Publications

Abstract Biofilm control is mainly based on chemical disinfection, without a clear understanding of the role of the biocides and process conditions on biofilm removal. This study aims to understand the effects of a biocide (benzyldimethyldodecyl ammonium chloride-BDMDAC) and mechanical treatment (an increase of shear stress -tau w) on single- and dual-species biofilms formed by Bacillus cereus and Pseudomonas fluorescens on high-density polyethene (HDPE). BDMDAC effects were initially assessed on bacterial physicochemical properties and initial adhesion ability. Then, mature biofilms were formed on a rotating cylinder reactor (RCR) for 7 days to assess the effects of chemical and mechanical treatments, and the combination of both on biofilm removal. The results demonstrated that the initial adhesion does not predict the formation of mature biofilms. It was observed that the dual-species biofilms were the most susceptible to BDMDAC exposure. The exposure to increasing tau w emphasised the mechanical stability of biofilms, as lower values of tau w (1.66 Pa) caused high biofilm erosion and higher tau w values (17.7 Pa) seem to compress the remaining biofilm. In general, the combination of BDMDAC and the mechanical treatment was synergic in increasing biofilm removal. However, these were insufficient to cause total biofilm removal (100%; an average standard deviation of 11% for the method accuracy should be considered) from HDPE.

Abstract Phytochemical antioxidants like gallic and caffeic acid are constituents of the normal human diet that display beneficial health effects, potentially via activating stress response pathways. Using primary human skin fibroblasts (PHSFs) as a model, we here investigated whether such pathways were induced by novel mitochondriatargeted variants of gallic acid (AntiOxBEN(2)) and caffeic acid (AntiOxCIN(4)). Both molecules reduced cell viability with similar kinetics and potency (72 h incubation, IC50 similar to 23 mu M). At a relatively high but non-toxic concentration (12.5 mu M), AntiOxBEN(2) and AntiOxCIN(4) increased ROS levels (at 24 h), followed by a decline (at 72 h). Further analysis at the 72 h timepoint demonstrated that AntiOxBEN(2) and AntiOxCIN(4) did not alter mitochondrial membrane potential (Delta psi), but increased cellular glutathione (GSH) levels, mitochondrial NAD(P) H autofluorescence, and mitochondrial superoxide dismutase 2 (SOD2) protein levels. In contrast, cytosolic SOD1 protein levels were not affected. AntiOxBEN(2) and AntiOxCIN(4) both stimulated the gene expression of Nuclear factor erythroid 2-related factor 2 (NRF2; a master regulator of the cellular antioxidant response toward oxidative stress). AntiOxBEN2 and ANtiOxCIN4 differentially affected the gene expression of the antioxidants Heme oxygenase 1 (HMOX1) and NAD(P)H dehydrogenase (quinone) 1 (NQO1). Both antioxidants did not protect from cell death induced by GSH depletion and AntiOxBEN(2) (but not AntiOxCIN(4)) antagonized hydrogen peroxide-induced cell death. We conclude that AntiOxBEN(2) and AntiOxCIN(4) increase ROS levels, which stimulates NRF2 expression and, as a consequence, SOD2 and GSH levels. This highlights that AntiOxBEN(2) and AntiOxCIN(4) can act as prooxidants thereby activating endogenous ROS-protective pathways.

Abstract Quorum sensing (QS) plays an essential role in the production of virulence factors, in biofilm formation and antimicrobial resistance. Consequently, inhibiting QS is being considered a promising target for antipathogenic/anti-virulence therapies. This study aims to screen 2-nitrovinylfuran derivatives structurally related to Furvina (a broad-spectrum antibiotic already used for therapeutic purposes) for their effects on QS and in biofilm prevention/control. Furvina and four 2-nitrovinylfuran derivatives (compounds 1-4) were tested to assess the ability to interfere with QS of Staphylococcus aureus using bioreporter strains (S. aureus ALC1742 and ALC1743). The activity of Furvina and the most promising quorum-sensing inhibitor (QSI) was evaluated in biofilm prevention and in biofilm control (combined with fusidic acid). The biofilms were further characterized in terms of biofilm mass, viability and membrane integrity. Compound 2 caused the most significant QS inhibition with reductions between 60% and 80%. Molecular docking simulations indicate that this compound interacts preferentially with the protein hydrophobic cleft in the LytTR domain of AgrA pocket. Metabolic inactivations of 40% for S. aureus ALC1742 and 20% for S. aureus ALC1743 were reached. A 24 h-old biofilm formed in the presence of the QSI increased the metabolic inactivation by fusidic acid to 80%, for both strains. The overall results highlight the effects of compound 2 as well as the potential of combining QSI with in-use antibiotics for the management of skin and soft tissues infections.

