Audrey De Jong

Thomas Sfara

Yvan Pouzeratte

Joris Pensier

Amélie Rolle

Gerald Chanques

Samir Jaber

Videolaryngoscopy as a first-intention technique for tracheal intubation in unselected surgical patients: a before and after observational study

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


Hoang Ngoc Tran

Frédéric Ott
email: frederic.ott@cea.fr

Jacques Darpentigny

Anthony Marchix

Alain Letourneau

Nicolas Chauvin

Fabien Prunes

Benoît Homatter

Burkhard Annighöfer

Alain Menelle

Jérôme Schwindling

Neutrons production on the IPHI accelerator for the validation of the design of the compact neutron source SONATE

We aim at building a compact accelerator-based neutron source (CANS) which would provide a thermal neutron flux on the order of 4x10 12 n.s -1 .cm -2 .sr -1 . Such a brilliance would put compact neutron sources on par with existing medium flux neutron research reactors for neutron scattering experiments. We performed the first neutron production tests on the IPHI proton accelerator at Saclay at a proton energy of 3 MeV. The thermal neutron flux were measured using gold foil activation and 3 He detectors. The measured flux were compared with GEANT4 Monte Carlo simulations (10.4) in which the whole experimental setup was modelled. There is a good agreement between the experimental measurements and the Monte-Carlo simulations. The available modelling tools will allow us to optimize the whole Target Moderator Reflector assembly together with the neutron scattering spectrometer geometries for the design of the neutron scattering facility SONATE.

Introduction

There is currently an interest in developing compact neutron sources based on low energy proton accelerators (10-100 MeV) [1]. Such sources could serve as neutron sources for neutron scattering to replace small ageing nuclear reactors [2] or in hospitals for Boron Neutron Capture Therapy [3][4]. There are already several such projects on-going around the world. They are generally referred to as CANS, Compact Accelerator-based Neutron Sources. The currently most advanced is LENS Low Energy Neutron Source at Indiana University [5]. The CANS operating or under construction have gathered into the UCANS, Union for Compact Accelerator-driven Neutron Sources [6]. At Saclay we are considering a similar possibility to replace the Orphée research reactor. Our final goal is to build a higher end facility SONATE which could serve a suite of about 10 neutron scattering instruments. The facility would operate at a proton energy around 20 MeV, in pulsed mode to use time-of-flight techniques with a typical power on the target of 80kW. Our first aim has been to experimentally validate the neutron production and moderation obtained by Monte Carlo simulations using GEANT4. Once reliable simulation tools are available we shall be able to reliably estimate the performances of a CANS for neutron scattering experiments (from the source to the spectrometers) and compare its performances to existing facilities (reactor or spallation based).

As a starting point we are using the IPHI proton accelerator at Saclay which is able to produce high current proton beams (up to 90 mA) [7] with proton energies of 3 MeV. The Monte Carlo simulation toolkit GEANT4 is a library for the simulation of the transport of particles through matter. This tool is widely used in various applications from high-energy to very low-energy physics, including astrophysics and space science, medical physics and radiation protection [8]. Within the neutron physics package of GEANT4, the particlehp (high precision) option has to be used for neutron energies below 20 MeV as it refers to the library G4NDL that is based on the evaluated nuclear data library ENDF/B-VII.0 [9]. Moreover, this latter includes the thermal scattering libraries that take into account the molecular effects occurring for neutron energies below 4 eV when considering moderator and reflector materials such as water, polyethylene, etc. Recently, the validation of the neutron transport treatment in GEANT4-10.3 for the thermal energy range was performed by inter-comparison with the standard Monte Carlo code MCNP-6.1 on microscopic and macroscopic observables [10]. It was pointed out some sampling issues for the data treatment of the thermal scattering libraries included in the G4NDL for the versions of GEANT4-10.4. These improvements concern the treatment of nuclear interactions involved during the propagation of neutrons in a complex geometry, in particular the moderation process. But, to ensure the reliability of simulation tools, the neutrons production has to be validated as the thermal neutron UCANS-8 distributions within the moderator are sensitive to the double differential cross sections in energy and angle of primary neutrons.

Within this framework, we have performed experiments in order to measure the primary neutron source characteristics associated to the reaction 9 Be(p,n), as well as the thermal neutron flux in various locations by using a simplified polyethylene (PE) moderator. This paper describes the experimental and simulation methods that was used to measure the thermal neutron flux inside and outside the moderator, including the thermal neutron spectra and the neutron beam divergences, as well as the fast-neutron angular distribution related to the reaction 9 Be(p,n). The method for calibrating the 3 He detector efficiency via wavelength is also discussed.

Experimental setup 2.1. Proton source

The experiments have been performed on the IPHI proton accelerator which is based at CEA Saclay, France. The accelerator consists of a proton source (SILHI) of energy 95 keV, a Low Energy Beam Transport Line and a Radio Frequency Quadrupole to accelerate the protons to an energy of 3 MeV. The accelerator is designed to operate in continuous mode with proton currents up to 100 mA which corresponds to a total power of 300 kW. For the current experiments dedicated to validations of neutron production and moderator Monte Carlo simulations we have operated the accelerator at a very low power of about 10 W, both to avoid any target damage and for radioprotection issues. The accelerator was operated in pulsed mode with proton pulses of length 100 µs and with a repetition rate of 1 Hz and a peak current of 30 mA. Rather narrow thermal neutron pulses are thus obtained so that precise time-of-flight measurements can be performed.

Fig. 1. Sketch of the experimental set-up. The target is installed on a deviator line and the neutron detectors are set at 8.4m from the target so as to be able to perform time-of-flight measurements.

Target and moderator assembly

In order to produce neutrons we opted for a beryllium target of thickness 0.5mm (99.0%) which stopped all incident protons. The target was attached on an aluminium support with titanium screws in order to minimize neutron activation. This support was air cooled. The proton beam size was limited to 16mm in diameter. The proton current incident on the target was continuously measured so as to have a precise value of the incident particle flux and be able to precisely estimate the neutron production. An electron repelling electrode set at a potential of 200 V was set in front of the beryllium target so as to prevent any bias in the target current measurement. The target was installed in a polyethylene (PE) moderator box (300x300x400 mm 3 ) so as to cool down the neutron to thermal energies (around 26meV). A 20 mm diameter hole was drilled through the moderator from the position where the thermal neutron density was expected to be the highest to the outside of the PE box (see Fig. 2). In order to change the moderator geometry, it was possible to insert PE "plugs" inside the exit hole so as to fill more or less the neutron extraction channel. The whole experimental setup (accelerator -targetmoderator -detectors) was installed in a 2 m thick concrete casemate.

Fig. 2. Target and moderator assembly

Neutron detection

Fast neutron detection

A technical difficulty arises from the fact that since we operate in pulsed mode (100µs proton pulses width), the instantaneous neutron flux is very high. This prevented using a regular Bonner sphere. A dedicated acquisition chain was mounted consisting of a very small 3 He detector (1cm in diameter) encased in a cubic polyethylene setup. We made sure that even though we operated in pulse mode we did not have events pile-up in the 3 He detector. This led to acquisition times on the order of 5-10 minutes to obtain a good statistics in the measurement of the fast neutrons around the source.

Thermal neutron detection

The thermal neutron flux was measured at 8.4m from the source using time-of-flight mode and a 3 He detector. The 3 He detector was surrounded with a shielding made of polyethylene, as shown in Fig. 3, and the detector was placed at 20 cm from the aperture of 6 cm in diameter. The solid angle covered by the detector was thus reduced to about 8° allowing to reduce the background noise from neutrons scattered and UCANS-8 moderated on the concrete walls of the casemate. Moreover, a layer of B4C was installed around the 3 He detector to capture the thermal neutrons coming from the polyethylene shielding. Even though the source was operated at a modest 10W power we were very close to the saturation limit of regular 3 He tubes (~20 kHz/tube) so that the detection area was limited to 20cm², corresponding to the front face of a single 2 inches 3 He detector tube (NH 50). Thermal neutrons (1.8 Å) travel at a speed of 2200m/s so that the travel time over 8.4m is 3.8 ms. For a proton pulse width of 100 µs, the energy resolution is thus on the order 0.1/3.8 = 2.6% which is good enough to measure a rather precise neutron energy distribution. Note that besides the proton pulse length, the moderation process leads to an intrinsic neutron pulse broadening which reduces the energy resolution. Monte-Carlo simulations provide an estimate of a broadening on the order of 100-150 µs in a PE moderator [11][12]. Fig. 3. Polyethylene (blue) moderator (left). 3 He detector system design (right).

The 3 He detector used in horizontal position suffered from an issue related to a dead zone at the front of the detector where neutrons are absorbed in the 3 He gas but not detected due to the absence of an amplification electric field at top end of the detector [13]. This dead zone bias the detection efficiency as a function of the wavelength. Short wavelengths penetrate deeply in the detector and are thus almost all detected while a large fraction of long wavelengths (>3Å) are absorbed in the front part of the 3 He gas but not detected. The "dead zone" of the detector was mapped with a pencil beam on the PRISM spectrometer at Orphée at a wavelength of 4Å (see Fig. 4a). The dead zone extends over 26mm from the top of the detector. The detector efficiency is deduced by integrating the ratio between the effective distribution and the ideal distribution to the detector length. Fig. 4b is provided the calculated detector efficiency as function of the neutron energy (via TOF). The measurement have been performed with a spectrometer of the Orphée reactor for a neutron beam of 4.3 ångström that confirms the calculated detector efficiency.

Gold foil activation

In order to validate the Monte-Carlo simulations inside the moderators, gold disks were positioned at various positions inside the moderator. Disks with a diameter of 6 mm and thickness 200 µm were used. The weight of these disks was 112 mg so that even when operating at 10W, measurable activation of the gold was achieved within 15-30 minutes of operation. Bare disks and disks enclosed in Cadmium were measured so as to be able to estimate the thermal and fast neutron flux [14]. The Cd absorption cut-off edge is at around 500 meV ( = 100 barn) so that the bare disks are activated by the whole neutron spectrum irradiation and the Cd covered disks are only activated by the fast neutrons. These gold activation measurements provide a simple and quantitative way of measuring the thermal neutron flux. However, while the measurement of the gold activation is quantitative and reliable, in order to calculate an absolute neutron flux, an a priori knowledge of the neutron energy distribution is necessary. Hence either an assumption of neutron energy distribution has to be made (Maxwellian distribution centered around 26meV for example) or the activation must be calculated using a simulated neutron energy distribution. Four gold disks with same size (with and without Cd casing) have been put into the moderator at positions 1 and 2 (see Fig. 3 Experimental results

Fast neutron yield and angular distribution

In order to use a reliable model for the fast neutron term an accurate knowledge of the fast neutron emission rate and angular distribution is required. Moreover, no data are available for 3 MeV protons, while they are needed for validating the models implemented in GEANT4 in the framework of the prototype tests at 3 MeV protons. In consequence, we performed a new measurement of the angular distribution of the fast neutrons produced by a 3 MeV proton beam impinging on a thick beryllium target.

