Lilian Bezard

Marie Debacq
email: marie.debacq@agroparistech.fr

Astrid Rosso

The Carnivorous Yoghurts: A "Serious" Escape Game for Stirring Labs

Keywords: Escape room, pedagogical game, chemical engineering, labs, autonomy

A serious escape game based on cards has been created for the preparation of stirring labs.

The implementation was successful: learners became autonomous, very efficient and were excited about the pedagogical game.

Introduction

"le Cnam" is a French institution dedicated to professional lifelong higher education. The learners are adults and workers (therefore with scheduling and availability constraints); their initial training, level and ability are diverse.

Starting in Japan, the United States and different places in Europe, escape rooms have flourished in the last decade, for fun adventure experiences with family or friends or for professional team-building activities. Players work together in a team to find clues around the room and solve enigmas, in order to escape from a locked room, in a given time (usually about one hour). The number of teachers implementing escape games in the classroom is gradually increasing. Two main reasons explain this trend: on the one hand, the institutions and the research community encourage pedagogical innovation to make learners more active; on the other, two main characteristics of escape games (enigmas and just 1h of play), make this kind of activity particularly well adapted to an educational context. So, there is nothing surprising in the rise of "serious escape games".

In the chemical engineering curriculum (Bachelor of Science level), stirring labs are important (they are one of the major unit operations in chemical, pharmaceutical, food… engineering), but boring (very simple activities and repetitive manipulations). In addition, learners find it difficult to obtain the right expressions to calculate the dimensionless numbers required to characterize this operation from the quantities measured on the pilot tank. Based on some studies about serious games in general [START_REF] Alvarez | Apprendre avec les Serious Games ? Réseau Canopé[END_REF][START_REF] Bodnar | Engineers at Play: Games as Teaching Tools for Undergraduate Engineering Students[END_REF] and serious escape games in particular [START_REF] Borrego | Room escape at class: Escape games activities to facilitate the motivation and learning in computer science[END_REF][START_REF] Guigon | A Model to Design Learning Escape Games: SEGAM[END_REF][START_REF] Vörös | Physics escape room as an educational tool[END_REF] and considering the benefits of such approaches, we thought that a serious escape game could be a good answer to these two issues with stirring labs. These pedagogical games are currently developing for two main reasons [START_REF] Duquesnoy | La pédagogie du jeu. PortailEduc[END_REF]: first, they present complex tasks allowing learners and their teachers to take stock of the integration of a particular aspect (or a whole set) of knowledge and know-how; second, they have a recreational dimension and, above all, they involve teamwork, which is a transversal competence to be acquired, whatever the level of education [START_REF] Azizan | Improving teamwork skills and enhancing deep learning via development of board game using cooperative learning method in Reaction Engineering course[END_REF].

When listing the main characteristics of a game (Bezard et al., 2019b;[START_REF] Lacombe | Écrire un scénario interactif : Jeux vidéo, escape games, serious games[END_REF], one realizes the very strong similarity to teaching. [START_REF] Bruckman | Can educational be fun? Game Developers Conference proceedings[END_REF] also invite to "make the learning inherently fun" and consider that "learning by making things is one useful approach that is both fun and educational"; she suggests moreover to "provide social support for learning", "whenever possible".

For [START_REF] Mcgonigal | Reality Is Broken: Why Games Make Us Better and How They Can Change the World[END_REF], games are omnipresent and engaging in a game is above all a turn of mind: by adopting this turn of mind we get good results in many areas, including learning. [START_REF] Chou | Actionable Gamification: Beyond Points, Badges and Leaderboards[END_REF] analyzed the different types of players, how a game adapts to the type of player and how during the game, the game itself evolves to maintain the pleasure of playing.

One very important feature in a game is progression [START_REF] Muletier | La Gamification: ou l'art d'utiliser les mécaniques du jeu dans votre business[END_REF]: we start by learning to play (rules and interactions, with others and/or with a medium); we then learn to master the game (first to win and therefore acquire skills; then we play it again to get a better result or to be more efficient, e.g. to win in less time or get more points); the next step is to learn how to "flout" the game (cheat, change the rules); finally we learn beyond the game (to communicate, to cooperate, to anticipate, to speak English, …). In this description there are again many parallels with teaching. [START_REF] Dehaene | Apprendre ! Les talents du cerveau, le défi des machines[END_REF] argued that the four pillars of learning are:

1. attention (focus to amplify): focusing your attention on an object of thought blinds you to other stimuli. We have all had the experience of being so absorbed in an activity (depending on the person, it may be playing, reading, sewing or debugging a computer code) that we didn't realize that it's long past lunchtime and that the feeling of hunger did not even distract us from what we were doing; this state of extreme concentration is as useful in the context of learning as in that of carrying out a task; 2. active engagement (engaging to produce): a passive organism does not learn; the person must be curious, go deeper and make an effort. Here we find the well-known notion of active learning: we retain much better what we have sought and constructed than what we have simply read or listened to passively; we remember the trapezium rule to calculate an area under a curve, not because we learned the formula of the area of a trapezoid by heart but because we applied this calculation method to obtain the yield of reactors; 3. feedback on errors (compare to correct): making mistakes is essential for progress; it must be de-dramatized. A student who made a big mistake in an intermediate assignment or exercise, if we explain to him exactly why what he did was wrong, will never make that mistake again: neither in the final exam nor in his professional life. It is also important to make clear that the errors made during learning are not that serious and that what needs to be remembered is not the error itself but the correct reasoning or the right approach acquired thanks to the error; 4. consolidation (automate to fluidize): the automation of mastered tasks frees up intellectual resources for other challenges. Once a methodology is well understood, it needs to be repeated until it becomes automatic, so that it can be applied without conscious thought and thus release the person's cognitive capacities for the resolution of new problems. When a chemical engineering student masters how to write the energy balance of a reactor, he can focus his attention on the subtleties of the precise calculation of the reaction enthalpy for example. This is in line with the concept of "flow" used in game design. In psychology, the "flow" state is the mental state in which a person performing an activity is fully immersed in a feeling of energized focus, full involvement, and enjoyment in the process of the activity, whatever the result.

In the "flow" state [START_REF] Chen | Flow in games (and everything else)[END_REF], the player, or the subject, loses all notion of time and of the environment around him; his capacities are totally focused on his activity which gives him a formidable efficiency. In the "flow" zone, the player must meet challenges in accordance with his abilities [START_REF] Koster | Theory of Fun for Game Design[END_REF]: if the challenge is too easy with respect to his abilities, he will rapidly be bored; if the challenge is too high in relation to his abilities, he will be discouraged; in both cases, he will quickly get lost and lose the remarkable efficiency benefits provided by the "flow" state.

Learning and playing are two complementary facets that meet the same objective: learning is part of a formal framework with a specific objective that one seeks to achieve; playing might be seen as a more informal learning activity, by developing things by accident, doing, exploring, making mistakes and going back on those mistakes. The main difficulty with educational games is to place the game in a more formal setting and to find the delicate balance between learning and fun [START_REF] Alvarez | Apprendre avec les Serious Games ? Réseau Canopé[END_REF].

A pedagogical game must have an educational specification: who is it developed for? to learn what? for which learning outcomes? The purpose of fun proposals is to integrate educational goals into a game. The construction of the educational game is then done in several phases: proposals; negotiations; prototyping or hijacking an existing game; tests; adjustments.

The chronological stages of an educational game can be summed up as follows [START_REF] Lhuillier | Concevoir un serious game pour un dispositif de formation[END_REF]Schell, 2014):

 a briefing to introduce a scenario (or at least a context): the immersion and the emotional register are mobilized at this stage; a quirky or wacky story is a good way to bring the players into the "flow" and at the same time to clearly play down the mistakes;  a presentation of the objectives: a problem to be solved, a challenge to be achieved;  to reach these objectives, the players have various resources and objects at their disposal , and can also call on their prior knowledge and background (some of them are used to play, some others not);  feedback (gratification; pass or fail; possibly aid) is essential so that players do not get stuck and are stimulated to move forward;  and a final debriefing, to link up the game and the targeted skills or objectives.

There are two main types of games: cooperative (which is better for most people) or competitive (some people get better results, but by "crushing" other types of personalities). The choice therefore depends on the context. In training, cooperative games are often the most suitable.

After an initial analysis of our context, considering the organization of the syllabus, learners' profiles, buildings and lab installations, time and budget constraints, we decided to develop a collaborative escape card game. The aim was to take advantage of the state of "flow" induced by the game to focus all the learners' attention on their learning.

Design

At the first meeting of the project, the team decided to use the method of "rapid prototyping for learning design" for the whole stirring lab session. There were three main reasons for this choice: 1) the course already existed; 2) the time and budgetary constraints of the project were considerable; 3) the authors were familiar with the course, the institutional context and the learners' profiles. The course, entitled "Chemical Engineering Labs: Unit operations", is part of the first year in the Chemical Engineering degree. At the end of the course the learners must be able to apply the theoretical approaches studied in another course entitled "Chemical Engineering: Unit operations" devoted to pilot plants. The course takes place in the second semester. This course involves 7 mandatory face-to-face lab days; 6 of them are labs. The lab sessions are usually conducted in small teams of two or three learners. Due to the learners' profiles (adults, mostly employed, with strong time constraints), all the lab sessions are usually done in a single week (therefore including Saturday). Most of the lab session subjects take 4 hours to be completed; two of them last 8 hours. As almost always for practical work on unit operations, only one pilot plant per subject is available, so there is a standard team rotation system.

