Person:
Gil Dones, Félix

First Name

Félix

Last Name

Gil Dones

URI

https://hdl.handle.net/20.500.14352/74303

Affiliation

Universidad Complutense de Madrid

Faculty / Institute

Ciencias Biológicas

Department

Genética, Fisiología y Microbiología

Area

Genética

Identifiers

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Now showing 1 - 10 of 12

Proteomic characterization of EPCs and CECs “in vivo” from acute coronary syndrome patients and control subjects
(Biochimica et Biophysica Acta (BBA) - General Subjects, 2013) Mourino-Alvarez, L.; Calvo, E.; Moreu, J.; Padial, L.R.; López, J.A.; Barderas, M.G.; Gil Dones, Félix
Background: Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) represent two scarce blood populations that are thought to play important roles in tissue vascularization. They have also been proposed as potential markers for more than a dozen pathologies. Moreover, EPCs have arisen as a new therapeutic option for cardiovascular disease. However nowadays there is certain controversy about their roles and a better understanding of EPC biology is required to develop new strategies for forthcoming therapies. Methods: Flow cytometry analysis was performed on freshly isolated mononuclear cells from control subjects and Acute Coronary Syndrome (ACS) patients. EPCs and CECs for both groups were isolated and quantified. Statistical analyses were performed to test the potential biomarker usefulness of both populations in ACS together with the first “in vivo” proteomic characterizations of these populations. Results: Our results do not show statistical differences in the quantification of CECs and EPCs in control subjects and ACS patients. The proteomic characterization allowed us to identify 673 proteins associated to CECs (389 in controls and 462 in ACS patients), and another 502 proteins in EPCs (350 in controls and 274 in ACS patients).
Development of an Optimal Protocol for the Proteomic Analysis of Stenotic and Healthy Aortic Valves
(Revista Española de Cardiologia, 2010) Martín-Rojas, Tatiana; López-Almodovar, Luis F.; Juárez-Tosina, Rocío; Gil Dones, Félix; Cuesta, Fernando de La; Álvarez-Llamas, Gloria; Alonso-Orgaz, Sergio; Vivanco, Fernando; Rodríguez-Padial, Luis; Barderas, María G.
Introduction and objectives: For many years, degenerative aortic stenosis was thought to be a passive process secondary to calcium deposition in aortic valves. Although its etiology remains unknown, several authors have pointed out that degenerative aortic stenosis is associated with the same risk factors as coronary artery disease. Furthermore, histological similarities have been found between aortic valve stenosis and atherosclerotic plaque, giving rise to the hypothesis that degenerative aortic stenosis is an inflammatory process similar to atherosclerosis. Nevertheless, some data do not fit with this hypothesis and, consequently, greater understanding of the condition is needed. The main aim of this study was to develop a practical protocol for extracting protein for use in proteomic analysis from both stenotic and healthy aortic valves. Methods: The study was carried out using a number of different proteomic methods: two-dimensional electrophoresis, mass spectrometry, and additional techniques. Results: We developed a simple and reproducible methodology in the laboratory for carrying out the proteomic analysis of human aortic valves and for identifying their component proteins. Conclusions: We developed a simple and reproducible method for extracting protein that can be used with mass spectrometry and that makes it possible to carry out large-scale proteomic analysis of stenotic aortic valves. Furthermore, the methodology will significantly increase our understanding of the valve proteome.
Analysis of the Plasma Proteome Associated with Acute Coronary Syndrome: Does a Permanent Protein Signature Exist in the Plasma of ACS Patients?
(Journal of protemoe research, 2010) Dardé, Veronica M.; Cuesta, Fernando de la; Gil Dones, Félix; Alvarez-Llamas, Gloria; Barderas, Maria G.; Vivanco, Fernando
Acute coronary syndrome (ACS) is triggered by the occlusion of a coronary artery usually due to the thrombosis caused by an atherosclerotic plaque. The identification of proteins directly involved in the pathophysiological events underlying ACS will enable more precise diagnoses and a more accurate prognosis to be determined. Accordingly, we have performed a longitudinal study of the plasma proteome in ACS patients by 2-DE and DIGE. Plasma samples from patients, healthy controls, and stable coronary artery disease (CAD) patients were immunodepleted of the six most abundant proteins, and they were analyzed in parallel at four different times: 0 (on admission) and after 4, 60, and 180 days. From a total of 1400 spot proteins analyzed, 33 proteins were differentially expressed in ACS patients when compared with control subjects/stable patients. A small group of seven proteins that appear to be altered at admission remain affected for 6 months and also in the stable CAD patients. Interestingly, the maximum number of altered proteins was observed in the stable CAD patients. Some of the proteins identified had been previously associated with ACS whereas others (such as Alpha-1-B-glycoprotein, Hakata antigen, Tetranectin, Tropomyosin 4) constitute novel proteins that are altered in this pathology.
