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Bicho, A, Roque ACA, Cardoso AS, Domingos P, Batalha ÍL.  2010.  In vitro studies with mammalian cell lines and gum arabic‐coated magnetic nanoparticles. Journal of Molecular Recognition. 23:536–542., Number 6 AbstractWebsite

Iron oxide magnetic nanoparticles {(MNPs)} were synthesized by the chemical co-precipitation method and coated with gum arabic {(GA)} by physical adsorption and covalent attachment. Cultures of mammalian cell lines {(HEK293}, {CHO} and {TE671)} were grown in the presence of uncoated and {GA-coated} {MNPs.} Cellular growth was followed by optical microscopy in order to assess the proportion of cells with particles, alterations in cellular density and the presence of debris. The in vitro assays demonstrated that cells from different origins are affected differently by the presence of the nanoparticles. Also, the methods followed for {GA} coating of {MNPs} endow distinct surface characteristics that probably underlie the observed differences when in contact with the cells. In general, the nanoparticles to which the {GA} was adsorbed had a smaller ability to attach to the cells' surface and to compromise the viability of the cultures. Copyright © 2010 John Wiley & Sons, Ltd.

Pina, AS, Roque ACA.  2009.  Studies on the molecular recognition between bioactive peptides and angiotensin-converting enzyme, apr. Journal of Molecular Recognition. 22:162–168., Number 2 AbstractWebsite

High blood pressure or hypertension is a condition affecting many individuals and represents a controllable risk factor for cardiovascular diseases such as coronary heart disease and stroke. A non-pharmacological approach to manage these includes the application of food components with antihypertensive activity. Milk protein-derived peptides have been exploited as natural hypotensive agents, namely the peptides {Val-Pro-Pro} {(VPP)} and {Ile-Pro-Pro} {(IPP)}, already commercialized in functional foods as a potential alternative to synthetic drugs. These bioactive peptides inhibit in vitro and in vivo the Angiotensin I-converting enzyme {(ACE)}, a protein with an important role in blood pressure regulation. In this work, we attempted to elucidate the possible mode of interaction between the peptides and {ACE}, including mechanisms of binding to the cofactor Zn2+, and further contrast this with the known mode of inhibition exerted by synthetic drugs {(Captopril}, Enalaprilat and Lisinopril). The bioactive peptide {Ala-Leu-Pro-Met-His-Ile-Arg} {(ALPMHIR)}, also known to inhibit the enzyme {ACE} but with a lower efficiency than {VPP} and {IPP}, was utilized in the docking studies for comparison. It was observed that the best docking poses obtained for {VPP} and {IPP} were located at the {ACE} catalytic site with very high resemblance to the drugs mode of interaction, including the coordination with Zn2+. As for {ALPMHIR}, the best docking poses were located in the narrow {ACE} channel outside the catalytic site, representing higher affinity energies and fewer resemblances with the interaction established by drugs.

Roque, ACA, Bispo S, Pinheiro ARN, Antunes JMA, Gonçalves D, Ferreira HA.  2009.  Antibody immobilization on magnetic particles. Journal of Molecular Recognition. 22:77–82., Number 2 AbstractWebsite

Magnetic particles {(MNPs)} offer attractive possibilities in biotechnology. {MNPs} can get close to a target biological entity, as their controllable sizes range from a few nanometres up to tens of nanometres, and their surface can be modified to add affinity and specificity towards desired molecules. Additionally, they can be manipulated by an external magnetic field gradient. In this work, the study of ferric oxide {(Fe3O4)} {MNPs} with different coating agents was conducted, particularly in terms of strategies for antibody attachment at the surfaces (covalent and physical adsorption) and the effects of blocking buffer composition and incubation times on the specific and non-specific interactions observed. The considered biological model system consisted of a coating antibody (goat {IgG)}, bovine serum albumin {(BSA)} as blocking agent, and a complementary antibody labelled with {FITC} (anti-goat {IgG).} The detection of antibody binding was followed by fluorescence microscopy and the intensity of the signals quantified. The ratio between the mean grey values of negative and positive controls, as well as the maximum intensity attainable in positive controls, were considered in the evaluation of the assays efficiency. The covalent immobilization of the coating antibody was more successful as opposed to protein adsorption. For covalent immobilization, silica-coated {MNPs}, a 5% (w/v) concentration of {BSA} in the blocking buffer and incubation times of 1 h produced the best results in terms of assay sensitivity. However, when conducting the assay for incubation periods of 10 min, the fluorescence signal was reduced by 44% but the assay specificity was maintained.

