Homo sapiens, which allow simulations that provide valuable insights into topics, including predictions of gene essentiality of pathogens, interpretation of genetic polymorphism in metabolic disease syndromes and suggestions for novel approaches to microbial metabolic engineering. These constraint-based simulations are being integrated with the functional genomics portals, an activity that requires efficient implementation of the constraint-based simulations in the web-based environment.Constraint-based approaches facilitate the prediction of cellular metabolic capabilities, based, in turn on predictions of the repertoire of enzymes encoded in the genome. Recently, genome annotations have been used to reconstruct genome scale metabolic reaction networks for numerous species, including E. coli, S. cerevisiae and M. tuberculosis.Here, we present Acorn, an open source (GNU GPL) grid computing system for constraint-based simulations of genome scale metabolic reaction networks within an interactive web environment. The grid-based architecture allows efficient execution of computationally intensive, iterative protocols such as Flux Variability Analysis, which can be readily scaled up as the numbers of models (and users) increase. The web interface uses AJAX, which facilitates efficient model browsing and other search functions, and intuitive implementation of appropriate simulation conditions. Research groups can install Acorn locally and create user accounts. Users can also import models in the familiar SBML format and link reaction formulas to major functional genomics portals of choice. Selected models and simulation results can be shared between different users and made publically available. Users can construct pathway map layouts and import them into the server using a desktop editor integrated within the system. Pathway maps are then used to visualise numerical results within the web environment. To illustrate these features we have deployed Acorn and created a web server allowing constraint based simulations of the genome scale metabolic reaction networks of Acorn is a free software package, which can be installed by research groups to create a web based environment for computer simulations of genome scale metabolic reaction networks. It facilitates shared access to models and creation of publicly available constraint based modelling resources. The acquisition of this information can be initiared by analysis of the genome sequence of interest, and this approach has been applied to the reconstruction of genome scale metabolic reaction networks (GSMNs) for major model organisms [Analysis of biochemical reaction networks, to enable predictions about the phenotype from a catalogue of molecular parts encoded in the genome, fulfils a major objective of Systems Biology. Due to the scarcity of detailed quantitative information about individual enzyme rate constants and intracellular concentrations, genome scale models have, so far, only been constructed for the special case of the metabolic reaction networks operating under steady state conditions. These models capitalize on the fact that metabolic conversions occur in the cell with very high rates and therefore, for the given set of genes active under experimental conditions of interest, the network of metabolic reactions can be considered to be in a quasi-steady state (where metabolite concentrations do not change). The system is then modelled by a set of linear relationships between the calculated reaction fluxes. Although, the linear system corresponding to genome scale models is underdetermined it can be exploited by Linear Programming (LP) based optimization of the objective function that represents the metabolic capability of interest. The objective function is defined as the flux through a specific reaction or the flux towards a specific metabolite. LP optimization results in a unique value for the objective function, but the corresponding flux distribution is not unique. The range of each individual reaction flux in all flux distributions that are consistent with the maximal objective function value can be exploited by the iterative LP protocols, described in the following sections. This approach is referred to as constraint based , becauserganisms -5, pathorganisms -8 and mirganisms ,10. The rganisms  and is crganisms ,13.The availability of full genome sequences has motivated the development of functional genomics internet portals, where a multiplicity of sequence-derived, high-throughput and traditional experimental data are integrated through appropriate comparative analysis protocols. The integration of constraint-based simulations of GSMNs with these portals will equip users, for the first time, with tools that will enable predictions of the metabolic phenotype of cells under defined experimental conditions, determined in terms of a set of nutrients present in the medium.The concept of gene essentiality provides a good illustration of these new capabilities. Currently, functional genomics portals describe each gene in terms of comparison to similar sequences in other organisms, direct experimental evidence about the function under particular experimental conditions and high throughput datasets in which the expression of the gene has been observed. A constraint-based analysis of the appropriate GSMN, integrated with this system, would allow this content to be dependent on experimental conditions, specified by the user. The user could specify the composition of the growth medium and then compute the function of the gene expressed as a set of metabolic capabilities for which this gene is essential under user-defined experimental conditions.Mycobacterium tuberculosis, the causative agent of Tuberculosis. In this case, the web interface allows the specification of the growth medium composition, and using this information, provides a calculation of the maximal theoretical flux towards biomass as well as a gene essentiality prediction. It also implements a reaction essentiality scan and Flux Variability Analysis (FVA) [Escherichia coli iJR904 model. The server displays metabolic pathway maps and overlays simulation results on these diagrams. Only a single evaluation of the objective function is provided and no iterative simulation approaches are available. We also note that none of the web servers described above are available as software that can be downloaded and used to create new web servers at the users institution. Recently, the BioMet Toolbox [So far, there have been the following attempts at creating web-based resources, allowing computation with GSMN models with subsequent linking of models and results to functional genomics resources. The GSMN-TB  is the fis (FVA) , which ais (FVA) , a functis (FVA)  system ais (FVA)  or Pathwis (FVA)  softwareis (FVA)  server a Toolbox  server hin silico experiments by specifying nutritional conditions and run virtual experiments in order to calculate the maximum theoretical value of the objective function, identify essential reactions, predict gene essentiality and perform Flux Variability Analysis (FVA) [Saccharomyces cerevisiae, E. coli and M. tuberculosis :In this work we present Acorn, a grid computing system, which can be deployed at the users institution and used to create web based resources for constraint based simulations of GSMN models. Research groups can install Acorn, set up user accounts and import models in the SBML format . Users cis (FVA) . Models http://sysbio3.fhms.surrey.ac.uk:8080/acorn/homepage.jsf.In this section we describe the implementation details of Acorn. We also explain and justify the technological and architectural choices made, and the design approaches applied to the implementation of the system.The Acorn system consists of three components Figure :1. The main application, which includes a web based user interface for defining and executing the required computational task as well a database for storing models, experiments and experimental results,2. The worker console application for performing the computations requested by users via the web interface,3. A graphical editor desktop application for defining which parts of the model are to be visualized and how.The user interface of the main application is web based, designed with ergonomics in mind and optimised for handling of large models. As an example of this latter feature, the user can quickly filter long lists with an AJAX (Asynchronous JavaScript and XML) based mechanism, i.e., without the delays that might be incurred in transferring data between the browser and the web server. An example of the ergonomic approach is the positioning of key control buttons such that they are always visible in the same place on the screen using specially defined CSS (Cascading Style Sheets). This facilitates working with long lists. A useful outcome of this approach is that the user does not have to scroll to start the simulation after accepting the choice of optimisation target on the reaction list.Since computations with Acorn can be time intensive, we have chosen an architecture Figure  that allhttps://glassfish.dev.java.net/. Acorn profits from the maturity and enterprise features of Java EE component technologies. For example, operations on the queue and database are subject to transaction control, so the computations can be consistently completed in situations like concurrent execution of many workers, failure of an active worker or restart of the main application. The GlassFish server is also equipped with necessary tools for monitoring and tuning. Furthermore, the flexibility of the enterprise programming environment allows the deployment of Acorn with any relational database management system providing JDBC drivers. The current version has been tested with Java Derby http://db.apache.org/derby/ and MySQL http://www.mysql.com databases.The system is implemented in Java with the use of the Java Enterprise Edition (Java EE) technology stack. The main application can be deployed on a Java EE 5.0 compatible application server as the Open Source GlassFish 2.1 http://www.gnu.org/software/glpk/. The linear programming engine is a binary executable file implemented in C++. The worker starts the linear programming engine using the operating system interface of Java, which sends the linear programming problem to its standard input and retrieves results from standard output without creating any temporary files. The engine is also available for users as a standalone command line tool, which can be used to implement other interfaces.Linear programming calculations are performed using the appropriate functions of the GLPK library http://platform.netbeans.org/. It runs on all platforms where the Java runtime environment is available. It accesses the main application to obtain the model data and to store defined visualisations for presentation in the web interface. This communication is performed though a Web Service provided by the main application, which, for the user, has the added advantage that it will not be blocked by firewalls.The graphical editor is a standalone Java desktop application built on the NetBeans Platform, which is a generic framework for Swing applications http://code.google.com/p/a-c-o-r-n/. The main application can be installed on any platform running Java VM, the Glassfish application server and a relational database management system. The binaries of the linear programming engine are available for Linux, MacOS × and Windows operating systems and, together with the Java VM, are necessary to run the worker. The graphical editor desktop application can be used on any system running Java VM. The current version of Acorn provides the following features.Distribution of Acorn can be implemented by downloading from Google Code repository The user logs into the system via web interface and can browse GSMN models in tabular format Figure . The rowThe following computational