Anaïs Lavoisier

Alejandro Avila-Sierra

Carsten Timpe

Peter Kuehl

Leonie Wagner

Carole Tournier

Marco Ramaioli
email: marco.ramaioli@inrae.fr

Anais Lavoisier

Addresses

A novel soft robotic pediatric in vitro swallowing device to gain insights into the swallowability of mini-tablets Authors

Keywords: Soft robotics, swallowing, food carrier, flexible solid oral dosage forms, minitablet, size, Highlights

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

INTRODUCTION

Children need age-appropriate pharmaceutical formats, specifically designed, developed, and evaluated for pediatric use (Mistry, Batchelor, and Uk 2017;[START_REF] Ternik | Assessment of Swallowability and Palatability of Oral Dosage Forms in Children: Report from an M-CERSI Pediatric Formulation Workshop[END_REF]. There, acceptability is particularly important to achieve better clinical outcomes, as well as improving the quality of life of young patients. According to the European Medicines Agency (EMA), acceptability assessment must be included in the pediatric pharmaceutical development program of new formulations and described in the pediatric investigation plan (Committee for Medical Products for Human Use and Paediatric Committee 2013). However, knowledge in this area is still limited, and different methodologies may be implemented due to uncertainties of regulatory requirements as no international consensus has been established yet [START_REF] Ternik | Assessment of Swallowability and Palatability of Oral Dosage Forms in Children: Report from an M-CERSI Pediatric Formulation Workshop[END_REF].

Acceptability can be defined as an overall ability of the patient and caregiver to use a medicinal product as intended or authorized [START_REF] Kozarewicz | Regulatory Perspectives on Acceptability Testing of Dosage Forms in Children[END_REF]. The acceptability of oral dosage forms depends mainly on their palatability and swallowability, which can be evaluated during clinical trials in relevant patient populations. Concerning solid oral dosage forms (SODF), size and shape, taste and after taste, dose volume, and ease of administration are generally considered as critical acceptability attributes [START_REF] Kozarewicz | Regulatory Perspectives on Acceptability Testing of Dosage Forms in Children[END_REF][START_REF] Walsh | Patient Acceptability, Safety and Access: A Balancing Act for Selecting Age-Appropriate Oral Dosage Forms for Paediatric and Geriatric Populations[END_REF].

Tablets and capsules are not recommended for children under 6 years old because of swallowing difficulties and risk of choking. However, mini-tablets (≤ 4 mm in diameter), considered suitable for children between 2 and 5 years (European Medicines Agency 2013; [START_REF] Mistry | Evidence of Acceptability of Oral Paediatric Medicines: A Review[END_REF], are particularly interesting as they combine the stability of SODF with the dosage flexibility of liquids [START_REF] Mistry | Evidence of Acceptability of Oral Paediatric Medicines: A Review[END_REF][START_REF] Van Riet-Nales | Safe and Effective Pharmacotherapy in Infants and Preschool Children: Importance of Formulation Aspects[END_REF]. Several clinical studies with placebos have shown that 2 years old children not only are able to swallow a mini-tablet but was also preferred over alternative formulations like powders, suspensions, or syrups [START_REF] Klingmann | Favorable Acceptance of Mini-Tablets Compared with Syrup: A Randomized Controlled Trial in Infants and Preschool Children[END_REF][START_REF] Klingmann | Acceptability of Uncoated Mini-Tablets in Neonates-A Randomized Controlled Trial[END_REF][START_REF] Musiime | The Pharmacokinetics and Acceptability of Lopinavir/Ritonavir Minitab Sprinkles, Tablets, and Syrups in African HIV-Infected Children[END_REF][START_REF] Riet-Nales | Acceptability of Different Oral Formulations in Infants and Preschool Children[END_REF][START_REF] Spomer | Acceptance of Uncoated Mini-Tablets in Young Children: Results from a Prospective Exploratory Cross-over Study[END_REF][START_REF] Thomson | Minitablets: New Modality to Deliver Medicines to Preschool-Aged Children[END_REF]). However, large volumes are often required, and it is still unclear how many mini-tablets can be administered at once to a 2-year-old child, even if early studies have shown that young children can swallow several mini-tablets at a time [START_REF] Klingmann | Acceptability of Multiple Uncoated Minitablets in Infants and Toddlers: A Randomized Controlled Trial[END_REF][START_REF] Kluk | Can Preschool-Aged Children Swallow Several Minitablets at a Time? Results from a Clinical Pilot Study[END_REF].

The administration method could also be determinant for the acceptability of such multiparticulate formulations. In clinical studies, single mini-tablets are generally placed on the child's tongue and accompanied by a drink of choice such as water, milk or juice [START_REF] Klingmann | Favorable Acceptance of Mini-Tablets Compared with Syrup: A Randomized Controlled Trial in Infants and Preschool Children[END_REF][START_REF] Spomer | Acceptance of Uncoated Mini-Tablets in Young Children: Results from a Prospective Exploratory Cross-over Study[END_REF][START_REF] Thomson | Minitablets: New Modality to Deliver Medicines to Preschool-Aged Children[END_REF], whilst larger amounts of minitablets ( 5to 100) are mixed with soft foods like jelly, yoghurt or mashed fruits, and administered on a spoon [START_REF] Klingmann | Acceptability of Multiple Uncoated Minitablets in Infants and Toddlers: A Randomized Controlled Trial[END_REF][START_REF] Kluk | Can Preschool-Aged Children Swallow Several Minitablets at a Time? Results from a Clinical Pilot Study[END_REF]. Among soft foods, applesauce, yoghurt, and puddings are often recommended as swallowing-assistive vehicles [START_REF] Freerks | Safe, Swallowable and Palatable Paediatric Mini-Tablet Formulations for a WHO Model List of Essential Medicines for Children Compound -A Promising Starting Point for Future PUMA Applications[END_REF][START_REF] Lee | Sprinkle Formulations-A Review of Commercially Available Products[END_REF][START_REF] Ternik | Assessment of Swallowability and Palatability of Oral Dosage Forms in Children: Report from an M-CERSI Pediatric Formulation Workshop[END_REF]), but literature demonstrating the suitability of these soft foods as carriers for young children is still limited. According to [START_REF] Kluk | Can Preschool-Aged Children Swallow Several Minitablets at a Time? Results from a Clinical Pilot Study[END_REF], the use of a jelly medium to swallow multiple mini-tablets avoided the spreading of units inside the oral cavity, helped deglutition, and protected children (2 to 3 years old) from choking. Similarly, [START_REF] Klingmann | Acceptability of Multiple Uncoated Minitablets in Infants and Toddlers: A Randomized Controlled Trial[END_REF] reported that the administration of high numbers of mini-tablets with soft foods instead of drinks improved their acceptability by children between 2 and 5 years of age.

Currently, the swallowability of multi-particulate formulations is evaluated during clinical studies by direct observation of the child's mouth after administration [START_REF] Bracken | Can Children Swallow Tablets? Outcome Data from a Feasibility Study to Assess the Swallowability and Acceptability of Different Sized Placebo Tablets in Children and Young People (Creating Acceptable Tablets -Cat)[END_REF][START_REF] Klingmann | Favorable Acceptance of Mini-Tablets Compared with Syrup: A Randomized Controlled Trial in Infants and Preschool Children[END_REF][START_REF] Klingmann | Acceptability of Uncoated Mini-Tablets in Neonates-A Randomized Controlled Trial[END_REF][START_REF] Klingmann | Acceptability of Multiple Uncoated Minitablets in Infants and Toddlers: A Randomized Controlled Trial[END_REF][START_REF] Kluk | Can Preschool-Aged Children Swallow Several Minitablets at a Time? Results from a Clinical Pilot Study[END_REF][START_REF] Münch | Acceptability of Small-Sized Oblong Tablets in Comparison to Syrup and Mini-Tablets in Infants and Toddlers: A Randomized Controlled Trial[END_REF][START_REF] Spomer | Acceptance of Uncoated Mini-Tablets in Young Children: Results from a Prospective Exploratory Cross-over Study[END_REF][START_REF] Thomson | Minitablets: New Modality to Deliver Medicines to Preschool-Aged Children[END_REF] or from parents reports about problems experienced during administration at home [START_REF] Musiime | The Pharmacokinetics and Acceptability of Lopinavir/Ritonavir Minitab Sprinkles, Tablets, and Syrups in African HIV-Infected Children[END_REF][START_REF] Riet-Nales | Acceptability of Different Oral Formulations in Infants and Preschool Children[END_REF]. Nevertheless, pharmaceutical companies need guidance to identify the key attributes that can improve swallowability before starting clinical trials in order to reduce iterations during product development and improve patient safety [START_REF] Kozarewicz | Regulatory Perspectives on Acceptability Testing of Dosage Forms in Children[END_REF][START_REF] Ternik | Assessment of Swallowability and Palatability of Oral Dosage Forms in Children: Report from an M-CERSI Pediatric Formulation Workshop[END_REF]. In this context, in vitro models of swallowing, and particularly soft robotics, could help to clarify the mechanisms involved during SODF swallowing by young children.

Different in vitro models have already been used to elucidate the relations between the physical properties of a bolus and its flow during the oral, pharyngeal, and oesophageal phases of swallowing in adults (Marconati et al. 2019;[START_REF] Qazi | In Vitro Models for Simulating Swallowing[END_REF]. Regarding pharmaceutical formulations, previous works used an experimental setup called the "artificial throat" [START_REF] Mowlavi | In Vivo Observations and in Vitro Experiments on the Oral Phase of Swallowing of Newtonian and Shear-Thinning Liquids[END_REF] to investigate the swallowing dynamics of a bolus with pellets (Marconati et al. 2019) and the dynamics of different combinations of liquid carriers and SODF, considering the impact on swallowing of both shear and extensional rheology [START_REF] Lavoisier | Effect of the Rheological Properties of the Liquid Carrier on the in Vitro Swallowing of Solid Oral Dosage Forms[END_REF], Marconati et al. Food Function, 2020). This in vitro model reproduces the peristaltic motion induced by the tongue during the oral phase of swallowing in adults, but it represents a strong simplification of the shape of the oral cavity and the tongue, using rigid materials. A more realistic adult in vitro swallowing model based on soft robotics has been recently developed (Marconati et al. 2020).

