Matthieu Dreher

Marc Piuzzi

Ahmed Turki

Matthieu Chavent

Marc Baaden

Nicolas Férey

Sébastien Limet

Bruno Raffin

Sophie Robert

Interactive Molecular Dynamics: Scaling up to Large Systems

Keywords: Interactive Molecular Dynamics, Computational Steering with Haptic Arm, High Performance Interactive Computing

Combining molecular dynamics simulations with user interaction would have various applications in both education and research. By enabling interactivity the scientist will be able to visualize the experiment in real time and drive the simulation to a desired state more easily. However, interacting with systems of interesting size requires significant computing resources due to the complexity of the simulation. In this paper, we propose an approach to combine a classical parallel molecular dynamics simulator, Gromacs, to a 3D virtual reality environment allowing to steer the simulation through external user forces applied with an haptic device to a selection of atoms. We specifically focused on minimizing the intrusion in the simulator code, on efficient parallel data extraction and filtering to transfer only the necessary data to the visualization environment, and on a controlled asynchronism between various components to improve interactivity. We managed to steer molecular systems of 1.7M atoms at about 25 Hz using 384 CPU cores. This framework allowed us to study a concrete scientific problem by testing one hypothesis of the transport of an iron complex from the exterior of the bacteria to the periplasmic space through the FepA membrane protein.

Introduction

The study of molecular assemblies provides important insights into their biological function and potential underlying mechanisms. To date, there are no experimental techniques simultaneously providing both atomicscale spatial and pico-to microsecond temporal precision.

Molecular dynamics (MD) simulations are commonly used to study the behavior of molecular assemblies. Molecular assemblies span a broad range of sizes, typically from several hundred to hundreds of millions of atoms, depending on their complexity. MD simulation packages such as Gromacs [START_REF] Hess | GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation[END_REF] or NAMD [START_REF] Phillips | Scalable molecular dynamics with NAMD[END_REF] are complex codes, often parallelized to enable the simulation of large systems. Result analysis also requires dedicated graphical tools like VMD [START_REF] Humphrey | VMD -Visual Molecular Dynamics[END_REF]. Visualization and analysis of the simulation are mainly carried out off-line, once the simulation has ended. In case a problem occurs or the system does not reach the desired state, the simulation must be restarted with a new set of parameters and/or constraints. As these simulations may run for several days, weeks or even

Lipid bilayer Enterobactin Iron Complex

Fig. 1. Schematic representation of the FepA iron transporter protein (gray) along with the lipid bilayer (blue) and the iron-enterobactin complex (red). The complex is known to pass through the channel but a plug (green) blocks the way in this static form (left). Interaction with a small peptide using an haptic device, a space navigator and a stereoscopic viewer (center). Interactive simulation of a large membrane protein system composed of 1.7 million atoms (right). The solvent atoms are filtered out of the image before rendering.

months on hundreds of processors, an erroneous combination of parameters may lead to the waste of numerous compute cycles. Current simulation techniques may fail to capture some biological processes because of the large amount of time needed for important conformational changes to take place. A common approach to accelerate the simulation consists in imposing external constraints to bias the simulation towards phenomena of interest. But, as these constraints are defined at starting-time, one simulation is required per constraint test set, which can be very time-consuming, requiring many trial-and-error cycles.

An alternative approach to solve these issues consists in coupling the simulation code with a set of input devices and a 3D visualization engine to enable interactive analysis and steering of the simulation behavior [START_REF] Stone | A system for interactive molecular dynamics simulation[END_REF][START_REF] Stone | Immersive molecular visualization and interactive modeling with commodity hardware[END_REF]. These systems, called Interactive Molecular Dynamics (IMD), allow the user to interact directly with the simulation by driving the experiment to a particular structural or thermodynamical state, while validating the whole process by live analysis. This direct dynamics 3D visualization helps comprehending structural changes. However, IMD use remains typically limited to demonstration and educational purposes. One of the possible reasons is the difficulty to scale to molecular systems large enough to match current simulations running in batch mode, while maintaining performance enabling interactivity and spanning biologically relevant time-scales.

In this paper we introduce the Fvnano numerical workbench. Fvnano focuses on pushing the limits of IMD to steer large systems. Fvnano is a data-flow oriented framework to harness a parallel MD simulator and augment it with interaction capabilities. Here we use the Gromacs simulation code [START_REF] Hess | GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation[END_REF]. The system is designed to limit intrusion in the simulation while getting close enough to the source of data production to minimize the overhead when extracting relevant data. These data are then forwarded asynchronously and processed up to the 3D visualization environment. An haptic device enables the user to select and apply constraints to a group of atoms.

Experiments with various system sizes and compute nodes show an interactive performance can be achieved for systems up to 1.7 million atoms running on 384 CPU cores. We also used the framework to tackle a first realistic problem: testing alternative hypotheses on the transport of an iron-siderophore complex through the lipid bilayer of a bacterial cell.

After analysing related work (Section 2), we present the framework (Section 3) and some of its algorithmic and system details. Experiments for testing the system scalability (Section 4) and a real use case (Section 5) precede the conclusion.

Related Work

For a long time, structural biologists have been using MD-type simulations to study the dynamics of biological complexes. Through simulation libraries, they pushed the system sizes that can be tackled, improving parallelization strategies and scalability. NAMD [START_REF] Phillips | Scalable molecular dynamics with NAMD[END_REF] and Gromacs [START_REF] Hess | GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation[END_REF] are two very common packages. Today these packages are also evolving to take advantage of new architectures, such as multi-core CPUs and GPUs.

Steered molecular dynamics (SMD) is a direct precursor to the development of IMDs, derived from regular MDs by applying external forces to an object (atom, residue, protein, etc ...) to probe its mechanical function as well as to accelerate a process that may be too slow to model otherwise. Such a system was proposed by [START_REF] Izrailev | Steered Molecular Dynamics[END_REF]. External forces are defined a priori, and applied at run-time but without the possibility for the user to change them.

The introduction of the user in the analysis loop has been highlighted in [START_REF] Van Liere | Computational Steering[END_REF]. SCIRun [START_REF] Parker | The SCIRun Computational Steering Software system[END_REF] proposes a generic system to construct large applications that can be steerable. Relying on the dataflow paradigm, the programmer can link the simulation to various modules including visualization to obtain a continuous feedback on the simulation and change its parameters when necessary. However, the system requires a global space memory; therefore it can not be deployed on clusters. More recent work has been carried out to mitigate the intrusion in the simulation code and create clear separations between the simulation and the visualization. [START_REF] Insley | Runtime Visualization of the Human Arterial Tree[END_REF] proposes an online visualization for artery studies. Each process of the parallel simulation also runs a data manager in charge of writing result files. The visualization module requests data from listeners that read back these files and pre-process the data. This approach limits the impact on the simulation but in turn the interactivity is limited due to the high latency introduced by accessing the intermediate files. [START_REF] Mazzeo | In situ ray tracing and computational steering for interactive blood flow simulation[END_REF] proposes a tighter coupling. The data are gathered on a master node by the simulation and subsequently forwarded to the visualization node. The system achieves high frequencies and a good interactivity, but incurs a significant overhead compared to the original simulation (50% performance drop).

Visualization of molecular assemblies classically relies on a variety of 3D representations, many of them available in common visualization tools like VMD [START_REF] Humphrey | VMD -Visual Molecular Dynamics[END_REF] or Pymol [START_REF] Schrödinger | The PyMOL Molecular Graphics System[END_REF]. Rendering large 3D models at interactive frame rates is still challenging though. Issues include performance as well as representations. Grottel et at. [START_REF] Grottel | Coherent Culling and Shading for Large Molecular Dynamics Visualization[END_REF] propose various levels of optimizations (culling, caching, ...) to interactively visualize time varying molecular datasets containing tens of millions of atoms. The quicksurf algorithm sinks the molecular system in a regular 3D grid, enabling to extract an iso-surface, an approach that is well suited to large assemblies [START_REF] Krone | Fast Visualization of Gaussian Density Surfaces for Molecular Dynamics and Particle System Trajectories[END_REF].

