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Pina, AS, Lowe CR, Roque ACA.  2014.  Challenges and opportunities in the purification of recombinant tagged proteins. Biotechnology Advances. 32(2):366-381. AbstractWebsite

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs “tag-ligand” combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established “tag-ligand” systems available for fusion protein purification and also explores current unconventional strategies under development.

Alves, R, Rodrigues J, Ramou E, Palma S, Roque A, Gamboa H.  2022.  Classification of Volatile Compounds with Morphological Analysis of e-nose Response, Feb. Proceedings of the 15th International Joint Conference on Biomedical Engineering Systems and Technologies - BIOSIGNALS. :31–39.: Scitepress AbstractPDF

Electronic noses (e-noses) mimic human olfaction, by identifying Volatile Organic Compounds (VOCs). This
work presents a novel approach that successfully classifies 11 known VOCs using the signals generated by
sensing gels in an in-house developed e-nose. The proposed signals’ analysis methodology is based on the
generated signals’ morphology for each VOC since different sensing gels produce signals with different shapes
when exposed to the same VOC. For this study, two different gel formulations were considered, and an average
f1-score of 84% and 71% was obtained, respectively. Moreover, a standard method in time series classification
was used to compare the performances. Even though this comparison reveals that the morphological approach
is not as good as the 1-nearest neighbour with euclidean distance, it shows the possibility of using descriptive
sentences with text mining techniques to perform VOC classification.

Pina, AS, Lowe CR, Roque ACA.  2010.  Comparison of Fluorescence Labelling Techniques for the Selection of Affinity Ligands from Solid-Phase Combinatorial Libraries. Separation Science and Technology. 45:2187–2193., Number 15 Abstract

This study reports the comparison of fluorimetric techniques (fluorescence microscopy and spectrofluorimetry on a 96-well format) for the on-bead screening of combinatorial libraries of affinity ligands for chromatographic separations. Two solid-phase libraries of synthetic ligands based on distinct scaffolds were synthesized by combinatorial chemistry. The libraries comprising ligands representing different hydrophobic/hydrophilic properties and sizes were tested for binding to randomly selected biomolecules (labelled with a fluorophore). Fluorescence microscopy was revealed to be a reliable and reproducible technique for the detection of lead ligands which strongly bound the target biomolecule. Results obtained by fluorescence intensity measurements in a 96-well format were less consistent, mainly due to challenges related with the accurate dispensing of the solid support.

Carvalho, HF, Roque ACA, Iranzo O, Branco RJF.  2015.  Comparison of the Internal Dynamics of Metalloproteases Provides New Insights on Their Function and Evolution, 2015/09/23. PLoS ONE. 10(9):e0138118-.: Public Library of Science AbstractWebsite

Metalloproteases have evolved in a vast number of biological systems, being one of the most diverse types of proteases and presenting a wide range of folds and catalytic metal ions. Given the increasing understanding of protein internal dynamics and its role in enzyme function, we are interested in assessing how the structural heterogeneity of metalloproteases translates into their dynamics. Therefore, the dynamical profile of the clan MA type protein thermolysin, derived from an Elastic Network Model of protein structure, was evaluated against those obtained from a set of experimental structures and molecular dynamics simulation trajectories. A close correspondence was obtained between modes derived from the coarse-grained model and the subspace of functionally-relevant motions observed experimentally, the later being shown to be encoded in the internal dynamics of the protein. This prompted the use of dynamics-based comparison methods that employ such coarse-grained models in a representative set of clan members, allowing for its quantitative description in terms of structural and dynamical variability. Although members show structural similarity, they nonetheless present distinct dynamical profiles, with no apparent correlation between structural and dynamical relatedness. However, previously unnoticed dynamical similarity was found between the relevant members Carboxypeptidase Pfu, Leishmanolysin, and Botulinum Neurotoxin Type A, despite sharing no structural similarity. Inspection of the respective alignments shows that dynamical similarity has a functional basis, namely the need for maintaining proper intermolecular interactions with the respective substrates. These results suggest that distinct selective pressure mechanisms act on metalloproteases at structural and dynamical levels through the course of their evolution. This work shows how new insights on metalloprotease function and evolution can be assessed with comparison schemes that incorporate information on protein dynamics. The integration of these newly developed tools, if applied to other protein families, can lead to more accurate and descriptive protein classification systems.

Palma, SICJ, Carvalho A, Silva J, Martins P, Marciello M, Fernandes AR, del Puerto Morales M, Roque ACA.  2015.  Covalent coupling of gum arabic onto superparamagnetic iron oxide nanoparticles for MRI cell labeling: physicochemical and in vitro characterization. Contrast Media & Molecular Imaging. 10:320–328., Number 4 AbstractWebsite

Gum arabic (GA) is a hydrophilic composite polysaccharide derived from exudates of Acacia senegal and Acacia seyal trees. It is biocompatible, possesses emulsifying and stabilizing properties and has been explored as coating agent of nanomaterials for biomedical applications, namely magnetic nanoparticles (MNPs). Previous studies focused on the adsorption of GA onto MNPs produced by co-precipitation methods. In this work, MNPs produced by a thermal decomposition method, known to produce uniform particles with better crystalline properties, were used for the covalent coupling of GA through its free amine groups, which increases the stability of the coating layer. The MNPs were produced by thermal decomposition of Fe(acac)3 in organic solvent and, after ligand-exchange with meso-2,3-dimercaptosuccinic acid (DMSA), GA coating was achieved by the establishment of a covalent bond between DMSA and GA moieties. Clusters of several magnetic cores entrapped in a shell of GA were obtained, with good colloidal stability and promising magnetic relaxation properties (r2/r1 ratio of 350). HCT116 colorectal carcinoma cell line was used for in vitro cytotoxicity evaluation and cell-labeling efficiency studies. We show that, upon administration at the respective IC50, GA coating enhances MNP cellular uptake by 19 times compared to particles bearing only DMSA moieties. Accordingly, in vitro MR images of cells incubated with increasing concentrations of GA-coated MNP present dose-dependent contrast enhancement. The obtained results suggest that the GA magnetic nanosystem could be used as a MRI contrast agent for cell-labeling applications. Copyright © 2015 John Wiley & Sons, Ltd.

Sandu, ICA, Schäfer S, Magrini D, Bracci S, Roque ACA.  2012.  Cross-Section and Staining-Based Techniques for Investigating Organic Materials in Painted and Polychrome Works of Art: A Review.. Microscopy and Microanalysis. 18(4):860-875. AbstractWebsite

The article presents a review of the use of cross-section and staining techniques for investigating natural organic materials (mainly proteinaceous and oil-based binders/varnishes) in painted and polychrome artworks, considering the requirements of conservation practice and routine diagnostics. The reviewed literature calls attention to the importance of using cross sections to prepare samples for optical microscopy and to different properties of embedding resins; the most appropriate instrumental conditions for optical microscopy; and the advantages and disadvantages of the most common staining techniques. A few case studies were selected to illustrate the use of autofluorescence (intrinsic fluorescence) and induced fluorescence (using specific staining tests and fluorophore-labeled antibodies) for mapping and identifying organic paint materials in cross sections. New directions of research in cross-section analyses and fluorescence-based techniques for the identification and mapping of artistic materials are presented. The complementary use of different stains on the same cross section, further exploration of intrinsic and induced fluorescence of aged versus fresh materials, and applicability of cross-section observation and staining as complementary methods for assessing the effectiveness of restoration treatments, such as cleaning and consolidation, are discussed in the last section of the article.