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2021
Polino, M, Rho HS, Pina MP, Mallada R, Carvalho AL, Romão MJ, Coelhoso I, Gardeniers JGE, Crespo JG, Portugal CAM.  2021.  Protein Crystallization in a Microfluidic Contactor with Nafion®117 Membranes. Membranes. 11, Number 8 AbstractWebsite

Protein crystallization still remains mostly an empirical science, as the production of crystals with the required quality for X-ray analysis is dependent on the intensive screening of the best protein crystallization and crystal’s derivatization conditions. Herein, this demanding step was addressed by the development of a high-throughput and low-budget microfluidic platform consisting of an ion exchange membrane (117 Nafion® membrane) sandwiched between a channel layer (stripping phase compartment) and a wells layer (feed phase compartment) forming 75 independent micro-contactors. This microfluidic device allows for a simultaneous and independent screening of multiple protein crystallization and crystal derivatization conditions, using Hen Egg White Lysozyme (HEWL) as the model protein and Hg2+ as the derivatizing agent. This microdevice offers well-regulated crystallization and subsequent crystal derivatization processes based on the controlled transport of water and ions provided by the 117 Nafion® membrane. Diffusion coefficients of water and the derivatizing agent (Hg2+) were evaluated, showing the positive influence of the protein drop volume on the number of crystals and crystal size. This microfluidic system allowed for crystals with good structural stability and high X-ray diffraction quality and, thus, it is regarded as an efficient tool that may contribute to the enhancement of the proteins’ crystals structural resolution.

Fernandes, TM, Morgado L, Turner DL, Salgueiro CA.  2021.  Protein Engineering of Electron Transfer Components from Electroactive Geobacter Bacteria. Antioxidants. 10, Number 6 AbstractWebsite

Electrogenic microorganisms possess unique redox biological features, being capable of transferring electrons to the cell exterior and converting highly toxic compounds into nonhazardous forms. These microorganisms have led to the development of Microbial Electrochemical Technologies (METs), which include applications in the fields of bioremediation and bioenergy production. The optimization of these technologies involves efforts from several different disciplines, ranging from microbiology to materials science. Geobacter bacteria have served as a model for understanding the mechanisms underlying the phenomenon of extracellular electron transfer, which is highly dependent on a multitude of multiheme cytochromes (MCs). MCs are, therefore, logical targets for rational protein engineering to improve the extracellular electron transfer rates of these bacteria. However, the presence of several heme groups complicates the detailed redox characterization of MCs. In this Review, the main characteristics of electroactive Geobacter bacteria, their potential to develop microbial electrochemical technologies and the main features of MCs are initially highlighted. This is followed by a detailed description of the current methodologies that assist the characterization of the functional redox networks in MCs. Finally, it is discussed how this information can be explored to design optimal Geobacter-mutated strains with improved capabilities in METs.

Fernandes, TM, Folgosa F, Teixeira M, Salgueiro CA, Morgado L.  2021.  Structural and functional insights of GSU0105, a unique multiheme cytochrome from G. sulfurreducens. Biophysical Journal. AbstractWebsite