The interest of haptic rendering to study molecular interactions has already been highlighted during the Grope project [START_REF] Brooks | Project GROPEHaptic displays for scientific visualization[END_REF], many years ago. In particular, the haptic feedback eases molecule manipulation and perception [START_REF] Bivall Persson | Evaluating the Effectiveness of Haptic Visualization in Biomolecular Education -Feeling Molecular Specificity in a Docking Task[END_REF]. IMD with haptics has been further developed in [START_REF] Stone | A system for interactive molecular dynamics simulation[END_REF][START_REF] Stone | Immersive molecular visualization and interactive modeling with commodity hardware[END_REF]. The framework is based on VMD for the visualization and NAMD for the molecular dynamics engine. Interaction is handled through a VRPN server, supporting 6 degrees of freedom input devices and haptic feedback. Initially focused on steering a simulation running on a parallel machine, the authors also recently experimented steering an MD simulated on a GPU. The advantage is to be able to simulate a reasonably complex system on a single GPU.

To further improve the user experience, visualization and steering of a scientific application can take place in an immersive environment. Renambot et al. [START_REF] Renambot | CAVEStudy: An Infrastructure for Computational Steering and Measuring in Virtual Reality Environments[END_REF] steered from a CAVE a molecular dynamics application running on the DAS parallel computer. Koutek et al. proposed a system to steer particles using a virtual workbench called MolDRIVE [START_REF] Koutek | Virtual spring manipulators for particle steering in molecular dynamics on the responsive workbench[END_REF]. MD simulations were launched on SGI or Cray supercomputers but, because of communication issues, the best results were achieved using only 8 nodes thus limiting the size of the studied system. Using VRDD [START_REF] Anderson | VRDD: applying virtual reality visualization to protein docking and design[END_REF] in a CAVE, users could experiment protein docking in an immersive way using both visual and auditory feedback.

To our knowledge, the largest published molecular systems simulated interactively reach a few tens of thousands of atoms so far. Our goal is here to significantly push this limit to a few million atoms.

The Fvnano Workbench

Architecture Overview

The FvNano application is composed of four main components: visualization, graphical user interface, device manager and simulation (Fig. 2(a)). The main pipeline steps are:

• The simulation outputs its current state toward the visualization.

• The visualization produces a graphical representation of the on-going simulation, the user being able to control asynchronously his viewpoint. • The user can select and apply forces to a set of atoms, using a combination of devices.

• The simulation updates its state taking into account user controlled parameters, such as the external forces to be applied on a selection of atoms. Secondary components include a selection module to select a subset of atoms from the visualization by clicking on atoms or through the user interface. This selection is mainly used to apply forces on the selected atoms. The forcegenerator defines the forces to be applied to the selected atoms, these forces being subsequently forwarded to the simulation, the visualization and the force feedback device. This framework targets clusters of hundreds of cores providing the necessary computing resources to simulate large systems interactively.

Performance is a key issue. Considering that the parallel simulation code is already highly optimized, our concern is to efficiently extract the data produced by the simulation to filter and forward them to the visualization. The simulation engine usually supports some mechanisms to save intermediate results in files. One approach consists in reading these files or grabbing the data on the master node that usually centralizes the data for building the file. We experimented these schemes with Gromacs, but the performance was low. The main reason is that while all the data are centralized on the master node, the simulation is suspended. We obtained the best performance relying on a different approach. The data are extracted directly from each Gromacs process. Their aggregation and filtering is performed in parallel and asynchronously, while the simulation runs.

Modularity was another concern. By relying on a data-flow and component oriented approach, we enforce the ability to change components with a limited impact on the remaining elements of the application. In this paper we rely on the Gromacs [START_REF] Hess | GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation[END_REF] MD simulation engine, but following a similar approach we could develop a component for other tools like NAMD [START_REF] Phillips | Scalable molecular dynamics with NAMD[END_REF] for instance.

Programming Environnement and Run-time System

Our implementation relies on the FlowVR framework [START_REF] Allard | Modularity for Large Virtual Reality Applications[END_REF]. FlowVR allows to describe an application as a dataflow graph where nodes (components) are operations on data and edges are communication channels. Here, we describe the main features relevant for our work. Refer to [START_REF] Allard | Modularity for Large Virtual Reality Applications[END_REF][START_REF] Lesage | A Hierarchical Component Model for Large Parallel Interactive Applications[END_REF] for more details.

A component (also called module) encapsulates an iterative code that can communicate with other components using input and output ports. When each input port has received a message, the module starts a new iteration. The data are processed and possibly new data are sent through the output ports. The strong point of this system is that the developer of a module does not have to know where the data comes from and where he has to send the produced data.

A communication channel is a link from one module output port to another module input port. By default, this channel is FIFO but special components can be installed along a data path to elaborate more complex communication policies (broadcast, gather, scatter, resampling, etc.). Such components will be described later.

This architecture allows a high level of modularity. Each component can be replaced independently without having to change anything in the code of other modules or in the overall application network.

The API to program a module is simple to limit the code refactoring necessary to turn an existing code into a module. Basically, a module is based on three main operations:

• wait(): block the execution of a module until each input port is filled with a message.

• get(): grab a message from an input port.

• put(): put a message on an output port (the actual data transmission is asynchronous) A FlowVR application can be executed on a cluster by distributing the different modules on different nodes. At runtime, one daemon runs on each machine node involved in the application. Each daemon is responsible for the modules hosted on its machine and for the associated communication channels. Modules never communicate with each other directly. To create and send a message, a module allocates memory into a shared memory space provided by the daemon and writes directly into that space. Through the put() instruction, a module posts a message to its daemon. If the destination module is located on the same node, the daemon simply passes a pointer on the message to the destination module, avoiding unnecessary copies. Otherwise, the daemon sends the message through the network to the daemon hosting the target module. The daemon stores the message in its local shared memory segment and forwards a pointer to the destination module.

To implement communication patterns between modules, we use specific FlowVR components called filters. These components are hosted directly by the daemons and are dedicated to light operations on messages. FlowVR comes with several filters, but the user can also develop custom filters. MergeFilter is an example of such a filter. This filter takes several input messages and produces one output message by concatenation of the input messages. We also used filters to bound the frequency of some modules or to extract the newest message waiting in a FIFO communication channel (resampling pattern), the older messages being discarded, for an improved latency.

Embedding a MPI parallel code into a FlowVR network is straight-forward. For each MPI process, a module is created encapsulating this process (done at the initialization). Then, each individual process can communicate using MPI and through its FlowVR ports.

Gromacs Integration

For the simulation module, we rely on Gromacs [START_REF] Hess | GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation[END_REF] to provide a molecular dynamics engine. Gromacs runs on desktop machines using a thread based parallelism or in a distributed context using MPI. A heterogeneous method using both MPI and OpenMP should be available in a future version of Gromacs but it is not yet released. In the remainder of this article, we assume that Gromacs only uses MPI.

Gromacs Internal Organization

Gromacs follows a master/slave organization. The first MPI process (the master) loads the simulation, configures it and propagates all necessary information to the slaves. During the simulation, only the master can access the global state of the simulation. The Algorithm-1 pseudo-code describes a usual Gromacs iteration. Each process is responsible only for the atoms contained inside its domain (referred to as the home atoms). Several communication steps take place between processes, to dynamically rebalance the work load as the atoms move in the simulation domain (dd partition system()), or to compute the forces that apply to each atom (do f orce()).

To write the trajectory and other backup files (write tra j()), a global state of the molecular system is gathered on the master node. This operation involves costly communications. To mitigate this cost, a common tradeoff is to write the state of the simulation only every x steps where x is supposed to be small enough to guarantee that the scientist will not miss anything important. Finally, the molecular system is updated and the atom states (positions, energies,etc) are modified (update()). Refer to [START_REF] Hess | GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation[END_REF] for details.

External Force Integration

The forces are produced by the forcegenerator module. A force is represented as a vector describing the intensity and direction of the force and an id indicating the atom the force applies to.

To add external forces to the simulation, we broadcast the array of forces to all the MPI processes through a FlowVR broadcast communication. The memory footprint is H times the size of the forces with H the number of different nodes (often smaller than the number of processes as several processes run per node). No copies are required inside the simulation because Gromacs gets a pointer to the message in shared memory. Then each process checks for each force whether it applies to a home atom. In this case the process adds the forces to the Gromacs force array.

We expect the user to apply forces on a few atoms and not on the full molecule. The size of the force message is thus small, making a full broadcast the best method. We could use a table linking the atom IDs and the hosts to route the atoms directly to the correct host. However, the cost of maintaining such a table does not compensate for the bandwidth gain if the force messages are too small.

Extracting Atom Positions from Gromacs

Each Gromacs process handles a set of home atoms. To extract these positions, Gromacs code was slightly modified to copy these atom positions into a FlowVR message. Gromacs proceeds on the current iteration as soon as the related put() call returns (Alg. 2). Then, the local FlowVR daemon asynchronously takes cares of the built message as detailled in the next section. In total, about 50 code lines have been added to Gromacs, mainly to initialize the FlowVR context and process messages. The only Gromacs function that has been modified is the saving of trajectories to files. We preserved the possibility to backup trajectories. However, to achieve the maximum performance, it is preferable to diminish or even disable the related communications and file writes, which can create some lags at run time and degrade the interactivity quality.

