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2020
Roma-Rodrigues, C, Rivas-García L, Baptista {PV}, Fernandes {AR}.  2020.  Gene therapy in cancer treatment: Why go nano?, mar Pharmaceutics. 12, Number 3: MDPI AG Abstract

The proposal of gene therapy to tackle cancer development has been instrumental for the development of novel approaches and strategies to fight this disease, but the efficacy of the proposed strategies has still fallen short of delivering the full potential of gene therapy in the clinic. Despite the plethora of gene modulation approaches, e.g., gene silencing, antisense therapy, RNA interference, gene and genome editing, finding a way to efficiently deliver these effectors to the desired cell and tissue has been a challenge. Nanomedicine has put forward several innovative platforms to overcome this obstacle. Most of these platforms rely on the application of nanoscale structures, with particular focus on nanoparticles. Herein, we review the current trends on the use of nanoparticles designed for cancer gene therapy, including inorganic, organic, or biological (e.g., exosomes) variants, in clinical development and their progress towards clinical applications.

Restani, {RB }, Pires {RF }, Baptista {PV}, Fernandes {AR}, Casimiro T, Bonifácio {VDB }, Aguiar-Ricardo A.  2020.  Nano-in-Micro Sildenafil Dry Powder Formulations for the Treatment of Pulmonary Arterial Hypertension Disorders: The Synergic Effect of POxylated Polyurea Dendrimers, PLGA, and Cholesterol, jun. Particle and Particle Systems Characterization. 37, Number 6: Wiley-VCH Verlag Abstract

POXylated polyurea dendrimer nanoparticles (PUREG4OOx48) are loaded with sildenafil (SDF) by a supercritical carbon dioxide–assisted (scCO2) impregnation. Further supercritical CO2-assisted spray drying (SASD) leads to hybrid nano-in-micro dry powder formulations that are investigated aiming at efficient pulmonary delivery of SDF in pulmonary arterial hypertension treatment. This is the first report of the production of poly(D,L-lactide-co-glycolide)-cholesterol (PLGA-Chol) microparticles processed by SASD. The optimized formulation of nano-in-microparticles is composed of PLGA, Chol, and PUREG4OOx48, loaded with SDF solutions in a 77:23 ratio (PLGA-Chol:dendrimer, w/w). The dry powders are fully characterized and found to be highly biodegradable and biocompatible, and the SDF release profile evaluates under different pH values. The median mass average diameter (MMAD) of the nano-in-micro systems varies between 2.57 and 5 µm and the fine particle fraction (FPF) between 36% and 29% for PUREG4OMeOx48[PLGA-Chol] and PUREG4OEtOx48[PLGA-Chol], respectively. The data validate the potential use of these new formulations in inhalation therapy. In vitro studies are also carried out in order to evaluate the effect of the free drug in cell viability and formulations cytotoxicity.

Roma-Rodrigues, C, Malta G, Peixoto D, Ferreira LM, Baptista P, Fernandes AR, Branco PS.  2020.  Synthesis of new hetero-arylidene-9(10H)-anthrone derivatives and their biological evaluation, JUN. BIOORGANIC CHEMISTRY. 99 Abstract
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Roma-Rodrigues, C, Malta G, Peixoto D, Ferreira {LM }, Baptista {PV}, Fernandes {AR}, Branco {PS }.  2020.  Synthesis of new hetero-arylidene-9(10H)-anthrone derivatives and their biological evaluation, jun. Bioorganic Chemistry. 99: Elsevier Science B.V., Amsterdam. Abstract

New hetero-arylidene-9(10H)-anthrone derivatives (1) were synthesized from reaction of 1,2-dimethyl-3-alkyl imidazolium salts (2) and 9-anthracenecarboxaldehyde. Ion exchange of the anion with dioctyl sulfosuccinate and lithium bis(trifluoromethanesulfonyl)imide led to the preparation of other derivatives. The antiproliferative effect of the compounds was evaluated in human ovarian (A2780) and colorectal (HCT116) carcinoma cell lines and in normal primary human fibroblasts. Compound 1 presented an antiproliferative effect related to the imidazolium pattern of substitution with compounds having a decyl group at the R-position (1c and 3c) showing the highest cytotoxic activities in all cell lines independently of the counter ion. Compounds 1b and 1c internalize A2780 cancer cells via a passive or an active transport, respectively, inducing A2780 cell death via an extrinsic apoptosis (1b) or intrinsic apoptosis and oncosis (1c). The localization of both compounds in the cytoplasm coupled to the absence of reactive oxygen species (ROS) induction suggest that the mechanisms of toxicity might be different than those of other anthracyclines currently used in chemotherapy.

