Gérard Demazeau

Patrick Maestro

Th Plante

Michel Pouchard

teaching and research institutions in France or abroad, or from public or private research centers.

Generation of high oxygen pressure by the decomposition of oxidizing acids or NH4Cl04 under hydrothermal conditions : application to the preparation of CrO2

ABSTRJ\CT A new method for generating high oxygen pressures has led to a novel oxidative process for the preparation of Cr0 2 . In order to improve the magnetic properties, in particular the coercive field , of such a ferromagnetic oxide a substitution by rhodium +IV led to the new interesting material cr 1 _ x Rh x o 2 (H � � 500 Oe).

THE HIGH OXYGEN PRESSURES

High oxygen pressures can be obtained by three methods : -by compressing the gas, this can be used onl î up to 5 kb due to the danger of explosive combustion in pure oxygen Ll], -in the solid phase by thermal dccomposition of an unstable oxide such as KCl0 3 in a "belt" apparatus, 'JP to 80 kb, -in hydrothermal conditions using oxidizing solutions such as HCl04 HCl0 3 , H 5 Io6 , HI0 3 or NH 4 Cl0 4 with a maximum pressure of 5 kb. The first and the second methods have been the subject cf numerous studies aiming, in particular, at the synthesis of oxides containing a transition element in a high oxidation state (cobalt +III , nickel +III , copper +III ... [2,3,4 ]. The last method recently developed, has led to a novel oxidative process for the preparation of Cro 2 • \ N)VEL OXICATIVE PROC'':SS FOR THE F:3H PRESSURE PREPARATI0N OF CHROMIUM DIOXIDE The structural and ma gnetic properties of Cro 2 , which is metastable at ordinary pressure, were first described by A. MICHEL and J. BENARD [s}. Chromium dioxide can be used for magnetic recording, in particular at high frequencies where its recording performance is better than the comp etitive material Fe 2 03ydopcd with cobalt [6]. For recording appli cations Cr0 2 must have a high coercive field (He> 300 Oe) and a rema nence ratio or/os of about 0.5 for a random assembly of particles. Such characteristics require Cro 2 to be obtained in needle-like parti cles of a single magnetic domain.

Methods for the preparation of this oxide çan be classified into two types, those based on reduction and those using oxidation. �or the first Cro 3 is water-soluble and the preparation of Cro 2 thus de pends on the conditions of the nucleation and growth of microcrystals in the solution phase. These conditions are particularly delicate if CrO 2 is to be obtained as needles of magnetic single domain. In th e oxidative method as the starting material cr 2 o 3 is almost insoluble in the reaction medium, the particle size of tne chromium dioxide obtained will depend sensitively on that of the cr 2 o 3 . As the latter is more easily controlled than the experimental condi tions governing the nucleation in solution we have chosen to develop the oxidative method. The choice of the oxidizing agent is of parti cular importance here as it determines both the shape and the size of the Cro 2 particles. Most of the industrial processes, and notably that of DUPONT DE NEMOURS, involve reduction of cro 3 [7]. This company has also paten ted several processes involving oxidation of cr 2 o 3 , anhydrous or hydrated, by compressed oxygen or by oxidizing agents such as CrO 3 or H2O2 (a), but these methods are difficult to industrialize. In our method the starting material is very finely divided Cr 2 O 3 obtained by the thermal decomposition (between 150 and 250 °C) of (NH 4 ) 2 cr 2 o 7 . Oxidative methods using oxidizerscontaining metallic cations such as KClO 3 or BaO 2 , yield simultaneously chromates (VI). The use of agents such as HClO 4 , HIO 3 , H 5 ro 6 or NH 4 c1O 4 at low tempc(ature (350<t< 4 00 °C) ann moderated pressure (2 kb) for short times leads to a � ood acicular Cro 2 with a coercive field of about 360 Oe (table I

--

A NEW FERROMAGNETIC MATERII\L Cr 1 _ x Rh :-:: O 2 For a significant improvement in the magnetic performance of the ma terial the acicularity and magnetic anisotropy must be increased. It is possible to accomplish this by doping with elements which fall, according to their mode of action, into one of the following catego ries: -the dopant acts as a crystallization nucleus for a Cro 2 microcrys tal in the solution � hase. This is the case of carbon in an aqueous solution of cro 3 [lOJ.

-the dopant favors ncedle-like growth of particles by crystal growth orientation. This is the case of some isostructural oxides such as ,. ). This solid io1&tion is ferromagnetic at room temperature for x � O.S. Since rhodium is expensive we have attempteù to obtain good magnetic performance at low rhodium concentration. Using as starting oxide Cr 2 O 3 finely divided and as oxidizing agent NH4ClO4 with addi tion of H 3 BO 3 as buffer agent it is possible to reach.in the same experimental conditions P, T as for pure CrO2 , coercive fields in excess of 500 Oe at very low rhodium contents (x = 0,001) [17]. The figure 1 shows the good acicularity of these particles and the absence of dendrites. ,. [17)

  Cr 2 WO 6 [11] or A 2 TeO 6 (A= Al, Cr, Mn, Fe) [1 2 , 1 3 ] bath phases of the trirutile type. -the dopant inhibits crystal growth of CrO2 particles by adsorptio n to the surface [14]. -the dopant can enter into the rutile lattice forming a solid solu tion and thus enhancesthe desired magnetic properties [1s]. We have chosen to prepare the solid solution Cr 1x Rh x O 2 , CrO2 and RhO 2 being isostructural (16]. The rhodium +IV in an ox S gen lattice and in octahedral sites has a low spin configuration (t 2 e O ) and exhi bits magnetic anisotropy(ground state 2 T 2 9

  lµ

Fig. 1

 1 Fig. 1 Scanning �lectron microaraphs of cr 1 _ x Rh x o 2 (x = 0,001) prerarations New development� in magnetic recording particles have brought about a new oxidative process for synthesizing CrO2 and the improvement of its mag netic characteristics by substitution of rhodium +IV into the rutile lattice of chromium dioxide.

GENERATION OF HIGH OXYGEN PRESSURE BY THE DECOMPOSITION OF OXIDIZING ACIDS OR NH4C 104 UNDER HYDROTHERMAL CONDITIONS: APPLICATION TO THE PREPARATION OF CrO2 G. Demazeau, P. Maestro, Th. Plante, M. Pouchard and P. Hagenmuller Laboratoire de Chimie du Solide du CNRS, Université de BDrdeaux I, 351 cours de la Libération, 33405 Talence, Cedex, France

  ) l9].

			TJ\BLf. I
	Variation of magnetic performancP w\.th rcaction time and tempcrature
	Starting material	oxid,rnt p(kb)	e < •c >	t (min.) H c COc)	Or os/3000 Oe)
	Cr 2 O 3 ,5H 2 O	NH 4 c1O 4	2	420	113	136
	Cr 2 o 3	NH 4 ClO 4	2	420	112	145	0.36
	Cr 2 o 3	NH 4 Cl0 4	2	400	98	205	0.41
	Cr 2 O 3	N11 4 c1O 4	2	370	65	270	0.42
	Cr 2 o 3	NH 4 c10 4	2	3'>0	48	360	0.54

Acknowledgments

The authors are grateful to D.G.R.S.T. and A.N.V.A.R. for their sup port. One of us (P.M.) wishes to thank Rhône-Poulenc.


Ben Béchir

L'enfant de remplacement entre le rural et l'urbain

Mlle S. est âgée de 53 ans, originaire d'un milieu rural (Nefza, Béja) et demeurant dans un milieu urbain. Elle est célibataire et travail en tant qu'infirmière dans un service de chirurgie pédiatrique. Motif de la consultation : Elle nous a été adressée du service de Neurologie de l'hôpital Razi pour : Troubles de la concentration et de la mémoire et une humeur dépressive. Une description du fonctionnement psychique était préconisée. Éléments biographiques : Elle est issue d'un mariage non consanguin et scolarisée jusqu'à la 6ème année secondaire. Arbre généalogique : À noter que juste avant la naissance de S : Une double perte de ses deux soeurs H et S La séparation de son père de sa 1ère femme Le décès de son grand père maternel Suite à ces événement dépression de la mère Grossesse et enfance : Grossesse non programmée mais désirée. Avant sa naissance décès de ses deux soeurs H âgée de 10 ans et S âgée de 3 ans par une insolation pendant la récolte. Père : est décédé à l'âge de 70 ans (S. avait 29 ans). Lui-même est un enfant de remplacement (il a remplacé son père). Il avait deux femmes et a divorcé de sa 1ère épouse pour stérilité (à la naissance de S). Il est proche de S au point qu'il la chargeait des affaires de l'agriculture dès son jeune âge. Mère : âgée de 86 ans. Elle est pratiquement absente du discours de la patiente. Fratrie/ relations familiales : S estime que ses soeurs ne s'occupent pas « bien » de leurs enfants Elle assume la charge de ses 4 neveux (tous les garçons de ses soeurs) dès l'âge de la puberté. Vie sociale : Elle n'a pas d'amis/es et ses liens sociaux se limitent à sa relation avec ses neveux. Vie amoureuse : Deux fiançailles rompues car elle mettait ses intérêts familiaux au dessus de sa vie amoureuse. Vie professionnelle : À 3/4 ans : Agriculture : la seule à s'occuper avec son père des affaires de l'agriculture familiale (rural) À 17 ans : Etudes d'infirmière tout en gérant la terre familiale (rural) À 21 ans : Infirmière à l'hôpital (urbain) À 24 ans : Arabie Saoudite : améliorer la situation financière de sa famille À 29 ans : Décès de son père : Chirurgie pédiatrique (urbain) S. n'a jamais pris un congé annuel pour se reposer et se décrit comme perfectionniste dans son travail au point qu'elle refuse de déléguer des taches à une autre personne

Introduction

Les "enfants de remplacement" sont des personnes qui ont été conçus après la mort d'un être proche des parents (enfants, parents…) ou dont le deuil n'est pas réalisé. Sa naissance est une « mise à la place de l'autre ». En effet, il n'est pas seulement conçu pour remplacer mais de se substituer à lui. L'enfant de remplacement est un bouc émissaire. Il est attendu d'abord pour porter le fardeau dont ses parents tentent de se décharger. Le poids des attentes, exprimées ou non, de ses parents peuvent l'empêcher de prendre en main sa vie, de choisir, de découvrir lui-même et pour lui-même le sens de sa vie. À l'extrême, il s'interdira de chercher son identité propre et envisagera d'autres identités [1]

Cas clinique

Discussion psychopathologie

[1] Alby, N., « L'enfant de remplacement », in Evolution Psychiatrique (1974) n°3, 557-566.

[2] Porot, M., (1996) L'enfant de remplacement, Frison-Roche, Paris.

[3] Mazet Ph., Lebovici S., (1996) Mort subite du nourrisson : un deuil impossible ? L'enfant suivant, PUF, Paris.

[4] Hadded, G., (2013) Tripalium. Pourquoi le travail est devenu une souffrance, Broché, Paris.

[5] Sabbadini, A., « L'enfant de remplacement », in La psychiatrie de l'enfant (1989) n°XXXII-2, 519-541.

