Alexander Bowring

Thomas Nichols

Camille Maumet

Same Data -Different Software -Different Results? Analytic Variability of Group fMRI Results

Introduction

A plethora of tools and techniques are now available to process and model fMRI data. However, this 'methodological plurality' has come with a drawback. Application of different analysis pipelines [START_REF] Carp | On the plurality of (methodological) worlds: estimating the analytic flexibility of fMRI experiments[END_REF], alterations in software version [START_REF] Glatard | Reproducibility of neuroimaging analyses across operating systems[END_REF], and even changes in operating system [START_REF] Gronenschild | The effects of FreeSurfer version, workstation type, and Macintosh operating system version on anatomical volume and cortical thickness measurements[END_REF] ) have all been shown to cause variation in the results of a neuroimaging study. This high analytic flexibility has been pinpointed as a key factor that can lead to increased false-positives [START_REF] Ioannidis | Why most published research findings are false[END_REF], and compounded with a lack of data sharing, irreproducible research findings [START_REF] Poldrack | Scanning the horizon: towards transparent and reproducible neuroimaging research[END_REF].

In this work, we seek to understand how choice of software package impacts analysis results. We reproduce the results of three published neuroimaging studies [START_REF] Schonberg | Decreasing ventromedial prefrontal cortex activity during sequential risk-taking: an fMRI investigation of the balloon analog risk task[END_REF][START_REF] Moran | Social-cognitive deficits in normal aging[END_REF]Padmanabhan, 2011 ) with publicly available data within the three main neuroimaging software packages: AFNI, FSL and SPM, using parametric and nonparametric inference. All information for how to process, analyze, and model each dataset we obtain from the publication. We make a variety of comparisons to assess the similarity of our results across both software packages and choice of inference method.

Methods

We reanalysed data from three published fMRI studies and attempted to replicate the result for the principal effect depicted in the main figure of each publication within the three packages. The dataset associated to each study was obtained from the OpenfMRI [START_REF] Poldrack | OpenfMRI: open sharing of task fMRI data[END_REF] database (ds000001, R: 2.0.4; ds000109, R:2.0.2; ds000120, R:2.0.4).

Prior to the analyses we determined a number of processing steps to be included in all of our reproductions, for example, inclusion of six motion regressors in the analysis design matrix to remove motion-related artefacts. Although this meant deviating from an exact reproduction of a publication's analysis, these steps were included to maximise comparability. Excluding these procedures, we endeavoured to choose the analysis pipeline within each package most consistent with the publication given the limitations of the software. Scripts were written to carry out the analyses in each package, and for FSL and SPM, export the group-level results as NIDM-Results packs.

For each study, the activation maps were uploaded to Neurovault [START_REF] Gorgolewski | NeuroVault. org: a web-based repository for collecting and sharing unthresholded statistical maps of the human brain[END_REF]. We applied three quantitative comparison methods: Bland-Altman plots, assessing differences in the magnitudes of activations between the unthresholded group T-statistic maps; Dice statistics, comparing the locations of activation in the FWE-thresholded maps. Finally, Euler Characteristics were computed for each software's group T-statistic map characterizing differences in the topological properties of the thresholded images. Comparisons were made both between software, as well as within software for the parametric and nonparametric inference results.

Results

Figures A-E present comparisons of the group-level results in each package for reproductions of the main contrast ' false belief > false photograph'

from the publication associated to the ds000109 dataset. Group-level inference was conducted using a cluster-forming threshold p < 0.005, FWE-corrected clusterwise threshold p < 0.05. While qualitatively the regions of activation determined in the thresholded images are similar, the comparisons display striking differences across software, as well as between parametric and nonparametric inferences within FSL.

Conclusions

We have found a disappointing level of agreement between software packages. While the general pattern of activations found was similar, the best inter-software Dice overlap was 54% (intra-software, parametric-vs-nonparametric, were better, e.g. 97% for SPM). This work supports the need for open sharing of data, and the importance of understanding the fragility of one's results under the choice of software used.


Eric Lichtfouse
email: eric.lichtfouse@dijon.inra.fr

Claire Chenu

François Baudin

Claudette Leblond

Martine Da Silva

Françoise Béhar

Sylvie Derenne

Claude Largeau

Patrick Wehrung

Pierre Albrecht

Éric Lichtfouse

Françoise Behar

A novel pathway of soil organic matter formation by selective preservation of resistant straight-chain biopolymers: chemical and isotope evidence

Keywords: 13 C of soil alkanes, NMR, resistant aliphatic biopolymer, pyrolysis of humin

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Soil organic matter is composed of a complex mixture of living organisms, dead organic debris and anthropogenic inputs. During the decay of plants, a major part of plant carbon (~ 99%) is biodegraded and recycled to the atmosphere as CO 2 . The organic matter remaining in soils is thus a carbon pool exhibiting high resistance to biodegradation. Various but minor classes of soil organic molecules including lipids, amino acids, phenols, and carbohydrates have so far been identified [START_REF] Schnitzer | Soil organic matter, the next 75 years[END_REF]. Indeed, despite numerous investigations, the bulk of soil organic matter is made up partly of macromolecular, brown yellow humic substances whose molecular structure is still a matter of debate [START_REF] Rice | A model of humin[END_REF]MacCarthy, 1990, de Leeuw andHatcher, 1992). Two main hypotheses have been advanced to explain how humification takes place : the selective preservation of plant biopolymers and the polycondensation of small molecules [START_REF] Bollag | Incorporation of xenobiotics into soil humus[END_REF]. The selective preservation of plant biopolymer residues should favour the accumulation of ligno-protein nuclei in humic substances. Nonetheless, 13 C isotope studies of various soil organic pools show that such a process should play only a minor part in the formation of humus from crop soils [START_REF] Lichtfouse | Unexpected 13 C-enrichment of organic components from wheat crop soils : evidence for the in situ origin of soil organic matter[END_REF]. Therefore in situ processes are more likely to be involved : 1) the polycondensation (or neogenesis) pathway [START_REF] Maillard | Synthèse des matières humiques par action des acides aminés sur les sucres réducteurs[END_REF][START_REF] Maillard | Identité des matières humiques de synthèse avec les matières humiques naturelles[END_REF], and 2) a pathway proposed by [START_REF] Lichtfouse | Unexpected 13 C-enrichment of organic components from wheat crop soils : evidence for the in situ origin of soil organic matter[END_REF][START_REF] Lichtfouse | Resistant ultralaminae in soils[END_REF] which involves selective preservation of resistant aliphatic biopolymers from microbes. While the polycondensation pathway is based upon polymerisation of rather small molecules such as amino acids, phenols, carbohydrates and various oligomers from biomass decomposition products as well as from root exudates, the new proposed pathway is based upon microbial biosynthesis of highly aliphatic cell walls which are chemically and physically resistant to biodegradation.

Several investigations have already detected the presence of aliphatic carbon in soils, including humic substances and resistant residues [START_REF] Hatcher | Aliphatic structure of humic acids; a clue to their origin[END_REF][START_REF] Schulten | Aliphatics in soil organic matter in fine-clay fractions[END_REF][START_REF] Kögel-Knabner | Nature and distribution of alkyl carbon in forest soil profiles : implications for the origin and humification of aliphatic biomacromolecules[END_REF][START_REF] Beyer | The chemical composition of soil organic matter in classical humic compound fractions and in bulk samples, a review[END_REF]. In a recent investigation, [START_REF] Lichtfouse | Unexpected 13 C-enrichment of organic components from wheat crop soils : evidence for the in situ origin of soil organic matter[END_REF] proposed on 13 C isotope grounds that soil organic matter may additionally be stored by selective preservation of resistant biopolymers from soil microorganisms. Moreover, in a recent note, we identified resistant ultralaminae [START_REF] Lichtfouse | Resistant ultralaminae in soils[END_REF] which are structurally similar to those found in aquatic sediments [START_REF] Largeau | Occurrence and origin of 'ultralaminar' structures in 'amorphous' kerogens of various source rocks and oil shales[END_REF][START_REF] Derenne | Chemical evidence of kerogen formation in source rocks and oil shales via selective preservation of thin resistant outer walls of microalgae : origin of ultralaminae[END_REF], thus strengthening our view. Here, using molecular and isotope techniques, we show that a part of the resistant carbon in crop soil is indeed composed of saturated straight-chain hydrocarbons, and we discuss implications for organic matter preservation in soils.

EXPERIMENTAL

Detailed procedures are reported elsewhere [START_REF] Lichtfouse | Resistant ultralaminae in soils[END_REF] : a sample of maize crop soil (290 g) was thoroughly extracted with CHCl 3 -MeOH 3/1 v/v to remove free lipids, then with NaOH 0.1 M to remove fulvic and humic acids. The remaining matter (humin and minerals) was treated with HCl and HF, then extracted with CHCl 3 -MeOH to yield 3.9 g of solid residue (31%C, 3% H, 2.5% N and 40% of uncombusted matter). Solid state 13 C NMR spectra of the resistant residue were recorded on a Bruker MSL 400 spectrometer at 100.6 Mhz for 13 C using the conventional Cross Polarization/Magic Angle Spinning (CP/MAS) technique with a contact time of 1 ms, a pulse delay of 5 s and dipolar dephasing delays T DD of 0 µs, 20 µs and 40 µs. The residue was pyrolyzed for 5 min at 600°C in a gold tube under continuous argon flow. The pyrolysate was fractionated into alkane-alkene and aromatic fractions by silica gel column chromatography eluting with pentane then pentane/methylene chloride (65/35 v/v). The alkanealkene fraction was analysed using a HP 5890 gas chromatograph coupled to a VG Autospec mass spectrometer (GC-MS). Conditions : splitless injection; helium carrier gas; 60 m x 0.2 mm capillary column coated with 0.2µm thickness methylpolysiloxane phase; oven temperature 50°C (1 min), 50°C to 140°C at 40°/min, 140°C to 320 °C at 2°/min then isothermal (60 min).; electronic ionisation 70 eV, 31-650 amu scan range. Isotopic analyses of individual n-alkanes from alkane-alkene fractions (3 to 4 replicates, deviation ≤ 0.3‰) were carried out under a continuous helium flow using a Hewlett Packard gas chromatograph coupled via a CuO furnace to a Finigan Mat 252 isotope ratio mass spectrometer. Isotope values were measured by comparison with CO 2 , 5α-androstane and n-tetracontane standards. Isotopic compositions are expressed in per mil as δ 13 C values relative to the Pee Dee Belemnite standard : δ 13 C = [( 13 C/ 12 Csample -13 C/ 12 Cstd)/( 13 C/ 12 Cstd)]x10 3

