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3. Client applications and Server Side docker for management of RNASeq and/or VariantSeq workflows and pipelines of the GPRO Suite

Author

Hafez, Ahmed, Soriano, Beatriz, Elsayed, Aya A., Futami, Ricardo, Ceprian, Raquel, Ramos-Ruiz, Ricardo, Martinez, Genis, Roig, Francisco J., Torres-Font, Miguel A., Naya-Català, Fernando, Calduch-Giner, Josep Alvar, Trilla-Fuertes, Lucia, Gamez-Pozo, Angelo, Arnau, Vicente, Sempere, Jose M., Perez-Sánchez, Jaume, Gabaldón, Toni, and Llorens, Carlos

Subjects
Quantitative Biology - Genomics
Abstract

The GPRO suite is an in-progress bioinformatic project for -omic data analyses. As part of the continued growth of this project, we introduce a client side & server side solution for comparative transcriptomics and analysis of variants. The client side consists of two Java applications called "RNASeq" and "VariantSeq" to manage workflows for RNA-seq and Variant-seq analysis, respectively, based on the most common command line interface tools for each topic. Both applications are coupled with a Linux server infrastructure (named GPRO Server Side) that hosts all dependencies of each application (scripts, databases, and command line interface tools). Implementation of the server side requires a Linux operating system, PHP, SQL, Python, bash scripting, and third-party software. The GPRO Server Side can be deployed via a Docker container that can be installed in the user's PC using any operating system or on remote servers as a cloud solution. The two applications are available as desktop and cloud applications and provide two execution modes: a Step-by-Step mode enables each step of a workflow to be executed independently and a Pipeline mode allows all steps to be run sequentially. The two applications also feature an experimental support system called GENIE that consists of a virtual chatbot/assistant and a pipeline jobs panel coupled with an expert system. The chatbot can troubleshoot issues with the usage of each tool, the pipeline job panel provides information about the status of each task executed in the GPRO Server Side, and the expert provides the user with a potential recommendation to identify or fix failed analyses. The two applications and the GPRO Server Side combine the user-friendliness and security of client software with the efficiency of front-end & back-end solutions to manage command line interface software for RNA-seq and variant-seq analysis via interface environments.

Published
2022

4. Silymarin-Enriched Extract from Milk Thistle Activates Thermogenesis in a Preclinical Model of High-Fat-Diet-Induced Obesity to Relieve Systemic Meta-Inflammation.

Author

Reguero, Marina, Reglero, Guillermo, Quintela, José Carlos, Ramos-Ruiz, Ricardo, Ramírez de Molina, Ana, and Gómez de Cedrón, Marta

Abstract

Background: Obesity and aging are associated with the progressive loss of brown adipose tissue (BAT), an increase in visceral white adipose tissue (vWAT), and a reduction in subcutaneous white adipose tissue (sWAT). The progressive expansion of visceral obesity promotes a low grade of systemic chronic inflammation (meta-inflammation), contributing to the onset of comorbidities such as type 2 diabetes mellitus (T2DM), metabolic syndrome, and even cancer. Thus, preserving the thermogenic activity of adipose tissue and improving the metabolic flexibility of sWAT could be an effective strategy to prevent the development of metabolic chronic diseases and promote healthy aging. Precision nutrition has emerged as a complementary approach to control the metabolic alterations associated with unhealthy obesity and aging. In a previous work, we described that a silymarin-enriched extract from milk thistle (Mthistle) increased markers of browning and thermogenesis in vitro in human differentiated adipocytes (SGBS). Objectives/Methods: Therefore, this study aims to evaluate the potential of Mthistle to activate thermogenesis in a preclinical model of high-fat diet (HFD)-induced obesity (DIO). Results: Our results demonstrate that Mthistle increases systemic energy expenditure (EE), preserves body temperature after cold exposure, improves insulin resistance, and reduces inflammatory markers in WAT. Conclusions: Based on these results, silymarin-enriched extract from Mthistle may be proposed as a nutraceutical for the management of metabolic chronic diseases and/or accelerated aging. [ABSTRACT FROM AUTHOR]

Published
2024
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7. Comprehensive Characterization of the Mutational Landscape in Localized Anal Squamous Cell Carcinoma

Author

Trilla-Fuertes, Lucía, Ghanem, Ismael, Maurel, Joan, G-Pastrián, Laura, Mendiola, Marta, Peña, Cristina, López-Vacas, Rocío, Prado-Vázquez, Guillermo, López-Camacho, Elena, Zapater-Moros, Andrea, Heredia, Victoria, Cuatrecasas, Miriam, García-Alfonso, Pilar, Capdevila, Jaume, Conill, Carles, García-Carbonero, Rocío, Heath, Karen E., Ramos-Ruiz, Ricardo, Llorens, Carlos, Campos-Barros, Ángel, Gámez-Pozo, Angelo, Feliu, Jaime, and Vara, Juan Ángel Fresno

Published
2020
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8. Genetic Profile and Functional Proteomics of Anal Squamous Cell Carcinoma: Proposal for a Molecular Classification

