Development and optimization of a new MALDI-TOF protocol for identification of the Sporothrix species complex

Accurate species identification of the Sporothrix schenckii complex is essential, since identification based only on phenotypic characteristics is often inconclusive due to phenotypic variability within the species. We used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification of 70 environmental and clinical isolates of the Sporothrix complex. A reference database was established for MALDI-TOF MS-based species identification according to minor adjustments in the manufacturer's guidelines. The MALDI-TOF MS clearly distinguished strains of Sporothrix brasiliensis, Sporothrix globosa, Sporothrix mexicana, S. schenckii, Sporothrix luriei and Sporothrix pallida, enabling identification of all isolates at the species level, as confirmed by partial calmodulin gene sequence analyses. The present methodology is simple, reliable, rapid and highly suitable for routine identification in clinical mycology laboratories and culture collections, particularly for updating and reclassifying of deposited Sporothrix isolates.The authors wish to thank the following international researchers for generously contributing strains to this study: Conchita Torrielo (EH194, EH252, EH253); Myrtha Arango (04015, 11029, 010221, 10036, 03017, 03022, 12013, 03003, 14879); and Masako Kawasaki (KMU975). Financial support was provided by FAPERJ/Rio de Janeiro, Brazil (grant proc. E-26/110.619/2012) and PAPES VI-Fiocruz/CNPq (Proc. 407693/2012-2) R. M. Z-O. is supported in part by CNPq 304976/2013-0 and FAPERJ E-26/103.157/2011. M. M. E. O. was supported by a grant from CAPES 2445/11-5 and PNPD/CAPES-Fiocruz/Pesquisa Clinica em Doencas Infecciosas. M. M. E. O., C. S. and N. L. thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and BioHealth-Biotechnology and Bioengineering approaches to improve health quality, Ref. NORTE-07-0124-FEDER-000027" co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER. Automated sequencing was done using the genomic platform/DNA sequencing platform at the Fundacao Oswaldo Cruz-PDTIS/FIOCRUZ (RPT01A), Brazil

Physiological characterization and transcriptome analysis of Pichia pastoris reveals its response to lignocellulose-derived inhibitors.

The negative efects of lignocellulose-derived inhibitors such as acetic acid and furaldehydes on microbial metabolism constitute a signifcant drawback to the usage of biomass feedstocks for the production of fuels and chemicals. The yeast Pichia pastoris has shown a great biotechnological potential for producing heterologous proteins and renewable chemicals. Despite its relevance, the performance of P. pastoris in presence of lignocellulose-derived inhibitors remains unclear. In this work, our results show for the frst time the dose-dependent response of P. pastoris to acetic acid, furaldehydes (HMF and furfural), and sugarcane biomass hydrolysate, both at physiological and transcriptional levels. The yeast was able to grow in synthetic media with up to 6 g.L-1 acetic acid, 1.75 g.L-1 furaldehydes or hydrolysate diluted to 10% (v/v). However, its metabolism was completely hindered in presence of hydrolysate diluted to 30% (v/v). Additionally, the yeast was capable to co-consume acetic acid and glucose. At the transcriptional level, P. pastoris response to lignocellulose-derived inhibitors relays on the up-regulation of genes related to transmembrane transport, oxidoreductase activities, RNA processing, and the repression of pathways related to biosynthetic processes and central carbon metabolism. These results demonstrate a polygenetic response that involves detoxifcation activi ties, and maintenance of energy and cellular homeostasis. In this context, ALD4, OYE3, QOR2, NTL100, YCT1, and PPR1 were identifed as target genes to improve P. pastoris tolerance. Altogether, this work provides valuable insights into the P. pastoris stress tolerance, which can be useful to expand its use in diferent bioprocesses

Extracellular vesicles from oral squamous carcinoma cells display pro- and anti-angiogenic properties

BackgroundA new intercellular communication mode established by neoplastic cells and tumor microenvironment components is based on extracellular vesicles (EVs). However, the biological effects of the EVs released by tumor cells on angiogenesis are not completely understood. Here, we aimed to understand the biological effects of EVs isolated from two cell lines of oral squamous cell carcinoma (OSCC) (SCC15 and HSC3) on endothelial cell tubulogenesis. MethodsOSCC-derived EVs were isolated with a polymer-based precipitation method, quantified using nanoparticle tracking analysis and verified for EV markers by dot blot. Functional assays were performed to assess the angiogenic potential of the OSCC-derived EVs. ResultsThe results showed that EVs derived from both cell lines displayed typical spherical-shaped morphology and expressed the EV markers CD63 and Annexin II. Although the average particle concentration and size were quite similar, SCC15-derived EVs promoted a pronounced tubular formation associated with significant migration and apoptosis rates of the endothelial cells, whereas EVs derived from HSC3 cells inhibited significantly endothelial cell tubulogenesis and proliferation. ConclusionThe findings of this study reveal that EVs derived from different OSCC cell lines by a polymer-based precipitation method promote pro- or anti-angiogenic effects.Peer reviewe

Efeito da superexpressão do fator de transcrição HAA1 em Komagataella phaffii produtora de ácido xilônico.

