Search

La metodología desarrollada en esta investigación fue patentada. A continuación se aportan datos relacionados con la misma: Título: Magnetic beads-based electrochemical biosensor Pub . No . : US 2019 / 0048402 A1 Pub . Date : Feb . 14 , 2019, Microribonucleic acids (miRNAs) have been linked with various regulatory functions and diseases and constitute important targets in future medical diagnostics and prognostics. We report here a novel sensitive and rapid bioelectrochemical strategy for miRNA determination. This strategy involves the development of a sensing approach making use of magnetic beads (MBs) modified with a specific DNA-RNA antibody as capture bioreceptor and amperometric detection implying the H2O2/hydroquinone (HQ) system at disposable screen-printed carbon electrodes (SPCEs). The developed biosensor exhibits a dynamic range from 8.2 to 250 pM and a detection limit of 2.4 pM (60 amol) of a synthetic target without any amplification step in 2 h. The usefulness of the approach was evaluated by analyzing total RNA (RNAt) extracted from metastatic cancer cell lines and human tumor tissues, which demonstrated its potential to perform determination of mature miRNAs in these complex samples. Moreover, the feasibility of the developed methodology to detect simultaneously the expression of two different miRNAs at dual SPCEs (SPdCEs) in one single experiment was also explored. The feasibility to capture and release target miRNAs make the developed methodology also an attractive tool to isolate, purify, and determine target miRNAs with great applicability in the clinical field., Spanish Ministerio de Economía y Competitividad, NANOAVANSENS, Spanish Ministry of Education, MINECO, Spanish Ministry of Education, Culture and Sports, CANNAN RESEARCH & INVESTMENT S.L, Depto. de Química Analítica, Fac. de Ciencias Químicas, TRUE, pub

CRUE-CSIC (Acuerdos Transformativos 2021), An electrochemical biosensing platform for serum autoantibodies (AAbs) detection is reported in this work, exploiting for the first time six Alzheimer's disease (AD)-specific phage-derived and frameshift aberrant HaloTag peptides as receptors, immobilized on magnetic microbeads (MBs) surface and captured on disposable electrodes to perform amperometric detection. Operational analytical characteristics and clinical diagnostic ability of the bioplatform were probed in optimized key experimental conditions by analysing serum AAbs of AD patients and healthy subjects. The value of 100% obtained for AUC, sensitivity, and selectivity from the all peptides combined ROC curve, indicate full AD-diagnostic capability of the methodology, which was further implemented, as proof of concept, in a POC multiplexing platform to detect the signature in a single test over clinically actionable times (1 h 15 min), opening great promise for the type of diagnosis and AD patients’ monitoring follow-up currently pursued., Ministerio de Ciencia e Innovación (MICINN), Instituto de Salud Carlos III (ISCIII), Comunidad de Madrid, Depto. de Química Analítica, Fac. de Ciencias Químicas, TRUE, pub

New biomarkers of Alzheimer's disease (AD) with a diagnostic value in preclinical and prodromal stages are urgently needed. AD-related serum autoantibodies are potential candidate biomarkers. Here, we aimed at identifying AD-related serum autoantibodies using protein microarrays and mass spectrometry-based methods. To this end, an untargeted complementary screening using high-density (42,100 antigens) and low-density (384 antigens) planar protein-epitope signature tag (PrEST) arrays and an immunoprecipitation protocol coupled to mass spectrometry analysis were used for serum autoantibody profiling. From the untargeted screening phase, 377 antigens corresponding to 338 proteins were selected for validation. Out of them, IVD, CYFIP1, and ADD2 seroreactivity was validated using 128 sera from AD patients and controls by PrEST-suspension bead arrays, and ELISA or luminescence Halotag-based bead immunoassay using full-length recombinant proteins. Importantly, IVD, CYFIP1, and ADD2 showed in combination a noticeable AD diagnostic ability. Moreover, IVD protein abundance in the prefrontal cortex was significantly two-fold higher in AD patients than in controls by western blot and immunohistochemistry, whereas CYFIP1 and ADD2 were significantly down-regulated in AD patients. The panel of AD-related autoantigens identified by a comprehensive multiomics approach may provide new insights of the disease and should help in the blood-based diagnosis of Alzheimer's disease. Mass spectrometry raw data are available in the ProteomeXchange database with the access number PXD028392., QC 20211201

