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- Proactive response to tackle the threat of emerging drugs: Synthesis and toxicity evaluation of new cathinonesPublication . Gaspar, Helena; Bronze, Soraia; Oliveira, Catarina; Victor, Bruno; Machuqueiro, Miguel; Pacheco, Rita; Caldeira, Maria João; Santos, SusanaThe emergence of potentially dangerous new psychoactive substances (NPS) imposes enormous challenges on forensic laboratories regarding their rapid and unambiguous identification. Access to comprehensive databases is essential for a quick characterization of these substances, allowing them to be categorized according to national and international legislations. In this work, it is reported the synthesis and structural characterization by NMR and MS of a library encompassing 21 cathinones, 4 of which are not yet reported in the literature, but with structural characteristics that make them a target for clandestine laboratories. This in-house library will be an important tool accessible to forensic laboratories, for the quick identification of seized NPS. The in vitro cytotoxicity of all cathinones was assessed in HepG2 cells, to have a preliminary but effective indication of their human hepatotoxicity potential. The two new cathinones DMB (8) and DMP (9) were the more cytotoxic, followed by the already seized mephedrone (2), 3,4-DMMC (3), 4-MDMC (7), NEB (12) with EC50 values ranging from 0.81 mM for (3) to 1.28 mM for (2). Results suggest an increase of cytotoxicity with the increase of the chain length of the acyl moiety and with the substitution (with one or two methyl groups) in the aromatic ring. The nature of the amine moiety seems to play only a minor role in the cytotoxic effect. Molecular dynamics simulations were performed to evaluate the molecular details related with the observed cytotoxicities. Although these studies indicated that cathinones are able to cross lipid bilayers with relative ease, when in their neutral forms, it was observed only a partial correlation between lipophilicity and cytotoxicity, indicating that membrane trafficking may not be the only key factor influencing the bioactivity of these compounds. This work is a valuable contribution to the forensic science field since a quick identification of novel cathinones is urgent to match their rapid increase in the market.
- Study of antioxidant and antitumoral activity of cork processing wastewaters components fractionated by ultrafiltration membranesPublication . Gomes, Luís; Borges, C.; Serralheiro, M.L.M.; Pacheco, Rita; Minhalma, MiguelCork processing wastewater present a severe environmental problem due to its high content in organic matter, such as sugars and non-biodegradable compounds like polyphenols (PPs), namely tannins. In this work, the assessment of the biological activity and identification of valuable polyphenolic compounds was envisaged. These compounds were obtained using membrane technology, leading to the valorisation of the wastewater components and simultaneously leading to the decrease of its pollutant content.
- BioMol4Health_Biological chemistry: longevity in a cup of teaPublication . RESSAISSI, Asma; Fale, Pedro; Pacheco, Rita; Serralheiro, Maria LuisaInfusions have been studied on what concerns Alzheimer Disease, digestive process, diet cholesterol absorption and its biosynthesis inhibition. In the first two cases the inhibition of acetylcholinesterase (AChE) has been addressed. In the last two situations, an in vitro intestinal barrier has been simulated and the inhibition of the regulator enzyme (HMGR) in cholesterol biosysnthesis pathway has been studied. AChE has been the target of infusions inhibitory activity as its inhibition has been seen to improve cognition and global functioning1 in AD suffering people and to improve the gastrointestinal motility2. Given to lab animals the compounds presente in the infusions were able to reach the brain and inhibit the enzyme3. The effect of infusions on cholesterol bioavailability pointed out that some infusions were able to reduce cholesterol permeation4 and also to have some inhibitory activity5. Studies have indicated that phenolics are able to modify the cell proteome6. The infusions have also been shown to modify the amount of cholesterol transporter proteins in cell membrane and this maybe one of possible explanations for the reduction in cholesterol transport detected under the effect of infusions, on some people ando n simulated intestinal barrier.
- Evaluation of the amount of nanoparticles emitted in welding fume from stainless steel using different shielding gasesPublication . Pacheco, Rita; Gomes, João; Miranda, R.; Quintino, M. L.The primary objective of this study was to correlate the emission of macro and nanoparticles released during the process of metal inert gas/metal active gas (MIG/MAG) of stainless steel with different gas mixtures. Using different gas mixtures with different heat inputs, it was possible to determine fume formation rates and surface areas of nanoparticles with alveolar lung deposition capacity. It was found, how the various transfer modes and the type of gas protection, in particular, the percentage of active elements in the in the chemical composition of the gas, affect the amount of fumes generated and also the generation of nanoparticles with a high capacity of deposition. The spray transfer mode always shows higher values of nanoparticles surface area, unlike the fume formation rates. Among the tested mixtures 82%Ar+18%CO2 generates higher emissions of nanoparticles as well as fume formation rates.