Processing Messages of Atom Positions

On each node, the local FlowVR daemon gets one handle per position message provided by each Gromacs process running on that node. These messages need to be gathered and forwarded to the visualization module. We adopt a three levels merge pattern that makes it possible to parallelize the merging, limit the amount of message copies and the number of messages transiting on the network. On each node the messages are gathered, sent to other nodes which don't host the simulation by bunch of ten, gathered and finally sent to the node running the visualization (See Fig. 2(b)). Received messages are merged and transfered to the visualization. This pattern is defined through a tree pattern where nodes are Merge filters and edges are communication channels for position messages. This pattern can flexibly be tuned to adapt to the target architecture (tree arity for instance).

The user may only be interested by some specific atoms. For that purpose we insert selection filters after the first merge operation. These filters can discard atoms based on their atom type or their residue. For large models, a common request from users is to filter the solvent, which represents a large number of atoms and may not be necessary to visualize. By removing these atoms -which commonly represent up to 80 percent of the total number of atoms -early in the process, we can save a lot of resources. By filtering out unused data as soon as possible, we reduce the amount of data transiting on the network and lighten the next operations along the pipeline (data conversion and visualization).

Rendering

Rendering performance is critical as we target an interactive rendering frame rate of large molecular systems. We rely on the Hyperball algorithm [START_REF] Chavent | GPU-accelerated atom and dynamic bond visualization using hyperballs: A unified algorithm for balls, sticks, and hyperboloids[END_REF], an improved ball-and-stick representation replacing cylinders linking the atom spheres by hyperboloids. Changing the hyperboloid parameters enables to sweep through different representations and in particular the classical ball-and-stick, licorice and space-filling van der Waals spheres. The implementation is optimized to take advantage of GPU capabilities and support large molecular systems. Refer to [START_REF] Chavent | GPU-accelerated atom and dynamic bond visualization using hyperballs: A unified algorithm for balls, sticks, and hyperboloids[END_REF] for details. We also support VMD [START_REF] Humphrey | VMD -Visual Molecular Dynamics[END_REF] for visualization. All interactive experiments presented here rely on the Hyperball algorithm.

Interaction

Our framework supports several commodity devices for interactions. The devicemanager module gets device events through VRPN [START_REF] Taylor | VRPN: A Device-Independent, Network-Transparent VR Peripheral System[END_REF], converts them to a standard representation and forwards them through FlowVR channels.

Our final implementation uses two devices : a SpaceNavigator 3D mouse to control the camera of the visualization and a Phantom haptic arm to select atoms and apply forces. The SpaceNavigator allows to translate and rotate the model. The Phantom allows to select atoms, apply forces and feel a force feedback. To help the user in this task, the visualization provides highlights of the atom that is selectable by the Phantom and a simple representation of the forces currently applied.

To help the user make the intended atom selection, we add a specific filter to control the speed of the simulation component. At more than 80 iterations per second, atoms are moving and vibrating quickly, making atom selection difficult without this filter. By changing the frequency parameter of this filter online through a GUI, the user can slow down the simulation for a comfortable atom selection (See the attached video 1 .) This filter broadcasts a short message to each Gromacs process at a given rate. The wait() call introduced in each process locks it as long as the signal is not received, constraining Gromacs to the maximum target frequency.

When the selection mode is activated, the selection module identifies the closest atoms to the Phantom and forwards this list to the visualization and ForceGenerator modules. Because the user selects atoms based on visual clues, the atoms the visualization highlights as selectable need to match the one the selection module computed. Otherwise, the atoms visually selected may be different from the actual selection. To avoid this pitfall, the visualization and selection modules are tightly coupled to run at the same speed. The visualization can not render the next frame unless the selection sends the selectable atom for the current position of the phantom.

Experimental Results

The hardware used for the visualization is a Xeon E5530 quad core CPU at 2.40GHz, a GeForce 680 GTX graphics card. The computational cluster used is composed of 72 nodes, each node running 2 Xeon E5520 quad core CPU at 2.27GHz, 24GB RAM. Communications between the computational cluster nodes go through a QDR Infiniband fabric (40Gbits/s) while we only have a DDR (20 Gbits/s) Infiniband interconnection between the computational cluster and the visualization node.

We launch 6 Gromacs processes per compute node (8 cores available). On each of these nodes runs one merge filter to gather the atom positions from the local Gromacs processes and one filter module (see Figure -2(b)). Every 10 nodes running Gromacs processes, an extra node is added to run one merge filter gathering the atoms positions from these 10 nodes. The remaining modules and filters, as well as the interaction devices are all executed on the visualization node.

Interactivity Evaluation on Large Molecular Models

We performed performance tests with models ranging from 500K atoms to 1.7M atoms (Fig. 3(c)). These models were built by replicating a base assembly composed of a Glic membrane protein along with a lipid bilayer and solvent. The 1.7M atoms system is composed of 12 juxtaposed Glic systems. The systems have been set up to use virtual sites and the following simulation parameters : AmberGS forcefield, 5 fs steps, Fast smooth Particle-Mesh Ewald (SPME) electrostatics and 1 nm cut-off. The performance is measured through 3 different metrics: simulation and visualization iterations per second, and the latency time it takes from the emission of forces by the forcegenerator module up to the receipt by the visualization module of the positions influenced by the emitted forces (Fig. 2(b)). As both modules run on the same machine, the measured latency is not affected by clock synchronization issues. The viewpoint is set to fit the whole molecule within the window (resolution 800x600). The visualization frequency can vary significantly depending on atom visibility, but even for the largest assembly the user can smoothly navigate through the molecular system.

Increasing the number of cores enables to improve performance significantly for all model sizes (not always linearly though). As detailled below this is consistent with the performance obtained when Gromacs runs standalone. Latency benefits not quite as much from additional cores. For small models (< 100K atoms), the latency is mainly dominated by the simulation time. For a system of 70K atoms running at 60 its on 64 cores, 17ms of the 24ms measured latency are due to the simulation time. For large models, even if the simulation times decrease significantly when involving more processors, the volume of data transmitted to the visualization is important and stays unchanged, dominating the latency time. For instance the 1.7M atoms assembly runs on 192 cores at 16 its, where 61ms out of 185ms measured latency are due to the simulation time.

The user experience is generally satisfactory from a latency and frequency point of view for mid size assemblies. Above 1M atoms, some spikes might occur during the user experiments causing discomfort for the user. Another limitation is related to the visual representation. Every individual atom of the scene is represented, cluttering the screen and making the selection of specific atoms difficult. We will experiment with alternative representations like surface-based ones to evaluate if they enable the user to better navigate through large molecular assemblies.

Cost of Interactivity

To evaluate the cost of interactivity, we compared the performance of running Gromacs standalone or in the Fvnano framework (Table - 1. Gromacs performance (iterations per second) when running standalone (first row), when coupled with FlowVR extracting atom positions, performing a per node merging but without sending out built messages (second row), when coupled with FlowVR and all Fvnano features enabled but (third row). Notice that for this last case all atom positions are sent on the network (no atom is filtered out). Molecular assembly of 1.1 million atoms simulated on various number of cores, using 6 cores per node for Gromacs processes.

According to the second row, the local treatment of the atom positions cause little arm to the simulation performance (5% in worst case). Compare to Gromacs standalone, 3 extra processes run on each simulation node : the flowvr daemon, an atom filter and a merge filter. Even if we expect these processes to run on separate cores than Gromacs ones, they can create contention during system calls or memory transfers. Moreover, each Gromacs process needs to handshake with the local daemon during the wait() operation, causing some delays on Gromacs execution. The resulting overhead is however very limited. Actually sending the messages on the network has a more import impact on Gromacs (row 3). These communications comes in addition to native Gromacs message exchanges and increase the load on the network cards and switches, causing most likely this performance drop. But a 17% performance drop stays a reasonable price to pay for interactivity.