Conclusion

La particularité des enfants de remplacement c'est qu'ils expriment leur difficulté à vivre dans l'ombre de l'enfant mort. De plus ils se confrontent à une lutte permanente contre la pulsion de mort. Notre patiente a du gérer cette instabilité entre l'urbain et le rural comme elle a fait avec la mort de son père et la maladie (Alzheimer) de sa mère. D'où la difficulté de sa prise en charge.

Références bibliographiques

Résultats des tests projectifs

  Représentation de soi : répercussion anxiogène/fragilité des contenants / Difficulté identificatoire Images parentales : mère archaïque dépressive et absente / père n'assume pas la position phallique Mode d'adaptation : marqué par l'inhibition / pensée conformiste et peu stéréotypée Mode perceptif : Tendance à saisir les choses dans leurs ensemble au détriment d'une pensée analytique / Difficultés à délimiter le dedans du dehors Mode d'expression : Utilisation excessive des affects (figure maternelle +++) Plus de pulsions agressives que libidinales / Manque d'empathie Problématique : anaclitique Angoisse : de perte d'objet Remplacer un mort : L'enfant, mort en plein développement, reste investi de nombreuses espérances qui ne pourront se réaliser. Il est fortement idéalisé par ses parents, qui gardent de lui le souvenir d'un être parfait. L'enfant à naître remplit une mission difficile : Dans l'inconscient des parents, il est confondu avec l'enfant mort. Cette identification à l'enfant perdu, idéalisé, est source de comparaison et de compétition impossible à dépasser (S. est idéalisée par son père) L'enfant de remplacement vit identifié à un objet mort. Il doit faire lui-même le deuil de l'enfant perdu par ses parents, deuil que ceux-ci ne sont pas parvenus à élaborer (S. déni la maladie d' Alzeimer de sa mère : maladie de l'oubli) Parmi les troubles psychologiques repérés chez la patiente nous évoquons : troubles de l'identité et des identifications /culpabilité / dépression [2] [3] Troubles structuraux et l'enfant de remplacement : Les troubles de l'identité vont du simple sentiment de n'être pas soi-même jusqu'à la confusion psychotique. L'enfant, conçu et élevé dans ces circonstances, est condamné à un non-être, puisqu'il n'a pas été conçu pour lui-même [2] Il risque d'éprouver des difficultés dans le domaine de l'identité de soi lors de la phase de séparation-individuation et durant l'adolescence [5] Le service de S. rapporte qu'elle « apporte la vie » à l'hôpital. La culpabilité chez elle s'exprime selon deux modes : Attitude dépressive, avec sentiment de dévalorisation et d'échec Attitude persécutrice avec l'impression que les autres vous accusent ou vous suspectent à propos de la mort de l'enfant précédent [2] Infirmière en chirurgie pédiatrique (urbain) : S. s'occupait particulièrement des enfants atteints de maladies rares et chez qui le risque de mort est élevé. C'est une défense contre l'angoisse de mort, la culpabilité et la mort enfant de remplacement. De plus, S. gardait les liens avec les familles des enfants malades et les investissait d'une manière excessive. Congé longue duré de 9 mois (rural) : Durant lequel la patiente a quitté sa maison et est allée à Nefza pour travailler la terre de sa famille. Un travail manuel qui lui rendait heureuse. Cela ressemble énormément aux bricoleurs, aux artisans et jardiniers du dimanche. D'où l'explosion que connaît le bricolage en France. [4] Elle tenait absolument à reprendre son travail au bout de 9 mois. Elle a appris la vente de la terre familiale faite à son insu (les 9 mois de grossesse puis la découverte du bébé) Le travail de la terre : l'agriculture : Depuis longtemps les psychanalystes ont vu dans les avatars de la sexualité, dans ses échecs, la racine principale de la douleur psychique. Freud avait bien pressenti l'existence d'une autre cause → le rapport du sujet à son labeur. (laborieux et amour : même racine → lăbrum) Le grand mystère, disait Lacan, n'est pas l'apparition de l'homo sapiens, c'est celle de l'homo faber, le premier primate qui s'est mis à travailler. S'il y a une essence de l'homme, ne faut-il pas la chercher de ce côté-là, dans son rapport au travail ? Pour S. le travail est une passion. Elle rapporte que depuis son jeune âge l'agriculture représentait une jouissance. C'est une activité partagée avec son père Le 1er acte du travail de l'agronome n'est que le fait d'ouvrir la terre pour déposer ensuite les semences. Et pour l'activité sexuelle le 1er acte est : ouvrir une femme puis déposer les semences [4] (lien avec son père et son congé)


domain: chim sde

text:
E Boullay

Guilmeau

Comment on the "Discovery

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

Comment on "Discovery of high-performance thermoelectric copper chalcogenide using modified diffusion-couple high-throughput synthesis and automated histogram analysis

technique" by T. Deng, T. Xing, M. K. Brod, Y. Sheng, P. Qiu, I. Veremchuk, Q. Song, T-R. Wei, J. Yang, G. J. Snyder, Y. Grin, L. Chen and X. Shi, Energy Environ. Sci., 2020,13, 3041-3053 P. Lemoine, [a,c] B. Raveau, [b] P. Boullay, [b] E. Guilmeau* [b] [a] Univ Rennes, CNRS, ISCR -UMR 6226, F-35000 Rennes, France [b] CRISMAT, CNRS, Normandie Univ, ENSICAEN, UNICAEN, 14000 Caen, France Email: emmanuel.guilmeau@ensicaen.fr [c] Institut Jean Lamour, UMR 7198 CNRS -Université de Lorraine, 2 allée André Guinier-Campus ARTEM, BP 50840, 54011, Nancy Cedex, France Abstract. The recent paper by Deng et al. 1 showed that a copper sulphide with Cu7Sn3S10 composition is a promising thermoelectric material reaching a figure of merit ZT of 0.8 at 750 K via Cl doping. However, structural reinvestigations of Cu7Sn3S10 sample (i.e. Cu2.10Sn0.90S3) prepared in the reported conditions show that the phase of interest is not tetragonal (space group 𝐼4 # 2𝑚, at = 5.4164 Å, ct = 10.832 Å), but crystallizes in the cubic symmetry (space group 𝑃4 # 3𝑛, ac = 10.832 Å) with a composition close to Cu22Sn10S32 similar to the results published by Pavan Kumar et al. 2 These observations, which indicate that the main phase around the composition Cu7Sn3S10 is cubic, impose a revision of the understanding of the relationships between the structure and thermoelectric properties in this family.

In a recent article, Deng et al. 1 discovered a high performance thermoelectric with the composition Cu7Sn3S10 and demonstrated that it is a promising material with ultralow thermal conductivity, moderate band gap and high electrical conductivity. A figure of merit ZT of 0.8 at 750 K was indeed reached via Cl doping. In the structural analysis of this material by powder X-ray diffraction, considering stannite-like structural models (i.e. tetragonal superstructure of the sphalerite type with the space group 𝐼4 # 2𝑚, a = 5.4164 Å, c = 10.832 Å), difficulties appeared in the refinement getting either chemical composition Cu39.3Sn10.7S50.0 far from the nominal one Cu35Sn15Sn50, also confirmed by EDXS (model 1), or large spread of the atomic ACCEPTED MANUSCRIPT / CLEAN COPY displacement parameters (model 2). A further structural model was mentioned (model 3) assuming chemically improbable mixed cation/anion occupancies of the positions, but yielding expected chemical composition and reasonable values of the atomic displacement parameters.

Finally, the authors decided to postpone the clarification of this issue to a separate study, and the structural model 2 was considered for the interpretation of the attractive thermoelectric properties of this material.

In posterior study of the Cu-Sn-S system, Pavan Kumar et al. 2 synthesized a low thermal conductivity degenerate semiconductor with a rather close composition, Cu22Sn10S32 (i.e. Cu2.0625Sn0.9375S3), and showed that this sulphide also exhibits attractive thermoelectric properties with a figure of merit ZT of 0.55 at 700 K via Sb for Sn substitution. For the structure solution in the tetragonal symmetry, the authors met in a first step difficulties similar to those encountered by Deng et al. like refined chemical composition far from the nominal one.

However, they noticed that, considering the fact that the c parameter is rigorously double of the a parameter in the tetragonal structure of 𝐼4 # 2𝑚 space group, may imply a cubic supercell (ac = 2at = ct). 2 Note that refinements reported in Deng et al. 1 article lead also to a cell parameters ratio ct/at of 2. Using a combination of synchrotron powder X-ray diffraction, single crystal Xray diffraction, 119 Sn Mössbauer spectroscopy and transmission electron microscopy, Pavan Kumar et al. 2 demonstrated that the phase of interest crystallizes in fact in a cubic superstructure sphalerite-type with space group 𝑃4 # 3𝑛 , and is characterized by a semi-ordered cationic distribution: the Cu-Sn disorder being localized on one crystallographic site (see Table 1).

Based on the comparison of these results showing strong similitudes between Cu7Sn3S10 and Cu22Sn10S32, we have analysed a sample provided by the group of X. Shi and prepared in the conditions reported in their paper (i.e. heat treatments at 723 K, 1223 K and 1073 K, followed by ball-milling process and SPS treatment at 923 K) to revisit the structural analysis of the materials around the composition Cu7Sn3S10 (i.e. Cu2.10Sn0.90S3). The aim of the present comment is to draw the attention on the crystal chemistry of the thermoelectric chalcogenides which requires detailed structural studies to elucidate complex order-disorder phenomena and on its impact on the understanding of their transport properties.

Rietveld refinements considering the tetragonal stannite-like (i.e. model 2) and the cubic Cu22Sn10S32 structural models, reported by Deng et al. 1 and Pavan Kumar et al. 2 respectively, have been performed on long duration (48 h) powder X-ray diffraction data collected at CRISMAT laboratory using a B ruker D8 Advance Vario A two-circle diffractometer (θ-2θ ACCEPTED MANUSCRIPT / CLEAN COPY Bragg-Brentano mode) equipped with a Ge(111) monochromator (Johansson type) and a Lynx Eye detector and a Cu X-ray tube (λ = 1.5406 Å). Similar to the description provided by Deng et al. 1 , the Rietveld refinement considering the tetragonal stannite-like structural model (𝐼4 # 2𝑚)

gives at first sight an apparent high-quality refinement and some correct reliability factors (Figure 1, top panel). However, a careful analysis of the refinement reveals the presence of additional non-indexed diffraction peaks of weak intensity as clearly evidenced in the top panel of Figure 2 with intensity in logarithmic scale. These weak intensity diffraction peaks can be indexed as superstructure diffraction reflexions in a twice larger cubic cell similar to that previously observed by Pavan Kumar et al. 2 for Cu22Sn10S32. The reinvestigation of the crystal structure of Cu7Sn3S10 sample was then carried out with the cubic structural model reported for Cu22Sn10S32, 2 assuming that an homogeneity range with rather close compositions may exist.