Author

Trilla-Fuertes, Lucía, Ghanem, Ismael, Gámez-Pozo, Angelo, Maurel, Joan, G-Pastrián, Laura, Mendiola, Marta, Peña, Cristina, López-Vacas, Rocío, Prado-Vázquez, Guillermo, López-Camacho, Elena, Zapater-Moros, Andrea, Heredia, Victoria, Cuatrecasas, Miriam, García-Alfonso, Pilar, Capdevila, Jaume, Conill, Carles, García-Carbonero, Rocío, Ramos-Ruiz, Ricardo, Fortes, Claudia, Llorens, Carlos, Nanni, Paolo, Fresno Vara, Juan Ángel, and Feliu, Jaime

Published
2020
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9. Description of the genetic variants identified in a cohort of patients diagnosed with localized anal squamous cell carcinoma and treated with panitumumab

Author

Trilla-Fuertes, Lucía, Gámez-Pozo, Angelo, Maurel, Joan, Garcia-Carbonero, Rocio, Capdevila, Jaume, G-Pastrián, Laura, Mendiola, Marta, Peña, Cristina, López-Vacas, Rocío, Cuatrecasas, Miriam, García-Alfonso, Pilar, Ramos-Ruiz, Ricardo, Llorens, Carlos, Ghanem, Ismael, Conill, Carles, Heredia-Soto, Victoria, Campos-Barros, Ángel, Fresno Vara, Juan Ángel, and Feliu, Jaime

Published
2021
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11. Vaccine breakthrough infections with SARS-CoV-2 Alpha mirror mutations in Delta Plus, Iota, and Omicron

Author

Martinez-Gonzalez, Brenda, Vazquez-Sirvent, Lucia, Soria, Maria E., Minguez, Pablo, Salar-Vidal, Llanos, Garcia-Crespo, Carlos, Gallego, Isabel, de Avila, Ana I., Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Esteban, Jaime, Fernandez- Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruiz-Hornillos, Javier, Perez-Jorge, Concepcion, Domingo, Esteban, and Perales, Celia

Subjects
Gene mutations -- Research, Immunological research, Health care industry
Abstract

Replication of SARS-CoV-2 in the human population is defined by distributions of mutants that are present at different frequencies within the infected host and can be detected by ultra-deep sequencing techniques. In this study, we examined the SARS-CoV-2 mutant spectra of amplicons from the spike-coding (S-coding) region of 5 nasopharyngeal isolates derived from patients with vaccine breakthrough. Interestingly, all patients became infected with the Alpha variant, but amino acid substitutions that correspond to the Delta Plus, Iota, and Omicron variants were present in the mutant spectra of the resident virus. Deep sequencing analysis of SARS-CoV-2 from patients with vaccine breakthrough revealed a rich reservoir of mutant types and may also identify tolerated substitutions that can be represented in epidemiologically dominant variants., Introduction SARS-CoV-2 continues its diversification worldwide, and a new variant termed Omicron (B.l.1.529), carrying a large number of mutations, was recently described in South Africa and classified as a potential [...]

Published
2022
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13. Multi-omics Characterization of Response to PD-1 Inhibitors in Advanced Melanoma

Author

Trilla-Fuertes, Lucía, primary, Gámez-Pozo, Angelo, additional, Prado-Vázquez, Guillermo, additional, López-Vacas, Rocío, additional, Soriano, Virtudes, additional, Garicano, Fernando, additional, Lecumberri, M. José, additional, Rodríguez de la Borbolla, María, additional, Majem, Margarita, additional, Pérez-Ruiz, Elisabeth, additional, González-Cao, María, additional, Oramas, Juana, additional, Magdaleno, Alejandra, additional, Fra, Joaquín, additional, Martín-Carnicero, Alfonso, additional, Corral, Mónica, additional, Puértolas, Teresa, additional, Ramos-Ruiz, Ricardo, additional, Dittmann, Antje, additional, Nanni, Paolo, additional, Fresno Vara, Juan Ángel, additional, and Espinosa, Enrique, additional

Published
2023
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14. SARS-CoV-2 Mutant Spectra at Different Depth Levels Reveal an Overwhelming Abundance of Low Frequency Mutations

Author

Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Agencia Estatal de Investigación (España), Comunidad de Madrid, Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Martínez-González, Brenda [0000-0002-4482-5181], Soria, María Eugenia [0000-0002-4719-3351], Lobo-Vega, Rebeca [0000-0002-4882-6763], Mínguez, Pablo [0000-0003-4099-9421], Llorens, Carlos [0000-0003-1402-4743], Ramos-Ruiz, Ricardo [0000-0002-6331-9786], Cortón, Marta [0000-0003-0087-1626], García-Crespo, Carlos [0000-0001-6561-5389], Durán-Pastor, Antoni [0000-0002-4248-0554], Delgado, Soledad [0000-0003-4868-3712], López-Galíndez, Cecilio [0000-0002-2324-9584], Enjuanes Sánchez, Luis [0000-0002-0854-0226], Salar-Vidal, Llanos [0000-0002-3052-0176], Esteban, Jaime [0000-0002-8971-3167], Ayuso, Carmen [0000-0002-9242-7065], Verdaguer, Núria [0000-0001-8826-7129], Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Somovilla, Pilar, Durán-Pastor, Antoni, Gallego, Isabel, Ávila, Ana Isabel de, Delgado, Soledad, Morán, Federico, López-Galíndez, Cecilio, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban-Muñoz, Mario, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, Perales, Celia, Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Agencia Estatal de Investigación (España), Comunidad de Madrid, Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Martínez-González, Brenda [0000-0002-4482-5181], Soria, María Eugenia [0000-0002-4719-3351], Lobo-Vega, Rebeca [0000-0002-4882-6763], Mínguez, Pablo [0000-0003-4099-9421], Llorens, Carlos [0000-0003-1402-4743], Ramos-Ruiz, Ricardo [0000-0002-6331-9786], Cortón, Marta [0000-0003-0087-1626], García-Crespo, Carlos [0000-0001-6561-5389], Durán-Pastor, Antoni [0000-0002-4248-0554], Delgado, Soledad [0000-0003-4868-3712], López-Galíndez, Cecilio [0000-0002-2324-9584], Enjuanes Sánchez, Luis [0000-0002-0854-0226], Salar-Vidal, Llanos [0000-0002-3052-0176], Esteban, Jaime [0000-0002-8971-3167], Ayuso, Carmen [0000-0002-9242-7065], Verdaguer, Núria [0000-0001-8826-7129], Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Somovilla, Pilar, Durán-Pastor, Antoni, Gallego, Isabel, Ávila, Ana Isabel de, Delgado, Soledad, Morán, Federico, López-Galíndez, Cecilio, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban-Muñoz, Mario, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, and Perales, Celia