The angular distribution of neutrons related to the reaction 9

Be(p,n) was measured by using a PE-3 He detector system that consists of 10 cm thick PE in front of a 3 He counter (4.5 mm radius, 200 mm length). The aim of the polyethylene was to moderate part of incoming neutrons aiming to be captured in the 3 He detector. The PE-3 He detector was located at a distance of 2 m from the target. The detector was shielded from parasitic thermal neutron scattered from the concrete wall with a Cd sheet. The Proton beam intensity was limited to 2.2 µA to avoid pileup effects in the 3 He counter. The fast neutron yield was measured for six angles around the target, from 0 to 150° by steps of 30°. Fig. 5 shows the measured angular distributions of neutrons produced in the reaction 9 Be(p,xn) for 3 MeV protons. Our new measurements confirms those from Howard et al. [15] performed with a proton beam of 3.7

MeV. The comparison is normalized to the neutron rate of each measurement (n0) which is adjusted by Howard's measurements [12]. The data were modelled with a fourth order polynomial to build the fast-neutron source used in the GEANT4 simulations.

Thermal neutron yield

Gold foil activation inside the moderator

Table 1 provides the data values obtained on the activity of the different gold foils. All the details have been fully described in Allen et al. [17]. The gold disks are irradiated for durations ranging from 15 to 22 minutes which lead to measurable gamma activity from 198 Au. It can be remarked that the Cd-layer strongly decreases the activity of each gold foils, especially for the furthest location (210 mm) that is below the detection limit. The measured activities of the gold foils have ~ 23% of incertitude due to determination of gold foils activation. Note that the measurements were performed by considering a 50 mm thick PE plug in the neutron extraction channel (see Fig. 2). The thermal neutron flux (showed in Table 1) associated to these measurements have been deduced based on the cross section of data library ENDF/B-VII.0. Table 1. The measured activation foils were used to reduce thermal neutron flux (less than 0.4 eV).

Thermal beam mapping

Outside the moderator, the thermal neutron flux was mapped with a 3 He detector by scanning the detector position in the X and Y directions to ensure we were aiming at the peak flux. Fig. 6 presents the thermal neutron beam divergences in both X and Y axis at the distance 8.4 m. We observe a fair agreement between the experimental data (red dots) and the calculation (dark line). The systematic underestimate between the experimental and calculated data may originate from secondary thermal neutron emission background in the experimental data. The result of the simulation performed with GEANT4 is also provided. A very good agreement is obtained between the measurements and the simulation result. In this simplified moderator geometry, it is possible to obtain a thermal neutron flux of about 1000 cm -2 .s -1 at the exit of the moderator.

Fig. 7. Thermal neutron flux (less than 0.4 eV) as function of the distance between the target and 3He counter given by Geant4 simulation and compared with measurements. Measured flux inside the moderator using the gold foil activation technics and outside the moderator using 3 He counter.

Thermal neutron energy spectrum

The comparison of the neutron time-of-flight spectrum requires a high statistic for the simulations, and consequently a high computing time, as the number of neutron events reaching the 3 He detector at 8.4m is extremely low in the simulation. To reduce the computational time, the GEANT4 simulation has been split into two steps:

 The first step: 10 9 neutrons were generated at the target position according to the measured angular distribution discussed above. This simulation performs the neutron transport within the polyethylene moderator. The neutron spectrum is stored at the entrance of the 3 He detector, as well as the neutron angular distribution and the neutron position in the transversal plane.

 The second step: based on the neutron distributions in energy, in angle and in position obtained from the first step, we generated 10 7 neutrons from the surface in front of the 3 He detector. The 3 He(n,p) reactions are tracked in the detector volume and the energy distribution of incident neutrons is recorded.

Considering the time of the moderation processes (less than 2 % in average) is negligible in comparison with the TOF in the large distance from the moderator to the detector (8.4 m), we convert the neutron energy spectrums to the travel time. The amount of attenuation due to air is about ~8% for the distance of 8.4 m. Fig. 8. shows the comparison of the neutron TOF spectrum obtained for the 50 mm thick PE plug configuration (see the geometry in Fig. 3a) between simulations and experimental data. A general agreement in the thermal Maxwell distribution with the measurement is showed in Fig. 7. An underestimation of simulation below 1 ms is due to the gamma background of the counter. Note that the calculation is corrected by the detector efficiency and the measurement is also corrected by the measured thermal neutron background in the casemate (figure 3).

The time-of-flight (ToF) measurements were performed by setting a single 3 He detector at a distance of 8.4m in the direction of the extraction hole. The incident neutron flux was measured with time channels of width 20 µs. The start of the acquisition was synchronized with the clock signal of the accelerator radio-frequency. Each ToF spectrum was acquired in 5 to 10 minutes. Figure 8 shows examples of ToF spectra measured in various conditions so that the contributions of various types of particles could be identified and quantified. The blue curve corresponds to the "reference signal" as measured with the box described on Fig. 3. The peak in the region between 1 and 6 ms corresponds to the thermal neutrons. The thermal spectrum is peaked around t = 2.2ms corresponding to a neutron speed of 3800m/s, i.e.  = 1.05Å. The neutron spectrum can be fitted with a Maxwellian distribution centred on a temperature of 350K. While this is higher

UCANS-8

than the actual moderator temperature, it is no surprise as neutron moderated with hydrogenated materials are always under-moderated contrary to thermal neutron produced in a D2O reactor vessel where the interaction time is much longer. Extra contributions are observed below TOF<1ms. In order to identify these contributions, a Cadmium sheet was put in front of the detector shielding box (Figure 8a, red dots). Above t = 1ms, there is no residual signal. t = 1ms corresponds to a neutron energy of 0.5eV which is the neutron Cd cut-off. Hence it is demonstrated that the measured spectrum actually corresponds only to thermal neutrons. In a third measurement, thermal neutrons were prevented from exiting the moderator with a Cd sheet at the moderator exit; fast neutron could still escape the moderator. The measured signal (purple dots) shows that a residual neutron signal is then observed for t > 1ms. It corresponds to the fast neutrons moderated into the casemate walls and eventually arriving into the detector. This allows telling that 85% of the measured thermal neutrons are actually the ones coming from the moderator extraction hole while 15% of the neutron are secondary emission thermal neutron originating from the moderation of fats neutron in the casemate wall. In further measurements, the collimation system should be improved. In a 4 th measurement the fast neutron contribution was stopped with a PE block followed by a Cd sheet in front of the detector shielding entrance. The data (green dots) shows that there is a remnant short time contribution of either epithermal neutrons or gammas. These different contributions can easily be discriminated using the ToF measurements. However, due to the finite proton pulse length (100µs), the finite moderation time (100µs) and the short flight path, no quantitative information can be retrieved for neutron energies above 30eV since above this energy, all ToF information is blurred in the first few time channels.

Conclusion

This paper describes the experimental and simulation methods to determine angular distribution of primary fast neutrons of 9 Be(p,n) and the thermal neutron flux inside and outside the PE moderator including thermal neutron energy spectra and neutron beam divergences based on 3 MeV proton beam of IPHI accelerator. The efficiency of the used 3 He detector for these measurements is estimated. Good agreements between GEANT4 simulation and measurement data are observed. This gives us confidence in the reliability of the GEANT4 for the quantitative description of neutron moderators.

Fig. 4 .

 4 Fig. 4. (a) Detection efficiency along the length of the 3 He tube. The dead zone is approximately 26mm long; (b) Calculated detector efficiency with GEANT4 together with a superimposed experimental calibration point (red) measured on Orphée. The average detection efficiency of the detector was 30%.

  2) at 70 mm and 210 mm from the intersection of the proton beam axis with the neutron extraction hole. Their activation has been calculated during the Monte Carlo simulations. The measured then been compared with the calculated activation values.

Fig. 5 .

 5 Fig. 5. Angular distributions of the neutrons from the 9 Be (p,n) reaction of 3 MeV proton beam compared with the results of Howard et al. [15] at 3.7 MeV. The angular resolution was ±2.8º.

Fig. 6 .

 6 Fig. 6. Thermal neutron beam divergences at 8.4 m from the target. The positive direction of Y axis is in the proton beam's side. The origin is at 8.4 m.

Fig. 7

 7 Fig.7summarizes the thermal neutron flux measurements obtained from gold foil activation and time of flight measurement along the neutron flight path (red dots). The result of the simulation performed with GEANT4 is also provided. A very good agreement is obtained between the measurements and the simulation result. In this simplified moderator geometry, it is possible to obtain a thermal neutron flux of about 1000 cm -2 .s -1 at the exit of the moderator.

Fig. 8 .

 8 Fig. 8. The comparison of the ToF spectrum between experimental data and Geant4 calculation.

  of 3He tube (PE 5cm + Cd) in front of 3He tube Cd at moderator exit

© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/).

EPJ Web of Conferences 231, 01007 (2020) https://doi.org/10.1051/epjconf/202023101007


Priyanka Singh

Krishnananda Samanta

Ndeye Mathy Kebe

Grégory Michel

Baptiste Legrand

Vera E Sitnikova

Andrey V Kajava

Michel Pagès

Patrick Bastien

Christelle Pomares

Olivier Coux
email: olivier.coux@crbm.cnrs.fr

Jean-François Hernandez
email: jean-francois.hernandez@umontpellier.fr

The C-Terminal Segment of Leishmania major HslU: toward Potential Inhibitors of LmHslVU Activity

Keywords: HlsV protease, Leishmania, medicinal chemistry, peptides, structure-activity relationships

Abstract.

It is urgent to develop less toxic and more efficient treatments for leishmaniases and trypanosomiases. We explore the possibility to target the parasite mitochondrial HslVU protease, which is essential for growth and has no analogue in the human host. For this, we develop compounds potentially inhibiting the complex assembly by mimicking the C-terminal (C-ter) segment of the ATPase HslU. We previously showed that a dodecapeptide derived from Leishmania major HslU C-ter segment (LmC12-U2, Cpd 1) was able to bind to and activate the digestion of a fluorogenic substrate by LmHslV. Here, we present the study of its structure-activity relationships. By replacing each essential residue with related nonproteinogenic residues, we obtained more potent analogues. In particular, a cyclohexylglycine residue at position 11 (cpd 24) allowed a more than three-fold gain in potency while reducing the size of compound 24 from twelve to six residues (cpd 50) without significant loss of potency, opening the way toward short HslU C-ter peptidomimetics as potential inhibitors of HslV proteolytic function. Finally, conjugates constituted of LmC6-U2 analogues and a mitochondrial penetrating peptide were found to penetrate into the promastigote form of L. infantum and to inhibit the parasite growth without showing toxicity toward human THP-1 cells at the same concentration (i.e. 30 µM).

Introduction.