Rietveld refinement of the powder X-ray diffraction data considering the cubic structural model (space group 𝑃4 # 3𝑛) of the Cu22Sn10S32 phase 2 also provides very low reliability factors (Figure 1, bottom panel). Moreover, on the opposite to the tetragonal unit cell, 1 the cubic structural model allows to perfectly fit all of the diffraction peaks, including the weak intensity superstructure reflexions (Figure 2, bottom panel). Importantly, it leads to refined structural parameters and chemical composition Cu22.2(1)Sn9.8(1)S32 (Table 1) in fair agreement with those reported for the cubic phase Cu22Sn10S32. 2 The refined composition (i.e. Cu2.08Sn0.92S3), slightly different from the nominal one Cu2.10Sn0.90S3, is supported by the existence of a broad diffraction peak, detected at 2θ = 26.5° (Figure 2), which may be assigned to a binary copper sulphide present in minor amount in the sample. Note that the refinement of the atomic parameters in the 923 K sample (Table 1) results in the composition Cu22.8(2)Sn9. As a consequence, the crystal chemistry results, reported by Deng et al., 1 cannot be used to explain the thermoelectric properties of the pristine Cu7Sn3S10 sample. The fact that the structure of the pristine sample is cubic and not tetragonal influence significantly the interpretation previously proposed for the lattice thermal conductivity of pristine and Clsubstituted SPS-treated Cu7Sn3S10-xClx materials which can no more be compared on the basis of a tetragonal-cubic conversion neither on the disappearance of the distorted non-cubic framework due to the Cl-substitution. 1 The fact that the pristine composition exhibits a lower lattice thermal conductivity than those of disordered Cl-substituted materials remains also still quite unexpected. Indeed, Cl-substituted samples exhibit a cubic fully-disordered sphalerite structure, while the pristine composition Cu7Sn3S10 (in fact cubic Cu22.2(1)Sn9.8(1)S32) has a partially ordered structure. The pristine compound should then exhibit a larger lattice thermal conductivity than the Cl-doped compounds. Full cationic disorder, induced by Cl-substitution, stoichiometry deviation or process effect, are well known to strongly enhance phonon scattering and to reduce the lattice thermal conductivity in copper-based sphalerite derivative compounds. 3,4 Attention must be drawn on the extremely low value of the lattice thermal conductivity of the pristine phase. A possible explanation may be in the estimation of the latter by the expression kL = k -LsT, where L is the Lorenz number calculated by the single parabolic band (SPB) model. [START_REF]Thermal Conductivity Theory, Properties, and Applications[END_REF][START_REF] May | Thermoelectrics and its energy harvesting[END_REF] Unfortunately, this model is inadequate for L calculation due to the multiband nature of the valence band structure of this highly metallic degenerate semiconductor. It results in too high L values leading to a substantial underestimation of kL.

ACCEPTED MANUSCRIPT / CLEAN COPY

ACCEPTED MANUSCRIPT / CLEAN COPY

This difficulty in determining the lattice contribution has been highlighted in the degenerate semiconductor Cu5Sn2S7, 3,[START_REF] Kumar | [END_REF] and several thermoelectric materials, such as SnTe, for which the ACCEPTED MANUSCRIPT / CLEAN COPY presence of several electronic bands tends to lower the L values despite the strongly degenerate nature of the compound. [8][9][10][11] Summarizing, our present structural investigations clearly demonstrate that the stable structure around the composition Cu2.1Sn0.9S3 is not tetragonal with composition Cu7Sn3S10 but cubic with space group 𝑃4 # 3𝑛 and the composition Cu22Sn10S32, requiring the revision of the understanding structure-transport properties relationships of this system. Moreover, we demonstrate once again that the order-disorder phenomena in copper sulphides are very complex, and very difficult to detect. In-depth structural analyses should be then systematically performed to elucidate the subtle influence of the crystal chemistry on the electrical and thermal properties of these materials.

  2(2)S32, which -in combination with the refined lattice parameter of 10.8234(2) Å and chemical composition of Cu21.8(1)Sn10.2(1)S32 reported by Pavan Kumar et al. 2 from synchrotron PXRD data -may indicate a small homogeneity range of the cubic phase. Moreover, it is worth pointing out that the local transmission electron microscopy analysis previously reported by Deng et al. (Fig. 4b in ref. 1) for "Cu7Sn3S10" is erroneous and incompatible with the proposed cell and space group 𝐼4 # 2𝑚. Indeed, several low intensity extra reflexions in the [110] zone axis pattern, for example between the transmitted beam (000) and the reflection indexed 1-10, cannot be indexed in the 𝐼4 # 2𝑚 group, but on the opposite, can be indexed with the cubic cell (a ~ 10.8 Å) and the space group 𝑃4 # 3𝑛 (Figure 3) Thus, these results demonstrate without any ambiguity that the samples of composition Cu7Sn3S10 prepared in the conditions reported by Deng et al. 1 (i.e. heat treatments at 723 K, ACCEPTED MANUSCRIPT / CLEAN COPY 4 1223 K and 1073 K, followed by ball-milling process and SPS treatment at 923 K) are biphasic and that the observed majority phase is not tetragonal with composition Cu7Sn3S10 but cubic with composition Cu22Sn10S32.

Figure 1 .

 1 Figure 1. Rietveld refinement of the Cu7Sn3S10 sample considering (i) the tetragonal structural model (𝐼4 # 2𝑚) proposed by Deng et al. 1 (top panel) and (ii) the cubic Cu22Sn10S32 structural model (𝑃4 # 3𝑛) proposed by Pavan Kumar et al. 2 (bottom panel).

Figure 2 .

 2 Figure 2. Zoom on the Rietveld refinement of the Cu7Sn3S10 sample, with intensity in logarithmic scale, considering (i) the tetragonal structural model (𝐼4 # 2𝑚) proposed by Deng et al. 1 (top panel) and (ii) the cubic Cu22Sn10S32 structural model (𝑃4 # 3𝑛) proposed by Pavan Kumar et al. 2 (bottom panel). * refers to copper sulphide impurity.

Figure 3 .

 3 Figure 3. Selected area electron diffraction (SAED) pattern of the sample Cu7Sn3S10 presented by Deng et al. 1 in Figure 4. The spots with high intensity (red) were indexed by Deng et al. in the tetragonal cell (𝐼4 # 2𝑚) with 2´at = ct while additional spots of weak intensity (blue) were not and cannot be indexed in this tetragonal cell. The present indexation, in blue, shows that the additional weak intensity spots are indexed in the cubic cell (𝑃4 # 3𝑛) 𝑤𝑖𝑡ℎ ac = 2´at.

Table 1 .

 1 Crystallographic data of the Cu7Sn3S10 sample from Rietveld refinement of the powder X-ray diffraction data considering the cubic Cu22Sn10S32 structural model2

			𝑃4 # 3𝑛, a = 10.8365(1) Å, Cu22.2(1)Sn9.8(1)S32
	Atom	Site	x	y	z	Biso (Å 2 )	SOF
	Sn1	6c	1/4	1/2	0	1.28(8)	1.00
	Cu2	6d	1/4	0	1/2	1.09(4)	1.00
	Cu3	8e	0.251(1)	x	x	1.23(5)	1.00
	Cu4/Sn4 12f 0.244(1)	0	0	0.95(7)	0.68(1)/0.32(1)
	S1	8e	0.127(1)	x	x	0.78(26)	1.00
	S2	24i 0.378(1)	0.370(1) 0.129(1) 0.72(9)	1.00

Acknowledgments

We thank the group of X. Shi for having provided a sample prepared in the conditions reported in their paper (i.e. heat treatments at 723 K, 1223 K and 1073 K, followed by ball-milling process and SPS treatment at 923 K). See their experimental section. 1


M Abidi

W Abou Saoud

A Bouzaza

A Hajjaji

B Bessais

D Wolbert

Aymen Amin Assadi
email: aymen.assadi@ensc-rennes.fr

S Rtimi
email: rtimi.sami@gmail.com

W Abou Saoud

Dynamics of VOCs degradation and bacterial inactivation at the interface of AgxO/Ag/TiO2 prepared by HiPIMS under indoor light

Keywords: HiPIMS coating, Photocatalysis, indoor air treatment, Redox catalysis, Bacterial inactivation

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

Introduction:

As it is largely connected to human health, indoor air quality has become a very important topic for the social, economic, and scientific community. It has been shown that modern society spends between 70% and 90% of their time indoors [START_REF] Bacaloni | Indoor Air Quality. Volatile Organic Compounds: Sources, Sampling and Analysis[END_REF], such as home, offices, cars, hospitals, and healthcare buildings… Besides, the world health organization declared that in 2012, 7 million premature deaths are due to air pollution. Amongst these 7 million deaths, 4.3 million were related to indoor contamination [START_REF]Public health, environmental and social determinants of health (PHE)[END_REF]. This is a complex problem containing different types of pollutants, such as particles (dust, and smoke…), radon, asbestos, biological agents (bacteria, viruses…), and gaseous contaminants such as ammonia [START_REF] Guieysse | Biological treatment of indoor air for VOC removal: Potential and challenges[END_REF][START_REF] Jones | Indoor air quality and health[END_REF][START_REF] Saoud | Integrated process for the removal of indoor VOCs from food industry manufacturing: Elimination of Butane-2, 3-dione and Heptan-2-one by cold plasma-photocatalysis combination[END_REF][START_REF] Saoud | Synergism between nonthermal plasma and photocatalysis: Implications in the post discharge of ozone at a pilot scale in a catalytic fixed-bed reactor[END_REF][START_REF] Assadi | Study of synergetic effect by surface discharge plasma/TiO 2 combination for indoor air treatment: Sequential and continuous configurations at pilot scale[END_REF][START_REF] Epa | Volatile Organic Compounds Impact on Indoor Air Quality[END_REF][START_REF] Montero-Montoya | Volatile organic compounds in air: sources, distribution, exposure and associated illnesses in children[END_REF][START_REF] Gu | Recent advances in convolutional neural networks[END_REF][START_REF] Lee | Investigation of indoor air quality at residential homes in Hong Kong-case study[END_REF]. Different sources are emitting VOCs such as outside pollution (food industry, swimming pool water treatment, hospital cleaning room….) and from everyday activities (e.g., cooking, smoking, or even using cleaning agents, and office equipment) [START_REF] Tsai | An overview of health hazards of volatile organic compounds regulated as indoor air pollutants[END_REF][START_REF] Lorimier | Adsorption of toluene onto activated carbon fibre cloths and felts: application to indoor air treatment[END_REF][START_REF] Squinazi | La pollution de l'air à l'intérieur des bâtiments (allergènes exclus)[END_REF][START_REF] Tucker | Volatile organic compounds[END_REF][START_REF] Chan | Commuter exposures to VOCs in Boston, Massachusetts[END_REF][START_REF] Lawryk | Concentrations of volatile organic compounds in the passenger compartments of automobiles[END_REF][START_REF] Dechow | Concentrations of selected contaminants in cabin air of airbus aircrafts[END_REF][START_REF] Chao | Quantification of indoor VOCs in twenty mechanically ventilated buildings in Hong-Kong[END_REF][START_REF] Abidi | Photocatalytic indoor/outdoor air treatment and bacterial inactivation on CuxO/TiO 2 prepared by HiPIMS on polyester cloth under low intensity visible light[END_REF]. To reduce the VOCs from air, various techniques were used, such as adsorption [START_REF] Matos | Combination of adsorption on activated carbon and oxidative photocatalysis on TiO 2 for gaseous toluene remediation[END_REF], condensation [START_REF] Wang | Post-combustion CO 2 capture with chemical absorption: a state-of-the-art review[END_REF], plasma combined with catalysis [START_REF] Saoud | Integrated process for the removal of indoor VOCs from food industry manufacturing: Elimination of Butane-2, 3-dione and Heptan-2-one by cold plasma-photocatalysis combination[END_REF][START_REF] Saoud | Synergism between nonthermal plasma and photocatalysis: Implications in the post discharge of ozone at a pilot scale in a catalytic fixed-bed reactor[END_REF][START_REF] Assadi | Study of synergetic effect by surface discharge plasma/TiO 2 combination for indoor air treatment: Sequential and continuous configurations at pilot scale[END_REF][START_REF] Assadi | A study of pollution removal in exhaust gases from animal quartering centers by combining photocatalysis with surface discharge plasma: from pilot to industrial scale[END_REF][START_REF] Assadi | Use of DBD plasma, photocatalysis, and combined DBD plasma/photocatalysis in a continuous annular reactor for isovaleraldehyde elimination-synergetic effect and byproducts identification[END_REF][START_REF] Maxime | Removal of gas-phase ammonia and hydrogen sulfide using photocatalysis, nonthermal plasma, and combined plasma and photocatalysis at pilot scale[END_REF], and photocatalysis [START_REF] Abidi | Photocatalytic indoor/outdoor air treatment and bacterial inactivation on CuxO/TiO 2 prepared by HiPIMS on polyester cloth under low intensity visible light[END_REF][START_REF] Assadi | Use of DBD plasma, photocatalysis, and combined DBD plasma/photocatalysis in a continuous annular reactor for isovaleraldehyde elimination-synergetic effect and byproducts identification[END_REF]. Notwithstanding their significant ability in the VOCs removal, these proposed techniques were shown to produce high amounts of dangerous intermediates, by-products, and waste. Furthermore, these processes exhibit a temporary pollution transfer from gaseous to solid phase or pollution allocation from one area to another [START_REF] Assadi | Isovaleraldehyde elimination by UV/TiO 2 photocatalysis: comparative study of the process at different reactors configurations and scales[END_REF].

