Experimental recreation of the evolution of lignin-degrading enzymes from the Jurassic to date

[Background] Floudas et al. (Science 336: 1715) established that lignin-degrading fungi appeared at the end of Carboniferous period associated with the production of the first ligninolytic peroxidases. Here, the subsequent evolution of these enzymes in Polyporales, where most wood-rotting fungi are included, is experimentally recreated using genomic information.[Results] With this purpose, we analyzed the evolutionary pathway leading to the most efficient lignin-degrading peroxidases characterizing Polyporales species. After sequence reconstruction from 113 genes of ten sequenced genomes, the main enzyme intermediates were resurrected and characterized. Biochemical changes were analyzed together with predicted sequences and structures, to understand how these enzymes acquired the ability to degrade lignin and how this ability changed with time. The most probable first peroxidase in Polyporales would be a manganese peroxidase (Mn3+ oxidizing phenolic lignin) that did not change substantially until the appearance of an exposed tryptophan (oxidizing nonphenolic lignin) originating an ancestral versatile peroxidase. Later, a quick evolution, with loss of the Mn2+-binding site, generated the first lignin peroxidase that evolved to the extant form by improving the catalytic efficiency. Increased stability at acidic pH, which strongly increases the oxidizing power of these enzymes, was observed paralleling the appearance of the exposed catalytic tryptophan.[Conclusions] We show how the change in peroxidase catalytic activities meant an evolutionary exploration for more efficient ways of lignin degradation by fungi, a key step for carbon recycling in land ecosystems. The study provides ancestral enzymes with a potential biotechnological interest for the sustainable production of fuels and chemicals in a biomass-based economy.We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI), and the EC OpenAIRE FP7 post-grant Open Access Pilot.This work was supported by the INDOX (KBBE-2013-613549) and EnzOx2 (H2020-BBI-PPP-2015-2-720297) EU projects and the NOESIS (BIO2014-56388-R) project of the Spanish Ministry of Economy and Competitiveness (MINECO). The work conducted by JGI was supported by the Office of Science of the U.S. Department of Energy under Contract DE-AC02-05CH11231.EUR 1,620 APC fee funded by the EC FP7 Post-Grant Open Access Pilo

Rational Enzyme Engineering Through Biophysical and Biochemical Modeling

Due to its importance in the pharmaceutical industry, ligand dynamic simulations have experienced a great expansion. Using all-atom models and cutting edge hardware, one can perform non-biased ligand migration, active site search and binding studies. In this letter we demonstrate (and validate by PCR mutagenesis) how these techniques, when combined with quantum mechanics, open new possibilities in enzyme engineering. We provide a complete analysis where: 1) biophysical simulations produce ligand diffusion and, 2) biochemical modeling samples the chemical event. Using such broad analysis we engineer a highly stable peroxidase activating the enzyme for new substrate oxidation after rational mutation of two non-conserved surface residues. In particular, we create a new surface-binding site, quantitatively predicting the in vitro change in oxidation rate obtained by mutagenic PCR and achieving a comparable specificity constant to active peroxidases.This work was supported by the INDOX (KBBE-2013-7-613549 to ATM) European project, and the CTQ2013-48287 (to VG) and BIO2014-56388-R (to FJR-D) projects of the Spanish Ministry of Economy and Competitiveness (MINECO). FJR-D acknowledges a MINECO Ramón&Cajal contract.Peer ReviewedPostprint (author's final draft

Evaluación del metabolismo colónico de un vino tinto mediante el empleo de un nuevo modelo de simulación gastrointestinal dinámico (SIMGI)

Este libro recoge el amplio y significativo elenco de estudios recientemente realizados por los grupos de investigación de la red GIENOL (Grupos de investigación enológica).Los polifenoles del vino pueden influir positivamente en la salud del hombre modificando la actividad metabólica y/o composición de la microbiota intestinal. El objetivo de este trabajo ha sido evaluar el metabolismo colónico de un vino tinto mediante el empleo de un nuevo modelo de simulación dinámico del tracto gastrointestinal (denominado SIMGI), utilizando heces humanas de donantes sanos (n=2). Para los tres compartimentos del colon ‐ascendente, transverso y descendente‐ se llevó a cabo la monitorización de diferentes parámetros metabólicos (compuestos fenólicos del vino y sus metabolitos, ion amonio y ácidos grasos de cadena corta (SCFA)) y microbiológicos (recuentos, qPCR), incluyendo la comparativa tras la alimentación del sistema con vino sintético (sin polifenoles). Los resultados mostraron que la ingesta moderada de vino activaba el metabolismo de la microbiota colónica. Se encontraron aumentos significativos para los ácidos gálico, protocatéquico, 3‐Ometilgálico, 4‐hidroxibenzoico, 3,4‐dihidroxifenilpropiónico, vainillínico, siríngico, y salicílico después de la alimentación con vino. Simultáneamente, se observó una disminución en la formación del ion amonio y un incremento en la proporción del ácido butanoico. A nivel microbiológico, los principales cambios tuvieron lugar en el colon ascendente. En conclusión, estos resultados ponen de manifiesto que el vino modula la actividad metabólica de la microbiota intestinal in vitro, y demuestran la utilidad del SIMGI como modelo de simulación gastrointestinal dinámico.Este trabajo ha sido financiado por el MINECO a través del proyecto AGL2012‐40172‐C02‐01, el Programa de la Comunidad Madrid ALIBIRD‐CM S2013/ABI ‐2728 y el Proyecto Intramural CSIC 201270E065.Peer Reviewe

