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

Full item page

Search Results

Now showing 1 - 10 of 23

Project number: 300
Laboratorio en abierto: aPrendiendo a CopiaR el ADN
(2021) Linacero de la Fuente, Rosario; Espino Nuño, Francisco Javier; Pradillo Orellana, Mónica; Gallego Rodríguez, Francisco Javier; González Sánchez, Mónica; Arana Montes, María del Pilar; Beroiz Remirez, Beatriz; Cuñado Rodríguez, Nieves; Figueiras Merino, Ana Margarita; Gil Dones, Félix; de la Peña Gómez, Alicia; Vega Melero, Juan Manuel; Barrios Díaz, José; Llanos Saavedra, Carolina; Moreno Ortiz, María del Carmen; Blasio, Francesco; Fernández Jiménez, Nadia; García García, Isabel; Lantero Bringas, Esther; Lira Garrido, Jaime; Martínez García, Marina; Méndez Cea, Belén; Matallanas Peñas, Beatriz
El objetivo principal del proyecto es la puesta a punto de recursos educativos en abierto (REA) dirigidos a los alumnos de secundaria. El punto de partida será plantear diferentes retos y situaciones que se pueden resolver utilizando distintos recursos científicos, para decidir qué recurso es el más adecuado y cómo se aplica. En esta propuesta la resolución de los problemas planteados estaría basada en la aplicación de una herramienta que ha revolucionado la genética y biología, la reacción en Cadena de la Polimerasa, conocida como PCR.
Oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus
(2000) Gómez, Nestor; Wigdorovitz, Andres; Castañón, Sonia; Gil Dones, Félix; Ordá, Ricardo; Borca, Manuel; Escribano, José
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.
A clinical perspective on the utility of alpha 1 antichymotrypsin for the early diagnosis of calcific aortic stenosis
(Clinical Proteomics, 2017) Martin-Rojas, Tatiana; Mourino-Alvarez, Laura; Gil Dones, Félix; Cuesta, Fernando de la; Rosello-Lleti, Esther; Laborde, Carlos ; Rivera, Miguel; Lopez-Almodovar, Luis Fernando; Lopez, Juan Antonio; Akerstrom, Finn ; Padial, Luis ; Barderas, Maria
Background: Calcific aortic stenosis (CAS) is the most common heart valve disease in the elderly, representing an important economic and social burden in developed countries. Currently, there is no way to predict either the onset or progression of CAS, emphasizing the need to identify useful biomarkers for this condition. Methods: We performed a multi-proteomic analysis on different kinds of samples from CAS patients and healthy donors: tissue, secretome and plasma. The results were validated in an independent cohort of subjects by immunohistochemistry, western blotting and selected reaction monitoring. Results: Alpha 1 antichymotrypsin (AACT) abundance was altered in the CAS samples, as confirmed in the validation phase. The significant changes observed in the amounts of this protein strongly suggest that it could be involved in the molecular mechanisms underlying CAS. In addition, our results suggest there is enhanced release of AACT into the extracellular fluids when the disease commences. Conclusions: The significant increase of AACT in CAS patients suggests it fulfils an important role in the physiopathology of this disease. These results permit us to propose that AACT may serve as a potential marker for the diagnosis of CAS, with considerable clinical value.
Watermelon Profilin: Characterization of a Major Allergen as a Model for Plant-Derived Food Profilins
(International Archives of Allergy Immunology, 2010) Cases, Bárbara ; Pastor Vargas, Carlos; Gil Dones, Félix; Perez-Gordo, Marina; Maroto, Aroa; las Heras, Manuel de las; Vivanco Martínez, Fernando; Cuesta-Herranz, Javier
Background: Plant profilins have been reported as minor allergens. They are a well-known pan-allergen family responsible for cross-reactivity between plant-derived foods and pollens. Watermelon profilin has been reported to be a major allergen in watermelon (Citrullus lanatus).The aim of this study was to characterize recombinant watermelon profilin, confirming its reactivity for diagnostic purposes and the development of immunotherapy. Methods: Native profilin was purified from watermelon extract by affinity chromatography using poly-L-proline. Recombinant His-tagged profilin was produced in Pichia pastoris yeast using pPICZαA vector and purified by metal chelate affinity chromatography. ELISA and immunoblot were carried out with sera from 17 watermelon-allergic patients. Biological activity was tested by the basophil activation test. Results: Native profilin and recombinant profilin were purified and identified by mass spectrometry. Both show similar IgE reactivity in vitro and are biologically active. Conclusions: Similarities were found in the IgE-binding patterns and biological activity of recombinant profilin and native profilin. Recombinant profilin may be a powerful tool for specific diagnosis.
A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants
(2002) Dus Santos, María ; Wigdorovitz, Andrés; Trono, Karina; Rı́os, Raúl ; Franzone, Pascual ; Gil Dones, Félix; Moreno, Javier; Carrillo, Consuelo; Escribano, José ; Borca, Manuel
The expression of antigens in transgenic plants has been increasingly used as an alternative to the classical methodologies for antigen expression in the development of experimental vaccines. However, an important limitation in most cases is the low concentration of the recombinant antigens in the plant tissues, which reduces the possibilities of practical applications. Because the site of insertion of the transferred DNA into the cellular chromosomal DNA is at random, different levels of foreign protein expression in independent transformants is expected. Strategies to allow the evaluation of a high number of the transgenic individuals, usually an expensive and very time consuming process, would permit the selection of those plants presenting the highest levels of recombinant protein expression. Here, we present the development of an experimental immunogen based in the expression of a highly immunogenic epitope from foot and mouth disease virus (FMDV) fused to the glucuronidase (gus A) reporter gene, which allows selection of the transgenic plants by the ß-glucuronidase (ßGUS) enzymatic activity. We produced transgenic plants