Alexandru Boţan
email: alexandru.botan@upmc.fr

Benjamin Rotenberg
email: benjamin.rotenberg@upmc.fr

Virginie Marry

Pierre Turq

Benoît Noetinger

Hydrodynamics in Clay Nanopores

à 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.

Introduction

The behavior of fluids confined in nanopores is of great interest both from the fundamental and the technological point of view. Surface effects play a central role in processes such as wetting, impregnation and drying in interface and colloid science. Understanding mass transfer through clay pores is necessary in many contexts, such as groundwater hydrology, petroleum and gas reservoir engineering, as well as carbon dioxide sequestration or geological disposal of radioactive waste.

Clays present a multiscale porosity, from the nanometer to the micrometer scale. Transport on large scales is controlled by the morphology of the material and the transport through the smallest pores. The upscaling from the pore-scale flow, usually described by the Navier-Stokes equation, to the macroscopic one, satisfying Darcy's law, can be obtained using the mathematical tools of homogenization. [1][2][3] However, the relevance of a continuous description of hydrodynamics in nanopores must be questioned. It is indeed well known that confining a fluid into pores not much larger than the molecular size may lead to dramatic changes in its phase diagram, structure and dynamics. Interactions of the surface with the fluid may shift the liquid-vapour coexistence. [START_REF] Brovchenko | Interfacial and confined water[END_REF] The molecular nature of a liquid results in a layered structure in the vicinity of any surface. [START_REF] Quirke | Adsorption and transport at the nanoscale[END_REF] Finally, diffusion and hydrodynamic flow are influenced by the interaction with the surface. [START_REF] Karniadakis | Fundamentals and Simulation[END_REF] Molecular simulation is the method of choice to describe a fluid confined in nanopores. For example, Grand canonical Monte-Carlo and molecular dynamics simulations have allowed to predict the adsorption isotherms of water in clay interlayers or the structure and diffusion of water in clay interlayer (see e.g. [START_REF] Auerbach | Handbook of Layered Materials[END_REF][START_REF] Skipper | [END_REF][9] for reviews) and in small interparticle pores. [10][11][12][13][14][15] Non-equilibrium molecular dynamics (NEMD) simulation has allowed to investigate the validity of continuous hydrodynamics of simple systems under confinement. [START_REF] Karniadakis | Fundamentals and Simulation[END_REF] Travis et al. simulated a Poiseuille flow of a Lennard-Jones fluid through slit pores with two different widths 16 and demonstrated that for channels smaller than 10 LJ diameters the velocity profiles are not well described by the Navier-Stokes prediction. Similar findings where reported by Di Leo and Marañón 17 for water in a 3-nm slit pore (about 10 times the size of a water molecule). Molecular simulations have also been used to address another crucial aspect of hydrodynamics, namely the choice of boundary condition at the solid/liquid interface. Since the pioneering work of Bocquet and Barrat on the derivation of a slip length from molecular simulations, [18][19][20] numerous work investigated its dependence on the solid roughness or on wall/fluid and fluid/fluid interactions. Sendner et al. reported the variation of the slip length for water in a model slit pore as a function of the wettability, by tuning the fluid/wall interaction and thereby changing the contact angle. 21 They found that a stick boundary condition could be used only for weak fluid/wall interactions. Huang et al. simulated Couette flows to investigate the dependence of the slip length on the surface charge density of a model surface 22 and found that the slip length decreassd with increasing surface charge density.

Voronov et al. reviewed studies of the effects of roughness on the slip length, and concluded that roughness can lead to both an increase or decrease slip, all other things being equal. 23 In all the above studies, the effect of the surfaces on the density of the confined fluid was not considered prior to the investigation of the hydrodynamic behaviour.

We report here a systematic molecular simulation study of water adsorption and transport in interparticle pores of sodium montmorillonite clays, with sizes ranging from 3 to 10 nm. For each pore size, we first determine the water density from Monte-Carlo simulations in the µ H 2 O V T en- semble. We then calculate the density profile and the radial distribution functions, as well as diffusion properties, via equilibrium molecular dynamics. Finally, we investigate using non-equilibrium molecular dynamics the hydrodynamics of water confined in clay nanopores. More precisely, we address the following questions : How far from the surface is the fluid velocity profile well described by the continuous prediction (e.g. Poiseuille flow)? Is the use of the bulk density and viscosity of the fluid appropriate to determine the properties of the confined fluid? Can the continuous description be improved using locally varying properties (density, viscosity) and/or boundary conditions (slip length) determined from molecular simulations?

Models and Methods

In the present calculations, we study the sodium-saturated Wyoming montmorillonite with unit formula

Na + 0.75 [Si 8 ](Al 3.25 Mg 0.75 )O 20 (OH) 4 ,
the atomic coordinates and force field are determined by Skipper 24 and Smith. 25 The single clay layer used in simulations consists of 8× 4 unit cells (totalling 1280 atoms) with a thickness 6.54

Å that corresponds 41.44 × 35.88 Å 2 in the xy plane. The clay layer is divided in two halflayers located at the top and bottom of the box, and forms a section of slit-like pore. The box is periodically replicated in all directions. The space between the half-layers contains water and Na + , as shown in Figure 1. Water is described by the extended simple point charge model SPC/E. 26 We perform the simulations for different pore widths: 3 nm, 4.5 nm, 6 nm, 8 nm and 10 nm, where the width is defined as the distance between the Al atoms in each half-layer.

All interactions between particles are described by Lennard-Jones (LJ) and Coulomb potentials:

U(r i j ) = 4ε i j [ ( σ i j r i j ) 12 - ( σ i j r i j ) 6 ] + q i q j 4πε 0 r i j ,
where r i j is the distance between sites i and j of different molecules, q i is the partial charge of the site, σ i j and ε i j are LJ parameters deduced from the conventional Lorentz-Berthelot mixing rules. [START_REF] Frenkel | Understanding molecular simulation: from algorithms to applications[END_REF] We use a cut-off radius of 10 Å for LJ interactions and Ewald summation to compute long-range electrostatic interactions.

Monte-Carlo simulations

The amount of water adsorbed for each pore width is determined from grand canonical Monte-Carlo (GCMC) simulations using configurational bias techniques. [START_REF] Frenkel | Understanding molecular simulation: from algorithms to applications[END_REF][START_REF] Hensen | [END_REF] Details of the GCMC al-gorithm and configurational bias can be found in. 29 In the present study, the pore water is in contact with a reservoir which sets its chemical potential. The temperature and pressure of the water in reservoir are fixed at 300 K and 1 atm, corresponding to a bulk density of 0.997 g/cm 3 . [START_REF] Lide | CRC Handbook of Chemistry and Physics[END_REF] The value of the chemical potential was first estimated from GCMC simulation of water bulk phase at T = 300 K in a cubic box of size L = 40 Å. (resulting in ≈ 2000 H 2 O molecules). For µ H 2 O = 49.0 kJ/mol the obtained density (ρ=0.994±0.006 g/cm 3 ) agrees with the experimental one within the error bar and we thus use this chemical potential for the subsequent GCMC simulations in clays. For the latter, the system is equilibrated for 10 8 steps and 5× 10 7 additional steps are used to compute the composition.

Equilibrium Molecular Dynamics

Once the water content is known, we use molecular dynamics (MD) ensemble to study the structure and diffusion of the confined fluid. All MD simulations (including non-equilibrium dynamics) are carried out with the LAMMPS [START_REF] Plimpton | [END_REF] simulation package in the canonical ensemble. Initial configurations are taken from Monte Carlo simulations. A constant temperature of 300 K is maintained with a Nose-Hoover 27 thermostat and the equations of motion for the rigid SPC/E are integrated using the SHAKE algorithm. 32 The trajectories of atoms for H 2 O and Na + are collected every 0.1 ps for 4.5 ns, after an equilibration period of 2 ns.

For the radial distribution functions of O-atoms, we divide the pore volume into layers, each of which is a slab parallel to the surface (xy plane) of thickness 0.5 Å. O-atoms in each layer contribute to the two-dimensional RDF of the layer:

g(z, r) = n(r, r + ∆r) ρ∆A ,
where ∆A = 2πr∆r is the area of a ring bounded by circles with r and r + ∆r radii, ρ is the twodimensional particle density. The value n denotes the number of O atoms with a distance to the center atom in [r : r + ∆r].

Self-diffusion coefficients are calculated according to the method introduced by Liu et al. 33 Due to symmetry of the system, the diffusion tensor contains two components: parallel (D || ) and perpendicular (D ⊥ ) to the clay surface. Since the properties of the confined fluid depend on the distance from the surface, we divide the pore into layers, in which we compute D || and D ⊥ . We calculate D || from the mean-square displacement of molecules in each layer, centered at a distance z i from the surface. To that end we introduce a function S i C whose value is 1 if the molecule remains continuously in the i-th layer during the interval [0;t] and 0 otherwise. Then, we have:

D || (z i ) = lim t→∞ ⟨[∆x 2 (t) + ∆y 2 (t)]S i C (t)⟩ 4t⟨S i C (t)⟩ .
In the direction perpendicular to the surface, the equation for diffusion coefficient is expressed through the correlation:

D ⊥ (z i ) = - ( L nπ ) 2 lim t→0 1 t ln ⟨Ψ i n (t)Ψ i n (0)S i C (t)⟩ ⟨S i C (0)⟩ , with Ψ i n (t) = sin(nπ(z(t) -z i min )/L),
where n is an integer, L = z i maxz i min and z i max and z i min are the boundaries of the i-th layer. Further details of the diffusion calculation are described elsewhere. 10,34 Non-Equilibrium Molecular Dynamics

Finally, we use non-equilibrium molecular dynamics to investigate the hydrodynamic properties of the confined fluid. More precisely, we want to assess the relevance of a continuous description to describe the hydrodynamic flow arising from a uniform pressure gradient. The usual ansatz in molecular simulation consists in applying a constant external force, parallel to the surface, to each fluid atom. We use a force F x = 0.75 10 -3 kcal/mol/Å, in the range corresponding to a linear response of the system (not shown). There are two additional differences compared to the equilibrium simulations. On the one hand, the thermostat is coupled only to the degree of freedom of the fluid in the y-direction, perpendicular to the force. On the other hand, periodic boundary conditions are applied only in the x and y directions. To that end we complete the two clay layers as indicated by the shaded atoms in Figure 1, to ensure that the thickness of the layers exceeds the cut-off radius R c = 10 Å. Steady state is reached in a time that depends on the pore size and does not exceed 2 ns. The velocities of the particles are then sampled for 6 ns in bins of width 0.5 Å.

The shear viscosity is estimated by two approaches. 35 The first one assumes a constant density and viscosity in the pore, a valid approximation sufficiently far from the surface. The steadystate Stokes equation predicts a parabolic (Poiseuille) velocity profile, whose curvature allows to determine the viscosity:

η = - F x ρ V ( d 2 v x dz 2 ) , ( 1
)
where ρ V is the number density. The second approach uses the definition of viscosity from the local shear rate γ(z) = dv x dz and the pressure tensor The comparison of water density profile among different types of surface of slit-like pore such as rutile, 36 laponite, 14 muscovite mica, 11 carbon 37 demonstrates that the nature of the surface modulates the position, width and intensity of the peaks. However, the distance from the surface over which the density oscillations decay, approximately equal to 12 Å (independently of the pore width), is almost independent of the surface. Further away from the surface, the density reaches a constant value: 0.982 g/cm 3 (H = 3 nm), 0.985 g/cm 3 (H = 4.5 nm) and 0.983 g/cm 3 for H=6, 8 and 10 nm. Within the accuracy of estimates (≈ 1 %), we conclude that the pore size does not influence the density of the fluid in the center of the pore. These values are close to the bulk one for the same chemical potential and temperature (0.994±0.004 g/cm 3 ). The results are given in Figure 4, which presents D || and D ⊥ as a function of the distance from the surface. The diffusivity close to the surface differs significantly from the values in the middle of the pore. This can be explained by the higher density of the fluid and H-bonds donated by water molecules to the surface oxygen atoms. 10 However, D || (z) reaches a minimum value only in the second layer, indicating that the hydration of Na + atoms has a large effect. In the absence of cations, the diffusivity monotonically increases from the surface to the center of the pore. 36 In the fourth and next layers both components are almost constant and D || > D ⊥ ≈ D bulk =2.80±0.05 10 -9 m 2 s -

P xz (z) = F x ∫ z 0 ρ V (z ′ )dz ′ : η(z) = -lim F x →0 P xz (z) γ(z) . ( 2
)

Results and Discussion

Static properties

The bulk value reported here corresponds to a simulation of 2000 molecules at the same density as in the center pore center. Note that the higher value compared to that often reported for the SPC/E model is not due to the slightly smaller density compared to the experimental one, but to the sizedependence of the diffusion coefficient. 38 Indeed, the latter are usually computed for small box sizes compared to the one simulated here. A study of the size-dependence for SPC/E by Kerisit et al. 39 concluded to an extrapolated value of 2.84 10 -9 m 2 s -1 for an infinite box size. The excess of D || over D ⊥ in the center of the pore, already observed with other systems 36,[START_REF] Rotenberg | Cosmochim[END_REF] is most likely an effect of the asymmetry of the simulation box, which is twice larger in the direction perpendicular to the surface than in the directions parallel to the surface.

Shear viscosity

We now turn to the hydrodynamic properties of water confined in clay nanopores and assess the relevance of continuous hydrodynamics on the nanoscale. In non-equilibrium Molecular Dynamics simulations, a constant force parallel to the surface is applied to each fluid atom, as described in section "Non-Equilibrium Molecular Dynamics", inducing steady-state velocity profiles reported in Figure 5 for pore widths H = 3, 4.5, 6, 8 and 10 nm. Assuming a uniform density and viscosity of the fluid, the Navier-Stokes equation predicts a parabolic (Poiseuille) velocity profile. The simulated profiles are indeed parabolic in the center of the pore and the curvature provides the viscosity of the confined fluid, according to Eq. ( 1). The resulting values are: η=0.70±0.07 cP for 4.5 nm, 0.68±0.02 cP for 6 nm, 0.709±0.005 cP for 8 nm and 0.685±0.003 cP for 10 nm. In the 3 nm pore, the region of constant density is too small (<3 Å) to provide an estimate of the viscosity. For comparison, we also computed the viscosity of bulk water with the same density (ρ m =0.983 g/cm 3 and T=300 K) and under normal conditions (ρ m =0.997 g/cm 3 and T=300 K) from the Green-Kubo equation, 41 with the result 0.66±0.02 cP and 0.67±0.02 cP, correspondingly.

Within the accuracy of these estimates, the viscosity of the confined fluid in the center of the pore is thus equal to that of the bulk fluid with the same density.

However, deviations from a parabolic flow profile are observed in the vicinity of the surface.

To analyze these deviations, we sampled more accurately the velocity profiles for two pore sizes, collecting the velocities for 40 ns instead of 6 ns in bins of width 0.1 Å instead of 0.5 Å. The results for H = 4.5 nm are reported in Figure 6a. The velocity profile is parabolic only in the center of the pore and oscillates closer to the surface. The velocity maxima coincide with that in the density.

Moreover, the larger the deviation from the bulk density, the larger the deviation from the parabolic velocity profile. Such oscillations arise from the discrete (molecular) nature of the fluid and have even been observed for a confined hard-sphere fluid. 42 A possible improvement of the continuous description consists in computing the local viscosity of the fluid as the ratio between the shear stress and the shear rate (Eq. ( 2)). To estimate the latter, we first fit the simulated density profile to a piecewise polynomial, shown in Figure 6a. The shear rate is then the derivative of this polynomial, presented in Figure 6b, together with the pressure

tensor P xz (z) = F x ∫ H/2 0 ρ V (z)dz.

Boundary condition

Since the velocity profile is parabolic at a distance larger than ≈ 1 nm from the surface, two questions arise. Firstly, to which boundary condition do the simulation results in the center of the pore correspond? Secondly, do the deviations from the parabolic profile in the vicinity of the surface contribute significantly to the overall flow? The usual "stick" boundary condition corresponds to a vanishing fluid velocity at the solid/fluid interface. When this is not the case, a slip boundary condition can be introduced, characterized by the slip length :

b = ± v x (z sur f ) ( dv x dz ) z sur f (3)
This requires the knowledge of the location z sur f of the surface where the hydrodynamic boundary condition is applied. Its definition for atomistically resolved surfaces requires some caution.

Several options can be considered.

(1) The first one was introduced by Bocquet and Barrat, 43 who derived a Green-Kubo expression for the location of the fluid-solid interface :

z sur f = ∫ ∞ 0 dt ⟨ F ∥ (t)P xz (0) ⟩ ∫ ∞ 0 dt ⟨ F ∥ (t)F ∥ (0) ⟩ , (4)
where F ∥ is the tangential force exerted by the wall on the fluid. A similar expression was also derived for the associated slip length. The numerical convergence of these integrals remains however a challenge even for a simple Lennard-Jones fluid, for which z sur f was found to be of the order of one atomic layer inside the liquid. 19,43 (2) A second point of view is to consider that z sur f corresponds to a partitioning of space between a region where the fluid is present and homogeneous and another one without any liquid. This is the definition of the Gibbs dividing surface, according to :

∫ z sur f 0 [ρ bulk -ρ(z)]dz = ∫ +∞ z sur f ρ(z)dz. (5)
From the density profiles we find z sur f at 1.2 Å closer to the center of the pore than the surface oxygen atoms.

(3) Finally, an intuitive definition of z sur f consists in splitting the pore into a region where the fluid behaves as in the bulk and another region where it behaves differently due to the proximity of the surfaces. The choice of z sur f is then rather arbitrary. From the previous sections, a possible choice would be z sur f = 1 nm, since this corresponds to a region where the structure and the dynamics differ significantly from the bulk. Because the "surface fluid" contained in this region is not immobile, such a model must account for a "surface flux", which can be determined from MD simulations in addition to z sur f and b. In the 4.5 nm pore, this flux contributes approximately 40 % of the total flux and 13 % in the 8 nm pore. Moreover, it should be stressed that the slip length defined by Eq. ( 3) then depends on the size of the pore, rendering its interpretation as an intrinsic property of the wall/fluid interface rather difficult.

Considering the numerical challenge to quantify properly z sur f and b with the first option and the arbitrariness of the last one, we restrict ourselves here to identifying the surface where the hydrodynamic boundary condition applies with the Gibbs dividing surface defined by Eq. ( 5). This "physical" definition also corresponds directly to the continuous model underlying the use of the Navier-Stokes equation in its simplest form, with a homogeneous fluid characterized by its bulk density and viscosity. In particular, this assumes that the parabolic velocity profile in the bulk region extends even within the region where the fluid is known from our molecular simulations to be inhomogenous and not well described by a purely viscous behaviour (see Figure 6a and Figure 6c).

Nevertheless, the parabolic fit to the simulated velocity profile near the center of the pore, already used to measure the viscosity in the previous section, describes reasonably well (see also below)

the simulation results closer to the surface, as can be seen on Figure 7.

From this parabolic profile, the slip length is then determined via Eq. ( 3) by computing the velocity and its derivative at z sur f . Comparing the result for different pore widths, we obtain the following estimate of the slip length: b = 2.1 ± 0.05 Å. A slip boundary condition in clays had already been introduced by Marry et al., 44 who investigated electro-osmosis in Na-montmorillonite with the same force field and model of water from equilibrium molecular simulations. However, the reported value of the slip length was 6 Å. Several reasons can explain this discrepancy: a different value of z sur f (2.4 Å vs 1.2 Å in this work), the determination of the Navier-Stokes profile using the experimental value of the viscosity (0.9 cP vs 0.67 cP for SPC/E water model) and the numerical issues related to the evalution of non-equilibrium quantities from equilibrium simulations using Green-Kubo relations. While there are no experimental determination of the slip length on montmorillonite clays, we can make a tentative comparison with experimental data using the connection proposed by Sender et al. for varying water/wall interactions between the slip length and the contact angle. 21 The contact angle corresponding to our value of the slip length does not exceed 60 • , in agreement with the experimental results of Lopez-Duran et al. 45 While the slip length may seem small, neglecting slip could result in small pores in a large error on the overall flow. To address this issue we compare the total fluid flow obtained from MD simulations to the one predicted by the parabolic profiles, solutions of the Stokes equation with stick (J stick ) and slip (J slip ) boundary conditions. The corresponding velocity profiles are illustrated on Figure 7. The relative errors with both models and two pore sizes are summarized in Table 1. Neglecting slip results in errors in the overall flow, which is the quantity of interest for upscaling, that exceed 10% even for the largest pores. However, our results also demonstrate that it is possible to correctly describe the flow in pores of a few nm only using a Poiseuille flow with slip boundary conditions, with the slip length determined from molecular simulation.

Conclusions

We demonstrated using Grand-Canonical Monte-Carlo simulations that the water density in the center of clay nanopores (3-10 nm) is similar to that of bulk water. The width of the region perturbed by the surface extends over approximately 1.2 nm. The Navier-Stokes equation, using the density and viscosity of the bulk fluid, provides a reasonable description of the hydrodynamic flow under an external force provided that the pore size is larger than 4 nm and that a slip boundary condition is used. We discussed the choice of the surface where the hydrodynamic boundary condition should be applied and determined a slip length of ≈2.1 Å at the clay surface. Although this value is small, neglecting slip in these nanopores results in large errors on the overall flow. In the vicinity of the surface, the deviations from the prediction of the Navier-Stokes equation cannot be captured by a local viscosity determined from MD simulations.

Table 1: Relative error on the overall flux, compared to molecular dynamics simulation, assuming a parabolic profile with stick or slip boundary conditions, with the same fluid viscosity (see also Figure 7).

H (nm) |J stick -J MD |/J MD |J slip -J MD |/J MD 4

Figure 2

 2 Figure 2 reports the number density profiles of water and ions as well as the mass density profile of the fluid, for a pore width H = 4.5 nm. Density oscillations near the surface for other pore widths are identical (not shown). The z-axis on this figure is limited from two sides by the location of the clay surface oxygens. The maxima of the mass density at 2.7, 4.3, 6.0 and 9.3 Å from the surface, coincide with that of the number density of water oxygen, except for the second one corresponding to the Na + ions.

Figure 3

 3 Figure3shows two-dimensional radial distribution functions (rdf) of O-atoms in layers parallel to the surface, computed from an 8-ns equilibrium MD simulation of the 10 nm pore. The inset of Figure3illustrates the location of the layers. The first 3 layers are centered at 2.7, 4.3 and 6.0 Å from the surface, i.e. the positions of the first 3 density maxima. Three additional layers are located in the region of constant density at 13, 24 and 33 Å from the surface. As expected, deviations in the rdf from bulk value are only observed in the three surface layers and the fluid further away from the surface is identical to that in bulk water.

  Figure6cshows the resulting viscosity profile. As expected, the viscosity is uniform in the middle of the pore and the value of 0.67 cP agrees with both the first estimate from Eq. (1) and the one obtained in the bulk fluid (0.66±0.02 cP). Closer to the surface the viscosity oscillates and even becomes negative. Such unphysical values indicate that the hydrodynamic behaviour of the fluid close to the surface cannot simply be described by a local viscosity defined by Eq. (2).

Figure 2 :

 2 Figure 2: (a) Mass density profiles for a pore size H = 4.5 nm. (b) Number density progiles for Na, O and H atoms. ρ Na was multiplied by a factor of 10 for better visualization. The extreme z values correspond to the location of surface oxygen atoms.

Figure 3 :

 3 Figure 3: Two-dimensional radial distribution functions for O-atoms calculated, in selected layers of the 10 nm pore. Each layer is a slab parallel to the clay surface. The gray stripes on the inset indicate the location of the layers with respect to the mass density profile. Results in the d) and all subsequent layers (not shown) almost coincide with the bulk one, shown for comparison.

Figure 4 :

 4 Figure 4: Water diffusion coefficient parallel D || and perpendicular D ⊥ to the surface as a function of the distance to the surface, for the largest pore (H=10 nm). The stripes indicate the location of the layers in which the diffusion coefficient are calculated.

Figure 5 :

 5 Figure 5: Steady-state velocity profile in the presence of a force parallel to the surface applied to each fluid atom. Note that the same force is applied in all cases. Symbols indicate the values obtained from NEMD simulations, solid lines are parabolic fits. The vertical dashed lines indicate the location of surface oxygen atoms.

Figure 6 :

 6 Figure 6: (a) Velocity and density profiles, (b) shear rate and shear stress, (c) viscosity and density profiles, in the 4.5 nm pore.

Figure 7 :

 7 Figure 7: Velocity profiles in the 4.5 nm pore: v MD is obtained from NEMD, v slip from fitting v MD to a parabolic profile and v stick by assuming the same viscosity and a vanishing velocity at the Gibbs dividing surface. The slip length computed from Eq. (3) is visible from the red short-dotted linear extrapolation of v slip beyond the Gibbs dividing surface (vertical solid line). The vertical dashed line indicates the location of surface oxygen atoms.

  Figure 3.

  Figure 4.

  Figure 5.

Figure 7 .

 7 Figure 7.

  Snapshot of the simulation box. Green: Al and Mg; yellow: Si; red: O; white: H; blue: Na. Bright atoms illustrate the simulation box for 3D periodic boundary conditions, both bright and shaded atoms -2D periodic boundary conditions.

	Figure 1:
	.5	27%	2.7%
	8.0	15%	0.8%

Acknowledgement

We thank Prof. J.-F. Dufrêche for valuable discussions. BR, VM and PT acknowledge financial support from the Agence Nationale de la Recherche under grant ANR-09-SYSC-012.


MD Nicolas Bertheuil
email: nbertheuil@gmail.com

MD Nicolas Isola

MD Damien Bergeat

MD Camille Mocquard

MD Eric Watier

MD Simon Rouze

MD Bernard Meunier

Thoracotomy and esophageal surgery: key points to preserve the possibilities of flaps

Thoracotomy and esophageal surgery: key points to preserve the possibilities

Introduction

Esophageal cancer is the eighth most common cancer worldwide and has a poor prognosis (5year survival < 51%) [START_REF] Lubbers | Totally minimally invasive esophagectomy after neoadjuvant chemoradiotherapy: Long-term oncologic outcomes[END_REF]. Surgery remains the best option when patients are operable.

Neoadjuvant radiotherapy improves long-term survival but is associated with higher perioperative morbidity and mortality than surgery alone. Anastomotic leakage is a common complication after surgery, and may trigger a tracheoesophageal fistula (TEF). A perioperative TEF is defined as one caused by major airway injury during esophagectomy or one that develops soon after operation. Different fistula locations have been reported, explaining the variety of treatment strategies. The complication is rare and difficult to treat; hence, no guideline is available. Indeed, only a few case reports and case series have described successful fistula management. Unlike the procedure used to treat a tracheobronchial fistula that develops after lung cancer surgery, tracheal or bronchial resection and anastomosis of a TEF does not appear to be feasible. For salvage surgerie, the plastic surgeon has a vital role within the multidisciplinary team. Surgical treatment using an interposition flap is an effective option that can be limited by the initial surgery. Indeed, if a mini-invasive esophagectomy via a thoracoscopic approach is performed, most flaps are workable. However, an open approach via a right thoracotomy is still frequently performed in clinical practice, restricting the choice of flap. We identified key points to be noted when performing thoracotomy, to preserve all flap possibilities.

Technique

Our clinical experience of re-operation for TEF suggests that some key points need to be observed during initial surgery.

During primary surgery, it is important to spare all flap possibilities (Fig. 1). When a miniinvasive procedure is chosen, thoracoscopic trocars should be carefully placed at the upper edge of the rib, avoiding the sixth, seventh, and eighth intercostal spaces. This would preserve all flap possibilities. Special care should be taken not to use these spaces for thoracic drainage, to avoid damage of the intercostal vascular pedicle. A thoracic drain placed through the abdominal wall will serve well [START_REF] Sun | Early oral feeding following McKeown minimally invasive esophagectomy: An open-label, randomized, controlled, noninferiority trial[END_REF]. Indeed, such drainage seems less painful than the traditional thoracic drain.

In practice, open thoracotomy is still routinely performed for tumor resection. Particularly when using an open approach, some key points must be borne in mind to maximize the possibilities for later flap harvesting if a complication develops (Fig. 2).

Firstly, the latissimus dorsi should always be preserved (Fig. 3) but that was rarely the case in our series [START_REF] Akcali | The effect of standard posterolateral versus musclesparing thoracotomy on multiple parameters[END_REF]. It reduces postoperative pain [START_REF] Akcali | The effect of standard posterolateral versus musclesparing thoracotomy on multiple parameters[END_REF] without increasing surgical morbidity. In our experience, with muscle conservation, esophagectomy is no more difficult than with standard posterolateral thoracotomy; the latissimus dorsi is moved forward and backward on demand during operation. If this muscle is preserved, a muscular flap can be harvested if a complication develops (Fig. 1).

The second key point concerns rib plane closure. Usually, three or four absorbable stitches are placed. These should be placed for the inferior rib to the transcostal plane and a manual perforator should be employed to preserve the intercostal vessels lying just under the thoracotomy site (Fig. 4 and5). This allows later creation of skin perforator flap pedicled by intercostal muscle (Fig. 6). Special attention should be brought to the placement of drains.

Results

Between June 2016 and August 2017, we treated five patients with postoperative TEFs by placing interposition skin perforator flaps pedicled by the intercostal muscle, as previously described [START_REF] Bertheuil | Skin perforator flap pedicled by intercostal muscle for repair of a tracheobronchoesophageal fistula[END_REF][START_REF] Bertheuil | Skin perforator flap pedicled with intercostal muscle: Additional data[END_REF]. Four patients underwent thoracotomy through the fifth intercostal space during initial surgery. When an emergency interposition flap is placed to treat a TEF, we seek to avoid complications associated with flap harvesting; we perform intercostal muscle dissection through the seventh intercostal space because the sixth intercostal vessels are systematically ligatured when the general surgeon closes the thoracotomy. In one patient, during flap harvesting, we discovered that the initial thoracotomy had been closed with a wire ligating the vessels of the seventh space in a region posterior to the axillary line. For this patient, we created a second skin perforator flap pedicled by the intercostal muscle in the underlying space and used tension to close the donor site.

Discussion

Gastric-airways fistulas after resection of esophageal cancer are rare and have been little reported in the literature which explains why there is no guideline currently for their management. Endoprosthesis placement is often chosen as the first-line therapy because it is less invasive, using of an interposition flap is often reserved for a trained team. Only a few relevant case reports have appeared. Various interposition flaps have been used to treat TEFs.

The choice depends on the location of the fistula. For an intrathoracic TEF, the most common options are latissimus dorsi [START_REF] Hayashi | Gastric tube-totracheal fistula closed with a latissimus dorsi myocutaneous flap[END_REF], pectoralis major [START_REF] Siu | Use of the pectoralis major muscle flap for repair of a tracheoesophageal fistula[END_REF], intercostal muscle flaps, and skin perforator flaps pedicled by an intercostal muscle flap [START_REF] Bertheuil | Skin perforator flap pedicled by intercostal muscle for repair of a tracheobronchoesophageal fistula[END_REF][START_REF] Bertheuil | Skin perforator flap pedicled with intercostal muscle: Additional data[END_REF]. Pericardial and pleural flaps have also been reported. If treating a cervical fistula, a neck muscle flap is also an option.

In our opinion, when treating an intrathoracic fistula, only two flaps can reach deep sites and thus cover all types of fistulae (in any area); these are a latissimus dorsi muscle flap and a skin perforator pedicled by an intercostal muscle flap (Fig. 6). Both flaps have a very long pedicle and a large surface. This is why it is essential to preserve the latissimus dorsi muscle during open surgery. The muscle is easily and rapidly harvested and can cover all types of fistulae (tracheal or bronchial). A disadvantage is that the flap is not readily malleable; waterproof closure is difficult. In addition, the flap is bulky, and may compress the gastric tract or the airway. In our experience, thoracotomy with preservation of the latissimus dorsi muscle can be performed without undermining, thus avoiding postoperative seroma formation [START_REF] Akcali | The effect of standard posterolateral versus musclesparing thoracotomy on multiple parameters[END_REF]. An intercostal muscle flap is principally used to reinforce the anastomosis created during thoracic surgery [8]. The aim is to place well-vascularized tissue permitting good revascularization of the tracheobronchial anastomosis, but this flap is not sufficiently wide to cover a defect. Currently, to solve this problem, we add a skin perforator flap to the intercostal muscle. We have been forced to use skin perforator flaps pedicled by the intercostal muscle to treat TEFs because the latissimus dorsi muscle is often cut during initial surgery without any note in the operative record. Otherwise, the flap can be harvested using a surgical revision approach, thus allowing direct closure of the donor site. This flap is very plastic; tracheoesophageal compression is not a concern and preservation of gastric plasty is possible. During primary thoracotomy, if rib closure preserves the underlying pedicle because a transcostal approach is used, the flap can be harvested in the underlying space without undermining subcutaneous tissue. tracheobronchial surgery. Ann Thorac Surg 1996;62:630-1.

Fig. 1 :Fig. 2 :

 12 Fig. 1: The two main flap interposition possibilities for surgical repair of a TEF.

Fig. 3 :Fig. 4 :Fig. 5 :Fig. 6 :

 3456 Fig. 3: Thoracotomy with preservation of the latissimus dorsi muscle

Figure 1 .

 1 Figure 1. The two main flap interposition possibilities for surgical repair of a TEF. The Latissimus dorsi muscle harvesting and the skin perforator flap pedicled by intercostal muscle.

Figure 2 .

 2 Figure 2. Thoracotomy errors. Thoracotomy without preservation of the latissimus dorsi muscle and without transcostal closure of the rib plane and consequently ligation of the subcostal pedicles.

Figure 3 .

 3 Figure 3. Thoracotomy with preservation of the latissimus dorsi muscle.

Figure 4 .

 4 Figure 4. Thoracotomy closure with handle perforator.

Figure 5 .

 5 Figure 5. Thoracotomy with preservation of the subcostal vessels.

Figure 6 .

 6 Figure 6. Skin perforator flap pedicled by intercostal muscle harvesting. A 62 years-old man with a TEF at day 6 postoperatively after a Lewis-Santy procedure. Primary surgery was performed by a thoracotomy in the 5th intercostal space with preservation of the latissimus dorsi muscle but without closure of the rib plane by transcostal points. The flap was harvested in the 7th intercostal plane.

In conclusion, we emphasize on some key points that thoracic or general surgeons should keep in mind when performing esophageal surgery. These optimally conserve flap options for the plastic surgeon who may later be required to create a transposition flap.

The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/Yyavne

Conflict of interest statement

The authors have no conflict of interest to disclose.


Simon Gonzalez

Florian Gallier

Sabrina Kellouche

Franck Carreiras

Ettore Novellino

Alfonso Carotenuto

Gérard Chassaing

Paolo Rovero

Jacques Uziel

Nadege Lubin-Germain

Nadège Lubin-Germain

Studies of membranotropic and fusogenic activity of two putative HCV fusion peptides

Keywords:

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

Introduction 1.Fusion peptide and cell-penetration

Since the discovery of the first cell-penetrating peptides (CPPs), Tat and penetratin in the 90s, membranotropic peptides have been of increasing interest for enhancing therapeutic agent cellular uptake. [START_REF] Bechara | Cell-Penetrating Peptides: 20 Years Later, Where Do We Stand?[END_REF][START_REF] Guo | Cell-penetrating peptides: Possible transduction mechanisms and therapeutic applications (Review)[END_REF][START_REF] Durzynska | Viral and Other Cell-Penetrating Peptides as Vectors of Therapeutic Agents in Medicine[END_REF][START_REF] Falanga | Membranotropic cell penetrating peptides: The outstanding journey[END_REF] Even if their mechanism of action is still controversial, the different sequences identified over the years, being CPPs or AMPs, shared particular properties. Indeed, these membranotropic sequences are generally amphipathic, composed of hydrophobic and cationic residues. Thus, they are usually classified as primary amphipathic peptides, where the amphipathicity is due to primary structure, and secondary amphipathic peptides, where a particular folding gives two distinct cationic and hydrophobic faces.

Another class of membrane-active peptides is fusion peptides. They differ from CPPs or AMPs as they are generally hydrophobic, thus allowing membrane insertion during the fusion process. This membrane activity could be interesting for enhancing cell penetration as described for gene delivery. [START_REF] Simeoni | Insight into the mechanism of the peptide-based gene delivery system MPG: Implications for delivery of siRNA into mammalian cells[END_REF] Hence, we propose to study different putative HCV fusion peptides as possible cell-penetrating agents, based on their propensity to interact with lipidic membranes (highlighted by DSC analyses), their conformations in membrane mimetic environments (CD and NMR) their membranotropic activity on liposomes as cell membrane models (studied by spectrofluorescence) and on epithelial cells (microscopy), and finally their ability to induce liposomes fusion (spectrofluorescence by FRET and DLS).

HCV fusion proteins

HCV is an enveloped RNA virus of the Flaviviridae family. The virus counts 10 different viral proteins: 4 structural proteins (Core, p7, E1 and E2) and 6 non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B). [START_REF] Moradpour | Hepatitis C Virus: From Molecular Virology to Antiviral Therapy[END_REF] While non-structural proteins have different roles during viral replication (protease, polymerase), structural proteins and particularly E1 and E2 are responsible for the first crucial steps of the infection being cells' receptors interaction (generally entailed by E2) as well as fusion. The two proteins E1 and E2 are thus called fusion proteins (FPs). [START_REF] Freedman | Structure and Function of the Hepatitis C Virus Envelope Glycoproteins E1 and E2: Antiviral and Vaccine Targets[END_REF] Several studies suggest that E1 and E2 could act as truncated class II FPs [START_REF] Garry | Proteomics computational analyses suggest that hepatitis C virus E1 and pestivirus E2 envelope glycoproteins are truncated class II fusion proteins[END_REF] which involves an E1/E2 heterodimer and a pHdependent mechanism, including fusion peptide exposure and insertion in cell membrane. [START_REF] Rodríguez-Rodríguez | Fusogenic properties of the Ectodomain of HCV E2 envelope protein[END_REF][START_REF] Vieyres | Characterization of the Envelope Glycoproteins Associated with Infectious Hepatitis C Virus[END_REF]

Selected sequences

A large number of studies have been carried out in order to identify the HCV fusion peptide, using mutagenesis and membrane behavior studies such as fusion or hemifusion experiments. For instance, Drummer et al. [START_REF] Drummer | Mutagenesis of a conserved fusion peptide-like motif and membrane-proximal heptad-repeat region of hepatitis C virus glycoprotein E1[END_REF] have demonstrated that the 284 VFLV 288 G sequence in E1 could be important for fusion since mutations in this region did not change protein production, dimer formation or receptor recognition but strongly impaired the viral fusion activity.

Pérez-Berná et al. [START_REF] Pérez-Berná | The membrane-active regions of the hepatitis C virus E1 and E2 envelope glycoproteins[END_REF] also investigated the fusogenic activity of different 11-mer peptides corresponding to E1 and E2 overlapping sequences. Each peptide was tested in fusion and hemifusion assays which allowed the authors to clearly identify several membranotropic regions, particularly E1 (276-286) and E2 (418-432). In another study using spectrofluorescence and 31 P-NMR, [START_REF] Pérez-Berná | Biophysical characterization of the fusogenic region of HCV envelope glycoprotein E1[END_REF] the same authors confirmed that E1 (274-291) is capable to interact and to modify lipidic membranes of different compositions through electrostatic and hydrophobic interactions. We opted for this consensual E1 (276-286) peptide. Moreover, more regions in the ectodomain of the hepatitis C virus envelope glycoprotein E2 (430-449, 543-560 and 603-624) were identified. [START_REF] Pacheco | Membrane-perturbing properties of three peptides corresponding to the ectodomain of hepatitis C virus E2 envelope protein[END_REF] But we have retained the (418-432) sequence in first intention. [START_REF] Lavillette | Characterization of Fusion Determinants Points to the Involvement of Three Discrete Regions of Both E1 and E2 Glycoproteins in the Membrane Fusion Process of Hepatitis C Virus[END_REF] Based on the literature, two HCV fusion peptides (Table 1) namely HCV1 and HCV2 (Entries 1 and 2) are putative fusion sequences. Both contain Cys residues which could disturb the membrane interaction study by side-product formation due to disulfide-bridge creation. Since these Cys residues are unlikely involved in intra or inter disulfide bridge within the protein, [START_REF] Li | Mutagenesis of the fusion peptide-like domain of hepatitis C virus E1 glycoprotein: Involvement in cell fusion and virus entry[END_REF] they were replaced by Ser residues in HCV1 and HCV2 giving these peptides named HCV3 and HCV7 (Entries 3 and 5). Finally, the HCV3 sequence was truncated in order to improve solubility and facilitate conjugation with a cargo, giving the sequence HCV6 (Entry 4). The Tat peptide (Entry 6), derived from the HIV Tat protein, was chosen as a reference since its membranotropic activity and its propensity to cross the plasma membrane have been studied in the literature. [START_REF] Schwarze | In Vivo Protein Transduction: Delivery of a Biologically Active Protein into the Mouse[END_REF][START_REF] Walrant | Membrane Crossing and Membranotropic Activity of Cell-Penetrating Peptides: Dangerous Liaisons?[END_REF]

Entry Name

Code Sequences The free peptides were also decorated with two different patterns (Table 2): a hydrophilic linker TTDS in order to increase solubility and a fluorescent tag NBD in order to perform spectrofluorescence assays and fluorescence microscopy. In the case of HCV6-NT and HCV7-N (Entries 3 and 4), the peptide was labeled on a Lys side-chain in order to keep the free amino-group for enhancing water solubility.

Entry

Peptide synthesis

All peptides were synthesized either on a 0.1, 0.05 or 0.025-mmol scale by a high-efficiency solidphase peptide synthesis (SPPS) strategy, using a Liberty Blue automated microwave synthesizer (CEM Corporation, Matthews, NC, USA) following the Fmoc/tBu methodology. The reactions were performed in a Teflon vessel and mixed by N 2 bubbling. Reaction temperatures were monitored by an internal fiber optic sensor. Usual amino-acid side chain's protecting groups were used and coupling steps were performed with DIC and Oxyma Pure in DMF.

After peptide elongation, pegylation was performed manually in a plastic syringe and continuously stirred by orbital shaking. The coupling step was performed using Fmoc-TTDS-OH (2.5 eq), TBTU (2.5 eq) and DIPEA (3.5 eq) for 30 min at room temperature. The reaction completion was confirmed by Kaiser Test and/or micro-cleavage.

Conjugation with the fluorophore was performed manually in a plastic syringe and continuously stirred by orbital shaking. The coupling step was performed using NBD (4 eq) and DIPEA (10 eq) for was confirmed by micro-cleavage.

In the case of HCV6-NT or HCV7-N, NBD was introduced on a Lys side-chain using a modified procedure. After peptide elongation, Fmoc-Lys(Dde)-OH (5 eq) was manually coupled on the N-ter position using TBTU (5 eq) and DIPEA (7 eq) for 30 min at room temperature. The Dde protecting group was selectively removed using a 3M/2.25M NH 2 OH.HCl/imidazole solution in NMP/DMF 5/1 (v/v) added to the resin and stirred at room temperature for 3h. After washing with DMF, the NBD was coupled on Lys side-chain as described above.

All peptides were then cleaved by a TFA/H 2 O/TIS (95/2.5/2.5) solution at room temperature for 2 to 4h. After precipitation in cold diethyl ether and freeze-drying, peptides were purified by semipreparative RP-HPLC and analyzed by UPLC-MS.

Circular dichroism

All CD spectra were recorded using a JASCO J715 spectropolarimeter at 25 °C with a cell of 1 mm path length. The CD spectra were acquired by the range from 260 nm to 190 nm, 1 nm bandwidth, 4 accumulations, and 100 nm/min scanning speed. The CD spectra of HCV6 and HCV7, at a concentration of 30 µM, were acquired in water (pH=7.4), and in DPC (20 mM) micellar solutions. HCV7 CD spectrum was also acquired in SDS solution (20 mM). Secondary structure content was predicted from CD spectra using the Jasco Secondary Structure Estimation tool (Jasco Inc.), the Contin-LL method [START_REF] Provencher | Estimation of globular protein secondary structure from circular dichroism[END_REF] and the BeStSel method [START_REF] Micsonai | Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy[END_REF][START_REF] Micsonai | BeStSel: A web server for accurate protein secondary structure prediction and fold recognition from the circular dichroism spectra[END_REF] (Table 3).

NMR

NMR Spectroscopy. 99.9% NMR spectra were recorded on a Varian INOVA 700 MHz spectrometer equipped with a z-gradient 5 mm tripleresonance probe head. All the spectra were recorded at a temperature of 25 °C. The spectra were calibrated relative to TSP (0.00 ppm) as internal standard. One-dimensional (1D) NMR spectra were recorded in the Fourier mode with quadrature detection. The water signal was suppressed by gradient echo. [START_REF] Hwang | Water Suppression That Works. Excitation Sculpting Using Arbitrary Wave-Forms and Pulsed-Field Gradients[END_REF] Two dimensional (2D) DQF-COSY, [START_REF] Piantini | Multiple quantum filters for elucidating NMR coupling networks[END_REF][START_REF] Marion | Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins[END_REF] TOCSY, [START_REF] Braunschweiler | Coherence transfer by isotropic mixing: Application to proton correlation spectroscopy[END_REF] and NOESY [START_REF] Jeener | Investigation of exchange processes by two dimensional NMR spectroscopy[END_REF] spectra were recorded in the phase-sensitive mode using the method from States. [START_REF] States | A two-dimensional nuclear overhauser experiment with pure absorption phase in four quadrants[END_REF] Data block sizes were 2048 addresses in t 2 and 512 equidistant t 1 values. Before Fourier transformation, the time domain data matrices were multiplied by shifted sin 2 functions in both dimensions. A mixing time of 70 ms was used for the TOCSY experiments. NOESY experiments were run with mixing time of 100 ms. The qualitative and quantitative analyses of DQF-COSY, TOCSY, and NOESY spectra, were obtained using the interactive program package XEASY. [START_REF] Bartels | The program XEASY for computersupported NMR spectral analysis of biological macromolecules[END_REF] Complete 1 H NMR chemical shift assignments were effectively achieved for HCV7 according to the Wüthrich procedure [START_REF] Wüthrich | NMR of Proteins and Nucleic Acids[END_REF] via the usual systematic application of DQF-COSY, TOCSY, and NOESY experiments with the support of the XEASY software package (Supporting Information, Table S1). 3 J αN coupling constants were measured by 1D proton and DQF-COSY spectra. The temperature coefficients of the amide proton chemical shifts were calculated from 1D 1 H NMR and 2D TOCSY experiments performed at different temperatures in the range 25-40°C by means of linear regression. Observed NOEs are reported in Figure S1 (Supporting Information).

Liposomes preparation

Differential Scanning Calorimetry

DSC experiments were performed on a MicroSC calorimeter (Setaram Instrumentation). For each analysis, three scans from 40°C to 5°C were performed with a scan rate of 1°C/min and waiting 15 min between sequential scans in a series to allow thermal equilibration. Data analysis was performed by the Calisto Software provided by Setaram Instrumentation and with Origin software. The MLVs suspensions were prepared as described above reaching a concentration of 5 mM in Tris Buffer (10 mM Tris, 100 mM NaCl, pH 7.3). Aliquots of peptide solution (concentration varying from 300 µM to 1 mM) in Tris buffer (with 5% DMSO for experiments with HCV6) were gradually added to the lipid MLVs to obtain peptide/lipid molar ratios of 1/100, 1/50, 1/25, and finally 1/10. For each addition, the exact same volume of Tris buffer solution was used as a reference.

Membranotropic and fusogenic studies 2.7.1. Internalization in LUVs

Time-course fluorescence measurements were recorded with a Jasco Fluorescence Spectrophotometer at the desired temperature (controlled by a Jasco MCB-100 mini circulation bath). As an excitation source, a Xenon lamp was used at 460 nm and emission was measured at 555 nm. The excitation bandwidth was set to 10 nm and the emission to 2.5 nm. Depending on the sequence, the PMT voltage was set between 650 and 750 V. To avoid peptide adsorption and subsequent fluorescence quenching, polymethacrylate cuvettes were used. During measurement, the solution was stirred at 800 rpm.

For each analysis, 3 mL of PBS buffer (10 mM phosphate, 100 mM NaCl, pH 7.4) and 30 µL of a 1 mM LUVs solution was introduced in a spectrophotometer cuvette. The emission measured at 555 nm was used as a blank value. 3 µL of a 100 µM solution of peptide bearing the NBD fluorescent tag (75 µL of a 4 µM solution for HCV6-NT and 18.5 µL of a 16 µM solution for HCV7-NT) was added into the cuvette and incubated at the desired temperature for 10-15 min. The solution was then cooled to 10°C and 30 µL of a concentrated (~1M) sodium dithionite (DT) solution was added for reduction.

Experiments with a diluted DT solution were performed following the same procedure but after cooling to 10°C, several aliquots of increasing volume [START_REF] Durzynska | Viral and Other Cell-Penetrating Peptides as Vectors of Therapeutic Agents in Medicine[END_REF][START_REF] Durzynska | Viral and Other Cell-Penetrating Peptides as Vectors of Therapeutic Agents in Medicine[END_REF][START_REF] Moradpour | Hepatitis C Virus: From Molecular Virology to Antiviral Therapy[END_REF][START_REF] Pérez-Berná | The membrane-active regions of the hepatitis C virus E1 and E2 envelope glycoproteins[END_REF][START_REF] Marion | Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins[END_REF][START_REF] Sabahi | Unexpected Structural Features of the Hepatitis C Virus Envelope Protein 2 Ectodomain[END_REF][START_REF] Sabahi | Unexpected Structural Features of the Hepatitis C Virus Envelope Protein 2 Ectodomain[END_REF] and 100 µL) of a 100 mM DT solution were added for reduction. Finally, several aliquots of 100 mM DT were added, until the blank value was reached.

Fusion assays by FRET

Fusion/hemifusion experiments using FRET were performed on a Jasco fluorescence spectrophotometer at 37°C for experiments with LM3 (POPC/POPE/POPS/sphingomyelin/cholesterol 10/5/2/2/10) LUVs (controlled by a Jasco MCB-100 mini circulation bath). A Xenon lamp was used at 450 nm and emission was measured at 530 nm for time-course fluorescence measurements. The excitation bandwidth was set to 10 nm and the emission to 2.5 nm and the PMT voltage was set at 600V. Analyses were performed using polymethacrylate cuvettes. During measurement, the solution was stirred at 800 rpm.

For each experiment, 1.7 mL of 10 mM phosphate buffer without NaCl (pH 7.4), 27 µL of a 1mM LM3 LUVs solution containing 2% mol Rho-DPPE and 2% mol NBD-DPPE, and 273 µL of a 1 mM LM3 LUVs solution (yielding a 1/10 ratio between fluorescent LUVs and non-fluorescent ones) were added into a spectrophotometer cuvette. The fluorescence emission measured at 530 nm was used as a blank value. 60 µL of a 100 µM peptide solution (77 µL of a 78 µM solution for HCV6 and 24 µL of a 1 mM solution for acetyl-penetratin Ac-Pen) was added yielding a peptide/lipid ratio of 1/50 (1/12 for Ac-Pen). The fluorescence emission at 530 nm was then recorded for 10 to 20 min. Cuvettes were then placed in a 37°C bath under stirring and fluorescence emission at 530 nm was measured after one night. A LUVs solution without peptide was studied following the same procedure as a control experiment.

Dynamic light scattering

Fusion/hemifusion experiments by DLS were performed using a Zeta-Sizer Nano (Malvern) considering the following parameters: n = 1.345 ; Absorption = 0.001 ; water as solvent. The samples were analyzed at 37°C.

For each sample, 1.7 mL of 10 mM phosphate buffer without NaCl (pH 7.4), and 300 µL of a 1mM LM3 LUVs solution (POPC/POPE/POPS/sphingomyelin/cholesterol 10/5/2/2/10) were poured into a screw-cap tube yielding a final concentration of 150 µM. 60 µL of a 100 µM peptide solution (77 µL of a 78 µM solution for HCV6 and 24 µL of a 1 mM solution for Ac-Pen) was added yielding a peptide/lipid ratio 1/50 (1/12 for Ac-Pen). The samples were placed in a 37°C bath under stirring (800 rpm), and polydispersity and vesicle size (in intensity) were measured after 20 min and one night.

After one night, the same volume of peptide solution was added to the samples, and polydispersity and size were again measured after another 20 min and one night.

A LUVs solution without peptide was studied following the same procedure as a control experiment.

Microscopy

Chinese Hamster Ovarian cells (CHO, ATCC) were grown in HAM F12 medium (Invitrogen) supplemented with 10% fetal calf serum in a humidified atmosphere with 5% CO 2 at 37 °C. For experiments, cells were harvested with 0.5% trypsin-EDTA (Invitrogen), then seeded onto glass coverslips at a density of 25 000 cells/cm 2 and incubated in culture medium for 24 h until subconfluence.

Cells were incubated with 400 µL of a (100 µM for Tat-NT and 25 µM for HCV7-N) peptide TFAfree solution (removed by several freeze-dryings with HCl 100 mM) in PBS buffer (10 mM phosphate, 100 mM NaCl, pH 7.4) for 30 min or 1 h at 37°C. Cells were fixed with 3% paraformaldehyde for 10 minutes and washed with PBS. After washing, cell nuclei were stained with DAPI (4,6-diamidino-2phenylindole dihydrochloride, D9542, Sigma Aldrich) at 0.3µg/ml. Coverslips were mounted in Prolong® Gold Antifade Reagent (P36930, Invitrogen). Staining was examined with fluorescence conventional microscopy (Leica MLB). All pictures were acquired using a x63 zoom and analyzed with Image J software.

Results

Structural analyses

Circular dichroism

To explore the conformational behavior of HCV6 and HCV7, we performed a CD study of these peptides in water, and DPC/water solutions. The last medium is commonly used as rough mimetic of zwitterionic membrane environment. [START_REF] Langham | How can a β-sheet peptide be both a potent antimicrobial and harmfully toxic? Molecular dynamics simulations of protegrin-1 in micelles, Biopolym[END_REF] CD spectrum of HCV6 in water (Figure 1a) reveals the prevalence of β-structures (β-turns and β-strands from about 35 to 50%) together with random conformations. Upon the addition of 20 mM of DPC micelles, the CD spectrum clearly changed indicating that the interactions of HCV6 with the micelle do occur. A single wide minimum at 208 nm could be observed in this environment. Secondary structure evaluation (Table 3) gave different results. ContinLL and BeStSel estimated an increase of the helical content, while Jasco tool estimated an increase of the β-strand structure. Unfortunately, we could not discern between the two conformations by NMR investigation (see below) due to the low solubility of HCV6. CD spectrum in water of HCV7 revealed the presence of disordered conformers with a minimum close to 198 nm (Figure 1b). In DPC micelles solution, the spectra of HCV7 showed a reduction and a shift towards higher wavelength of the minimum (from 198 to 201 nm) with the appearance of a wide shoulder around 220 nm. This increase of ordered structure content as confirmed by the secondary structure estimations reported in Table 3. HCV7 was also investigated in SDS micelles as mimetic of negatively charged membranes, [START_REF] Saviello | New insight into the mechanism of action of the temporin antimicrobial peptides[END_REF][START_REF] Grieco | Alanine scanning analysis and structure-function relationships of the frog-skin antimicrobial peptide temporin-1Ta[END_REF] since DSC results highlighted its propensity to interact with negative membranes. CD spectrum acquired in SDS is very similar to that obtained in DPC with minimal differences pointing to a further stabilization of the turn/strand structures.

NMR

Conformational preferences of HCV7 in DPC micelles were also investigated by solution NMR.

Unfortunately, low water solubility (< 50 µM) of HCV6 prevented the NMR analysis of this peptide. NMR parameters of HCV7 (Table S1) indicated a high degree of flexibility of this peptide in solution.

In particular, the observed 3 J αN couplings range between 6.3 and 7.7 Hz except for Alanine residue and the Wishart-Sykes chemical shift indexes [START_REF] Wishart | The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy[END_REF] based on the observed Hα chemical shifts predict no regular α-helix or β-sheet structures. A few NOEs between Hα and HN protons of residues i and i+2 (Figure S1) indicate the presence of β-turn structures mainly in the central and C-terminal part of the peptide (residues 6-13) as confirmed by the relatively low HN temperature coefficients of residues 9, 10, and 13. However, the exiguous number of the overall NOE correlations (Figure S1), again indicating structural flexibility, prevented the calculation of an NMR-based 3D structure of the peptide.

Differential scanning calorimetry

In order to characterize the effect of HCV6 and HCV7 on lipidic membranes, DSC analyses with zwitterionic (PC) and anionic (PG) MLVs were performed. Indeed, PC is one of the most representative membrane lipids in the biological membrane (more than 50%) [START_REF] Van Meer | Membrane lipids: Where they are and how they behave[END_REF] and therefore constitute an appropriate model. The cell membrane is also composed of anionic lipids such as PI or PS, but at lower levels. PG is rarely found in eukaryotic membranes but has been considered as a good representative for PI, PS or other anionic membrane components (such as GAGs) in order to study electrostatic interactions. The biological membrane shows a high diversity in the lipid's chains composition with a majority of unsaturated fatty acids. However, since the phase transition of a membrane composed of unsaturated lipids is below 0°C, such lipids are irrelevant for DSC analyses. Hence, we chose to work with C 14 lipids such as dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG) since their phase transition occurs near room temperature thus facilitating the experiments.

Thermograms illustrating the effect of Tat, HCV6, and HCV7 on DMPG are represented in Figure 2 and thermodynamic parameters for MLVs with 1/100 and 1/25 P/L ratios are presented in Table 4.

The Tat peptide was studied as a reference considering its well-known membranotropic activity, particularly on membranes composed of anionic lipids due to a bidentate interaction between guanidinium groups of arginine residues and phosphate. [START_REF] Rothbard | Role of Membrane Potential and Hydrogen Bonding in the Mechanism of Translocation of Guanidinium-Rich Peptides into Cells[END_REF] The obtained results for Tat are in accordance with the literature since the thermograms show an endothermic event around 23°C corresponding to the transition from the P β ' ripple phase to the L α fluid phase (also called the main transition). The first peptide additions, yielding to P/L ratios of 1/100 and 1/50, did not affect the main transition which was still measured around 23°C. The other two additions, yielding to a P/L ratio of 1/25 and 1/10 respectively, lead to an important decrease in T m indicating that Tat interacts more efficiently with the fluid phase than the gel phase and that the peptide is able to promote fluid domains within the lipidic membrane. The enthalpy modification induced by Tat demonstrates its propensity to insert in the membrane hydrophobic core and to disturb lipids acyl chain packing. The same experiment performed with HCV6 (Table 4) gives unexpected results. Indeed, because of its supposed overall negative charge, HCV6 should not be able to interact with a membrane composed of anionic lipids due to electrostatic repulsion, but the thermograms obtained show an increase in T m at a P/L ratio of 1/50. This increase implies a modification of the thermotropic membrane behavior and that the peptide is able to enhance gel domain formation in the lipidic layer. Nevertheless, one should note that the experiments were performed using 5% DMSO peptide solution because of the low solubility of HCV6 in water. An experiment performed with a DMSO-free solution at a P/L ratio of 1/50 did not exhibit any modification in T m (data not shown) indicating that the observed thermotropic behavior could be largely imputed to the presence of an organic solvent or its combined effect with HCV6. For this reason, modification in enthalpy induced by HCV6 will not be discussed here.

Peptide

On the contrary, HCV7 (Table 4 and Figure 2) seems to be more active as a membranotropic peptide. Indeed, the thermograms obtained with MLVs composed of DMPG show a significant decrease in T m at a P/L ratio of 1/25 and 1/10 (to 19.92°C and 18.17°C respectively). As described for Tat, these observations highlight the propensity of HCV7 to interact with a membrane composed of DMPG (probably due to electrostatic interactions resulting from the arginine residue) and its ability to promote fluidic behavior. Interestingly, thermograms also display a second endothermic transition, with a more important width, corresponding to the initial main transition temperature. This observation could indicate that HCV7 is able to interact with DMPG in a cooperative manner, yielding fluidic peptide-rich domains and unmodified peptide-poor domains. The overall enthalpy (corresponding to both phase transitions combined) is only slightly modified (0.51 kJ/mol) which implies that if HCV7 is able to interact with lipid head groups and to modify membrane fluidity, it may not be able to efficiently insert in the membrane and to disturb the lipid acyl chains packing.

Because of HCV6 poor solubility in water and HCV7 promising activity, further experiments were only performed with HCV7, considered as our "lead". Thus, its interaction with a membrane composed of zwitterionic lipids such as DMPC was also investigated by DSC analyses (Figure 3). The resulting thermograms did not show any significant effect on the membrane behavior (either in terms of T m or ΔH as shown in Table 5), even after a peptide addition yielding to a P/L ratio of 1/5. Considering the previous results, it appears that electrostatic interactions are crucial for HCV7 membranotropic activity, probably because of the arginine residue. In order to characterize the peptide behavior in more biologically relevant conditions, DSC analyses were also performed with a membrane containing less anionic lipids: MLVs composed of a mixture DMPC/DMPG 9/1. The thermograms (data not shown) did not display any modification in T m or ΔH implying that HCV7 needs more than 10 % anionic residues in the membrane in order to efficiently disturb its properties. Overall, the results indicate that HCV7 is able to interact with the lipidic membrane, more effectively with anionic lipids than zwitterionic ones. The data also show that this sequence could act in a cooperative way in contact with the membrane, which creates fluidic domains.

Peptide

Spectrofluorescence

Internalization in liposomes

In order to characterize the membrane behavior of HCV6 and HCV7, internalization assays with LUVs were performed and monitored by spectrofluorescence. This technique, described by Swiecicki et al., [START_REF] Swiecicki | Accumulation of cell-penetrating peptides in large unilamellar vesicles: A straightforward screening assay for investigating the internalization mechanism, Biopolym[END_REF] involves peptides conjugated to a fluorescent tag NBD, which is chemically reduced during the experiment. The reduction performed with sodium dithionite (DT) at 10°C transforms the NBD nitro group into an amino group, which leads to complete loss of fluorescence. Monitoring the fluorescence intensity by time-course measurement displays a decrease which could be directly related to the percentage of peptide in solution. Since the reductive agent is unlikely able to cross the membrane at low temperature, the remaining fluorescence corresponds to peptides either internalized or inserted in LUVs membrane (Figure 4). In order to prevent possible perturbations of the peptide/membrane interaction and to increase the solubility in water, the fluorescent tag NBD was initially coupled on a hydrophilic linker (TTDS), introduced at peptide N-terminus. The first result obtained with LUVs composed of DMPC/DMPG 9/1 and the Tat peptide as a reference (Table 6) confirms the ability of such cationic sequence to interact with anionic membrane and to penetrate the intravesicular medium.

Peptide

A B

Interestingly, the ability of Tat to cross the lipidic bilayer seems higher at T m (24°C) than in fluid phase (37°C). Since the membrane is composed of several fluid and gel domains at T m , separated by so-called phase-defects, this observation could be related to the importance of such permeabilized domains for peptide insertion and/or internalization as it has already been described. [START_REF] Murayama | Loosening of Lipid Packing Promotes Oligoarginine Entry into Cells[END_REF][START_REF] Yang | HIV gp41-mediated membrane fusion occurs at edges of cholesterol-rich lipid domains[END_REF] HCV6-NT and HCV7-NT did not exhibit any membranotropic activity since all fluorescence was quenched after DT addition indicating that the peptide is only present in solution. However, this result could also be explained by the presence of the hydrophilic TTDS linker. Indeed, due to its length, the NBD fluorophore may no longer be protected from the reduction, even though the peptide is inserted in the membrane. The fluorescence would then be quenched just as if it was in solution.

To investigate the influence of TTDS linker, we have also tested the Tat peptide with NBD directly introduced at the peptide N-terminus (Tat-N, Entry 1 Table 2). The result obtained with LUVs of DMPC/DMPG 9/1 at 24°C (Table 7) show a better peptide internalization which could be consistent with our hypothesis. Since TTDS is a very hydrophilic linker, it could also interfere with the peptide internalization. For these reasons, HCV7-N (Entry 4 Table 2) was redesigned without the TTDS linker. The first results obtained with the new designed fluorescent HCV7-N and LUVs of different compositions demonstrate its propensity to interact with the lipidic membrane (Table 7). Hence, the fusion peptide HCV7-N membranotropic activity seems influenced by the proportion of anionic lipid DMPG since no activity could be observed with pure DMPC while membrane insertion and/or internalization are observed with different content of DMPG. This conclusion is consistent with the DSC analyses, showing a significant membrane modification induced by HCV7 on vesicles composed of DMPG.

Peptide

However, the reproducibility of these results remains problematic and can be explained by several hypotheses (Figure 5) : (i) the peptide could be adsorbed on the vesicle surface through electrostatic interaction, rather than inserted in the lipidic membrane, explaining why the NBD becomes accessible to reduction by large quantities of DT which are then able to diffuse more easily through the electrical double layer (Case A); (ii) the peptide could exist in several conformations in contact with the membrane, as it has already been demonstrated by structural analyses, and some conformations could be more sensitive to reduction than others (Case B); (iii) the peptide could be involved in a partition equilibrium between the membrane and solution indicating that, depending on the time, a different amount of inserted/internalized peptide could be measured after reduction (Case C). We are unable to distinguish one of the hypotheses and certainly a critical concentration of DT close to the membrane, the accessibility of the NBD (conformations, equilibrium) are additional factors. In order to investigate the influence of DT concentration, we performed a new experiment with several additions of diluted DT solution (Figure 6) performed at 10°C. The obtained result on HCV7-N showed no fluorescence intensity decrease during the first additions until the effective concentration (244 µL of diluted DT solution in total) is reached. A significant decline after the threshold is observed up to a value corresponding to 8% of the initial intensity that could correspond to 8% of peptide insertion/internalization. This could imply that a certain DT concentration is needed to observe the proper NBD reduction. Further DT addition results in complete loss of fluorescence which confirms our hypotheses as an important excess of reductive agent could explain discrepancies in results observed with this sequence. Therefore, this experiment seems consistent with the influence of DT concentration on the diffusion to achieve a complete reduction (Figure 5 case A).

Fusion by FRET

In order to characterize the fusogenic activity of modified HCV fusion peptides, we also performed spectrofluorescence assays involving FRET. In this technique, first described by Struck et al. and applied in several studies, [START_REF] Struck | Use of resonance energy transfer to monitor membrane fusion[END_REF][START_REF] Wadhwani | Antimicrobial and cell-penetrating peptides induce lipid vesicle fusion by folding and aggregation[END_REF] LUVs are labeled by two fluorescent tags (one donor, NBD, and one acceptor, the rhodamine B (Rho)) allowing the FRET between them. After peptide addition, if fusion or aggregation is observed, the FRET efficiency decrease and an increase in NBD fluorescence intensity is observed. The peptide fusion activity could then be observed by following the fluorescence intensity at 530 nm.

In order to use biologically relevant lipid composition [START_REF] Yang | Solid-State Nuclear Magnetic Resonance Evidence for an Extended β Strand Conformation of the Membrane-Bound HIV-1 Fusion Peptide[END_REF] and to compare our results with the literature, we performed our analysis with LM3 LUVs (POPC/POPE/POPS/SM/Chol 10/5/2/2/10) and in the conditions described by Wadhwani et al. [START_REF] Wadhwani | Antimicrobial and cell-penetrating peptides induce lipid vesicle fusion by folding and aggregation[END_REF] This composition reflects the cholesterol and lipid headgroup composition of the plasma membranes of H9 cells.

As already described, the addition of Tat peptide leads to spontaneous LUVs aggregation or even precipitation, indicating that this sequence is not a suitable reference to follow the fusion phenomenon.

For this reason, we also used a sequence described as fusogenic by Wadhani et al., the acetylated penetratin (Ac-Pen). [START_REF] Wadhwani | Antimicrobial and cell-penetrating peptides induce lipid vesicle fusion by folding and aggregation[END_REF][START_REF] Soleymani-Goloujeh | Effects of N-terminal and C-terminal modification on cytotoxicity and cellular uptake of amphiphilic cell penetrating peptides[END_REF] The different P/L ratios tested resulted in no or low fluorescence intensity increase until reaching the 1/12 value (Figure 7). The intensity measured after 1h30 was 842, much higher than the value corresponding to LUVs alone (330), thus demonstrating the fusion/aggregation induced by Ac-Pen. Interestingly, the observed phenomenon occurred at low rates. Indeed, the fluorescence intensity continues to rise after peptide addition until reaching a stable value (1280) after 4h. The different HCV fusion sequences, HCV6 and HCV7, were then tested in the same conditions and no fluorescence increase was observed 10 min after peptide addition. As the phenomenon observed with Ac-Pen seems to be relatively slow, the fluorescence was also measured after one night (Figure 8). For the two HCV sequences, the value (565 for HCV7) was higher than LUVs alone but lower than the intensity reached with Ac-Pen. This observation demonstrates that HCV7 and HCV6 could act as a fusogenic sequence involving a very slow phenomenon. In order to check if the fluorescence increase observed after one night at 37°C was not due to experimental conditions or artifacts, a control experiment involving LUVs alone was carried out in the same conditions. The results obtained show no fluorescence intensity increase after 10 min but a significantly higher value after one night. This observation could imply a slow fusion and/or aggregation phenomenon in these conditions, even without peptide. Interestingly, HCV6 and HCV7 lead to slightly higher intensity values than the control experiment after one night (*After 10 min, ** after 1 night Table 8). Even if the difference may not be significant, this observation could imply that these two sequences have a low fusogenic activity in these conditions. This phenomenon should thus be confirmed by another technique such as dynamic light scattering.

Sequence

Dynamic light scattering

The fusogenic activity was also characterized by DLS using LM3 LUVs and free peptides in the same conditions than FRET assays. After a first peptide addition reaching a P/L ratio of 1/50, vesicle size is measured by DLS after 20 min and one night. Another addition is then performed (reaching a 1/12 ratio) and two more measurements are performed (corresponding to 24 h and two days since the first addition).

As expected, Ac-Pen induces a significant size increase 20 min after the first peptide addition (reaching 180.4 ± 11.5 nm instead of 136.5 ± 10.3 nm for LUVs alone), demonstrating a possible fast fusion/aggregation phenomenon (Figure 9A). Interestingly, the control experiment performed with LUVs alone did not display any vesicle size increase, indicating that the liposomes are unlikely able to fuse or aggregate in these conditions (Figure 9B) conversely to what was observed during FRET assays. Among the HCV sequences, only HCV6 shows a significant vesicle size increase from 141.6 ± 8.1 nm for LUVs alone to 220.3 ± 14.7 nm (Figure 10). This observation demonstrates that the HCV6 peptide is able to induce vesicle fusion or aggregation and is consistent with the behavior described with FRET spectrofluorescence. Indeed, the only significant size increase is noticed after the second peptide addition and 2 days after the first one, indicating a low and slow fusogenic or aggregation activity.

A B

Fluorescence Microscopy

In order to characterize the peptide activity on the biological membrane, sequences labeled by fluorescent NBD were incubated with subconfluent epithelial cells (CHO) at 37°C and the peptide behavior was observed by fluorescence microscopy after 30 min or 1h. Considering the low solubility and membranotropic activity of HCV6, these experiments were only performed with Tat-NT as the reference and HCV7-N.

As already demonstrated by several groups, Tat-NT is able to cross the plasma membrane since NBD fluorescence (yellow) could be observed in the intracellular media after 30 min incubation (Figure 1). The punctiform staining observed in the cytoplasm shows vesicles formation, demonstrating that internalization by endocytosis could be involved as it has already been described [START_REF] Kaplan | Cationic TAT peptide transduction domain enters cells by macropinocytosis[END_REF][START_REF] Richard | Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors[END_REF]. The experiments with HCV7-N led to the same conclusions. Indeed, NBD fluorescence and punctiform staining are observed in the cytoplasm after 30 min incubation (Figure 12). This observation demonstrates the propensity of HCV7-N to cross a plasma membrane, probably involving endocytosis.

Discussion

HCV peptides structure in micelles

Conformational analysis performed on HCV6 and HCV7 by CD (Figure 1 and Table 3) pointed to a prevalence of unstructured states in water solution for both peptides. In the presence of DPC micelles as a mimetic of zwitterionic (mammalian) membranes, HCV6 structure becomes more ordered (Figure 1a). Depending on the estimation method used, an increase of the α-helix or β-strand content is provided. The last suggestion, i.e. a high propensity of HCV6 to adopt a β-sheet conformation could explain the observed low solubility of HCV6 which, in that case, would be due to the formation of amyloid-like β-sheet aggregates. Unfortunately, this possibility could not be confirmed by NMR investigations because the peptide solubility is too low.

HCV7 showed a lower degree of structuration upon DPC micelle interaction. An increase of the βstrand content is provided by only one of the methods used for the estimation (Table 3). HCV7 was also investigated in SDS micelles as mimetic of negatively charged membranes since DSC results clearly indicate that electrostatic interactions are crucial for HCV7 membranotropic activity. CD spectrum acquired in SDS is very similar to that obtained in DPC (Figure 1b). From this result, it can be stated that the different membranotropic behavior of HCV7 toward zwitterionic or anionic membranes is not driven by a conformational modification.

HCV7 conformation in the presence of DPC micelle was also investigated by solution NMR. We used a DPC micelle solution as mimetic of zwitterionic membranes since these, i.e. mammalian membranes, are the putative target of the peptide. Since CD analysis demonstrated minimal conformational differences of the peptide interacting with negatively charged SDS micelles compared to DPC, NMR analysis was limited to the last solution. The NMR analysis demonstrated the presence of β-turn structures along the central and C-terminal regions of the peptide in equilibrium with unordered conformations. There is no evidence of the β-strand structures estimated by the CD spectra. This is probably to attribute to the largely overlapping dichroic bands arising from β-turn and β-sheet structures.

HCV peptides membranotropic behavior

HCV6 membrane behavior was difficult to assess since its low solubility in water did not allow performing relevant DSC analyses. The solubility also seems to be a limiting factor for studying the HCV6 membranotropic activity by spectrofluorescence since no insertion and/or internalization could be observed (Table 6) either with DMPC/DMPG 9/1 and DMPG LUVs. This lack of activity could be related to a low local concentration in contact with the membrane.

On the contrary, HCV7 clearly interacts with lipids in the membrane as it has been demonstrated by DSC and structural analyses. This interaction seems to be more important with anionic lipids such as PG, creating fluid peptide-rich domains within the membrane. The absence of membrane modification with zwitterionic lipids such as PC suggests an electrostatic interaction, probably between the arginine residue and the phosphate group.

Spectrofluorescence assays show that HCV7 is a membranotropic sequence, able to insert into the lipid bilayer and/or to cross it. Again, this behavior seems to be influenced by the presence of anionic residues in membrane since its activity could not be observed with pure DMPC vesicles.

Unfortunately, the lack of reproducibility does not allow clearly defining the peptide membranotropic behavior. This low activity could be related to experimental conditions (DT concentration, peptide concentration) and/or to peptide properties (weak membrane interaction, secondary structure, partition equilibrium).

However, the fluorescence microscopy did demonstrate the HCV7's propensity to cross the plasma membrane thus indicating its potential as a cell-penetrating agent. The peptide intake seems to involve endocytosis which could also explain the difficulties faced during internalization experiments with LUVs. Interestingly, the peptide seems to induce cytotoxic activity or cell's adhesion inhibition. A possible effect of NBD could not be excluded, thus it should be interesting to study the peptide internalization within cells using a different fluorescent probe [START_REF] Yang | Cell-penetrating peptide induces leaky fusion of liposomes containing late endosome-specific anionic lipid[END_REF] or immunofluorescence.

HCV peptides fusogenic activity

FRET assays and DLS analyses both state that HCV6 is a fusogenic sequence whereas HCV7 is not. The fusion or aggregation induced by HCV6 seems weak and occurs at low rates. This low activity could be explained by an insufficient structuration in contact with membranes. Indeed, Wadhwani et al. [START_REF] Wadhwani | Antimicrobial and cell-penetrating peptides induce lipid vesicle fusion by folding and aggregation[END_REF] have shown that peptide fusogenic activity is directly correlated to its propensity to adopt a particular structure during the fusion process (being α-helix or β-structures). Thus, the lack of fusogenic activity for HCV7 could be imputed to its prevailing random coil conformation in contact with lipids as it has been demonstrated by structural analyses.

In the case of HCV6, the low membranotropic activity could be explained by the too short sequence or the absence of important residues for the fusion process, remaining to be determined. Moreover the peptide membranotropic properties should certainly be correlated to combined effects of sequences rather than to the presence of a specific amino acid. This phenomenon could be associated to a cooperative type behavior, where the fusion peptide structuration occurs in contact with the membrane as the same time as the interactions of others sequences with the membrane or/and receptors. This hypothesis has been ascertained by Banerjee et al, [START_REF] Banerjee | Folded Monomers and Hexamers of the Ectodomain of the HIV gp41 Membrane Fusion Protein: Potential Roles in Fusion and Synergy Between the Fusion Peptide, Hairpin, and Membrane-Proximal External Region[END_REF] studying the synergetic relation between the MPER domain (C-terminal membrane proximal external region) and the folding of gp41 ectodomain monomers into an hairpin conformation followed by assembly into a hexamer. Concerning the HCV, a cooperative cascade of foldings and interactions involving several peptide sequences domains has been also postulated. [START_REF] Sabahi | Unexpected Structural Features of the Hepatitis C Virus Envelope Protein 2 Ectodomain[END_REF] However, from a molecular point of view, the role of important residues remains less explained even if the presence of hydrophobic (leucine) or aromatic (histidines) aminoacids and glycosylation are involved in the membranotropic properties.

Conclusion

Altogether, the techniques involved in this study allowed us to identify a new HCV membranotropic sequence (HCV7). This peptide seems to interact preferentially with anionic membrane components due to its arginine residue as it has been demonstrated for several CPPs. Even if its membrane behavior on LUVs has not been clearly assessed, experiments performed with cells did show its propensity to cross plasma membrane by endocytosis, thus confirming its potential as a cellpenetrating agent.