IMD Real Use case

Membrane proteins are essential to the transport of molecules through the lipid bilayer of the cell. Transporters may work in very different ways and, in some cases, the transport mechanism is yet to be discovered. This is the case with the FepA protein, which uptakes an iron atom complexed with a siderophore, a bacterial molecule called enterobactin, and transports it through the lipid bilayer. The structure of the FepA protein has been solved by X-Ray crystallography and shows a barrel shape formed by β strands with a globule folding inside it (Figure-1(a)). This particular folded globule forms a plug which is known to block the passage of the iron complex in this static form. To date, two hypotheses exist for the transport mechanism : the plug (i) undergoes structural rearrangement to open a passage, (ii) is dislodged towards the periplasm (outside the channel). In a recent work [START_REF] Esque | A case study of protein topology: how enterobactin modifies the water network through fepA[END_REF], two water channels inside the protein have been identified. They may be precursors for transport, which would tend to confirm the first hypothesis. It was observed that these channels are wide enough to let water molecules pass through the protein, thanks to a small structural rearrangement of the plug. However, the iron-enterobactin complex is bigger than those channels and we wanted to determine if the structural rearrangement may be large enough for the entire complex to pass through. As SMDs would be difficult to use in this particular case (the supposed trajectories do not follow a straight line) we decided to use the FvNano IMD framework. The goal was to lead the iron-enterobactin complex through the channel, based on the hypotheses formulated in the earlier study, using the haptic device and the visual feedback.

The simulation parameters are strictly identical to those used for regular simulations. The total system size is about 72K atoms and contains the FepA protein, a small portion of a lipid bilayer, the iron-enterobactin complex and the solvent. This simulation was executed on a computational cluster using 128 cores and a separate node for visualization and interaction. The IMD allowed us to confirm two distinct paths along which the plug only undergoes light structural changes (mainly on flexible loops) when pulling the complex through. To ensure the reproducibility of the results, the simulation was run several times and each time equivalent trajectories were found. These trajectories are compatible with the two water channels that have been previously identified. In this experiment, it is important to track the amount of force applied to the system, as a high level of force would limit the biological meaning. In this case, the mean value of the forces applied to each atom of the iron-enterobactin complex was about 3735 pN per step (2 fs) during the simulations. This value is quite high in regards to SMD simulations. However, this is the first step of the search process as these simulations allowed us to identify potential trajectories for the complex which will be further studied. Decreasing the forces applied per step requires more iterations, i.e. more time, to follow a similar trajectory, but increasing the overall simulation time impairs interactivity.

This experiment showed us that it is possible to drive large simulations in an interactive session. However, for a 15 min interactive session, only nano second reactions are observable interactivly. This is a major obstacle as a large number of chemical reactions take place in micro seconds or more. In the case of the FepA, we chose to use important forces allowing us to speed up the reaction process at the price of realism. Using more CPU should help us to run longer simulation but the micro second simulation seems unreachable in interactive time. We are currently looking at intermediate solutions between IMD and SMD. The goal is to provide the user a way to describe a trajectory for selected atoms, run the simulation for several days and, from time to time, connect the interactive tools to the simulation to check the state of the simulation and adjust the trajectory if necessary.

Conclusion

We presented an effort to enable interactive, haptic based, steering of large molecular dynamics simulations. Our main contribution is the efficient integration of a parallel Gromacs simulation within a virtual reality frame-work associating haptics and high performance 3D rendering. This framework enables us to simulate molecular systems of 1.7M atoms at 25 iterations per second relying on 384 computing cores. This framework was used to explore an open biology problem, enabling us to find two plausible paths for the transport of an iron-siderophore complex through a FepA channel. This is one of the few examples of a virtual reality application that directly contributes to solving an open problem in structural biology. However, the FepA experiment remains a mid-size molecular system compared to the capabilities of our framework. Future work will explore its effectiveness for tackling molecular systems at an even larger scale.

Fig. 2 .

 2 Fig. 2. The FvNano network is composed of four main components (blue boxes) with a few secondary modules (white boxes). The red arrows represent the main bandwidth consuming data-flows issued from the simulation. The green arrows represent light communications between modules.

Fig. 3 .

 3 Fig. 3. Simulation (iterations per second), visualization (frames per second) frequencies and latency measures for various molecular assemblies, Gromacs running on an increasing number of cores (from 48 up to 384).

  [START_REF] Allard | Modularity for Large Virtual Reality Applications[END_REF]. Gromacs performance is affected by the extra computations performed on each node at FlowVR level as well as the extra communications the network needs to perform. The overhead is about 17.74% of Gromacs standalone performance when running on 384 cores.

	number of cores	96	192	288	384
	Gromacs standalone	13.67	25.02	35.71	42.90
	Gromacs & Fvnano w/o com. (% overhead) 13.29 (2.77%) 24.67 (1.32%) 33.80 (5.35%)	41.05 (4.30%)
	Gromacs & Fvnano (% overhead)	12.76 (6.62%) 23.00 (8.07%) 30.16 (15.55%) 35.29 (17.74%)

Table

http://moais.imag.fr/membres/matthieu.dreher/FvNanoICCS1080p.mp4

Acknowledgments

This work has been partly funded by the ANR, project FVNANO ANR-07-CIS7-003 and EXAVIS ANR-11-MONU-003. Experiments were performed on the Grid'5000 experimental testbed (https://www.grid5000.fr).


Boyang Ji

Gregory Gimenez

Valérie Barbe

Benoit Vacherie

Zoé Rouy

Amira Amrani

Marie-Laure Fardeau

Philippe Bertin

Didier Alazard

Sabine Leroy

T Boyang

Benoît Vacherie

Emmanuel Talla

Bernard Ollivier

Alain Dolla

Nathalie Pradel
email: nathalie.pradel@ird.fr.

Complete Genome Sequence of the Piezophilic, Mesophilic, Sulfate-Reducing Bacterium Desulfovibrio hydrothermalis AM13

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

D eep-sea sulfate-reducing microorganisms must be consid- ered as key actors in Earth's sulfur cycles. Metagenomic studies have revealed that sulfate-reducing bacteria (SRB) are largely represented among the microorganisms that inhabit deep-sea environments [START_REF] Hu | Construction and preliminary analysis of a deep-sea sediment metagenomic fosmid library from Qiongdongnan basin, south China sea[END_REF][START_REF] Martín-Cuadrado | Metagenomics of the deep Mediterranean, a warm bathypelagic habitat[END_REF][START_REF] Stokke | Integrated metagenomic and metaproteomic analyses of an ANME-1dominated community in marine cold seep sediments[END_REF]. However, only a few SRB have been isolated from deep-sea environments (4-9). Among them, the Desulfovibrio genus is well-represented, with Desulfovibrio profundus, Desulfovibrio piezophilus, and Desulfovibrio hydrothermalis having been isolated previously [START_REF] Alazard | Desulfovibrio hydrothermalis sp. nov., a novel sulfate-reducing bacterium isolated from hydrothermal vents[END_REF][START_REF] Bale | Desulfovibrio profundus sp. nov., a novel barophilic sulphate-reducing bacterium from deep sediment layers in the Japan Sea[END_REF][START_REF] Khelaifia | Desulfovibrio piezophilus, sp. nov., a novel piezophilic sulfate-reducing bacterium isolated from wood falls in Mediterranean Sea[END_REF]. Here, we report the genome sequence of D. hydrothermalis, a piezo-mesophilic, hydrogenotrophic SRB isolated from a deep-sea hydrothermal chimney sample collected at a depth of 2,600 m (13°N on the East Pacific Rise) [START_REF] Alazard | Desulfovibrio hydrothermalis sp. nov., a novel sulfate-reducing bacterium isolated from hydrothermal vents[END_REF]. It represents, with D. piezophilus [START_REF] Pradel | The first genomic and proteomic characterization of a deepsea sulfate reducer: insights into the piezophilic lifestyle of Desulfovibrio piezophilus[END_REF], the second known genome of piezophilic bacteria in the genus Desulfovibrio, and thus gives the opportunity to specify the systems that are involved in the piezophilic lifestyles of Desulfovibrio species.

Genome sequencing was performed by the Genoscope (CEA, Évry, France), using 454-sequencing method (Roche) and Solexa technology. A mate-paired genomic library with 8-kb insert size was prepared according to the manufacturer's protocols (454 Life Sciences Corporation, Branford, CT). Approximately 19-fold coverage of GS FLX Titanium (http://www.roche.com/) reads was assembled using Newbler. Gaps between contigs were closed with 1,038 PCR amplifications and the Consed editing software (http: //www.phrap.org/). For the quality assessment, approximately 1,000-fold coverage of paired-end Illumina reads (36 bp) was mapped onto the whole-genome sequence.

The genome consists of two contigs of 284,269 bp and 3,418,665 bp, for a total of 3,702,934 bp, and of a circular plasmid of 5,328 bp. The average GϩC contents for the chromosome and the plasmid DNA are 45.1% and 51.1%, respectively. A total of 3,543 coding DNA sequences (CDSs) were predicted, as well as 7 pseudogenes, 6 miscellaneous RNAs (misc-RNA), 5 disrupted rRNA operons, and 77 tRNA genes.

Phylogenetic analysis based on 16S rRNA shows that the clos-est sequenced genomes to D. hydrothermalis are those of Desulfovibrio salexigens and Desulfovibrio aespoensis. Reciprocal best BLAST analysis indicated that D. hydrothermalis shares 2,824 and 2,069 orthologous proteins with D. salexigens and D. aespoensis, respectively, and 1,974 proteins are common to the three species. Like other Desulfovibrio species, D. hydrothermalis possesses all the genes necessary for the sulfate-reduction pathway. Fatty acid membrane composition has been shown to play an important role in hydrostatic pressure adaptation [START_REF] Bartlett | Pressure effects on in vivo microbial processes[END_REF]. In. D. hydrothermalis, all fatty acid biosynthesis pathways are present. Interestingly, three copies of the fabF gene (DESAMv2_20244, DESAMv2_21138, and DESAMv2_21153), encoding a ␤-ketoacyl-acyl carrier protein (ACP) synthase II involved in monounsaturated fatty acid production and suggested to be involved in hydrostatic pressure adaptation [START_REF] Bartlett | Pressure effects on in vivo microbial processes[END_REF], are found in the genome. Thus, the three copies of this gene might play a pivotal role in the adaptation strategy of D. hydrothermalis to hydrostatic pressure.

Comparative genomics, including the use of other Desulfovibrio species, will give insights into the adaptation of Desulfovibrio species to the piezophilic lifestyle that they encounter in the deepsea biosphere.

Nucleotide sequence accession numbers. The final annotated genome of D. hydrothermalis is available in EMBL under the accession no. FO203522 (for the chromosome) and FO203523 (for the plasmid).

Genome Announcements January/February 2013 Volume 1 Issue 1 e00226-12 genomea.asm.org 1

Genome Announcements 2 genomea.asm.org January/February 2013 Volume 1 Issue 1 e00226-12

ACKNOWLEDGMENTS

This work was supported by CNRS and IRD grants. B.J. is a PhD student with a China Scholarship Council fellowship.


H Louche
email: herve.louche@univ-montp2.fr

V Delobelle
email: vincent.delobelle@hmg.inpg.fr

D Favier
email: denis.favier@hmg.inpg.fr

EXPERIMENTAL VALIDATION OF HEAT SOURCE FIELD ESTIMATIONS

INTRODUCTION

Thermal field measurements are interesting tools to study thermomechanical couplings often associated with deformation of materials. Intensity and spatio-temporal distribution of the temperature variations are depending on the material behaviour (elasticity, plasticity, viscosity, damage, phase transformation,…) but also on the different characteristics of the thermal problem (geometry, thermal properties, boundary conditions, …). Previous work [START_REF] Chrysochoos | An infrared image processing to analyse the calorific effects accompanying strain localization[END_REF] has shown the interest to study the local heat sources, causes of the temperature variations. Different methods have been proposed to get such heat source fields [START_REF] Andre | A two-step regularized inverse solution for 2-D heat source reconstruction[END_REF]. The method used in this work to estimate heat sources is based on approximations of derivative operators involved in the heat diffusion equation [START_REF] Chrysochoos | An infrared image processing to analyse the calorific effects accompanying strain localization[END_REF]. It was applied to study Lüders bands and necking in steels, PLC bands in AlMg alloys, plasticity in Al olygocrystals and stress induced phase transformation in Nickel-Titanium (NiTi) shape memory alloys [START_REF] Schlosser | Image processing to estimate the heat sources related to phase transformations during tensile tests of NiTi tubes[END_REF]. Few works have been proposed to validate heat source field estimations. A first approach, qualified as "theoretical", uses analytical or numerical examples of temperature and heat source distributions [START_REF] Chrysochoos | An infrared image processing to analyse the calorific effects accompanying strain localization[END_REF][START_REF] Schlosser | Image processing to estimate the heat sources related to phase transformations during tensile tests of NiTi tubes[END_REF]. The objective of our presentation is to show a second approach, qualified as "experimental" and based on thermal induced phase transformation observed in a NiTi shape memory alloy. After a description of the experimental benchmark, typical experimental results will be presented to discuss the validity of heat source estimations in homogeneous and one-dimensional cases.

METHODOLOGY

Fig. 1 shows the different steps of the method. An infrared camera observes the natural temperature decrease (or increase) at the surface of a NiTi sample. Depending on the boundary conditions the temperature distribution is homogeneous or axially heterogeneous (Fig. 2). Temperature variation fields are then processed to estimate the heat source fields qualified as local differential scanning calorimetry and noted DSCTFM (TFM: Temperature Field Measurement). These DSCTFM fields are then compared to a reference DSC obtained by standard DSC (noted DSCEXP) in order to evaluate the performance of the heat source estimations. These kinds of results will be analysed and discussed in regard to the validation of the method of heat source estimation. 4.

Figure 1 :Figure 2 :

 12 Figure 1: scheme showing the different steps of the experimental validation of heat source estimations

Figure 3 :

 3 Figure 3: b) Spatio-temporal representation of the temperature variations along an axial profile plotted in Fig. a. c) Same representation for the specific heat sources (W/g). d) Local DSCTFM, in each point (pixel) of the profile, compared with the reference (DSCEXP). e) Close-up around the second peak.


Catherine Deneux-Tharaux
email: catherine.deneux-tharaux@inserm.fr

Loic Sentilhes

Françoise Maillard

Emmanuel Closset

Delphine Vardon

Jacques Lepercq

François Goffinet

Should routine controlled cord traction be part of the active management of third stage of labour? The Tracor multicenter randomized controlled trial

Objectives Active management of the third stage of labour is recommended for preventing postpartum haemorrhage (PPH). However, the specific effects of each of its components have not been adequately evaluated. The TRACOR Study aimed to assess the impact of controlled cord traction on the incidence of PPH and other characteristics of the third stage of labour, in a high-resource setting.

Design Randomized controlled trial conducted between January 1, 2010, and January 31, 2011.

Setting Five French university hospital maternity units

Participants Women aged ≥18, with a planned vaginal delivery, at a gestational age ≥ 35 weeks, with a singleton fetus.

Interventions Women were randomly assigned to have third stage of labor managed either by controlled cord traction (CCT), or by standard placenta expulsion (SPE) i.e. awaiting the spontaneous placental separation before facilitating its expulsion. Prophylactic oxytocin just after birth of the baby was administered in the 2 arms.

Main outcome measures The primary outcome was the incidence of PPH ≥ 500 mL as measured in a collector bag.

Results

The incidence of PPH was not different in the CCT group (9. 8% (196/2005) and in the SPE group (10.3% (206/2008), RR 0.95, 95% CI (0.79 to 1.15). The need for manual removal of placenta was significantly less frequent in the CCT than in the SPE group (4.2%(85/2033) and 6.1%(123/2024), RR 0.69, 95%CI (0.53 to 0.90)); as was third stage > 15 minutes (4.5% (91/2030) and 14.3% (289/2020), RR 0.31, 95%CI 0.25 to 0.39)). Women in the CCT group reported a significantly lower intensity of pain and discomfort during the third stage than those in the SPE group. No uterine inversion occurred in either arm.

Conclusions

In a high-resource setting, the use of CCT for the management of placenta expulsion had no significant effect on the incidence of PPH and other markers of postpartum blood loss. Therefore, there is no evidence to recommend routine CCT for the management of placenta expulsion in order to prevent PPH.

Trial registration ClinicalTrials.gov NCT01044082.

"What this paper adds " box

What is already known on this subject  Active management of the third stage of labour includes the administration of an uterotonic drug immediately after child birth and controlled cord traction (CCT), and is recommended for the prevention of PPH.

 The management of third stage of labour without CCT does not increase the risk of severe PPH in low and middle income countries, and therefore could be omitted in non-hospital settings.

 However, the impact of CCT on PPH incidence and other characteristics of third stage in the context of high-resource settings is unknown.

What this study adds

 In a high-resource setting, the use of CCT for the management of placenta expulsion has no significant effect on the incidence of PPH and other markers of postpartum blood loss.

Therefore, there is no evidence to recommend routine CCT for the management of placenta expulsion in order to prevent PPH.  However, CCT is safe, reduces the length of third stage, the need for manual removal of placenta and for additional uterotonics after placenta delivery, and results in a better experience of the third stage for women.