Leishmaniases and trypanosomiases are worldwide spreading diseases caused by the parasitic protozoa Leishmania and Trypanosoma, respectively. These Trypanosomatid parasites mainly affect sub-tropical and tropical zones and significantly contribute to human misery in these regions. About 350 million people are at risk for leishmaniases. With 1.5-2 million new cases per year and annual mortality of about 60,000 mainly due to visceral leishmaniasis, the rise in the number of cases is alarming [START_REF] Maxfield | Leishmaniasis. In: StatPearls, Treasure Island (FL)[END_REF]. A recent concern is the sharp increase in the overlapping of HIV infection and visceral leishmaniasis. Particularly worrying in Africa, this co-infection also caused an increased incidence of this disease in southern Europe (Spain, France, Italy, Portugal) [START_REF] Lindoso | Visceral leishmaniasis and HIV coinfection: current perspectives[END_REF]. About 70 million persons are at risk for African (sleeping sickness, T. brucei) and 25 million for American (Chagas' disease, T. cruzi) trypanosomiases [3,4]. The African disease has largely regressed during the last decade due to control programs but the risk of major epidemic remains. T. cruzi infects 6-7 million people and kills 10,000 persons every year [4][5][6].

There is as yet no effective treatment against any form of these human diseases. Current drugs generally suffer of several drawbacks, including toxicity, administration complexity, resistance, inability to treat all forms of the diseases, and cost [7]. New safe and effective drugs are thus urgently needed. Whereas the interest of pharmaceutical companies to develop new drugs has been traditionally scarce as these diseases are mainly prevalent in the poorest areas of the world, important actions have been implemented since the beginning of the century to fight these neglected diseases. For instance, leishmaniases and trypanosomiases are the main targets of the not-for-profit organization "Drugs for Neglected Diseases initiative" (DNDi, dndi.org), which enables research and development networks between academic and private partners.

Ideally these new drugs should be effective against both types of parasites and in both stages of the parasite life cycle (i.e. promastigote and amastigote for Leishmania, trypomastigote and amastigote for Trypanosoma).

The biology of trypanosomatids has been intensively studied and their genomes fully sequenced [8][START_REF] El-Sayed | The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease[END_REF][10]. These organisms use many unique and atypical molecular and biochemical pathways, which may be exploited as potential drug targets [11].

The ATP-dependent HslVU protease complex is one of these interesting targets. First characterized in eubacteria [12][13][START_REF] Yoo | Purification and characterization of the heat shock proteins HslV and HslU that form a new ATP-dependent protease in Escherichia coli[END_REF], absent in higher eukaryotes, HslVU was found in ancestral eukaryotes like Plasmodium [START_REF] Mordmüller | Plasmodia express two threoninepeptidase complexes during asexual development[END_REF][START_REF] Jain | The prokaryotic ClpQ protease plays a key role in growth and development of mitochondria in Plasmodium falciparum[END_REF] and Trypanosomatids [17] with a mitochondrial localization [START_REF] Li | Identification of a bacterial-like HslVU protease in the mitochondria of Trypanosoma brucei and its role in mitochondrial DNA replication[END_REF][START_REF] Tschan | Mitochondrial localization of the threonine peptidase PfHslV, a ClpQ ortholog in Plasmodium falciparum[END_REF][START_REF] Mbang | The bacterial-like HslVU protease complex subunits are involved in the control of different cell cycle events in trypanosomatids[END_REF]. Studies by us [START_REF] Mbang | The bacterial-like HslVU protease complex subunits are involved in the control of different cell cycle events in trypanosomatids[END_REF] and others [START_REF] Li | Identification of a bacterial-like HslVU protease in the mitochondria of Trypanosoma brucei and its role in mitochondrial DNA replication[END_REF] have shown that HslVU plays an essential role for cell growth and survival in Leishmania and Trypanosoma. Thus, inhibiting HslVU may be a promising approach to cure these diseases. Because HslV and the human proteasome have the same catalytic mechanism and binding sites with wide specificity, targeting their active sites appears a risky approach to identify selective inhibitors. An alternative consists in the inhibition of HslV/HslU association, which is essential for activity of the protease [12].

HslVU is the assembly of two stacked hexameric rings of the threonine protease HslV, which are capped on both side by an hexameric ring of the ATPase HslU. HslU recognizes and unfolds protein substrates and translocates them into the internal proteolytic chamber of the HslV dodecamer. X-ray structures of bacterial HslVU complexes [START_REF] Sousa | Crystal and solution structures of an HslUV protease-chaperone complex[END_REF][START_REF] Wang | Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism[END_REF] and functional studies [23][24][START_REF] Sousa | Crystal structure of HslUV complexed with a vinyl sulfone inhibitor: corroboration of a proposed mechanism of allosteric activation of HslV by HslU[END_REF] have evidenced the role of the C-terminal segment (C-ter) of HslU in protease activation. By inserting into the HslV complex, HslU C-ters induce a conformational rearrangement of HslV that propagates to the active sites [START_REF] Shi | Tracing an allosteric pathway regulating the activity of the HslV protease[END_REF]. This allosteric activation mechanism differs from that of the eukaryotic proteasome, for which binding of activators opens a gated channel that controls access of substrates to the proteolytic chamber. These functional studies also showed the importance of HslU C-ter in the stability of HslVU and hexameric HslU complexes. A synthetic octapeptide derived from Haemophilus HslU Cterminus was shown to bind HslV and to potently activate the digestion of a small fluorogenic substrate but not proteins, which necessitate prior unfolding by HslU [23]. A similar result was obtained with Leishmania, for which the activation of HslV is accompanied by large conformational rearrangements of the complex [START_REF] Kebe | The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides[END_REF]. This interaction is specific as substitution of the strictly conserved C-terminal leucine residue by an alanine abolishes HslV activation.

HslU C-ter from eubacteria and protozoan parasites is highly conserved, strongly supporting that an identical mechanism operates. It is noteworthy that HslU and HslV have only a moderate affinity (Kd = 1 µM) for each other in the absence of substrate [28] probably because the HslU C-ter is buried in the HslU complex in these conditions [START_REF] Bochtler | The structures of HsIU and the ATP-dependent protease HsIU-HsIV[END_REF]. This low affinity is an advantage for developing competing molecules based on the C-terminal segment of HslU. In support, a synthetic dodecapeptide derived from P. falciparum HslU C-ter was shown to compete with PfHslU for binding to PfHslV [START_REF] Rathore | Disruption of a mitochondrial protease machinery in Plasmodium falciparum is an intrinsic signal for parasite cell death[END_REF].

Intriguingly, in Trypanosomatids only, two HslU proteins, termed U1 and U2 co-exist. U1 and U2 are highly homologous in sequence except in the so-called I-domain thought to be involved in substrate recognition. The respective roles of U1 and U2 are not known. We previously found that inhibition of the expression of HslU1 and HslU2 from Trypanosoma brucei yielded different phenotypes and that only HslU1 was indispensable to parasite survival [START_REF] Mbang | The bacterial-like HslVU protease complex subunits are involved in the control of different cell cycle events in trypanosomatids[END_REF]. In this context, Sung et al. reported that only a C-terminal octapeptide derived from HslU2 of T. brucei (TbHslU2) and not from TbHslU1 was able to activate the peptidolytic activity of TbHslV [START_REF] Sung | Structural and biochemical analyses of the eukaryotic heat shock locus V (HslV) from Trypanosoma brucei[END_REF].

We recently described the production, purification and biochemical characterization of HslV from Leishmania major (LmHslV) [START_REF] Kebe | The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides[END_REF]. In contrast to Sung's result, we showed that octapeptides as well as dodecapeptides derived from both LmHslU1 and LmHslU2 were able to activate LmHslV, although the activation was indeed more efficient with LmHslU2 peptides. In addition, we found that the corresponding dodecapeptides from EcHslU, TbHslU2 and PfHslU were also able to activate LmHslV, sometimes with higher efficiency than the LmHslU2 peptide itself. This result suggested that a same HslU Cter mimetic could target HslV of all microorganisms. Finally, based on a C-terminal U2-derived dodecapeptide chosen as a reference (i.e. LmC12-U2, H-arg-O2Oc-Leu-Gln-Lys-Asn-Val-Asn-Leu-Ala-Lys-Tyr-Leu-Leu-OH, Cpd 1, Figure 1), we found that five out of its six C-terminal residues were essential for HslV binding and activation and that side-chain to side-chain cyclisation allowed reducing the peptide size from 12 to 9 residues without loss of potency.

H-arg-O2Oc-Leu-Gln-Lys-Asn-Val-Asn-Leu 7 -Ala-Lys 9 -Tyr 10 -Leu 11 -Leu 12 -OH Here, based on these results, we present an extended and extensive study of the structureactivity relationships of LmC12-U2 with the objective to obtain more potent analogues and to significantly reduce the size of HslU Cter mimetics. Finally, having identified potent LmC6-U2 analogues, conjugates with mitochondrial penetrating peptides (MPP) as well as fluorescently-labelled analogues were prepared and tested on Leishmania infantum cultures.

Results and Discussion

All peptides were assembled on a 2-chloro-trityl resin with Fmoc as the Na-protecting group and HBTU or HATU as coupling agents as already described [START_REF] Kebe | The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides[END_REF].

Their ability to activate LmHslV was assessed using the fluorogenic substrate Z-EVNL-AMC (JMV4482) [START_REF] Kebe | The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides[END_REF], which we found much more efficient than the Z-GGL-AMC substrate classically used to study HslVU [12]. As previously published, LmHslV displayed no activity in the absence of peptides [START_REF] Kebe | The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides[END_REF]. It gained activity in the presence of C-ter HslU peptides as already reported for bacterial HslV and TbHslV [23,[START_REF] Sung | Structural and biochemical analyses of the eukaryotic heat shock locus V (HslV) from Trypanosoma brucei[END_REF]. Peptides were compared based on the initial velocity of Z-EVNL-AMC digestion by LmHslV and results are reported as percentage of the initial velocity measured in the presence of the reference LmC12-U2 (Cpd 1) in most experiments (Tables 123).

Structure-activity relationships of LmC12-U2

Five out of the six C-terminal residues of LmC12-U2 (Cpd 1, Figure 1) were found to be essential for binding to and activation of LmHslV [START_REF] Kebe | The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides[END_REF]. We considered that the essential nature of these residues was related to their tight interaction with well-defined pockets at the interface of two HslV subunits. With the objective to better define the structural requirements of LmHslU2 binding to LmHslV and to possibly obtain more efficient analogues, we individually substituted each essential position by structurally close and/or bulkier and mostly non-natural amino acids (Figure 2, Tables 123).

In addition, as even structurally close residues display slightly different physico-chemical properties, hydrophobicity and bulkiness, these changes may lead to a gain in affinity.

Leucine residues (positions 7, 11 and 12) were mainly replaced by bulkier and more hydrophobic aliphatic amino acids such as cyclohexylalanine (Cha) or cyclohexylglycine (Chg). Lysine at position 9 was replaced by other basic residues or isosteric neutral ones.

