domain: chim sdv

text:
Kristaps Jaudzems

Tatyana Polenova

Guido Pintacuda

Hartmut Oschkinat

Anne Lesage

DNP NMR of Biomolecular Assemblies

Keywords: Solid-state NMR, Dynamic Nuclear Polarization, Biological assemblies, Protein structure and dynamics, Magic Angle Spinning

Dynamic Nuclear Polarization (DNP) is an effective approach to alleviate the inherently low sensitivity of solid-state NMR (ssNMR) under magic angle spinning (MAS) towards large-sized multi-domain complexes and assemblies. DNP relies on a polarization transfer at cryogenic temperatures from unpaired electrons to adjacent nuclei upon continuous microwave irradiation. This is usually made possible via the addition in the sample of a polarizing agent. The first pioneering experiments on biomolecular assemblies were reported in the early 2000s on bacteriophages and membrane proteins. Since then, DNP has experienced tremendous advances, with the development of extremely efficient polarizing agents or with the introduction of new microwaves sources, suitable for NMR experiments at very high magnetic fields (currently up to 900 MHz). After a brief introduction, several experimental aspects of DNP enhanced NMR spectroscopy applied to biomolecular assemblies are discussed. Recent demonstration experiments of the method on viral capsids, the type III and IV bacterial secretion systems, ribosome and membrane proteins are then described.

Introduction

High-resolution solid-state NMR (ssNMR) under magic angle spinning (MAS) has recently emerged as a powerful structural tool for studying structure and dynamics of noncrystalline macromolecular complexes. The inherently low sensitivity of this spectroscopy limits however its applicability, and as a result large-sized multi-domain complexes and assemblies still often remain inaccessible to site-specific structural studies.

In the last two decades, Dynamic Nuclear Polarization (DNP) has emerged as a key method to increase the sensitivity of MAS solid-state NMR spectroscopy. The first proofs of principle on biomolecular assemblies were reported in the early 2000s by Griffin and coworkers on bacteriophages and membrane proteins [START_REF] Bajaj | Dynamic nuclear polarization at 9T using a novel 250GHz gyrotron microwave source[END_REF][START_REF] Rosay | Sensitivityenhanced NMR of biological solids: dynamic nuclear polarization of Y21M fd bacteriophage and purple membrane[END_REF][START_REF] Rosay | High-frequency dynamic nuclear polarization in MAS spectra of membrane and soluble proteins[END_REF]. Since then, thanks to great progress in instrumentation operating at higher and higher frequencies [START_REF] Barnes | Dynamic nuclear polarization at 700 MHz/460 GHz[END_REF][START_REF] Ni | High frequency dynamic nuclear polarization[END_REF] and to the introduction of tailor-designed polarizing agents [START_REF] Sauvee | Highly efficient, water-soluble polarizing agents for dynamic nuclear polarization at high frequency[END_REF][START_REF] Sauvee | Tailoring of Polarizing Agents in the bTurea Series for Cross-Effect Dynamic Nuclear Polarization in Aqueous Media[END_REF][START_REF] Song | TOTAPOL: a biradical polarizing agent for dynamic nuclear polarization experiments in aqueous media[END_REF], DNP has established itself as a key tool for the investigation of a wide range of biological solids, from fibrils [START_REF] Bayro | Intermolecular Structure Determination of Amyloid Fibrils with Magic-Angle Spinning and Dynamic Nuclear Polarization NMR[END_REF][START_REF] Debelouchina | Dynamic nuclear polarization-enhanced solid-state NMR spectroscopy of GNNQQNY nanocrystals and amyloid fibrils[END_REF][START_REF] Frederick | Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register[END_REF][START_REF] Potapov | Successive Stages of Amyloid-beta Self-Assembly Characterized by Solid-State Nuclear Magnetic Resonance with Dynamic Nuclear Polarization[END_REF] to biomaterials (Geiger et al., 2016b;[START_REF] Jantschke | Insight into the Supramolecular Architecture of Intact Diatom Biosilica from DNP-Supported Solid-State NMR Spectroscopy[END_REF][START_REF] Ravera | Biosilica-Entrapped Enzymes Studied by Using Dynamic Nuclear-Polarization-Enhanced High-Field NMR Spectroscopy[END_REF], membrane embedded proteins [START_REF] Becker-Baldus | Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy[END_REF], intrinsically disordered proteins [START_REF] Uluca | DNP-Enhanced MAS NMR: a Tool to Snapshot Conformational Ensembles of a-Synuclein in Different States[END_REF] or cells [START_REF] Frederick | Sensitivity-enhanced NMR reveals alterations in protein structure by cellular milieus[END_REF][START_REF] Renault | Solid-State NMR Spectroscopy on Cellular Preparations Enhanced by Dynamic Nuclear Polarization[END_REF]Albert el al., 2018) to name but a few, thus gaining more and more importance in structural biology.

Several landmark review articles have already discussed the potential of DNP MAS NMR in this area [START_REF] Akbey | Structural biology applications of solid state MAS DNP NMR[END_REF][START_REF] Su | Magic angle spinning NMR of proteins: high-frequency dynamic nuclear polarization and (1)H detection[END_REF]). Throughout the present review, we will focus more specifically on recent advances on biomolecular assemblies. Experimental aspects of DNP enhanced NMR spectroscopy will first be discussed. The scope of the method will then be illustrated within the context of the structural investigation of viral capsids, the type III and IV bacterial secretion systems, ribosome and membrane proteins. Fibrillar assemblies are not included in this article.

Experimental aspects of DNP MAS NMR for biomolecular assemblies

Principle

DNP relies on a transfer of polarization between unpaired electron spins and surrounding nuclei. The samples are usually doped with stable radicals (the polarizing agents) and the unpaired electrons are saturated by continuous micro-wave (MW) irradiation. This is accomplished using gyrotrons that operate in the required frequency range (140-600 GHz) and produce high-power microwaves. The relaxation times of the unpaired electrons must be sufficiently long to allow saturation of the transitions, which is achieved by performing the experiments at low temperatures (usually ~100 K) using cryogenic MAS probes. Continuous wave DNP can proceed via several mechanisms that dictate the choice of the polarizing agent. Currently, the most efficient polarization transfer scheme for high-field MAS DNP experiments is the cross-effect mechanism, which requires a dipolar-coupled system of two electrons and a nucleus, and which is thus particularly effective with chemically stable organic biradicals [START_REF] Can | Mechanisms of dynamic nuclear polarization in insulating solids[END_REF]. This mechanism dominates when the resonance frequencies of the two electrons of the biradical are separated by the nuclear Larmor frequency. To date, the most successful biradicals for biomolecular studies are the water soluble binitroxides TOTAPOL [START_REF] Song | TOTAPOL: a biradical polarizing agent for dynamic nuclear polarization experiments in aqueous media[END_REF] and AMUPol [START_REF] Sauvee | Highly efficient, water-soluble polarizing agents for dynamic nuclear polarization at high frequency[END_REF]. Like all nitroxides, they have an inhomogeneously broadened EPR line width that is comparable to the 1 H Larmor frequencies of contemporary NMR spectrometers (hundreds of MHz), allowing polarization transfer from the electrons to neighboring 1 H nuclei. The DNPenhanced 1 H polarization is then transported in the sample by 1 H-1 H spin diffusion and subsequently transferred to heteronuclei via cross-polarization. The sensitivity enhancement by DNP, referred to as ε, can be measured as the ratio of signal intensities in NMR spectra acquired with and without microwave irradiation.

2.2.

Water-soluble polarizing sources TOTAPOL was the first popular water-soluble biradical polarizing agent and was shown to yield enhancements of up to ~80 at 400 MHz at 100 K. AMUPol was designed later to be more rigid and to possess longer electron relaxation times and stronger electron-electron couplings, improving by more than a factor of 3 the enhancement factors under identical conditions (typically 235 at 400 MHz, 128 at 600 MHz and 35 at 800 MHz for frozen bulk aqueous solutions). A direct comparison between the overall sensitivity gains obtained using TOTAPOL and AMUPol has been recently reported, confirming the overperformance of the latter [START_REF] Mentink-Vigier | Nuclear depolarization and absolute sensitivity in magic-angle spinning cross effect dynamic nuclear polarization[END_REF]. Only these two radicals have been used for the DNP studies of molecular assemblies discussed in this article (Table 1).

We note that several new biradicals, specifically designed for biomolecular DNP studies, have recently been introduced. Notable examples include new families of binitroxides like bcTol [START_REF] Jagtap | bcTol : a highly water-soluble biradical for efficient dynamic nuclear polarization of biomolecules[END_REF] or PyPolPEG2OH [START_REF] Sauvee | Tailoring of Polarizing Agents in the bTurea Series for Cross-Effect Dynamic Nuclear Polarization in Aqueous Media[END_REF] as well as trityl-nitroxide biradicals TEMTriPols [START_REF] Mathies | Efficient Dynamic Nuclear Polarization at 800 MHz/527 GHz with Trityl-Nitroxide Biradicals[END_REF]. bcTol delivers similar sensitivity enhancements as AMUPol, but exhibits unparalleled solubility in water and glycerol/water mixtures as well as somewhat improved temperature dependence. PyPolPEG2OH is a derivative of AMUPol that provides better anchoring in the glycerol/water matrix through additional hydrogen bonds resulting in longer T 1e and ca. 1.2 times higher enhancement factor. All binitroxides suffer from an unfavourable magnetic field dependence, as their EPR line width increases linearly with B 0 , which in turn decreases the efficiency of the cross-effect mechanism (Maly et al., 2008). Conversely, TEMTriPols show unprecedented performance at high magnetic fields with a record 1 H NMR signal enhancement of 65 at 800 MHz and 100 K, almost a factor of two higher than AMUPol under similar experimental conditions.

The search for better-performing cross-effect DNP polarizing agents is still ongoing as the parameters of the current biradicals are far from optimal, and as the enhancements achieved today are still far from the theoretical maximum (658 for 1 H). Thus, it remains to be seen if any of the recently introduced or forthcoming biradicals will replace today's gold standard AMUPol in future biomolecular DNP studies. It should also be noted that in parallel, efforts are made to develop radicals for high-field DNP applications exploiting the Overhauser effect which scales favorably with the magnetic field [START_REF] Can | Overhauser effects in insulating solids[END_REF], instead of the cross-effect.

Obtaining a large enhancement requires that the radicals are well dispersed in the sample to avoid inter-radical interactions, which is conventionally achieved by using a glycerol/water or a DMSO/water glass-forming solvent (see the next paragraph). An alternative method, which also ensures a homogeneous dispersion of the radical, is "targeted DNP". In this approach, the radical is covalently attached either directly to the molecule of interest or to its binding partner using a linker (e.g. via cysteine mutagenesis and sulfhydryl chemistry). Nitroxide monoradicals as well as the biradicals TOTAPOL and AMUPol have been attached to peptides, [START_REF] Vitzthum | Fractional spin-labeling of polymers for enhancing NMR sensitivity by solvent-free dynamic nuclear polarization[END_REF] to membrane proteins, (van der [START_REF] Van Der Cruijsen | Biomolecular DNP-Supported NMR Spectroscopy using Site-Directed Spin Labeling[END_REF][START_REF] Voinov | Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR[END_REF][START_REF] Wylie | Dynamic nuclear polarization of membrane proteins: covalently bound spinlabels at protein-protein interfaces[END_REF], to lipids [START_REF] Fernandez-De-Alba | Matrix-free DNP-enhanced NMR spectroscopy of liposomes using a lipid-anchored biradical[END_REF][START_REF] Smith | A method for dynamic nuclear polarization enhancement of membrane proteins[END_REF] and to an interaction partner of the target protein [START_REF] Rogawski | Dynamic Nuclear Polarization Signal Enhancement with High-Affinity Biradical Tags[END_REF][START_REF] Viennet | Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization[END_REF]. In parallel, complexes of paramagnetic metal ions like Gd 3+ and Mn 2+ have been attached to proteins for site-selective DNP experiments [START_REF] Kaushik | Gd(III) and Mn(II) complexes for dynamic nuclear polarization: small molecular chelate polarizing agents and applications with site-directed spin labeling of proteins[END_REF]. While the DNP enhancements provided by these approaches are generally comparable to those obtained by untargeted means, they offer the additional benefits of increased the filling factor removing the need of a glassy matrix, of exploiting a site-directed specificity in terms of enhancement and of using paramagnetic relaxation enhancement (PRE) effects from the radicals to obtain structural information. Notably, these methods have shown a great potential for studies of membrane proteins, where uniform dispersion of the radical in the membranes may otherwise be difficult. No targeted DNP studies have been reported so far on molecular assemblies.

Sample preparation

Multiple studies have investigated the impact of different sample preparation protocols, composition of the polarizing medium, protein and matrix deuteration levels and concentration of the polarizing agents on the DNP enhancement. Table 1 lists the experimental conditions used in most of the recent DNP-enhanced NMR studies of various molecular assemblies.

Insert Table 1 here.

A common way to perform DNP studies of proteins is to use frozen solutions [START_REF] Siemer | Protein Linewidth and Solvent Dynamics in Frozen Solution NMR[END_REF]. Upon freezing, pure water forms ice crystals, which may spatially sequester the different sample components (protein, polarizing agent and water) in different domains, and lead to aggregation of the radical and poor enhancement. Therefore, most studies are performed in mixtures of water and glycerol, which are known to form a glass at low temperatures. A drawback of frozen solutions is that the filling factor and consequently the absolute sensitivity is relatively low as the vast majority of the sample volume is taken by the solvent. To avoid this, the protein samples can be first precipitated by crystallization (Geiger et al., 2016a;[START_REF] Van Der Wel | Dynamic nuclear polarization of amyloidogenic peptide nanocrystals: GNNQQNY, a core segment of the yeast prion protein Sup35p[END_REF] or sedimentation [START_REF] Ravera | DNP-Enhanced MAS NMR of Bovine Serum Albumin Sediments and Solutions[END_REF][START_REF] Ravera | Dynamic Nuclear Polarization of Sedimented Solutes[END_REF], and then immersed in a solution containing a cryo-protectant before freezing. In favorable cases the samples can be measured in the absence of a glass forming agent. An early investigation on apoferritin by the group of Bertini [START_REF] Ravera | Dynamic Nuclear Polarization of Sedimented Solutes[END_REF], for example, used sedimented proteins (via ultracentrifugation) that were very concentrated and obtained an enhancement of ε> 40 at 212 MHz and 90 K. This was achieved without adding glycerol as glass-forming agent, suggesting the formation of a glass-like state when solutions of such a high concentration are frozen. A 'normal', dilute solution showed only an enhancement of 2 under the same conditions.

Note that only one of the studies reported in Table 1 was done using a frozen solution. In all other cases, the sample was first precipitated, which is often relatively easy for molecular assemblies, due to their high molecular weight. This highlights the importance of obtaining the highest possible absolute sensitivity, even in DNP conditions.

Another approach, which is greatly facilitated by DNP, is the investigation of biomolecules in their native cellular environments. While this is an attractive approach, it is also associated with several experimental difficulties, which is the reason why only a few examples of such DNP-supported studies of cellular preparations have been reported. This includes a membrane protein [START_REF] Renault | Solid-State NMR Spectroscopy on Cellular Preparations Enhanced by Dynamic Nuclear Polarization[END_REF], a membrane-embedded protein complex [START_REF] Kaplan | Probing a cell-embedded megadalton protein complex by DNP-supported solid-state NMR[END_REF], and a yeast prion protein [START_REF] Frederick | Sensitivity-enhanced NMR reveals alterations in protein structure by cellular milieus[END_REF], overproduced in cells. The main difficulty of this approach is the complexity of the experimental NMR data, arising from overlaps with "background" signals from other components of the cell. This usually requires that either a dedicated labelling scheme [START_REF] Kaplan | Probing a cell-embedded megadalton protein complex by DNP-supported solid-state NMR[END_REF] or a targeted approach [START_REF] Frederick | Sensitivity-enhanced NMR reveals alterations in protein structure by cellular milieus[END_REF][START_REF] Viennet | Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization[END_REF] is employed. The obtained DNP enhancements of such cellular preparations are generally inferior to those for conventionally prepared samples. For these reasons, this methodology is not yet widely used, but is well suited to answer specific questions in comparison to in vitro data, or to validate an in vitro model in a cellular setting.

The composition and particularly the deuteration level of the cryo-protectant matrix has a significant effect on spin diffusion and consequently on the DNP enhancement. The most commonly used solvent composition is 60% glycerol-d 8 /30% D 2 O/10% H 2 O (v/v/v, a.k.a "DNP juice"). However, many precipitated samples are not compatible with a high glycerol content, which results in re-dissolution. In these cases, the lowest possible glycerol concentration needs to be determined, which still provides cryo-protection and does not result in sample re-dissolution. Alternatively, other glass-forming solvents (such as mixtures of water and DMSO-d 6 ) may be explored. The deuteration level of the cryo-protectant matrix is usually adjusted by adding an appropriate amount of H 2 O in otherwise deuterated cryoprotectant/D 2 O background to yield the highest enhancement. For the molecular assembly studies reviewed here, the highest enhancements were obtained by using a fully deuterated matrix: ε H of 65 at 600 MHz and 21 at 800 MHz using 30/70 % glycerol-d 8 /D 2 O and 70/30 % glycerol-d 8 /D 2 O, respectively [START_REF] Sergeyev | Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization[END_REF][START_REF] Fricke | High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles[END_REF]. These results indicate that the small number of protons introduced with the protein is sufficient for an effective spin diffusion in samples of molecular assemblies.

The level of protein deuteration is another factor that affects the DNP enhancement by extending the longitudinal relaxation times, and it has been shown that protein perdeuteration provides superior DNP enhancements as compared to the same type of experiments in fully protonated samples [START_REF] Akbey | Dynamic nuclear polarization of deuterated proteins[END_REF]. This method requires that the protein of interest is expressed in a fully deuterated culture medium and that protons, which would serve as a magnetization source for cross-polarization, are re-introduced at exchangeable sites during protein purification by refolding in a protonated buffer. However, this approach has not been explored for molecular assemblies so far, due to difficulties in preparing perdeuterated samples of protein assemblies.

Finally, the radical concentration affects longitudinal and transverse relaxation as well as the DNP enhancement, which together have a dramatic effect on the intensity of NMR signals. Although some guidelines exist, the optimal concentration of the radical is system dependent, and is usually adjusted to provide the maximum signal-to-noise observed per unit time without additional line broadening [START_REF] Lange | The effect of biradical concentration on the performance of DNP-MAS-NMR[END_REF]. It is worth mentioning that in some cases the radical may bind to the protein, requiring the use of very low radical concentrations [START_REF] Nagaraj | Surface Binding of TOTAPOL Assists Structural Investigations of Amyloid Fibrils by Dynamic Nuclear Polarization NMR Spectroscopy[END_REF]. For molecular assemblies, the employed radical concentrations are between 8 and 28 mM (Table 1), with the majority of studies done using around 8-10 mM AMUPol.

Spectral resolution

Impressive sensitivity enhancement factors (ε H ), as high as ~210 (at 400 MHz, 100 K and 8.8 kHz MAS), have been reported on microcrystalline protein samples [START_REF] Jagtap | bcTol : a highly water-soluble biradical for efficient dynamic nuclear polarization of biomolecules[END_REF]. This signal enhancement comes, however, at the expense of a substantial signal broadening, which compromises spectral resolution. The observed linewidths under various DNP conditions are also summarized in Table 1. Several studies have shown that this broadening is primarily inhomogeneous and associated with the low temperature required for DNP experiments, which results in freezing-out of different side chain conformers (Linden et al., 2011a;[START_REF] Siemer | Protein Linewidth and Solvent Dynamics in Frozen Solution NMR[END_REF]. Additional factors that contribute to this are the presence of paramagnetic radicals and the typically low magnetic field strength (400 MHz) and MAS rate (below 20 kHz) at which the experiments are usually performed.

Several directions have been explored to alleviate the resolution problem in DNP spectra of proteins. [START_REF] Akbey | High-Temperature Dynamic Nuclear Polarization Enhanced Magic-Angle-Spinning NMR[END_REF] introduced high-temperature DNP, which aims at increasing spectral resolution by performing experiments at temperatures of around 180 K instead of 100 K. At this temperature only modest enhancements of 11-18 were obtained (at 400 MHz). However, the resolution was shown to be similar to room temperature spectra for some favorable sites in deuterated microcrystalline SH3. More recently, Barnes and coworkers developed an alternative approach, aimed at removing the deleterious effects on NMR signals produced by paramagnetic DNP polarizing agents. They notably demonstrated that electron decoupling produces a significant reduction in 13 C line widths of [ 13 C, 15 N]urea mixed with Trityl experiments under magic angle spinning [START_REF] Saliba | Electron Decoupling with Dynamic Nuclear Polarization in Rotating Solids[END_REF][START_REF] Sesti | Electron decoupling with cross polarization and dynamic nuclear polarization below 6 K[END_REF].

Another possibility to partly restore spectral resolution of biological solids is to perform the DNP experiments at high magnetic fields (600 and 800 MHz), where the homogeneous component of the line broadening is reduced (Lopez del [START_REF] Lopez Del Amo | Cryogenic solid state NMR studies of fibrils of the Alzheimer's disease amyloid-beta peptide: perspectives for DNP[END_REF]. Lange, Baldus and co-workers showed that a significant increase in resolution can be attained by doubling the magnetic field strength from 400 to 800 MHz, thereby allowing detailed structural investigation of the membrane protein KcsA in different functional states and of the type III secretion needles (T3SS) [START_REF] Fricke | High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles[END_REF][START_REF] Koers | NMRbased structural biology enhanced by dynamic nuclear polarization at high magnetic field[END_REF]. However, this approach is also accompanied by a reduction in DNP enhancements and a maximum enhancement of 21 at 800 MHz was obtained on the T3SS. A smaller reduction in the DNP enhancement paralleled with smaller gains in resolution has been obtained at 600 MHz magnetic field strength suggesting that this magnetic field might be a good compromise for DNP of biomolecular assemblies, yielding increased resolution with reasonably high enhancement [START_REF] Fricke | Studies on the MxiH Protein in T3SS Needles Using DNP-Enhanced ssNMR Spectroscopy[END_REF][START_REF] Gupta | Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies[END_REF][START_REF] Sergeyev | Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization[END_REF].

The benefits of faster sample spinning (up to 40 kHz) were recently investigated. [START_REF] Sergeyev | Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization[END_REF] showed on the coat protein of Pf1 virus that DNP at 600 MHz and 25 kHz MAS results in peaks sharper than in the slow spinning regime and in improvements in signal-to-noise due to better microwave penetration, enabling novel experiments with multiple inter-nuclear transfers and long mixing times for efficient backbone and side chain assignment. More recently, Jaudzems and coworkers reported that high quality spectra of the Acinetobacter phage 205 (AP205) nucleocapsid could be obtained by combining 800 MHz magnetic field strength with 40 kHz MAS and high power proton decoupling [START_REF] Jaudzems | Dynamic nuclear polarization enhanced biomolecular NMR spectroscopy at high magnetic field with fast magic-angle spinning[END_REF]. DNP enhanced carbon-13 NMR linewidths of less than 1 ppm were reported on AP205, and the authors additionally showed that a significant reduction of the homogeneous contribution to the line broadening was obtained by increasing the MAS rates (from 10 to 40 kHz) and the proton decoupling field strengths (from 100 to 150 kHz). This effect was explained as the result of an improved averaging of the anisotropic interactions and of the paramagnetic couplings with the radical. While this improvement is masked by inhomogeneous broadening in the observed linewidths, it does nonetheless guarantee long coherence lifetimes, allowing in turn the acquisition of scalar-based experiments.

In summary, DNP can enhance protein NMR signals by up to two orders of magnitude. However, a major bottleneck, which prevents its widespread application to protein structural studies is the deterioration of spectral resolution at the DNP experimental conditions. Although the resolution can be improved by performing experiments at higher temperatures and higher magnetic fields, these approaches yield today reduced enhancements. In this sense, increasing the MAS frequency seems to be appealing as it provides both improved resolution and high signal-to-noise ratios in the case of densely packed multi-component assemblies. High temperature DNP has not shown its potential so far for molecular assemblies.

Application to structure and dynamics characterization of molecular assemblies

The increased sensitivity provided by DNP has allowed one to perform structural investigations on molecular assemblies that would otherwise have been impossible due to prohibitively long experimental times, often yielding unprecedented structural information, missing in conventional room temperature MAS NMR measurements. In this section, we recapitulate these findings and highlight the additional benefits obtained by DNP in the structural studies of viral capsids, and of the type III and IV bacterial secretion systems.

Viral capsids

To date three different types of viral assemblies have been successfully characterized using DNP-enhanced solid-state NMR, suggesting that DNP is uniquely positioned for viral capsid structural studies. This concerns the tubular assemblies of the HIV-1 capsid protein (CA) and its maturation intermediate (CA-SP1) [START_REF] Gupta | Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies[END_REF], the filamentous bacteriophage Pf1 formed by helical assemblies of the coat protein wrapping up the viral DNA [START_REF] Sergeyev | Chemical Shifts for the Unusual DNA Structure in Pf1 Bacteriophage from Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR Spectroscopy[END_REF][START_REF] Sergeyev | Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization[END_REF] and the icosahedral capsid assembly of the Acinetobacter phage 205 (AP205) coat protein [START_REF] Jaudzems | Dynamic nuclear polarization enhanced biomolecular NMR spectroscopy at high magnetic field with fast magic-angle spinning[END_REF].

The DNP experiments on HIV-1 CA and CA-SP1 assemblies allowed to directly observe functionally important residues that were invisible in room-temperature dipolarbased MAS NMR spectroscopy, cryo-EM and X-ray measurements because of dynamic disorder. This included the observation of most of the resonances of the SP1 peptide residues in the CA-SP1 maturation intermediate, multiple side-chain conformers for several residues and unique correlations arising from intermolecular interactions (Figure 1). The SP1 peptide resonances could be assigned based on a single DARR measurement and their chemical shifts indicated a helical conformation. While similar indications were obtained previously based on cryo-EM data of the in vitro assembled CA-SP1 capsids from Rous sarcoma virus, where electron density associated with SP1 could not be seen, the DNP NMR spectra provided a direct experimental evidence of the presence of the minor helical conformer. In addition, residues in the hinge region of CA, which play a critical role in capsid assembly and are difficult to detect at 253-277 K due to millisecond timescale dynamics, were readily observed and assigned in the DNP spectra. An intermolecular correlation between a key hinge residue, Y145 and R162, was also identified illustrating how DNP can provide critical restraints for structure determination of capsid assemblies. Two detailed DNP studies of the Pf1 bacteriophage have been reported in the literature. The first aimed at assigning the chemical shifts of the unusual DNA structure in the Pf1 virion [START_REF] Sergeyev | Chemical Shifts for the Unusual DNA Structure in Pf1 Bacteriophage from Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR Spectroscopy[END_REF], while the second proposed a novel DNP-enabled backbone assignment strategy [START_REF] Sergeyev | Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization[END_REF]. The DNA peaks were faintly observable in ssNMR spectra acquired at 213-243 K, and their assignment had been nearly impossible. With a 22-fold enhancement from DNP, these peaks could be clearly resolved and assigned for the first time. This assignment revealed extreme chemical shift values indicative of an unusual DNA structure (Figure 2), lacking hydrogen bonding but involving some type of aromatic ring interaction. The complete de novo assignment of the coat protein of Pf1 virus in the frozen state near 100 K has been achieved by combining the sensitivity benefits of DNP, fast MAS and non-uniform sampling, with the introduction of a new sequential side-chain-side-chain correlation method. Comparison of DNP and conventional NMR data collected near 100 and 273 K respectively showed 13 C and 15 N chemical shift perturbations into distinct regions of the protein, concomitant with a broadening of the lines. Correlations could be established between the temperature-induced perturbations and the hydration maps of the viral assembly, allowing rationalization in terms of solvation phenomena.

More recently, high quality DNP-enhanced NMR spectra of the Acinetobacter phage 205 (AP205) nucleocapsid could be obtained at high magnetic field (800 MHz) and fast MAS (40 kHz). These spectra enabled the assignment of aromatic resonances of the coat protein and its packaged RNA, which are not observed at room temperature. In particular, several new aromatic-to-aliphatic and aromatic-to-aromatic contacts were detected, which could be used to refine the 3D structure of the protein [START_REF] Jaudzems | Dynamic nuclear polarization enhanced biomolecular NMR spectroscopy at high magnetic field with fast magic-angle spinning[END_REF]. Although site-specific internuclear proximities had been already measured under DNP conditions in assemblies reconstituted from differently labelled components, [START_REF] Bayro | Intermolecular Structure Determination of Amyloid Fibrils with Magic-Angle Spinning and Dynamic Nuclear Polarization NMR[END_REF][START_REF] Maciejko | Visualizing Specific Cross-Protomer Interactions in the Homo-Oligomeric Membrane Protein Proteorhodopsin by Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR[END_REF] this study represents the first example where data extracted from DNP-enhanced spectra were constructively used in a structure determination on a uniformly labelled system.

Other large protein complexes

In recent years, exciting applications of DNP-enhanced solid-state NMR to large molecular assemblies other than viruses appeared. DNP has thus been applied to bacterial secretion systems (T3SS or T4SS) by the groups of Lange [START_REF] Fricke | Studies on the MxiH Protein in T3SS Needles Using DNP-Enhanced ssNMR Spectroscopy[END_REF][START_REF] Fricke | High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles[END_REF] and Baldus [START_REF] Kaplan | Probing a cell-embedded megadalton protein complex by DNP-supported solid-state NMR[END_REF]. As previously mentioned, the seminal work by Fricke and co-workers was very instructive concerning factors that determine line width in DNP NMR spectra [START_REF] Fricke | Studies on the MxiH Protein in T3SS Needles Using DNP-Enhanced ssNMR Spectroscopy[END_REF]. Here, the T3SS needles investigated are formed by rings of the protein MxiH whose structure shows two helices, helix one being located at the outer surface of the filamentous needle, and helix two at the inside of the ring. It turns out that under DNP conditions at 14.1 T and 104 K, using TOTAPOL (ε = 23), a sub-spectrum of sharper lines is observed that stems from the inner ring. The favorable line width observed for signals of residues in the inner ring is influenced by a locking of their side chains due to space restrictions, avoiding the formation of conformers and thus the appearance of heterogeneous broadening at the measurement temperatures used, and presumably also a larger distance to radicals. Later, the same system was investigated at a field strength of 18 T at 95 K and using AMUPol as radical, yielding an enhancement of ε = 21 [START_REF] Fricke | High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles[END_REF]. On the whole, a very similar spectrum was observed, however the resolved signals showed improved resolution (the line width was reduced by 22%), demonstrating thus a contribution of homogeneous broadening due to the presence of the radicals. The application of DNP enabled an investigation of a T4 secretion system core complex in vivo [START_REF] Kaplan | Probing a cell-embedded megadalton protein complex by DNP-supported solid-state NMR[END_REF], where it was found that it acquires a more compact structure than in vitro.

As an example of a large molecular machine that would defy NMR investigations under non-DNP conditions, aspects of ribosome structure were investigated. A first seminal investigation [START_REF] Gelis | Solid-state NMR enhanced by dynamic nuclear polarization as a novel tool for ribosome structural biology[END_REF] demonstrated the feasibility of DNP applications to the ribosome (ε = 25), and showed suitable line width by means of selectively labelled samples. A second investigation was focused on the structure of the nascent chain inside the exit tunnel [START_REF] Lange | Structural analysis of a signal peptide inside the ribosome tunnel by DNP MAS NMR[END_REF]. Here, the DsbA signal peptide was fused to the SecM stalling signal to generate ribosomes with the same nascent chain, and the expression protocol ensured its exclusive labelling with 13 C and 15 N. 13 C-double-quantum versus single-quantum spectra were recorded and the observed chemical shifts for isoleucine, valine, proline and serine residues interpreted with respect to the indicated secondary structure. It was shown that the DsbA signal peptide as nascent chain does not form a helix inside the ribosome tunnel and in consequence very unlikely to be influencing the surface of the ribosome with regards to promoting binding of factors co-translationally binding to the nascent chain.

Membrane proteins

A surprisingly large number of fruitful DNP MAS NMR investigations of membrane proteins appeared in the last few years, covering retinal-containing photoreceptors [START_REF] Bajaj | Functional and shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarizationenhanced solid-state NMR[END_REF][START_REF] Becker-Baldus | Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy[END_REF][START_REF] Maciejko | Visualizing Specific Cross-Protomer Interactions in the Homo-Oligomeric Membrane Protein Proteorhodopsin by Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR[END_REF][START_REF] Mehler | Chromophore Distortions in Photointermediates of Proteorhodopsin Visualized by Dynamic Nuclear Polarization-Enhanced Solid-State NMR[END_REF][START_REF] Stöppler | Dynamic Nuclear Polarization Provides New Insights into Chromophore Structure in Phytochrome Photoreceptors[END_REF] a G-protein-coupled receptor [START_REF] Joedicke | The molecular basis of subtype selectivity of human kinin G-protein-coupled receptors[END_REF], neurotoxin II bound to a ligand-gated ion channel (Linden et al., 2011b), and transporters [START_REF] Lehnert | Antigenic Peptide Recognition on the Human ABC Transporter TAP Resolved by DNP-Enhanced Solid-State NMR Spectroscopy[END_REF][START_REF] Ong | Detecting substrates bound to the secondary multidrug efflux pump EmrE by DNP-enhanced solid-state NMR[END_REF][START_REF] Spadaccini | The effect of drug binding on specific sites in transmembrane helices 4 and 6 of the ABC exporter MsbA studied by DNP-enhanced solid-state NMR[END_REF], illustrating the potential of DNP MAS NMR to the wide field of membrane proteins. Since such proteins may be investigated in frozen detergent solutions, after incorporation into liposomes, or when still embedded in native membranes, a variety of approaches were tested. In some cases, the application of DNP appeared natural and did not bring about additional complications, because the respective preparations needed to be investigated in the frozen state anyways for reasons of unfavourable protein dynamics interfering with MAS NMR requirements (especially those dissolved in detergent solutions and some samples involving proteoliposomes). With regards to the DNP-specific measurement conditions, the glycerol content was often optimized to an appropriately low amount, choosing mostly 30%, or sometimes even less. In individual cases, radicals were added without glycerol to the membrane protein samples that were equilibrated in an appropriate H 2 O/D 2 O mixture after preparation. In investigations of the photoactive proteins, signal enhancements in the range of 30-60 are reported at 9.4 T when using AMUPol together with a reduced amount of glycerol. The highest enhancement was reported in an investigation of the bradykinin receptor in frozen detergent solutions (ε = 169, AMUPol).

In a seminal paper on bacteriorhodopsin within intact purple membranes, the group of Griffin reported additional sets of signals occurring upon trapping photointermediates at low temperatures [START_REF] Bajaj | Functional and shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarizationenhanced solid-state NMR[END_REF], in particular four sets for the L state. In this case, a field strength less than 9 T and TOTAPOL were employed, yielding enhancements in the range of 90 at 90 K. The very stable purple membranes were kept in 60% d 8 -glycerol for the measurements. Investigations on channelrhodopsins [START_REF] Becker-Baldus | Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy[END_REF][START_REF] Stöppler | Dynamic Nuclear Polarization Provides New Insights into Chromophore Structure in Phytochrome Photoreceptors[END_REF] in proteoliposomes of phosphatidylcholine or POPC/POPG/Cholesterol aimed at determining the isomer composition in the long-term dark state, finding more than 95% alltrans retinal in contrast to earlier biochemical investigations, and to detect intermediates of the photocycle. The group of Glaubitz was successful in determining intermolecular contacts within proteorhodopsin pentamers [START_REF] Maciejko | Visualizing Specific Cross-Protomer Interactions in the Homo-Oligomeric Membrane Protein Proteorhodopsin by Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR[END_REF], using mixed samples of 15 N and 13 C labelled protein in DMPC/DMPA proteoliposomes, and applying 30% v/v glycerol as cryoprotectant. Measurements of such heteronuclear interactions in mixed or diluted samples require sufficient signal-to-noise, and DNP was critical to this end. In a second investigation on proteorhodopsin, deviations of retinal structure from an ideal planar arrangement were detected with the help of DNP [START_REF] Mehler | Chromophore Distortions in Photointermediates of Proteorhodopsin Visualized by Dynamic Nuclear Polarization-Enhanced Solid-State NMR[END_REF].

Several transporters were studied by DNP NMR with a focus on mechanism. An investigation on the secondary transporter EmrE was set out to test the effects of substrate binding under DNP conditions [START_REF] Ong | Detecting substrates bound to the secondary multidrug efflux pump EmrE by DNP-enhanced solid-state NMR[END_REF], using DMPC proteoliposomes, reporting an enhancement of 19 at 9 T. Conformational changes in the ABC transporter MsbA embedded in DMPC/DMPA liposomes, in particular a movement of helices 4 and 6, were reported in connection with ligand binding and ATP hydrolysis [START_REF] Spadaccini | The effect of drug binding on specific sites in transmembrane helices 4 and 6 of the ABC exporter MsbA studied by DNP-enhanced solid-state NMR[END_REF]. A landmark investigation concerned peptide binding to the detergent-solubilized transporter associated with antigen processing (TAP) [START_REF] Lehnert | Antigenic Peptide Recognition on the Human ABC Transporter TAP Resolved by DNP-Enhanced Solid-State NMR Spectroscopy[END_REF], and included the derivation of an atomic-resolution structural model of a bound peptide, revealing interactions critical for TAP binding and allowing to explain substrate selection rules.

An example for the investigation of a membrane protein embedded in whole membrane patches is given by the Reif laboratory [START_REF] Jacso | Characterization of Membrane Proteins in Isolated Native Cellular Membranes by Dynamic Nuclear Polarization Solid-State NMR Spectroscopy without Purification and Reconstitution[END_REF]. There, the protein mistic contained 13 C-labelled glycine, serine, threonine, and valine, and was expressed into E. coli membranes to demonstrate that it adopts α-helical structure in this environment. Membrane patches were purified and directly subjected to DNP MAS NMR measurements with no glycerol added, yielding spectra that enabled an analysis of the backbone and Cβ chemical shifts with regards to the adopted secondary structure. The application of 20-40 mM TOTAPol as radical yielded enhancements in the range of 15-30.

Recently, the conformations of ligands bound to certain subtypes of the bradykinin receptor were determined by solid-state NMR under DNP conditions [START_REF] Joedicke | The molecular basis of subtype selectivity of human kinin G-protein-coupled receptors[END_REF]. Dilute but frozen detergent solutions of the receptor were employed, containing 50% v/v d 8glycerol, hence the application of DNP was critical and the large enhancement of ε = 169 was absolutely welcome. To obtain resonance assignments, peptides were labelled at individual sites, employing labelling of one or two amino acids, and structures calculated on the basis of dihedral angles derived from the chemical shifts of backbone atoms and Cβ.

Conclusion:

With the introduction of highly efficient polarization sources and fast MAS cryogenic probes, MAS DNP enhanced biomolecular NMR spectroscopy has recently greatly matured. As exemplified by the applications described above, it has become an effective and relevant technique for the structural investigation of biological assemblies, in the (several) cases where conventional room temperature NMR lacks sensitivity. The broadening of the NMR lines observed systematically at cryogenic temperatures (together with the today's scarcity of very high-field DNP NMR spectrometers) so far hamper, however, an optimal and therefore broader use of this technique. In this context, the future development of new formulations or experimental approaches that will allow one to maintain a high signal enhancement factor with no penalty in spectral resolution appear highly strategic.

Figure 1 .

 1 Figure 1. Amino acid sequence of the SP1 peptide and an overlay of DNP-enhanced 13 C-13 C DARR spectra at 109 K and 600 MHz of tubular assemblies of U-13 C, 15 N CA (blue) and CA-SP1 (black). The MAS frequency was 12.5 kHz, and the mixing time was 40 ms. Assigned SP1 resonances are labeled by residue name and number. Adapted with permission from Gupta et al., 2016. Copyright 2016 American Chemical Society.

Figure 2 .

 2 Figure 2. DNP-enhanced SSNMR spectra of Pf1 showing (a) the assignments of the dC/dT base resonances, (b) the dA/dG base resonances, and (c) the sugar spin systems. Data were collected using a 400 MHz wide-bore AVANCE III spectrometer at cryogenic temperatures (100 K), with a mixing time of 22 ms. Adapted with permission from Sergeyev 2011. Copyright 2011 American Chemical Society.

Figure 3 .

 3 Figure 3. POST-C7 DQ-SQ spectra of the ribosome embedded nascent chain S16A/S18A-DsbA-SecM (left panel) and S22A/S28A-DsbA-SecM (right panel). Averaged chemical shifts for helical (red squares), strand-like (blue squares), or coil-like (green triangles) geometries are shown (35). Well-separated cross peak patterns of amino acids that do not strongly overlap with other residue types are labeled accordingly. Acquisition in the direct and indirect dimension was 20 ms (1582 points) and 2 ms (128 points), respectively. Adapted with permission from Lange 2016.

Table 1 .

 1 Experimental conditions used for DNP-enhanced NMR studies of various molecular assemblies

	Sample	Preparation	Matrix/cryo-	Radical	Labeling	B0	MAS	T (K)	ε ( 1 H-	13 C	Source
			protectant			(MHz)	(kHz)		13 C	linewidths
			composition						CP)	(ppm)
	Pf1	PEG-8000	40/50/10 %	20 µM	uniform	400		100	17/22	1.8,	(Sergeyev
		precipitate	(v/v/v)	TOTAPOL	13C-and					average	et al.,
			DMSO-		15N					2.4	2011)
			d6/D2O/H2O,
			and 20%
			w/v PEG-
			8000.
	Pf1	PEG-8000	30/70 %	10 mM	uniform	600	11	98-106	35	1.3	(Sergeyev
		precipitate	glycerol-	AMUPol	13C-and						et al.,
			d8/D2O		15N						2017)
	Pf1	PEG-8000	30/70 %	10 mM	uniform	600	25	98-108	65		(Sergeyev
		precipitate	glycerol-	AMUPol	13C-and						et al.,
			d8/D2O		15N						2017)
	HIV-1	Frozen protein	50/37.5/12.5	10 mM	uniform	400	8.9	110/182 20/2		(Gupta et
	CA-SP1-	solution	% (v/v/v)	TOTAPOL	13C-and						al., 2016)
	NC		glycerol-		15N
	protein		d8/D2O/H2O
	HIV-1 CA	Sedimented	20/80 % (v/v)	8 mM	uniform	600	12.5	109/180 64/20		(Gupta et
	protein	tubular	glycerol-	AMUPol	13C-and						al., 2016)
		assemblies	d8/H2O		15N
	HIV-1	Sedimented	20/80 % (v/v)	8 mM	uniform	600	12.5	109	20	(0.5) 1.5-	(Gupta et
	CA-SP1	tubular	glycerol-	AMUPol	13C-and					2.0	al., 2016)
	protein	assemblies	d8/H2O		15N
	HIV-1 CA	Sedimented	20/80 % (v/v)	8 mM	uniform	800	10	110	4		(Gupta et
	protein	tubular	glycerol-	TOTAPOL	13C-and						al., 2016)
		assemblies	d8/H2O		15N
	HIV-1 CA	Sedimented	20/80 % (v/v)	8 mM	uniform	800	10	110	4		(Gupta et
	protein	tubular	glycerol-	AMUPol	13C-and						al., 2016)
		assemblies	d8/H2O		15N
	HIV-1	Sedimented	20/72/8 %	8 mM	uniform	800	10	110	7-11		(Gupta et
	CA-SP1-	tubes and cones	(v/v) glycerol-	AMUPol	13C-and						al., 2016)
	NC		d8/D2O/H2O		15N
	protein
	AP205	Microcrystalline 16% (w/v)	10 mM	uniform	400	12.5	107	82	1.9-2.8	(Jaudzems
			deuterated	AMUPol	13C-and						et al.,
			PEG-4600		15N						2018)
	AP205	Microcrystalline 16% (w/v)	10 mM	uniform	800	12	103	16	1.1-1.8	(Jaudzems
			deuterated	AMUPol	13C-and						et al.,
			PEG-4600		15N						2018)
	AP205	Microcrystalline 16% (w/v)	10 mM	uniform	800	40	115	17	1.0-1.7	(Jaudzems
			deuterated	AMUPol	13C-and						et al.,
			PEG-4600		15N						2018)
	MxiH	In vitro	72/25/3 %	28 mM	uniform	600	11	104	23	1.1-2.2	(Fricke et
	needles	polymerized	(v/v/v)	TOTAPOL	13C-and						al., 2014)
		needles	glycerol-		15N
			d8/D2O/H2O
	MxiH	In vitro	70/30 % (v/v)	14 mM	uniform	800	8	95	21	0.9-1.8	(Fricke et
	needles	polymerized	glycerol-	AMUPol	13C-and						al., 2016)
		needles	d8/D2O		15N
	T4SScc	Cell-embedded	1)	20 mM	selective	400	8	100	60		(Kaplan et
		protein complex	45/32.5/22.5	AMUPol	13C,15N-						al., 2015)
			% (v/v/v)		Gly, Ser,
			glycerol-		Leu, Val
			d8/D2O/H2O		or
			2)		selective
			22.5/32.5/42.5		13C,15N-
			% (v/v/v)		Thr, Val
			glycerol-
			d8/D2O/H2O
	T4SScc	Cell-embedded				800	8	100	15	1.5	(Kaplan et
		protein complex									al., 2015)

Acknowledgements: KJ is supported by a MC incoming fellowship (REA grant agreement n°661175 "virus-DNP-NMR").


Claude Daubert

Claire Bouleti

Nicolas Danchin

Bernard Iung
email: bernard.iung@aphp.fr

Evolution of scientific activities and production over the last decade

Keywords: CVD, cardiovascular disease, EHJ: European Heart Journal, HPCR, Hospital

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

Background

In France, as in most countries, cardiovascular disease (CVD) is a leading cause of death, along with cancer, and is responsible for more than 1 in 4 deaths (approximately 140,000 deaths per year) [START_REF]Maladies cardiovasculaires[END_REF][START_REF] Tzoulaki | Worldwide Exposures to Cardiovascular Risk Factors and Associated Health Effects: Current Knowledge and Data Gaps[END_REF]. Cardiovascular research, diagnostic tool refinement and prevention have improved survival in Europe [START_REF] Nichols | Trends in age-specific coronary heart disease mortality in the European Union over three decades: 1980-2009[END_REF] and the USA, with CVD deaths decreasing from 771,169 in 1985 to 596,577 in 2011, whereas the number of deaths from cancer has nearly tripled over the last 60 years [START_REF] Heron | Changes in the Leading Cause of Death: Recent Patterns in Heart Disease and Cancer Mortality[END_REF]. This decrease in CVD mortality has led to a need for clinical trials with very large sample sizes and longer periods of follow-up to demonstrate any further improvement in survival [START_REF] Calvo | Large streamlined trials in cardiovascular disease[END_REF][START_REF] Collins | Reliable assessment of the effects of treatment on mortality and major morbidity, I: clinical trials[END_REF][START_REF]MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20,536 high-risk individuals: a randomised placebo-controlled trial[END_REF].

In parallel, financial investment in cardiovascular research, particularly funding by industry, has declined because of the many challenges involved, including regulatory requirements, extensive clinical trial management bureaucracy and the complexity of the healthcare system [START_REF] Jackson | Improving clinical trials for cardiovascular diseases: a position paper from the Cardiovascular Round Table of the European Society of Cardiology[END_REF].

The aim of this paper was to analyse scientific production and vitality in France, and its evolution over the last decade. We focused mainly on clinical research, as the fundamental aspects of CVD research have already been addressed in a recent paper [START_REF] Boulanger | Cardiovascular Research in France[END_REF], although some barriers are common between basic and clinical research.

Methods

For global analysis, we used Lab Times online data (labtimes.org) obtained through the Web of Science (Thomson-Reuters, Toronto, ON, Canada) for articles published from 2007 to 2013 (quoted from 2009 to 2015) to analyse original articles. This analysis dealt with the 352 specialty or subspecialty journals listed in the database of the SCImago Journal & Country Rank portal. However, these data do not take into account cardiovascular studies published in multidisciplinary medical journals or the precise rank of the authors. To overcome these limitations, we further analysed all the studies published between 2005 and 2015 in cardiovascular or multidisciplinary journals with high impact factors (IFs). This analysis was restricted to the three multidisciplinary journals with the highest The number of cardiovascular clinical research projects was analysed by using the ClinicalTrials.gov database and the French healthcare site for government-funded projects (https://solidarites-sante.gouv.fr/systeme-de-sante-et-medico-social/recherche-et-innovation/linnovation-et-la-recherche-clinique/appels-a-projets/article/les-appels-a-projets-de-la-dgos-les-projetsretenus). For the ClinicalTrials.gov site, the projects list was obtained by requesting: ("Aortic disease"or "Heart disease"or "Cardiac disease"or "Cardiovascular disease"or "Valvular disease") between 01 January 2008 and 31 December 2017, for each country of interest.

For the government-funded projects, all data between 2008 and 2017 were obtained via the dedicated website. All projects were classified by the research topic, with 19 topic categories identified (Appendix B). In case of doubt regarding the best category for classification of a project (frontiers form between two specialties), the evaluation was performed by two reviewers (B. I. and C. B.), and the final classification was reached by consensus.

Results

French publications in the CVD field

Rank in Europe and worldwide

Based on the Lab Times evaluation obtained through the Web of Science portal, France was ranked fifth in Europe for articles in the CVD field published between 2007 and 2013 (citations from 2009 to 2015), as illustrated in Table 1.

Evolution of French publications in the journals with the highest IFs, from 2005 to 2015

Multidisciplinary journals

We found 52 French publications during the 10-year period between 2005 and 2015 in the NEJM, Lancet and JAMA; an average of 5 publications per year. These French publications accounted for 0.2% of all publications in Lancet and 0.3% of all publications in NEJM or JAMA. Over the years, there was a trend for more publications in Lancet and JAMA, while French participation remained stable in NEJM. Overall, French production in these journals remains low. These results are detailed in Table 3 and Fig. 1A.

Cardiology journals

When focusing on the top three cardiology journals (EHJ, Circulation and JACC), French publications accounted for about 2% of all published articles, with 423 papers. The majority of the articles were published in the EHJ (202 articles; 3.2%), while they accounted for < 2% of publications in journals from the USA: 118 articles (1.9%) in JACC and 103 articles (1.2%) in Circulation. Over time, publication remained stable in Circulation and slightly decreased in JACC, but decreased in EHJ, with an average number of publications of 21 articles/year between 2005 and 2010, and 15 articles/year between 2011 and 2015. These results are illustrated in Table 3 and Fig. 1B.

Subspecialty journals

Regarding the four main subspecialties of cardiology (interventional cardiology, electrophysiology, imaging and heart failure), we studied the journals derived from EHJ, JACC and Circulation, as detailed in Appendix A. The results are detailed in Table 4 for each subspecialty.

Interventional cardiology

published in the European Journal of Heart Failure, which has the highest IF of the three journals.

These results are detailed in Table 4 and Fig. 2D.

French abstracts at the ESC congress

Participation in the ESC congress is another reflection of research activity. Over the last decade, we have observed a steady decrease in submissions and acceptances of French abstracts at the ESC congress (Fig. 3). From 2009 to 2013, approximately 5% of submitted abstracts were French; this percentage was around 3.5% from 2016 to 2018. Consequently, the percentage of accepted French abstracts at the ESC congress decreased in proportion, from over 4% to 2.5%.

Regarding the position of France compared with other countries, detailed information was available for the 2017 ESC congress, and is illustrated in Fig. 4. French cardiologists submitted 274 abstracts, which placed France in eighth position in Europe, behind Germany, Italy, Spain, the UK, Portugal, Poland and Greece. The acceptance rate for French abstracts was 58% (159 accepted abstracts), which was better than the rates for Germany (52%), the UK (51%), Italy (50%), Spain (41%), Greece (36%), Poland (34%) and Portugal (32%). France therefore ranked fifth for accepted abstracts (Fig. 4A). However, France ranked thirteenth when the number of accepted abstracts was divided by the number of cardiologist members of the ESC, and fifteenth when the number of accepted abstracts was divided by the population of the country (Fig. 4B)

Dynamism of French cardiovascular research regarding new projects

Analysis of the ClinicalTrials.gov database

The ClinicalTrials.gov database lists the registries and clinical trials launched in each country.

Between 2008 and 2017, there were 23,725 studies in the cardiovascular field, including both interventional and observational studies. The evolution of the number of studies submitted per year is illustrated in Fig. 5A for the world and for France.

There has been an increase in the annual number of declared studies on ClinicalTrials.gov over the past 10 years, from 1797 in 2008 to 2721 in 2017 worldwide; this evolution was similar for French data, with 173 studies registered in 2008 and 245 in 2017. French participation accounted for approximately 9% of cardiovascular research, with a minimal rate of 7.6% in 2012 and a maximal rate of 10.1% in 2015 (Fig. 5A). This increase was also observed for interventional studies and registries, both worldwide and for France (Fig. 5B and Fig. 5C, respectively). Over the last 10 years, the USA registered 1747 observational studies out of 7848 studies (22%), whereas France submitted 696 observational studies out of 2070 studies (34%). Moreover, as illustrated in Fig. 5C, the proportion of registries has actually increased in France over time, accounting for 40% of total submitted research projects over the last 3 years.

Compared with non-European countries, the results for France were good, as the country was ranked second behind the USA -the uncontested leaders, with 7848 submitted studies over the last decade. France ranked better than Canada, China and Japan (Fig. 6A).

Only six European countries had > 1000 registered studies over the 10 years of interest: Germany, France, the UK, Italy, Spain and the Netherlands. With 2070 registered studies, France ranked second, just after Germany (2160 studies) and before the UK (1669 studies), Italy, Spain and the Netherlands (Fig. 6B). More importantly, the analysis of temporal trends in the number of studies registered for the top three European countries (Germany, the UK and France), showed that France has been ranked first, before Germany, in recent years (Fig. 6C). Indeed, Germany has had stable production over time (207 studies in 2008 and 193 in 2017), while France has had a steady increase in study promotion over the last decade (from 173 studies in 2008 to 245 in 2017). France was ranked first in 2013, and has remained the leader in the cardiovascular field over the last 3 years. This positive evolution of CVD research in France compared with Germany and the UK was observed for both interventional and observational studies; France has ranked first in these two categories over the last 3 years (Fig. 6D and Fig. 6E).

Analysis of accepted French government-funded clinical research projects

The Hospital Programme of Clinical Research (HPCR) was created in 1992 in France to promote medical progress, dynamize hospital clinical research and improve patient care. Since 1993, the government has launched the HPCR project call once a year, and has provided financial support for accepted projects (total funding of €130,000 for 2016) Between 2008 and 2017, 1231 research projects were accepted by the HPCR. The maximal number of accepted projects was reached between the years 2009 and 2011 (average of 174 projects/year), and the minimum was reached in 2013 (86 projects), which corresponded with the launch of another research programme (the medicoeconomical research programme). The number of projects per year and per pathology domain are detailed in Table 5, for the eight medical specialties with ≥ 60 accepted projects over the study period.

The number of accepted projects was highest in clinical neurosciences, with 183 projects (15%).

Heart ranked second, with 124 accepted projects, followed by psychiatry, digestive and liver diseases, kidney-urinary diseases, respiratory diseases and vascular diseases. When considering CVD instead of heart and vascular diseases separately, CVD was ranked first over the decade, with 184 accepted projects, just before neurosciences (Fig. 7A). CVD has ranked first since 2014, and the evolution of the rate of accepted projects over the total number of projects has increased over time (Fig. 7B). Indeed, until 2013, the rate of accepted CVD projects was 0-15%, then 15-20% in 2014-2015 and 20-25% in the last 2 years.

Discussion

Clinical research data in the CVD field over the last decade in France show contrasting results. France is very well represented in terms of new CVD projects, whether government-funded projects (HPCR) or worldwide registered projects (ClinicalTrials.gov database). Conversely, actual French scientific production scored poorly and has tended to decline, as illustrated by abstracts presented to the world's largest cardiovascular congress (the ESC congress), and especially by published articles in top journals. Scientific production is mainly assessed by the publication of original articles, in highranking international journals. Although IFs have been vigorously challenged in recent years [START_REF] Abbasi | Let's dump impact factors[END_REF], and may indeed be an imperfect metric, they remain the means by which most journals are judged [START_REF] Fuster | Impact factor versus impact to readers: not necessarily at odds[END_REF]. In the three multidisciplinary journals and three cardiovascular journals with the highest IFs, France is now ranked fifth in Europe and eighth worldwide. On the other hand, France ranks in the top three and is often a leader in Europe, as for project submissions. These discrepancies should be further analysed to reverse the current trends. Several explanations for the current situation can be proposed.

Too much mediatized success for cardiology in France ?

France is widely considered as an important country for medical advances in the field of CVD.

Tremendous progress has been achieved in the management of coronary artery diseases, electrophysiology and heart valve diseases, with French researchers being involved or pioneers in these fields [START_REF] Carpentier | Cardiac valve surgery--the "French correction[END_REF][START_REF] Cazeau | Multisite pacing for end-stage heart failure: early experience[END_REF][START_REF] Cribier | Percutaneous transcatheter implantation of an aortic valve prosthesis for calcific aortic stenosis: first human case description[END_REF][START_REF] Daubert | Past, present and future of cardiac resynchronization[END_REF][START_REF] Haissaguerre | Spontaneous initiation of atrial fibrillation by ectopic beats originating in the pulmonary veins[END_REF][START_REF] Latremouille | Animal studies with the Carmat bioprosthetic total artificial heart[END_REF][START_REF] Puel | Early and late assessment of stenosis geometry after coronary arterial stenting[END_REF][START_REF] Steg | Bivalirudin started during emergency transport for primary PCI[END_REF][START_REF] Vahanian | Results of percutaneous mitral commissurotomy in 200 patients[END_REF]. For example, the mortality rates for myocardial infarction have achieved their lowest level in France, with rates as low as 3% at 30 days and 5.3% at 6-month follow-up, with a further 22% reduction in standardized mortality from 2010 to 2015 [START_REF] Puymirat | Acute Myocardial Infarction: Changes in Patient Characteristics, Management, and 6-Month Outcomes Over a Period of 20 Years in the FAST-MI Program (French Registry of Acute ST-Elevation or Non-ST-Elevation Myocardial Infarction) 1995 to 2015[END_REF][START_REF] Puymirat | Association of changes in clinical characteristics and management with improvement in survival among patients with ST-elevation myocardial infarction[END_REF]. This decrease in coronary disease mortality and the longer global average life span also rely on the validation of new therapeutic concepts supported by large trials and active research [START_REF] Nichols | Trends in age-specific coronary heart disease mortality in the European Union over three decades: 1980-2009[END_REF]. However, these advances should not overshadow the fact that CVD remains the most common cause of morbimortality worldwide [START_REF] Murphy | Deaths: final data for 2010[END_REF][START_REF] Nichols | Cardiovascular disease in Europe: epidemiological update[END_REF].

In France, a study concerning French health insurance beneficiaries showed that the most common causes of deaths in 2013 were cardiovascular/neurovascular diseases (56%), cancers (42%) and neurological and degenerative diseases (25%) [START_REF] Poulalhon | Deaths in France: Characteristics, place of death, hospitalisations and use of palliative care during the year before death[END_REF]. However, cancer and neurodegenerative diseases seem to be considered to be of greater concern to the general population in France, and the financial incentives from the government are much higher than those offered for CVD [START_REF] Boulanger | Cardiovascular Research in France[END_REF].

Perhaps CVD research is a victim of its own success and, despite major breakthroughs during the last decades, a reminder is needed of the large burden of CVD. It is indeed in the patients' interest to benefit from adequate levels of clinical research and translation into new prevention and therapeutic strategies. However, there are limitations to CVD research in France, both at the financial and institutional levels.

Financial difficulties and an institutional brake to CVD research

As showed by Boulanger et al., the research and development budget has not increased over the past 20 years, placing France below the average Organization for Economic Cooperation and Development (OECD) countries for research financial support [START_REF] Boulanger | Cardiovascular Research in France[END_REF]. Nonetheless, financial support is a key issue for current CVD research, particularly given the decline in cardiovascular mortality, which leads to a need for larger population studies with very large sample sizes to bring to light incremental risk reduction [START_REF] Calvo | Large streamlined trials in cardiovascular disease[END_REF][START_REF] Collins | Reliable assessment of the effects of treatment on mortality and major morbidity, I: clinical trials[END_REF][START_REF] Jackson | Improving clinical trials for cardiovascular diseases: a position paper from the Cardiovascular Round Table of the European Society of Cardiology[END_REF]. Larger population studies actually translate into greater budgets. Moreover, publication standards have increased over time, and publishing in top journals now requires much more data and time, thereby increasing costs [START_REF] Boulanger | Cardiovascular Research in France[END_REF].

In parallel to the government's financial participation, the pharmaceutical industry's willingness to invest in CVD has also declined. Advocated reasons are, in particular, increasing regulatory requirements, the bureaucracy associated with conducting clinical trials and the difficulties in guaranteeing access to newly-approved medications [START_REF] Calvo | Large streamlined trials in cardiovascular disease[END_REF][START_REF] Boulanger | Cardiovascular Research in France[END_REF][START_REF] Reith | Randomized clinical trials--removing unnecessary obstacles[END_REF]. However, despite the lower level of funding, CVD research remains active and productive in France, and still competes with research in the neurosciences field, as illustrated by the results of the HPCR proposals. Indeed, after many years of dominance by neurosciences research projects, cardiology -especially CVD research -has become the leader in HPCR-accepted projects. However, this may not be enough to translate these promising projects into high-ranking publications, given the limited funding associated with HPCR. The question is to know how long CVD researchers will maintain this high quality of research without appropriate funding, and how to prevent a decline in competitiveness compared with other specialties and other countries that are increasing their financial support for CVD research [START_REF] Heusch | Cardiovascular Research in Germany[END_REF].

The place of France in CVD research has been great and, with some changes in public policies, more golden years are yet to come. Indeed, as presented in this study, France has the ideas and the energy to produce high quality research, and even ranks first in Europe in terms of the number of cardiovascular projects registered online on the ClinicalTrials.gov website.

French culture: French language and underexploitation of databases

The vitality of CVD research, as illustrated by the number of projects launched, does not translate sufficiently enough into high-ranking publications. In addition to the funding limitations detailed in the previous section, French cardiologists do not have a culture of intensive database exploitation. Indeed, whereas large databases in the USA and Sweden, for example, lead to high numbers of publications, most French studies give rise to only one to five publications. The FAST-MI registry should be regarded as exemplary, with more than 50 publications in international journals [START_REF] Puymirat | Association of changes in clinical characteristics and management with improvement in survival among patients with ST-elevation myocardial infarction[END_REF]. This approach should be taught to researchers and championed by the leaders of CVD research in France.