Hussain, A, Pina AS, Roque ACA.  2009.  Bio-recognition and detection using liquid crystals. Biosensors and Bioelectronics. 25:1–8., Number 1 AbstractWebsite

Liquid crystals {(LCs)} are used extensively by the electronics industry as display devices. Advances in the understanding of the liquid crystalline phase and the chemistry therein lead to the development of {LC} exhibiting faster switching speed with greater twist angle. This in turn lead to the emergence of liquid crystal displays, rendering dial-and-needle based displays (such as those used in various meters) and cathode ray tubes obsolete. In this article, we review the history of {LC} and their emergence as an invaluable material for display devices and the more recent discovery of their use as sensing elements in biosensors. This new application of {LC} as tools in the development of fast and simple biosensors is envisaged to gain more importance in the foreseeable future.

Roque, ACA, Bicho A, Batalha IL, Cardoso AS, Hussain A.  2009.  Biocompatible and bioactive gum Arabic coated iron oxide magnetic nanoparticles. Journal of Biotechnology. 144:313–320., Number 4 AbstractWebsite

The surface modification of iron oxide magnetic nanoparticles {(MNPs)} with gum Arabic {(GA)} via adsorption and covalent coupling was studied. The adsorption of {GA} was assessed during {MNP} chemical synthesis by the co-precipitation method {(MNP\_GA)}, and after {MNP} synthesis on both bare magnetite and {MNP\_GA.} The covalent immobilization of {GA} at the surface of aldehyde-activated {(MNP\_GAAPTES)} or aminated {MNPs} {(MNP\_GAEDC)} was achieved through free terminal amino and carboxylate groups from {GA.} The presence of {GA} at the surface of the {MNPs} was confirmed by {FTIR} and by the quantification of {GA} by the bicinchoninic acid test. Results indicated that the maximum of {GA} coating was obtained for the covalent coupling of {GA} through its free carboxylate groups {(MNP\_GAEDC)}, yielding a maximum of 1.8&\#xa0;g of {GA} bound/g of dried particles. The hydrodynamic diameter of {MNPs} modified with {GA} after synthesis resulted in the lowest values, in opposition to the {MNPs} co-precipitated with {GA} which presented the tendency to form larger aggregates of up to 1&\#xa0;μm. The zeta potentials indicate the existence of negatively charged surfaces before and after {GA} coating. The potential of the {GA} coated {MNPs} for further biomolecule attachment was assessed through anchorage of a model antibody to aldehyde-functionalized {MNP\_GA} and its subsequent detection with an {FITC} labeled anti-antibody.

Pina, AS, Hussain A, Roque ACA.  2009.  An historical overview of drug discovery. Ligand-Macromolecule Interactions in Drug Discovery. (Roque, A. C. A., Ed.).:3-12., USA: Humana Press Inc. Abstract

Drug Discovery in modern times straddles three main periods. The first notable period can be traced to the nineteenth century where the basis of drug discovery relied on the serendipity of the medicinal chemists. The second period commenced around the early twentieth century when new drug structures were found, which contributed for a new era of antibiotics discovery. Based on these known structures, and with the development of powerful new techniques such as molecular modelling, combinatorial chemistry, and automated high-throughput screening, rapid advances occurred in drug discovery towards the end of the century. The period also was revolutionized by the emergence of recombinant DNA technology, where it became possible to develop potential drugs target candidates. With all the expansion of new technologies and the onset of the "Omics" revolution in the twenty-first century, the third period has kick-started with an increase in biopharmaceutical drugs approved by FDA/EMEA for therapeutic use.