RESULTS AND DISCUSSION

A resistant organic residue was isolated from a maize crop soil by removal of lipids, humic acids, fulvic acids, and minerals. The solid state 13 C-NMR of this residue exhibits a relatively high signal at 32 ppm which shows the occurrence of aliphatic, saturated carbon (Fig. 1). The dephasing experiment shows a strong decrease in the 32 ppm peak, which indicates that the saturated carbon is mainly composed of secondary (R-CH 2 -R') and tertiary carbon (R-CH-R'R'') with few methyl groups (15 ppm) and few quaternary carbons. This finding suggests the occurrence of straight-chain skeletons with few branched and cyclic alkane skeletons, and is in agreement with IR analyses which give peaks at 2850 and 2920 cm -1 (unpublished data). GC-MS analyses of the alkane-alkene fraction from the pyrolysate residue show a major contribution of linear alkanes and alk-1-enes along with very few branched and cyclic molecules (Figure 2). The hydrocarbons can be classified into two main categories. First, n-alkanes extending from C 27 to C 33 with a strong odd predominance, which are typical plant wax products (e.g. [START_REF] Lichtfouse | Molecular, 13 C, and 14 C evidence for the allochthonous and ancient origin of C 16 -C 18 n-alkanes in modern soils[END_REF]. These alkanes have somehow been trapped in the organic network despite exhaustive CHCl 3 -MeOH extraction of the residue prior to pyrolysis. Nonetheless, this finding is in agreement with the occurrence of bonded alkanes and fatty acids in humic substances [START_REF] Schnitzer | Alkanes and fatty acids in humic substances[END_REF], and further suggests that this physical mode of sequestration of apolar molecules may play a role in the preservation of soil organic matter. Second, n-alkane/n-alkene doublets extending from C 10 to C 25 , are present with no odd/even predominance, which are 'true' cleavage products of the macromolecular soil matrix. Such a distribution, with very few branched and cyclic species is typical of kerogens and resistant aliphatic biopolymers [START_REF] Behar | Geochemistry of asphaltenes[END_REF][START_REF] Derenne | Comparison of torbanites of various origins and evolutionary stages. Bacterial contribution to their formation. Cause of the lack of botryococcane in bitumens[END_REF][START_REF] Schnitzer | Soil organic matter, the next 75 years[END_REF], 1992). Together with 13 C-NMR data, these findings show clearly that a part of resistant organic matter from soil is made of straight-chain saturated hydrocarbons. The δ 13 C values of n-alkanes and n-alkenes from the soil residue pyrolysate are reported in Fig. 3. The 13 C-depleted values for the C 29 and C 31 n-alkanes are in agreement with the derivation of these compounds from plant waxes of maize and wheat grown previously at the site [START_REF] Lichtfouse | Heterogeneous turnover of molecular organic substances from crop soils as revealed by 13 C labeling at natural abundance with Zea mays[END_REF]. The C 27 n-alkane derives also from plant waxes, despite a 13 C enrichement which can be explained by a higher quantitative input of this molecule from maize waxes [START_REF] Lichtfouse | Isotope and molecular evidence for direct input of maize leaf wax n-alkanes into crop soils[END_REF]. In contrast, C 15 -C 19 n-alkenes are significantly enriched in 13 C relative to n-alkane homologues. Furthermore, the δ 13 C values of C 15 -C 19 n-alkenes are closer to the δ 13 C value of the total organic carbon. Such an isotopic feature gives two pieces of information. First, it shows that these cleavage products are more representative of the bulk carbon than the longer chain nalkanes. Second, it indicates that the carbon source of the organisms synthesizing the n-alkene precursors is enriched in 13 C. Therefore, this latter observation confirms our previous hypothesis on the formation of a substantial part of soil organic matter by 'preservation of resistant aliphatic biopolymers from microorganisms consuming 13 C-enriched plant polysaccharides' [START_REF] Lichtfouse | Unexpected 13 C-enrichment of organic components from wheat crop soils : evidence for the in situ origin of soil organic matter[END_REF]. This novel, additional pathway contrasts with the current hypothesis involving recondensation of rather small molecules such as amino acids, phenols, carbohydrates and various oligomers. Nonetheless, a recent investigation on archaeological plant remains has brought some molecular evidence for the Maillard (or browning) reaction in buried organic matter [START_REF] Evershed | Volatile compounds in archaeological plant remains and the Maillard reaction during decay of organic matter[END_REF]. Further work is now in progress to evaluate the significance of these two processes in various fossil material.

  The other main NMR peaks have been tentatively assigned to α carbons of C-O or C-N functions (56 ppm), protonated C-O or C-N functions (73 ppm), glycosidic C-O (104 ppm), olefinic and/or aromatic C=C (120 ppm), phenols (148 ppm), and quaternary ester or amide C (173 ppm).

Figure 1 .

 1 Figure 1. Solid-state 13 C-NMR spectra of a resistant organic residue from a maize crop soil. Note the major peak at 32 ppm showing the occurrence of saturated carbon. The dipolar dephasing experiments with delays of 20 µs and 40 µs show that this saturated carbon is mainly composed of secondary (R-CH2-R') and tertiary carbon (R-CH-R'R'').

Figure 2 .

 2 Figure 2. GC-MS total ion current of the alkane-alkene fraction of the soil organic residue pyrolysate. Note the occurrence of C 10 -C 25 alkane-alkene doublets, typical of straight-chain macromolecular material.

Figure 3 .

 3 Figure 3. δ 13 C values of n-alkanes and n-alkenes from the pyrolysate of a soil residue. Note the 13 C enrichment in the short-chain n-alkenes


Andras Kemeny
email: andras.kemeny@renault.com

Emmanuel Icart
email: eicart@scale1portal.com

Alexandre Sepchat
email: alexandre.sepchat@scale1portal.com

Florent Colombet
email: florent.colombet@renault.com

Stéphane Espié
email: stephane.espie@ifsttar.fr

Jean-Rémy Chardonnet
email: jean-remy.chardonnet@ensam.eu

Large scale collaborative autonomous vehicle simulation and analysis using smartphones

Keywords: Driving Simulation, Autonomous Vehicle Simulation, Virtual Reality, Traffic Simulation, Smartphone

Simulation appears as an indispensable tool for automotive manufacturers to validate autonomous vehicle development, this latter reported to require billions of miles of driving. To involve the end users in this validation process, a project of large scale public application on smartphone allowing them to test and submit specific use cases, is presented in this paper. A first version of this application "3DAV Explore" has been released

and is already available, allowing users to see in Virtual Reality traffic scenarios in subjective view from the autonomous vehicle and/or from an exterior view. This paper details more deeply the development steps and the libraries used for the second version of this application. Further development steps and implications are also detailed in the conclusion.

Introduction

Autonomous vehicles development is one of the key challenges for automotive manufacturers. A large number of studies put forward billions of miles to run with different scenarios for an acceptable road safety level [Koo16]. Driving simulation may play a key role in its development, allowing to simulate a wide range of possible scenarios and automated behaviors. But even with cutting-edge software and infrastructure, running all the tests takes a lot of time, and often does not involve sufficiently man-in-the-loop simulation. The objective of this research project consists in evaluating how large scale public applications deployed on major smartphone platforms could help with autonomous vehicles simulation and development. With hundreds of millions of capable devices being used daily by consumers, we estimate a massive gain into using even a fraction of this available computing power for the benefits of simulation, through dedicated applications. The very first step of this project consists in the release of the 3DAV Explore smartphone application [3DA17] (Figure 1), which allows users to observe in virtual reality, in monoscopic or stereoscopic with VR glasses conditions (Figures 2 and3), a running traffic made of autonomous vehicles into a lane merging situation. This application sets the basis for the main core components of driving simulation: a road network definition, a traffic model to manage autonomous cars, a rendering engine, a user interface, all in real-time and running in an interactive way. Moreover, a second version was developed, adding more interactions as well as networking functionalities. The users drive a car in an American city using a mobile device. The car's steering wheel is controlled by the device's tilt while two touchcontrolled buttons serves as the brake and throttle (Figure 4). Two different modes of transportation are available to the user in this second application. By default, the user is placed outside of any vehicle, flying above the road. In this mode, he can move around the city by targeting the pink dots scattered all around the scene (Figure 5). In order to enter the second mode the user must target one of the red cars and tap the screen. He will then be transported to the driver seat of said car and takes control of it. A blue diamond above the vehicle signals to other users than the car is already being used and can't be entered (Figure 6). It is possible to exit the vehicle to go back to the outside view at any moment by simply tapping the center of the screen. The previously occupied vehicle will automatically return to auto mode and continue its own.

In both modes a button on the bottom of the screen allows to switch between 2D and 3D view to be used in a VR headset. While in 3D mode, the vehicle is controlled by the head's movements. Leaning forward and backward will accelerate and decelerate respectively and left/right movements will allow to turn the wheel and look around. The menu is opened by tapping the top left icon. Among the features available in this menu are (Figure 7): Resetting the gyroscope to adapt to the user's position, turning shadow off/on and data regarding the current state of the network and scene (Number of users...).

Tapping the "Auto" button on the bottom left will instruct the vehicle to switch into autonomous mode and start driving on its own. However, because of the limitations of the traffic system currently used it is possible for a vehicle to get stuck while trying to reconnect to its driving path.

Application Development

On the technical side, 3DAV explore has been developed using the Unity3D software.

