TY - JOUR AB - The activity of hippocampal pyramidal cells reflects both the current position of the animal and information related to its current behavior. Here we investigated whether single hippocampal neurons can encode several independent features defining trials during a memory task. We also tested whether task-related information is represented by partial remapping of the place cell population or, instead, via firing rate modulation of spatially stable place cells. To address these two questions, the activity of hippocampal neurons was recorded in rats performing a conditional discrimination task on a modified T-maze in which the identity of a food reward guided behavior. When the rat was on the central arm of the maze, the firing rate of pyramidal cells changed depending on two independent factors: (1) the identity of the food reward given to the animal and (2) the previous location of the animal on the maze. Importantly, some pyramidal cells encoded information relative to both factors. This trial-type specific and retrospective coding did not interfere with the spatial representation of the maze: hippocampal cells had stable place fields and their theta-phase precession profiles were unaltered during the task, indicating that trial-related information was encoded via rate remapping. During error trials, encoding of both trial-related information and spatial location was impaired. Finally, we found that pyramidal cells also encode trial-related information via rate remapping during the continuous version of the rewarded alternation task without delays. These results suggest that hippocampal neurons can encode several task-related cognitive aspects via rate remapping. AU - Allen, Kevin AU - Rawlins, J Nick AU - Bannerman, David AU - Csicsvari, Jozsef L ID - 2958 IS - 42 JF - Journal of Neuroscience TI - Hippocampal place cells can encode multiple trial-dependent features through rate remapping VL - 32 ER - TY - JOUR AB - We study maximum likelihood estimation in Gaussian graphical models from a geometric point of view. An algebraic elimination criterion allows us to find exact lower bounds on the number of observations needed to ensure that the maximum likelihood estimator (MLE) exists with probability one. This is applied to bipartite graphs, grids and colored graphs. We also study the ML degree, and we present the first instance of a graph for which the MLE exists with probability one, even when the number of observations equals the treewidth. AU - Uhler, Caroline ID - 2959 IS - 1 JF - Annals of Statistics TI - Geometry of maximum likelihood estimation in Gaussian graphical models VL - 40 ER - TY - JOUR AB - Background: The outcome of male-male competition can be predicted from the relative fighting qualities of the opponents, which often depend on their age. In insects, freshly emerged and still sexually inactive males are morphologically indistinct from older, sexually active males. These young inactive males may thus be easy targets for older males if they cannot conceal themselves from their attacks. The ant Cardiocondyla obscurior is characterised by lethal fighting between wingless (" ergatoid" ) males. Here, we analyse for how long young males are defenceless after eclosion, and how early adult males can detect the presence of rival males.Results: We found that old ergatoid males consistently won fights against ergatoid males younger than two days. Old males did not differentiate between different types of unpigmented pupae several days before emergence, but had more frequent contact to ready-to-eclose pupae of female sexuals and winged males than of workers and ergatoid males. In rare cases, old ergatoid males displayed alleviated biting of pigmented ergatoid male pupae shortly before adult eclosion, as well as copulation attempts to dark pupae of female sexuals and winged males. Ergatoid male behaviour may be promoted by a closer similarity of the chemical profile of ready-to-eclose pupae to the profile of adults than that of young pupae several days prior to emergence.Conclusion: Young ergatoid males of C. obscurior would benefit greatly by hiding their identity from older, resident males, as they are highly vulnerable during the first two days of their adult lives. In contrast to the winged males of the same species, which are able to prevent ergatoid male attacks by chemical female mimicry, young ergatoids do not seem to be able