No in vitro models have been used yet to gain insights on SODF swallowing by young children, due to the specific anatomical and physiological features.

To investigate the swallowability of multi-particulate pediatric oral formulations, this study developed a soft-robotic in vitro device that, adapting the soft-robotic tongue proposed by Marconati et al., (2020), reproduces the key features of the anatomy and swallowing physiology of a 2-year-old child. This novel in vitro model has been used to investigate the swallowability of mini-tablets under different scenarios: (i) testing the effect of semi-solid carriers (apple puree, stirred yoghurt, and xanthan gum solutions), (ii) the effect of the size of the particles, (iii) of their volume fraction in the bolus and finally, (iv) comparing different administration methods (e.g., on the tongue, mixed with the carrier).

MATERIALS & METHODS

Materials

Carriers and insalivation ratio

This study considered two food carriers, apple puree ("Pomme nature", 100 g, Andros France SNC, Biars-sur-Cère, France) and stirred yoghurt ("Velouté yaourt nature brassé", 125 g, Danone SA, Paris, France). Three different concentrations of xanthan gum (0.25, 0.5, and 1 % w/v) in mineral water (Vittel) were also used (43708, xanthan from Xanthomonas campestris, Sigma-Aldrich, St. Louis, MO, USA). Each food carrier was diluted with artificial saliva [START_REF] Brodkorb | INFOGEST Static in Vitro Simulation of Gastrointestinal Food Digestion[END_REF][START_REF] Minekus | A Standardised Static in Vitro Digestion Method Suitable for Food -an International Consensus[END_REF] to mimic saliva incorporation during bolus formation in the mouth before swallowing. In this study the artificial saliva did not contain αamylase. No information about saliva incorporation during bolus formation by toddlers was found in the literature, but salivary flows seem to be similar between 2 years old and adults (i.e., basal salivary flow rate (0.2-1 mL/min) and stimulated salivary flow rate (0.5-5 mL/min)) [START_REF] Wollmer | Review of Paediatric Gastrointestinal Physiology Relevant to the Absorption of Orally Administered Medicines[END_REF]. Therefore, the amount of saliva incorporated when eating semi-solid food products was estimated with adult volunteers, and calculated according to [START_REF] Drago | Relationships between Saliva and Food Bolus Properties from Model Dairy Products[END_REF]. Briefly, five healthy volunteers (31 ± 10 years old) took a teaspoon of apple puree or yoghurt, kept the product in mouth for 30 s, and spat it in a container. The ratio of saliva added in the bolus with respect to the wet food sample (hw) was 0.23 ± 0.10, meaning that approx. 0.2 g of saliva were incorporated /g of semi-solid food. Based on these preliminary tests, the dilution ratio (carrier: artificial saliva) was fixed at 5:1.

Minitablets

All formulations were placebos. Minitablets of 1.8, 2.3, 2.5, and 3 mm in diameter were produced and provided by F. Hoffmann La Roche AG (Basel, CH). Minitablets were coated with Surelease ® (E-7-7050, Colorcon, Darford, Kent, UK) to avoid swelling and solubilization during swallowing experiments. The density of the coated minitablets was 1.3 g/mL.

Rheological properties

The rheological properties of the carriers were assessed with a Modular Compact Rheometer 301 (Anton Paar GmbH, Graz, Austria) at 20°C. A concentric cylinder geometry (CC27) was used to measure the properties of the apple puree and XG samples, and a parallel-plate system (PP50, 1 mm gap) with a rough surface was used for yoghurt samples. Shear viscosity of the carriers was evaluated by steady shear tests in a range of shear rates between 1 and 500 reciprocal seconds, and their yield stress was estimated through steady stress tests by increasing the shear stress from 0.01 to 30 Pa for all samples.

In vitro swallowing with the Pediatric Soft Robotic Tongue (PSRT)

A pediatric version of the soft robotic tongue proposed by Marconati et al., (2020) was developed to simulate the oral phase of swallowing of 2-year-old children. This model relies on physiological parameters from human studies available in the literature, as detailed in the following sections.

Oral cavity

The design of the oral cavity was adapted from a model of the oral airway constructed by [START_REF] Xi | Transport and Deposition of Micro-Aerosols in Realistic and Simplified Models of the Oral Airway[END_REF] based on CT scans of a healthy adult and measurements reported in the literature. This model was modified to include the functionality of the tongue (Marconati et al. 2020). A smaller version of this 3D model was designed to stimulate the morphology of the oral cavity at 24 months by selecting relevant anatomical features and measurements from clinical studies on healthy children around this age reported in the literature (Figure 1a).

First, the distance between the inner side of the lips to the posterior wall of the pharynx reported by [START_REF] Bickmann | In Vitro Determination of Respimat® Dose Delivery in Children: An Evaluation Based on Inhalation Flow Profiles and Mouth-Throat Models[END_REF] based on NMR/CT scans of children between 2 and 3 years old in Germany was considered as the length of the oral cavity (i.e., 58 mm). Then, the width of the back of the oral cavity (i.e., 35 mm) was considered as the width of the dental arch between the mandibular 2 nd molars measured by [START_REF] Foster | Dentition and Dental Arch Dimensions in British Children at the Age of 2 to 3 Years[END_REF] on the primary dentition of British children between 2 ½ and 3 years old. Finally, the values extracted by [START_REF] Vorperian | Development of Vocal Tract Length during Early Childhood: A Magnetic Resonance Imaging Studya)[END_REF] from observations by MRI of the hard and soft tissue vocal tract structures of 2 years old children in Wisconsin (USA), and particularly the hard and soft palate lengths, were considered as a representative measure of the arc length of the oral cavity (ie., 66 mm).

The top of the oral cavity was pierced with 4 holes located at 1, 5.8, 22.8 and 44.8 mm from the tongue tip along the sagittal direction. Two large holes with 1/8" metallic nuts glued on top were used to tightly screw pressure transducers to record the dynamic evolution of palatal pressure during swallowing tests of liquid samples. The last two were used as feeding holes: a large one (14 mm diameter) to feed small solids like mini-tablets or foods containing large particles (hermetically sealed with a rubber stopper during the swallowing tests), and a small one (4 mm diameter) where a plastic tube was fitted and used to fed liquids from a syringe pump. The oral cavity was 3D printed in a transparent material (VeroClear® resin) to allow bolus movement observation during swallowing.

Soft robotic tongue

A soft actuator was designed by Marconati et al., (2020) to reproduce the peristaltic movement of the tongue. This soft robotic tongue was made up of two air chambers that can be inflated and deflated independently to reproduce key lingual functions: bolus containment prior to swallowing, and bolus propulsion during the oral phase of swallowing. The shape obtained during a swallowing test has been compared qualitatively against in vivo ultrasound imaging of the tongue [START_REF] Mowlavi | In Vivo Observations and in Vitro Experiments on the Oral Phase of Swallowing of Newtonian and Shear-Thinning Liquids[END_REF]. The tongue was produced by casting silicone rubber (Smooth-On Eco-flex 00-30) mixed with 0.5% w/w of a nonionic surfactant (sorbitan mono-oleate, Span 80, CAS: 1338-43-8, from Sigma Aldrich) in a mold. Mechanical properties and wettability were similar to the human tongue (Marconati et al. 2020). This soft actuator was adapted to mimic the tongue of a 2-year-old child by scaling down the adult model to obtain a pediatric soft tongue of 46 mm length and 18 to 32 mm, proportional to the pediatric oral cavity previously designed (Figure 1b).

Swallowing pattern

The soft robotic tongue control system previously developed by Marconati et al., (2020) was used, with slight modifications to better simulate relevant physiological features of the swallowing pattern at 24 months. According to [START_REF] Potter | Tongue Strength in Children With and Without Speech Sound Disorders[END_REF] the maximum tongue strength of 3 years old children is approx. 20 kPa (no data has been reported in the literature for younger children). Since the tongue strength used during swallowing is around 40% of the maximum tongue strength [START_REF] Ferris | Pressure-Flow Analysis for the Assessment of Pediatric Oropharyngeal Dysphagia[END_REF][START_REF] Park | Comparison of Maximal Tongue Strength and Tongue Strength Used during Swallowing in Relation to Age in Healthy Adults[END_REF][START_REF] Rommel | Videomanometry Reveals Clinically Relevant Parameters of Swallowing in Children[END_REF], the maximal pressure applied against the palate should be around 8 kPa in the PSRT. The target inflation pressures for the anterior and posterior chambers of the tongue were adjusted accordingly. Various studies have shown that swallowing coordination and oropharyngeal transit time are similar between healthy adults and young children [START_REF] Almeida | Assessment of Swallowing Sounds by Digital Cervical Auscultation in Children[END_REF][START_REF] Frakking | Acoustic and Perceptual Profiles of Swallowing Sounds in Children: Normative Data for 4-36 Months from a Cross-Sectional Study Cohort[END_REF][START_REF] Rommel | Videomanometry Reveals Clinically Relevant Parameters of Swallowing in Children[END_REF][START_REF] Rommel | Objective Assessment of Swallow Function in Children With Suspected Aspiration Using Pharyngeal Automated Impedance Manometry[END_REF], therefore, the actuation sequence described by Marconati et al., (2020) with no delay between the deflation of the posterior chamber and the inflation of the anterior chamber (tA = 0 ms) was chosen.

