Fatiha El

Marjorie Grzeskowiak
email: marjorie.grzeskowiak@univ-mlv.fr

David Delcroix
email: david.delcroix@univ-mlv.fr

Thierry Alves
email: talves@univ-mlv.fr

Stéphane Protat
email: protat@univ-mlv.fr

Shermila Mostarshedi
email: shermila.mostarshedi@univ-mlv.fr

Member, IEEE Odile Picon
email: odile.picon@univ-mlv.fr

A Multilayered Coil Antenna for Ingestible Capsule: Near-Field Magnetic Induction Link

Keywords: Magnetic induction link budget, multilayered stacked coil antenna, on-body spiral coil antenna, wireless ingestible capsule

A compact multilayered stacked ingestible coil antenna is investigated for medical systems. The inductive link, comprising a 5-layer transmitter coil antenna and a 3-turn receiver spiral coil, is modeled through a tissue-simulating liquid modeling the human body. The diameter and the thickness of the transmitter coil are respectively equal to 1 cm and 5 mm, while the dimensions of the receiver coil are equal to 7 cm . The variations of the position and the orientation of the capsule antenna are taken into account to evaluate the coupling response between the two magnetically coupled coils. We found that the inductive link presents an attractive option for improving the lifetime of ingestible capsules.

I. INTRODUCTION

W IRELESS ingestible capsule systems have garnered significant interest in medical applications and have been widely used in diagnoses and medical treatments of gastrointestinal (GI) tract diseases such as stomach and colon cancers [START_REF] Picon | Wireless robotic capsule endoscopy: State-of-the-art and challenges[END_REF]. The introduction of the noninvasive ingestible capsule in telemedicine is aimed at offering thorough coverage in terms of time and area [START_REF] Wang | In situ characterization of two wireless transmission schemes for ingestible capsules[END_REF] and thus at reducing pain and discomfort of the patient [START_REF] Guozheng | Design of battery-less and realtime telemetry system for gastrointestinal applications[END_REF], [START_REF] Lee | Fat arm spiral antenna for wideband capsule endoscope systems[END_REF]. The ingestible electronic capsule, which integrates sensors, batteries, transmission circuit, antennas, and so on, is used for monitoring physiological parameters of the GI tract including temperature, pressure, pH, and oxygen concentration [START_REF] Wang | In situ characterization of two wireless transmission schemes for ingestible capsules[END_REF], [START_REF] Aydin | Design and implementation of a spread spectrum based communication system for an ingestible capsule[END_REF], [START_REF] Cao | An implantable, batteryless, and wireless capsule with integrated impedance and pH sensors for gastroesophageal reflux monitoring[END_REF]. It can also provide the drug delivery to patients [START_REF] Rajagopalan | Novel ingestible capsule antenna designs for medical monitoring and diagnostics[END_REF] or the internal image transmission of the GI tract through video capsule [START_REF] Lee | Fat arm spiral antenna for wideband capsule endoscope systems[END_REF], [START_REF] Rajagopalan | Link budget analysis and characterization for ingestible capsule antenna[END_REF]. The capsule must be small enough to be swallowed by patients. From the antenna designer's perspective, it is crucial to consider some factors that affect the in-body antenna performances such as miniaturization and electrical properties of biological tissues as the link path is no longer air but human body.

The magnetic field power falls off as the inverse sixth power of the distance [START_REF] Agbinya | Size and characteristics of the "cone of silence" in near-field magnetic induction communications[END_REF], while the electromagnetic (EM) wave power drops off only with the inverse square of the distance. However, the magnetic field depends on the relative permeability of the media, which is equal to one for biological tissues. Therefore, the magnetic field is not influenced by such media, unlike the electric field, which depends on the permittivity of the dissipative media. The radiation efficiency of a meandered capsule antenna is found to be equal to 0.1% in [START_REF] Rajagopalan | Link budget analysis and characterization for ingestible capsule antenna[END_REF] and hardly reaches 0.9% for a spiral multilayered antennas in a 3-layer cylindrical body model [START_REF] Merli | Design, realization and measurements of a miniature antenna for implantable wireless communication systems[END_REF]; this is due to the high permittivity of biological tissues, which leads to steep attenuation of the EM wave especially at high frequencies.

Magnetic induction is a promising alternative for capsule antennas operating inside the human body composed of various dissipative tissue layers.

Under a strict size limitation of the in-body systems, the magnetic induction has been used to wirelessly transmit power to ingestible [START_REF] Guozheng | Design of battery-less and realtime telemetry system for gastrointestinal applications[END_REF], [START_REF] Kobayashi | A batteryless wireless communication circuit for measurement of gastric acid[END_REF] or implanted [START_REF] Tikka | Secure wireless powering and interrogation of an implantable microvalve[END_REF] antennas in order to avoid the use of batteries that limits the lifetime of such systems. The implanted antenna does not move inside the human body contrarily to the ingestible capsule, which slides down into the GI tract. Hence, in wireless link modeling, it is necessary to take into account the position and orientation variations of the ingestible capsule to guarantee maximum power transfer between the two magnetically coupled coils [START_REF] Hatmi | Link budget of magnetic antennas for ingestible capsule at 40 MHz[END_REF].

In this letter, an inductive link is established between a multilayered stacked coil capsule antenna and a receiving on-body spiral coil. The capsule antenna generates an alternating magnetic field at 40.68 MHz in the ISM band and is mutually coupled to the receiving on-body antenna located in its near field. Moreover, the orientation and the position of the moving capsule coil antenna are taken into account in the inductive link modeling.

II. COIL ANTENNA DESIGN AND MEASUREMENT SETUP

Several antenna parameters as well as the distance between the two coils representing the coupling elements of the wireless link govern the efficiency of the inductive link. The coupling response [START_REF] Picon | Wireless robotic capsule endoscopy: State-of-the-art and challenges[END_REF], given by the ratio of the received power to the transmitted power [START_REF] Agbinya | Near field magnetic induction communication link budget: Agbinya-Masihpour model[END_REF], depends on the quality factors and and the efficiencies and of the transmitter and the receiver coils and the coupling coefficient between the two coils. The latter coefficient is a function of the distance between the transmitter and the receiver and their respective coil radii and as presented in (2)

(

(2) where , , , and can be given as

(3) (4)
and are the resistances of the transmitter and the receiver coils, and are the source and the load resistances, and are the inductances of the transmitter and the receiver coils that along with the mutual inductance give . The dimensions of the transmitter antenna, operating inside the GI tract, are limited by the size of the capsule in which it will be enclosed. Therefore, the in-body antenna diameter must be smaller than 10.1 mm, which corresponds to the capsule inner diameter [START_REF] Lee | Fat arm spiral antenna for wideband capsule endoscope systems[END_REF]. To increase the magnetic field strength while reducing the dimensions of the in-body transmitter, the capsule antenna is chosen to be a multilayered coil. The advantages of the multilayered printed coil compared to the classic wired coil are essentially the robustness and the solidity.

As it is depicted by Fig. 1(a)-(c), the structure of the capsule antenna is made of five turns printed on five stacked dielectric substrate separated by four superstrates. The thickness and the diameter of the substrate are 0.4 mm and 1 cm, respectively, while the thickness of the superstrate is 0.76 mm, and its diameter is equal to that of the substrate. The superstrate layers are used on the one hand to ensure a stable distance between the coil turns and on the other hand to make the antenna more solid. The built loop in every layer has a diameter of 9.5 mm and a line width of 1 mm to increase the quality factor of the transmitter coil. If the manufacturing process of the antenna allows metallized holes, the superstrate layers can be removed, and the total antenna height can be reduced from 5 to 2 mm. Hence, the turn number can be increased to guarantee maximum power transfer. The diameter of via hole used to connect each layer to the other is equal to 0.7 mm.

The fifth layer is connected to the feed terminal by a connector that runs through all substrates of the antenna. In order to increase the received power through the inductive link, the on-body receiver coil needs to have large dimensions, and its input impedance must be matched with that of the transmitter coil. As it is shown by Fig. 1(d), the on-body antenna is a 3-turn spiral coil with outer size of 7 8 cm , a line width and spacing between lines of 1.5 mm, making it possible to increase the quality factor of the magnetic receiver coil. Both in-body and on-body coils are built, by mechanical etching, on the FR4 epoxy substrate with relative dielectric constant of 4.4, dielectric loss tangent of 0.02, and metallic thickness of 35 m. The substrate thickness of the receiver antenna is equal to 0.76 mm.

A matching circuit including one series capacitance followed by one parallel capacitance is used to match both coil input impedances close to 50 . The antennas are designed with the commercial simulator HFSS, and the structure is simulated in a homogeneous tissue-simulating liquid available in our laboratory, which represents the human body, using its measured characteristics. The tissue-simulating liquid is characterized using the Agilent Dielectric Probe Kit, and the measured permittivity and conductivity at 40.68 MHz are equal to 64 and 0.78 S/m, respectively. Fig. 2 (Link 1: tube along ) summarizes the measurement setup of the inductive link; the in-body coil is in front of the on-body antenna, every coil being parallel to the -plane, and the path is along the -direction perpendicular to the antenna planes. The capsule antenna is isolated from the body liquid by a Plexiglass tube, whose diameter and thickness are respectively equal to 1.4 cm and 0.5 mm. As the small intestine is located at almost 5 cm from the surface of the skin [START_REF] Rajagopalan | Link budget analysis and characterization for ingestible capsule antenna[END_REF], the transmitter antenna is placed 5 cm away from the receiver antenna in the -direction including 4.6 cm of body liquid and 0.4 cm of air. This air gap between the receiver coil and the back side of the plastic container is due to the thickness of the SMA connector. The dimensions of the plastic container are cm . The emitting and receiving coils are designed separately, and their respective matching networks are calculated.

When the inductive link is established, there is a coupling effect between the two coils: Both antenna input impedances are mismatched, and there is a frequency shift of a few hundred kilohertz. Therefore, the antenna matching networks have to be adjusted in order to get the resonant frequencies close to 40.68 MHz. As in the measurement setup, the capsule coil antenna is surrounded by air contained in the isolated tube; the input impedance and the operating frequency of the transmitting antenna immersed in the tissue-simulating liquid are slightly shifted compared to the free space. Therefore, when the transmission channel changes, the capacitances of the transmitter Fig. 3. Simulated and measured coupling response , according to the frequency, between the capsule coil and the receiver coil separated by a distance of 5 cm when the path is the tissue-simulating liquid. antenna do not vary; the series capacitance is equal to 180 pF, and the parallel capacitance is equal to 800 pF. However, the receiver antenna operates otherwise; the input impedance of the receiver coil changes dramatically when the transmission channel is no longer free space. Therefore, it is necessary to adjust the capacitances of the receiver coil in each media. Hence, the series capacitance of the receiver coil is equal to 8.2 pF in free space and to 7.6 pF in the tissue-simulating liquid, while the parallel capacitance is equal to 213 pF in free space and 56 pF in the tissue-simulating liquid. Moreover, we had to use ferrites to limit the influence of leakage current in the cable that connects the receiver coil to the network analyzer.