Consequently, the lab sessions are independent of one another and they can be done in any order. The stirring lab session is one of these mandatory lab sessions. The project presented here focuses on the stirring lab session. The engineering objective of this session is to assimilate the notion of standard tank, visualize and quantify the effect of the different types of impellers and of baffles, be able to calculate the characteristic agitation quantities from simple measurements (rotation speed and engine torque) and try to trace the standard power curves, with or without baffles.

Originally, the 4h-labs comprised four experimental stages:

1. assemble the device: this is fairly straightforward, but it is necessary to do things in the right order, while strictly respecting the dimensional rules (standard tank) and with some safety precautions; 2. plotting the power curves point by point for different impellers (marine propeller, Rushton turbine, pitched blade turbine, anchor) in various configurations (with or without 1 or 4 baffles): learners must find out how to calculate the Reynolds number and the power number from the elements provided by the stirring motor plus a measurement of the size of the impeller; it is these calculations (in particular the power number) that the learners found difficult to establish; 3. study of the vortex at the same time as the trials of stage 2 without baffles and comparison with the predictions of correlations from the literature; 4. total homogeneous suspension of solid particles, based on the observations during stages 2 and 3.

During the first three years after the introduction of this stirring lab session in the curriculum, the teacher noticed that the students did not prepare the lab session properly and that they had difficulties during assembly (stage 1 mentioned above). She also observed that they took note of the measurements provided by the instruments (during the long and boring process) but did not know how to use then in the subsequent calculations. In addition, since this was one of the last steps and the assembly had taken too long, the teacher often had to be too directive. Over the following years, young novice teachers were in charge of the stirring labs and the organization of the session remained unchanged. When she was again put in charge of this lab, the teacher decided that it had to be redesigned.

The goal of the project was to make the lab more meaningful, make the learners autonomous in the lab session and calculations, and make the stirring lab session less boring. So, we decided to modify the session by introducing the preparation of the lab session with the learners. However, the teacher also had to supervise the other groups working on other pilot plants at the same time. The total time of the session (preparation + lab session) has to be basically the same (four hours). Consequently, we had to find a strategy to prepare the lab session with the learners (in teams) so that it would be meaningful but not boring, not very long and without the teacher constantly by their side, not expensive and quickly accomplished. So, we decided to design a serious escape game based on cards. This strategy is in line with the four pillars of learning in that the game to prepare for the lab session represents the first pillar (capturing attention), the second pillar (active engagement) is the lab session itself, the third one (feedback on errors) the debriefing by the teacher during the last ten minutes of the lab session, and the fourth one (consolidation) takes place when writing up the lab report.

Building the game

To build the game (Bezard et al., 2019a), we first read the literature about pedagogical games, which highlights their benefits. We rapidly decided to build an escape game because we wanted a cooperative game, a level of complexity that would mobilize various skills and the possibility to prohibit learners from entering the laboratory before having solved all the puzzles of the game.

Then we played card-based domestic escape games (Exit and Unlock) and explored a French website dedicated to pedagogical escape games (S'CAPE): this step was very useful to understand the game mechanics and identify different types of puzzle. Concurrently, the aim of the game was defined, related to the features described in the introduction.

After that we scripted the game (first simply on paper, see Fig. 1), clarifying the order of the main enigmas, and the simultaneity of events and places. Each enigma was divided into several steps, each one of them becoming a new enigma or a piece of the puzzle. After this first script, we began producing the cards and other material (with standard office software) and beta-testing it. Then, we decided where to include more steps or clues to guide the learners, to change the nature of some of the enigmas and to make some help cards. We repeated this process twice. And finally, we tested the game with a "crash-test" group of learners, modified it for the second group, and adjusted it again for the third group.

The game was in "perfect" running order for the fourth and last group of learners.

"The carnivorous yoghurts" game is composed of several things:  1 sheet with the rules of the game;  7 "place" cards, which are actually photos of a room with visible and hidden card numbers leading to various other cards;

 80 "object" or "puzzle" cards or sometimes "penalty" cards (see examples of cards on Fig. 2 and3);  7 help cards for hidden numbers;

 other stuff: a Lab Notebook, a "ConcentrX" (circular device where it is necessary to adjust superimposed discs by turning them, to reveal a sentence or a drawing), punch cards that are placed over another card to solve one of the puzzles, and red-blue-green-yellow plastic folders.

The cost of this serious escape game was nearly zero euros: only usual office supplies and a very widely used software were needed to produce the cards and the board game. However, the design of the game took some time to mature and its production was very time-consuming. We estimate that the maturation phase took between two and three months (but not full time) and the production phase one month (with other activities in parallel, but intensive work on this project).

The scenario

The preparation phase of the labs, formerly in the form of a PDF document that students were required to read and a data sheet to study , was replaced by a fifteen-minute video presenting the fundamental principles of agitation (with no particular connection to the labs): https://youtu.be/y_IK-AVOa4s. The theoretical part of the PDF support remained available for the learners. The experimental part of the session was reduced from 4 to 3 hours. The game takes place at the beginning of the session and it has three successive levels. To limit the game to about one hour, the vortex phenomenon is not mentioned at all (but it is explained and illustrated in the video). The game master stays with the players throughout the game. The three levels of play include:

1. setting up the tank (10 points): this level of the game is based on finding and associating objects; it makes it possible to dissect all the stages of assembly, to review the ratio of the standard tank dimensions and to emphasize the safety precautions; it is deliberately simple, to help learners enter the "flow" and gain confidence; 2. establishing the expression of the Reynolds number for stirring (20 points): this level is based essentially on logic and observation; this puzzle is quite complex and may require some assistance from the game master with certain groups, in order not to exceed one hour of play; 3. establishing the expression of the power number, including the factor taking into account the units of quantities that will be measured during the practical session (30 points): this level is also based on logic and observation, but in addition, includes calculation; it is also complex, but the learners who have accomplished level 2 pass level 3 a little more easily.

The game begins with this message: Hi, My name's Ford PERFECT, I was the foreman in charge of our yoghurt production. Rats left their droppings in the lactic acid culture tank. At first, we pretended nothing had happened and we carried on producing as before. But very soon the attacks started in supermarkets. A few days after having been produced, the yoghurts in our "Yogiyog" range became carnivorous. They started devouring the shoppers.

We recalled all the yoghurts in this range, it came to 250,000 units altogether. But the factory manager threatened me: he said he'd blame me for everything if I didn't quickly find a solution. He wants the yoghurts back to normal again, to avoid having to throw them all away. So time is short... especially as there's a press conference later today. There'll be Sue CROOKS, you know who I mean? the journalist... Anyway, the manager wants a solution, and fast.

I've been working on it for the last 30 hours, non-stop, but I've had enough, I really need a break. I'm going to lie down for an hour or so: I'll leave you to carry on looking for a solution... For future engineers in process engineering like you, that shouldn't be too difficult. You'll find my lab notebook on the desk.

And now, it's up to you. Start the timer and turn over the card marked zero. Get moving, there are killer yoghurts in the lab fridge! Good luck! At the end of the game, when the learners have passed the three levels, the game master gives them a bottle containing the "miracle substance" which eliminates the effects of rat droppings and which will have to be suspended in the tank to purify the yogurts. Ultimately this bottle will be placed in a cryptographer; the first attempt to 3D-print it was fairly conclusive, but there are some details that need to be improved and we did not have enough time to complete this during the first trial of the game. A quick debriefing is done with the teacher, and the learners are then allowed to enter the laboratory to carry out the experiments.

Fifteen minutes before the end of the practical session, the factory manager (actually the teacher) inspects the total homogeneous suspension of the particles, then leaves (normally, he should be satisfied with what he sees) to go to his press conference.

The learners must then empty, dismantle and clean all the experimental device. They also complete a MCQ, as for all the other labs, but the last questions of this MCQ concern their perception about the game.

Playing the game

The game was first experimented in April-May of 2019 at Cnam in Paris (Fig. 4):

1. First the rules of the game were explained to participants and they were reminded that it doesn't matter if they make mistakes! 2. Then the game was run in pairs or groups of three learners for usually an hour, in the presence of the game master (this role was taken by the teacher) and as often as possible one of the pedagogical engineers.

3. At the end of the game, the debriefing and remediation were conducted by the teacher with the learners. 4. After a short break (needed because players were often exhausted after an hour of intense concentration; and also a good moment for the pedagogical engineer to try to get more or different feedback), the players/learners were allowed to enter the laboratory to perform the experiments; in this sense the game can be considered as a flipped escape game: solving puzzles allows you to enter the laboratory, rather than leaving the game room. With the skills acquired during the game, they proved to be sufficiently efficient to carry out all the manipulations (formerly carried out in four hours), in just three hours.

To succeed in the development of an educational escape game, the É S'CAPE website (http://scape.enepe.fr/), a resource pooling space, offers guidance and many tools, as well as examples of implementation. For the development of "The Carnivorous Yoghurts", we used this website many times and an article on the game is now published on it [START_REF] Nadam | Les yaourts carnivores. É S'CAPE[END_REF].

