M Abidi

A A Assadi
email: aymen.assadi@ensc-rennes.fr

A Bouzaza

A Hajjaji

B Bessais

S Rtimi
email: sami.rtimi@epfl.ch

Photocatalytic indoor/outdoor air treatment and bacterial inactivation on CuxO/TiO2 prepared by HiPIMS on polyester cloth under low intensity visible light

Keywords: Photocatalysis, HiPIMS coating, VOC elimination, bacterial inactivation, redox catalysis

In this study, photoactive CuxO/TiO2 coatings were prepared using magnetron sputtering operated at different powers and modes (Direct-current magnetron sputtering and High Power Impulse magnetron sputtering). Indoor/outdoor air pollutions (Volatile Organic Compounds, VOCs) degradation have been dramatically reduced on sputtered CuxO/TiO2 on polyester (PES) cloth under low intensity visible light. The Low intensity visible light was used to irradiate the sputtered photocatalysts, which degraded various VOC molecules concomitant with a bacterial inactivation ability. The VOC removal kinetics were studied in the dark and under visible light as a function of the deposition time and applied peak currents during the coating by HiPIMS. High concentrations of chloroform and butyraldehyde were shown to be degraded (90% and 85%, respectively) on CuxO/TiO2-PES fabrics under daylight irradiation. The repetitive reuse of the sputtered coatings was also investigated showing the long operational lifetime of the prepared fabrics. The deposition rates of Ti and Cu atoms and their atomic distribution along the sputtered film have been investigated by Transmission Electronic Microscopy (TEM). Redox catalysis was shown to happen within the VOC degradation time as detected by X-ray Photoelectron Spectroscopy (XPS). Deconvolution of the Cu2p3/2 peak in the CuxO/TiO2 on PES samples showed changes in the Cu2O and CuO within the VOCs degradation and the bacterial inactivation. The interfacial charge transfer (IFCT) between CuxO induced under light leading to VOCs oxidation path seemed to require a low photons energy and were able to oxidize the pollutants even at high concentrations. The mechanisms involving the VOCs degradation on CuxO/TiO2 catalysts are suggested in which the holes generated by Cu2O transfer to TiO2 in the TiO2(n)/CuxO(p) in the hetero-junction. This transfer is favored by the electrostatic interaction between both semiconductors. The photogenerated ROS, mainly ⦁ OH-radicals were determined by fluorescence method. The possible contribution of the ⦁ OH-radicals in the bacterial inactivation on the sputtered CuxO/TiO2 catalysts was discussed. DRS, TEM, contact angle (CA) and XPS were used to characterize the catalyst optical and structural properties.

Introduction

Volatile Organic Compounds (VOCs) became of a huge concern to the social, economic and scientific community as it presents a huge issue that threats the sustainability of ecosystems and the well-being of both fauna and flora [1,[START_REF]Qualification énergétique et sanitaire des systèmes d'épuration intégrés aux réseaux de ventilation[END_REF]. European Environmental Agency estimated that Europeans spend 90% of their time indoor [START_REF]2284 du Parlement Européen et du Conseil du 14 décembre 2016 concernant la réduction des émissions nationales de certains polluants atmosphériques, modifiant la directive 2003/35/CE et abrogeant la directive 2001[END_REF]. Americans and Canadians spend 87% of time indoor and an additional 6% in a vehicle [START_REF] Klepeis | The National Human Activity Pattern Survey (NHAPS): a resource for assessing exposure to environmental pollutants[END_REF]. In fact, many reports have emphasized on the hazardous aspect of these compounds, since their presence in the atmosphere could lead to serious respiratory problems for humans and animals [START_REF]Qualification énergétique et sanitaire des systèmes d'épuration intégrés aux réseaux de ventilation[END_REF][START_REF] Parmar | Emerging control technologies for volatile organic compounds[END_REF][START_REF] Gharib-Abou Ghaida | Association of surface dielectric barrier discharge and photocatalysis in continuous reactor at pilot scale: Butyraldehyde oxidation, by-products identification and ozone valorization[END_REF], significantly decreases crops production yield and causes an unpleasant mist especially in mega-cities and industrial areas [START_REF]2284 du Parlement Européen et du Conseil du 14 décembre 2016 concernant la réduction des émissions nationales de certains polluants atmosphériques, modifiant la directive 2003/35/CE et abrogeant la directive 2001[END_REF][START_REF] Klepeis | The National Human Activity Pattern Survey (NHAPS): a resource for assessing exposure to environmental pollutants[END_REF][START_REF] Parmar | Emerging control technologies for volatile organic compounds[END_REF][START_REF] Gharib-Abou Ghaida | Association of surface dielectric barrier discharge and photocatalysis in continuous reactor at pilot scale: Butyraldehyde oxidation, by-products identification and ozone valorization[END_REF][START_REF]United States Environmental Protection Agency, NOx-How nitrogen oxides affect the way we live and breathe[END_REF].

