Phospholipid arachidonic acid remodeling in macrophages: role of plasmalogen species

El ácido araquidónico (cis-5,8,11,14-eicosatetraenoico; AA) es el precursor de los eicosanoides, una gran familia de mediadores con papeles fundamentales en las fases de iniciación y resolución de la inflamación. En la presente tesis doctoral se persiguen los siguientes objetivos específicos: a) conocer los procesos que regulan la disponibilidad celular de AA, lo cual incluye estudiar la influencia de la compartimentalización del AA y examinar la dependencia de su remodelación del contenido de plasmalógenos; b) descubrir nuevas características reguladoras de las reacciones de transacilación independientes de CoA en la homeostasis del AA y revelar el papel de las fosfolipasas A2 en las respuestas de remodelación; c) estudiar el proceso del primado con LPS de macrófagos para la liberación de AA y caracterizar el papel de las especies plasmalógenas en los mecanismos implicados en la disponibilidad de AA.Departamento de Biología Celular, Histología y FarmacologíaDoctorado en Investigación Biomédic

Generación de líneas celulares humanas KNOCKOUT mediante el uso del sistema CRISPR/Cas9

La terapia génica consiste en la introducción de material genético en la célula con el fin de sustituir o reparar un gen defectuoso. Existen tres formas de emplear dicha terapia: sustitución génica (que consiste en la sustitución del gen defectuoso por el gen normal mediante recombinación homóloga), inserción génica (es decir, introducción de una copia del gen funcional para compensar el defectivo, sin modificar dicho gen endógeno), o modificación génica (donde el gen defectuoso es reparado mediante mutagénesis dirigida). Esta terapia puede llevarse a cabo in vivo o ex vivo. Las terapias in vivo se basan en la administración directa del gen terapéutico al paciente. Frente a ellas, las terapias ex vivo cobran importancia al permitir el tratamiento de las células aisladas en cultivo, lo que ofrece la posibilidad de la manipulación in vitro con el transgén. El vehículo encargado de introducir el material genético deseado en el interior de la célula diana se denomina vector, el cual puede ser transferido mediante un método de transducción (vectores virales) o de transfección (mediante métodos químicos o físicos). Entre los métodos de transferencia génica basados en vectores no virales se encuentran los métodos químicos, como la lipofección y el empleo de fosfato cálcico o de DEAE-dextran; y los métodos físicos como la electroporación, la micro-inyección y la micro-balística. Por otro lado, los vectores virales, que provienen de virus modificados (recombinantes) como Retrovirus, Lentivirus, Adenovirus, Virus Adeno-asociados o Herpes Simplex Virus entre otros, son muy eficientes en la transferencia de material genético en las células hospedadoras y pueden dirigirse a las mismas de forma específica. Sin embargo, a excepción de los Adenovirus, Herpes Simplex Virus y Virus Adeno-asociados (cuya localización es extracromosómica), el resto presentan la desventaja de insertarse en el genoma del huésped al azar e incluso ser capaces de insertar varias copias. Estos inconvenientes pueden ser causa de mutagénesis y conducir a la muerte celular. Además, la estabilidad de la expresión del transgén es impredecibleInstituto de Biologí­a y Genética Molecular (IBGM)Máster en Investigación Biomédic

Seasonal variation of biogas upgrading coupled with digestate treatment in an outdoors pilot scale algal-bacterial photobioreactor

Producción CientíficaThe yearly variations of the quality of the upgraded biogas and the efficiency of digestate treatment were evaluated in an outdoors pilot scale high rate algal pond (HRAP) interconnected to an external absorption column (AC) via a conical settler. CO2 concentrations in the upgraded biogas ranged from 0.7% in August to 11.9% in December, while a complete H2S removal was achieved regardless of the operational month. CH4 concentrations ranged from 85.2% in December to 97.9% in June, with a limited O2 and N2 stripping in the upgraded biogas mediated by the low recycling liquid/biogas ratio in the AC. Biomass productivity ranged from 0.0 g m−2 d−1 in winter to 22.5 g m−2 d−1 in summer. Finally, microalgae diversity was severely reduced throughout the year likely due to the increasing salinity in the cultivation broth of the HRAP induced by process operation in the absence of effluent.INCOVER - Innovative Eco-Technologies for Resource Recovery from Wastewater (689242

Essential Role for Ethanolamine Plasmalogen Hydrolysis in Bacterial Lipopolysaccharide Priming of Macrophages for Enhanced Arachidonic Acid Release

