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How reliable is the evaluation of DNA binding constants?
Publication . Dömötör, Orsolya; Binacchi, Francesca; Ribeiro, Nádia; Busto, Natalia; Gonzalez-García, Jorge; Garcia-España, Enrique; Correia, Isabel; Enyedy, Éva A.; Hamacek, Josef; Terenzi, Alessio; Basílio, Nuno; Barone, Giampaolo; Cavaco, Isabel; Biver, Tarita; LAQV@REQUIMTE; DQ - Departamento de Química; Elsevier Science Publisher B.V.
In all experimental sciences, the precision and reliability of quantitative measurements are paramount. This is particularly true when examining the interactions between small molecules and biomolecules/polyelectrolytes, such as DNAs/RNAs, and yet it is overlooked in most publications of thermodynamic binding parameters. This paper presents findings from COST Action 18202 “Network for Equilibria and Chemical Thermodynamics Advanced Research,” which assessed the consistency of data derived from the interactions of calf-thymus DNA (CT-DNA) with the fluorescent intercalator ethidium bromide (EB) through spectrofluorimetric titrations. We first discuss critical experimental aspects and propose a reference experimental protocol which can be used to calibrate procedures for the determination of nucleic acid binding equilibrium constants. We then fit the experimental points according to different procedures and analyse the results focusing on the statistical dispersion of the data, aiming at enlightening the strong and weak points of different fitting procedures. The implications of this work are significant, demonstrating how the statistical dispersion of experimental data can influence the interpretation of biochemical coordination mechanisms. Our study reveals that, despite rigorous protocol standardization, the determination of binding parameters remains sensitive to the choice of data fitting method, with deviations in the logarithmic stability constant (logK) values not falling below 5 % relative standard deviation (RSD), or ± 0.5 logK units for 95 % confidence. This variability evidences the critical need for standardized best practices in data treatment as well as experimental procedures. Although our study focuses on the EB/CT-DNA system through fluorescence titrations, the broader implications for other methodologies across various biochemical systems highlight the importance of this first-of-its-kind inter-laboratory comparison in advancing our understanding of biochemical coordination processes.
Exploring the catalytic and anticancer activity of gold(I) complexes bearing 1,3,5-triaza-7-phosphaadamantane (PTA) and related ligands
Publication . Conceição, Nuno Reis; Mahmoud, Abdallah G.; Dietl, Martin C.; Caligiuri, Isabella; Rizzolio, Flavio; Carabineiro, Sónia A. C.; Rudolph, Matthias; Silva, M. Fátima C. Guedes da; Pombeiro, Armando J. L.; Hashmi, A. Stephen K.; Scattolin, Thomas; LAQV@REQUIMTE; DQ - Departamento de Química; DCEA - Departamento de Ciências e Engenharia do Ambiente; RSC - Royal Society of Chemistry
A series of water-soluble gold(i) complexes bearing phosphine ligands, [AuCl(L)] {where L = 1,3,5-triaza-7-phosphaadamantane, PTA (1); 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane, DAPTA (2); or 1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane-3,7-diylbisphenylmethanone, DBPTA (3)} and [AuCl(L)]X {where L is either PTA-CH2-C6H4-p-COOH and X = Br (4) or PTA-CH2-C6H3-p-OH-m-CHO and X = Cl (5)}, were synthesized under mild conditions and characterized with multinuclear (1H, 13C and 31P) nuclear magnetic resonance (NMR) spectroscopy, attenuated total reflection - Fourier transform infrared (ATR-FTIR) spectroscopy, matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) and elemental analysis. The catalytic activity of the complexes was evaluated in the peroxidative oxidation of cyclohexane to cyclohexanol and cyclohexanone. Homogeneous reactions were conducted in aqueous media, while heterogeneous reactions were performed after immobilizing the complexes on porous carbon supports, including activated carbon (AC), carbon nanotubes (CNT) and their oxidized derivatives (AC-ox, AC-ox-Na, CNT-ox and CNT-ox-Na). The results demonstrated a better catalytic performance, in terms of yields and selectivity, under heterogeneous conditions depending on the nature of the carbon support. Finally, complexes 1-5 showed remarkable cytotoxicity against a selection of ovarian, lung and colon cancer cell lines, with IC50 values comparable to (or even better than) those of cisplatin. Interestingly, the most promising complexes exhibited good to excellent cytotoxicity against cancer cells while demonstrating substantial inactivity against normal ones.
