Confidence in the Beef Production System as a Key Factor to Mitigate the Impact of BSE on Beef Consumption

Recent food scares in the food market has caused a reduction in consumer's confidence in the food system that it has induced a significant reduction in consumption in a sector, the beef sector that was already characterized by a saturated trend in quantity terms. In this context, all participants in the beef production system are facing to a great challenge, to retrieve consumer's confidence in the food chain and to mitigate the reduction in beef consumption. The aim of the paper is to analyse the impact of consumer's confidence in the food system as well as other factors on the explanation of food consumption reduction. A structural modelling approach has been used to analyse factors affecting the reduction in beef consumption in two different regions characterised by different production systems and different marketing strategies (PGI beef label). Results indicate that main factor explaining the reduction in beef consumption is the confidence in the beef and a positive relation has been found. Moreover, confidence in a product is directly related to the perceived quality offered by farmers and other decision makers on the beef chain, and to the consumer involvement with the product. Therefore, the main implication is that participants in the food chain has to develop adequate communication strategies such as quality labelling in order to increase consumers perceive quality because, higher quality perception will recover consumers' confidence in beef, and therefore, it will mitigate beef consumption reduction.food confidence, consumer behaviour, structural equation modelling, beef sector, quality label, Food Consumption/Nutrition/Food Safety,

Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling

[EN] Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca2+ are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca2+ signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca2+-dependent recruitment of the pyrabactin resistance 1/PYR1like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca2+ sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca2+-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress.A.A. and J.A.M. thank the European Syncrotron Radiation Facility and EMBL for access to the synchrotron radiation source. This work was funded by Ministerio de Economia y Competitividad (MINECO) Grants BFU2014-59796-R (to A.A.), BFU2011-28184-C02 (to M.J.S.-B.), and BIO2014-52537-R (to P.L.R.) and Comunidad de Madrid Grant S2010/BMD-2457 (to A.A and M.M.). M.J.S.-B. is supported by Ramon y Cajal Contract RYC-2008-03449 from MINECO and M.D. by a fellowship from Senacyt-Ifarhu. Access to the High Throughput Crystallization facility at European Molecular Biology Laboratory (EMBL) Grenoble was supported by the European Community's Seventh Framework Programme through the Protein Production Platform project (P-CUBE) Grant 227764.Diaz, M.; Sanchez-Barrena, MJ.; Gonzalez Rubio, JM.; Rodríguez Solovey, LN.; Fernández, D.; Antoni-Alandes, R.; Yunta, C.... (2016). Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling. Proceedings of the National Academy of Sciences. 113(3):E396-E405. https://doi.org/10.1073/pnas.1512779113SE396E4051133Serrano, R., & Rodriguez-Navarro, A. (2001). Ion homeostasis during salt stress in plants. Current Opinion in Cell Biology, 13(4), 399-404. doi:10.1016/s0955-0674(00)00227-1Bassil, E., & Blumwald, E. (2014). The ins and outs of intracellular ion homeostasis: NHX-type cation/H + transporters. Current Opinion in Plant Biology, 22, 1-6. doi:10.1016/j.pbi.2014.08.002Batistič, O., & Kudla, J. (2012). Analysis of calcium signaling pathways in plants. Biochimica et Biophysica Acta (BBA) - General Subjects, 1820(8), 1283-1293. doi:10.1016/j.bbagen.2011.10.012Cutler, S. R., Rodriguez, P. L., Finkelstein, R. R., & Abrams, S. R. (2010). Abscisic Acid: Emergence of a Core Signaling Network. Annual Review of Plant Biology, 61(1), 651-679. doi:10.1146/annurev-arplant-042809-112122McAinsh, M. R., Brownlee, C., & Hetherington, A. M. (1990). Abscisic acid-induced elevation of guard cell cytosolic Ca2+ precedes stomatal closure. Nature, 343(6254), 186-188. doi:10.1038/343186a0Maierhofer, T., Diekmann, M., Offenborn, J. N., Lind, C., Bauer, H., Hashimoto, K., … Hedrich, R. (2014). Site- and kinase-specific phosphorylation-mediated activation of SLAC1, a guard cell anion channel stimulated by abscisic acid. Science Signaling, 7(342), ra86-ra86. doi:10.1126/scisignal.2005703Allen, G. J., Kwak, J. M., Chu, S. P., Llopis, J., Tsien, R. Y., Harper, J. F., & Schroeder, J. I. (1999). Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells. The Plant Journal, 19(6), 735-747. doi:10.1046/j.1365-313x.1999.00574.xLee, S. C., Lan, W.-Z., Kim, B.-G., Li, L., Cheong, Y. H., Pandey, G. K., … Luan, S. (2007). A protein phosphorylation/dephosphorylation network regulates a plant potassium channel. Proceedings of the National Academy of Sciences, 104(40), 15959-15964. doi:10.1073/pnas.0707912104Sánchez-Barrena, M., Martínez-Ripoll, M., & Albert, A. (2013). Structural Biology of a Major Signaling Network that Regulates Plant Abiotic Stress: The CBL-CIPK Mediated Pathway. International Journal of Molecular Sciences, 14(3), 5734-5749. doi:10.3390/ijms14035734Quan, R., Lin, H., Mendoza, I., Zhang, Y., Cao, W., Yang, Y., … Guo, Y. (2007). SCABP8/CBL10, a Putative Calcium Sensor, Interacts with the Protein Kinase SOS2 to Protect Arabidopsis Shoots from Salt Stress. The Plant Cell, 19(4), 1415-1431. doi:10.1105/tpc.106.042291Ma, Y., Szostkiewicz, I., Korte, A., Moes, D., Yang, Y., Christmann, A., & Grill, E. (2009). Regulators of PP2C Phosphatase Activity Function as Abscisic Acid Sensors. Science. doi:10.1126/science.1172408Park, S.-Y., Fung, P., Nishimura, N., Jensen, D. R., Fujii, H., Zhao, Y., … Cutler, S. R. (2009). Abscisic Acid Inhibits Type 2C Protein Phosphatases via the PYR/PYL Family of START Proteins. Science. doi:10.1126/science.1173041Santiago, J., Rodrigues, A., Saez, A., Rubio, S., Antoni, R., Dupeux, F., … Rodriguez, P. L. (2009). Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs. The Plant Journal, 60(4), 575-588. doi:10.1111/j.1365-313x.2009.03981.xNishimura, N., Sarkeshik, A., Nito, K., Park, S.