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

Pectinolytic activity of Aspergillus section Nigri strains

Pectinases are a heterogeneous group of related enzymes that hydrolyze pectic substances present mostly in plants. Pectinases are produced by plants, fungi, yeasts and bacteria. Filamentous fungi are good producers of pectinolytic enzymes (e.g., exopolygalacturonase (exo-PG) and endopolygalacturonase (endo-PG) and Aspergillus niger is the most commonly used fungal species for industrial production of pectinolytic enzymes. The application of pectinolytic enzymes plays an important role in food technology. In juice production, these enzymes have been used to improve the yield, decrease the viscosity, clarify the juices and make them more stable. In this context, the concept of using filamentous fungi and row and cheaper materials for pectinase production is an important parameter in technological development. In the present study a microplate method was developed for a rapid screening of Aspergillus strains. Fifty-tree strains of Aspergillus section Nigri obtained from the University of Recife Mycology (URM) culture collection and 8 of the Micoteca da Universidade do Minho (MUM) culture collection were used. Orange peel was the unique carbon source in the composition of the culture medium. The samples were incubated at 25 ºC for 120 h. After 24, 48, 72, 96 and 120 h the exo-PG and endo-PG were assessed using absorbance colorimetric and decrease in viscosity methods, respectively. The utilization of orange peel allowed the detection of exo-PG and endo-PG activity for all strains studied. The maximum exo-PG and endo-PG activity was obtained by strain URM5162 to the values 4.37 U and 2.13 U, respectively. This method and substrate may be useful to reduce the time for selecting promising strains and in reducing the enzyme production costs. The strain is now being used in a bioreactor and the enzymes and their mechanisms are also under further investigation

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

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

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

Partitioning and purification of polygalacturonases produced by Aspergillus niger URM 5162 using PEG-phosphate in an aqueous two-phase system

Pectinases, or pectinolytic enzymes, are naturally produced by plants, filamentous fungi, bacteria and yeasts. The pectinases are of great importance to clarify and reduce viscosity in fruit juices, improving and increasing tbe filtration efficiency. When used in the crushing of grapes or wine must improve juice extraction, reduce the time to clarify and enhance tbe content ofterpenes in wine. The filamentous fungi most frequently used fur industrial purposes because as much as 90% ofthe enzyme can be excreted into the culture medium. The partitioning and purification of polygalacturonases (PG) produced by Aspergillus niger URM 5162 were investigated in aqueous two-phase systems (ATPS), furmed by polyetbylene glycol and phosphate salts (PE(ijlhosphate). To evaluate the effect oftbe 4 independent variables- molar mass ofpolyetbylene glycol (PEG) (400-8000 g1nol MPEG), PEG concentration (12.5-17.5%, w/w- CPEG), phosphate concentration (15-25%, ...W, CPHOS) and pH (6.0, 8.0) - on the 3 response variables: partition coefficient (K), activity yield (Y) and purification fàctor (PF), a fuctorial design (24) was used. The endo-polygalacturonases (endo-PG) were prefurentially partitioned in tbe top phase. For endo-PG, the highest values for the response variables K, Y and PF of 1.23, 74.04% and 8.18, respectively, were obtained for a CPEG of 12.5% (...W), MPEG of8000 g1nol, and CPHOS of25% (w/w) at pH 6.0. Also, exo-polygalacturonases (exo-PG) were preferentially partitioned in the top phase. ln tbis case, the highest values ofK (2.40), Y (33.33%), and PF (1.98) were obtained with a MPEG of 8000 g1nol, CPEG of 12.5% (...W), and CPHOS of25% (...W) at pH 6.0. ln both cases, MPEG had a positive influence on K, Y and PF. The conditions ofMPEG 8000 g1nol, CPEG of 12.5% (...W), and CPHOS of25% (...W) at pH 6.0 were considered the most suitable for tbe purification of PG produced by A. niger URM 5162. Furtbermore, MPEG and CPHOS were the most important independent variables. The PEG/phosphate system is a useful cost-effective altemative for PG purification

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

Bezerromycetales and Wiesneriomycetales ord. nov. (class Dothideomycetes), with two novel genera to accommodate endophytic fungi from Brazilian cactus

During a survey of endophytic fungi from the cactus Tacinga inamoena in a Brazilian tropical dry forest (Caatinga) some undescribed ascomycetous fungi were isolated. These fungi are characterized by superficial and immersed, globose to subglobose, smooth or hairy ascomata, bitunicate asci, and muriformly septate, ellipsoidal ascospores. Multigene phylogenetic analyses using sequences from partial ITS, SSU and LSU nrDNA and the translation elongation factor 1-alpha gene (tef1) demonstrated a monophyletic clade accommodating these endophytic fungi in the class Dothideomycetes, closely related to the order Tubeufiales. Based on morphological features and phylogenetic analyses, these fungi could not be placed in the order Tubeufiales, in the new order Wiesneriomycetales, or any other known genus in the class Dothideomycetes. Thus, two new genera (Bezerromyces, with B. brasiliensis and B. pernambucoensis, and Xiliomyces with X. brasiliensis), a new family (Bezerromycetaceae) and a new order (Bezerromycetales) are introduced to accommodate these novel taxa. Our phylogenetic analyses also demonstrated that the clade accommodating Wiesneriomycetaceae represents a new order, here introduced as Wiesneriomycetales.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Process 203132/2014-9), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE) of Brazil.http://link.springer.com/journal/115572018-04-30hb2017Microbiology and Plant Patholog