Abstract Despite the international guidelines on the containment of the coronavirus disease 2019 (COVID-19) pandemic, the European scientific community was not sufficiently prepared to coordinate scientific efforts. To improve preparedness for future pandemics, we have initiated a network of nine European-funded Cooperation in Science and Technology (COST) Actions that can help facilitate inter-, multi-, and trans-disciplinary communication and collaboration.

Abstract The effect that the hydrodynamic conditions under which biofilms are formed has on their persistence is still unknown. This study assessed the behaviour of Pseudomonas fluorescens biofilms, formed on stainless steel under different shear stress (tau w) conditions (1, 2 and 4 Pa), to chemical (benzalkonium chloride - BAC, glutaraldehyde - GLUT and sodium hypochlorite - SHC) and mechanical (20 Pa) treatments (alone and combined). The biofilms formed under different tau w showed different structural characteristics. Those formed under a higher tau w were invariably more tolerant to chemical and mechanical stresses. SHC was the biocide which caused the highest biofilm killing and removal, followed by BAC. The sequential exposure to biocides and mechanical stress was found to be insufficient for effective biofilm control. A basal layer containing biofilm cells mostly in a viable state remained on the surface of the cylinders, particularly for the 2 and 4 Pa-generated biofilms.

Abstract Neurotransmitter depletion and mitochondrial dysfunction are among the multiple pathological events that lead to neurodegeneration. Following our previous studies related with the development of multitarget mitochondriotropic antioxidants, this study aims to evaluate whether the pi-system extension on the chemical scaffolds of AntiOXCIN2 and AntiOXCIN3 affects their bioactivity and safety profiles. After the synthesis of four triphenylphosphonium (TPP+) conjugates (compounds 2-5), we evaluated their antioxidant properties and their effect on neurotransmitter-metabolizing enzymes. All compounds were potent equine butyrylcholinesterase (eqBChE) and moderate electric eel acetylcholinesterase (eeAChE) inhibitors, with catechols 4 and 5 presenting lower IC50 values than AntiOXCIN2 and AntiOXCIN3, respectively. However, differences in the inhibition potency and selectivity of compounds 2-5 towards non-human and human cholinesterases (ChEs) were observed. Co-crystallization studies with compounds 2-5 in complex with human ChEs (hChEs) showed that these compounds exhibit different binging modes to hAChE and hBChE. Unlike AntiOXCINs, compounds 2-5 displayed moderate human monoamine oxidase (hMAO) inhibitory activity. Moreover, compounds 4 and 5 presented higher ORAC-FL indexes and lower oxidation potential values than the corresponding AntiOXCINs. Catechols 4 and 5 exhibited broader safety windows in differentiated neuroblastoma cells than benzodioxole derivatives 2 and 3. Compound 4 is highlighted as a safe mitochondria-targeted antioxidant with dual ChE/MAO inhibitory activity. Overall, this work is a contribution for the development of dual therapeutic agents addressing both mitochondrial oxidative stress and neurotransmitter depletion.