III. RESULTS (LINK 1)

The measured input resistance of the receiver coil increases when the transmission channel changes from the air to the tissue-simulating liquid ( and ). Thus, the quality factor decreases and becomes equal to 7.1 instead of 9.1 in the air. Moreover, the bandwidth of the receiver coil in the tissue-simulating liquid increases; the measured bandwidth is 0.2 MHz in the air, while 0.9 MHz in the tissue-simulating liquid. However, because the transmitter coil is inserted in the Plexiglass tube filled with air, the bandwidth of the capsule antenna hardly varies when the transmission channel changes and the measured bandwidth is around 0.47 MHz in both cases. Similarly, the quality factor of the capsule antenna does not change and remains around 0.61. From the plots of Fig. 3, it is observed that the simulated and the measured maximum transmission coefficient , which occur around 40.68 MHz in the tissue-simulating liquid, are equal to 18.1 and 20.7 dB, respectively. Thus, the difference between the simulated and the experimental results is 2.6 dB.

To evaluate the influence of the dissipative media on the received power, we measured the coupling response in the air; it is found to be equal to 16.8 dB at 40.68 MHz. Therefore, because the in-body link uses the magnetic coupling between the coils, the difference between the maximum coupling response measured in the air and in the tissue-simulating liquid around 40.68 MHz is only equal to 3.9 dB. The data corresponding to the simulated and the measured antenna parameters, when the transmission channel is the tissue-simulating liquid, are listed in I. The quality factors and the efficiencies of the coils are deduced when the resistances and are set to 50 . We also analyze the analytical link budget through the tissue-simulating model at 40.68 MHz based on the Agbinya-Masihpour model proposed in [START_REF] Agbinya | Near field magnetic induction communication link budget: Agbinya-Masihpour model[END_REF]. The antenna parameters are deducted from HFSS simulation (Table I) when and is set to 1 W. The coupling response in the tissue-simulating model obtained by analytical formula (1) at 40.68 MHz is equal to 14.2 dB, while it is found to be equal to 18.1 dB in simulation. Furthermore, before choosing the 5-layer structure, several simulations according to the number of layers of the transmitter coil were made in the tissue-simulating model to evaluate the effect of this parameter on the link budget. The data listed in Table II show the coupling response according to the number of layers and the corresponding quality factor of the transmitter coil. Thus, when the number of layers increases from 1 to 8, the response can be improved from 22.4 to 14.5 dB. It is important to note that for this parametric study, the dimensions of the 3-turn receiver antenna are 8 5 cm , its quality factor is 44, and the distance between the two coils is set to 5.2 cm. Later in practice, we have slightly increased the width of the spiral coil (8 7 cm ) to improve the magnetic flux.

IV. CAPSULE ANTENNA POSITION AND ORIENTATION VARIATION IN THE TISSUE-SIMULATING LIQUID (LINK 2)

In Section III, the capsule coil was at 5 cm from the receiver antenna. In this paragraph, at first, we assume that the distance between the two coils in the -direction varies. Fig. 4 shows the parameter between the ingested and the on-body coils when the transmission channel is the tissue-simulating liquid and the distance increases from 1 to 8 cm. The measured coupling response varies between 9 and 27.5 dB, and there is a marginal drop that varies from 1 to 2.6 dB between the simulated and the experimental results.

As the swallowable capsule slides down into the GI tract, an effective inductive link can only be established if the receiver on-body coil detects the transmitted signal regardless of the transmitter position and orientation. The purpose of the second study in this paragraph is to verify that the received signal remains at an acceptable level when the capsule antenna is located randomly. In this way, we varied the position of the ingestible antenna along the -axis and then, for every -position, we changed the angle of the transmitter coil from 0 to 80 in the -plane while keeping the -distance constant and equal to 5 cm as it is shown by Fig. 2 (Link 2).

It is not possible to reach the angle of 90 because the in-body antenna is not enclosed in the capsule and the tissue-simulating liquid could flow into the isolated tube in this orientation. The plastic container dimensions in this case are equal to cm to allow a maximum variation of the angle by reducing the height of the container. The experimental results presenting the parameter according to the angle, where the -position is equal to 0, 1, 2, and 3 cm, are depicted by Fig. 5. It is clear from these plots that the maximum received signal level for each -position occurs when . This configuration is the most optimal because the H-field lines perpendicular to the on-body antenna surface are maximal. We can also conclude that for every capsule antenna position, the parameter decreases when the angle increases. Hence, the response always varies between 20.5 and 35.5 dB; if a threshold of 35 dB is fixed, we can say that the received signal remains efficient regardless of the orientation and the position of the ingested coil.

V. CONCLUSION

In wireless ingestible capsule systems, magnetic induction is a promising alternative technique because the path loss depends on the permeability of biological tissues, which is equal to that of the air. Although the magnetic field power falls off as the inverse sixth power of the distance, magnetic induction technique has been adopted in this letter to design a short-range wireless link through a tissue-simulating liquid since the dielectric properties of biological tissues do not perturb the magnetic field. Through HFSS simulation that is validated by measurements, the proposed multilayered stacked coil antenna is shown to be suitable as a compact robust ingestible transmitter. An analytical model is also applied to evaluate the factors affecting the link budget. The measured coupling response of the system, which depends on the position and the orientation of the capsule antenna, remains effective and varies between 20.5 dB (0.89%) and 35.5 dB (0.03%) in the tissue-simulating liquid.

As the capsule antenna should transmit physiological parameter data, it is necessary to design a receiver coil antenna array to find the accurate position of this data source inside the GI tract. It will be the future work of this study.

Fig. 1 .

 1 Fig. 1. Transmitter and receiver coils: (a) multilayered capsule antenna structure; (b) five layers of the capsule antenna arranged from left to right; (c) fabricated 5-layer capsule antenna; (d) fabricated 3-turn receiver spiral coil.

Fig. 2 .

 2 Fig. 2. Measurement setup: (a) side view; (b) top view.

Fig. 4 .

 4 Fig. 4. Simulated and measured coupling response around 40.68 MHz in the tissue-simulating liquid between the two coils according to the -distance.

Fig. 5 .

 5 Fig. 5. Measured parameter around 40.68 MHz in the tissue-simulating liquid according to the angle at different -positions when cm.

TABLE I MEASURED

 I IN-BODY AND ON-BODY ANTENNAS CHARACTERISTICS IN THE PRESENCE OF THE TISSUE-SIMULATING LIQUID AT 40.68 MHz TABLE II COUPLING RESPONSE ACCORDING TO THE NUMBER OF LAYERS OF THE TRANSMITTER COIL IN THE TISSUE-SIMULATING MODEL AROUND 40.68 MHz

	Table


Nassima Oumata

Phu Hai Nguyen

Vincent Beringue

Flavie Soubigou

Yanhong Pang

Nathalie Desban

Catherine Massacrier

Yannis Morel

Carine Paturel

Marie-Astrid Contesse

Serge Bouaziz

Suparna Sanyal

Hervé Galons

Marc Blondel
email: marc.blondel@univ-brest.fr

Cécile Voisset
email: cecile.voisset@univ-brest.fr

The Toll-Like Receptor Agonist Imiquimod Is Active against Prions

Using a yeast-based assay, a previously unsuspected antiprion activity was found for imiquimod (IQ), a potent Tolllike receptor 7 (TLR7) agonist already used for clinical applications. The antiprion activity of IQ was first detected against yeast prions [PSI + ] and [URE3], and then against mammalian prion both ex vivo in a cell-based assay and in vivo in a transgenic mouse model for prion diseases. In order to facilitate structure-activity relationship studies, we conducted a new synthetic pathway which provides a more efficient means of producing new IQ chemical derivatives, the activity of which was tested against both yeast and mammalian prions. The comparable antiprion activity of IQ and its chemical derivatives in the above life forms further emphasizes the conservation of prion controlling mechanisms throughout evolution. Interestingly, this study also demonstrated that the antiprion activity of IQ and IQderived compounds is independent from their ability to stimulate TLRs. Furthermore, we found that IQ and its active chemical derivatives inhibit the protein folding activity of the ribosome (PFAR) in vitro.

Introduction

Prions are infectious agents that have been affecting human and animal health for centuries. Prion diseases, also named transmissible spongiform encephalopathies (TSEs), are invariably fatal and no treatment is currently available. Today, the ability of the new variant of Creutzfeldt-Jakob disease to be transmitted by blood transfusion is well documented [START_REF] Lefrère | From mad cows to sensible blood transfusion: the risk of prion transmission by labile blood components in the United Kingdom and in France[END_REF]. In face of potential new TSE outbreaks due to this or other secondary infection routes [START_REF] Langevin | Marked influence of the route of infection on prion strain apparent phenotype in a scrapie transgenic mouse model[END_REF], our current therapeutics are limited. More efficient therapies are urgently needed.

As a result of the life span increase, age-related neurodegenerative diseases impose a considerable burden on our societies. Many such diseases are proteinopathies, defined as conditions in which accumulation of a protein in a misfolded form leads to neuronal degeneration. The misfolded proteins present in these neurodegenerative diseases as well as in related systemic (non-neuronal) diseases, assemble into highly ordered supramolecular assemblies called amyloids. Amyloids are partially resistant to proteolytic degradation and can template the further recruitment of protein or peptide monomers into the polymers. These templating abilities are reminiscent of those of prion diseases and suggest that propagation of neurodegenerative disorders may occur in a "prion-like" manner through mechanisms similar to those that underlie prion pathogenesis [START_REF] Brundin | Prion-like transmission of protein aggregates in neurodegenerative diseases[END_REF][START_REF] Frost | The expanding realm of prion phenomena in neurodegenerative disease[END_REF][START_REF] Frost | Prion-like mechanisms in neurodegenerative diseases[END_REF][START_REF] Goedert | The propagation of prionlike protein inclusions in neurodegenerative diseases[END_REF][START_REF] Miller | Neurodegeneration. Could they all be prion diseases?[END_REF][START_REF] Soto | Amyloids, prions and the inherent infectious nature of misfolded protein aggregates[END_REF]. A better understanding of the mechanisms of prion propagation is therefore crucial for the development of therapeutic strategies to prevent the propagation of misfolded proteins in the brain and in other organs.