Geobacter sulfurreducens possesses over 100 cytochromes that assure an effective electron transfer to the cell exterior. The most abundant group of cytochromes in this microorganism is the PpcA family, composed of five periplasmic triheme cytochromes with high structural homology and identical heme coordination (His-His). GSU0105 is a periplasmic triheme cytochrome synthetized by G. sulfurreducens in Fe(III)-reducing conditions but is not present in cultures grown on fumarate. This cytochrome has a low sequence identity with the PpcA family cytochromes and a different heme coordination, based on the analysis of its amino acid sequence. In this work, amino acid sequence analysis, site-directed mutagenesis, and complementary biophysical techniques, including ultraviolet-visible, circular dichroism, electron paramagnetic resonance, and nuclear magnetic resonance spectroscopies, were used to characterize GSU0105. The cytochrome has a low percentage of secondary structural elements, with features of α-helices and β-sheets. Nuclear magnetic resonance shows that the protein contains three low-spin hemes (Fe(II), S = 0) in the reduced state. Electron paramagnetic resonance shows that, in the oxidized state, one of the hemes becomes high-spin (Fe(III), S = 5/2), whereas the two others remain low-spin (Fe(III), S = 1/2). The data obtained also indicate that the heme groups have distinct axial coordination. The apparent midpoint reduction potential of GSU0105 (−154 mV) is pH independent in the physiological range. However, the pH modulates the reduction potential of the heme that undergoes the low- to high-spin interconversion. The reduction potential values of cytochrome GSU0105 are more distinct compared to those of the PpcA family members, providing the protein with a larger functional working redox potential range. Overall, the results obtained, together with an amino acid sequence analysis of different multiheme cytochrome families, indicate that GSU0105 is a member of a new group of triheme cytochromes.

2020
Mirante, F, Alves AC, Juliao D, Almeida PL, Gago S, Valenca R, Ribeiro JC, de Castro B, Granadeiro CM, Balula SS.  2020.  Large-pore silica spheres as support for samarium-coordinated undecamolybdophosphate: Oxidative desulfurization of diesels, {JAN 1}. Fuel. 259:116213. AbstractWebsite

A novel composite has been prepared through the immobilization of the Keggin sandwich-type {[}Sm (PMo11O39)(2)](11-) anion (SmPOM) on large-pore silica spheres previously functionalized with trimethylammonium groups (TMA). The resulting SmPOM@TMA-LPMS material has been evaluated as heterogeneous catalyst in a biphasic desulfurization 1:1 diesel/extraction solvent system using H2O2 as oxidant. Preliminary experiments were conducted with different extraction solvents, acetonitrile and {[}BMIM]PF6 ionic liquid. The optimized extractive and catalytic oxidative desulfurization system (ECODS) with {[}BMIM]PF6 was able to reach complete sulfur removal from a model diesel containing 2100 ppm S in just 60 min (10 min of initial extraction + 50 min of catalytic step). The reutilization of catalyst and extraction phase has been successfully performed without loss of desulfurization efficiency in consecutive cycles, turning the process more sustainable and cog-effective. The remarkable results with simulated diesel have motivated the application of the catalyst in the desulfurization of untreated real diesel and 74% of efficiency was achieved after only 2 h for three consecutive cycles.

Martins, CF, Neves LA, Chagas R, Ferreira LM, Afonso CAM, Crespo JG, Coelhoso IM.  2020.  CO2 removal from anaesthesia circuits using gas-ionic liquid membrane contactors, NOV 1. SEPARATION AND PURIFICATION TECHNOLOGY. 250 Abstract
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Oliveira, B, Veigas B, Fernandes {AR}, Águas H, Martins R, Fortunato E, Baptista {PV}.  2020.  Fast prototyping microfluidics: Integrating droplet digital lamp for absolute quantification of cancer biomarkers, mar. Sensors. 20, Number 6: MDPI - Multidisciplinary Digital Publishing Institute Abstract

Microfluidic (MF) advancements have been leveraged toward the development of state-of-the-art platforms for molecular diagnostics, where isothermal amplification schemes allow for further simplification of DNA detection and quantification protocols. The MF integration with loop-mediated isothermal amplification (LAMP) is today the focus of a new generation of chip-based devices for molecular detection, aiming at fast and automated nucleic acid analysis. Here, we combined MF with droplet digital LAMP (ddLAMP) on an all-in-one device that allows for droplet generation, target amplification, and absolute quantification. This multilayer 3D chip was developed in less than 30 minutes by using a low-cost and extremely adaptable production process that exploits direct laser writing technology in “Shrinky-dinks” polystyrene sheets. ddLAMP and target quantification were performed directly on-chip, showing a high correlation between target concentration and positive droplet score. We validated this integrated chip via the amplification of targets ranging from five to 500,000 copies/reaction. Furthermore, on-chip amplification was performed in a 10 µL volume, attaining a limit of detection of five copies/µL under 60 min. This technology was applied to quantify a cancer biomarker, c-MYC, but it can be further extended to any other disease biomarker.