Tyrosine at position 10 was replaced by numerous aromatic residues more or less bulky and hydrophobic (Figure 2). We assumed that a higher activation level would reflect a higher affinity, although we cannot rule out that it could result from a more efficient substrate binding or catalytic activity.

Table 1. LmHslU2 C-ter peptide mono-and multisubstituted analogues 1-35 and their

LmHslV activation potency at 500 µM.

Cpd

Monosubstituted analogues.

Substitutions at positions 7 or 9.

Table 1 shows the activity of analogues monosubstituted at positions 7 or 9 relative to the reference 1. The substitution of Leu 7 by its closest analogue Cha (2) or the shorter Chg (3) had no effect on the activation level. In contrast, Oic (4), which is a proline analogue of Cha, led to a five-fold decrease in potency. This effect could be due to side-chain constraining and/or to the absence of a potential hydrogen bond between the peptide and HslV. In the case of Lys 9 , changes showed that this residue binds to a large pocket in HslV and that a positive charge could be spared. Indeed, a minimal size is required as replacing Lys by its one-methylene shorter analogue Orn (5) decreased the activating potency by more than six-fold, while replacing it with the bulkier paminophenylalanine (6) yielded similar potency. As expected, the other natural basic residue Arg yielded a similarly efficient analogue (7). In HiHslVU (1G3I.pdb [START_REF] Sousa | Crystal and solution structures of an HslUV protease-chaperone complex[END_REF]), the residue equivalent to Lys 9 is HiHslU Arg 441 , whose side-chain faces an acidic environment formed by HiHslV Glu 61 and Glu 58 residues, which are conserved in LmHslV, suggesting important ionic interaction. In contrast, replacing Lys 9 by the isosteric but uncharged arginine analogues citrulline (8) and thiocitrulline [START_REF] El-Sayed | The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease[END_REF] only slightly decreased the activating potency (relative potencies of 53% and 83%, respectively), suggesting that the urea group and in particular the bulkier thiourea group could compensate the absence of charge by their ability to establish several hydrogen bonds.

2.1.1.2. Substitutions at positions 10, 11, or 12. More dramatic variations were observed when substituting the last three residues Tyr-Leu-Leu. Substitution of Tyr 10 led to disparate results (Table 1). As already reported, the replacement of this residue by Phe (10) led to a eight-fold decrease in activity [START_REF] Kebe | The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides[END_REF]. In addition, introducing the constrained pipecolic analogue of Phe, 1,2,3,4-tetrahydroisoquinoline-3-carboxyl (Tic) (11), completely abolished HslV activation.

Introducing various substituents on Phe (p-fluoro-12, 3,4-difluoro-13, p-nitro-14) only slightly increased the potency compared to 10 (up to less than 30% relative potency), but all three analogues were three-to five-fold less potent than the reference peptide, suggesting an important interaction between the tyrosine phenol group and HslV that could not be compensated by fluorine or nitro group. In fact, structural data obtained for TbHslV [START_REF] Sung | Structural and biochemical analyses of the eukaryotic heat shock locus V (HslV) from Trypanosoma brucei[END_REF] and HiHslVU [START_REF] Sousa | Crystal and solution structures of an HslUV protease-chaperone complex[END_REF] suggest that the phenol group establishes H-bond(s) with Ala 79 main chain atoms and/or Lys 80 in HslV. In agreement with this, introducing substituents on Tyr led to less important changes and could be favourable to potency in some cases. Whereas the [m-nitroTyr 10 ]-LmC12-U2 analogue 15 was about half as potent as the parent peptide 1, the DOPA (or m-hydroxyTyr)-containing compound 16 was 1.4-fold more potent. Overall, these results support the importance of the tyrosine hydroxyl group in HslV activation. However, we found that the absence of the hydroxyl group can be balanced by bulky bicyclic aromatic residues. Indeed, the three analogues 17 ([1-Nal 10 ]), 18 ([2-Nal 10 ]) and 19 ([Trp 10 ]) were as or significantly more efficient than the reference peptide. In particular, 17 was the most potent of this series with a 1.6-fold increase in activity. 20 ([Bip 10 ]-) was an exception, indicating that the second phenyl group at the para position of Phe might not be well accommodated by the corresponding HslV interaction pocket. More striking results were obtained when exploring position 11 (Table 1). First, replacing the leucine residue by its closest analogue Cha (21) led to an approximately 2.7-fold increase in potency compared to LmC12-U2 1. Interestingly, the corresponding aromatic residue Phe in compound 22 was much less favourable (2-fold less potent than LmC12-U2), indicating that the larger volume and/or higher adaptability of the cyclohexyl ring allowed a better recognition by HslV. Then, increasing the side-chain by one methylene (i.e. a homoCha residue) yielded the less potent 23, which nevertheless still proved better than compound 1 (1.6-fold more efficient). In contrast, shortening it by the same unit (i.e. a Chg residue) was more favourable to activity, with 24 being about 3.5-fold more potent than LmC12-U2.

Replacing Cha by octahydroindolecarboxyl (Oic, compound 25) abolished the activity, indicating that the Cha-like side-chain of Oic was blocked in an orientation not compatible with binding and/or that an essential hydrogen bond was lost. Finally, it is noteworthy that replacing the Tyr 10 residue by Phe in the [Chg 11 ] analogue [START_REF] Shi | Tracing an allosteric pathway regulating the activity of the HslV protease[END_REF] did not lead to a lower level of activation compared to 24. It is also striking that, although substitution of the C-terminal Leu residue by Ala ( 27) led to a ten-fold decrease in potency compared to 24, the activity was not abolished in contrast to what was previously observed for [Ala 12 ]-LmC12-U2 [START_REF] Kebe | The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides[END_REF]. 27 showed about 35% activity as compared with compound 1. Overall, these results showed that the presence of a cyclohexyl ring at position 11 is of high interest but the structural basis of this particular behaviour is unclear.

Finally, as expected [23], the C-terminal Leu residue was confirmed to be critical to activate LmHslV. Indeed, introducing the b-amino acid residue b-homoLeu (28) abolished the activity. However, whereas substitution by the closest analogue Cha (29) caused a four-fold decrease in potency, replacing Leu 12 by the shorter Chg (30) was fully tolerated. 7, 9, 10, 11. The first part of the study showed that it was possible to modulate the activation potency of the reference peptide 1 LmC12-U2 by replacing essential residues with closely related ones. Moreover, more potent analogues were obtained with substitutions at positions 10 and 11, which could help the design of peptidomimetics. To check if the combination of several of these favourable substitutions could yield a synergetic effect, we prepared a few di- [START_REF] Sung | Structural and biochemical analyses of the eukaryotic heat shock locus V (HslV) from Trypanosoma brucei[END_REF][START_REF] Marshal | Angiotensin II. Studies on the biologically active conformation[END_REF][START_REF] Burgess | An obligatory alpha-helical amino acid residue[END_REF] and tri-substituted [START_REF] Sudha | Stabilization of beta-turn conformations in enkephalins. alpha-Aminoisobutyric acid analogs[END_REF][START_REF] Benedetti | Structural versatility of peptides from C α,α dialkylated glycines: Linear Ac3c homooligopeptides[END_REF] analogues of LmC12-U2 diversely combining the neutral (i.e. no individual effect) [Cha 7 ],

Combined substitutions at positions

[Arg 9 ], and two of the most favourable mono-substitutions [1Nal 10 ] and [Cha 11 ] (Table 1).

Unfortunately, compared to the corresponding mono-substituted peptides, most analogues showed lower activation potency. In particular, compound 33, which contained two favorable substitutions (1Nal 10 and Cha 11 ) showed intermediate activity compared to the corresponding mono-substituted 17 and 21. Interestingly, compound 35, which differed from 33 by the presence of the neutral Cha at position 7 was 1.2-fold more potent than 33. In addition, the trisubstituted analogue 34 ([Cha 7 , Arg 9 , Cha 11 ]), which combined two neutral and one favourable substitution was found to be slightly more potent (303% vs 278%) than the [Cha 11 ]-LmC12-U2 analogue 21.

Our observation that most of the combined substitutions do not have a synergic effect can be explained by the assumption that each individual substitution causes slight adjustment of the peptides within the binding pocket. The synergy will be observed only in the cases when the adjustment induced by two or more combined substitutions occurs in the same direction.

Constrained analogues.

When interacting with HslV, the C-terminal hexapeptide segment of HslU in Hemophilus influenzae forms a short helical structure, the C-terminal leucine residue being distended from this helix [START_REF] Sousa | Crystal and solution structures of an HslUV protease-chaperone complex[END_REF]. Various local and global conformational constraints have been introduced in order to favour this conformation (Table 2). First, the three leucine residues at positions 7, 11 and 12 as well as Ala 8 have been individually replaced by their Ca-methyl analogue (CaMethyl-Leucine (CaMeLeu) and aaminoisobutyric acid (Aib), respectively). These amino acids have been shown to induce local constraints favouring either b-turn or helix secondary structures [START_REF] Marshal | Angiotensin II. Studies on the biologically active conformation[END_REF][START_REF] Burgess | An obligatory alpha-helical amino acid residue[END_REF][START_REF] Sudha | Stabilization of beta-turn conformations in enkephalins. alpha-Aminoisobutyric acid analogs[END_REF][START_REF] Benedetti | Structural versatility of peptides from C α,α dialkylated glycines: Linear Ac3c homooligopeptides[END_REF]. In addition, they could also provide the peptides with a higher resistance to enzyme degradation [START_REF] Payne | The primary structure of alamethicin[END_REF][START_REF] Leduc | Differentiation of bradykinin receptors and of kininases with conformational analogues of bradykinin[END_REF].

Unfortunately, the analogues 38 (CaMeLeu 11 ) and 39 (CaMeLeu 12 ) were devoid of activity, while analogue 37 (Aib 8 ) was half as potent as the reference peptide and a CaMeLeu at position 7 (36) did not induce any change (Table 2). To check the influence of these substitutions on peptide conformation, we studied compounds 1, 36 and 37 by circular dichroism (CD) in aqueous buffer without or with increasing concentrations of trifluoroethanol (TFE), a co-solvent with helix-promoting properties (Figure S1 and Table S4). As already observed in our previous study [START_REF] Kebe | The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides[END_REF], all peptides display random conformation with very low helix content (< 5% in the absence of TFE). Addition of TFE only slightly increased it further, reaching a maximum of 12% helix content at 50% TFE.

Overall, all three peptides behaved quite identically (Figure S1 and Table S4), indicating that the substitution by Ca-methyl aminoacids in compounds 36 and 37 had no effect on the conformation when compared to the parent peptide 1. These results may explain why no improvement in activity was observed following this approach. In addition, the decreased activity observed for compounds 37-39 indicated that the CaMethyl group might hinder some important interaction with HslV and confirmed the essential role of the last two C-terminal leucine residues.