Abstract

Populations of RNA viruses are composed of complex and dynamic mixtures of variant genomes that are termed mutant spectra or mutant clouds. This applies also to SARS-CoV-2, and mutations that are detected at low frequency in an infected individual can be dominant (represented in the consensus sequence) in subsequent variants of interest or variants of concern. Here we briefly review the main conclusions of our work on mutant spectrum characterization of hepatitis C virus (HCV) and SARS-CoV-2 at the nucleotide and amino acid levels and address the following two new questions derived from previous results: (i) how is the SARS-CoV-2 mutant and deletion spectrum composition in diagnostic samples, when examined at progressively lower cut-off mutant frequency values in ultra-deep sequencing; (ii) how the frequency distribution of minority amino acid substitutions in SARS-CoV-2 compares with that of HCV sampled also from infected patients. The main conclusions are the following: (i) the number of different mutations found at low frequency in SARS-CoV-2 mutant spectra increases dramatically (50- to 100-fold) as the cut-off frequency for mutation detection is lowered from 0.5% to 0.1%, and (ii) that, contrary to HCV, SARS-CoV-2 mutant spectra exhibit a deficit of intermediate frequency amino acid substitutions. The possible origin and implications of mutant spectrum differences among RNA viruses are discussed.

Published
2022

15. SARS-CoV-2 Point Mutation and Deletion Spectra and Their Association with Different Disease Outcomes

Author

Ramos-Ruiz, Ricardo [0000-0002-6331-9786], Enjuanes Sánchez, Luis [0000-0002-0854-0226], Esteban, Jaime [0000-0002-8971-3167], Verdaguer, Núria [0000-0001-8826-7129], Domingo, Esteban [0000-0002-0573-1676], Perales, Celia [0000-0003-1618-1937], Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Gallego, Isabel, Ávila, Ana Isabel de, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, Perales, Celia, Ramos-Ruiz, Ricardo [0000-0002-6331-9786], Enjuanes Sánchez, Luis [0000-0002-0854-0226], Esteban, Jaime [0000-0002-8971-3167], Verdaguer, Núria [0000-0001-8826-7129], Domingo, Esteban [0000-0002-0573-1676], Perales, Celia [0000-0003-1618-1937], Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Gallego, Isabel, Ávila, Ana Isabel de, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, and Perales, Celia

Abstract

Mutant spectra of RNA viruses are important to understand viral pathogenesis and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultradeep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of 30 nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low-frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype, and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three-dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19.

Published
2022

16. SARS-CoV-2 Point Mutation and Deletion Spectra, and Their Association with Different Disease Outcome

Author

Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Instituto de Salud Carlos III, Comunidad de Madrid, Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Ramos-Ruiz, Ricardo [0000-0002-6331-9786], Enjuanes Sánchez, Luis [0000-0002-0854-0226], Esteban, Jaime [0000-0002-8971-3167], Verdaguer, Núria [0000-0001-8826-7129], Domingo, Esteban [0000-0002-0573-1676], Perales, Celia [0000-0003-1618-1937], Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Gallego, Isabel, Ávila, Ana Isabel de, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, Perales, Celia, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Instituto de Salud Carlos III, Comunidad de Madrid, Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Ramos-Ruiz, Ricardo [0000-0002-6331-9786], Enjuanes Sánchez, Luis [0000-0002-0854-0226], Esteban, Jaime [0000-0002-8971-3167], Verdaguer, Núria [0000-0001-8826-7129], Domingo, Esteban [0000-0002-0573-1676], Perales, Celia [0000-0003-1618-1937], Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Gallego, Isabel, Ávila, Ana Isabel de, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, and Perales, Celia

Abstract

Mutant spectra of RNA viruses are important to understand viral pathogenesis, and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultra-deep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of thirty nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19.