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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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.

Ferreira, D, Fontinha D, Martins C, Pires D, Fernandes {AR}, Baptista {PV}.  2020.  Gold nanoparticles for vectorization of nucleic acids for cancer therapeutics, aug. Molecules. 25, Number 15: MDPI - Multidisciplinary Digital Publishing Institute Abstract

Cancer remains a complex medical challenge and one of the leading causes of death worldwide. Nanomedicines have been proposed as innovative platforms to tackle these complex diseases, where the combination of several treatment strategies might enhance therapy success. Among these nanomedicines, nanoparticle mediated delivery of nucleic acids has been put forward as key instrument to modulate gene expression, be it targeted gene silencing, interference RNA mechanisms and/or gene edition. These novel delivery systems have strongly relied on nanoparticles and, in particular, gold nanoparticles (AuNPs) have paved the way for efficient delivery systems due to the possibility to fine-tune their size, shape and surface properties, coupled to the ease of functionalization with different biomolecules. Herein, we shall address the different molecular tools for modulation of expression of oncogenes and tumor suppressor genes and discuss the state-of-the-art of AuNP functionalization for nucleic acid delivery both in vitro and in vivo models. Furthermore, we shall highlight the clinical applications of these spherical AuNP based conjugates for gene delivery, current challenges, and future perspectives in nanomedicine.

Maximo, P, Ferreira LM, Branco PS, Lourenco A.  2020.  Invasive Plants: Turning Enemies into Value, AUG. MOLECULES. 25, Number 15 Abstract

In this review, a brief description of the invasive phenomena associated with plants and its consequences to the ecosystem is presented. Five worldwide invasive plants that are a threat to Portugal were selected as an example, and a brief description of each is presented. A full description of their secondary metabolites and biological activity is given, and a resume of the biological activity of extracts is also included. The chemical and pharmaceutical potential of invasive species sensu lato is thus acknowledged. With this paper, we hope to demonstrate that invasive species have potential positive attributes even though at the same time they might need to be controlled or eradicated. Positive attributes include chemical and pharmaceutical properties and developing these could help mitigate the costs of management and eradication.

Mendes, MJ.  2020.  Colloidal lithography for transparent electronics and light trapping in thin film flexible solar cells, 3-4 Nov.. Encontro Ciência 2020. , Lisbon, Portugal: Fundação para a Ciência e a Tecnologia
Oliveira, B, Veigas B, Fernandes AR, Aguas H, Martins R, Fortunato E, Baptista PV.  2020.  Fast Prototyping Microfluidics: Integrating Droplet Digital Lamp for Absolute Quantification of Cancer Biomarkers, 2020. Sensors (Basel). 20(6) AbstractWebsite

Microfluidic (MF) advancements have been leveraged toward the development of state-of-the-art platforms for molecular diagnostics, where isothermal amplification schemes allow for further simplification of DNA detection and quantification protocols. The MF integration with loop-mediated isothermal amplification (LAMP) is today the focus of a new generation of chip-based devices for molecular detection, aiming at fast and automated nucleic acid analysis. Here, we combined MF with droplet digital LAMP (ddLAMP) on an all-in-one device that allows for droplet generation, target amplification, and absolute quantification. This multilayer 3D chip was developed in less than 30 minutes by using a low-cost and extremely adaptable production process that exploits direct laser writing technology in "Shrinky-dinks" polystyrene sheets. ddLAMP and target quantification were performed directly on-chip, showing a high correlation between target concentration and positive droplet score. We validated this integrated chip via the amplification of targets ranging from five to 500,000 copies/reaction. Furthermore, on-chip amplification was performed in a 10 microL volume, attaining a limit of detection of five copies/microL under 60 min. This technology was applied to quantify a cancer biomarker, c-MYC, but it can be further extended to any other disease biomarker.