methods are available:i) Flux Balance Analysis (FBA) - calculation of the maximal theoretical value of the objective function under nutritional conditions specified by the user;iI) Gene essentiality prediction - the fluxes through all reactions which require the functionality of a selected gene are constrained to 0 and the maximal theoretical value of the objective function is calculated. If the objective function value equals 0, the gene is declared to be essential for particular metabolic function;iii) Reaction essentiality scan - each reaction, in turn, is inactivated and the FBA optimisation is run. The result lists the maximal value of the objective function for each single reaction knock-out;iv) Flux Variability Analysis (FVA)  - the flin silico experiment. Users can share in silico experiments and visualise their results on pathway diagrams.To run a simulation via the Acorn web interface the user first selects one of the methods and then specifies the objective function and nutritional conditions. The objective function can be specified by a metabolite name  or reaction name . Nutritional conditions are defined by constraining lower and upper bounds of transport reaction fluxes. The Acorn interface allows the specification of bounds for all reactions in the system, allowing for more complex analysis scenarios such as incorporation of internal metabolic flux measurements or simulations of multiple gene knockouts. The keyword searches can be used to quickly find reactions and metabolites of interest. Simulations are scheduled and the user can either wait for the completion of computational tasks, or log out and return to the system later to examine results. The simulation scenario, together with the simulation results are saved into the database as an A single execution of FBA is very computationally efficient. For example, calculation of the maximal growth rate for yeast GSMN model iND750 takes 0.73s on 2.13 GHz Intel Core 2 Duo processor CPU when calculated with the linear programming engine of the Acorn system. However, iterative protocols of reaction essentiality scans and FVA require one or two (respectively) evaluations of the objective function for each reaction in the system. Therefore, execution of FVA for the yeast GSMN model containing 1265 reactions takes about 15 minutes of CPU time. This computational time can be substantial for web-based applications, especially if multiple users are simultaneously using the server. The Acorn server, on the other hand, is using grid-based solutions to distribute computational load between multiple computers. The worker program is installed on each computer in the cluster. The worker connects to the Acorn server and receives a set of FBA objective function evaluation tasks to execute. Results of these tasks are sent back to the server (see Implementation section for more details). Therefore, FBA calculations from the Acorn server can be distributed over an arbitrary number of computers in the local area network or the internet, rendering the system scalable to large number of users and computational tasks.The user can import GSMN models in the SBML format Figure . The reain silico experiments with a particular model initially belong the user who created the model. The user may chose to share models and in silico experiments with other users in the system. This allows for the creation of online communities where users share models and results of numerical simulations. The models and experiments can be also made available to a publicly accessible account, which enables creation of web based resources for constraint based simulations of GSMN models.Every new user of Acorn has to register via the web interface. The first user registered, after the software installation step, is given administration privileges. The models are created during SBML file upload, and initially can be only used by the user who uploaded the SBML file. Likewise, Acorn provides a desktop editor for metabolic pathway diagrams Figure . DiagramNumerical results of FBA and FVA calculations can be visualised on pathway diagrams Figure . The patThe computational engine of the Acorn software is available as a stand-alone tool. The tool can be used to evaluate the objective function of the GSMN model written in simple text file format. The command line tool interprets boolean gene-reaction association formulas and can be therefore used to perform evaluate the objective function for a single gene knockout version of the model. Availability of the Acorn computational engine in the form of a command line tool binary is useful for the users who intend to develop their own interfaces. The tool could be easily integrated with the script languages used by the bioinformatics community and then become a part of an annotation pipeline. It can be also used to create graphics user interfaces other than Acorn. Finally, users of the Acorn web server can also use the command line tool to run some of their computations on their local machines if the resources of the web server are limited or constrained.M. tuberculosis [E. coli iAF1260 [S. cerevisiae iND750 [To demonstrate the features of Acorn, we have constructed a web resource for constraint-based simulation of the GSMNs of interest to our research group. The resource contains the GSMN-TB model of rculosis , reconste iND750 . The iAFe iND750 . In the E. coli model is linked to KEGG database, and the S. cerevisiae model is linked to the SGD database. These implementations have demonstrated that the model upload mechanism of Acorn can be used to link reaction lists and results files of GSMN models to major functional genomics resources, thus facilitating interpretation of computer simulations in the context of the wealth of knowledge and comparative analysis results available within these knowledge bases.Each of the models available within the web resource is linked to the relevant functional genomics portal. The gene names in GSMN-TB model are linked to the Tuberculist database, the The user can use our web resource to run all simulation protocols implemented in Acorn on the models mentioned above. To demonstrate the visualisation functionality of Acorn, we have used the desktop editor to create a layout of glycolytic pathway in Yeast and uploaded this layout into the iND750 model. Users can now visualise fluxes calculated by computer simulation on the pathway layouts of their choice. The fluxes are obtained by the calculations involving all reactions of the entire GSMN, but only the reactions of interest to the users are displayed in a format, which is the most informative for them.In this paper, we present the Acorn software package, which can be deployed at the user's institution and used to create web-based resources for constraint based simulations of GSMNs. Our software can be used to make a collection of GSMNs available to a community of users who hold accounts within the system. Users can upload models and run constraint-based simulation methods to make predictions about the metabolic capabilities of organisms under investigation. The models and results are linked to the functional genomic resources through the gene names. Therefore, the results of constraint based calculations can be examined in the context of genome sequences, annotations and sequence similarity-based predictions available in these portals. The grid-based architecture of Acorn allows for the implementation of computationally expensive simulation protocols requiring the execution of thousands of linear programming evaluations for the entire model. Users can visualise numerical results of simulation methods on pathway maps loaded into the system. These maps can represent tthe users view, a part of the model of specific interest to the user concerned, while numerical data visualised on the map are calculated by constraint-based modelling of the entire network. Pathway diagrams are constructed with the desktop tool, which supports rapid assignment of the graphical symbols to substances and reactions present in the model.One of the most important features of Acorn is user management and model sharing. Select users can upload starting models and share them with others. All other users can create variations of those starting models by modifying their parameters and set their own simulations without affecting the models and simulations run by other users. They may then choose to render the models, simulation scenarios and results visible to other users in the system and, through the guest user account, available to the research community. Therefore, Acorn can be used to create research community websites dedicated to the annotation and investigation of metabolic reaction network models.S. cerevisiae, E. coli, and M. tuberculosis. Users can either use a guest account or register and gain access to these models without the need of installing and learning modelling environments such as, for example, MATLAB based COBRA toolkits. Users can also use our server to upload their models, if they are represented in the SBML format, and use computational tools implemented within the system to study those models (and share the results with other users).In addition to the software that can be used to create a constraint-based modelling server, we also provide a web resource giving access to the GSMN models of We believe that Acorn provides several advantages and alternative solutions compared to existing web servers for constrained-based modelling of metabolic networks. It provides advantages when compared to the GSMN-TB server with respect to visualisation and implements a more convenient interface for model specification. Its main advantage over CycSim and WEbcoli is the implementation of the FVA and reaction essentiality scans. FVA is currently the only method, which allows the exploration of internal flux distribution in GSMN.s It is efficiently implemented in Acorn, due to its grid based architecture. Acorn also provides an alternative mechanism for visualising metabolic pathway maps. While CyCsim uses standard KEGG and BioCyc maps, the Acorn server allows the individual user to create their own views, which are linked with the model. Another major advantage of the Acorn environment over CycSim and WEbcoli is that it provides source code distribution, which can be used to create collaborative model development environments and public web resources in other laboratories.in silico experiments. This will facilitate improvement of metabolic models used in the identification of novel drug targets in microbial pathogens and metabolic engineering of industrially valuable microbial strains.To summarise, we believe that the Acorn server will be very useful for research groups reconstructing and studying GSMNs. Its application will result in establishment of online communities sharing models and results of Project name: Acornhttp://code.google.com/p/a-c-o-r-n/Project home page: http://sysbio3.fhms.surrey.ac.uk:8080/acorn/homepage.jsfSurrey GSMN server: Operating systems(s): Platform independentProgramming language: Java, C++Other requirements: Java 6.0 or higher, GlassFish Java EE Server v2 or higherLicense: GNU GPL v2Any restrictions to use by non-academics: The use of constraint based modelling results obtained by using iAF1260 and iND750 models on Surrey FBA resource is subjected to the BiGG database license.AMK and JS designed software functionality. JS designed software architecture, supervised software development and participated in software implementation. LBK, SG, DL, JL implemented database and worker modules. MM implemented pathway visualisation. JL implemented command line computational engine. CAR developed case studies for visualisation of yeast pathways. MB and JMF tested software functionality and participated in writing of the manuscript. AMK and JS wrote the manuscript. All authors read and approved the final manuscript.