Exploring the Hypocholesterolemic Potential of a Fucus vesiculosus Extract
Publication . André, Rebeca; Pacheco, Rita; Santos, Hugo M.; Serralheiro, Maria Luísa; LAQV@REQUIMTE; DQ - Departamento de Química; MDPI - Multidisciplinary Digital Publishing Institute
High blood cholesterol levels are a major risk factor for cardiovascular diseases. A purified aqueous extract of Fucus vesiculosus, rich in phlorotannins and peptides, has been described for its potential to inhibit cholesterol biosynthesis and intestinal absorption. In this work, the effect of this extract on intestinal cells’ metabolites and proteins was analysed to gain a deeper understanding of its mode of action on lipids’ metabolism, particularly concerning the absorption and transport of exogenous cholesterol. Caco-2 cells, differentiated into enterocytes, were exposed to the extract, and analysed by untargeted metabolomics and proteomics. The results of the metabolomic analysis showed statistically significant differences in glutathione content of cells exposed to the extract compared to control cells, along with an increased expression of fatty acid amides in exposed cells. A proteomic analysis showed an increased expression in cells exposed to the extract compared to control cells of FAB1 and NPC1, proteins known to be involved in lipid metabolism and transport. To the extent of our knowledge, this study is the first use of untargeted metabolomics and a proteomic analysis to investigate the effects of F. vesiculosus on differentiated Caco-2 cells, offering insights into the molecular mechanism of the extract’s compounds on intestinal cells.
Molecular landscape of therapeutic deep eutectic systems selective toxicity towards colorectal cancer
Publication . Oliveira, Filipe; Pinto, Joana; Amaro, Filipa; Pereira, Joana; Ferreira, Inês; Diniz, Mário S.; Pinho, Paula Guedes de; Duarte, Ana Rita C.; DQ - Departamento de Química; LAQV@REQUIMTE; UCIBIO - Applied Molecular Biosciences Unit; Elsevier BV
The foremost modern medicine challenges, such as cancer's high mortality rates, have emphasized the growing need for improved therapeutic agents. Following eutectic systems spotlight due to their remarkable physicochemical and biological properties, while mostly in compliance with the Green Chemistry Principles and sustainability metrics as the Sustainable Development Goals (SDG) from United Nations, we aimed to push forward their understanding as anticancer agents. Considering therapeutic deep eutectic systems (THEDES) promising reports of anticancer activity towards colorectal cancer (CRC) cells, we sought to fill the knowledge gap on these eutectics impact on these cancer cells molecular landscape. For that, an integrated approach was used to study how combining a terpene – as menthol (Me), thymol (Thy), perillyl alcohol (POH) and limonene (Lim) – with ibuprofen (Ibu) as a THEDES, affects permeability, cellular transport, cell viability, reactive oxygen species (ROS), cell metabolome and systemic toxicity. THEDES exposure resulted in increased Ibu cellular uptake, depletion of ROS, and cell dead induction via apoptosis. Moreover, CRC cells experienced alterations in their metabolite landscape with deleterious effects on essential metabolic pathways, such as lipid and anaerobic glycolysis energy production pathways. Furthermore, non-relevant systemic toxicity of these THEDES, within the range of concentrations tested, was observed. These findings underscore THEDES potential as selective anticancer agents, while offering a promising path for the development of therapeutics aligned with the sustainability metrics.
Innovative approach in sustainable agriculture
Publication . Ferreira, Alice; Vladić, Jelena; de Oliveira Corrêa, Diego; Butzke, Valéria Louzada Leal; Martins, Pedro L.; Ribeiro, Belina; Marques-dos-Santos, Cláudia; Acién, F. Gabriel; Gouveia, Luisa; DQ - Departamento de Química; Faculdade de Ciências e Tecnologia (FCT); Elsevier BV
Microalgae can contribute to sustainable agriculture and wastewater treatment. This study investigated Tetradesmus obliquus, grown in piggery wastewater (To-PWW), as a biostimulant/biofertilizer compared to biomass grown in synthetic medium (To-B). Subcritical water extraction was tested for disruption/hydrolysis of wet biomass, at three temperatures (120, 170, and 220 °C) and two biomass loads (1:10 and 1:80 (g dry biomass/mL water)). Extracts were evaluated for germination, and root formation/expansion. Residues were quantified for nutrient composition to assess their biofertilizer potential and tested for their affinity to oil compounds for bioremediation. The best germination was achieved by To-B extracts at 170 °C (1:10: 148 % at 0.2 g/L, 1:80: 145 % at 0.5 g/L). Only To-PWW extracts at 0.2 g/L had a significant germination effect (120 °C: 120–123 % for both loads; 170 °C: 115 % for 1:80). To-PWW extract at 120 °C and 1:10 significantly affected cucumber and mung bean root formation (224 and 268 %, respectively). Most extracts significantly enhanced root expansion, with all To-B extracts at 1:10 showing the best results (139–181 %). The residues contained essential nutrients (NPK), indicating their biofertilizer potential, helping decrease synthetic fertilizers demands. To-B residues had high affinity to toluene and diesel but lower to used cooking and car oils. To-PWW showed very low affinity to all oil compounds. Finally, all residues were only able to form stable emulsions with the used car oil. This study fully exploits the use of microalgal biomass in sustainable agriculture, producing biostimulant extracts, and residues for biofertilizer and bioremediation, from a low-cost wastewater source.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
Concurso de avaliação no âmbito do Programa Plurianual de Financiamento de Unidades de I&D (2017/2018) - Financiamento Base
Número da atribuição
UIDB/50006/2020