They are greatly resistant to decomposition or being broken down in digestion, so they remain in the food chain in meat and dairy products and even temperature treatments, such as cooking and freezing, do not destroy mycotoxins. Aspergillus niger aggregate strains are commonly found on soil and are pathogenic to several crops. This group is formed by a series of morphologically indistinguishable species. Aspergillus niger is one of the species in the aggregate and, apart from its economic value (it is used for industrial purposes), it is also an important mycotoxin producer, such as ochratoxin A (OTA) and more recently fumonisin B2 (FB2). Both mycotoxins were evaluated by the International Agency for Research on Cancer (IARC) as “Group 2B carcinogens”, i.e., probably carcinogenic to humans. The continued exposure to these mycotoxins can cause chronic toxicity which is characterized by low-dose exposure over a long time period, resulting in cancers and other generally irreversible effects. Hence, a proper diagnosis is important, which will allow correct treatment. Fast identification of fungi is, therefore, a must needed necessity. Matrix-assisted laser desorption ionization-time (MALDI-TOF MS) allows rapid and reliable identification of microorganisms, being sensitive and accurate for the discrimination between species and strains of Aspergillus. This work consisted in the identification of Aspergillus niger strains through MALDI-TOF, with known mycotoxigenic profile. For that about 250 strains belonging to Aspergillus niger aggregate were analysed and compared with type strains deposited in Micoteca da Universidade do Minho (MUM). Results showed that all strains were Aspergillus niger

Polyphasic identification and typing Trichophytum rubrum strains of clinical origin

The dermatophyte Trychophytum rubrum is the most frequent aetiological agent of dermatophytosis in humans around the world representing a major public health problem, not just for European countries but also for tropical countries where climate conditions allow major propagation for this ascomycete. For instance, in Portugal, T. rubrum was the dermatophyte most frequently isolated (83.3%) in a toenail onychomycosis geriatric population survey [1]. The identification, pathogenicity, biology, and epidemiology of T. rubrum, is of interest for both dermatologists and medical mycologists [1,2]. Currently, in many countries and clinical laboratories, the T. rubrum strains isolated from lesions are primarily identified by conventional culture-based methods, including colony morphology and slide culture only. This approach does not provide evidence of intraspecific variations with a lack of information to track infections, determine common sources of infections and recurrence or reinfection after treatment, and analyse their virulence and drug resistance [3]. The aim of this work is to use a polyphasic approach to study T. rubrum from different geographic origins in order to identify intraspecific characteristics with clinical interest. Materials and Methods About 40 European and South American T. rubrum and reference strains were used. Macro and micro morphological techniques, urease assay, dermatophyte milk agar test and hair perforation test (HPT) where combined with molecular biology techniques, such as the analysis of internal transcribed spacer (ITS) region, Trubrum specific primers for species differentiation among close related species, mating type MAT1-1 -box characterisation and DNA fingerprinting (e.g., (GACA)4). Results and conclusions Culturally T. rubrum strains showed on the plates white and cottony colonies on the obverse and blood-red pigment on the reverse. T. rubrum strains were urease negative and inhibited in dermatophyte milk agar. In the HPT, which is useful to differentiate T. rubrum from T. interdigitale, any strain was able to perforate the hair despite normal growth being observed. The analysis of ITS region confirmed all the strains as a T. rubrum species as well as the Trubrum primers generate a typical amplicon of 200 bp. The DNA fingerprinting is now explored in order to find the best approach to differentiate intraspecific variations and/or geographic differences. In conclusion, there are several techniques that can be applied to identify and characterise T. rubrum from different origins depending of the technologies available in each clinical laboratory or country.info:eu-repo/semantics/publishedVersio