Due to their high content in esterified arachidonic acid (AA), macrophages provide large amounts of eicosanoids during innate immune reactions. Bacterial lipopolysaccharide (LPS) is a poor trigger of AA mobilization in macrophages but does have the capacity to prime these cells for greatly increased AA release upon subsequent stimulation. In this work, we have studied molecular mechanisms underlying this phenomenon. By using mass spectrometry-based lipidomic analyses, we show in this work that LPS-primed zymosan-stimulated macrophages exhibit an elevated consumption of a particular phospholipid species, i.e., the ethanolamine plasmalogens, which results from reduced remodeling of phospholipids via coenzyme A-independent transacylation reactions. Importantly however, LPS-primed macrophages show no changes in their capacity to directly incorporate AA into phospholipids via CoA-dependent acylation reactions. The essential role for ethanolamine plasmalogen hydrolysis in LPS priming is further demonstrated by the use of plasmalogen-deficient cells. These cells, while responding normally to zymosan by releasing quantities of AA similar to those released by cells expressing normal plasmalogen levels under the same conditions, fail to show an LPS-primed response to the same stimulus, thus unambiguously demonstrating a cause–effect relationship between LPS priming and plasmalogen hydrolysis. Collectively, these results suggest a hitherto unrecognized role for ethanolamine plasmalogen hydrolysis and CoA-independent transacylation reactions in modulating the eicosanoid biosynthetic response

Influence of the seasonal variation of environmental conditions on biogas upgrading in an outdoors pilot scale high rate algal pond

Producción CientíficaThe influence of the daily and seasonal variations of environmental conditions on the quality of the upgraded biogas was evaluated in an outdoors pilot scale high rate algal pond (HRAP) interconnected to an external absorption column (AC) via a conical settler. The high alkalinity in the cultivation broth resulted in a constant biomethane composition during the day regardless of the monitored month, while the high algal-bacterial activity during spring and summer boosted a superior biomethane quality. CO2 concentrations in the upgraded biogas ranged from 0.1% in May to 11.6% in December, while a complete H2S removal was always achieved regardless of the month. A limited N2 and O2 stripping from the scrubbing cultivation broth was recorded in the upgraded biogas at a recycling liquid/biogas ratio in the AC of 1. Finally, CH4 concentration in the upgraded biogas ranged from 85.6% in December to 99.6% in August.2019-12-312019-12-31INCOVER - Innovative Eco-Technologies for Resource Recovery from Wastewater (689242

Seasonal variation of biogas upgrading coupled with digestate treatment in an outdoors pilot scale algal-bacterial photobioreactor

[EN] The yearly variations of the quality of the upgraded biogas and the efficiency of digestate treatment were evaluated in an outdoors pilot scale high rate algal pond (HRAP) interconnected to an external absorption column (AC) via a conical settler. CO2 concentrations in the upgraded biogas ranged from 0.7% in August to 11.9% in December, while a complete H2S removal was achieved regardless of the operational month. CH4 concentrations ranged from 85.2% in December to 97.9% in June, with a limited O2 and N2 stripping in the upgraded biogas mediated by the low recycling liquid/biogas ratio in the AC. Biomass productivity ranged from 0.0 g m−2 d−1 in winter to 22.5 g m−2 d−1 in summer. Finally, microalgae diversity was severely reduced throughout the year likely due to the increasing salinity in the cultivation broth of the HRAP induced by process operation in the absence of effluentSIThis work was supported by the project INCOVER. The project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the grant agreement No. 689242. The financial support from MINECO (Red Novedar) and the Regional Government of Castilla y León (UIC 71) is also gratefully acknowledge

Essential Role for Ethanolamine Plasmalogen Hydrolysis in Bacterial Lipopolysaccharide Priming of Macrophages for Enhanced Arachidonic Acid Release