Heterogeneous photocatalysis is deemed as a promising method for air purification owing to its ability to remove VOCs even at relatively elevated concentrations [START_REF] Saoud | Integrated process for the removal of indoor VOCs from food industry manufacturing: Elimination of Butane-2, 3-dione and Heptan-2-one by cold plasma-photocatalysis combination[END_REF][START_REF] Assadi | Study of synergetic effect by surface discharge plasma/TiO 2 combination for indoor air treatment: Sequential and continuous configurations at pilot scale[END_REF][START_REF] Binas | Modified TiO 2 based photocatalysts for improved air and health quality[END_REF][START_REF] Zhong | Photocatalytic air cleaners and materials technologies-Abilities and limitations[END_REF][START_REF] Saoud | Study of synergetic effect, catalytic poisoning and regeneration using dielectric barrier discharge and photocatalysis in a continuous reactor: Abatement of pollutants in air mixture system[END_REF]. This technique was able to degrade a wide range of pollutants and their by-products [START_REF] Mo | Photocatalytic purification of volatile organic compounds in indoor air: a literature review[END_REF][START_REF] Farhanian | Photocatalytic oxidation air cleaner: identification and quantification of by-products[END_REF][START_REF] Sleiman | Photocatalytic oxidation of toluene at indoor air levels (ppbv): Towards a better assessment of conversion, reaction intermediates and mineralization[END_REF]. Titanium dioxide (TiO 2 ) is widely employed photocatalyst for pollutants' removal/abatement due to its high stability, excellent activity, inexpensive to build and operate, and its non-toxicity [START_REF] Das | Photocatalytic degradation of volatile pollutants[END_REF][START_REF] Shah | A Review on Catalytic Nanomaterials for Volatile Organic Compounds VOC Removal and Their Applications for Healthy Buildings[END_REF]. TiO 2 supported on glass fiber tissue [START_REF] Saoud | Study of synergetic effect, catalytic poisoning and regeneration using dielectric barrier discharge and photocatalysis in a continuous reactor: Abatement of pollutants in air mixture system[END_REF][START_REF] Shan | Immobilisation of titanium dioxide onto supporting materials in heterogeneous photocatalysis: a review[END_REF], cellulosic paper [START_REF] Assadi | Photocatalytic oxidation of trimethylamine and isovaleraldehyde in an annular reactor: Influence of the mass transfer and the relative humidity[END_REF], polymer support [START_REF] Saoud | Study of synergetic effect, catalytic poisoning and regeneration using dielectric barrier discharge and photocatalysis in a continuous reactor: Abatement of pollutants in air mixture system[END_REF], and TiO 2 on PES fabrics [START_REF] Abidi | Photocatalytic indoor/outdoor air treatment and bacterial inactivation on CuxO/TiO 2 prepared by HiPIMS on polyester cloth under low intensity visible light[END_REF][START_REF] Zeghioud | Photocatalytic performance of TiO 2 impregnated polyester for the degradation of Reactive Green 12: Implications of the surface pretreatment and the microstructure[END_REF]. However, the main limiting factor affecting the photocatalytic application of TiO 2 in real settings is its wide band gap (3.2 eV) allowing only the absorption of UV photons [START_REF] Ozimek | Optical Properties of TiO 2 Nanopowder Doped by Silver (Copper) during Synthesis or PVD Method[END_REF][START_REF] Hajjaji | Bacterial adhesion and inactivation on Ag decorated TiO 2 -nanotubes under visible light: Effect of the nanotubes geometry on the photocatalytic activity[END_REF]. To extend the spectral absorption of TiO 2 in the visible light range, ionic (cationic or anionic) doping was seen to tighten its band gap allowing solar/visible light absorption [START_REF] Ribeiro | Band-Gap engineering for photocatalytic applications: Anionic and cationic doping of TiO 2 anatase[END_REF][START_REF] Delegan | Bandgap tailoring of insitu nitrogen-doped TiO 2 sputtered films intended for electrophotocatalytic applications under solar light[END_REF][START_REF] Albiter | Photocatalytic deposition of Ag nanoparticles on TiO2: Metal precursor effect on the structural and photoactivity properties[END_REF][START_REF] Pandiyan | Probing the electronic surface properties and bandgap narrowing of in situ N, W, and (W, N) doped magnetron-sputtered TiO 2 films intended for electro-photocatalytic applications[END_REF] Other researchers adopted the engineering of tailored heterojunctions with other metal oxides allowing visible light absorption [START_REF] Hajjaji | Bacterial adhesion and inactivation on Ag decorated TiO 2 -nanotubes under visible light: Effect of the nanotubes geometry on the photocatalytic activity[END_REF][START_REF] Gim | RuO 2 -doping into high-aspect-ratio anodic TiO 2 nanotubes by electrochemical potential shock for water oxidation[END_REF][START_REF] Rtimi | TiON and TiON-Ag sputtered surfaces leading to bacterial inactivation under indoor actinic light[END_REF][START_REF] Lee | Surface plasmon-driven hot electron flow probed with metal-semiconductor nanodiodes[END_REF][START_REF] Pearson | Decoration of TiO 2 nanotubes with metal nanoparticles using polyoxometalate as a UV-switchable reducing agent for enhanced visible and solar light photocatalysis[END_REF].

In this study, we chose to combine TiO 2 with AgO to enhance the photocatalytic activity in the visible spectral range. Ag x O was chosen due to its strong plasmon resonance occurring under visible light excitation. The choice of Ag x O is favorable for energy levels' alignments that authorize the silver plasmonic electrons to be transferred into the TiO 2 . Both oxides were sputtered on textile fabrics to overcome the labor and time required to recover the catalyst after use. This work addresses the synthesis of Ag x O-TiO 2 on polyester cloth by two sputtering modes (DCMS and HiPIMS). Odorous compounds such as chloroform (Trichloromethane, CHCl 3 ) and Diacetyl (butane-2,3-dione, (CH 3 CO) 2 ) were chosen as indoor VOCs models. Chloroforms are model pollutants commonly encountered in hospital settings. Diacetyl is normally emitted during food processing and preparation; they are dangerous to health [START_REF] Abidi | Photocatalytic indoor/outdoor air treatment and bacterial inactivation on CuxO/TiO 2 prepared by HiPIMS on polyester cloth under low intensity visible light[END_REF]. In addition, the E. coli bacteria was chosen as a microorganism model. The effect of the applied sputtering mode on the kinetics of COVs removal (chloroform and butadione) and bacterial inactivation was studied and is presented in details in this work. The surface properties of the sputtered samples were examined by up-to-date surface science techniques. The prepared films will open the doors to potential applications in hospital settings to reduce bacterial infections and to remove malodors.

Experimental

Catalyst synthesis: Ag/TiO 2 sputtered PES

Ag and Ti were sputtered on PES using magnetron sputtering in the (HiPIMS and DCMS modes). The HiPIMS deposition was assisted with using a HiP3 5 kW Solvix generator at a time-average power of 100 W (5 Wcm -2 , pulse-width of 50 µs and a frequency of 1 kHz, gas phase 95% Ar -5% O 2 . The applied power to the Ti-target was 90 W for the first 30 s then at 50 W to regulate the relative content of Ti in the film as it will be shown later by transmission electron microscopy (TEM). The distance between the sputtered target and the sample holder was 10 cm as previously optimized [START_REF] Zhou | Preparation and studies of Ag-TiO 2 hybrid nanoparticles of core-shell structure[END_REF]. The visual perception of the sputtered PES fabrics is shown in Figure 1.

Thickness of the sputtered AgxO-TiO 2 -PES, TEM, spectral absorption and XPS

The thicknesses of TiO 2 , Ag, and AgO-TiO 2 deposited on Si-wafer we measured using a Profilometer (Alphastep-500, Tencor, USA). To carry out TEM observations, the PES cloths were embedded in epoxy resin (Embd 812) and cross-sectioned with an ultra-microtome (Ultratcut E) and then imaged using FEI Osiris operated in 200kV.

With Spot size of 5, dwell time 50 μs, real time 600 s.

Diffuse reflectance spectroscopy (DRS) was carried out by means of a Perkin

Elmer Lambda 900 UV-VIS-NIR spectrometer within 250-700 nm wavelength range.

In order to determine the existing species on the sputtered Ag x O-TiO 2 film, Xray photoelectron spectroscopy (XPS) was performed using an AXIS NOVA photoelectron spectrometer (Kratos Analytical, Manchester, UK, monochromatic Al-K  with h =1486.6 eV).

In order to correct the charging effect at the interface of the sputtered samples, the carbon C1s line having position at 284.6 eV was used as reference. The surface atomic concentration was determined from the peak areas considering the sensitivity factors for each element. The XPS spectral peaks were deconvoluted using the CasaXPS V2 software.