Our previous studies have detected compounds active against yeast and mammalian prions [START_REF] Bach | Isolation of drugs active against mammalian prions using a yeast-based screening assay[END_REF][START_REF] Bach | A yeast-based assay to isolate drugs active against mammalian prions[END_REF][START_REF] Tribouillard-Tanvier | Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs[END_REF][START_REF] Voisset | Procedure for identification and characterization of drugs efficient against mammalian prion: from a yeast-based antiprion drug screening assay to in vivo mouse models[END_REF]. Briefly, compounds from chemical libraries have been tested for their ability to cure primarily [PSI + ] (primary screening) and then [URE3] (secondary screening) yeast prions. Compounds displaying an antiprion activity against both [PSI + ] and [URE3] yeast prions have been further tested for their ability to clear pathogenic mammalian prion protein PrP Sc in a cell-based assay using chronically prion infected MovS6 cells [START_REF] Voisset | Procedure for identification and characterization of drugs efficient against mammalian prion: from a yeast-based antiprion drug screening assay to in vivo mouse models[END_REF][START_REF] Voisset | Tools for the study of ribosome-borne protein folding activity[END_REF]. Drugs that have a potent antiprion activity in both yeast-and ex vivo cell-based assays, and that display a limited or no toxicity ex vivo, have then been tested in vivo in a mouse model for prion-based diseases. These compounds have also been used to characterize their cellular targets [START_REF] Tribouillard-Tanvier | Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs[END_REF]. The initial steps of the screening strategy are based on two yeast prions that are related neither in sequence nor function and have no link with PrP. Thus, isolated compounds active against yeast and mammalian prions should most probably target cellular prionisation mechanisms and not the prion proteins themselves. We have indeed isolated compounds able to inhibit prion propagation in both yeast and mammalian systems [START_REF] Bach | Isolation of drugs active against mammalian prions using a yeast-based screening assay[END_REF][START_REF] Bach | A yeast-based assay to isolate drugs active against mammalian prions[END_REF][START_REF] Tribouillard-Tanvier | Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs[END_REF][START_REF] Voisset | Procedure for identification and characterization of drugs efficient against mammalian prion: from a yeast-based antiprion drug screening assay to in vivo mouse models[END_REF], indicating that at least part of prionisation mechanisms are preserved from yeast to mammals. Thus, the use of such a screening process as a complement to animal-based models is highly fruitful and has been successfully used for other pathologies [START_REF] Couplan | A yeast-based assay identifies drugs active against human mitochondrial disorders[END_REF]. This screening procedure has allowed the identification of two active drugs, 6-aminophenanthridine (6AP) and Guanabenz (GA), that have proven to be valuable tools to identify a novel cellular mechanism potentially involved in the prion replication cycle, i.e. the protein folding activity of the ribosome (PFAR, [START_REF] Tribouillard-Tanvier | Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs[END_REF][START_REF] Voisset | Tools for the study of ribosome-borne protein folding activity[END_REF][START_REF] Reis | Mode of action of the antiprion drugs 6AP and GA on ribosome assisted protein folding[END_REF][START_REF] Tribouillard-Tanvier | Antihypertensive drug guanabenz is active in vivo against both yeast and mammalian prions[END_REF]).

Here, by testing a library of drugs already on the market, we found Imiquimod (IQ), a potent Toll-like receptor agonist, to be active against both yeast and mammalian prions. We also set up a new and efficient synthesis pathway of IQ to ease its production and to initiate the synthesis of its new derivatives so far difficult to produce. The antiprion activity of prepared IQ chemical derivatives as well as other TLR7/8 agonists such as gardiquimod was evaluated against yeast and mammalian prions. The potential of IQ chemical derivatives to stimulate TLR7/8 was also assessed. These observations revealed that antiprion activity of the tested compounds do not overlap with their agonist activity on TLR7/8 receptors. Furthermore, we found that IQ and its chemical derivatives, acting as antiprions, were specific inhibitors of the protein folding activity of the ribosome, in addition to the already known TLR7 agonist activity of IQ.

Results and Discussion

Imiquimod is active against yeast and mammalian prions

In order to identify new antiprion compounds, we used the yeast-based screening method we previously described [START_REF] Bach | Isolation of drugs active against mammalian prions using a yeast-based screening assay[END_REF][START_REF] Bach | A yeast-based assay to isolate drugs active against mammalian prions[END_REF]. Briefly, [PSI + ] or [URE3] yeast cells were spread on Petri dishes containing solid agar-based rich medium. Compounds were loaded onto individual filters placed on top of agar surface and dishes were further incubated for three days at 25°C. [PSI + ] or [URE3] yeast strains form white colonies. Upon the addition of the compound to the filter, compound's antiprion activity was detected readily and rapidly when a halo of red [psi -] or [ure3-0] colonies appeared around the filter. The strength of this method lies on its ability to produce in one simple step, a full gradient of the compounds from active at sub-toxic concentrations to toxic at high concentrations. The BIOMOL's FDA Approved Drug Library was chosen because it allows drug repositioning. Twelve positive compounds active against [PSI + ] and [URE3] yeast prions were identified, among which some compounds have previously been described for their antiprion activity, e.g. guanabenz [START_REF] Tribouillard-Tanvier | Antihypertensive drug guanabenz is active in vivo against both yeast and mammalian prions[END_REF] and amiodarone [START_REF] Klingenstein | Tricyclic antidepressants, quinacrine and a novel, synthetic chimera thereof clear prions by destabilizing detergent-resistant membrane compartments[END_REF]. One of the original hits identified was Imiquimod (1-isobutyl-1H-imidazo[4,5-c] quinolin-4-amine, IQ, Figure 1A). IQ has potent antiviral and antitumor properties and is used in a large diversity of cutaneous diseases like warts on the skin of the genital and anal areas [START_REF] Schöfer | Evaluation of imiquimod for the therapy of external genital and anal warts in comparison with destructive therapies[END_REF], and is also used to treat actinic keratoses, a skin condition of the face and scalp, and superficial basal cell carcinoma a type of skin cancer [START_REF] Lacarrubba | Successful treatment and management of large superficial basal cell carcinomas with topical imiquimod 5% cream: a case series and review[END_REF][START_REF] Serra-Guillen | A randomized pilot comparative study of topical methyl aminolevulinate photodynamic therapy versus imiquimod 5% versus sequential application of both therapies in immunocompetent patients with actinic keratosis: clinical and histologic outcomes[END_REF]. IQ does not show a direct activity against the viruses that cause warts [START_REF] Torres | Immune-mediated changes in actinic keratosis following topical treatment with imiquimod 5% cream[END_REF]. It appears to act via activation of the local innate immune response, through Toll-like receptor 7 (TLR7) stimulation. However its precise molecular mechanism of action remains unclear [START_REF] Lee | Accessory molecules for Tolllike receptors and their function[END_REF]. IQ displayed a strong activity against [PSI + ] prion, and was also active against [URE3] yeast prion (Figure 1B). Then we verified whether IQ was readily curing [PSI + ] and [URE3] prions and did not interfere with the colorimetric assay [START_REF] Bach | A yeast-based assay to isolate drugs active against mammalian prions[END_REF]. For that purpose, cells from red halos, induced by IQ loading, were streaked onto drug-free YPD medium. They mainly formed red colonies, which emphasized IQ is readily able to eliminate [PSI + ] and [URE3] yeast prions, similar to the GuHCl positive control (Figure S1A).

IQ was then tested for its ability to clear PrP Sc in the mammalian MovS6 cell-based assay [START_REF] Archer | Cultured peripheral neuroglial cells are highly permissive to sheep prion infection[END_REF]. MovS6 cells are murine peripheral neuroglial cell line expressing the ovine PrP gene under the control of its endogenous promoter. These cells are chronically infected by the 127S sheep scrapie prion [START_REF] Archer | Cultured peripheral neuroglial cells are highly permissive to sheep prion infection[END_REF][START_REF] Vilotte | Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp[END_REF] and therefore allows rapid ex vivo testing of molecules against mammalian prions. After 6 days of treatment of prioninfected MovS6 cells with a 0 to 20 µM range of concentration of IQ, PrP Sc was detected on the basis of its proteinase K (PK) resistance. For this purpose, a fraction of cell lysates was treated by PK to discriminate PrP Sc proteins, i.e. partially resistant to PK treatment, from total proteins (PrP tot ). PK treated and untreated cell lysates were separated by SDS-PAGE and immunostained using specific anti-PrP antibodies. Upon IQ treatment, PrP Sc band intensity was greatly reduced, in a dose-dependent manner, with an IC 50 around 5 µM (Figure

Activity of IQ in a Mouse Model for Prion Diseases

We next evaluated the antiprion activity of IQ in vivo in a mouse model for prion disease. IQ has been authorized for topical use as the lesions treated are external and bounded, but several in vivo trials have previously been performed on various animal species in which IQ exhibited only moderate toxicity (reviewed in 25). To evaluate the in vivo potential of IQ against prions, we used transgenic mice expressing ovine PrP [START_REF] Beringue | Opposite effects of dextran sulfate 500, the polyene antibiotic MS-8209, and Congo red on accumulation of the protease-resistant isoform of PrP in the spleens of mice inoculated intraperitoneally with the scrapie agent[END_REF]. 30 min after the onset of intraperitoneal inoculation of prions, 12 mice were intraperitoneally injected with DMSO (control group) and 12 mice were injected with 25 mg/kg of IQ solubilised in DMSO (IQ group). Mice were then treated 6 days per week for 30 days and then every 3 days from day 31 at the same dose. Mice from both groups were treated until the appearance of the first clinical signs in mice from the control group at day 90, at which time treatment was stopped for mice of both groups. The effect of IQ on mice survival time was assessed on 9 mice per group, as 3 mice from both group were euthanized when still healthy at mid-infection (at 56 days postinoculation, Figure 2B) for further analysis of the PrP Sc content of the spleen. IQ treatment was beneficial compared to DMSO treatment, as the mean survival time of the IQ group mice increased by approximately 10%, with a good statistical significance of the difference observed between the two types of regimen (9 mice per group, p<0.0001, Kruskal-Wallis test; Figure 2A). This effect is quite noticeable given the negative results obtained following treatment of mice with quinacrine (QC) and chlorpromazine (CPZ), two compounds already used for treatment of malaria and psychotic disorders, respectively. Indeed, despite being active against mammalian prion in cellbased assays, QC and CPZ turned out to be inactive both in mouse models for prion diseases and in patients in compassionate treatments of CJD [START_REF] Collins | Quinacrine does not prolong survival in a murine Creutzfeldt-Jakob disease model[END_REF][START_REF] Barret | Evaluation of quinacrine treatment for prion diseases[END_REF]. IQ thus presents a significant effect on the survival time of Tg338 mice, an animal model that might be difficult to cure, due to the high infectious load inoculated and to the high level of expression of PrP. We expect that IQ effects should be more noticeable in a less stringent model [START_REF] Dirikoc | Nonpsychoactive cannabidiol prevents prion accumulation and protects neurons against prion toxicity[END_REF]. PrP Sc accumulation in the spleen was assessed in mice euthanized at mid-infection (3 mice per group) (Figure 2B) and at terminal stage (4 mice per group) (Figure 2C) [START_REF] Beringue | Opposite effects of dextran sulfate 500, the polyene antibiotic MS-8209, and Congo red on accumulation of the protease-resistant isoform of PrP in the spleens of mice inoculated intraperitoneally with the scrapie agent[END_REF]. IQ did not affect significantly spleen PrP Sc level, although it showed a beneficial effect on mouse survival, suggesting that IQ may delay 127S prion neuroinvasion phase. Hence, this study suggests a potential novel therapeutic indication of imidazoquinolines compounds such as IQ for the treatment of prion-based diseases in mammals, including humans. (Aldara®, Zyclara® and Beselna®). The imiquimod series continues to be the subject of intensive drug discovery studies in regard to the broad therapeutic potencies of immune enhancers. IQ contains an imidazoquinoline moiety which is found in other biologically active compounds such as modulators of the A3 adenosine receptor [START_REF] Kim | Novel: 2 and 4-substituted 1H-imidazo[4,5-c]quinolin-4-amine derivatives as allosteric modulators of the A3 adenosine receptor[END_REF] and anticancer drugs used for the treatment of melanomas [START_REF] Khier | Pharmacology of EAPB0203, a novel imidazo[1,2a]quinoxaline derivative with anti-tumoral activity on melanoma[END_REF]. IQ analogues such as Gardiquimod, R-848 and Loxoribine have recently entered clinical trials. Several routes to synthesize IQ have been previously reported and patented. The simplest one was proposed by V. Nakkada [START_REF] Naddaka | Process for preparing Imiquimod[END_REF]. Briefly, the process consisted of heating of 4-chloro-1-isobutyl-1H-imidazole[4,5-c] quinoline with formamide. In most of the previously described methods, the imidazole ring is formed during the last steps of the process precursered by quinolines derivatives. In the presented method, the central pyridine ring of the tricyclic system is formed during the last step. This new simple synthesis route allows to obtain IQ and other aminoimidazoquinolines, as well as structurally related amino pyrazoloquinoline by allowing a Suzuki-Miyaura coupling between a 2-aminoarylboronic ester and a iodocyanoimidazole (Figure 3). The biarylic intermediate was easily converted into a tricyclic aromatic compound under basic conditions. This method, a model for short synthesis pathway of IQ (Figure 3A), was further extended to pyrazoloquinolines (Figure 3B). The cyanoaminoimidazole 1 was obtained using a known procedure [START_REF] Peinador | A Short and Facile Synthesis for Heteromine A[END_REF]: Aminomalononitrile was reacted with triethylformate followed by the addition of iso-butylamine (Figure 3A). In the second step, the iodination was achieved using diiodomethane containing Iso-amylnitrite (iso-pentylnitrite) leading to iodoimidazonitrile 2 (Figure 3A). The Suzuki coupling was then performed under classical reaction conditions using orthoaminoboronic acid to obtain birarylaminonitriles 3. Several approaches were investigated for the final cyclisation step. It was efficiently achieved upon heating sodium amide in toluene. The final compounds 4 could be isolated as free bases or hydrochlorides salts (Figure 3A). The same approach was also used for the synthesis of the previously unknown amino pyrazoloquinolines. Aminopyrazolonitrile 5 can be efficiently prepared by the reaction of hydrazines with ethoxymethylene aminonitrile (Figure 3B). They were converted into the corresponding iodopyrazolonitrile 6 under the same conditions as for the synthesis of 3. The coupling of 6 with 2-aminoboronic ester led to the intermediate biaryl 7 which was then converted into aminopyrazolo [4,3-c] quinolines. The new synthesis pathway described here now allows easy synthesis of IQ and efficient synthesis of IQ derivatives to date hard to achieve.