In this context, an important attention has been paid for the treatment of industrial emitted gases in order to carry on with the important growth that this sector has witnessed during the last decades in terms of goods production and resources consumption [START_REF]United States Environmental Protection Agency, NOx-How nitrogen oxides affect the way we live and breathe[END_REF]. A variety of techniques was highlighted in order to reduce the amount of organic pollution in the air, namely adsorption [START_REF] Matos | Combination of adsorption on activated carbon and oxidative photocatalysis on tio2 for gaseous toluene remediation[END_REF], condensation [START_REF] Wang | Post-combustion CO2 capture with chemical absorption: a state-of-the-art review[END_REF], plasma combined with catalysis [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] Guillerm | Removal of gas-phase ammonia and hydrogen sulfide using photocatalysis, non-thermal plasma, and combined plasma and photocatalysis at pilot scale[END_REF], incineration and thermic combustion [START_REF] Wang | Post-combustion CO2 capture with chemical absorption: a state-of-the-art review[END_REF] and biological degradation [START_REF] Palau | Effect of pre-treatments based on UV photocatalysis and photo-oxidation on toluene biofiltration performance[END_REF]. Despite their considerable efficiency in the elimination of VOCs, these techniques were shown to generate high amounts of hazardous by-products and sludge and in some cases only presents a temporary pollution transfer from gaseous to solid phase [START_REF] Assadi | Isovaleraldehyde elimination by UV/TiO2 photocatalysis: comparative study of the process at different reactors configurations and scales[END_REF] or from one place to another. Among these techniques, heterogeneous photocatalysis was considered as a clean technology for air depollution due to its capacity to eliminate VOCs with high efficiency [START_REF] Binas | Modified TiO2 based photocatalysts for improved air and health quality[END_REF][START_REF] Zhong | Photocatalytic air cleaners and materials technologies -Abilities and limitations[END_REF], leading to their complete mineralization without a significant production of possibly harmful oxidation by-products [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] Farhanian | Photocatalytic oxidation air cleaner: Identification and quantification of by-products[END_REF]. However, the majority of the elaborated studies reported the exorbitant amount of energy that has been consumed during the run of the photocatalytic processes [START_REF] Assadi | Isovaleraldehyde elimination by UV/TiO2 photocatalysis: comparative study of the process at different reactors configurations and scales[END_REF][START_REF] Assadi | Photocatalytic oxidation of Trimethylamine and Isovaleraldehyde in an annular reactor: Influence of the Mass Transfer and the relative humidity[END_REF][START_REF] Assadi | Abatement of 3-methylbutanal and trimethylamine with combined plasma and photocatalysis in a continuous planar reactor[END_REF][START_REF] Bouzaza | photocatalytic degradation of some VOCs in the gas phase using an annular flow reactor: determination of the contribution of mass transfer and chemical reaction steps in the photo-degradation process[END_REF][START_REF] Assadi | A continuous air reactor for photocatalytic degradation of 3-methylbutanal: effect of different operating parameters and chemical degradation pathway[END_REF] as well as the unsuitability of these techniques for a certain industrial requirement [START_REF] Bouzaza | photocatalytic degradation of some VOCs in the gas phase using an annular flow reactor: determination of the contribution of mass transfer and chemical reaction steps in the photo-degradation process[END_REF]. Furthermore, the classically used catalyst, TiO2 on glass fiber tissue [START_REF] Abou 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 | Immobilization of titanium dioxide onto supporting materials in heterogeneous photocatalysis: a review[END_REF], TiO2 on 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], TiO2 on polymer support [START_REF] Abou 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 TiO2 on PES fabrics [START_REF] Zeghioud | Photocatalytic performance of TiO2 impregnated polyester for the degradation of Reactive Green 12: implications of the surface pretreatment and the microstructure[END_REF][START_REF] Shan | Immobilization of titanium dioxide onto supporting materials in heterogeneous photocatalysis: a review[END_REF][START_REF] Pachoalino | Gas phase photocatalytic decontamination using polymer supported TiO2[END_REF] did not offer a pertinent purification capacity of the effluent under visible light and needed to be further improved in order to reach higher mineralization levels [START_REF] Assadi | Photocatalytic oxidation of Trimethylamine and Isovaleraldehyde in an annular reactor: Influence of the Mass Transfer and the relative humidity[END_REF][START_REF] Abou 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] Elfalleh | Innovative And Stable TiO2 Supported Catalytic Surfaces Removing Aldehydes Under UV-Light Irradiation[END_REF]. Hence, the shifting of photocatalytic processes to the use of visible light on metal-doped semiconductors was highlighted by recent studies reporting encouraging results in the treatment of gas emissions and dye-loaded industrial wastewater effluents [START_REF] Assadi | A continuous air reactor for photocatalytic degradation of 3-methylbutanal: effect of different operating parameters and chemical degradation pathway[END_REF][START_REF] Abou 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 | Immobilization of titanium dioxide onto supporting materials in heterogeneous photocatalysis: a review[END_REF][START_REF] Pachoalino | Gas phase photocatalytic decontamination using polymer supported TiO2[END_REF][START_REF] Elfalleh | Innovative And Stable TiO2 Supported Catalytic Surfaces Removing Aldehydes Under UV-Light Irradiation[END_REF][START_REF] Zeghioud | Photocatalytic performance of TiO2 impregnated polyester for the degradation of Reactive Green 12: implications of the surface pretreatment and the microstructure[END_REF]. Indeed, the use of visible light in presence of metal-doped semiconductors can lead to improve the efficiency of VOCs degradation and microorganisms [START_REF] Rtimi | Growth of TiO2/Cu films by HiPIMS for accelerated bacterial loss of viability[END_REF][START_REF] Rtimi | ZrNO-Ag co-sputtered surfaces leading to E. coli inactivation under actinic light: Evidence for the oligodynamic effect[END_REF][START_REF] Shayegan | TiO2 photocatalyst for removal of volatile organic compounds in gas phase-A review[END_REF][START_REF] Pham | Advanced removal of toluene in aerosol by adsorption and photocatalytic degradation of silver-doped TiO2/PU under visible light irradiation[END_REF].