Martins, C, Rodrigo {AP }, Madeira C, D'Ambrosio M, Goncalves C, Parola {AJ }, Grosso {AR }, Baptista {PV }, Fernandes {AR }, Costa {PM }.  2020.  Porphyrin Pigments in Polychaeta: Explorations on the Evolution of Haem Metabolism in Marine Eumetazoans, jan. 18 Abstract
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Rippel, R, Pinheiro L, Lopes M, Lourenco A, Ferreira LM, Branco PS.  2020.  Synthetic Approaches to a Challenging and Unusual Structure-An Amino-Pyrrolidine Guanine Core, FEB 2. MOLECULES. 25, Number 4 Abstract
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Kordestani, N, Rudbari {HA}, Fernandes {AR}, Raposo {LR }, Baptista {PV}, Ferreira {DA }, Bruno G, Bella G, Scopelliti R, Braun {JD }, Herbert {DE }, Blacque O.  2020.  Antiproliferative Activities of Diimine-Based Mixed Ligand Copper(II) Complexes, feb. ACS Combinatorial Science. 22:89–99., Number 2: ACS - American Chemical Society Abstract

A series of Cu(diimine)(X-sal)(NO3) complexes, where the diimine is either 2,2′-bipyridine (bpy) or 1,10-phenanthroline (phen) and X-sal is a monoanionic halogenated salicylaldehyde (X = Cl, Br, I, or H), have been synthesized and characterized by elemental analysis and X-ray crystallography. Penta-coordinate geometries copper(II) were observed for all cases. The influence of the diimine coligands and different halogen atoms on the antiproliferative activities toward human cancer cell lines have been investigated. All Cu(II) complexes were able to induce a loss of A2780 ovarian carcinoma cell viability, with phen derivatives more active than bpy derivatives. In contrast, no in vitro antiproliferative effects were observed against the HCT116 colorectal cancer cell line. These cytotoxicity differences were not due to a different intracellular concentration of the complexes determined by inductively coupled plasma atomic emission spectroscopy. A small effect of different halogen substituents on the phenolic ring was observed, with X = Cl being the most highly active toward A2780 cells among the phen derivatives, while X = Br presented the lowest IC50 in A2780 cells for bpy analogs. Importantly, no reduction in normal primary fibroblasts cell viability was observed in the presence of bpy derivatives (IC50 > 40 μM). Mechanistically, complex 1 seems to induce a stronger apoptotic response with a higher increase in mitochondrial membrane depolarization and an increased level of intracellular reactive oxygen species (ROS) compared to complex 3. Together, these data and the low IC50 compared to cisplatin in A2780 ovarian carcinoma cell line demonstrate the potential of these bpy derivatives for further in vivo studies.

Branco, S, Mateus EP, Richter Gomes da Silva MD, Mendes D, Araujo Pereira MM, Schutz S, Paiva MR.  2020.  Identification of pheromone candidates for the eucalyptus weevil, Gonipterus platensis (Coleoptera, Curculionidae), FEB. JOURNAL OF APPLIED ENTOMOLOGY. 144:41-53., Number 1-2 Abstract

The eucalyptus weevil, Gonipterus platensis (Coleoptera, Curculionidae), is a major pest of eucalyptus plantations worldwide. To date, no pheromones have been identified for this species, despite their valuable potential as tools in monitoring or control strategies. Here we report the detection and identification of pheromones candidates of G. platensis. The weevil's volatile compounds were collected by solid phase micro extraction (SPME) and monolithic material sorption extraction (MMSE). Using Gas Chromatography coupled to Mass Spectrometry (GC/MS) analysis, eleven insect specific compounds were detected and identified: verbenene, cis-verbenol, trans-verbenol, verbenone, 2-oxo-1,8-cineole, 9-hydroxy-1,8-cineole, 2-alpha-hydroxy-1,8-cineole, 3-oxo-1,8-cineole, 2-beta-hydroxy-1,8-cineole, 3-alpha-hydroxy-1,8-cineole and 7-hydroxy-1,8-cineole. Three of these compounds, verbenene, cis-verbenol and trans-verbenol, were shown to be male-specific. Antennal sensitivity towards ten compounds emitted by G. platensis was detected using Gas Chromatography-Mass Spectrometry/Electroantennographic Detection (GC-MS/EAD). Extracts from virgin males proved to be attractive to virgin females in olfactometer bioassays. Further behavioural bioassays showed that both virgin females and virgin males were attracted to the male-specific compound cis-verbenol and that virgin females were attracted to trans-verbenol. Verbenone was attractive to mated females. Regarding 2-alpha-hydroxy-1.8-cineole and 2-oxo-1,8-cineole, which are produced by both sexes, the alcohol was attractive to virgin males and both the alcohol and the ketone were repellant to mated females. This is, to our knowledge, the first identification of pheromones candidates in Gonipterus spp. and also the first evidence of cineole metabolites acting as semiochemicals.