-Y., Wang, A., Carvalho, P. C., … Schroeder, J. I. (2009). PYR/PYL/RCAR family members are major in-vivo ABI1 protein phosphatase 2C-interacting proteins in Arabidopsis. The Plant Journal, 61(2), 290-299. doi:10.1111/j.1365-313x.2009.04054.xWang, P., Xue, L., Batelli, G., Lee, S., Hou, Y.-J., Van Oosten, M. J., … Zhu, J.-K. (2013). Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action. Proceedings of the National Academy of Sciences, 110(27), 11205-11210. doi:10.1073/pnas.1308974110Umezawa, T., Sugiyama, N., Takahashi, F., Anderson, J. C., Ishihama, Y., Peck, S. C., & Shinozaki, K. (2013). Genetics and Phosphoproteomics Reveal a Protein Phosphorylation Network in the Abscisic Acid Signaling Pathway in Arabidopsis thaliana. Science Signaling, 6(270), rs8-rs8. doi:10.1126/scisignal.2003509Kollist, H., Nuhkat, M., & Roelfsema, M. R. G. (2014). Closing gaps: linking elements that control stomatal movement. New Phytologist, 203(1), 44-62. doi:10.1111/nph.12832Lind, C., Dreyer, I., López-Sanjurjo, E. J., von Meyer, K., Ishizaki, K., Kohchi, T., … Hedrich, R. (2015). Stomatal Guard Cells Co-opted an Ancient ABA-Dependent Desiccation Survival System to Regulate Stomatal Closure. Current Biology, 25(7), 928-935. doi:10.1016/j.cub.2015.01.067Geiger, D., Scherzer, S., Mumm, P., Stange, A., Marten, I., Bauer, H., … Hedrich, R. (2009). Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair. Proceedings of the National Academy of Sciences, 106(50), 21425-21430. doi:10.1073/pnas.0912021106Imes, D., Mumm, P., Böhm, J., Al-Rasheid, K. A. S., Marten, I., Geiger, D., & Hedrich, R. (2013). Open stomata 1 (OST1) kinase controls R-type anion channel QUAC1 in Arabidopsis guard cells. The Plant Journal, 74(3), 372-382. doi:10.1111/tpj.12133Ishitani, M., Liu, J., Halfter, U., Kim, C.-S., Shi, W., & Zhu, J.-K. (2000). SOS3 Function in Plant Salt Tolerance Requires N-Myristoylation and Calcium Binding. The Plant Cell, 12(9), 1667-1677. doi:10.1105/tpc.12.9.1667Grefen, C., & Blatt, M. R. (2012). Do Calcineurin B-Like Proteins Interact Independently of the Serine Threonine Kinase CIPK23 with the K+ Channel AKT1? Lessons Learned from a Ménage à Trois. Plant Physiology, 159(3), 915-919. doi:10.1104/pp.112.198051Qiu, Q.-S., Guo, Y., Dietrich, M. A., Schumaker, K. S., & Zhu, J.-K. (2002). Regulation of SOS1, a plasma membrane Na+/H+ exchanger in Arabidopsis thaliana, by SOS2 and SOS3. Proceedings of the National Academy of Sciences, 99(12), 8436-8441. doi:10.1073/pnas.122224699Quintero, F. J., Martinez-Atienza, J., Villalta, I., Jiang, X., Kim, W.-Y., Ali, Z., … Pardo, J. M. (2011). Activation of the plasma membrane Na/H antiporter Salt-Overly-Sensitive 1 (SOS1) by phosphorylation of an auto-inhibitory C-terminal domain. Proceedings of the National Academy of Sciences, 108(6), 2611-2616. doi:10.1073/pnas.1018921108Núñez-Ramírez, R., Sánchez-Barrena, M. J., Villalta, I., Vega, J. F., Pardo, J. M., Quintero, F. J., … Albert, A. (2012). Structural Insights on the Plant Salt-Overly-Sensitive 1 (SOS1) Na+/H+ Antiporter. Journal of Molecular Biology, 424(5), 283-294. doi:10.1016/j.jmb.2012.09.015Ohta, M., Guo, Y., Halfter, U., & Zhu, J.-K. (2003). A novel domain in the protein kinase SOS2 mediates interaction with the protein phosphatase 2C ABI2. Proceedings of the National Academy of Sciences, 100(20), 11771-11776. doi:10.1073/pnas.2034853100Xu, J., Li, H.-D., Chen, L.-Q., Wang, Y., Liu, L.-L., He, L., & Wu, W.-H. (2006). A Protein Kinase, Interacting with Two Calcineurin B-like Proteins, Regulates K+ Transporter AKT1 in Arabidopsis. Cell, 125(7), 1347-1360. doi:10.1016/j.cell.2006.06.011Geiger, D., Scherzer, S., Mumm, P., Marten, I., Ache, P., Matschi, S., … Hedrich, R. (2010). Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+affinities. Proceedings of the National Academy of Sciences, 107(17), 8023-8028. doi:10.1073/pnas.0912030107Brandt, B., Brodsky, D. E., Xue, S., Negi, J., Iba, K., Kangasjarvi, J., … Schroeder, J. I. (2012). Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action. Proceedings of the National Academy of Sciences, 109(26), 10593-10598. doi:10.1073/pnas.1116590109Rodriguez, L., Gonzalez-Guzman, M., Diaz, M., Rodrigues, A., Izquierdo-Garcia, A. C., Peirats-Llobet, M., … Rodriguez, P. L. (2014). C2-Domain Abscisic Acid-Related Proteins Mediate the Interaction of PYR/PYL/RCAR Abscisic Acid Receptors with the Plasma Membrane and Regulate Abscisic Acid Sensitivity in Arabidopsis. The Plant Cell, 26(12), 4802-4820. doi:10.1105/tpc.114.129973Martens, S., & McMahon, H. T. (2008). Mechanisms of membrane fusion: disparate players and common principles. Nature Reviews Molecular Cell Biology, 9(7), 543-556. doi:10.1038/nrm2417Martens, S., Kozlov, M. M., & McMahon, H. T. (2007). How Synaptotagmin Promotes Membrane Fusion. Science, 316(5828), 1205-1208. doi:10.1126/science.1142614Jahn, R., Lang, T., & Südhof, T. C. (2003). Membrane Fusion. Cell, 112(4), 519-533. doi:10.1016/s0092-8674(03)00112-0Sutter, J.