Target compounds and tested bacteria

Two target organic compounds were used, Butadione (C 4 H 6 O 2 ) and Chloroform (CHCl 3 ), existing particularly in the food and hospital sectors, respectively. Butadione (99% purity) and Chloroform (99.4% purity) were used as VOCs probes. Escherichia coli (DSM 10198-0307-001) was used as a model microorganism to test the antibacterial activity. The preparation protocol and the testing method have been detailed before [START_REF] Abidi | Photocatalytic indoor/outdoor air treatment and bacterial inactivation on CuxO/TiO 2 prepared by HiPIMS on polyester cloth under low intensity visible light[END_REF].

Reactor design for VOC removal

The used batch reactor has approximately 10 mm as upper diameter and 50 mm of working height contains two septa that are able to contain up to 50 mL of solution.

The first one is used for samples extraction and the second one is used for VOCs injection. The catalytic samples were fixed to the internal wall of the batch reactor to be in direct contact with the studied VOC and light radiation. For all experiments, 20 cm 2 of coated Ag x O-TiO 2 -PES were used. A fluorescent visible lamp (Sylvania CF-L 24 W/840) with emission in the visible (400 -720 nm) has been inserted inside the reactor. This light is a typical indoor light like those used for houses, hospitals and schools. To prevent extra-light from, the hall reactor was covered with Al-foil during the photocatalytic experiments. The homogenization of the gas phase was allowed by a magnetic stirrer and barrel.

VOCs analysis system and antibacterial testing

To examine the performance of our catalysts for VOCs removal C 4 H 6 O 2 and CHCl 3 analysis was carried out using gas chromatography (Fisons GC9000) equipped with a FID. The mobile phase (carrier gas) was Nitrogen. We used a syringe of 500 µL for all injection. All injections were carried out in triplicates. Once the flow inside the reactor gets stabilized (at equilibrium), the pollutants analysis starts.

The antibacterial activity of Ag x O-TiO 2 -PES, Escherichia coli (E. coli) was studied as reported in our previous study [START_REF] Abidi | Photocatalytic indoor/outdoor air treatment and bacterial inactivation on CuxO/TiO 2 prepared by HiPIMS on polyester cloth under low intensity visible light[END_REF]. In this study, the initial bacterial concentration is∼ 2.6 10 6 colony-forming unit per milliliter (CFU/mL).

Results and discussion

Thickness calibration and TEM of sputtered Ag x O-TiO 2 -PES

Figure 2a presents the high-angle annular dark field (HAADF) mapping of elements in the Ag x O-TiO 2 -PES samples sputtered by HiPIMS at 30 A. Figures 2b,2c and 2d show the mapping of Ti-, Ag-, and O-element, respectively. The Ag-atoms distribution (red image) appears to be dense and uniform. This likewise provides evidence for the presence of different oxidation states of Ag-NPs (AgO) in the topmost layers. In addition, many kinds of research studied in a detailed way the properties of Ag-coated PES [START_REF] Rtimi | New Evidence for Ag-Sputtered Materials Leading to Bacterial Inactivation by Surface-Contact without the Release of Ag-ions: End of a Long Controversy[END_REF][START_REF] Fratoddi | Hydrophobic and hydrophilic Au and Ag nanoparticles[END_REF][START_REF] Yuranova | Antibacterial textiles prepared by RF-plasma and vacuum-UV mediated deposition of silver[END_REF][START_REF] Yuranova | Performance and characterization of Ag-cotton and Ag/TiO 2 loaded textiles during the abatement of E. coli[END_REF][START_REF] Kelly | Comparison of the tribological and antimicrobial properties of CrN/Ag, ZrN/Ag, TiN/Ag, and TiN/Cu nanocomposite coatings[END_REF]. The EDX mapping of oxygen clearly shows (Fig. 2d) relatively large amounts of oxygen species (purple image). This is due to TiO 2 (EDX mapping of Ti is shown in figure 2b) and AgO nanoparticles, which exhibit a higher O-content compared to the content of oxygen species in the film bulk. In the context of a comparison between the samples sputtered by DCMS and those sputtered by HiPIMS, Rtimi et al reported that the distribution of oxygen species in the samples sputtered by DCMS shows more oxidized copper (in this case Ag) species in the bulk than the samples prepared by HiPIMS, while the latter has shown more oxidized copper species at the upper layers in contact with bacteria [START_REF] Rtimi | Extracellular bacterial inactivation proceeding without Cu-ion release: Drastic effects of the applied plasma energy on the performance of the Cu-polyester (PES) samples[END_REF]. edge. This was related to the interfacial charge transfer (IFCT) between AgxO and TiO 2 semiconductors [START_REF] Ozimek | Optical Properties of TiO 2 Nanopowder Doped by Silver (Copper) during Synthesis or PVD Method[END_REF]. A wide absorption band from 500 to 750 nm is noted for AgxO/TiO 2 photocatalysts sputtered by HiPIMS at 30A and DCMS at 0.3A. These bands can be ascribed to surface plasmon resonance (SPR) of the Ag species [START_REF] Ozimek | Optical Properties of TiO 2 Nanopowder Doped by Silver (Copper) during Synthesis or PVD Method[END_REF][START_REF] Albiter | Photocatalytic deposition of Ag nanoparticles on TiO2: Metal precursor effect on the structural and photoactivity properties[END_REF]. In order to better analyze the light absorption of our coatings, we applied a spectral analysis based on high derivatives. The method of higher derivatives has been known

for decades [START_REF] Butler | Higher derivative analysis of complex absorption spectra[END_REF], but it has not previously been applied to the analysis of photocatalytic materials' spectra. Analysis of extrema in the representation of the second and fourth derivatives allows the determination of the UV-Vis absorption maxima giving a clear idea about the appropriate wavelengths to be used with this system. The signal-tonoise ratio in modern spectrometers is high enough to reliably determine higher derivatives. The fourth derivative of the absorption spectra allows to reliably determining the position of significant peaks. If these peaks overlap slightly, the second derivative becomes enough to locate the significant peaks. Figure 4 shows the second derivatives of the absorption spectra of (a) TiO 2 (using DCMS at 0.3 A) and Ag x O-TiO 2

(using DCMS at 0.3A and HiPIMS at 30 A).

It is readily seen from the 2 nd derivative of the Figure 4a (AgxO/TiO 2 (0.3 A)) that the thin film is absorbing at 350 nm and at 410 and 730 nm with a lower intensity. However, the thin AgxO/TiO 2 film prepared by HiPIMS exhibited higher spectral absorption peak at 350 nm than the film prepared by DCMS. In addition, the HiPIMS prepared film showed peaks at 420, 480 and 740 nm. The TiO 2 thin film showed only spectral absorption in the UV range (320 and 365 nm). From Figure 4b, the HiPIMS thin film absorb in the UVA, violet and blue. This is attributed to the Ag surface plasmon. The weak peak at 730-740 nm refers to the far-red absorption. Figure 5 shows the oxidative states of Ag and Ti sputtered on the polyester cloth as determined by XPS. Figure 5 shows the Ti2p and Ag3d peaks from AgxO-TiO 2 deposited by HiPIMS and DCMS. The found peaks were asymmetric. AgO peaks at 367.6 eV were detected in both samples (HiPIMS and DCMS). The spectra showed also the presence of lower peaks at 368 eV characteristics of metallic Ag. The presence of Ti3+ in the HiPIMS samples could arise from the AgO incorporation on the TiO 2 . The peak at 367.6 eV is characteristic of Ag-ions adsorbed on hydroxylated TiO 2 [START_REF] Arabatzis | Silver-modified titanium dioxide thin films for efficient photodegradation of methyl orange[END_REF].

Photocatalytic experiments: COVs removal and Bacterial inactivation tests

Effect of deposition of Ag-NPs TiO 2 -PES by HiPIMS and DCMS on the photocatalytic activity of AgxO-TiO 2 -PES photocatalyst

The photocatalytic performances of AgxO-TiO 2 -PES catalysts were evaluated by photocatalytic removal of COVs (Chloroform) under visible light irradiation at ambient temperature. Figure 6 shows the chloroform removal using AgxO-TiO 2 -PES sputtered by HiPIMS for 10, 20, 30 A and sputtered by DCMS at 0.3A. Figure 6 shows that the chloroform removal is pertinently affected by the different sputtering current. In this test, it is readily seen that the initial concentrations of Chloroform decreased over time under visible light irradiation. Also, an increase in sputtering current from 10A until 30A increases the chloroform removal to about 98% of the initial concentration of 10 g.m -3 .

This enhancement is due to the availability of active sites generated at the interface of the sample deposited at 30A [START_REF] Elfalleh | Innovative and stable TiO 2 supported catalytic surfaces removing aldehydes under UV-light irradiation[END_REF]. However, at low current values the chloroform degradation reaches a limit; in fact, these current values are rather small to form a catalyst layer leading to perfect and complete removal of chloroform. Previously S.

Rtimi et al reported [START_REF] Rtimi | Extracellular bacterial inactivation proceeding without Cu-ion release: Drastic effects of the applied plasma energy on the performance of the Cu-polyester (PES) samples[END_REF] that the HiPIMS deposition current generates different ions and charge species, leading to distinguish charges at the interfaces of the thin film. In addition, compared to the AgxO-TiO 2 -PES sputtered by direct current magnetron sputtering (DCMS) at 0.3A, AgxO-TiO 2 -PES (HiPIMS at 30A) exhibits a high photocatalytic activity under visible light. This is due to the generation of a higher number of Ag-ions at the catalysts surface when using HiPIMS (30A) than DCMS [START_REF] Rtimi | New Evidence for Ag-Sputtered Materials Leading to Bacterial Inactivation by Surface-Contact without the Release of Ag-ions: End of a Long Controversy[END_REF][START_REF] Rtimi | Extracellular bacterial inactivation proceeding without Cu-ion release: Drastic effects of the applied plasma energy on the performance of the Cu-polyester (PES) samples[END_REF].

Mixtures of pollutants (VOCs and bacteria)

Effect of the presence of two Kinds of VOCs on AgxO-TiO 2 -PES performance

The removal performance of two kinds of indoor air pollutants, with equimolar concentrations, was investigated. Figure 7 shows that at 30A HiPIMS and low visible light illumination, the degradation of Chloroform is quite similar to that of Butadione (BUT). Firstly, before switching on the lamp and during the adsorption stage, it was found that the adsorbed Butadione amount is slightly greater than that of chloroform.