to produce a protective chemical profile. Conflicts in male-male competition between ergatoid males of different age thus seem to be resolved in favour of the older males. This might represent selection at the colony level rather than the individual level. © 2012 Cremer et al.; licensee BioMed Central Ltd. AU - Cremer, Sylvia AU - Suefuji, Masaki AU - Schrempf, Alexandra AU - Heinze, Jürgen ID - 2966 JF - BMC Ecology TI - The dynamics of male-male competition in Cardiocondyla obscurior ants VL - 12 ER - TY - JOUR AB - The choice of summary statistics is a crucial step in approximate Bayesian computation (ABC). Since statistics are often not sufficient, this choice involves a trade-off between loss of information and reduction of dimensionality. The latter may increase the efficiency of ABC. Here, we propose an approach for choosing summary statistics based on boosting, a technique from the machine learning literature. We consider different types of boosting and compare them to partial least squares regression as an alternative. To mitigate the lack of sufficiency, we also propose an approach for choosing summary statistics locally, in the putative neighborhood of the true parameter value. We study a demographic model motivated by the re-introduction of Alpine ibex (Capra ibex) into the Swiss Alps. The parameters of interest are the mean and standard deviation across microsatellites of the scaled ancestral mutation rate (θanc = 4 Ne u), and the proportion of males obtaining access to matings per breeding season (ω). By simulation, we assess the properties of the posterior distribution obtained with the various methods. According to our criteria, ABC with summary statistics chosen locally via boosting with the L2-loss performs best. Applying that method to the ibex data, we estimate θanc ≈ 1.288, and find that most of the variation across loci of the ancestral mutation rate u is between 7.7×10−4 and 3.5×10−3 per locus per generation. The proportion of males with access to matings is estimated to ω ≈ 0.21, which is in good agreement with recent independent estimates. AU - Aeschbacher, Simon AU - Beaumont, Mark AU - Futschik, Andreas ID - 2962 IS - 3 JF - Genetics TI - A novel approach for choosing summary statistics in approximate Bayesian computation VL - 192 ER - TY - JOUR AB - Dieser Artikel soll die sechs verschiedenen Creative Commons Lizenzen erläutern und ihre Bedeutung im Rahmen des wissenschaftlichen Publizierens und des Open Access erklären (CC-BY, CC-BY-SA, CC-BY-NC, CC-BY-ND, CC-BYNC-SA, CC-BY-NC-ND). AU - Danowski, Patrick ID - 2965 IS - 2 JF - Mitteilungen der Vereinigung Österreichischer Bibliothekarinnen & Bibliothekare TI - Kontext Open Access: Creative Commons VL - 65 ER - TY - JOUR AB - Zebra finches are an ubiquitous model system for the study of vocal learning in animal communication. Their song has been well described, but its possible function(s) in social communication are only partly understood. The so-called ‘directed song’ is a high-intensity, high-performance song given during courtship in close proximity to the female, which is known to mediate mate choice and mating. However, this singing mode constitutes only a fraction of zebra finch males’ prolific song output. Potential communicative functions of their second, ‘undirected’ singing mode remain unresolved in the face of contradicting reports of both facilitating and inhibiting effects of social company on singing. We addressed this issue by experimentally manipulating social contexts in a within-subject design, comparing a solo versus male or female only company condition, each lasting for 24 hours. Males’ total song output was significantly higher when a conspecific was in audible and visible distance than when they were alone. Male and female company had an equally facilitating effect on song output. Our findings thus indicate that singing motivation is facilitated rather than inhibited by social company, suggesting that singing in zebra finches might function both in inter- and intrasexual communication. AU - Jesse, Fabienne AU - Riebel, Katharina ID - 2963 IS - 3 JF - Behavioural Processes TI - Social facilitation of male song by male and female conspecifics in the zebra finch, Taeniopygia guttata VL - 91 ER - TY - CONF AB - We construct a perfectly binding string commitment scheme whose security is based on the learning parity with noise (LPN) assumption, or equivalently, the