Second, we further explored global conformational constraint through side-chain to side-chain cyclisation between positions 4 (i) and 8 (i + 4). We previously reported that cyclization between Asp 4 and Lys 8 through a lactam bridge in LmC12-U2 was well tolerated and that it was possible to decrease the size to the corresponding cyclic C9-U2 (40, see Table 2) without loss of potency [START_REF] Kebe | The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides[END_REF]. We first substituted Asp 4 to Glu 4 to slightly increase the cycle size (compound 41), and, as observed when decreasing it [START_REF] Kebe | The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides[END_REF], this change led to a ten-fold loss in potency. These results indicated that the 20-atom ring formed by the (Asp i , Lys i+4 ) bridge is optimal for activity. The fact that it is also optimal for helix stabilization [START_REF] Taylor | The synthesis and study of side-chain lactam-bridged peptides[END_REF] suggests that it stabilizes at least one helix turn in LmC9-U2. We also reversed Asp and Lys residues (compound 42) and decreased activity was also observed. This result was not unexpected as it has been reported that the orientation and/or the position of a lactam bridge could be critical for helix stability [START_REF] Houston | Lactam bridge stabilization of alpha-helical peptides: ring size, orientation and positional effects[END_REF]. (Table 1). The two first analogues were found to be slightly less potent than the nonsubstituted cyclic C9-U2 peptide. In contrast, the [Cha 11 ] analogue 45 was found slightly more potent, but the observed 1.3-fold increase in activity was less notable than in the case of the linear LmC12-U2 reference peptide. More interestingly, the presence of a Chg residue at position 11 [START_REF] Horton | Tuning the activity of mitochondria-penetrating peptides for delivery or disruption[END_REF] strongly increased potency, being about three-fold more active than the parent cyclic peptide. In addition, the analogue 47 with a reverse lactam bridge was found as potent as 46, in contrast to what was observed for compound 42. These results confirmed the exceptional interest of Chg at this position. where the dipeptide Leu 7 -Ala 8 was replaced by a gLeu residue displayed significant activity (about 50% that of LmC12-U2). Overall, Chg was much more efficient than Cha, and 50 was approximately three-fold and seventeen-fold more potent than LmC12-U2 and LmC6-U2, respectively.

Analogues of compound 50 were prepared where Tyr 10 and/or Leu 12 were replaced by close residues (1-Nal ( 53), 2-Nal (54), DOPA [START_REF]H-arg-O2Oc-Leu-Ala-Lys-DOPA-Chg-Leu-OH [DOPA 10[END_REF] and m-nitroTyr (56) at position 10; Chg residue (57) at position 12). Table 3 showed that no analogue was found significantly more potent than 50, while substituting Leu 12 with an Ala residue (58) abolished activity as expected.

Assuming that a better activation potency relates to stronger binding, we tried to understand the higher activity of Chg 11 -containing analogues by molecular modelling. The compounds LmC8-U2 and [Chg 11 ]-LmC8-U2 were modelled into the HslU C-ter-binding site situated between two subunits of the HslV complex from Leishmania. The Leishmania-based models were generated using the known structure of the HslVU complex from Haemophilus influenza (i.e. HiHslVU, PDB code 1G3I [START_REF] Sousa | Crystal and solution structures of an HslUV protease-chaperone complex[END_REF]) and INSIGHT II program [START_REF] Dayringer | Interactive program for visualization and modelling of proteins, nucleic acids and small molecules[END_REF]. The modelling showed that both Chg 11 and Leu 11 fit similarly the binding pocket (see Figure S2 for the Chg 11containing segment) and no clear difference in binding energy could be observed in this study. One hypothesis is that Chg imposes a higher rigidity to the peptide leading to a difference in the binding kinetics, which could impact the activation mode. This hypothesis is in line with the fact that Chg is a b-branched residue (not the case of Leu) and its lateral chain is further constrained by cyclisation and thus less mobile, which in turn could be an advantage to rapidly fit in the binding site, provided that is has the right orientation.

In vitro activity against the promastigote form of Leishmania infantum

We studied whether the most potent hexapeptide compound found here, 50 ([Chg 11 ]-LmC6-U2), could impair parasites' survival in culture.

It is generally admitted that such peptides are not able to cross cell membranes. In addition, to reach its target HslVU, the compound has to cross both the cell and mitochondrion membranes. Thus, we considered to conjugate the peptide to a cell penetrating peptide (CPP).

This approach has already been reported to target the HslVU complex of Plasmodium falciparum (PfHslVU) [START_REF] Rathore | Disruption of a mitochondrial protease machinery in Plasmodium falciparum is an intrinsic signal for parasite cell death[END_REF]. In this study, the Authors attached the well-known 16-residueslong CPP penetratin to the PfHslU C-terminal dodecapeptide (i.e. PfC12-U), and the resulting conjugate was shown to inhibit parasite growth in a dose-dependent manner. However, this conjugate is long (28 residues) and therefore highly exposed to proteolytic degradation. In addition, the C-terminal HslU segment was not optimized and penetratin is not considered to preferentially target the mitochondrion. We therefore conjugated the hexapeptidic segment of 50 to another CPP belonging to a family of mitochondrial penetrating peptides (MPPs), which have been shown to specifically localize and to deliver small peptidic cargoes to mitochondria in human cell [START_REF] Zhao | Cellpermeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury[END_REF][START_REF] Horton | Mitochondirapenetrating peptides[END_REF][START_REF] Yousif | Mitochondria-penetrating peptides: sequence effects and model cargo transport[END_REF][START_REF] Kelley | Development of novel peptides for mitochondrial drug delivery: amino acids featuring delocalized lipophilic cations[END_REF]. First, we checked if MPPs could indeed enter in the parasite cell and mitochondrion. In contrast to higher eukaryotes, parasitic protozoa (e.g. Leishmania, Trypanosoma, Plasmodium) often exhibit a single large mitochondrion with a membrane composition similar to that of higher eukaryote mitochondria. We synthesized several fluorescentlylabelled MPP's (Table 4). Two fluorophores were studied, i.e. thiazole orange (TO) [START_REF] Carreon | Cyanine dye conjugates as probes for live cell imaging[END_REF] and fluorescein (FITC), which were conjugated at the N-terminus of MPPs. In the case of fluorescein, an aminohexanoyl spacer had to be inserted. Promastigotes of L. infantum were incubated with the compounds at a concentration of 60 µM for 1h and, after washings, observed by confocal microscopy. The fluorescein-labelled 63 analogue was found to not significantly penetrate into the parasites, while the two fluorescent versions of the longest peptide (i.e. 64 and 65 = doubling of the 63 sequence) were toxic, yielding rounded and immotile cells. However, all other fluorescent MPPs labelled cells in a more or less homogeneous fashion, indicating cytoplasmic as well as possible mitochondrial localization (see Figure S3). To get more insight into their localization, cell labelling by 67, composed of three units of the dipeptide Cha-arg, was compared to that obtained with mitotracker (Figure 3). Compound 67 was shown to enter the parasite and merged images indicate that it could at least partially localize to the mitochondrion. Having checked that MPPs could indeed penetrate the parasite, we prepared several conjugates containing the (Cha-arg)n MPP (n = 2, 3) (Table 5).

We first assessed their ability to activate isolated LmHslV (Table 5). The LmC6-U2

conjugates with a (Cha-arg)2 (69) or a (Cha-arg)3 (72) MPP were found to be about 67% and 48% as potent as 1 (LmC12-U2), and three and two-fold more potent than LmC6-U2 (48, Table 1), respectively. Thus, N-terminal lengthening increased potency despite the presence of several D-amino acid residues in the MPP sequence. However, 72 with the longest MPP was slightly less potent than 69. As expected, the corresponding [Ala 12 ] analogues 70 and 73

were inactive. The [Chg 11 ]-LmC6-U2 conjugates behaved differently. Whereas 71 with a (Cha-arg)2 MPP was only slightly less potent than the corresponding hexapeptide 50 (Table 3), adding a Cha-arg unit (74) led to a significant drop in activity with 74 being about 50% as active as [Chg 11 ]-LmC6-U2 [START_REF]H-arg-O2Oc-Leu-Ala-Lys-Tyr-Chg-Leu-OH[END_REF], suggesting that the MPP segment significantly counteracted the favourable effect of Chg 11 . In any case, 74 was found about 1.5-, 7-and 3-fold more potent than LmC12-U2 (1), LmC6-U2 [START_REF]H-arg-O2Oc-Leu-Ala-Lys-Tyr-Leu-Leu-OH LmC6-U2 17 ± 4[END_REF], and 72 respectively. Finally, as already observed for 24 and 27 (Table 1), 75, the [Ala 12 ] analogue of 74, showed significant activating potency, being 30% as active as 74, and as active as 72. Therefore, in contrast to 70 and 73, 75 could not be used as a negative control in the following experiments. Finally, the same compounds were tested in cultures of the promastigote form of L. infantum (Figure 4B). As expected, no non-conjugated peptides (i.e. 1, 24, 48, not shown in Figure 4B for the sake of clarity) and no vector alone (i.e. 76, 77) were found toxic for the parasite after 48h incubation at a 60 µM concentration. The LmC6-U2 conjugate with two Cha-arg units (69) was also inactive, as of course its [Ala 12 ] analogue 70 (not shown in Figure 4B). The more potent [Chg 11 ] analogue 71 showed some activity with about 35% growth inhibition at 60 µM. More significant effects were found for conjugates containing three Cha-arg units.

The corresponding LmC6-U2 conjugate 72 inhibited parasite growth by 65% at 30 µM and maximally (about 80% inhibition) at 60 µM. As expected, its [Ala 12 ] analogue 73 showed no significant toxicity. Finally, the more potent [Chg 11 ] analogue 74 showed higher activity with 65% inhibition at 15 µM and maximally at 30 µM. Overall, these results suggested that a C-terminal HslU peptide conjugated to an MPP may indeed inhibit parasite growth, and that a minimum of three Cha-arg units is necessary for efficient vectorization. A fluorescently labelled analogue of 74, 78, was shown to penetrate into L. infantum promastigotes with similar localization as the fluorescent vector 67 (Figure 3). Compared to 67, the parasite appeared damaged after 3h incubation with 78, suggesting that some toxicity occurred in agreement with the observed toxicity of the non-fluorescent analogue 74 (Figure 4B).

Conclusion

We performed the first steps toward the development of peptidomimetics potentially able to inhibit the interaction between the LmHslU and LmHslV complexes. By substituting essential residues of the reference peptide LmC12-U2 1 (i.e. 5 out of the 6 C-terminal residues) with structurally close amino acids, we identified up to three-fold more potent analogues. In particular, substitution at positions 10 and 11 afforded the largest improvements. More particularly, a Chg at position 11 (Cpd 24) resulted in the most dramatic effect. This particular behaviour was also observed in the LmC6-U2 and cyclic LmC9-U2 series. Indeed the [Chg 11 ]-LmC6-U2 analogue 50 was found only slightly less potent than the corresponding dodecapeptide 24, in strong contrast with LmC6-U2 48 whose activity was about six times lower than that of LmC12-U2 1. The reason why the Chg residue is so favourable is not clear but its higher rigidity could lead to more favourable binding kinetics. Finally, we found that the conjugate 74, derived from compound 50 and incorporating a known mitochondrial penetrating peptide (i.e. (Cha-arg)3) inhibited the growth of the parasite promastigote form with a maximal effect at 30 µM, while it did not show any toxicity toward human THP-1 cells at the same concentration. 78, a fluorescent analogue of 74, confirmed the ability of the conjugate to penetrate the parasite. Future efforts will focus on the design of C-ter HslU peptidomimetics with enhanced metabolic stability.