The road scenery was built using visual 3D tiles from the Highway Construction Set, from the Visual Imagination Software company (http://www.visgames.de/). This consists into a 4 lanes highway section, with a lane merging area, of about 600 meters in length.

The city used for the latter scene was created using assets from the Modern City pack (https://www.assetstore.unity3d.com/en/#!/content/1 8005). It contains a selection of building meshes (FBX format) as well as various props (Bus stop, signs etc…) made with the Autodesk software series, optimized for use on a mobile platform (Figure 8). The layout was designed to form a closed road network. On the graphical side, the full road scene weight is about 300 000 polygons, depending on the selected viewpoint. A car-paint shader is used to provide better rendering on cars, offering environment reflections on the vehicles (see Figure 7). It runs at 60 Hz on an iPhone 6 device, in stereoscopic mode.

Even though the application was designed to run on middle-class devices (from iPhone 5C and above), the rendering engine can handle real-time soft shadows, and supports dynamic reflection probes, to get surrounding cars reflections in the car bodies and rear-view mirrors.

The city scene weights about 1.3 million polygons, depending on the selected viewpoint. It runs at 60Hz on the same iPhone 6 device. For low-class devices, an option to disable real-time shadows is available in order to increase performances.

In addition and also to further increase performances, the application uses the occlusion culling feature (see Figure 11). Every object not in direct view of the user's camera is not displayed, reducing drastically the number of polygons to compute on the screen. This functionality is especially effective in a city layout such as the one used in our application as the buildings hide most of the surroundings at any given time. The rendering engine used depends on the device the application is built on: Metal for iOS devices, OpenGL ES for Android and DirectX for Windows The next steps of the project are consisting in adding more interactivity: allowing the end-user to drive cars of the application; or allowing him to add obstacles and events that will challenge the autonomous vehicles AI. By involving end-users, and adding connectivity that will allow to turn this application into a true companion tool for driving simulation software, we will be able to introduce massive man-in-the-loop based scenario generation and simulation. There are already several realistic driving simulation tools using internet and different levels of driving and visualization and/or VR equipment on the market (The Good Drive, 2017 [Goo17]) and the trend is to see new more and more participative applications developed.

Network infrastructure & messaging

In order to allow for third-party software to inject their own vehicle behaviors inside the existing traffic the second application was created with an integrated networking infrastructure. The Photon Engine (https://www.photonengine.com/en/PUN) was used to handle synchronization between devices through client to server messaging. For this document, the third-party software used as an example will be SCANeR™ Studio 1.7 [Cla99].

Figure 12 illustrates the Photon Cloud working principle. The messages are sent through the Internet to the Photon cloud which hosts the server. The server then conveys the message to the other clients. On average, if 4 users are connected simultaneously, the server sends 200 messages (~200 bytes each) by second. The free version of the Photon engine allows up to 20 users connected at the same time. This maximum number can be upgraded to 5000. One user, designated as the "Master client" (by default the first to start the application), handle all calculations regarding the iTS traffic as well as the ones regarding its own vehicle. The resulting positions of the cars is sent to the server which will transmit to every other client. If the current master client disconnects, one of the remaining client is designated to take its place.

Application scenario & Conclusion

The application was designed to tackle a large variety of scenarios an autonomous vehicle might encounter. One example of such potential scenarios are the 25 cut-in scenarios as part of the NFI's research objectives "Autonomous vehicle" (http://pole-moveo.org/wpcontent/uploads/2015/07/Objectifs_de_recherche-Veh_Auton_V1.2.pdf). 3D AV Explore will provide engineers and managers a unique tool they will be able to use while reviewing high-end simulation running, analysis and reporting, especially for worst case scenario, in a complementary way of more traditional AD simulation software tools such as CarCraft [Mad17] or SCANeR Studio™ [SCA18]. Smartphone with the latest virtual, augmented and mixed realities technologies may thus be used to provide natural and collaborative review tools (Figures 1 and13). To complete the use of 3D AV Explore for extensive simulation trials integration in SCANeR Studio™ is planned in the next future as well as an open software implementation. Then, in-depth user studies should be performed to validate our approach of highly multi-collaborative simulation and check whether results could be transferred to real situations. Especially, as third-parties softwares will be able to communicate, it is possible to imagine collaborative driving not only between smartphones but also with heterogeneous platforms such as dynamic driving simulators.

Figure 1 -

 1 Figure 1 -Screenshot of the first version of 3DAV Explore

Figure 4 -

 4 Figure 4 -Driver's view and UI

Figure 5 -

 5 Figure 5 -Outside view of vehicle and pink dots

Figure 6 -

 6 Figure 6 -Vehicle currently under user's control

Figure

  Figure 7 -Menu options

Figure 8 -

 8 Figure 8 -American city model top down viewTo handle the road network definition and the autonomous driving vehicles behavior, the iTS (Intelligent Traffic System) software component (http://www.dagagames.com) has been used. The component includes the road network definition, with parameters for maximum lane speed, width, car type and occupation density, as well as priority pass for each lane connector. The traffic component also offers a simple car spawning component, which handles car addition during the simulation in real-time. It was configured to spawn cars in a 250 meters radius from the main point of interest of the scene: the lane merging point (see Figure9).

Figure 9 -

 9 Figure 9 -iTS Traffic component in action in the first version of 3DAV explore The car behavior is handled through the "iTS Traffic AI" script component, which uses an algorithm based on waypoints and car sensing distance to decide when it should accelerate, slowdown or change lane. This component allows developers to handle all the

Figure 10 -

 10 Figure 10 -"iTS Traffic AI" component parameters

Figure 11 -

 11 Figure 11 -Illustration of the Occlusion culling in the city scene

Figure 12 -

 12 Figure 12 -Illustration of the Photon Cloud working principle Each client, beside the master client, only handles calculations regarding its own vehicle and sends its position and rotation to the Photon Server. The positions of the other cars are received from the server and updated in real time on the local client. A script smoothens the motion between consecutives positions. The next step of development will consist in creating the Vhcom client interface to permit communication between SCANeR™ Studio and the application. This will allow the injection of autonomous cars into the scene as well as intelligent traffic. The third-party software will serve as substitute for a human user, controlling the car through the VhCom client interface. In return, it will receive the information regarding the positions of other vehicles in the scene to react accordingly.

Figure 13 -

 13 Figure 13 -A user of 3D AV explore, running the virtual reality mode


domain: math shs

text:
Charlotte Derouet

Alain Kuzniak

Dominique Laval

Elizabeth Montoya Delgadillo

Laurent Moutet

Assia Nechache

Bernard Parzysz

Laurent Vivier
email: laurent.vivier@univ-paris-diderot.fr

Modeling tasks and mathematical work

Keywords: Mathematical Working Space (MWS), modeling tasks, modeling cycle

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

In the Mathematical Working Space (MWS) model, an epistemological plane and a cognitive plane are introduced with a focus on their interactions related to semiotic, instrumental and discursive dimensions (Figure 1a). The model is devoted to the analysis of mathematical work with, specifically, paradigms guiding and orienting the work. Numerous researches are based on the MWS model and the reader may refer to special issues in journals such as Bolema 30(54) and ZDM-Mathematics Education 48(6) in which an introduction to the model is given in the survey paper. Nevertheless, until now, few studies on modeling tasks have been based on the MWS model and we want to highlight recent researches, in particular coming from PhD studies within our team.

Based on the modeling cycle (figure 1b) proposed by [START_REF] Blum | Filling Up"the problem of independance-preserving teacher interventions in lessons with demanding modeling tasks[END_REF], we suggest, in the poster, some adaptations which help to understand how MWSs can be used. The whole modeling process is not taken in its whole, and we focus more on how the analysis can be refined, mostly between phases 3 and 5 of the cycle, in relation to activity in different mathematical domains. Nechache ( 2016) suggests describing the modeling work in probability situations, with the MWS framework. She identifies the importance of the theoretical referential of the MWSProba in the constitution of the real model. Then, for the analysis of the mathematical part, she fully uses the MWSProba. In the same way, the MWS model can be used for studying other mathematical domains. [START_REF] Derouet | La fonction de densité au carrefour entre probabilités et analyse. Étude de la conception et de la mise en oeuvre de tâches d'introduction articulant lois à densité et calcul intégral[END_REF] proposes a similar type of use for the mathematical part, but she associates subphases to the stages of the cycle in order to investigate the progress of the modeling process. She isolates a part of the cycle containing "real model" and "real results" that she names pseudoconcrete. It allows her to identify, in a modeling situation related to continuous probability, a work within the MWSProba in various working paradigms.

In these studies, the MWS model allows to refine the analysis of the mathematical part by taking into account a first horizontal mathematization followed by a second vertical mathematization allowing to strengthen the mathematical model. Other types of change, or transition, are possible, like the change of MWS or mathematical domains. In his study in relativist kinematics, [START_REF] Moutet | Diagrammes et théorie de la relativité restreinte : Une ingénierie didactique[END_REF] suggests an extension of the MWS model to take into account a change of matters. He considers a second epistemological plane for physics, and he studies the interactions between these two planes and the cognitive level.

In these studies, simulation associated with digital models can also be considered as an important stage of the modeling process. It plays two different roles. The first one is in relation to the development of the real model with a simulation close to the initial situation (urn model or a calculator which proposes rolls of dice or coin, in probability). The second role presupposes a stronger mathematical expertise in the MWS of the domain at stake as, for example, the implementation of an algorithm of dichotomy in analysis.

Hence, the use of the MWS framework can enrich and strengthen the analysis of the modeling process based on the study of a cycle (figure 1b) in connection with a first resolution of the problem. It constitutes a first interaction between MWS and the modeling cycle, as a first cycle1 : The modeling problem has been mathematized and it is possible to identify the epistemological and cognitive components of the MWS in relation to the student's activity and realization in the different domains and paradigms. But we can also, in a more didactic way, think of a second cycle aiming at a better understanding of the model and of the mathematical objects introduced to solve the problem by students. In that case, the modeling task proposed by a teacher aims not only at solving a real problem but more deeply at exploring and understanding the numerous uses of a mathematical notion, enriching the MWS, in particular the theoretical referential. This is what we are developing in a work on progress on the exponential function.