hardness of decoding random linear codes. Our scheme not only allows for a simple and efficient zero-knowledge proof of knowledge for committed values (essentially a Σ-protocol), but also for such proofs showing any kind of relation amongst committed values, i.e. proving that messages m_0,...,m_u, are such that m_0=C(m_1,...,m_u) for any circuit C. To get soundness which is exponentially small in a security parameter t, and when the zero-knowledge property relies on the LPN problem with secrets of length l, our 3 round protocol has communication complexity O(t|C|l log(l)) and computational complexity of O(t|C|l) bit operations. The hidden constants are small, and the computation consists mostly of computing inner products of bit-vectors. AU - Jain, Abhishek AU - Krenn, Stephan AU - Pietrzak, Krzysztof Z AU - Tentes, Aris ED - Wang, Xiaoyun ED - Sako, Kazue ID - 2974 TI - Commitments and efficient zero knowledge proofs from learning parity with noise VL - 7658 ER - TY - JOUR AB - The coupling between presynaptic Ca^(2+) channels and Ca^(2+) sensors of exocytosis is a key determinant of synaptic transmission. Evoked release from parvalbumin (PV)-expressing interneurons is triggered by nanodomain coupling of P/Q-type Ca^(2+) channels, whereas release from cholecystokinin (CCK)-containing interneurons is generated by microdomain coupling of N-type channels. Nanodomain coupling has several functional advantages, including speed and efficacy of transmission. One potential disadvantage is that stochastic opening of presynaptic Ca^(2+) channels may trigger spontaneous transmitter release. We addressed this possibility in rat hippocampal granule cells, which receive converging inputs from different inhibitory sources. Both reduction of extracellular Ca^(2+) concentration and the unselective Ca^(2+) channel blocker Cd^(2+) reduced the frequency of miniature IPSCs (mIPSCs) in granule cells by ~50%, suggesting that the opening of presynaptic Ca^(2+) channels contributes to spontaneous release. Application of the selective P/Q-type Ca^(2+) channel blocker ω-agatoxin IVa had no detectable effects, whereas both the N-type blocker ω-conotoxin GVIa and the L-type blocker nimodipine reduced mIPSC frequency. Furthermore, both the fast Ca^(2+) chelator BAPTA-AM and the slow chelator EGTA-AM reduced the mIPSC frequency, suggesting that Ca^(2+)-dependent spontaneous release is triggered by microdomain rather than nanodomain coupling. The CB_(1) receptor agonist WIN 55212-2 also decreased spontaneous release; this effect was occluded by prior application of ω-conotoxin GVIa, suggesting that a major fraction of Ca^(2+)-dependent spontaneous release was generated at the terminals of CCK-expressing interneurons. Tonic inhibition generated by spontaneous opening of presynaptic N- and L-type Ca^(2+) channels may be important for hippocampal information processing. AU - Goswami, Sarit AU - Bucurenciu, Iancu AU - Jonas, Peter M ID - 2969 IS - 41 JF - Journal of Neuroscience TI - Miniature IPSCs in hippocampal granule cells are triggered by voltage-gated Ca^(2+) channels via microdomain coupling VL - 32 ER - TY - JOUR AB - Morphogen gradients regulate the patterning and growth of many tissues, hence a key question is how they are established and maintained during development. Theoretical descriptions have helped to explain how gradient shape is controlled by the rates of morphogen production, spreading and degradation. These effective rates have been measured using fluorescence recovery after photobleaching (FRAP) and photoactivation. To unravel which molecular events determine the effective rates, such tissue-level assays have been combined with genetic analysis, high-resolution assays, and models that take into account interactions with receptors, extracellular components and trafficking. Nevertheless, because of the natural and experimental data variability, and the underlying assumptions of transport models, it remains challenging to conclusively distinguish between cellular mechanisms. AU - Kicheva, Anna AU - Bollenbach, Mark Tobias AU - Wartlick, Ortrud AU - Julicher, Frank AU - Gonzalez Gaitan, Marcos ID - 2970 IS - 6 JF - Current Opinion in Genetics & Development TI - Investigating the principles of morphogen gradient formation: from tissues to cells VL - 22 ER - TY - CONF AB - We study the task of interactive semantic labeling of a segmentation hierarchy. To this end