Experimental

Reagents and resins for peptide synthesis (Fmoc-protected amino acids, HATU, DIEA, TFA, piperidine, 2-chlorotrityl chloride (1.6 mmol/g) and Fmoc-Rink amide (0.56 mmol/g) polystyrene resins, solvents and other reagents) were purchased from Iris-Biotech, Merck/Novabiochem, Bachem, Riedel-de Haën, Carlo-Erba, Merck/Sigma-Aldrich or Alfa-Aesar, and used without further purification. Solvents used for RP-HPLC and LC-MS were of HPLC grade.

After parasite counting by nephelometry, an adequate volume of parasite culture (10 7 parasites per mL for one condition) was placed in a sterile 50 mL tube. After centrifugation (3 min at 3600 rpm), the supernatant was gently removed and re-suspended in complete Schneider's medium. One mL suspension per condition was introduced in an Eppendorf tube and peptide solution in DMSO was added to a 60 µM final concentration. After homogenisation, the tubes were incubated at 26 °C and in the dark for 1h. The tubes were then centrifuged (3 min at 3600 rpm) and the supernatant gently removed. For washing, 1 mL of 1X PBS was added and the resulting suspension again centrifuged (3 min at 3600 rpm) and the supernatant removed. For cell fixation, 1 mL of 1X PBS containing 4% paraformaldehyde was added and the tubes were incubated at 26 °C for 1h30. After centrifugation (3 min at 3600 rpm) and gentle removal of the supernatant, 1 mL of 1X PBS was added (washing) and a portion of the suspension was placed on a microscope slide, which was examined with a Zeiss LSM 510-META confocal laser scanning microscope. To observe the fluorescence of thiazole orange-and fluorescein-labelled peptides, lexc/em were 512/533 nm and 488/525 nm, respectively. To observe DAPI and Mitotracker, lexc/em were 405/460 nm and 488/525, respectively.

Toxicity assays toward THP-1 cells.

After cell counting by nephelometry, a defined volume of cell suspension was centrifugated

(5 min at 10,000 rpm), the supernatant removed and the pellet was diluted to a concentration of 5.10 5 cells/mL with THP-1 medium containing 20 ng/mL of PMA (phorbol 12-myristate-13-acetate) to activate THP-1. After homogenization, the suspension was distributed in a 96wells plate (100 µL per well) and the cultures incubated at 37 °C under 5% CO2 atmosphere for 24h. A serial dilution (3.75, 7.50, 15, 30 and 60 µM) of compounds was applied on the cultures, which were then incubated for 48h in an incubator at 37 °C in a humidified atmosphere containing 5% CO2. Cell viability was finally assessed by Alamar Blue assay. 10 µL of Alamar Blue solution was added to each well and reading was performed using a plate reader after 3h (lexc/em = 530/590 nm). Each experiment was repeated three times.

Toxicity assays toward L. infantum (promastigote form).

After cell counting by nephelometry, a defined volume of cell suspension to have 2.10 5 parasites per well was centrifugated (5 min at 10,000 rpm), the supernatant removed, and the pellet was diluted to a concentration of 2.10 6 cells/mL with parasite medium. After homogenization, the suspension was distributed in a 96-wells plate (100 µL per well). A serial dilution (3.75, 7.50, 15, 30 and 60 µM) of compounds was applied on the cultures, and plates were sealed with parafilm and incubated for 48h at 26 °C. Cell viability was finally assessed by a bioluminescence assay. 20 µL of a 30 mg/mL solution of luciferin was added to each well and the bioluminescence was rapidly measured using a plate reader Synergy 2 (Bio Tek).

Each experiment was repeated three times.

Table of Contents graphical abstract.

Highlights

-The HslVU protease of Trypanosomatids is a potential drug target.

-We developed LmHslU C-terminal-derived peptides binding to LmHslV and potentially inhibiting HslVU assembly.

-A SAR study of the C-terminal LmHslU dodecapeptide identified a potent hexapeptide.

-Conjugated to a penetrating peptide, it was found to be toxic to parasites in culture.

-The fluorescently-labelled conjugate entered the promastigote form of L. infantum.

Figure 1 .

 1 Figure 1. Sequence of the reference activating peptide, LmC12-U2. Essential residues are in bold. The Nterminal segment arg-O2Oc provides better aqueous solubility.

Figure 2 .

 2 Figure 2. Structure of the non-proteinogenic amino acid residues used in this study. The positions (7, 8, 9, 10, 11 or 12) where they have been individually introduced are indicated.

18 a

 18 -O2Oc-Leu 1 -Gln-Lys-Asn-Val-Asn-Leu 7 -Ala-Lys 9 -Tyr 10 -Leu 11 -Leu 12 -O2OC-Leu-Gln-Lys-Asn-Val-Asn-Leu-Ala--O2OC-Leu-Gln-Lys-Asn-Val-Asn-Leu-Ala--O2OC-Leu-Gln-Lys-Asn-Val-Asn-Leu-Ala-Lys-1Nal-Cha-Leuarg-O2OC-Leu-Gln-Lys-Asn-Val-Asn-Cha-Alaarg-O2OC-Leu-Gln-Lys-Asn-Val-Asn-Cha-Ala-Lys-1Nal-Cha-Leu-OH [Cha 7 , 1Nal 10 , Cha 11 ]-LmC12-U2 242 ± Percentage of initial velocity relative to compound 1 (100%). LmHslV (2 µg, 75.2 nM) was incubated at 37 °C with 100 µM JMV4482 (Z-EVNL-AMC) in the presence of 500 µM peptides. The fluorescence of the free AMC group, proportional to the activity of the sample, was measured at 2 min. intervals. b NA: no activity (<10%). Bip = Biphenylalanine (para-Phenyl-Phenylalanine); Cha = CyclohexylAlanine; Chg = CyclohexylGlycine; Cit = Citrulline; diFPhe = 3,4-DifluoroPhenylalanine; DOPA = 3,4-DihydroxyPhenylalanine; pFPhe = para-FluoroPhenylalanine; hCha = homoCyclohexylAlanine; bhLeu = b-homoLeucine; 1Nal, 2Nal = 1-, 2-Naphthylalanine; pNH2Phe = para-AminoPhenylalanine; pNO2Phe = para-NitroPhenylalanine; mNO2Tyr = meta-NitroTyrosine; O2Oc = [2-(2-aminoethoxy)ethoxy]acetyl; Oic = Octahydroindole-2-carboxyl; Orn = Ornithine; Tci = Thiocitrulline; Tic = Tetrahydroisoquinoline-3-carboxyl.

36 a

 36 -O2Oc-Leu 1 -Gln-Lys-Asn-Val-Asn-Leu 7 -Ala-Lys 9 -Tyr 10 -Leu 11 -Leu12 -OH LmC12-U2 100 Ca-Methyl-substituted analogues of LmC12-U2 36 H-arg-O2Oc-Leu-Gln-Lys-Asn-Val-Asn--O2Oc-Leu-Gln-Lys-Asn-Val-Asn-Leu-Ala-Lys-Tyr-Ca-MeLeu-Leu-OH [CaMeLeu 11 ]-LmC12-U2 NA 39 H-arg-O2Oc-Leu-Gln-Lys-Asn-Val-Asn-Leu-Ala-Lys-Tyr-Leu--O2OC-c[Asp-Val-Asn-Leu-Lys]-Arg-Tyr-Leu-Leu-OH c[Asp 4 , Lys 8 , Arg 9 ]-LmC9-U2 59 ± 2 44 H-arg-O2OC-c[Asp-Val-Asn-Leu-Lys]-Lys-Percentage of initial velocity relative to compound 1 (100%). LmHslV (2 µg, 75.2 nM) was incubated at 37 °C with 100 µM JMV4482 (Z-EVNL-AMC) in the presence of 500 µM peptides. The fluorescence of the free AMC group, proportional to the activity of the sample, was measured at 2 min. intervals. b NA: no activity (<10%). Aib = a-aminoisobutyryl; CaMeLeu = Ca-Methyl-Leucine; Cha = CyclohexylAlanine; Chg = CyclohexylGlycine; 1Nal = 1-Naphthylalanine; O2Oc = [2-(2-aminoethoxy)ethoxy]acetyl.

  The next step was to combine the cyclic constraint with the most favourable amino acid substitutions described above. Several analogues of the cyclic C9-U2 peptide were prepared, including [Arg 9 ] (43), [1Nal 10 ] (44), [Cha 11 ] (45) and [Chg 11 ] (46) substituted compounds

  -arg-Phe-Lys-NH2 FITC-Ahx-Phe-arg-Phe-Lys-NH2 [41] 62 63 TO-Cha-arg-Cha-Lys-NH2 FITC-Ahx-Cha-arg-Cha-Lys-NH2 [42] 64 65 TO-Cha-arg-Cha-Lys-Cha-arg-Cha-Lys-NH2 FITC-Ahx-Cha-arg-Cha-Lys-Cha-arg-Cha-Lys--arg-Cha-arg-Cha-arg-NH2 FITC-Ahx-Cha-arg-Cha-arg-Cha-arg-NH2 [46] a Axh = 6-aminohexanoyl; Cha = cyclohexylalanine; FITC = fluorescein-isothiocyanate; TO = thiazole orange.

Figure 3 .

 3 Figure 3. Penetration of 67 (top) and of the conjugate 78 (bottom) in promastigotes of L. infantum. Parasites were re-suspended in complete Schneider's medium and incubated with compounds (60 µM) at 26 °C for 3h in the dark. Cells were then washed, fixed, stained for DAPI and Mitotracker as described in Experimental Procedures and observed under confocal microscopy. Selected images are shown.