- Investigation of structural effects and behaviour of pseudomonas aeruginosa amidase encapsulated in reserved micellesPublication . Fragosa, Ana; Pacheco, Rita; Karmali, AminThe acetohydroxamic acid synthesis reaction was studied using whole cells, cell-free extract and purified amidase from the strains of Pseudomonas aeruginosa L10 and A13 entrapped in a reverse micelles system composed of cationic surfactant tetradecyltrimethyl ammonium bromide. The specific activity of amidase, yield of synthesis and storage stability were determined for the reversed micellar system as well as for free amidase in conventional buffer medium. The results have revealed that amidase solutions in the reverse micelles system exhibited a substantial increase in specific activity, yield of synthesis and storage stability. In fact, whole cells from P. aeruginosa L10 and AI3 in reverse micellar medium revealed an increase in specific activity of 9.3- and 13.9-fold, respectively, relatively to the buffer medium. Yields of approximately 92% and 66% of acetohydroxamic acid synthesis were obtained for encapsulated cell free extract from P. aeruginosa L10 and A13, respectively. On the other hand, the half-life values obtained for the amidase solutions encapsulated in reverse micelles were overall higher than that obtained for the free amidase solution in buffer medium. Half-life values obtained for encapsulated purified amidase from P. aeruginosa strain L10 and encapsulated cell-free extract from P. aeruginosa strain AI3 were of 17.0 and 26.0 days, respectively. As far as the different sources biocatalyst are concerned, the data presented in this work has revealed that the best results, in both storage stability and biocatalytic efficiency, were obtained when encapsulated cell-free extract from P. aeruginosa strain AI3 at 14/0 of 10 were used. Conformational changes occurring upon encapsulation of both strains enzymes in reverse micelles of TAB in heptane/octanol were additionally identified by FTIR spectroscopy which clarified the biocatalysts performances.
- Pseudomonas aeruginosa amidase: Aggregation in recombinant Escherichia coliPublication . Borges, Patrícia; Pacheco, Rita; Karmali, AminThe effect of cultivation parameters such as temperature incubation, IPTG induction and ethanol shock on the production of Pseudomonasaeruginosa amidase (E.C.3.5.1.4) in a recombinant Escherichia coli strain in LB ampicillin culture medium was investigated. The highest yield of solubleamidase, relatively to other proteins, was obtained in the condition at 37 degrees C using 0.40 mM IPTG to induce growth, with ethanol. Our results demonstrate the formation of insoluble aggregates containing amidase, which was biologically active, in all tested growth conditions. Addition of ethanol at 25 degrees C in the culture medium improved amidase yield, which quantitatively aggregated in a biologically active form and exhibited in all conditions an increased specific activity relatively to the soluble form of the enzyme. Non-denaturing solubilization of the aggregated amidase was successfully achieved using L-arginine. The aggregates obtained from conditions at 37 degrees C by Furier transform infrared spectroscopy (FTIR) analysis demonstrated a lower content of intermolecular interactions, which facilitated the solubilization step applying non-denaturing conditions. The higher interactions exhibited in aggregates obtained at suboptimal conditions compromised the solubilization yield. This work provides an approach for the characterization and solubilization of novel reported biologically active aggregates of this amidase.
- Phenolic compounds from Actinidia deliciosa leaves: Caco-2 permeability, enzyme inhibitory activity and cell protein profile studiesPublication . Henriques, Joana; Fale, Pedro; Pacheco, Rita; Florêncio, Maria Helena; Serralheiro, Maria LuisaChemical compounds from leaves of fruit-producing trees, a waste from agricultural activity can be isolated and used as a source of natural bioactive chemicals. Boiling water was used as an extractant of bioactive compounds from Actinidia deliciosa leaves and co-extracted fibres were removed with ethanol precipitation. Rutin and quercitrin were the main flavonoids identified and quantified by RP-HPLC-DAD. No cytotoxicity was detected for any of the extracts towards Caco- 2 cell line. A permeation of approx. 14% of extract components through the cells monolayer was determined. The cell protein profile of Caco-2 cells was modified when in the presence of the fibre-free extract and transketolase was the protein over-expressed in the presence of polyphenols. Acetylcholinesterase inhibitory activity was also studied, IC50 of 0.56 mg/mL was obtained with the fibre-free extract. A. deliciosa leaves are a good source of phenolic compounds and, therefore, some advantage may be taken of this agricultural residue, due to their biological activity.
