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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.
Innovative colorimetric detection of clinical Gram-negative bacteria using low-cost bacteriostatic barbiturate polymers
Publication . Galhano, Joana; Kurutos, Atanas; Dobrikov, Georgi M.; Duarte, Maria Paula; Santos, Hugo M.; Capelo-Martínez, José Luis; Lodeiro, Carlos; Oliveira, Elisabete; DQ - Departamento de Química; LAQV@REQUIMTE; MEtRICS - Centro de Engenharia Mecânica e Sustentabilidade de Recursos; Elsevier
This study introduces the synergistic antibacterial activity derived from the combination of barbituric acid derivatives with PVC, PMMA and PMMMA polymers. The barbiturates - C1, C2, and C3, exhibit potent Minimum Inhibitory Concentration (MIC) values and clear selectivity towards Gram-positive bacteria in solution, resulting in inhibitory activity at exceptionally low concentrations (0.19–0.0015 μg/mL). By integrating these barbiturates within polymeric matrices, an advanced antibacterial polymer was obtained. This novel material facilitates the dual benefit of detecting Gram-negative bacterial colonies via colorimetric alterations, while simultaneously providing a broad-spectrum antibacterial approach, effective against both Gram-positive and Gram-negative bacteria. The potential of these barbiturate-enhanced polymers is substantial, not least because of their cost-effective nature. Their inherent feature of enabling naked-eye selective alterations, positions them as an efficient, onsite tool for monitoring contamination by E. coli and P. aeruginosa in clinical environments. Thus, these polymers open a new horizon for innovative, rapid, and low-cost strategies in microbial contamination control and patient safety.
Assessment of in situ product recovery techniques to enhance 2-phenylethanol production by Acinetobacter soli ANG344B
Publication . Bernardino, Ana R.S.; Torres, Cristiana A.V.; Crespo, João G.; Reis, Maria A.M.; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; LAQV@REQUIMTE; Elsevier Science B.V., Inc
The 2-phenylethanol (2-PE) production process by the newly isolated Acinetobacter soli ANG344B is limited by product toxicity. To overcome this limitation and enhance 2-PE production process, various alternatives based in in situ product removal (ISPR) approaches were evaluated. The approaches selected for assessment were gas stripping using the air supplied to the bioreactor, liquid-liquid extraction and adsorption. Adsorption was found to be the most promising approach to increase 2-PE production. Amberlite XAD 4 was chosen from the different adsorbents tested since it has high affinity for 2-PE, being able to adsorb 205.8 ± 8.1 mg2-PE/gdry resin. In a batch cultivation process, in presence of 3 % (dry w/v) of Amberlite XAD 4, A. soli ANG344B was able to produce 6.99 ± 0.06 g/L of 2-PE with a volumetric productivity of 0.17 ± 0.00 g/L.h, which represents an improvement of 3.3-fold. To the best of our knowledge, this is the highest 2-PE production reported for a wild-type bacteria. These findings highlight the potential of Acinetobacter soli ANG344B as 2-PE producer, contributing to the development of natural 2-PE production process.
Hydrophobic Eutectogels for the Removal of Contaminants of Emerging Concern from Water
Publication . Gabirondo, Elena; Araújo, Joao M. M.; Pereiro, Ana B.; Tomé, Liliana C.; LAQV@REQUIMTE; DQ - Departamento de Química; Wiley | Wiley-VCH Verlag
Five hydrophobic eutectic solvents based on menthol and thymol were prepared and incorporated into a poly(ethylene glycol) diacrylate (PEGDA) network to form eutectogel membranes. The two most promising eutectogels were further optimized by adding ethyl hexylacrylate (HA) to the eutectogel formulation to improve the compatibility between the eutectic solvent and polymer network and to enhance the water resistance of the resulting membranes. Thermal analysis confirmed the successful formation and integration of eutectic solvents within the polymer network. Rheological studies demonstrated the rubber-like behavior of the prepared hydrophobic eutectogels, with menthol-based variants exhibiting superior mechanical properties. Finally, sorption experiments were conducted using the optimized octanoic acid:menthol PEGDA-HA eutectogel to evaluate its efficiency in removing various contaminants of emerging concern (CECs), including diclofenac, iopromide, cefazolin, bisphenol A, and dichlorophenol. The results revealed high sorption capacities for bisphenol A (3213 mg⋅kg−1) and dichlorophenol (2981 mg⋅kg−1), followed by diclofenac (1490 mg⋅kg−1), whereas minimal sorption capacities were observed for iopromide and cefazolin. Overall, this study demonstrates the potential of hydrophobic eutectogels as efficient and tunable materials for water purification, paving the way for their application in the environmental remediation of different emerging pollutants related to global change and human activities.
Dual-site engineering of N vacancies and K single-atoms in C3N4
Publication . Xu, Xiao; Xiao, Yao; Xu, Xuelian; Carabineiro, Sónia A. C.; Zhu, Junjiang; LAQV@REQUIMTE; DQ - Departamento de Química; Chinese Ceramic Society
Graphitic carbon nitride (C3N4) is a promising photocatalyst due to its suitable band gap and polymer properties, but its efficiency is limited by the poor separation of photoinduced electron/hole (e–/h+) pairs. To address this issue, we propose creating N vacancies within the layers and bridging K single-atoms between the C3N4 layers through the self-assembly of potassium citrate and melamine–urea monomers. The introduction of N vacancies disrupts the symmetry of C3N4, promoting electron transfer along the delocalized π-conjugated network, while the presence of K atoms provides channels for electron transfer between the layers by forming N[sbnd]K[sbnd]N bridges, thereby leading to significant enhancement in the separation and transfer of e–/h+ pairs across spatial dimension. As expected, the co-modified C3N4, with N vacancies and K single-atoms (designated as CN-K-VN), exhibits excellent photocatalytic performance, with reaction rate constant of 9.69 × 10−2 min−1 (7.39 × 10−2 min−1 in real water environment) for tetracycline, achieving 80% degradation of tetracycline within 20 min. The reaction mechanism, as well as the toxicity of the degradation intermediates, is deeply discussed. This study provides a strategy to enhance the spatial separation of electrons for photocatalyst, highlighting its significance role in photocatalysis.
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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 Programático
Número da atribuição
UIDP/50006/2020