Once adsorption of both species reaches saturation, the lamp is switched on. We found (Fig. 7) that that the photocatalytic degradation kinetics of BUT is faster than that of chloroform. This may be attributed to the chemical composition of the pollutants (e.g., carbon number), their affinities to the catalyst and the amounts of their intermediate by-products on the catalyst surface, which in turn leads to a competitive effect of the pollutants towards the active catalytic sites. This result is similar to our previous work [START_REF] Abidi | Photocatalytic indoor/outdoor air treatment and bacterial inactivation on CuxO/TiO 2 prepared by HiPIMS on polyester cloth under low intensity visible light[END_REF] where we used Cu x O/TiO 2 -PES for the removal of VOCs. To further understand the chemical transformation of the heterogeneous photocatalytic degradation of Butadione over AgxO-TiO 2 -PES, the Langmuir-Hinshelwood (L-H) model was employed to fit the experimental results [START_REF] Zhou | Preparation and studies of Ag-TiO 2 hybrid nanoparticles of core-shell structure[END_REF][START_REF] Arabatzis | Silver-modified titanium dioxide thin films for efficient photodegradation of methyl orange[END_REF]. The L-H reaction rate equation shown in the following equation is employed to explain the adsorption and degradation of Butadione:

𝑟 0 = - 𝑑[𝑉𝑂𝐶] 𝑑𝑡 = 𝑘 𝑐 𝐾[𝑉𝑂𝐶] 0 1 + 𝐾[𝑉𝑂𝐶] 0
Where r 0 (mmol/g cat m 3 s) is the initial photocatalytic degradation rate, [VOC] 0 is the initial BUT concentration (mmol/m 3 ), K is the adsorption constant (m 3 /mmol) and k c is the kinetic constant (mmol.m -3 s g TiO2 ) at maximum coverage for the experimental conditions.

The plot of 1/r 0 versus 1/[COV] 0 enables determining the values of k c and K.

According to Figure 9, we estimate the values of the kinetic and adsorption constants (Table 1) using the linearized L-H equation ( ).

1

𝑟 0 = 1 𝑘 𝑐 𝐾 × 1 [𝑉𝑂𝐶] 0 + 1 𝑘 𝐶
Table 1 shows Butadione shows a slow degradation rate regarding the use of AgxO-TiO 2 -PES sputtered at 30A; this may be due to the physicochemical properties of the pollutant and to the interfacial charge transfer mechanism (IFCT), which will be shown in a later section. Table 1 shows that AgxO-TiO 2 -PES leads to a more efficient degradation of butadione compared to PES-TiO 2 , which in turn leads to an almost twice faster degradation of butyraldehyde under UV irradiation [START_REF] Elfalleh | Innovative and stable TiO 2 supported catalytic surfaces removing aldehydes under UV-light irradiation[END_REF]. This result may be due to the Ag amount present on the photocatalyst surface and to the chemical properties of the target pollutants.

Simultaneous removal of butadione and bacteria

After studying the effect of the existence of two kinds of indoor air pollutants on AgxO-TiO 2 -PES performance, we investigate the bacterial (E. coli) inactivation activity of AgxO-TiO 2 -PES together with simultaneous removal of BUT and E. coli. Many experiments were done with our composites with initial bacterial concentration and inlet BUT concentration of ~ 2.6 10 6 (CFU/mL) and 5 mg/m 3 , respectively. In fact, the goal of mixing target molecules with bacteria is to validate the effectiveness of the advanced oxidation process (AOP) in a case closer to real conditions where large amounts of pollutants must be removed simultaneously, and to see that there is always a degradation of pollutants during mixing. (Light source: Sylvania CF-L 24 W/840; 400 -720 nm).

Identification of the VOCs' degradation by-products

The by-products emanating from the photocatalytic degradation of butadione in presence of bacteria using the AgxO-TiO 2 -PES catalyst were determined by Gaseous Chromatography-Mass Spectroscopy (GC-MS). Figure 11

Catalyst recyclability

The reusability of the catalyst is an important factor to take into account since it concerns the possibility of its potential application in environments polluted by toxic gases [START_REF] Abidi | Photocatalytic indoor/outdoor air treatment and bacterial inactivation on CuxO/TiO 2 prepared by HiPIMS on polyester cloth under low intensity visible light[END_REF]. The re-use of the catalyst was examined for four successive cycles. The AgxO-TiO 2 -PES was washed well after each photodegradation cycle and reused.

Figure 12 shows no loss of sample photoactivity after four cycles of use. These results

prove the overall potential of the AgxO-TiO 2 -PES to be used as photocatalyst for air treatment applications. Furthermore, the same slope is noted in each cycle; these results can confirm the non-deactivation of the surface sites of the catalyst and its excellent catalytic stability.

Photocatalytic degradation mechanism

To understand the possible photocatalytic mechanism of VOC removal and E. coli interface, which has been reported in Ag/ZnO and Ag/TiO 2 systems [START_REF] Vaiano | Enhanced photocatalytic removal of phenol from aqueous solutions using ZnO modified with Ag[END_REF][START_REF] Awazu | A plasmonic photocatalyst consisting of silver nanoparticles embedded in titanium dioxide[END_REF]. This could be due to the fact that the heterojunction in Ag-AgO/TiO 2 assisted the transfer of photogenerated electrons and holes. Thus, these electrons can quickly be transferred into the TiO 2 interface and then undergo a transformation as described below:

AgO/TiO 2 + hʋ → e -+ h + (1)
e -+ h + /AgO → e -/Ag → e -/TiO 2 (2)

O 2 + e -→ O 2 •- (3)
O 2 •-+ 2H 2 O + 2e -→ 3OH -+ • OH (4)
In reaction (4), one can describe it as a multi-electron transfer process. However, the activation energy of such reactions, as follows from the Marcus theory [START_REF] Marcus | Nobel Lecture: Electron Transfer Reactions in Chemistry: Theory and Experiment[END_REF], is directly proportional to the square of the number of transferred electrons. The proposed mechanism is then described as the classical chain degenerated-branched oxidation of organic materials (eqs.5-11) [START_REF] Nadtochenko | Inactivation of Pathogenic Microorganisms in the Photocatalytic Process on Nanosized TiO2 Crystals[END_REF][START_REF] Rtimi | Update on Interfacial Charge Transfer (IFTC) Processes on Films Inactivating Viruses/Bacteria under Visible Light: Mechanistic Considerations and Critical Issues[END_REF][START_REF] Nadtochenko | Photoinduced mineralization of xylidine by the Fenton reagent. 2. Implications of the precursors formed in the dark[END_REF]:

h + + OH -→ • OH (-1.9 eV) (5) h + + RH → R • + H + (6) O 2 -+ H + → HO 2 • (7) HO 2 • + RH → HOOH + R • (8) R • +O 2 → RO 2 • (9) RO 2 • + RH → ROOH + R • (10)
HOOH, ROOH + e-[M (possible participation of the reduced metal ion)] →

• OH, RO • + HO-HO • , RO • + RH → H 2 O, ROH + R • (11)
Reaction can be simplified as seen in eq.12:

VOC/Bacteria + O 2

Figure 1 .

 1 Figure 1. Visual perception of the Ag x O-TiO 2 sputtered on PES.

Figure 2 .

 2 Figure 2. TEM images of Ag x O-TiO 2 -PES as sputtered by HiPIMS at a current of 30 A for 20s: a) High-angle annular dark-field imaging (HAADF), b) EDXS mapping of Ti, c) EDXS mapping of Ag, and d) EDXS mapping of O.

3. 2 .

 2 Figure 3 shows the spectral absorption of the sputtered Ag x O-TiO 2 -PES by HiPIMS (at 30 A) and DCMS (at 0.3 A). Analysis of the absorption spectra show that TiO 2 thin film do not absorb in the visible and presented an absorption onset (λ) at 260 nm. In contrast, the light absorption of Ag x O/TiO 2 sputtered by HiPIMS and DCMS extends to the visible region. In addition, a significant phenomenon was observed related to the interaction of light with The Ag incorporated into Titanium dioxide near the absorption

Figure 3 .

 3 Figure 3. Diffuse reflectance spectroscopy (UV-Vis) of sputtered TiO 2 (using DCMS at 0.3 A) and Ag x O-TiO 2 (using DCMS at 0.3 A and HiPIMS at 30 A).

Figure 4 .

 4 Figure 4. Analysis of spectral absorption extrema through second derivatives of light absorption of (a) AgxO/TiO 2 (0.3 A), (b) AgxO/TiO 2 (30 A) with respect to (c) sputtered TiO 2 .

Figure 5 .

 5 Figure 5. XPS deconvolution of Ag3d and Ti2p peaks from AgxO-TiO 2 deposited by HiPIMS and DCMS.

Figure 6 .

 6 Figure 6. Removal of chloroform with catalyst AgxO-TiO2-PES at different HiPIMS sputtering currents deposited for 20 s. Light source: fluorescent visible lamp (Sylvania CF-L 24 W/840; 400 -720 nm)

Figure 7 .Figure 8 Figure 8 .

 788 Figure 7. Two kinds of Indoor air pollutants removal on AgxO-TiO 2 -PES HiPIMS-sputtered 30 A for 20 s under a fluorescent visible lamp (Sylvania CF-L 24 W/840; 400 -720 nm)

Figure 9 .

 9 Figure 9. Linear regression using L-H model for the BUT degradation on AgxO-TiO 2 -PES sputtered at 30A for 20s.

Figure 10 .

 10 Figure 10. Simultaneous removal of E. coli and BUT on AgxO-TiO 2 -PES catalyst sputtered at a peak current of 30A. The initial concentration of E. coli is ~2.6 10 6 CFU/mL.

  shows GC-MS spectra of by-products originating from AgxO-TiO 2 -PES -based Butadione photo degradation. The detected degradation by-products are: (i) acetone (C 3 H 5 O), (ii) acetaldehyde (C 2 H 4 O) and (iii) acetic acid (C 2 H 4 O 2 ). Butadione (C 4 H 6 O 2 ) is also present after 4.83 minutes. These results are similar to those reported by Abou Saoud et al. [5],subsequent to the use of TiO 2 -GFT (Glass Fiber Tissue) as photo-catalyst for butadione degradation under photocatalytic/plasma processes.

Figure 11 .

 11 Figure 11. GC-MS of products observed during Butadione photo degradation by AgxO-TiO 2 -PES.

Figure 12 .

