13 research outputs found
Engineered fluorescent strains of cryptococcus neoformans : a versatile toolbox for studies of host-pathogen interactions and fungal biology, including the viable but nonculturable state
Cryptococcus neoformans is an opportunistic fungal pathogen known for its remarkable ability to infect and subvert phagocytes. This ability provides survival and persistence within the host and relies on phenotypic plasticity. The viable but nonculturable (VBNC) phenotype was recently described in C. neoformans, whose study is promising in understanding the pathophysiology of cryptococcosis. The use of fluorescent strains is improving host interaction research, but it is still underexploited. Here, we fused histone H3 or the poly(A) binding protein (Pab) to enhanced green fluorescent protein (eGFP) or mCherry, obtaining a set of C. neoformans transformants with different colors, patterns of fluorescence, and selective markers (hygromycin B resistance [Hygr
] or neomycin resistance [Neor]). We validated their similarity to the parental strain in the stress response, the expression of virulence-related phenotypes, mating, virulence in Galleria mellonella, and survival within murine macrophages. PAB-GFP, the brightest transformant, was successfully applied for the analysis of phagocytosis by flow cytometry and fluorescence microscopy. Moreover, we demonstrated that an engineered fluorescent strain of C. neoformans was able to generate VBNC cells. GFP-tagged Pab1, a key regulator of the stress response, evidenced nuclear retention of Pab1 and the assembly of cytoplasmic stress granules, unveiling posttranscriptional mechanisms associated with dormant C. neoformans cells. Our results support that the PAB-GFP strain is a useful tool for research on C. neoformans
Anais da XX SemaBioMetamorfoses : respostas da ciência aos momentos de crise
Sobre o evento - Imagine a Terra bilhões de anos atrás, ela certamente era diferente dos dias atuais. Foi nela, todavia, que compostos orgânicos simples começaram a se agrupar, crescer e evoluir para formar todas as formas de vida que podemos ver hoje, uma das quais é o Homo sapiens. Este, por gerações, viveu sem que nada verdadeiramente o destacasse dentre as outras diversas espécies com as quais compartilhava o habitat. Foi apenas com o advento da Revolução Cognitiva, que o ser humano se diferenciou, tornando-se consciente deles mesmos, da vida e da natureza. Porém, ao mesmo tempo que esta consciência os permitiu lançar luz sobre a própria vida, a capacidade de aprender e alterar o ambiente à sua volta trouxe consigo um acúmulo de problemas fundamentais: crises a serem solucionadas. Hoje, em particular, o avanço tecnológico e a forma como nos relacionamos com a Terra faz do tempo em que vivemos um momento sem precedentes na história da humanidade, que será decisivo para o futuro da vida. A crise sanitária que o mundo experienciou com a pandemia de Covid-19 – e as marcas profundas que esta deixará em nossas memórias pelas tantas vidas perdidas – é certamente um exemplo emblemático do quão insustentável está nossa situação, o quão perigoso pode ser o negacionismo e o quão crucial a ciência é para nós hoje. Da mesma forma, ao enxergar como lá fora o colapso ecológico se anuncia, a desigualdade cresce cada vez mais sob formas históricas e sistemáticas de opressão e emergem novamente ideias extremistas, devemos buscar o que podemos aprender com as tantas rupturas. Assim, esperançosa por um futuro melhor, a XX Semana da Biologia decidiu abordar o tema “Metamorfoses: respostas da Ciência aos momentos de crise”. Acreditamos que a ciência seja a chave para resolver os problemas que afligem a nossa sociedade, portanto cabe à edição comemorativa da SemaBio fomentar o sonho de uma nova realidade: uma que seja mais sustentável, igualitária e científica. Em síntese, nas palavras de Carl Sagan, “a ciência nos esclarece sobre as questões mais profundas das origens, naturezas e destinos – de nossa espécie, da vida, de nosso planeta, do universo”. Fonte: https://www.even3.com.br/semabiounb/.Instituto de Ciências Biológicas (IB)Departamento de Genética e Morfologia (IB GEM
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Insights into the Lignocellulose-Degrading Enzyme System of Humicola grisea var. thermoidea Based on Genome and Transcriptome Analysis.