A biomassa lignocelulósica se apresenta como uma matéria?prima promissora para produção de compostos químicos de base renovável. Porém a plena utilização dos açúcares presentes em hidrolisados lignocelulósicos exige microrganismos capazes de tolerar os compostos inibidores presentes neles. Dentre os compostos inibidores, destaca?se o ácido acético, o qual pode inibir completamente o metabolismo microbiano. Visando à produção de ácido xilônico (AX) a partir de hidrolisados lignocelulósicos, foi construída uma linhagem de Komagataella phaffii expressando a enzima xilose desidrogenase (XDH) ? Kp?XDH. Para aumentar a tolerância dessa linhagem a ácido acético e, possivelmente, ao AX, foi superexpresso na levedura o fator de transcrição HAA1 ? Kp?XDH?HAA1. Uma terceira linhagem expressa somente o HAA1 ? Kp?HAA1 e não é capaz de produzir AX porque não possui a enzima XDH. Este trabalho tem por objetivo avaliar os efeitos da superexpressão de Haa1 na produção de AX. Para tanto, o desempenho das três linhagens de K. phaffii contendo XDH, Haa1, XDH?Haa1 foi comparado em diferentes condições de cultivo com a adição de ácido acético e em reciclo de suas células. Os resultados demonstram que o fator de transcrição promove aumento da tolerância da levedura ao ácido acético, sendo que a sua melhor produção de ácido xilônico é em concentrações de 2 g/L de ácido acético. Além disso, o reciclo das células pode ser uma importante chave para o aumento da produção de ácido xilônico, já que as linhagens de levedura se adaptam ao meio de cultivo

Multicenter, International Study of MIC/ MEC Distributions for definition of epidemiological cutoff values for sporothrix species identified by molecular methods

Clinical and Laboratory Standards Institute (CLSI) conditions for testing the susceptibilities of pathogenic Sporothrix species to antifungal agents are based on a collaborative study that evaluated five clinically relevant isolates of Sporothrix schenckii sensu lato and some antifungal agents. With the advent of molecular identification, there are two basic needs: to confirm the suitability of these testing conditions for all agents and Sporothrix species and to establish species-specific epidemiologic cutoff values (ECVs) or breakpoints (BPs) for the species. We collected available CLSI MICs/minimal effective concentrations (MECs) of amphotericin B, five triazoles, terbinafine, flucytosine, and caspofungin for 301 Sporothrix schenckii sensu stricto, 486 S. brasiliensis, 75 S. globosa, and 13 S. mexicana molecularly identified isolates. Data were obtained in 17 independent laboratories (Australia, Europe, India, South Africa, and South and North America) using conidial inoculum suspensions and 48 to 72 h of incubation at 35°C. Sufficient and suitable data (modal MICs within 2-fold concentrations) allowed the proposal of the following ECVs for S. schenckii and S. brasiliensis, respectively: amphotericin B, 4 and 4 /ml; itraconazole, 2 and 2 μg/ml; posaconazole, 2 and 2 μg/ml; and voriconazole, 64 and 32 μg/ml. Ketoconazole and terbinafine ECVs for S. brasiliensis were 2 and 0.12 μg/ml, respectively. Insufficient or unsuitable data precluded the calculation of ketoconazole and terbinafine (or any other antifungal agent) ECVs for S. schenckii, as well as ECVs for S. globosa and S. mexicana. These ECVs could aid the clinician in identifying potentially resistant isolates (non-wild type) less likely to respond to therapy.A. Espinel-Ingroff, D. P. B. Abreu, R. Almeida-Paes, R. S. N. Brilhante, A. Chakrabarti, A. Chowdhary, F. Hagen, S. Córdoba, G. M. Gonzalez, N. P. Govender, J. Guarro, E. M. Johnson, S. E. Kidd, S. A. Pereira, A. M. Rodrigues, S. Rozental, M. W. Szeszs, R. Ballesté Alaniz, A. Bonifaz, L. X. Bonfietti, L. P. Borba-Santos, J. Capilla, A. L. Colombo, M. Dolande, M. G. Isla, M. S. C. Melhem, A. C. Mesa-Arango, M. M. E. Oliveira, M. M. Panizo, Z. Pires de Camargo, R. M. Zancope-Oliveira, J. F. Meis, J. Turnidge