A new synthesis pathway for IQ and its chemical derivatives

Activity of chemical derivatives of IQ

The new synthesis pathway described above enabled the production of a first set of IQ chemical derivatives, i.e. a first corpus of structure-activity relationship (SAR). The five original chemical derivatives of IQ obtained (Figure 4A) were tested against both yeast and mammalian prions using respectively the yeast-based ([PSI + ] and [URE3] prions) and MovS6-based assays described above (Figure 4B &4C). We also checked that the red halos induced by IQ's chemical derivatives corresponded to [PSI + ] and [URE3] cured cells (Figure S1B).

This structure/activity relationship study allowed the identification of four active (4b, 4c, 8a, 8b) as well as one inactive (4d) derivatives which constitutes a valuable negative control (Figure 4B &4C). Interestingly, activity of the various derivatives of IQ in the MovS6 cell-based assay paralleled their activity against yeast prions. For example, the replacement of a phenyl or substitution of phenyl by a pyridine completely abolished the antiprion activity (4d) whereas a methyl in position 8 was associated with a slight increase of antiprion activity (4b) (Figure 4B &4C). On the contrary, a chlorine group in the same position slightly reduced the antiprion activity of the bioactive compounds (4c & 8b). These parallels in SAR in both yeast and mammalian cells strongly support the idea that these drugs target a conserved pathway.

Antiprion activity of IQ does not involve its agonist activity on Toll-like receptors 7/8

In order to determine if the antiprion activity of IQ and its chemical derivatives could be linked to their agonist activity on Toll-like receptors (TLRs), we first tested the potential antiprion activity of commercially available TLR7 and TLR8 agonists which are pharmacologically and chemically close to IQ such as Gardiquimod, Loxoribine and R-848 (TLR7 agonists), and CL-075 and CL-097 (TLR7/8 agonists) (Figure 5A). Indeed, like IQ, these TLR7 or TLR7/8 agonists are used in various preclinical tests to stimulate the innate and acquired immune responses. As shown in Figure 5B, only one of the five TLR7/8 agonists tested, CL-075, exhibited some antiprion activity against both [PSI + ] and [URE3] yeast prions. CL-097 was slightly active only against [URE3]. In addition, only Gardiquimod and CL-075 displayed a very modest activity against PrP Sc in MovS6 cells (Figure 5C). Next, the capacity of IQ and its chemical derivatives 4b and 4c to activate TLR7 and TLR8 was evaluated. As shown on the left panel of Figure 5D, IQ/4as and the TLR7/8 agonist R-848 activated cells through TLR7 whereas 4b, 4c, and 4d had no agonist activities on TLR7. Contrary to R-848, neither IQ nor 4as, 4b, 4c, and 4d were able to activate TLR8 receptor (Figure 5D, right panel). Thus the antiprion activity of IQ and its chemical derivatives does not parallel their agonist activity on TLR7/8. In addition, the antiprion activity of IQ against S. cerevisiae prions cannot be due to its agonist activity on TLR7 since yeast does not contain this type of receptors. Altogether, these results suggest that the antiprion activity of IQ does not involve the same mechanism than the one responsible for its capacity to trigger immune responses.

Recent data suggested that IQ could exert its effect by increasing the level of the opioid growth factor receptor (OGFr) [START_REF] Zagon | Imiquimod upregulates the opioid growth factor receptor to inhibit cell proliferation independent of immune function[END_REF]. Our data showed that the antiprion activity of IQ and its chemical derivatives is independent from their ability to stimulate TLRs. This provides another example where IQ's beneficial effects may be independent of its capacity to trigger the innate immune system.

IQ and its chemical derivative 4b inhibit the protein folding activity of the ribosome

IQ is structurally quite close to 6AP, an antiprion compound we engineered in the course of a structure/activity relationship study we led around phenanthridine [START_REF] Bach | Isolation of drugs active against mammalian prions using a yeast-based screening assay[END_REF]. 6AP is a specific inhibitor of the protein folding activity of the ribosome (PFAR), which is an RNA-based activity borne by the domain V of the large rRNA of the large subunit of the ribosome [START_REF] Voisset | Tools for the study of ribosome-borne protein folding activity[END_REF][START_REF] Chattopadhyay | Reactivation of denatured proteins by 23S ribosomal RNA: role of domain V[END_REF][START_REF] Das | In vitro protein folding by ribosomes from Escherichia coli, wheat germ and rat liver: the role of the 50S particle and its 23S rRNA[END_REF][START_REF] Das | Role of the ribosome in protein folding[END_REF][START_REF] Kudlicki | Ribosomes and ribosomal RNA as chaperones for folding of proteins[END_REF][START_REF] Sanyal | The folding of dimeric cytoplasmic malate dehydrogenase. Equilibrium and kinetic studies[END_REF]. This activity of the ribosome is still poorly understood but is preserved throughout evolution, from prokaryotes to eukaryotes. Since IQ antiprion activity is not linked to its TLR7 agonist activity, we wondered if this drug might, like 6AP, modulate PFAR. Human Carbonic Anhydrase (hCA) was used as a substrate for in vitro assisted folding experiments. Denatured hCA was diluted in native buffer either alone (to determine self-folding efficiency) or in the presence of preparations of E. coli 70S ribosome. The correct refolding of hCA was assessed by following the reappearance of its enzymatic activity in comparison to the native hCA. Self-folding restored about 25% of hCA activity. PFAR borne by E. coli ribosomes restored about 55% of hCA activity in the absence or presence of DMSO (Figure 6A). In the presence of IQ and its chemical derivative 4b, the protein folding activity of 70S ribosomes was greatly inhibited (Figure 6A & 6B) whereas the self-folding of hCA was not affected (Figure 6C). This inhibition was specific since 4d compound, which presented no antiprion activity, also showed no inhibition of PFAR (Figure 6A). As the main function of the ribosome is protein synthesis, the effect of IQ and 4b on translation was also assessed in vivo in yeast. None of the tested compounds exhibited a significant effect on global protein synthesis, in contrast with cycloheximide (CHX), a known inhibitor of global translation, which completely inhibited protein synthesis at the tested concentration (Figure 6D). Therefore, at concentrations at which they exhibit antiprion activity, neither IQ nor 4b affected protein synthesis in vivo in yeast whereas, like 6AP [START_REF] Tribouillard-Tanvier | Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs[END_REF], they are specific inhibitors of the protein folding activity of the ribosome. Taken together, these results indicate that IQ and 4b are specific inhibitors of PFAR, and that their ability to inhibit PFAR paralleled their antiprion activity. They also confirm that PFAR might be involved in prion propagation both in yeast and mammals. Our recent data confirm that PFAR is involved in prion propagation in yeast (CV, PN, FS & MB unpublished data). In addition to their potential therapeutic application, 6AP, GA and IQ are currently the sole PFAR specific inhibitors. They can be regarded as precious tools for explaining the biological role of PFAR which has only been partially explored to date, in particular, in prion diseases, and more generally in amyloid-based diseases [START_REF] Voisset | Tools for the study of ribosome-borne protein folding activity[END_REF].

Materials and Methods

Yeast strains and culture media

Yeast strains used in this study were STRg6 (74-D694, Mata, erg6::TRP1, ade1-14, trp1-289, his3Δ200, ura3-52, leu2-3,112, [PSI + ]) and SB34 (Mata, erg6::TRP1, dal5::ADE2, ade2-1, trp1-1, leu2-3,112, his3-11,15, ura2::HIS, [URE3]) and were grown and used as previously described [START_REF] Bach | Isolation of drugs active against mammalian prions using a yeast-based screening assay[END_REF].

Commercially available compounds

BIOMOL's FDA-Approved Drug Library (Enzo Life Sciences) is a collection of 640 FDA-approved molecules all in use in the clinics selected to maximize chemical and pharmacological diversity. The compounds are supplied as 2 mg/ml DMSO solutions. IQ and GuHCl were purchased from Sigma Aldrich. CL-075, CL-097, Gardiquimod and Loxoribine powders were purchased from InvivoGen. R-848 was purchased from IMGENEX (1 mg/ml in water).

