Role of surface tryptophan for peroxidase oxidation of nonphenolic lignin

Background: Despite claims as key enzymes in enzymatic delignification, very scarce information on the reaction rates between the ligninolytic versatile peroxidase (VP) and lignin peroxidase (LiP) and the lignin polymer is available, due to methodological difficulties related to lignin heterogeneity and low solubility.Results: Two water-soluble sulfonated lignins (from Picea abies and Eucalyptus grandis) were chemically characterized and used to estimate single electron-transfer rates to the H2O2-activated Pleurotus eryngii VP (native enzyme and mutated variant) transient states (compounds I and II bearing two- and one-electron deficiencies, respectively). When the rate-limiting reduction of compound II was quantified by stopped-flow rapid spectrophotometry, from fourfold (softwood lignin) to over 100-fold (hardwood lignin) lower electron-transfer efficiencies (k 3app values) were observed for the W164S variant at surface Trp164, compared with the native VP. These lignosulfonates have ~20–30 % phenolic units, which could be responsible for the observed residual activity. Therefore, methylated (and acetylated) samples were used in new stopped-flow experiments, where negligible electron transfer to the W164S compound II was found. This revealed that the residual reduction of W164S compound II by native lignin was due to its phenolic moiety. Since both native lignins have a relatively similar phenolic moiety, the higher W164S activity on the softwood lignin could be due to easier access of its mono-methoxylated units for direct oxidation at the heme channel in the absence of the catalytic tryptophan. Moreover, the lower electron transfer rates from the derivatized lignosulfonates to native VP suggest that peroxidase attack starts at the phenolic lignin moiety. In agreement with the transient-state kinetic data, very low structural modification of lignin, as revealed by size-exclusion chromatography and two-dimensional nuclear magnetic resonance, was obtained during steady-state treatment (up to 24 h) of native lignosulfonates with the W164S variant compared with native VP and, more importantly, this activity disappeared when nonphenolic lignosulfonates were used.Conclusions: We demonstrate for the first time that the surface tryptophan conserved in most LiPs and VPs (Trp164 of P. eryngii VPL) is strictly required for oxidation of the nonphenolic moiety, which represents the major and more recalcitrant part of the lignin polymer

Improving the Oxidative Stability of a High Redox Potential Fungal Peroxidase by Rational Design

Ligninolytic peroxidases are enzymes of biotechnological interest due to their ability to oxidize high redox potential aromatic compounds, including the recalcitrant lignin polymer. However, different obstacles prevent their use in industrial and environmental applications, including low stability towards their natural oxidizing-substrate H2O2. In this work, versatile peroxidase was taken as a model ligninolytic peroxidase, its oxidative inactivation by H2O2 was studied and different strategies were evaluated with the aim of improving H2O2 stability. Oxidation of the methionine residues was produced during enzyme inactivation by H2O2 excess. Substitution of these residues, located near the heme cofactor and the catalytic tryptophan, rendered a variant with a 7.8-fold decreased oxidative inactivation rate. A second strategy consisted in mutating two residues (Thr45 and Ile103) near the catalytic distal histidine with the aim of modifying the reactivity of the enzyme with H2O2. The T45A/I103T variant showed a 2.9-fold slower reaction rate with H2O2 and 2.8-fold enhanced oxidative stability. Finally, both strategies were combined in the T45A/I103T/M152F/M262F/M265L variant, whose stability in the presence of H2O2 was improved 11.7-fold. This variant showed an increased half-life, over 30 min compared with 3.4 min of the native enzyme, under an excess of 2000 equivalents of H2O2. Interestingly, the stability improvement achieved was related with slower formation, subsequent stabilization and slower bleaching of the enzyme Compound III, a peroxidase intermediate that is not part of the catalytic cycle and leads to the inactivation of the enzyme.This work was funded by the Commission of the European Communities through the INDOX project (KBBE-2013-7-613549, "Optimized oxidoreductases for medium and large scale industrial biotransformations"), and by the Spanish Ministerio de Economía y Competitividad (MINECO) through the HIPOP project (BIO2011-26694, “Screening and engineering of new high-redoxpotential peroxidases”). VS-J and FJR-D thank the financial support of a research fellowship (Formación de Personal Investigador, FPI) and a Ramón y Cajal contract of the Spanish MINECO, respectively. The authors have no conflict of interest to declare. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer ReviewedPostprint (published version

