Thierry M Faure
email: thierry.faure@defense.gouv.fr

Vincent Drouet

Olivier Montagnier

Olivier Montagnier Discrete

Discrete vortex method for a detached flow on a two-airfoil configuration at high angle of attack

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

INTRODUCTION

Unsteady aerodynamics of airfoils in incompressible flow, operated on moderate Reynolds numbers in the range 10 4 to 10 5 , has gained in importance, for unmanned air vehicles (UAV) and micro air vehicles (MAV) applications [START_REF] Mueller | Aerodynamics of small vehicles[END_REF]. Fixed-wing MAV need to fly at conditions close to the stall point, and post-stall flight would occur during maneuvers. Hence, delaying stall and improving performance for large values of the angle of attack is necessary. To do so, two-wing configurations are proposed as a means of generating large enough lift coefficient and delaying stall by controlling the flow. The present investigation focuses on the understanding of two-dimensional effects. The interaction between two airfoils is a key mechanism of flow control, with wakes and vortex shedding effects. Computational fluid dynamics is a classical way to address that issue, but low-order unsteady methods are useful and reliable options for engineering purpose. These methods have been developed some forty years ago, with limited application because of the small computer performance at that time [START_REF] Sarpkaya | An inviscide model of twodimensional vortex shedding for transient and asymptotically steady separated flow over an inclined plate[END_REF]. However, with the advance of computing power, low-order numerical methods are a good balance between fidelity and cost. The present method is an unsteady airfoil theory based on potential flows, initially developed for flapping airfoil motion but applied hereafter to predict the unsteady detached flow on a static airfoil. After validating the algorithm on a single airfoil from low to high values of the angle of attack, the periodic characteristic of the vortex shedding are compared with experimental results. The method is then adapted to the case of two airfoils in interaction. The lift efficiency ratios are compared for different stagger and gap between the airfoils.

LEADING EDGE SUCTION PARAMETER DISCRETE VORTEX METHOD

The Leading edge suction parameter Discrete Vortex Method (LDVM) is based on the potential thin airfoil theory in unsteady flows, applicable for large values of the angle of attack [START_REF] Ramesh | Discrete-vortex method with novel shedding criterion for unsteady aerofoil flows with intermittent leading-edge vortex shedding[END_REF]. It is built on the time-stepping approach of [START_REF] Katz | Low-Speed Aerodynamics[END_REF] with the addition of a criterion for the leading edge detachment. In figure 1, the aerodynamic frame (X,Z) is fixed and the airfoil frame (x,z) moves with a velocity U ∞ opposite to the X axis with a timedependant angle of attack α(t). No motion along the Z axis is considered hereafter. The fluid velocity components are respectively U and W in the aerodynamic frame and w is the velocity along z in the airfoil frame. The local circulation distribution over the airfoil is obtained from a Fourier series:

( ) ( ) ( )         + + = ∑ ∞ = ∞ 1 0 sin sin cos 1 2 , n n n t A t A U t θ θ θ θ γ (1)
with A 0 (t), … , A n (t) the time-dependant Fourier coefficients, c the airfoil chord and where:

( ) θ cos 1 2 - = c x (2)
Figure 1. Airfoil motion and frames of reference.

The Kutta condition is enforced through the form of the Fourier series. The Fourier coefficients are determined from the instantaneous local downwash w(θ,t) by enforcing the potential flow boundary condition that the velocity is tangential to the airfoil surface:

( ) ( ) ∫ ∞ - = π θ θ π 0 0 , 1 d U t w t A (3) ( ) ( ) ∫ ∞ = π θ θ θ π 0 cos , 2 d n U t w t A n ( 4
)
where w(θ,t) is the velocity normal to the airfoil calculated from the motion kinematics:

( )

x x x U x x x U x t w LEV TEV LEV TEV B ∂ ∂ - ∂ ∂ - - -       ∂ ∂ + ∂ ∂ + ∂ ∂ + ∂ ∂ = ∞ ∞ Φ Φ α α Φ Φ Φ α η θ  sin cos , (5)
with Φ B , Φ TEV , Φ LEV the velocity potentials associated with the bound, leading edge and trailing edge circulations and η the airfoil mean camber line. At each time step, if the flow around the airfoil is attached, a trailing edge vortex (TEV) is released and advected by the flow at the following time step. However, a separation model is needed for large values of the angle of attack. The separation on the airfoil is obtained with an inviscid parameter developed by [START_REF] Ramesh | Discrete-vortex method with novel shedding criterion for unsteady aerofoil flows with intermittent leading-edge vortex shedding[END_REF]. The Leading Edge Suction Parameter (LESP) is a non-dimensional measure of the suction at the leading edge, and equates the first Fourier coefficient:

( ) ( ) t A t 0 LESP = (6)
The critical value LESP crit corresponds to the A 0 value associated with the angle of attack for which a peak of pressure coefficient is reached at the leading edge. It is a measure of the maximum suction that a given airfoil can bear before separation and is independent of the motion. Beyond that LESP crit value, the airfoil suction side boundary layer separates from the leading edge, which corresponds to the release of a leading edge vortex (LEV). In that case, there is shedding of both a TEV and a LEV. This situation is illustrated in figure 1 for the iteration step i, which is the first one with the LESP exceeding its critical value, and with the release of two vortices. These ones, at every time step, must enforce Kelvin's circulation theorem:

( ) 0 1 , 1 , = + + ∑ ∑ = = i l l LEV i k k TEV B t Γ Γ Γ (7)
With the airfoil bound circulation:

( ) ( ) ( ) ( )       + = = ∞ ∫ 2 , 1 0 0 t A t A c U d t t B π θ θ γ Γ π (8)
The previous variables are written in a non-dimensional form as:

c U c Z Z c X X U W W U w U U w w ∞ * * * ∞ * ∞ * ∞ * = = = = = = Γ Γ (9)
The velocity induced by a given vortex is described with the model of Vatistas which incorporates a finite core radius r c [START_REF] Vatistas | A simpler model for concentrated vortices[END_REF]:

( ) ( ) ( ) ( ) 4 2 2 2 4 2 2 2 2 2 * * * * * * * * * * * * * * * * * * +       - + - - - = +       - + - - = c k k k k k c k k k k k r Z Z X X X X W r Z Z X X Z Z U π Γ π Γ (10)
The non-dimensional time step is:

015 . 0 = = ∞ * c tU t δ δ (11)
The vortex core radius is taken to be 1.3 times the average spacing between the vortices [START_REF] Leonard | Vortex methods for flow simulations[END_REF]:

02 . 0 3 . 1 = = = * * t c r r c c δ (12)
The last shed vortex is placed at one-third of the distance from the shedding edge to the previously shed vortex [START_REF] Ansari | Nonlinear unsteady aerodynamic model for insectlike flapping wings in the hover. Part 1 methodology and analysis[END_REF]:

( ) ( ) ( ) ( ) * * - * * * * - * * * * - * * * * - * * - + = - + = - + = - + = LE l LEV LE l LEV LE l LEV LE l LEV TE k TEV TE k TEV TE k TEV TE k TEV Z Z Z Z X X X X Z Z Z Z X X X X 1 , , 1 , , 1 , , 1 , , 3 1 3 1
3 1 3 1 (13)
For each time-step, the unknown parameters are the circulations corresponding to the newly shed vortices, advected by the velocity field. Firstly, if there is only a TEV shed at the iteration i, equation ( 5) is reduced to:

( ) 2 , 1 , T T t w i TEV i * * * + = Γ θ (14)
where T 1 and T 2 are terms depending on the angle of attack and the summation of the influence of the previously shed vortices. The only unknown parameter is the circulation of the TEV shed at iteration i. The airfoil bound circulation is obtained from [START_REF] Bentley | Multidimensional binary search trees used for associative searching[END_REF] substituting the Fourier coefficients:

( ) ( ) * * * + = - + - = ∫ ∫ i TEV i TEV B I I d T d T , 2 1 0 2 , 0 1 1 cos 1 cos Γ θ θ Γ θ θ Γ π π (15)
Substituting ( 15) into Kelvin's theorem (7):

2 1 1 , 1 1 , 1 , 1 I I i l l TEV i k k TEV i TEV + + + - = ∑ ∑ - = * - = * * Γ Γ Γ (16)
Secondly, if both a TEV and LEV are shed at iteration i, then equation ( 5) is reduced to:

( ) 3 , 2 , 1 , T T T t w i LEV i TEV i * * * * + + = Γ Γ θ
(17) where T 1 , T 2 and T 3 are terms depending on the angle of attack and the summation of the influence of the previously shed vortices. There is two unknown parameters which are the circulations of the TEV and LEV shed at iteration i, requiring two equations. The airfoil bound circulation is obtained from [START_REF] Bentley | Multidimensional binary search trees used for associative searching[END_REF] substituting the Fourier coefficients:

* * * + + = i LEV i TEV B J J J , 3 , 2 1 Γ Γ Γ (18)
where J 1 , J 2 and J 3 are terms resulting of the integrals of T 1 , T 2 and T 3 . Kelvin's theorem [START_REF] Ansari | Nonlinear unsteady aerodynamic model for insectlike flapping wings in the hover. Part 1 methodology and analysis[END_REF] and the criterion on the critical LESP provide:

0 LESP 0 0 1 1 , 1 1 , , , = - = + + + + ∑ ∑ - = * - = * * * * crit i l l LEV i k k TEV i LEV i TEV B A Γ Γ Γ Γ Γ (19)
Substituting the bound circulation and the value of A 0 , equation ( 19) is:

( ) ( ) 0 LESP 0 1 1 , 3 , 2 1 1 1 , 1 1 , 3 , 2 , 1 =
- + + = + + + + + + * * - = * - = * * * ∑ ∑ crit i LEV i TEV i l l LEV i k k TEV i LEV i TEV Γ Γ Γ Γ (20)
Note that this is a linear system, and no iteration scheme is required as previously mentioned in [START_REF] Ramesh | Discrete-vortex method with novel shedding criterion for unsteady aerofoil flows with intermittent leading-edge vortex shedding[END_REF] and [START_REF] Katz | Low-Speed Aerodynamics[END_REF]. Therefore, a significant gain in calculation time is expected. As only the shed LEV and TEV are computed with the method, it is worth noticing that the simulation time is increasing with the number of vortices. In order to reduce that number, the vortices located four chords downstream of the leading edge can be amalgamated into larger structures [START_REF] Sarpkaya | An inviscide model of twodimensional vortex shedding for transient and asymptotically steady separated flow over an inclined plate[END_REF]. That clustering can be realized with a multidimensional binary search tree or k-d tree [START_REF] Bentley | Multidimensional binary search trees used for associative searching[END_REF], for all the vortices downstream of four chord of the airfoil.

AERODYNAMIC COEFFICIENTS

The unsteady form of the Bernoulli theorem is used to calculate the pressure distribution along the airfoil:

( )         ∂ ∂ - ∂ ∂ + - = - t t V V p p
x x x U V x x x U V       ∂ ∂ +       ∂ ∂ +       ∂ ∂ + =       ∂ ∂ +       ∂ ∂ +       ∂ ∂ + = ∞ ∞ Φ Φ Φ α Φ Φ Φ α cos cos , , (23)
From the thin airfoil theory:

( ) ( ) 2 , 2 , t x x t x x ps B ss B γ Φ γ Φ - =         ∂ ∂ =         ∂ ∂ (24)
Then:

( )

t x x x U V V LEV TEV ps t ss t , cos 2 2 , 2 , γ Φ Φ α       ∂ ∂ + ∂ ∂ + = - ∞ (25)
The potential functions time derivatives are:

( ) ∫ ∂ ∂ = ∂ ∂ - ∂ ∂ x ps ss d t t t t 0 , ξ ξ γ Φ Φ (26)
Hence (21) becomes:

( ) ( )    ∂ ∂ +    ∂ ∂ +       ∂ ∂ + = - ∫ ∞ x LEV TEV ss ps d t t x x x U p p 0 , cos ξ ξ γ γ Φ Φ α ρ (27)
The normal force on the airfoil is obtained:

( ) ( )    ∂ ∂ +    ∂ ∂ +       ∂ ∂ + = ∫ ∫ ∫ ∞ c x LEV c TEV N dx d t t d t x x x U F 0 0 0 , , cos ξ ξ γ ξ γ Φ Φ α ρ (28)
Using the Fourier coefficients, it is reduced to:

( )           ∂ ∂ + ∂ ∂ +     + +         +       + = ∫ ∞ ∞ c LEV TEV N dx t x x x A A A c A A U c U F 0 2 1 0 1 0 , 8 4 4 3 2 cos γ Φ Φ ρ α π ρ    (29)
The axial force is given by the Blasius formula [START_REF] Katz | Low-Speed Aerodynamics[END_REF]:

2 0 2 A c U F A π ρ ∞ =
(30) Similarly, the moment about the position x ref on the airfoil is:

( )          ∂ ∂ + ∂ ∂ -         - + + +          - + - = ∫ ∞ ∞ c LEV TEV N ref xdx t x x x A A A A c A A A U c U F x M

UNSTEADY MOTION

The present algorithm is tested for unsteady motions on the cases 1, 5A and 5B of [START_REF] Ramesh | Discrete-vortex method with novel shedding criterion for unsteady aerofoil flows with intermittent leading-edge vortex shedding[END_REF]. They are respectively a pitch-hold-return motion of a SD7003 airfoil at a Reynolds number Re = 30000 (case 1) and pitch-up maneuvers of a flat plate at Re = 1000 (cases 5A and 5B). Figure 2 shows the results for case 1, where each LEV or TEV is represented by a color dot in blue (respectively red) for a clockwise (respectively counterclockwise) circulation. The time development of the shedding vortices is exactly identical as in [START_REF] Ramesh | Discrete-vortex method with novel shedding criterion for unsteady aerofoil flows with intermittent leading-edge vortex shedding[END_REF]. The aerodynamic coefficients are given in figure 3. The variation with time of the angle of attack is an increasing and decreasing ramp between 0° and 25° (figure 3-a). The lift, drag and moment about the quarter chord coefficients are compared with LDVM computations of [START_REF] Ramesh | Discrete-vortex method with novel shedding criterion for unsteady aerofoil flows with intermittent leading-edge vortex shedding[END_REF], experiments and CFD (figure 3-b to d). An excellent agreement is observed between the two LDVM, and the deviation between the present computation and the experiment or CFD is not larger than what is found in [START_REF] Ramesh | Discrete-vortex method with novel shedding criterion for unsteady aerofoil flows with intermittent leading-edge vortex shedding[END_REF]. The same comments are valid for cases 5A and 5B.

SINGLE AIRFOIL CONFIGURATION

The present study applies the LDVM to the unsteady flow developing around a steady airfoil. In order to get the critical value of the LESP, a SD7003 airfoil is chosen because the development of that parameter with Reynolds number is published in [START_REF] Ramesh | Theoretical analysis of perching and hovering maneuvers[END_REF], and equals 0.149 for the selected value Re = 10 5 . For each run, corresponding to a given value of the angle of attack, 15000 iterations are performed. Figure 4 shows the lift, drag and pitching moment about the quarter-chord coefficients versus angle of attack. The agreement of the lift coefficient obtained with the LDVM and the thin airfoil theory is excellent as long as the flow is attached between -10° and 10°. Experimental results at Re = 10 5 [START_REF] Selig | Summary of low-speed airfoil data[END_REF] and CFD performed at Re = 6×10 4 [START_REF] Lian | Laminar-turbulent transition of a low-Reynolds number rigid of flexible airfoil[END_REF] are also plotted in the figure. The agreement is good up to the stall angle, which is around 11°. Then, the plateau observed in the experiment and CFD is found, but with values greater by 30%. A good agreement is observed on the drag coefficient. Note that the LDVM is able to predict the aerodynamic coefficients far beyond the stall point, where available data are usually limited to.

LEV i k k TEV i LEV i TEV Γ Γ Γ Γ Γ (36) 0 1 1 , 2 , 1 1 , 2 , , 2 , , 2 , 2 , = +
+ + + ∑ ∑ - = * - = * * * * i n n LEV i m m Γ Γ Γ Γ Γ (37)
Thus, the circulation generation is decoupled in the dual airfoil arrangement by the initial condition imposed by the starting vortex, but the global flow and the advection of the vortices are calculated with all the circulations present at a given time. The linear system of four unknown parameters for four equations is solved with no iterative scheme. The computation of the aerodynamic coefficients for each airfoil is then performed in the same way as for a single airfoil. The velocity field used for the advection is obtained considering the influence of the bound airfoil circulations and all the shed vortices.

AERODYNAMIC EFFICIENCY RATIO

In order to quantify the control induced by potential effects or vortex shedding between the airfoils, a systematic study of the lift coefficient is carried out for different gaps and staggers. The lift efficiency ratio is defined, comparing the average lift coefficient for the dual airfoil arrangement (C L,1 +C L,2 )/2 with the lift coefficient of the single airfoil configuration C L,s , and it is defined from time-averaged values as: Preliminary results obtained for a larger gap ∆X/c = 3 seems to prove that the lift efficiency presents a similar behavior with an increase of the efficiency of the dual airfoil arrangement between -5° and 5°.

s L L L L C C C R , 2 , 1 , 2 + = (

CONCLUSION

The LDVM is implemented successively for the investigation of the unsteady flow around a steady airfoil for large values of the angle of attack. The method is very similar to the one developed by [START_REF] Ramesh | Discrete-vortex method with novel shedding criterion for unsteady aerofoil flows with intermittent leading-edge vortex shedding[END_REF], but without the iteration scheme which is increasing the computing time, since the relevant equations are written as a linear system. The method is validated for three unsteady motions published in [START_REF] Ramesh | Discrete-vortex method with novel shedding criterion for unsteady aerofoil flows with intermittent leading-edge vortex shedding[END_REF], and the very same results are found for the vortex shedding morphology and the time development of aerodynamic coefficients.

For the steady airfoil case, the results of lift, drag and moment coefficients are compared with the existing data for the SD7003 airfoil. The vortex shedding Strouhal number is found in good agreement with data concerning the flow developing downstream of a detached airfoil. The method is then adapted to the case of two airfoils of equal chord in interaction. To solve the problem, an additional condition is necessary, that is the initial starting vortex condition is written for each airfoil, while the global flow depends on the whole set of shedding vortices. A lift efficiency ratio, comparing lift coefficient resulting from the control between the two airfoils relative to the single airfoil configuration, shows an increase for attached flow conditions between -5° and 5°. These results are in good agreement with the experiments performed with two Wortmann FX63-137 airfoils, in the same Reynolds number range, for a gap equal to zero. A systematic investigation, varying the gap and stagger, is needed to obtain general conclusions on the effective control induced by a dual airfoil configuration. The airfoil shape is also an important parameter. As long as the flow is attached, flow control resulting from the aerodynamic interaction between the airfoils seems to be a good way of improving airfoil performances. For angles of attack larger than 20°, the increase of the lift efficiency ratio observed by [START_REF] Jones | Aerodynamics of biplane and tandem wings at low Reynolds numbers[END_REF] seems probably due to three dimensional effects.

  ps and ss respectively for the pressure side and suction side and V t the tangential velocity. As the flow potential function is the sum of the potential functions of the bound circulation, TEV and LEV:

Figure 2 .

 2 Figure 2. Flow features for case 1 (airfoil SD7003, Re = 30000, pitch-hold-return motion).

Figure 3 .

 3 Figure 3. Case 1 of the unsteady motion [3], variation with t * of : a) angle of attack, b) lift coefficient from LDVM, experiment and CFD, c) drag coefficient from LDVM, experiment and CFD, d) pitching-moment about the quarter chord from LDVM and CFD.

Figure 4 .

 4 Figure 4. Lift, drag and moment about the quarter-chord coefficients for a SD7003 airfoil.

Figure 5

 5 Figure 5 presents the flow development for an angle of attack of 35° at a simulation time t * = 220, with no vortex clustering. Note that the airfoil suction side is completely detached, with a well-defined detachment line from the leading edge. Periodic shedding is observed downstream of the airfoil, each individual vortex core developing into larger structures. That vortex shedding is also inducing time modulation of the aerodynamic coefficients.Figure 6 is the time

Figure 6

 6 Figure 5 presents the flow development for an angle of attack of 35° at a simulation time t * = 220, with no vortex clustering. Note that the airfoil suction side is completely detached, with a well-defined detachment line from the leading edge. Periodic shedding is observed downstream of the airfoil, each individual vortex core developing into larger structures. That vortex shedding is also inducing time modulation of the aerodynamic coefficients.Figure 6 is the time development of the lift coefficient for α = 35°. After a transient region from 0 ≤ t * ≤ 25, corresponding to the development and advection of the starting vortex, a periodic modulation of the lift coefficient is observed.

Figure 5 .

 5 Figure 5. Vortex shedding downstream of a SD7003airfoil with an angle of attack of 35°.

Figure 6 .

 6 Figure 6. Time development of the lift coefficient of a SD7003 airfoil with an angle of attack of 35°.A power spectral density function of the lift coefficient is obtained using segment length of 3000 samples and a discrete Fourier transform length of 3000 points. A fundamental peak is observed and its frequency is used to define a Strouhal number associated with the angle of attack as: α α

Figure 7 .

 7 Figure 7. Vortex shedding Strouhal number versus angle of attack for NACA 0012 [12], NACA 65 4 -421 [13] and SD7003. The development of that Strouhal number with angle of attack is plotted in figure 7. Comparison with the

Figure 9 .

 9 Figure 9. Dual airfoil configuration for gap ∆X/c = 0, stagger ∆Z/c = 4 and angle of attack α = 35°. An example of a LDVM simulation is provided in figure 9 for a Reynolds number Re = 10 5 , an angle of attack of 35°, a gap ∆X/c = 0 and a stagger ∆Z/c = 4, without vortex clustering downstream of X/c = 4. A vortex shedding is developing downstream of each airfoil, but a phase shift is observed in the creation of larger structures, probably due to the potential interaction between the airfoils. Figure 10 is the time development of the flow for a Reynolds number Re = 10 5 , an angle of attack of 30°, a gap ∆X/c = 0.5 and a stagger ∆Z/c = 1.5. Shortly after starting the flow, at t * = 1.5 (figure 10-a), the suction sides of both airfoils are detached with LEV generation, and the starting vortices are advected downstream. The flows are similar for airfoils 1 and 2. At t * = 3 (figure 10-b), the LEV are developing into large vortices on the suction side, and small discrepancies appear between the airfoils. At t * = 4.5 (figure 10-c), the starting vortices are leaving the field of observation, and a new large TEV is generated. Note the flow discrepancy between the

Figure 10 .

 10 Figure 10. Dual airfoil configuration for gap ∆X/c = 0.5, stagger ∆Z/c = 1.5 and α = 30° for times: a) t * = 1.5, b) t * = 3, c) t * = 4.5, d) t * = 217.5, e) t * = 219, f) t * = 220.5.

  Figure 11 presents the lift efficiency ratio for a Reynolds number of 10 5 , a gap ∆X/c = 1.5 and a stagger -1 ≤ ∆Z/c ≤ 1, versus the angle of attack. Comparison is made with the only two-dimensional data available [14] concerning two Wortmann FX63-137 airfoils for a Reynolds number of 8.5×10 4 , a gap ∆X/c = 1.5 and a stagger ∆Z/c = 0. A general agreement is found between LDVM and the experiment, although the airfoils considered are different. First of all, a global invariance of the lift efficiency ratio is found with ∆Z/c. The development with the angle of attack is similar, with an increase of the lift efficiency between -5° and 5°, corresponding to attached flow conditions. A very different behavior is found comparing the present results with [15], with an increased lift efficiency for α ≥ 20° and ∆Z/c ≥ 0.5, but these authors used flat

Figure 11 .

 11 Figure 11. Lift efficiency ratio versus angle of attack for ∆X/c = 1.5 and -1 ≤ ∆Z/c ≤ 1.

I I I J J J Γ Γ

i LEV i TEV B B Γ Γ Γ Γ Γ Γ

i LEV i TEV B

TEV i LEV i TEV B

experimental results of [START_REF] Alam | The ultra-low Reynolds number airfoil wake[END_REF] conducted with a NACA 0012 airfoil, and [START_REF] Sicot | Unsteady characteristics of the static stall of an airfoil subjected to freestream turbulence level up to 16%[END_REF] conducted with a NACA 65 4 -421 airfoil with various upstream turbulence levels are provided. Note that for large values of the angle of attack, the suction side flow is completely detached, and the actual form of the airfoil has little importance, as it is shown by the relative agreement between the points, converging toward a plateau around St α = 0.13.

DUAL-AIRFOIL ARRANGEMENT

A dual airfoil arrangement is considered hereafter, with a gap ∆X and a stagger ∆Z between two airfoils of equal chord c (figure 8). The wakes and vortex shedding created by the airfoils and the potential effects between them modifies the flow and the aerodynamic performance of the configuration. The arrangement is investigated with the LDVM adapted for two airfoils. For each time step, circulations associated to the TEV and LEV are created, providing four new variables. To solve the problem, the available equations are the LESP criteria for airfoil 1 and 2 and Kelvin's theorem, that is three equations for four unknown parameters:

where the indices 1 and 2 are relative to airfoils 1 and 2. An additional condition on Kelvin's theorem is obtained by the initial condition, that is the starting vortex around each airfoil equals zero, replacing (35) by: 0


Richard Rouil
email: richard.rouil@nist.gov

Fernando J Cintrón
email: fernando.cintron@nist.gov

Aziza Ben Mosbah
email: aziza.benmosbah@nist.gov

Samantha Gamboa Quintiliani
email: samantha.gamboaquintiliani@nist.gov

A Long Term Evolution (LTE) Device-to-Device module for ns-3

Keywords: CCS Concepts, Networks ➝ Network performance evaluation, Networks ➝ Network types ➝ Mobile networks 3GPP, Long Term Evolution, Device-to-Device communication, network modeling, ns-3

In this paper, we provide an overview of our on-going implementation of a 3 rd Generation Partnership Program (3GPP) Proximity Services (ProSe) module in ns-3 to enable the performance evaluation of device-to-device (D2D) discovery and communication in Long Term Evolution (LTE) networks.

INTRODUCTION

Device-to-device (D2D) communication is a common feature in technologies such as Bluetooth and WiFi. However, this is a fairly new concept for 3GPP where historically access to the radio resources has been controlled by the network. If the network is not available, the devices are not able to communicate. With Release 12 and the introduction of ProSe [START_REF]Proximity-based services (ProSe); Stage 2[END_REF], 3GPP provides the ability for User Equipments (UEs) to discover each other and, in some cases, communicate with each other without eNodeB intervention. The discovery service provided by ProSe responds to the increasing use of social media where users want to know what is happening around them. Some of the use cases include advertisement broadcast in shopping malls or notifications of nearby people with similar interests. While discovery is available to all UEs when the network supports it, direct communication is currently restricted to Public Safety users, which have identified this feature as critical in order to fully transition to LTE. Furthermore, D2D communication has been identified as one of the enablers for 5G communications [START_REF] Bertenyi | 3GPP system standards heading into the 5G era[END_REF] indicating that further research is needed in that domain and that the current restrictions may have to be removed. The rest of this document provides an overview of the ProSe module implementation followed by validation results, open issues and future work.

IMPLEMENTATION

Our ProSe module is extending the ns-3 LTE model [START_REF] Baldo | The ns-3 LTE module by the LENA project[END_REF] by adding UE to UE communication capabilities. In 3GPP specifications, the term sidelink refers to the D2D communication in contrast to downlink and uplink communication between the eNodeB and the UE.

Architecture

In our implementation, the structure of the LTE UE nodes, shown in Figure 1, has not been changed. However, the extension involved some modifications at all levels of the LTE protocol stack, from the Non Access Stratum (NAS) down to the physical layer. The main design alteration, shown in Figure 2, was the addition of a new instance of the SpectrumPhy class in the UE in order to allow the reception of packets sent by other UEs on the uplink channel.

Summary of modifications

Radio Resource Control (RRC) Protocol

The RRC layer provides signaling between the eNodeB and the UE to perform attachment and setup radio bearers. Extensions to the protocol have been made so the UE can indicate its interest in performing D2D when in coverage of an eNodeB. It also stores the preconfigured radio resources when the UEs are out of coverage.

Packet Data Convergence Protocol (PDCP) and Radio Link Control (RLC) Protocol

The sidelink radio bearers use the Unacknowledged Mode (UM) transmission mode that is already available in ns-3. However, the identifier for the logical channels has been extended to include the source L2 ID and destination L2 ID that identifies the transmitter UE and the group to which the packets must be

Medium Access Control (MAC) Protocol

The MAC protocol responsible for allocating radio resources had to be modified at both the eNodeB and the UE. The changes include the processing of sidelink Buffer Status Request (BSR) to indicate how much D2D traffic needs to be transmitted and the schedulers to handle the new type of resource allocations. The concept of scheduling algorithm is new to the UE implementation as it normally just follows the information provided by the eNodeB at each subframe. With D2D, the UE has to decide how to allocate resources when it is out of coverage or when it is in coverage but the allocation mode is UE selected. The initial scheduler implementation provides a static allocation, with the same number of resource blocks and subframes used in each sidelink period, that is configurable from the scenario.

Physical Layer Protocol

The physical layer was extended to support additional physical channels such as the Physical Sidelink Discovery Channel (PSDCH) to send and receive discovery messages, Physical Sidelink Control Channel (PSCCH) to carry the allocation information, and Physical Sidelink Shared Channel (PSSCH) to carry the D2D data. The model also supports scanning and transmission of sidelink synchronization messages so UEs can detect and synchronize with each other. Finally, a half-duplex constraint has also been added so UEs cannot send and receive uplink/sidelink transmissions as the same time.

Propagation and Error Modeling

Several D2D propagation models have been implemented as specified by 3GPP [START_REF]Study on LTE device to device proximity services; Radio aspects[END_REF] to model outdoor to outdoor, outdoor to indoor, and indoor to indoor environments. A new error model was introduced to handle interference from multiple sources on the same resource blocks and to properly characterize uplink modulations.

VALIDATION

A critical step for the adoption of a new model is to compare the results obtained using well defined scenarios. This section describes the performance evaluation of the sidelink shared (data) channel and shows that our results are within range of other models documented in [START_REF]Study on LTE device to device proximity services; Radio aspects[END_REF].

Modeling assumptions

The assumptions contained in Table 1 were used to perform the evaluation of the sidelink shared channel. Outdoor' uniform Outdoor' hotspot Indoor' Outdoor

Topologies

The validation scenarios are based on a 19 macro-cell topology. The UEs are deployed following one of three options: in outdoor uniform, all UEs are uniformly and randomly deployed within each sector; in outdoor hotspot, some of the UEs are concentrated in random locations; finally, for indoor outdoor, buildings are randomly placed in the topology and UEs are deployed either indoor or outdoor. Random transmitters are selected throughout the topology and receiver UEs are associated to the transmitters if the RSRP is above the preconfigured threshold. For legibility, Figure 3 illustrates those deployments for a 7 macro-cell deployment.

Simulation results

In this section we present the performance results obtained with our model side by side with the results provided in [START_REF]Study on LTE device to device proximity services; Radio aspects[END_REF]. The first performance metric used is the fraction of successful VoIP links, which is defined by a link with less than 2 % packet loss. As shown in Figure 4, our results are comparable to the other companies.

Figure 4: Fraction of successful VOIP links for various deployments

The second metric is the number of successful links per transmitter. As with the fraction of successful links, Figure 5 shows that our results are in the same range as the other companies.

OPEN ISSUES

Among the model limitations, we can mention the lack of idle mode in the default LTE implementation. Workarounds had to be made to simulate the out of network scenarios. In addition, we would like to create an UE scheduler interface as currently done in the eNodeB so that researchers can easily test their own algorithms. Finally, traces will need to be extended to support the D2D packet transmissions.

CONCLUSION AND FUTURE WORK

In this extended abstract, we presented an overview of the extensions made to the ns-3 LTE implementation to support D2D discovery and communication. The model presented is under active development and several new features may be added such as UE-to-network relay or priority queues, which are features added in 3GPP Release 13.

Figure 1 :Figure 2 :

 12 Figure 1: LTE radio protocol stack architecture for the UE on the data plane

Figure 3 :

 3 Figure 3: Topologies used for model validation

Figure 5 :

 5 Figure 5: Number of successful links per transmitter for various deployments

Table 1 : Simulation parameters Parameter Value

 1

	Transmission BW (RBs)	2.0
	Packet size including CRC (bits)	328.0
	Modulation and Coding Scheme	QPSK
	Coding	Turbo
	Number of
	symbols/Transmissions	12.0
	Tx Power (dBm)	23.0
	Num HARQ transmissions	4.0
	Frequency Diversity (across
	transmissions)	Yes [per period]
	Time Diversity (across
	transmissions)	Yes [per period]
	Number of TX/cell	3.0
	RSRP threshold (dBm)	-112.0
		On/off Voice over IP
	Traffic	(VoIP)


domain: chim sdv

text:
Ph.D Pierre Stocker
email: p.stocker@univ-amu.fr

Mathieu Cassien

Nicolas Vidal

Sophie Thétiot-Laurent

Sylvia Pietri

A fluorescent homogeneous assay for myeloperoxidase measurement in biological samples. A positive correlation between myeloperoxidasegenerated HOCl level and oxidative status in STZ-diabetic rats

Keywords: Myeloperoxidase, hypochlorous acid, 7-hydroxy-2-oxo-2H-chromene-8-carbaldehyde oxime, fluorimetric assay myeloperoxidase, MPO, protein carbonyls, PC, glutathione, GSH, oxidized glutathione, GSSG, streptozotocin, STZ, 7-hydroxy-2-oxo-2H-chromene-8

Myeloperoxidase (MPO) is a key enzyme derived from leucocytes which is associated with the initiation and progression of many inflammatory diseases. Increased levels of MPO may contribute to cellular dysfunction and tissues injury by producing highly reactive oxidants such as hypochlorous acid (HOCl). Myeloperoxidase-generated HOCl is therefore considered as a relevant biomarker of oxidative stress-related damage and its quantitation is of great importance to the study of disease progression.

In this context, the current study describes a rapid, sensitive and homogeneous fluorescencebased method for detecting the MPO chlorination activity in biological samples. This assay utilizes 7-hydroxy-2-oxo-2H-chromene-8-carbaldehyde oxime as a selective probe for HOCl detection, and is adapted to 96-well microplates to allow high-throughput quantitation of active MPO.

The ability of the method to monitor HOCl release was further investigated in hyperglycemic streptozotocin-treated diabetic rats. The data proved that the present assay has a reliable performance when quantitating the active MPO in the plasma of diabetic animals, a feature of inflammatory disease found concomitant with an elevation of protein carbonyls levels and lipid peroxidation and which was negatively correlated with the ratio of reduced-to-oxidized glutathione.

Introduction

Myeloperoxidase (MPO) is a mammalian pro-oxidant enzyme mainly released by activated neutrophils, which catalyzes the conversion of hydrogen peroxide (H2O2) and chloride to hypochlorous acid (HOCl) which in turn readily oxidizes key molecules, such as proteins and lipids. It is well established that MPO promotes endothelium dysfunction leading to various vascular damages and elevated blood MPO and HOCl levels could be predictive for increased risk for cardiovascular events [START_REF] Brennan | Emerging role of myeloperoxidase and oxidant stress markers in cardiovascular risk assessment[END_REF][START_REF] Podrez | Myeloperoxidase-generated oxidants and atherosclerosis[END_REF][START_REF] Kettle | Myeloperoxidase and protein oxidation in the airways of young children with cystic fibrosis[END_REF][START_REF] Wiersma | Diabetes mellitus type 2 is associated with higher levels of myeloperoxidase[END_REF]. Among the numerous pathologies associated with increased cardiovascular risk, high-glucose stimulated H2O2 production contributes to MPOmediated atherosclerosis progression in type 2 diabetes mellitus [START_REF] Shetty | Variations in serum myeloperoxidase levels with respect to hyperglycemia, duration of diabetes, Bmi, sex and aging in type 2 diabetes mellitus[END_REF][START_REF] Zhang | Association between myeloperoxidase levels and risk of coronary artery disease[END_REF][START_REF] Askari | Myeloperoxidase and plasminogen activator inhibitor 1 play a central role in ventricular remodeling after myocardial infarction[END_REF][START_REF] Song | Association of plasma myeloperoxidase level with risk of coronary artery disease in patients with type 2 diabetes[END_REF]. MPO actions in human health and disease have been widely recognized as specific biomarkers for disease progression, therefore the MPO quantitation in blood and tissues is of great importance in both the experimental and clinical setting.

In clinical practice the most widely used method for MPO detection and quantitation is the enzyme-linked immunosorbent assay (ELISA) method which uses MPO-specific antibodies and which are not relevant for the measurement of the active enzyme. Since then immunological methods have been developed for the measurement of the active content of the enzyme in complex media such as the specific immunological extraction followed by enzymatic detection (SIEFED [START_REF] Pulli | Measuring Myeloperoxidase Activity in Biological Samples[END_REF][START_REF] Franck | A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts[END_REF]. The immunological methods have been found to be very sensitive and satisfactorily specific but are expensive and time-intensive. In the research field, few alternative methods have been developed for the detection of chlorination or peroxidase activity of the enzyme using chromogenic and fluorogenic substrates [START_REF] Odobasic | Endogenous myeloperoxidase promotes neutrophil-mediated renal injury, but attenuates T cell immunity inducing crescentic glomerulonephritis[END_REF][START_REF] Marquez | Mechanism of the oxidation of 3,5,3′,5′ tetramethylbenzidine by myeloperoxidase determined by transient-and steady-state kinetics[END_REF][START_REF] Setsukinai | Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species[END_REF][START_REF] Kettle | Assays for the chlorination activity of myeloperoxidase[END_REF][START_REF] Dypbukt | A sensitive and selective assay for chloramine production by myeloperoxidase, Free Rad[END_REF]. However, chlorination and peroxidase activities of the enzyme cannot be measured simultaneously and could be strongly affected by pH, hydrogen peroxide, superoxide, chloride and endogenous inhibitors such as ceruloplasmin, lipoproteins and antioxidants like ascorbic acid [START_REF] Klebanoff | Inflammation: Basic Principles and Clinical Correlates[END_REF][START_REF] Winterbourn | Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid[END_REF][START_REF] Frei | Ascorbate is an outstanding antioxidant in human blood plasma[END_REF][START_REF] Chapman | Ceruloplasmin is an endogenous inhibitor of myeloperoxidase[END_REF].

There are four distinct peroxidase superfamilies that evolved independently in evolution that can oxidize chloride (e.g. chloroperoxidase) [START_REF] Zamocky | Independent evolution of four heme peroxidase superfamilies[END_REF]. But MPO is the only human peroxidase that produces HOCl at reasonable rates; note that eosinophil peroxidase is able to oxidize chloride only at low pH values [START_REF] Furtmüller | Reaction of myeloperoxidase compound I with chloride, bromide, iodide, and thiocyanate[END_REF]. Thus, for the measurement of myeloperoxidase activity, assays based on measurement of chlorination activity are considered more specific than those based on the detection of peroxidase substrates such as 0-dianisidine hydrochloride or tetramethylbenzidine [START_REF] Odobasic | Endogenous myeloperoxidase promotes neutrophil-mediated renal injury, but attenuates T cell immunity inducing crescentic glomerulonephritis[END_REF][START_REF] Marquez | Mechanism of the oxidation of 3,5,3′,5′ tetramethylbenzidine by myeloperoxidase determined by transient-and steady-state kinetics[END_REF]. Therefore, investigating chlorination activity appears more attractive in distinguishing myeloperoxidase from other heme proteins with peroxidase activity. For this purpose, several assays have been developed to measure the chlorination activity of myeloperoxidase and most of them are based on the detection of chloramines which are the HOCl oxidation products of proteins or amino acids [START_REF] Setsukinai | Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species[END_REF][START_REF] Dypbukt | A sensitive and selective assay for chloramine production by myeloperoxidase, Free Rad[END_REF][START_REF] Shepherd | A novel fluorescent probe for the detection of myeloperoxidase activity in atherosclerosisassociated macrophages[END_REF]. However some weaknesses in the reliability, specificity or sensitivity were reported, rendering these assays hardly applicable to complex biological samples in vivo [START_REF] Kettle | Assays for the chlorination activity of myeloperoxidase[END_REF].

With the aim to overcome some of these later issues, the present study describes a sensitive homogeneous assay allowing the high-throughput MPO quantitation and rapid processing of multiple samples simultaneously in a short period of time. This method was developed to measure the chlorination activity based on MPO-derived HOCl detection which is an endpoint of the actual oxidative capacity of the enzyme. First, a convenient procedure is reported to synthesize 7-hydroxy-2-oxo-2H-chromene-8-carbaldehyde oxime (7-HCCO, Scheme1) as a highly specific fluorescent probe for HOCl detection since the double bond of the oxime could be selectively cleaved by HOCl leading to a strong increase in fluorescence [START_REF] Yu | A hypochlorous acid turn-on fluorescent probe based on HOCl-promoted oxime oxidation and its application in cell imaging[END_REF].

Second, the current study reports an accurate method to monitor and quantitate the reaction of HOCl on 7-HCCO, adapted to 96 wells microplate, which is cost-effective and does not require time-consuming steps when compared to ELISA techniques or other methods. The reliability of this method was then investigated by measuring MPO levels in various biologically-relevant milieus such as rat plasma and HL60 cells medium. Finally, the method was successfully applied in vivo (i) to demonstrate MPO-derived HOCl release in streptozotocin (STZ)-treated diabetic rats. (ii) To investigate the relationship between the MPO level and biomarkers of oxidative stress such as protein carbonyls and plasma malondialdehyde or antioxidant defense such as glutathione.

Experimental

Reagents and materials

All chemicals and reagents were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France) and were of the highest grade available. The protein carbonyl assay kit (Carbofax ® ) based on 7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole (NBDH) was purchased from Yelen (Ensuès-la-Redonne, France; http://www.yelen-analytics.com). Analytical NMR spectra were recorded using a Bruker Avance III Nanobay spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) relative to internal Me4Si ( 1 H and 13 C) and coupling constants (J) are given in hertz (Hz). Splitting patterns are reported as follows: s = singlet; d = doublet; dd = doublet of doublets; br = broad peak. The screening assay was performed with a microplate spectrofluorimeter TECAN Infinite 200 (TECAN Männedorf, Switzerland). Doubly distilled deionized water filtered through a 0.2-µm Millipore filter was used throughout. Purified MPO (ref MY176) was supplied by Elastin Products Co (Owensville, USA).

Synthesis of 7-HCCO

The synthesis of probe 7-HCCO was performed according to modifications of a previous procedure [START_REF] Yu | A hypochlorous acid turn-on fluorescent probe based on HOCl-promoted oxime oxidation and its application in cell imaging[END_REF]. 7-acetylcoumarine (1): 7-hydroxycoumarin (3.09 g, 19.1 mmol) was dissolved in dichloromethane (50 mL). Then acetic anhydride (2 mL, 21.2 mmol) and pyridine (2 drops) were added and the mixture stirred overnight at 38°C. The mixture was then allowed to cool at room temperature and concentrated under vacuum. The residue was dissolved in water (50 mL) and ethyl acetate (100 mL). The aqueous layer was extracted with ethyl acetate (3 × 50 mL). The organic layers were gathered, washed with an aqueous saturated solution of K2CO3 (3 × 50 mL), dried over MgSO4, filtered and concentrated to afford 1 as a white powder (3.9 g, quant.). 8-formyl-7-hydroxycoumarine (2): Compound 1 (3.9 g, 19.1 mmol) was dissolved in trifluoroacetic acid (26 mL) and the solution was cooled at 0°C. Hexamethylenetetramine (3.8 g, 27.5 mmol) was then added; the resulting solution was allowed to warm at room temperature and then heated under reflux overnight. The mixture was then concentrated under vacuum and the residue was suspended in water (52 mL) and heated at 60°C for 30 min. The mixture was then cooled at 0°C to afford 2 as an orange powder that was filtered and dried (1.35 g, 37 %). 1

7-hydroxy-2-oxo-2H-chromene-8-carbaldehyde oxime (7-HCCO):

Hydroxylamine hydrochloride (750 mg, 10.7 mmol) and triethylamine (1.5 mL, 10.7 mmol) were added to a solution of compound 2 (1.35 g, 7.11 mmol) in absolute ethanol (20 mL). The mixture was heated under reflux overnight, and then concentrated under a vacuum. The residue was dissolved in water (40 mL) and ethyl acetate (25 mL). The aqueous layer was extracted with ethyl acetate (4 × 20 mL). The organic layers were gathered, dried over MgSO4, filtered and concentrated to afford 7-HCCO as an orange powder (1.0 g, 69 %). 1

Synthesis of oxidized 7-HCCO

NaOCl (1 mL, 10 % dissolved in water, 16.5 mmol) was added to a solution of compound 7-HCCO (50 mg, 0.244 mmol) dissolved in methanol (6 mL). The mixture was stirred at room temperature for 30 min and concentrated. The residue was purified by column chromatography (SiO2, gradient AcOEt/MeOH from 100/0, v/v to 90/10, v/v) to afford oxidized-7-HCCO as a yellow powder (20 mg, 44 %). 1

High-performance liquid chromatography analysis

Chromatographic analyses were performed with a Merck Hitachi System consisting of a LaChrom L-7000 interface module and a L-7480 fluorescence detector controlled by the EZChrome Chromatography manager software. Reverse phase HPLC was carried out at room temperature on a Nucleodur RP-18 column (125 mm × 4.6 mm; 3 µm; Macherey Nagel) using a gradient with a flow rate of 0.8 mL.min -1 . Gradient: solvent A (phosphate buffer 10 mM, pH 7.4), solvent B (acetonitrile): 0-15 min, 10-30% B; 16-24 min, 10% B. Derivatives were measured at excitation and emission wavelengths of 350 nm and 470 nm, respectively.

HOCl production by isolated myeloperoxidase

Myeloperoxidase was mixed at 37°C in 1X PBS pH 6.5 with 7-HCCO probe at 150 µM in 96 wells microplate and H2O2 (100 µM) was added to start the reaction. The fluorescence was measured every 3 min at 470 nm, exciting at 350 nm. Fluorescence intensity change (∆F) was directly proportional to the quantity of active MPO and related to a standard curve to determine the concentration of hypochlorous acid produced. One unit (U) of the enzyme was the activity which formed 1 µmol HOCl per min at 37°C calculated from the initial rate of reaction with peroxide as the substrate.

HOCl production by HL-60 cells

The characteristics and origin of HL60 cell line used in the present study was purchased from ATCC (Rockville, MD). HL60 (acute promyelocytic leukemia) is a promyelocytic cell line derived by S.J. Collins et al. [START_REF] Collins | The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression[END_REF]. Peripheral blood leukocytes were obtained by leukopheresis from a 36-year-old Caucasian female with acute promyelocytic leukemia. Cells were grown in DMEM(1X)+GlutaMAX TM containing a stabilized form of L-glutamine, Lalanyl-L-glutamine and supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C in 5% carbon dioxide atmosphere. Cells (5 × 10 6 /mL) were lysed by a freeze/thaw treatment and sonicated on ice for 10 min. Cell lysates were centrifuged for 10 min at 15,000 × g at 4°C. Two hundred µL of supernatant 10 times diluted in 1X PBS pH 6.5 was preincubated 30 min at 37°C with 20 µL PBS 1X or 20 µL of sodium azide (10 mM) in 96 wells microplate. After 10 min of incubation at 37°C, 7-HCCO at 100 µM and H2O2 at 100 µM were added and fluorescence was measured every 3 min at 470 nm, exciting at 350 nm.

Animals and biological samples preparation

A total of twenty-four Sprague-Dawley male rats weighing 120-150 g were purchased from CERJ (Le Genest St Isle, France) and fed ad libitum with a standard Teklad 2016 diet (Harlan Laboratories, Gannat, France). Animals were maintained in the local animal house under conventional conditions, including an enriching structural and social environment. To induce diabetes, streptozotocin (STZ) was administered to twelve overnight fasted rats (STZ-diabetic group) by a single intraperitoneal injection (final dose 60-70 mg/kg in 0.4-0.5 mL). STZ was dissolved beforehand in citrate buffer (1 mM, pH 4.5) and maintained on ice before use. Seventy-two hours after STZ injection, the blood glucose concentration was checked using a commercial glucose auto analyser (AlphaTRAK®, Abbott Laboratories).

Blood samples (6 μL/sample) were collected from the tail vein under local anaesthesia induced by 0.25% lidocaine application in ointment (B Braun, France). In the study, a blood glucose level higher than 350 mg.dL -1 was found in 100% of animals which were considered to be diabetic and used in the experiments. During the 3 weeks following STZ injection blood samples were collected weekly and the blood glucose level was measured in fasting (5-6 h, morning fast) conditions. A second group of twelve untreated rats was used for control (non diabetic control group). All animals were observed daily for clinical signs of suffering, weight loss and moribundity. At the end of the protocol, animals were euthanized by intraperitoneal injection of sodium pentobarbital (120 mg.kg -1 ; Ceva Santé Animale, Libourne, France) before thoratocomy. The blood was withdrawn from the heart cavity and immediately added to serine borate buffer and processed for GSH and GSSG determination (see below) or into heparinized glass tubes for plasma preparation by centrifugation (1,650 × g for 15 min at 3°C) and kept at -80°C for further analysis of MPO, protein carbonyls and MDA-TBA levels.

Chlorination activity of myeloperoxidase in rat plasma

Plasma samples from control normal rats were diluted ten-fold in 1X PBS pH 6.5. One hundred µL of diluted plasma was placed in 96 wells microplate. Then 7-HCCO at 100 µM and H2O2 at 100 µM were added and fluorescence was measured every 3 minutes at 470 nm, exciting at 350 nm during 15 minutes. MPO content was calculated from the relative linear fluorescence intensity and related to a calibration curve constructed in plasma with purified human MPO. The precision of the method was estimated by the coefficients of variation (CV) calculated for several determinations made in triplicate.

GSH and GSSG quantitation

GSH and GSSG were determined in the blood of diabetic and control rats according to the method previously described by N'Guessan et al. [START_REF] N'guessan | Absence of tumor suppressor Tumor Protein 53-Induced Nuclear Protein 1 (TP53INP1) sensitizes mouse thymocytes and embryonic fibroblasts to redox-driven apoptosis[END_REF] with modifications. Blood was collected and immediately mixed with the equivalent volume of serine borate buffer (pH 8.5) containing: L-serine (100 mM), boric acid (80 mM), sodium tetraborate (20 mM). This buffer was associated with bathophenanthroline disulfonate sodium salt (2 mM), iodoacetic acid (10 mM) and Na-heparin (2020 USP units) to form the collecting solution. The association of blood and collecting solution was plunged rapidly on liquid nitrogen and kept at -80°C.

Protein was precipitated by adding 300 µl of 1 M perchloric acid in 0.

Protein Carbonyls

Protein carbonyls were determined in plasma samples from diabetic and control rats after derivatization with the probe NBDH according to a recently published fluorometric method adapted to 96-microplates (TECAN Infinite 200) [START_REF] Stocker | Fluorimetric screening assay for protein carbonyl evaluation in biological samples[END_REF]. Plasma samples were diluted with 4 vol of PBS 0.1X before analysis. Then 100 µL of diluted samples were placed in 96-well microplates and reacted for 15 min at room temperature with an aliquot (100 µL) of the NBDH assay solution (Carbofax ® , Yelen Analytics). Concentration of NBDH-reacted carbonyls was determined by fluorescence (emission determined at 560 nm using an excitation wavelength at 480 nm) and reduced and oxidized bovine serum albumin was used for calibration. The amount of protein carbonyls was calculated with respect to total protein content obtained using the Pierce BCA protein assay kit (Fisher Scientific, France) and is expressed in nanomoles/mg protein. Data are means of twelve independent experiments in each group made in duplicate.

Lipid peroxidation measurement

In test tubes aliquots (50 µL) of plasma were added to a mixture of 450 µL of thiobarbituric acid (TBA) solution in orthophosphoric acid (0.3 M, pH 3.5) and 10 µL of butylated hydroxytoluene solution (5 mM) in ethanol. The samples were heated at 95 °C for 30 min, cooled to room temperature, centrifuged (1800 × g) for 10 min and the malondialdehyde (MDA)-TBA adduct content of the supernatants were assayed by HPLC. Separation was carried out at room temperature using the HPLC system described above with fluorescence emission determined at 553 nm using an excitation wavelength at 532 nm, and a C18-bonded Macherey-Nagel silica column (250 mm × 4.6 mm, 5 µm). HPLC analyses used solvent A: 20 mM phosphate buffer (pH 6.9) and solvent B: methanol. Samples (20 µL) underwent an isocratic elution (A:B, 50:50) at a flow rate of 1 mL/min for 10 min, with peak area quantification. MDA-TBA levels were calculated against a standard curve of MDA tetrabutylammonium and are the means of twelve independent experiments in each group made in duplicate. Data are expressed in nanomoles/mg protein.

Statistical analysis

Data are presented as mean ± SD or SEM for the indicated number of independent experiments performed in duplicate. Evaluation of statistical significance was conducted using an unpaired Student's t test or Mann-Whitney when appropriate (GraphPad Prism Software). Differences were considered significant when P < 0.05.

Results

Synthesis and characterization of probe 7-HCCO

Synthesis of probes 7-HCCO and oxidized 7-HCCO is reported in Scheme 1. Acetylation of 7-hydroxycoumarine, which afforded compound 1 in a quantitative yield. The formulation of compound 1 was performed through a Duff reaction inspired from the synthesis described by Lee et al. [START_REF] Lee | Fluorescent chemodosimeter for selective detection of cyanide in water[END_REF] to give compound 2 in a moderate yield. Reaction of aldehyde with hydroxylamine gave the probe 7-HCCO with a 26 % overall yield. To verify the dehydration of the probe into the nitrile, reaction of 7-HCCO with sodium hypochlorite was performed.

The dehydration was proved by the disappearance of the signal attributed to the oxime function (δ 8.53 ppm in 7-HCCO) a result which positively correlates to the previously reported spectra [START_REF] Yu | A hypochlorous acid turn-on fluorescent probe based on HOCl-promoted oxime oxidation and its application in cell imaging[END_REF].

Chemosensor 7-HCCO is not fluorescent but its oxidation by HOCl gives highly fluorescent derivative. Other reactive oxygen and nitrogen species such as OH, H2O2, 1 O2, NO2 -, NO3 -, NO, ONOO -only induced negligible fluorescence enhancement [START_REF] Yu | A hypochlorous acid turn-on fluorescent probe based on HOCl-promoted oxime oxidation and its application in cell imaging[END_REF]. The probe 7-HCCO is then highly selective and sensitive towards HOCl. Fig. 1 shows the absorbance and fluorescence spectra of 7-HCCO after reacting with HOCl in PBS 1X pH 6.5. The absorption spectrum of derivative shows a maximum at 350 nm and the emission spectrum presents a maximum at 470 nm. Fig. 2 shows an HPLC analysis of 7-HCCO and 7-HCCOox. HOCl and 7-HCCO alone did not present a fluorescent signal contrary to the derivative mixture prepared with 7-HCCO and HOCl identified as oxidized 7-HCCOox. The fluorescence emission recorded with 7-HCCO (100 µM in 1X PBS pH 6.5) is proportional to HOCl from 0 to 150 µM and showed a linear regression with good correlation coefficient ( F(a.u.) = 10.485c, R 2 =0.9992 ). Under these conditions, the limit of detection for HOCL is estimated to be 8 µM.

Chlorination activity of purified MPO

It is known that the optimum pH of the MPO-catalyzed chlorination reaction varies with chloride and H2O2 concentrations. Under usual experimental conditions the activity of the MPO-H2O2-halide system has an optimum pH varying from 4 to 7.4 depending on the [H2O2]/[Cl -] ratio. The H2O2 concentration dependences of the human purified MPO activity in 1X PBS pH 6.5 at 37°C are shown in Fig. 3. The MPO activity has a maximum at 100 µM and decreases dramatically at 1 mM. The results of MPO inactivation by hydrogen peroxide are in agreement with previous data [START_REF] Ohno | Diffusion of extracellular hydrogen peroxide into intracellular compartments of human neutrophils. Studies utilizing the inactivation of myeloperoxidase by hydrogen peroxide and azide[END_REF][START_REF] Gorudko | New approaches to the measurement of the concentration and peroxidase activity of myeloperoxidase in human blood plasma[END_REF]. Myeloperoxidase inactivation by hydrogen peroxide was not prevented by chloride-free medium (data not shown). The chlorination specific activity of the native human myeloperoxidase measured by this method was 180 units/mg of protein. Purified human MPO produced HOCl in a concentration-dependent manner. Fluorescence intensity change (∆F) was recorded in 1X PBS pH 6.5 with 7-HCCO at 100µM is proportional for a range of MPO content between 0 to 120 µg/l. The following relationship was obtained: y = 6.959x ; R 2 =0.9978, were y is in µmol/min and x is µg/l).

Under the conditions of the assay, concentrations as low as 5 µg/L myeloperoxidase could be reliably detected.

MPO chlorination activity in HL-60 cells

To study whether the 7-HCCO probe can detect HOCl generated by MPO in biological samples, HL-60 cells were first used for the experiments. Fig. 4 shows the kinetic curves for 7-HCCO oxidation in HL-60 cell lysate. A fluorescence increase is clearly recorded without the heme enzyme inhibitor sodium azide. The inhibition of MPO activity by azide was greater than that observed in the absence of H2O2, suggesting that azide action goes beyond simple inhibition of MPO activity.

MPO activity in rat plasma

Having established that the probe 7-HCCO can detect MPO activity in isolated cells, the probe was then used to provide direct evidence of HOCl formed by MPO in rat plasma.

Samples were diluted 10 fold in order to reduce the effects of native MPO inhibitors such as ceruloplasmin or ascorbic acid [START_REF] Goiffon | A rapid bioluminescence assay for measuring myeloperoxidase activity in human plasma[END_REF]. At a higher dilution, the limit of the sensitivity is reached by samples with a low content of active MPO. The precision of the method was estimated by repeatedly measuring the same assay with five different concentrations of active MPO. These MPO concentrations were added to the same sample and ranged from 8 to 125 µg/L. Using the current assay, a calibration curve was built (15 µM of 7-HCCO and 100 µM H2O2) with twenty different rat plasma samples that were supplemented with incremental purified human MPO concentrations (standard curve ∆F = 0.7633c, R 2 =0.9981). Intra-assay CV ranged from 4% to 9.8% and the inter-assay CV ranged from 6.9% to 13.9%. The MPO concentrations were chosen to reach common ranges found in other studies or commercial kits [START_REF] Gorudko | New approaches to the measurement of the concentration and peroxidase activity of myeloperoxidase in human blood plasma[END_REF][START_REF] Goiffon | A rapid bioluminescence assay for measuring myeloperoxidase activity in human plasma[END_REF][31]. In each sample MPO content was calculated from the calibration curve.

Comparative measurements of MPO to multiple oxidative stress status biomarkers in diabetic rats

With the aim to test the ability of the method in a relevant physiopathological model known to trigger oxidative stress and inflammation, MPO levels were measured in plasma samples from rats allocated to control or STZ-diabetic groups (n=12/group). It was checked beforehand that all rats of the STZ-diabetic group have developed hyperglycemia within 3-5 days after STZ injection, as indicated by increased blood glucose concentrations (231 ± 8 mg.dL -1 in STZ-treated rats versus 79 ± 2 mg.dL -1 in controls, P<.0.01). The content of protein carbonyls, glutathione and oxidized glutathione and the malondialdehyde levels was determined for each animal 3 weeks after STZ injection, and the results are reported in Fig. 5.

It was shown that the values of MPO level (± SEM) in diabetic rat plasma samples (433 ± 19 µg/L) were significantly higher (P<0.001) than the values obtained in control animals (106 ± 6 µg/L). The values of GSH blood content in the STZ group (87 ± 11 µmol.L -1 ) were found to be significantly lower than that of the controls (264 ± 19 µmol.L -1 ). In addition, a significant increase (by 65%) in the GSSG content was observed in the blood of diabetic rats when compared to controls (26 ± 5 µmol.L -1 ). As expected, these changes were accompanied by a significant 80% decrease of the ratio GSH/GSSG in the STZ group when compared to normal rats (Fig. 5). This drop in GSH/GSSG was found concomitant with a dramatically increased blood glucose level (498 ± 15 mg.dL -1 ) when compared to controls. As described in Fig. 5, a significant increase in both levels of protein carbonyls (P<0.005) and MPO values (ranging 330-557 µg/L) was determined in diabetic rats and a positive correlation was obtained (R 2 = 0.8906; Fig. 6A). As a consequence, an inverse correlation (R 2 = 0.8863) between GSH/GSSG and MPO could be drawn (Fig. 6B), which supports the hypothesis that the MPO-derived HOCl may contribute to oxidative stress in the STZ-induced model of experimental diabetes. In addition, Figure 5 shows that the lipid peroxidation degree (MDA-TBA levels) in the plasma of the diabetic group (7.61 ± 0.34 nmol/mg protein) was significantly higher than that of the controls (4.26 ± 0.31 nmol/mg protein; P<0.001).

Discussion

The MPO level could be a representative marker of neutrophil activation [START_REF] Lau | Myeloperoxidase mediates neutrophil activation by association with CD11b/CD18 integrins[END_REF] and it is well established that myeloperoxidase-derived HOCl plays a crucial role in several pathologies and diseases. Therefore, the determination of the enzyme concentration is essential for the understanding of its role as one of the major components of the inflammatory process. The ELISA assay is considered to be robust and sensitive, but it is time intensive, costly and allows the evaluation of the total content of the enzyme and not the active one. Despite other methods based on the quantitation of MPO enzymatic activity that have been proposed, no consensus has been found regarding their ability to specifically measure MPO activity in biological samples. Most of them are based on the properties of chemical probes such as odianisidine, guaiacol, luminol, tetramethylbenzidine (TMB) or acetyl-dihydroxyphenoxazine (ADPH) for measuring the peroxidase activity. The ADPH and luminol assays were found to be sensitive enough but not specific to MPO activity since the activity of other peroxidases, including eosinophil and hemoproteins such as hemoglobin and myoglobin present in the tissues, can interfere with the results. In contrast the probes 3'-(p-aminophenyl) fluorescein (APF) and 5-thio-2-nitrobenzoic acid (TNB) have the ability to detect the chlorination activity of MPO but were found to lack sensitivity or required long incubation steps. Another assay of MPO activity has been developed based on the detection of 3-chlorotyrosine, the HOCl oxidation product of protein tyrosyl residues, that can be measured with chromatography/mass spectrometry [START_REF] Whiteman | Loss of 3-chlorotyrosine by inflammatory oxidants: implications for the use of 3-chlorotyrosine as a bio-marker in vivo[END_REF][START_REF] Delporte | Simultaneous measurement of protein-bound 3-chlorotyrosine and homocitrulline by LC-MS/MS after hydrolysis assisted by microwave: Application to the study of myeloperoxidase activity during hemodialysis[END_REF]. Although 3-chlorotyrosine is specifically produced under MPO stimulation, it can be quickly degraded in an inflammatory environment or markedly reduced by thiocyanate ions [START_REF] Wu | Eosinophils generate brominating oxidants in allergen-induced asthma[END_REF]. The bromide-dependent chemiluminescence has also been shown to be specific towards MPO but this technique can only be used for the quantification of polymorphonuclear leukocytes in selected tissues with an appropriate control [START_REF] Haqqani | A myeloperoxidase-specific assay based upon bromide-dependent chemiluminescence of luminol[END_REF][START_REF] Ormrod | Inhibition of neutrophil myeloperoxidase activity by selected tissues[END_REF]. All together, these assays are dependent on the tissue microenvironment and cannot be specific towards MPO without the use of an antibody-capture step. Some authors found a good compromise to measure in complex media the activity of MPO after its capture by specific antibodies by using for example the SIEFED technique [START_REF] Pulli | Measuring Myeloperoxidase Activity in Biological Samples[END_REF][START_REF] Franck | A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts[END_REF].

In the present study, a homogeneous assay that uses a new fluorimetric method in order to measure MPO in plasma and other biological samples was proposed as an alternative to ELISA and other assays. This method allows the direct determination of chlorination activity of myeloperoxidase based on a synthetic probe designed to selectively detect HOCl, and has been used to measure active MPO level for the first time in STZ-diabetic rats. This assay has many practical advantages over other methods. The most obvious is that it saves time as it requires only 10 minutes of incubation in order to observe a linear increase in fluorescence intensity. Also, it is homogeneous and requires no labor-intensive sample preparation; biological samples were only diluted in order to reduce the interactions between MPO and other biomolecules. Since it uses a fluorogenic and selective probe for HOCl detection, the technique disclose the MPO activity and allows an easy measurement of the active MPO level with a high specificity and sensitivity. It is adapted to 96 wells microplate allowing highthroughput quantitation of MPO in multiple samples concurrently.

One limitation of our assay is the lower sensitivity compared to ELISA or SIEFED techniques [START_REF] Zhang | Association between myeloperoxidase levels and risk of coronary artery disease[END_REF][START_REF] Franck | A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts[END_REF]. The assay's detection limit is based on a 3-sigma criterion that corresponds to approximately 8 µg/L, which is about 10 fold higher than immunological methods. However, as the mean MPO concentrations in human plasma of healthy donors were reported to range 18 to 49 µg/L for total level [START_REF] Franck | A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts[END_REF][START_REF] Goiffon | A rapid bioluminescence assay for measuring myeloperoxidase activity in human plasma[END_REF][START_REF] Mocatta | Plasma concentrations of myeloperoxidase predict mortality after myocardial infarction[END_REF][START_REF] Brennan | Prognostic value of myeloperoxidase in patients with chest pain[END_REF], or 14 -15 µg/l for active level [START_REF] Franck | A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts[END_REF][START_REF] Goiffon | A rapid bioluminescence assay for measuring myeloperoxidase activity in human plasma[END_REF], the sensitivity of the probe should be therefore sufficient to measure MPO level in vivo, even when in a low grade of oxidative stress and could serve for investigation of inflammatory pathologies.

In rats, STZ treatment lead to an activation and degranulation of polymorphonuclear neutrophils as revealed by the percentage increase (380 %) of MPO level in plasma of diabetic group when compared to the control. Our results are in accordance with a previous murine study that shows an increase of 192 % of plasma MPO concentration in diabetic animals [START_REF] Nishat | Increased A3AR-dependent vasoconstriction in diabetic mice is promoted by myeloperoxidase[END_REF]. A significant increase was also observed (178 % to 342 %) in some tissues of STZ-induced diabetic rats [START_REF] Di Filippo | Hyperglycemia in streptozotocin-induced diabetic rat increases infarct size associated with low levels of myocardial HO-1 during ischemia/reperfusion[END_REF][START_REF] Ojha | Withania coagulans Fruit extract reduces oxidative stress and inflammation in kidneys of streptozotocin-induced diabetic rats[END_REF][START_REF] Maheshwari | Effect of coenzyme Q10 alone and its combination with metformin on streptozotocin-nicotinamide-induced diabetic nephropathy in rats[END_REF][START_REF] Uluoglu | The effect of experimental diabetes on the circadian pattern of adenosine deaminase and myeloperoxidase activities in rat liver[END_REF][START_REF] Olukman | Fenofibrate treatment enhances antioxidant status and attenuates endothelial dysfunction in streptozotocininduced diabetic rats[END_REF]. Taken together all these results confirm the assumption of increased inflammation in diabetic animals in this model.

Myeloperoxidase-derived HOCl can react readily with a number of biological molecules. In a complex system such as plasma, proteins are important targets for HOCl, leading to an increase of protein-bound carbonyl levels [START_REF] Himmelfarb | Plasma protein thiol oxidation and carbonyl formation in chronic renal failure[END_REF]. Many studies have demonstrated a significant increase of protein carbonyl content in plasma and different cells of diabetic patients resulting from increased myeloperoxidase activity [START_REF] Piwowar | Advanced oxidation protein products. Part I. Mechanism of the formation, characteristics and property[END_REF][START_REF] Suzuki | Carbonyl stress in the pathogenesis of diabetic nephropathy[END_REF][START_REF] Pennathur | Mechanisms for Oxidative Stress in Diabetic Cardiovascular Disease[END_REF]. A correlation between protein carbonyl levels and MPO concentrations has been reported previously in preterm infants [START_REF] Buss | Elevated protein carbonyls and lipid peroxidation products correlating with myeloperoxidase in tracheal aspirates from premature infants[END_REF]. Released HOCl is able to penetrate the cell membrane and oxidize intracellular thiols. Glutahione oxidation in erythrocyte by HOCl is considered to be a highly selective process and most of the GSH is converted into GSSG [START_REF] Vissers | Oxidation of intracellular glutathione after exposure of human red blood cells to hypochlorous acid[END_REF]. Glutathione is the most abundant cellular non-protein thiol which plays an efficient role against oxidative stress. It is present in almost all mammalian tissues, at millimolar concentrations in liver, and less than 10% exists in the oxidized form in a normal situation. The ratio GSH/GSSG is considered as a major biomarker of blood redox state or overall health [START_REF] Lu | Glutathione synthesis[END_REF]. As a consequence of an increased oxidative status, alterations of GSH and GSSG concentrations are rapidly observed. A decreased GSH level plays a role in the oxidative damage and many works have shown that the GSH/GSSG ratio was much lower in the diabetic subjects than in controls [START_REF] Calabrese | Oxidative stress, glutathione status, sirtuin and cellular stress response in type 2 diabetes[END_REF][START_REF] Sekhar | Glutathione synthesis is diminished in patients with uncontrolled diabetes and restored by dietary supplementation with cysteine and glycine[END_REF][START_REF] Dincer | Assessment of DNA base oxidation and glutathione level in patients with type 2 diabetes[END_REF]. The reduction in GSH concentration observed after STZ treatment is due to its increased utilization and reduced regeneration, and is presumably associated with an increased production of peroxides [START_REF] Raza | Glutathione metabolism and oxidative stress in neonatal rat tissues from streptozotocin-induced diabetic mothers[END_REF]. In accordance with these findings, we observed in the present study that after 3 weeks of STZinduced hyperglycemia a strong correlation could be established between MPO concentrations and both protein carbonyls and GSH/GSSG ratio, suggesting that neutrophil activation is one of the major contributors to the variations of these markers. The increase of lipid peroxidation evaluated by the MDA content and observed in diabetic rats strongly supports these finding and showed a clear unbalance between oxidative stress status and antioxidant defense. Similarities are found between this study's results and those reported earlier in the literature, showing an enhanced oxidative status in diabetes mellitus involving MPO activation [START_REF] Calabrese | Oxidative stress, glutathione status, sirtuin and cellular stress response in type 2 diabetes[END_REF][START_REF] Unlucerci | Testis glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase activities in aminoguanidine-treated diabetic rats[END_REF][START_REF] Bhor | Oxidative damage and altered antioxidant enzyme activities in the small intestine of streptozotocin-induced diabetic rats[END_REF][START_REF] Miyata | Increased carbonyl modification by lipids and carbohydrates in diabetic nephropathy[END_REF][START_REF] Díaz-Flores | Effect of an aqueous extract of Cucurbita ficifolia Bouché on the glutathione redox cycle in mice with STZ-induced diabetes[END_REF][START_REF] Saravanan | Ameliorative potential of S-allyl cysteine on oxidative stress in STZ induced diabetic rats[END_REF].

Conclusions

In summary, it can be concluded that 7-HCCO could be used as a convenient and sensitive fluorogenic probe to detect HOCl generation in biological fluids that allow a reliable highthroughput assessment of active MPO levels in vivo. All reactions were performed in one 96well microplate by the sequential addition of reagents, and the results obtained were in accordance to those obtained by other methods. In addition, the presence of an inflammatory process and some variations in oxidant-antioxidant balance in STZ diabetic rats is demonstrated.

  1 H RMN (CDCl3, 300 MHz) δ 7.69 (d, J = 9.5Hz, 1H, 4-H), 7.49 (d, J = 8.4Hz, 1H, 5-H), 7.11 (d, J = 2 Hz, 1H, 8-H), 7.05 (dd, J = 8.4 and 2.0 Hz, 1H, 6-H), 6.39 (d, J = 9.5Hz, 1H, 3-H), 2.34 (s, 3H, CH3); 13 C RMN (CDCl3, 75 MHz) δ 168.8 (CO-CH3), 160.4 (2-CO), 154.7 (7-C), 153.2 (8a-C), 142.9 (4-C), 128,6 (5-C), 118.4 (6-C), 116.7 (4a-C), 116.1 (3-C), 110.4 (8-C), 21.1 (CH3).

  H RMN (DMSO-d6, 300 MHz) δ 11.92 (bs, 1H, OH), 10.40 (s, 1H, CHO), 8.01 (d, J = 9.54 Hz, 1H, 4-H), 7.85(d, J = 8.6 Hz, 1H, 3-H), 6.94 (d, J = 8.6 Hz, 1H, 6-H), 6.4 (d, J = 9.5Hz, 1H, 3-H); 13 C RMN (DMSO-d6, 75 MHz) δ 190.8 (CHO), 163.9 (2-C), 159.1 (8a-C), 155.7 (7-C), 144.5 (4-C), 136.3 (5-C), 114.0 (6-C), 112.6 (3-C), 111.2 (4a-C), 109.2 (8-C).

  H RMN (DMSO-d6, 300 MHz) δ 7.72 (d, J = 9.2 Hz, 1H, 4-H), 7.37 (d, J = 8.5 Hz, 1H, 5-H), 6.53 (d, J = 8.5 Hz, 1H, 6-H), 5.97 (d, J = 9.2 Hz, 1H, 3-H).

  Physical and cognitive activity was promoted in a room with a controlled temperature (22 ± 3°C) and a reverse 12h light/dark cycle with food and water available ad libitum. All animal care and experimental procedures were performed according to the rules of the Directive 2010/63/EU of the European Parliament. The protocol was approved by the local Animal Research Ethics Committee (2010-2012) and the National Research Committee for the project FEDER-AdiabaOx (2008, N° 13851). The CNRS and Aix-Marseille University have valid licences for animal housing and experimentation (agreement C13-055-06) delivered by the French Government. Among coauthors M.C. and S.P are graduated for the Certificate in Small Animal Surgery (Aix-Marseille University, Faculté de Pharmacie and Centre de Formation Permanente CNRS DR12, Marseille).

  2 M boric to 150 µl sample. After centrifugation (2000 × g, 10 min at 4°C), 200 µL supernatants were derivatized with 40 µL of iodoacetic acid at 40 mM for at least 10 min at room temperature. The pH was adjusted to 9.0 with 300 µL of NaOH/tetraborate solution at pH 11. Then, dansyl chloride (200 µL at 50 mM) was added and the samples were kept at room temperature overnight in the dark to form S-carboxymethyl-N-dansyl-GSH and N,N-9-bis-dansyl-GSSG. Unreacted dansyl chloride was removed by chloroform extraction (500µl). Prior to analysis, the aqueous phase was diluted 5-fold in 100 mM Borate Buffer pH 9. The GSH and GSSG adducts were separated by HPLC using a Merck Hitachi/Lachrom HPLC system: autosampler, pump L-7100, fluorescence detector L-7480 (excitation wavelength, 335 nm; emission wavelength, 515 nm), interface D-7000, degasser L-7612, controlled by the Agilent EZChrom Elite Compact software (Pleasanton, USA). Separation was achieved at room temperature on a 3aminopropyl column (250 mm × 4.6 mm; 5 µm; Macherey-Nagel, Hoerdt, France) with an isocratic (Solvent A/Solvent B, 90/10, vol/vol) flow rate of 1.2 mL.min -1 during 35 min. Solvent A is a 0.2 M sodium acetate buffer (pH 4.6) in methanol (35/65, vol/vol) and solvent B is a methanol/water solution (80/20, vol/vol). Quantification was based on peak area and relative to standards from a calibration curve obtained with commercial GSH and GSSG. Data are expressed in µM and are the means of twelve independent experiments in each group made in duplicate.

Figures captions Scheme 1 .

 1 Figures captions

Figure 1 .

 1 Figure 1. Normalized absorbance ( ) and fluorescence ( ) spectra of 7-HCCO (100 µmol/L) after reaction with HOCl (50 µmol/L) in PBS 1X pH 6.5. The absorption spectrum shows a minimum at 350 nm and the fluorescence spectrum presents a maximum at 470 nm.

Figure 2 .

 2 Figure 2. Chromatograms of : (a) HOCl (100 µM) ; (b) 7-HCCO (100 µM) ; (c) 7-HCCOox (10 µM); (d) 7-HCCO (100 µM) after 10 minutes incubation at 37°C with HOCl (100 µM) in PBS 1X pH 6.5. 7-HCCOox was prepared as described in section 2.3.

Figure 3 .

 3 Figure 3. Effect of various amount of H2O2 on specific MPO activity in PBS 1X pH 6.5 with 7-HCCO at 100 µM. The specific MPO activity (As) was expressed as U/mg protein. One unit (U) of the enzyme was the activity which formed 1 µmol HOCl per min at 37°C. Data are the means ± standard deviations from 3 independent experiments made in triplicate. Statistics: one-way ANOVA followed by Mann-Whitney test *p < 0.05 compared to control without H2O2.

Figure 4 . 1 ,

 41 Figure 4. Kinetic curves for oxidation of 100 µM 7-HCCO with 100 µM H2O2 at 37°C: 1, Oxidation by HL-60 cell lysate 10 times diluted in 1X PBS pH 6.3; 2, HL-60 cell lysate without H2O2 ; 3, HL-60 cell lysate treated with 1 mM sodium azide. Data are the means ± standard deviations from 2 independent experiments made in triplicate.

Figure 5 .

 5 Figure 5. Measurements of oxidative stress in streptozotocin-treated hyperglycemic rats (STZ group) compared with non-diabetic controls (A) Myeloperoxidase (MPO) activity in plasma (B) Blood reduced-to-oxidized glutathione ratio (GSH/GSSG) (C) Protein carbonylation (D) Lipid peroxidation measured by the MDA-TBA assay. Data represent means ± SEM of 12 independent experiments made in duplicate or triplicate (MPO assay). One-way ANOVA followed by Mann Whitney test: ** P<0.01 and *** P<0.001 versus controls.

Figure 6 .

 6 Figure 6. Myeloperoxidase activity correlates with oxidative stress biomarkers in STZ diabetic rats (A) Plasma protein carbonyl levels and MPO concentrations (A) Blood reducedto-oxidized glutathione ratio GSH/GSSG and MPO concentrations.

Aknowledgements

The authors acknowledge Sebastien Combes for his advice in cell culture and Amy Cope for her kind help. The authors gratefully acknowledge the support of the "French National Research Agency" (ANR) within the framework "Contaminant, Ecosystème et Santé 2008" through the integrated project INTOX. For the in vivo experiments, this work has been carried out thanks to the support of the A*MIDEX project (n° ANR-11-IDEX-0001-02, PESTOX) funded by the «Investissements d'Avenir» French Government program, managed by the French National Research Agency and grants of the project FEDER-AdiabaOx (2008, N° 13851).