Fernandes, {AR}, c}a-Martins IM{\c, Santos {MFA }, Raposo {LR }, Mendes R, Marques J, Romão {CC }, Romão {MJ}, Santos-Silva T, Baptista {PV}.  2020.  Improving the Anti-inflammatory Response via Gold Nanoparticle Vectorization of CO-Releasing Molecules, feb. ACS Biomaterials Science and Engineering. 6:1090–1101., Number 2: ACS - American Chemical Society Abstract

CO-releasing molecules (CORMs) have been widely studied for their anti-inflammatory, antiapoptotic, and antiproliferative effects. CORM-3 is a water-soluble Ru-based metal carbonyl complex, which metallates serum proteins and readily releases CO in biological media. In this work, we evaluated the anti-inflammatory and wound-healing effects of gold nanoparticles-CORM-3 conjugates, AuNPs@PEG@BSA·Ru(CO)x, exploring its use as an efficient CO carrier. Our results suggest that the nanoformulation was capable of inducing a more pronounced cell effect, at the anti-inflammatory level and a faster tissue repair, probably derived from a rapid cell uptake of the nanoformulation that results in the increase of CO inside the cell.

s}il{\u a}, MB{\c, a}b{\u a}caru AT{\u, s}sat VM{\c, Vasile {BS}tefan}{\c, Nea{\c s}u {IA}, Pinheiro T, Roma-Rodrigues C, Baptista {PV}, Fernandes {AR}, Matos {AP}, Marques {FM}.  2020.  Size-Dependent Biological Activities of Fluorescent Organosilane-Modified Zinc Oxide Nanoparticles, feb. Journal of biomedical nanotechnology. 16:137–152., Number 2: American Scientific Publishers Abstract

Surface modification of zinc oxide nanoparticles (ZnO NPs) is a strategy to tune their biocompatibility. Herein we report on the synthesis of a series of fluorescent ZnO NPs modified with 2-10% (3-glycidyloxypropyl)trimethoxysilane (GPTMS) to investigate the fluorescence properties and to explore their applications in microbiology and biomedicine. The obtained ZnO NPs were characterized by X-ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM) and Fourier transform infrared spectroscopy (FTIR). Size reduction occurred from ca. 13 nm in unmodified ZnO to 3-4 nm in silane-modified samples and fluorescence spectra showed size-dependent variation of the photoemission bands' intensity. The antibacterial and cytotoxic activities were investigated on Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria, and in ovarian (A2780) and prostate (PC3) cancer cells by tetrazolium/formazan-based methods. The antibacterial effect was higher for E. coli than S. aureus, while the cytotoxic activity was similar for both cancer cells and varied with the particle size. Cell death by apoptosis, and/or necrosis versus autophagy, were explored by flow cytometry using an Annexin V based-method and transmission electron microscopy (TEM). The main mechanism of ZnO NPs toxicity may involve the generation of reactive oxygen species (ROS) and the induction of apoptosis or autophagy. This work revealed the potential utility of GPTMS-modified ZnO NPs in the treatment of bacterial infection and cancer.

Kordestani, N, Rudbari HA, Fernandes AR, Raposo LR, Baptista PV, Ferreira D, Bruno G, Bella G, Scopelliti R, Braun JD, Herbert DE, Blacque O.  2020.  Antiproliferative Activities of Diimine-Based Mixed Ligand Copper(II) Complexes, 2020. ACS Comb Sci. 22(2):89-99. AbstractWebsite