In vitro swallowing conditions

The volume of food typically swallowed by 2-year-old children is considered to be 5 mL [START_REF] Jones | Volume of a Swallow[END_REF][START_REF] Vaiman | Surface Electromyographic Studies of Swallowing in Normal Children, Age 4-12 Years[END_REF]. MT were therefore administered with 5 mL of carrier. To study the effect of the administration method on the swallowability of the formulations three different configurations were used: (1) MT were mixed with the carrier in the oral cavity (2.5 mL of the carrier fed first and MT spread on top before feeding the last 2.5 mL), (2) MT were directly spread on the tongue before adding the 5 mL of carrier, and (3) MT were mixed with the 5 mL of carrier in a beaker and fed together to ensure an homogeneous distribution of MT in the bolus. A first swallow, with the studied carrier only, was performed before to lubricate the oral cavity. Swallowing tests were done at room temperature (ca. 20°C).

Measured variables

Swallowed MT (%)

The number of particles successfully swallowed during the in vitro swallowing test was monitored. MT were counted by visual examination of the swallowed bolus. Results are expressed in terms of percentage of total MT initially fed.

Bolus velocity (s)

The velocity of the bolus was measured during the swallowing test. A high-speed camera (model ac A2040-120 um, Basler, Ahrensburg, Germany) was used to record the bolus transit at 150 frames per seconds. The characteristic oral transit time was defined as the time required for the bolus front to exit the oral cavity (bolus FO).

Carrier residues (%)

Residues left in the oral cavity after the swallowing test were also monitored. The container receiving the sample propelled out of the PSRT was weighted before and after the test. The amount of carrier residues left in the oral cavity was calculated as follow:

𝑚 𝑚 𝑚 (1) 𝑚 𝑚 𝑚 (2)
Results are expressed in terms of percentage of total carrier initially fed.

Palatal pressure (kPa)

The dynamic evolution of palatal pressure (mid and post-palate) during swallowing tests was recorded by two piezoresistive pressure sensors (model PX2AG2XX002BAAAX from Honeywell, MN, USA) housed in the holes of the rigid palate. These sensors were used to determine the pressures involved in the in vitro oral transit of the carriers. Results are expressed as a relative pressure in kPa.

Adhesion measurements

A TAHD Texture Analyzer (Stable Micro Systems, Surrey, UK) with a 500 g load cell was used to measure the adhesion between a MT coated with Surelease and the surface of the silicon tongue described in section M&M 3.2. The MT was fixed to a 2 cm diameter compression platen probe using double-sided adhesive tape, while the tongue was fixed on the lower platform of the instrument.

To study the effect of the carriers on the adhesion between the MT and the tongue, 30 µL droplets of the target carrier were placed on the top of the tongue surface before testing; this amount of liquid fully covered the MT during testing.

The 'hold until time' mode was used with five seconds of holding time making contact, a testspeed of 1 mm/s, a maximum compression force Fmax of 0.05 N, and a trigger force of 0.005 N (Figure 2). The study was carried out at room temperature. Adhesion force was quantified as the hysteresis upon retraction of the MT from the tongue. Adhesion measurements were carried out over three different locations on the silicon tongue per sample, with at least three compressions per location.

Statistical analysis

Results are shown in terms of the mean ± SD. The Kruskall-Wallis test on ranks was used to study differences among samples. Conover-Iman test was then used to determine the significant differences between samples (p < 0.05). All analyses were performed with XLSTAT statistical software (version 2020.3.1.27, Microsoft Excel, Adinsoft, Paris, France).

The rheological properties of the semi-solid carriers were investigated since they influence the ability of maintaining particles in suspension, the palatability (or mouthfeel), and the swallowability of multi-particulates [START_REF] Kluk | Application Properties of Oral Gels as Media for Administration of Minitablets and Pellets to Paediatric Patients[END_REF][START_REF] Lopez | Effect of Formulation Variables on Oral Grittiness and Preferences of Multiparticulate Formulations in Adult Volunteers[END_REF][START_REF] Steele | The Influence of Food Texture and Liquid Consistency Modification on Swallowing Physiology and Function: A Systematic Review[END_REF]. Flow curves obtained in steady shear are presented in Figure 3a.

In adults, the shear rates (𝛾 during human swallowing have been estimated to be in a range between 1 s -1 in the mouth, and up to 1000 s -1 in the pharynx [START_REF] Gallegos | Rheology and Dysphagia: An Overview[END_REF][START_REF] Nishinari | The Food Colloid Principle in the Design of Elderly Food: FOOD COLLOID PRINCIPLE[END_REF]. The shear rheology of texture modifiers and food products is commonly reported at 𝛾 = 50 s -1 . Oral shear rates for 2-year-old are unknown, and there is no consensus on the shear rates most representative of food oral processing in infants and young children [START_REF] Makame | Nutrient Density of Common African Indigenous/Local Complementary Porridge Samples[END_REF][START_REF] Steele | The Influence of Food Texture and Liquid Consistency Modification on Swallowing Physiology and Function: A Systematic Review[END_REF][START_REF] Sukkar | Optimizing Texture Modified Foods for Oro-Pharyngeal Dysphagia: A Difficult but Possible Target?[END_REF].

All the samples showed a shear thinning behaviour (i.e., viscosity decreased rapidly with increasing shear rate) in the whole range of shear rates studied (1 -500 s -1 ). The XG suspensions had a more pronounced shear thinning behavior than the food carriers. The apple puree diluted with artificial saliva and XG 1 % had similar shear viscosities at 𝛾 = 50 s -1 (587 ± 14 and 460 ± 59 mPa.s, respectively); the yoghurt diluted with artificial saliva and XG 0.5 % had similar shear viscosities at 𝛾 = 50 s -1 (209 ± 5 and 167 ± 1 mPa.s, respectively). Compared to Newtonian fluids like syrups, shear thinning fluids require less efforts in oral processing and swallowing [START_REF] Steele | The Influence of Food Texture and Liquid Consistency Modification on Swallowing Physiology and Function: A Systematic Review[END_REF]) but could also be less effective in "masking" the presence of particles in the mouth as their viscosity decreases under the relatively high shear rates experiences during oral processing [START_REF] Steele | The Influence of Food Texture and Liquid Consistency Modification on Swallowing Physiology and Function: A Systematic Review[END_REF].

The yield stress values estimated for the different carriers are presented in Figure 3b. The yield stress is the force required to break down the internal structure of a material for flowing [START_REF] Cichero | Thickened Liquids for Children and Adults with Oropharyngeal Dysphagia: The Complexity of Rheological Considerations[END_REF][START_REF] Zargaraan | Rheological Aspects of Dysphagia-Oriented Food Products: A Mini Review[END_REF]). It provides valuable information about the effort needed to swallow a bolus: when τ0 increases the tongue force necessary to make the carrier flow will also increase [START_REF] Cichero | Thickened Liquids for Children and Adults with Oropharyngeal Dysphagia: The Complexity of Rheological Considerations[END_REF][START_REF] Malouh | Are Medication Swallowing Lubricants Suitable for Use in Dysphagia? Consistency, Viscosity, Texture, and Application of the International Dysphagia Diet Standardization Initiative (IDDSI) Framework[END_REF]). The XG suspension 1 % had the highest estimated τ0 (12.2 ± 0.9 Pa) and XG 0.25 % the lowest (0.9 ± 0.2 Pa).

Intermediate τ0 were found for XG 0.5 % and the food carriers diluted with artificial saliva (between 2 and 5 Pa).

Mini-tablets swallowability in vitro

Effect of the type of semi-solid carrier on mini-tablets swallowability in vitro

The carriers detailed in section 1.2 were used to swallow 64 MT of 3 mm in diameter, which corresponds to a particle volume fraction of 0.20. Here, MT were mixed with the carrier in the oral cavity (i.e., administration method (1) cf. section M&M 3.4). The in vitro swallowing results are presented in Figure 4, and images of the PSRT containing the bolus (MT + carrier) before triggering the swallow are shown in Figure 5.

Overall, high success rates were reached with more than 75 % of the MT successfully swallowed (Fig. 4a) but differences were observed depending on the rheological properties of the carriers. A lower number of MT were swallowed with XG 0.25 % in these conditions (77 ± 7 %). This carrier had a low shear viscosity (ca. 60 mPa.s at 𝛾 = 50 s -1 ), and a low yield stress (0.9 Pa). Consequently, MT were able to sediment in the bolus during the initial part of the test and were positioned on the tongue when the swallow was triggered (Fig. 5c). Interactions between the artificial tongue and MT, such as adhesion, may affect the swallow of the particles.

Moreover, it has been shown in vitro that low viscosity fluids are not the most efficient carriers for SODF as they tend to flow faster than the particles which lag behind the liquid bolus [START_REF] Lavoisier | Effect of the Rheological Properties of the Liquid Carrier on the in Vitro Swallowing of Solid Oral Dosage Forms[END_REF][START_REF] Marconati | An in Vitro Experiment to Simulate How Easy Tablets Are to Swallow[END_REF].

With XG 0.5 % and the yoghurt diluted with artificial saliva, MT also sedimented on the tongue (Fig. 5d) but the effect was much reduced, and almost all the MT were successfully swallowed (≥ 90 %). These two carriers had higher shear viscosities and yield stresses (η 167 and 209 mPa.s at 𝛾 = 50 s -1 and τ0 of 4.1 and 2.4 Pa, respectively) than XG 0.25 %.

When swallowed with XG 1 % and the apple puree diluted with artificial saliva, very high success rates were also reached (≥ 90 %). In this case, the shear viscosity of the carriers was too high for the MT to sediment in the bolus (η between 460 and 590 mPa.s at 𝛾 = 50 s -1 ) (Fig. 5e). Interestingly, the apple puree gave better results than XG 1% (96 ± 2 % against 90 ± 2 %, respectively), which may be caused by the XG 1% high yield stress (4.5 for the apple puree vs.

