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- Brewing spent yeast as a sustainable solution for Tannin adsorptionPublication . Vieira, Elsa F.; Amaral, Tomás; Moreira, Jorge; Brandão, Tiago; Delerue-Matos, CristinaThis study explored the potential of brewing spent yeast (BSY) as an adsorbent for tannins from a chestnut shell extract (CS-tannin extract). The extract was obtained through an alkaline treatment (5% NaOH (v/v)) to recover cellulosic material from chestnut shells, which requires further valorisation. Various BSY treatments, including lyophilization, immobilization in calcium alginate beads, and alkaline and acid treatments, were tested to determine the best tannin adsorption capacity. The BSY material underwent characterization before and after the experiments, including point of zero charge (pHPZC) determination, Fourier transform infrared (FT-IR) analysis, and scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS). Equilibrium was achieved within 10 minutes, with the highest biosorption capacity of CS-tannin extract observed in lyophilized BSY, which showed a value of 31.81 ± 3.08 mg Tannic Acid Equivalents g⁻¹ yeast. The Sips isotherm model fitted well to the data, suggesting that tannin biosorption onto residual yeast cells is a chemisorption process. FT-IR analysis revealed several functional groups in the BSY, particularly carboxyl, amino/hydroxyl, and amide groups, which play a key role in tannin biosorption. These results demonstrate that BSY, a valuable by-product of the brewing industry, is an effective biosorbent for tannins from the disposal solution resulting from chestnut shell cellulosic material extraction. Further research is needed to better understand the specific interaction mechanisms and explore the practical applications of tannin-enriched BSY.
- Innovative use of spent Brewer’s yeast for tannin adsorption from treatment solutionPublication . Vieira, Elsa Marisa; Amaral, Tomás; Delerue-Matos, Cristina; Ferraz, Ricardo; Ferraz, RicardoThis study aimed to evaluate the capacity of spent brewer’s yeast (BSY) to adsorb tannins and other phenolic compounds from an alkaline-extracted chestnut shell tannin solution (CS tannin extract). The alkaline extraction process used 5% NaOH (v/v), a method commonly employed to extract cellulosic material from chestnut shells (CSs). The findings of this research contribute to the development of more sustainable laboratory practices and enhance the economic viability of cellulosic material extraction from CS. Various treatments—lyophilization, immobilization in calcium alginate beads, and both alkaline and acid treatments—were applied to BSY to determine which method resulted in the highest tannin adsorption capacity from the CS tannin extract. Kinetic and equilibrium adsorption studies were performed to identify the best adsorption approach. The tannin content was analyzed using the Folin–Ciocalteau method, with results expressed as milligrams of tannic acid equivalents (TAEs) per milliliter of extract solution. The adsorbent material was characterized before and after the experiments; the characterization methods included the determination of the point of zero charge (pHPZC), Fourier Transform Infrared (FT-IR) Spectroscopy, and Scanning Electron Microscopy with Energy Dispersive Spectroscopy (SEM/EDS). Equilibrium was reached within 10 min, with the highest (p < 0.05) biosorption capacity of tannins from the CS tannin extract observed in lyophilized BSY (35.51 ± 0.97 mg TAE per gram of BSY). The Sips models provided an adequate description of the adsorption process, indicating that tannin biosorption by BSY is driven by chemisorption. FTIR analysis identified various functional groups in BSY, with carboxyl, amino/hydroxyl, and amide groups playing a significant role in the biosorption process. Overall, these findings suggest that BSY has potential as a delivery system for the valorization of tannins from treatment solutions.
