Percorrer por autor "Almeida, Maria Gabriela"
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- Cell-to-cell contact and antimicrobial peptides play a combined role in the death of Lachanchea thermotolerans during mixed-culture alcoholic fermentation with Saccharomyces cerevisiaePublication . Kemsawasd, Varongsiri; Branco, Patrícia; Almeida, Maria Gabriela; Caldeira, Jorge; Albergaria, Helena Albergaria; Arneborg, NilsThe roles of cell-to-cell contact and antimicrobial peptides in the early death of Lachanchea thermotolerans CBS2803 during anaerobic, mixed-culture fermentations with Saccharomyces cerevisiae S101 were investigated using a commercially available, double-compartment fermentation system separated by cellulose membranes with different pore sizes, i.e. 1000 kDa for mixed- and single-culture fermentations, and 1000 and 3.5-5 kDa for compartmentalized-culture fermentations. SDS-PAGE and gel filtration chromatography were used to determine an antimicrobial peptidic fraction in the fermentations. Our results showed comparable amounts of the antimicrobial peptidic fraction in the inner compartments of the mixed-culture and 1000 kDa compartmentalized-culture fermentations containing L. thermotolerans after 4 days of fermentation, but a lower death rate of L. thermotolerans in the 1000 kDa compartmentalized-culture fermentation than in the mixed-culture fermentation. Furthermore, L. thermotolerans died off even more slowly in the 3.5-5 kDa than in the 1000 kDa compartmentalized-culture fermentation, which coincided with the presence of less of the antimicrobial peptidic fraction in the inner compartment of that fermentation than of the 1000 kDa compartmentalized-culture fermentation. Taken together, these results indicate that the death of L. thermotolerans in mixed cultures with S. cerevisiae is caused by a combination of cell-to-cell contact and antimicrobial peptides.
- Identification of novel GAPDH-derived antimicrobial peptides secreted by Saccharomyces cerevisiae and involved in wine microbial interactionsPublication . Branco, Patrícia; Francisco, Diana; Chambon, Christophe; Hébraud, Michel; Arneborg, Nils; Almeida, Maria Gabriela; Caldeira, Jorge; Albergaria, HelenaSaccharomyces cerevisiae plays a primordial role in alcoholic fermentation and has a vast worldwide application in the production of fuel-ethanol, food and beverages. The dominance of S. cerevisiae over other microbial species during alcoholic fermentations has been traditionally ascribed to its higher ethanol tolerance. However, recent studies suggested that other phenomena, such as microbial interactions mediated by killer-like toxins, might play an important role. Here we show that S. cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against a wide variety of wine-related yeasts (e.g. Dekkera bruxellensis) and bacteria (e.g. Oenococcus oeni). Mass spectrometry analyses revealed that these AMPs correspond to fragments of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. The involvement of GAPDH-derived peptides in wine microbial interactions was further sustained by results obtained in mixed cultures performed with S. cerevisiae single mutants deleted in each of the GAPDH codifying genes (TDH1-3) and also with a S. cerevisiae mutant deleted in the YCA1 gene, which codifies the apoptosis-involved enzyme metacaspase. These findings are discussed in the context of wine microbial interactions, biopreservation potential and the role of GAPDH in the defence system of S. cerevisiae.
