Small electron transfer proteins as mediators in enzymatic electrochemical biosensors

The final publication is available at Springer."Electrochemical mediators transfer redox equivalents between the active sites of enzymes and electrodes and, in this way, trigger bioelectrocatalytic redox processes. This has been very useful in the development of the so-called second generation biosensors, where they are able to transduce the catalytic event into an electrical signal. Among other pre-requisites, redox mediators must be readily oxidized/reduced at the electrode surface and easily interact with the biorecognition component. Small chemical compounds (e.g. ferrocene derivatives, ruthenium or osmium complexes and viologens) are frequently used for this purpose, but lately, small redox proteins (e.g. horse heart cytochrome c) have also played the role of redox partners in biosensing applications. In general, the docking between two complementary proteins introduces a second level of selectivity to the biosensor and enlarges the list of compounds targeted for analysis. Moreover, electrochemical interferences are frequently minimized owing to the small overpotentials achieved. This paper aims to provide an overview of enzyme biosensors that are mediated by electron transfer proteins. The article begins with a few considerations on mediated electrochemistry in biosensing 2 systems and proceeds with a detailed description of relevant works concerning the cooperative use of redox enzymes and biological electron donors/acceptors.

A comprehensive view of nitrite reductases

The authors acknowledge the support of the research centers Applied Molecular Biosciences Unit-UCIBIO, which is co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). CMS acknowledges support from Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI). Publisher Copyright: © 2022 Elsevier B.V.The last years have witnessed a steady increase of social and political awareness for the need of studying, monitoring, and controlling several anthropological activities that are dramatically impacting the environment and human health. The increasing turnover rates of the nitrogen cycle across the Planet are of major concern, so the understanding of the biological, chemical, and physical processes associated with the biogeochemical nitrogen cycle has been attracting the attention of several scientific disciplines. For many years, the primary focus has been the so-called “dissimilatory reduction of nitrate”, which refers to the stepwise conversion of nitrate into molecular nitrogen, closely followed by the assimilatory nitrate reduction pathway, which allow nitrogen incorporation into biomolecules. The contribution of bioinorganic chemists to better understand the enzymology underlying these two branches of the N-cycle has been remarkable. The constant development of mechanistic, structural, and biological tools has been keeping this bioinorganic chemistry field very active, making it a highly relevant research area still today. In this paper, we review the state-of-the-art in both dissimilatory and assimilatory nitrite reducing enzymes, highlighting the structural peculiarities of the different metalloenzymes involved in this step.publishersversionpublishe

Development of new analytical tools for monitoring of cardiovascular disease markers – towards the detection of homocysteine-thiolactone

Poster presented at the 4th International Conference on Bio-Sensing Technology, 10-13 May 2015, Lisbon, Portuga

New PON1-based biosensor for the detection of homocysteine-thiolactone in human plasma

Poster presented at the XXIII International Symposium on Bioelectrochemistry and Bioenergetics, 14-18 June 2015, Malmo, Sweden

Inhibition-based biosensor for cyanide detection – a preliminary study

Abstract in proceedings of the Fourth International Congress of CiiEM: Health, Well-Being and Ageing in the 21st Century, held at Egas Moniz’ University Campus in Monte de Caparica, Almada, from 3–5 June 2019.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.info:eu-repo/semantics/publishedVersio

Clitocybe alexandri extract induces cell cycle arrest and apoptosis in a lung cancer cell line: identification of phenolic acids with cytotoxic potential

