Desenvolvimento tecnológico e inovação no Brasil: desafios na área de biotecnologia

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Paracoccidioides species complex : ecology, phylogeny, sexual reproduction, and virulence

Paracoccidioidomycosis (PCM) is a deep systemic mycosis caused by human fungal pathogens of the Paracoccidioides genus. The disease is geographically restricted to subtropical areas of Latin America (from south of Mexico to north of Argentina) with a high prevalence in Brazil, Colombia, Venezuela, and Argentina [1]. The annual incidence rate in Brazil is 10–30 infections per million inhabitants, and the mean mortality rate is 1.4 per million inhabitants per year, making this disease the highest cause of mortality among systemic mycoses [2]. PCM is endemic in rural populations and mainly affects individuals engaged in agricultural activities, who inhale aerosols containing fungal material during manipulation of the soil. Molecular evolutionary studies place the genus Paracoccidioides in the thermodimorphic fungal pathogen clade related to the family Ajellomycetaceae (Ascomycetes), which includes the Blastomyces, Histoplasma, and Emmonsia genera, and with which it shares a common ancestor, Lacazia loboi. PCM can be caused by two species Paracoccidioides brasiliensis and P. lutzii [3]. P. brasiliensis has been considered a single species since its discovery, although several studies including molecular and morphological data support the split of P. brasiliensis into two species [3,4]. P. lutzii is composed of a single monophyletic and recombining population so far found in central, southwest, and north Brazil and Ecuador [3–5]. On the other hand, P. brasiliensis contains a complex of at least four different cryptic species (S1, PS2, PS3 and PS4; Figure 1A [6]). P. brasiliensis S1 represents a monophyletic and recombining population widely distributed in South America and has been associated with the majority of cases of PCM detected up until the present time. Strains belonging to P. brasiliensis S1 have previously been recovered from armadillos, soil, and penguin feces [6]. P. brasiliensis PS2 is a paraphyletic and recombining population identified so far only in Brazil and Venezuela [6]. P. brasiliensis PS3 is comprised of a monophyletic and clonal population that has been recovered in humans and armadillos in endemic regions of Colombia [6]. P. brasiliensis PS4 was recently identified and is composed of a monophyletic population of clinical isolates from Venezuela [5,7]. Besides the typical bicorn cocked hat– and barrel-shaped conidia produced by both species, P. lutzii frequently produces elongated rod-shaped conidia, a characteristic feature that may be used for species identification [3]. Because of the difficulties of conidia production in the laboratory and slight morphological differences among species, molecular identification of Paracoccidioides species has become the most common tool of choice. Several molecular markers have already been applied in population studies of the Pararacoccidioides genus, and for multilocus sequencing typing, gp43, arf, b-tub, and hsp70 loci are the best choices for species delineation [4,6]

Extracellular Paracoccidioides brasiliensis phospholipase B involvement in alveolar macrophage interaction

Background: Phospholipase B (PLB) has been reported to be one of the virulence factors for human pathogenic fungi and has also been described as necessary for the early events in infection. Based on these data, we investigated the role of PLB in virulence and modulation of the alveolar pulmonary immune response during infection using an in-vitro model of host-pathogen interaction, i.e. Paracoccidioides brasiliensis yeast cells infecting alveolar macrophage (MH-S) cells. Results: The effect of PLB was analyzed using the specific inhibitor alexidine dihydrochloride (0.25 μM), and pulmonary surfactant (100 μg mL-1), during 6 hours of co-cultivation of P. brasiliensis and MH-S cells. Alexidine dihydrochloride inhibited PLB activity by 66% and significantly decreased the adhesion and internalization of yeast cells by MH-S cells. Genes involved in phagocytosis (trl2, cd14) and the inflammatory response (nfkb, tnf-α, il-1β) were down-regulated in the presence of this PLB inhibitor. In contrast, PLB activity and internalization of yeast cells significantly increased in the presence of pulmonary surfactant; under this condition, genes such as clec2 and the pro-inflammatory inhibitor (nkrf) were up-regulated. Also, the pulmonary surfactant did not alter cytokine production, while alexidine dihydrochloride decreased the levels of interleukin-10 (IL-10) and increased the levels of IL-12 and tumor necrosis factor-α (TNF-α). In addition, gene expression analysis of plb1, sod3 and icl1 suggests that P. brasiliensis gene re-programming is effective in facilitating adaptation to this inhospitable environment, which mimics the lung-environment interaction. Conclusion: P. brasiliensis PLB activity is involved in the process of adhesion and internalization of yeast cells at the MH-S cell surface and may enhance virulence and subsequent down-regulation of macrophage activation