Published
2022

17. Client Applications and Server-Side Docker for Management of RNASeq and/or VariantSeq Workflows and Pipelines of the GPRO Suite

Author

European Commission, Ministerio de Asuntos Económicos y Transformación Digital (España), Ministerio de Ciencia e Innovación (España), Agencia Estatal de Investigación (España), Generalitat Valenciana, Hafez, Ahmed, Soriano, Beatriz, Elsayed, Aya Allah, Futami, Ricardo, Ceprian, Raquel, Ramos-Ruiz, Ricardo, Martínez, Genis, Roig, Francisco J., Torres-Font, Miguel Ángel, Naya-Català, Fernando, Calduch-Giner, Josep A., Trilla-Fuertes, Lucía, Gamez-Pozo, Angelo, Arnau, Vicente, Sempere-Luna, Jose María, Pérez-Sánchez, Jaume, Gabaldón, Toni, Llorens, Carlos, European Commission, Ministerio de Asuntos Económicos y Transformación Digital (España), Ministerio de Ciencia e Innovación (España), Agencia Estatal de Investigación (España), Generalitat Valenciana, Hafez, Ahmed, Soriano, Beatriz, Elsayed, Aya Allah, Futami, Ricardo, Ceprian, Raquel, Ramos-Ruiz, Ricardo, Martínez, Genis, Roig, Francisco J., Torres-Font, Miguel Ángel, Naya-Català, Fernando, Calduch-Giner, Josep A., Trilla-Fuertes, Lucía, Gamez-Pozo, Angelo, Arnau, Vicente, Sempere-Luna, Jose María, Pérez-Sánchez, Jaume, Gabaldón, Toni, and Llorens, Carlos

Abstract

The GPRO suite is an in-progress bioinformatic project for -omics data analysis. As part of the continued growth of this project, we introduce a client- and server-side solution for comparative transcriptomics and analysis of variants. The client-side consists of two Java applications called “RNASeq” and “VariantSeq” to manage pipelines and workflows based on the most common command line interface tools for RNA-seq and Variant-seq analysis, respectively. As such, “RNASeq” and “VariantSeq” are coupled with a Linux server infrastructure (named GPRO Server-Side) that hosts all dependencies of each application (scripts, databases, and command line interface software). Implementation of the Server-Side requires a Linux operating system, PHP, SQL, Python, bash scripting, and third-party software. The GPRO Server-Side can be installed, via a Docker container, in the user’s PC under any operating system or on remote servers, as a cloud solution. “RNASeq” and “VariantSeq” are both available as desktop (RCP compilation) and web (RAP compilation) applications. Each application has two execution modes: a step-by-step mode enables each step of the workflow to be executed independently, and a pipeline mode allows all steps to be run sequentially. “RNASeq” and “VariantSeq” also feature an experimental, online support system called GENIE that consists of a virtual (chatbot) assistant and a pipeline jobs panel coupled with an expert system. The chatbot can troubleshoot issues with the usage of each tool, the pipeline jobs panel provides information about the status of each computational job executed in the GPRO Server-Side, while the expert system provides the user with a potential recommendation to identify or fix failed analyses. Our solution is a ready-to-use topic specific platform that combines the user-friendliness, robustness, and security of desktop software, with the efficiency of cloud/web applications to manage pipelines and workflows based on command line interface softw

Published
2023

18. Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing

Author

Instituto de Salud Carlos III, European Commission, Ministerio de Economía y Empresa (España), Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Fundació La Marató de TV3, Ministerio de Ciencia e Innovación (España), Comunidad de Madrid, CSIC - Plataforma Temática Interdisciplinar del CSIC Salud Global (PTI Salud Global), Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, Llorens-Revull, Meritxell, Martínez-González, Brenda, Quer, Josep, Esteban, Juan Ignacio, Núñez-Moreno, Gonzalo, Mínguez, Pablo, Burgui, Idoia, Ramos-Ruiz, Ricardo, Soria, María Eugenia, Rico, Angie, Riveiro-Barciela, Mar, Sauleda, Silvia, Piron, María, Corrales, Irene, Borràs, Francesc E., Rodríguez-Frías, Francisco, Rando, Ariadna, Ramírez-Serra, Clara, Camós, Silvia, Domingo, Esteban, Bes, Marta, Perales, Celia, Costafreda, María Isabel, Instituto de Salud Carlos III, European Commission, Ministerio de Economía y Empresa (España), Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Fundació La Marató de TV3, Ministerio de Ciencia e Innovación (España), Comunidad de Madrid, CSIC - Plataforma Temática Interdisciplinar del CSIC Salud Global (PTI Salud Global), Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, Llorens-Revull, Meritxell, Martínez-González, Brenda, Quer, Josep, Esteban, Juan Ignacio, Núñez-Moreno, Gonzalo, Mínguez, Pablo, Burgui, Idoia, Ramos-Ruiz, Ricardo, Soria, María Eugenia, Rico, Angie, Riveiro-Barciela, Mar, Sauleda, Silvia, Piron, María, Corrales, Irene, Borràs, Francesc E., Rodríguez-Frías, Francisco, Rando, Ariadna, Ramírez-Serra, Clara, Camós, Silvia, Domingo, Esteban, Bes, Marta, Perales, Celia, and Costafreda, María Isabel

Abstract

MicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies in miRNA patterns and their validation are still frequent due to differences in sample origin, EV isolation, and miRNA sequencing methods. The aim of the present study is to find a reliable EV isolation method for miRNA sequencing, adequate for clinical application. To this aim, two comparative studies were performed in parallel with the same human plasma sample: (i) isolation and characterization of EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), and its combination (SEC+GRAD) and (ii) evaluation of the yield of miRNA sequences obtained using NextSeq 500 (Illumina) and three miRNA library preparation protocols: NEBNext, NEXTFlex, and SMARTer smRNA-seq. The conclusion of comparison (i) is that recovery of the largest amount of EVs and reproducibility were attained with SEC, but GRAD and SEC+GRAD yielded purer EV preparations. The conclusion of (ii) is that the NEBNext library showed the highest reproducibility in the number of miRNAs recovered and the highest diversity of miRNAs. These results render the combination of GRAD EV isolation and NEBNext library preparation for miRNA retrieval as adequate for clinical applications using plasma samples.