12.2 for XG 1 %) or the slightly higher viscosity of apple puree at shear rates above 100 s -1 .

These results are consistent with a previous study (Marconati et al. 2019) in which a critical viscosity threshold for smooth swallowing was observed both in vivo and in vitro. Differences between carriers above η = 45 mPa.s at 𝛾 = 50 s -1 did not result in a significant improvement of multi-particulates palatability and oral transport. Similarly, [START_REF] Lopez | The Effect of Administration Media on Palatability and Ease of Swallowing of Multiparticulate Formulations[END_REF] observed in vivo that multi-particulates were easier to swallow when they were dispersed in polymeric hydrogels compared to water. No significant differences were found between hydrogels with different rheological properties (η between 70 and 1150 mPa.s at 𝛾 = 50 s -1 ), even if participants reported differences in terms of mouthfeel perception and tended to prefer samples with thin and middlerange consistencies as opposed to thicker samples [START_REF] Lopez | The Effect of Administration Media on Palatability and Ease of Swallowing of Multiparticulate Formulations[END_REF].

Bolus velocity and post-swallow residues were not influenced by the type of carrier used. Bolus FO was measured between 0.16 and 0.19 s (Fig. 4b) which is coherent with the physiological duration of the oral phase of swallowing (i.e., < 0.5 s) [START_REF] Almeida | Assessment of Swallowing Sounds by Digital Cervical Auscultation in Children[END_REF][START_REF] Frakking | Acoustic and Perceptual Profiles of Swallowing Sounds in Children: Normative Data for 4-36 Months from a Cross-Sectional Study Cohort[END_REF][START_REF] Rommel | Videomanometry Reveals Clinically Relevant Parameters of Swallowing in Children[END_REF][START_REF] Rommel | Objective Assessment of Swallow Function in Children With Suspected Aspiration Using Pharyngeal Automated Impedance Manometry[END_REF]. Carrier post-swallow residues were low, between 5 and 10 % of the initial amount of carrier fed (Fig. 4c), which indicates that all sample boluses were effectively transported and ejected during the swallowing tests. Finally, the maximum relative pressures in the oral cavity were measured mid-palate between 8 and 10 kPa and were not influenced by the type of carrier used (Appendix Fig. A).

According to these results, the in vitro swallowing tests performed with the novel Pediatric Soft Robotic Tongue (PSRT) showed physiologically relevant oral transit times and palatal pressures for 2-year-old children. Regarding multiple MT swallowability, results suggest that increasing the shear viscosity of the carrier above ~ 150 mPa.s at 𝛾 = 50 s -1 , and its yield stress up to ~ 5 Pa, can improve MT transport without decreasing the bolus velocity nor increasing post-swallow residues. Semi-solid foods such as apple puree and yoghurt therefore appear as suitable carriers to help MT swallowing in young children.

Effect of the size of the mini-tablets on their swallowability in vitro

Swallowing of multi-particulates as well as the feeling of residual particles in the mouth seem to increase with particle size (Marconati et al., 2019). In this section, diluted yoghurt with artificial saliva was used as a carrier to swallow MTs of different sizes (1.8, 2.5, and 3 mm in diameter), keeping a particle volume fraction constant of 0.20. MT were mixed with the carrier in the oral cavity (i.e., administration method (1) cf. section M&M 3.4). Results of these swallowing tests are presented in Figure 6.

Overall, neither the percentage of MTs swallowed nor the post-swallow residues were influenced by the diameter of the MTs (FIG. 6a and6c, respectively), yet bolus front out time decreased from 0.19 to 0.16 s when the MTs size increased from 1.8 to 3 mm (Fig. 6b). The inclusion of a large number of solid particles in the liquid carrier modifies the rheological properties of the resulting suspension [START_REF] Mueller | The Rheology of Suspensions of Solid Particles[END_REF], which can influence the dynamics of swallowing [START_REF] Marconati | An in Vitro Experiment to Simulate How Easy Tablets Are to Swallow[END_REF].

According to these results in vitro, reducing the size of MT did not improve swallowability for a fixed particle volume fraction. Furthermore, when using smaller MT, a higher number of particles is needed to reach a specific volume/dose which may be more complicated to manipulate for parents or caregivers. Therefore, MT with an intermediate diameter (i.e., 2 to 3 mm) seem to be a good compromise to conciliate swallowability and practicability.

with 2.5 mm MT and yoghurt diluted with artificial saliva. MT were mixed with the carrier in the oral cavity (i.e., administration method (1) cf. section M&M 3.4). The different particle volume fractions used in this section are presented in Table 1. Results of these swallowing tests are presented in Figure 7. As expected, increasing the volume fraction of MT (and the total volume of bolus swallowed) resulted in a decrease in the percentage of MT successfully swallowed, and in an increase of post-swallow residues (FIG. 7a and7c, respectively). From 0.05 to 0.20 particle volume fraction, a very high success rate was observed (≥ 90 %), whilst bolus velocity was not impacted (bolus FO between 0.18 and 0.20 s) and post-swallow residues were low (≤ 15 %). From 0.35 particle volume fraction (3.49 g of MT, 7.70 mL of total bolus), the percentage of MT swallowed decreased strongly and success rate fall under 75 %.

According to these results, it would be possible to increase the MT volume fraction up to 0.20 without influencing swallowability. However, the acceptability threshold should be evaluated by a sensory panel to evaluate mouthfeel sensations such as grittiness [START_REF] Imai | Oral Perception of Grittiness: Effect of Particle Size and Concentration of the Dispersed Particles and the Dispersion Medium[END_REF]. Data in published literature demonstrating the acceptability of large quantities of multi-particulates administered with semi-solid foods with a calibrated dosing spoon are lacking. [START_REF] Klingmann | Acceptability of Multiple Uncoated Minitablets in Infants and Toddlers: A Randomized Controlled Trial[END_REF] studied the acceptability and swallowability of multiple uncoated MT in toddlers (i.e., 2 to 5 years old). They administered an entire dose of a maximum of 400 MT with a soft food or a drink of the child's choice on a teaspoon. This number of MT would equate with a drug dose of approx. 500 mg of a drug, allowing for the administration of up to 80 mg/kg per day of the active drug. If we considered that the 400 MT of 2 mm diam.

were administered with 5 mL of carrier, it corresponds to a 0.25 particle volume fraction. The authors reported that this was the upper limit of acceptability for toddlers, which is quite consistent with our results, obtained with 2.5 mm MT.

Effect of the administration method on mini-tablets swallowability in vitro

The effect of the administration method on MT swallowability was studied with 2.5 mm MT, a particle volume fraction of 0.20, and yoghurt diluted with artificial saliva as carrier. Three different configurations were used: MT were either (1) spread on the carrier in the oral cavity,

(2) spread on the tongue before feeding the carrier, or (3) mixed with the carrier before feeding (cf. section M&M 3.4). Results of these swallowing tests are presented in Figure 8.

The percentage of MT swallowed decreased significantly when the MT were placed on the tongue (from 90 % with the administration method 1 to 61 % with method 2, Fig. 8a), whilst both bolus velocity and post-swallow residues were not influenced (Fig. 8b and8c, respectively). This may be due to adhesion between the MT and the artificial tongue impeding their flow with the carrier (cf. section R&D 2.5) or to an unfavorable position of the MT in the bolus (i.e., carrier flowing on top of the MT). The percentage of MT swallowed was even lower when the MT were previously mixed with the carrier (50 % with the administration method 3, Fig. 8a), decreasing bolus velocity (Fig. 8b) and increasing the amount of post-swallow carrier residues left in the in vitro oral cavity (Fig. 8c). This fact could be likely related to the homogeneous distribution of the MT in the carrier that would increase the contact area available between MT and the in vitro oral surfaces, increasing adhesive interactions, and reducing swallowability consequently. These results suggest that MT should not be placed on the tongue of the child first, and that MT should rather be spread on top of a spoonful of carrier than mixed with the carrier in its container. Clinical studies are however necessary to confirm these recommendations based on in vitro observations.

Adhesion between mini-tablets and the artificial tongue

Unintended adhesion of SODF to oral surfaces (e.g., mucosal tissue, tongue, teeth) is an important aspect that should be considered during pediatric drug development to improve swallowability [START_REF] Drumond | Polymer Adhesion Predictions for Oral Dosage Forms to Enhance Drug Administration Safety. Part 3: Review of in Vitro and in Vivo Methods Used to Predict Esophageal Adhesion and Transit Time[END_REF]. The results presented in the previous paragraph suggest that adhesive interactions between the MT and the artificial tongue may affect the percentage of MT swallowed in vitro (cf. section R&D 2.1 & 2.4). Therefore, adhesion phenomena between a MT coated with Surelease ® and the surface of the silicone tongue in the presence of different carriers were further investigated (air and water were used as a reference). Adhesion results are presented in Figure 9.

The strongest adhesion forces between the MT and the artificial tongue were measured in air (4.3 ± 0.5 mN) and water (3.1 ± 1.3 mN). The presence of the artificial saliva reduced the adhesion force between the MT and the artificial tongue (1.2 ± 0.6 mN). [START_REF] Pailler-Mattei | Ex Vivo Approach to Studying Bio-Adhesive and Tribological Properties of Artificial Salivas for Oral Dryness (Xerostomia)[END_REF] studied the adhesive interactions involved between a rigid indenter and ex vivo tongues of young pigs (ca. 1 year) in the presence of human saliva and salivary substitutes. They reported that bio-adhesive properties of the salivary substitutes were similar to human saliva, and they were ranged between 0.2 and 1 mN depending on the type of saliva used. Those values are fairly close to the adhesion force measured in this work, however, direct comparison is not possible due to differing testing conditions (e.g., applied force, probe speed, probe size, volume of wetting fluid, contact time).