Figure 1a :

 1a Figure 1a: MWS diagram; Figure 1b: modeling cycle[START_REF] Blum | Filling Up"the problem of independance-preserving teacher interventions in lessons with demanding modeling tasks[END_REF]

Proceedings of CERME10

Even if, of course, one should not understand the cycle as requiring to go through every stage, in a linear way.Thematic Working Group 06Proceedings of CERME10

Acknowledgment

Study supported by ECOS-Sud project C13H03.


Peter Lercher

MD Maurizio Lunati

MD Roberto Rordorf

MD Maurizio Landolina

MD Nima Badie

PhD ; Fujian Qu

DSc Cyrille Casset

MEng Kyungmoo Ryu

PhD Stefano Ghio

MD Jagmeet P Singh

PhD, FHRS Christophe Leclercq
email: christophe.leclercq@chu-rennes.fr

Badie

Long-term Reverse Remodeling by Cardiac Resynchronization Therapy with MultiPoint Pacing: A Feasibility Study of Noninvasive Hemodynamics Guided Device Programming Short Title: Noninvasive Hemodynamics to Guide CRT and MPP Programming

Keywords: Cardiac resynchronization therapy, heart failure, reverse remodeling, noninvasive hemodynamics, Multipoint Pacing, end-systolic volume, finger arterial pressure

Background: Cardiac resynchronization therapy (CRT) with multipoint left ventricular (LV) pacing (MultiPoint™ Pacing [MPP]) improves acute hemodynamics and chronic outcomes over conventional biventricular pacing (BiV), though MPP programming questions persist.

Objective: In this multi-center feasibility study, we evaluated the feasibility of using noninvasive systolic blood pressure (SBP) to guide MPP programming, and assessed the chronic 6-month echocardiographic CRT response.

Methods: Patients implanted with MPP-enabled CRT defibrillator (CRT-D) devices underwent noninvasive hemodynamic assessment (finger arterial pressure) during a pacing protocol that included atrial-only pacing and various BiV and MPP configurations. Each configuration was repeated 4 times, alternating with reference, to calculate the SBP difference relative to reference (ΔSBP). CRT configurations with the greatest ΔSBP were programmed. An independent core lab analyzed baseline and 6-month echocardiography, with CRT response defined as a 6-month reduction in LV end-systolic volume (ΔLVESV) ≥15%.

Results: Forty-two patients (71% male, LV ejection fraction 30.3±7.5%, QRS duration 161±19ms, 26% ischemic) were enrolled in 4 European centers. Relative to atrial-only pacing, the best BiV and best MPP configurations produced significant SBP elevations of 3.1±4.2 (p<0.01) and 4.1±4.1mmHg (p<0.01), respectively (BiV vs MPP, p<0.01). Greater SBP elevations were associated with the best MPP compared to the best BiV configurations in 29/37 (78%) of patients completing the pacing protocol. Of MPPprogrammed patients completing the 6-month follow-up, 23/27 (85%) were classified as CRT responders (ΔLVESV = 31±20%).

Conclusion:

Acute noninvasive hemodynamics after CRT implant predominantly favored MPP over BiV programming. MPP programming guided by noninvasive hemodynamics resulted in positive LV structural remodeling.

Introduction

Conventional cardiac resynchronization therapy (CRT) involves simultaneous pacing of the right ventricle (RV) and the left ventricle (LV). A coronary sinus lead, typically in a lateral or posterolateral coronary vein, is placed for LV pacing in addition to RV endocardial lead. Results from randomized, controlled clinical trials have consistently demonstrated that CRT improves cardiac structure and function while significantly reducing the risk of worsening heart failure. [START_REF] Abraham | Cardiac resynchronization therapy for heart failure[END_REF] However, up to 40% of the patients fail to respond to this therapy. [START_REF] Fornwalt | Agreement is poor among current criteria used to define response to cardiac resynchronization therapy[END_REF] In an effort to improve CRT response, methods to deliver pacing at multiple LV sites have been developed. Although attractive from a physiological perspective, dual-vein multisite LV pacing is hindered by technical complexities and several clinical issues. [START_REF] Leclercq | Triple Resynchronization In Paced Heart Failure Patients) Study Group. A randomized comparison of triple-site versus dual-site ventricular stimulation in patients with congestive heart failure[END_REF][START_REF] Lenarczyk | Mid-term outcomes of triple-site vs. conventional cardiac resynchronization therapy: a preliminary study[END_REF][START_REF] Lenarczyk | Implantation feasibility, procedure-related adverse events and lead performance during 1-year follow-up in patients undergoing triple-site cardiac resynchronization therapy: a substudy of TRUST CRT randomized trial[END_REF][START_REF] Rogers | A randomized double-blind crossover trial of triventricular versus biventricular pacing in heart failure[END_REF][START_REF] Anselme | Safety, feasibility, and outcome results of cardiac resynchronization with triple-site ventricular stimulation compared to conventional cardiac resynchronization[END_REF] A recently introduced technique to achieve multisite LV pacing without the need for two LV leads makes use of a quadripolar LV lead paired with a pulse generator capable of pacing from two LV electrodes in the same coronary vein (MultiPoint™ Pacing [MPP] technology, Abbott, Abbott Park, IL).

Initial clinical experience has shown that MPP, compared with conventional BiV pacing, provides acute benefits to LV dP/dt , LV dyssynchrony [START_REF] Rinaldi | Acute effects of multisite left ventricular pacing on mechanical dyssynchrony in patients receiving cardiac resynchronization therapy[END_REF] , LV peak radial strain [START_REF] Rinaldi | Improvement in acute contractility and hemodynamics with multipoint pacing via a left ventricular quadripolar pacing lead[END_REF][START_REF] Osca | The use of multisite left ventricular pacing via quadripolar lead improves acute haemodynamics and mechanical dyssynchrony assessed by radial strain speckle tracking: initial results[END_REF] , and LV electrical activation [START_REF] Menardi | Characterization of ventricular activation pattern and acute hemodynamics during multipoint left ventricular pacing[END_REF] , and improves long-term CRT response [START_REF] Pappone | Improving cardiac resynchronization therapy response with multipoint left ventricular pacing: Twelve-month follow-up study[END_REF][START_REF] Zanon | Optimization of left ventricular pacing site plus multipoint pacing improves remodeling and clinical response to cardiac resynchronization therapy at 1 year[END_REF][START_REF] Forleo | Multipoint pacing via a quadripolar left-ventricular lead: preliminary results from the Italian registry on multipoint left-ventricular pacing in cardiac resynchronization therapy (IRON-MPP)[END_REF] . However, questions persist on how to program MPP in a routine clinical setting. Noninvasive methods have the advantage of being convenient to acquire, free from potential complications associated with invasive measurements, and allow multiple measurements to be made to increase the confidence of measurements. In this multi-center feasibility study, we evaluated the feasibility of using noninvasive systolic blood pressure (SBP) to guide MPP programming, and assessed the chronic echocardiographic CRT response at 6 months.

Methods

This study was conducted at 4 European centers. All patients provided written informed consent, and the study protocol was reviewed and approved by the Human Research Ethics Committee of each participating institution. The patient population included in this study was characterized by NYHA functional class II or III and indicated by ESC/EHRA guidelines to undergo de novo implant of a CRT defibrillator (CRT-D) system. Patients were excluded for any of the following conditions: age <18 years, pregnancy or planned pregnancy within 12 months, persistent or permanent atrial fibrillation, recent myocardial infarction within 40 days, cardiac surgery or coronary revascularization procedure within 3 months or scheduled for such procedure in the following 12 months, recent cerebrovascular accident or transient ischemic attack within 3 months, on intravenous inotropic support in the last 30 days, or either scheduled or considered for heart transplantation over the next 12 months. The study was registered on ClinicalTrials.gov (NCT02064751).

Procedures and Follow-up Visits

Abbott CRT-D devices with the MPP feature (Quadra Assura MP™, models 3371-40Q/QC) and Quartet® quadripolar LV pacing lead were used in this study. Patients underwent device implant according to standard practice. Prior to discharge, each patient underwent noninvasive hemodynamic assessment

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under a study-specific pacing protocol and was programmed to the CRT configuration that yielded the greatest increase in systolic blood pressure. Noninvasive hemodynamic assessment was repeated at the 6-month follow-up visit.

Echocardiographic evaluation was performed at the pre-implant baseline visit and again at 6 months post-implant. LV end-systolic volume (LVESV) and LV ejection fraction (LVEF) were analyzed by a core lab. Patients were considered to be responders to CRT at the 6-month follow-up visit if they demonstrated a reduction in LVESV of at least 15% relative to baseline (ΔLVESV≥15%). Clinical evaluations including NYHA functional class, 6 minute hall walk distance (6MWD), and Minnesota living with heart failure questionnaire (MLWHF) were collected at baseline and every follow-up visit.

Selection of Pacing Configurations

Atrial-only pacing, conventional biventricular pacing (BiV) configurations, and MPP configurations were evaluated at the post-implant hospital discharge (pre-discharge) visit, as shown in Table 1. The cathodeanode combinations that comprised the LV pacing vectors of interest included the 4 quadripolar LV lead electrodes (D1, M2, M3, P4: distal-to-proximal) along with the RV coil. LV pacing vectors with a capture threshold higher than 3.5 V or presence of phrenic nerve stimulation (PNS) at twice the capture threshold were excluded. The 4 BiV configurations tested included each of the 4 quadripolar LV lead electrodes as cathodes, with the RV coil as anode.

Two MPP vector combinations ("anatomically-defined MPP" and "electrically-defined MPP") were selected for testing. The anatomically-defined MPP configuration was chosen to pace first using a distal electrode as cathode (LV1) and second using a proximal electrode as cathode (LV2) with maximum separation between the two cathodes along the quadripolar lead body. The electrically-defined MPP configuration was selected to pace first using the cathode with the earliest electrical activation (LV1) and second using the cathode with the latest electrical activation (LV2), as measured from RV pacing to LV sensing at each of the 4 quadripolar LV lead electrodes. RV coil was used as the anode of the MPP LV pacing vectors. An additional MPP configuration was tested, using D1-M2 as the LV1 pacing vector and M3-RV coil as the LV2 pacing vector with LV1 LV2 and LV2 RV delays of 10 ms each.