Ole e 7 allergen from Olea europaea pollen possesses a major clinical relevance because it produces severe symptoms, such as anaphylaxis, in allergic patients exposed to high olive pollen counts. Ole e 7 is a non-specific lipid transfer protein (nsLTP) characterized by the presence of a tunnel-like hydrophobic cavity, which may be suitable for hosting and, thus, transporting lipids -as it has been described for other nsLTPs-. The identification of the primary amino acid sequence of Ole e 7, and its production as a recombinant allergen, allowed characterizing its lipid-binding properties and its effect at air-liquid interfaces. Fluorescence and interferometry experiments were performed using different phospholipid molecular species and free fatty acids to analyse the lipid-binding ability and specificity of the allergen. Molecular modelling of the allergen was used to determine the potential regions involved in lipid interaction. Changes in Ole e 7 structure after lipid interaction were analysed by circular dichroism. Changes in the IgE binding upon ligand interaction were determined by ELISA. Wilhelmy balance measurements and fluorescence surfactant adsorption tests were performed to analyse the surface activity of the allergen. Using these different approaches, we have demonstrated the ability of Ole e 7 to interact and bind to a wide range of lipids, especially negatively charged phospholipids and oleic acid. We have also identified the protein structural regions and the residues potentially involved in that interaction, suggesting how lipid-protein interactions could define the behaviour of the allergen once inhaled at the airways., Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, Ministerio de Educación, Cultura y Deporte (España), Instituto de Salud Carlos III, Junta de Andalucía. Consejería de Salud, Fondo Europeo de Desarrollo Regional (España), Sección Deptal. de Bioquímica y Biología Molecular (Biológicas), Fac. de Ciencias Biológicas, TRUE, pub

Received 19 September 2019, Revised 2 January 2020, Accepted 5 January 2020, Available online 7 January 2020., Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer related death worldwide. Its diagnosis at early stages would significantly improve the survival of CRC patients. The humoral immune response has been demonstrated useful for cancer diagnosis, predating clinical symptoms up to 3 years. Here, we employed an in-depth seroproteomic approach to identify proteins that elicit a humoral immune response in CRC patients. The seroproteomic approach relied on the immunoprecipitation with patient-derived autoantibodies of proteins from CRC cell lines with different metastatic properties followed by LC-MS/MS. After bioinformatics, we focused on 31 targets of CRC autoantibodies. After WB and IHC validation, ERP44 and TALDO1 showed potential to discriminate disease-free and metastatic CRC patients, and time to recurrence of CRC patients in stage II. Using plasma samples of 30 healthy individuals, 28 premalignant individuals, and 32 CRC patients, nine out of 13 selected targets for seroreactive analysis showed significant diagnostic ability to discriminate either CRC patients or premalignant subjects from controls. Our results suggest that the here defined panel of CRC autoantibodies and their target proteins should be included in CRC blood-based biomarker panels to get a clinically useful blood-based diagnostic signature for CRC detection. Significance: Colorectal cancer is one of the deadliest cancer types mainly due to its late diagnosis. Its early diagnosis, therefore, is of great importance since it would significantly improve the survival of CRC patients. In our work, the in-depth seroproteomic analysis of colorectal cancer using isolated IgGs from colorectal cancer patients and controls and protein extract of colorectal cancer cells provide the identification of valuable biomarkers with diagnostic and prognostic ability of the disease., Ministerio de Economía y Empresa de España (MINECO) - Programa Ramon y Cajal, Instituto de Salud Carlos III (ISCIII), Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) (España), Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid (España), Fondo Social Europeo, Ministerio de Educación, Cultura y Deporte de España, The Flanders Research Foundation (FWO), Unidad Docente de Bioquímica y Biología Molecular, Fac. de Óptica y Optometría, TRUE, pub

