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Gene reactivation upon erosion of X chromosome inactivation in female hiPSCs is predictable yet variable and persists through differentiation
Publication . Raposo, Ana Cláudia; Caldas, Paulo; Jeremias, Joana; Arez, Maria; Mateus, Francisca Cazaux; Barbosa, Pedro; Sousa-Luís, Rui; Água, Frederico; Oxley, David; Mupo, Annalisa; Eckersley-Maslin, Melanie; Casanova, Miguel; Grosso, Ana Rita; Rocha, Simão Teixeira da; UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; Elsevier
Female human induced pluripotent stem cells frequently undergo X-chromosome inactivation (XCI) erosion, marked by X-inactive specific transcript (XIST) RNA loss and partial reactivation of the inactive X (Xi). This overlooked phenomenon limits our understanding of its impact on stem cell applications. Here, we show that XCI erosion is frequent and heterogeneous, leading to the reactivation of several X-linked genes. These are primarily located on the short arm of the X chromosome, particularly near escape genes and within H3K27me3-enriched domains, with reactivation linked to reduced promoter DNA methylation. Interestingly, escape genes further increase their expression from Xi upon XCI erosion, highlighting the critical role of XIST in their dosage regulation. Importantly, global (hydroxy)methylation levels and imprinted regions remain unaffected, and analysis of trilineage commitment and cardiomyocyte formation reveals that XCI erosion persists across differentiation. These findings underscore the need for greater awareness of the implications of XCI erosion for stem cell research and clinical applications.
Evaluation of Biotechnological Active Peptides Secreted by Saccharomyces cerevisiae with Potential Skin Benefits
Publication . Maurício, Elisabete Muchagato; Branco, Patrícia; Araújo, Ana Luiza Barros; Roma-Rodrigues, Catarina; Lima, Katelene; Duarte, Maria Paula; Fernandes, Alexandra R.; Albergaria, Helena; UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; Faculdade de Ciências e Tecnologia (FCT); DQ - Departamento de Química; MEtRICS - Centro de Engenharia Mecânica e Sustentabilidade de Recursos; MDPI - Multidisciplinary Digital Publishing Institute
Biotechnological active peptides are gaining interest in the cosmetics industry due to their antimicrobial, anti-inflammatory, antioxidant, and anti-collagenase (ACE) effects, as well as wound healing properties, making them suitable for cosmetic formulations. The antimicrobial activity of peptides (2–10 kDa) secreted by Saccharomyces cerevisiae Ethanol-Red was evaluated against dermal pathogens using broth microdilution and challenge tests. ACE was assessed using a collagenase activity colorimetric assay, antioxidant activity via spectrophotometric monitoring of nitrotetrazolium blue chloride (NBT) reduction, and anti-inflammatory effects by quantifying TNF-α mRNA in lipopolysaccharides (LPS)-exposed dermal fibroblasts. Wound healing assays involved human fibroblasts, endothelial cells, and dermal keratinocytes. The peptides (2–10 kDa) exhibited antimicrobial activity against 10 dermal pathogens, with the Minimum Inhibitory Concentrations (MICs) ranging from 125 µg/mL for Staphylococcus aureus to 1000 µg/mL for Candida albicans and Streptococcus pyogenes. In the challenge test, peptides at their MICs reduced microbial counts significantly, fulfilling ISO 11930:2019 standards, except against Aspergillus brasiliensis. The peptides combined with MicrocareⓇ SB showed synergy, particularly against C. albicans and A. brasilensis. In vitro, the peptides inhibited collagenase activity by 41.8% and 94.5% at 250 and 1000 µg/mL, respectively, and demonstrated antioxidant capacity. Pre-incubation with peptides decreased TNF-α expression in fibroblasts, indicating anti-inflammatory effects. The peptides do not show to promote or inhibit the angiogenesis of endothelial cells, but are able to attenuate fibrosis, scar formation, and chronic inflammation during the final phases of the wound healing process. The peptides showed antimicrobial, antioxidant, ACE, and anti-inflammatory properties, highlighting their potential as multifunctional bioactive ingredients in skincare, warranting further optimization and exploration in cosmetic applications.