Yeast-based antiprion screening assay

This assay was performed as previously described [START_REF] Bach | Isolation of drugs active against mammalian prions using a yeast-based screening assay[END_REF][START_REF] Bach | A yeast-based assay to isolate drugs active against mammalian prions[END_REF]. Briefly, an aliquot of an exponentially growing culture (340 µl of 0.55 OD 600 culture of STRg6 or 170 µl of 0.55 OD 600 culture of SB34 strains) was spread homogeneously using sterile glass beads on square plates (12 x 12 cm) containing YPD solid medium supplemented with 200 µM (for [PSI + ] STRg6 strains) or 800 µM Guanidine hydrochloride (for SB34 [URE3] strain). Small sterile filters (Thermo-Fisher) were then placed on the agar surface and individual compounds were applied to each filter. DMSO, the vehicle compound, was applied to the top left filter as a negative control, and 10 µl of 300 mM GuHCl solubilized in DMSO was applied to the bottom right filter as a positive control. 2 µl of each of the 640 compounds of the The effect of the various drugs on ribosome-assisted folding of GuHCl-denatured hCA was evaluated. Correct refolding was assessed by measuring the recovery of hCA enzymatic activity as a function of time in comparison to that of the native enzyme stored undiluted on ice (normalized to 100%). Self-refolding of hCA was about 25%, the refolding with 70S ribosome increased to 55% due to PFAR. In the presence of 250 µM of IQ and 4b, PFAR was fully inhibited, whereas 250 µM 4d showed no inhibitory effect. 250 µM 6AP was used as a positive control [START_REF] Tribouillard-Tanvier | Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs[END_REF]. B. IQ inhibits PFAR in a dose-dependent manner. C. IQ and its chemical derivatives (250 µM) do not affect the selffolding of GuHCl-denatured hCA. D. The effect of the indicated compounds (100 µM) on global in vivo translation in living yeast cells was evaluated. Briefly, various drugs or DMSO alone were added to yeast cells in exponential growth in YPD rich medium at a final concentration of 100 µM. After 20 minutes, radiolabelled [ 35 S] methionine/cystein was added for 20 minutes. The cells were lysed and analyzed by SDS-PAGE followed by autoradiography. Error bars correspond to standard deviations. BIOMOL chemical library, or 50 nmoles of IQ chemical derivatives 4b-d, or 50 nmoles of TLR7 and TLR7/8 agonists Gardiquimod, R-848, Loxoribine and CL-097, or 5 nmoles of CL-075 were applied on each of the remaining filters. Plates were then incubated 5 days at 25°C and scanned using a Snap Scan121s2 (Agfa).

PrP Sc clearance assay in MovS6 cells

Experiments were performed as previously described [START_REF] Archer | Cultured peripheral neuroglial cells are highly permissive to sheep prion infection[END_REF]. Briefly, MovS6 cells chronically infected with ovine 127S prion strain were treated for six days with the indicated concentrations of compounds and then lysed (0.5% Na deoxycholate, 0.5% Triton X-100, 5 mM Tris-HCl pH 7.4). To distinguish between cellular (PrP C ) and pathological forms of PrP Sc , 250 µg of cell lysates were digested by proteinase K (PK) as PrP Sc is partially resistant to proteolysis. The detection of PrP tot was performed on 25 µg of crude cell lysate. Proteins were analyzed by 10% SDS-PAGE (Invitrogen) and transferred to 0.45 µm nitrocellulose membranes (Whatman). Membranes were incubated for two hours with 1:40 000 anti-PrP antibody (Sha31, Bertin pharma). The membranes were then washed with fresh PBS 1X / 0.1% Igepal and incubated for 45 min with 1:3000 secondary HRP-conjugated antibodies (Biorad), and analyzed by Enhanced Chemiluminescence (GE Healthcare) using a Vilber-Lourmat Fusion SL image acquisition system which allows precise quantification of the signals.

Ethics Statement

Animal experiments were carried out in strict accordance with EU directive 2010/63 and were approved by the author's institution local ethics committee (Comethea, INRA Agroparitech ethics committee, permit number 12/034). All efforts were made to minimize suffering.

Mouse model for prion-based disease

Experiments were performed as previously described [START_REF] Dur | A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes[END_REF]. 24 mice overexpressing ovine PrP (tg338 line [START_REF] Vilotte | Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp[END_REF]) were infected intraperitoneally with 100 µl of the 127S scrapie strain at 0.02% (w/v) dose. This strain has an intracerebral infectious titre of 10 9 ID 50 U/g of brain [START_REF] Vilotte | Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp[END_REF]. 12 mice (IQ group) were then intraperitoneally injected with a dose of 100 µl of 2.5 mg/ml IQ solubilized in 8% DMSO (25 mg/kg) 30 min after 127S injection, and 12 infected mice were intraperitoneally injected with 100 µl of 8% DMSO (control group). Mice were then treated 6 days per week for 30 days and then every 3 days from day 31 at the same dose. The treatment was stopped around day 90 post-infection, when the first symptoms appeared in mice from the control group. Mice were euthanized at terminal stage according to ethics rules. At mid-treatment (56 days post-infection), 3 of 12 mice from both control and IQ groups were euthanized. Spleens were also collected from 4 mice euthanized at terminal stage of disease. Spleens collected at mid-treatment and at terminal stage of disease from euthanized mice were analyzed for PrP Sc content, as previously described [START_REF] Langevin | Marked influence of the route of infection on prion strain apparent phenotype in a scrapie transgenic mouse model[END_REF][START_REF] Tribouillard-Tanvier | Antihypertensive drug guanabenz is active in vivo against both yeast and mammalian prions[END_REF]. Briefly, spleen tissues were homogenized at 20% (w/v) in 5% glucose with a Rybolyser (Hybaid). PrP res was extracted according to the Biorad test protocol, by using 200 mg/ml PK for 10 min. at 37°C. After denaturation in Laemmli buffer, proteins were analyzed by SDS-PAGE (Invitrogen) and transferred to nitrocellulose membranes and immunoblotted with 0.1 mg/ml Sha31 anti-PrP antibody. The equivalent of 1 mg of spleen tissue was loaded onto the gels. Immunoreactivity was visualized by chemiluminescence (GE Healthcare).

Chemical Methods

The synthesis methods for the compounds 1, 2, 3a, 3b, 3c, 3d, 4a, 4b, 4c, 4d, 5, 6, 7a, 7b, 8a and 8b are described in Methods S1. 1 H-NMR, 13 C-NMR spectra, IR spectra and microanalyses are available upon request.

TLR7 and TLR8-based assays

293XL-hTLR7 cells and 293XL-hTLR8 cells (Invivogen) were transiently transfected with an NF-κB inducible reporter plasmid (Clontech). On day 0, cells were seeded overnight at 5.10 5 cells/mL on 6 well plates. On day 1, cells were transfected for 6 hours by pNFkB-luc plasmid using FuGENE® 6 Reagent (Roche) as recommended by the manufacturer. Transfected cells were further incubated overnight at 37°C, followed by a 24h incubation with different compounds at the indicated concentrations. The presence of Luciferase was revealed using Steady Glo Luciferase assay system (Promega). The light emitted by Luciferase activation in each well was quantified as "Count Per Second" (CPS) on a Victor apparatus (Perkin-Elmer). Fold increase was quantified as the ratio between experimental CPS (stimulus) and spontaneous CPS (medium).

Translation assay

A yeast culture grown at 29°C in YPD (OD 600 nm = 0.6 in exponential phase of growth) was incubated with the indicated compounds (100 µM) or the corresponding volume of DMSO for 20 min at 29°C at which time [ 35 S] methionine and cystein were added for 20 min (PerkinElmer Life Sciences). Cells were then harvested and lysed (lysis buffer: 25 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.2% Triton X-100, antiproteases cocktail (Roche), 1 mM phenyl-methylsulfonyl fluoride). Crude extracts were analyzed by 10% SDS-PAGE (Invitrogen). The gel was dried and analyzed using a Typhoon 9400 Phosphorimager (GE Healthcare).

In vitro ribosome assisted protein folding assay 70S E. coli ribosomes were prepared using sucrose gradient zonal ultracentrifugation as described previously [START_REF] Ederth | A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli[END_REF]. For the in vitro refolding experiments described in [START_REF] Reis | Mode of action of the antiprion drugs 6AP and GA on ribosome assisted protein folding[END_REF], human Carbonic Anhydrase (hCA) at a concentration of 30 µM was denatured by an overnight treatment at room temperature with 6 M GuHCl and 30 nM EDTA. To allow refolding, hCA was diluted 100 times at a final concentration of 300 nM in a buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl and 5 mM magnesium acetate for 30 min with or without 70S ribosomes at a final concentration of 300 nM, in the absence or presence of compounds. The refolding of hCA, as a function of the native enzyme activity (normalized to 100%), was followed by a colorimetric assay measuring the increase of OD 400 with time when hCA substrate, para-nitrophenyl acetate (pNPA), was added directly to the refolding mix at a final concentration of 500 µM. To check if drugs may affect hCA self-folding, denatured hCA was diluted 100 times at a final concentration of 300 nM in a buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl and 5 mM magnesium acetate for 30 min in the presence of compounds, in the absence of 70 ribosomes.

  PLOS ONE | www.plosone.org 1 August 2013 | Volume 8 | Issue 8 | e72112

Figure 1 .

 1 Figure 1. Imiquimod (IQ) is active against both yeast and mammalian prions. A. Molecular structure of IQ. B. [PSI + ] (left panel) and [URE3] (right panel) strains were spread on YPD medium supplemented with 200 and 800 µM GuHCl, respectively. Small filters were then placed on the agar surface and increasing amounts of IQ were applied to each filter, except for the top left filter where DMSO, the compounds vehicle was added (negative control) and for the bottom left filter where GuHCl was added (positive control). The apparition of a halo of red colonies around a filter indicates that a compound active against [PSI + ] or [URE3] prions has been spotted, whereas colonies remain white in case of inactive compounds. C. Scrapie-infected MovS6 cells were treated for six days with increasing amounts of IQ and then lysed. Cell lysates were then subjected to PK digestion to specifically reveal PrP Sc by immunoblot. The effect of IQ on the steadystate level of PrP (PrP tot ) was determined on the same MovS6treated cell lysates in the absence of PK treatment (lower panel). Ratios of Western blot PrP Sc /PrP tot signals are indicated below each lane. The blot shown is representative of three independent experiments which all produced similar results. doi: 10.1371/journal.pone.0072112.g001

  IQ belongs to the imidazoquinolines class of bioactive compounds. It is commercialized under different brand names

Figure 2 .

 2 Figure 2. IQ extends survival time in a mouse model for prion diseases. Transgenic mice overexpressing ovine PrP were intraperitoneally infected with scrapie. 12 mice were treated with DMSO (control group) and 12 mice were treated with IQ solubilised in DMSO (IQ group) until the appearance of the first clinical signs in mice from the control group at day 90. A. Survival time of mice treated with DMSO (control group) or mice treated with IQ (IQ group). B, C. Effects of IQ on PrP Sc accumulation in scrapie-infected mouse spleens. The spleen of 3 mice euthanized at 56 days post-inoculation (B) and the spleen of 4 mice euthanized at terminal stage (C) from both control and IQ groups were analyzed for PrP Sc content. The PrP Sc content of spleens was determined by Western blot. The same amount of spleen tissue proteins was loaded on each well. doi: 10.1371/journal.pone.0072112.g002

Figure 3 .