-U., Sieben, C., Hartel, A., Eisenach, C., Thiel, G., & Blatt, M. R. (2007). Abscisic Acid Triggers the Endocytosis of the Arabidopsis KAT1 K+ Channel and Its Recycling to the Plasma Membrane. Current Biology, 17(16), 1396-1402. doi:10.1016/j.cub.2007.07.020Bueso, E., Rodriguez, L., Lorenzo-Orts, L., Gonzalez-Guzman, M., Sayas, E., Muñoz-Bertomeu, J., … Rodriguez, P. L. (2014). The single-subunit RING-type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling. The Plant Journal, 80(6), 1057-1071. doi:10.1111/tpj.12708Larsen, J. B., Jensen, M. B., Bhatia, V. K., Pedersen, S. L., Bjørnholm, T., Iversen, L., … Stamou, D. (2015). Membrane curvature enables N-Ras lipid anchor sorting to liquid-ordered membrane phases. Nature Chemical Biology, 11(3), 192-194. doi:10.1038/nchembio.1733Demir, F., Horntrich, C., Blachutzik, J. O., Scherzer, S., Reinders, Y., Kierszniowska, S., … Kreuzer, I. (2013). Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3. Proceedings of the National Academy of Sciences, 110(20), 8296-8301. doi:10.1073/pnas.1211667110Guerrero-Valero, M., Ferrer-Orta, C., Querol-Audi, J., Marin-Vicente, C., Fita, I., Gomez-Fernandez, J. C., … Corbalan-Garcia, S. (2009). Structural and mechanistic insights into the association of PKC -C2 domain to PtdIns(4,5)P2. Proceedings of the National Academy of Sciences, 106(16), 6603-6607. doi:10.1073/pnas.0813099106Guillen, J., Ferrer-Orta, C., Buxaderas, M., Perez-Sanchez, D., Guerrero-Valero, M., Luengo-Gil, G., … Corbalan-Garcia, S. (2013). Structural insights into the Ca2+ and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1. Proceedings of the National Academy of Sciences, 110(51), 20503-20508. doi:10.1073/pnas.1316179110Corbalan-Garcia, S., & Gómez-Fernández, J. C. (2014). Signaling through C2 domains: More than one lipid target. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1838(6), 1536-1547. doi:10.1016/j.bbamem.2014.01.008CHO, W., & STAHELIN, R. (2006). Membrane binding and subcellular targeting of C2 domains. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1761(8), 838-849. doi:10.1016/j.bbalip.2006.06.014Verdaguer, N., Corbalan-Garcia, S., Ochoa, W. F., Fita, I., & Gómez-Fernández, J. C. (1999). Ca2+ bridges the C2 membrane-binding domain of protein kinase Cα directly to phosphatidylserine. The EMBO Journal, 18(22), 6329-6338. doi:10.1093/emboj/18.22.6329Honigmann, A., van den Bogaart, G., Iraheta, E., Risselada, H. J., Milovanovic, D., Mueller, V., … Jahn, R. (2013). Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment. Nature Structural & Molecular Biology, 20(6), 679-686. doi:10.1038/nsmb.2570Ausili, A., Corbalán-García, S., Gómez-Fernández, J. C., & Marsh, D. (2011). Membrane docking of the C2 domain from protein kinase Cα as seen by polarized ATR-IR. The role of PIP2. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1808(3), 684-695. doi:10.1016/j.bbamem.2010.11.035Hermoso, J. A., Lagartera, L., González, A., Stelter, M., García, P., Martínez-Ripoll, M., … Menéndez, M. (2005). Insights into pneumococcal pathogenesis from the crystal structure of the modular teichoic acid phosphorylcholine esterase Pce. Nature Structural & Molecular Biology, 12(6), 533-538. doi:10.1038/nsmb940Thompson, D., Pepys, M. B., & Wood, S. P. (1999). The physiological structure of human C-reactive protein and its complex with phosphocholine. Structure, 7(2), 169-177. doi:10.1016/s0969-2126(99)80023-9Ochoa, W. F., Garcia-Garcia, J., Fita, I., Corbalan-Garcia, S., Verdaguer, N., & Gomez-Fernandez, J. C. (2001). Structure of the C2 domain from novel protein kinase Cϵ. A membrane binding model for Ca2+-independent C2 domains. Journal of Molecular Biology, 311(4), 837-849. doi:10.1006/jmbi.2001.4910Fuson, K., Rice, A., Mahling, R., Snow, A., Nayak, K., Shanbhogue, P., … Sutton, R. B. (2014). Alternate Splicing of Dysferlin C2A Confers Ca2+-Dependent and Ca2+-Independent Binding for Membrane Repair. Structure, 22(1), 104-115. doi:10.1016/j.str.2013.10.001Radhakrishnan, A., Stein, A., Jahn, R., & Fasshauer, D. (2009). The Ca2+Affinity of Synaptotagmin 1 Is Markedly Increased by a Specific Interaction of Its C2B Domain with Phosphatidylinositol 4,5-Bisphosphate. Journal of Biological Chemistry, 284(38), 25749-25760. doi:10.1074/jbc.m109.042499Schapire, A. L., Voigt, B., Jasik, J., Rosado, A., Lopez-Cobollo, R., Menzel, D., … Botella, M. A. (2008). Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability. The Plant Cell, 20(12), 3374-3388. doi:10.1105/tpc.108.063859Wang, J., Bello, O., Auclair, S. M., Wang, J., Coleman, J., Pincet, F., … Rothman, J. E. (2014). Calcium sensitive ring-like oligomers formed by synaptotagmin. Proceedings of the National Academy of Sciences, 111(38), 13966-13971. doi:10.1073/pnas.1415849111Jaenicke, R., & Rudolph, R. (1986). [12]Refolding and association of oligomeric proteins. Enzyme Structure Part L, 218-250. doi:10.1016/0076-6879(86)31043-7Goni, G. M., Epifano, C., Boskovic, J., Camacho-Artacho, M., Zhou, J., Bronowska, A., … Lietha, D. (2014). Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes. Proceedings of the National Academy of Sciences, 111(31), E3177-E3186. doi:10.1073/pnas.1317022111Wilkie, A. O. (1994). The molecular basis of genetic dominance. Journal of Medical Genetics, 31(2), 89-98. doi:10.1136/jmg.31.2.89Saez, A., Apostolova, N., Gonzalez-Guzman, M., Gonzalez-Garcia, M. P., Nicolas, C., Lorenzo, O., & Rodriguez, P. L. (2003). Gain-of-function and loss-of-function phenotypes of the protein phosphatase 2CHAB1reveal its role as a negative regulator of abscisic acid signalling. The Plant Journal, 37(3), 354-369. doi:10.1046/j.1365-313x.2003.01966.xSimons, K., & Gerl, M. J. (2010). Revitalizing membrane rafts: new tools and insights. Nature Reviews Molecular Cell Biology, 11(10), 688-699. doi:10.1038/nrm2977McMahon, H. T., & Boucrot, E. (2015). Membrane curvature at a glance. Journal of Cell Science, 128(6), 1065-1070. doi:10.1242/jcs.114454Tapken, W., & Murphy, A. S. (2015). Membrane nanodomains in plants: capturing form, function, and movement. Journal of Experimental Botany, 66(6), 1573-1586. doi:10.1093/jxb/erv054Lingwood, D., & Simons, K. (2007). Detergent resistance as a tool in membrane research. Nature Protocols, 2(9), 2159-2165. doi:10.1038/nprot.2007.294Schuck, P. (2000). Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation Modeling. Biophysical Journal, 78(3), 1606-1619. doi:10.1016/s0006-3495(00)76713-0Bensmihen, S., To, A., Lambert, G., Kroj, T., Giraudat, J., & Parcy, F. (2004). Analysis of an activated ABI5 allele using a new selection method for transgenic Arabidopsis seeds. FEBS Letters, 561(1-3), 127-131. doi:10.1016/s0014-5793(04)00148-6Deblaere, R., Bytebier, B., De Greve, H., Deboeck, F., Schell, J., Van Montagu, M., & Leemans, J. (1985). Efficient octopine Ti plasmid-derived vectors forAgrobacterium-mediated gene transfer to plants. Nucleic Acids Research, 13(13), 4777-4788. doi:10.1093/nar/13.13.4777Clough, S. J., & Bent, A. F. (1998). Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana. The Plant Journal, 16(6), 735-743. doi:10.1046/j.1365-313x.1998.00343.xHua, J. (2001). Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genes. Genes & Development, 15(17), 2263-2272. doi:10.1101/gad.918101Kabsch, W. (2010). XDS. Acta Crystallographica Section D Biological Crystallography, 66(2), 125-132. doi:10.1107/s0907444909047337Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., … Wilson, K. S. (2011). Overview of theCCP4 suite and current developments. Acta Crystallographica Section D Biological Crystallography, 67(4), 235-242. doi:10.1107/s0907444910045749Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W., Echols, N., … Zwart, P. H. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D Biological Crystallography, 66(2), 213-221. doi:10.1107/s0907444909052925Emsley, P., & Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallographica Section D Biological Crystallography, 60(12), 2126-2132. doi:10.1107/s0907444904019158Dimasi, N., Flot, D., Dupeux, F., & Márquez, J. A. (2007). Expression, crystallization and X-ray data collection from microcrystals of the extracellular domain of the human inhibitory receptor expressed on myeloid cells IREM-1. Acta Crystallographica Section F Structural Biology and Crystallization Communications, 63(3), 204-208. doi:10.1107/s1744309107004903Emsley, P., Lohkamp, B., Scott, W. G., & Cowtan, K. (2010). Features and development ofCoot. Acta Crystallographica Section D Biological Crystallography, 66(4), 486-501. doi:10.1107/s0907444910007493DeLano (2002) The PyMOL Molecular Graphics System, 1.5.0.4 (DeLano Scientific, San Carlos, CA