9 70 26 72 6 73 20 75 5 76

 9266205 Cha-arg)2-Leu-Ala-Lys-Tyr-Leu-Leu-OH 67 ± Ac-(Cha-arg)2-Leu-Ala-Lys-Tyr-Leu-Ala-OH NA b 71 Ac-(Cha-arg)2-Leu-Ala-Lys-Tyr-Chg-Leu-OH 269 ± Ac-(Cha-arg)3-Leu-Ala-Lys-Tyr-Leu-Leu-OH 48 ± Ac-(Cha-arg)3-Leu-Ala-Lys-Tyr-Leu-Ala-OH NA 74 Ac-(Cha-arg)3-Leu-Ala-Lys-Tyr-Chg-Leu-OH 148 ± Ac-(Cha-arg)3-Leu-Ala-Lys-Tyr-Chg-Ala-OH 47 ± Ac-Cha-arg-Cha-arg-NH2 ND 77 Ac-Cha-arg-Cha-arg-Cha-arg-NH2 ND 78 TO-(Cha-arg)3-Leu-Ala-Lys-Tyr-Chg-Leu-OH ND c a Percentage of initial velocity relative to compound 1 (100%). LmHslV (2 µg, 75.2 nM) was incubated at 37 °C with 100 µM JMV4482 (Z-EVNL-AMC) in the presence of 500 µM peptides. The fluorescence of the free AMC group, proportional to the activity of the sample, was measured at 2 min. intervals. b NA = No activity (≤5%). c ND = Not determined. Next, the toxicity toward the human monocytic cell line THP-1 of all conjugates (excepted 75), the MPP's alone 76 and 77, and the peptides LmC12-U2 (1), LmC6-U2 (48) and [Chg 11 ]-LmC12-U2 (24), was assessed after 48h incubation. As presented in Figure 4A (not shown for 1, 24, 48 and 70), no compound showed toxicity at 60 µM concentration.

Figure 4 .

 4 Figure 4. A) Toxicity of compounds toward human THP-1 cells after 48h and at concentrations up to 60 µM. B) Toxicity of compounds toward the promastigote form of L. infantum after 48h and at concentrations up to 60 µM.

Table 2 . LmHslU2 C-ter peptide constrained analogues 36-47 and their LmHslV activation potency at 500 µM.

 2

Table 3 . LmHslU2 C-ter hexapeptide analogues 48-58 and their LmHslV activation potency at 500 µM

 3 .

	Cpd	Structure	Name	Relative
				potency
				(%) a

Acknowledgments.

We thank the Infectiopole Sud Foundation (IHU Méditerranée Infection, Marseille, France) for allocating post-doctoral fellowships to P.S. and K.S. N.M.K was supported by a doctoral contract from Montpellier University, a stipend from the CRBM and a fellowship from the FRM (Fondation pour la Recherche Médicale). We also thank Pierre Sanchez for mass spectrometry analyses.

Appendix A. Supplementary material

Synthesis, Analysis and purification, characteristics of synthetic compounds (Tables S1-S3), CD experiments (Fig. S1, Table S4), molecular modelling (protocol, model of [Chg 11 ]-C8-U2 binding to HslV (Fig. S2)), confocal microscopy study of labelled MPPs in L. infantum (Fig. S3),


Dayong Wang
email: wangdayong@cqupt.edu.cn

Xin Wang

Yu Sun
email: yusun@uca.edu

Weisheng Li

Xin Lu
email: xin.lu@dmu.ac.uk

Frederic Dufaux
email: frederic.dufaux@l2s.centralesupelec.fr

GAUSSIAN DISTRIBUTION-BASED MODE SELECTION FOR INTRA PREDICTION OF SPATIAL SHVC

Keywords: SHVC, ILR mode, Bayes decision rule, Gaussian distribution

Due to the diversity of terminal devices, Spatial Scalable High Efficiency Video Coding (SSHVC) is an efficient solution to meet this requirement. However, its coding process is very complex, which seriously prevents its wide applications. Therefore, it is very crucial to reduce coding complexity and improve coding speed. In this paper, we propose a Gaussian Distribution-based Mode Selection for Intra Prediction of SSHVC. We show that the rate distortion costs of Inter-layer Reference (ILR) mode and Intra mode are significantly different, and both follow a Gaussian distribution. Based on this discovery, we propose to use a Bayes decision rule to determine whether ILR is the best mode so as to skip Intra mode. Experimental results demonstrate that the proposed algorithm can significantly improve coding speed with negligible coding efficiency losses.

INTRODUCTION

Video applications are becoming increasingly popular in recent years. These applications often involve different terminal devices with various display resolutions. Therefore, video streaming must be compatible with different screen resolutions. Spatial Scalable High Efficiency Video Coding (SSHVC) is an efficient solution to meet the requirement [START_REF] Boyce Y. Yan | Overview of shvc: Scalable extensions of the high efficiency video coding standard[END_REF]. Video sequences with the same content but different spatial resolutions are encoded at a base layer (BL) and one or more enhancement layers (ELs) in SSHVC. SSHVC permits to decode the appropriate layers to adapt to a specific display resolution.

In SSHVC, video sequences at different layers have various resolutions, but the underlying video content presented are the same. In order to improve the coding efficiency, SSHVC allows the coding unit (CU) in EL to be predicted from the upsampled co-located CU in the BL. The mechanism is referred to as inter-layer prediction, and the coding mode involved is denoted as Inter-layer reference (ILR) mode. For the base layer, the input video is encoded by a HEVC encoder. Apart from the regular coding mode, additional ILR modes are required to be evaluated in the EL. Since the coding process of HEVC is already very complex, the encoding process of SSHVC is even more complex, which seriously limits its wide usage, especially for wireless and real-time applications. Consequently, there is a urgent need to develop fast SSHVC encoders to speed up the coding process.

A novel fast intra prediction algorithm is therefore proposed for SSHEVC in this paper. It is observed that the ratedistortion (RD) costs of the ILR mode and those of the Intra mode are significantly different, but both follow a Gaussian distribution. Upon these observations, a Bayes decision rule is proposed to early determine whether the ILR mode is optimal, thus skipping the evaluation of unnecessary Intra modes.

RELATED WORK

In order to accelerate the processing speed, commonly-used techniques include skipping of unlikely modes and early termination of evaluations. Some representative methods are reviewed and discussed as follows.

In general, the current CU and its spatial neighbours in the same EL and it co-locate CU in the BL are highly correlated. Therefore, the modes of relevant CUs are often used to predict the mode candidates for the current CU. To reduce the computational complexity, Tohidypour et al. [START_REF] Tohidypour | Content adaptive complexity reduction scheme for quality/fidelity scalable hevc[END_REF] use the relevant CUs to predict possible coding mode and skip unlikely modes for CUs in EL. The algorithm proposed in [START_REF] Tohidypour | Fast mode assignment for quality scalable extension of the high efficiency video coding (hevc) standard: A bayesian approach[END_REF] combines depth and the co-located CU's mode in BL to predict likely modes, and then jointly use Inter-layer and spatial correlations to eliminate unlikely modes. The algorithms in [START_REF] Tohidypour | Content adaptive complexity reduction scheme for quality/fidelity scalable hevc[END_REF] and [START_REF] Tohidypour | Fast mode assignment for quality scalable extension of the high efficiency video coding (hevc) standard: A bayesian approach[END_REF] are developed based on the Inter-layer and spatial correlations only. In fact, the texture complexity can also be used to assist the mode decision process. Lu et al. [START_REF] Yu | A fast intra coding algorithm for spatial scalability in shvc[END_REF] and [START_REF] Yu | Fast intra-and intercoding algorithms for the spatially scalable extension of h.265/hevc[END_REF] jointly use the temporal-spatial correlation and the texture complexity to predict the mode candidates and exclude unlikely modes.

In order to reduce encoding time, RD costs and residual coefficients are also widely used to early terminate coding process. Given the fact that the RD costs of neighbouring CUs are similar, Tohidypour et al. [START_REF] Tohidypour | An encoder complexity reduction scheme for quality/fidelity scalable hevc[END_REF] have consequently determined whether the current mode is optimal according to RD cost hence avoid the unnecessary evaluations. In our previous work [START_REF] Sun | Fast inter mode predictions for shvc[END_REF], [START_REF] Sun | Fast depth and inter mode prediction for quality scalable high efficiency video coding[END_REF], the difference of RD costs for both ILR mode and the merge mode was compared to early terminate the mode selection process. RD costs formed the basis for the above algorithms.

Generally, most of the residual coefficients for the best mode are small and the distribution often obey a certain pattern, e.g. Gaussian distribution [START_REF] Pao | Modeling dct coefficients for fast video encoding[END_REF] or Laplacian distribution [START_REF] Hu | Fast motion estimation based on confidence interval[END_REF]. Wang et al. [START_REF] Zhu | Efficient multi-strategy intra prediction for quality scalable high efficiency video coding[END_REF] make decisions on whether to skip the Intra mode or not by checking the distribution of the residual coefficients of ILR mode. Since the probabilities of modes are strongly relative to mode selection, Wang et al. [START_REF] Sun | Probability-based fast intra prediction algorithm for spatial shvc[END_REF] combine the probability of ILR mode with its residual coefficients to early skip Intra mode. The works in [START_REF] Sun | Fast depth and mode decision in intra prediction for quality shvc[END_REF] [START_REF] Kwong | Early merge mode decision based on motion estimation and hierarchical depth correlation for hevc[END_REF] adopt zero blocks to early terminate mode selection.

Although the aforementioned algorithms can reduce the encoding time to some extent, the underlying mechanisms of mode selection have not yet been investigated. This limits the reduction in computational complexity of the SSHVC encoder. In this paper, a further investigation on mode selection between ILR mode and Intra mode is presented. Consequently, a fast mode selection algorithm for SSHVC is developed.

THE FUNDAMENTALS OF MODE SELECTION BETWEEN ILR MODE AND INTRA MODE

In order to investigate the fundamentals of mode selection be- 1.

As shown in Table 1, the average RD Cost of ILR mode is greater than that of Intra mode for depths 0, 1, and 2. It is the opposite for depth 3, but the difference in average RD costs is insignificant. The way to generate the predicted CU in EL explains this observation. To be specific, ILR mode uses the collocated CU in the BL to predict the current CU in the EL, while Intra mode refers to the reference pixels in the same layer. For CUs containing complex textures at depth levels [0,2], Intra mode is often unable to provide an accurate prediction, as the predicted pixels and the reference pixels are a great distance apart due to the large CU sizes. In this case, ILR mode which uses the co-located pixels in BL as reference usually performs better than Intra mode. On the contrary, when the current CU contains simple textures, Intra mode often makes a better prediction than ILR mode, as neighbouring CUs tend to contain similar textures. For CUs at depth level 3, the predicted pixels are very close to the reference pixels, as the size of the CUs is small. Consequently. the predicted pixels are similar to the reference pixels, even if the current CU contains rich details. Intra mode outperforms ILR mode in such a case. When the texture is simple, both Intra mode and ILR mode perform well in prediction. There is a margin difference only between the RD costs incurred by Intra mode and ILR mode.

In addition, the RD cost distributions of both ILR mode and Intra mode are further investigated. The RD cost distribution of ILR mode for the 'Ducks' sequence at depth levels 2 and 3 are shown in Fig. 1. (a) and Fig. 1 (b), respectively. In Fig. 1, the x-axis is the RD cost, and the y-axis represents the histograms, i.e., the number of CUs in each bin.