 12 Figure 12. Repetitive degradation of Butadione on Ag x O-TiO 2 -PES. VOC concentration = 10g/m 3 .

  inactivation in Ag x O/TiO 2 composite, Figure13was proposed. Under UV-vis light, electron-hole pairs generate in the valence bands (VB) of AgO and TiO 2 , then migrate to the conduction bands (CB). The AgO conduction band potential was 0.72 eV and the Ag Fermi level was 0.99 eV[START_REF] Sun | Photocatalytic degradation of gaseous o-xylene over M-TiO 2 (M= Ag, Fe, Cu, Co) in different humidity levels under visible-light irradiation: Activity and kinetic study[END_REF]. As a result, and because the Fermi level of Ag was more positive than the conduction band potential of AgO, the electrons that are in the AgO conduction band can move to the Fermi level of Ag. At the same time, electrons on the Fermi level of Ag can be rapidly transferred to the TiO 2 semiconductor

( 12 )

 12 At the same time, the photo-generated holes in the Valence band of TiO 2 are easily transferred to the Valence band of AgO. This result could be due to the fact that the Valence band of AgO (1.84 eV) is more negative than that of TiO 2 (~3eV). Therefore, while the photo-generated electrons flow on the TiO 2 surfaces, the holes are gathered in the AgO Valence band. Therefore, it was constructed an inter-semiconductor transfer flows for electrons (AgO→Ag→TiO 2 ) and holes (TiO 2 →AgO), which significantly limited the charge recombination and improved the charge separation efficiency. The fact that there was effective charge separation resulted in an increase in the lifetime of photogenerated charges, which increased the photocatalytic performance of our photocatalyst. Indeed, adsorbed oxygen (O 2 ) and water (H 2 O) reacted with electrons (e -) and holes (h + ) to produce the superoxide anion (O 2 .-), hydroxyl radical (OH • ), OH and H + . The superoxide anion 0 2 •-and the hydroxyl radical (OH•) were considered to be highly oxidizing reactive oxygen species (ROS). They can decompose VOCs, bacteria and other organic materials adsorbed on the active sites of the nanocomposites into CO 2 , H 2 O and other products, as shown in eq.[START_REF] Bacaloni | Indoor Air Quality. Volatile Organic Compounds: Sources, Sampling and Analysis[END_REF][START_REF]Public health, environmental and social determinants of health (PHE)[END_REF][START_REF] Guieysse | Biological treatment of indoor air for VOC removal: Potential and challenges[END_REF][START_REF] Jones | Indoor air quality and health[END_REF][START_REF] Saoud | Integrated process for the removal of indoor VOCs from food industry manufacturing: Elimination of Butane-2, 3-dione and Heptan-2-one by cold plasma-photocatalysis combination[END_REF][START_REF] Saoud | Synergism between nonthermal plasma and photocatalysis: Implications in the post discharge of ozone at a pilot scale in a catalytic fixed-bed reactor[END_REF][START_REF] Assadi | Study of synergetic effect by surface discharge plasma/TiO 2 combination for indoor air treatment: Sequential and continuous configurations at pilot scale[END_REF][START_REF] Epa | Volatile Organic Compounds Impact on Indoor Air Quality[END_REF][START_REF] Montero-Montoya | Volatile organic compounds in air: sources, distribution, exposure and associated illnesses in children[END_REF][START_REF] Gu | Recent advances in convolutional neural networks[END_REF][START_REF] Lee | Investigation of indoor air quality at residential homes in Hong Kong-case study[END_REF][START_REF] Tsai | An overview of health hazards of volatile organic compounds regulated as indoor air pollutants[END_REF].

Figure 13 .

 13 Figure 13. Proposed mechanism of photocatalysis by Ag-AgO/TiO 2 nanocomposite under light.

Acknowledgement: SR would like to thank Profs. H. Hofmann and C. Pulgarin. This study was partly supported by NanoScreen CCMX Materials Challenge Project (EPFL-STI-IMXLTP).


Sonya Nassari

Mickael Orgeur

Cédrine Blavet

Sigmar Stricker

Claire Fournier- Thibault

Delphine Duprez
email: delphine.duprez@upmc.fr

In addition to being a marker for muscle connective tissue, Odd skipped-related 2 (OSR2) is expressed in differentiated muscle cells during chick development

Keywords: chick, limb, head, muscle, connective tissue, OSR2

The zinc finger transcription factor, Odd skipped-related 2 (OSR2) is a recognized marker of connective tissue in chick embryos. OSR2 gain-and loss-of-function experiments indicate a role in irregular connective tissue differentiation in chick limb undifferentiated cells. Reinvestigation of OSR2 transcript location during chick development with in situ hybridization experiments showed that OSR2 was also expressed in differentiated muscle cells in limbs and head. OSR2 expression was also observed in differentiated myotubes in chick foetal myoblast cultures. This shows that in addition to being a marker of connective tissue, OSR2 is also expressed in muscle fibres during chick development.

INTRODUCTION

Connective tissue (CT) is an important component of the body supporting and connecting organs. During development, reciprocal interactions between CT and myogenic cells are required to form a functional muscular system (reviewed in Nassari et al., 2017a). In limbs, CT cells and myogenic cells are intermingled in muscle masses but have different embryological origins. CT cells originate from lateral plate, while myogenic cells originate from somites [START_REF] Bourgeois | Stable and bicistronic expression of two genes in somite-and lateral platederived tissues to study chick limb development[END_REF][START_REF] Chevallier | Limb-somite relationship: origin of the limb musculature[END_REF][START_REF] Kardon | Muscle and tendon morphogenesis in the avian hind limb[END_REF]. The muscle differentiation program is under the control of the Myogenic Regulatory Factor (MRFs), including MYF5, MYOD, MRF4 and MYOG (reviewed in Tajbakhsh, 2009). The differentiation program of limb muscle CT cells is not known (reviewed in Nassari et al., 2017a). To date, three types of transcription factors have been identified to regulate muscle formation in a non cell-autonomous manner during limb development: TCF4, a member of the TCF/LEF family [START_REF] Kardon | A Tcf4-positive mesodermal population provides a prepattern for vertebrate limb muscle patterning[END_REF][START_REF] Mathew | Connective tissue fibroblasts and Tcf4 regulate myogenesis[END_REF], the T-box transcription factors, TBX4 and TBX5 [START_REF] Hasson | Tbx4 and Tbx5 Acting in Connective Tissue Are Required for Limb Muscle and Tendon Patterning[END_REF] and the zinc finger transcription factors, Odd skipped-related 1 and 2, OSR1 and OSR2 [START_REF] Stricker | Odd-Skipped Related Genes Regulate Differentiation of Embryonic Limb Mesenchyme and Bone Marrow Mesenchymal Stromal Cells[END_REF][START_REF] Vallecillo-García | Odd skipped-related 1 identifies a population of embryonic fibro-adipogenic progenitors regulating myogenesis during limb development[END_REF] The odd gene coding for the zinc finger transcription factor odd-skipped was first identified in a screen for gene mutations in Drosophila as pair-rule genes involved in body segmentation [START_REF] Coulter | Gene activities and segmental patterning in Drosophila: analysis of odd-skipped and pair-rule double mutants[END_REF][START_REF] Nüsslein-Volhard | Mutations affecting segment number and polarity in Drosophila[END_REF]. In vertebrates, two odd-skipped-related genes, Osr1 and Osr2, have been identified [START_REF] Lan | Osr2, a new mouse gene related to Drosophila odd-skipped, exhibits dynamic expression patterns during craniofacial, limb, and kidney development[END_REF][START_REF] So | Cloning and expression analysis of a mouse gene related to Drosophila odd-skipped[END_REF]. During mouse and chick embryonic development and organogenesis, Osr1 and Osr2 exhibit partially overlapping expression domains in intermediate and lateral mesoderm and at later stages in mesonephros, branchial arches, limbs, mandibular and maxillary prominences [START_REF] Lan | Osr2, a new mouse gene related to Drosophila odd-skipped, exhibits dynamic expression patterns during craniofacial, limb, and kidney development[END_REF][START_REF] So | Cloning and expression analysis of a mouse gene related to Drosophila odd-skipped[END_REF][START_REF] Stricker | Comparative expression pattern of Odd-skipped related genes Osr1 and Osr2 in chick embryonic development[END_REF]. Osr1 and Osr2 genes display functional equivalence during mouse development, indicating that the distinct functions of Osr genes rely on their expression domains [START_REF] Gao | Functional equivalence of the zinc finger transcription factors Osr1 and Osr2 in mouse development[END_REF]. Consistent with Osr expression sites, phenotypes of Osr mutant mice indicate a role for Osr1 in heart and urogenital development [START_REF] James | Odd-skipped related 1 is required for development of the metanephric kidney and regulates formation and differentiation of kidney precursor cells[END_REF][START_REF] Wang | Odd-skipped related 1 (Odd1) is an essential regulator of heart and urogenital development[END_REF] and a role for Osr2 in palate and tooth development [START_REF] Lan | Oddskipped related 2 (Osr2) encodes a key intrinsic regulator of secondary palate growth and morphogenesis[END_REF][START_REF] Zhang | Antagonistic Actions of Msx1 and Osr2 Pattern Mammalian Teeth into a Single Row[END_REF].

Osr1 and Osr2 are recognized markers for irregular CT during chick and mouse development (reviewed in Nassari et al., 2017a). During chick limb development, the expression of both genes labels CT cells and is excluded from myogenic cells, labelled with PAX3 or MYOD [START_REF] Stricker | Comparative expression pattern of Odd-skipped related genes Osr1 and Osr2 in chick embryonic development[END_REF][START_REF] Stricker | Odd-Skipped Related Genes Regulate Differentiation of Embryonic Limb Mesenchyme and Bone Marrow Mesenchymal Stromal Cells[END_REF]. However, OSR1/2-positive cells display partial overlap with tendon SCX-positive cells and CT TCF4-positive cells in limb buds of embryonic day 4, E4, (HH24) chick embryos. However, OSRs are not expressed in tendons when they are formed [START_REF] Vallecillo-García | Odd skipped-related 1 identifies a population of embryonic fibro-adipogenic progenitors regulating myogenesis during limb development[END_REF]bioRxiv posted 20 July 2017doi:10.1101/165837). Moreover, the overexpression of either OSR1 or OSR2 promotes irregular CT differentiation, while preventing cartilage, tendon and muscle differentiation in chick limb undifferentiated progenitors [START_REF] Stricker | Odd-Skipped Related Genes Regulate Differentiation of Embryonic Limb Mesenchyme and Bone Marrow Mesenchymal Stromal Cells[END_REF][START_REF] Vallecillo-García | Odd skipped-related 1 identifies a population of embryonic fibro-adipogenic progenitors regulating myogenesis during limb development[END_REF]bioRxiv posted 20 July 2017doi:10.1101/165837). Conversely, the blockade of OSR1 or OSR2 activity decreases the expression of CT markers, while promoting cartilage marker expression in chick limb undifferentiated progenitors [START_REF] Stricker | Odd-Skipped Related Genes Regulate Differentiation of Embryonic Limb Mesenchyme and Bone Marrow Mesenchymal Stromal Cells[END_REF]. Recently, Osr1 has been shown to identify a population of embryonic fibro-adipogenic progenitors that has a non cellautonomous effect on developmental myogenesis [START_REF] Vallecillo-García | Odd skipped-related 1 identifies a population of embryonic fibro-adipogenic progenitors regulating myogenesis during limb development[END_REF]. In chick limbs, when the final muscle pattern is set, OSR2 appears mainly associated with individual muscles, while OSR1 is expressed in irregular CT within and outside muscles [START_REF] Nassari | The chemokines CXCL12 and CXCL14 differentially regulate connective tissue markers during limb development[END_REF].

In this study, we re-investigated the expression pattern of OSR2 transcripts during chick development. We showed that in addition to the already known expression in muscle CT, OSR2 was expressed in myofibres in limbs and head during chick development and in myotubes in muscle cell cultures.