Modification of the Secretion Pattern of Proteases, Inflammatory Mediators, and Extracellular Matrix Proteins by Human Aortic Valve is Key in Severe Aortic Stenosis
(Molecular and cellular proteomics, 2013) Alvarez-Llamas, Gloria; Martín-Rojas, Tatiana; Cuesta, Fernando de la; Calvo, Enrique; Gil Dones, Félix; Dardé, Veronica M.; Lopez-Almodovar, Luis F; Padial, Luis R.; Lopez, Juan-Antonio; Vivanco, Fernando; Barderas, Maria G.
One of the major challenges in cardiovascular medicine is to identify candidate biomarker proteins. Secretome analysis is particularly relevant in this search as it focuses on a subset of proteins released by a cell or tissue under certain conditions. The sample can be considered as a plasma subproteome and it provides a more direct approximation to the in vivo situation. Degenerative aortic stenosis is the most common worldwide cause of valve replacement. Using a proteomic analysis of the secretome from aortic stenosis valves we could identify candidate markers related to this pathology, which may facilitate early diagnosis and treatment. For this purpose, we have designed a method to validate the origin of secreted proteins, demonstrating their synthesis and release by the tissue and ruling out blood origin. The nLC-MS/MS analysis showed the labeling of 61 proteins, 82% of which incorporated the label in only one group. Western blot and selective reaction monitoring differential analysis, revealed a notable role of the extracellular matrix. Variation in particular proteins such as PEDF, cystatin and clusterin emphasizes the link between aortic stenosis and atherosclerosis. In particular, certain proteins variation in secretome levels correlates well, not only with label incorporation trend (only labeled in aortic stenosis group) but, more importantly, with alterations found in plasma from an independent cohort of samples, pointing to specific candidate markers to follow up in diagnosis, prognosis, and therapeutic intervention.
Inside human aortic stenosis: a proteomic analysis of plasma
(Journal of Proteomics, 2012) Gil Dones, Félix; Darde, Verónica M.; Alonso-Orgaz, Sergio; Lopez-Almodovar, Luis F.; Mourino-Alvarez, Laura; Padial, Luis R.; Vivanco, Fernando; Barderas, Maria G.
Valvular aortic stenosis (AS) produces a slowly progressive obstruction in left ventricular outflow track. For this reason, aortic valve replacement is warranted when the valvular stenosis is hemodinamically significant, becoming the most common worldwide cause of aortic valve surgery. Recent epidemiologic studies have revealed an association between degenerative AS and cardiovascular risk factors for atherosclerosis, althought reducing the exposure to such factors and statin therapies both fail to delay or reverse the pathology. Hence, a deeper understanding of the pathophysiology of this disease is required to identify appropriate preventive measures. A proteomic analysis of plasma will permit to know and identify the changes in protein expression induced by AS in this tissue. Using two-dimensional difference gel electrophoresis (2D-DIGE) followed by mass spectrometry (MS), we compared the crude (not pre-fractioned) and pre-fractioned plasma from AS patients and control subjects. We sought to identify plasma proteins whose expression is modified in AS. In addition we investigated if crude plasma presented some alterations in the more abundant proteins since to date, has never been studied before. We also further investigated the link between this disease and atherosclerosis with a view to identifying new potential markers and therapeutic targets.
Multimerization of peptide antigens for production of stable immunogens in transgenic plants
(Journal of Biotechnology, 2007) Gil Dones, Félix; Reytor, Edel; Pérez-Filgueira, Daniel Mariano; Escribano, José M.
Previous literature addressing the production of recombinant proteins in heterologous systems has consistently shown that proteins capable of forming complex structures tend to accumulate within host cells at relatively higher levels than monomeric forms. In this report, we translationally fused a 21-aminoacids long highly immunogenic peptide (2L21), derived from canine parvovirus (CPV) VP2 protein to a 41-aminoacid long tetramerization domain (TD) from the transcriptional factor p53. The chimerical DNA construction 2L21-TD was cloned in a binary plant transformation vector and used to transform Arabidopsis thaliana plants. Fifteen of the 25 transgenic lines obtained in the experiment showed detectable 2L21-TD RNA accumulation and from these we chose 4 to study 2L21-TD protein accumulation. Non-denaturing immunoblotting assays revealed that 2L21-TD chimeras effectively formed tetrameric complexes with yields reaching up to 12 μg/mg of soluble protein. Mice immunized by oral or intraperitoneal routes with crude protein extracts containing 2L21-TD protein were able to detect both 2L21-synthetic peptide and CPV VP2 proteins, with titers similar to those elicited by a previously reported fusion between 2L21 and the β-glucuronidase protein. These results demonstrate that multimerization directed by the small TD domain contributed to the stabilization and consequently to the accumulation of the 2L21 peptide in transgenic plants, without altering its native antigenicity and immunogenicity.