A series of Cu(diimine)(X-sal)(NO3) complexes, where the diimine is either 2,2'-bipyridine (bpy) or 1,10-phenanthroline (phen) and X-sal is a monoanionic halogenated salicylaldehyde (X = Cl, Br, I, or H), have been synthesized and characterized by elemental analysis and X-ray crystallography. Penta-coordinate geometries copper(II) were observed for all cases. The influence of the diimine coligands and different halogen atoms on the antiproliferative activities toward human cancer cell lines have been investigated. All Cu(II) complexes were able to induce a loss of A2780 ovarian carcinoma cell viability, with phen derivatives more active than bpy derivatives. In contrast, no in vitro antiproliferative effects were observed against the HCT116 colorectal cancer cell line. These cytotoxicity differences were not due to a different intracellular concentration of the complexes determined by inductively coupled plasma atomic emission spectroscopy. A small effect of different halogen substituents on the phenolic ring was observed, with X = Cl being the most highly active toward A2780 cells among the phen derivatives, while X = Br presented the lowest IC50 in A2780 cells for bpy analogs. Importantly, no reduction in normal primary fibroblasts cell viability was observed in the presence of bpy derivatives (IC50 > 40 muM). Mechanistically, complex 1 seems to induce a stronger apoptotic response with a higher increase in mitochondrial membrane depolarization and an increased level of intracellular reactive oxygen species (ROS) compared to complex 3. Together, these data and the low IC50 compared to cisplatin in A2780 ovarian carcinoma cell line demonstrate the potential of these bpy derivatives for further in vivo studies.

Ferreira, P, Cerqueira NSMFA, Fernandes PA, Romão MJ, Ramos MJ.  2020.  Catalytic Mechanism of Human Aldehyde Oxidase, 2020. ACS CatalysisACS Catalysis. 10(16):9276-9286.: American Chemical Society AbstractWebsite

The mechanism of oxidation of N-heterocycle phthalazine to phthalazin-1(2H)-one and its associated free energy profile, catalyzed by human aldehyde oxidase (hAOX1), was studied in atomistic detail using QM/MM methodologies. The studied reaction was found to involve three sequential steps: (i) protonation of the substrate’s N2 atom by Lys893, (ii) nucleophilic attack of the hydroxyl group of the molybdenum cofactor (Moco) to the substrate, and (iii) hydride transfer from the substrate to the sulfur atom of the Moco. The free energy profile that was calculated revealed that the rate-limiting step corresponds to hydride transfer. It was also found that Lys893 plays a relevant role in the reaction, being important not only for the anchorage of the substrate close to the Moco, but also in the catalytic reaction. The variations of the oxidation state of the molybdenum ion throughout the catalytic cycle were examined too. We found out that during the displacement of the products away from the Moco, the transfer of electrons from the catalytic site to the FAD site was proton-coupled. As a consequence, the most favorable and fastest pathway for the enzyme to complete its catalytic cycle was that with MoV and a deprotonated SH ligand of the Moco with the FAD molecule converted to its semiquinone form, FADH•.The mechanism of oxidation of N-heterocycle phthalazine to phthalazin-1(2H)-one and its associated free energy profile, catalyzed by human aldehyde oxidase (hAOX1), was studied in atomistic detail using QM/MM methodologies. The studied reaction was found to involve three sequential steps: (i) protonation of the substrate’s N2 atom by Lys893, (ii) nucleophilic attack of the hydroxyl group of the molybdenum cofactor (Moco) to the substrate, and (iii) hydride transfer from the substrate to the sulfur atom of the Moco. The free energy profile that was calculated revealed that the rate-limiting step corresponds to hydride transfer. It was also found that Lys893 plays a relevant role in the reaction, being important not only for the anchorage of the substrate close to the Moco, but also in the catalytic reaction. The variations of the oxidation state of the molybdenum ion throughout the catalytic cycle were examined too. We found out that during the displacement of the products away from the Moco, the transfer of electrons from the catalytic site to the FAD site was proton-coupled. As a consequence, the most favorable and fastest pathway for the enzyme to complete its catalytic cycle was that with MoV and a deprotonated SH ligand of the Moco with the FAD molecule converted to its semiquinone form, FADH•.