Discovery of first-in-class reversible dual small molecule inhibitors against G9a and DNMTs in hematological malignancies

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.We particularly acknowledge the Biobank of the University of Navarra for its collaboration. We thank Dr Edorta Martínez de Marigorta and Dr Francisco Palacios from Departamento de Química Orgánica I, Facultad de Farmacia, Universidad del Pais Vasco for 13C NMR determination and Angel Irigoyen Barrio and Dr Ana Romo Hualde, from University of Navarra, for HRMS determination. Dr. Irene de Miguel Turrullols from Small Molecule Discovery Platform, CIMA, University of Navarra is acknowledged for NMR data interpretation. This work was funded by grants from Instituto de Salud Carlos III (ISCIII) PI10/01691, PI13/01469, PI14/01867, PI10/2983, TRASCAN (EPICA), CIBERONC, cofinanciacion FEDER, RTICC RD12/0036/0068, Fundació La Marató de TV3 (20132130-31-32) and ‘Fundación Fuentes Dutor’. B.P. is supported by a Sara Borrell fellowship CD13/00340 and X.A. is a Marie Curie researcher under contract ‘LincMHeM-330598’.S

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Confidence in the Beef Production System as a Key Factor to Mitigate the Impact of BSE on Beef Consumption

Recent food scares in the food market has caused a reduction in consumer's confidence in the food system that it has induced a significant reduction in consumption in a sector, the beef sector that was already characterized by a saturated trend in quantity terms. In this context, all participants in the beef production system are facing to a great challenge, to retrieve consumer's confidence in the food chain and to mitigate the reduction in beef consumption. The aim of the paper is to analyse the impact of consumer's confidence in the food system as well as other factors on the explanation of food consumption reduction. A structural modelling approach has been used to analyse factors affecting the reduction in beef consumption in two different regions characterised by different production systems and different marketing strategies (PGI beef label). Results indicate that main factor explaining the reduction in beef consumption is the confidence in the beef and a positive relation has been found. Moreover, confidence in a product is directly related to the perceived quality offered by farmers and other decision makers on the beef chain, and to the consumer involvement with the product. Therefore, the main implication is that participants in the food chain has to develop adequate communication strategies such as quality labelling in order to increase consumers perceive quality because, higher quality perception will recover consumers' confidence in beef, and therefore, it will mitigate beef consumption reduction

La certificacion como estrategia para la recuperacion de la confianza del consumidor en la adquisicion de la carne de ternera