- Physicochemical properties of gutta-percha cones before and after a rapid disinfection protocol and its clinical relevancePublication . Martsynyshyn, Mar’yana; Brito, José; Proença, Luís; Barroso, Helena; Neves, João Albernaz; Almeida, Maria GabrielaThis study aimed to evaluate the chemical composition, radiopacity, and antimicrobial activity of five standardized (#25) commercially available gutta-percha points (ProTaper Gold, Zarc, Cerkamed, Autofit, and Reciproc R25), before and after a rapid disinfection protocol. The cones were divided into three groups: untreated (control), treated with sodium hypochlorite, and treated with sodium hypochlorite followed by ethanol rinsing. Significant compositional differences, as assessed by a solvent extraction method, were observed among brands, with gutta-percha content ranging from 11.6 to 17.5%, wax and/or resin from 1.2 to 4%, and inorganic fraction between 74.0 and 83.0%, with ZnO as the predominant component. Radiopacity determined via digital radiography varied among brands and showed a stronger correlation with ZnO content than with BaSO₄, challenging the common assumption that barium is the principal radiopacifying agent; Zarc exhibited the highest radiopacity, whereas Cerkamed showed the lowest. None of the gutta-percha points exhibited antimicrobial activity against Enterococcus faecalis and Staphylococcus aureus under standard clinical conditions, underscoring the necessity of proper disinfection procedures in clinical practice. The disinfection protocols produced minor changes in the wax/resin content of Cerkamed, Reciproc, Autofit, and ProTaper, with the effect being more pronounced in the latter two. In contrast, the metal oxide composition analyzis with wavelength dispersive X-ray fluorescence (WDXRF), showed that only Zark exhibits slight variations in ZnO, BaSO₄, SiO₂, and CuO. However, these changes remain insufficient to be considered clinically relevant. Importantly, no effect on the radiographic integrity of the gutta-percha cones was observed, confirming the safety and adequacy of the NaOCl-based disinfection protocols for clinical purposes.
- Point-of-care testing of nitrite in oral medicine : application and validation of an enzymatic biosensor in human salivaPublication . Gaspar, Sara Rodrigues; Proença, Luís; Alves, Ricardo; Almeida, Maria GabrielaPeriodontal disease (PD) is a chronic inflammatory condition triggered by bacterial biofilms and progresses through two main stages: gingivitis and periodontitis. The existing diagnostic methods remain time-consuming and require clinical expertise. Salivary nitrite, a stable end-product of nitric oxide produced during inflammation, has emerged as a promising biomarker for PD. However, its reliable quantification in saliva typically relies on laboratory-based assays that are unsuited to clinical workflows. This study aimed to test and validate a new nitrite point-of-care test (POCT) for oral medicine. Building on previous research, we developed a biosensing platform based on screen-printed carbon electrodes modified with a selective enzyme, the cytochrome c nitrite reductase. To adapt bioelectrodes for salivary analysis, they were further modified with a poly(vinyl) alcohol coating and a biochemical oxygen scavenger system (ascorbate oxidase and ascorbate). The nitrite biosensor achieved a suitable linear range of 5–300 μM, with a sensitivity of 0.015 μM−1, validated against the gold-standard Griess method. Unlike the Griess reaction, the biosensor was unaffected by sample turbidity, rendering centrifugation unnecessary. Additionally, we observed that sample freezing altered nitrite concentrations, increasing levels in non-centrifuged samples, while decreasing them in previously clarified samples. These findings highlight the need for real-time analysis and call into question the reliability of previously published data that overlooked these variables. Therefore, our results demonstrate the potential of the nitrite biosensor as a novel salivary POCT and emphasize the critical need for standardized sample-handling protocols.
- A quasi-reagentless point-of-care test for nitrite and unaffected by oxygen and cyanidePublication . Monteiro, Tiago; Gomes, Sara; Jubete, Elena; Añorga, Larraitz; Silveira, Célia M.; Almeida, Maria GabrielaThe ubiquitous nitrite is a major analyte in the management of human health and environmental risks. The current analytical methods are complex techniques that do not fulfil the need for simple, robust and low-cost tools for on-site monitoring. Electrochemical reductase-based biosensors are presented as a powerful alternative, due to their good analytical performance and miniaturization potential. However, their real-world application is limited by the need of anoxic working conditions, and the standard oxygen removal strategies are incompatible with point-of-care measurements. Instead, a bienzymatic oxygen scavenger system comprising glucose oxidase and catalase can be used to promote anoxic conditions in aired environments. Herein, carbon screen-printed electrodes were modified with cytochrome c nitrite reductase together with glucose oxidase and catalase, so that nitrite cathodic detection could be performed by cyclic voltammetry under ambient air. The resulting biosensor displayed good linear response to the analyte (2–200 µM, sensitivity of 326 ± 5 mA M−1 cm−2 at −0.8 V; 0.8–150 µM, sensitivity of 511 ± 11 mA M−1 cm−2 at −0.5 V), while being free from oxygen interference and stable up to 1 month. Furthermore, the biosensor’s catalytic response was unaffected by the presence of cyanide, a well-known inhibitor of heme-enzymes.