Terao, M, Garattini E, Romão MJ, Leimkühler S.  2020.  Evolution, expression, and substrate specificities of aldehyde oxidase enzymes in eukaryotes, 2020. Journal of Biological ChemistryJournal of Biological Chemistry. 295(16):5377-5389.: Elsevier AbstractWebsite

Aldehyde oxidases (AOXs) are a small group of enzymes belonging to the larger family of molybdo-flavoenzymes, along with the well-characterized xanthine oxidoreductase. The two major types of reactions that are catalyzed by AOXs are the hydroxylation of heterocycles and the oxidation of aldehydes to their corresponding carboxylic acids. Different animal species have different complements of AOX genes. The two extremes are represented in humans and rodents; whereas the human genome contains a single active gene (AOX1), those of rodents, such as mice, are endowed with four genes (Aox1-4), clustering on the same chromosome, each encoding a functionally distinct AOX enzyme. It still remains enigmatic why some species have numerous AOX enzymes, whereas others harbor only one functional enzyme. At present, little is known about the physiological relevance of AOX enzymes in humans and their additional forms in other mammals. These enzymes are expressed in the liver and play an important role in the metabolisms of drugs and other xenobiotics. In this review, we discuss the expression, tissue-specific roles, and substrate specificities of the different mammalian AOX enzymes and highlight insights into their physiological roles.Aldehyde oxidases (AOXs) are a small group of enzymes belonging to the larger family of molybdo-flavoenzymes, along with the well-characterized xanthine oxidoreductase. The two major types of reactions that are catalyzed by AOXs are the hydroxylation of heterocycles and the oxidation of aldehydes to their corresponding carboxylic acids. Different animal species have different complements of AOX genes. The two extremes are represented in humans and rodents; whereas the human genome contains a single active gene (AOX1), those of rodents, such as mice, are endowed with four genes (Aox1-4), clustering on the same chromosome, each encoding a functionally distinct AOX enzyme. It still remains enigmatic why some species have numerous AOX enzymes, whereas others harbor only one functional enzyme. At present, little is known about the physiological relevance of AOX enzymes in humans and their additional forms in other mammals. These enzymes are expressed in the liver and play an important role in the metabolisms of drugs and other xenobiotics. In this review, we discuss the expression, tissue-specific roles, and substrate specificities of the different mammalian AOX enzymes and highlight insights into their physiological roles.

Roma-Rodrigues, C, Rivas-Garcia L, Baptista PV, Fernandes AR.  2020.  Gene Therapy in Cancer Treatment: Why Go Nano?, 2020 Pharmaceutics. 12(3) AbstractWebsite

The proposal of gene therapy to tackle cancer development has been instrumental for the development of novel approaches and strategies to fight this disease, but the efficacy of the proposed strategies has still fallen short of delivering the full potential of gene therapy in the clinic. Despite the plethora of gene modulation approaches, e.g., gene silencing, antisense therapy, RNA interference, gene and genome editing, finding a way to efficiently deliver these effectors to the desired cell and tissue has been a challenge. Nanomedicine has put forward several innovative platforms to overcome this obstacle. Most of these platforms rely on the application of nanoscale structures, with particular focus on nanoparticles. Herein, we review the current trends on the use of nanoparticles designed for cancer gene therapy, including inorganic, organic, or biological (e.g., exosomes) variants, in clinical development and their progress towards clinical applications.

Roma-Rodrigues, C, Pombo I, Fernandes AR, Baptista PV.  2020.  Hyperthermia Induced by Gold Nanoparticles and Visible Light Photothermy Combined with Chemotherapy to Tackle Doxorubicin Sensitive and Resistant Colorectal Tumor 3D Spheroids, 2020. Int J Mol Sci. 21(21) AbstractWebsite

Current cancer therapies are frequently ineffective and associated with severe side effects and with acquired cancer drug resistance. The development of effective therapies has been hampered by poor correlations between pre-clinical and clinical outcomes. Cancer cell-derived spheroids are three-dimensional (3D) structures that mimic layers of tumors in terms of oxygen and nutrient and drug resistance gradients. Gold nanoparticles (AuNP) are promising therapeutic agents which permit diminishing the emergence of secondary effects and increase therapeutic efficacy. In this work, 3D spheroids of Doxorubicin (Dox)-sensitive and -resistant colorectal carcinoma cell lines (HCT116 and HCT116-DoxR, respectively) were used to infer the potential of the combination of chemotherapy and Au-nanoparticle photothermy in the visible (green laser of 532 nm) to tackle drug resistance in cancer cells. Cell viability analysis of 3D tumor spheroids suggested that AuNPs induce cell death in the deeper layers of spheroids, further potentiated by laser irradiation. The penetration of Dox and earlier spheroid disaggregation is potentiated in combinatorial therapy with Dox, AuNP functionalized with polyethylene glycol (AuNP@PEG) and irradiation. The time point of Dox administration and irradiation showed to be important for spheroids destabilization. In HCT116-sensitive spheroids, pre-irradiation induced earlier disintegration of the 3D structure, while in HCT116 Dox-resistant spheroids, the loss of spheroid stability occurred almost instantly in post-irradiated spheroids, even with lower Dox concentrations. These results point towards the application of new strategies for cancer therapeutics, reducing side effects and resistance acquisition.

