Publications

Abstract Imidazopyrazinone is a typical scaffold present in marine bioluminescence, in which thermal energy is converted into excitation energy in an enzyme-catalyzed reaction. In fact, the imidazopyrazinone scaffold is a common link among organisms of eight phyla. The characterization of the light emission mechanism is essential for the development of future applications in bioimaging, bioanalysis and biomedicine. Herein, we have studied the chemiluminescent reaction of three commercially-available imidazopyrazinones (Cypridina luciferin, Coelenterazine and Coelenterazine-e) in several aprotic solvents at different pH. We have found that at acidic pH only DMF and DMSO consistently present high light emission, while chemiluminescence in other solvents is negligible. We have attributed this to the inability of most solvents to allow for the deprotonation of the imidazopyrazinone core, thereby preventing the oxygenation step. We have also observed that increasing the pH of the solution leads to the inhibition of chemiluminescence, which we attributed to the deprotonation of the dioxetanone intermediate, as the neutral species is the one associated with efficient chemiexcitation. We have also observed that the pK(a) of dioxetanone increases with the dielectric constant of the medium. Finally, our work indicated that the chemiexcitation yield increases with increasing polarity of the medium, due to a reduced transition dipole moment associated with S-0 -> S-1 transition.

Abstract The human immunodeficiency virus (HIV) uses the brain as reservoir, which turns it as a promising target to fight this pathology. Nanoparticles (NPs) of poly(lactic-co-glycolic) acid (PLGA) are potential carriers of anti-HIV drugs to the brain, since most of these antiretrovirals, as efavirenz (EFV), cannot surpass the blood-brain barrier (BBB). Forasmuch as the conventional production methods lack precise control over the final properties of particles, microfluidics emerged as a prospective alternative. This study aimed at developing EFV-loaded PLGA NPs through a conventional and microfluidic method, targeted to the BBB, in order to treat HIV neuropathology. Compared to the conventional method, NPs produced through microfluidics presented reduced size (73 nm versus 133 nm), comparable polydispersity (around 0.090), less negative zeta-potential (-14.1 mV versus -28.0 mV), higher EFV association efficiency (80.7% versus 32.7%) and higher drug loading (10.8% versus 3.2%). The microfluidics-produced NPs also demonstrated a sustained in vitro EFV release (50% released within the first 24 h). NPs functionalization with a transferrin receptor-binding peptide, envisaging BBB targeting, proved to be effective concerning nuclear magnetic resonance analysis (delta = -0.008 ppm; delta = -0.017 ppm). NPs demonstrated to be safe to BBB endothelial and neuron cells (metabolic activity above 70%), as well as non hemolytic (1-2% of hemolysis, no morphological alterations on erythrocytes). Finally, functionalized nanosystems were able to interact more efficiently with BBB cells, and permeability of EFV associated with NPs through a BBB in vitro model was around 1.3-fold higher than the free drug.

Abstract Antibiotic resistance has been increasingly reported for a wide variety of bacteria of clinical significance. This widespread problem constitutes one of the greatest challenges of the twenty-first century. Faced with this issue, clinicians and researchers have been persuaded to design novel strategies in order to try to control pathogenic bacteria. Therefore, the discovery and elucidation of the mechanisms underlying bacterial pathogenesis and intercellular communication have opened new perspectives for the development of alternative approaches. Antipathogenic and/or antivirulence therapies based on the interruption of quorum sensing pathways are one of several such promising strategies aimed at disarming rather than at eradicating bacterial pathogens during the course of colonization and infection. This review describes mechanisms of bacterial communication involved in biofilm formation. An overview of the potential of marine bacteria and their bioactive components as QS inhibitors is further provided.