Strandings of cetaceans and sea turtles in the Alboran Sea and Strait of Gibraltar: a long-term glimpse at the north coast (Spain) and the south coast (Morocco)

., 2011. Strandings of cetaceans and sea turtles in the Alboran Sea and Strait of Gibraltar: a long-time glimpse of the north coast (Spain) and the south coast (Morocco). Animal Biodiversity and Conservation, 34.1: 151-163. Abstract Strandings of cetaceans and sea turtles in the Alboran Sea and Strait of Gibraltar: a long-term glimpse at the north coast (Spain) and the south coast (Morocco) .-A total of 13 species of cetaceans and three species of marine turtles were found in this study. Data were collected by eight independent and self-regulated stranding networks, providing information about 1,198 marine mammal (10 odontocetii, three mysticetii and one phocidae) and 574 sea turtle stranding events between 1991 and 2008. Trends in the strandings were analysed in relation to species composition and abundance, and their geographic and seasonal distribution. The most abundant species recorded were the striped dolphin and the loggerhead turtle. Some of the strandings, such as the humpback whale, harbour porpoise, hooded seal and olive ridley turtle, were considered 'rare' because their distribution did not match the pattern of the study. When the north and south coasts in the study area were compared, pilot whales stranded more frequently in the north, while delphinid species stranded more in the south coast, and loggerhead turtles stranded more frequently in the north while leatherback turtles stranded more in south coast. Key words: Strandings, South-western Mediterranean, Distribution, Marine turtle, Cetacean, Conservation. Resumen Varamientos de cetáceos y tortugas marinas en el mar de Alborán y el Estrecho de Gibraltar: un vistazo a largo plazo de la costa norte (España) y la costa sur (Marruecos) .-En este estudio se registraron un total de 13 especies de cetáceos y tres especies de tortugas marinas, proviniendo los datos de redes de voluntarios que prestan asistencia en los varamientos. Se recogió información de 1.198 mamiferos marinos (10 odontocetos, tres misticetos y un fócido) y 574 tortugas marinas entre los años 1991 y 2008. Se analizaron las tendencias de los varamientos en relación a la composición de especies, su abundancia y su distribución geográfica y estacional. Las especies más comunes fueron el delfín común y la tortuga boba. Algunos de los varamientos, como la ballena jorobada, la marsopa común, la foca de casco o la tortuga olivácea, pueden considerarse "anómalos" puesto que su distribución se escapa a los patrones del estudio. Comparando la costa norte del área de estudio con la sur, los calderones y tortugas bobas vararon con mayor frecuencia en la costa norte, mientras que las especies de delfines y las tortugas laúd vararon con mayor frecuencia en la costa sur

Dietary intake and major food sources of polyphenols in a Spanish population at high cardiovascular risk: The PREDIMED study