Humicola grisea var. thermoidea is a thermophilic ascomycete and important enzyme producer that has an efficient enzymatic system with a broad spectrum of thermostable carbohydrate-active (CAZy) enzymes. These enzymes can be employed in lignocellulose biomass deconstruction and other industrial applications. In this work, the genome of H. grisea var. thermoidea was sequenced. The acquired sequence reads were assembled into a total length of 28.75 Mbp. Genome features correlate with what was expected for thermophilic Sordariomycetes. The transcriptomic data showed that sugarcane bagasse significantly upregulated genes related to primary metabolism and polysaccharide deconstruction, especially hydrolases, at both pH 5 and pH 8. However, a number of exclusive and shared genes between the pH values were found, especially at pH 8. H. grisea expresses an average of 211 CAZy enzymes (CAZymes), which are capable of acting in different substrates. The top upregulated genes at both pH values represent CAZyme-encoding genes from different classes, including acetylxylan esterase, endo-1,4-β-mannosidase, exoglucanase, and endoglucanase genes. For the first time, the arsenal that the thermophilic fungus H. grisea var. thermoidea possesses to degrade the lignocellulosic biomass is shown. Carbon source and pH are of pivotal importance in regulating gene expression in this organism, and alkaline pH is a key regulatory factor for sugarcane bagasse hydrolysis. This work paves the way for the genetic manipulation and robust biotechnological applications of this fungus. IMPORTANCE Most studies regarding the use of fungi as enzyme producers for biomass deconstruction have focused on mesophile species, whereas the potential of thermophiles has been evaluated less. This study revealed, through genome and transcriptome analyses, the genetic repertoire of the biotechnological relevant thermophile fungus Humicola grisea. Comparative genomics helped us to further understand the biology and biotechnological potential of H. grisea. The results demonstrate that this fungus possesses an arsenal of carbohydrate-active (CAZy) enzymes to degrade the lignocellulosic biomass. Indeed, it expresses more than 200 genes encoding CAZy enzymes when cultivated in sugarcane bagasse. Carbon source and pH are key factors for regulating the gene expression in this organism. This work shows, for the first time, the great potential of H. grisea as an enzyme producer and a gene donor for biotechnological applications and provides the base for the genetic manipulation and robust biotechnological applications of this fungus
Comparative isolation protocols and characterization of stem cells from human primary and permanent teeth pulp
Aim: This study was developed to compare the morphological,
proliferative and immunophenotypic profiles of pulp cells from
permanent and primary teeth, obtained by two isolation methods.
Methods: Normal human impacted third molars and exfoliated primary
teeth were collected and cut around the cementoenamel junction. Pulp
cells cultures were established by two approaches: enzyme digestion (3
mg/mL type I colagenase and 4 mg/mL dispase), or culture of the tissue
explants in cell culture dishes. Morphological and proliferative
analyses, as well as immunophenotype characterization with monoclonal
antibodies against CD117, CD34 and CD45 surface receptors were
performed. Results: For the permanent teeth, on the 4th day of culture,
the cell number was significantly higher for the outgrowth method. By
the end of the studied period (14th day), the enzymatic method was more
efficient in promoting culture growth. On the other hand, for primary
teeth, enzymatic digestion always promoted a higher cell proliferation.
The immunophenotypic profiles were CD117+/ CD34-/ CD45- and CD117+/
CD34+/ CD45- for cells from permanent and primary teeth, respectively.
Conclusions: The findings of this study indicate that both isolation
methods can be efficiently used. The cell population displayed an
immunophenotype compatible to the one of stem cells, with remarkable
positive expression of CD117
Comparative isolation protocols and characterization of stem cells from human primary and permanent teeth pulp
is study was developed to compare the morphological, proliferative and immunophenotypic profiles of pulp cells from permanent and primary teeth, obtained by two isolation methods. Methods: Normal human impacted third molars and exfoliated primary teeth were collected and cut around the cementoenamel junction. Pulp cells cultures were established by two approaches: enzyme digestion (3 mg/mL type I colagenase and 4 mg/mL dispase), or culture of the tissue explants in cell culture dishes. Morphological and proliferative analyses, as well as immunophenotype characterization with monoclonal antibodies against CD117, CD34 and CD45 surface receptors were performed. Results: For the permanent teeth, on the 4th day of culture, the cell number was significantly higher for the outgrowth method. By the end of the studied period (14th day), the enzymatic method was more efficient in promoting culture growth. On the other hand, for primary teeth, enzymatic digestion always promoted a higher cell proliferation. The immunophenotypic profiles were CD117+/ CD34-/ CD45- and CD117+/ CD34+/ CD45- for cells from permanent and primary teeth, respectively. Conclusions: The findings of this study indicate that both isolation methods can be efficiently used. The cell population displayed an immunophenotype compatible to the one of stem cells, with remarkable positive expression of CD117