INTRODUCTION

Postpartum haemorrhage (PPH) remains a major complication of childbirth worldwide. [START_REF] Khan | WHO analysis of causes of maternal death: a systematic review[END_REF] Population-based studies in high-resource countries report a prevalence of severe PPH from 0.5% to 1% of deliveries, (2)[START_REF] Zhang | Incidence of severe pre-eclampsia, postpartum haemorrhage and sepsis as a surrogate marker for severe maternal morbidity in a European population-based study: the MOMS-B survey[END_REF][START_REF] Zwart | Severe maternal morbidity during pregnancy, delivery and puerperium in the Netherlands: a natiowide populayionbased study of 371 000 pregnancies[END_REF][START_REF] Brace | Quantifying severe maternal morbidity: a Scottish population study[END_REF] making it the main component of severe maternal morbidity. Uterine atony is the leading cause of PPH, accounting for 60 to 80% of cases [START_REF] Oyelese | Postpartum hemorrhage: epidemiology, risk factors, and causes[END_REF]. Prevention of atonic PPH is thus crucial, and preventive measures are recommended for all women giving birth, given that individual risk factors are poor predictors.

Active management of the third stage of labour (AMTSL) has been proposed for the prevention of PPH [START_REF]Recommandations for the prevention of postpartum haemorhage[END_REF]. Its standard definition combines the following three procedures: administration of an oxytocic drug immediately after child birth, early cord clamping and cutting, and controlled cord traction.

Several trials [START_REF] Begley | A comparison of 'active' and 'physiological' management of the third stage of labour[END_REF][START_REF] Khan | Controlled cord traction versus minimal intervention techniques in delivery of the placenta: a randomized controlled trial[END_REF][START_REF] Prendiville | The Bristol third stage trial: active versus physiological management of third stage of labour[END_REF][START_REF] Rogers | Active versus expectant management of third stage of labour: the Hinchingbrooke randomised controlled trial[END_REF] combined in a meta-analysis [START_REF] Begley | Active versus expectant management for women in the third stage of labour[END_REF] showed that AMTSL is associated with a 60% reduction in the incidence of PPH compared with expectant management. Given its efficacy, AMTSL has been included in international [START_REF] Lalonde | Postpartum hemorrhage today: ICM/FIGO initiative 2004-2006[END_REF][START_REF]WHO recommendations for the prevention of postpartum haemorrhage[END_REF] and national [START_REF] Goffinet | Postpartum haemorrhage: recommendations for clinical practice by the French College of Obstetricians and Gynecologists (December 2004)[END_REF][START_REF] Leduc | Active management of the third stage of labour: prevention and treatment of postpartum hemorrhage[END_REF][START_REF]Green Top Guideline n°52. Prevention and management of postpartum haemorrhage[END_REF] guidelines for the prevention of PPH.

However, an adequate evaluation of the specific efficacy of each of its components has not been done.

The independent efficacy of preventive oxytocics has been shown with a good level of evidence [START_REF] Cotten | Prophylactic use of oxytocin for the third stage of labour[END_REF] and it is therefore often considered the essential component of AMTSL. This is not the case for controlled cord traction (CCT). [START_REF] Bonham | Intramuscular Oxytocics and Cord Traction in Third State of Labour[END_REF] Although most guidelines for prevention of PPH include CCT, its actual implementation is highly variable, varying in Europe from 12% in Hungary to 95% in Ireland. [START_REF] Winter | Variations in policies for management of the third stage of labour and the immediate management of postpartum haemorrhage in Europe[END_REF] In countries, such as France, where CCT is not recommended, pulling the cord in the absence of any sign of placenta separation is considered poor practice because of the potential risk of uterine inversion. [START_REF] Goffinet | Inversion uterine[END_REF] The variation in use of CCT may be explained by the paucity of available evidence for assessing either the efficacy of CCT for the prevention of PPH or its potential risks. Until recently, only two trials conducted in the 1960's [START_REF] Bonham | Intramuscular Oxytocics and Cord Traction in Third State of Labour[END_REF][START_REF] Kemp | A review of cord traction in the third stage of labour from 1963 to 1969[END_REF] and with important limitations had assessed the specific effect of CCT and they had conflicting results. Very recently, a large randomized controlled trial conducted in eight low and middle income countries reported that the omission of CCT as part of AMTSL did not increase the risk of severe PPH [START_REF] Gulmezoglu | Active management of the third stage of labour with and without controlled cord traction: a randomised, controlled, non-inferiority trial[END_REF]. The authors concluded that CCT could be omitted in non-hospital settings. However, the results of this trial may be relevant for low/middle income countries and not applicable to other countries.

The TRACOR Study aimed to assess the impact of controlled cord traction on the incidence of PPH and other characteristics of the third stage of labour, in a high-resource setting.

METHODS

Trial design

The TRACOR (TRAction of the CORd) trial was a multicenter randomized controlled trial with two parallel groups and took place in five French university hospitals between January 1, 2010, and January 31, 2011.

CCT was not a standard part of third-stage management in any of the participating units before the trial. Before the beginning of the trial, all staff likely to recruit women into the trial (midwives and obstetricians) were trained in the trial procedures and more particularly in the technique of CCT. In each participating centre, several training meetings were led by a team from the steering committee pairing a midwife with an obstetrician. Films demonstrated the placement of the collector bag and the practice of CCT. Following this initial training, a period of one month was devoted to using CCT in actual practice. Before the inclusion period, a meeting was organized in each unit to verify the attendants' adherence to the protocol and their ease in practicing the relevant procedures.

Participants

Women aged 18 or more, with a planned vaginal delivery, at a gestational age ≥ 35 weeks, with a singleton foetus, were eligible for inclusion. We excluded women with a severe haemostasis disease, those with placenta praevia, in utero foetal death, and multiple gestations. We also excluded women who did not understand French. Eligible women were approached and offered information about the study during a prenatal visit in the third trimester of pregnancy by a midwife or an obstetrician. This information was repeated after their arrival in delivery room for the planned vaginal delivery; the women then confirmed their participation and provided informed written consent.

Interventions

We compared CCT with standard management of placental expulsion.

In the intervention arm, CCT was implemented immediately after the delivery of the baby upon obtaining a uterine contraction, as initially described, [START_REF] Bonham | Intramuscular Oxytocics and Cord Traction in Third State of Labour[END_REF] according to the following instructions: 1) after birth, CCT is started with a firm uterine contraction without waiting for placenta separation; 2) with one hand, grasp the lower segment between the thumb and index finger and exert steady pressure upwards; 3)at the same time, hold the cord in the other hand, and exert steady cord traction downwards and backwards, exactly countered by the upwards pressure of first hand, so that the position of the uterus remains unchanged. 4) If the placenta is not expelled on the first attempt, repeat CCT with counter-pressure with the next uterine contraction.

In the control arm, the attendant awaited the signs of spontaneous placental separation and descent into the lower uterine segment. Once the placenta was separated, it was then delivered through the mother's efforts (helped by fundal pressure or soft tension on the cord to facilitate placental expulsion through the vagina if needed). This standard placental expulsion (SPE) is the usual management in France, as taught in university hospitals and midwifery schools, and it was the routine procedure in the five participating centers before the trial.

All other aspects of third stage management were identical in both arms: IV injection of 5 IU oxytocin within 2 minutes after birth; clamping and cutting the cord within the 2 minutes following birth; placement of a graduated collection bag (manufactured by MVF Merivaara France, 100 mL graduation) just after birth, left in place until the birth attendant judged that the postpartum bleeding had stopped and that there was no reason to further monitor it [START_REF] Zhang | Effect of a collector bag for measurement of postpartum blood loss after vaginal delivery: cluster randomised trial in 13 European countries[END_REF], and always at least for 15 minutes; manual removal of the placenta at 30 minutes after birth if not expelled. A blood sample was taken from all women on the second day after delivery for the measurement of haemoglobin (Hb) and haematocrit (Ht).

Outcomes

The primary outcome of the trial was the incidence of PPH defined by blood loss ≥ 500 ml, measured with a graduated collector bag [START_REF] Rath | Postpartum hemorrhage--update on problems of definitions and diagnosis[END_REF]. The main secondary outcomes were other objective measures of postpartum bleeding: measured blood loss ≥ 1000 mL at bag removal; mean measured blood loss at 15 minutes after birth (the bag had to be left in place at least 15 minutes to have one measure of blood loss at the same time point in all women); mean measured postpartum blood loss at bag removal; mean changes in peripartum haemoglobin and haematocrit (difference between Hb/Ht before delivery and D2). Other secondary outcomes included: supplementary uterotonic treatment, postpartum transfusion (until discharge), arterial embolization or emergency surgery for PPH; other characteristics of the third stage, including its duration, manual removal of the placenta; and women's experience of the third stage, assessed by a self-administered questionnaire on D2 postpartum. Safety outcomes included uterine inversion, cord rupture, and pain.