 3 Figure 3. Reagents and conditions for the preparation of IQ and its chemical derivatives. A. (i) Step 1: CH(OEt) 3 , CH 3 CN 30 min reflux; Step 2: (iso-butylamine) 12h 20°C; (ii) CH 2 I 2, iso-pentylnitrite; (iii) 2N Na 2 CO 3 , Pd[(P(C 6 H 5 ) 3 ] 4 , dioxane; (iv) NaNH 2 , toluene, 2h at 80 °C. B. (i) CH 3 NHNH 2 , NEt 3 , THF, 60 °C; (ii) CH 2 I 2 , iso-pentylnitrite; (iii) 2N Na 2 CO 3 , Pd](P(C 6 H 5 ) 3 ] 4 , dioxane, 80 °C; (iv) NaNH 2 , toluene. doi: 10.1371/journal.pone.0072112.g003

Figure 4 .

 4 Figure 4. Activity of the chemical derivatives of IQ. A. Chemical structures of the 5 derivatives of IQ. 4as corresponds to the hydrochloride of IQ. B. STRg6 [PSI + ] and SB34 [URE3] strains were spread on YPD medium supplemented with 200 and 800 µM GuHCl, respectively. 50 nmoles of IQ and of its 5 chemical derivatives were spotted on filters as described in Figure 1B. The top left filter correspond to DMSO and the bottom right filter was spotted with GuHCl (positive control). C. Activity of IQ and its chemical derivatives against PrP Sc in the MovS6 cell-based system described in Figure 1C. Ratios of Western blot PrP Sc /PrP C signals are indicated below each lane. The blot shown is representative of three independent experiments which all produced similar results. doi: 10.1371/journal.pone.0072112.g004

Figure 5 .

 5 Figure 5. Antiprion activity of some TLR7/8 agonists related to IQ. A. Structures of Gardiquimod, R-848, Loxoribine, CL-075 and CL-097, five other known TLR7/8 agonists. B. These five TLR7/8 agonists were tested against both [PSI + ] and [URE3] yeast prions (50 nmoles of compounds per filter, 5 nmoles for CL-075) as described in Figure 1B. C. Evaluation of the activity of Gardiquimod (Gardi), R-848, Loxoribine (Loxo), CL-075 and CL-097 against mammalian prion using the MovS6 cell-based assay. Ratios of Western blot PrP Sc /PrP tot signals are indicated below each lane. The blot shown is representative of two independent experiments which all produced similar results. D. The potential of IQ chemical derivatives 4b, 4c and 4d as TLR7 (left panel) and TLR8 (right panel) agonists was also evaluated ex vivo. Briefly, 293XL-hTLR7 and 293XL-hTLR8 cells transiently transfected with an NF-κB inducible reporter plasmid were incubated with compounds at indicated concentrations and Luciferase production, which reflects the activation of the receptor, was quantified. Experiments were performed two (TLR8) or three (TLR7) times in duplicates. doi: 10.1371/journal.pone.0072112.g005

Figure 6 .

 6 Figure 6. in vitro specific anti-PFAR activity of IQ and its chemical derivative 4b. A. The effect of the various drugs on ribosome-assisted folding of GuHCl-denatured hCA was evaluated. Correct refolding was assessed by measuring the recovery of hCA enzymatic activity as a function of time in comparison to that of the native enzyme stored undiluted on ice (normalized to 100%). Self-refolding of hCA was about 25%, the refolding with 70S ribosome increased to 55% due to PFAR. In the presence of 250 µM of IQ and 4b, PFAR was fully inhibited, whereas 250 µM 4d showed no inhibitory effect. 250 µM 6AP was used as a positive control[START_REF] Tribouillard-Tanvier | Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs[END_REF]. B. IQ inhibits PFAR in a dose-dependent manner. C. IQ and its chemical derivatives (250 µM) do not affect the selffolding of GuHCl-denatured hCA. D. The effect of the indicated compounds (100 µM) on global in vivo translation in living yeast cells was evaluated. Briefly, various drugs or DMSO alone were added to yeast cells in exponential growth in YPD rich medium at a final concentration of 100 µM. After 20 minutes, radiolabelled [ 35 S] methionine/cystein was added for 20 minutes. The cells were lysed and analyzed by SDS-PAGE followed by autoradiography. Error bars correspond to standard deviations.

PLOS ONE | www.plosone.org

August 2013 | Volume 8 | Issue 8 | e72112

doi: 10.1371/journal.pone.0072112.g006

Acknowledgements

We thank Gaëlle Friocourt and Olivier Billant for critical reading of the manuscript, Zarrin Alavi, INSERM CIC 0502, CHRU of Brest, for proof-reading of this manuscript and for her precious advice, Hélène Simon for her excellent technical assistance, Laetitia Guedeville, Laetitia Herzog, Fabienne Reine and Emilie Jaumain for expert animal care and excellent technical assistance, Blandine Baratte for help with radioactivity handling and Vincent Guen for help with the Typhoon.

This work was supported by an Agence Nationale de la Recherche "blanche" from the French government, a Contrat Jeune Chercheur from Institut National de la Santé et de la Recherche Médicale and the SSF-Dalen (Sweden-France Bilateral Collaboration) Program (to MB & SS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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Daru ! Ehsan

Structure-selectivity relationship of a zirconia-based heterogeneous acid catalyst in the production of green mono-and dioleate product

A novel catalytic technique is vital to produce mono-and dioleate (GMO and GDO) from bioglycerol: a renewable resource and by-product of biodiesel. The advantage of this invention is the direct production of GMO and GDO through catalytic approach compared to the conventional method that requires transesterifcation and distillation processes. In this paper, glycerol esterifi cation with oleic acid using a catalyst was experimented. The process was carried out over a hydrophobie mesoporous zirconia-silica heterogeneous acid catalyst (Zr0 2 -Siû,Me&Et-PhSO 3 H) with three types of sulphated zirconia catalysts (So/ /Zr0 2 ) to produce high-selectivity GMO and GDO products. The catalytic performance of the hydrophobie Zr0 2 -SiOrMe&Et-PhSO 3 H catalyst was benchmarked with that of so/-!Zr0 2 which was developed from three zirco nium precursors. Results showed that the pore volume and hydrophobicity of the designed catalyst greatly could influence the product selectivity, thus enabling smaller substrates GMO and GDO to be dominated in the synthesis. This finding was supported by characterisation data obtained through N 2 adsorption-desorption, X-ray diffraction and scanning electron microscopy. In addition, a good correlation was found between pore volume (pore size) and product selectivity. High pore volume catalyst favoured GDO production under identical reaction conditions. Pore volume and size can be used to control product sensitivity. The hydrophobicity of the catalyst was found to improve the initial reaction rate effectively.

structures due to its amphiphilic nature [START_REF] Kulkarni | Monoolein: a magic lipid?[END_REF]. The robust expanding industries such as nutraceuticals, pharmaceuticals and lubricants are the major channels for GMO. In the health care industry, it is reported that GMOs and GDO are considered as appealing lipids for the formulation of potential vehicles of drugs and to improve the poor solubilisation of bioactive compounds. GMO and GDO lipids could enhance their bio-accessibility at intestinal level or to protect against their degradation under gastrointestinal conditions, which eventually achieve the most efficient bioactivity compounds in a selfemulsifying lipid delivery system. The need for GMOs is associated with the requirement from the health care or lubricant market owing to the slow market of food and plastics industries (Frost and Sullivan Research Service 2014). At the moment, GDO is widely employed in drug delivery and applied as a safe plasticiser in the polymer industry [START_REF] Barauskas | Sponge" nanoparticle dispersions in aqueous mixtures of diglycerol monooleate, glycerol dioleate, and polysorbate 80[END_REF][START_REF] Zhang | Production of diacylglycerols by esterification of oleic acid with glycerol catalyzed by diatomite loaded SO 4 2-/TiO 2[END_REF].

To date, heterogeneous acid catalysts, such as ion exchange resins (Åkerman et al. 2011), zeolites [START_REF] Singh | Esterification of oleic acid with glycerol in the presence of supported zinc oxide as catalyst[END_REF], double-metal cyanide complexes [START_REF] Kotwal | Esterification of fatty acids with glycerol over Fe-Zn double-metal cyanide catalyst[END_REF]), heteropolyacid-supported catalysts (Wee et al. 2013), hydrotalcite [START_REF] Hamerski | LDH-catalyzed esterification of lauric acid with glycerol in solvent-free system[END_REF][START_REF] Hamerski | Kinetics of layered double hydroxide catalyzed esterification of fatty acids with glycerol[END_REF]) and sulphated metal oxide catalysts [START_REF] Kong | Catalytic esterification of bioglycerol to value-added products[END_REF], have been explored for catalytic glycerol esterification with OA. On the contrary, the drawbacks of homogeneous acid catalysts are dark product colour and burnt taste and require additional refining steps. Homogenous acid catalyst can disperse into the reactants rapidly but resulted in poor product selectivity. In a recent study, it was demonstrated that the application of the Fe-Zn DMC complex is able to achieve GMO conversion with 63.4% and GMO selectivity of 67.3% under optimised conditions of 180 °C reaction temperature, 8 wt.% catalyst loading and 8 h of reaction time [START_REF] Kotwal | Esterification of fatty acids with glycerol over Fe-Zn double-metal cyanide catalyst[END_REF]. Meanwhile, titanium silicate-type catalyst (Ti-SBA-16) featuring a hydrophobic surface attained a conversion of 72.8% at 180 °C, equimolar ratio, 3 wt % catalyst concentration and 3 h of operation time. The presence of catalyst sites in Ti-SBA-16 catalyst is not the only reason contributing to the excellent catalytic performance, but also the catalyst structure further helps in maximising the selectivity of the anticipated products. It was reported that the hydrophobicity characteristic of Ti metal is able to enhance the glycerol esterification rate and selectivity. However, the high reaction temperature (180 °C) and long reaction time (10 h) are several of the limitations that could be improved to synthesise promising solid acid catalysts.

Therefore, this work aims to examine the relationship of the structural and hydrophobicity surface of a heterogeneous catalyst with the product selectivity over four zirconia-based heterogeneous acid catalysts, for the industrially important glycerol catalyst-assisted esterification with OA. To the best of our knowledge, this study is the first to provide insights into the impact of pore size, pore volume and hydrophobicity of catalyst surface towards the product selectivity via various zirconia-precursor-developed catalysts. The comparative catalytic activity for the ZrO 2 -SiO 2 -Me&Et-PhSO 3 H catalyst (higher hydrophobicity) with three types of SO 4 2-/ZrO 2 catalysts [less hydrophobic and developed from different zirconium precursors, Zr(OCH 2 CH 3 ) 4 , ZrOCl 2 •8H 2 O and commercial Zr(OH) 4 )] was investigated. The catalyst synthetic methods for SO 4 2-/ZrO 2sol-gel , SO 4 2-/ZrO-2precipitation , SO 4 2-/ZrO 2commercial and highly hydrophobic ZrO 2 -SiO 2 -Me&Et-PhSO 3 H and their textural properties were characterised. The esterification reactions are performed at a steady amount of acidity, and the reaction conditions are optimised to assess the effect of the textural properties on catalytic performance.