During the last few years, many research groups focused on the preparation of innovative catalysts for bacterial inactivation [START_REF] Lin | Iodine-modified nanocrystalline titania for photo-catalytic antibacterial application under visible light illumination[END_REF]. Wang et al., reported on the antibacterial behavior of CuI-BiOI/Cu films prepared by water bath method and their enhanced photo-induced charge separation under visible-light [START_REF] Zhang | CuI-BiOI/Cu film for enhanced photo-induced charge separation and visible-light antibacterial activity[END_REF]. The same research group reported on the role of the photogenerated h + , e -and O2 -playing synergistic effect for the photocatalytic disinfection over Ag@AgI/Bi-BiOI three-dimensional nano-architectures [START_REF] Ning | Dual couples Bi metal depositing and Ag@AgI islanding on BiOI 3D architectures for synergistic bactericidal mechanism of E. coli under visible light[END_REF]. Many efforts are running today for the design of Lotus effect self-cleaning/self-sterilizing coatings for hospital textile [START_REF] Rtimi | TiON and TiON-Ag sputtered surfaces leading to bacterial inactivation under indoor actinic light[END_REF][START_REF] Rtimi | Growth of TiO2/Cu films by HiPIMS for accelerated bacterial loss of viability[END_REF][START_REF] Rtimi | ZrNO-Ag co-sputtered surfaces leading to E. coli inactivation under actinic light: Evidence for the oligodynamic effect[END_REF].

The aim of the present work is to explore the removal efficiency of indoor air pollutants such butyraldehyde (from now as BUTY) and chloroform in batch reactor on CuxO/TiO2 sputtered on PES. The influence of inlet concentration and catalyst on the pollutant removal are investigated in details. In addition to the indoor air pollutants, the sputtered fabrics were tested for the chloroform removal. Moreover, an interesting challenge of this work is to study also the effect of the preparation conditions on the photocatalytic inactivation of E. coli. The photocatalyst were characterized by up-to-date surface science techniques.

Material and methods

CuxO/TiO2 sputtered PES

The CuxO/TiO2 coatings were sputtered using combined reactive DCMS/HiPIMS process. Ti target was operated under DCMS mode and Cu target (99.99% pure) was operated using reactive HIPIMS mode. The sputtering chamber was operated at a high vacuum with a residual pressure < 4 x 10 -5 Pa equipped with two confocal targets 5 cm in diameter (Lesker. Hastings, East Sussex pure). A HiP3 5KW Solvix generator was used for the HiPIMS deposition and was operated at an average power of 100 W (5 Wcm -2 ) with a pulse-width of 50 µs and a frequency of 1 KHz. The gas phase inside the sputtering chamber was 95% Ar : 5% O2. The DC power in the second target was performed at 90 W for the first 30 seconds then at 50 W to adjust the relative content of Ti in the film as it will be shown later by high-resolution transmission electron microscopy. A TiO2 under layer was puttered before the CuxO deposition. The target-to-substrate distance was fixed at 10 cm as shown in scheme 1 below. Scheme 1. Illustration of the sputtering unit used to prepare the photocatalyst used in the present study.