Results and discussion

The question of the efficiency of the game is obviously crucial. It should be remembered that the objective of the game was not that the learners get better marks: the aim was to make them more autonomous and more involved.

Initially, the teacher was apprehensive that Cnam learners would not be very receptive to an educational game, or that some would refuse this non-serious activity. It was just the opposite in fact; all the learners were delighted to play the game and very satisfied after this new organizational structure of labs.

The fact that the learners were exhausted but smiling after the game is an indicator that they had indeed entered the "flow".

The game was conducted in 3 steps: the briefing (we explain the game, the rules of the game, the role of each person, the place); the game itself; the debriefing (we come back to the learning objectives of the activity and the skills).

In a serious escape game, the successive puzzles must enable both entry into the game and achievement of the educational objectives. We therefore divided the game into 3 levels:

1. entering the game, which corresponds to the start of the activity: it involves giving learners easy puzzles to solve, in order to encourage them to continue their efforts; in this stage we therefore favored observation and manipulation; this phase serves as a boost; 2. recreational consolidation: during this part, the puzzles are more diverse and increase in difficulty; the enigmas of the game intersect the educational objectives (how to set up the experimental device, what are the measurement units, how to implement the device safely); 3. pedagogical consolidation: at this point the game stages the most complex elements of learning through complex puzzles (in this case, the equations). This is the most sensitive stage to manage in order to keep the learner / player in the "flow": he must not drop out because the task is too difficult and must maintain intense concentration to successfully solve a complex problem.

The evaluation of the pedagogical game was carried out in several stages: informally, on the spot just after the game (before entering the laboratory) with one of the pedagogical engineers who co-designed the game and without the presence of the teacher; in the end-of-session paper MCQ; in the end-of-course survey two months later; and finally by an open questionnaire seven months later.

Considering the small number of learners involved in this first experiment (nine), it does not make sense to present statistics, but we can note the following points:

The on-the-spot discussions confirmed that the learners reacted positively. Some even suggested ideas for improvement.

Several encouraged us to push the wacky story further.

In the end-of-course survey, to which five learners replied, all described the game as "attractive"; the other most frequently used qualifiers were "pleasant" and "useful for learning". Learners found that the value of the game lay more in carrying out the lab session than in preparing it. On the balance between fun and learning, 4 out of 5 found the game "well balanced" and 1 "too playful". The terms most often used to qualify the scenario of the game were "relevant" and "enlightening". None of the negative qualifiers proposed to evaluate the game were checked. Concerning the duration and the level of difficulty of the game, learners considered that the duration was "appropriate" to "a bit too long" and that the level of difficulty was "appropriate" to "a bit too difficult". The presence of the game master (here the teacher) was judged, by order of occurrence among the responses: "necessary"; "caregiver"; "reassuring". In the free comments, many positive and encouraging comments were made. The interest of cooperation in the group and the complementarity of skills were mentioned several times.

The question of activity perception is important in assessing the impact of the involvement of learners. Here the game brings together the two impacts commonly sought in this type of activity: pleasure and skill. Pleasure is important because it promotes memorization. Unfortunately, only two learners answered the questionnaire seven months later, but both correctly answered the question aimed at checking whether the skill required to understand dimensionless numbers had been durably acquired. One learner who was particularly mature and sensitive to pedagogical questions, explained that he appreciated the construction of knowledge during the game by association of ideas and collaborative construction step by step.

As mentioned above, given the small number of learners concerned, it would not make sense to present quantitative results, for example by comparing the scores obtained by the 4 groups in the stirring labs the year the game was deployed with the average of the grades from previous years. It turns out that their marks were better, but we were also dealing with very good students overall that year, and it would not be honest to claim that their better marks are the effect of the game. This is why we prefer here to stick to the comments and reactions reported above. This is in line with Kirkpatrick's Four Levels of Training Evaluation (2016), first published in 1959 by Professor Donald Kirkpatrick and recently updated by his son and his wife. In this model, level 1 of evaluation is "Reaction" i.e. measuring how engaged the learners were, how actively they contributed, and how they reacted to the training, helps to understand how well they received it. We tried to apprehend this level 1 of the evaluation thanks to the "on the spot" discussions and the end-of-session paper MCQ. Kirkpatrick's level 2 "Learning" measures what learners think they'll be able to do differently after training, how confident they are that they can do it, and how motivated they are to make changes. We asked questions along these lines in the end-of-course survey. Kirkpatrick's level 3 "Behavior" helps to understand how well people apply their training. This was the purpose of our open questionnaire seven months later; unfortunately, only two responded and even if their answers are satisfactory, it is not enough to affirm the long-term effect of the game. Kirkpatrick's level 4 "Results" is the only one where one tries to quantify the effects of training. This level cannot objectively be claimed in our case. Reusing the game for another 1 or 2 years and/or transferring it to another institution could give more quantitative results in the future.

We can claim that the goal has been achieved: this lab session is less boring; the learners are totally autonomous and very efficient. This is because at the end of the game, they know exactly how to implement the tank and have the equations to calculate the dimensionless numbers from the measurements (with the right units and coefficients). The game has the desired effect of any pedagogical game, namely immersing the learner so that he focuses all his cognitive abilities on the purpose of the game. The practical work is still performed, but is reduced from 4 to 3 hours. Since the learners have gained autonomy and efficiency thanks to the game, this time is more than enough to carry out the same experiments as in the past. Thus engineering skills specific to the topic stirring are acquired (standard tank, effect of types of impellers and baffles, power curves), but also transversal scientific skills (in particular the use of units to guide reasoning on physical quantities) and soft skills (working and communicating as a team, making joint or shared decisions).

Learners unanimously state that the game master should be present throughout the game: his/her attendance seems necessary to help learners during the game; but this probably inhibits players-learners who do not dare to try (and sometimes fail). And above all, other groups are working in parallel on other pilot-plants and the teacher also needs to teach and help them out at times. It would be great to have a different person to master the game.

The scenario could have been a simple simulation, or something very serious. A wacky topic was used to design a scenario and therefore an activity with no other stake than the game itself: learners do not receive a mark for the game; they can fail with no consequences in real life; they are under no outside pressure to succeed in the game. They are in a blurry area which is a teaching moment, but which is not entirely so. The wackier the scenario, the further the metaphor is from the subject, that is to say the further the story of the game is from the concrete context of the skills, and the longer the debriefing will be to bring the experience of the game back to reality and pedagogical objectives.

The feedback on the wacky scenario at the end of the lab session (when the factory manager inspects the tank before going to his press conference) was welcomed by the editor of the article presenting our game on the É S'CAPE website, who is also the co-author of a very recent work on pedagogical escape games [START_REF] Fenaert | S'capade pédagogique avec les jeux d'évasion -Apprendre grâce aux escape games -De la maternelle à la formation d'adultes[END_REF].

Conclusions and perspectives

As a conclusion, one should always remember that a pedagogical game is a tricky balance between an educational scenario and having fun. Some keywords should be highlighted here: immersion and emotions are very important in such an experience; true gamification is required for a pedagogical game, which should not be a mere simulator; finally, feedback is crucial.

The objective of an educational game is often to intervene on part of the syllabus that is problematic, either because it is too complex, too conceptual, or too boring. The game brings a dimension that solves a difficulty. Through pleasure and commitment, learners overcome this difficulty.

To summarize some guidelines for developing a pedagogical game: clear goals must be well defined upstream, explained at the beginning of the game and validated at the end; the screenplay is essential, so as not to forget the learning objectives: the first stages of the game must be easy (so that learners get immersed in the game) and the scenario must allow them to remain in the "flow"; all the material (the cards and other stuff) should be designed to help players stay in the "flow"; debriefing should not be neglected and must reconnect the game to goals and skills (and the rest of the teaching); the atmosphere of the game should lead learners to try things out: the principle of trial/error is very important in the process of learning with a game; always iterate to adjust the level of difficulty: if no one is available to test upstream, it will be necessary to inform the first groups that they might be in "crash-test" mode; teachers tend to include too many concepts in a game, which is harmful; the sustainability of the game should be considered, because some game mechanics can quickly become obsolete; and last but not least, have versatility in mind from the beginning, that is: try to design a game concept that can be applied to other cases, in the hope of saving time in the development of future games.

In our experience, educational games present six main advantages: capturing the attention of the learner; playing down mistakes; promoting peer exchange; increasing learner involvement through playing; leaving a lasting effect on the learner's memory; allowing the learner to self-assess and have feedback with the teacher.

One can find a very useful guide for the design, the animation and the evaluation of a fun-educational activity, based on Alvarez's work, at the following address: https://urlz.fr/aBn1

The main risks when developing an educational game are:

 producing an ineffective game (too complex, with too many things in the game, too far from the educational objectives, too boring, too old-fashioned);

 not having been able to create the appropriate "atmosphere" for the game to develop, i.e. to be in a situation where the game cannot take place properly: the learners do anti-game (because they do not join this educational form) or even firmly refuse to play. In any case it must be said to the learners that they are going to play; when we assume that this kind of behavior can happen with learners (who refuse to become players), we must forecast a fallback plan.

While building the game, it is necessary to keep in mind: the importance of the atmosphere (so that learners do not hesitate to try, and make mistakes); iterations to adjust the difficulty of the game (one or more crash-test group(s) are essential to test the game); durability (some game mechanics can quickly become dated); if possible, a game concept that can be reused on other cases to make the long development time cost-effective.