[Background and aims]: Epidemiological data have shown an inverse association between the consumption of polyphenol-rich foods and the risk of cardiovascular disease or overall mortality. A comprehensive estimation of individual polyphenol intake in nutritional cohorts is needed to gain a better understanding of this association. The aim of this study was to estimate the quantitative intake of polyphenols and the major dietary sources in the PREDIMED (PREvención con DIeta MEDiterránea) cohort using individual food consumption records. [Methods and results]: The PREDIMED study is a large, parallel-group, multicentre, randomised, controlled 5-year feeding trial aimed at assessing the effects of the Mediterranean diet on the primary prevention of cardiovascular disease. A total of 7200 participants, aged 55–80 years, completed a validated 1-year food frequency questionnaire (FFQ) at baseline. Polyphenol consumption was calculated by matching food consumption data from the FFQ with the recently developed Phenol-Explorer database on polyphenol content in foods. The mean total polyphenol intake was 820 ± 323 mg day−1 (443 ± 218 mg day−1 of flavonoids and 304 ± 156 mg day−1 of phenolic acids). Hydroxycinnamic acids were the phenolic group with the highest consumption and 5-caffeoylquinic acid was the most abundantly ingested individual polyphenol. The consumption of olives and olive oil was a differentiating factor in the phenolic profile of this Spanish population compared with other countries. [Conclusion]: In Mediterranean countries, such as Spain, the main dietary source of polyphenols is coffee and fruits, but the most important differentiating factor with respect to other countries is the consumption of polyphenols from olives and olive oil.The authors would like to express their gratitude for financial support from CICYT (AGL2009-13906-C02-02 and AGL2010-22319-C03), RETICS-RD06/0045, and PI11/02505 from the Spanish Ministry of Economy and Competitiveness (MEC) and ACOMP/2012/190 from the Generalitat Valenciana. The CIBERobn-CB06/03 is an initiative of the Instituto de Salud Carlos III, Spain. A.T-R would like to thank the ISCIII for granting her a predoctoral fellowship (FI10/00265) and J.P-J would like to thank the ISCIII for a Sara Borrell postdoctoral contract (CD09/00068)