The presence of food carriers reduced the adhesion between the MT and the artificial tongue, except for XG 0.25%, which showed adhesion forces similar to those measured in water (Fig. 9). Adhesive interactions may therefore have hindered the swallowability of MT with XG 0.25% (cf. section R&D 2.1). The stronger MT sedimentation with XG 0.25% may also contribute to this poor swallowing performance.

Finally, we observed that the apple puree tended to reduce the adhesion force slightly more than the yoghurt (Fig. 9). This could justify the trend in swallowability presented in Figure 4a, although the differences observed for these two carriers were not significant. This may be related to the different composition and structure of these two products, which can influence their adhesion and spread on the tongue surface [START_REF] Dresselhuis | Fat Retention at the Tongue and the Role of Saliva: Adhesion and Spreading of "protein-Poor" versus "Protein-Rich" Emulsions[END_REF][START_REF] Fan | 3D Enabled Facile Fabrication of Substrates with Human Tongue Characteristics for Analysing the Tribological Behaviour of Food Emulsions[END_REF], as well as affect the specific energy of the solid-liquid interface.

Limitations and perspectives for future studies

In this paragraph the limitations of this study will be described, also in view of identifying interesting future research directions. In this study, the yoghurt and apple puree were diluted with a relevant amount of water to imitate the insalivation ratio measured in adults in vivo. It was also verified that the rheology of these two soft foods is not affected by the contact with salivary amylase (unpublished data). However, the presence of a salivary lubrication layer was not considered, nor its peculiar rheological properties. Considering these aspects is certainly an interesting research direction for the future. Similarly, the PSRT could be improved by considering the natural roughness induced by the tongue's papillae, taking inspiration by some recent in vitro tribology studies (Andablo-Reyes et al. 2020;Mantelet et al. 2020;Srivastava et al. 2021;Wang, Zhu, and Chen 2021).

CONCLUSIONS

AFigure 1 :

 1 Figures captions :

Figure 2 :

 2 Figure 2: Test used to measure the adhesion between a MT coated with Surelease and the surface of the soft robotic tongue.

Figure 3 :

 3 Figure 3: (a) Shear viscosity as a function of shear rate for the different carriers and (b) yield stress of the different carriers.

Figure 4 :

 4 Figure 4: (a) % of MT swallowed, (b) bolus velocity (s), (c) % of carrier residues depending on the semi-solid carrier used. Red letters indicate significant differences (p < 0.05).

Figure 5 :

 5 Figure 5: Initial position of the MT (64 MT , 3 mm in diameter) before an in vitro swallow. Arrows indicate the position of the MT in the bolus.

Figure 6 :

 6 Figure 6: (a) % of MT swallowed, (b) bolus velocity (s), (c) % of carrier residues depending on the size of the MT. Red letters indicate significant differences (p < 0.05).

Figure 7 :

 7 Figure 7: (a) % of MT swallowed, (b) bolus velocity (s), (c) % of carrier residues depending on the particle volume fraction. Red letters indicate significant differences (p < 0.05).

Figure 8 :

 8 Figure 8: (a) % of MT swallowed, (b) bolus velocity (s), (c) % of carrier residues depending on the administration method. Red letters indicate significant differences (p < 0.05).

Figure 9 :

 9 Figure 9: Adhesion force between the MT and the artificial tongue as a function of the carrier used. Adhesion measurements in air and water are used as reference. Red letters indicate significant differences (p < 0.05).

  Figure 1 (a)

Table 1 .

 1 MT volume fraction and bolus mass used in the present study.

		MT number	MT mass (g)	Total bolus volume
	MT volume fraction
		(d. 2.5 mm)	(d. 2.5 mm)	(mL)
	0.05	22	0.34	5.26
	0.10	46	0.72	5.55
	0.15	74	1.15	5.89
	0.20	104	1.62	6.25
	0.25	139	2.17	6.67
	0.30	178	2.78	7.14
	0.35	224	3.49	7.70
	0.40	278	4.34	8.34

This study was funded by F. Hoffmann-La Roche


Chandan Yadav
email: chandan@nitc.ac.in

Sebastien Fregonese

Marco Cabbia

Marina Deng

Magali De Matos

Thomas Zimmer

Sebastien Fregonese $

Marco Cabbia $

Marina Deng $

Magali De Matos $

S-Parameter Measurement and EM Simulation of Electronic Devices towards THz frequency range

Keywords: Calibration kit Design, On-wafer S-parameter Measurement, TRL, Sub-THz, THz, EM Simulation, Electronic Devices

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

I. INTRODUCTION

Recent development in high frequency characterization equipment has enabled S-parameter measurement beyond 1 THz [START_REF] Bauwens | A 1.1 THz micromachined on-wafer probe[END_REF]. Over time, the high frequency performance of transistors fabricated on silicon substrate has also improved significantly [START_REF] Chevalier | A 55 nm triple gate oxide 9 metal layers SiGe BiCMOS technology featuring 320 GHz f T / 370 GHz f MAX HBT and high-Q millimeter-wave passives[END_REF], [START_REF] Heinemann | SiGe HBT with f T /fmax of 505 GHz/720 GHz[END_REF]. The process design kit (PDK) is one essential requirement for analog and RF circuit designs and it is usually prepared by foundries [START_REF] Fujishima | 98 mW 10 Gbps Wireless Transceiver Chipset With D-Band CMOS Circuits[END_REF]. Accurate characterization and modeling of the electronic devices in upper sub-THz and THz frequency range is essential for circuit and system design in these very high frequency ranges [START_REF] Fujishima | 98 mW 10 Gbps Wireless Transceiver Chipset With D-Band CMOS Circuits[END_REF]. It is reported that onwafer measurements of electronic devices are more accurate compared to off-wafer measurement in upper sub-THz [START_REF] Fregonese | Comparison of On-Wafer TRL Calibration to ISS SOLT Calibration With Open-Short De-Embedding up to 500 GHz[END_REF]. In order to perform on-wafer S-parameter measurement of a device under test (DUT), on-wafer calibration kit design is an essential requirement.

II. ON-WAFER CALIBRATION KIT DESIGN

To perform the on-wafer TRL calibration, an on-wafer calibration kit is designed (see Fig. 1) and fabricated on the silicon substrate [START_REF] Deng | Design of Silicon On-Wafer Sub-THz Calibration Kit[END_REF]. The kit consists of a long Line for measurement below 110 GHz and a short Line for beyond 110 GHz. In the kit, designed Thru standard length is 35 µm and designed open type Reflect standard is used in this study. A detailed description about the designed on-silicon on-wafer TRL calibration kit can be found in [START_REF] Deng | Design of Silicon On-Wafer Sub-THz Calibration Kit[END_REF]. In this work, meander line and transistor-open shown in Fig. 2 are used as DUTs. The error-terms are calculated using the designed on-wafer TRL calibration kit standards. The position of the reference plane after applying the on-wafer TRL is shown in see Fig. 2 by green dashed line. The step-by-step procedure adopted to apply the on-wafer TRL calibration on DUTs is shown in Fig. 3. To know suitability of the designed on-wafer calibration kit in the on-wafer measurement beyond 500 GHz, a close EM simulator model of a RF probe reported in [START_REF] Reck | Micromachined Probes for Submillimeter-Wave On-Wafer Measurements-Part II: RF Design and Characterization[END_REF] is developed in HFSS (see Fig. 4).

III. RESULTS AND DISCUSSIONS

The terminal capacitances of transistor-open and Sparameters of meander line are shown in Fig. 5 and Fig. 6, respectively. The terminal capacitances of transistor-open are calculated using S-parameters. In Fig. 5 and Fig. 6, orange color solid lines shows on-wafer measurements obtained using four sets of RF Picoprobe probes [START_REF] Yadav | Investigation of Variation in On-Si On-Wafer TRL Calibration in Sub-THz[END_REF]. To assess accuracy of the on-wafer measurements 'intrinsic EM simulation' prediction is also included in both figures. The 'intrinsic EM simulation' predictions are obtained by considering only intrinsic part of the device during the EM simulation. In this work, intrinsic part of the device refers to part of the device located between the two reference planes (i.e. between green dashed lines in Fig. 2). The on-wafer measurements shown in orange color are roughly following the 'intrinsic EM simulation' results, but a frequency dependent difference between both results is visible. The observed frequency dependent differences between both are possibly due to the contribution of coupling from the probe heads (coupling from probe heads to wafer, as well as coupling between probe heads) which is not completely corrected by the on-wafer calibration [START_REF] Yadav | Investigation of Variation in On-Si On-Wafer TRL Calibration in Sub-THz[END_REF], [START_REF] Cabbia | Silicon Test Structures Design for Sub-THz and THz Measurements[END_REF].

The EM simulations with RF probe models mimicking onwafer measurements of DUTs are shown with solid blue lines in Fig. 5 and Fig. 6. In the EM simulation, four different RF Picoprobe probe models (not shown in this paper) are used up to 500 GHz [START_REF] Yadav | Importance and Requirement of Frequency Band Specific RF Probes EM Models in Sub-THz and THz Measurements up to 500 GHz[END_REF] and the EM model shown in Fig. 4 is used for 500 -750 GHz. The predictions obtained using EM simulations with RF picoprobe probe models are consistent with measurement up to 500 GHz. In 500 -750 GHz, EM simulation with RF probe predictions are following the 'intrinsic EM simulation' trend but a frequency dependent variation between both the results can also be observed. To find a possible reason behind the variations, electric field distribution in transistor-open shown in Fig. 5b and Fig. 5c are studied. The electric field distribution shows undesired coupling of the DUTs with RF probes, and the substrate which may be a possible reason behind the observed variation. In Fig. 6, S 21 obtained from the EM simulation with the RF probe model is very close to measurements up to 500 GHz; comparing it to intrinsic EM simulation, a discrepancy above 625 GHz is notable between the intrinsic EM simulation and the simulation with probe. Behind these frequency dependent differences, the undesired frequency dependent coupling of the DUT (see Fig. 6e and Fig. 6f) with substrate and RF probe can be a reason. The EM simulation based discussion in 500 -750 GHz highlights challenges in accurate on-wafer S-parameters measurement of silicon based DUTs in THz frequency range.