of alfalfa expressing the immunogenic site between amino acid residues 135–160 of structural protein VP1 (VP135–160), fused to the ßGUS protein. Plants expressing the highest levels of the immunogenic epitope VP135–160, analyzed by Western blot, were efficiently selected based on their levels of ßGUS enzymatic activity. The FMDV epitope expressed in plants was highly immunogenic in mice which developed, after immunization, a strong anti-FMDV antibody response against a synthetic peptide representing the region VP135–160, to native virus VP1, and to purified FMDV particles. Additionally, these mice were completely protected against experimental challenge with the virulent virus. To our knowledge, this constitutes the first report of a peptide-based vaccine produced in transgenic plants that induces a protective immune response when used in experimental hosts. Also, these results demonstrated the possibility of using a novel and simple methodology for obtaining transgenic plants expressing high levels of foreign immunogenic epitopes, which could be directly applied in the development of plant-based vaccines.
Targeting antigens to an invariant epitope of the MHC Class II DR molecule potentiates the immune response to subunit vaccines
(Virus Research, 2011) Pérez-Filgueira, Mariano; Barderas, María G.; Alonso, Covadonga; José M, Escribano; Gil Dones, Félix; Pastor Vargas, Carlos; Vivanco Martínez, Fernando
Recombinant subunit and peptidic vaccines in general present a reduced immunogenicity in vaccinated individuals with respect to the whole pathogen from which they derived. The generation of strong immune responses to these vaccines requires the use of potent adjuvants, high antigen doses and repetitive vaccinations. In this report, we document the enhanced antibody response obtained against two recombinant subunit vaccines by means of targeting to antigen-presenting cells by a recombinant single chain antibody. This antibody, named APCH1, recognizes an epitope of MHC Class II DR molecule preserved in different animal species, including humans. We showed that vaccinal antigens translationally fused to APCH1 antibody and produced by recombinant baculoviruses in insect larvae (Trichoplusia ni), elicited an increased antibody response in comparison with the same antigens alone or fused to a carrier molecule. These results suggest that targeting of antigens to this invariant MHC Class II epitope has immunopotentiating effects that could circumvent the reduced potency of peptidic or subunit vaccines, opening the possibility of widespread application of APCH1 as a new adjuvant antibody of general use.
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) Álvarez Llamas, Gloria; Martín Rojas, Tatiana; Cuesta, Fernando De La; Calvo, Enrique; Gil Dones, Félix; Dardé, Veronica ; López Almodovar, Luis ; Padial, Luis ; Lopez, Juan Antonio; Vivanco Martínez, Fernando; Barderas, Maria
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.
Proteomic characterization of human coronary thrombus in patients with ST-segment elevation acute myocardial infarction
(Journal of Proteomics, 2014) Alonso-Orgaz, Sergio; Moreno-Luna, Rafael; López, Juan ; Gil Dones, Félix; Padial, Luis ; Moreu, Jose; Cuesta, Fernando de la; Barderas, Maria
Abstract Acute myocardial infarction with ST-segment elevation (STEMI) initiates with intraluminal thrombosis and results in total occlusion of the coronary artery. To date, characterization of the coronary thrombus proteome in STEMI patients has not been yet accomplished. Therefore, we aimed to perform an in-depth proteomic characterization of the human coronary thrombus by means of three different approaches: 2-DE followed by mass spectrometry (MALDI MS/MS), 1-DE combined either with liquid chromatography coupled to mass spectrometry in a MALDI TOF/TOF (LC–MALDI-MS/MS), or in a LTQ-Orbitrap (LC–ESI-MS/MS). This approach allowed us to identify a total of 708 proteins in the thrombus. Expression in coronary thrombi (n = 20) of 14 proteins was verified, and the expression of fibrin and 6 cell markers (platelets, monocytes, neutrophils, eosinophils, T-cells and B-cells) quantified by selected reaction monitoring (SRM). A positive correlation of 5 proteins (fermitin homolog 3, thrombospondin-1, myosin-9, beta parvin and ras-related protein Rap-1b) with CD41 was found, pointing out the potential activation of a focal adhesion pathway within thrombus platelets. DIDO1 protein was found to correlate negatively with thrombus fibrin, and was found up-regulated in the plasma of these STEMI patients, which may constitute a starting point for further analyses in the search for biomarkers of thrombosis. Biological significance The proteomic characterization of the human coronary thrombus may contribute to a better understanding of the mechanisms involved in acute coronary syndrome, and thus pave the road for the identification of new therapeutic targets that may help addressing this and other thrombotic diseases. A novel methodology to characterize thrombus composition and expression of a sub-group of proteins is hereby described, which allowed linking protein expression with cellular and ECM matrix composition of the thrombus. Five proteins (fermitin homolog 3, thrombospondin-1, myosin-9, beta parvin and ras-related protein Rap-1b) co-express within the human coronary thrombus with CD41, pointing out the potential activation of a focal adhesion pathway within thrombus platelets during thrombus formation. Besides, the protein death-inducer obliterator 1, found to be expressed within the human coronary thrombus, has been proved to increase in the plasma of STEMI patients, which constitutes an important starting point for further analyses in the search for biomarkers of thrombosis.
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, Laura; Calvo, Enrique; Moreu, José; Padial, Luis; López, Juan; Barderas, María; 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).
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, Ángeles ; Bolaños-Gutiérrez, Amada; Pérez Gil, Jesús; Gil Dones, Félix; Estrada Díaz, María Del Pilar
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.