The game "Carnivorous Yoghurts" is now adopted for stirring labs and will be replayed every year. Several colleagues from other universities have expressed interest in transposing the game with their students.

The game material is therefore fully available for anybody in French and in English: https://hal.archives-ouvertes.fr/hal-02396374

The instructions for use of the game material ask the teachers who use it to give feedback about this educational innovation for stirring labs. We are looking forward to reading this feedback.

FiguresFig. 1 .

 1 Figures

Fig. 2 .

 2 Fig. 2. Example of how cards are combined in order to find new objects and puzzles.

Fig. 3 .

 3 Fig. 3. Examples of puzzle cards mixing fun and scientific purpose.

Fig. 4 .

 4 Fig. 4. Learners during a session.

Acknowledgements

We would like to thank the nine learners who experimented this game in spring 2019: for their enthusiasm, their comments, suggestions and encouragement.


M Azib
email: azib@laplace.univ-tlse.fr

F Baudoin

C Villeneuve-Faure

G Teyssedre

N Binaud

F Bugarin

S Segonds

Numerical Simulations for Sensitivity Analysis of the Electrostatic Force Curve on Charge Localization in 3D

Keywords: AFM, space charge localization, thin dielectric films, numerical simulations

In the present work we present a model for Atomic Force Microscopy (AFM) tip interacting electrostatically with a thin dielectric layer. Numerical simulations enabled us to estimate the electrostatic force induced by a half-ellipsoid charge distribution. The sensitivity of the shape of the Electrostatic Force Distance Curves (EFDC) to charge localization is investigated for the charge parameters as radius, depth and density of charges.

I. INTRODUCTION

The electrostatic force due to the interaction of electric charges is the fundamental force that is best understood, and it has been studied and verified experimentally with highest precision. As electrostatic forces govern most physical and chemical processes, their control at sub micrometer scale is of relevant importance for the development of nanotechnology, so as to build operative devices such as electronic components destined to the computer modern industry. Part of the progress achieved over the last decades has been driven by the development of Scanning Probe Microscopy (SPM) [START_REF] Binnig | Surface Studies by Scanning Tunneling Microscopy[END_REF] and in particular Atomic Force Microscopy (AFM) [START_REF] Binnig | Atomic Force Microscope[END_REF]. Dielectric materials regularly take part of nanotechnology engineering as important components of electronic devices with thicknesses of the order 10 to 500 nm. Understanding the behavior of dielectric materials under different kinds of stress (electrical, thermal, etc.) is required to guarantee the reliability of the electronic devices. This is why; electrical modes derived from AFM [START_REF] Marchi | Characterisation of trapped electric charge carriers behaviour at nanometer scale by electrostatic force microscopy[END_REF][START_REF] Morita | Microscale contact charging on a silicon oxide[END_REF][START_REF] Terris | Contact electrification using force microscopy[END_REF][START_REF] Rezende | Detection of charge distribution in insulator surfaces[END_REF][START_REF] Ishii | Static states and dynamic behaviour of charges: observation and control by scanning probe microscopy[END_REF][START_REF] Borowik | Calculating Kelvin force microscopy signals from static force fields[END_REF] have been more and more used to probe the electrical properties of insulating materials at small scale. In these techniques, a metalized silicon probe composed by tip and cantilever is used to scan a thin dielectric fixed on a conducting surface. When a potential difference appears between the tip and the underlying metallic surface, an electrostatic force appears between the both. Moreover, as electrostatic force has a long-range character and it is stronger than the short-range forces such as the Van der Waals forces, topographic and local electrical properties as surface potential or permittivity could be measured simultaneously.

Charge injected in dielectric layer induced electrostatic force and consequently could be probed by electric modes derived from AFM. The first mode which was used is the Electrostatic Force Microscopy (EFM) [START_REF] Marchi | Characterisation of trapped electric charge carriers behaviour at nanometer scale by electrostatic force microscopy[END_REF][START_REF] Morita | Microscale contact charging on a silicon oxide[END_REF]. In general cases of EFM, a bias voltage is applied between the AFM probe and the sample, this bias voltage induces an electric field which is related to charge distribution in the dielectric by the first Maxwell's equation. The tip is submitted to an electrostatic force proportional to the capacitance and depends critically on tip-sample distance. By applying this method, it is possible to image charges with sub-micrometric lateral resolution [START_REF] Terris | Contact electrification using force microscopy[END_REF]. However, the real issue of the EFM method is sensitivity to image charges which is critical to evaluate the amount of injected charge. To overcome this issue, the Kelvin Probe Force Microscopy (KPFM) [START_REF] Rezende | Detection of charge distribution in insulator surfaces[END_REF][START_REF] Ishii | Static states and dynamic behaviour of charges: observation and control by scanning probe microscopy[END_REF][START_REF] Borowik | Calculating Kelvin force microscopy signals from static force fields[END_REF] is used. KPFM measures with sub-micrometric lateral resolution the surface potential induced by charges stored in dielectrics. According to, hypothesis about charge distribution in surface or volume charge density can be estimated from KPFM measurements [START_REF] Borowik | Calculating Kelvin force microscopy signals from static force fields[END_REF]. However, a lack of information about the in-depth position of charges limits its application for space charge detection.

For space charge detection in thin dielectric layers a new approach called EFDC (Electrostatic Force Distance Curve) [START_REF] Villeneuve-Faure | Towards 3D charge localization by a method derived from atomic force microscopy: the electrostatic force distance curve[END_REF] attempts to exploit the electrostatic force tip-sample distance curve obtained by the Force Distance Curve (FDC) [START_REF] Cappella | Force-distance curves by atomic force microscopy[END_REF] technique to get accurate information about the distribution in 3D of charges stored in a dielectric. As for KPFM and EFM, modelling of the electrostatic force between the AFM probe and the sample as a function of tip-sample distance is necessary. Various methods were developed, not always analytical [START_REF] Morita | Microscale contact charging on a silicon oxide[END_REF][START_REF] Hu | Scanning polarization force microscopy: A technique for imaging liquids and weakly adsorbed layers[END_REF][START_REF] Beladi | Electrostatic forces acting on the tip in atomic force microscopy: Modelization and comparison with analytic expressions[END_REF][START_REF] Gómez-Moñivas | Electrostatic force between sharp tips and metallic and dielectric sample[END_REF], even for highly simplified geometry, and numerical methods [START_REF] Colchero | Resolution enhancement and improved data interpretation in electrostatic force microscopy[END_REF][START_REF] Blaidi | Finite element simulations of the resolution in electrostatic force microscopy[END_REF][START_REF] Gros | Two-dimensional, electrostatic finite element study of tip-substrate interactions in electric force microscopy of high density interconnect structure[END_REF][START_REF] Makasheva | Dielectric Layers for RF-MEMS Switches: Design and Study of Appropriate structure Preventing Electrostatic Charging[END_REF] are preferred. The limitations of these methods come from the simplification of the geometry of the system as well as hypotheses on the position of charges. These models are consistent with experimental result in restricted ranges of distances. A sophisticated model with an accurate geometry of the tip was proposed [START_REF] Boularas | 3D modelling of electrostatic interaction between AFM probe and dielectric surface: Impact of tip shape and cantilever contribution[END_REF] in which a three dimensional model was developed: the electrostatic tip model provides an estimation of the electrostatic force between the tip and a dielectric layer free from charges. However, in this approach the tilt angle of the AFM tip was not considered. Recently, we have proposed a model [START_REF] Azib | Numerical simulations for quantitative analysis of electrostatic interaction between atomic force microscopy probe and an embedded electrode within a thin dielectric: meshing optimization, sensitivity to potential distribution and impact of cantilever contribution[END_REF] using the same geometry with taking into accounts the tilted angle and optimizing the mesh to cut down the computational time. To give more credit to this approach, the model estimates the electrostatic force over a buried electrode embedded in thin dielectric layer. Hence, the electrostatic force sensibility to potential distribution was investigated. The EFDC sensitivity to the potential distribution in 3D is highlighted.

The objective in the present work is to propose a model based on the same geometry introduced in previous studies [START_REF] Boularas | 3D modelling of electrostatic interaction between AFM probe and dielectric surface: Impact of tip shape and cantilever contribution[END_REF][START_REF] Azib | Numerical simulations for quantitative analysis of electrostatic interaction between atomic force microscopy probe and an embedded electrode within a thin dielectric: meshing optimization, sensitivity to potential distribution and impact of cantilever contribution[END_REF], in order to study theoretically the electrostatic force sensibility to charge localization. The next sections present the tip-dielectric model and the sensibility of the tip to charge localization.

II. ELECTROSTATIC TIP MODEL

A. Geometry construction

Our general approach is to calculate the electrostatic force exerted on a stationary tip as a function of its vertical position over an infinite dielectric layer. The modeled layer is silicon nitride (SiNx) of thickness z = 500 nm and relative permittivity = 7.5. The injected charges into the dielectric layer are described as a half-ellipsoid cloud of uniform charge density ρ, with a radius r and a depth d, cf. Fig. 1. The tip is modelled by the most representative shape [START_REF] Azib | Numerical simulations for quantitative analysis of electrostatic interaction between atomic force microscopy probe and an embedded electrode within a thin dielectric: meshing optimization, sensitivity to potential distribution and impact of cantilever contribution[END_REF]: a sphericaltetrahedral shape with tip apex of 25 nm, tip tetrahedron halfangle of 20° and tip height of 10 µm, in order to match to the shape of a real tip. An air domain with dimensions large enough to avoid edge effects is surrounding the tip and is in contact with the dielectric surface.

