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- Impact of simulated digestion on microplastics: Surface changes and intestinal in vitro exposurePublication . Lamas, Mariana; Fernandes, Virgínia Cruz; Oliveira, Marta; Rodrigues, Francisca; Fernandes, VirgíniaMicroplastics (MP) have been widely detected in a variety of food products1, and ingestion is one of the main pathways for human exposure to these contam-inants. Once ingested, MP might interact with the gastrointestinal (GI) environment, yet it remains un-clear whether digestive processes alter their chemical structure or mainly influence their surface properties, and possibly their interaction capacity3. This study in-vestigates digested-induced modifications in MP and establishes suitable conditions for intestinal in vitroassays. Simulated digestion of six polymers (PTFE; PMMA, PA6, uPVC, PET, and LDPE) followed the IN-FOGEST model2. Following digestion, particles were analysed using Fourier-transform infrared spectros-copy (FTIR) to assess structural and surface changes. Cell viability was evaluated, by MTT, in Caco-2 and HT29-MTX intestinal cell lines exposed to different dilutions (1:10, 1:20, and 1:40) of digestion fluids, with respective MP. FTIR results indicated that all polymers maintained their fundamental chemical structure after digestion. Instead, consistent surface-related alter-ations were observed, including reduced intensity of characteristic polymer bands and enhanced signals in regions associated with proteins and hydroxyl groups, suggesting biomolecules adsorption and increased hy-dration. Cell studies demonstrated that 1:20 dilution of digestive fluids with MP maintained the cell viability above 70%, representing the most suitable condition tested. Simulated GI digestion appears to preserve the intrinsic chemical identity of MP, while promoting surface modifications that may affect their biological interactions. The identification of 1:20 digestion fluid dilution supports its application in future studies using advanced intestinal models, including three-dimen-sional systems and assessment of cellular oxidative stress responses.
- Profiling airway microbiome composition through volatilomicsPublication . Neto, Filipa; Ferraz, Ricardo; Vieira, Mónica; Prudêncio, Cristina; Rufo, João; Almeida Vieira, Mónica Andreia; Cavaleiro Rufo, JoãoThe airway microbiome is known to mediate respi-ratory health. However, current available methods for microbiome analysis are time-consuming and/or rep-resent significant costs for a generalized application in clinical practice. Volatilomics has been suggested as a rapid and low-cost approach to screen microbial pro-files in human samples. Therefore, we aimed to study the efficacy of volatilomics in discriminating microbi-al isolates collected from human breath condensate samples. Bacterial strains showing significant growth under conditions simulating the respiratory environ-ment were isolated. Each strain was standardised to an inoculum of 10⁸ CFU/mL and analysed using an elec-tronic nose equipped with a six-sensor matrix. Data were explored through principal component analysis, cluster analysis, pattern analysis, random forests and recursive partitioning regression. One sensor was re-moved from the analysis due to high collinearity. Prin-cipal component analysis was able to separate strains and the control mainly through the second principal component (p = 0.024), characterized by high MQ3 and MQ8 sensor responses. Sensor profile maps showed distinct volatile patterns across strains (Fig-ure 1), suggesting the presence of distinct microbial signatures. However, reproducibility was low between replicas and time since culture. Recursive partitioning for separating sterile controls from inoculated sam-ples showed the highest accuracy (AUC = 0.73). These results show the potential of separation of microbial strains based on volatilomics. Nonetheless, relative robustness was only achieved for the discrimination of sterile vs inoculated samples.
- Enhancing antifungal activity against Candida albicans using novel organic compoundsPublication . Alves, Beatriz; Silva, Rui; Vieira, Mónica; Ferraz, Ricardo; Fernandes, Silvia; Teixeira, Vitor; Pereira, Clara; Almeida Vieira, Mónica Andreia; Fernandes, SílviaCandida albicans is the predominant cause of inva-sive candidiasis, a major global health threat [1 The increasing emergence of resistance to commonly used antifungals (ex.:iazoles and polyenes) demonstrates an urgent need for alternative therapeutic strategies. The objective of this study was to evaluate the antifungal activity of novel organic derivatives from bioactive natural compound, including Naphthalene, Betulinic Acid (BA) [2]; Protocatechuic Acid (PCA) and Gallic Acid (GA), alone, or in combination with conventional antifungals to investigate synergistic effects that en-hance their efficacy. Antifungal susceptibility assays were performed in C. albicans ATCC 10231 using the broth microdilution method, according to CLSI M27 guidelines, to determine Minimum Inhibitory Concen-trations (MICs) and Minimum Fungicidal Concentra-tions (MFC). The interaction between BA derivatives and amphotericin B was assessed by checkerboard assay and fractional inhibitory concentration index (FICI). Naphthalene, PCA and GA derivatives showed low antifungal activity, falling within the moderate to weak bioactivity range (MIC ≥ 100 μg mL−1; [3]). Among BA derivatives, BA-4 and BA-5 exhibited the best antifungal activity, with MIC values of 0,95 and 1,91 μg mL−1, respectively, achieving 99% fungicidal activity at the same concentration. Furthermore, in combination assays, both compounds enhanced Am-photericin B activity, showing synergistic effects (FICI = 0.50), resulting in a decrease in MIC. Although most newly synthesized compounds exhibited limited anti-fungal potential, BA-4 and BA-5 demonstrated very strong bioactivity and fungicidal effects against C. albicans. The observed synergy with amphotericin B highlights their potential role as antifungal adjuvants and supports the further exploration of betulinic acid derivatives as viable approaches to overcoming anti-fungal resistance.