Abstract Hypothesis: Amphiphilic molecules spontaneously adsorb to fluid polar-nonpolar interfaces. The time scale of such adsorption depends on the molecular size and structure of the solute. This process should be accompanied by a power heat exchange that could be detected by commercial isothermal calorimeters. Experiments: Air is injected in the bulk of different aqueous solutions contained in the sample cell of an isothermal titration calorimeter. The formation of the resulting bubbles leads to a liquid/air interface to which the solute molecules spontaneously adsorb. Continuous injection experiments to produce multiple bubbles as well as experiments with static bubbles stand from the capillary tip, aiming to observe slow adsorption processes, were performed. Findings: The power associated with the formation, growth and release of air bubbles in different liquids was measured. Different independent contributions that can be associated to the pressure change in the gas phase, the evaporation-condensation of the solvent, the increase of interfacial area, the change in the heat capacity of the sample cell content, and the release of the bubble were observed. The periodic pattern produced by the continuous injection of air at a constant rate is used to determine the surface tension of different liquids, including solutions of different molecules and (bio)macromolecules.

Abstract Equilibrium binding constants (Kb) between chemical compounds and target proteins or between interacting proteins provide a quantitative understanding of biological interaction mechanisms. Reported uncertainties of measured experimental parameters are critical for decision-making in many scientific areas, e.g., in lead compound discovery processes and in comparing computational predictions with experimental results. Uncertainties in measured Kb values are commonly represented by a symmetric normal distribution, often quoted in terms of the experimental value plus–minus the standard deviation. However, in general, the distributions of measured Kb (and equivalent Kd) values and the corresponding free energy change ΔGb are all asymmetric to varying degree. Here, using a simulation approach, we illustrate the effect of asymmetric Kb distributions within the realm of isothermal titration calorimetry (ITC) experiments. Further we illustrate the known, but perhaps not widely appreciated, fact that when distributions of any of Kb, Kd and ΔGb are transformed into each other, their degree of asymmetry is changed. Consequently, we recommend that a more accurate way of expressing the uncertainties of Kb, Kd, and ΔGb values is to consistently report 95% confidence intervals, in line with other authors’ suggestions. The ways to obtain such error ranges are discussed in detail and exemplified for a binding reaction obtained by ITC. © 2021, European Biophysical Societies' Association.

Abstract Purpose: AntiOxCIN(3) is a novel mitochondriotropic antioxidant developed to minimize the effects of oxidative stress on neurodegenerative diseases. Prior to an investment in preclinical in vivo studies, it is important to apply in silico and biophysical cell-free in vitro studies to predict AntiOxCIN(3) biodistribution profile, respecting the need to preserve animal health in accordance with the EU principles (Directive 2010/63/EU). Accordingly, we propose an innovative toolbox of biophysical studies and mimetic models of biological interfaces, such as nanosystems with different compositions mimicking distinct membrane barriers and human serum albumin (HSA). Methods: Intestinal and cell membrane permeation of AntiOxCIN(3) was predicted using derivative spectrophotometry. AntiOxCIN(3)-HSA binding was evaluated by intrinsic fluorescence quenching, synchronous fluorescence, and dynamic/electrophoretic light scattering. Steady-state and time-resolved fluorescence quenching was used to predict AntiOxCIN(3)-membrane orientation. Fluorescence anisotropy, synchrotron small- and wide-angle X-ray scattering were used to predict lipid membrane biophysical impairment caused by AntiOxCIN(3) distribution. Results and Discussion: We found that AntiOxCIN(3) has the potential to permeate the gastrointestinal tract. However, its biodistribution and elimination from the body might be affected by its affinity to HSA (>90%) and by its steady-state volume of distribution (VDSS=1.89 +/- 0.48 L.Kg(-1)). AntiOxCIN(3) is expected to locate parallel to the membrane phospholipids, causing a bilayer stiffness effect. AntiOxCIN(3) is also predicted to permeate through blood-brain barrier and reach its therapeutic target - the brain. Conclusion: Drug interactions with biological interfaces may be evaluated using membrane model systems and serum proteins. This knowledge is important for the characterization of drug partitioning, positioning and orientation of drugs in membranes, their effect on membrane biophysical properties and the study of serum protein binding. The analysis of these interactions makes it possible to collect valuable knowledge on the transport, distribution, accumulation and, eventually, therapeutic impact of drugs which may aid the drug development process.