The device can or an LV electrode was substituted for the RV coil as the anode in BiV or MPP configurations when high capture threshold or PNS was observed.

Pacing Protocol and Noninvasive Hemodynamic Assessment

Each of the above pacing configurations defined a Test Pacing Intervention (TPI). To minimize the effect of intrinsic blood pressure variation, a fixed pacing configuration was used as the Reference Pacing Intervention (RPI) between all repeated TPIs. In order to minimize abrupt changes in hemodynamics between pacing transitions and better characterize benefit of MPP over BiV pacing, the RPI for each patient was defined as BiV pacing using the distal LV electrode as cathode and RV coil as anode. Each TPI alternated with the RPI four times to yield eight pacing transitions between TPI and RPI. All pacing interventions were delivered for 20 sec at a time, with a pacing constant rate above the patient's

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intrinsic rate, and a fixed atrioventricular (AV) delay (between 100-150ms) to ensure consistent ventricular capture.

Noninvasive hemodynamics were measured using a finger arterial pressure system (Finometer MIDI, Finapres Medical Systems, Netherlands). The relative change in systolic blood pressure (ΔSBP) was calculated between each TPI and neighboring RPI by comparing the mean SBP 10 seconds immediately before and after a pacing transition. The mean and standard deviation of ΔSBP from the eight pacing transitions were used to rank each TPI relative to the RPI. The use of ΔSBP to guide AV delay programming has been previously described by Whinnett et al [START_REF] Whinnett | Determination of optimal atrioventricular delay for cardiac resynchronization therapy using acute non-invasive blood pressure[END_REF] .

Prior to hospital discharge, patients were programmed to the BiV or MPP TPI associated with the greatest mean ΔSBP. If no MPP vector combination was available, the patient was programmed at the discretion of the investigator and withdrawn from the study.

Statistical Analysis

All measurements were expressed as mean ± standard deviation. Comparisons of different pacing interventions within each patient were made by using paired t tests. Comparisons of patient subgroups were made using unpaired t tests. P values <0.05 were considered significant.

Results

Study Population

Forty-five patients scheduled to undergo de novo implantation of a CRT-D device (Quadra Assura MP™ with Quartet™ LV lead, Abbott) were enrolled in 4 European centers. Of those patients, 42 completed the baseline visit and implant (withdrawn: 1 presented in persistent AF, 1 with baseline LVEF > 35%, 1 had an unknown paracardiac mass identified by MRI), 39 patients completed the pre-discharge data collection visit (withdrawn: 3 presented high capture thresholds), and 34 patients participated through the 6 month follow-up (withdrawn: 2 exhibited PNS, 2 LV lead dislodgement, 1 patient request). Patients were implanted between May, 2014 and June, 2015. The distributions of the implanted RV and LV lead locations are shown in Figure 1. Pre-implant baseline patient demographics, comorbidities, and HF metrics for all patients with complete 6 month data are shown in Table 2.

Acute Hemodynamic Evaluation: Pre-discharge

Noninvasive finger arterial blood pressure measurements at pre-discharge demonstrated a greater acute response to MPP compared to BiV configurations. The BiV and MPP configurations listed in Table 1 were ranked for each patient according to SBP elevation relative to atrial-only pacing. As shown in the top-left panel of Figure 2, the best BiV and best MPP configurations produced significant SBP elevations of 3.2±4.3 (p<0.001) and 4.1±4.2 mmHg (p<0.001), relative to atrial-only pacing, respectively with a significant difference between best MPP and best BiV (MPP-BiV: 0.9 mmHg, p<0.001).

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ACCEPTED MANUSCRIPT As shown in Figure 5, clinical assessment at 6 months showed reduced NYHA functional class (-0.53±0.66), improved quality of life (MLWHF score, -16.7±22.5), and improved 6MWD (+37.6±91.7 meters) from baseline in all patients. No significant differences between responders and non-responders were observed.

The 6 CRT non-responders at 6 months underwent 9-and 12-month follow-up visits. Devices in these patients were not reprogrammed at 6 months. At 12 months, 2/6 non-responders became responders, 3/6 remained non-responders but showed ESV improvement, and 1 was unknown due to echocardiography errors.

Discussion

CRT device programming is important in improving patient outcomes. Routine optimization techniques have included echocardiography, device-based algorithms, and other noninvasive techniques, such as blood pressure measurements. To our knowledge, this is the first multi-center study to prospectively evaluate the feasibility of using noninvasive systolic blood pressure measurements to guide MPP programming and subsequently assess the chronic CRT response.

The main findings are (1) the acute noninvasive hemodynamics after CRT implant predominantly favored MPP over BiV programming, and (2) MPP programming guided by systolic blood pressure assessments resulted in positive LV structural remodeling at 6 months post device implant.

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Acute and Chronic Hemodynamics Post-implant

During the acute hemodynamic assessments after device implant, the best BiV and best MPP configurations (i.e., those yielding the greatest blood pressure elevation) produced significant SBP elevations relative to atrial-only pacing. Greater SBP elevations were associated with the best MPP setting in 79% of the patients. The evaluated MPP pacing configurations showed a wider range of hemodynamic variation, suggesting acute optimization and individualized programming should definitely be considered to maximize the benefit of MPP. Fortunately, such individualization can be achieved efficiently by noninvasive hemodynamic measurements. The repeated hemodynamic assessment at the 6-month follow-up visit showed a sustained benefit of MPP over BiV configurations.

The achieved SBP elevation in acute optimization protocol was quite small in amplitude and difficult to speculate the clinical implications of the magnitude of response. In our opinion, it is probably more important to compare the SBP elevations by the tested CRT configurations and select the optimal setting. In another word, use it as a metric to identify the CRT setting that results in the highest acute change in SBP rather than link the magnitude of SBP change to long term CRT response.

BiV vs. MPP

In this study, devices were predominantly programmed to MPP due to superior acute hemodynamics. The 6-month echocardiographic and clinical results in these BiV patients were almost as good as MPP patients. Although statistical significance was not achieved due to sample size limitations, patients programmed to BiV had longer baseline QRS durations and less RV-to-LV conduction dyssynchrony compared to patients programmed to MPP. This observation further underscores the importance of patient-individualized CRT optimization and selective use of MPP in different clinical scenarios.

Importance of Repeated Measurements

Hemodynamic measurements used to compare CRT configurations are susceptible to confounding factors, such as respiration and baseline signal drift. Previous validation studies [START_REF] Whinnett | Haemodynamic effects of changes in atrioventricular and interventricular delay in cardiac resynchronisation therapy show a consistent pattern: analysis of shape, magnitude and relative importance of atrioventricular and interventricular delay[END_REF][START_REF] Thibault | Design of an acute dP/dt hemodynamic measurement protocol to isolate cardiac effect of pacing[END_REF] have shown that repeated comparisons of test and reference interventions can reduce the impact of confounding factors, allowing the specific effects of the test interventions to be distinguished. Similarly, this study employed a pacing protocol that included four repeated alternations between each TPI and the fixed RPI, resulting in eight replicates of pacing transitions. Although this pacing protocol may seem exhaustive, a recent report showed proof of concept of a simple, fully-automated photoplethysmographic method for AV optimization [START_REF] Kyriacou | Fully automatable, reproducible, noninvasive simple plethysmographic optimization: proof of concept and potential for implantability[END_REF] . Such a system may be applied to automate the pacing protocol used in this study to guide MPP programming.

Dependence of CRT Response on HF Etiology

In this study, we observed similar improvement in acute noninvasive hemodynamics and long-term reverse remodeling regardless of HF etiology and QRS duration (greater or less than 150 ms). It is well known that HF etiology and lack of dyssynchrony are independent predictors of non-response to conventional CRT. [START_REF] Rickard | Predictors of response to cardiac resynchronization therapy: A systematic review[END_REF][START_REF] Brignole | ESC Guidelines on cardiac pacing and cardiac resynchronization therapy: the Task Force on cardiac pacing and resynchronization[END_REF] In the CARE-HF and MADIT-CRT study, reverse remodeling was more pronounced

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in the non-ischemic population without a significant impact on clinical outcomes. [24][25] Conceivably, MPP may provide more benefit in patients with ischemia and heterogeneous patterns of dyssynchrony by providing better synchronization of viable LV tissue and normalizing electrical propagation patterns around the scar tissue. Our observations highlight the potential of further studies to elucidate the impact of MPP with respect to LV scar presence and scar distribution in HF patients.

Chronic Response of MPP Programming Guided by Noninvasive Hemodynamics

In this study, the CRT response rate and super-response rate (defined by ∆LVESV ≥ 30%) at 6 months were 82.4% and 50%, respectively. This high response rate is consistent with previous single-center studies in which acute invasive hemodynamic assessments have been used to guide MPP programming.

In a previous study by Pappone et al [START_REF] Pappone | Improving cardiac resynchronization therapy response with multipoint left ventricular pacing: Twelve-month follow-up study[END_REF] , CRT response rate and super-response rate based on clinical response at 12 months was 76% and 33% in the MPP group, respectively (vs. 57% and 14% in the BiV group). Another study by Zanon et al [START_REF] Zanon | Optimization of left ventricular pacing site plus multipoint pacing improves remodeling and clinical response to cardiac resynchronization therapy at 1 year[END_REF] reported that CRT response at nearly 1 year post-implant was 90% when LV pacing site optimization was used with MPP. The results from the current study demonstrate that superior chronic response can be achieved by MPP programming guided by systolic blood pressure measurement using a noninvasive system.