Abstract While photodynamic therapy is known for significant advantages over conventional cancer therapies, its dependence on light has limited it to treating tumors on or just under the skin or on the outer lining of organs/cavities. Herein, we have developed a single-molecule photosensitizer capable of intracellular self-activation and with potential tumor-selectivity due to a chemiluminescent reaction involving only a cancer marker. Thus, the photosensitizer is directly chemiexcited to a triplet excited state capable of generating singlet oxygen, without requiring either a light source or any catalyst/co-factor. Cytotoxicity assays involving the photosensitizer show significant toxicity toward tumor cells, even better than reference drugs, while not inducing toxicity toward normal cells. This work provides a proof-of-concept for a novel type of photosensitizer that eliminates the current restrictions that photodynamic therapy presents regarding tumor size and localization.

Abstract Cancer is a very challenging disease to treat, both in terms of treatment efficiency and side-effects. To overcome these problems, there have been extensive studies regarding the possibility of improving treatment by employing combination therapy, and by exploring therapeutic modalities with reduced side-effects (such as photodynamic therapy (PDT)). Herein, this work has two aims: (i) to develop self-activating photosensitizers for use in light-free photodynamic therapy, which would eliminate light-related restrictions that this therapy currently possesses; (ii) to assess their co-treatment potential when combined with reference chemotherapeutic agents (Tamoxifen and Metformin). We synthesized three new photosensitizers capable of self-activation and singlet oxygen production via a chemiluminescent reaction involving only a cancer marker and without requiring a light source. Cytotoxicity assays demonstrated the cytotoxic activity of all photosensitizers for prostate and breast tumor cell lines. Analysis of co-treatment effects revealed significant improvements for breast cancer, producing better results for all combinations than just for the individual photosensitizers and even Tamoxifen. By its turn, co-treatment for prostate cancer only presented better results for one combination than for just the isolated photosensitizers and Metformin. Nevertheless, it should be noted that the cytotoxicity of the isolated photosensitizers in prostate tumor cells was already very appreciable.

Abstract Persistent bacterial infections and increased resistance to multidrugs are mostly related with biofilm formation which constitute a global concern in nowadays society. Therefore, studies must be conducted in order to discover more efficient antibiofilm agents. Nine compounds with a catecholic moiety (catechol (CAT), veratrol (VER), guaiacol (GUA), 2-ethoxphenol (ETH), 4-methylcatechol (MEC), 4-tert-butylcatechol (TEB), pyrogallol (PYR), 3-methoxycatechol (MET), and o-phenylene-phosphochloridite (OPP)) were investigated for their potential to prevent and eradicate Escherichia coil biofilms. Their action was assessed on biomass and metabolic activity, sessile cells' membrane integrity and culturability, motility and cell surface hydrophobicity (CSH). ETH, MEC, TEB, PYR and OPP exhibited the best antibiofilm activities among the tested catechols, inducing biofilm removal and metabolic inactivation, sessile cells' membrane disruption and death. CAT, TEB and PYR significantly inhibited E. coil swimming motility. Likewise, ETH, MEC, TEB and PYR significantly reduced E. coil CSH. An additive interaction of combined catechols with ciprofloxacin was attained in both control and preventive strategies. From a structure activity relationship (SAR) study, an increase in the hydrocarbon side chain and lipophilicity using ETH and TEB as scaffolds is suggested for increased activity. Hydroxyl groups are also suggested to be particularly related to their antibiofilm activity. All the catechols fulfil the Lipinski's "rule of five" requisites and thus are promising scaffolds in the development of new formulations for therapeutic purposes.

Abstract The comprehension of molecular recognition phenomena demands the understanding of the energetic and kinetic processes involved. General equations valid for the thermodynamic analysis of any observable that is assessed as a function of the concentration of the involved compounds are described, together with their implementation in the AFFINImeter software. Here, a maximum of three different molecular species that can interact with each other to form an enormous variety of supramolecular complexes are considered. The corrections currently employed to take into account the effects of dilution, volume displacement, concentration errors and those due to external factors, especially in the case of ITC measurements, are included. The methods used to fit the model parameters to the experimental data, and to generate the uncertainties are described in detail. A simulation tool and the so called kinITC analysis to get kinetic information from calorimetric experiments are also presented. An example of how to take advantage of the AFFINImeter software for the global multi-temperature analysis of a system exhibiting cooperative 1:2 interactions is presented and the results are compared with data previously published. Some useful recommendations for the analysis of experiments aimed at studying molecular interactions are provided.