Roque, ACA.  2009.  Ligand-Macromolecule Interactions in Drug Discovery. , U.S.A.: Methods in Molecular Biology, Humana Press Inc.Website
Roque, ACA, Wilson OC.  2008.  Adsorption of gum Arabic on bioceramic nanoparticles. Materials Science & Engineering C.- Biomimetic and Supramolecular Systems. 28:443–447., Number 3 Abstract


Roque, ACA, Lowe CR.  2007.  Affinity chromatography: History, Perspectives, Limitations and Prospects. Affinity Chromatography: Methods and Protocols. (M. Zachariou, Ed.).:1-23., U.S.A.: Humana Press Inc. Abstract

Biomolecule separation and purification has until very recently steadfastly remained one of the more empirical aspects of modern biotechnology. Affinity chromatography, one of several types of adsorption chromatography, is particularly suited for the efficient isolation of biomolecules. This technique relies on the adsorbent bed material that has biological affinity for the substance to be isolated. This review is intended to place affinity chromatography in historical perspective and describe the current status, limitations and future prospects for the technique in modern biotechnology.

Roque, ACA, Silva CSO, Taipa ÂM.  2007.  Affinity-based methodologies and ligands for antibody purification: Advances and perspectives. Journal of Chromatography A. 1160:44–55., Number 1-2 AbstractWebsite

Many successful, recent therapies for life-threatening diseases such as cancer and rheumatoid arthritis are based on the recognition between native or genetically engineered antibodies and cell-surface receptors. Although naturally produced by the immune system, the need for antibodies with unique specificities and designed for single application, has encouraged the search for novel antibody purification strategies. The availability of these products to the end-consumer is strictly related to manufacture costs, particularly those attributed to downstream processing. Over the last decades, academia and industry have developed different types of interactions and separation techniques for antibody purification, affinity-based strategies being the most common and efficient methodologies. The affinity ligands utilized range from biological to synthetic designed molecules with enhanced resistance and stability. Despite the successes achieved, the purification “paradigm” still moves interests and efforts in the continuous demand for improved separation performances. This review will focus on recent advances and perspectives in antibody purification by affinity interactions using different techniques, with particular emphasis on affinity chromatography.

Ferreira, IMPLVO, Eça R, Pinho O, Tavares P, Pereira A, Roque AC.  2007.  Development and Validation of an HPLC/UV Method for Quantification of Bioactive Peptides in Fermented Milks. Journal of Liquid Chromatography & Related Technologies. 30:2139–2147., Number 14 Abstract

The simultaneous separation and quantification of two casein peptides {(IPP}, {VPP)} presenting potent inhibitory activity of angiotensin-converting-enzyme {(ACE)} and casein in fermented milks was developed. Gradient elution was carried out at a flow-rate of 1 {mL/min}, using a mixture of two solvents. Solvent A was 0.1% {TFA} in water and solvent B was acetonitrile-water-trifluoracetic acid 95:5:0.1. The effluent was monitored by {UV} detector at 214 nm. Calibration curves were constructed in the interval of 0.01-1.0 {mg/mL} for {VPP}, 0.005-1.0 {mg/mL} for {IPP}, and 0.05-3.0 {mg/mL} for casein. R2 invariably exceeded 0.999. The detection limits were 0.004 for {VPP}, 0.002 {mg/mL} for {IPP}, and 0.02 {mg/mL} for casein. Repeatability of the method was evaluated by six consecutive injections of two standard solutions containing {VPP}, {IPP}, and casein. The {RSD} values for concentration were all below 5.08%. Recovery studies were carried out to determine the accuracy of the method. Recoveries ranged between 88 and 98.2%. The methodology was applied, not only, for the monitorization of {VPP}, {IPP}, and casein in commercial fermented milks labeled as presenting antihypertensive properties, but also, in milk with different degrees of fermentation by L. Helveticus, and in other commercial functional fermented milks, such as, those presenting cholesterol lowering properties.

Roque, ACA, Lowe CR.  2007.  Rationally designed ligands for use in Affinity Chromatography: An artificial Protein L. Affinity Chromatography: Methods and Protocols. (M. Zachariou, Ed.).:93-110., U.S.A.: Humana Press Inc. Abstract

Synthetic affinity ligands can circumvent the drawbacks of natural immunoglobulin (Ig)-binding proteins by imparting resistance to chemical and biochemical degradation and to in situ sterilization, as well as ease and low cost of production. Protein L (PpL), isolated from Peptostreptococcus magnus strains, interacts with the Fab (antigen-binding fragment) portion of Igs, specifically with kappa light chains, and represents an almost universal ligand for the purification of antibodies. The concepts of rational design and solid-phase combinatorial chemistry were used for the discovery of a synthetic PpL mimic affinity ligand. The procedure presented in this chapter represents a general approach with the potential to be applied to different systems and target proteins.