Mushrooms are a possible rich source of biologically active compounds with potential for drug discovery. The aim of this work was to gain further insight into the citotoxicity mechanism of action of Clitocybe alexandri ethanolic extract against a lung cancer cell line (NCI-H460 cells). The effects on cell cycle profile and levels of apoptosis were evaluated by flow cytometry, and the effect on the expression levels of proteins related to cellular apoptosis was also investigated by Western blot. The extract was characterized regarding its phenolic composition by HPLC-DAD, and the identified compounds were studied regarding their growth inhibitory activity, by sulforhodamine B (SRB) assay. The effect of individual or combined compounds on viable cell number was also evaluated using the Trypan blue exclusion assay. It was observed that the Clitocybe alexandri extract induced an S-phase cell cycle arrest and increased the percentage of apoptotic cells. In addition, treatment with the GI50 concentration (concentration that was able to cause 50% of cell growth inhibition; 24.8 µg/ml) for 48h caused an increase in the levels of wt p53, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). The main components identified in this extract were protocatechuic, p-hydroxybenzoic and cinnamic acids. Cinnamic acid was found to be the most potent compound regarding cell growth inhibition. Nevertheless, it was verified that the concomitant use of the individual compounds provided the strongest decrease in viable cell number. Overall, we found evidence for alterations in cell cycle and apoptosis, involving p53 and caspase-3. Furthermore, our data suggests that the phenolic acids identified in the extract are at least partially responsible for the cytotoxicity induced by this mushroom extract

In vitro growth inhibitory activity of the Portuguese wild mushroom Clitocybe alexandri in human tumour cell lines

QC 20190812</p

Respiratory versatility in Desulfovibrio desulfuricans ATCC 27774 – a proteomic approach

Poster presented at the Bacterial Electron Transfer Processes and their Regulation Meeting, European Federation of Biotechnology Microbial Physiology Section, 15-18 March 2015, Vimeiro, Portugal

A study of the antitumour potential of three Portuguese wild mushrooms

Natural matrixes such as mushrooms represent a rich source of biologically active compounds with recognized potential in drug discovery and development [1,2]. Indeed, many pre-clinical studies have been conducted in human tumour cell lines and in some cases, a number of compounds extracted from mushrooms have entered clinical trials [3]. Our previous results showed that extracts from Agaricus arvensis, Suillus collinitus and Clytocibe alexandri are promising sources of low molecular weight bioactive compounds [4]. The aim of the present work was to study the antitumour potential of the extracts and isolated compounds from three Portuguese wild mushrooms by verifying their effect on various human tumour cell lines in what concerns effect on cell growth, cell cycle profile and programmed cell death. Wild mushrooms were collected from the Northeast of Portugal and classified as Agaricus arvensis, Suillus collinitus and Clitocybe alexandri. Phenolic (methanolic and ethanolic) and polissacharidic extracts were prepared. The effect of the extracts on tumour cell growth inhibition was verified with the SRB assay and the GI50 of each extract was determined for each of the cell lines studied (NCI-H460, MCF-7, AGS and HCT-15). Our preliminary results revealed that all the extracts from Clitocybe alexandri are capable of causing cell growth inhibition and provided GI50 concentrations bellow 60µg/ml in all the cell lines tested [4]. Regarding the effect of the Agaricus arvensis extracts, they all caused an inhibition of cell growth in all cell lines, particularly the methanolic extract which revealed to be a very potent inhibitor of cell growth, especially in the MCF-7 cell line. The evaluation of the effect of the Suillus collinitus extracts will be carried out as well as cell cycle and apoptosis analyses, by flow cytometry. Finally, the isolation and characterization of compounds from these extracts will also be carried out, using HPLC-DAD or HPLC-RI. The structures of the compounds will be established by NMR spectral analysis (1H, 13C, DEPT, COSY, HSQC and HMBC).FCT and COMPETE/QREN/UE- project PTDC/AGR-ALI/110062/200

Caracterização fenólica e avaliação da atividade antiproliferativa de Fistulina hepatica e Suillus collinitus em linhas celulares tumorais humanas

Neste trabalho, avaliou-se a atividade antiproliferativa de extratos metanólicos, etanólicos e em água fervente de Fistulina hepatica e Suillus collinitus, dois cogumelos comestíveis do Nordeste de Portugal, em linhas celulares tumorais humanas. Determinaram-se ainda, cromatograficamente, os ácidos fenólicos presentes nos extratos metanólicos. Ao contrário dos extratos de Fistulina hepatica que não revelaram nenhuma atividade à máxima concentração testada (400 μg/mL), o extrato metanólico de Suillus collinitus foi bastante potente, particularmente em células MCF-7 (GI50 25,2±0,2 µg/mL), induzindo alterações no seu ciclo celular, aparentemente por um efeito mediado pela p53, e aumentando a percentagem de células apoptóticas