Abstract Bioseparation processes are a relevant part of modern biotechnology, particularly regarding the development of efficient and biocompatible methods for the separation and purification of added-value biologically active compounds. In this field, ionic liquids (ILs) have been proposed, either in liquid–liquid extractions, in which non-water miscible ILs or aqueous biphasic systems (ABS) formed by ILs can be used, or in solid–liquid extractions, in which they are covalently attached to create supported IL phases (SILPs). Aprotic ILs possess unique properties, such as non-volatility and designability, which are valuable in their use in bioseparation processes. In this chapter, we summarize and discuss bioseparation processes based on ILs, including both liquid–liquid and solid–liquid extractions, applied to amino acids and proteins. The most recent and remarkable advances in this area are emphasized, and improvements brought by the use of ILs properly discussed. New insights and envisaged directions with IL-based bioseparation processes are suggested. © Springer International Publishing AG, part of Springer Nature 2018.

Abstract The effects of essential oils (EOs) and their eight major components extracted from five aromatic and medicinal plant (AMP) species on the development of Fusarium oxysporum f. sp. albedinis, the phytopathogenic fungus responsible for fusarium wilt (referred to as Bayoud in Morocco) on date palm in Moroccan oases (Errachidia region), were studied. The EOs were extracted from the plants by hydrodistillation and chemically analyzed by gas chromatography-mass spectrometry. The major chemical components extracted from the EOs of each AMP were: Origanum compactum (carvacrol 29.26%, thymol 18.52%, O-cymene 11.89%, gamma-terpinene 10.77%); Thymus satureioides (borneol 22.73%, carvacrol 16.96%, alpha-terpineol: 12.20%); Rosmarinus officinalis (eucalyptol 26.45%, camphor 15.51%, alpha- and beta-pinene 21.31%); Lavandula dentata (linalool 30.22%, linalyl anthranilate 23.47%, eucalyptol 9.17%); Myrtus communis (myrtenyl acetate 50.28%, eucalyptol 15.01%). The antifungal activity of the isolated EOs and their major components in comparison to that of two synthetic fungicides was evaluated by calculating the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of each antifungal product. The results of this analysis showed that the EOs of O. compactum and T. satureioides were able to inhibit the growth of F. oxysporum at very low concentrations (MFC 5 and 13 mu l ml(-1), respectively); these values were relatively better than those of the mancozeb synthetic fungicide (MFC 6 mu l ml(-1)) and very much better than the EOs of R. officinalis, L. dentata and M. communis (MFC 40 mu l ml(-1)). Thymol and carvacrol were effective fungicides at a very low concentration (MFC 0.94 and 2.08 mu l ml(-1), respectively), followed in increasing concentration by alpha-terpineol (MFC 10 mu l ml(-1)), linalool (MFC 13.00 mu l ml(-1)) and borneol (MFC 26.67 mu l ml(-1)). alpha-Pinene, eucalyptol and myrtenyl acetate were shown to have comparable fungicide properties, but at high concentrations ranging from 20 to 80 mu l ml(-1). The effectiveness of the EO of Origanum and Thymus species would therefore appear to be linked to the abundant presence of carvracrol and thymol compounds. Given the damage caused by F. oxysporum on date palm trees and other plants in Morocco, as well as the environmental and health impacts of fungicides, the use of O. compactum and T. satureioides EOs or their major components (thymol and carvacrol) against this pathogen could be an alternative to commercially available synthetic fungicides.