To compute the electrostatic force exerted on the tip induced by the charge distribution stored in the dielectric layer, we need to determine the electrostatic potential distribution V in the air box and the dielectric layer. This requires the resolution of the Poisson's equation (Eq. 1) over the domain Ω, taking into account the boundary conditions on the interface Γ (see Fig. 1). Γ is composed of three parts Γ0, Γ1 and Γ2. The problem is written as follows:

div (-ε grad(V)) = ρ in Ω (1)
V = 0 on Γ0 and Γ1

(2)

= 0 on Γ2 ( 3
)
where ρ is the charge density distribution, n is the normal vector to the surface and ε is the permittivity of the dielectric or the air. The tip surface and the back side of the dielectric are set to the ground. In order to solve numerically the Poisson's equation in the domain Ω and to determine the electrostatic force, the Finite Element Method (FEM) has been applied for discretizing the geometry. As explained about mesh construction and optimization in [START_REF] Azib | Numerical simulations for quantitative analysis of electrostatic interaction between atomic force microscopy probe and an embedded electrode within a thin dielectric: meshing optimization, sensitivity to potential distribution and impact of cantilever contribution[END_REF], the regions where the electrostatic interactions are very high (around the apex) need to be meshed finely and the ones representing weak interactions are meshed more coarsely. Thus, we have added a domain without any boundary condition around the apex to mesh very finely this area.

The electrostatic force F acting on the tip surface was computed by the integration of the built-in Maxwell-stresstensor, Mt, over all faces of the tip:

Mt = ‖ ‖ (4) = Mt. . ds (5)
Where E is the electric field.

In order to study the electrostatic force sensitivity to charge localization we have modified successively the values of each charge parameters: the density ρ, the radius r and the depth d, then determine their variation impact on the force curve shape. The force curve is characterized by two parameters: (i) a maximum of the electrostatic force Fmax, which is reached on D = 0 nm (contact point between tip and dielectric); (ii) a curvature which is representative to curve shape [START_REF] Villeneuve-Faure | Towards 3D charge localization by a method derived from atomic force microscopy: the electrostatic force distance curve[END_REF].

B. Electrostatic force sensitivity to charge density

Fig. 2a represents the electrostatic force distance curves computed for different densities of charge ρ (r and d are fixed). This figure shows clearly that the electrostatic force is strongly influenced by the density of charges stored in the dielectric layer. Indeed, when the charge density increases the maximum of electrostatic force increases too. A variation by a factor of 1.5 on the charge density, i.e. from 10 000 C.m -3 to 15 000 C.m -3 , leads to a variation on the maximum force by a factor of 2, from 0.39 nN to 0.81 nN. Fig. 2b plots out the ratio of the electrostatic force over the maximum of force for each density. The results reveal that the charge density variation does not modify the curvature of the EFDC curves. The shape of the EFDC is rigorously independent from the charge amount.

C. Electrostatic force sensitivity to radius of charge

Fig. 3a presents the variation of the EFDC as a function of radius r (ranging from 100 nm to 550 nm) at charge depth d fixed to 100 nm and density ρ fixed to 10 5 C.m -3 . From this figure, it can be readily seen that when the radius increases, the maximum of force increases too. For example, the maximum force for radius 100 nm and 500 nm is respectively 0.115 nN and 0.47 nN. Fig. 3b shows the normalized force curves for different charge cloud radii. The lateral spreading of charges affects not only the maximum of force but also the curvature. The curvature is more important for smaller values of charge cloud radius. Higher values of the curvature correspond to situations where the charges are highly localized (small radius) to the surface. With charges localized on small spots, a sharp distance dependence of the electrostatic force is obtained. Contrarily, when the charges spread laterally over the surface (larger values of r), a smooth curve is obtained and the baseline is far from the maximum of force, due to the contribution of charges at different points from the surface and accordingly the curvature is lower.

D. Electrostatic force sensitivity to in-depth position of charges

Fig. 4a and Fig. 4b represent the influence of the in-depth distribution of charges on the force curve shape. The in-depth distribution of charges influences only the maximum force of the EFDC curve. The maximum force increases with increasing the height of the charge cloud, while the curvature remains nearly the same. Up to now it has not been possible to identify the relationship between the in-depth position of charges and the shape of the electrostatic force curve. One of the reasons in the computations reported here can be that the EFDC shape is dominated by the upper charge layers. Also, the sports are relatively broad in respect to the height in such a way that the effect of the in-depth position is smoothed. Certainly working with smaller charge clusters positioned at different depths could unravel some of the trends. However, our results are highly relevant in the context of the sensitivity of electrostatic force toward the in-depth of charge in the sample, as they demonstrate qualitatively the direct link between the both. The result of this work adds some precise information about the electrostatic force curve sensitivity to charge localization against the experiment work [START_REF] Villeneuve-Faure | Towards 3D charge localization by a method derived from atomic force microscopy: the electrostatic force distance curve[END_REF] but relative contribution of density and spatial distribution of charge was not clearly identified. However, from experimental data, it was conjectured that the curvature of the EFDC is modified by the quantity of charges. However, the quantity of charge depends on the charge density, and on the width and depth of the charge cloud. With the ellipsoid shape taken here, the mathematical relation which links the quantity of charge Q to those parameters is described by the following equation:

Q = ρ× ( × × × ) (6)
Therefore, saying that the curvature is modified by the quantity of charge Q is futher generalised, while our results are very consistent in a way we could extract the individual effect of each parameter on the force curve. This is why special care should be given to the measurement interpretations, and numerical simulations are necessary steps in providing a full understanding of EFDC, identifying the influence of parameters and evaluating the performances of EFDC towards space charge probing.

III. CONCLUSION AND PERSPECTIVE

In summary, the sensitivity of electrostatic force interactions between the AFM tip and a dielectric thin film, to charge distribution has been studied accurately using numerical simulations. A Finite Element Model is developed to simulate the electrostatic force exerted on the tip, which is induced by a half-ellipsoid charge pattern stored in the dielectric layer. Results bring useful information regarding this sensitivity. We demonstrated that the electrostatic force is sensitive to the density of charges and the in-depth spreading of charges, a variation of the density or the in-depth leads only to a variation of the maximum force while the curvature of the electrostatic force curve remain unchanged. On the other hand, the lateral spreading of charges (radius) variation affects at the same time the maximum of force and the curvature. Therefore, these results open up opportunities for possibility to localize the charge distribution in thin dielectric layers i.e. quantify the density of charge, the lateral spreading of charge and reaching an in-depth resolution. To further progress we need to develop an inverse model, which takes an experimental force curve as the input and its output will be the charge's parameter: the radius r, the density ρ and the depth d. For this reason, we have undertaken to develop an inverse model which using directly the tip model as a forward model.

Fig. 1 .

 1 Fig. 1.Description of the geometry used for tip-dielectric modelling.

Fig. 2 .

 2 Fig. 2. (a) The electrostatic force F as function of tip-sample distance D, (b) the ratio of F over the maximum force (Fmax) as a function of D, for different values of charge density ρ, the depth d and the radius r of the charge cloud were respectively fixed at 100 nm and 250 nm.

Fig. 3 .

 3 Fig. 3. (a) The electrostatic force F as function of the tip-sample distance D, (b) the ratio of F over the maximum force (Fmax) as a function of D, for different values of the charge's radius, the depth and the density of charges were respectively fixed at 100 nm and 10 5 C.m -3 .

Fig. 4 .

 4 Fig. 4. (a) The electrostatic force F vs the tip-sample distance D (b) the ratio of F over the maximum force (Fmax) as a function D, for different values of the charge cloud height, the radius and the amount of charges were respectively fixed at 250 nm and 10 5 C.m -3 .


Arnaud Leroy
email: arnaud.leroy@chru-lille.fr

Ali Amad

Fabien D'hondt

Delphine Pins

Nematollah Jaafari

Pierre Thomas

Renaud Jardri

Reward anticipation in schizophrenia: a coordinate-based meta-analysis

Keywords: reward, salience, schizophrenia, antipsychotics, fMRI, meta-analysis

Reward processing impairments have been linked with positive and negative symptoms of schizophrenia. Here, we performed a coordinate-based meta-analysis that combined eleven BOLD-fMRI studies comparing reward anticipation signals between schizophrenia patients and healthy controls. We observed a reduced difference in activation in schizophrenia patients within a frontal-striatal network. Meta-regressions revealed that this functional signature was linked to the severity of psychotic symptoms and persisted even after controlling for the dose of antipsychotic medications.