Who leads research productivity growth? Guidelines for R&D policy-makers

[EN] This paper evaluates to what extent policy-makers have been able to promote the creation and consolidation of comprehensive research groups that contribute to the implementation of a successful innovation system. Malmquist productivity indices are applied in the case of the Spanish Food Technology Program, finding that a large size and a comprehensive multi-dimensional research output are the key features of the leading groups exhibiting high efficiency and productivity levels. While identifying these groups as benchmarks, we conclude that the financial grants allocated by the program, typically aimed at small-sized and partially oriented research groups, have not succeeded in reorienting them in time so as to overcome their limitations. We suggest that this methodology offers relevant conclusions to policy evaluation methods, helping policy-makers to readapt and reorient policies and their associated means, most notably resource allocation (financial schemes), to better respond to the actual needs of research groups in their search for excellence (micro-level perspective), and to adapt future policy design to the achievement of medium-long term policy objectives (meso and macro-level).Jiménez Saez, F.; Zabala Iturriagagoitia, JM.; Zofio, JL. (2013). Who leads research productivity growth? Guidelines for R&D policy-makers. Scientometrics. 94(1):273-303. doi:10.1007/s11192-012-0763-0S273303941Abbring, J. H., & Heckman, J. J. (2008). Dynamic policy analysis. In L. Mátyás & P. Sevestre (Eds.), The econometrics of panel data (3rd ed., pp. 795–863). Heidelberg: Springer.Acosta Ballesteros, J., & Modrego Rico, A. (2001). Public financing of cooperative R&D projects in Spain: the concerted projects under the national R&D plan. Research Policy, 30, 625–641.Arbel, A. (1981). Policy evaluation in the dynamic input–output model. International Journal of Systems Science, 12, 255–260.Arnold, E. (2004). Evaluation research and innovation policy: A systems world needs systems evaluations. Research Evaluation, 13, 3–17.Arrow, J. K. (1962). Economic welfare and the allocation of resources for inventions. In R. Nelson (Ed.), The rate and direction of inventive activity: Economic and social factor (pp. 609–625). Princeton: Princeton University Press and NBER.Autio, E. (1997). New, technology-based firms in innovation networks symplectic and generative impacts. Research Policy, 26, 263–281.Balk, B. (2001). Scale efficiency and productivity change. Journal of Productivity Analysis, 15, 153–183.Balzat, M., & Hanusch, H. (2004). Recent trends in the research on national innovation systems. Journal of Evolutionary Economics, 14, 197–210.Berg, S. A., Førsund, F. R., & Jansen, E. S. (1992). Malmquist indices of productivity growth during the deregulation of Norwegian banking. Scandinavian Journal of Economics, 94, S211–S228.Bergek, A., Carlsson, B., Lindmark, S., Rickne, A., & Jacobsson, S. (2008). Analyzing the functional dynamics of technological innovation systems: A scheme of analysis. Research Policy, 37, 407–429.Bonaccorsi, A., & Daraio, C. (2005). Exploring size and agglomeration effects on public research productivity. Scientometrics, 63(1), 87–120.Buisseret, T. J., Cameron, H., & Georghiou, L. (1995). What difference does it make? Additionality in the public support of R&D in large firms. International Journal of Technology Management, 10, 587–600.Bustelo, M. (2006). The potential role of standards and guidelines in the development of an evaluation culture in Spain. Evaluation, 12, 437–453.Chavas, J. P., & Cox, T. M. (1999). A generalized distance function and the analysis of production efficiency. Southern Economic Journal, 66, 295–318.CICYT. (1987). Programa Nacional de Tecnología de los Alimentos. Madrid: Ministerio de Educación y Ciencia.CICYT (1988). Plan Nacional de Investigación Científica y Desarrollo Tecnológico 1988–1991. Ministerio de Educación y Ciencia, Secretaría de Estado de Universidades e Investigación, Madrid.Cooper, W. W., Seiford, L. M., & Tone, K. (2000). Data envelopment analysis: A comprehensive text with models, applications, references and DEA-software. Boston: Kluwer Academic Publishers.David, P., Mowery, D., & Steinmueller, W. E. (1994). Analyzing the economic payoffs from basic research. In D. Mowery (Ed.), Science and technology policy in interdependent economies (pp. 57–78). Boston: Kluwer Academic Publishers.Dopfer, K., Foster, J., & Potts, J. (2004). Micro-meso-macro. Journal of Evolutionary Economics, 14, 263–279.Edquist, C., & Hommen, L. (2008). Comparing national systems of innovation in Asia and Europe: Theory and comparative framework. In C. Edquist & L. Hommen (Eds.), Small country innovation systems: Globalisation, change and policy in Asia and Europe (pp. 1–28). Cheltenham: Edward Elgar.Färe, R., Grosskopf, S., Norris, M., & Zhang, Z. (1994). Productivity growth, technical progress, and efficiency change in industrialized countries. American Economic Review, 84, 66–83.Farrell, M. (1957). The measurement of productive efficiency. Journal of the Royal Statistical Society, Series A, General, 120(3), 253–281.Førsund, F. R. (1993). Productivity growth in Norwegian ferries. In H. O. Fried, C. A. K. Lovell, & S. S. Schmidt (Eds.), The measurement of productive efficiency: Techniques and applications (pp. 352–373). New York: Oxford University Press.Førsund, F. R. (1997). The Malmquist productivity index, TFP and scale. University of Oslo, Oslo: Working Paper, Department of Economics and Business Administration.Freeman, C. (1987). Technology policy and economic performance: Lessons from Japan. London: Printer Publishers.García-Martínez, M., & Briz, J. (2000). Innovation in the Spanish food & drink industry. International Food and Agribusiness Management Review, 3, 155–176.Gibbons, M., Limoges, C., Nowotny, H., Schwartzman, S., Scott, P., & Trow, M. (1994). The