It is observed from Fig. 1 that both the RD costs of ILR mode at depth levels 2 and 3 follow a Gaussian distribution. Kolmogorov-Smirnov (KS) test, as a non-parametric distribution estimation method, is robust and widely used in distribu- It can be concluded that the average RD costs for both ILR mode and Intra mode are distinct, but they both follow a Gaussian distribution for all depth levels.

THE PROPOSED MODE SELECTION-BASED FAST INTRA PREDICTION ALGORITHM

Compared with the other coding modes, ILR mode is more likely to be selected as the best mode [START_REF] Zhu | Efficient multi-strategy intra prediction for quality scalable high efficiency video coding[END_REF] [13], we therefore firstly evaluate this mode's RD cost to determine whether it is the best mode, thus skipping Intra mode as early as possible. Suppose m 0 and m 1 refer to ILR mode and Intra mode respectively, x is the RD cost of the current CU, the probability of ILR mode being selected as the best mode can be derived by [START_REF] Cho | Fast cu splitting and pruning for suboptimal cu partitioning in hevc intra coding[END_REF] p

(m 0 |x) = p (x|m 0 ) p (m 0 ) p (x) , (1)
where p(x|m 0 )is the conditional probability of x when ILR mode is chosen for the current CU, p(m 0 ) is the probability of ILR mode being chosen, p(x) is the probability of the RD cost being equal to x.

As there are two modes only, i.e. ILR mode and Intra mode, p(x) is calculated by

p (x) =p (x|m 0 ) p (m 0 ) +p (x|m 1 ) p (m 1 ) . ( 2
)
As the current CU is usually very similar to its relevant CUs, the latters can therefore be used to predict the current CU. As shown in Fig. 2, U 0 represents the current CU, U 1 , U 2 , U 3 and U 4 are its neighboring CUs, U 5 , U 6 , U 7 , U 8 and U 9 are the co-located CUs of U 0 , U 1 , U 2 , U 3 and U 4 in the previous frame. Let k be the number of relevant CUs using ILR mode, since there are nine relevant CUs, from U 0 to U 9 , p(m 0 ) and p(m 1 ) are k 9 and 1-k 9 , respectively. As mentioned above, the RD costs of both ILR and Intra modes follow a Gaussian distribution. For CUs at depth levels [0,2], the average RD costs of ILR mode is greater than that of Intra mode, the corresponding RD cost distributions are shown in Fig. 3(a). Conversely, for CUs at depth level 3, the average RD costs of ILR mode is less than that of Intra mode, the corresponding RD cost distributions are shown in Fig. 3(b). In Fig. 3, the x-axis represents the RD cost, the y-axis represents the probability density function. The blue curve is the probability density function of RD cost when using Intra mode, the yellow curve is the probability density function of RD cost when using ILR mode. T 0 and T 1 are the thresholds used in the mode decision process in Fig. 3(a) and Fig. 3(b), respectively. In Fig. 3(a), if x ≥ T 0 , ILR mode is selected as the best one. While in Fig. 3(b), if x ≤ T 1 , ILR mode is selected as the best one. Therefore, the early termination condition for ILR mode can be written as

U7 U8 U9 U6 U5

x ≥ T 0 x ≤ T 1 (3)
Consequently, if one of the conditions is satisfied, Intra mode can be directly skipped. The key here is to determine suitable T 0 and T 1 . Suppose f 0 (x|w 0 ) is the conditional probability density function of x when ILR mode is chosen, the corresponding probability of T 0 can be calculated by

p (T 0 |m 0 ) = ∞ T0 f 0 (x|w 0 )dx. (4)
Eq. ( 4) can be rewritten as

p (T 0 |m 0 ) =1- T0 -∞ f 0 (x|m 0 )dx. (5)
Suppose f 1 (x|m 1 ) is the conditional probability density function of x when Intra mode is chosen, we can calculate the corresponding probability of T 0 as shown below

p (T 0 |m 1 ) =1- T0 -∞ f 1 (x|m 1 )dx. ( 6
)
Suppose α is the probability of using ILR mode, according to Eq. ( 1), it can be derived as

α= p (m0) 1- T 0 -∞ f0 (x|m0)dx p (m0) 1- T 0 -∞ f0 (x|m0)dx + p (m1) 1- T 0 -∞ f1 (x|m1)dx . (7)
Obviously, if α ≥ 0.95, the probability of using ILR mode is very high, so ILR is very likely to be chosen as the best mode. Therefore, α is set to 0.95. The current CU often contains highly-similar video content with the relevant CUs. When checking ILR mode, the expectation µ 0 and variance σ 0 of the RD costs of the relevant CUs using ILR mode are used to estimate the corresponding values of the current CU. Similarly, when checking Intra mode for the current CU, the expectation µ 1 and variance σ 1 of the RD costs generated from the relevant CUs using Intra mode are used for estimations. By checking the cumulative standard normal distribution table, we can obtain upper bound of the integral of the cumulative standard normal distribution z, then T 0 can be calculated as

T 0 =µ 0 -z • σ 0 . (8)
In a similar way, we can obtain a threshold T 1 for depth level 3.

EXPERIMENTAL RESULTS

To test the performance of the proposed fast Intra prediction algorithm for SSHVC, we implement our algorithm on the reference software (SHM 11.0) with AI configuration and carry out testing on a server with Intel (R) 2.0 GHz CPU and 30 GB memory. Commonly-used performance measures are coding efficiency and coding speed. Coding efficiency in EL is measured by Bjontegaard Delta Bit Rate (BDBR) [START_REF] Bjontegaard | Calculation of average psnr difference between rd-curves[END_REF], which represents the bitrate difference at an equal PSNR compared with the reference software. Coding speed is denoted by TS, which is the percentage of encoding run-time savings only for EL.

In order to demonstrate the performance of the proposed algorithm, we compare the performance of our algorithm with FIICA [START_REF] Yu | Fast intra-and intercoding algorithms for the spatially scalable extension of h.265/hevc[END_REF] and PBFIP (%) [START_REF] Sun | Probability-based fast intra prediction algorithm for spatial shvc[END_REF]. According to common SHM test conditions (CSTC) [START_REF]Common shm test conditions and software reference configurations[END_REF], only one QP set is used for BL: (22, 26, 30, 34) and two QP sets for EL: (22, 26, 30, 34) and (24, 28, 32, 36). We denote QP sets of (22, 26, 30, 34) and (24, 28, 32, 36) for EL as case 1 and case 2. The corresponding performance comparisons are listed in Table 2 (case 1) and Table 3 (case 2), respectively.

In Table 2 (case 1), the average BDBRs of the proposed algorithm, FIICA and PBFIP are 0.26%, 0.38% and 0.58%, 3 (case 2), the average BDBRs of the proposed algorithm, FIICA and PBFIP are 0.24%, 0.40% and 0.43%, respectively, while the average TS of the proposed algorithm, FIICA and PBFIP are 58.91%, 43.25% and 47.81% correspondingly. In both case 1 and case 2, the proposed algorithm achieve the lowest BDBR increment and significantly better encoding time reduction than the other two algorithms.

CONCLUSION

In this paper, we first investigate the fundamentals of mode selection, and then propose to use a Bayes decision rule to speed up the coding process of Intra prediction algorithm for SSHVC. Experimental results show that the proposed algorithm can reduce the encoding time effectively with negligible coding efficiency losses.

Fig. 1 .

 1 Fig. 1. The RD cost distribution of ILR mode for sequence "Ducks".

Fig. 2 .

 2 Fig. 2. The current CU and its relevant CUs.

3 Fig. 3 .

 33 Fig. 3. RD cost distributions of ILR and Intra modes at different depth levels.

Table 1 .

 1 The RD Costs of ILR mode and Intra mode in All Depths The average values of RD Costs of both ILR mode and Intra mode at four coding depth levels are listed in Table

	Sequence	Depth 0 ILR Intra ILR Intra ILR Intra ILR Intra Depth 1 Depth 2 Depth 3
	Blue sky 23285 9296 5806 2243 1504 581 398 430
	Ducks 91351 24112 22537 11784 5662 4848 1438 1512
	Pedestrian 26302 10307 6534 5057 1646 1497 416 483
	Tractor 35746 15127 8992 3386 2309 1396 606 615
	Town 76524 57183 19207 14526 4850 3778 1138 1221
	Station2 29918 10248 7498 3334 1898 1366 487 543
	Average 47187 21045 11762 6722 2978 2244 747 801
	produce similar performance. In SHVC, Coding Tree Unit
	(CTU) can be encoded at four coding depth levels 0, 1, 2 and
	3, corresponding to CU sizes of 64×64, 32×32, 16×16, and
	8×8.

tween ILR mode and Intra mode, extensive experiments have been carried out. Video sequences including Blue sky, Ducks, Pedestrian, Tractor, Town and Station2 are processed in our experiments. These sequences are representative since they cover different motion and texture from simple to complex. According to common SHM test conditions (CSTC)

[START_REF]Common shm test conditions and software reference configurations[END_REF]

, we use QP with 22 in BL and 24 in EL in conducting experiments. Extensive experimental results show other QPs

Table 2 .

 2 Overall performance comparisons with case 1

	Sequence	Proposed (%)FIICA(%) [5]PBFIP(%) [12] BDBR TS BDBR TS BDBR TS
	Traffic	0.20 60.5 0.41 36.37 0.08 44.61
	PeopleOnStreet -0.20 58.58 0.10 39.43 0.03 40.30
	Kimono	-0.20 61.80 -0.13 60.27 -0.22 53.30
	ParkScene -0.10 59.97 0.22 36.49 -0.16 32.32
	Cactus	0.50 55.57 0.89 37.92 0.91 39.41
	BasketballDrive 0.80 48.85 0.71 41.48 2.09 45.61
	BQTerrace	0.80 45.66 0.50 43.56 1.30 33.30
	Average	0.26 55.85 0.38 42.22 0.58 41.27

Table 3 .

 3 Overall performance comparisons with case 2 Sequence Proposed (%)FIICA(%) [5]PBFIP(%) [12] BDBR TS BDBR TS BDBR TS Traffic -0.10 64.19 -0.30 37.89 0.01 45.82 PeopleOnStreet -0.30 63.70 -0.23 40.15 -0.23 42.65 Kimono -0.30 63.69 0.19 60.18 -0.21 59.21 ParkScene -0.10 62.10 0.11 38.13 0.11 41.81 Cactus 0.60 57.99 0.70 39.29 0.52 48.09 BasketballDrive 1.00 51.04 1.72 42.74 1.72 50.45 BQTerrace 0.90 49.68 0.61 44.37 1.09 46.62 Average 0.24 58.91 0.40 43.25 0.43 47.81 respectively, while the average TS of the proposed algorithm, FIICA and PBFIP are 55.85%, 42.22% and 41.27% correspondingly. In Table

This work was supported in part by the Natural Science Foundation of Chongqing under Grant cstc2020jcyj-msxmX0766; in part by the Science and Technology Research Program of Chongqing Municipal Education Commission under Grant KJZD-K202100604; in part by the Jiangxi Provincial Natural Science Foundation under Grant 20202BABL202006.