INTRODUCTION

Motivation is considered to be one of the major aspects of salience in humans (Winton-Brown et al., 2014). Furthermore, motivational impairments are considered to be cardinal in schizophrenia and can be observed from the earliest stages of the disorder (Foussias and Remington, 2010;Schlosser et al., 2014). These impairments are related to deficits in anticipating future rewards (Juckel et al., 2006;Simon et al., 2010) and were shown to be associated with specific clinical features of schizophrenia. On the one hand, reward anticipation seems to be involved in the genesis of positive symptoms, i.e., hallucinations and delusions (Kapur, 2003;Murray et al., 2008), while suppressed reward processing could contribute to the reinforcement of negative symptoms, such as anhedonia (Barch and Dowd, 2010;Gold et al., 2008). Dopamine plays a central role in the pathophysiology of schizophrenia (Howes et al., 2015), and it has also been implicated in reward signaling since dopamine neurons exhibit phasic firing during reward tasks in animals (Schultz, 2001). For several decades, D2 antagonists have been used as the reference pharmacological treatment for schizophrenia. However, their exact role in reward processing in this disorder, especially in reward anticipation, is still subject to debate (Juckel, 2016;Nielsen et al., 2018Nielsen et al., , 2012;;Schlagenhauf et al., 2014).

Recent meta-analyses have addressed the question of the neural bases of reward signalling in schizophrenia and revealed activation changes in the ventral striatum (Chase et al., 2018;Radua et al., 2015). Despite the great interest these studies attract, some questions remain unanswered: (a) the inclusion of a significant number of region-of-interest (ROI) studies has exposed these meta-analyses to a bias towards the striatum. In this context, it remains difficult to fully exclude the possibility that some parts of the network involved in reward may have been missed; (b) the impact of symptom severity or medication on the resulting maps has not been directly explored.

To further these findings, we conducted a new coordinate-based meta-analysis of functional Magnetic Resonance Imaging (fMRI) studies comparing appetitive anticipation signals between patients with schizophrenia and healthy controls. Because salience and reward are heterogeneous concepts, we chose to focus only on anticipation rather than on more learning-dependent signals (such as reward outcome or prediction error). The aim of this work was twofold: (i) determining the whole-brain bases of reward anticipation in schizophrenia; and (ii) clarifying the impact of potential moderators on the main findings (i.e. severity, antipsychotics dosage).

METHODS

Literature Selection, Data collection and preparation

We conducted systematic MEDLINE searches until September 2018 to identify taskbased functional brain imaging studies on reward in schizophrenia. We used the following algorithm: ("schizophren* OR psychosis" "fMRI OR PET" "salienc* OR reward), complemented by the "related articles" function of the PubMed database and the reference list of studies found. To avoid bias towards the striatum, we only considered primary-data articles reporting whole-brain analyses or more than three ROIs. Among the 367 initial hits, eleven met our inclusion/exclusion criteria (see the PRISMA Flowchart in Figure S1). Selected activation studies are listed in Table 1 (Chung and Barch, 2016;da Silva Alves et al., 2013;Gradin et al., 2011;Koch et al., 2010;Potvin et al., 2016;Reckless et al., 2015;Richter et al., 2015;Schlagenhauf et al., 2009;Smieskova et al., 2015;Subramaniam et al., 2015;Walter et al., 2009). They referred to a Monetary Incentive Delay (MID) or similar task and reported the following fMRI contrasts: [reward anticipation vs. neutral cue] in [controls vs. schizophrenia patients]. Both controls > schizophrenia and schizophrenia > controls contrasts are reported, because the SDM meta-analysis software accounts for peaks' effect size and sign to counteract positive and negative differences. Stereotaxic coordinates of the 44 foci of interest for these contrasts were extracted and tagged by study and sample size. We considered only foci reported as significant at an uncorrected p-value < 0.001 or p-value < 0.05 corrected for multiple comparisons in the source studies.

Meta-Analysis Procedure

Data were analyzed using the SDM (seed-based d mapping) algorithm (Radua et al., 2012;Radua and Mataix-Cols, 2009). Effect sizes were calculated using (i) t-scores in the voxels containing a peak and (ii) a Gaussian kernel applied to the surrounding voxels (Radua et al., 2014). Estimated statistical maps were then included in a random effects meta-analytic model that weighted the contribution of each study according to its sample size and intra/interstudy heterogeneity. The statistical significance of the resulting SDM Z-maps was estimated through 50 permutation tests. Following standard criteria, intergroup comparisons were limited to (i) p <0.005 (Rauch et al., 2002); (ii) an SDM Z-score >1; and (iii) a cluster extent >10 voxels (Radua et al., 2012;Radua and Mataix-Cols, 2009). A jackknife sensitivity analysis was conducted to assess the robustness of the findings by iteratively repeating the analysis and by excluding one dataset at a time, while a potential publication bias was assessed using Egger's tests and funnel plots. We verified that when results were changed in a particular dataset combination in the jackknife analysis, the discarded study did not drive the results using a visual inspection of funnel plots. We also verified that Egger's test was not significant (p > 0.05).

Finally, we performed voxelwise meta-regressions to assess the influence of potential moderators on the Group x Reward effect. For symptom severity, we used the average Positive and Negative Syndrome Scale (PANSS) total and subscores from each study. When necessary, a conversion from other scales to the PANSS was performed (van Erp et al., 2014).

We also investigated the impact of the medication dosage converted to chlorpromazine equivalents (Gardner et al., 2010) on group differences. The moderating role of antipsychotics was then controlled for the mean symptom severity. Meta-regressions were limited to p<0.0005 (Thorsen et al., 2018), while findings in regions other than those detected in the main analyses were discarded (Radua and Mataix-Cols, 2009).

RESULTS

Meta-analytical estimates of brain responses during reward anticipation

We collected data for 488 subjects (254 schizophrenia patients and 234 controls) and 44 foci (Table 1). When comparing controls with patients for the [reward anticipation vs.

neutral cue] contrast, we demonstrated an increased difference in activation, i.e., [reward anticipation > neutral cue] in [controls > schizophrenia patients], within a network involving the left striatum, the right median cingulate/paracingulate gyri, the left thalamus, the left postcentral gyrus, the left middle frontal gyrus, the cerebellum and the superior temporal gyrus.

After applying a jackknife analysis (testing for heterogeneity between clusters), Egger's tests and funnel plots (testing for the heterogeneity between studies), only the left striatum and the right median cingulate/paracingulate gyri remained reliable regions for consideration in the [controls>schizophrenia patients] contrast (Figure 1A). No significant difference was demonstrated for the [schizophrenia patients>controls] contrast (Table S1). that the meta-regression SDM value is derived from the proportion of studies that reported BOLD change near the voxel, so it is expected that the values of some of the studies are at 0 or near +1 (instead of being close to the line). "PANSS": Positive and Negative Syndrome Scale;

"SDM": signed d mapping.

Potential effect of symptoms severity and medication

Voxel-wise meta-regressions revealed that the left striatum was more affected in the contrast [reward anticipation > neutral cue] in [controls > schizophrenia patients] in severe schizophrenia patients (i.e. with higher total PANSS scores), independent of medication (Figure 1B). More precisely, less striatal activation was demonstrated in patients exhibiting more severe psychotic symptoms (PANSS positive sub-scores), even after controlling for the dosage of antipsychotics (Figure 1B).

DISCUSSION

In the present paper, we produced robust whole-brain meta-analytic maps associated with reward anticipation in schizophrenia. We showed that during reward anticipation, schizophrenia patients exhibit a reduced difference in activation in two key nodes of the putative salience network, the left striatum and the cingulate/paracingulate gyri. These findings support the idea that reward anticipation extends beyond the ventral striatum (VS) (Radua et al., 2015). Furthermore, our results are fully compatible with recent exploration in the healthy brain underscoring the role played by these nodes in reward anticipation (Wilson et al., 2018). Critically, we showed that within this salience network, VS hypoactivation was more pronounced in schizophrenia patients exhibiting more severe positive symptoms. These results remained significant even after controlling for the effect of antipsychotic dosage, suggesting that they are not driven by medication (Juckel, 2016).

Despite some methodological differences, our findings nicely complement three previous meta-analyses addressing similar questions: one taking a strict region-of-interest approach (Radua et al., 2015); another using a transdiagnosis approach, beyond schizophrenia (Zhang et al., 2016); and a last one that explored wider and more heterogeneous rewardrelated brain activation (Chase et al., 2018). We made the choice to focus only on anticipation of reward studies to favor a homogeneous selection of activation studies. Even if this remains a point of debate, some studies have suggested that anticipation of reward and prediction error may only partially share their neural bases. First, at the behavioral level (as in MID tasks), processes related to anticipation, outcome and prediction error can be separated (Knutson et al., 2000). Furthermore, electrophysiological studies have reported that reward signals of dopamine neurons were influenced by reward predictability (Hollerman and Schultz, 1998).

Finally, recent fMRI studies (Cao et al., 2019) have shown that different reward processing stages during the MID task were associated with distinct patterns of activation and connectivity. By performing a new prediction-error meta-analysis (based on 11 studies and using similar selection quality criteria), we were also able to provide evidence for differences with anticipation of reward, notably a nonrobust significant result in the cingulate for the [controls>schizophrenia patients] contrast (Table S2). What can initially appear as a power issue (i.e., 11 selected studies) was in fact balanced by the quality and homogeneity of the papers used (Müller et al., 2018). We further ensured the robustness of our findings by (i)

taking into account covariates such as symptom severity and medications, (ii) applying a restrictive threshold (Thorsen et al., 2018), and (iii) conducting a sensitivity analysis. However, the small number of studies makes it impossible to examine other issues, such as whether the results differed as a function of paradigms (e.g., MID vs. other types of tasks) or stages of illness (early vs chronic).