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular matrix proteins and interaction with cells

The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. the P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. the recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.Univ Fed Goias, Inst Ciencias Biol, Mol Biol Lab, BR-74001970 Goiania, Go, BrazilUniv Brasilia, BR-70910900 Brasilia, DF, BrazilUniv Estadual Julio Mesquita Filho, Araraquara, SP, BrazilUniversidade Federal de São Paulo, São Paulo, BrazilUniversidade Federal de São Paulo, São Paulo, BrazilWeb of Scienc

MCIBiotec: solução tecnológica para o planejamento pedagógico de aulas de biotecnologia

A Educação precisa ser pautada pela geração de estudantes que se fazem presentes na sala de aula, assim como pelo uso de inovações tecnológicas pelos docentes, o que precisa estar presente no planejamento pedagógico. A Base Nacional Comum Curricular (BNCC) (BRASIL, 2018), que apresenta o novo Ensino Médio, enfatiza a importância da Biotecnologia para o componente curricular de Ciências da Natureza e suas Tecnologias. Neste cenário este artigo tem como objetivo apresentar o aplicativo (App) desenvolvido para assessoramento pedagógico de docentes deste componente curricular, especificamente na temática Biotecnologia, com ênfase ao uso das cores, na concepção de Kafarski (2012). Denominado MCIBiotec, o App contribuirá para o planejamento pedagógico da escolha do tema até a avaliação, passando pelas competências a serem desenvolvidas e pela metodologia a ser desenvolvida; ao final está apresentado um template que pode ser compartilhado e reeditado pelo responsável. O desenvolvimento do App ocorreu baseado em pesquisas com docentes das áreas de Biologia, Química e Física, do Ensino Médio, bem como com Professores Doutores de Programas de Pós-graduação em Biotecnologia que referendaram sua aplicabilidade

Semi-nested PCR para a detecção molecular de Paracoccidioides brasiliensis em amostras de tecido

INTRODUCTION: Paracoccidioidomycosis is a systemic infection caused by Paracoccidioides brasiliensis. METHODS: In this study, a semi-nested PCR for paracoccidioidomycosis diagnosis was developed. The primers ITS1 and ITS4 were used in the first reaction, while the primers MJ03 and ITS1 primer were used in the second reaction. The semi-nested PCR was used to investigate biopsies of five patients with oral lesions that resembled paracoccidioidomycosis. RESULTS: The semi-nested PCR was positive for four samples and negative for a sample from a patient later diagnosed with leishmaniasis. CONCLUSIONS: The new semi-nested PCR describe is useful for aracoccidioidomycosis diagnosis. ________________________________________________________________________________________________________________ RESUMOINTRODUÇÃO: A paracoccidioidomicose é uma infecção sistêmica causada pelo Paracoccidioides brasiliensis. MÉTODOS: Neste estudo, uma semi-nested PCR foi desenvolvida para o diagnóstico da paracoccidioidomicose. Os oligonucleotídeos iniciadores ITS1 e ITS4 foram usados na primeira reação, enquanto os oligonucleotídeos iniciadores MJ03 e ITS1 foram usados na segunda reação. A semi-nested PCR foi usada para investigar biopsias de cinco pacientes com lesões orais que se assemelhavam a paracoccidioidomicose. RESULTADOS: A semi-nested PCR foi positiva para quatro amostras e negativa para a amostra de um paciente, posteriormente diagnosticado com leishmaniose. CONCLUSÕES: A semi-nested PCR descrita aqui é útil para o diagnóstico da paracoccidioidomicose

Identification and characterization of expressed retrotransposons in the genome of the Paracoccidioides species complex