RESULTS AND DISCUSSION

OSR2 has been described as being expressed in muscle CT, a subpopulation of irregular CT cells during chick limb development [START_REF] Nassari | The chemokines CXCL12 and CXCL14 differentially regulate connective tissue markers during limb development[END_REF][START_REF] Stricker | Comparative expression pattern of Odd-skipped related genes Osr1 and Osr2 in chick embryonic development[END_REF][START_REF] Stricker | Odd-Skipped Related Genes Regulate Differentiation of Embryonic Limb Mesenchyme and Bone Marrow Mesenchymal Stromal Cells[END_REF]. We re-investigated OSR2 expression with in situ hybridization experiments to sections at different developmental stages. We used the chick probe of 361 kb length located between the nucleotides 127 to 487 of OSR2 gene (Figure 1). This probe has been used previously for gene expression analysis [START_REF] Stricker | Comparative expression pattern of Odd-skipped related genes Osr1 and Osr2 in chick embryonic development[END_REF][START_REF] Stricker | Odd-Skipped Related Genes Regulate Differentiation of Embryonic Limb Mesenchyme and Bone Marrow Mesenchymal Stromal Cells[END_REF]. Moreover, this OSR2 probe did not crossreact/blast with any gene in chicken genomic databases and did not harbour the zinc finger domain of the OSR2 gene. At E3, corresponding to HH20 (40 somites), OSR1 was expressed in limb mesenchyme, while no OSR2 expression was observed in chick limb buds (Figure 2A,B). OSR2 was observed in the mesonephros (Figure 2B, arrow), as previously described [START_REF] Stricker | Comparative expression pattern of Odd-skipped related genes Osr1 and Osr2 in chick embryonic development[END_REF]. Muscle progenitors migrate from the hypaxial lips of the dermomyotomes towards the forelimb buds from E2 (HH17, 30 somites) in chick embryos [START_REF] Chevallier | Limb-somite relationship: origin of the limb musculature[END_REF][START_REF] Tozer | Involvement of vessels and PDGFB in muscle splitting during chick limb development[END_REF]. Migrating muscle progenitors assessed with MYOR expression did not express OSR2 at E3/HH20 (Figure 2B,C), showing that OSR2 was not expressed in muscle progenitors at this stage. At E5 (HH26), the OSR2 expression domains were similar to that of the myogenic transcription factor MYOD in dorsal and ventral limb muscle masses (Figure 2D,E), however it was not clear whether they were expressed in the same cells. From E5 to E8, in addition to being expressed in irregular CT between cartilage elements in distal limb regions and in dermis regions, OSR2 displayed a strong expression in developing muscles [START_REF] Nassari | The chemokines CXCL12 and CXCL14 differentially regulate connective tissue markers during limb development[END_REF][START_REF] Stricker | Odd-Skipped Related Genes Regulate Differentiation of Embryonic Limb Mesenchyme and Bone Marrow Mesenchymal Stromal Cells[END_REF]. At E9, when the final muscle pattern is set, OSR2 expression was mostly expressed in muscles regions, in addition to feather buds (Figure 2F). Within muscles, OSR2 was expressed in a subset of MF20-positive muscle fibres (Figure 2G,arrowheads) and in between MF20-positive cells (Figure 2G, arrows). These results show that in addition to being expressed in irregular and muscle CT during chick limb development, OSR2 is expressed in differentiated muscle fibres.

Previous studies have highlighted a key role for OSR2 in the development of specific craniofacial regions [START_REF] Lan | Oddskipped related 2 (Osr2) encodes a key intrinsic regulator of secondary palate growth and morphogenesis[END_REF]. During craniofacial development of mouse embryos, Osr2 expression is restricted to specific mesenchymal tissues, including the mesenchyme of the developing palatal shelves and the tongue [START_REF] Lan | Osr2, a new mouse gene related to Drosophila odd-skipped, exhibits dynamic expression patterns during craniofacial, limb, and kidney development[END_REF]. In chick embryos, OSR2 has been described to be expressed in branchial arches from E3 [START_REF] Stricker | Comparative expression pattern of Odd-skipped related genes Osr1 and Osr2 in chick embryonic development[END_REF]. In order to determine whether OSR2 was also expressed in differentiated muscle cells during chick craniofacial development, we performed in situ hybridization to head sections of chick embryos at different developmental stages. At E3 (HH20), OSR1 and OSR2 were expressed in distinct regions of the first branchial arches outside the muscle regions labelled by MYOR (Figure 3A-C). At E4 (HH22), OSR2 expression was detected in mesenchymal cells of the third branchial arches and did not overlap with that of MYOR (Figure 3D-G), which labelled the core of muscle progenitor cells in branchial arches [START_REF] Grenier | Relationship between Neural Crest Cells and Cranial Mesoderm during Head Muscle Development[END_REF]. This indicated that OSR2 was restricted to neural-crest-derived mesenchymal cells in branchial arches before E4. At E7, OSR2 was strongly expressed in muscle regions in addition to displaying an expression in the head mesenchyme (Figure 3H-J). High magnifications of muscles visualised with transverse (Figure 3K-M) and longitudinal (Figure 3N-P) sections showed that OSR2 expression was observed in MF20-positive differentiated muscle cells (Figure 3K-P We also analysed OSR2 expression in vitro, in primary cell cultures of foetal myoblasts isolated from limbs of E10 chicken embryos. Chicken foetal myoblasts were cultured in differentiation culture medium for 2 days. OSR2 expression was assessed by in situ hybridization (Figure 4A-D) and muscle cell differentiation with MF20 staining (Figure 4A,B,E,F). We observed OSR2 transcripts in MF20-positive cells (Figure 4A-F, arrows), showing that OSR2 was expressed in myotubes in chick foetal myoblast cultures

In addition to being a marker of CT and to promoting CT differentiation from chick mesenchymal stem cells [START_REF] Nassari | The chemokines CXCL12 and CXCL14 differentially regulate connective tissue markers during limb development[END_REF][START_REF] Stricker | Comparative expression pattern of Odd-skipped related genes Osr1 and Osr2 in chick embryonic development[END_REF][START_REF] Stricker | Odd-Skipped Related Genes Regulate Differentiation of Embryonic Limb Mesenchyme and Bone Marrow Mesenchymal Stromal Cells[END_REF][START_REF] Vallecillo-García | Odd skipped-related 1 identifies a population of embryonic fibro-adipogenic progenitors regulating myogenesis during limb development[END_REF] bioRxiv posted 20 July 2017 doi:10.1101/165837), we identified OSR2 as being also expressed in myosin-positive differentiated muscle cells during development of chick embryos. OSR2 expression was also observed in myotubes of chick foetal myoblast cultures. This unexpected expression of OSR2 in chick differentiated muscle fibres has never been described before and could bring new insights for OSR2 function during development. It is not clear whether Osr2 displays similar muscle expression in mice. X-gal staining of forelimb sections of E13.5 and E15.5 Osr2 Lacz/-embryos did not show any obvious expression in limb muscles [START_REF] Gao | The zinc finger transcription factors Osr1 and Osr2 control synovial joint formation[END_REF]. Moreover, Osr2 IresCre mice displayed specific expression (of reporter gene) in mandibular mesenchyme and did not display any obvious expression in muscle areas in branchial arches of E12.5 mice [START_REF] Lan | A unique mouse strain expressing Cre recombinase for tissue-specific analysis of gene function in palate and kidney development[END_REF]. Since previous expression analyses rely on genetic tools, double labelling experiments are nevertheless required to exclude an Osr2 expression in myotubes during mouse development.

MATERIALS AND METHODS

Chick embryos

Fertilized chick eggs from commercial sources (JA57 strain, Institut de Sélection Animale, Lyon, France) were incubated at 38°C in a humidified incubator until appropriate stages.

Embryos were staged according to the number of days in ovo. Staging using Hamburger and Hamilton, HH [START_REF] Hamburger | A series of normal stages in the development of the chick embryo[END_REF] or somite numbers was also used for young stages.

Chick myoblast primary cultures

Primary muscle cell cultures were prepared from skeletal muscles of forelimbs of E10 chick foetuses. Muscle cells were mechanically dissociated and seeded in plastic dishes coated with 0.1% gelatine. Myoblast primary cultures were first incubated in a proliferation medium (2/3 Minimum Essential Eagle Medium (MEM), 1/3 Hanks' salts medium 199, 10% foetal calf serum, 1% penicillin streptomycin and 1% glutamine). At 80% of confluence, differentiation was induced using a differentiation medium (2/3 Minimum Essential Eagle Medium (MEM), 1/3 Hanks' salts medium 199, 2% foetal calf serum, 1% penicillin streptomycin and 1% glutamine).

In situ hybridization to embryo sections and cells

Embryos were fixed in 4% paraformaldehyde PBS solution, rinsed successively in 4% and 15% sucrose solution, embedded in a gelatine-sucrose solution (7.5% gelatine, 15% sucrose, 50% Phosphate buffer), and frozen in chilled isopentane. Cryostat sections of 10 to 14 µm were collected on Superfrost/Plus slides (CML, France). Primary muscle cell cultures were fixed in 4% paraformaldehyde PBS solution and rinsed with PBS. Sections and cell cultures were processed for in situ hybridization as previously described [START_REF] Escot | Misregulation of SDF1-CXCR4 Signaling Impairs Early Cardiac Neural Crest Cell Migration Leading to Conotruncal Defects[END_REF][START_REF] Nassari | The chemokines CXCL12 and CXCL14 differentially regulate connective tissue markers during limb development[END_REF] using digoxigenin-labelled mRNA probes for chick OSR1, chick OSR2 [START_REF] Nassari | The chemokines CXCL12 and CXCL14 differentially regulate connective tissue markers during limb development[END_REF][START_REF] Stricker | Comparative expression pattern of Odd-skipped related genes Osr1 and Osr2 in chick embryonic development[END_REF], chick MYOR and chick MYOD [START_REF] Grenier | Relationship between Neural Crest Cells and Cranial Mesoderm during Head Muscle Development[END_REF][START_REF] Von Scheven | Protein and genomic organisation of vertebrate MyoR and Capsulin genes and their expression during avian development[END_REF].

Immunohistochemistry

Differentiated muscle cells were detected on limb and head sections and chick foetal myoblast cultures after in situ hybridization using the monoclonal antibody against sarcomeric myosin heavy chain, MF20 (Developmental Hybridoma Bank, non-diluted supernatant).

Immunohistochemistry was processed after in situ hybridization, as previously described [START_REF] Grenier | Relationship between Neural Crest Cells and Cranial Mesoderm during Head Muscle Development[END_REF][START_REF] Tozer | Involvement of vessels and PDGFB in muscle splitting during chick limb development[END_REF].

Image capturing

Images of labelled sections and cultured cells were obtained using a Leica DMI6000 B microscope or a Nikon microscope equipped for epifluorescence. Images were processed using Adobe Photoshop software.

FIGURE LEGENDS

  , arrowheads) in addition to muscle CT (Figure 3K-P, arrows).

Figure 1

 1 Figure 1

Figure 2 OSR2

 2 Figure 2

Figure 3 OSR2

 3 Figure 3

Figure 4 OSR2

 4 Figure 4

staining. (B,D,F) represent high magnifications of the boxed regions in (A,C,E), respectively.

Arrows indicate OSR2 expression in MF20-positive cells.

FUNDING

This work was supported by the Fondation pour la Recherche Médicale (FRM) DEQ20140329500, Association Française contre les Myopathies (AFM) N•16826. S.N. and M.O. were part of the MyoGrad International Research Training Group for Myology and received financial support from the AFM (AFM 20150532272) and FRM (FDT20150532272) and from the Université Franco-Allemande (CDF1-06-11 and CT-24-16).

COMPETING INTERESTS

The authors declare no competing or financial interests.