Experimental

Materials

The chemicals used in this study are similar to our previous paper [START_REF] Kong | Development of a novel hydrophobic ZrO 2 -SiO 2 based acid catalyst for catalytic esterification of glycerol with oleic acid[END_REF]. For the synthesis of ZrO 2 -SiO 2 -Me&Et-PhSO 3 H hydrophobic-enhanced catalyst, chemicals utilised are zirconium hydroxide powder, ethanol, ammonia solution (25%), tetraethyl orthosilicate, trimethoxymethylsilane (TMMS) and dry toluene that were obtained from Sigma-Aldrich. 2-(4-chlorosulphonylphenyl) ethyltrimethoxysilane (CSPETS, 50% in dichloromethane and sulphuric acid solution (H 2 SO 4 , 99.99%) were obtained from Fisher Scientific. Other chemicals that include zirconium (IV) propoxide, Zr(OCH 2 CH 2 CH 3 ) 4 precursor, 1-propanol and 0.5 M aqueous H 2 SO 4 were obtained from Sigma-Aldrich. These chemicals are used to create sol-gel method-prepared catalyst (SO 4 2-/ZrO-2sol-gel ). As for the (SO 4 2-/ZrO 2precipitation ) catalyst synthesis, the materials can be found in the paper [START_REF] Kong | Development of a novel hydrophobic ZrO 2 -SiO 2 based acid catalyst for catalytic esterification of glycerol with oleic acid[END_REF]. Reactants glycerol and technical grade OA were acquired from Sigma-Aldrich. Monoolein, diolein and triolein that were used for quantitatively product analysis were obtained from Sigma-Aldrich. Analytical-grade solvents purchased from Sigma-Aldrich, such as acetonitrile (ACN), methanol (MeOH) and tetrahydrofuran (THF), were used as the mobile phase. Trifluoroacetic acid (TFA) was used as a mobile phase additive due to its high resolving power.

Catalyst preparations

A catalyst featuring hydrophobic surface was synthesised by using modified volume of TMMS hydrophobic agent (ZrO 2 -SiO 2 -Me&Et-PhSO 3 H) as described in the previous study [START_REF] Kong | Development of a novel hydrophobic ZrO 2 -SiO 2 based acid catalyst for catalytic esterification of glycerol with oleic acid[END_REF]. Three zirconium precursors were used to synthesise three SO 4 2-/ZrO 2 catalysts: (i) a SO 4 2-/ZrO 2sol-gel catalyst was prepared using the Zr(OCH 2 CH 2 CH 3 ) 4 precursor. Zr(OCH 2 CH 2 CH 3 ) 4 (5 mL) was first mixed with 6.6 mL of 1-propanol. Subsequently, 9.7 mL of 0.5 M aqueous H 2 SO 4 was added dropwise into the prepared mixed solution and was stirred vigorously at ambient temperature for 6 h. The formed gels were filtered, dried at 100 °C for overnight and were calcined at 625 °C for 4 h. (ii) A SO 4 2-/ZrO 2precipitation catalyst was prepared through precipitation using the ZrOCl 2 •8H 2 O precursor. About 21 mL aliquot of the 1 M NaOH solution was added slowly into 5 mL of ZrOCl 2 •8H 2 O under mild stirring at ambient temperature until pH 8 was reached. The formed catalyst was washed thoroughly with distilled water, filtered and then dried at 100 °C (12 h). Subsequently, 5.2 g of prepared powder was mixed with 4.6 mL of 0.5 M H 2 SO 4 at room temperature and stirred overnight. The final form SO 4 -/ZrO 2 was filtered, dried at 100 °C and then calcined at 625 °C for 4 h (Oh et al. 2013a). (iii) A SO 4 2-/ZrO 2commercial was synthesised by commercially available Zr(OH) 4 . Zr(OH) 4 (5.2 g) was mixed with 4.6 mL of 0.5 M H 2 SO 4 at room temperature and stirred overnight. The resulting SO 4 -/ZrO 2 catalyst was filtered, dried at 100 °C and then calcined at 625 °C for 4 h.

Catalyst characterisations

The catalyst properties that focus on the texture were analysed using the N 2 physisorption method [BELSORP-max analyser (Japan)]. The samples were degassed for a period of 5 h under vacuum condition at a temperature of 200 °C. The sample particle size was measured using a Malvern MS3000 particle sizer (dry) with a working pressure of 2 bar. By using the water contact angle method of KRUSS DSA100, the different hydrophobicity levels of the catalyst were measured. Acid-base titration method was used in determining catalyst acidity (mmol/g). Degassing of the catalyst sample was done at a temperature of 120 °C with a reaction time of 3 h. Following that, the sample is mixed in 25 mL of NaCl (2 M) and stirring was done at room temperature for about 24 h to attain equilibrium. Subsequently, the resultant was titrated with 8.38 × 10 -3 M of sodium hydroxide solution [START_REF] Kong | Development of a novel hydrophobic ZrO 2 -SiO 2 based acid catalyst for catalytic esterification of glycerol with oleic acid[END_REF]. The X-ray diffraction (XRD) patterns of the catalysts were recorded with a Bruker D8 Da Vinci diffractometer in Bragg-Brentano configuration by using Cu K Alpha 1,2 radiation (λ = 1.54056 Å) over the 2Ɵ range of 10°-80°. Fourier-transform infrared (FTIR) spectra were performed using a PerkinElmer, Spectrum BXII spectrometer ranging from 200 to 4000 cm -1 to identify functional groups on the catalyst surface. A field-emission scanning electron microscope (Model JSM-7100F) was used to analyse the surface morphology of the catalyst at an acceleration voltage of 1-30 kV. Thermogravimetric analysis was conducted to determine the catalyst thermal stability by applying the Mettler Toledo model at a rate of 10 °C/min and operating temperature from 25 to 900 °C.

Catalytic reaction

Using a batch reactor with a working volume of 250 mL, the catalytic esterification was accomplished. The equipment is connected with a temperature gauge, condenser and a vacuum system. The esterification process is performed at a temperature of 160 °C utilising an equimolar ratio of reactant and constant acidity (1.55 mmol H + ) for 8 h. The analysis was performed utilising HPLC. The type of column and the solvent employed for this particular study can be found in our previous study [START_REF] Kong | Development of a novel hydrophobic ZrO 2 -SiO 2 based acid catalyst for catalytic esterification of glycerol with oleic acid[END_REF]. By using Eqs. ( 1)-(3), respectively, the conversion, yield and selectivity are calculated:

Results and discussion

Catalyst characterisations

The synthesised catalyst (ZrO 2 -SiO 2 -Me&Et-PhSO 3 H) and three SO 4 2-/ZrO 2 catalysts were evaluated at a constant 1.55 mmol H + catalyst acidity and identical reaction conditions (160 °C reaction temperature, an equimolar ratio of reactant and 8 h). Those optimal reaction conditions were obtained from the previous optimisation study [START_REF] Kong | Development of a novel hydrophobic ZrO 2 -SiO 2 based acid catalyst for catalytic esterification of glycerol with oleic acid[END_REF]. The textural properties and hydrophobicity of each catalyst are summarised in Table 1. The acidity of SO 4 2-/ZrO 2 catalysts ranged from 0.35 mmol/g to 0.62 mmol/g. With regard to the influence of catalyst acidity on the reaction activity and selectivity, comparative studies were carried out at a constant concentration of 1.55 mmol H + to investigate the effect of textural property on the reaction performance. As it was reported, factors affecting the total performance of catalysts such as the acidity of catalyst ( 1)

Conversion (C) = mol OA consumed mol OA initial × 100%
(2) Yield total GMO,GDO,GTO (Y) = mol total esters mol OA initial × 100%

(3) Selectivity GMO (S) = mol GMO mol total GMO+GDO+GTO × 100%.

should be eliminated, especially in studying the effect of complex catalyst structure [START_REF] Ogino | Esterification of levulinic acid with ethanol catalyzed by sulfonated carbon catalysts: Promotional effects of additional functional groups[END_REF].

The surface areas of SO 4 2-/ZrO 2 catalysts prepared using different precursors (SO 4 2-/ZrO 2 sol-gel , SO 4 2-/ZrO 2 precipitation and SO 4 2-/ZrO 2 commercial ) were 85, 44 and 60 m 2 /g, respectively. These results corresponded with those obtained by Oh et al. (Oh et al. 2013b). In addition, the hydrophobicity of each catalyst was examined. The results showed that all SO 4 2-/ZrO 2 catalysts, regardless of the presence of Zr precursors, were less hydrophobic (as the obtained contact angle degree was low) compared with that of the designed catalyst ZrO 2 -SiO 2 -Me&Et-PhSO 3 H. The acidity level of SO 4 2-/ZrO 2 sol-gel was identical with ZrO 2 -SiO 2 -Me&Et-PhSO 3 H (0.6 mmol/g), while SO 4 2-/ZrO 2 commercial and SO 4 2-/ZrO 2 precipitation demonstrated lower acidity level, 0.44 mmol/g and 0.35 mmol/g, respectively. FTIR spectra were employed to characterise and clarify the composition of the synthesised materials. For ZrO 2 -SiO 2 -Me&Et-PhSO 3 H, a visible band at 1061 cm -1 was observed, which was ascribed to the elongating vibration of Si-O-Si, Si-O and Si-O-Zr bonds [START_REF] Chen | Efficient and stable PS-SO3H/SiO2 hollow nanospheres with tunable surface properties for acid catalyzed reactions[END_REF][START_REF] Kong | Development of a novel hydrophobic ZrO 2 -SiO 2 based acid catalyst for catalytic esterification of glycerol with oleic acid[END_REF][START_REF] Saravanan | Esterification of stearic acid with methanol over mesoporous ordered sulfated ZrO 2 -SiO 2 mixed oxide aerogel catalyst[END_REF]. Meanwhile, the bands at 988 cm -1 , 1054 cm -1 , 1125 cm -1 and 1207 cm -1 indicated the presence of sulphate group, for all types of SO 4 2-/ZrO 2 catalysts [START_REF] Chen | Efficient and stable PS-SO3H/SiO2 hollow nanospheres with tunable surface properties for acid catalyzed reactions[END_REF][START_REF] Saravanan | Esterification of stearic acid with methanol over mesoporous ordered sulfated ZrO 2 -SiO 2 mixed oxide aerogel catalyst[END_REF].

The thermal stability of the catalysts was measured by TGA. The curves of ZrO 2 -SiO 2 -Me&Et-PhSO 3 H and different types of SO 4 2-/ZrO 2 catalysts are displayed in Fig. 1. About 4 wt.% of mass loss was detected for ZrO 2 -SiO 2 -Me&Et-PhSO 3 H catalyst at the temperature ranging from 260 to 300 °C which attributed to the decomposition of sulphate moiety [START_REF] Fang | Silica-immobilized Aquivion PFSA superacid: application to heterogeneous direct etherification of glycerol with n-butanol[END_REF]; nevertheless, at a high product yield. Therefore, the correlation between the structure/property of catalysts and the catalytic performance was investigated in the following section. Understanding the correlation characteri stics of catalyst to reaction is vital in developing effective catalysts [START_REF] Diao | Optimizing the structure of supported Pd catalyst for direct oxidative esterification of methacrolein with methanol[END_REF].