Germano, GCM, Machado YDR, Martinho L, Fernandes SN, Costa AMLM, Pecoraro E, Gomes ASL, Carvalho ICS.  2020.  Flexible random lasers in dye-doped bio-degradable cellulose nanocrystalline needles, 2020. Journal of the Optical Society of America BJournal of the Optical Society of America B. 37(1):24-29.: OSA AbstractWebsite

In this work, we developed and investigated a random laser based on rhodamine6G (Rh6G) in ethylene glycol (EG) solution with varying cellulose nanocrystalline (CNC) needles as scatterers in the lasing media. Besides the suspension-in-cuvette scheme, an alternative configuration was also employed: a dye-CNC flexible self-supported thick-film (70 µm) random laser made by drop casting of the ${\rm CNCs}+{\rm Rh6G}+{\rm hydroxypropyl}$CNCs+Rh6G+hydroxypropyl cellulose suspension. In relation to conventional scatterers, the biodegradable cellulose nanocompounds showed a comparable reduction in both the spectral full width at half-maximum and the energy threshold values, with an optimal concentration of 5 mg [CNC]/ml[EG] in suspension. Its performance was also compared with other cellulose-based random lasers, presenting advantages for some parameters. The flexible film configuration showed similar results, but contained 10% less Rh6G than the suspension.

Vidossich, P, Castañeda Moreno LE, Mota C, de Sanctis D, Miscione GP, De Vivo M.  2020.  Functional Implications of Second-Shell Basic Residues for dUTPase DR2231 Enzymatic Specificity, 2020. ACS CatalysisACS Catalysis. 10(23):13825-13833.: American Chemical Society AbstractWebsite

Nucleotide-processing enzymes are key players in biological processes. They often operate through high substrate specificity for catalysis. How such specificity is achieved is unclear. Here, we dealt with this question by investigating all-α dimeric deoxyuridine triphosphate nucleotidohydrolases (dUTPases). Typically, these dUTPases hydrolyze either dUTP or deoxyuridine diphosphate (dUDP) substrates. However, the dUTPase enzyme DR2231 from Deinococcus radiodurans selectively hydrolyzes dUTP only, and not dUDP. By means of extended classical molecular dynamics simulations and quantum chemical calculations, we show that DR2231 achieves this specificity for dUTP via second-shell basic residues that, together with the two catalytic magnesium ions, contribute to properly orienting the γ-phosphate of dUTP in a prereactive state. This allows a nucleophilic water to be correctly placed and activated in order to perform substrate hydrolysis. We show that this enzymatic mechanism is not viable when dUDP is bound to DR2231. Importantly, in several other dUTPases capable of hydrolyzing either dUTP or dUDP, we detected that active site second-shell basic residues are more in number, anchoring the β-phosphate of the nucleotide substrate too, in contrast to what is observed in DR2231. Thus, strategically located basic second-shell residues mediate precise reactant positioning at the catalytic site, determining substrate specificity in dUTPases and possibly in other structurally similar nucleotide-processing metalloenzymes.Nucleotide-processing enzymes are key players in biological processes. They often operate through high substrate specificity for catalysis. How such specificity is achieved is unclear. Here, we dealt with this question by investigating all-α dimeric deoxyuridine triphosphate nucleotidohydrolases (dUTPases). Typically, these dUTPases hydrolyze either dUTP or deoxyuridine diphosphate (dUDP) substrates. However, the dUTPase enzyme DR2231 from Deinococcus radiodurans selectively hydrolyzes dUTP only, and not dUDP. By means of extended classical molecular dynamics simulations and quantum chemical calculations, we show that DR2231 achieves this specificity for dUTP via second-shell basic residues that, together with the two catalytic magnesium ions, contribute to properly orienting the γ-phosphate of dUTP in a prereactive state. This allows a nucleophilic water to be correctly placed and activated in order to perform substrate hydrolysis. We show that this enzymatic mechanism is not viable when dUDP is bound to DR2231. Importantly, in several other dUTPases capable of hydrolyzing either dUTP or dUDP, we detected that active site second-shell basic residues are more in number, anchoring the β-phosphate of the nucleotide substrate too, in contrast to what is observed in DR2231. Thus, strategically located basic second-shell residues mediate precise reactant positioning at the catalytic site, determining substrate specificity in dUTPases and possibly in other structurally similar nucleotide-processing metalloenzymes.