new production of knowledge: The dynamics of science and research in contemporary societies. London: Sage Publications.Grammatikopoulos, V., Kousteiios, A., Tsigilis, N., & Theodorakis, Y. (2004). Applying dynamic evaluation approach in education. Studies in Educational Evaluation, 30, 255–263.Grifell-Tatjé, E., & Lovell, C. A. K. (1999). A generalized Malmquist productivity index. Top, 7(1), 81–101.Grimpe, C., & Sofka, W. (2007). Search patterns and absorptive capacity: A comparison of low- and high-technology firms from thirteen European countries. Discussion paper no. 07-062. Centre for European Economic Research (ZEW), Mannheim, Germany.Guan, J., & Wang, J. (2004). Evaluation and interpretation of knowledge production efficiency. Scientometrics, 59(1), 131–155.Hekkert, M. P., Suurs, R. A. A., Negro, S. O., Kuhlmann, S., & Smits, R. E. H. M. (2007). Functions of innovation systems: A new approach for analysing technological change. Technological Forecasting and Social Change, 74, 413–432.Jiménez-Sáez, F. (2005). Una Evaluación del Programa Nacional de Tecnología de Alimentos: análisis de la articulación fomentada sobre el Sistema Alimentario de Innovación en España. PhD dissertation, Servicio de Publicaciones de la Universidad Politécnica de Valencia, Valencia.Jiménez-Sáez, F., Zabala-Iturriagagoitia, J. M., Zofío, J. L., & Castro-Martínez, E. (2011). Evaluating research efficiency within National R&D Programmes. Research Policy, 40, 230–241.Kao, C. (2008). Efficiency analysis of university departments: An empirical study. OMEGA, 36, 653–664.Kuhlmann, S. (2003). Evaluation of research and innovation policies: A discussion of trends with examples from Germany. International Journal of Technology Management, 26, 131–149.Laitinen, E. K. (2002). A dynamic performance measurement system: Evidence from small Finnish technology companies. Scandinavian Journal of Management, 18, 65–99.Laranja, M., Uyarra, E., & Flanagan, K. (2008). Policies for science, technology and innovation: Translating rationales into regional policies in a multi-level setting. Research Policy, 37(5), 823–835.Lee, T.-L., & von Tunzelman, N. (2005). A dynamic analytic approach to national innovation systems: The IC industry in Taiwan. Research Policy, 34, 425–440.Lipsey, R., & Carlaw, K. (1998). A structuralist assessment of technology policies: Taking Schumpeter seriously on policy. Ottawa: Industry Canada Research Publications Program.Lipsey, R., Carlaw, K., & Bekar, C. (2005). Economic transformations: General purpose technologies and long term economic growth. Oxford: Oxford University Press.Lundvall, B. Å. (1992). National systems of innovation: Toward a theory of innovation and interactive learning. London: Printer Publishers.Lundvall, B. Å., Johnson, B., Andersen, E. S., & Dalum, B. (2002). National systems of production, innovation and competence building. Research Policy, 31, 213–231.Markard, J., & Truffer, B. (2008). Actor-oriented analysis of innovation systems: Exploring micro-meso level linkages in the case of stationary fuel cells. Technology Analysis & Strategic Management, 20, 443–464.Metcalfe, J. S. (2002). Equilibrium and evolutionary foundations of competition and technology policy: New perspectives on the division of labour and the innovation process. CRIC Working Papers series, University of Manchester.Miettinen, R. (1999). The riddle of things. Activity theory and actor network theory as approaches of studying innovations. Mind, Culture and Activity, 6, 170–195.Molas-Gallart, J., & Davies, A. (2006). Toward theory-led evaluation: The experience of European science, technology, and innovation policies. American Journal of Evaluation, 27, 64–82.Mytelka, L. K., & Smith, K. (2002). Policy learning and innovation theory: An interactive and co-evolving process. Research Policy, 31, 1467–1479.Olazarán, M., Lavía, C., & Otero, B. (2004). ¿Hacia una segunda transición en la ciencia? Política científica y grupos de investigación. Revista Española de Sociología, 4, 143–172.Potts, J. (2007). The innovation system & economic evolution. Productivity commission submission, public support for science & innovation, productivity commission, Camberra.Ray, S., & Desli, E. (1997). Productivity growth, technical progress, and efficiency change in industrialized countries: Comment. American Economic Review, 87(5), 1033–1039.Rip, A., & Nederhof, A. J. (1986). Between dirigism and laissez-faire: Effects of implementing the science policy priority for biotechnology in the Netherlands. Research Policy, 15, 253–268.Schmidt, E. K., Graversen, E. K., & Langberg, K. (2003). Innovation and dynamics in public research environments in Denmark: A research-policy perspective. Science and Public Policy, 30, 107–116.Schmoch, U., & Schubert, T. (2009). Sustainability of incentives for excellent research—The German case. Scientometrics, 81(1), 195–218.Shephard, R. (1970). Theory of cost and production functions. New Jersey: Princeton University Press.Simar, L., & Wilson, P. W. (1998). Productivity growth in industrialized countries. Discussion paper 9810, Universite Catholique de Louvain, Belgium.Van Raan, A. F. J. (2000). R&D evaluation at the beginning of the new century. Research Evaluation, 8, 81–86.Zofio, J. L. (2007). Malmquist productivity index decompositions: A unifying framework. Applied Economics, 39, 2371–2387.Zofio, J. L., & Lovell, C. A. K. (1998). Yet another Malmquist productivity index decomposition. Working paper, Department of Economics, University of Georgia, Athens, GA 30602, USA.Zofio, J. L., & Lovell, C. A. K. (2001). Graph efficiency and productivity measures: An application to US agriculture. Applied Economics, 33(10), 1433–1442.Zofio, J. L., & Prieto, A. M. (2006). Return to dollar, generalized distance function and the Fisher productivity index. Spanish Economic Review, 8, 113–138