- Inhibition of HMG-CoA reductase activity and cholesterol permeation through Caco-2 cells by caffeoylquinic acids from Vernonia condensata leavesPublication . Arantes, Ana A.; Fale, Pedro L.; Costa, Larissa C. B.; Pacheco, Rita; Ascensão, Lia; Serralheiro, Maria LuísaThe aim ofthis study was to provide scientific knowledge to supportthe use of Vernonia condensata Baker, Asteraceae, beverages for their alleged hypocholesterolemic properties by testing their action as HMGCoA reductase inhibitors and their capacity to lower dietary cholesterol permeation. Chlorogenic acid, and other caffeoylquinic acids derivatives were identified as the main components of these beverages by LC–MS/MS. No changes in the composition were notice after the in vitro gastrointestinal digestion and no toxicity against Caco-2 and HepG2 cell lines was detected. Cholesterol permeation through Caco-2 monolayers was reduced in 37% in the presence of these herbal teas, and the caffeoylquinic acids permeated the monolayers in 30–40% of their initial amount in 6 h. HMG-CoA reductase activity was reduced with these beverages, showing an IC50 of 217 g ml−1. It was concluded that caffeoylquinic acids, the major components, justified 98% of the enzyme inhibition measured.
- Optimized Production of Hydroxamic Acid Derivatives with Antioxidant and Anticholinergic Potential by Immobilized Pseudomonas aeruginosa CellsPublication . Bernardo, Marisa; Reis, Telma; Minhalma, Miguel; Karmali, Amin; Serralheiro, Maria Luísa; Pacheco, RitaIn this study were investigated, the synthesis, acetylcholinesterase inhibition and antioxidant activity of a series of hydroxamic acid derivatives (HAD), with different chemical group characteristics, such as aliphatic (acetohydroxamic acid and butyryl hydroxamic acid), aromatic (benzohydroxamic acid and phenylalanine hydroxamic acid) and amino acid (glycine hydroxamic acid and alanine hydroxamic acid). It was observed that these HAD compounds present very promising activity as acetylcholinesterase (AChE) inhibitors and as antioxidants. The aliphatic HAD demonstrated to have a higher inhibitory activity of AChE than amino acid or aromatic HAD. As for the antioxidant activity, a high antioxidant potential was found for all the compounds with EC50 values ranging from 0.19 µM to 1.65 µM. Aiming these applications, a biocatalysis approach was used to obtain these HADs with optimal reactional conditions. In this study, reverse micelles with immobilized Pseudomonas aeruginosa intact cells containing amidase were used as a biocatalyst to catalyze the acyltransferase reaction of the corresponding substrate amide and hydroxylamine to obtain various HAD and this was achieved for the first time with yields of approximately 100 %.
- Action of euptox A from Ageratina adenophora juice on human cell lines: a top-down study using FTIR spectroscopy and protein profilingPublication . André, Rebeca; Catarro, Joana; Freitas, Dalia; Pacheco, Rita; Oliveira, Maria Conceição; Serralheiro, Maria Luisa; Fale, PedroEuptox A, from Ageratina adenophora juice, is a toxin associated with the plant's resistance to infections, invasiveness and traditional use in cancer treatment. We used FTIR spectroscopy and protein profiling of cell lines to study the impact of euptox A on human cells, to clarify its mechanism of action in a top-down approach. Euptox A was extracted from the juice of A. adenophora. Its stability in the gastrointestinal tract was evaluated, as the compound/juice is generally taken orally. Cytotoxicity was determined in HeLa, Caco-2 and MCF7 cells, and the mechanism of action analyzed by protein and metabolite profiles using electrophoresis and FTIR spectroscopy. Euptox A resisted gastrointestinal digestion and was the most cytotoxic component of the extract for all cell lines tests. Euptox A-treated HeLa cells showed changes in protein profile, especially on 40S ribosomal protein S8 (RP), generally associated with cancer cells. FTIR profiles of treated cells diverged in the same metabolites as cells treated with cisplatin, both in metabolite directed analysis and in multivariate analysis (principal component analysis). In conclusion, euptox A in this top-down study showed a cellular impact that suggests a strong potential against cancer, acting on cancer targeted cellular characteristics.
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