Ferreira, D, Fontinha D, Martins C, Pires D, Fernandes AR, Baptista PV.  2020.  Gold Nanoparticles for Vectorization of Nucleic Acids for Cancer Therapeutics, 2020. Molecules. 25(15) AbstractWebsite

Cancer remains a complex medical challenge and one of the leading causes of death worldwide. Nanomedicines have been proposed as innovative platforms to tackle these complex diseases, where the combination of several treatment strategies might enhance therapy success. Among these nanomedicines, nanoparticle mediated delivery of nucleic acids has been put forward as key instrument to modulate gene expression, be it targeted gene silencing, interference RNA mechanisms and/or gene edition. These novel delivery systems have strongly relied on nanoparticles and, in particular, gold nanoparticles (AuNPs) have paved the way for efficient delivery systems due to the possibility to fine-tune their size, shape and surface properties, coupled to the ease of functionalization with different biomolecules. Herein, we shall address the different molecular tools for modulation of expression of oncogenes and tumor suppressor genes and discuss the state-of-the-art of AuNP functionalization for nucleic acid delivery both in vitro and in vivo models. Furthermore, we shall highlight the clinical applications of these spherical AuNP based conjugates for gene delivery, current challenges, and future perspectives in nanomedicine.

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.

Grey, P, Fernandes SN, Gaspar D, Deuermeier J, Martins R, Fortunato E, Godinho MH, Pereira L.  2020.  Ionically Modified Cellulose Nanocrystal Self-Assembled Films with a Mesoporous Twisted Superstructure: Polarizability and Application in Ion-Gated Transistors, 2020. ACS Applied Electronic MaterialsACS Applied Electronic Materials. 2(2):426-436.: American Chemical Society AbstractWebsite
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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

Machado, JF, Sequeira D, Marques F, Piedade MFM, Villa de Brito MJ, Helena Garcia M, Fernandes AR, Morais TS.  2020.  New copper(I) complexes selective for prostate cancer cells, 2020. Dalton Trans. 49(35):12273-12286. AbstractWebsite

A new family of eighteen Cu(i) complexes of the general formula [Cu(PP)(LL)][BF4], where PP is a phosphane ligand and LL represents an N,O-heteroaromatic bidentate ligand, has been synthesized and fully characterized by classical analytical and spectroscopic methods. Five complexes of this series were also characterized by single crystal X-ray diffraction studies. The cytotoxicity of all compounds was evaluated in breast (MCF7) and prostate (LNCap) human cancer cells and in a normal prostate cell line (RWPE). In general, all compounds showed higher cytotoxicity for the prostate cancer cells than for the breast cells, with IC50 values in the range 0.2-2 muM after 24 h of treatment. The most cytotoxic compound, [Cu(dppe)(2-ap)][BF4] (16), where dppe = 1,2-bis(diphenylphosphano) ethane and 2-ap = 2-acetylpyridine, showed a high level of cellular internalization, generation of intracellular ROS and activation of the cell death mechanism via apoptosis/necrosis. Owing to its high cytotoxic activity for LNCap cells, being 70-fold higher than that for normal prostate cells (RWPE), complex (16) was found to be the most promising for further research in prostate cancer models.

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