Published
2023

19. Client Applications and Server-Side Docker for Management of RNASeq and/or VariantSeq Workflows and Pipelines of the GPRO Suite

Author

Hafez, Ahmed Ibrahem, primary, Soriano, Beatriz, additional, Elsayed, Aya Allah, additional, Futami, Ricardo, additional, Ceprian, Raquel, additional, Ramos-Ruiz, Ricardo, additional, Martinez, Genis, additional, Roig, Francisco Jose, additional, Torres-Font, Miguel Angel, additional, Naya-Catala, Fernando, additional, Calduch-Giner, Josep Alvar, additional, Trilla-Fuertes, Lucia, additional, Gamez-Pozo, Angelo, additional, Arnau, Vicente, additional, Sempere-Luna, Jose Maria, additional, Perez-Sanchez, Jaume, additional, Gabaldon, Toni, additional, and Llorens, Carlos, additional

Published
2023
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21. Vaccine breakthrough infections with SARS-CoV-2 Alpha mirror mutations in Delta Plus, Iota, and Omicron

Author

Instituto de Salud Carlos III, Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Comunidad de Madrid, European Commission, Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Soria, María Eugenia, Mínguez, Pablo, Salar-Vidal, Llanos, García-Crespo, Carlos, Gallego, Isabel, Ávila, Ana Isabel de, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Pérez-Jorge, Concepción, Domingo, Esteban, Perales, Celia, Instituto de Salud Carlos III, Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Comunidad de Madrid, European Commission, Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Soria, María Eugenia, Mínguez, Pablo, Salar-Vidal, Llanos, García-Crespo, Carlos, Gallego, Isabel, Ávila, Ana Isabel de, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Pérez-Jorge, Concepción, Domingo, Esteban, and Perales, Celia

Abstract

Replication of SARS-CoV-2 in the human population is defined by distributions of mutants that are present at different frequencies within the infected host and can be detected by ultra-deep sequencing techniques. In this study, we examined the SARS-CoV-2 mutant spectra of amplicons from the spike-coding (S-coding) region of 5 nasopharyngeal isolates derived from patients with vaccine breakthrough. Interestingly, all patients became infected with the Alpha variant, but amino acid substitutions that correspond to the Delta Plus, Iota, and Omicron variants were present in the mutant spectra of the resident virus. Deep sequencing analysis of SARS-CoV-2 from patients with vaccine breakthrough revealed a rich reservoir of mutant types and may also identify tolerated substitutions that can be represented in epidemiologically dominant variants.

Published
2022

22. SARS-CoV-2 mutant spectra reveal differences between COVID-19 severity categories

Author

Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Agencia Estatal de Investigación (España), Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, European Commission, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Somovilla, Pilar, Durán-Pastor, Antoni, Gallego, Isabel, Ávila, Ana Isabel de, Delgado, Soledad, Morán, Federico, López-Galíndez, Cecilio, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban-Muñoz, Mario, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, Perales, Celia, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Agencia Estatal de Investigación (España), Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, European Commission, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Somovilla, Pilar, Durán-Pastor, Antoni, Gallego, Isabel, Ávila, Ana Isabel de, Delgado, Soledad, Morán, Federico, López-Galíndez, Cecilio, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban-Muñoz, Mario, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, and Perales, Celia

Abstract

RNA virus populations are composed of complex mixtures of genomes that are termed mutant spectra. SARS-CoV-2 replicates as a viral quasispecies, and mutations that are detected at low frequencies in a host can be dominant in subsequent variants. We have studied mutant spectrum complexities of SARS-CoV-2 populations derived from thirty nasopharyngeal swabs of patients infected during the first wave (April 2020) in the Hospital Universitario Fundación Jiménez Díaz. The patients were classified according to the COVID-19 severity in mild (non-hospitalized), moderate (hospitalized) and exitus (hospitalized with ICU admission and who passed away due to COVID-19). Using ultra-deep sequencing technologies (MiSeq, Illumina), we have examined four amplicons of the nsp12 (polymerase)-coding region and two amplicons of the spike-coding region. Ultra-deep sequencing data were analyzed with different cut-off frequency for mutation detection. Average number of different point mutations, mutations per haplotype and several diversity indices were significantly higher in SARS-CoV-2 isolated from patients who developed mild disease. A feature that we noted in the SARS-CoV-2 mutant spectra from diagnostic samples is the remarkable absence of mutations at intermediate frequencies, and an overwhelming abundance of mutations at frequencies lower than 10%. Thus, the decrease of the cut-off frequency for mutation detection from 0.5% to 0.1% revealed an increasement (50- to 100 fold) in the number of different mutations. The significantly higher frequency of mutations in virus from patients displaying mild than moderate or severe disease was maintained with the 0.1% cut- off frequency. To evaluate whether the frequency repertoire of amino acid substitutions differed between SARS-CoV-2 and the well characterized hepatitis C virus (HCV), we performed a comparative study of mutant spectra from infected patients using the same bioinformatics pipelines. HCV did not show the deficit of intermediat

Published
2022

23. Hypoxia classifier for transcriptome datasets

Author

Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Comunidad de Madrid, Puente-Santamaría, Laura, Sánchez-González, Lucía, Ramos-Ruiz, Ricardo, Peso, Luis del, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Comunidad de Madrid, Puente-Santamaría, Laura, Sánchez-González, Lucía, Ramos-Ruiz, Ricardo, and Peso, Luis del

Abstract

Molecular gene signatures are useful tools to characterize the physiological state of cell populations, but most have developed under a narrow range of conditions and cell types and are often restricted to a set of gene identities. Focusing on the transcriptional response to hypoxia, we aimed to generate widely applicable classifiers sourced from the results of a meta-analysis of 69 differential expression datasets which included 425 individual RNA-seq experiments from 33 different human cell types exposed to different degrees of hypoxia (0.1–5%O2) for 2–48 h. The resulting decision trees include both gene identities and quantitative boundaries, allowing for easy classification of individual samples without control or normoxic reference. Each tree is composed of 3–5 genes mostly drawn from a small set of just 8 genes (EGLN1, MIR210HG, NDRG1, ANKRD37, TCAF2, PFKFB3, BHLHE40, and MAFF). In spite of their simplicity, these classifiers achieve over 95% accuracy in cross validation and over 80% accuracy when applied to additional challenging datasets. Our results indicate that the classifiers are able to identify hypoxic tumor samples from bulk RNAseq and hypoxic regions within tumor from spatially resolved transcriptomics datasets. Moreover, application of the classifiers to histological sections from normal tissues suggest the presence of a hypoxic gene expression pattern in the kidney cortex not observed in other normoxic organs. Finally, tree classifiers described herein outperform traditional hypoxic gene signatures when compared against a wide range of datasets. This work describes a set of hypoxic gene signatures, structured as simple decision tress, that identify hypoxic samples and regions with high accuracy and can be applied to a broad variety of gene expression datasets and formats.

Published
2022

29. Validation and clinical application of a targeted next-generation sequencing gene panel for solid and hematologic malignancies

Author

Prieto-Potin, Iván, primary, Carvajal, Nerea, additional, Plaza-Sánchez, Jenifer, additional, Manso, Rebeca, additional, Aúz-Alexandre, Carmen Laura, additional, Chamizo, Cristina, additional, Zazo, Sandra, additional, López-Sánchez, Almudena, additional, Rodríguez-Pinilla, Socorro María, additional, Camacho, Laura, additional, Longarón, Raquel, additional, Bellosillo, Beatriz, additional, Somoza, Rosa, additional, Hernández-Losa, Javier, additional, Fernández-Soria, Víctor Manuel, additional, Ramos-Ruiz, Ricardo, additional, Cristóbal, Ion, additional, García-Foncillas, Jesús, additional, and Rojo, Federico, additional

Published
2020
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31. Draft Genome Sequences of Sporulation-Impaired Bacillus pumilus Strain NRS576 and Its Native Plasmid p576

Author

Ministerio de Economía y Competitividad (España), Agencia Estatal de Investigación (España), European Commission, Fundación Ramón Areces, Banco Santander, Val-Calvo, Jorge, Miguel-Arribas, Andrés, Gago-Córdoba, César, López-Pérez, Arancha, Ramachandran, Gayetri, Praveen, K. Singh, Ramos-Ruiz, Ricardo, Meijer, Wilfried J. J., Ministerio de Economía y Competitividad (España), Agencia Estatal de Investigación (España), European Commission, Fundación Ramón Areces, Banco Santander, Val-Calvo, Jorge, Miguel-Arribas, Andrés, Gago-Córdoba, César, López-Pérez, Arancha, Ramachandran, Gayetri, Praveen, K. Singh, Ramos-Ruiz, Ricardo, and Meijer, Wilfried J. J.

Abstract

Bacillus pumilus spores can cause foodborne poisonings. B. pumilus strain NRS576 forms spores with a very reduced efficiency due to the presence of a plasmid, named p576. Here, we report the genome sequence of strain B. pumilus NRS576 and its plasmid p576.

Published
2019

32. Genes and variants underlying human congenital lactic acidosis—from genetics to personalized treatment

Author

Fundación Isabel Gemio, Fundación la Caixa, European Commission, Comunidad de Madrid, Ministerio de Economía y Competitividad (España), Bravo-Alonso, Irene, Navarrete, Rosa, Vega, Ana Isabel, Ruíz-Sala, Pedro, García Silva, María Teresa, Martín-Hernández, Elena, Quijada-Fraile, Pilar, Belanger-Quintana, Amaya, Stanescu, Sinziana, Bueno, María, Vitoria, Isidro, Toledo, Laura, Couce, María Luz, García-Jiménez, Inmaculada, Ramos-Ruiz, Ricardo, Martín, Miguel Ángel, Desviat, Lourdes R., Ugarte, Magdalena, Pérez-Cerdá, Celia, Merinero, Begoña, Pérez, Belén, Rodríguez-Pombo, Pilar, Fundación Isabel Gemio, Fundación la Caixa, European Commission, Comunidad de Madrid, Ministerio de Economía y Competitividad (España), Bravo-Alonso, Irene, Navarrete, Rosa, Vega, Ana Isabel, Ruíz-Sala, Pedro, García Silva, María Teresa, Martín-Hernández, Elena, Quijada-Fraile, Pilar, Belanger-Quintana, Amaya, Stanescu, Sinziana, Bueno, María, Vitoria, Isidro, Toledo, Laura, Couce, María Luz, García-Jiménez, Inmaculada, Ramos-Ruiz, Ricardo, Martín, Miguel Ángel, Desviat, Lourdes R., Ugarte, Magdalena, Pérez-Cerdá, Celia, Merinero, Begoña, Pérez, Belén, and Rodríguez-Pombo, Pilar

Abstract

Congenital lactic acidosis (CLA) is a rare condition in most instances due to a range of inborn errors of metabolism that result in defective mitochondrial function. Even though the implementation of next generation sequencing has been rapid, the diagnosis rate for this highly heterogeneous allelic condition remains low. The present work reports our group’s experience of using a clinical/biochemical analysis system in conjunction with genetic findings that facilitates the taking of timely clinical decisions with minimum need for invasive procedures. The system’s workflow combines different metabolomics datasets and phenotypic information with the results of clinical exome sequencing and/or RNA analysis. The system’s use detected genetic variants in 64% of a cohort of 39 CLA-patients; these variants, 14 of which were novel, were found in 19 different nuclear and two mitochondrial genes. For patients with variants of unknown significance, the genetic analysis was combined with functional genetic and/or bioenergetics analyses in an attempt to detect pathogenicity. Our results warranted subsequent testing of antisense therapy to rescue the abnormal splicing in cultures of fibroblasts from a patient with a defective GFM1 gene. The discussed system facilitates the diagnosis of CLA by avoiding the need to use invasive techniques and increase our knowledge of the causes of this condition.

Published
2019

33. Genes and Variants Underlying Human Congenital Lactic Acidosis—From Genetics to Personalized Treatment

Author

Bravo-Alonso, Irene, primary, Navarrete, Rosa, additional, Vega, Ana Isabel, additional, Ruíz-Sala, Pedro, additional, García Silva, María Teresa, additional, Martín-Hernández, Elena, additional, Quijada-Fraile, Pilar, additional, Belanger-Quintana, Amaya, additional, Stanescu, Sinziana, additional, Bueno, María, additional, Vitoria, Isidro, additional, Toledo, Laura, additional, Couce, María Luz, additional, García-Jiménez, Inmaculada, additional, Ramos-Ruiz, Ricardo, additional, Martín, Miguel Ángel, additional, Desviat, Lourdes R., additional, Ugarte, Magdalena, additional, Pérez-Cerdá, Celia, additional, Merinero, Begoña, additional, Pérez, Belén, additional, and Rodríguez-Pombo, Pilar, additional

Published
2019
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34. Genetic profile and functional proteomics of anal squamous cell carcinoma: proposal for a molecular classification

Author

Trilla-Fuertes, Lucía, primary, Ghanem, Ismael, additional, Gámez-Pozo, Angelo, additional, Maurel, Joan, additional, G-Pastrián, Laura, additional, Mendiola, Marta, additional, Peña, Cristina, additional, López-Vacas, Rocío, additional, Prado-Vázquez, Guillermo, additional, López-Camacho, Elena, additional, Zapater-Moros, Andrea, additional, Heredia, Victoria, additional, Cuatrecasas, Miriam, additional, García-Alfonso, Pilar, additional, Capdevila, Jaume, additional, Conill, Carles, additional, García-Carbonero, Rocío, additional, Ramos-Ruiz, Ricardo, additional, Fortes, Claudia, additional, Llorens, Carlos, additional, Nanni, Paolo, additional, Fresno Vara, Juan Ángel, additional, and Feliu, Jaime, additional

Published
2019
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35. Comprehensive characterization of the mutational landscape in localized anal squamous cell carcinoma

Author

Trilla-Fuertes, Lucía, primary, Ghanem, Ismael, additional, Maurel, Joan, additional, G-Pastrián, Laura, additional, Mendiola, Marta, additional, Peña, Cristina, additional, López-Vacas, Rocío, additional, Prado-Vázquez, Guillermo, additional, López-Camacho, Elena, additional, Zapater-Moros, Andrea, additional, Heredia, Victoria, additional, Cuatrecasas, Miriam, additional, García-Alfonso, Pilar, additional, Capdevila, Jaume, additional, Conill, Carles, additional, García-Carbonero, Rocío, additional, Heath, Karen E., additional, Ramos-Ruiz, Ricardo, additional, Llorens, Carlos, additional, Campos-Barros, Ángel, additional, Gámez-Pozo, Angelo, additional, Feliu, Jaime, additional, and Vara, Juan Ángel Fresno, additional

Published
2019
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37. In-depth resistome analysis by targeted metagenomics

Author

European Commission, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, Comunidad de Madrid, European Society of Clinical Microbiology and Infectious Diseases, Agence Nationale de la Recherche (France), Lanza, Val F., Baquero, Fernando, Martínez, José L., Ramos-Ruiz, Ricardo, Gonzalez-Zorn, B., Andremont, Antoine, Sánchez-Valenzuela, Antonio, Dusko Ehrlich, Stanislav, Kennedy, Sean, Ruppé, Etienne, Schaik, Willem van, Willems, Rob J., Cruz, Fernando de la, Coque, Teresa M., European Commission, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, Comunidad de Madrid, European Society of Clinical Microbiology and Infectious Diseases, Agence Nationale de la Recherche (France), Lanza, Val F., Baquero, Fernando, Martínez, José L., Ramos-Ruiz, Ricardo, Gonzalez-Zorn, B., Andremont, Antoine, Sánchez-Valenzuela, Antonio, Dusko Ehrlich, Stanislav, Kennedy, Sean, Ruppé, Etienne, Schaik, Willem van, Willems, Rob J., Cruz, Fernando de la, and Coque, Teresa M.

Abstract

[Background]: Antimicrobial resistance is a major global health challenge. Metagenomics allows analyzing the presence and dynamics of “resistomes” (the ensemble of genes encoding antimicrobial resistance in a given microbiome) in disparate microbial ecosystems. However, the low sensitivity and specificity of available metagenomic methods preclude the detection of minority populations (often present below their detection threshold) and/or the identification of allelic variants that differ in the resulting phenotype. Here, we describe a novel strategy that combines targeted metagenomics using last generation in-solution capture platforms, with novel bioinformatics tools to establish a standardized framework that allows both quantitative and qualitative analyses of resistomes. [Methods]: We developed ResCap, a targeted sequence capture platform based on SeqCapEZ (NimbleGene) technology, which includes probes for 8667 canonical resistance genes (7963 antibiotic resistance genes and 704 genes conferring resistance to metals or biocides), and 2517 relaxase genes (plasmid markers) and 78,600 genes homologous to the previous identified targets (47,806 for antibiotics and 30,794 for biocides or metals). Its performance was compared with metagenomic shotgun sequencing (MSS) for 17 fecal samples (9 humans, 8 swine). ResCap significantly improves MSS to detect “gene abundance” (from 2.0 to 83.2%) and “gene diversity” (26 versus 14.9 genes unequivocally detected per sample per million of reads; the number of reads unequivocally mapped increasing up to 300-fold by using ResCap), which were calculated using novel bioinformatic tools. ResCap also facilitated the analysis of novel genes potentially involved in the resistance to antibiotics, metals, biocides, or any combination thereof. [Conclusions]: ResCap, the first targeted sequence capture, specifically developed to analyze resistomes, greatly enhances the sensitivity and specificity of available metagenomic methods and offers t

Published
2018

41. Mobility of the Native Bacillus subtilis Conjugative Plasmid pLS20 Is Regulated by Intercellular Signaling

Author

Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), Wellcome Trust, Singh, Praveen Kumar, Ramachandran, Gayetri, Ramos-Ruiz, Ricardo, Peiró-Pastor, Ramón, Abia, David, Wu, Ling Juan, Meijer, Wilfried J. J., Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), Wellcome Trust, Singh, Praveen Kumar, Ramachandran, Gayetri, Ramos-Ruiz, Ricardo, Peiró-Pastor, Ramón, Abia, David, Wu, Ling Juan, and Meijer, Wilfried J. J.

Abstract

Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default >OFF> state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.

Published
2013

42. Population structure of OXA-48-producing Klebsiella pneumoniaeST405 isolates during a hospital outbreak characterised by genomic typing

Author

López-Camacho, Elena, Paño-Pardo, José Ramón, Ruiz-Carrascoso, Guillermo, Wesselink, Jan-Jaap, Lusa-Bernal, Silvia, Ramos-Ruiz, Ricardo, Ovalle, Susana, Gómez-Gil, Rosa, Pérez-Blanco, Verónica, Pérez-Vázquez, María, Gómez-Puertas, Paulino, and Mingorance, Jesús

Abstract

•Genomes of ten OXA-48-producing Klebsiella pneumoniae(KpO48) ST405 isolates were sequenced.•SNPs were identified and analysed in 54 ST405 isolates from a single hospital.•No spatial clustering of haplotypes was found.•The temporal distribution of haplotypes was sequential.•A ST405 KpO48 outbreak was sustained by a dispersed population of colonised patients.

Published
2018
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44. Genome Sequence of OXA-48 Carbapenemase-Producing Klebsiella pneumoniae KpO3210

Author

Wesselink, Jan-Jaap, primary, López-Camacho, Elena, additional, de la Peña, Santiago, additional, Ramos-Ruiz, Ricardo, additional, Ruiz-Carrascoso, Guillermo, additional, Lusa-Bernal, Silvia, additional, Fernández-Soria, Victor M., additional, Gómez-Gil, Rosa, additional, Gomez-Puertas, Paulino, additional, and Mingorance, Jesús, additional

Published
2012
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47. Mobility of the Native Bacillus subtilis Conjugative Plasmid pLS20 Is Regulated by Intercellular Signaling.

Author

Singh, Praveen K., Ramachandran, Gayetri, Ramos-Ruiz, Ricardo, Peiró-Pastor, Ramón, Abia, David, Wu, Ling J., and Meijer, Wilfried J. J.

Subjects
BACTERIAL transformation, HEREDITY, PHYSIOLOGICAL effects of antibiotics, GRAM-positive bacteria, PLASMID genetics
Abstract

Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default “OFF” state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed. [ABSTRACT FROM AUTHOR]

Published
2013
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