Background: Species from the Paracoccidioides complex are thermally dimorphic fungi and the causative agents of paracoccidioidomycosis, a deep fungal infection that is the most prevalent systemic mycosis in Latin America and represents the most important cause of death in immunocompetent individuals with systemic mycosis in Brazil. We previously described the identification of eight new families of DNA transposons in Paracoccidioides genomes. in this work, we aimed to identify potentially active retrotransposons in Paracoccidioides genomes.Results: We identified five different retrotransposon families (four LTR-like and one LINE-like element) in the genomes of three Paracoccidioides isolates. Retrotransposons were present in all of the genomes analyzed. P. brasiliensis and P. lutzii species harbored the same retrotransposon lineages but differed in their copy numbers. in the Pb01, Pb03 and Pb18 genomes, the number of LTR retrotransposons was higher than the number of LINE-like elements, and the LINE-like element RtPc5 was transcribed in Paracoccidioides lutzii (Pb01) but could not be detected in P. brasiliensis (Pb03 and Pb18) by semi-quantitative RT-PCR.Conclusion: Five new potentially active retrotransposons have been identified in the genomic assemblies of the Paracoccidioides species complex using a combined computational and experimental approach. the distribution across the two known species, P. brasiliensis and P. lutzii, and phylogenetics analysis indicate that these elements could have been acquired before speciation occurred. the presence of active retrotransposons in the genome may have implications regarding the evolution and genetic diversification of the Paracoccidioides genus.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed Minas Gerais, Inst Ciencias Biol, Dept Microbiol, BR-31270901 Belo Horizonte, MG, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Programa Posgrad Bioinformat, BR-31270901 Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, SP, BrazilUniv Brasilia, Inst Ciencias Biol, Lab Biol Mol, BR-70910900 Brasilia, DF, BrazilUniv Fed Goias, Inst Ciencias Biol, Lab Biol Mol, BR-74001970 Goiania, Go, BrazilFIOCRUZ Minas, Ctr Pesquisas Rene Rachou, Grp Informat Biossistemas, BR-30190002 Belo Horizonte, MG, BrazilUniv Fed Minas Gerais, Dept Biol Geral, BR-31270901 Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, SP, BrazilFAPEMIG: APQ-01661-13CNPq: 301652/2012-0CNPq: 486618/2013-7Web of Scienc

Bioinformatics of the Paracoccidioides brasiliensis EST Project

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, an endemic mycosis of Latin America. This fungus presents a dimorphic character; it grows as a mycelium at room temperature, but it is isolated as yeast from infected individuals. It is believed that the transition from mycelium to yeast is important for the infective process. The Functional and Differential Genome of Paracoccidioides brasiliensis Project - PbGenome Project was developed to study the infection process by analyzing expressed sequence tags - ESTs, isolated from both mycelial and yeast forms. The PbGenome Project was executed by a consortium that included 70 researchers (professors and students) from two sequencing laboratories of the midwest region of Brazil; this project produced 25,741 ESTs, 19,718 of which with sufficient quality to be analyzed. We describe the computational procedures used to receive process, analyze these ESTs, and help with their functional annotations; we also detail the services that were used for sequence data exploration. Various programs were compared for filtering and grouping the sequences, and they were adapted to a user-friendly interface. This system made the analysis of the differential transcriptome of P. brasiliensis possible

Molecular epidemiology and antimicrobial susceptibility of enterococci recovered from Brazilian intensive care units

ABSTRACT: We studied the antimicrobial resistance and the molecular epidemiology of 99 enterococcal surveillance isolates from two hospitals of Brasília, Brazil. Conventional biochemical tests were used to identify the enterococcal species and the disk diffusion method was used to determine their resistance profiles. Enterococcus faecalis (76%) and E. faecium (9%) were the most prevalent species. No enterococci showed the vanA or vanB vancomycin resistance phenotypes or genotypes. Only the intrinsically resistant species E. gallinarum (n=2) and E. casseliflavus (n=3) harbored the vancomycin-resistance genes vanC1 and vanC2/3, respectively. We found E. faecalis isolates with high-level resistance to gentamicin (22%) and streptomycin (8%) and both E. faecalis and E. faecium isolates with resistance to more than two antimicrobials (84% and 67%, respectively). Nine E. faecalis isolates (12%) were resistant to ampicillin; the minimal inhibitory concentration (MIC) values were 16µg/mL (n=6) and 32µg/mL (n=3). Among these ampicillin-resistant E. faecalis, seven were also resistant to gentamicin, ciprofloxacin, rifampin, penicillin, chloramphenicol, tetracycline and erythromycin. Pulsed-field gel electrophoresis classified those isolates in three different genotypes, suggesting dissemination of genetically related ampicillin-resistant E. faecalis strains among different patients