Proyecto de Investigación: DESARROLLO Y CARACTERIZACION FUNCIONAL DE APTAMEROS COMO HERRAMIENTAS BIOTECNOLOGICAS FRENTE A VIRUS RNA PATOGENOS
Cargando...
Colaboradores
Financiadores
ID
BIO2016-79618-R
Autores
Publicaciones
The Complex Molecules Detector (CMOLD): A Fluidic-Based Instrument Suite to Search for (Bio)chemical Complexity on Mars and Icy Moons
(Mary Ann Liebert Publishers, 2020-09-15) Fairén, Alberto G.; Gómez Elvira, J.; Briones, C.; Prieto Ballesteros, O.; Rodríguez Manfredi, J. A.; López Heredero, R.; Belenguer, T.; Moral, A.; Moreno Paz, M.; Parro García, V.; European Research Council (ERC); Agencia Estatal de Investigación (AEI); Briones, C. [0000-0003-2213-8353]; Prieto Ballesteros, O. [0000-0002-2278-1210]; López Heredero, R. [0000-0002-2197-8388]; Unidad de Excelencia Científica María de Maeztu Centro de Astrobiología del Instituto Nacional de Técnica Aeroespacial y CSIC, MDM-2017-0737
Organic chemistry is ubiquitous in the Solar System, and both Mars and a number of icy satellites of the outer Solar System show substantial promise for having hosted or hosting life. Here, we propose a novel astrobiologically focused instrument suite that could be included as scientific payload in future missions to Mars or the icy moons: the Complex Molecules Detector, or CMOLD. CMOLD is devoted to determining different levels of prebiotic/biotic chemical and structural targets following a chemically general approach (i.e., valid for both terrestrial and nonterrestrial life), as well as their compatibility with terrestrial life. CMOLD is based on a microfluidic block that distributes a liquid suspension sample to three instruments by using complementary technologies: (1) novel microscopic techniques for identifying ultrastructures and cell-like morphologies, (2) Raman spectroscopy for detecting universal intramolecular complexity that leads to biochemical functionality, and (3) bioaffinity-based systems (including antibodies and aptamers as capture probes) for finding life-related and nonlife-related molecular structures. We highlight our current developments to make this type of instruments flight-ready for upcoming Mars missions: the Raman spectrometer included in the science payload of the ESAs Rosalind Franklin rover (Raman Laser Spectrometer instrument) to be launched in 2022, and the biomarker detector that was included as payload in the NASA Icebreaker lander mission proposal (SOLID instrument). CMOLD is a robust solution that builds on the combination of three complementary, existing techniques to cover a wide spectrum of targets in the search for (bio)chemical complexity in the Solar System.
Amino acid substitutions associated with treatment failure for Hepatitis C virus infection
(American Society for Microbiology, 2020-12) Soria, María Eugenia; García Crespo, Carlos; Martínez González, Brenda; Vázquez Sirvent, L.; Lobo Vega, Rebeca; Ávila, Ana Isabel de; Gallego, Isabel; Chen, Qian; García Cehic, Damir; Llorens Revull, Meritxell; Briones, C.; Gómez, Jordi; Ferrer Orta, Cristina; Verdaguer, Nuria; Gregori, Josep; Rodríguez Frías, Francisco; Buti, María; Ignacio Esteban, Juan; Domingo, Esteban; Quer, Josep; Perales, Celia; Ministerio de Economía y Competitividad (MINECO); Agencia Estatal de Investigación (AEI); Instituto de Salud Carlos III (ISCIII); Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas, España; Centro para el Desarrollo Tecnológico Industrial (CDTI); Fundación Ramón Areces; Banco de Santander; CSIC-INTA - Centro de Astrobiología, CAB; Unidad de Excelencia Científica María de Maeztu Centro de Astrobiología del Instituto Nacional de Técnica Aeroespacial y CSIC, MDM-2017-0737
Despite the high virological response rates achieved with current directly acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. The identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultradeep sequencing (UDS) methods, for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitutions (RAS), from 220 patients who failed therapy. They were present frequently in basal and posttreatment virus of patients who failed different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. They also have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value for treatment response, are discussed.