The detail of procedures used to manage the third stage, as well as all clinical outcomes identified during the immediate postpartum, were prospectively collected by the midwife or the obstetrician in charge of the delivery and recorded in the woman's electronic form in the labour room. Other data were collected by a research assistant, independent of the local medical team. An independent Data Monitoring Committee, which met monthly, was responsible for reviewing adherence to the trial procedures, the recruitment and safety data; the quality of collected outcome data was checked in each centre for 10% of the included women, randomly selected, and in all PPH cases.

Sample size

We assumed a 7% incidence of PPH in the absence of CCT. This incidence is that found in the cohort as a whole in the Pithagore6 trial in 6 French perinatal networks in 2006 from a total of approximately 147 000 births. [START_REF] Deneux-Tharaux | Multifaceted intervention to decrease the rate of severe postpartum haemorrhage: the PITHAGORE6 clusterrandomised controlled trial[END_REF] We hypothesized that CCT might explain half of the 60% reduction in PPH incidence described in the meta-analysis measuring the overall effect of active management. To show a reduction of at least 30% in the incidence of PPH in the CCT arm -that is, a PPH incidence of 4.9% or less in this arm , with  =0.05, 1-=0.80, and a bilateral test, the study required 1990 women with vaginal deliveries in each group, for a total of 3980 patients.

Given the expected proportion of women with a caesarean delivery in labour after randomization (estimated at 5% to 10%), a higher number of women needed to be randomized to include the needed number of women with vaginal deliveries. The decision to stop inclusions was made by the independent Data Monitoring Committee, which was able to access the electronic inclusion system to determine the real-time cumulative number of randomized women and their mode of delivery. This sample size provided a 70% statistical power to detect a reduction in the incidence of severe PPH (defined by blood loss ≥ 1000 ml) from 2% to 1% or less of deliveries.

Randomization

Randomization took place after women completed the participation form, during labour and before delivery. It was performed centrally through an automated web-based system, which ensured allocation concealment. Allocation was stratified by centre.

Statistical methods

The two groups were compared for main and secondary outcomes in an intention-to-treat analysis. The effects of CCT were expressed as mean differences with their 95% confidence intervals for quantitative outcomes, and as relative risks with their 95% confidence intervals for categorical outcomes. To test the consistency of the primary outcome across centres, we used the Mantel-Haenszel homogeneity test. The incidence of each adverse event (cord rupture and uterine inversion) was expressed as a proportion with binomial exact confidence intervals.

An analysis including women who had a caesarean delivery after randomization (for a total of 2172 and 2180 women in the CCT and SPE groups, respectively), for secondary outcomes available in these women (mean change in Hb, mean change in Ht, postpartum transfusion, arterial embolization or emergency surgery) was conducted.

A post-hoc "per protocol" analysis was conducted among women who were managed in accordance with the protocol and the allocation, i.e who had all the following procedures: prophylactic oxytocin administration at birth, cord clamping and cutting within 2 minutes, management of placenta expulsion in accordance with the allocation group (CCT or SPE) and blood collection bag left in place at least 15 minutes. Software used for analysis was Stata 10.1 (Stata Corporation, USA).

Ethics

The trial protocol was approved by the Paris-Ile de France III Committee for the Protection of Research Subjects (Ethics Committee) in September 2009 (n°B90885-20).

Registration

This trial is registered (ClinicalTrials.gov), number NCT01044082.

The full trial protocol can be accessed at: http://www.u953.idf.inserm.fr/page.asp?page=5211 RESULTS

Figure 1 shows the trial profile. The trial was carried out in all five hospitals between 1 January 2010 and 31 January 2011. In all, 4355 women in labour were enrolled and randomly assigned. After randomization and before delivery, 294 (6.8%) women became ineligible because an intrapartum caesarean was performed, and 3 others declined to participate. Thus 4058 randomized participants delivered vaginally: 2034 assigned to CCT and 2024 to SPE. Baseline demographic and obstetric characteristics were similar in the two groups (Table1).

The management of the third stage of labour is described in Table 2. Overall, the adherence to the protocol was high in both groups. The reasons for deviating from the allocated intervention are detailed in Figure1.

Primary outcome data were collected for 4013 (98.9%) participants. The proportion of women with a measured postpartum blood loss of 500mL or more at bag removal did not differ between the 2 groups (196/2005, 9.8% in the CCT group and 206/2008, 10.3% in the SPE group, relative risk 0.95, 95% CI (0.79;1.15))(Table 3). There was no significant heterogeneity between centres for this result (Table3).

Similarly, the incidence of PPH ≥ 1000mL at bag removal did not differ between the 2 groups; nor did the mean measured blood loss at 15 minutes and at removal of the bag (Table3).

Outcome data related to blood count indicators before and after delivery were available for 1963/2034(96.5%) women in the CCT group and 1953/2024 (96.5%) in the SPE group (at least one peripartum change in Hb or Ht available). Twenty women (11 in the CCT group and 9 in the SPE group) had transfusion before day 2 and were excluded from this analysis. There was no significant difference in the mean peripartum change in Hb or Ht (Table3). The proportion of women with a peripartum drop in Hb of 4g/dl or more did not differ between the 2 groups, 2.1% (41/1961) in the CCT group and 1.8% (35/1953) in the SPE group, (RR 1.17, 95%CI (0.75;1.82)).

Women in the CCT group had fewer manual removals of the placenta than those in the SPE group (RR 0.69, 95%CI (0.53; 0.90)) (Table 3). Third stage was shorter in the CCT group.

Regarding safety, no uterine inversion occurred among the 1943 women who had controlled cord traction (incidence 0.0%, one sided 97.5%CI (0.0%-0.18%)). Cord rupture occurred in 89 (incidence 4.6%, 95% CI (3.6%; 5.5%); among those 89 women, manual removal of the placenta was needed in 43 (48%). No other adverse events occurred in the two groups.

Women in the CCT group reported a significantly lower intensity of pain and discomfort during the third stage than those in the SPE group; they were less likely to have felt tired and anxious and to report that the duration of third stage was long (Table 4).

The per-protocol analysis was conducted in 1437/1999(71.9%) women in the CCT arm, and 1574/1990 (79.1%) in the SPE arm. The proportion of women with a measured postpartum blood loss of 500mL or more at bag removal did not differ between the two groups (11.7% (168/1431) in the CCT group and 10.7% (168/1570) in the SPE group, relative risk 1.10, 95% CI (0.90 ;1.34 )).

Finally, the analysis including women who had a caesarean delivery after randomization provided results similar to those of the main analysis (data not shown).

DISCUSSION

In this large multicenter randomized trial, we found that the use of controlled cord traction as one component of the active management of third stage of labour had no significant effect on the incidence of PPH. However, CCT reduced the duration of the third stage and the need for manual removal of placenta. Moreover, women in the CCT group reported a significantly lower intensity of pain and discomfort as well as less fatigue and anxiety. This trial included a large population of pregnant women with few exclusion criteria. Hence, the results are likely to be generalizable to women with vaginal deliveries in similar contexts of care.

Moreover, the adherence to the allocated intervention and other standardized aspects of third stage management was high, making it possible to isolate the effect of CCT.

It was not possible to blind this intervention as the procedures being tested require different actions by the attendants. However, the trial primary and main secondary outcomes (change in peripartum Hb and Ht ) were objective measures of postpartum blood loss as opposed to other definitions of PPH based on visual estimation or interventions, influenced by caregiver decisions. Although the quality of the CCT technique was not formally evaluated, a real difference in the management of placenta expulsion between the 2 groups is very likely given the emphasis on the initial training. Moreover, the attendants in the two groups clearly reported different procedures, and the length of the third stage was significantly shorter and the incidence of cord rupture higher in the CCT group.

Two small trials in the 1960's [START_REF] Bonham | Intramuscular Oxytocics and Cord Traction in Third State of Labour[END_REF][START_REF] Kemp | A review of cord traction in the third stage of labour from 1963 to 1969[END_REF][START_REF] Althabe | Controlled cord traction in the third stage of labor. Systematic review[END_REF] assessed the specific effects of CCT during the third stage.