Correlation between SO 4 2 -1zro 2 catalyst properties and selectivity/activities

Two correlations were successfully established. Firstly, the correlation between structural properties was made. between textural properties and selectivity has been proven by XRD and BET results, whereby the finding showed that the thermodynamically stable monoclinic phase catalyst can contribute to smaller pore size; consequently, lower selectivity of the product can be dominated in the product synthesis. In addition, the correlation of catalyst's hydrophobicity with selectivity/initial reaction rate was also proven. The higher hydrophobicity of catalyst leads to a higher initial rate, indicating that the initial reaction rate can be accelerated by the hydrophobicity surface of the catalyst. All SO 4 2-/ZrO 2 catalysts possessed a highly tetragonal phase and less monoclinic phase. The acidic tetragonal phase may be stabilised by sulphated group (Oh et al. 2013b). XRD results also clearly revealed that Zr0 2 -SiOi-Me&Et-PhSO 3 H catalyst was composed of a more thermodynamically stable monoclinic phase than the other so/-/ZrO 2 catalysts. The monoclinic diffractions at 16 °, 26 °, 28 ° and 32 ° were more intense than those of so/-lZr02preci pit at ion (c) and so/-lZr02 commercia1 (d). Nota bly, no monoclinic peak was observed at so/-/Zr0 2 so l-ge l (b), but a crystalline tetragonal phase was mostly detected. This result reasonably corresponds with the highest pore volume (0.475 cm 3 /g) and pore size (4.85 nm) of so/-/Zr0 2 s o l-ge l obtained in the BET results (refer to Table 1). The tetragonal phase was highly attributed to the different precursors used with identical calcination temper ature and calcined time. Crystallinity was also proven to affect the type of zirconia precursor [START_REF] Rashad | Effect of thermal treatment on the crystal structure and morphology of zirconia nanopowders produced by three different routes[END_REF].

Correlation between structural properties and selectivity

Consequently, the relationship between the texturai prop erties of each catalyst is related to the selectivity and con version obtained from the reaction. Figure 4 displays the correlation between pore volume versus conversion and selectivity. The experimental results fitted with the curve representing selectivity and conversion. This work proved the importance of pore volume in controlling product selec tivity. Notably, increasing the pore volume of a catalyst can increase the formation of GDO and GTO with large molecu lar sizes. Furthermore, GMO formation was unfavourable when catalysed by catalyst with a large pore volume. The conversion rate increased gradually with the increased cata lyst pore volume.

The pore size (average pore diameter (nm); Table 1) was correlated with product selectivity, but the outcome was irrational because the obtained average pore diam eter mostly ranged at the same peak (3.77 nm). Neverthe less, a remarkable phenomenon was observed in the BJH plot of different catalysts (Fig. 5). The high selectivity of GDO and GTO detected in the reaction mixtures using the so/-/Zr0 2 s o lge l and so/-/Zr0 2 commerci al catalysts was attributed to their macroporous sizes, which exceeded 50 nm, and mesoporous catalyst (20-50 nm). Nonethe less, the pore sizes of ZrO i-SiO i-Me&Et-PhSO 3 H and so/ -!Z r02prec i pitat ion were highly uniform and< 15 nm. The BJH plots of each catalyst clarifed the influence of pore size on product selectivity.

The FESEM images for Zr0 2 -SiO 2 -Me&Et-PhSO 3 H, so / -!Zr02so�1 , so/ -!Zr02precipitation and so / -!Zr02oommerci are presented in Fig. 6. Zr0 2 -SiO 2 -Me&Et-PhSO 3 H cata lyst (Fig. 6a) demonstrates the formation of monodis persed spherical silica particles on zirconia support which resulted from TEOS hydrolysis and succeeding condensation [START_REF] Kong | Development of a novel hydrophobic ZrO 2 -SiO 2 based acid catalyst for catalytic esterification of glycerol with oleic acid[END_REF][START_REF] Rahman | Synthesis of silica nanoparticles by sol-gel: size-dependent properties, surface modification, and applications in silica-polymer nanocomposites: a review[END_REF]. Unlike Zr0 2 -SiO y-Me&Et-PhSO 3 H, all so/-!Z r0 2 catalysts pre sented typical rough and irregular surface morphology. It can be observed that so/-lZr02 prec ipi l a t i o n exhibited rougher and higher pore space. Meanwhile, the crystallinity and morphology for SO/-/Z r02 sol-!}! I and SQ/-/Zr0 2 commertial are almost identi cal. The morphology evidenced that the nature of the zirconium precursor influences the crystalline and porous structure of the catalyst.

Correlation between hydrophobicity and selectivity/initial reaction rate

The correlation of catalyst hydrophobicity with the selec ti vity of reactions was evaluated. Figure 7 shows that the increase in hydrophobicity of the catalyst decreased the conversion rate. Linear regression with R-squared (R 2 ) of 1.0 was obtained for conversion as a fonction of hydropho bicity. It is shown that increasing catalyst hydrophobic ity causes a slight decrease in the conversion. In addition, good regression was obtained for selectivity of GMO, GDO and GTO using equation Y= y 0 + a(le-bx), which was obtained using Sigma Plot 11 software. The R 2 for GMO and GDO selectivity is 1 as experimental data are well fit ted in the equation curve. It is shown that the selectivity for GMOs increased exponentially from 8 to 13 degrees of hydrophobicity value. Meanwhile, the selectivity of GDO decreased gradually by increasing the hydrophobicity of the catalyst. In of the GTO selectivity, the R 2 was lower (0.7886), but the trend clearly shows that the selectivity GTO decreased when the hydrophobicity value of catalyst was increased. It is similar to the finding of the previous work, and higher hydrophobicity of a catalyst's surface can reduce GTO formation selectivity, which is very beneficial for GMO and GDO production. Unlike the pore textural properties, the increased hydrophobicity enhanced the GMO formation. The GDO and GTO selectivity decreased with higher hydrophobicity. Despite the decreased hydrophobicity and the total conversion of reaction at 8 h, the initial reaction rate or turnover frequency, which was calculated during the first 15-min reaction, indicated that a catalyst with improved hydrophobicity displayed an increased turnover frequency value (Table 2). Surface hydrophobicity plays an important role in esterification reactions with polyols (glycerol) [START_REF] Kotwal | Three-dimensional, mesoporous titanosilicates as catalysts for producing biodiesel and biolubricants[END_REF]. The importance of the catalytic system is vital to enhance production on GMO and GDO due to its biphasic properties; it acts as important emulsifiers in the personal care and food industry. The increased selectivity of GTO resulted in poor dispersion of emulsion.

Conclusions

This study proved that ZrO 2 -SiO 2 -Me&Et-PhSO 3 H catalyst with a hydrophobic surface feature is a potential heterogeneous catalyst for glycerol esterification. In brief, the comparative study on ZrO 2 -SiO 2 -Me&Et-PhSO 3 H with different SO 4 2-/ZrO 2 catalysts validated the following points: (i) the structural crystallinity of SO 4 2-/ZrO 2 is affected by the type of zirconia precursor and (ii) the importance of catalyst pore volume or high active sites accessibility for the organic reactants. The catalyst pore volume was correlated with the selectivity of the reaction; a large pore volume enabled the formation of large-molecular-size GDO and GTO. It is confirmed that highly uniform pores measuring < 5 nm attained high GMO selectivity. In addition, this study has proven that the activity and selectivity of catalytic reactions are dependent on the catalyst properties such as pore volume and pore size, catalyst acidity and hydrophobicity. Nevertheless, this work proved that ZrO 2 -SiO 2 -Me&Et-PhSO 3 H with consistent narrow pore volume (0.0247 cm 3 /g) and the hydrophobic property is the potential acid catalyst for the production of GMO, whereas SO 4 2-/ZrO 2 sol-gel is more suited for GDO production. This study has proven that designated catalysts with specific pore volume and hydrophobic characteristics can produce the increasingly important GMO and GDO lipids, particularly in the health care and wellness sector, in order to formulate potential vehicles of drugs and useful bioactive compound. The thermodynamics of the catalytic reaction and modelling of the correlation between structure and selectivity of the product can be framed in the future context of the study.

Fig. 1

 1 Fig. 1 TGA curves for a ZrO 2 -SiO 2 -Me&Et-PhSO 3 H b SO 4 2-/ZrO 2 sol-gel c SO 4 2-/ZrO precipitation d SO 4 2-/ZrO 2 commercial

Figure 3

 3 Figure 3 demonstrates the XRD patterns for various catalysts. The XRD results indicated that ZrO 2 -SiO 2 -Me&Et-PhSO 3 H catalyst mainly comprises monoclinic phase and a minor tetragonal phase. A comparison of the XRD results of different types of SO 4 2-/ZrO 2 catalysts with ZrO 2 -SiO 2 -Me&Et-PhSO 3 H can provide a correlation between the structural properties and the selectivity of the product mixture.All SO 4 2-/ZrO 2 catalysts possessed a highly tetragonal phase and less monoclinic phase. The acidic tetragonal phase may be stabilised by sulphated group(Oh et al. 2013b). XRD results also clearly revealed that

Fig. 5 Fig. 6 Fig. 7 •

 567 Fig. 5 BJH plots of a ZrO 2 -SiO 2 -Me&Et-PhSO 3 H, b SO 4 2-/ ZrO 2 sol-gel , c SO 4 2-/ZrO 2 precipitation and d SO 4 2-/ZrO 2 commercial

Table 1

 1 Textural properties of the different types of SO 4

			2-	/ZrO 2 catalysts and ZrO 2 -SiO 2 -Me&Et-PhSO 3 H catalyst
	Catalysts	BET Area a (m 2 /g)	Pore volume b (cm 3 /g)	Average pore diameter (nm)	Acidity (mmol/g)	Particle size distribution c (µm)	Contact angle analysis d (degree)
	ZrO 2 -SiO 2 -Me&Et-PhSO 3 H	79.75	0.0247	3.77	0.62	5.01	41.5
	SO 4 2-/ZrO 2 sol-gel	85.29	0.475		4.85	0.61	11.3	9.0
	SO 4 2-/ZrO 2 precipitation	44.91	0.079		3.77	0.35	4.36	12.1
	SO 4 2-/ZrO 2 commercial	60.06	0.240		3.77	0.44	12.5	10.8

a Total surface area was calculated using Brunauer-Emmett-Teller (BET) equation b Barrett-Joyner-Halenda (BJH) method was employed in determining pore volume and average pore diameter c Particle size distribution of catalyst was measured by Malvern particle sizer d Contact angle analysis was employed for measuring catalyst hydrophobicity

Acknowledgement The authors acknowledge the financial support provided by INCREASE CNRS France and supported by the Fundamental Research Grant Scheme (Malaysia, FRGS/1/2019/STG05/UNIM/02/2). Dual PhD scholarship (University of Malaya) and French government scholarship (Embassy France of Kuala Lumpur) are gratefully acknowledged. The technical support and facilities from Laboratoire de Génie Chimique and Sime Darby Plantation Research Sdn. Bhd. are highly appreciated.