Broad and dynamic diversification of infectious hepatitis c virus in a cell culture environment
(American Society for Microbiology, 2020-02-28) Gallego, Isabel; Eugenia Soria, M.; García Crespo, Carlos; Chen, Q.; Martínez Barragán, P.; Khalfaoui, S.; Martínez González, B.; Sánchez Martín, I.; Palacios Blanco, I.; Isabel de Ávila, A.; García Cehic, Damir; Ignacio Esteban, J.; Gómez, Jordi; Briones, C.; Gregori, Josep; Quer, J.; Perales, C.; Domingo, Esteban; Instituto de Salud Carlos III (ISCIII); Comunidad de Madrid; Ministerio de Economia y Competitividad (MINECO); Fundación Ramón Areces; Banco Santander; Agencia Estatal de Investigación (AEI); Eugenia Soria, M. [0000-0003-1755-6382]; García Crespo, C. [0000-0001-6561-5389]; García Cehic, D. [0000-0002-0009-038X]; Briones, C. [0000-0003-2213-8353]; Domingo, E. [0000-0002-0573-1676]; Martínez González, B. [0000-0002-4482-5181]; Perales Viejo, C. B. [0000-0003-1618-1937]; García Crespo, C. [0000-0001-6561-5389]; Gregori Font, J. [0000-0002-4253-8015]; Gómez, J. [0000-0002-7806-1503]; Quer, J. [0000-0003-0014-084X]
Previous studies documented that long-term hepatitis C virus (HCV) replication in human hepatoma Huh-7.5 cells resulted in viral fitness gain, expansion of the mutant spectrum, and several phenotypic alterations. In the present work, we show that mutational waves (changes in frequency of individual mutations) occurred continuously and became more prominent as the virus gained fitness. They were accompanied by an increasing proportion of heterogeneous genomic sites that affected 1 position in the initial HCV population and 19 and 69 positions at passages 100 and 200, respectively. Analysis of biological clones of HCV showed that these dynamic events affected infectious genomes, since part of the fluctuating mutations became incorporated into viable genomes. While 17 mutations were scored in 3 biological clones isolated from the initial population, the number reached 72 in 3 biological clones from the population at passage 200. Biological clones differed in their responses to antiviral inhibitors, indicating a phenotypic impact of viral dynamics. Thus, HCV adaptation to a specific constant environment (cell culture without external influences) broadens the mutant repertoire and does not focus the population toward a limited number of dominant genomes. A retrospective examination of mutant spectra of foot-and-mouth disease virus passaged in cell cultures suggests a parallel behavior here described for HCV. We propose that virus diversification in a constant environment has its basis in the availability of multiple alternative mutational pathways for fitness gain. This mechanism of broad diversification should also apply to other replicative systems characterized by high mutation rates and large population sizes. IMPORTANCE The study shows that extensive replication of an RNA virus in a constant biological environment does not limit exploration of sequence space and adaptive options. There was no convergence toward a restricted set of adapted genomes. Mutational waves and mutant spectrum broadening affected infectious genomes. Therefore, profound modifications of mutant spectrum composition and consensus sequence diversification are not exclusively dependent on environmental alterations or the intervention of population bottlenecks.
