102 research outputs found
Bioprospection of antibiotics and biofilm inhibitors from under-exploited filamentous fungi
In the past century, millions of lives were saved by antibiotics, which treat some of the
historically more deadly diseases. However, bacteria may quickly develop antibiotic
resistance and the speed of development of new drugs has decreased significantly in the last
decades. Filamentous fungi have a great potential for novel antibiotic discovery, given their
vast, largely unexplored metabolome. The One Strain MAny Compounds (OSMAC) approach
serves as a tool to obtain a wide range of metabolites by varying various culture conditions.
In this study, eight under-explored fungal species from Micoteca da Universidade do Minho
(MUM) were used, aiming to identify compounds with antibiotic or antibiofilm properties
produced by them: Coprinopsis spilospora, Penicillium tunisiense, Trichoderma aestuarinum,
Colletotrichum coccodes, Talaromyces saxoxalicus, Diaporthe phillipsii, Cladosporium
rubrum, and Neopestalotiopsis scalabiensis. They grew in different culture media, under
submerged fermentation for 7 and 14 days under varying conditions of agitation and
aeration, and the resulting supernatants were tested for their antimicrobial activity against
Staphylococcus aureus and Escherichia coli using the disk diffusion method. Almost all fungi
grew on the different media and under the diverse process conditions. It was found that
Coprinopsis spilospora metabolites inhibited S. aureus growth and demonstrated antibiofilm
properties. They reduced the biofilm by 74% in crystal violet staining, metabolic activity by
100% in Alamar blue test, and viable cell counts by 98% in CFU counting. The effect against
E. coli was more modest, although still reduced CFU counts by 96%. High-pressure liquid
chromatography/high-resolution mass spectrometry (HPLC/HRMS) showed that these
results are likely due to the presence of compounds in the illudin family. The pioneer results
obtained in the present study highlight the potential of filamentous fungi for bioprospection
for antibiotic discovery.info:eu-repo/semantics/publishedVersio
A secreted serine protease of Paracoccidioides brasiliensis and its interactions with fungal proteins
<p>Abstract</p> <p>Background</p> <p><it>Paracoccidioides brasiliensis </it>is a thermodimorphic fungus, the causative agent of paracoccidioidomycosis (PCM). Serine proteases are widely distributed and this class of peptidase has been related to pathogenesis and nitrogen starvation in pathogenic fungi.</p> <p>Results</p> <p>A cDNA (<it>Pb</it>sp) encoding a secreted serine protease (<it>Pb</it>SP), was isolated from a cDNA library constructed with RNAs of fungal yeast cells recovered from liver of infected mice. Recombinant <it>Pb</it>SP was produced in <it>Escherichia coli</it>, and used to develop polyclonal antibodies that were able to detect a 66 kDa protein in the <it>P. brasiliensis </it>proteome. <it>In vitro </it>deglycosylation assays with endoglycosidase H demonstrated that <it>Pb</it>SP is a <it>N</it>-glycosylated molecule. The <it>Pb</it>sp transcript and the protein were induced during nitrogen starvation. The <it>Pb</it>sp transcript was also induced in yeast cells infecting murine macrophages. Interactions of <it>Pb</it>SP with <it>P. brasiliensis </it>proteins were evaluated by two-hybrid assay in the yeast <it>Saccharomyces cerevisiae</it>. <it>Pb</it>SP interacts with a peptidyl prolyl cis-trans isomerase, calnexin, HSP70 and a cell wall protein PWP2.</p> <p>Conclusions</p> <p>A secreted subtilisin induced during nitrogen starvation was characterized indicating the possible role of this protein in the nitrogen acquisition. <it>Pb</it>SP interactions with other <it>P. brasiliensis </it>proteins were reported. Proteins interacting with <it>Pb</it>SP are related to folding process, protein trafficking and cytoskeleton reorganization.</p
The amino acids motif-32GSSYN36-in the catalytic domain of E. coli flavorubredoxin NO reductase is essential for its activity
Funding Information: Funding: This study was financially supported by the Portuguese Fundação para a Ciência e Tec-nologia (FCT), grants PTDC/BIA-BQM/27959/2017 and PTDC/BIA-BQM/0562/2020, and Project MOSTMICRO-ITQB with references UIDB/04612/2020 and UIDP/04612/2020. This project has also received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement 810856. MCM is the recipient of FCT grant SFRH/BD/143651/2019. BAS is the recipient of FCT grant DFA/BD/8066/2020. Funding Information: This study was financially supported by the Portuguese Funda??o para a Ci?ncia e Tecnologia (FCT), grants PTDC/BIA-BQM/27959/2017 and PTDC/BIA-BQM/0562/2020, and Project MOSTMICRO-ITQB with references UIDB/04612/2020 and UIDP/04612/2020. This project has also received funding from the European Union?s Horizon 2020 research and innovation program under grant agreement 810856. MCM is the recipient of FCT grant SFRH/BD/143651/2019. BAS is the recipient of FCT grant DFA/BD/8066/2020. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif,-G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.publishe