Furthermore, emotionally salient stimuli could have been an issue in the generation of reproducible findings because of their greater dependence on the patient's history (Winton-Brown et al., 2014), and we excluded studies using these kinds of stimuli. In contrast, reward stimuli, such as monetary rewards, seem more reliable (Daniel and Pollmann, 2010). The brain responses were also shown to vary between different types of salience, such as emotional salience or salience linked to novelty (Knolle et al., 2018). At this stage, it remains difficult to fully exclude the possibility that alternative procedures may have resulted in different rewardrelated functional results in the VS and, more globally, in the salience network. For instance, we did not replicate left VS hypoactivation in patients with more negative symptoms, which was a result observed in a previous striatum-focused meta-analysis (Radua et al., 2015).

Overall, we demonstrated a robust frontal-striatal signature of impaired reward anticipation in schizophrenia patients. In this study, we aimed to analyze a very homogeneous selection of studies, to reflect on paper selection from the reward literature and to perform meta-regression on symptom severity and dosage of treatment, which has not been done before. Within this functional network, VS hypoactivation was linked to the severity of psychotic symptoms, even when controlling for medication dosage.

SUPPLEMENTARY MATERIAL

Figure 1 :

 1 Figure 1: Coordinate-based meta-analytic findings for reward anticipation in schizophrenia. 1A. Inter-group effect size on signed differential maps during reward anticipation in schizophrenia. The right median cingulate/paracingulate gyri (1) and left striatum (2) were significant for the contrast [controls > schizophrenia patients (SCZ)] and survived (a) jackknife analysis, (b) Egger's tests and (c) funnel plots analyses. 1B. Meta-regression analysis testing for an association between symptom severity in the left striatum. Larger circles indicate studies with a larger sample size. The meta-regression SDM slope after controlling for the dosage of antipsychotics is presented as a straight line for average PANSS global scores (left panel) and PANSS positive sub-scores (right panel). Note

Table 1 : Characteristics of the activation studies included in the meta-analysis.

 1 Mixed studies recruited patients corresponding to (a) first-episode psychosis and (b) chronic schizophrenia categories.

	Study	Number of patients Type of patients	Number of controls Number of foci Design
	Alves et al.	10	First episode	12	2	Monetary incentive Delay Task
	Gradin et al.	15	Chronic	20	5	Instrumental reward learning task
	Koch et al.	44	Chronic	44	21	Monetary incentive Delay Task
	Walter et al.	16	Chronic	16	1	Monetary incentive Delay Task
	Schlagenhauf et al.	15	First episode and chronic	15	2	Monetary incentive Delay Task
	Smieskova et al.	29	First episode	19	3	Salience Attribution Task
	Subramaniam et al.	37	Chronic	20	1	Monetary incentive Delay Task
	Richter et al.	16	Chronic	16	5	Desire-reason-dilemma paradigm
	Chung et al.	36	Chronic	27	1	Variant of a response conflict task
	Potvin et al.	18	Chronic	24	2	Cue associated with smoking
	Reckless et al	18	Chronic	21	1	Rewarded perceptual decision-making task

Table S1 . Coordinate-based meta-analytic complementary results.

 S1

						Brain regions with
		significant activations during reward anticipation for the contrast [controls > patients with
		schizophrenia] or [patients with schizophrenia > controls]. MNI: Montreal Neurological
		Institute; "*" means nearest grey matter.
	Clusters Coordinate (MNI) Number of voxels	p	SDM-Z	Label	Jacknife	p Egger's test
	1	-16,6,-4	606	0.000045538	-2.137	Left striatum	9/11	0.553
	2	6,8,36	110	0.001922309	-1.635	Right median cingulate/paracingulate gyri	9/11	0.268
	3	-2,-14,6	80	0.001346052	-1.691	Left thalamus	10/11	0.016
	4	-42,-16,34	35	0.001594365	-1.666	Left postcentral gyrus*	10/11	0.063
	5	-38,50,0	24	0.002998114	-1.56	Left middle frontal gyrus	1/11	0.028
	6	4,-60,-8	22	0.003202200	-1.548	Cerebellum, vermic lobule, BA 18	10/11	0.054
	7	56,12,-12	11	0.002559066	-1.587	Right temporal pole, superior temporal gyrus, BA 38	9/11	0.023
	Clusters Coordinate (MNI) Number of voxels	p	SDM-Z	Label	Jacknife	p Egger's test
	1	0, -42, 36	659	0,000508249	-1,82	Left median cingulate/paracingulate gyri	6/10	0.09
	2	0, -6, -8	148	0,000837684	-1,754	undefined	2/10	0.20
	3	-2,-14,6	36	0,003093004	-1,556	Right precuneus	7/10	0.14

Table S2. Coordinate-based meta-analytic complementary results. Brain regions with significant activations during prediction error for the contrast [controls > patients with schizophrenia] or [patients with schizophrenia > controls]. MNI: Montreal Neurological Institute; "*" means nearest grey matter.

Figure S1. PRISMA Flow diagram of article selection process.

	Initial review process MEDLINE database search (N=376)	Key words (schizophren* OR psychosis) AND (fMRI OR PET) AND (salienc* OR reward OR aversive)
	)
		Not applicable
		Systematic literature review
		No fMRI or PET analysis
	Studies included for full-	No Schizophrenia
	text review (N=120)	No reward
		Excluded
		Studies without normalized coordinates
		No comparison SCZ vs control
		Partial brain coverage
	Studies included in the meta-analysis (N=25) 1076 subjects	Connectivity analysis (5 seed-based, 1 PLS, 6 ICA, 1 Granger causality, 1 structural, 1 DCM connectivity) Mono-ROI
		No Reward analysis
		No Prediction error analysis
		Overlaping studies
	Reward anticipation
	(N=11)

the SDM map from this study will be made available on the NeuroVault repository (https://identifiers.org/neurovault.collection:6255


Vincent Frochot
email: vincent.frochot@aphp.fr

Vincent Castiglione

Ivan T Lucas

Jean-Philippe Haymann

Emmanuel Letavernier Abc

Dominique Bazin

Giovanni B Fogazzi

Michel Daudon

Advances in the identification of calcium carbonate urinary crystals

INTRODUCTION

Crystalluria is the result of urine supersaturation with different types of salts the most frequent of which are calcium oxalate, calcium phosphate, and uric acid [1,[START_REF] Daudon | Intérêt clinique de l'étude de la cristallurie[END_REF]. In normal subjects, supersaturation can transiently occur due to non-pathological high solute concentration, while in stone formers supersaturation is frequent and persistent over time [1,[START_REF] Daudon | Intérêt clinique de l'étude de la cristallurie[END_REF]. Various conditions can cause crystalluria, including infection [START_REF] Flamigan | Renal. Struvite stones -pathogenesis, microbiology, and management strategies[END_REF], acquired or inherited metabolic diseases [START_REF] Frochot | Clinical value of crystalluria and quantitative morphoconstitutional analysis of urinary calculi[END_REF] or, more often, dietary habits [START_REF] Bazin | La place dela physicochimie en urologie et en néphrologie : le bilan de 10ans de collaboration entre physiciens, chimistes et praticiens hospitaliers[END_REF]. In all these conditions a correct identification of crystals is mandatory, this being achieved with the combined and matched knowledge of: urinary pH, crystal morphology, and birefringence features under polarized light [START_REF] Verdesca | Crystalluria: prevalence, type of crystals and the role of infrared spectroscopy[END_REF]. When this approach is insufficient, more sophisticated techniques, such as infrared spectroscopy [START_REF] Verdesca | Crystalluria: prevalence, type of crystals and the role of infrared spectroscopy[END_REF][START_REF] Bazin | Characterization and some physicochemical aspects of pathological microcalcifications[END_REF][START_REF] Bazin | Some advances in the field of physico-chemical characterization of pathological microcrystals[END_REF], scanning infra-red and Raman vibrational spectroscopy [START_REF] Rouziere | In-lab X-ray fluorescence and diffraction techniques for pathological calcifications[END_REF] are needed.

In this paper, we report the case of a woman whose urine contained three different types of crystals with unusual morphology which, with the use of advanced infrared spectroscopy techniques, were identified as variants of calcium carbonate (Ca-carb) crystals.

CASE REPORT

On July 4, 2016 a 64-year-old woman was seen in the outpatient clinic of the Rheumatology Department of the University Hospital at Liège (Belgium) for a periodic evaluation of her psoriatic arthritis, for which she was under treatment with methotrexate. Co-morbidities included arterial hypertension, hypothyroidism and dyslipidemia, which were treated with nifedipine, L-thyroxine, and rosuvastatine respectively. Laboratory investigation was normal but for a reduced e-GFR-MDRD (51 ml/min/1.73 m 2 , normal value: >90 mL/min/1.73 m 2 ). Urinalysis, performed with dipstick 30 minutes after micturition on the second urine of the morning, collected 2 hours after getting up with the patient under fasted conditions, showed: pH 7.5, specific gravity 1.025, and negative albumin, hemoglobin, leukocyte esterase, and nitrites.