- Study of the relevance of antioxidant enzymes in thyroid cancerPublication . Freitas, Sílvia; Peixoto, Joana; Máximo, Valdemar; Soares, PaulaThyroid cancer(TC) is the most common endocrine malignancy, arising from follicular and parafollicular cells. Oxidative stress, caused by excess reactive oxygen species(ROS) and metabolites, influences TC development and progression, as the thyroid is highly exposed to ROS. The redox balance is maintained by antioxidant enzymes (SOD, GPX) and non-enzymatic antioxidants, which limit ROS formation and detoxify metabolites. Dysregulation of this balance is observed in various TC types(papillary, follicular, medullary, anaplastic). However, the role of antioxidants in TC progression, prognosis, and therapy remains under study. This project aims to explore the correlation between antioxidant enzyme expression and clinicopathological factors to address their role in TC. A series of 90 TC samples were collected, corresponding to benign and malignant lesions. The protein expression pattern of superoxide dismutase 1(SOD1), superoxide dismutase 2(SOD2) and glutathione peroxidase 1(GPX1) was optimized and assessed by immunohistochemistry in formalin-fixed paraffin-embedded samples for this cohort. In addition, the mRNA expression of SOD1, SOD2 and GPX1 are underway by quantifying mRNA expression using real-time quantitative PCR(qPCR). Our series comprise the following histotypes: Multinodular Follicular Disease(2.2%), follicular adenoma(17.8%), fetal adenoma(7.8%), papillary thyroid carcinoma (PTC)(31.1%) - classic PTC(5.6%) and follicular variant of PTC(11.1%) - follicular thyroid carcinoma(12.2%), oncocytic carcinoma(1.1%), medullary thyroid carcinoma(3.3%) and others(26.7%). QuPath evaluation of immunohistochemical analyses are underway being validated by a specialized pathologist. In parallel, qPCR analysis is ongoing. Clinicopathological parameters will be correlated with the expression patterns of the analyzed molecules. We expect to disclose the expression of antioxidant enzymes in TC and assess their potential as diagnostic and prognostic biomarkers.
- Determining the impact of impaired acylation on the nervous tissue using a novel mutant micePublication . Torres, Guilherme; Alves, Nygell; Brites, PedroNormal cellular function is tightly controlled by various post-translational modifications. ZDHHC14, a member of the zinc-finger DHHC motif-containing enzymatic family, is predicted to mediate S-acylation of proteins, a process that greatly influences protein localization, association to membranes and stability. ZDHHC14 is highly expressed in the nervous system and its deficiency is often associated with the 6q25 microdeletion syndrome in patients with microencephaly, developmental delay and cognitive impairment. However, there is a knowledge gap with regards to ZDHHC14 activity, function and targets. To address this, a Zdhhc14 KO mice model was generated, using CRISPR-Cas9 technology, by deletion of exon 5. In this work, we characterized the expression of Zdhhc14 in WT and Zdhhc14 KO mice. Using cDNA prepared from brains of WT and Zdhhc14 KO mice, PCR analysis showed that deletion of exon 5 with its ensuing mutation, i.e., R235Lfs1*, leads to nonsense mRNA decay and virtually undetected Zdhhc14 mRNA in Zdhhc14 KO mice. We also determined that brain regions including corpus callosum, hippocampus and cortex, display the highest expression of Zdhhc14 mRNA. Following on preliminary neuropathological data, we investigated the effects of Zdhhc14 on central nervous system myelination. Using electron microscopy on optic nerves from WT and Zdhhc14 KO mice, we unraveled a defect in myelination characterized by reduced myelin thickness and increased number and length of myelin outfoldings. Moreover, measurement of myelin components, using western blot, revealed decreased expression of some myelin markers that could be related to impaired Zdhhc14 activity and may modulate myelin defects. Currently, our aim is to identify the substrates of Zdhhc14 activity (by comparing the palmitoylome of WT mice with that of Zdhhc14 KO mice) in order to better understand the biological processes and cellular functions of Zdhhc14, and the implications of its deficiency towards neuropathology and disease presentation.