Limitations

With MPP, the additional LV pacing vector and programmable LV-to-LV delay result in many additional programming options. All of these options were not tested in this study due to the time limitations of an acute pacing protocol. Instead, we prioritized the options based on "anatomically-guided" and "electrically-guided" MPP vector selection methods, both of which are easy to understand and straightforward for clinical practice. In each individual patient, a single AV delay was used in all pacing interventions. Furthermore, BiV pacing interventions were performed with simultaneous BiV pacing, as it is considered standard programming for CRT in routine clinical practice. Whether the benefits of MPP over BiV persist across the physiological range of AV and interventricular delays remains to be addressed. Another limitation of this feasibility study was the small sample size and relatively high dropout rate for final analysis, which should be supplemented by larger-scale clinical studies.

Conclusion

This study showed that acute noninvasive hemodynamic evaluation after CRT implant predominantly favored MPP over BiV programming. In addition, MPP programming guided by noninvasive hemodynamics, specifically systolic blood pressure, resulted in positive LV structural remodeling and improved chronic outcomes. These findings have important clinical implications for device programming and CRT optimization using MPP to maximize patient outcomes.

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Figure 1 .

 1 Figure 1. Distributions of implanted RV (left) and LV (center, right) lead locations, shown by percent of patients.

Figure 2 :

 2 Figure 2: Acute hemodynamic assessment at Pre-discharge (left) and 6-month (right) visits. Change in noninvasive systolic blood pressure, relative to atrial-only pacing, shown for the BiV and MPP settings of each patient that elicited the "worst" (smallest change) and "best" (greatest change) acute response. Top panel shows all patients; middle panels group patients by heart failure etiology; bottom panels group patients by QRS duration.

Figure 3 :

 3 Figure 3: Distribution of 6-month response across all 34 patients. Left pie-chart shows proportion of responders (ΔLVESV ≥ 15%) vs. non-responders. Right pie-chart further distinguishes super-responders (ΔLVESV ≥ 30%) and negative non-responders (ΔLVESV < 0%).

Figure 4 : 6 -

 46 Figure 4: 6-month LV remodeling outcomes. Mean LVESV and LVEF response of all patients, responder patients (ΔLVESV ≥ 15%), and non-responder patients (ΔLVESV < 15%). Responder and super-responder (ΔLVESV ≥ 30%) thresholds are indicated as dashed lines. Response for each patient is also shown: super-responder (upward triangle), responder (square), and non-responder (downward triangle).

Figure 5 :

 5 Figure 5: Clinical parameters at baseline visit and 6-month follow-up. After 6 months, patients had significantly improved NYHA functional class, MLWHFQ score, and 6 minute walk distance (*p < 0.05).

6

  Furthermore, the SBP changes elicited by the best BiV and best MPP configurations, relative to atrialonly pacing, were slightly greater for non-ischemic (BiV: 3.5±4.1, MPP: 4.4±4.1 mmHg) vs. ischemic patients (BiV: 2.0±4.9, MPP: 3.0±4.4 mmHg), as shown in the middle-left panels (∆BiV: 1.5, ∆MPP: 1.4 mmHg). Similarly, these changes were also slightly greater for patients with QRS duration > 150 ms (The SBP changes were highly dependent on the specific CRT settings, with a broad range of ΔSBP magnitudes observed. Differences in ΔSBP between the best and worst settings were 1.6±1.3 mmHg for BiV configurations and 4.4±1.9 mmHg for MPP configurations, indicating the importance of MPP setting selection.Greater acute SBP elevations were associated with the best MPP configuration compared to the best BiV configuration in 31/39 (79%) of patients who completed the pacing protocol. Each patient was ultimately programmed to the BiV or MPP configuration associated with the highest acute SBP elevation. Patients programmed to BiV, in comparison to those programmed to MPP, had longer baseline QRS duration (169±29 vs. 158±29 ms) and less RV-to-LV electrical conduction dyssynchrony (13±12 vs. 21±18 ms, measured by the difference in activation times among the 4 LV electrodes during RV pacing). There was no difference in heart failure etiology between patients programmed to MPP and BiV.The distribution of pacing electrodes (cathodes) associated with the best BiV and best MPP configurations is provided in Table3. Although a single cathode was not particularly favored in BiV configurations, the best MPP configurations favored D1 and either P4 or M3 as cathodes. In terms of LV1 LV2 RV pacing delays in the best MPP configurations, 18/31 (58%) included delays >20 ms, while 13/31 (42%) included only delays ≤20 ms.Repeated noninvasive finger blood pressure measurements 6 months post-implant revealed a sustained acute benefit of MPP over BiV configurations, as shown in the right panels of Figure2. Relative to atrialonly pacing, the best BiV and best MPP configurations at 6 months post-implant produced significant SBP elevations of 2.9±5.1 (p<0.01) and 3.9±4.7 mmHg (p<0.01), respectively (MPP-BiV: 1.0 mmHg, p<0.01).

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	months post-implant were 2.3±2.4 mmHg for BiV configurations and 5.6±2.5 mmHg for MPP
	configurations.
	Chronic Outcomes at 6 and 12 Months Post-implant	BiV:
	4.1±4.4, MPP: 4.9±4.4) vs. those with QRS duration ≤ 150 ms (BiV: 1.2±3.2, MPP: 2.3±2.9), as shown in In terms of ΔLVESV≥15% at 6-months relative to pre-implant, 4/5 BiV patients and 24/29 MPP patients
	the bottom-left panels (∆BiV: 2.9, ∆MPP: 2.6 mmHg). M were classified as CRT responders, for a total response rate of 28/34 (82.4%), shown in Figure 3. All 6 non-responders were male, 2 had ischemic cardiomyopathy, 4 had LV leads in lateral and 2 in posterolateral vein branches, and RV leads were septal in 2 patients and apical in 4 patients. The single BiV non-responder was programed to BiV simultaneous pacing using the D1 LV electrode. Among the 5 MPP non-responders, 3 were programmed with D1 and P4 LV electrodes, 1 with D1 and M3, and 1 with M2 and P4. CRT responder rate was 9/11 (81.8%) in ischemic patients and 19/23 (82.6%) in non-ischemic patients. Chronic 6-month LV remodeling outcomes assessed using echocardiography are shown in Figure 4. CRT responders and non-responders demonstrated relative LVESV changes of 36.3±13.0% and 0.9±14.6%, respectively, with a collective ΔLVESV of 30.1±18.9%. In addition, CRT responders and non-responders A N U S C R I P T demonstrated absolute elevations in LV ejection fraction of 11.3±5.3% and 9.0±6.6%, respectively, with
	Acute Hemodynamic Evaluation: 6 Months Post-implant Similar to pre-discharge measurements, the SBP changes elicited by the best BiV and best MPP configurations were slightly greater for non-ischemic (BiV: 3.5±5.7, MPP: 4.8±5.1 mmHg) vs. ischemic patients (BiV: 1.7±3.2, MPP: 2.1±2.9 mmHg), as shown in the middle-right panels of Figure 2 (∆BiV: 1.8, A C C E P T E D
	∆MPP: 2.7 mmHg). These changes were also slightly greater for patients with QRS duration > 150 ms
	(BiV: 3.7±5.8, MPP: 4.7±5.2) vs. those with QRS duration ≤ 150 ms (BiV: 1.0±1.7, MPP: 2.1±2.2), as
	shown in the bottom-right panels (∆BiV: 2.7, ∆MPP: 2.6 mmHg).
		7

Also consistent with pre-discharge measurements, the best and worst BiV and MPP configurations resulted in a broad range of SBP changes. Differences in ΔSBP between the best and worst settings 6 a collective ΔLVEF of 10.9±5.6%. MPP-programmed responder patients, specifically, experienced a relative LVESV change of 37.0±13.6% and an absolute LVEF elevation of 12.0±5.2%. ΔLVESV was 31.8±15.0% in ischemic patients and 29.2±20.8% in non-ischemic patients. ΔLVEF was 11.7±6.1% in ischemic patients and 10.5±5.3% in non-ischemic patients.

Table 1 :

 1 Pacing configurations tested.The LV-LV-RV timing for MPP configurations is displayed as "vector 1 -(delay 1-2 ) vector 2 -(delay 2-3 ) vector 3 ."

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Table 2 : Baseline patient characteristics.

 2

		Patients completed	Patients completed
	Characteristic	Implant	6 Month Follow-up
		(N = 42)	(N = 34)
	Age (yr)	63 ± 11	63 ± 11
	Gender (% male)	74%	76%
	Baseline Ischemic Etiology	74% non-ischemic	68% non-ischemic
	NYHA Functional Class	2.3 ± 0.5	2.4 ± 0.5
	QRS Duration (ms)	160 ± 20	161 ± 20
	6 min Walk Test Distance (m)	409 ± 126	408 ± 134
	LV Ejection Fraction (%)	30 ± 7	30 ± 7
	LV End-systolic Volume (mL)	183 ± 85	188 ± 87
	LV End-diastolic Volume (mL)	256 ± 97	261 ± 98
	Medication (% of patients)
	ACE Inhibitors / ARBs	100%	100%
	Beta Blockers	93%	94%
	Diuretics	90%	88%
	Antiplatelets	50%	50%
	Anticoagulants	29%	29%
	Antiarrhythmics Class III	24%	24%

Table 3 : Pacing electrodes associated with greatest ΔSBP.

 3

	BiV Cathode	# Patients (N = 8 BiV)	MPP Cathode Combination (LV1/LV2)	# Patients (N = 31 MPP)
	D1	2	D1/P4	10
	M2	3	D1/M3	7
	M3	1	D1/M2	2
	P4	2	P4/D1	3
			M3/D1	3
			M2/D1	2
			M2/P4	2
			P4/M3	1
			M2/M3	1

The study was funded by Abbott. Maurizio Lunati received funding from Medtronic, Abbott, Sorin, , Boehringer-Ingelheim, and Bayer. Roberto Rordorf received funding from Medtronic, Abbott, and Boston Scientific. Maurizio Landolina received funding from Medtronic. Nima


Rémi Grenier

Caroline Costedoat

Rémi Chappaz

Vincent Dubut
email: vincent.dubut@imbe.fr

P C Alves

: R Chappaz

Two multiplexed sets of 21 and 18 microsatellites for Phoxinus phoxinus (L.) and Gobio gobio (L.) developed by cross-species amplification

Keywords: Microsatellite, Cyprinid, Population genetics, Conservation, Management

A total of 68 cyprinid microsatellites were screened for cross-species amplification in order to develop three PCR multiplex kits for the Eurasian minnow (Phoxinus phoxinus L.) and the gudgeon (Gobio gobio L.). The multiplex kits cluster 21 and 18 microsatellites for P. phoxinus and G. gobio, respectively. All loci were evaluated using ~80 individuals sampled from three distinct populations. By multiplexing the markers, we aim to decrease genotyping costs and increase time efficiency. These two microsatellite sets represent a valuable tool for ecological, evolutionary, conservation and management issues.