Fernandes, AR, Mendonça-Martins I, Santos MFA, Raposo LR, Mendes R, Marques J, Romão CC, Romão MJ, Santos-Silva T, Baptista PV.  2020.  Improving the Anti-inflammatory Response via Gold Nanoparticle Vectorization of CO-Releasing Molecules, 2020. ACS Biomaterials Science & EngineeringACS Biomaterials Science & Engineering. 6(2):1090-1101.: American Chemical Society AbstractWebsite

CO-releasing molecules (CORMs) have been widely studied for their anti-inflammatory, antiapoptotic, and antiproliferative effects. CORM-3 is a water-soluble Ru-based metal carbonyl complex, which metallates serum proteins and readily releases CO in biological media. In this work, we evaluated the anti-inflammatory and wound-healing effects of gold nanoparticles–CORM-3 conjugates, AuNPs@PEG@BSA·Ru(CO)x, exploring its use as an efficient CO carrier. Our results suggest that the nanoformulation was capable of inducing a more pronounced cell effect, at the anti-inflammatory level and a faster tissue repair, probably derived from a rapid cell uptake of the nanoformulation that results in the increase of CO inside the cell.CO-releasing molecules (CORMs) have been widely studied for their anti-inflammatory, antiapoptotic, and antiproliferative effects. CORM-3 is a water-soluble Ru-based metal carbonyl complex, which metallates serum proteins and readily releases CO in biological media. In this work, we evaluated the anti-inflammatory and wound-healing effects of gold nanoparticles–CORM-3 conjugates, AuNPs@PEG@BSA·Ru(CO)x, exploring its use as an efficient CO carrier. Our results suggest that the nanoformulation was capable of inducing a more pronounced cell effect, at the anti-inflammatory level and a faster tissue repair, probably derived from a rapid cell uptake of the nanoformulation that results in the increase of CO inside the cell.

Fernandes, AR, Mendonça-Martins I, Santos MFA, Raposo LR, Mendes R, Marques J, Romão CC, Romão MJ, Santos-Silva T, Baptista PV.  2020.  Improving the Anti-inflammatory Response via Gold Nanoparticle Vectorization of CO-Releasing Molecules, 2020. ACS Biomaterials Science & Engineering. 6(2):1090-1101. AbstractWebsite
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Choroba, K, Raposo LR, Palion-Gazda J, Malicka E, Erfurt K, Machura B, Fernandes AR.  2020.  In vitro antiproliferative effect of vanadium complexes bearing 8-hydroxyquinoline-based ligands - the substituent effect, 2020. Dalton Trans. 49(20):6596-6606. AbstractWebsite

This is the first comprehensive study demonstrating the antiproliferative effect of vanadium complexes bearing 8-hydroxyquinoline (quinH) ligands, including the parent and -CH3 (Me), -NO2, -Cl and -I substituted ligands, on HCT116 and A2780 cancer cell lines. To determine the structure-cytotoxicity relationships seven six-coordinate oxovanadium(v) complexes [VO(OMe)(5,7-(Me)2-quin)2] (1), [VO(OMe)(5,7-Cl2-quin)2] (2), [VO(OMe)(5,7-Cl,I-quin)2] (3), [VO(OMe)(5,7-I2-quin)2] (4), [VO(OMe)(5-NO2-quin)2] (5), [VO(OMe)(5-Cl-quin)2] (6), and [VO(OMe)(quin)2] (7) were investigated. The cytotoxicity of 8-hydroxyquinoline oxovanadium(v) complexes is higher in the A2780 cell line (lower IC50) than that observed for the widely used chemotherapeutic agent, cisplatin, while displaying low cytotoxicity for normal human primary fibroblasts. Substituents introduced into the 8-hydroxyquinoline backbone reduced the antiproliferative effect of the vanadium complexes, and the complexes with the ligand substituted only in the 5 position (5 and 6) were more cytotoxic than those with substituents in the 5,7 positions of the quin backbone (1-4). Depending on the substituent type, the cytotoxicity of 1-4 followed the trend: -Cl > -CH3 > -I. Incubation of A2780 cancer cells with IC50 concentrations of complexes 5, 6 and 7 promoted cellular detachment, possibly through membrane destabilization, and triggered apoptosis and necrosis. ROS production might be responsible for the cell death mechanism observed particularly in the A2780 cells exposed to complexes 5 and 6.