Both had important methodological weaknesses including inadequate method of randomization, visual estimation of blood loss for determining outcome measures and limited sample sizes. Very recently, a large randomized controlled trial conducted in eight low and middle income countries compared CCT with "hands-off " management of third stage [START_REF] Gulmezoglu | Active management of the third stage of labour with and without controlled cord traction: a randomised, controlled, non-inferiority trial[END_REF] . The results showed that the omission of CCT did not result in an increased risk of measured blood loss of 1000 mL or more. However, heterogeneity between centres in other components of third stage management (type of uterotonic used, combination with uterine massage), absence of report on the actual duration of blood loss measurement in each arm and absence of outcomes based on blood counts, may limit the interpretation of the results. In addition, although it is of major importance to conduct research studies in low and middle income countries, the generalizability of their results for high income settings needs to be tested. Indeed, characteristics of women, management of labour, resources and organization of care in the labour ward clearly differ between low and high resource countries, and these differences may impact the risk and the characteristics of PPH. Mechanisms of PPH and effective preventive procedures may differ between settings. It is noteworthy that the incidence of PPH ≥ 500 mL in the reference group of the previous CCT trial was about 30% higher than the incidence found in the Tracor trial, which might indicate higher exposure to the risk of PPH. For these reasons, our results provide valuable additional evidence that CCT is not an essential component of management of the third stage of labour for prevention of PPH, in high resource countries.

Cord rupture occurred in about 1 in 22 women who had CCT. This rate may appear notable at first. However, in the majority of cases (52%), delivery of the placenta occurred without any extra intervention; and overall, the rate of manual removal of placenta was lower in women who had CCT.

In consequence, cord rupture should not be considered an important adverse effect of CCT and does not imply manual removal of placenta.

The 30% reduction in the need for manual removal of placenta found in the CCT arm may provide a meaningful decrease in morbidity considering the need for analgesia and antibiotics, separation of mother and baby, and the risk of infection associated with this intervention [START_REF] Chongsomchai | Prophylactic antibiotics for manual removal of retained placenta in vaginal birth[END_REF]. However, we cannot exclude the possibility that such a difference may have been less important (or even not significant) if the French policy was more conservative, allowing a duration of third stage greater than 30 minutes before manually removing the placenta, in particular in the SPE group. Our finding of a lower risk of manual removal of placenta when its expulsion is managed with CCT is in contrast with the conclusions of the trial cited above [START_REF] Gulmezoglu | Active management of the third stage of labour with and without controlled cord traction: a randomised, controlled, non-inferiority trial[END_REF]. However, in this study, manual removal of placenta was performed in less than 1% of deliveries in both arms, which is low in comparison with previous reports from high resource countries [START_REF] Cheung | The retained placenta: historical and geographical rate variations[END_REF][START_REF] Weeks | The retained placenta[END_REF], and may actually illustrate the variations in policies for the management of the third stage of labour between settings [START_REF] Deneux-Tharaux | Policies for manual removal of placenta at vaginal delivery: variations in timing within Europe[END_REF]. Our trial also showed that CCT significantly reduced the duration of third stage. This result may have implications for optimizing the organization of postpartum surveillance and care, in particular in hospitals where the number of midwives or birth attendants in labour wards is limited. In addition, the shorter third stage and lesser need for manual removal of placenta associated with CCT are likely to be the main reasons why women reported a better experience of the third stage of labour in the CCT arm, although we cannot exclude a patient preference bias since the study was not blinded.

Another controversial aspect of the management of the third stage of labour is the timing of cord clamping. Recent results from a trial conducted in Sweden showed that, even in a region with low prevalence of iron deficiency, delayed cord clamping reduced the prevalence of neonatal anaemia and improved iron status at 4 months of age in term deliveries [START_REF] Andersson | Effect of delayed versus early umbilical cord clamping on neonatal outcomes and iron status at 4 months: a randomised controlled trial[END_REF], confirming the findings of previous trials conducted in low and middle income populations [START_REF] Hutton | Late vs early clamping of the umbilical cord in full-term neonates: systematic review and meta-analysis of controlled trials[END_REF]. CCT, as it is classically performed, is not compatible with delayed cord clamping. Our finding that CCT has no significant effect on maternal postpartum haemorrhage constitutes reassuring information for clinicians willing to implement a policy of delayed cord clamping, from both maternal and neonatal perspectives.

In a high-resource setting, the use of CCT for the management of placenta expulsion has no significant effect on the incidence of PPH and other markers of postpartum blood loss. Therefore, there is no evidence to recommend routine CCT for the management of placenta expulsion in order to prevent PPH.

Figure 1 :

 1 Figure 1: Trial flow diagram

Table 1 .

 1 Baseline characteristics of women p for Mantel-Haenszel test of homogeneity across centers b Prepartum Hb measured within 8 th month of gestation and arrival in labour ward in 1778 (90.7%) and 1760 (90.1%), at arrival in labour ward in 95 (4.8%) and 99 (5.1%) and between the 5 th -7 th months of gestation in 88 (4.5%) and 94 (4.8%), in the CCT and SPE groups, respectively ; postpartum Hb measured at Day 2 in 1793 (91.4%) and 1787 (91.5%), and on another day between 1 and 8 days in 168 (8.6%) and 166 (8.5%) , in the CCT and SPE groups, respectively. Prepartum Ht measured within 8 th month of gestation and arrival in labour ward in 1724 (90.5%) and 1707 (90.3%), at arrival in labour ward in 95 (5.0%) and 99 (5.2%) and between the 5 th -7 th months of gestation in 85 (4.5%) and 84 (4.4%), in the CCT and SPE groups, respectively; postpartum Ht measured at Day 2 in 1737 (91.2%) and 1725 (91.3%), and on another day between 1 and 8 days in 167 (8.8%) and 165 (8.7%) , in the CCT and SPE groups, respectively.

	Characteristics	Controlled Cord Traction (N= 2034)	Standard Placenta Expulsion (N= 2024)
	Hospital

a c

Table 4 .

 4 Women's experience of third stage

			Controlled Cord Traction	Standard Placenta Expulsion	P a
	Completed forms		1838/2034 (90.4)	1844/2024 (91.2)	0.41
	Felt tired	Not at all	466/1829 (25.5)	426/1838 (23.2)
		A little	656/1829 (35.9)	621/1838 (33.8)
		Moderately	378/1829 (20.6)	445/1838 (24.2)	0.017

ACKNOWLEDGEMENTS

The authors want to thank the independent data monitoring committee chaired by Pr JM Treluyer from the Unité de Recherche Clinique Paris Centre.

The authors also thank the women who participated in the trial, the staff from the participating maternity units for including women, and the members of the Tracor Study Group (see list in appendix).

Inserm Unit 953 has received a grant from the Bettencourt Foundation (Coups d'élan pour la Recherche française) in support of its research activities.

Funding

The Tracor trial was funded by the French Ministry of Health under its Clinical Research Hospital Program (contract n° P081206). The Ministry of Health had no role in the design and conduct of the study, no role in the collection, management, analysis, or interpretation of the data, and no role in the preparation, review and approval of the manuscript or in the decision to submit for publication.

Data sharing: no additional data available.

Contribution of authors

CDT participated in the design of the study, obtained funding participated in the central monitoring of data collection, supervised the cleaning, analysis, and interpretation of the data and the drafting and revision of the paper, and has seen and approved the final version. She had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

As the Corresponding Author, she has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in BMJ editions and any other BMJPGL products and sublicences to exploit all subsidiary rights. LS participated in the design of the study, supervised the inclusion of women and the running of the trial in his hospital, participated in the revision of the paper, and has seen and approved the final version.

FM participated in the central monitoring of data collection, supervised the cleaning of the data, conducted the analysis and participated in the drafting and the revision of the paper and has seen and approved the final version.

EC participated in the design of the study, supervised the inclusion of women and the running of the trial in his hospital, participated in the revision of the paper, and has seen and approved the final version.

DV participated in the design of the study, supervised the inclusion of women and the running of the trial in her hospital, participated in the revision of the paper, and has seen and approved the final version.

JL participated in the design of the study, supervised the inclusion of women and the running of the trial in his hospital, participated in the revision of the paper, and has seen and approved the final version.

FG is the principal investigator of the trial; he participated in the design of the study, obtained funding for it, participated in the central monitoring of data collection, supervised the cleaning, analysis, and interpretation of the data and the drafting and revision of the paper, and has seen and approved the final version. He had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare: had support from the French Ministry of Health for the submitted work; LS had relationships (board membership, consultancy and lectures) with Ferring; other authors had no financial relationships with any organisations that might have an interest in the submitted work in the previous 3 years; no other relationships or activities that could appear to have influenced the submitted work.