The bright field microscopic examination of the centrifuged urine sample showed a few bacteria, a few squamous epithelial cells, and a massive crystalluria. With the additional use of polarized light, three different types of crystals could be seen: 1. Flattened rectangles slightly birefringent under polarized light; 2. Non birefringent "daisy-like" crystals, identical to those described elsewhere [START_REF] Fogazzi | An unusual type of crystalluria (appearing only once every 130 years?)[END_REF][START_REF] Fogazzi | Daisy-like" crystals: a rare and unknown type of urinary crystal[END_REF]; 3. Strongly birefringent sticks and needles with welldefined edges (Figures 1 and2).

Due to the unclear nature of crystals, the remaining aliquot of urine was filtered and dried and the sediment was submitted to advanced infrared sprectroscopy investigation in the Service des Explorations Fonctionnelles Multidisciplinaires, Hôpital Tenon, Paris (see below).

Of note, crystalluria was not found in any of the urinary samples examined subsequently.

Infrared microspectroscopy and attenuated total reflectance (ATR) imaging allowed to identify the flattened rectangles as calcite (Figure 3). Raman spectroscopy with spatial resolution under 1 micron identified "daisy-like" crystals as vaterite, and sticks/needles crystals as aragonite (Figure 4). Energy dispersive X-ray spectroscopy (EDX), performed on glass slide, showed a homogeneous spatial repartition of calcium in all crystals (Figure 5). Thus, we could identify the observed crystals as polymorphs of anhydrous Ca-carb.

DISCUSSION

Different types of Ca-carb exist. The most stable ones are: the anhydrous (CaCO 3 ) i.e., calcite, aragonite, and vaterite; the hydrated i.e., monohydrocalcite (CaCO 3 , H 2 O), and ikaite (CaCO 3 , 6H 2 O); the amorphous, which may contain water molecules [START_REF]Powder diffraction file[END_REF][START_REF] Yong-Jin Han | Calcium carbonate storage in amorphous form and its template-induced crystallization[END_REF]. Such biominerals are the most common types present in mollusk shells or pearls, eggshells, corals [START_REF] Politi | Transformation mechanism of amorphous calcium carbonate into calcite in the sea urchin larval spicule[END_REF][START_REF] Lowenstam | Transformation of Amorphous Calcium Phosphate to Crystalline Dahillite in the Radular Teeth of Chitons[END_REF] and cyanobacteria [START_REF] Benzerara | Intracellular Ca-carbonate biomineralization is widespread in cyanobacteria[END_REF].

Ca-carb has a pivotal role in biomineralisation processes in a large number of animal species [START_REF] Nwaokorie | Risk factors for calcium carbonate urolithiasis in goats[END_REF] and microorganisms, while it is infrequent in humans, with the exception of physiological crystals in the inner ear and pathological calcifications in pancreas and gallbladder, where the three polymorphs of Ca-carb calcite, aragonite and vaterite are often observed [START_REF] Cros | Investigation at the micrometer scale of pancreatic calcifications in chronic pancreatitis by mFTIR spectroscopy and field emission scanning electron microscopy[END_REF]. Moreover, it has been reported that, in the thin descending limb of the Henle's loop, urine is supersaturated not only with calcium phosphate but also with Ca-carb [START_REF] Asplin | Evidence of calcium phosphate supersaturation in the loop of Henle[END_REF]. The clinical meaning of Ca-carb crystals in the urine of humans is unclear at the moment, even though a possible association with a diet rich in vegetables has been hypothesized for some subjects whose urine contained daisy-like crystals [START_REF] Fogazzi | An unusual type of crystalluria (appearing only once every 130 years?)[END_REF][START_REF] Fogazzi | Daisy-like" crystals: a rare and unknown type of urinary crystal[END_REF].

The Ca-carb described in this paper are very unusual and differ from those reported in textbooks and atlases mostly as dumbbells, granules and, less frequently, as ovoids, spheres, or tetrads [START_REF] Haber | The surface ultrastructure of urinary casts[END_REF][START_REF] Graff | A Handbook of Routine Urinalysis[END_REF][START_REF] Piccoli | Atlas of Urinary Sediments. Diagnosis and Clinical Correlations in Nephrology[END_REF][START_REF] Strasinger | Urinalysis and Body Fluids. A self-Instructional Text[END_REF][START_REF] Landy | Textbook of urinalysis and body fluids : a clinical approach[END_REF][START_REF] Barlough | Canine and feline urinalysis: value of macroscopic and microscopic examinations[END_REF][START_REF] Fogazzi | Urinary sediment findings in acute interstitial nephritis[END_REF][START_REF] Haber | Color Atlas of the Urinary Sediment. An Illustrated Field Guide Based on Proficiency Testing[END_REF][START_REF] Brunzel | Fundamentals of Urine & Body Fluid Analysis Fourth Edition[END_REF], which are birefringent under polarized light [START_REF] Haber | The surface ultrastructure of urinary casts[END_REF][START_REF] Fogazzi | Urinary sediment findings in acute interstitial nephritis[END_REF][START_REF] Haber | Color Atlas of the Urinary Sediment. An Illustrated Field Guide Based on Proficiency Testing[END_REF][START_REF] Brunzel | Fundamentals of Urine & Body Fluid Analysis Fourth Edition[END_REF]. Microscopic infrared spectroscopy and Raman spectroscopy show that these crystals are anhydrous Cacarb polymorphs and EDX analysis confirmed the high calcium content. Similarly to previous cases with "daisy-like" crystals [10-11] also in the present subject Ca-carb crystals were not found in subsequent urine samples. This fact suggests that supersaturation with Ca-carb, the cause of which is unclear, was transient.

The difference in morphology between these three Ca-carb crystals is due to their crystalline structure: trigonal for calcite, orthorhombic for aragonite and hexagonal for vaterite. We can explain the differences between the three polymorphs from the different arrangement of the carbonate ions in their crystal structures. Many force fields have been developed to describe the Ca-carb interactions [START_REF] Devilliers | Crystal structures of aragonite, strontianite, and witherite[END_REF][START_REF] De Leeuw | Surface structure and morphology of calcium carbonate polymorphs calcite, aragonite, and vaterite: an atomistic approach[END_REF]. Other empirical models have been used to explore the bulk and surface properties of Ca-carb at ambient conditions [START_REF] De Leeuw | Surface structure and morphology of calcium carbonate polymorphs calcite, aragonite, and vaterite: an atomistic approach[END_REF][START_REF] Fisler | A shell model for the simulation of rhombohedral carbonate minerals and their point defects[END_REF]. Environment factors influence the structure that the crystal lattice takes during its formation: humic acid, presence and/or association of magnesium, sulfate, potassium, sodium and the rate of calcium [START_REF] Angel | Automated profile analysis for single-crystal diffraction data[END_REF]. The complexity of the urinary environment and its ionic balance could explain the presence of these three types of Ca-carb in this patient. Furthermore, it has been underlined that several kinds of amorphous carbonate exist, their stability depending on the presence of other compounds such as phosphates [START_REF] Levi-Kalisman | Structural differences between biogenic amorphous calcium carbonate phases using X-ray absorption spectroscopy[END_REF].

In conclusion, with the use of advanced infrared spectroscopy techniques we have identified for the first time with certainty three different types and morphologies of calcium carbonate in human urine. However, the relationship between urinary environment, concentration between different ions and the formation of calcite, aragonite and vaterite, cannot be still answered in this study. Moreover, our patient and all patients except one in the series reported by Fogazzi et al. were females [START_REF] Fogazzi | An unusual type of crystalluria (appearing only once every 130 years?)[END_REF][START_REF] Fogazzi | Daisy-like" crystals: a rare and unknown type of urinary crystal[END_REF]. No clear explanation can be provided to explain the high prevalence of female gender and alkaline urine among patients who form vaterite in urine.

Further investigation is required.

Legends to figures

Figure 1 .

 1 Figure 1. Crystals as seen with bright field microscopy: 1. flattened rectangles; 2. "daisylike"; 3. stick/needle -like; 4. Agglomerate of "daisy-like" crystals.

Figure 2 .

 2 Figure 2. Crystals as seen with polarized light microscopy: 1. slightly birefringent flattened rectangles; 2. non birefringent "daisy-like"; 3. strongly birefringent stick-needle like.

Figure 3 .

 3 Figure 3. Infrared spectroscopy peaks (1797, 1413, 875 and 711 cm-1) of calcium carbonate (calcite).

Figure 4 .

 4 Figure 4. Typical RAMAN spectra of vaterite (daisy like crystals) and aragonite (sticks and needles).

Figure 5 .

 5 Figure 5. Energy dispersive X-Ray spectroscopy (EDX) spectra showing a presence of calcium in all crystals (calcite; aragonite and vaterite). The urine sediment is deposited on a glass slide.

  Figure 1

Figure 3 Figure 4 Fig ur e 5

 345 Figure 3

KEY WORDS

Urinary sediment

Calcium carbonate crystals

Infrared microspectroscopy

Raman spectroscopy

Energy dispersive X-Ray spectroscopy

ABSTRACT

The examination of the urinary sediment of a 64-year-old woman showed the presence of three different types of crystals, all with unusual morphology, which could not be identified with bright field microscopy, polarized light, and the knowledge of urine pH (7.5). The use of microscopic infrared spectroscopy, Raman spectroscopy and energy dispersive X-ray spectroscopy led to the identification of the three types of crystals as calcite, vaterite and aragonite, which are all variants of calcium carbonate crystals. This paper confirms the complex morphology and nature that urinary crystals may at times have and the utility of advanced infrared spectroscopy techniques for their identification.