A pilot study combining ultrafiltration with ozonation for the treatment of secondary urban wastewater: organic micropollutants, microbial load and biological effects
Ozonation followed by ultrafiltration (O3 + UF) was employed at pilot scale for the treatment of secondary urban wastewater, envisaging its safe reuse for crop irrigation. Chemical contaminants of emerging concern (CECs) and priority substances (PSs), microbial load, estrogenic activity, cell viability and cellular metabolic activity were measured before and immediately after O3 + UF treatment. The microbial load was also evaluated after one-week storage of the treated water to assess potential bacteria regrowth. Among the organic micropollutants detected, only citalopram and isoproturon were not removed below the limit of quantification. The treatment was also effective in the reduction in the bacterial loads considering current legislation in water quality for irrigation (i.e., in terms of enterobacteria and nematode eggs). However, after seven days of storage, total heterotrophs regrew to levels close to the initial, with the concomitant increase in the genes 16S rRNA and intI1. The assessment of biological effects revealed similar water quality before and after treatment, meaning that O3 + UF did not produce detectable toxic by-products. Thus, the findings of this study indicate that the wastewater treated with this technology comply with the water quality standards for irrigation, even when stored up to one week, although improvements must be made to minimise microbial overgrowth.info:eu-repo/semantics/publishedVersio
The second International Symposium on Fungal Stress: ISFUS
The topic of ‘fungal stress’ is central to many important disciplines, including medical mycology, chronobiology, plant and insect pathology, industrial microbiology, material sciences, and astrobiology. The International Symposium on Fungal Stress (ISFUS) brought together researchers, who study fungal stress in a variety of fields. The second ISFUS was held in May 8-11 2017 in Goiania, Goiás, Brazil and hosted by the Instituto de Patologia Tropical e Saúde Pública at the Universidade Federal de Goiás. It was supported by grants from CAPES and FAPEG. Twenty-seven speakers from 15 countries presented their research related to fungal stress biology. The Symposium was divided into seven topics: 1. Fungal biology in extreme environments; 2. Stress mechanisms and responses in fungi: molecular biology, biochemistry, biophysics, and cellular biology; 3. Fungal photobiology in the context of stress; 4. Role of stress in fungal pathogenesis; 5. Fungal stress and bioremediation; 6. Fungal stress in agriculture and forestry; and 7. Fungal stress in industrial applications. This article provides an overview of the science presented and discussed at ISFUS-2017.Sao Paulo Research Foundation (FAPESP) 2010/06374-1, 2013/50518-6, 2014/01229-4Brazilian National Council for Scientific and Technological Development (CNPq) PQ2 302312/2011-0, PQ1D 308436/2014-8Coordenação de Aperfeiçoãmento de Pessoal de Nível Superior (CAPES) PAEP 88881.123209/2016-01Fundação de Amparo à Pesquisa do Estado de Goiás Brazil 20171026700011
A review of mycotoxins in food and feed products in Portugal and estimation of probable daily intakes
Mycotoxins are toxic secondary metabolites produced by filamentous fungi that occur naturally in agricultural commodities worldwide. Aflatoxins, ochratoxin A, patulin, fumonisins, zearalenone, trichothecenes and ergot alkaloids are presently the most important for food and feed safety. These compounds are produced by several species that belong to the Aspergillus, Penicillium, Fusarium and Claviceps genera and can be carcinogenic, mutagenic, teratogenic, cytotoxic, neurotoxic, nephrotoxic, estrogenic and immunosuppressant. Human and animal exposure to mycotoxins is generally assessed by taking into account data on the occurrence of mycotoxins in food and feed as well as data on the consumption patterns of the concerned population. This evaluation is crucial to support measures to reduce consumer exposure to mycotoxins. This work reviews the occurrence and levels of mycotoxins in Portuguese food and feed to provide a global overview of this issue in Portugal. With the information collected, the exposure of the Portuguese population to those mycotoxins is assessed, and the estimated dietary intakes are presented.Lu ıs Abrunhosa, H ector Morales, C elia Soares, and Thalita
Calado received support through grants SFRH/BPD/43922/
2008, SFRH/BPD/38011/2007, SFRH/BD/37264/2007 and
SFRH/BD/79364/2011, respectively, from the Fundac¸ ao para ~
a Ciencia e Tecnologia ^ –FCT, Portugal
A selective p53 activator and anticancer agent to improve colorectal cancer therapy