Resumen El objetivo del trabajo se centra en analizar el efecto de la presencia de etiqueta en un producto con el fin de minimizar el riesgo percibido o la incertidumbre asociada al producto. El análisis ha sido realizado tomando como ejemplo la carne fresca de ternera, producto que ha presentado en Europa, en general, y en España, en particular, importantes problemas de salubridad o seguridad alimentaria que han generado una importante pérdida de confianza por parte de los consumidores e, indirectamente, una disminución de su consumo, lo que ha supuesto, a su vez, serias implicaciones sobre la oferta. Teniendo en cuenta el objetivo planteado, se especifica un modelo para analizar la disposición del consumidor a aumentar el consumo de carne de ternera si se le garantiza su salubridad. Se considera que el consumidor realiza una doble decisión: 1) consumir más o no; y 2) una vez que ha decidido incrementar su consumo, determina en qué cantidad, considerándose, adicionalmente, que ambas decisiones se adoptan de forma simultánea. Los resultados obtenidos en tres Comunidades Autónomas españolas (Comunidad de Aragón, Comunidad Foral de Navarra y Comunidad Valenciana) muestran la idoneidad de las etiquetas para mitigar los efectos que sobre la demanda tienen estos problemas surgidos en la oferta, siendo los resultados especialmente relevantes en el mercado que ha presentado mayores problemas de salubridad. Palabras Clave: comportamiento del consumidor, modelos de decisión, etiqueta, carne de ternera, seguridad alimentaria. Summary The objective of the paper is to analyse the effect of the label product presence in the reduction of the health hazard perceived or in the uncertainty associated to a product. The market selected like an example was the fresh beef, because in Europe, in general, and in Spain in particular, has been serious problems related with the food safety. These problems had influenced in the lost confidence of the consumers with important consequences in the supply. We propose a consumer decision model with two steps: 1) to consume more or no, 2) if the consumer decided to increase the consume analyse the factors which affect the quantity to increment; these decisions are considered simultaneous. The results obtained in the three Spanish regions (Comunidad de Aragón, Comunidad Foral de Navarra and Comunidad Valenciana) showed the suitability of the utilisation of the labels in the product to reduce or mitigate the demand effects of these supply complications, specially in the market which major food safety problems

La certificacion como estrategia para la recuperacion de la confianza del consumidor en la adquisicion de la carne de ternera

Resumen El objetivo del trabajo se centra en analizar el efecto de la presencia de etiqueta en un producto con el fin de minimizar el riesgo percibido o la incertidumbre asociada al producto. El análisis ha sido realizado tomando como ejemplo la carne fresca de ternera, producto que ha presentado en Europa, en general, y en España, en particular, importantes problemas de salubridad o seguridad alimentaria que han generado una importante pérdida de confianza por parte de los consumidores e, indirectamente, una disminución de su consumo, lo que ha supuesto, a su vez, serias implicaciones sobre la oferta. Teniendo en cuenta el objetivo planteado, se especifica un modelo para analizar la disposición del consumidor a aumentar el consumo de carne de ternera si se le garantiza su salubridad. Se considera que el consumidor realiza una doble decisión: 1) consumir más o no; y 2) una vez que ha decidido incrementar su consumo, determina en qué cantidad, considerándose, adicionalmente, que ambas decisiones se adoptan de forma simultánea. Los resultados obtenidos en tres Comunidades Autónomas españolas (Comunidad de Aragón, Comunidad Foral de Navarra y Comunidad Valenciana) muestran la idoneidad de las etiquetas para mitigar los efectos que sobre la demanda tienen estos problemas surgidos en la oferta, siendo los resultados especialmente relevantes en el mercado que ha presentado mayores problemas de salubridad. Palabras Clave: comportamiento del consumidor, modelos de decisión, etiqueta, carne de ternera, seguridad alimentaria. Summary The objective of the paper is to analyse the effect of the label product presence in the reduction of the health hazard perceived or in the uncertainty associated to a product. The market selected like an example was the fresh beef, because in Europe, in general, and in Spain in particular, has been serious problems related with the food safety. These problems had influenced in the lost confidence of the consumers with important consequences in the supply. We propose a consumer decision model with two steps: 1) to consume more or no, 2) if the consumer decided to increase the consume analyse the factors which affect the quantity to increment; these decisions are considered simultaneous. The results obtained in the three Spanish regions (Comunidad de Aragón, Comunidad Foral de Navarra and Comunidad Valenciana) showed the suitability of the utilisation of the labels in the product to reduce or mitigate the demand effects of these supply complications, specially in the market which major food safety problems.consumer behaviour, decision models, label, beef, food safety, Consumer/Household Economics, M31, R11, Q13,

Structural basis of the regulatory mechanism of the plant CIPK family of protein kinases controlling ion homeostasis and abiotic stress

Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases

The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

The neuronal calcium sensor 1 (NCS-1), an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Gα have revealed how Ric-8A phosphorylation promotes Gα recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Gα subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Gα. Our data show that the binding of NCS-1 and Gα to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to casein kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A guanine nucleotide exchange factor (GEF) activity toward Gα when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity