A Belinger

P Hardy

P Barricau

J P Cambronne

D Caruana
email: daniel.caruana@onera.fr

Influence of the energy dissipation rate in the discharge of a Plasma Synthetic Jet Actuator

Keywords: Plasma actuator, Synthetic Jet, Spark discharge

A promising actuator for high-speed flow control, referred to as a Plasma Synthetic Jet (P.S.J), is being studied by the DMAE department of the ONERA, and the Laplace laboratory of the CNRS, in France. This actuator was inspired by the "Sparkjet" device developed by the Johns Hopkins University Applied Physics Laboratory. The PSJ, which produces a synthetic jet with high exhaust velocities, no active mechanical components and no mass flow admission, holds the promise of enabling high-speed flows to be manipulated. With this high velocity jet it is possible to reduce fluid phenomena such as transition and turbulence, thus making it possible to increase an aircraft's performance whilst at the same time reducing its environmental impact. A thermal plasma discharge was created in a micro cavity, causing the gas to be expelled. It is relevant that the velocity and momentum depend on the energy dispersed by the electric discharge. To control the frequency and energy dispersed in the plasma, the Laplace laboratory has developed two high voltage power supply systems. These allow two different types of discharge to be produced, with energy being supplied to the discharge in two different manners. In this paper, we focus on the impact of the power supply on the Plasma Synthetic Jet, and in particular on the role of the rate of energy dissipation in the discharge. In order to estimate the influence of the power supply on the machinery of the actuator, specific experimental techniques were used to investigate the electrical (voltage, current), thermal (Infra-red camera) and aerodynamic (jet duration, isentropic pressure, jet velocity) characteristics. These data sets were used to determine which of the two power supplies was more effective, thus allowing us to reach several conclusions concerning the importance of the energy dissipation rate on the PSJ actuator.

1.

• Flow control 2.

Introduction The development of flow control applications with active devices requires efficient, robust, easy to integrate and low energy consumption actuators. In recent years, various studies have been conducted on electrostatic and electromagnetic actuators. Actuators making use of piezoelectric materials [START_REF] Smith | The formation and evolution of synthetic jets[END_REF][START_REF] Mane | Experimental design and analysis for piezoelectric circular actuators in flow control applications Smart[END_REF][START_REF] Wiltse | Direct excitation of small-scale motions in free shear flows[END_REF] or plasma [START_REF] Roth | Aerodynamic flow acceleration using paraelectric and peristaltic electrohydrodynamic effects of a One Atmosphere Uniform Glow Discharge Plasma[END_REF][START_REF] Moreau | Airflow control by non-thermal plasma actuators[END_REF][START_REF] Hyun | The wake flow control behind a circular cylinder using ion wind[END_REF][START_REF] Corke | Single dielectric barrier discharge plasma enhanced aerodynamics: physics, modeling and applications[END_REF][START_REF] Boeuf | Electrohydrodynamic force and aerodynamic flow acceleration in surface dielectric barrier discharge[END_REF][START_REF] Benard | A large-scale multiple dielectric barrier discharge actuator based on an innovative three-electrode design[END_REF][START_REF] Benard | Aerodynamic plasma actuators: A directional micro-jet device[END_REF][START_REF] Cybyk | Performance Characteristics of the sparkjet flow control actuator 2nd AIAA Flow Control Conference[END_REF][START_REF] Cybyk | Single-Pulse Performance of the sparkjet flox control actuator 43rd[END_REF][START_REF] Ko | Analysis of flow distribution from high-speed flow actuator using particle image velocimetry and digital speckle tomography Flow[END_REF][START_REF] Narayanaswamy | Characterization of a High-Frequency Pulsed-Plasma Jet Actuator for Supersonic Flow Control[END_REF][START_REF] Caruana | The "Plasma Synthetic Jet" Actuator: Aero-thermodynamic Characterization and first Flow Control[END_REF] are also being developed. There are several possible configurations for piezoelectric actuators, including: a synthetic jet [START_REF] Smith | The formation and evolution of synthetic jets[END_REF][START_REF] Mane | Experimental design and analysis for piezoelectric circular actuators in flow control applications Smart[END_REF] and a flat plat actuator [START_REF] Wiltse | Direct excitation of small-scale motions in free shear flows[END_REF]. Wilste and Glezer have shown that a flat plate actuator is able to control a shear layer [START_REF] Wiltse | Direct excitation of small-scale motions in free shear flows[END_REF]. Smith and Glezer [START_REF] Smith | The formation and evolution of synthetic jets[END_REF] presented modifications which can be achieved in the jet, by means of a piezoelectric actuator. Mane analyzed [START_REF] Mane | Experimental design and analysis for piezoelectric circular actuators in flow control applications Smart[END_REF] the influence of the sawtooth, the orifice diameter, and the cavity height on the performance of the actuator, for three different diaphragm materials.

Various aspects of plasma actuators have been extensively studied. Moreau [START_REF] Moreau | Airflow control by non-thermal plasma actuators[END_REF] provides a general overview of the subject. This paper describes different types of actuator: surface corona discharge and surface dielectric barrier discharge actuators, and several types of airflow control for different configurations (cylinder, flat plate …). Surface corona actuators have shown the ability to control external flow in a flat plate [START_REF] Roth | Aerodynamic flow acceleration using paraelectric and peristaltic electrohydrodynamic effects of a One Atmosphere Uniform Glow Discharge Plasma[END_REF], or in a cylinder [START_REF] Hyun | The wake flow control behind a circular cylinder using ion wind[END_REF]. In [START_REF] Corke | Single dielectric barrier discharge plasma enhanced aerodynamics: physics, modeling and applications[END_REF], Corke and Orlov provide a general review of the Surface Dielectric Barrier Discharge actuator, and of different applications such as boundary layer separation control. In [START_REF] Boeuf | Electrohydrodynamic force and aerodynamic flow acceleration in surface dielectric barrier discharge[END_REF], Boeuf and Pitchford calculated the electrodynamic force created by a DBD actuator. The authors concluded that SDBD and surface corona discharges create the same electrohydrodynamic effect. An original SDBS with multiple electrodes is presented in [START_REF] Benard | A large-scale multiple dielectric barrier discharge actuator based on an innovative three-electrode design[END_REF]. This actuator extends the plasma discharge and thus the control surface, when compared with a single SDBD. The SDBD actuator has also influenced new plasma actuator designs, for example a directional micro jet is presented in [START_REF] Benard | Aerodynamic plasma actuators: A directional micro-jet device[END_REF]. The velocity of all of these actuators is less than 10 m/s. A new plasma actuator referred to as the "sparkjet" has been proposed by the Johns Hopkins University Applied Physics Laboratory [START_REF] Cybyk | Performance Characteristics of the sparkjet flow control actuator 2nd AIAA Flow Control Conference[END_REF][START_REF] Cybyk | Single-Pulse Performance of the sparkjet flox control actuator 43rd[END_REF]. This actuator is able to produce high-speed jet velocities, with a peak velocity measured by the PIV technique in excess of 50 m/s [START_REF] Ko | Analysis of flow distribution from high-speed flow actuator using particle image velocimetry and digital speckle tomography Flow[END_REF]. A pulsed plasma jet inspired by the sparkjet actuator is presented in [START_REF] Narayanaswamy | Characterization of a High-Frequency Pulsed-Plasma Jet Actuator for Supersonic Flow Control[END_REF], with a jet velocity of the order of 300 m/s. The ONERA, in collaboration with the CNRS Laplace laboratory (France), is also involved in research into a PSJ actuator [START_REF] Caruana | The "Plasma Synthetic Jet" Actuator: Aero-thermodynamic Characterization and first Flow Control[END_REF]. The Laplace laboratory is more particularly interested in the interactions between the power supply and the PSJ actuator. Indeed, the impact of the power supply on the plasma can modify the processes which occur, for example, in dielectric barrier discharge lamps [START_REF] Mildren | Enhanced performance of a dielectric barrier discharge lamp using short-pulsed excitation[END_REF][START_REF] Mildren | Visible and VUV emission from a xenon dielectric barrier discharge using pulsed and sinusoidal voltage excitation waveforms Plasma Science[END_REF][START_REF] Liu | Double discharges in unipolar-pulsed dielectric barrier discharge xenon excimer lamps[END_REF] or ozone generator [START_REF] Williamson | Comparison of high-voltage ac and pulsed operation of a surface dielectric barrier discharge[END_REF][START_REF] Koudriavtsev | A novel pulse-densitymodulated high-frequency inverter for silent-discharge-type ozonizer[END_REF][START_REF] Ordiz | Development of a high-voltage closed-loop power supply for ozone generation APEC[END_REF]. In [START_REF] Mildren | Enhanced performance of a dielectric barrier discharge lamp using short-pulsed excitation[END_REF], Mildren showed that the efficiency and light intensity (between 175 and 200 nm) are improved when the lamps are supplied by a pulsed power supply (rather than an AC power supply), and photographs of the light emission are presented in [START_REF] Mildren | Visible and VUV emission from a xenon dielectric barrier discharge using pulsed and sinusoidal voltage excitation waveforms Plasma Science[END_REF]. When using an ozone generator, Williamson obtained the same results [START_REF] Williamson | Comparison of high-voltage ac and pulsed operation of a surface dielectric barrier discharge[END_REF], with the pulsed power supply producing more ozone than an AC power supply. Consequently, various researchers have investigated the role of the power supply for ozone generation. For example, in [START_REF] Ordiz | Development of a high-voltage closed-loop power supply for ozone generation APEC[END_REF] Ordiz presents a buck converter, and in [START_REF] Koudriavtsev | A novel pulse-densitymodulated high-frequency inverter for silent-discharge-type ozonizer[END_REF] Koudriavtsev et al. present a resonant power supply. The supply strategy influences the process and radically changes the efficiency and performance of the application. The influence of the power supply on the plasma has also been studied for flow control applications. Jolibois showed that the high voltage waveform changes the electric wind produced by SDBD [START_REF] Jolibois | Enhancement of the Electromechanical Performances of a Single Dielectric Barrier Discharge Actuator Dielectrics and Electrical Insulation[END_REF]. Indeed, a negative sawtooth leads to a higher velocity than a positive sawtooth [START_REF] Balcon | Positive and negative sawtooth signals applied to a DBD plasma actuator -influence on the electric wind[END_REF], since the former produces a high current peak, and thus a more intense filament, whereas a positive sawtooth produces a current peak of lower amplitude. The frequency of the high voltage modulation also influences the velocity of the electric wind produced by the SDBD [START_REF] Benard | Capabilities of the dielectric barrier discharge plasma actuator for multi-frequency excitations[END_REF].

The influence of the power supply on the plasma, and consequently on the process, have not yet been studied on the PSJ actuator. In the present paper, we propose to compare two power supplies, in particular the influence of the energy dissipation rate on the jet velocity. We thus compare the jet velocity, and the dynamic pressure produced by the PSJ actuator supplied by two different power supplies: an inductive power supply (IPS), and a capacitive power supply (CPS). The CPS produces a pulsed plasma, whose energy is rapidly dissipated by the discharge, whereas the IPS causes the discharge energy to dissipate more slowly. In the first section of this paper, the discharge created by each of these power supplies is described, and an electrical model of the discharges is presented. In the second section, the dissipation rate of the discharge energy is analyzed for both power supplies.

Finally, the velocity, gas expulsion time and total pressure of both power supplies are compared. indeed the diameter and the height of the cavity are both equal to 4 mm. As shown in Figure 1, the two electrodes are enclosed in the ceramic, and an orifice is positioned at the top of the cavity. At the beginning of the cycle, a high voltage is applied between the two electrodes, by means of the high voltage power supply described in the following subsection. When the voltage reaches the disruptive voltage of air (approximately 4700 V for this actuator), air is ionized and an electric arc is created. This discharge leads to a strong increase in the internal energy. Because the air is confined, its pressure and temperature increase rapidly. Relaxation of the fluid through the circular orifice creates a hot air jet. When temperature in cavity drops, ambient air is then sucked up into the cavity (recovery stage during some hundred of microsecond), and the actuator is ready for a new cycle.

3.

Experimental set-up

Plasma synthetic Jet

Power supply

In order to create the discharge, a high voltage power supply is essential. Moreover, this power supply must control the frequency of, and the energy dissipated in, the discharge. Two solutions can be used to create a high voltage:

• A high voltage power supply, associated with an IGBT switch, to commute the high voltage directly during the discharge,

• A low voltage power supply, associated with a high voltage transformer and a transistor.

The first solution would lead to very high costs, since it requires the use of a high voltage transistor.

We thus chose the second approach, with a flyback converter [START_REF] Redondo | Flyback Versus Forward Switching Power Supply Topologies For Unipolar Pulsed-Power Applications[END_REF]. In association with the flyback converter architecture, we proposed two different types of power supply. In the first of these, a PSJ is directly connected to the secondary voltage of the transformer; this solution is referred to as IPS it is inspired to the spark engine power supply [START_REF] Hnatiuc | Experimental analysis of a double-spark ignition system[END_REF]. The second solution is referred to as a CPS [START_REF] Zaepffel | Experimental study of an electrical discharge used in reactive media ignition[END_REF], and involves the use of a capacitor, inserted between the transformer and the PSJ. The supply mode is totally different for these two power supplies. In the first, the inductance transfers the energy to the discharge, such that the PSJ is powered by a current source. In the second, the capacitor transfers the energy to the discharge, such that the PSJ is powered by a voltage source. The functioning of these two power supplies is analyzed in the following section.

Measurements

Electrical Measurement.

The actuator was supplied by a high voltage power supply, as described in the following section. The high voltage was measured by a 20 MHz high voltage probe, and the current probe measured the AC current only, with a pass-band limited to 100 MHz. The data was recorded with a 100 MHz LECROY oscilloscope.

Stagnation Pressure Measurements.

For flow control applications (especially separation), the jet diameter of the active vortex generator is close to 1 mm. Since it is difficult to achieve direct measurements of the PSJ characteristics, we used a dedicated instrumentation setup mounted on a calibration bench, thus enabling the dynamic pressure and velocity of the jet to be measured: this consisted in a stagnation pressure probe, with a differential unsteady KULITE ±5 psi sensor. A MACOR tube was fitted to the sensor. This device allows the jet to be channeled towards the sensitive element of the sensor. The probe was mounted on a micrometric translation plate, to ensure its accurate positioning in front of the actuator orifice. The Schlieren setup used a conventional Z-type mirror system as seen on Figure 2. The knife-edge was oriented orthogonally to the jet axis. This allowed the density gradient to be visualized in the streamwise direction. The actuator was mounted on an electrically isolated flat plane, parallel to the optical axis of the schlieren bench. Image frames were acquired by means of a MotionPro X5 camera.

The camera CCD had a 2352 x 1728 pixel imaging matrix with a spatial resolution of 64 µm. For the purposes of the very short integration times needed for each frame, the illumination was produced by the flash from a Flash lamp (HSPS FX-Xenon),with typical durations of approximately 20 ns.

The control signal of the power supply was used to synchronize the spark lamp with the camera acquisitions. A Standford Research Systems DG535 delay generator was triggered by the signal from the probe, allowing it to generate the TTL signals for the spark lamp and the camera. The position of the jet was thus a function of the phase difference between the spark of the PSJ and the frame acquisition. The jet's velocity could then be computed from the ratio between the distance it covered in the images, and the corresponding time delay. The jet's position was determined from the average value of several acquisitions.

The time difference used to determine the exhaust velocity varied between 5 and 10 µs, depending on which power supply was used. The maximum working frequency of the spark lamp limits the velocity measurement bench. The measurements are made with an actuator frequency of 10 Hz.

3.3.4.Temperature measurement

It is too interesting to know the temperature of the gas expel by the PSJ actuator. However, the jet duration is too short to measure his temperature. That is the reason why we have measured the temperature of the top of the cap of the actuator with an infrared camera ThermoVision SC 6000 FLIR. This measurement allows us to quantify the temperature in the cavity before the breakdown. So we can know which power supply heats over the actuator.

An image of the cap is taken at a time interval of 5s. At each image, the cap temperature is uniform throughout the cap; it is certainly due to the small time of the device. As the emissivity of the brass is low, the infrared radiation is strongly attenuated. To have a better precision in this measurement, we recover the cap with high emissivity paint ( =0.92). To estimate the temperature we use the Plank law.

4.

Power supply The magnetization stage is identical for both power supplies. The high-voltage transformer is powered by the low-voltage power supply during the on-state of the IGBT transistor. During this phase, the primary current increases linearly and energy is accumulated in the transformer. This energy is determined by the duration of the magnetization and depends on the square of the primary current (I p )

2 1 . . 2 1 p m I L E = ( 1 )
where L 1 represents the magnetizing inductance of the transformer. Moreover, the working frequency of the actuator is imposed by the IGBT frequency control. The IGBT can be easily controlled to adjust the frequency and the energy accumulated in the transformer.

Energy transfer to the higher voltage.

The magnetization phase ends with the opening of the IGBT transistor. The energy accumulated in the transformer is transferred to the secondary coil and thus to the discharge. The energy dissipated in the discharge (E d ) can be calculated from the measurement of the discharge voltage (Vs) and the discharge current (Id):

- = e disch e disch t t d wire d s dt t i R dt t i t v E arg arg . ) ( . ). ( ). ( 2 ( 2 )
Where R wire represents the resistance of the wire: 0.5 in this work.

Inductive Power supply

The magnetization phase ends with the opening of the IGBT. After switch-off of the IGBT, the secondary voltage increases until the breakdown voltage is reached. Figure 4 shows the ignition of the discharge. The current increases to 30 A in 50 ns. During the ignition of the discharge, the voltage decreases rapidly to 400 V (this voltage is the same in every case).

Figure 4. Ignition of the IPS discharge

Whatever the energy dissipated in the discharge, the ignition of the discharge is the same, and the current rapidly increases to 30 A. This peak current value depends on the equivalent capacitance of the transformer wire, as described in section 3.3.2.

Following ignition, the duration and the maximum value of the discharge current varies with the energy accumulated in the transformer, as seen in Figure 5.

and the constant voltage of the discharge is equal at: U discharge =400V

As seen in [START_REF] Granda-Gutiérrez | Modelling and optimization of a low-pressure DC glow discharge in stable regime Surf[END_REF][START_REF] Takaki | Voltage-current characteristics of high-current glow discharges[END_REF][START_REF] Staack | Characterization of a dc atmospheric pressure normal glow discharge Plasma Sources[END_REF], the glow discharge has the same electric characteristics, a constant voltage, and a variable current. Takaki [START_REF] Takaki | Voltage-current characteristics of high-current glow discharges[END_REF] observed the same discharge voltage (400V), and the current varied between 1 and a few milliamperes.

Moreover, the current and voltage waveforms are similar to those of the discharge of an inductance in series with a constant voltage source: since the current decreases linearly, it is controlled by the inductance and as a consequence the discharge behaves like a constant source voltage.

Capacitive power supply

In the CPS, the magnetization stage is terminated by the opening of the IGBT transistor. In an initial phase, the energy accumulated in the transformer, then transferred to the capacitor, is greater than that transferred to the discharge. The remainder of the energy is dissipated into a series resistance (500 ).

If this resistance is disconnected, the terminal voltage of the transformer is non-zero when the IGBT is closed. A considerable increase in the IGBT current is thus induced by the energy transfer, which remains in the transformer. Unfotunately this resistance is required to protect the IGBT transistor. If the discharge is not initiated, the energy accumulated in the transformer is dissipated into the resistance. When the capacitor voltage on the actuator terminals reaches the electrical breakdown voltage (about 4700 V), the capacitor delivers its energy through the discharge. The strong scattering of the breakdown voltage value (about ± 500 V) imposes the storage of a greater quantity of energy in the transformer than in the capacitor. This large scattering in the breakdown voltage is certainly caused by the erosion of the electrode. Indeed this phenomenon can create some point in the electrode.

This point increase locally the electric field and then diminish the breakdown voltage. These parameters allow the discharge voltage to be calculated (Vs in Figure 6). Vs correspond at the real voltage between the electrode and Vc to the capacitor voltage. The difference between these two voltages corresponds to the wire voltage. Moreover, the wire is strongly inductive; consequently Vs and Vc are not in phase. During the ignition time, the discharge voltage falls to zero in a few nanoseconds. Following ignition, the current and the voltage discharge are sinusoidal and in phase.

Furthermore, the maximum current is proportional to the breakdown voltage. In the same manner, the maximum value of the current increases as a function of the energy stored in the capacitor. Equation [START_REF] Roth | Aerodynamic flow acceleration using paraelectric and peristaltic electrohydrodynamic effects of a One Atmosphere Uniform Glow Discharge Plasma[END_REF] shows the relation between capacitance, wire inductance and breakdown voltage.

max

.

wire s b L I V C ≈ ( 4 )
For a mean value of disruptive voltage (4700 V), the maximum current is plotted as a function of capacitance, as shown in Figure 7. The experimental points are in good agreement with the theoretical curve (the dotted line) , the dispersion seen in Figure 7 being the consequence of variations in the breakdown voltage. The first experimental point represents the peak current of the IPS. Indeed, the capacitance of the wire in the transformer is equal to 100 pF, which explains the peak current of 30 A. With both power supplies, the maximal current of the ignition of the discharge is influenced by the capacitor, such that the ignition mechanism is identical in both cases. Although the ignition is in principle only initiated by the capacitor, if the stored energy of the latter is very high, it may completely control the discharge.

The temporal variation of the current and the voltage indicates that the discharge behaves like a spark discharge [START_REF] Greason | Investigation of the optical and electrical characteristics of a spark gap[END_REF][START_REF] Greason | Methodology to study the resistance of spark discharges[END_REF][START_REF] Randeberg | A new method for generation of synchronised capacitive sparks of low energy[END_REF][START_REF] Papadopoulos | Investigation of the electrical characteristics of charge transfer circuits used in gas laser excitation[END_REF][START_REF] Persephonis | The inductance of the discharge in a spark gap[END_REF][START_REF] Persephonis | The time evolution of the resistances and inductances of the discharges in a pulsed gas laser through its current waveforms[END_REF].

5.

Influence of each power supply on the discharge behavior

Discharge Duration

We measured the discharge duration between two instants in time:

• Ignition of the discharge, marked by the instant at which voltage breakdown occurs

• Termination of the discharge, marked by the current's fall to zero

The two power supplies applied the same energy to the discharge. Nevertheless, the energy was not dissipated in the same manner. supplies. In the case of the IPS, this is not affected by the frequency. However, the discharge duration increases when the energy accumulated by the transformer is increased. This phenomenon leads to an increase in the magnetization time. On the one hand, the magnetization time increases the energy accumulated in the transformer and, consequently, the energy dissipated during the discharge. On the other hand, the discharge behaves like a constant voltage source, whatever the energy dissipated in the discharge. The maximum current thus increases with increasing energy. Since the discharge current decreases linearly, the discharge duration increases with the energy dissipated in the discharge. For the CPS, the discharge duration is also increased by the energy dissipated in the plasma. After 40 mJ, the discharge duration is only weakly increased by the arc energy. Indeed, the arc duration is limited to 8 µs. The IPS produces a more extended discharge than the CPS.

Energy dissipation

Figure 9 presents the time evolution of the discharge energy dissipation, for a total discharge energy of 55 mJ. This energy is dissipated more slowly by the IPS than by the CPS. With the CPS, we observe that the energy dissipation oscillates in time. Indeed, the energy is calculated from the capacitor voltage and the current of the discharge. Half of the total energy is dissipated during the ignition, in a period of 500 nano-seconds. The rest of the energy is then dissipated over a period of some microseconds.

In the case of the IPS, the energy is dissipated gradually, and ignition of the IPS consumes a small quantity of energy. Indeed, the maximum current is lower than in the CPS.

Figure 9. Temporal evolution of the energy dissipation

The stored energy is thus not transmitted in the same manner by the two power supplies.

Consequently, the gas is not heated by the plasma at the same rate: heating of the gas is obtained more rapidly with the CPS. It is thus likely that the jet velocity and pressure are greater with this power supply. Indeed, the heating is more rapid, since the energy is dissipated in a shorter time. For the high energy of the IPS, the discharge lasts for 1 millisecond, such that gas expulsion begins before the electric discharge has finished. In this case, the heating effect is slower, and the resulting overpressure is certainly lower than in the case of the CPS.

Influence of the energy on the temperature of the cap

The air in the cavity is not heated in the same way by the two power supply. Before the expulsion, air presents on the cavity heat the ceramic and the brass cap. Consequently, the temperature of the cap is representative of the heating of the air present in the cavity by the discharge. Figure 10 present the evolution of the cap temperature with the energy for the both power supply, frequency of the discharge is equal to 100Hz. The cap's temperature is stabilized after few min of functioning, so we plot this temperature versus energy dissipated in the discharge. impossible to operate the actuator with this power supply at high frequency or at high energy dissipated in the discharge. Indeed, the cap's temperature depends directly of the power, which is directly linked to the frequency and to the energy dissipated in the discharge. The temperature of the cap is less hot with the CPS, but still reached 100°C for a power of 20 W.

The energy dissipation rate influences strongly the temperature of the cap and therefore the heating of the gas present of the cavity before the breakdown voltage. With the IPS power supply the gas present in the cavity is inevitably warmer than for the CPS before the discharge.

Effect of the rate of energy dissipation on the breakdown voltage

According to Paschen's theory, the breakdown voltage depends on the product of the electrode gap and the pressure, but also on the temperature. In our actuator, the breakdown voltage depends only on the temperature and on the pressure; indeed the electrode gap is constant. In order to show this assumption, we measured the breakdown voltage for different value of pressure or temperature inside the PSJ actuator. To change these parameters, the actuator without this cap is placed in a climatic chamber. In this chamber, the pressure can vary between 0.1 and 1 bar and the temperature between 20

°C and 180 °C. The cap of the actuator is removed in order to control the temperature and the pressure inside the cavity. In one experiment, the pressure is maintained to the atmospheric pressure and we change the temperature inside the climatic chamber. In another experiment the temperature is maintained at ambient temperature and we change the pressure in the climatic chamber. The evolution of the breakdown for this both experiment is plotted in function of the ratio temperature over pressure in Figure 11. This ratio corresponds to an equivalent temperature at atmospheric pressure and corresponds to the evolution of the gas density. The evolution of V b is the same for these two experiments because the two curves are stacked up.

Consequently, these two parameters have the same influence in the breakdown voltage. Figure 12 present the evolution of the breakdown voltage for the two power supplies with the frequency. The measurement is carried out few minutes after the beginning of the actuator functioning. Frequency (Hz)

Figure 12. Breakdown voltage of the two power supplies

We want now to know if the drop of V b is due to the pressure or temperature. So we show the evolution of the disruptive voltage and of the cap temperature during the beginning of the functioning of the PSJ, indeed the thermal effect is negligible during this phase. The cap temperature measured with the infra-red camera is equal to the ambient temperature. So the gas temperature before the discharge is equal to the ambient air, because the cavity wall heat the air sucked during the recovery stage. Moreover the breakdown voltage remains unchanged for the first breakdown which corresponds to the beginning of the functioning of the PSJ. Consequently, at the end of the recovery stage, the pressure in the chamber is equal to the atmospheric pressure. Then, the drop of the breakdown voltage with the frequency and with the energy dissipated in the cavity is caused by the increase of the temperature in the cavity before the breakdown. Indeed if the temperature increases, the density of the gas decreases together with the breakdown voltage.

On the one hand, at the same frequency and for the same equivalent energy, the breakdown voltage of the IPS is smaller than the breakdown voltage of the CPS. On the other hand, the decrease in breakdown voltage as a function of accumulated energy is more significant for the IPS. The breakdown voltage for the two power supplies decreases with the frequency of the actuator, and with the energy accumulated by the transformer or the capacitor, as shown in Figure 12.

Consequently, the IPS heats the cavity more strongly than the CPS. This phenomenon can be understood from the energy dissipation rate and from the temperature cap. Indeed, with the IPS, the energy is dissipated over a longer period of time and the cap temperature is higher. The gas present in the cavity is thus ejected before the electric discharge has ended as shown in Figure 13.

Figure 13: Evolution of the current with the pressure signal during the gas expulsion

The remaining energy heats only a small quantity of gas, such that most of this heat is dissipated in the ceramic of the actuator, which becomes hotter, before reaching the recovery stage. In summary, the air sucked into the cavity is hotter with the IPS. For the CPS, the discharge is too short (some microseconds) for the gas to be ejected before the discharge has ended.

For these reasons the breakdown voltage of the IPS is lower than that of the CPS.

6.

Influence of power supplies on the jet characteristics

The dynamic pressure is measured by a pressure sensor tube, which is located a few millimeters in front of the PSJ orifice.

Figure 14. Signal of the dynamic pressure sensor

With the CPS, for which the typically bell-shaped signal is shown in Figure 14, the jet is developed some micro seconds after the discharge. After the bell, the pressure signal is lower than 1 bar, this phase correspond to a violent suction of air by the PSJ actuator. This depression affects the pressure sensor and sucked the air present in the MACOR tube into the cavity. Indeed the expulsion phase ends very abruptly. This perturbation is not present in the signal corresponding to the IPS because the expulsion is more gradual and ends slowly, so the transition from the expulsion to the recovery stage is more progressive.

With the IPS, the pressure signal does not have exactly the same shape because the discharge continues during the gas expulsion. For the both power supply we can see the electromagnetic interference due to the discharge.

Maximum dynamic pressure

The maximum dynamic pressure, as a function of frequency, for different energies accumulated in the transformer or in the capacitor, is shown in Figure 15. The CPS produces a more powerful jet than the IPS. This can be explained by the brevity of the CPS discharge, and by the fact that it is not complete when the gas starts to be expelled by the IPS (Figure 13): during expulsion of the gas, the heating is less efficient, because the gas's concentration in the cavity is low. This has the effect of heating the ceramic cavity which will warm the air during the suction. We have seen this effect in the cap temperature in Figure 10. Before the breakdown, the gas temperature in the cavity is then lower for the ISP than the CPS. This temperature is directly linked to the quantity of matter inside the cavity. The gas density before breakdown is then lower for the IPS than the CPS. Moreover, lower the density is, lower the quantity of gas that can be expelled by the PSJ is. The resulting pressure is then reduced for the IPS.

For the both power supplies, the dynamic pressure decreases at higher energies and higher frequency and then when the power dissipated in the PSJ is important. This drop is caused by the heating of the cavity. In the case of the CPS, the heating of the cavity is not the only reason for the fall in total pressure. Indeed, the breakdown voltage, and thus the energy dissipated during the discharge, is reduced by the heating of the cavity because of the relationship between capacitor energy and breakdown voltage.

. . 2 1 b c V C E ≈ (5)
The drop-off in total pressure is thus also due to the decline in energy dissipated during the discharge.

The CPS power supply is somehow self regulated because the heating of the cavity induces a fall in the breakdown voltage which decreases the energy dissipated during the discharge. The temperature in the cavity is then lower which induces a diminution of the heating of the cavity.

6.2.

Gas expulsion time

The gas expulsion time is estimated as the period during which the total pressure is greater than ten percent of the maximum over pressure. The over pressure correspond to pressure measured by the sensor which we subtract the atmospheric pressure.

Figure 16 shows the gas expulsion time as a function of the energy dissipated in the discharge. For the CPS, this time is constant (220 µs), whatever the energy dissipated in the discharge. For the IPS, the gas expulsion time increases with energy (until 25 mJ). Beyond 25 mJ, the gas expulsion time is almost constant, and equal at 800 µs.

0,00 0,05 0,10

IPS CPS

Expulsion gas duration (s) Energy (J)

Figure 16. Gas expulsion time

The jet created by the IPS is expelled from the PSJ actuator during a relatively long time interval, whereas the CPS produces a short, pulsed jet.

Indeed, in the latter case, the gas expulsion time remains constant as a function of the energy or frequency. Consequently, if the discharge is too short, the discharge time will simply influence the maximum total pressure, without affecting the gas expulsion time.

However, in the case of the IPS, the gas expulsion time is influenced by the discharge time. Indeed, the jet is maintained because the IPS continues to discharge energy during expulsion of the gas. With the IPS at high energies, the discharge time is longer than the gas expulsion time, such that when the jet has been completely expelled, the discharge has still not come to an end. This phenomenon explains the stagnation of the gas expulsion time at higher energies.

6.3.

Velocity calculation

The jet velocity was also calculated, on the basis of the total pressure measurements for frequency equal to 10 Hz. If the jet flow is considered to be isentropic: and T is the temperature of the jet, M is the mach number, and is the isentropic coefficient of air. The air present in the cavity is heated by the discharge but during the expulsion it is cooled by the cavity.

As it is impossible to measure the temperature during the expulsion phase, the temperature is assumed to be equal to 293 K, such that the isentropic coefficient is equal to 1.4. The expression for the velocity is then:

- - = - 1 1 . . . 2 1 max max γ γ γ γ a t P P T R V ( 8
)
The curves resulting from this calculation are shown in Figure 17. With both power supplies, the velocity increases as a function of the energy dissipated in the discharge. The PSJ jet is produced by heating of the air in the cavity, involving an increase in the static pressure. The pressure gradient between the heated air in the cavity and the external air leads to the creation of the jet. The stronger the pressure gradient is, the higher the jet Mach number is. The jet produced by the PSJ actuator supplied by the IPS has a lower velocity than the jet produced by the PSJ actuator supplied by the CPS (as seen in the schlieren measurement). We have observed than the cap temperature is higher for IPS than for the CPS, the thermal loss is then more important for the IPS.

Comparison between schlieren velocity measurements and the velocity calculation

The part of the energy involved in the gas expulsion is lower. IPS dissipates too much energy in heating PSJ the ceramic cavity and enough in the heating of the gas present in the cavity before breakdown, because of the expulsion begins when the discharge is not finished as seen on Figure 13.

Moreover, as seen in Figure 17, there are differences between the velocities determined from the schlieren images and those calculated from pressure measurements, indicating that the modeled velocity, based on these measurements, is not perfect. This outcome shows that the assumption of an isentropic flow of the jet is incorrect. The hypothesis of an isentropic flow does not take heat losses into account, which lead to heating of the cavity by the discharge. The consequence of the heat loss is an increase in the temperature of the cavity, and thus an increase in the gas temperature.

Moreover, it can be seen that the differences between the schlieren image velocities and the pressure model velocities are greater for the inductive than for the CPS. This suggests that the heat loss or temperatures are greater when the jet is created by the IPS. This explains why the temperature of the cap is higher for the IPS than the CPS. This conclusion is supported by the fact that the breakdown voltage obtained with the IPS is lower than that obtained with the CPS, meaning that the jet created by the IPS is hotter than that created by the CPS.

Conclusions

The influence of the two power supplies on the plasma is very different:

• In the case of the IPS, the discharge is equivalent to a constant voltage. The current is thus controlled by the transformer, and depends on the energy accumulated in its magnetizing inductance. Consequently, the discharge duration increases with the energy dissipated on the plasma. Moreover, the discharge is relatively long, such that the energy is dissipated relatively slowly.

• The CPS creates a pulsed discharge, which is controlled by a capacitor. This discharge resembles a spark discharge and behaves like a small and constant resistance from the energetic point of view, such that the energy is dissipated very quickly.

The energy dissipation rate influences the warming of the gas present in the cavity, and consequently the gas expulsion time. This time interval is longer for the IPS, and is influenced by the energy dissipated in the cavity.

Moreover, the front jet velocity is influenced by the energy dissipation rate in the cavity, as shown by the velocities measured with the schlieren bench, which are lower with the IPS. The energy dissipation rate also influences the temperature of the gas, and thus the heat loss, as can be seen from the breakdown voltage and the velocity calculation. Indeed, the breakdown voltage decreases with the temperature of the gas before breakdown, and is lower for the IPS than for the CPS. Furthermore, the differences between the velocities derived from the pressure model, and those determined from the schlieren images, also indicate that the IPS heats the gas and the cavity of the PSJ actuator more strongly than does the CPS.

The energy dissipation rate is a very important parameter, because it influences the velocity and heating of the PSJ actuator. In order to increase the efficiency of the PSJ actuator, it is very important to diminish the energy dissipation rate in the discharge. The CPS thus produces a more powerful jet (higher velocity, shorter expulsion time), and probably heats the gas and the cavity less significantly than the IPS. In order to further increase the velocity of the jet, and decrease the heat losses, it would be beneficial to develop a ns-pulse power supply, able to supply 1 mJ in an interval of a few nanoseconds [START_REF] Pai | Transitions between corona, glow, and spark regimes of nanosecond repetitively pulsed discharges in air at atmospheric pressure[END_REF][START_REF] Pai | Nanosecond repetitively pulsed discharges in air at atmospheric pressure-the spark regime[END_REF].

Aknowledgments

The research leading to these results has received funding from the European Community's Seventh

Framework Programme FP7/2007-2013 under the PLASMAERO grant agreement n°234201.

Figure 1 .

 1 Figure 1. Plasma Synthetic Jet actuator : (a) sectional view; and (b) view of the actuator without

3. 3 . 3 .

 33 Velocity Measurement. Velocity measurements are performed by means of the Schlieren technic. Since the jet is produced by a thermal source (electric arc), the resulting density (hence refractive index) gradients allow the position of the jet to be easily measured.

Figure 2 .

 2 Figure 2. Schlieren setup

4. 1 .

 1 Energy transfer 4.1.1. Magnetization stage. Figure3shows the circuit diagrams of the two power supplies. Both power supplies consist of a high-voltage transformer, an IGBT, a DC power supply (60 V -500W) and a capacitor for the second power supply. The magnetizing inductance has a value of 10mH, and the transformer's rated voltage ratio is equal to 10.

Figure 3 .

 3 Figure 3. Inductive (a) and capacitive (b) power supplies

Figure 5 .

 5 Figure 5. Discharge of the IPS

Figure 6 .

 6 Figure 6. Discharge created by the CPS

Figure 7 .

 7 Figure 7. Maximal current as a function of capacitor value

Figure 8 .Figure 8

 88 Figure 8. Discharge duration as a function of the energy dissipated during the discharge

Figure 10 .

 10 Figure 10. Evolution of the cap temperature with the energy dissipated on the discharge

Figure 11 :

 11 Figure 11: Evolution of the breakdown voltage with an equivalent temperature

Figure 15 .

 15 Figure 15. Maximum Total pressure of the two power supplies: ( a ) inductive power supply; and

2

 2

Figure 17 .

 17 Figure 17. Computed and measured maximum jet velocities as a function of the discharge


N Kondori

L Baltzer

G T Dolphin

I Mattsby-Baltzer
email: inger.mattsby-baltzer@microbio.gu.se

Fungicidal activity of human lactoferrin-derived peptides based on the antimicrobial  region

Keywords:

A c c e p t e d M a n u s c r i p t

INTRODUCTION

Candida albicans is an opportunistic pathogen that causes mucosal and systemic infections in immunosuppressed patients. Because of the rising number of failures in the treatment of mycoses in severely immunosuppressed individuals owing to resistant strains of Candida, there is a need to develop new therapeutic agents that either on their own exert antifungal activity or in combination with conventional antimycotics enhance their activity.

Lactoferrin (LF) is a single-chain, iron-binding glycoprotein found in milk, mucosal secretions and the secondary granules of neutrophils. It is an innate defence factor expressing antimicrobial, anti-inflammatory and immunoregulatory activities [START_REF] Brogden | Antimicrobial peptides in animals and their role in host defences[END_REF][START_REF] Nuijens | Structure and biological actions of lactoferrin[END_REF][START_REF] Tollin | Antimicrobial peptides in the first line defence of human colon mucosa[END_REF].

Pepsin digestion of LF releases a fragment, lactoferricin, which is several times more bactericidal than the parent molecule [START_REF] Bellamy | Identification of the bactericidal domain of lactoferrin[END_REF][START_REF] Yamauchi | Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment[END_REF]. Human lactoferricin consists of amino acid residues 1-46 from the N-terminal end of LF, including two disulphide bonds [START_REF] Bellamy | Identification of the bactericidal domain of lactoferrin[END_REF][START_REF] Baker | Molecular structure, binding properties and dynamics of lactoferrin[END_REF].

Amino acid residues 14-31 form an -helix in the human LF (hLF) molecule, of which the latter half is amphipathic [START_REF] Anderson | Structure of human lactoferrin at 3.2-Å resolution[END_REF][START_REF] Odell | Antibacterial activity of peptides homologous to a loop region in human lactoferrin[END_REF]; antibacterial activity resides in this part. A short peptide fragment comprising amino acid residues 21-31 was shown to retain bactericidal activity, but was less active against C. albicans than a peptide fragment containing amino acid residues 1-11 from the N-terminal end of hLF [START_REF] Odell | Antibacterial activity of peptides homologous to a loop region in human lactoferrin[END_REF][START_REF] Chapple | Structurefunction relationship of antibacterial synthetic peptides homologous to a helical surface region on human lactoferrin against Escherichia coli serotype O111[END_REF][START_REF] Lupetti | Candidacidal activities of human lactoferrin peptides derived from the N terminus[END_REF].

In an earlier study on synthetic peptide fragments derived from the antibacterial helix region of hLF, it was found that peptides comprising amino acid residues 17-31, 18-31, 19-31 and 20-31 from the N-terminal end expressed stronger microbicidal activity than the peptide fragment 21-31 [START_REF] Håversen | Structure-microbicidal activity relationship of synthetic fragments derived from the antibacterial -helix of human lactoferrin[END_REF]. By changing two neutral amino acid The aim of this study was to explore the antifungal activity of HLopt2 against Candida spp. and Cryptococcus neoformans and to compare its activities with the double-sized lactoferricin-like synthetic fragment HLBD1 (amino acids . In addition, the antifungal effects of these two peptides were investigated in an experimental C. albicans skin infection model.

Materials and methods

Fungal strains

Lactoferrin and peptides

LF from human milk was purchased from Sigma (St Louis, MO). hLF peptides were synthesised using a 9-fluoryl-mehoxycarbonyl continuous-flow strategy on a Pioneer TM automated peptide synthesizer (Applied Biosystems, Foster City, CA) and have been described previously [START_REF] Håversen | Structure-microbicidal activity relationship of synthetic fragments derived from the antibacterial -helix of human lactoferrin[END_REF].

Synthesis of lactam-bridged HLBD1

Lactam bridges were introduced into HLBD1 by changing the amino acids to Glu (E) and Lys (K) four amino acids apart (Fig. 1). Lactam formation between the side chains was achieved by treatment with a six-fold excess of benzotriazole-1-yl-oxy-trispyrrolidino-phosphoniumhexafluorophosphate-N-hydroxybenzotriazolediisopropylethylamine (PyBOP-HOBt-DIPEA) (1:1:2) in dimethylformamide (DMF) for 16 h. The resin was washed with DMF and dichloromethane and was dried under vacuum. The peptide was cleaved from the resin as described previously [START_REF] Håversen | Structure-microbicidal activity relationship of synthetic fragments derived from the antibacterial -helix of human lactoferrin[END_REF]. Lactam bridge formation induces some helical content in short peptide fragments that are random coiled. Helix formation is readily detected by circular dichroism (CD) spectroscopy, and increased helical content upon lactam bridge formation was concluded from the increased negative value of the mean residue ellipticity at 208 nm and 222 nm.

The peptide sequences used in this study are shown in Fig. 1.

A c c e p t e d M a n u s c r i p t 7

Antifungal drugs

Amphotericin B (AmB) and flucytosine (5-FC) were purchased from Sigma. Fluconazole (FLZ) was obtained from Pfizer (Illertissen, Germany). FLZ and 5-FC were dissolved in sterile water and physiological saline (1 mg/mL), respectively, whereas AmB was first dissolved in dimethyl sulphoxide (DMSO) (20 mg/mL) before diluting it further.

Minimum microbicidal concentrations (MMCs) and minimum inhibitory concentrations (MICs)

The microplate assay has been described elsewhere [START_REF] Håversen | Structure-microbicidal activity relationship of synthetic fragments derived from the antibacterial -helix of human lactoferrin[END_REF]. In short, peptides were serially diluted in 0.03% BHI (BHI dil ) or 1% Bacto TM Peptone (BP) in two-fold steps and were incubated with yeast cells at a concentration of 2  10 5 cells/mL for 2 h at 37 C.

Viability of the inoculum was analysed by culturing 5 L of the suspension of each well on blood agar plates for 20 h at 37 C. The concentration of hLF or peptide corresponding to the dilution at which 99.9 % killing of the inoculum was obtained was defined as the MMC value. A four-fold difference in MMC values (two-fold titre steps) was regarded as significantly different. In some experiments, the incubation time of the microplate was extended to 20 h.

Moreover, in some experiments the microplate was incubated for 20-44 h at 37 C in order to analyse MIC values of the peptides or hLF. The MIC value was defined as the concentration of hLF or peptide corresponding to the dilution at which the absorbance at 650 nm was reduced to at least 50% of that of the untreated inoculum. Absorbance was read in an EMax ® Microplate Reader (Molecular Devices, Sunnyvale, CA).

A c c e p t e d M a n u s c r i p t 8

Candida membrane permeability

The DNA-binding fluorescent probe propidium iodide (PI) (Sigma) was used to analyse changes in membrane permeability of Candida upon exposure to hLF or peptides. A fluorescence-activated cell sorter (FACS) was used to monitor permeability changes. Briefly, yeast cells were grown overnight and were then transferred to new BHI medium for another 2 h of growth.

Cells were washed and diluted in 10 mM phosphate buffer (pH 7.2). Yeast cells plus hLF or peptide at various concentrations were incubated for 2 h at 37 C. The final concentration of yeast cells was 2  10 6 cells/mL. The cells were then re-incubated at room temperature with 1 g of PI for 5 min followed by analysis on a FACScan Flow Cytometer (BD Biosciences, San José, CA).

Candida mitochondrial membrane permeability

The fluorescent probe rhodamine 123 (Sigma) was used to study mitochondrial permeability [START_REF] Clark | Correlation between rhodamine 123 accumulation and azole sensitivity in Candida species: possible role for drug efflux in drug resistance[END_REF] since it accumulates in this cell compartment. In short, C. albicans in log phase was suspended in potassium phosphate buffer (1 mM, pH 7.0) and was incubated with rhodamine 123 (5 M) for 15 min at 37 C in a water-bath. After 3 washes with K-phosphate buffer, the cells were treated for 10 min at 37 C with hLF or peptide at final concentrations of 2000 g/mL and 25 g/mL, respectively. The distribution of rhodamine 123 in Candida cells was examined using a fluorescence microscope (Leica Microsystems Cambridge Ltd., Cambridge, UK).

A c c e p t e d M a n u s c r i p t 9

Electron microscopy

Candida albicans ATCC 64549 and C. neoformans CCUG 37833 were incubated overnight. Fresh BHI medium was inoculated with the cultures, which then was incubated for another 2 h at 37 C. After washing, cells were suspended in 10 mM phosphate buffer at a final concentration of 2  10 7 cells/mL. hLF or HLopt2 at final concentrations of 4000 g/mL and 200 g/mL, respectively, was added to the cell suspension and was incubated for 2 h at 37 C. High concentrations of hLF and the peptide were chosen in order to achieve killing of a high number of yeast cells. Cells were separated from the suspensions by centrifugation and were fixed with an aldehyde mixture (2% paraformaldehyde, 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer, pH 7.2) diluted 1:1 with phosphate-buffered saline (PBS). The suspension was then washed with PBS.

Yeast cells (250 L) were transferred to poly-L-lysine-treated, gold-coated Thermanox TM coverslips (Nunc A/S, Roskilde, Denmark) for 30 min to allow the cells to adhere to the surface. After rinsing with PBS, cells were prepared for scanning electron microscopy (SEM) using the OTOTO method comprising repeated treatments with osmium tetroxide and thiocarobhydrazide [START_REF] Friedman | Enhanced visualization of peripheral nerve and sensory receptors in the scanning electron microscope using cryofracture and osmium-thiocarbohydrazide-osmium impregnation[END_REF]. The coverslips were dehydrated in ethanol and were infiltrated with hexamethyldisilazane, which was allowed to evaporate in a fume hood.

. Comparison with conventional antimycotics

The modified 12-amino acid long peptide HLopt2 and its natural counterpart HL10 as well as the double-sized lactoferricin-like HLBD1 (Fig. 1) were compared with the parental hLF molecule as well as with several conventional antimycotics using BHI dil as medium in the antifungal microplate assay (Table 1). All peptides had significantly greater fungicidal activity than hLF. Comparison with AmB showed that HLopt2 was in the same range as this antimycotic, whilst all three peptides had stronger fungicidal activity in comparison with 5-FC and FLZ. Regarding growth inhibition, only AmB expressed a significantly lower MIC value compared with its MMC value.

In line with hLF, the presence of pseudohyphae did not alter the antifungal activity of one tested peptide (HLBD1) against C. albicans ATCC 64549 after 2 h of incubation compared with yeast form (data not shown).

Kinetics of the fungicidal activity of HLopt2 and HLBD1

The antimicrobial activity of cationic peptides is strongly influenced by the assay broth used. Since earlier studies had shown that HLBD1 and hLF expressed significantly reduced fungicidal activity in the 2-h microplate assay when replacing BHI dil with BP, we investigated whether the difference in fungicidal activity was due to a slower killing rate in the presence of BP medium. Thus, the fungicidal activity of HLopt2, HLBD1

A c c e p t e d M a n u s c r i p t 12 and hLF was followed over 1 min to 20 h of incubation in BHI dil and was compared with that in BP.

In BHI dil the shortest peptide HLopt2 reduced the number of yeast cells to 5% of the inoculum within the first minute, whilst no yeast cells could be detected at 5 min (Fig. 2). It took 1 h for HLBD1 and 2 h for hLF to reduce the number of cells below the detection limit (99.9%). However, in BP the corresponding time for ≥99.9% killing for HLopt2 and HLBD1 was 30 min and 20 h, respectively, whilst no killing was observed with hLF.

Fungicidal activity against Candida spp. and Cryptococcus neoformans

Changing the culture medium to 1% BP affected the killing capacity of HLBD1 and HL10 dramatically (Table 2). Thus, MMC values for C. albicans ATCC 64549 increased 16-fold for HLBD1 and 8-fold for HL10 in order to achieve the same killing activity as in BHI dil (Table 1). However, the activity of HLopt2 did not change significantly. Furthermore, HLopt2 had significantly greater fungicidal activity compared with the other two peptides and hLF against all Candida and C. neoformans, except for C. glabrata (Table 2) (HLopt2 vs. HL10, P = 0.0020, Wilcoxon signed rank test). Neither the peptides nor hLF killed C. glabrata at the highest concentration tested.

Effects of some preformed helical content in HLBD1

The fungicidal activity of HLBD1 was dramatically reduced in BP medium (Table 1 and 2; Fig. 2). Thus, it was investigated whether any preformed helical content in HLBD1

A c c e p t e d M a n u s c r i p t 13 would improve its fungicidal activity in BP. According to the results of a previous report [START_REF] Helmerhorst | Roles of cellular respiration, CgCDR1, and CgCDR2 in Candida glabrata resistance to histatin 5[END_REF], positions 22, 26 and 30, in addition to position 18 (Thr), were suitable for lactam bridges (Fig. 1). The lactam bridges induced some helical content as confirmed by CD spectroscopy. In contrast to HLBD1 lactam bridge 18-22, lactam bridges between positions 22-26 and 26-30 lowered the MMC values significantly against C. albicans ATCC 64549 at 2 h compared with the native sequence of HLBD1 (Table 3). The activities of the two lactam-bridged HLBD1 peptides were comparable to HLopt2 (Table 2). After 20 h of incubation, MMC values of HLBD1 lactam bridges 22-26 and 26-30 against C. albicans did not change from those at 2 h, whilst MMC values for HLBD1 lactam bridge 18-22 and HLBD1 were significantly lower (Table 3).

Permeabilisation of fungal membranes by HLopt2 and HLBD1

Permeabilisation of the cytoplasmic membrane of C. albicans ATCC 64549 induced by the peptides after 2 h of incubation was demonstrated by the percentage of PIpositive cells (Fig. 3). The percentage of PI-positive cells, which ranged between 96% and 98% at 20 g/mL of peptides, reflected the killing shown by the MMC assay.

Thus, killing at 20 g/mL was 99.9% by the MMC assay except for hLF (data not shown). hLF at 4 mg/mL gave rise to 16% PI-positive cells (Fig. 3), whilst no reduction was recorded by the MMC assay (data not shown).

Rhodamine 123 fluorescence was found localised to subcellular compartments presumed to be mitochondria in the yeast cells treated with buffer (data not shown).

HLopt2 changed this pattern of fluorescence in the cells by showing an even distribution all over the cell. hLF-treated cells remained unaffected after 10 min of A c c e p t e d M a n u s c r i p t 14 incubation. This result was expected since no fungicidal effect of hLF on C. albicans ATCC 64549 could be recorded at this time point (Fig. 2).

The effects of HLopt2 and hLF on the cell wall of C. albicans and C. neoformans were studied by SEM using 10 mM phosphate buffer as incubation medium. HLopt2 treatment changed the surface of the yeasts greatly (Fig. 4). The C. albicans surface appeared punctured. Deep pits were seen all over the surface in combination with scattered blebs or knobs. An even rougher surface was observed for C. neoformans in addition to leakage of material between the cells. In addition to the pits, the surface was heavily covered with blebs or knobs (Fig. 4). More than 99.9% of the yeast cells were dead following incubation with HLopt2.