Introduction

The Eurasian minnow (Phoxinus phoxinus L. 1758) and the gudgeon (Gobio gobio L. 1758) are both widespread and common freshwater cyprinid species in Europe. They are commonly stocked and are used by anglers as fry, bait or forage species [START_REF] Kottelat | The genus Gobio in France, with redescription of G. gobio and description of two new species (Teleostei: Cyprinidae)[END_REF]. Phoxinus phoxinus and G. gobio are therefore prone to recurrent translocation and are considered as invasive species in some European areas where they are not native [START_REF] Crivelli | Are fish introductions a threat to endemic freshwater fishes in the northern Mediterranean region?[END_REF][START_REF] Elvira | Freshwater fish introductions in Spain: facts and figures at the beginning of the 21st century[END_REF][START_REF] Hesthagen | Non-native freshwater fishes in Norway: history, consequences and perspectives[END_REF][START_REF] Museth | The history of the minnow Phoxinus phoxinus (L.) in Norway: from harmless species to pest[END_REF]). In their native range however, they proved to be valuable models either for studying the impact of water pollution on freshwater fishes [START_REF] Knapen | Historical metal pollution in natural gudgeon populations: inferences from allozyme, microsatellite and condition factor analysis[END_REF] or for investigating the processes involved in local adaptive variation [START_REF] Collin | Evidence for morphological and adaptive genetic divergence between lake and stream habitats in European minnows (Phoxinus phoxinus, Cyprinidae)[END_REF]. Furthermore, it was shown that anthropogenic habitat fragmentation impacts the genetic diversity and structure of these two species differently [START_REF] Blanchet | Species-specific responses to landscape fragmentation: implications for management strategies[END_REF]). Together, P. phoxinus and G. gobio thus represent a valuable system for an array of management, conservation and evolutionary purposes. Additionally, molecular and morphological studies recently suggested revisions of the taxonomy of both P. phoxinus and G. gobio [START_REF] Kottelat | The genus Gobio in France, with redescription of G. gobio and description of two new species (Teleostei: Cyprinidae)[END_REF][START_REF] Kottelat | Three new species of Phoxinus from Greece and southern France (Teleostei: Cyprinidae)[END_REF][START_REF] Mendel | Molecular phylogeny of the genus Gobio Cuvier, 1816 (Teleostei: Cyprinidae) and its contribution to taxonomy[END_REF], implying critical conservation and management issues.

To date, no specific microsatellites have been developed for P. phoxinus and only a limited set of markers is available for G. gobio (six reliable markers; [START_REF] Knapen | Isolation and characterization of polymorphic microsatellite loci in the gudgeon, Gobio gobio (Cyprinidae)[END_REF][START_REF] Knapen | Historical metal pollution in natural gudgeon populations: inferences from allozyme, microsatellite and condition factor analysis[END_REF]. The development of additional markers would permit the evolutionary history of these species to be deciphered and would allow a straightforward evaluation of the genetic diversity and structure in both evolutionary ecology and conservation frameworks. From this perspective, crossspecies amplification represents a cost-and time-effective approach (e.g. [START_REF] Wang | Polymorphic microsatellites in the Reeves's pheasant developed by cross-species amplification[END_REF][START_REF] Rosenbom | Cross-species genetic markers: a useful tool to study the world's most threatened wild equid-Equus africanus[END_REF]) and has proven especially valuable for cyprinid species [START_REF] Hamilton | Identification of microsatellite loci for parentage analysis in roach Rutilus rutilus and eight other cyprinid fish by cross-species amplification, and a novel test for detecting hybrids between roach and other cyprinids[END_REF][START_REF] Dubut | Cross-species amplification of 41 microsatellites in European cyprinids: a tool for evolutionary, population genetics and hybridization studies[END_REF]. Crossspecies amplification was previously used for P. phoxinus and G. gobio in order to identify amplifiable and polymorphic microsatellites [START_REF] Holmen | Crossspecies amplification of zebrafish and central stoneroller microsatellite loci in six other cyprinids[END_REF][START_REF] Holmen | Crossspecies amplification of 36 cyprinid microsatellite loci in Phoxinus phoxinus (L.) and Scardinius erythrophthalmus (L.)[END_REF]. Nevertheless, the screening was conducted on a limited number of individuals for most of the markers, which prevented full validation and characterisation. Using crossspecies amplification, our study aimed to (1) identify a sufficient number of loci that would allow for discriminating populations at a microgeographic scale (see [START_REF] Nikolic | A set of 37 microsatellite DNA markers for genetic diversity and structure analysis of Atlantic salmon Salmo salar populations[END_REF]), (2) standardise PCR conditions (one single PCR protocol) and (3) multiplex loci in order to minimise the genotyping time and cost.

Materials and methods

Fish were caught by electrofishing, and caudal fin samples were collected before the fish were returned to their original sampling site. Two distinct locations from the Rhone River system (Isere River and Saone River, Eastern France) and one location from the Meuse River (Northern France) were sampled. At each location, 25 to 33 individuals of P. phoxinus and G. gobio were collected (see Table 1). Additional samples were collected in the Garonne and Loire River systems (France) for preliminary amplifiability and polymorphism testing. DNA was extracted from ~0.25 cm 2 of fin using the Gentra® Puregene™ Tissue Kit (QIAGEN).

To identify amplifiable loci, 56 published primer sets developed for Cyprinidae species were tested: BL1-2b, BL1-30, BL1-36, BL1-44, BL1-61, BL1-84, BL1-98, BL1-153, BL1-158, BL1-T2 and BL2-114 (Dubut et al. 2009a[START_REF] Dubut | From late Miocene to Holocene: processes of differentiation within the Telestes genus (Actinopterygii: Cyprinidae)[END_REF]; Ca1 and Ca3 [START_REF] Dimsoski | Microsatellite characterization in central stoneroller Campostoma anomalum (Pisces: Cyprinidae)[END_REF]; CnaB-030, CnaD-112, CnaF-177, CtoA-254, CtoE-249, CtoF-172, CtoG-075 and CtoG-216 (Dubut et al. 2010); CypG9, CypG24 and CypG30 [START_REF] Baerwald | Characterization of microsatellite loci for five members of the minnow family Cyprinidae found in the Sacramento-San Joaquim Delta and its tributaries[END_REF]; IV04 [START_REF] Salgueiro | Microsatellite analysis of genetic population structure of the endangered cyprinid Anaecypris hispanica in Portugal: implications for conservation[END_REF]; LC27 [START_REF] Vyskočilová | Isolation and characterization of microsatellites in Leuciscus cephalus (Cypriniformes, Cyprinidae) and cross-species amplification within the family Cyprinidae[END_REF]); LceC1 and LceA149 [START_REF] Larno | Isolation and characterization of microsatellite loci from chub Leuciscus cephalus (Pisces: Cyprinidae)[END_REF]; LleA-029, LleA-071, LleA-131, LleA-150, LleA-191, LleB-048, LleB-071, LleB-072, LleC-049, LleC-090 andLleC-184 (Dubut et al. 2009b, 2010); Lco1, Lco3 and Lco5 [START_REF] Turner | Variable microsatellite markers amplify across divergent lineages of cyprinid fishes (subfamily Leuciscinae)[END_REF]); Lid8 and Rru4 [START_REF] Barinova | Identification and characterization of microsatellite DNA markers developed in ide Leuciscus idus and Siberian roach Rutilus rutilus[END_REF]); Lsou5, Lsou8, Lsou10, Lsou19, Lsou29 and Lsou34 [START_REF] Muenzel | Microsatellites from the vairone Leuciscus souffia (Pisces: Cyprinidae) and their application to closely related species[END_REF]); MFW1 [START_REF] Crooijmans | Microsatellite markers in common carp (Cyprinus carpio L.)[END_REF]; N7K4 [START_REF] Mesquita | Isolation and characterization of polymorphic microsatellite loci in the endangered Portuguese freshwater Squalius aradensis (Cyprinidae)[END_REF]; Ppro132 [START_REF] Bessert | Microsatellite loci for paternity analysis in the fathead minnow, Pimephales promelas (Teleostei: Cyprinidae)[END_REF]; Rhca20 [START_REF] Girard | Characterization of microsatellite loci in longnose dace (Rhinichthys cataractae) and interspecific amplification in five other Leuciscinae species[END_REF]; Rser10 [START_REF] Dawson | Isolation of microsatellite loci in the freshwater fish, the bitterling Rhodeus sericeus (Teleostei: Cyprinidae)[END_REF]; and Z21908 [START_REF] Shimoda | Zebrafish genetic map with 2000 microsatellite markers[END_REF]). These primer pairs were initially tested for amplification using four individuals, stemming from Garonne, Loire, Meuse and Rhone drainages (France). Thermocycling was performed using one single protocol: 95 °C for 15 min, followed by 30 cycles (94 °C for 30 s, 56 °C for 90 s, 72 °C for 60 s) and 60 °C for 45 min. PCR were conducted in a total volume of 10 μL containing ~20 ng of DNA and using the QIAGEN Multiplex PCR Kit (QIAGEN). Visualisation of the amplicons was performed by electrophoresis on 1.5 % agarose gels. We retained loci that amplified in all individuals and that gave clean PCR products, i.e. discrete single bands or at most two bands when a large size difference between alleles existed.