Recently, the heterogeneity that arises from stochastic fate decisions has been reported for several types of cancer-derived cell lines and several types of clonal cells grown under constant environmental conditions. However, the relation between this stochasticity and the responsiveness to extracellular stimuli remains largely unknown. Here we focused on the fate decisions of the PC12 cell line, which was derived from rat pheochromocytoma, and is a model system to study differentiation into sympathetic neurons. Whereas epidermal growth factor (EGF) stimulates the proliferation of populations of PC12 cells, nerve growth factor (NGF) promotes the differentiation of neurites to neuron-like cells. We found that phenotypic heterogeneity increased with time at several surrounding serum concentrations, suggesting stochastic cell-fate decisions in single cells. We made a simple mathematical model assuming Markovian transitions of the cell fates, and estimated the transition rates based on Bayes' theorem. The model suggests that depending on the serum concentration, EGF (NGF) even directs differentiation (proliferation) at the single-cell level. The maximum effects of the growth factors were ensured when the transition rates were appropriately controlled by the serum concentration to produce a nonextremal, moderate amount of cell-fate heterogeneity. Our model was validated by the experimental finding that the means and variances of the local cell densities obey a power-law relationship. These results suggest that even when efficient responses to growth factors are observed at the population level, the growth factors stochastically direct the cell-fate decisions in different directions at the single-cell level. Elucidation of the mechanisms that regulate cell fate has become one of the primary goals of research in cell biology and regenerative medicine. Growth factors are often used to regulate cell fate. However, stochastic cellular responses to growth regulators have prevented precise control of cell fate. We report our investigation of the relationship between heterogeneity and responsiveness in cell fate decisions by both single cells and populations of cells. Our study involved PC12, a cultured cell line for which cell-fates are affected by exposure to growth factors and culture conditions. Computational methods using a mathematical model enabled us to determine the cell-fate decisions rate in single PC12 cells and analyze the population responses to growth factors from experimental data. Our findings reveal that growth factors control cell-fate decisions rate in single PC12 cells, and suggest distinct differences in the mechanisms of actions of growth factors under different culture conditions. In addition, we observed maximum effects of growth factors when a nonextremal, moderate amount of cell-fate heterogeneity exists. Our results give several insights into stochastic cell responses, including the effects of anticancer agents on cancer cells and the optimization of methods to induce the differentiation of stem cells. Escherichia coli or competence in Bacillus subtilisPhenotypic heterogeneity, which has been thoroughly discussed for tumor cells, is not a unique property of cancerous cells but has also been observed in normal clonal cells in culture Although heterogeneity was observed in several types of cells, the effects of extracellular stimuli on those heterogeneous populations have not been fully clarified. Here, we used PC12 cells to attempt to address this issue. The rat pheochromocytoma clone PC12, which was developed from an adrenal medullary tumor derived from the adrenergic neural crest The response of PC12 cells to growth factors is heterogeneous on the level of individual cells and is affected by the surrounding serum concentration, but the relationship between cell heterogeneity and cell responsiveness to growth factors has not been measured quantitatively. Here, we report the effects of the growth factors EGF and NGF under three different serum conditions, in which PC12 cells show different degrees of heterogeneity in their fate decisions. We measured the time courses of the numbers of cells in three states  and constructed a mathematical model. By definition, we regard concentrations of surrounding serum as an environmental condition, and regard cell responses as meaning changes in cell fate triggered by stimuli, such as EGF and NGF, at that particular serum concentration.In this study, we first used entropy values to define the heterogeneity of a population of cells containing a large fraction of proliferative cells, and found that the entropy values increased as a function of time, suggesting that stochastic cell-fate decisions in single PC12 cells increased the heterogeneity of the population, regardless of the surrounding serum. This heterogeneity decreased as the concentration of the surrounding serum increased. We made a simple mathematical model assuming that cells determine their fates with constant probabilities, and estimated the probabilities for which the model can explain the experimental results, based on Bayes' theorem. This method enabled us to use experimental data collected using populations of cells to estimate the rates with which single cells made cell-fate decisions. Based on the model and the parameter values, we redefined the effects of the growth factors: EGF increased the proliferation rate at the single-cell level, although the effect could be covered and was affected by the surrounding serum at the population level, as shown in previous experiments. At some serum concentrations, EGF (NGF) even directed differentiation (proliferation) at the single-cell level. Even when efficient responses to the growth factors were observed at the population level, the growth factors only stochastically directed single cells to different cell fates. We evaluated the strength of the responses to the growth factors at the population level on a phase portrait using the Malthus coefficient and the fluxes of the three phenotypes, and examined the relationship between cell heterogeneity and cell responsiveness. The strength of the responses to EGF and NGF as a function of entropy (cell-fate heterogeneity) peaked at a moderate entropy value. Finally, we showed that the relationship between the means and variances of the local cell densities obeyed a power-law relationship, which could be explained by a stochastic simulation that supported the idea that the cells have a single set of transition rates with constant values under each condition.In this section, we first report experimental results that describe dynamic changes in the number of cells at the population level. Next, we propose the use of a mathematical model to calculate transition rates at the single cell level from experimental results.Three typical states of PC12 cells are defined by their morphologies ; These agle dish .We compared the cell-fate processes under control conditions, in which the cells were cultured without additional growth factors but in the presence of three different concentrations of serum . In the The growth factor EGF was added to these control conditions to evaluate how cell-fate decisions change in response to this stimulus . A saturWhen we added the growth factor NGF, differentiation was accelerated under all of the serum conditions tested . A saturTherefore, in our experiments, cell death and differentiation were not completely suppressed under conditions that support logarithmic growth, and cells differentiate without any growth factors even under serum-free conditions. Thus, the clonal PC12 cells became a heterogeneous population under each of the serum conditions tested. In addition, the effects of EGF and NGF depended on the environmental serum conditions, with both of these growth factors affecting multiple transition pathways. To further validate this issue, we generated a mathematical model to capture these stochastic state transitions of PC12 cells. This model is discussed in the section that follows.It is difficult to evaluate and compare the processes that determine the fates of single cells quantitatively among different conditions directly from monitoring changes in the number of cells over time. For quantification, we constructed a mathematical model that uses ordinary differential equations to capture the state transitions of cells . TherefoWe carried out four independent sets of experiments and predicted parameter values for each experiment. The mean parameter values enabled us to estimate typical characteristics of cell-fate decisions in single PC12 cells. State transition rates at the single-cell level were compared among control conditions . In the py value . At the py value . Under spy value . Each paIn It has usually been suggested that NGF has an antimitogenic effect on PC12 cells We also found that EGF stimulated the differentiation rate The responses of the de-differentiation rate Suppression of cell death by NGF in serum-free medium at the cell-population level was reported previously Furthermore, using a mathematical model of differential equations, portrait . The numnditions . As predThe parameter values estimated in the previous section denote single-cell-level transition rates. However, at the population level, the effects of these parameters are very complex (see Models section), and it is difficult to evaluate the effects of the growth factors on the population dynamics from the estimated parameter values. In this section, we introduce a method to use a phase portrait to capture dynamic changes in the number of cells at the population level, and to compare the responses to growth factors under three serum conditions. We use this portrait to quantitatively characterize the responses to growth factors. Finally, we evaluate the relationships between the heterogeneity of a population and the response indexes.We now introduce some indexes to quantify the direction of cell fate decision processes derived from a mathematical model. In the following section, we evaluate the extent of responses to growth factors compared with the control conditions that use these indexes. We propose three indices. The first is the Malthusian coefficient on occur . Thus, c2/dayAs the second and the third indices, we used the initial flux rocesses . The uninditions . For theThe effects of growth factors are summarized in 12 cells . As has We next compare the strength of responses to growth factors among three serum conditions . In ordentration . An explntration  or minimntration  peaks inntration  or minimntration  for all As shown here, we found the optimal condition for the maximal responses to the growth factors to be a nonextremal, moderate amount of cell-fate heterogeneity. An explanation of this observation is that when entropy is low, most cell fates are controlled by the materials contained in the serum, and additional growth factors have only limited effects. When entropy is high, most cell fates are not controlled by the serum. Instead, they are determined spontaneously, and the high level of spontaneity prevents the cells from effectively responding to external stimuli. The flux responses  were constant, as defined in l result . Other ml result . Furtherribution , which sibutions .As shown here, a stochastic model with constant parameter values is sufficient to explain the observed power-law relation. Therefore, it is highly probable that a cell fate is decided by stochastic fluctuations in intracellular reactions. However, what decides the rates of fate transitions is still unknown. Interpretations of power-law relations using mathematical models have been performed in several contexts We analyzed the time courses of the cell fate decision processes of PC12 cells after the addition of either of the growth factors EGF or NGF under three different environmental serum conditions. The results are summarized in Rat PC12 pheochromocytoma cells from the Riken Cell Bank  were cultured and maintained at To count the densities of proliferating, differentiated, and dead cells, thirty images of living PC12 cells within thirty areas of In addition to using the Analysis of variance was used when more than three population averages were available for comparison. We assumed that there were 2To estimate the mean transition rates between the cell states, we generated a mathematical model that describes processes that decide cell fates. In this model, we assumed that (i) each cell independently determines cell fates with no cell–cell interaction, (ii) cells do not proliferate when they are differentiated, and (iii) de-differentiation occurs. The second assumption is based on reports indicating that upon treatment with NGF, cells appeared to stop dividing and the number of surviving cells was saturated The velocities In our model, the parameter values are always nonnegative. Therefore, the number of cells converges asymptotically to the following equation, with a long time limit The rates of responses to external signals compared with a control condition were defined in one of three ways: (i) the Malthusian parameter, (ii) the initial response speeds, or (iii) the time average of response speeds. The Malthusian parameter To estimate parameter values for our three-state model, we applied Bayesian inference to our experimental data. In addition to the differential The posterior probability density function ribution . In our Here, we seek to make a stochastic model. A scheme of cell fate decision processes is as followsLognormal distributions  were calWhen we define the number of cells at time Figure S1Averages of the estimated parameter values for the control serum conditions from four sets of independent experiments. We applied a simple one-sided (TIF)Click here for additional data file.Figure S2Phase portraits of cell fate transitions under different serum conditions or growth factor conditions. Phase portraits for the high serum (A–C), the low serum (D–F), and the serum free (G–I) conditions are shown. Parameters of Experiment 1 in (TIF)Click here for additional data file.Figure S3Dependency of the initial conditions on the dynamics of the number of cells in a phase portrait. (A) The dynamics of the number of cells under the low serum condition (HS (TIF)Click here for additional data file.Figure S4Dependency of response rates on initial conditions. The initial response speeds (TIF)Click here for additional data file.Figure S5Log-normal distributions of the simulated cell density. Histograms of the cell densities (cells/mm2(TIF)Click here for additional data file.Figure S6Power-law relations under various parameter values and initial conditions. To examine the generality of the result in As shown here, power-law relation did not depend on the specific conditions of parameter values and the initial number of cells when the initial distribution was lognormal and exponential. For Poisson distribution, the slope (TIF)Click here for additional data file.Figure S7Dependency of parameter estimations on the initial conditions. To show the typical process of parameter estimations under different initial conditions, these figures plotted selected parameter values (TIF)Click here for additional data file.Table S1Estimated parameter values in four independent experiments. High serum: (PDF)Click here for additional data file.


The last two decades have seen a tremendous development in high resolution microscopy techniques giving rise to acronyms such as TIRFM, SIM, PALM, STORM, and STED. The goal of all these techniques is to overcome the physical resolution barrier of light microscopy in order to resolve precise protein localization and possibly their interaction in cells. Neuroendocrine cell function is to secrete hormones and peptides on demand. This fine-tuned multi-step process is mediated by a large array of proteins. Here, we review the new microscopy techniques used to obtain high resolution and how they have been applied to increase our knowledge of the molecular mechanisms involved in neuroendocrine cell secretion. Further the limitations of these methods are discussed and insights in possible new applications are provided. It is u2+, that mediate several steps of exocytosis. However, the results of these studies become more and more complex to interpret as we investigate the role of proteins that appear to be involved throughout exocytosis, e.g., Synaptotagmin. The question is whether super-resolution microscopy can help in this quest by providing a link between morphological (EM) and functional data .Taken together, using a combination of membrane capacitance recording, carbon fiber amperometry and EM very effectively uncovered the function of several proteins, such as the SNARE proteins or Munc13, which play a role at only one step of exocytosis in chromaffin cells. TIRFM helped to examine the function of certain proteins or substances, such as Munc18 or Can and converges with an angle θ).The aim of super-resolution microscopy is to provide similar resolution as EM but with light microscopy. In light microscopy the wave-like nature of light limits spatial resolution to half the wavelength of the observed light. This so called diffraction barrier was established by Ernst Abbé 140 years ago and is expressed by the formula:All microscopy technologies developed at the end of the twentieth century, such as confocal microscopy or TIRFM, improve the signal-to-noise ratio and thus produced highly contrasted and crisp images that showed a wealth of unprecedented details, but the resolution was still diffraction limited. An early approach was to use deconvolution algorithms to subtract the predicted point spread function (PSF) of individual fluorophores from the image and thus to reduce the contribution of diffracted light. This technique produces images with even better signal-to-noise ratio, but the resolution improvement is modest. Furthermore, due to the use of flawed PSFs, this technique is prone to generate artifacts like non existing signal patterns. The new super-resolution microscopy methods have addressed most of these problems and can achieve a resolution down to 10 nm Hell, .SIM achieves super-resolution by extracting fine structural details from the interference of a structure with predetermined illumination patterns. When a periodic illumination pattern, such as stripes, is applied to a fluorescent sample, an interference pattern is generated. The diffraction-limited fringes of this interference pattern, called moiré fringes, contain information about underlying structural pattern of the sample that cannot be observed with conventional light microscopy. By shifting and rotating the illumination pattern, sub-diffraction-limited structural information of the sample can be extracted from Fourier transformations of the resulting interference pattern (Gustafsson, In PALM or STORM the resolution has been improved to much greater extent since they provide a lateral resolution of 10–30 nm and about 50 nm axial resolution (Betzig et al., In contrast to the aforementioned super-resolution microscopy techniques, STED microscopy does not rely on post processing of blurry raw images but rather uses a specific illumination method and photo-physics to generate directly highly resolved images. In STED microscopy the excited fluorescent dye molecules return to the ground state (S0, Figure The impact of super-resolution microscopy in the field of neuroendocrinology is just about to spark. STED microscopy has been used to verify that the size of LDCVs was unaltered upon overexpression of a mutated Munc18-1 in neuroendocrine cells (Jorgacevski et al., Super-resolution microscopy will help us to understand the detailed molecular interactions in chromaffin cells function. As discussed in the introduction, one crucial issue is to clearly demonstrate the correlation between the functional and the morphological data on docking. Another important aspect in chromaffin cell research is to uncover at which time point during the exocytotic process, docking and priming factors bind to the release machinery of LDCVs. FRET is the tool of choice to demonstrate protein interactions in living cells. However, with the remarkable exceptions of Lam et al.  and ZhaoThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We propose phase-like characteristics in scale-free broadband processes and consider fluctuation synchrony based on the temporal signature of significant amplitude fluctuation. Using wavelet transform, successful captures of similar fluctuation pattern between such broadband processes are demonstrated. The application to the financial data leading to the 2008 financial crisis reveals the transition towards a qualitatively different dynamical regime with many equity price in fluctuation synchrony. Further analysis suggests an underlying scale free “price fluctuation network” with large clustering coefficient. There is an extensive literature on the subject, from Huygen's clock One area where FIS likely occurs is the market dynamics. Equity price is known to exhibit broadband fluctuation and fractal property WTMML roots. Huang and Mallet proved the seminal result on the convergence of Our approach relies on the so-called wavelet transform modulus maxima line (WTMML). For square-integrable function or bounded signal in practice, wavelet transform can effectively describe the local fluctuation according to the scale of resolution isfying   below. Ocomplete. As the FIS level drops, the WTMML roots are more scattered and the shape of When all WTMML roots of In the numerical analysis, the We will first apply the above ideas to demonstrate FIS using artificial time series. Consider multifractal measures ions at  . They leredicted .For a fixedWhile We now apply these ideas to analyze the fluctuation in the equity price series. The daily closing price, are used . This seTo analyze the potential FIS in the price series, we take into account the market nonstationarity by processing the data in window segments of Given A large transition year8 regime . The risThese FIS characteristics, the market transition at ameters  , and usiameters  . The rob of 2009  suggestsIt is reasonable to assume the action from traders has an immediate impact on the observed FIS in the price series. To examine this potential link, we also consider FIS from the daily returns. In particular, let Hence, in terms of the fluctuation pattern, the after-hour Similar to the price series, our result shows the same ascending trend in elopment . FIG. 4bdegree of In light of the above, we take a network approach to further characterize the FIS in the closing price. Here, script ) . The expectively , indicatthrough  . It follThe notion of synchronized broadband processes in general should rest on the statistical ground of certain phase variable, and possibly be supported by the observation of similar fluctuation pattern. In this work, we adopt these premises to establish the preliminary ideas for synchrony in broad-band processes showing scale free characteristics. In particular, we propose using the wavelet transform to associate the phase to the large amplitude jump characterized as the singularity. We then suggest the state of FIS with the assumption of a common mechanism underlying the singular fluctuation. While these ideas are successfully demonstrated using artificial examples, we stress the importance of the “selectiveness” of the analyzing wavelet in wavelet transform. When it is too sensitive, many WTMMLs can emerge, which may be unrelated to the phase setting event leading to the large amplitude jump. We suggest using the wavelet that has the lowest number of vanishing moment, so as to associate the “most singular” fluctuation to the phase event.The application of the proposed ideas to the market data leading to the 2008 financial crisis reveals several nontrivial results. The main implication of the findings is the significance of the market synchrony and its variability. The match of the rising FIS indicators Lillo and collaborators also suggest synchronous market activities based on the persistent bid-ask spread in the market limit orders Finally, we suggest that similar price fluctuation in a group of stocks without obvious business link reflects the herding or collective behavior of the traders File S1The ticker symbols for the price series(DOCX)Click here for additional data file.


RFamide-related peptide (rfrp) neurons, present in the mediobasal hypothalamus, is strongly down-regulated by melatonin in short day conditions, but not altered by circulating levels of sex steroids. The role of rfrp and its product RFRP-3 in the regulation of reproductive activity has been extensively studied in mammals, and our recent findings indicate that this peptide is a potent stimulator of the reproductive axis in the Syrian hamster. It induces a marked increase in GnRH neuron activity and gonadotropin secretion, and it is able to rescue reproductive activity in short day sexually inactive hamsters. Little is known about the localization of the RFRP-3 receptor, GPR147, in the rodent brain. Accumulating evidence suggests that RFRP-3 could be acting via two intermediates, the GnRH neurons in the preoptic area and the Kiss1 neurons in the arcuate nucleus, but future studies should aim at describing the localization of Gpr147 in the Syrian hamster brain. Altogether our data indicate that the rfrp neuronal population within the mediobasal hypothalamus might be a serious candidate in mediating the photoperiodic effects of melatonin on the regulation of the reproductive axis.Seasonally breeding mammals rely on the photoperiodic signal to restrict their fertility to a certain time of the year. The photoperiodic information is translated in the brain via the pineal hormone melatonin, and it is now well-established that it is the variation in the duration of the nocturnal peak of melatonin which synchronizes reproduction with the seasons. The Syrian hamster is a long day breeder, and sexual activity is therefore promoted by exposure to a long day photoperiod and inhibited by exposure to a short day photoperiod. Interestingly, in this species electrolytic lesion of the mediobasal hypothalamus abolishes the short day-induced gonadal regression. We have shown that the expression of a recently discovered neuronal population, namely  A large number of species restrict their fertility to a particular time of the year to ensure that the birth of the offspring occurs during the most favorable season. To determine the time of the year and synchronize their reproductive activity accordingly, mammals use the highly reproducible annual variations in light duration (or photoperiod). Photoperiod is transduced by a photoneuroendocrine system composed of the retina, the suprachiasmatic nucleus (seat of the master circadian clock) and the pineal gland which releases the hormone melatonin exclusively at night, so that the duration of the secretion varies according to night length. Therefore, the photoperiodic variations in circulating levels of melatonin throughout the year provide the body with a robust and reproducible representation of the seasons . It has Syrian hamsters are long day breeders, meaning that they are sexually active in long day (LD: 14 light/10 h dark) conditions. Upon exposure to short day (SD: 10 light/14 h dark) conditions, they undergo a dramatic inhibition of reproductive activity within 8–10 weeks manifested by a marked atrophy of the gonads and accessory organs resulting in low levels of circulating sex steroids. Surgical removal of the pineal gland before exposure to SD conditions prevents hamsters from undergoing sexual inactivation. Conversely, exogenous melatonin injections mimicking SD conditions in hamsters raised in LD conditions induce sexual inactivation. In contrast to small rodents, large mammals with a longer gestation time like sheep are SD-breeders; they are sexually active in SD conditions and become quiescent after transfer to LD conditions. Although the reproductive timing is opposite in hamsters and sheep, in both cases the circulating levels of melatonin synchronize reproduction with photoperiod. However, why the reproductive systems of LD- and SD-breeders respond in opposite ways to the same melatonin signal is not known.It is clear that melatonin does not act directly on GnRH neurons and responsiveness to GnRH does not change with photoperiod . MelatonAltogether these observations point to the pars tuberalis as a key site for the integration of the endocrine melatoninergic message for the seasonal regulation of reproductive activity. However, the central reproductive site(s) actually controlled by the melatonin/TSH system is(are) still unknown. Various structures in the mediobasal hypothalamus have been proposed to be direct or indirect sites of action for melatonin, particularly in the sheep  and hamsRFamide-related peptide (rfrp) gene in a photoperiodic-dependent manner in Siberian and Syrian hamsters  motif. In the quail, GnIH was shown to act directly at the level of the pituitary to inhibit gonadotropin release . We have recently found a similar SD-induced inhibition of rfrp expression in other LD-breeders, notably the European hamster  and the jerboa  and SD- (sheep) breeders, it is remarkable to find that while the expression of the peptide in increased in LD conditions in both kinds of breeders, the effect of the peptide on the gonadotropic axis is opposite, as it is stimulatory in hamsters and inhibitory in sheep. This suggests a non-conserved role and/or site of action for RFRP-3 across seasonally breeding species. It is tempting to speculate that RFRP neurons may play a key role in discriminating between long and short day breeders because RFRP expression is down-regulated by a short day profile of melatonin in both kinds of seasonal breeders but the peptide appears to have an opposite effect on the reproductive axis, being stimulatory in long day and inhibitory in short day breeders. Complementary experiments will have to be carried out in other species to test this hypothesis and fully understand the role of RFRP-3 in the seasonal control of reproduction.Interestingly, the RFRP peptides were shown to have a modulatory action on feeding behavior  thus it The complex involvement of RFRP peptides in the regulation of the hypothalamic–pituitary–gonadal axis has raised a number of questions regarding the sites of action of the peptides. In various mammalian species including humans, RFRP fiber networks are found in multiple brain regions including the preoptic area, the arcuate nucleus, the lateral septum, the anterior hypothalamus, and the bed nucleus of the stria terminalis . NotablyThere is still uncertainty as to whether RFRP-3 also exerts a hypophysiotropic effect in mammals as reported in birds. A large body of evidence now reports the absence of fibers in the median eminence of rodents . Howeveri3 and Gαs proteins  and with a lower affinity to GPR74 , which were first identified as neuropeptide FF receptors . The GPRproteins  suggestiproteins .in situ hybridization facilitated precise localization and made it possible to show that about 25% of GnRH neurons express Gpr147, but not Gpr74, in various rodent species  suggesting that the effects observed on the reproductive axis are mediated via these neurons  in humans and rodents  preventeIn the male Syrian hamster, we demonstrated that kisspeptin expression in the arcuate nucleus is down-regulated by melatonin in SD conditions, despite a negative feedback effect of testosterone on these neurons . In the To make things more complicated, other parameters also influenced by seasons regulate kisspeptin expression. This is particularly true for sex steroids which inhibit kisspeptin expression in the arcuate nucleus and increase it in the anteroventro-periventricular nucleus in various species including the Syrian  and the Figure 1), in the jerboa . We propose that in LD conditions, RFRP-3 would activate GnRH neuronal activity directly and/or indirectly via the kisspeptinergic neurons. The former pathway is supported by the report of RFRP-3 fibers apposed to subpopulations of GnRH neurons (Kiss1 expression together with a reactivation of testicular function in the Syrian hamster (These observations have led us to propose a working model for the seasonal control of reproduction in rodents ( neurons  whereas  neurons  and by o hamster . In SD c hamster .In regard to the seasonal regulation of reproductive activity, recent data have shed light on the involvement of two hypothalamic peptides of the RFamide family, RFRP-3 and kisspeptin. Clearly both peptides are regulated by the photoperiodic melatoninergic signal but may also be sensitive to other seasonally regulated factors. Our current hypothesis is that RFRP expression undergoes a conserved inhibition in short photoperiod but the peptide may be stimulatory or inhibitory according to the reproductive physiology of the species; on the other hand, kisspeptin has a conserved stimulatory action on the gonadotropic axis but its seasonal regulation shows species differences. Whether RFRP-3 and kisspeptin act on each other’s expression or independently to regulate reproductive activity has yet to be clarified in different species. In the years to come, we believe that thorough comparative analyses on the effects and sites of action of both peptides between LD- and SD-breeders should help to resolve the yet unanswered question of why the same photoperiodic cue induces opposite behavioral responses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest


Genetic variation in pathogen populations may be an important factor driving heterogeneity in disease dynamics within their host populations. However, to date, we understand poorly how genetic diversity in diseases impact on epidemiological dynamics because data and tools required to answer this questions are lacking. Here, we combine pathogen genetic data with epidemiological monitoring of disease progression, and introduce a statistical exploratory method to investigate differences among pathogen strains in their performance in the field. The method exploits epidemiological data providing a measure of disease progress in time and space, and genetic data indicating the relative spatial patterns of the sampled pathogen strains. Applying this method allows to assign ranks to the pathogen strains with respect to their contributions to natural epidemics and to assess the significance of the ranking. This method was first tested on simulated data, including data obtained from an original, stochastic, multi-strain epidemic model. It was then applied to epidemiological and genetic data collected during one natural epidemic of powdery mildew occurring in its wild host population. Based on the simulation study, we conclude that the method can achieve its aim of ranking pathogen strains if the sampling effort is sufficient. For powdery mildew data, the method indicated that one of the sampled strains tends to have a higher fitness than the four other sampled strains, highlighting the importance of strain diversity for disease dynamics. Our approach allowing the comparison of pathogen strains in natural epidemic is complementary to the classical practice of using experimental infections in controlled conditions to estimate fitness of different pathogen strains. Our statistical tool, implemented in the R package StrainRanking, is mainly based on regression and does not rely on mechanistic assumptions on the pathogen dynamics. Thus, the method can be applied to a wide range of pathogens. Development of epidemiological models has been driven by the need to understand and predict the dynamics, invasion, and persistence of plant and animal diseases Until recently, the scarcity of suitable genetic markers has impeded the study of variation in pathogen populations Plantago lanceolata in the archipelago of Finland. A recently developed protocol for field sampling of pathogen strains and Single Nucleotide Polymorphism (SNP) genotyping panel Linking genetic pathogen data to epidemiological dynamics allows unraveling the role of pathogen intraspecific diversity in disease dynamics but this is currently limited by the availability of suitable analytical tools. Among the challenges to overcome in the development of these tools are: (i) The scarcity of data required to fit multi-strain dynamical models; (ii) The change of scale in the resolution from epidemiological data to the resolution of pathogen genetic data; and (iii) The ambiguity in the effect of the heterogeneity in pathogen strains, as discussed above. Here, we present an exploratory analysis tool based on data transformation, linear regression and kernel smoothing to assign ranks to different pathogen strains with respect to epidemiological spread, and we analyze whether this ranking is significant. The linear regression links epidemiological data (response variable) to genetic data (explanatory variables), and the kernel smoothing allows the scale of genetic data to be matched with the scale of epidemiological data. We apply our model to (i) data obtained under a statistical stochastic model, (ii) data obtained under a multi-strain mechanistic model , and (iii) fine-scale within-season epidemiological data and genetical characterization of pathogen samples collected for the powdery mildew naturally infecting Podosphaera plantaginis  is a fungal pathogen specific to the ribwort plantain Plantago lanceolata. This species has been studied in the Åland archipelago (southwestern Finland), with long-term data evidencing metapopulation dynamics Po. plantaginis belongs to the family of powdery mildews . These obligate pathogens develop conspicuous white-greyish mycelia on the surface of their host leaves and only penetrate the host tissue through feeding structures named haustoria. Continuous production of asexual spores named conidia leads to the succession of various overlapping asexual cycles during the summer (generation time varying between one and two weeks under controlled conditions). The powdery mildew population crashes during the winter due to a lack of living host tissue, but re-initiation of the epidemics in the following spring is enabled by the germination of sexual resting structures named chasmothecia.Pl. lanceolata meadow (ID 609) located in the Eckero part of the Åland archipelago (Neither the host plant nor the pathogen is protected species and Finnish legislation (Jokamiehenoikeus) allows the sampling of wild species to everyone). This Pl. lanceolata population (approx. 2400 m2) was visited weekly between July 18 and August 25 (week 29 to 34), except on week 33 . Pattern of infection within host populations is known to be highly aggregated in this pathosystem 2. Every week, each cell was visually inspected, and the number of host leaves infected by the powdery mildew was recorded.Small-scale data were collected during the summer 2011 within one On the last day of the survey (last week of August), 45 infected leaves were sampled for genetic characterization. Samples were chosen from the different infected cells so that, in each cell, the number of collected samples was approximately related to the disease intensity. Genotyping of the fungal pathogen for 27 SNP markers was performed as described in Consider a host population covering a spatial domain divided into The model (2) can be viewed as a regression linear model where In model (2), the explanatory variables In kernel smoothing, the choice of the bandwidth lm function of the R statistical software) to obtain point estimates We use the approximate regression model (3) to rank the pathogen strains in their contributions to natural epidemics. In this model, To assess whether the ranking in the coefficients Let Because several pairwise tests can be carried out when there are more than two strains, the usual significance level 0.05 for a Our method consists of performing a linear regression with noisy explanatory variables. Thus, the convergence results of classical linear regression do not hold generated under mechanistic processes that are more complex than model (2). To assess the effect of using a regression model for the analysis instead of the true mechanistic model, we applied the method to simulated data sets obtained under an original mechanistic model detailed in In practice, our method will be applied to data We carried out 1,600 simulations of the mechanistic model; 800 with the dispersal parameter An R package entitled StrainRanking containing the ranking method, the real data and generators of data under the regression and mechanistic models is available in the CRAN package repository. In this package, the ranking is carried out with the function entitled ranking.strains.The application of the method to simulations performed under the regression model is shown in simulation model, and with the coefficients analysis model. The rankings of Then, the method was applied to simulations under the mechanistic model. In this case, the model used to analyze the data is definitely different from the model used to simulate the data, but, with our ranking method, we expect to detect a signature of the variation in strain fitness (the signature is the ranking). The contributions of the pathogen strains to the epidemics are measured with the coefficients The application of the method to three simulations performed under the mechanistic model . Nevertheless, strain 5 tends to have a higher fitness than the other strains and, to be able to conclude in future studies about differences in strain fitness, more genetic samples should be gathered.The method presented here was developed to assign ranks to different pathogen strains with respect to their contribution to natural epidemics. As shown with a simulation-based study, the method can achieve this aim. Importantly, the success of the method depends on the design of the field survey as the statistical power of our approach depends on the sampling size  and on the sampling scale .b and the rejection threshold could be developed using cross-validation or Monte Carlo simulations performed under the fitted regression model.To reach more accurate ranking, the method could be improved as follows. (i) Other growth variables Disease ecologists commonly use experimental infections in controlled conditions to estimate the fitness of different pathogen strains/genotypes Click here for additional data file.


Nedcolbertia justinhofmanni, the therizinosauroid Falcarius utahensis, the troodontid Geminiraptor suarezarum, and the dromaeosaurid Utahraptor ostrommaysorum. Recent excavation has uncovered three new dromaeosaurid specimens. One specimen, which we designate the holotype of the new genus and species Yurgovuchia doellingi, is represented by a partial axial skeleton and a partial left pubis. A second specimen consists of a right pubis and a possibly associated radius. The third specimen consists of a tail skeleton that is unique among known Cedar Mountain dromaeosaurids.The Yellow Cat Member of the Cedar Mountain Formation  of Utah has yielded a rich theropod fauna, including the coelurosaur Y. doellingi resembles Utahraptor ostrommaysorum in that its caudal prezygapophyses are elongated but not to the degree present in most dromaeosaurids. The specimen represented by the right pubis exhibits a pronounced pubic tubercle, a velociraptorine trait that is absent in Y. doellingi. The specimen represented by the tail skeleton exhibits the extreme elongation of the caudal prezygapophyses that is typical of most dromaeosaurids. Here we perform a phylogenetic analysis to determine the phylogenetic position of Y. doellingi. Using the resulting phylogeny as a framework, we trace changes in character states of the tail across Coelurosauria to elucidate the evolution of the dromaeosaurid tail.Y. doellingi in a clade with Utahraptor, Achillobator, and Dromaeosaurus. Character state distribution indicates that the presence of intermediate-length caudal prezygapophyses in that clade is not an evolutionarily precursor to extreme prezygapophyseal elongation but represents a secondary shortening of caudal prezygapophyses. It appears to represent part of a trend within Dromaeosauridae that couples an increase in tail flexibility with increasing size.The new specimens add to the known diversity of Dromaeosauridae and to the known diversity within the Yellow Cat paleofauna. Phylogenetic analysis places  Acrocanthosaurus is known from fragmentary material from the Ruby Ranch Member Nedcolbertia justinhofmanniFalcarius utahensisGeminiraptor suarezarumUtahraptor ostrommaysorumYurgovuchia doellingi. Another specimen is represented by a pubis (UMNH VP 21752) and possibly also by a radius (UMNH VP 21751) found near it. The third specimen (UMNH VP 20209) is a tail skeleton.A diversity of theropods is found in the fauna of the Cedar Mountain Formation (Lower Cretaceous) of Utah. A large carnosaur similar to Dromaeosauridae is a diverse family of predatory dinosaurs with a plethora of species that have been discovered within the last two decades cauda (tail) and theca , was recently introduced In most members of the family the centra of the tail are encased in a bundle of bony rods that lie parallel to each other across the lengths of several centra, binding the tail into a rod Achillobator and Utahraptor. In both cases, distal caudal vertebrae have elongated prezygapophyses, but the prezygapophyses are not known to extend much farther than the length of one caudal centrum There are exceptions to the presence of the caudotheca in Dromaeosauridae. It is absent in the basal dromaeosaurid subfamily Unenlagiinae Microraptor than to Velociraptor or DromaeosaurusSaurornitholestes langstoni, Deinonychus antirrhopus, Dromaeosaurus albertensis, and Velociraptor mongoliensisTo facilitate understanding of the results of this study, we must address the definitions of three relevant terms: Microraptoria, Eudromaeosauria, and transition point. The name Microraptoria was introduced in 2004 for the clade of dromaeosaurids more closely related to Russell introduced the term “transition point” in 1972 in reference to ornithomimid tails All three specimens described here are from the Yellow Cat Member of the Cedar Mountain Formation, which is divided into upper and lower parts . The uppYurgovuchia doellingi and the isolated pubis (UMNH VP 21752) and radius (UMNH VP 21751) are from a portion of the Doelling’s Bowl bone bed (Gr 300v) that is designated as Don’s Place  and polacanthine ankylosaurs  The bone bed is a relatively low diversity, multitaxic bone bed of associated, if not articulated, skeletons, and while there is some orientation of long bones the close association of elements from the same individuals suggests low energy The new dromaeosaurid tail skeleton (UMNH VP 20209) is from Andrew’s Site (Gr 290v), a small but highly significant dinosaur site near the top of the Yellow Cat Member of the Cedar Mountain Formation , 5. The Scipionyx Operational Taxonomic Unit (OTU) and other compsognathid OTUs have been updated with new information from a recent study Chirostenotes OTU has been updated by removal of data that came from the holotype of the newly recognized genus EpichirostenotesChirostenotesOrnithomimus OTU has been updated with information from a recent study Ornithomimus and other advanced ornithomimosaurs.For this analysis we used the phylogenetic data matrix of a recent study The data matrix for the phylogenetic analysis is given in Phylogenetic analysis was performed with PAUP 4.0 for Windows We used specimens and literature to compare caudal character states across Coelurosauria for taxa in which articulated tail skeletons are known or for which enough of the caudal series is known to be able to deduce the relevant character states . We thenThe electronic version of this document does not represent a published work according to the International Code of Zoological Nomenclature (ICZN), and hence the nomenclatural acts contained in the electronic version are not available under that Code from the electronic edition. Therefore, a separate edition of this document was produced by a method that assures numerous identical and durable copies, and those copies were simultaneously obtainable (from the publication date noted on the first page of this article) for the purpose of providing a public and permanent scientific record, in accordance with Article 8.1 of the Code. The separate print-only edition is available on request from PLoS by sending a request to PLoS ONE, 1160 Battery Street, Suite 100, San Francisco, CA 94111, USA along with a check for $10 (to cover printing and postage) payable to “Public Library of Science”.http://zoobank.org/”. The LSID for this publication is: urn:lsid:zoobank.org:pub:4D1838FD-10BB-4754-8127-9B881D33D004.In addition, this published work and the nomenclatural acts it contains have been registered in ZooBank, the proposed online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “Systematic hierarchy:Dinosauria Owen, 1841 Saurischia Seeley, 1887 Theropoda Marsh, 1881 Coelurosauria von Huene, 1914 Dromaeosauridae Colbert and Russell, 1969 Yurgovuchia gen. nov.urn:lsid:zoobank.org:act:110C8550-8718-40C6-8AA8-8A534A33535BYurgovuchia doellingi sp. nov.urn:lsid:zoobank.org:act:8C781C52-96FF-4377-A337-82FC875DC24EThe holotype specimen is UMNH VP 20211. It includes cervical, dorsal, and caudal vertebrae; and the proximal end of a left pubis.yurgovuch, meaning “coyote,” a predator of similar size to Y. doellingi that currently inhabits the same region.The genus name honors the Ute Tribe of northeastern Utah. It is derived from the Ute word The species name honors Helmut Doelling in recognition of his 50-plus years of geological research and mapping of Utah for the Utah Geological Survey. The Doelling’s Bowl dinosaur sites were first discovered as a result of his providing taped-together color photocopies of his then-unpublished geological maps of the Arches National Park region The specimen comes from Don’s Place, part of the Doelling’s Bowl bone bed in Grand County, Utah . The bonDromaeosaurid theropod; centrum of axis with a single pneumatopore on each side; cranial end of centrum of third cervical vertebra not beveled; cervical prezygapophyses flexed; epipophyses of cervical vertebrae above postzygapophyseal facets; cervico-dorsal vertebrae with hypapophyses and without pneumatopores; cranial faces of centra of proximal caudal vertebrae round; caudal prezygapophyses elongated distal to transition point, but not over the length of a centrum. Pubis without pubic tubercle.Below, specimen numbers in parentheses indicate personal observation by PS of features not recorded in the literature.Utahraptor ostrommaysorum but instead represents a much smaller species. Measurements are given in Preserved vertebrae and parts of vertebrae include the left neural arch of the atlas, a nearly complete axis, three postaxial cervical vertebrae, partial neural arches of two more postaxial cervicals, two cervico-dorsal vertebrae, the neural arch of one dorsal vertebra, and seven caudal vertebrae . In all DeinonychusThe postzygapophysis of the atlantal neural arch is bulbous . A deep DeinonychusBambiraptor . The neural spine is laterally compressed and was at least as tall as the centrum. Its tip and the diapophyses and parapophyses are eroded off. A single pneumatopore is present at mid-length on each side of the centrum, as in DeinonychusBambiraptor (AMNH FR 30556). A pneumatopore is absent in the axis of TsaaganDeinonychusBambiraptor (AMNH FR 30556), the centrum of the axis lacks the beveling that is characteristic of dromaeosaurid mid-cervical centra.The intercentrum and odontoid process of the axis  resembleTsaagan mangas, the first ten cervicals of which were found articulated in situ T. mangas, but the shapes of the postzygapophyses and the laminae connecting them to the prezygapophyses are more similar to those of the third than the fourth cervical of T. mangas. The cranial surface of its centrum is not strongly beveled as are those of most other coelurosaurs, including other dromaeosaurids One cervical vertebra is completely preserved except that its neural spine is broken off . On the Two nearly complete posterior cervical vertebrae are present. The more cranial of the two  has beenThe other nearly complete posterior cervical vertebra is missing the neural spine, the left cranial part of the neural arch, and the left dorsolateral part of the centrum . The cenPartial neural arches from two other cervical vertebrae are present. On one  the leftTwo cervico-dorsal vertebrae are preserved, each with a hypapophysis. Neither has pneumatopores and both are amphiplatyan. In both vertebrae the parapophysis is on the anterodorsal edge rather than the anteroventral edge of the centrum, which indicates that both are from the caudal section of the series of vertebrae with hypapophyses. The vertebra with the larger hypapophysis is therefore the more cranial of these two vertebrae. The more cranial of the two  is missiThe more posterior cervico-dorsal vertebra is missing the diapophyses, the prezygapophyses, the right postzygapophysis, and all but the bases of the parapophyses . Its hypThe neural arch of the dorsal vertebra appears to be broken off the vertebra rather than simply unfused to the centrum, because of the rough texture of its ventral surfaces. It is missing all four zygapophyses . The neuAchillobatorY. doellingi have subquadrangular central faces.The cranial six of the seven preserved caudal vertebrae appear to form a consecutive series . All areThe first  caudal vertebra in the series  is from The prezygapophyses are vertical on the first vertebra of the series and upswept in lateral view  but are On the fifth caudal vertebra in the series , a prongVelociraptorOne other caudal vertebra is preserved . It is sYurgovuchia exhibits the eudromaeosaurian condition. In Velociraptor prezygapophyseal elongation begins with the tenth vertebra Yurgovuchia, then the series of six caudals consists of caudals 6–11.In unenlagiine and microraptorian dromaeosaurids the middle and distal caudals are at least twice the lengths of those cranial to the transition point The proximal end of the left pubis is preserved . The pubY. doellingi. The pubis (UMNH VP 21752) was at the opposite end of the quarry from the Y. doellingi bones, and the radius (UMNH VP 21751) was about halfway between the two . A pubic tubercle as a discrete, prominent process is absent in other dromaeosaurids The shaft and proximal end of the right pubis are preserved . The preThe pubic shaft is craniocaudally flattened. As in other dromaeosaurids Y. doellingi holotype. The pubis of the latter is much larger and lacks the pubic tubercle.This pubis definitely comes from an individual other than the The discrete pubic tubercle and the close morphological match between the pubis and those of velociraptorines Utahraptor ostrommaysorum. We do not consider the new tail skeleton to represent U. ostrommaysorum, because the latter exhibits a hemicaudotheca, whereas the new tail skeleton exhibits a caudotheca.UMNH VP 20209 consists of a proximal caudal vertebra  and a seThe proximal caudal vertebra  has a ceThe distal section of tail is sharply bent at approximately a right angle, 218 mm from its cranial end . The taiCaudal to the bend the caudal end of a centrum  and four more consecutive centra  are visible. Only centrum 2 can be measured with certainty. It is 41 mm long and is cranially 16 mm tall. Centra 3–5 are estimated to be 40, 43, and 46 mm long respectively. All are of similar height.In a few cases elongated prezygapophyses can be traced to their respective vertebrae. Their lengths cannot be measured with certainty because their tips may be broken off. The longest visible section of a prezygapophysis is from centrum 5. It is bifurcated at its base, and the two tines remain close to each other, nearly appressed, through half their preserved lengths. The longer (as preserved) of the two tines extends 122 mm beyond the centrum (approximately 2.7 times the length of the centrum). One tine of a prezygapophysis is traceable to centrum 3. As preserved, it extends 106 mm beyond the centrum. In contrast, the postzygapophysis of centrum 3 extends only approximately 17 mm beyond the centrum. That of centrum 4 extends only approximately 11 mm beyond the centrum.A detached hemal arch lies on the right side of the tail, with its body on the anterodorsal corner of centrum 3 . It has In the new specimen, all the proximal caudals with transverse processes are missing. Using a conservative estimate of four missing caudals with transverse processes, we see that the first caudal vertebra contributing to the caudotheca is no farther cranial than the eighth caudal vertebra and the caudotheca extends no farther forward than the seventh.The phylogenetic analysis recovered 1217 most-parsimonious trees with 1304 steps. For these trees, the consistency index is 0.3758, the homoplasy index is 0.6242, the retention index is 0.8124, and the rescaled consistency index is 0.3053.Xiaotingia is placed at the base of Dromaeosauridae, whereas a previous analysis placed it with Anchiornis and Archaeopteryx in a clade that formed the sister taxon to Deinonychosauria Shanag is placed at the base of the sister clade of Unenlagiinae, whereas previous analyses placed it at the base of Unenlagiinae itself Mahakala is placed within Unenlagiinae, whereas a previous analysis placed it at the base of Dromaeosauridae The phylogeny of Coelurosauria recovered here  matches Yurgovuchia is found to be part of a clade within Dromaeosaurinae that includes Utahraptor, Achillobator, and Dromaeosaurus but excludes Deinonychus. Relationships within this clade are uncertain. Synapomorphies of Deinonychosauria and its sub-clades that are confirmed to be present on Yurgovuchia are given in Bambiraptor is no longer at the base of Eudromaeosauria in the consensus tree but is part of an unresolved trichotomy with Eudromaeosauria and Microraptoria.When deinonychosaurian OTUs known only from fragmentary material are deleted from the analysis, the analysis yields 54 most-parsimonious trees of 1265 steps. For these trees, the consistency index is 0.3866, the homoplasy index is 0.6134, the retention index is 0.8083, and the rescaled consistency index is 0.3125. Decay indices for several deinonychosaurian clades are increased, in some cases dramatically, in comparison to the analysis that included fragmentary taxa . The topComparison of data from a phylogenetically broad spectrum of coelurosaurian theropod taxa  reveals In Microraptoria transverse processes are present only up to Cd  6, the first caudal vertebra contributing to the caudotheca is no farther cranial than Cd 6, and the caudotheca extends forward to Cd 3 or 4. In Eudromaeosauria transverse processes are present up to Cd 10, the first caudal vertebra contributing to the caudotheca is no further cranial than Cd 10, and the caudotheca extends no farther forward than Cd 6. The new specimen described here that is represented only by a tail skeleton exhibits the eudromaeosaurian condition.Falcarius utahensisGeminiraptor suarezarumYurgovuchia doellingi and the velociraptorine dromaeosaurid represented by the new pubis. From the upper Yellow Cat the large dromaeosaurid Utahraptor ostrommaysorum has previously been described Nedcolbertia justinhofmanniThe three new dromaeosaurid specimens described here add to the known dinosaurian diversity of the upper and lower Yellow Cat Member of the Cedar Mountain Formation. The therizinosauroid Y. doellingi and U. ostrommaysorum are both members of the subfamily Dromaeosaurinae. The new pubis represents the subfamily Velociraptorinae. During the Late Cretaceous Asia was also simultaneously inhabited by dromaeosaurines (Achillobator) Tsaagan, Velociraptor) According to our phylogenetic results, Archaeopteryx (at the base of Aves), Xiaotingia (at the base of Dromaeosauridae), and Anchiornis (at the base of Troodontidae). The analysis that placed these three genera together in a clade of their own Jeholornis, Sapeornis, Confuciusornis, and Yanornis; the troodontids Anchiornis, Sinusonasus, Mei, and Sinornithoides; and the dromaeosaurids Buitreraptor, Sinornithosaurus, Microraptor zhaoianus, M. gui, Bambiraptor, Tsaagan, Velociraptor, Deinonychus, and Utahraptor. We therefore consider the matrix used here particularly reliable for placement of paravian taxa and therefore doubt the placement of Archaeopteryx, Xiaotingia, and Anchiornis in a clade of their own. The results of the phylogenetic analysis with fragmentary OTUs deleted yields a high decay index for Avialae (with Archaeopteryx therein), which indicates strong support for a close relationship between Archaeopteryx and other birds (Anchiornis and Xiaotingia therein), which indicates strong support for phylogenetic separation of Archaeopteryx from Anchiornis and Xiaotingia Click here for additional data file.Appendix S2Phylogenetic Data Matrix for Phylogenetic Analysis of Coelurosauria.(DOC)Click here for additional data file.


Measurements on gene level are widely used to gain new insights in complex diseases e.g. cancer. A promising approach to understand basic biological mechanisms is to combine gene expression profiles and classical clinical parameters. However, the computation of a correlation coefficient between high-dimensional data and such parameters is not covered by traditional statistical methods.p-value of the identified correlations. Therefore, the microarray data is clustered by hierarchical agglomerative clustering using standard settings. In a second step, the computed cluster tree is evaluated. For each label, a NTI is computed measuring the correlation between that label and the clustered microarray data.We propose a novel index, the Normalized Tree Index (NTI), to compute a correlation coefficient between the clustering result of high-dimensional microarray data and nominal clinical parameters. The NTI detects correlations between hierarchically clustered microarray data and nominal clinical parameters (labels) and gives a measurement of significance in terms of an empiric The NTI successfully identifies correlated clinical parameters at different levels of significance when applied on two real-world microarray breast cancer data sets. Some of the identified highly correlated labels confirm the actual state of knowledge whereas others help to identify new risk factors and provide a good basis to formulate new hypothesis.p-value measures the level of significance of the detected correlations.The NTI is a valuable tool in the domain of biomedical data analysis. It allows the identification of correlations between high-dimensional data and nominal labels, while at the same time a  The primary data is obtained in the main experiment whereas the secondary data includes all supplementary data about the analyzed subjects. In the context of gene expression analysis, the primary data is the gene expression data from the microarray experiments. The corresponding secondary data consists of clinical data, disease outcome, information about the applied treatments and therapies, as well as gene annotations. It is common practice to visualize the computed cluster tree in combination with the clustered microarray data (the primary data) and the secondary data available for the clustered samples  and every single gene (second variable), and to combine the results in a final correlation coefficient. Depending on the type of variables, statistics provides various methods to compute the correlation between two variables. For interval data, Pearson's correlation coefficient r  computesr  is still unknown. We propose to empirically calculate TImin and (TImax) for each considered label by using a Monte Carlo simulation  computes a normalized correlation coefficient between nominal parameters and hierarchically clustered data. In order to avoid biases with respect to the number of classes and the distribution of cluster sizes ,15, the n Figure :min, the labels are permuted r ≥ 10000 times  and the biological stability index (BSI) . Let xst be the scaled logarithm of sample s and gene t. The expression profiles xs are clustered by hierarchical agglomerative clustering using average linkage and a distance metric (dissimilarity measure) based on the correlation between a pair of subjects. This correlation distance metric ij d∈  of two expression profiles xi and xj of length g is defined asIn this paper, the entire molecular expression signature is used to demonstrate the merits of the NTI and its p-value are computed for each clinical parameter listed in Tables intended operation rather reveals an unexpected relationship than any insight into a biomedical process.By applying the NTI on cluster trees obtained from real-world data sets, we simulate the scenario where a biomedical researcher is looking for clinical parameters that are correlated with the microarray data. The NTI and H0 (no correlation) is statistically significant at the 5% level, two asterisks at the 1% level, and three asterisks at the 0.1% level. No asterisk means that H0 cannot be rejected.A summary of all results is shown in Figure estrogen receptor (ESR1). The correlation between the clinical parameters metastasis, event death, and estrogen receptor (ESR1) and the microarray data is statistically significant at the 0.1% level. The StGallen consensus criteria is statistically significant at the 1% level. On the right of Figure estrogen receptor IHC. The correlations between age, BMI, native country, grading, progesterone receptor IHC, and estrogen receptor IHC and the microarray data are statistically significant at the 0.1% level. The correlations of the parameters menopause, smoking, T (tumor dimension), and intended operation are statistically significant at the 1% level. The correlations of the parameters ethnic group, nursing period, alcohol, sleep, N (lymph nodes), Her2-new, inspection, tumor size (mammogramm) and chemoregime are statistically significant at the 5% level.On the left of Figure positive lymph nodes and estrogen receptor (ESR1). No correlation is detected between the microarray data and positive lymph nodes. In contrast to that, the microarray data is highly correlated with estrogen receptor (ESR1), since the rejection of H0 is statistically significant at the 0.1% level. The cluster tree of the BBCP data set is displayed in Figure estrogen receptor IHC, progesterone receptor IHC, grading, and nursing. The p-values < 0.001 indicate that the microarray data is highly correlated with the first three parameters, whereas there is no correlation with nursing.The cluster tree of the van de Vijver data set is displayed in Figure p-value is derived which measures the level of significance of the detected correlations between labels and primary data.A novel index, the Normalized Tree Index (NTI), is developed to compute a normalized correlation coefficient between hierarchically clustered primary data (microarray data) and nominal labels of secondary data . The NTI is an extension to the TI as described in , but it estrogen receptor and the progesterone receptor is unquestioned [metastasis and event death is shown in [grading is reported in [age, BMI, native country, menopause, smoking, T (tumor dimension) are highly correlated with the genomic data. These high correlations indicate that there might be common underlying mechanisms or pathways. The linkages between the phenotypes  and the genotypes (the microarray data) help to formulate new hypothesis and aid to obtain new insights into the complex mechanisms of diseases. Some of the detected correlations are harder to interpret: The correlations between intended operation, and chemoregime and the microarray data are probably not based on direct causal relationships. Interestingly, no significant correlation is reported between the parameters familial breast cancer, histology, lateral acoustical shadow, dorsal acoustic attenuation and the microarray data - an indicator that the genomic information offers a new approach to access and thus improve the diagnoses of breast cancer.Some of the detected correlations reflect common knowledge: The clinical relevance of the estioned -25. The estioned -28. Alsoshown in , and a corted in . For othEven though applied to microarray data in a medical setting, the NTI can be applied to any complex data, in whose context a cluster analysis of the primary data is reasonable. Whenever there is the slightest assumption that the internal structure of the primary data might be correlated with a given label of the secondary data, the NTI provides an objective measure for this structural relationship.progesterone receptor IHC and estrogen receptor IHC have been transformed to nominal labels with three categories: negative (0 to 1), intermediate (2 to 8) and high positive (9 to 12). Nevertheless, strategies for a direct application of the NTI on ordinal and interval labels need to be developed.The Normalized Tree Index (NTI) is developed to compute a correlation coefficient between primary data and nominal labels of secondary data. Ordinal and interval labels have to be converted to nominal labels by label-specific transformations Table . The corHierarchical agglomerative clustering and the computation of the NTI are advantageous compared to the following method that is sometimes used to obtain a correlation coefficient: A classifier is trained on the microarray data. A selected label is used to rate the correlation depending on the ability of a classifier to predict the correct label in a leave-one-out setting. The higher the classification rate, the higher the correlation between the primary data and the label. The major drawback of this approach is that a visualization is not provided this way. A high classification rate indicates a high correlation, but there is no way to retrace how the specific classification rate has been obtained. Homogeneous clusters, outliers, and other significant patterns cannot be identified this way.p-value measures the level of significance of the detected correlations. Even though applied to microarray data in a medical setting, the NTI can be applied to any complex data. This general applicability makes it a powerful tool in diverse domains.The Normalized Tree Index (NTI) is the first cluster index that uses the structure of the hierarchical clustering tree to compute a normalized correlation coefficient between nominal labels and high-dimensional primary data. Its normalization feature enables the easy identification of labels that are correlated with the primary data, while at the same time a The authors declare that they have no competing interests.CM conceived of and carried out the study and the statistical analysis and drafted the manuscript. AT carried out the expression profiling experiments and the primary data analysis. AB contributed to the design of the experiments and constructed the microarray. TN participated in the design and coordination of the study and in drafting the manuscript. All authors read and approved the final manuscript.


Neurology is a fast advancing discipline in which professional development and improved patient care are much influenced by information technology. An important component of this is the ever increasing use of the internet for clinical, educational, and research activities, which also contributes to the requirement for health practitioners to keep their knowledge up-to-date Masters, . The youThere is a variety of electronic information resources for health practitioners to update their professional knowledge. A greater insight into the current use of web-based learning would not only shed light on the approach individuals take, but also help in determining necessary measures to improve the provision of electronic content. The objective of this study was to explore some aspects of the e-learning activity of European junior neurologists.An anonymous multiple choice question based survey was conducted using a real time interactive voting system among the participants of a focused workshop organized by the European Association of Young Neurologists and Trainees (EAYNT) at the 2011 Meeting of the European Neurological Society (ENS). The questions addressed the frequency of internet use to specifically learn neurology, the most commonly assessed resources, the verification of credibility, and the opinions about websites aimed for patients. Seventy attendees participated.Demographic data revealed that 84% of the 70 participants were aged between 25 and 35 years. Most participants reported that, internet is used specifically to learn neurology at either at an average frequency of once weekly or more than once a week Figure . The mosprior to the launch in late 2011, awareness was still relatively low just a few months before going live. Interestingly, although two-third were aware of patient websites, many would not recommend them to their patients.E-learning includes the use of Internet-based approaches to broaden knowledge and improve performance. There is evidence for the effectiveness and acceptance of e-learning within the medical community (Ruiz et al., While this study has several limitations, the findings underscore the importance e-learning has gained over recent years. Training to improve skills in assessing the credibility of web-based resources as well as better access to valuable high-end pay sites need to be considered as next steps in this development. It would also be relevant to further analyze the cause of negative perceptions about patient-oriented web content.