After 2 h of incubation of the yeast cells in the presence of hLF, the cells were agglutinated and cell surface alterations were observed. A more ruffled cell surface was noticed compared with untreated cells (Fig. 4). Killing activity of hLF during this incubation period was not observed as determined by the antifungal assay (data not shown).

Membrane damaging activity on erythrocytes

The membrane destabilising effect of HLBD1, HLopt2 and AmB was assessed by haemolysis of human red blood cells (Fig. 5). The peptides induced ca. 8% haemolysis at 100 g/mL, whilst no further significant increase in haemolysis was recorded at the highest concentration tested (200 g/mL). The 50% haemolysis value of the membrane-damaging detergent CTAB was 37 g/mL.

A c c e p t e d

M a n u s c r i p t 15

Anticandidal activity of HLopt2 and HLBD1 in vivo

In an experimental fungal infection, mice were treated twice with 100 g or 400 g of peptide per spot. A significant reduction in colony-forming units in the washings from the infected area was achieved with both peptides at a dose of 400 g/spot (Table 4).

No significant reduction was observed with the lower dose.

Discussion

In a previous report on the structure-antimicrobial activity relationship of hLF-derived peptides, the modified peptide HLopt2 comprising 12 amino acids (Fig. 1) expressed the strongest antimicrobial activity against the three test organisms Escherichia coli, Staphylococcus aureus and C. albicans [START_REF] Håversen | Structure-microbicidal activity relationship of synthetic fragments derived from the antibacterial -helix of human lactoferrin[END_REF]. In the present study, the antifungal activity of HLopt2 was further explored. HLopt2 was shown to be fungicidal against C. glabrata.

The enhanced antifungal activity of HLopt2 (4-8-fold) compared with its natural counterpart was due to additional charged and hydrophobic amino acids, substituting the neutral hydrophilic amino acid residues Asp and Glu in the natural peptide sequence of HL10 [START_REF] Håversen | Structure-microbicidal activity relationship of synthetic fragments derived from the antibacterial -helix of human lactoferrin[END_REF]. Moreover, HLopt2 was more than 100-fold more active (>3fold on a molar basis) against several Candida strains and C. neoformans compared with intact hLF, which is in line with an earlier report on lactoferricin and hLF [START_REF] Bellamy | Identification of the bactericidal domain of lactoferrin[END_REF].

Although the lactoferricin-like and double-sized HLBD1 was less effective than HLopt2, induction of some helical content by lactam bridge formation (positions 22-26 or 26-30) (Fig. 1) in the part of the molecule that corresponds to the amphipathic half However, after 20 h of incubation the MMC values were almost identical for HLBD1 with or without lactam bridges. One explanation for the enhancing effect of some helical content may be that pre-organisation of part of an amphiphilic conformation could speed up the binding processes of the peptide to targets on the fungal membrane. Recently, human lactoferricin was suggested to display a nascent helix from residue 14 to 29 in aqueous solution as a result of the hydrophobic side chains [START_REF] Hunter | Human lactoferricin is partially folded in aqueous solution and is better stabilized in a membrane mimetic solvent[END_REF].

The different killing kinetics of C. albicans by the peptides and hLF in the two nutrient broths indicated possibly increased concentrations of metallic salts in 1% BP compared with 0.03% BHI. In a previous report on the sensitivity of HLopt2 to metallic salts, particularly Ca 2+ , concentrations of 2.5 mM reduced its candidacidal activity [START_REF] Håversen | Structure-microbicidal activity relationship of synthetic fragments derived from the antibacterial -helix of human lactoferrin[END_REF].

In this study, HLopt2 appeared to have induced both large openings/pits in the outer part of the yeast cell wall of C. albicans as well as loss of membrane integrity as indicated by SEM and PI staining, respectively [START_REF] Gerhardt | Porosity of isolated cell walls of Saccharomyces cerevisiae and Bacillus megaterium[END_REF]. Furthermore, loss of mitochondrial membrane integrity suggests that the peptide also accumulated in the cytoplasm and targeted intracellular organelles. Thus, the effects of HLopt2 resembled the direct interaction of bovine lactoferricin with the cell surface of C. albicans as well as the membrane permeabilising activity of the human lactoferricin-like peptide sequence 18-40 on C. albicans cells [START_REF] Bellamy | Killing of Candida albicans by lactoferricin B, a potent antimicrobial peptide derived from the N-terminal region of bovine lactoferrin[END_REF][START_REF] Viejo-Diaz | Different anti-Candida activities of two human lactoferrin-derived peptides, Lfpep and kaliocin-1[END_REF].

A c c e p t e d M a n u s c r i p t

In general, the antifungal mechanism of antimicrobial peptides appears to involve rupture of the fungal membrane and suppression of mitochondrial respiration [START_REF] Tollin | Antimicrobial peptides in the first line defence of human colon mucosa[END_REF][START_REF] Okamoto | Regulation of fungal infection by a combination of amphotericin B and peptide 2, a lactoferrin peptide that activates neutrophils[END_REF].

However, less is known about the targets for peptide binding in the cytoplasmic membrane and the following events of the killing mechanism.

The synthetic peptide fragment comprising amino acid residues 1-11 from the Nterminal end of the hLF molecule (hLF11) was suggested to interact with structural elements of the plasma membrane of C. albicans and to be taken up by an energydependent way. Thus, candidacidal activity was dependent on mitochondrial respiration and a subsequent rise in extracellular ATP [START_REF] Lupetti | Candidacidal activities of human lactoferrin peptides derived from the N terminus[END_REF]. Moreover, the hLF11induced PI uptake was associated with non-lytic ATP release of C. albicans. A bovine lactoferricin-derived peptide comprising amino acid residues 12-21 was shown to require the Trk1p potassium transporter for fungicidal activity, but not the additional cell wall proteins ssA1/2p (heat shock 70 family) as shown for histatin 5 [START_REF] Vylkova | Distinct antifungal mechanisms: -defensins require Candida albicans Ssa1 protein, while Trk1p mediates activity of cysteine-free cationic peptides[END_REF].

The reason for the lack of effect of the peptides against C. glabrata is not known.

Candida glabrata has repeatedly been identified as being the least sensitive among the Candida spp. to many azoles. Helmerhorst et al. [START_REF] Helmerhorst | Candida glabrata is unusual with respect to its resistance to cationic antifungal proteins[END_REF] showed that C. glabrata is also insensitive to several cationic antifungal proteins. Investigation of the resistance of C. glabrata to histatin 5 suggested that the cellular characteristics providing resistance to azoles were distinct from those of cationic antifungal peptides [START_REF] Helmerhorst | Roles of cellular respiration, CgCDR1, and CgCDR2 in Candida glabrata resistance to histatin 5[END_REF][START_REF] Bennett | Mechanism of increased fluconazole resistance in Candida glabrata during prophylaxis[END_REF][START_REF] Vermitsky | Azole resistance in Candida glabrata: coordinate upregulation of multidrug transporters and evidence for a Pdr1-like transcription factor[END_REF].

Comparison of HLBD1 and HLopt2 with conventional antimycotics such as 5-FC, FLZ and AmB showed that the peptides were comparable with AmB in their fungicidal activity. This is to be expected since AmB also causes impairment of membrane barrier function and cell death, in contrast to the other two antimycotics that are a Mann-Whitney.

  increasing number of infections in hospitalised patients caused by resistant strains of fungi, there is a need to develop new therapeutic agents for these infections. Naturally occurring antimicrobial peptides may constitute models for developing such agents. A modified peptide sequence (CFQWKRAMRKVR; HLopt2) based on amino acid residues 20-31 of the N-terminal end of human lactoferrin (hLF) as well as a double-sized human lactoferricin-like peptide (amino acid residues 16-40; HLBD1) were investigated for their antifungal activities in vitro and in vivo. By in vitro assay, HLopt2 was fungicidal at concentrations of 12.5-25 g/mL against Cryptococcus neoformans, Candida albicans, Candida krusei, Candida kefyr and Candida parapsilosis, but not against Candida glabrata. HLopt2 was demonstrated to have 16-fold greater killing activity than HLBD1. By inducing some helical formation caused by lactam bridges or by extending the assay time (from 2 h to 20 h), HLBD1 became almost comparable with HLopt2 in its fungicidal activity. Killing of C. albicans yeast cells by HLopt2 was rapid and was accompanied by cytoplasmic and mitochondrial membrane permeabilisation as well as formation of deep pits on the yeast cell surface. In a murine C. albicans skin infection model, atopic treatment with the peptides resulted in significantly reduced yields of Candida from the infected skin areas. The antifungal activities of HLopt2 in vitro and in vivo suggest possible potential as a therapeutic agent against most Candida spp. and C. neoformans. The greatly improved antifungal effect of the lactam-modified HLBD1 indicates the importance of amphipathic helix formation for lethal activity.

  natural peptide sequence 20-31 (HL10) to one charged (Lys) and one hydrophobic (Ala) amino acid (forming HLopt2), fungicidal activity was enhanced.