we propose a framework interleaving two components: an automatic labeling step, based on a Conditional Random Field whose dependencies are defined by the inclusion tree of the segmentation hierarchy, and an interaction step that integrates incremental input from a human user. Evaluated on two distinct datasets, the proposed interactive approach efficiently integrates human interventions and illustrates the advantages of structured prediction in an interactive framework. AU - Zankl, Georg AU - Haxhimusa, Yll AU - Ion, Adrian ID - 2971 TI - Interactive labeling of image segmentation hierarchies VL - 7476 ER - TY - JOUR AB - Growth and development are coordinated by an array of intercellular communications. Known plant signaling molecules include phytohormones and hormone peptides. Although both classes can be implicated in the same developmental processes, little is known about the interplay between phytohormone action and peptide signaling within the cellular microenvironment. We show that genes coding for small secretory peptides, designated GOLVEN (GLV), modulate the distribution of the phytohormone auxin. The deregulation of the GLV function impairs the formation of auxin gradients and alters the reorientation of shoots and roots after a gravity stimulus. Specifically, the GLV signal modulates the trafficking dynamics of the auxin efflux carrier PIN-FORMED2 involved in root tropic responses and meristem organization. Our work links the local action of secretory peptides with phytohormone transport. Root growth factor (RGF) or GOLVEN (GLV) secreted peptides have previously been implicated in meristem regulation. Whitford et al. now show that RGF/GLV peptides induce rapid relocalization of the auxin efflux regulator PIN2, regulate auxin gradients, and modulate auxin-dependent root responses to specific stimuli. AU - Whitford, Ryan AU - Fernandez, Ana AU - Tejos, Ricardo AU - Pérez, Amparo Cuéllar AU - Kleine-Vehn, Jürgen AU - Vanneste, Steffen AU - Drozdzecki, Andrzej AU - Leitner, Johannes AU - Abas, Lindy AU - Aerts, Maarten AU - Hoogewijs, Kurt AU - Pawel Baster AU - De Groodt, Ruth AU - Lin, Yao-Cheng AU - Storme, Véronique AU - Van de Peer, Yves AU - Beeckman, Tom AU - Madder, Annemieke AU - Devreese, Bart AU - Luschnig, Christian AU - Jirí Friml AU - Hilson, Pierre ID - 3105 IS - 3 JF - Developmental Cell TI - GOLVEN secretory peptides regulate auxin carrier turnover during plant gravitropic responses VL - 22 ER - TY - JOUR AB - Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin-and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane. AU - Irani, Niloufer G AU - Di Rubbo, Simone AU - Mylle, Evelien AU - Van Den Begin, Jos AU - Schneider-Pizoń, Joanna AU - Hniliková, Jaroslava AU - Šíša, Miroslav AU - Buyst, Dieter AU - Vilarrasa-Blasi, Josep AU - Szatmári, Anna-Maria AU - Van Damme, Daniël AU - Mishev, Kiril AU - Codreanu, Mirela-Corina AU - Kohout, Ladislav AU - Strnad, Miroslav AU - Caño-Delgado, Ana I AU - Jirí Friml AU - Madder, Annemieke AU - Russinova, Eugenia ID - 3109 IS - 6 JF - Nature Chemical Biology TI - Fluorescent castasterone reveals BRI1 signaling from the plasma membrane VL - 8 ER - TY - JOUR AB - Gradients of the plant hormone auxin, which depend on its active intercellular transport, are crucial for the maintenance of root meristematic activity. This directional transport is largely orchestrated by a complex interaction of specific influx and efflux carriers that mediate the auxin flow into and out of cells, respectively. Besides these transport proteins, plant-specific polyphenolic compounds knownasflavonols have beenshownto act as endogenous regulators of auxin transport. However, only limited information is available on how flavonol synthesis is developmentally regulated. Using reduction-of-function and overexpression approaches in parallel, we demonstrate that the WRKY23 transcription factor is needed for proper root growth and development by stimulating the local biosynthesis of flavonols. The expression of WRKY23 itself is controlled by auxin through the AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 transcriptional response pathway. Our results suggest a model in which WRKY23 is part of a transcriptional feedback loop of auxin on its own transport through local regulation of flavonol biosynthesis. AU - Grunewald, Wim AU - De Smet, Ive AU - Lewis, Daniel R AU - Löfke, Christian AU - Jansen, Leentje AU - Goeminne, Geert AU - Vanden Bossche, Robin AU - Karimi, Mansour AU - De Rybel, Bert AU - Vanholme, Bartel AU - Teichmann, Thomas AU - Boerjan, Wout AU - Van Montagu, Marc C AU - Gheysen, Godelieve AU - Muday, Gloria K AU - Jirí Friml AU - Beeckman, Tom ID - 3104 IS - 5 JF - PNAS TI - Transcription factor WRKY23 assists auxin distribution patterns during Arabidopsis root development through local control on flavonol biosynthesis VL - 109 ER - TY - JOUR AB - The phytohormone auxin acts as a prominent signal, providing, by its local accumulation or depletion in selected cells, a spatial and temporal reference for changes in the developmental program. The distribution of auxin depends on both auxin metabolism (biosynthesis, conjugation and degradation) and cellular auxin transport. We identified in silico a novel putative auxin transport facilitator family, called PIN-LIKES (PILS). Here we illustrate that PILS proteins are required for auxin-dependent regulation of plant growth by determining the cellular sensitivity to auxin. PILS proteins regulate intracellular auxin accumulation at the endoplasmic reticulum and thus auxin availability for nuclear auxin signalling. PILS activity affects the level of endogenous auxin indole-3-acetic acid (IAA), presumably via intracellular accumulation and metabolism. Our findings reveal that the transport machinery to compartmentalize auxin within the cell is of an unexpected molecular complexity and demonstrate this compartmentalization to be functionally important for a number of developmental processes. AU - Barbez, Elke AU - Kubeš, Martin AU - Rolčík, Jakub AU - Béziat, Chloe AU - Pěnčík, Aleš AU - Wang, Bangjun AU - Rosquete, Michel Ruiz AU - Zhu, Jinsheng AU - Dobrev, Petre I AU - Lee, Yuree AU - Zašímalová, Eva AU - Petrášek, Jan AU - Geisler, Markus AU - Jirí Friml AU - Kleine-Vehn, Jürgen ID - 3108 IS - 7396 JF - Nature TI - A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants VL - 485 ER - TY - JOUR AB - Cell polarization via asymmetrical distribution of structures or molecules is essential for diverse cellular functions and development of organisms, but how polarity is developmentally controlled has been poorly understood. In plants, the asymmetrical distribution of the PIN-FORMED (PIN) proteins involved in the cellular efflux of the quintessential phytohormone auxin plays a central role in developmental patterning, morphogenesis, and differential growth. Recently we showed that auxin promotes cell interdigitation by activating the Rho family ROP GTPases in leaf epidermal pavement cells. Here we found that auxin activation of the ROP2 signaling pathway regulates the asymmetric distribution of PIN1 by inhibiting its endocytosis. ROP2 inhibits PIN1 endocytosis via the accumulation of cortical actin microfilaments induced by the ROP2 effector protein RIC4. Our findings suggest a link between the developmental auxin signal and polar PIN1 distribution via Rho-dependent cytoskeletal reorganization and reveal the conservation of a design principle for cell polarization that is based on Rho GTPase-mediated inhibition of endocytosis. AU - Nagawa, Shingo AU - Xu, Tongda AU - Lin, Deshu AU - Dhonukshe, Pankaj AU - Zhang, Xingxing AU - Jirí Friml AU - Scheres, Ben AU - Fu, Ying AU - Yang, Zhenbiao ID - 3106 IS - 4 JF - PLoS Biology TI - ROP GTPase-dependent actin microfilaments promote PIN1 polarization by localized inhibition of clathrin-dependent endocytosis VL - 10 ER - TY - GEN AU - Vanneste, Steffen AU - Friml, Jirí ID - 3107 IS - 5 T2 - Nature Chemical Biology TI - Plant signaling: Deconstructing auxin sensing VL - 8 ER - TY - CONF AB - We present an approach for artist-directed animation of liquids using multiple levels of control over the simulation, ranging from the overall tracking of desired shapes to highly detailed secondary effects such as dripping streams, separating sheets of fluid, surface waves and ripples. The first portion of our technique is a volume preserving morph that allows the animator to produce a plausible fluid-like motion from a sparse set of control meshes. By rasterizing the resulting control meshes onto the simulation grid, the mesh velocities act as boundary conditions during the projection step of the fluid simulation. We can then blend this motion together with uncontrolled fluid velocities to achieve a more relaxed control over the fluid that captures natural inertial effects. Our