Due to their high content in esterified arachidonic acid (AA), macrophages provide large amounts of eicosanoids during innate immune reactions. Bacterial lipopolysaccharide (LPS) is a poor trigger of AA mobilization in macrophages but does have the capacity to prime these cells for greatly increased AA release upon subsequent stimulation. In this work, we have studied molecular mechanisms underlying this phenomenon. By using mass spectrometry-based lipidomic analyses, we show in this work that LPS-primed zymosan-stimulated macrophages exhibit an elevated consumption of a particular phospholipid species, i.e., the ethanolamine plasmalogens, which results from reduced remodeling of phospholipids via coenzyme A-independent transacylation reactions. Importantly however, LPS-primed macrophages show no changes in their capacity to directly incorporate AA into phospholipids via CoA-dependent acylation reactions. The essential role for ethanolamine plasmalogen hydrolysis in LPS priming is further demonstrated by the use of plasmalogen-deficient cells. These cells, while responding normally to zymosan by releasing quantities of AA similar to those released by cells expressing normal plasmalogen levels under the same conditions, fail to show an LPS-primed response to the same stimulus, thus unambiguously demonstrating a cause–effect relationship between LPS priming and plasmalogen hydrolysis. Collectively, these results suggest a hitherto unrecognized role for ethanolamine plasmalogen hydrolysis and CoA-independent transacylation reactions in modulating the eicosanoid biosynthetic response.This work was supported by grants SAF2016-80883-R and SAF2015-73000-EXP from the Spanish Ministry of Economy, Industry and Competitiveness, and grant CSI073U16 from the Education Department of the Regional Government of Castile and Leon. PL was supported by a predoctoral fellowship from the University of Valladolid. CIBERDEM is an initiative of Instituto de Salud Carlos III.Peer reviewe

Occurrence and biological activity of palmitoleic acid isomers in phagocytic cells

Recent studies have highlighted the role of palmitoleic acid [16:1n-7 (cis-9-hexadecenoic acid)] as a lipid hormone that coordinates cross-talk between liver and adipose tissue and exerts anti-inflammatory protective effects on hepatic steatosis and insulin signaling in murine models of metabolic disease. More recently, a 16:1n-7 isomer, cis-7-hexadecenoic acid (16:1n-9), that also possesses marked anti-inflammatory effects, has been described in human circulating monocytes and monocyte-derived macrophages. By using gas chromatographic/mass spectrometric analyses of dimethyl disulfide derivatives of fatty acyl methyl esters, we describe in this study the presence of a third 16:1 isomer, sapienic acid [16:1n-10 (6-cis-hexadecenoic acid)], in phagocytic cells. Cellular levels of 16:1n-10 appear to depend not only on the cellular content of linoleic acid, but also on the expression level of fatty acid desaturase 2, thus revealing a complex regulation both at the enzyme level, via fatty acid substrate competition, and directly at the gene level. However, unlike 16:1n-7 and 16:1n-9, 16:1n-10 levels are not regulated by the activation state of the cell. Moreover, while 16:1n-7 and 16:1n-9 manifest strong anti-inflammatory activity when added to the cells at low concentrations (10 μM), notably higher concentrations of 16:1n-10 are required to observe a comparable effect. Collectively, these results suggest the presence in phagocytic cells of an unexpected variety of 16:1 isomers, which can be distinguished on the basis of their biological activity and cellular regulation.This work was supported by Ministry of Economy and Competitiveness Grants SAF2016-80883-R; and SAF2015-73000-EXP and Department of Education, Government of Castile-Leon Grant CSI073U16. CIBERDEM is an initiative of Instituto de Salud Carlos III.Peer reviewe

Quantitative analysis of methane monooxygenase (MMO) explains process robustness in continuous and feast-famine bioreactors treating methane

Producción CientíficaThe ability of methanotrophs to rapidly respond to intentional or accidental stress conditions caused by operational failures or process fluctuations is of utmost importance to guarantee the robustness of CH4 abatement biotechnologies. In this study, the performance of a continuous and two feast-famine (5:5 days feast-famine cycles) stirred tank reactors treating diluted CH4 emissions (4–5% v/v) was comparatively assessed for 149 days. The robustness of the three bioreactors towards a 5 days CH4 deprivation episode was thoroughly evaluated at a molecular level (pmoA gene expression level) and correlated to macroscopic process performance. The bioreactors recovered their steady-state abatement performance (in terms of CH4 elimination capacity and CO2 production rate) within 1.5–2 h following CH4 supply resumption concomitantly with a maximum in pmoA gene expression, regardless of the previous operational mode. However, while methanotrophs from the continuous unit maintained higher basal levels of pmoA expression as a strategy for a rapid CH4 metabolism initiation, the strategy of the feast-famine adapted-methanotrophs consisted on a more accurate regulation of their pmoA transcripts levels along with a higher and/or more rapid induction of the pmoA gene by CH4 availability.Ministerio de Economía, Industria y Competitividad- FEDER (Proyect CTM2015-70442-R and Red Novedar)Juan de la Cierva Program (IJCI-2014-19432)Junta de Castilla y León (programa de apoyo a proyectos de investigación - Ref. Project UIC71