Beola, L, Asin L, Roma-Rodrigues C, Fernandez-Afonso Y, Fratila RM, Serantes D, Ruta S, Chantrell RW, Fernandes AR, Baptista PV, de la Fuente JM, Grazu V, Gutierrez L.  2020.  The Intracellular Number of Magnetic Nanoparticles Modulates the Apoptotic Death Pathway after Magnetic Hyperthermia Treatment, 2020. ACS Appl Mater Interfaces. 12(39):43474-43487. AbstractWebsite

Magnetic hyperthermia is a cancer treatment based on the exposure of magnetic nanoparticles to an alternating magnetic field in order to generate local heat. In this work, 3D cell culture models were prepared to observe the effect that a different number of internalized particles had on the mechanisms of cell death triggered upon the magnetic hyperthermia treatment. Macrophages were selected by their high capacity to uptake nanoparticles. Intracellular nanoparticle concentrations up to 7.5 pg Fe/cell were measured both by elemental analysis and magnetic characterization techniques. Cell viability after the magnetic hyperthermia treatment was decreased to <25% for intracellular iron contents above 1 pg per cell. Theoretical calculations of the intracellular thermal effects that occurred during the alternating magnetic field application indicated a very low increase in the global cell temperature. Different apoptotic routes were triggered depending on the number of internalized particles. At low intracellular magnetic nanoparticle amounts (below 1 pg Fe/cell), the intrinsic route was the main mechanism to induce apoptosis, as observed by the high Bax/Bcl-2 mRNA ratio and low caspase-8 activity. In contrast, at higher concentrations of internalized magnetic nanoparticles (1-7.5 pg Fe/cell), the extrinsic route was observed through the increased activity of caspase-8. Nevertheless, both mechanisms may coexist at intermediate iron concentrations. Knowledge on the different mechanisms of cell death triggered after the magnetic hyperthermia treatment is fundamental to understand the biological events activated by this procedure and their role in its effectiveness.

Vale, TM, Leitão J, Preguiça N, Rodrigues R, Dias RJ, Lourenço JM.  2020.  Lazy State Determination: More Concurrency for Contending Linearizable Transactions, 2020. , Lisboa: FCT-NOVAvl20_-_lsd.pdf
dos Santos, R, Iria I, Manuel AM, Leandro AP, Madeira CAC, Goncalves J, Carvalho AL, Roque AC.  2020.  Magnetic Precipitation: A New Platform for Protein Purification, 2020. Biotechnology JournalBiotechnology Journal. n/a(n/a):2000151.: John Wiley & Sons, Ltd AbstractWebsite

One of the trends in downstream processing comprises the use of ?anything-but-chromatography? methods to overcome the current downfalls of standard packed-bed chromatography. Precipitation and magnetic separation are two techniques already proven to accomplish protein purification from complex media, yet never used in synergy. With the aim to capture antibodies directly from crude extracts, a new approach combining precipitation and magnetic separation was developed and named as affinity magnetic precipitation. A precipitation screening, based on the Hofmeister series, and a commercial precipitation kit were tested with affinity magnetic particles to assess the best condition for antibody capture from human serum plasma and clarified cell supernatant. The best conditions were obtained when using PEG3350 as precipitant at 4°C for 1h, reaching 80% purity and 50% recovery of polyclonal antibodies from plasma, and 99% purity with 97% recovery yield of anti-TNFα mAb from cell supernatants. These results show that the synergetic use of precipitation and magnetic separation can represent an alternative for the efficient capture of antibodies. This article is protected by copyright. All rights reserved

Leisico, F, Godinho LM, Gonçalves IC, Silva SP, Carneiro B, Romão MJ, Santos-Silva T, de Sá-Nogueira I.  2020.  Multitask ATPases (NBDs) of bacterial ABC importers type I and their interspecies exchangeability, 2020. 10(1):19564. AbstractWebsite

ATP-binding cassette (ABC) type I importers are widespread in bacteria and play a crucial role in its survival and pathogenesis. They share the same modular architecture comprising two intracellular nucleotide-binding domains (NBDs), two transmembrane domains (TMDs) and a substrate-binding protein. The NBDs bind and hydrolyze ATP, thereby generating conformational changes that are coupled to the TMDs and lead to substrate translocation. A group of multitask NBDs that are able to serve as the cellular motor for multiple sugar importers was recently discovered. To understand why some ABC importers share energy-coupling components, we used the MsmX ATPase from Bacillus subtilis as a model for biological and structural studies. Here we report the first examples of functional hybrid interspecies ABC type I importers in which the NBDs could be exchanged. Furthermore, the first crystal structure of an assigned multitask NBD provides a framework to understand the molecular basis of the broader specificity of interaction with the TMDs.