Population Disequilibrium as Promoter of Adaptive Explorations in Hepatitis C Virus
(Multidisciplinary Digital Publishing Institute (MDPI), 2021-04-03) García Crespo, Carlos; Gallego, Isabel; Eugenia Soria, M.; Isabel de Ávila, A.; Martínez González, B.; Vázquez Sirvent, L.; Lobo Vega, Rebeca; Moreno, E.; Gómez, Jordi; Briones, C.; Gregori, Josep; Quer, J.; Domingo, Esteban; Perales, C.; Banco Santander; Fundación Ramón Areces; Instituto de Salud Carlos III (ISCIII); Agencia Estatal de Investigación (AEI); Ministerio de Economía y Competitividad (MINECO); García Crespo, C. [0000-0001-6561-5389]; Martínez González, B. [0000-0002-4482-5181]; Moreno, E. [0000-0002-2301-4558]; Briones, C. [0000-0003-2213-8353]; Quer, J. [0000-0003-0014-084X]; Unidad de Excelencia Científica María de Maeztu Centro de Astrobiología del Instituto Nacional de Técnica Aeroespacial y CSIC, MDM-2017-0737
Replication of RNA viruses is characterized by exploration of sequence space which facilitates their adaptation to changing environments. It is generally accepted that such exploration takes place mainly in response to positive selection, and that further diversification is boosted by modifications of virus population size, particularly bottleneck events. Our recent results with hepatitis C virus (HCV) have shown that the expansion in sequence space of a viral clone continues despite prolonged replication in a stable cell culture environment. Diagnosis of the expansion was based on the quantification of diversity indices, the occurrence of intra-population mutational waves (variations in mutant frequencies), and greater individual residue variations in mutant spectra than those anticipated from sequence alignments in data banks. In the present report, we review our previous results, and show additionally that mutational waves in amplicons from the NS5A-NS5B-coding region are equally prominent during HCV passage in the absence or presence of the mutagenic nucleotide analogues favipiravir or ribavirin. In addition, by extending our previous analysis to amplicons of the NS3- and NS5A-coding region, we provide further evidence of the incongruence between amino acid conservation scores in mutant spectra from infected patients and in the Los Alamos National Laboratory HCV data banks. We hypothesize that these observations have as a common origin a permanent state of HCV population disequilibrium even upon extensive viral replication in the absence of external selective constraints or changes in population size. Such a persistent disequilibrium—revealed by the changing composition of the mutant spectrum—may facilitate finding alternative mutational pathways for HCV antiviral resistance. The possible significance of our model for other genetically variable viruses is discussed
Morphology Clustering Software for AFM Images, Based on Particle Isolation and Artificial Neural Networks
(Institute of Electrical and Electronics Engineers, 2019-11-04) Delgado, A.; Moreno, M.; Vázquez, L. F.; Martín Gago, J. A.; Briones, C.; Ministerio de Economía y Competitividad (MINECO); Comunidad de Madrid; Agencia Estatal de Investigación (AEI); Delgado, A. [0000-0003-4868-3712]; Moreno, M. [0000-0002-6065-4095]; MartínGago, J. A. [0000-0003-2663-491X]; Briones, C. [0000-0003-2213-8353]; Unidad de Excelencia Científica María de Maeztu Centro de Astrobiología del Instituto Nacional de Técnica Aeroespacial y CSIC, MDM-2017-0737
Advanced microscopy techniques currently allow scientists to visualize biomolecules at high resolution. Among them, atomic force microscopy (AFM) shows the advantage of imaging molecules in their native state, without requiring any staining or coating of the sample. Biopolymers, including proteins and structured nucleic acids, are flexible molecules that can fold into alternative conformations for any given monomer sequence, as exemplified by the different three-dimensional structures adopted by RNA in solution. Therefore, the manual analysis of images visualized by AFM and other microscopy techniques becomes very laborious and time-consuming (and may also be inadvertently biased) when large populations of biomolecules are studied. Here we present a novel morphology clustering software, based on particle isolation and artificial neural networks, which allows the automatic image analysis and classification of biomolecules that can show alternative conformations. It has been tested with a set of AFM images of RNA molecules (a 574 nucleotides-long functional region of the hepatitis C virus genome that contains its internal ribosome entry site element) structured in folding buffers containing 0, 2, 4, 6 or 10 mM Mg 2+ . The developed software shows a broad applicability in the microscopy-based analysis of biopolymers and other complex biomolecules.