- Evaluation of behavioral and neuroprotective effects of losartan in diabetic ratsPublication . Silva, Joana; Teixeira, Fábio; Esteves-Monteiro, Marisa; Araújo, Margarida; Magalhães, AnaDiabetes is known to negatively impact the brain, leading to memory loss and attention issues [1], as well as an increased incidence of depression, cognitive impairment, and behavioral changes. Recent studies indicate that angiotensin II AT1 receptor antagonists (ARAs) may reduce depressive symptoms, however, the mechanism of action is not yet fully understood [2]. The aim of the present study was to verify whether losartan, an ARA, has antidepressant and neuroprotective properties, with an emphasis on its effect on anxious behavior and cognition in type 1 diabetic rats. The study included 32 adults male Wistar rats divided into 3 groups: the Control group (non-diabetic rats), the STZ group (type 1 diabetic rats, induced by streptozotocin, 55mg/kg IP, under analgesia with tramadol 20mg/kg PO) and the STZ + LOS group (diabetic rats voluntarily orally treated with Losartan, 20mg/kg/day mixed in peanut butter, for 2 weeks). The effects on the animals' anxiety were evaluated using the Marble test and the Open field test, performed 10-13 days after diabetes induction. Statistical analysis revealed no significant changes in behavior among the control and treated groups. While no behavioral differences have been observed between control and diabetic animals, it is imperative to conduct histological studies by addressing regional brain networks to further understand and complement the observed functional analysis.
- Development of an in vitro skin cell model and comparison with ex vivo models: the case study of green cosmetic active ingredientsPublication . Marques, Mariana; Teixeira, Filipa; Vieira, Mónica; Rodrigues, Francisca; Vieira, MónicaSkin is the first physical barrier against pathogens and mechanical injuries, and stratum corneum, the outermost layer of the skin, is the primary barrier to therapeutic delivery. Skin is divided into the epidermis, which consists mainly of keratinocytes and the dermis, composed of fibroblasts, and extracellular matrix components. Skin models are crucial for assessing the safety and efficacy of cosmetic ingredients, especially under European Regulation (CE) No. 1223/2009 that bans animal testing in cosmetics [1]. While commercial skin models approved by the Organization for Economic Co-operation and Development (OECD) exist, they often lack physiological complexity, limiting their relevance for regulatory compliance [2]. With this work, we aim to develop and characterize a 3D in vitro skin model to evaluate the effect of green cosmetic active ingredients—catechin, epicatechin, chlorogenic acid, and neochlorogenic – due to their antioxidant and anti-aging properties. A keratinocyte (HaCaT)-fibroblast (HDF) coculture and a hydrogel matrix were established to mimic native skin properties, with TEER measurements assessing barrier integrity and MTT assays evaluating cytotoxicity and viability. Permeability studies, performed using Franz cells and HPLC-MS analysis, compared compound penetration in the developed 3D model, ex vivo skin explants, and the commercial EpiSkinTM model. Preliminary results indicate that the developed 3D in vitro skin model successfully supports keratinocyte-fibroblast co-culture, with TEER values suggesting the establishment of a functional barrier. The development of a physiologically relevant 3D in vitro skin model represents a significant step toward improving in vitro testing of green cosmetic active ingredients. This work is an advancement on sustainable and ethical cosmetic testing, bridging the gap between traditional models and human physiology.