Restani, RB, Pires RF, Baptista PV, Fernandes AR, Casimiro T, Bonifácio VDB, Aguiar-Ricardo A.  2020.  Nano-in-Micro Sildenafil Dry Powder Formulations for the Treatment of Pulmonary Arterial Hypertension Disorders: The Synergic Effect of POxylated Polyurea Dendrimers, PLGA, and Cholesterol, 2020. 37(6):1900447. AbstractWebsite

Abstract POXylated polyurea dendrimer nanoparticles (PUREG4OOx48) are loaded with sildenafil (SDF) by a supercritical carbon dioxide–assisted (scCO2) impregnation. Further supercritical CO2-assisted spray drying (SASD) leads to hybrid nano-in-micro dry powder formulations that are investigated aiming at efficient pulmonary delivery of SDF in pulmonary arterial hypertension treatment. This is the first report of the production of poly(D,L-lactide-co-glycolide)-cholesterol (PLGA-Chol) microparticles processed by SASD. The optimized formulation of nano-in-microparticles is composed of PLGA, Chol, and PUREG4OOx48, loaded with SDF solutions in a 77:23 ratio (PLGA-Chol:dendrimer, w/w). The dry powders are fully characterized and found to be highly biodegradable and biocompatible, and the SDF release profile evaluates under different pH values. The median mass average diameter (MMAD) of the nano-in-micro systems varies between 2.57 and 5 µm and the fine particle fraction (FPF) between 36% and 29% for PUREG4OMeOx48[PLGA-Chol] and PUREG4OEtOx48[PLGA-Chol], respectively. The data validate the potential use of these new formulations in inhalation therapy. In vitro studies are also carried out in order to evaluate the effect of the free drug in cell viability and formulations cytotoxicity.

Busila, M, Tabacaru A, Mussat V, Vasile BS, Neasu IA, Pinheiro T, Roma-Rodrigues C, Baptista PV, Fernandes AR, Matos AP, Marques F.  2020.  Size-Dependent Biological Activities of Fluorescent Organosilane-Modified Zinc Oxide Nanoparticles, 2020. J Biomed Nanotechnol. 16(2):137-152. AbstractWebsite

Surface modification of zinc oxide nanoparticles (ZnO NPs) is a strategy to tune their biocompatibility. Herein we report on the synthesis of a series of fluorescent ZnO NPs modified with 2-10% (3-glycidyloxypropyl)trimethoxysilane (GPTMS) to investigate the fluorescence properties and to explore their applications in microbiology and biomedicine. The obtained ZnO NPs were characterized by X-ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM) and Fourier transform infrared spectroscopy (FTIR). Size reduction occurred from ca. 13 nm in unmodified ZnO to 3-4 nm in silane-modified samples and fluorescence spectra showed size-dependent variation of the photoemission bands' intensity. The antibacterial and cytotoxic activities were investigated on Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria, and in ovarian (A2780) and prostate (PC3) cancer cells by tetrazolium/formazan-based methods. The antibacterial effect was higher for E. coli than S. aureus, while the cytotoxic activity was similar for both cancer cells and varied with the particle size. Cell death by apoptosis, and/or necrosis versus autophagy, were explored by flow cytometry using an Annexin V based-method and transmission electron microscopy (TEM). The main mechanism of ZnO NPs toxicity may involve the generation of reactive oxygen species (ROS) and the induction of apoptosis or autophagy. This work revealed the potential utility of GPTMS-modified ZnO NPs in the treatment of bacterial infection and cancer.

Gomes, AS, Ramos H, Gomes S, Loureiro JB, Soares J, Barcherini V, Monti P, Fronza G, Oliveira C, Domingues L, Bastos M, Dourado DFAR, Carvalho AL, Romão MJ, Pinheiro B, Marcelo F, Carvalho A, Santos MMM, Saraiva L.  2020.  SLMP53-1 interacts with wild-type and mutant p53 DNA-binding domain and reactivates multiple hotspot mutations, 2020. 1864(1):129440. AbstractWebsite

BackgroundHalf of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1.
Methods and results
By cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced.
Conclusions
SLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53.
General Significance
This work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status.

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