The Eurasian Modern Pollen Database (EMPD), version 2

The Eurasian (née European) Modern Pollen Database (EMPD) was established in 2013 to provide a public database of high-quality modern pollen surface samples to help support studies of past climate, land cover, and land use using fossil pollen. The EMPD is part of, and complementary to, the European Pollen Database (EPD) which contains data on fossil pollen found in Late Quaternary sedimentary archives throughout the Eurasian region. The EPD is in turn part of the rapidly growing Neotoma database, which is now the primary home for global palaeoecological data. This paper describes version 2 of the EMPD in which the number of samples held in the database has been increased by 60 % from 4826 to 8134. Much of the improvement in data coverage has come from northern Asia, and the database has consequently been renamed the Eurasian Modern Pollen Database to reflect this geographical enlargement. The EMPD can be viewed online using a dedicated map-based viewer at https://empd2.github.io and downloaded in a variety of file formats at https://doi.pangaea.de/10.1594/PANGAEA.909130 (Chevalier et al., 2019)Swiss National Science Foundation | Ref. 200021_16959

The management of acute venous thromboembolism in clinical practice. Results from the European PREFER in VTE Registry

Venous thromboembolism (VTE) is a significant cause of morbidity and mortality in Europe. Data from real-world registries are necessary, as clinical trials do not represent the full spectrum of VTE patients seen in clinical practice. We aimed to document the epidemiology, management and outcomes of VTE using data from a large, observational database. PREFER in VTE was an international, non-interventional disease registry conducted between January 2013 and July 2015 in primary and secondary care across seven European countries. Consecutive patients with acute VTE were documented and followed up over 12 months. PREFER in VTE included 3,455 patients with a mean age of 60.8 ± 17.0 years. Overall, 53.0 % were male. The majority of patients were assessed in the hospital setting as inpatients or outpatients (78.5 %). The diagnosis was deep-vein thrombosis (DVT) in 59.5 % and pulmonary embolism (PE) in 40.5 %. The most common comorbidities were the various types of cardiovascular disease (excluding hypertension; 45.5 %), hypertension (42.3 %) and dyslipidaemia (21.1 %). Following the index VTE, a large proportion of patients received initial therapy with heparin (73.2 %), almost half received a vitamin K antagonist (48.7 %) and nearly a quarter received a DOAC (24.5 %). Almost a quarter of all presentations were for recurrent VTE, with >80 % of previous episodes having occurred more than 12 months prior to baseline. In conclusion, PREFER in VTE has provided contemporary insights into VTE patients and their real-world management, including their baseline characteristics, risk factors, disease history, symptoms and signs, initial therapy and outcomes