Lastly, in addition to the different barriers listed previously to France being better represented in global CVD research, the French face another limitation compared with other European countries: the inability to speak fluent English. The French were ranked recently as the worst English speakers in Europe, according to the Education First English Proficiency Index [START_REF] Mitchell | Study Offers Snapshot of Global English-Language-Learner Trends[END_REF]. These results may lead to changes in public policies regarding the English teaching system in France, and to reform our dubbing industry, which provides French language on most national television channels. This may seem a minor detail, but the low involvement of French cardiologists in the ESC congress may be the result, in part, of this limitation. Once this language limitation is overcome, we can reasonably expect that French cardiologists will be more present and active at the ESC congress, especially given the good acceptance rate of French abstracts. The quality of our research is good, but incentivizing public policies are needed to overcome language and funding limitations in the years to come.

Young cardiologists are strongly involved in France, with one-third of French Society of Cardiology members aged < 40 years. This new generation of cardiovascular researchers should not be denied the possibility to improve patient care through high quality research in their field. Investing in CVD research seems therefore to be a priority, to increase the level of publication and to compete with other leading countries in Europe and worldwide.

  IFs (New England Journal of Medicine [NEJM], The Lancet and Journal of the American Medical Association [JAMA]) and the three cardiology journals with the highest IFs (Circulation, Journal of the American College of Cardiology [JACC] and European Heart Journal [EHJ]). We also analysed the four main cardiology subspecialty (interventional cardiology, cardiovascular imaging, electrophysiology and heart failure) journals derived from the three cardiology journals with the highest IFs. The 12 subspecialty journals are listed in Appendix A. The publication list was obtained from the PubMed database (National Center for Biotechnology Information [NCBI], Bethesda, MD, USA) by requesting (("Circulation"[Journal]) AND "journal article"[Publication Type]) AND ("2005/01/01"[Date -Publication] : "2005/12/31"[Date -Publication]). Only original articles, reviews, recommendations and consensus or position papers in the CVD field were included. The articles were considered as French only if the first or last author had a French affiliation. European Society of Cardiology (ESC) congress abstract publications were evaluated. Data on the number of French abstracts submitted and accepted were obtained directly from the ESC.

  The detailed analysis of temporal trends showed that clinical neurosciences dominated the HPCR acceptance rate, particularly during the years 2008-2011. Heart ranked second most of the time(2008, 2011, 2014, 2015 and 2017), but came first in 2016.

Figure 1 .

 1 Figure 1. A. Evolution of the proportion of French publications in the top three multidisciplinary journals. B. Evolution of the proportion of French publications in the top three cardiology journals. Eur Heart J: European Heart Journal; JACC: Journal of the American College of Cardiology; JAMA: Journal of the American Medical Association; NEJM: New England Journal of Medicine.

Figure 2 .

 2 Figure 2. Evolution of the proportion of French publications in subspecialty journals. A. Interventional cardiology. B. Cardiovascular imaging. C. Electrophysiology. D Heart failure. Arrhy Electr: Arrhythmia and Electrophysiology; Circ: Circulation; CV Imaging: Cardiovascular Imaging; CV Int: Cardiovascular Interventions; EHJ: European Heart Journal; Eur J: European Journal; HF: Heart Failure; JACC: Journal of the American College of Cardiology.

Figure 3 .

 3 Figure 3. French abstracts submitted to and accepted by the European Society of Cardiology congress between 2009 and 2018. A. Numbers. B. Percentages.

Figure 4 .

 4 Figure 4. A. Abstracts submitted to and accepted by the 2017 European Society of Cardiology congress among European countries. B. Ratio of accepted abstracts to total French population.

Figure 5 .

 5 Figure 5. Evolution of the number of cardiovascular studies registered on ClinicalTrials.gov between 2008 and 2017. A. All studies worldwide and in France. B. Interventional and observational studies worldwide. C. Interventional and observational studies in France.

Figure 6 .

 6 Figure 6. A. Number of registered cardiovascular studies worldwide. B. Number of registered cardiovascular studies in Europe. C-E. Temporal trends over the last decade in the top three European countries. C. All studies. D. Interventional studies. E. Observational studies.

Figure 7 .

 7 Figure 7. A. Number of accepted projects according to medical specialty over the decade 2008-2017. B. Evolution of cardiovascular disease (CVD) representation over the decade 2008-2017.

Table 1

 1 Ranking of the top 10 European countries according to the number of citations and published articles in cardiovascular disease field.

		Number of citations	Number of articles
	Europe	1,136,157	80,984
	European ranking
	1. Germany	279,208	14,980
	2. UK	237,984	12,397
	3. Italy	222,467	13,165
	4. Netherlands	187,910	9369
	5. France	153,515	8369
	6. Spain	81,769	5052
	7. Sweden	77,444	4050
	8. Switzerland	73,760	3883
	9. Belgium	72,213	3266
	10. Denmark	49,396	2583

Table 2

 2 Ranking of the top 10 countries worldwide according to the number of citations and published articles in the cardiovascular disease field.

		Number of citations	Number of articles
	Europe	1,136,157	80,984
	World ranking
	1. USA	1,049,838	59,915
	2. Germany	279,208	14,980
	3. UK	237,984	12,397
	4. Italy	222,467	13,165
	5. Netherlands	187,910	9369
	6. Canada	167,114	8640
	7. Japan	160,840	13,981
	8. France	153,515	8369
	9. Australia	85,306	4963
	10. Spain	81,769	5052

Table 3

 3 French cardiovascular disease publications in major multidisciplinary and cardiology journals between 2005 and 2015.

	Journals	IF	Number	%	Number/year	%/year	Trend
	Major multidisciplinary journals
	NEJM	79.3	21	0.3	1-4	0.1-0.4	Stable
	Lancet	53.2	17	0.2	0-4	0-0.4
	JAMA	47.7	14	0.3	0-3	0-0.4
	Major cardiology journals
	Circulation	18.9	103	1.2	4-13	0.5-2	Stable
	JACC	16.8	118	1.9	4-16	0.9-2.7
	EHJ	23.4	202	3.2	14-26	1.9-6.4
	Total		475	1.8

EHJ: European Heart Journal; IF: impact factor; JACC: Journal of the American College of Cardiology; JAMA: Journal of the American Medical Association; NEJM: New England Journal of Medicine.

Table 4

 4 French cardiovascular disease publications in major cardiology subspecialty journals between 2005 (or the year of the journal creation) and 2015. European Heart Journal; IF: impact factor; JACC: Journal of the American College of Cardiology.

	Journals

Table 5

 5 Evolution of number of accepted projects by medical specialty over the decade 2008-2017.

	Number of accepted projects/specialty	2008	2009	2010	2011	2012	2013	2014	2015	2016	2017	Total
	All	134	174	167	174	112	86	89	103	95	97	1231
	Heart	17	9	8	20	11	9	7	13	16	14	124
	Digestive and liver diseases	9	14	9	10	7	8	5	6	5	15	88
	Gynaecology	7	9	10	10	6	5	8	9	3	2	69
	Infectious diseases	5	10	13	10	6	5	2	7	6	2	66
	Clinical neurosciences	25	25	30	34	16	14	8	15	11	5	183
	Respiratory diseases	1	10	13	8	8	11	8	6	9	2	76
	Psychiatry	11	20	12	18	11	8	8	8	10	9	115
	Kidney/urinary diseases	9	9	11	14	11	8	4	4	7	7	84
	Vascular diseases	4	6	7	6	4	4	8	7	5	9	60

The evolution was not available for the whole 10-year period for JACC Cardiovascular Interventions and Circulation Cardiovascular Interventions, which were created in 2008 and 2009, respectively.

In total, 164 French articles were published between 2005 and 2015, which accounted for 3.9% of all published articles: 2.1% in JACC Cardiovascular Interventions, 2.9% in Circulation Cardiovascular Interventions and 5.6% in Eurointervention. Overtime, there was a trend toward increased production in Circulation Cardiovascular Interventions, while the number of French publications decreased in the two other journals. These results are detailed in Table 4 and Fig. 2A.

Acknowledgments

We thank Christine Gouillard and Isabel Bardinet from the ESC for sharing their data regarding French involvement in the ESC congress.

Sources of funding

None.

Cardiovascular imaging

The main specialist journals on cardiovascular imaging are EHJ Cardiovascular Imaging (which replaced the European Journal of Echocardiography in 2013), and JACC Cardiovascular Imaging and Circulation Cardiovascular Imaging, both of which were created in 2008.

In total, 72 French articles were published between 2005/2008 and 2015, which accounts for 1.8% of all published articles: 0.9% in JACC Cardiovascular Imaging, 1.7% in Circulation Cardiovascular Imaging and 1.8% in EHJ Cardiovascular Imaging. Over time, there was an increase in the proportion of French papers published in EHJ Cardiovascular Imaging. These results are detailed in Table 4 and Fig. 2B.

Electrophysiology

Circulation Arrhythmias and Electrophysiology was created in 2008; JACC Clinical Electrophysiology was created in 2015 and does not appear in the analyses. In total, 111 French articles were published between 2005 and 2015 in these journals, which accounted for 2.2% of all published articles: 1.3% in Circulation Arrythmias and Electrophysiology and 2.5% in Europace. Over time, the proportion of French publications remained stable. These results are detailed in Table 4 and Fig. 2C.

Disclosure of interest

The authors declare that they have no conflicts of interest concerning this article.