The selected loci were then amplified using forward primers labelled with fluorescent dye 6-FAM (Eurogentec), PET, NED or VIC (Applied Biosystems) with PCR conditions as described above and using eight individuals (two individuals from each of the Garonne, Loire, Meuse and Rhone River systems). Visualisation of the amplicons was conducted on an ABI 3130xl Genetic Analyzer (Applied Biosystems). Allele sizes were scored against an internal GeneScan-500 LIZ® Size Standard (Applied Biosystems) and genotypes were obtained using GeneMapper® 3.7 (Applied Biosystems). We retained loci that (1) amplified in all individuals, (2) had unambiguous genotype profiles and (3) were polymorphic.

At this stage, 22 loci were retained for P. phoxinus. However, only 10 loci were selected for G. gobio. Therefore, 12 additional primers pairs for loci previously detected as polymorphic for G. gobio [START_REF] Holmen | Crossspecies amplification of zebrafish and central stoneroller microsatellite loci in six other cyprinids[END_REF][START_REF] Vyskočilová | Isolation and characterization of microsatellites in Leuciscus cephalus (Cypriniformes, Cyprinidae) and cross-species amplification within the family Cyprinidae[END_REF][START_REF] Hamilton | Identification of microsatellite loci for parentage analysis in roach Rutilus rutilus and eight other cyprinid fish by cross-species amplification, and a novel test for detecting hybrids between roach and other cyprinids[END_REF] were tested: Ca13 and Ca14 [START_REF] Dimsoski | Microsatellite characterization in central stoneroller Campostoma anomalum (Pisces: Cyprinidae)[END_REF]; LC293 [START_REF] Vyskočilová | Isolation and characterization of microsatellites in Leuciscus cephalus (Cypriniformes, Cyprinidae) and cross-species amplification within the family Cyprinidae[END_REF]); Lco4 [START_REF] Turner | Variable microsatellite markers amplify across divergent lineages of cyprinid fishes (subfamily Leuciscinae)[END_REF]); Lid1 [START_REF] Barinova | Identification and characterization of microsatellite DNA markers developed in ide Leuciscus idus and Siberian roach Rutilus rutilus[END_REF]; and Z1206, Z6804, Z8249, Z10362, Z11841, Z14008 and Z15401 [START_REF] Shimoda | Zebrafish genetic map with 2000 microsatellite markers[END_REF]). These 12 markers were tested on G. gobio, permitting the selection of three additional loci using the protocols and criteria described above. Furthermore, five specific microsatellites (Gob12, Gob15, Gob16, Gob22, Gob28; [START_REF] Knapen | Isolation and characterization of polymorphic microsatellite loci in the gudgeon, Gobio gobio (Cyprinidae)[END_REF]) were included, resulting in a total of 18 polymorphic loci for G. gobio. Finally, the primer pairs selected for P. phoxinus and G. gobio were combined into three PCR multiplex kits for each species (Table 1).

The microsatellites were evaluated using three distinct populations of P. phoxinus and G. gobio (Table 1).

GENEPOP 4.0 [START_REF] Rousset | GENEPOP'007: a complete reimplementation of the GENEPOP software for Windows and Linux[END_REF]) was used to (1) test Hardy-Weinberg (HW) equilibrium, (2) estimate the observed (H o ) and expected (H e ) heterozygosities for all loci and populations and (3) test for linkage disequilibrium (LD) among loci within populations. MICRO-CHECKER 2.2.3 (van [START_REF] Van Oosterhout | MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data[END_REF]) was used to analyse the causes of departures from HW equilibrium. In order to graphically prospect the discrimination power of the two sets of markers at population scale, factorial correspondence analyses (FCA) were conducted on genotypes using GENETIX 4.05 [START_REF] Belkhir | GENETIX 4.05: logiciel sous Windows TM pour la génétique des populations, Laboratoire Génome Populations Interactions[END_REF]) (Fig. 1). For FCA, the locus Ca3 was discarded from the P. phoxinus dataset since null alleles were detected in all three populations (see below).

Results and discussion

Based on three population samples, the total number of alleles ranged from 3 to 42 for P. phoxinus (excluding locus Ca3) and from 2 to 33 for G. gobio, and the expected heterozygosity of polymorphic loci ranged from 0.04 to 0.95 for P. phoxinus and from 0.09 to 0.90 for G. gobio (Table 1). For G. gobio, LC293 was monomorphic in the three population samples, although polymorphic (including heterozygotes) in the eight individual samples used for the preliminary tests. After applying the false discovery rate (FDR) controlling procedure [START_REF] Benjamini | Controlling the false discovery rate: a practical and powerful approach to multiple testing[END_REF], no significant HW disequilibrium was detected for G. gobio. On the contrary, significant HW disequilibrium was detected for P. phoxinus in one out of three populations: loci BL1-44 (Meuse population), BL1-153 (Saone), CtoG-075 (Meuse), LleA-071 (Saone), Lsou19 (Isere) and Rru4 (Isere). Additionally, Ca3 exhibits HW disequilibrium in all three P. phoxinus populations (Table 1). For loci at HW disequilibrium, MICRO-CHECKER detected neither evidence for scoring error due to stuttering nor evidence for large allele dropout. However, null alleles may be present at all loci (which present heterozygotes deficiency), except BL1-153 and Rru4. After FDR correction, no LD was detected in P. phoxinus. In G. gobio, Lco4 and CypG24 showed significant LD in the Isere River population only, likely indicating a local demographic event or local selection. Furthermore, using 18 microsatellites and 21 microsatellites, FCA permitted the unambiguous discrimination of the populations of G. gobio and P. phoxinus, respectively. Although some population samples were collected from the same drainage, any overlap was observed between the distinct populations (Fig. 1).

Using cross-species amplification and five specific markers, we were able to develop 18 microsatellites for G. gobio and 21 microsatellites (excluding Ca3) for P. phoxinus. To catch the widest allelic range and to minimise the retention of loci with null alleles, individuals from four different hydrographical systems (Garonne, Loire, Rhone and Meuse Rivers) were included in the microsatellite development protocol (see [START_REF] Sinama | Challenges of microsatellite development in Lepidoptera: Euphydryas aurinia (Nymphalidae) as a case study[END_REF]. For P. phoxinus however, punctual occurrences of null alleles were detected at loci BL1-44, CtoG-075, LleA-071 and Lsou19. This suggests that these four loci must be used with caution and should be further evaluated depending on the geographic location of samples and the objectives of the study.

Finally, standardising the PCR protocol and multiplexing the microsatellite loci make their use more time-and costeffective and minimise errors during the compilation of genotypes (see [START_REF] Guichoux | Current trends in microsatellite genotyping[END_REF]. Both sets of loci also proved to correctly discriminate populations. These two sets of microsatellites therefore represent a useful tool for an array of ecological, evolutionary, conservation and management applications. For instance, these markers could be useful in developing molecular tools for relatedness and parentage analyses (as initiated by [START_REF] Bernhardt | Are shoals of minnow Phoxinus phoxinus formed by close kin?[END_REF]).

Fig. 1

 1 Fig. 1 Factorial correspondence analyses (FCA) of microsatellite genotypes of three populations of P. phoxinus (21 loci) and G. gobio (18 loci)

Table 1

 1 Microsatellites developed by cross-species amplification for P. phoxinus and G. gobio: characteristics and polymorphism

		Reference
			Meuse	River	(n=25)
			Saone	River	(n=27)
		H o /H e	Isere	River	(n=25)
		Multiplex PCR	Kit Primer	conc.	(nM)
		Allele size	range (N A )
		Core motif			(ATC)
	Phoxinus phoxinus	Locus Primers (5′-3′)	(GenBank ID)		CtoA-247 F-VIC: TGCAAACATATAAACTGAAACAAGG	(GU254031)

Table 1

 1 (continued)

			Vyskočilová et al. (2007)		Dubut et al. (2009b)		Muenzel et al. (2007)		Crooijmans et al. (1997);	Tong et al. (2005)		Reference
			0.32/0.33		0.88/0.95		0.88/0.85		0.28/0.29				Meuse	River	(n=33)
			0.04/0.04		0.48/0.89*		0.67/0.76		0.85/0.91				Saone	River	(n=32)
			0.00/0.00		0.88/0.86		0.80/0.78		0.76/0.89			H o /H e	Isere	River	(n=30)
			50		100		50		400			Multiplex PCR	Kit Primer	conc.	(nM)
			Pp3		Pp3		Pp3		Pp3
			141-153 (3)		340-432 (33)		178-202 (13)		164-215 (19)			Allele size	range (N A )
			(CT) 3 22 (CACT)		(CA) 6 T(AC) 10		(GT) 17		4 (GT) 14 N 3 (GA)			Core motif			(CA)
	R: GGACAGGTAAATGGATGAGGAGATA		F-6FAM: TCCAGTTCTTCCTTCCTAATT	R: GCGGAGGGAGAGTATGTCAA	F-PET: GTCTTAGATTGTGTAGCGGG	R: ACTTCAGTTACTAAGAGATTAGTGA	F-NED: GCGGTGAACAGGCTTAACTC	R: TAGGAACGAAGAGCCTGTGG	F-VIC: GTCCAGACTGTCATCAGGAG	R: GAGGTGTACACTGAGTCACGC		Primers (5′-3′)			F-NED: AAGACGATGCTGGATGTTTAC
	CypG30	(AY439148)	LC27	(EF362792)	LleA-071	(FJ601715)	Lsou8	(EF209003)	MFW1	(AY703054 )	Gobio gobio	Locus	(GenBank ID)		Ca1	(AF277573)

Table 1

 1 (continued)

			(AC)
	R: CAGTGAGGTATAAAGCAAGG		F-6FAM: GCACAGCTCTAATCGGTCACT
	Rhca20	(DQ106915)	BL1-153	(FJ468350)

Acknowledgments Data used in this work were partly produced through molecular genetic analysis technical facilities of the Plateforme Génomique Environnementale of LabEx CeMEB (Montpellier, France). We are very grateful to the technicians from the Office National de l'Eau et des Milieux Aquatiques (ONEMA, France) for their very valuable help with sample collection. This work was supported by the ONEMA (project NeoTaxon).