  Three strains of C. albicans (ATCC 64549, CCUG 90008 and CCUG 5594), three strains of Candida glabrata (ATCC 90030, CCUG 85247 and CCUG 42987), three strains of Candida parapsilosis (ATCC 22019, CCUG 37233 and patient isolate C. parapsilosis Ir1), one strain of Candida krusei (ATCC 6258), one patient isolate of Candida kefyr Ir2 and two strains of C. neoformans (CCUG 37833 and patient isolate C. neoformans Ir3) were studied. Candida albicans ATCC 64549 was used as the main test organism for the fungicidal activities of the peptides. For susceptibility tests, all strains were cultivated in brain-heart infusion (BHI) medium at 37 C overnight and part of the culture was transferred to fresh BHI medium for another 2 h of growth. Conversion of yeast to germ tube and hyphal forms of C. albicans was carried out by transferring the C. albicans yeast cells to RPMI 1640 followed by cultivation at 37 C for 24 h.

3 . 1 .

 31 Antifungal activity of HLopt2, HL10 and HLBD1 3.1.1

  neoformans, C. albicans, C. parapsilosis, C. kefyr and C. krusei, but not against C.

A c c e p t e d M a n u

  s c r i p t of the -helix of hLF promoted a more rapid killing of C. albicans, approaching the MMC value of HLopt2.

A c c e p t e d M a n u s c r i p t 18 fungistatic.

 18 The mechanism of action of AmB involves binding to ergosterol, which may explain why AmB compared with HLopt2 and HLBD1 has a somewhat weaker haemolysis effect than the peptides.To prevent candidal infection in immunosuppressed patients, colonisation with Candida spp. should be avoided. However, preventive treatment with conventional antimycotics is not recommended owing to resistance development. The risk is lower with naturally derived charged peptides since these rarely induce microbial resistance[START_REF] Devine | Cationic peptides: distribution and mechanisms of resistance[END_REF]. In the present study, an experimental model of cutaneous Candida infection was used to investigate the antifungal effects of Hlopt2 and HLBD1. Both peptides showed protective activities by significantly reducing the number of C. albicans recovered from the infected skin area. These results agree with the earlier reported anti-infectious activity of HLBD1 in E. coli-induced urinary tract infection in mice[START_REF] Haversen | Human lactoferrin and peptides derived from a surface-exposed helical region reduce experimental Escherichia coli urinary tract infection in mice[END_REF]. Whether the antifungal activity of HLopt2 was superior to HLBD1 in vivo could not be ruled out in this study.In summary, HLopt2, originally based on a part of the antimicrobial -helix region of the hLF molecule, exerts fungicidal activity against several Candida spp. as well as C. neoformans. The antifungal effect is suggested to involve disarrangement of the cell wall and a mode of action that results in disruption of the cytoplasmic and mitochondrial membranes. By introducing some pre-organised helix conformation into the double-sized HLBD1, a killing activity almost comparable with that of HLopt2 could be observed in vitro. The antifungal effect of Hlopt2 against experimental Candida skin infection indicates a potential therapeutic use.

Fig. 1 .

 1 Fig. 1. Peptides derived from the antimicrobial region of human lactoferrin (hLF).

Fig. 2 .

 2 Fig. 2. Killing kinetics of the peptides HLBD1 (200 g/mL) and HLopt2 (100 g/mL)

Fig. 3 .Fig. 4 .

 34 Fig. 3. Propidium iodide (PI)-staining of Candida albicans incubated for 2 h at 37 C

Fig. 5 .

 5 Fig. 5. Haemolysis in the presence of peptides (HLopt2) and HLBD1), amphotericin B

  Human red blood cells were washed and diluted in PBS to a concentration of 2%. A volume of 25 L of the erythrocyte suspension was added to the wells of a microplate.

	2.8. Haemolysis
	Peptides and AmB solutions were diluted in two-fold steps (25-100 g/mL) and were
	M added in 50 L volumes to the wells. All concentrations were run in triplicate. The microplate was incubated for 1 h at 37 C. The optical density at 650 nm was then measured in a microplate reader and was compared with the detergent hexadecyltrimethylammonium bromide (CTAB) (Sigma) that is known to lyse a n u s c r i p t erythrocytes.
	The dried specimens were mounted on aluminium SEM A c c e p t e d
	stubs and were examined without thin film metal coating in a Zeiss 982 Gemini
	scanning electron microscope (Carl Zeiss, Oberkochen, Germany).
	10

2.9. Anticandidal activity of HLopt2 and HLBD1 in vivo 2.9.1. Cutaneous candidiasis Candida albicans ATCC 64549 was cultured overnight in Sabouraud broth at 37 C.

Yeast cells were then washed and diluted in phosphate buffer (10 mM, pH 7.4) to 2  10 10 cells/mL. BALB/c mice (8-10 weeks old) were treated with C. albicans on a shaved area by gently rubbing a volume of 10 L (2  10 8 yeast cells per 12-mm diameter spot) with a blunt plastic rod during isoflurane anaesthesia

[START_REF] Sohnle | Epidermal proliferation and the neutrophilic infiltrates of experimental cutaneous candidiasis in mice[END_REF]

. The next day, anaesthetised mice were treated with 10 L of a peptide solution or vehicle (phosphate buffer) by applying the volume onto the infected spots. After 4 h the treatment was repeated. Animals were killed the following day. Viable counts were determined from washings obtained from the treated areas using a metal ring placed over the spot and gentle rubbing in the presence of 70 L of phosphate buffer containing Triton X-100 (0.01%). The procedure was repeated once giving a total sample volume of ca. 100 L for culturing.

Table 1

 1 Fungicidal and fungistatic activity of the peptides HLBD1, HL10 and Hlopt2 as well as human lactoferrin (hLF) and conventional antimycotics against Candida albicans

	ATCC 64549 in BHI dil . Two-fold serial dilutions starting from 200 g/mL of peptide or
	antimycotic and from 2000 g/mL of hLF were used in the assay	a
	Peptide	MMC ( g/mL)	b MIC ( g/mL)
	HLBD1	12.5	6.2
	HL10	12.5	12.5
	HLopt2	6.2	N/D¤
	hLF	1000	500
	AmB	3.1	0.4
	Flucytosine	>200	200
	Fluconazole	>200	≥200
	BHI

dil , 0.03% brain-heart infusion; MMC, minimum microbicidal concentration; MIC, minimum inhibitory concentration; AmB, amphotericin B; N/D, not determined. a All samples were analysed in triplicate in two separate experiments. b The MMC values of the two experiments were identical.

Table 2

 2 Fungicidal effects of the peptides HLBD1, HL10 and HLopt2 compared with human lactoferrin (hLF) in 1% Bacto TM Peptone. Two-fold serial dilutions starting from 2000 g/mL for hLF and 400 g/mL for the peptides were used All samples were run in duplicate, giving identical MMC values.

	Edited Table 2
	Organism	MMC ( g/mL)	a
			hLF	HL10	HLopt2	HLBD1
	Candida albicans
	ATCC 64549	>2000	100	12	>200
	CCUG 90008	>2000	200	25	N/D
	CCUG 5594	>2000	100	25	N/D
	Candida glabrata
	ATCC 90030	>2000	>400	>400	N/D
	CCUG 85247	>2000	>400	>400	N/D
	CCUG 42987	>2000	>400	>400	N/D
	Candida parapsilosis
	Ir1	b	>2000	100	25	N/D
	CCUG 37233	>2000	200	25	N/D
	CCUG 22019	>2000	100	12	N/D
	Candida kefyr
	Ir2	b	>2000	100	12	N/D
	Candida krusei
	CCUG 6258	>2000	100	12	N/D
	Cryptococcus neoformans
	Ir3	b	>2000	50	12	N/D
	CCUG 37833	>2000	50	12	N/D
	MMC, minimum microbicidal concentration; N/D, not determined.

a b Patient isolate.

Table 3

 3 Effect on microbicidal activity of some helical content in HLBD1 caused by the introduction of a lactam bridge. Candida albicans ATCC 64549 was incubated with the peptides in 1% Bacto TM Peptone and killing activity was assessed at 2 h and 20 h.

	Peptides were serially diluted in two-fold steps starting at 200 g/mL
	Substitution of amino acid residue for lactam bridge	MMC ( g/mL)	a
	formation	2 h	20 h
	HLBD1 lactam bridge
	18-22	>200	50
	22-26	25	25
	26-30	25	25
	HLBD1	>200	50

a All samples were run in duplicate, giving identical MMC values.

Table 4

 4 Experimental skin infection by Candida albicans ATCC 64549 in mice. Peptide (400 g/spot) or vehicle alone (phosphate buffer) was applied twice, 4 h apart, onto a shaved area on the back of animals infected with C. albicans 24 h previously. The next day viable counts of washings from each spot were determined by culturing on

	Sabouraud plates
	Peptid	No. of	CFU (mean S.E.)		P-value	a
	e	experiments	Peptide	Vehicle
			treatment	alone
	HLBD1 2	191 57	414 60	0.0148
	HLopt2 2	160 60	570 251	0.0070
	S.E., standard error.

Funding

This study was financially supported by A+ Science AB, Göteborg, Sweden, and the Scandinavian Society for Antimicrobial Chemotherapy.