Structural and functional studies of the drug metabolizing enzyme, human aldehyde oxidase
Publication . Videira, Carolina Dias; Mota, Cristiano; Franco, Irina
Human aldehyde oxidase (hAOX1; EC 1.2.3.1) is a cytosolic molybdo-flavoenzyme, mainly expressed in liver and adipose tissue. Due to its well-established role in phase I of drug metabolism, this enzyme has gained significant interest to pharmaceutical companies. hAOX1 presents a broad substrate specificity, catalysing the oxidation of various aldehydes and N-heterocycles, the hydrolysis of amide bonds, and the reduction of nitro and S/N-oxides. Despite extensive studies, the mechanism by which this enzyme catalyses substrate reduction remains poorly understood, and the exact location where the reaction occurs is still uncertain, although the FAD site has been proposed as a candidate. Moreover, the physiological role of hAOX1 is still unknown, even though some studies have shown its relevance in certain conditions and pathways, such as adipogenesis. In this work, a Yeast Two-Hybrid (Y2H) screening was performed to search for interacting partners of hAOX1 and unveil potential physiological roles. 17 potential interacting partners were identified, one involved in fat metabolism. hAOX1 harbours a flexible loop that contains two pairs of highly conserved vicinal cysteines and whose function is not acknowledged. To understand the function of the conserved loop, a variant of hAOX1 with six cysteines mutated by alanines (hAOX1_6A) was constructed and fluorescence microscopy experiments were performed to analyse its subcellular localisation. The cytosolic nature was confirmed for both variants. Finally, a new crystallisation condition using Tris(2-carboxyethyl)phosphine (TCEP) instead of Dithiothreitol (DTT) as the reducing agent was optimised and successful co-crystallisation of the enzyme with inhibitors and substrates was achieved. TCEP, contrary to DTT, was found to preserve the enzyme’s activity. Three new ternary complexes in the presence of thioridazine were obtained: two with the reduction substrates, 5-nitroquinoline and ziprasidone, and one with the oxidation substrate, phenanthridine. These data suggest that the reduction substrates have affinity to the molybdenum centre.
Evaluation of Novel Platinum-Based Combination Therapies in 2D and 3D Colorectal Cancer Models
Publication . Salas, Beatriz Filipa Bengala; Fernandes, Maria Alexandra
With rapidly increasing numbers, cancer continues to be a major cause of death globally, with colorectal cancer ranking as the second deadliest. Although chemotherapy continues to play a central role, its lack of selectivity and frequent resistance development highlight the urgent need for new therapeutic approaches. Thus, four platinum compounds and one manganese compound, along with their respective free ligands, were evaluated for their antiproliferative potential in doxorubicin-sensitive (HCT116) and resistant (HCT116-DoxR) colorectal carcinoma cell lines, as well as in non-tumorigenic human fibroblasts. Preliminary stability and solubility studies identified Pt-Methoxy and Mn-Methoxy as the most stable compounds, while the ligands showed poor solubility. Cytotoxicity assays in 2D cultures revealed that all tested metal compounds exhibited activity in the low micromolar range, with all but one platinum derivative showing enhanced potency against resistant cells. Selectivity analysis highlighted Pt-diF-terpy-OTf and the Methoxy ligand as particularly promising candidates, combining strong anti-cancer activity with low fibroblast toxicity. In 3D spheroid cultures, the three platinum complexes maintained their cytotoxicity for the HCT116-DoxR cell line and were selected for further evaluation. Mechanistic studies demonstrated that the compounds primarily induced cell death via apoptosis and autophagy, associated with high ROS production, residual mitochondrial depolarization, and cell cycle disruption. Furthermore, combination treatments with FOLFOX, shown to induce extrinsic apoptosis, significantly potentiated cytotoxic and pro-apoptotic effects. In vivo analysis revealed strong antiangiogenic activity for FOLFOX and contrasting vascular responses for Pt-Methoxy. Collectively, these findings support these platinum-based compounds, especially Pt-diF-terpy-OTf, as promising candidates for further preclinical development and highlight the therapeutic potential of combinatorial approaches.
Recovery and fractionation of volatile fatty acids from fermented solutions by electrodialysis
Publication . Barros, Kayo Santana; Marreiros, Bruno C.; Reis, Maria A.M.; Crespo, João Goulão; Pérez-Herranz, Valentín; Velizarov, Svetlozar; DQ - Departamento de Química; LAQV@REQUIMTE; Faculdade de Ciências e Tecnologia (FCT); UCIBIO - Applied Molecular Biosciences Unit; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); Elsevier BV
Electrodialysis can be used to recover charged precursors, such as volatile fatty acids (VFAs), of the biopolymers polyhydroxyalkanoates (PHAs). In general, all VFAs are recovered in a receiver solution, despite their advantageous partial fractionation. Herein, the separation of acetic, propionic, butyric, and valeric acids by electrodialysis was evaluated using three anion-exchange membranes (namely, Ralex AMH-PES, Fumasep FAS-PET-130 and PC200D) applying cell voltages from 0 V to 2.88 V. The mass transfer mechanisms were evaluated by linear sweep voltammetry and chronopotentiometry. Fumasep showed a slightly greater VFAs fractionation capacity than Ralex, while it was much greater for PC200D due to the presence of tertiary amines in its fixed functional groups. Overall, increasing the operating voltage and/or time reduced the degree of VFAs fractionation with all membranes. The higher percent extraction values and greater VFAs fractionation degrees obtained with the PC200D membrane could enhance PHAs storage efficiency.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
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Concurso para Atribuição do Estatuto e Financiamento de Laboratórios Associados (LA)
Número da atribuição
LA/P/0140/2020