IV. CONCLUSION

We presented on-wafer measurements of silicon based devices in the sub-THz and THz frequency range. The EM simulation study with and without RF probe models are presented up to 750GHz. The frequency dependent difference between intrinsic EM simulation i.e. without RF probe and onwafer measurements shows requirement of efforts to improve on-wafer S-parameters measurement of DUTs in the sub-THz and THz frequency range. One factor behind these observed differences can be presence of different undesirable couplings.

Fig. 1 .

 1 Fig. 1. Top view of on-wafer TRL calibration standards designed and fabricated on Si substrate. The thru and lines have conventional straight line design. In the on-wafer TRL calibration, Line 100GHz is used only up to 110 GHz and Pad Open is used as reflect.

Fig. 2 .Fig. 3 .

 23 Fig. 2. Top view of DUTs 'Line 500 GHz meander' and 'Transistor-Open'. The 'Ref. Plane' shows the measurement reference planes location after applying the on wafer TRL calibration on the DUTs. For the transistor-open, terminal capacitances C 11 , C 21 and C 22 are also shown.

Fig. 4 .

 4 Fig. 4. (a) Top view of a RF probe reported in [7] and reproduced here ©[2011] IEEE. A RF probe model similar to RF probe shown in panel (a) is designed in EM simulator HFSS and its back and front view is shown in (b) and (c), respectively.

Fig. 5 .

 5 Fig. 5. (a) The capacitance behaviour obtained from on-wafer TRL calibrated S-parameters of transistor-open (open-M1). The electric field distribution in transistor-open at 700 GHz is shown in (b) and (c). The panel (b) shows top view while (c) shows the side view. Note that the electric field scale shown in (c) is used for all the electric field distribution presented in this work.

Fig. 6 .

 6 Fig. 6. The S-parameter behaviour of the 500 GHz meander line after applying the on-wafer TRL calibration and obtained from intrinsic EM simulation. The electric field distribution is shown in panel (e) and (f) at 700 GHz and 650 GHz, respectively.

ACKNOWLEDGMENT

This work is partly funded by the French Nouvelle-Aquitaine Authorities through the SUBTILE and FAST project. The authors also acknowledge financial support from the EU under Project Taranto (No. 737454). The authors would like to thank STMicroelectronics for supplying Silicon wafer.


domain: spi info math phys

text:
Gaston Lenczner
email: gaston.lenczner@alteia.com

Adrien Chan-Hon-Tong

Nicola Luminari

Bertrand Le Saux

WEAKLY-SUPERVISED CONTINUAL LEARNING FOR CLASS-INCREMENTAL SEGMENTATION

Transfer learning is a powerful way to adapt existing deep learning models to new emerging use-cases in remote sensing. Starting from a neural network already trained for semantic segmentation, we propose to modify its label space to swiftly adapt it to new classes under weak supervision. To alleviate the background shift and the catastrophic forgetting problems inherent to this form of continual learning, we compare different regularization terms and leverage a pseudo-label strategy. We experimentally show the relevance of our approach on three public remote sensing datasets. (a) Initial prediction (b) New class annotations (c) New prediction (d) Ground-truth Fig. 1. Add building in the {background, road} label space with clicked annotations. Initial prediction (a) leads to newclass annotations (b) to collect the new prediction (c).

INTRODUCTION

Semantic segmentation, or pixel-wise classification of an image, is a challenging computer vision task with various remote sensing applications such as land-cover mapping, building recognition or change detection. Nowadays, deep neural networks (DNNs) are commonly applied for such purposes. They are usually trained once on a given training dataset and then deployed to automatically process potentially large volumes of data.

This approach outcomes frozen algorithms that can lead to excellent outputs in ideal conditions. However, there are still many open questions, such as domain adaptation or limited training data, for which such a static approach is often not suitable. Another important issue of these algorithms is their lack of flexibility due to their frozen output space fixed before training. Many works now use semi-supervised learning [START_REF] Castillo-Navarro | Semi-Supervised Semantic Segmentation in Earth Observation: The MiniFrance suite, dataset analysis and multi-task network study[END_REF] and weakly supervised learning [START_REF] Hua | Semantic segmentation of remote sensing images with sparse annotations[END_REF] to address limited training data issues and domain adaptation [START_REF] Lucas | A Bayesian-inspired, deep learningbased, semi-supervised domain adaptation technique for land cover mapping[END_REF][START_REF] Wang | Weakly supervised adversarial domain adaptation for semantic segmentation in urban scenes[END_REF]. Another promising approach to handle these problems comes with interactive learning [START_REF] Lenczner | Interactive learning for semantic segmentation in earth observation[END_REF], where the algorithm learns to adapt to user inputs. To improve the neural networks plasticity for semantic segmentation, recent works propose customized losses for class-incremental segmentation.

Even though fine-tuning a deep network on a small set of samples of new classes seems quite risky due to both catastrophic forgetting and lack of convergence threats, we show in this work that this is possible within the correct framework. Precisely, we combine weak and class-incremental segmentation works to propose to increment neural network label space using sparse clicked annotations, as showed by Figure 1. This set-up is suited for the interactive learning of new classes since these annotations are particularly handy for an annotator [START_REF] Hua | Semantic segmentation of remote sensing images with sparse annotations[END_REF]. Currently, it might be easier to ask an operator to annotate training data with the new segmentation classes to fully retrain the algorithm. Yet, the purpose of the proposed framework is precisely to be able to learn a new class on new data. This method is therefore suitable for use-cases where initial training data is unavailable due to privacy concerns or where accurate predictions are needed quickly on small datasets.

To this purpose, we first compare different regularizations to fully benefit from the sparse annotations. We also explore a pseudo-label strategy to alleviate catastrophic forgetting. To summarize, the contributions of this paper are:

• A framework to increase the label space of a DNN for semantic segmentation with sparse annotations.

• A pseudo-label strategy and a comparison of different regularizations for this weakly supervised fine-tuning.

• An experimental validation of the proposed approach on three public remote sensing datasets.

RELATED WORK

Semi and weak supervision in semantic segmentation

When labels are scarce or flawed, the common training paradigm is to learn to perform the segmentation task with the available labels while leveraging unlabeled data to learn a better inner representation as support. In semantic segmentation, weak labels can take various forms like incomplete ground-truth maps or bounding boxes. Recently, different works in remote sensing explore these different aspects.

In [START_REF] Castillo-Navarro | Semi-Supervised Semantic Segmentation in Earth Observation: The MiniFrance suite, dataset analysis and multi-task network study[END_REF], the authors learn from a mix of fully annotated images and unannotated ones with auxiliary tasks while [START_REF] Lucas | A Bayesian-inspired, deep learningbased, semi-supervised domain adaptation technique for land cover mapping[END_REF] designs a custom regularization weighted by the proportion of labeled data to address semi-supervised domain adaptation. In weakly supervised learning, semantic segmentation can be achieved from bounding-boxes [START_REF] Wang | Weakly supervised adversarial domain adaptation for semantic segmentation in urban scenes[END_REF], rough polygons [START_REF] Daudt | Weakly supervised change detection using guided anisotropic diffusion[END_REF] or image-level labels [START_REF] Li | On the effectiveness of weakly supervised semantic segmentation for building extraction from high-resolution remote sensing imagery[END_REF]. Closely related to our kind of supervision, DISCA [START_REF] Lenczner | Interactive learning for semantic segmentation in earth observation[END_REF] and FESTA [START_REF] Hua | Semantic segmentation of remote sensing images with sparse annotations[END_REF] leverage point annotations either as user input clicks or partial labels. We here use similar point annotations to add a new segmentation class to a pretrained DNN.

Class-incremental semantic segmentation

The goal of class incremental learning is to modify the output space of DNNs to add label classes. This problem was already considered two decades ago [START_REF] Bruzzone | An incremental-learning neural network for the classification of remote-sensing images[END_REF] and two main pitfalls are identified. First, like in other class-incremental tasks, it is important to prevent the catastrophic forgetting of the previously learned knowledge. Second, specific to semantic segmentation, the background-shift first tackled in [START_REF] Cermelli | Modeling the background for incremental learning in semantic segmentation[END_REF] has to be dealt with. Indeed, the new class comes from the background, which causes a discrepancy with the previously learned background class. Many methods addressed these two problems by simply storing previous examples [START_REF] Tasar | Incremental learning for semantic segmentation of large-scale remote sensing data[END_REF] but more recent works consider this constraint to be too restrictive due to limited storage or security reasons. Hence, we draw inspiration from [START_REF] Douillard | PodNet: Pooled outputs distillation for small-tasks incremental learning[END_REF][START_REF] Michieli | Continual semantic segmentation via repulsion-attraction of sparse and disentangled latent representations[END_REF] which design customized regularization to address the two identified pitfalls. [START_REF] Douillard | PodNet: Pooled outputs distillation for small-tasks incremental learning[END_REF] proposes distillation losses which enforce statistical matches between the networks. Inspired by few-shot and contrastive learning, [START_REF] Michieli | Continual semantic segmentation via repulsion-attraction of sparse and disentangled latent representations[END_REF] relies on prototypes representing the different semantic classes to distinguish them.