Strategies for installing authentic ADP-ribosylation (ADPr) at desired positions are fundamental for creating the tools needed to explore this elusive post-translational modification (PTM) in essential cellular processes. Here, we describe a phospho-guided chemoenzymatic approach based on the Ser-ADPr writer complex for rapid, scalable preparation of a panel of pure, precisely modified peptides. Integrating this methodology with phage display technology, we have developed site-specific as well as broad-specificity antibodies to monoADPr. These recombinant antibodies have been selected and characterized using multiple ADP-ribosylated peptides and tested by immunoblotting and immunofluorescence for their ability to detect physiological ADPr events. Mono-ADPr proteomics and poly-to-mono comparisons at the modification site level have revealed the prevalence of mono-ADPr upon DNA damage and illustrated its dependence on PARG and ARH3. These and future tools created on our versatile chemical biology-recombinant antibody platform have broad potential to elucidate ADPr signaling pathways in health and disease.

Dada la importancia del inglés y de las tecnologías móviles hoy día, los estudiantes utilizarán dichas tecnologías para reforzar el aprendizaje de Biología, y motivarlos a estudiar, mediante dos actividades: crear test Kahoot en inglés y concursar.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer related death worldwide. Its diagnosis at early stages would significantly improve the survival of CRC patients. The humoral immune response has been demonstrated useful for cancer diagnosis, predating clinical symptoms up to 3years. Here, we employed an in-depth seroproteomic approach to identify proteins that elicit a humoral immune response in CRC patients. The seroproteomic approach relied on the immunoprecipitation with patient-derived autoantibodies of proteins from CRC cell lines with different metastatic properties followed by LC-MS/MS. After bioinformatics, we focused on 31 targets of CRC autoantibodies. After WB and IHC validation, ERP44 and TALDO1 showed potential to discriminate disease-free and metastatic CRC patients, and time to recurrence of CRC patients in stage II. Using plasma samples of 30 healthy individuals, 28 premalignant individuals, and 32 CRC patients, nine out of 13 selected targets for seroreactive analysis showed significant diagnostic ability to discriminate either CRC patients or premalignant subjects from controls. Our results suggest that the here defined panel of CRC autoantibodies and their target proteins should be included in CRC blood-based biomarker panels to get a clinically useful blood-based diagnostic signature for CRC detection. SIGNIFICANCE: Colorectal cancer is one of the deadliest cancer types mainly due to its late diagnosis. Its early diagnosis, therefore, is of great importance since it would significantly improve the survival of CRC patients. In our work, the in-depth seroproteomic analysis of colorectal cancer using isolated IgGs from colorectal cancer patients and controls and protein extract of colorectal cancer cells provide the identification of valuable biomarkers with diagnostic and prognostic ability of the disease.

The characterization of the humoral response in Alzheimer’s disease (AD) patients might aid in detecting the disease at early stages. We have combined phage display and protein microarrays to identify AD autoantibodies and their target biomarkers. After enrichment of the T7 phage display libraries from AD and healthy brain tissue mRNA in AD-specific phages, 1536 monoclonal phages were printed on microarrays to probe them with 8 AD and 8 healthy control sera. A total of 57 phages showed higher seroreactivity in AD. In total, 13 out of the 44 unique sequences displayed on the phages were selected for validation using 68 AD and 52 healthy control sera. Peptides from Anthrax toxin receptor 1, Nuclear protein 1, Glycogen phosphorylase, and Olfactory receptor 8J1 expressed in bacteria as HaloTag fusion proteins showed a statistically significant ability to discriminate between AD patients and controls. The identified panel of AD autoantibodies might provide new insights into the blood-based diagnosis of the disease.