Striatal dopamine D2-muscarinic acetylcholine M1 receptor-receptor interaction in a model of movement disorders

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor control deficits, which is associated with the loss of striatal dopaminergic neurons from the substantia nigra. In parallel to dopaminergic denervation, there is an increase of acetylcholine within the striatum, resulting in a striatal dopaminergic-cholinergic neurotransmission imbalance. Currently, available PD pharmacotherapy (e.g., prodopaminergic drugs) does not reinstate the altered dopaminergic-cholinergic balance. In addition, it can eventually elicit cholinergic-related adverse effects. Here, we investigated the interplay between dopaminergic and cholinergic systems by assessing the physical and functional interaction of dopamine D2 and muscarinic acetylcholine M1 receptors (D2R and M1R, respectively), both expressed at striatopallidal medium spiny neurons. First, we provided evidence for the existence of D2R-M1R complexes via biochemical (i.e., co-immunoprecipitation) and biophysical (i.e., BRET1 and NanoBiT®) assays, performed in transiently transfected HEK293T cells. Subsequently, a D2R-M1R co-distribution in the mouse striatum was observed through double-immunofluorescence staining and AlphaLISA® immunoassay. Finally, we evaluated the functional interplay between both receptors via behavioral studies, by implementing the classical acute reserpine pharmacological animal model of experimental parkinsonism. Reserpinized mice were administered with a D2R-selective agonist (sumanirole) and/or an M1R-selective antagonist (VU0255035), and alterations in PD-related behavioral tasks (i.e., locomotor activity) were evaluated. Importantly, VU0255035 (10 mg/kg) potentiated the antiparkinsonian-like effects (i.e., increased locomotor activity and decreased catalepsy) of an ineffective sumanirole dose (3 mg/kg). Altogether, our data suggest the existence of putative striatal D2R/M1R heteromers, which might be a relevant target to manage PD motor impairments with fewer adverse effects

Fusion of 8He with 206Pb around Coulomb barrier energies

The experimental study of the fusion of light neutron-rich nucleus 8He with 206Pb is reported in this work. A fusion stack of 206Pb targets has been used for this study. The most prominent evaporation residue (210Po), which has half-life of 138 days and decays by alpha emission, is populated in the reaction. Radiochemical analysis technique is used to extract the yield of this evaporation residue.Ministerio de Ciencia e Innovavión FPA2007-63074European Union 21269

Age determination procedures for benthic fish in Spanish Institute of Oceanography (IEO)

This handbook presents a summary of the age estimation procedures used in the Spanish Institute of Oceanography (IEO) for some of the main commercial benthic species of fish for the Spanish fleet: megrim (Lepidorhombus whiffiagonis), four spot megrim (Lepidorhombus boscii), white anglerfish (Lophius piscatorius), black anglerfish (Lophius budegassa). It provides information about the sampling program, the morphology of hard parts (otoliths and illicia), their extraction, preparation, and the age estimation criteria. A summary of information related to the accuracy, validation and corroboration of age of each species is also presented, as well as that related to the precision, quality control and verification of age

Scattering of 8He on 208Pb at Energies Around the Coulomb Barrier

We have measured the angular distributions of elastic scattering and 6,4He fragments produced in the collisions of an exotic beam of 8He on a 208Pb target at laboratory energies of 18 and 22 MeV, just around the Coulomb barrier (19 MeV). The measurements were performed at the SPIRAL/GANIL facility in Caen, France. In this paper, we present preliminary data on elastic cross sections and discuss the results using optical model and coupled reaction channel calculations.Spanish Research Council FPA-2010-22131-C02-01Ministry of Science and Higher Education of Poland No. N202 03363

Age determination procedures for pelagic and benthic species from ICES area in Spanish Institute of Oceanography (IEO)

This handbook presents a summary of the age estimation procedures used in Spanish Institute of Oceanography (IEO) for some of the main commercial small and medium pelagic species and benthic species of the Spanish fleet. It provides information about the sampling program, otolith extraction and preparation, and the age estimation criteria. A summary of the information related to the age accuracy, validation and corroboration of each species is also presented, as well as that related to the age precision, quality control and verification