- Modulation of brain structure and motor function by safinamide multimodal actions in a pre-clinical model of Parkinson’s DiseasePublication . Araújo, Bruna; Campos, Jonas; Silva, Rita Caridade; Pinheiro, Bárbara Mendes; Marques, Raquel; Barata, Sandra; Lima, Rui; Macedo, Joana Martins; Gomes, Eduardo; Larrat, Benoit; Salgado, António; Mériaux, Sébastien; Domingues, Sofia; Teixeira, Fábio; Gomes, EduardoTo date, no neuroprotective/disease-modifying strategy has been approved as a Parkinson’s Disease (PD) therapy, because of the‘one-disease-one-target’ view that has been followed. New drug-based therapeutic routes, namely Safinamide, have been introduced as a promising multimodal drug combining dopaminergic and non-dopaminergic (neuroprotective) actions, representing a new potential alternative therapy to prevent or delay PD progression. Thus, the present work addressed Safinamide's impact on PD, relying on the possibility of potentiating dopaminergic neurons (DAn) survival by tackling cellular/molecular impairments responsible for its failure. Safinamide (10mg/kg) was given by oral gavage to a 6-OHDA pre-clinical rat model. DAn survival, neuroinflammation, and redox system homeostasis were assessed by histological and molecular analysis. Additionally, to overpass the selective blood-brain barrier (BBB) permeability, which reduces drug bioavailability reaching PD brain regions, we conducted magnetic resonance imaging (MRI)-guided focused ultrasound (FUS) to transiently open the BBB to precisely deliver Safinamide in PD-affected areas. Results revealed that Safinamide monotherapy was able to potentiate the densities of DAn and fibers, revealing a protective effect when compared to the untreated group. To understand possible pathways associated with this improvement, we found that Safinamide appears to be a modulator of the antioxidant and autophagy systems since an increase in the expression levels of DJ-1, SOD-1, and LC3B was observed when compared to the non-treated group. Furthermore, Safinamide presents a potential modulatory activity on neuroinflammation and astrogliosis, as a decrease in microglia (CD11b+) and astrocytic (GFAP+) cells number was observed when compared to 6-OHDA group. Additionally, the anatomical and functional MRI analysis exhibited connectivity and metabolite alterations. Collectively, these data demonstrate the promising therapeutic potential of Safinamide as a neuroprotection strategy for PD, which may open new therapeutic opportunities for individuals in prodromal stages, potentially delaying clinical manifestation in high-risk patients.
- Low skeletal muscle function, but not mass, is associated with the presence of type 2 DiabetesPublication . Rigor, Joana; Barbosa, João Portugal; Luís, Carla; Fernandes, Rúben; Barata, Pedro; Martins-Mendes, DanielaThe pathophysiology of type 2 Diabetes mellitus (T2DM) is intimately connected to the skeletal muscle (SkM). SkM affects insulin resistance and is, in turn, affected by the metainflammation, microvascular disease and ectopic fat deposition of T2DM. SkM mass can be inferred by the waist-to-calf ratio (WCR) and its function by the Short Physical Performance Battery (SPPB). The aim of this study was to determine the association between SkM mass and function with T2DM in patients with Metabolic Syndrome (MetS). Patients with MetS, aged 18 to 75 years-old, attending an outpatient clinic from April 15th to September 30th 2019, were consecutively included. Exclusion criteria comprised type 1 Diabetes, secondary hypertension, active neoplasia, autoimmune disease, HIV or hepatitis virus B or C infection and end-stage renal disease and/or liver disease. History and anthropometric data were collected, including weight, height, waist circumference (WC) and WCR; the SPPB was applied. A total of 81 patients were included, of which 58.0% had T2DM; most patients were female (55.6%) and the median age was 65 (interquartile range 16.5) years. Patients with T2DM were older (64.1 vs. 56.5 years, p=0.001) and more likely to have concurrent hypertension (96% vs. 65%, p<0.001) and dyslipidemia (96% vs. 56%, p<0.001). In univariate analysis, WC [odds ratio (OR) 1.1, 95% confidence interval (CI) 1.0-1.1), WCR (OR 146.2, 95% CI 9.9-2159.0) and SPPB (0.6, 95% CI 0.4-0.8) were associated with T2DM. In multivariate analysis, only SPPB maintained its association (OR 0.65, 95% CI 0.44-0.97). Poorer muscle function, as determined by the SPPB, was associated with the presence of T2DM, even when considering body composition, per WCR. Longitudinal and mechanistic studies are warranted to best characterize this relationship.