This study also confirms the HCV6 fusogenic activity described in the literature thus supporting the hypothesis that this sequence corresponds to the HCV fusion peptide.

However, the two peptides' activity is lower than expected. This observation could be explained by the short sequences used, their lack of secondary structure and the absence of possibly important residues. Thus, it should be interesting to extend our study to longer HCV sequences folding into stable β-turns, which is generally the fusion peptide structure adopted within the protein, by keeping the cysteine residues for disulfide bond formation, or by lactamisation in order to constrain the sequences.

Figure 1 :

 1 Figure 1: CD spectra of HCV6 (a) and HCV7 (b) in water (black line), DPC (blue line), and SDS (red line). HCV peptides concentration 30 µM in water (pH=7.4) and in DPC (20 mM) micellar solutions. HCV7 CD spectrum was also acquired in SDS solution (20 mM)

Figure 2 :

 2 Figure 2: Thermograms illustrating the effect of Tat, HCV6, and HCV7 on MLVs 5 mM membranes composed of DMPG. Aliquots of peptide solution (concentration varying from 300 µM to 1 mM) in Tris buffer (with 5% DMSO for experiments with HCV6) were gradually added to the lipid MLVs to obtain peptide/lipid molar ratios of 1/100, 1/50, 1/25, and finally 1/10.

Figure 3 :

 3 Figure 3: Thermogram illustrating the effect of HCV7 on MLVs membrane composed of DMPC. The initial main transition is represented by a dashed line. Aliquots of peptide solution (concentration varying from 300 µM to 1 mM) in Tris buffer were gradually added to the lipid MLVs to obtain peptide/lipid molar ratios of 1/100, 1/50, 1/25, and finally 1/5.

Figure 4 :

 4 Figure 4: (A) Monitoring NBD fluorescence intensity (555 nm) during internalization assays. (1) Intensity at 10°C; (2) addition of reductive agent DT and decrease in fluorescence intensity; (3) Residual fluorescence corresponding to peptide internalized and/or inserted in LUVs membrane. (B) Experimental time-course measurement for Tat-NT and DMPC/DMPG 9/1 LUVs.

Table 6 :

 6 Results of internalization experiments with pegylated peptides Tat, HCV6-NT, and HCV7-NT, and LUVs of DMPC/DMPG 9/1 at 555nm. 3 µL of a 100 µM solution of peptide bearing the NBD fluorescent tag (75 µL of a 4 µM solution for HCV6-NT and 18.5 µL of a 16 µM solution for HCV7-NT) was added into the cuvette containing LUV (10 mM phosphate, 100 mM NaCl, pH 7.4 and 30 µL of a 1 mM LUVs solution) and incubated at the desired temperature for 10-15 min. The solution was then cooled to 10°C and 30 µL of a concentrated (~1M) sodium dithionite (DT) solution was added for reduction.

Table 7 :

 7 Results obtained during internalization assays using tagged peptides incubated at 24°C, 555nm. 3 µL of a 100 µM solution of peptide bearing the NBD fluorescent tag (75 µL of a 4 µM solution for HCV6-NT and 18.5 µL of a 16 µM solution for HCV7-NT) was added into the cuvette containing LUV (10 mM phosphate, 100 mM NaCl, pH 7.4 and 30 µL of a 1 mM LUVs solution) and cooled to 10°C for 10-15 min.before adding 30 µL of a concentrated (~1M) sodium dithionite (DT) solution.

Figure 5 :

 5 Figure 5: Different hypotheses explaining the lack of reproducibility in fluorescence assays: (A) Influence of DT concentration on diffusion through the electrical double bilayer; (B) Different peptide structures in contact with the membrane; (C) Partition equilibrium between the solution and the membrane

Figure 6 :

 6 Figure 6: Experiment HCV7/LUVs DMPC/DMPG 7/3 with diluted DT solution. First additions were performed with increasing volumes of a 100 mM DT solution (3, 3, 6, 12, 24, 48, 48, 100 µL) at 10°C. Finally, several aliquots of the same solution were added, until the blank value was reached.

Figure 7 :

 7 Figure 7: Fusion induced by Ac-Pen monitored by FRET To a solution of 1.7 mL of 10 mM phosphate buffer without NaCl (pH 7.4), 27 µL of a 1mM LM3 LUVs solution containing 2% mol Rho-DPPE and 2% mol NBD-DPPE, and 273 µL of a 1 mM LM3 LUVs solution (yielding a 1/10 ratio between fluorescent LUVs and non-fluorescent ones) were added. 60 µL of a 100 µM peptide solution (24 µL of a 1 mM solution for acetyl-penetratin Ac-Pen (peptide/lipid ratio of 1/12). The fluorescence emission at 530 nm was then recorded for 10 to 20 min at 37°C under stirring and fluorescence emission at 530 nm was measured after one night.

Figure 8 :

 8 Figure 8: Fusion induced by HCV7 monitored by FRET. To a solution of 1.7 mL of 10 mM phosphate buffer without NaCl (pH 7.4), 27 µL of a 1mM LM3 LUVs solution containing 2% mol Rho-DPPE and 2% mol NBD-DPPE, and 273 µL of a 1 mM LM3 LUVs solution (yielding a 1/10 ratio between fluorescent LUVs and non-fluorescent ones) were added. 60 µL of a 100 µM peptide solution (77 µL of a 78 µM solution for HCV7 (peptide/lipid ratio of 1/50). The fluorescence emission at 530 nm was then recorded for 10 to 20 min at 37°C under stirring and fluorescence emission at 530 nm was measured after one night.

Table 8 :

 8 Fluorescence intensity at 530 nm for each experiment performed at 37°C for experiments with LM3 (POPC/POPE/POPS/sphingomyelin/cholesterol 10/5/2/2/10) LUVs (controlled by a Jasco MCB-100 mini circulation bath). A Xenon lamp was used at 450 nm and emission was measured at 530 nm for time-course fluorescence measurements. The excitation bandwidth was set to 10 nm and the emission to 2.5 nm and the PMT voltage was set at 600V.

Figure 9 :

 9 Figure 9: DLS analysis of LM3 LUVs in presence of Ac-Pen (A); and alone as a control experiment (B). At 1.7 mL of 10 mM phosphate buffer without NaCl (pH 7.4), and 300 µL of a 1mM LM3 LUVs solution (POPC/POPE/POPS/sphingomyelin/cholesterol 10/5/2/2/10) with a final concentration of 150 µM was added 24 µL of a 1 mM solution for Ac-Pen yielding a peptide/lipid ratio 1/12. At 37°C under stirring (800 rpm), polydispersity and vesicle size (in intensity) were measured after 20 min and one night.

Figure 10 :

 10 Figure 10: DLS analysis of LM3 LUVs in presence of HCV6. At 1.7 mL of 10 mM phosphate buffer without NaCl (pH 7.4), and 300 µL of a 1mM LM3 LUVs solution (POPC/POPE/POPS/sphingomyelin/cholesterol 10/5/2/2/10) with a final concentration of 150 µM was added 77 µL of a 78 M solution of HCV6 yielding a peptide/lipid ratio 1/50. At 37°C under stirring (800 rpm), polydispersity and vesicle size (in intensity) were measured after 20 min and one night.

Figure 11 :

 11 Figure 11: Pictures acquired by fluorescence microscopy for Tat-NT after 30 min incubation with CHO cells. Cells were incubated with 400 µL of a 100 µM for Tat-NT peptide TFA-free solution in PBS buffer for 30 min or 1 h at 37°C. Cells were fixed with 3% paraformaldehyde for 10 minutes and washed with PBS. After washing, cell nuclei were stained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride) at 0.3µg/ml. Coverslips were mounted in Prolong® Gold Antifade Reagent

Figure 12 :

 12 Figure 12: Pictures acquired by fluorescence microscopy for HCV7 after 30 min incubation with CHO cells. Cells were incubated with 400 µL of a 25 µM for HCV7-N peptide TFA-free solution in PBS buffer for 30 min or 1 h at 37°C. Cells were fixed with 3% paraformaldehyde for 10 minutes and washed with PBS. After washing, cell nuclei were stained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride) at 0.3µg/ml. Coverslips were mounted in Prolong® Gold Antifade Reagent..

Table 1 : Selected HCV fusion sequences and Tat as a reference. Underlined residues correspond to Ser in place of Cys in native sequences.

 1

	1	HCV-E1(265-304)	HCV1 LVGSATLCSALYVGDLCGSVFLVGQLFTFSPRRHWTTQDC
	2	HCV-E2(418-432)	HCV2 SWHINRTALNCNDS
	3	[ 272 Ser, 281 Ser, 304 Ser] HCV-E1(265-304)	HCV3 LVGSATLSSALYVGDLSGSVFLVGQLFTFSPRRHWTTQDS
	4	[ 281 Ser]HCV-E1(276-286)	HCV6 YVGDLSGSVFL
	5	[ 429 Ser]HCV-E2(418-432)	HCV7 SWHINRTALNSNDS
	6	HIV-Tat(47-57)	Tat	YGRKKRRQRRR

Table 2 : Modified Tat and HCV peptide sequences 2. Experimental 2.1. Materials

 2

HPLC-MS analyses were performed on an Alliance Chromatography (Waters) apparatus with a Phenomenex Kinetex C18 column (2.6μm, 3.0 × 100 mm) using a 0.6 mL/min flowrate, with the indicated linear gradients (see supporting information), coupled to a single quadrupole ESI-MS (Micromass ZQ). The solvent systems used were: A (0.1% TFA in H 2 O) and B (0.1 % TFA in ACN). UPLC-MS analyses were performed on a Waters Acquity UPLC apparatus, which was equipped with a Waters Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 50 mm) using a 0.6 mL/min flowrate, with the indicated linear gradients (see supporting information), coupled to a single quadrupole ESI-MS (Waters 3100 Mass Detector). The solvent systems used were: A (0.1% TFA in H 2 O) and B (0.1 % TFA in ACN).

Table 3 : Percentage of secondary structure from CD spectra Secondary structure content was predicted from CD spectra using a the Jasco Secondary Structure Estimation tool (Jasco Inc.), b the Contin-LL method and c the BeStSel method

 3

	Peptide (medium) α-helix	β-strand	β-turn	Random coil
	HCV6 (water)	9.3 a /11.1 b /14.7 c	31.2 a /17.0 b /18.4 c	22.2 a /18.5 b /15.6 c	37.3 a /53.3 b /51.3 c
	HCV6 (DPC)	12.9 a /32.5 b /33.5 c 42.3 a /4.2 b /17.4 c	13.0 a /18.3 b /6.6 c	31.9 a /45.0 b /42.5 c

Table 4 : Thermodynamic results obtained by DSC with MLVs of DMPG. MLV concentration 5 mM in Tris Buffer. Experiments with HCV6 were performed using 5% peptide DMSO solution. Data analysis was performed by the Calisto Software provided by Setaram Instrumentation and with Origin software

 4

Table 5 : Thermodynamic results obtained by DSC with MLVs of DMPC and HCV7. Data analysis was performed by the Calisto Software provided by Setaram Instrumentation and with Origin software

 5

		P/L ratio	ΔH (kcal/mol)	T m (°C)
	HCV7	1/100 1/25	-5.79 ± 0.04 -5.78 ± 0.24	23.93 ± 0.01 23.89 ± 0.03