METHODOLOGY

Scenario, baseline and constraints

A DNN pre-trained for semantic segmentation to detect N -1 classes is applied on a new image I. The goal is to add a new possible segmentation class and to provide an accurate segmentation map with respect to these N classes. To this aim, M annotations are provided to make the network interactively learn the N th segmentation class. In this work, we follow findings from [START_REF] Hua | Semantic segmentation of remote sensing images with sparse annotations[END_REF] and use point annotations as they appear to be particularly handy for a potential user. In practice, the last layer of the DNN is modified to increment its output space.

As we observed that freezing parts of the network lowered its accuracy, the entire network is then retrained with a crossentropy (CE) loss on the annotated pixels. However, different problems arise with this simple baseline:

• Catastrophic forgetting since the new provided labels only belong to the new class.

• Background shift since the new class was previously classified as background. So, the network has to both learn to detect a new class and to modify the learned representation of the background class.

• Poor class representation since the network has to learn the new class representation from sparse annotations (i.e. highly incomplete ground-truth).

To address these problems, we notably rely on a pseudolabel strategy and compare different regularizations added to the cross-entropy loss.

Following notation from [START_REF] Tasar | Incremental learning for semantic segmentation of large-scale remote sensing data[END_REF], we refer through the rest of this paper to the previous network with the N -1 class prediction as the memory network and to the new one as the updated network. We also refer to the set of semantic classes except the background as the classes of interest.

Learning from pseudo-labelling

The prediction of the memory network is used to sample old classes labels. This ensures that the user does not have to provide such old-class annotations while preventing the network from forgetting them. In addition, the interactive user inputs provide both the new class and the background labels. These background labels avoid the aforementioned background-shift issue since background predictions from the memory network could actually belong to the new class. To emphasize the N annotations of the new class, only a maximum of N of the most confident old-classes pixels are considered as annotations.

Regularization

In order to make the most of the sparse annotations, we consider the following regularization schemes.

Ground-truth

Control prediction

Memory prediction Baseline prediction SDR prediction Fig. 2. Building and road segmentation with 300 building annotations and road pseudo-labels on an example from Vaihingen compared to ground-truth and control prediction.

DISCA. Since the new class was previously part of the background, the predictions over the other classes of interest should remain similar. To enforce this property, we follow [START_REF] Lenczner | Interactive learning for semantic segmentation in earth observation[END_REF] and consider adding a cross entropy regularization term over the pixels which are predicted as belonging to classes of interest by the memory network. This can be seen as an outputlevel knowledge distillation loss.

PodNet. Following PodNet [START_REF] Douillard | PodNet: Pooled outputs distillation for small-tasks incremental learning[END_REF], we enforce a statistic match at the encoder level over the channels between the memory network and the updated one with a L 2 intermediatelevel knowledge distillation loss.

SDR. Inspired by few-shots and contrastive learning, we follow [START_REF] Michieli | Continual semantic segmentation via repulsion-attraction of sparse and disentangled latent representations[END_REF] to regularize the DNN latent space. This aims to reduce forgetting whilst improving the recognition of the new class. Concretely, we add a prototype-based regularization at the encoder level of the DNN. Prototypes are vectors representative of each segmentation class and are computed on the fly at each new learning step. This regularization is composed of three terms: a matching term enforces that the updated prototypes stay close to the previous ones, a repulsive term ensures that the prototypes are far from each other and an attracting term pushes the pixels to remain close to their associated prototype.

FESTA. To deal with sparse annotations for segmentation in remote sensing, [START_REF] Hua | Semantic segmentation of remote sensing images with sparse annotations[END_REF] introduces an unsupervised loss that accounts for neighbourhood structures both in spatial and feature domains as it assumes that nearby pixels share labels.

EXPERIMENTS

Experimental set-up

We evaluate our approach on the ISPRS Potsdam & Vaihingen datasets [START_REF] Rottensteiner | The ISPRS benchmark on urban object classification and 3D building reconstruction[END_REF] respectively composed of 38 (6000 × 6000 pxls, 5cm res.) and 33 (various sizes, 9cm res.) images and on SemCity Toulouse [START_REF] Roscher | SemCity Toulouse: A benchmark for building instance segmentation in satellite images[END_REF] composed of 4 images (3504 × 3452 pxls, 50 cm res.). We pretrain the network for old class (road 1 ) segmentation using dense ground-truth maps on 30 (Potsdam), 26 (Vaihingen) and 2 (Toulouse) images. We finetune for new class (building 2 ) using 3003 clicked new-class and background annotations simulated from the ground-truth on the remaining data. To compare to a potential upper born, we consider a control network directly pretrained on dense {road, building} ground-truth maps (and not fine-tuned).

The considered metric is the Intersection over Union (IoU) averaged image-wise. Except when we analyze the impact of the number of annotations, we simulate 300 new-class and background annotations.

For all experiments, we use a LinkNet [START_REF] Chaurasia | LinkNet: Exploiting encoder representations for efficient semantic segmentation[END_REF] architecture trained using Adam optimizer with a learning rate of 10 -4 during 10 pseudo-epochs. Each pseudo-epoch consists in 10000 256 × 256 labeled samples randomly chosen from training data. We infer using a 256 × 256 sliding window with an overlap of 50%. Due to the stochastic nature of the optimization process and the simulation of the annotations, all experiments are averaged on 3 runs to obtain statistically significant results.

During fine-tuning, we use an Adam optimizer with a learning rate of 2 • 10 -5 . We fine-tune for 30 steps and select the best performances obtained in the last 15 steps. Each step consists of 10 back-propagation iterations. -3.4 74.8 +-1.8 74.1 +-5.4 79.7 +-1.6 Toul. 74 63.2 +-3.2 62.4 +-1.5 54.0 +-0.9 65.4 +-1.0 67.7 +-0.6 As indicates Table 1, the DNN is able to learn a new class with clicked annotations. Even without additional regularizations, it reaches an IoU over the three classes of 68.7% on Potsdam, 76.2% on Vaihingen and 63.2% on Toulouse.

Approach assessment

Appropriate regularization further improves the performances up to 4%. Indeed, SDR consistently improves the results on the three datasets: 3.3% on Potsdam, 3.5% on Vaihingen and 4.5% on Toulouse. Moreover, SDR tends to stabilize the results compared to the baseline, as testifies the standard deviation measures (e.g. the std on Toulouse is of 0.6 for SDR and of 3.2 for the baseline). As shows Figure 2, it visually translate into sharper contours, even though the baseline already produces visually accurate results. However, the other regularizations under-perform in this setting in regards to SDR and can even lead to worse results than the baseline on Vaihingen and Toulouse. Hence, the distillation losses and FESTA seem to be less relevant for this problem than the latent space regularization proposed by SDR. To better apprehend our approach, we analyze the influence of the number of annotations over the performances. According to the results of the previous section, we fine-tune the network using SDR regularization. As we can observe on Figure 3, less than one hundred annotations leads to sub-optimal results and the performances then greatly improve with respect to the number of annotations. After 100 annotations for the two ISPRS datasets and 300 annotations for the SemCity dataset, the DNN almost catches up with the control network performances and then stabilizes on a plateau.

Influence of the number of annotations

CONCLUSION

To conclude, we have shown that it is possible to make a DNN learn on the fly new segmentation classes after the initial training with point supervision using pseudo-labelling and relevant regularizations. We have assessed the method efficiency with experiments on three public remote sensing datasets. Moreover, with a sufficient amount of annotations, the performances even almost catch up with a DNN trained on the classes of interest in a fully supervised way.

Fig. 3 .

 3 Fig. 3. Metric evolution on the three datasets w.r.t. the number of annotations. Control network performances are in dots.

Table 1 .

 1 Comparison of the different regularizations. Contr. stands for Control and Bas. for Baseline.

		Contr.	Bas.	FESTA DISCA PodNet	SDR
	Pot. 76.4 68.7 +-2.7 66.5 +-8.4 68.2 +-4.1 71.7 +-2.3 72.0 +-0.3
	Vai.	84 76.2 +-2.3 75.1 +

Except when specified otherwise.


Afaf Mikou
email: afaf.mikou@parisdescartes.fr

Alexandre Cabayé

Anne Goupil

Hugues-Olivier Bertrand

Jean-Pierre Mothet

Francine C Acher

Computational Study of the Asc-1 Transporter (SLC7A10) Insights into the first steps of the transport mechanism

The alanine-serine-cysteine transporter Asc-1 regulates the synaptic availability of D-serine and glycine (the two co-agonists of the NMDA receptor) and is regarded as an important drug target. To shuttle the substrate from the extracellular space to the cytoplasm, this transporter undergoes multiple distinct conformational states. In this work, homology modeling, substrate docking and molecular dynamics simulations were carried out to learn more about the transition between the "outward-open" and "outward-open occluded" states.

We identified a transition state involving the highly-conserved unwound TM6 region in which the Phe243 flips close to the D-serine substrate. This feature and those of other key residues are proposed to control the binding site and substrate translocation. Competitive inhibitors ACPP, LuAE00527 and their binding modes at the substrate binding site corroborated the key role played by Phe243 of TM6. For ACPP and LuAE00527, strong hydrophobic interactions with this residue hinder its mobility and prevent the uptake and the efflux of substrates. Overall, we propose a molecular basis for the inhibition of substrate translocation of the Asc-1 transporter that should be valuable for rational drug design.

Introduction

The population nowadays is globally aging, and around 9% of the population is over 65 years old with an expected value of 16% by 2050 which means that more people every year are diagnosed with an age related neurological disorder. This drives the scientific research to find new compounds, as the number of patients will increase over the next years.

However not all neurological disorders are age related and some disorders may affect people under 30 years old, like Schizophrenia. In fact, more than 0.5% of the global population is concerned by this disease and the symptoms are usually associated with social exclusion and shorter life expectancy. Therefore, the question arises as to how can we provide a way to treat the schizophrenia symptoms in order to improve the patients' life?