- Development of a stable melanoma dual reporter cell line expressing Luciferase and GFPPublication . Aguiar, Gonçalo; Torres, Sílvia; Prudêncio, Cristina; Soares, Raquel; Coelho, Pedro; Prudêncio, Cristina; Coelho, PedroMelanoma is the most aggressive and lethal form of skin cancer, with a high risk of metastatic spread. Obesity is recognized as a risk factor for various types of cancer. However, regarding melanoma, this association remains controversial. Obesity might act as a double-edged sword in melanoma, promoting primary tumour growth but at the same time limiting metastatic spread - the "obesity paradox”. Herein, we aimed to create a stable murine B16F10 melanoma cell line expressing both firefly luciferase (Luc) and green fluorescent protein (GFP), which will later be engrafted into diet induced-obesity animal model for future in vivo studies. B16F10-Luc-GFP cells were generated by transfection with premade lentiviral particles, featuring a construct with Luc and GFP under a cytomegalovirus promoter and mediated by a F2A element. The antibiotic selection marker (puromycin) is expressed under a Rous sarcoma virus promoter. Afterwards, the transfected cells were selected with 1 μg/ml of puromycin. The clones with the highest levels of GFP-positive cells and GFP fluorescence were purified by two rounds of cell sorting and submitted to fluorescence and bioluminescence quantification, morphology, injury, BrdU incorporation, 7-AAD, and PI cell cycle assays and compared to the parental cell line. B16F10-Luc-GFP were successfully generated, and both GFP fluorescence and D-luciferin bioluminescence are present and proportional to cell density. As expected, the parental cell line didn’t display GFP or Luc activities. Moreover, transduced cells exhibit similar morphology, motility, proliferation, viability, and cell cycle progression as B16F10 cells. Conclusions: Altogether, the future engraftment of B16F10-LucGFP in obese mice, will improve melanoma research models, enabling the in vivo and ex vivo visualization of primary tumours and metastasis, providing a better understanding of the underlying molecular mechanisms, to clarify the “obesity paradox” in melanoma.
- Exploring actinobacterial diversity in Ruta graveolens: Phylogenetic identification and bioactive potential investigationPublication . Ferreira, Sílvia; Ribeiro, Inês; Oliveira, Rui S.; Carvalho, M. FátimaMedicinal plants and their components have been utilized in traditional medicine for centuries and have significantly influenced the development of modern medicine. Ruta graveolens, a Rutaceae medical plant, is known for its antibacterial, anti-inflammatory and cytotoxic properties. Actinobacteria are a rich source of compounds exhibiting diverse biological activities and potential therapeutic applications. The aim of this study was to perform the phylogenetic identification of a collection of actinobacterial strains previously isolated from R. graveolens and to investigate their bioactive potential. Actinobacterial strains previously isolated from stem, roots and leaves of R. graveolens were grown in Actinomycete Isolation Agar (AIA) or Starch-Casein-Nitrate-Agar (SCN). DNA from grown cultures was extracted and phylogenetically identified through 16S rRNA gene sequencing. For each strain, organic extracts were performed and used for the screening of antimicrobial activity, using the disk diffusion test, against four reference bacteria (Staphylococcus aureus, Bacillus subtilis, Salmonella typhimurium, Escherichia coli) and one yeast (Candida albicans). Thirty-two actinobacterial isolates were so far identified. Most of the strains was identified as Tsukamurella tyrosinosolvens, constituting 13 out of 32 isolates, followed by 8 Streptomyces sp., 7 Brevibacterium sediminis, 3 Microbacterium ginsengiterrae, and one Gordonia hydrophobica. The organic extracts obtained from each isolate were tested for their antimicrobial activity. Up to moment, no significant bioactivity was detected in the reference strains screened in this study. A collection of 32 actinobacterial strains was obtained from various parts of the medicinal plant R. graveolens. Though no relevant antimicrobial activity was yet found, extracts of these actinobacteria open new opportunities to explore their bioactive potentials with therapeutic applications.