Impairment of the p53 pathway is a critical event in cancer. Therefore, reestablishing p53 activity has become one of the most appealing anticancer therapeutic strategies. Here, we disclose the p53-activating anticancer drug (3S)-6,7-bis(hydroxymethyl)-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole (MANIO). MANIO demonstrates a notable selectivity to the p53 pathway, activating wild-type (WT)p53 and restoring WT-like function to mutant (mut)p53 in human cancer cells. MANIO directly binds to the WT/mutp53 DNA-binding domain, enhancing the protein thermal stability, DNA-binding ability, and transcriptional activity. The high efficacy of MANIO as an anticancer agent toward cancers harboring WT/mutp53 is further demonstrated in patient-derived cells and xenograft mouse models of colorectal cancer (CRC), with no signs of undesirable side effects. MANIO synergizes with conventional chemotherapeutic drugs, and in vitro and in vivo studies predict its adequate drug-likeness and pharmacokinetic properties for a clinical candidate. As a single agent or in combination, MANIO will advance anticancer-targeted therapy, particularly benefiting CRC patients harboring distinct p53 status.We thank PT national funds (FCT/MCTES, Fundação para a Ciência e a Tecnologia, and Ministério da Ciência, Tecnologia e Ensino Superior) through grants UIDB/50006/2020, UID/BIO/04469/2019, UIDB/04539/2020, and
UIDP/04539/2020 (CIBB); BioTecNorte operation (NORTE-01-0145-FEDER000004) and Porto Neurosciences and Neurologic Disease Research Initiative at I3S (Norte-01-0145-FEDER-000008) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional
Regional do Norte; Masaryk University (Project MUNI/A/1127/2019) and Ministry of Education, Youth and Sports of the Czech Republic (project nos. LQ1605 and LM2018125); FCT financial support through the fellowships SFRH/BD/119144/2016 (H.R.) and SFRH/BD/117949/2016 (L.R.); Fondazione
AIRC (IG#18985, A.I.); and the Programa Operacional Potencial Humano
(POCH), specifically the BiotechHealth Programme (Doctoral Programme on
Cellular and Molecular Biotechnology Applied to Health Sciences, PD/00016/2012). We thank Dario Rizzotto for assistance in preparing the libraries for RNA sequencing. Funding: This work was supported by PT National Funds (FCT/MCTES, Fundação para a Ciência e Tecnologia, and Ministério da Ciência, Tecnologia e Ensino Superior) via the projects UIDB/50006/2020 (LAQV/REQUIMTE), UIDB/00313/2020, and UIDP/00313/2020, co-funded by COMPETE2020-UE.info:eu-repo/semantics/publishedVersio
Making the invisible visible: searching for human T-cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2) in Brazilian patients with viral hepatitis B and C
Configuração e recuperação da informação em documentos de ciência e tecnologia: estudo tipológico em arquivo pessoal no arquivo pessoal do físico Bernhard Gross
Proteomic Analysis Reveals That Iron Availability Alters the Metabolic Status of the Pathogenic Fungus Paracoccidioides brasiliensis
Paracoccidioides brasiliensis is a thermodimorphic fungus and the causative agent of paracoccidioidomycosis (PCM). The ability of P. brasiliensis to uptake nutrients is fundamental for growth, but a reduction in the availability of iron and other nutrients is a host defense mechanism many pathogenic fungi must overcome. Thus, fungal mechanisms that scavenge iron from host may contribute to P. brasiliensis virulence. In order to better understand how P. brasiliensis adapts to iron starvation in the host we compared the two-dimensional (2D) gel protein profile of yeast cells during iron starvation to that of iron rich condition. Protein spots were selected for comparative analysis based on the protein staining intensity as determined by image analysis. A total of 1752 protein spots were selected for comparison, and a total of 274 out of the 1752 protein spots were determined to have changed significantly in abundance due to iron depletion. Ninety six of the 274 proteins were grouped into the following functional categories; energy, metabolism, cell rescue, virulence, cell cycle, protein synthesis, protein fate, transcription, cellular communication, and cell fate. A correlation between protein and transcript levels was also discovered using quantitative RT-PCR analysis from RNA obtained from P. brasiliensis under iron restricting conditions and from yeast cells isolated from infected mouse spleens. In addition, western blot analysis and enzyme activity assays validated the differential regulation of proteins identified by 2-D gel analysis. We observed an increase in glycolytic pathway protein regulation while tricarboxylic acid cycle, glyoxylate and methylcitrate cycles, and electron transport chain proteins decreased in abundance under iron limiting conditions. These data suggest a remodeling of P. brasiliensis metabolism by prioritizing iron independent pathways
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