Self-aggregation of a recombinant form of the propeptide NH2-terminal of the precursor of pulmonary surfactant protein SP-B: a conformational study
(Journal of Industrial Microbiology & Biotechnology, 2008) Bañares-Hidalgo, A.; Bolaños-Gutiérrez, A.; Pérez Gil, Jesús; Gil Dones, Félix; Estrada, P.; Estrada, P.; Society for Industrial Microbiology
A recombinant form of the peptide N-terminally positioned from proSP-B (SP-BN) has been produced in Escherichia coli as fusion with the Maltose Binding Protein, separated from it by Factor Xa cleavage and purified thereafter. This protein module is thought to control assembly of mature SP-B, a protein essential for respiration, in pulmonary surfactant as it progress through the progressively acidified secretory pathway of pneumocytes. Self-aggregation studies of the recombinant propeptide have been carried out as the pH of the medium evolved from neutral to moderately acid, again to neutral and finally basic. The profile of aggregation versus subsequent changes in pH showed differences depending on the ionic strength of the medium, low or moderate, and the presence of additives such as L-arginine (a known aggregation suppressor) and Ficoll 70 (a macromolecular crowder). Circular dichroism studies of SP-BN samples along the aggregation process showed a decrease in α-helical content and a concomitant increase in β-sheet. Intrinsic fluorescence emission of SP-BN was dominated by the emission of Trp residues in neutral medium, being its emission maximum shifted to red at low pH, suggesting that the protein undergoes a pH-dependent conformational change that increases the exposure of their Trp to the environment. A marked increase in the fluorescence emission of the extrinsic probe bis-ANS indicated the exposure of hydrophobic regions of SP-BN at pH 5. The fluorescence of bis-ANS decreased slightly at low ionic strength, but to a great extent at moderate ionic strength when the pH was reversed to neutrality, suggesting that self-aggregation properties of the SP-BN module could be tightly modulated by the conditions of pH and the ionic environment encountered by pulmonary surfactant during assembly and secretion.
Oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus
(2000) Gómez, N.; Wigdorovitz, A.; Castañón, S.; Gil Dones, Félix; Ordá, R.; Borca, M. V.; Escribano, J. M.
Transgenic plants represent an inexpensive alternative to classical fermentation systems for production of recombinant subunit vaccines. Transgenic potato plants were created that express the N-terminal domain of the glycoprotein S (N-gS) from Transmissible gastroenteritis coronavirus (TGEV), containing the major antigenic sites of the protein. Extracts from potato tubers expressing N-gS were inoculated intraperitoneally to mice, and the vaccinated mice developed serum IgG specific for TGEV. Furthermore, when potato tubers expressing N-gS were fed directly to mice, they developed serum antibodies specific for gS protein, demonstrating the oral immunogenicity of the plant derived spike protein from TGEV.
Potential blood biomarkers for stroke
(Expert Review of Proteomics, 2012) Laborde, Carlos M.; Mourino–Alvarez, Laura; Akerstrom, Finn; Padial, Luis R.; Vivanco, Fernando; Gil Dones, Félix; Barderas, Maria G.
Stroke is one of the most common causes of death worldwide and a major cause of acquired disability in adults. Despite advances in research during the last decade, prevention and treatment strategies still suffer from significant limitations, and therefore new theoretical and technical approaches are required. Technological advances in the proteomic and metabolomic areas, during recent years, have permitted a more effective search for novel biomarkers and therapeutic targets that may allow for effective risk stratification and early diagnosis with subsequent rapid treatment. This review provides a comprehensive overview of the latest candidate proteins and metabolites proposed as new potential biomarkers in stroke.
Successful oral prime‐immunization with VP60 from rabbit haemorrhagic disease virus produced in transgenic plants using different fusion strategies
(Plant Biotechnology Journal, 2005) Elena Titarenko; Estela Terrada; Elsa Arcalís; José M. Escribano; Gil Dones, Félix
Expression levels of vaccine antigens in transgenic plants have important consequences in their use as edible vaccines. The major structural protein VP60 from the rabbit haemorrhagic disease virus (RHDV) has been produced in transgenic plants using different strategies to compare its accumulation in plant tissues. The highest expressing plants were those presenting stable, complex, high-density structures formed by VP60, suggesting the importance of multisubunit structures for the stability of this protein in plant cells. Mice fed with leaves of transgenic plants expressing VP60 were primed to a subimmunogenic baculovirus-derived vaccine single dose. This indicates that plants expressing VP60 antigen may be a new means for oral RHDV immunization.