Olive pollen is a major allergenic source worldwide due to its extensive cultivation. We have combined available genomics data with a comprehensive proteomics approach to get the annotated olive tree ( Olea europaea L.) pollen proteome and define its complex allergenome. A total of 1907 proteins were identified by LC-MS/MS using predicted protein sequences from its genome. Most proteins (60%) were predicted to possess catalytic activity and be involved in metabolic processes. In total, 203 proteins belonging to 47 allergen families were found in olive pollen. A peptidyl-prolyl cis-trans isomerase, cyclophilin, produced in Escherichia coli, was found as a new olive pollen allergen (Ole e 15). Most Ole e 15-sensitized patients were children (63%) and showed strong IgE recognition to the allergen. Ole e 15 shared high sequence identity with other plant, animal, and fungal cyclophilins and presented high IgE cross-reactivity with pollen, plant food, and animal extracts.

El proyecto Innova-Docencia 70 es una continuación del proyecto Innova-Docencia 30 que se desarrolló durante el curso académico 2016-2017 y su objetivo principal es implementar una nueva estrategia de enseñanza/aprendizaje que motive a los estudiantes hacia el estudio de Biología, favorezca la comprensión de los contenidos y les permita conectar los nuevos conocimientos adquiridos con el mundo real que les rodea. Motivación, comprensión e interdisciplinaridad son tres grandes retos a los que se enfrenta el profesorado de la asignatura de Biología del Grado en Química, que se imparte durante el primer cuatrimestre y que es de carácter obligatorio para los estudiantes de primero (6 ECTS), dado el escaso interés que despierta esta asignatura en los estudiantes de esta disciplina (además de otras, como física, arquitectura etc.), al considerarla una asignatura difícil de aprender y de poca utilidad. Esta estrategia docente tiene como eje metodológico principal la Biomímesis, pero a su vez se apoya en otras dos metodologías: (1) La enseñanza/aprendizaje basada en Proyectos y (2) Los Museos como espacios de enseñanza/aprendizaje. Todas ellas comparten un mismo principio: motivar al estudiante a aprender, implicándole en su propio proceso de aprendizaje. El estudiante ha de planificar, desarrollar y evaluar un proyecto (de carácter interdisciplinar) que le interesa, al ser elegido por él, y que le permite conectar lo académico con la vida.

Uno de los principales problemas con los que se enfrentan los profesores de Biología es el escaso interés que despierta esta asignatura en los estudiantes universitarios de algunas disciplinas -como química, física, arquitectura o ingeniería- al considerarla una asignatura difícil de aprender y de poca utilidad. Así, motivación, comprensión e interdisciplinaridad son tres retos a los que se enfrenta el profesorado de la asignatura de Biología del Grado en Química. El objetivo general del proyecto ha sido contribuir a la mejora de la enseñanza/aprendizaje de la asignatura de Biología. Para ello el profesorado ha implementado en el aula, y de forma combinada, dos metodologías: La "Biomímesis" y "La enseñanza/aprendizaje basada en Proyectos", que han demostrado ser herramientas muy útiles en el aula, para despertar la motivación y el interés de los estudiantes por el aprendizaje de Biología. Por un lado, al implicarles en su propio proceso de aprendizaje, los estudiantes han tenido un papel protagonista al ser los responsables del desarrollo de proyectos interesantes y retadores, elegidos por ellos mismos. Por otro lado, les ha permitido ver la aplicación o transferencia sostenible de lo aprendido en el aula a la sociedad y el medio ambiente. A estas dos metodologías didácticas se ha unido una tercera: Los Museos de Ciencia Naturales como espacios de enseñanza/aprendizaje de ciencia por investigación. Un total de 2 grupos de teoría han participado en esta experiencia docente. Los trabajos realizados han sido expuestos a otros estudiantes (en el aula) y al público general (en el Museo Nacional de Ciencias Naturales).