One of the solutions recently found is to analyze the motion and structure of Asc1, that is, the Alanine Serine Cysteine transporter in order to be able to regulate it. Thus, one of the solutions to reduce symptoms of schizophrenia would be to regulate this alanine-serine-cysteine transporter. This transmembrane protein has high affinity for neutral amino acids, such as l-serine, l-alanine, l-cysteine and especially glycine and D-serine (Fig. 1) which are positive allosteric modulators of the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors. Indeed, NMDA receptors hypofunction was shown to be associated with Schizophrenia symptoms. Moreover, a direct activation with small therapeutic molecules of this receptor can generate strong side effects. This is why, an indirect control of its activation through a regulation of the efflux of D-Serine by the Asc-1 transporter would be a better option.

However, up to now, no structure has been solved, neither by X-ray nor cryo electron microscopy for Asc-1.

The transport Mechanism of Asc-1 : Simulations and Results

The transport mechanism of Asc-1 can be described roughly through three main states as shown in Figure 2 As I mentioned before, no structures are available for Asc1 transporter. Indeed, membrane proteins are well known to be extremely difficult to solved by X-Ray. But hopefully, we found structures of other transporters that are quite similar in terms of amino acid sequence identity with our transporter.

One is the bacterial transporter, AdiC, in the Outward-Open state, with approximatly 20% sequence identity

The other is LAT1, a human large amino-acid transporter, in the Inward-Open state, with almost 50% sequence identity.

By using a homology modelling MODELLER which is integrated in BIOVIA Discovery Studio software, we were able to generate models from the template structures of Asc-1 in OO state and IO state respectively.

Then we docked several compounds into these models, including D-Serine, Glycine and two competitive inhibitors (ACPP and LuAE00527) (Fig. 3). These docking studies allowed us to well define the binding site and the key interactions involved between the protein and the ligands or the competitive inhibitors.

The results of the docking study showed good interactions between D-Serine and the central helices, TM1 and TM6. In the case of the inhibitors (ACPP and LuAE00527), it showed that they were blocking any potential rotation of the side chain of F243, located on TM6 (Fig. 3).

This was in good agreement with an inhibition effect by preventing any potential conformation changes. To know more about the Asc-1 transport mechanism in the presence of D-serine, we tried to perform more realistic simulations in the presence of explicite solvent. Indeed, we built an explicit membrane around our models with water and ions on each side. So, from those full explicit systems we ran molecular dynamics simulations. The results show that during the simulations, the F243 side chain flipped from the "out" conformation, to the "in" conformation only after only 30 ns of simulation with D-serine stuck within the binding site pocket. In order to have a better insight of the event we monitored the torsion angle of the first bond of the side chain, which allows its rotation (Fig. 5). It appeared clear that the F243 side chain has two preferential states, controlling the accessibility of the binding pocket. We also followed the bending angle of the TM6 and noticed that it was strongly correlated with the state of the F243. It appears that the end of TM6 shifted more than 8 Å with the flip of the F243, which initiates the opening of the inward side of the Asc-1 transporter.

In conclusion, during this study we were able to determine the first steps of the transport mechanism of the Asc-1 transporter. However, we couldn't simulate the complete transport of D-Serine in that time scale. Also, we propose a molecular basis for the inhibition of substrate translocation of the Asc-1 transporter that should be valuable for rational drug design.

Asc-1 Transporter (SLC7A10): homology models and molecular dynamics insights into the first steps of the transport mechanism. Mikou, A.; Cabaye, A.; Goupil, A.; Bertrand, H. O.; Mothet, J. P.; Acher, F. C. Sci Rep 2020, 10 (1), 3731.

Fig. 1 :

 1 Fig. 1 : Afinity of Asc-1 transmembrane protein for neutral amino acids, such as l-serine, l-alanine, lcysteine and especially glycine and D-serine.

  (see reference Mikou at al. 2020). An Outward-Open (OO) state which allows the ligand binding from the extracellular part, The second state is an Occluded (O) conformation of the transporter, and the third state is described by an Inward-Open (IO) conformation which prepare the release of the ligand in the intracellular part.

Fig. 2 :

 2 Fig. 2 : The three different conformations of Asc-1 during the transport of amino acids (in red).

Fig. 3 :

 3 Fig. 3: The interactions between D-Serine and the central helices, TM1(yellow), and TM6 (pink) of The Asc-1 transporter and the blocking of the side chain of F243 (TM6) by both inhibitors ACPP and LuAE00527.

Fig. 4 :

 4 Fig. 4: The flipping of the side chain of F243 (TM6) during the Molecular Dynamic simulations

Fig. 5 :

 5 Fig. 5: The variation of the torsion angle of the first bond of the F243 side chain of TM6, which allows its rotation and therefore the bending of this helix.


A Mikou
email: afaf.mikou@parisdescartes.fr

Raising scientific awareness for better quality of life

There is an urgent need for rural populations worldwide to better understand scientific concepts to spark ideas for finding solutions to their daily problems. Club "Science Citoyenne"

is to transfer scientific thinking to large audiences, and thus have a positive impact on citizen's general awareness, values and decision-making. The Club has organized numerous activities including interactive exhibitions, films, debates, web radio programs and thematic outings. One set of activities is especially noteworthy. The Club organized a large program targeting rural area in Morocco to raise scientific awareness of children. Workshops were organized to teach concepts in chemical sciences to focus attention on environmental education and sustainable development. The Club's on-site strategy proved to be effective and valuable. The team helped the population to identify potential solutions to their daily issues such as water quality, hygiene, nutrition and environment. This strategy has been awarded international funding and impacted more than 10,000 children. For this, the program has been awarded an international prize We are also taking part in other international programs such as, "Imagine Project" for indigenous communities in Brazil and recently the "European PISEA Program" for Intercultural Science Education. In conclusion, such efforts and programs have proven to have significant positive impacts on individuals and societies. Thus, we are actively searching for further funding opportunities to continue to provide these and related programs.

There is a serious rise in interest for promoting scientific awareness of the public population.

For example, the recent COVID crisis shows that there is an urgent need for the populations worldwide to better understand scientific concepts to avoid being trapped by fake news and other forms of misinformation. Thus, well executed scientific mediation is needed to better achieve the following points:

-communicate scientific concepts and findings -develop a critical thinking especially for the younger generations -encourage equal distribution of knowledge -demystify the image of science and scientists -develop future disseminators of knowledge -find solutions to daily issues and situations increase citizen's value of their surroundings -respect the environment of course, this can be easily be expanded to many other benefits… An important question is how can we achieve the above benefits? Thus far, our scientific mediation efforts have focused on implementing various programs to improve scientific awareness of the general population such as:

• Conferences and meetings that promote interactions with scientific experts This program was awarded international funding and fortunately benefited over 10,000 children. Workshops were organized to teach concepts in chemical sciences to focus attention on environmental education and sustainable development. The Club's on-site strategy proved to be effective and valuable. The team helped the population to identify potential solutions to their daily issues such as water quality, hygiene, nutrition and environment. For instance, this workshop (see below) entitled, « Let's compare the salt from the mountain to the salt from the sea», was carried out to prevent diseases due the iodine deficiency. We therefore conducted an experiment in Chemistry to reveal the iodine in different salts and clearly demonstrated the main message that « Iodine is only present in the salt of the sea or the commercial salt» and not in their daily salt secured via mountain sources.

Next, we organized another workshop entitled, « Let's build a wastewater treatment plant » (see below) was conducted in order to prevent the disease caused by contaminated water from wells and the need to chlorinate it. So the main message delivered was « Clear water is not a potable water ». These project attracted international attention which resulted in funding opportunities; and they were awarded a prestigious prize from the French Academy of Sciences for which I was the recipient (1). Since then, other projects were created. We also took part in other international programs such as, "Imagine Project" for indigenous communities in Brazil (2) and recently the "European PISEA Program" for Intercultural Science Education.

In conclusion, such efforts and programs have proven to have significant positive impacts on individuals and societies. Thus, we are actively searching for further funding opportunities to continue to provide these and related programs. Moreover, we encourage others to create their own strategies for raising scientific awareness, especially those that are adapted to their surroundings and environment.

(1) Mikou, A. was awarded the Purkwa Prize (improving the scientific literacy of children) in 2010 by the "Académie Française des Sciences".

(2) Ramos A. & Razzera G., "Education: Funding Plea for Rural Outreach". Nature 515, 198 (2014).

•

  Film projections and debates that engage critical thinking • Interactive exhibitions • Scientific workshops • On-line activities • Web radio, podcasts … A recent example in 2021 took place during the "Fête de la science" at our Laboratory Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques at Université Paris Cité. This event welcomed the public into our laboratories for a unique experience in our NMR plateform MetaboSanté.Here, people were shown a new technique to diagnose rare diseases. This "medicine for tomorrow" approach excited the attendees. Their curiosity was impressive, and many relevant questions were asked. Overall, there was a clear exchange between the public and scientists! Our experience with scientific mediation, in fact, started approximately 15 years ago at the faculty of science -University Hassan II Casablanca Morocco when my colleague and I founded the Club "Science Citoyenne" (Citizenship Science Club). One of the Club's goals was to transfer scientific thinking to large audiences aimed at producing positive impacts on citizen's general awareness, values and decision-making. The Club organized numerous activities including interactive exhibitions, films, debates, web radio programs and thematic outings.One set of activities was especially noteworthy. The Club organized a large program targeting rural Morocco between the years 2008 and 2010 to raise scientific awareness in children. On a monthly basis, we created and organized scientific workshops targeted mainly for young people to improve their quality of life by promoting the hygiene and encouraging the respect for their environment.