method can produce highly detailed liquid surfaces with control over sub-grid details by using a mesh-based surface tracker on top of a coarse grid-based fluid simulation. We can create ripples and waves on the fluid surface attracting the surface mesh to the control mesh with spring-like forces and also by running a wave simulation over the surface mesh. Our video results demonstrate how our control scheme can be used to create animated characters and shapes that are made of water. AU - Raveendran, Karthik AU - Thuerey, Nils AU - Wojtan, Christopher J AU - Turk, Greg ID - 3119 T2 - Proceedings of the ACM SIGGRAPH/Eurographics Symposium on Computer Animation TI - Controlling liquids using meshes ER - TY - JOUR AB - We present a method for recovering a temporally coherent, deforming triangle mesh with arbitrarily changing topology from an incoherent sequence of static closed surfaces. We solve this problem using the surface geometry alone, without any prior information like surface templates or velocity fields. Our system combines a proven strategy for triangle mesh improvement, a robust multi-resolution non-rigid registration routine, and a reliable technique for changing surface mesh topology. We also introduce a novel topological constraint enforcement algorithm to ensure that the output and input always have similar topology. We apply our technique to a series of diverse input data from video reconstructions, physics simulations, and artistic morphs. The structured output of our algorithm allows us to efficiently track information like colors and displacement maps, recover velocity information, and solve PDEs on the mesh as a post process. AU - Bojsen-Hansen, Morten AU - Li, Hao AU - Wojtan, Christopher J ID - 3118 IS - 4 JF - ACM Transactions on Graphics TI - Tracking surfaces with evolving topology VL - 31 ER - TY - JOUR AB - Since Darwin's pioneering research on plant reproductive biology (e.g. Darwin 1877), understanding the mechanisms maintaining the diverse sexual strategies of plants has remained an important challenge for evolutionary biologists. In some species, populations are sexually polymorphic and contain two or more mating morphs (sex phenotypes). Differences in morphology or phenology among the morphs influence patterns of non-random mating. In these populations, negative frequency-dependent selection arising from disassortative (intermorph) mating is usually required for the evolutionary maintenance of sexual polymorphism, but few studies have demonstrated the required patterns of non-random mating. In the current issue of Molecular Ecology, Shang (2012) make an important contribution to our understanding of how disassortative mating influences sex phenotype ratios in Acer pictum subsp. mono (painted maple), a heterodichogamous, deciduous tree of eastern China. They monitored sex expression in 97 adults and used paternity analysis of open-pollinated seed to examine disassortative mating among three sex phenotypes. Using a deterministic 'pollen transfer' model, Shang et al. present convincing evidence that differences in the degree of disassortative mating in progeny arrays of the sex phenotypes can explain their uneven frequencies in the adult population. This study provides a useful example of how the deployment of genetic markers, demographic monitoring and modelling can be integrated to investigate the maintenance of sexual diversity in plants. AU - Field, David AU - Barrett, Spencer ID - 3122 IS - 15 JF - Molecular Ecology TI - Disassortative mating and the maintenance of sexual polymorphism in painted maple VL - 21 ER - TY - JOUR AB - Voltage-activated Ca(2+) channels (VACCs) mediate Ca(2+) influx to trigger action potential-evoked neurotransmitter release, but the mechanism by which Ca(2+) regulates spontaneous transmission is unclear. We found that VACCs are the major physiological triggers for spontaneous release at mouse neocortical inhibitory synapses. Moreover, despite the absence of a synchronizing action potential, we found that spontaneous fusion of a GABA-containing vesicle required the activation of multiple tightly coupled VACCs of variable type. AU - Williams, Courtney AU - Chen, Wenyan AU - Lee, Chia AU - Yaeger, Daniel AU - Vyleta, Nicholas AU - Smith, Stephen ID - 3121 IS - 9 JF - Nature Neuroscience TI - Coactivation of multiple tightly coupled calcium channels triggers spontaneous release of GABA VL - 15 ER -