- Optimization of SPE-HPLC-FLD methodologies for the extraction and detection of steroid hormones in plasmaPublication . Bracchi, Isabella; Paíga, Paula; Delerue-Matos, Cristina; Pestana, Diogo; Negrão, Rita; Keating, Elisa; Fernandes, VirgíniaThe quantification of steroid hormones, such as es-tradiol and ethinylestradiol, in plasma requires sensi-tive and reliable analytical methodologies1. This study presents the optimization of a bioanalytical method based on Solid-Phase Extraction (SPE) coupled with High-Performance Liquid Chromatography with Flu-orescence Detection (HPLC-FLD). For sample ex-traction, Strata-X (polymeric reversed-phase) cartridg-es were used, where critical SPE parameters, including cartridge conditioning, matrix washing, and analyte elution, were evaluated. Chromatographic separation was achieved on a C18 stationary phase using a mobile phase of acetonitrile and 0.1% formic acid in ultrapure water. Both isocratic and gradient elution programs were tested to maximize resolution and peak shape. The method was validated for linearity, recovery, and precision (intra- and inter-day), meeting all established criteria for bioanalytical applications. This combined optimization resulted in improved selectivity and sen-sitivity for the determination of estradiol and ethinyl-estradiol in plasma (Figure 1), demonstrating a robust bioanalytical tool for monitoring these compounds in complex biological matrices.
- Betulinic acid modulates adipocyte differentiation and secretome activity: Downstream effects on Glioblastoma cell migrationPublication . Gouveia, Afonso; Ferraz, Ricardo; Prudêncio, Cristina; Fernandes, Silvia; Fernandes, SílviaBetulinic acid (BA) has been recognized for its po-tential to modulate tumor progression and adipocyte function, yet its effects on adipocyte secretome re-modeling and the downstream consequences for glio-blastoma behavior remain incompletely understood1-4. In this study, we investigated the influence of BA on adipocyte secretory activity and its functional impact on glioblastoma cell migration. Direct exposure of U251 glioblastoma cells to BA significantly reduced cell migration, confirming its intrinsic anti-migratory potential. To further explore microenvironmental in-teractions, adipocyte-conditioned media (ACM) de-rived from BA-treated adipocytes was evaluated for its effects on glioblastoma behavior. Secretome anal-ysis demonstrated that BA altered matrix metallopro-teinase (MMP) expression and reduced gelatinolytic activity, indicating significant modulation of extracel-lular matrix–related proteolytic pathways. Functional assessment using wound healing assays revealed that ACM from BA-treated adipocytes did not consistent-ly suppress glioblastoma migration. Intermediate BA concentrations were associated with increased migratory behavior relative to basal conditions, although differences compared with control ACM were not statistically significant. Additionally, transwell migra-tion assays did not demonstrate significant differ-ences between treatment conditions, suggesting that BA-induced alterations in adipocyte secretome com-position do not uniformly translate into measurable changes in glioblastoma migratory capacity across dif-ferent experimental models. These findings indicate that modulation of gelatinolytic pathways alone is insufficient to predict glioblastoma cell migration and support the contribution of additional adipocyte-de-rived signalling mechanisms in regulating tumor be-havior. Overall, BA induces substantial remodelling of adipocyte secretory function, producing complex and non-linear downstream effects on glioblastoma cells. This study highlights the importance of tumor micro-environment crosstalk and underscores the need to evaluate multiple regulatory pathways when consid-ering the therapeutic potential of BA and related bio-active compounds.
- Evaluation of the functional properties of kombucha-derived Brettanomyces yeastsPublication . Areal-Hermida, Lara; Coelho, Pedro; Pichardo-Gallardo, Ángeles; Prudêncio, Cristina; Sieiro, Carmen; Prudêncio, Cristina; Coelho, PedroKombucha is a fermented beverage traditionally produced from tea and widely associated with benefi-cial health effects. These properties have been mainly attributed to the fermentable substrate and, to a less-er extent, to the microorganisms involved in the fer-mentation process. Therefore, the characterization of kombucha-associated microorganisms is essential to better understand and validate their potential health benefits. In this study, four probiotic yeast strains isolated from kombucha tea were identified, by the PCR-RFLP analysis of the ribosomal ITS region and the sequence of the D1/D2 domain of the 26S rDNA, as Brettanomyces bruxellensis (UVI55 and UVI56) and B. anomalus (UVI57 and UVI58). The evaluated strains exhibited remarkable antioxidant capacity, the abil-ity to hydrolyze bile salts, and antimicrobial activity against several key pathogens. Additionally, they displayed significant cytotoxic effects across various tumor cell lines. Notably, in the Caenorhabditis ele-gans in vivo model, strain UVI56 significantly extend-ed the nematode’s lifespan and conferred protection against S. enterica infection. Although additional in vivo studies are necessary to further elucidate the un-derlying biological mechanisms and validate the ther-apeutic potential of these yeasts, the present findings highlight the relevance of Brettanomyces species as key members of the kombucha microbial consortium. These strains exhibit multiple biological activities of potential biomedical interest and may contribute syn-ergistically to the health-promoting properties tradi-tionally associated with kombucha beverages.
- Sustainable development: an educational laboratory exercise using bacteriophagesPublication . Teixeira, Dulce; Vieira, Mónica; Prudêncio, Cristina; Almeida Vieira, Mónica Andreia; Prudêncio, CristinaSustainable development is a model that seeks to balance growth, social inclusion, and environmental protection, ensuring present and future well-being. The United Nations 17 Sustainable Development Goals (SDGs) guide this goal until 2030. Sustainable biotechnology is a branch that refers to the set of tech-niques and processes that use living organisms, their parts, or derivatives to develop products and solutions that provide economic, social, and environmental ben-efits. The growing development of the techniques that use living organisms, alongside the multidisciplinary nature of a career in medicinal biotechnology, under-scores the need for students in this sector to acquire a robust understanding of sustainable biotechnology as a fundamental component of their education. To this end, we propose a class focused on bacteriophages and their added value, covering five laboratory ses-sions: (i) design of a strategy for the recovery, enrich-ment, and isolation of a bacteriophage; (ii) extraction and recovery of bacteriophages from an environ-mental water sample; (iii) bacteriophage enrichment (Staphylococcus aureus ATCC 6538P, Staphylococcus epidermidis ATCC 1457, Escherichia coli ATCC 25922, and E. coli ATCC 8739); (iv) isolation and propagation of bacteriophages; and (v) research on the applicabili-ty of bacteriophages in the field of sustainable devel-opment. The major goals of the present work were (i) to provide students with the necessary tools to adapt protocols in the context of a laboratory class; (ii) to assess the students’ skills to carry out the assays in accordance with the expected results, and (iii) to fos-ter awareness for sustainable development. All assays were performed by students of the B.Sc in Medicinal Biotechnology. The results showed that Escherichia coli ATCC 8739 was the only strain that allowed the isolation of bacteriophages. Overall, these laboratory sessions demonstrated the practical implementation of sustainability concepts while also expanding the knowledge of biotechnological laboratory techniques.
- Blood group influence in umbilical cord blood immune cell profilePublication . Pina, Joana; Oliveira, Melissa; Ferraz, Ricardo; Gomes, Andreia; Ferraz, RicardoStem cells, characterised by their remarkable re-generative potential and ability to differentiate into different cell types, hold immense promise in thera-peutic applications. The umbilical cord is one source of stem cells which is easy, safe, and non-invasive for the mother and infant. BebéVida, a cryopreservation laboratory in Porto, served as the pivotal setting for this study. Beyond the therapeutic potential of stem cells, this study focuses on the intricate relationship between blood groups and the immune system. Vari-ations in immune cell numbers across different blood groups suggest a potential influence of ABO antigens on early immune cell composition. The human immune system undergoes a remarkable journey from embry-onic stages to infancy, marked by a gradual transition from reliance on innate immunity, which is genetically encoded, to the development of adaptive responses. ABO blood group antigens, determined by genetic in-heritance, further shape immune characteristics, with antibodies passed from the mother providing tempo-rary protection until the infant’s immune system ma-tures. This intricate interplay between genetic factors, maternal influence, and blood group genetics lays the foundation for lifelong immune health and disease susceptibility. As it has been demonstrated in differ-ent studies, there are potential links between ABO blood groups and cancer, cardiovascular, infectious (bacteria, viruses, and parasites), metabolic, and aller-gic diseases, and that is why this study was conducted, to understand the influence of blood groups on the umbilical cord blood immune cell profile. The parame-ters analysed were the nucleated cells, WBC, neutro-phils, monocytes, lymphocytes, NRBC, and platelets. In fact, this study underscores the dual significance of umbilical cord stem cells in therapeutics and the intri-cate interplay between blood groups and immune sys-tem dynamics. By elucidating these relationships, we can advance personalized medicine approaches and improve our understanding of disease predisposition across diverse population.
- Evaluation of deep learning models for Aptamer Modelling in SilicoPublication . Dias, Félix; Martins, Fábio; Sousa, Sérgio F.Aptamers are single-stranded DNA or RNA oligo-nucleotides that fold into complex three-dimensional structures to bind molecular targets with high affinity and specificity (1). Often termed “chemical antibodies,” these biomaterials are vital for diverse applications in therapeutics, diagnostics, bioassays, and in vitro and in vivo imaging (2). Historically, aptamer discovery has relied on the Systematic Evolution of Ligands by EXponential Enrichment (SELEX), an in vitro process involving iterative selection, amplification and enrich-ment rounds. However, SELEX is time-consuming and often results in low success rates (3). Consequently, an in silico approach is essential to streamline the discov-ery process, reduce experimental costs, and provide insights into aptamer-target interactions. Recently, various deep learning models capable of predicting the 3d structure of aptamer-target complexes have become available. In this work, we evaluated gener-al biomolecular prediction models, like Alphafold-3, Protenix-v2, Chai-1, Boltz-2, Openfold-3, RoseTTA-Fold2NA, and two specialized aptamer modelling tools, AptaTrans and AptaBLE. Among the evaluated models, AlphaFold-3 stands as the current state-of-the-art general model for aptamer modelling, with Protenix-v2, Chai-1, Boltz-2 and RoseTTAFold2NA, accuracies falling short of AlphaFold-3. The only mod-el that rivals Alphafold-3 in aptamer-target bench-marks is OpenFold-3. On the other hand, specialized tools such as AptaTrans and AptaBLE, significantly outperform AlphaFold3 on validated DNA and RNA aptamers, which can be attributed to their optimiza-tion for sequence-based patterns. Overall, this work highlights the importance of an in silico approach for aptamer discovery and evaluates deep learning mod-els capable of predicting the 3D structure of aptam-er-target complexes. While general models provide a robust baseline for aptamer-target complex structure prediction, specialized tools offer enhanced perfor-mance for aptamer discovery. Future work will include a systematic comparison of the evaluated models.
- Glioma behavior under the influence of Cinnamic Acid: a cellular approachPublication . Moreira, Ana; Almeida, Joana; Ferraz, Ricardo; Ferraz, RicardoCancer is one of the biggest health problems worldwide, characterized by uncontrolled proliferation, rapid growth and the ability to invade other tissues (1). Gliomas are highly challenging tu-mours from a therapeutic perspective, so new and more effective therapeutic strategies have been in-vestigated, with particular focus on the therapeu-tic potential of plant-derived compounds, such as Cinnamic Acid (2,3). Cinnamic Acid, extracted from Cinnamomum cassia, has proved to be a promising therapeutic agent, as it exhibits antitumor properties and low cytotoxicity toward healthy cells (4–6). This phenolic compound has the ability to cross the blood-brain barrier and affect tumour cell survival by reduc-ing their proliferation rate, inducing apoptosis and inhibiting key signalling pathways involved in tumour growth (7–9). This study aims to evaluate the glioma behaviour under the influence of cinnamic acid, using a GL261 cell model. To this end, cells were cultured and treated with different concentrations of cinnamic acid (1x10-3 mol/L to 4x10-3 mol/L), followed by in vi-tro assays to assess its impact on cell viability and cell migration. The MTT assay performed on the GL261 cell line demonstrated that Cinnamic Acid is capable of reducing cell viability at all tested concentrations, with a cytotoxic effect that tends to be dose-dependent. At lower concentrations (1x10-3 mol/L and 2x10-3 mol/L), cell viability was approximately 83% (SD=13,3% and 13,9%, respectively) and the most pronounced reduc-tion in cell viability was detected at the highest tested concentration (4x10-3 mol/L), with a viability of 76% ± 13,9%, corresponding to an approximately 24% re-duction relative to the control (viability of 100%).