The low surface energy of PES cloth can lead to poor sputtered nanoparticles' adhesion. In order to increase the uniform distribution of Cu on PES, an under layer of TiO2 was sputtered for 20 second followed by the Cu deposition (from a separate target). The latter plays the role of stabilizer to Cu species as previously reported [START_REF] Rtimi | Insight on the photocatalytic bacterial inactivation by co-sputtered TiO2-Cu in aerobic and anaerobic conditions[END_REF][START_REF] Mathews | Epitaxial Growth Part B[END_REF]. In addition, the presence of TiO2 reduces the aggregation of Cu on the substrate surface [START_REF] Mejia | Preparation, testing and performance of TiO2/Polyester for the degradation of gaseous methanol[END_REF]. Furthermore, the PES fabrics were pretreated by UVC-light (15 min) to induce a higher density of functional polar groups able to bind the sputtered Ti-species [START_REF] Rtimi | TiON and TiON-Ag sputtered surfaces leading to bacterial inactivation under indoor actinic light[END_REF][START_REF] Rtimi | Growth of TiO2/Cu films by HiPIMS for accelerated bacterial loss of viability[END_REF][START_REF] Ochiai | Fabrication of a TiO2 nanoparticles impregnated titanium mesh filter and its application for environmental purification[END_REF][START_REF] Rtimi | Insight on the photocatalytic bacterial inactivation by co-sputtered TiO2-Cu in aerobic and anaerobic conditions[END_REF]. UVC irradiation was carried out using a 185/254 nm low-pressure mercury lamp (Ebara Corp., Tokyo, Japan). The UVC lamp (25 W) presented emission lines at 185 nm and 254 nm in a ratio 1:4.

Materials and chemicals

Photocatalytic experiments were performed using a cylindrical batch reactor, 10 mm as upper diameter and 50 mm of working height, thus accepting up to 50 mL of the studied pollutant. A fluorescent visible lamp could be inserted inside the reactor from above. The reactor contains two septum; one of them is used for samples extractions (Figure 1). The photon excitation was provided by a Sylvania CF-L 24W/840 source with a spectral emission in visible range (400-720 nm). This light source is similar to light used for hospital facilities, schools and houses. This is the reason why we are calling it as "indoor light". The spectral emission of the lamp is illustrated in Figure 1. The whole micro-reactor was covered with an aluminum thin layer to prevent visible emission. A magnetic stirrer and barrel allows the gas phase to be homogenized.

The CuxO/TiO2-PES samples were placed in the inner space of the tube to be in direct contact with the studied gas and the light radiation. The surface of the catalyst was constant of about 20 cm² for all the carried experiments.

Photocatalytic tests and analytical tools

In order to test the photocatalytic performance, a common volatile organic compound was used, chloroform. Analysis of Chloroform/BUTY was performed using a gas chromatography gas phase (Fisons GC9000) equipped with a flame ionization detector (FID). Nitrogen is the carrier gas and constitutes the mobile phase. All injections were performed manually with a syringe of 500 μL and repeated at least three times.

The pollutants analysis can only start once the airflow in the reactor reaches equilibrium. A certain time is required after the pollutant is injected in the reactor, so that its concentration stabilizes.

Once the outlet concentration is stabilized and the catalyst is loaded, light is on and the photocatalytic oxidation starts.

Bacterial inactivation tests and • OH-radical identification

The evaluation of the bacterial inactivation was carried out by plate counting agar method. The Escherichia coli (E. coli K12) was obtained from the Deutsche Sammlung von Mikro-organismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany to test the sputtered samples bacterial inactivation. The sputtered PES fabrics were sterilized keeping them at 70°C overnight. Aliquots of 50 µL bacterial culture suspended in NaCl/KCl (8 g/l NaCl and 0.8 g/l KCl) solution with a concentration of 8x10 5 colony forming unit per milliliter (CFU.mL -1 ) were placed on sputtered and unsputtered (control) PES samples. The samples were placed in Petri dishes provided with a lid to prevent evaporation. At preselected times, the samples were transferred into a sterile 2 mL Eppendorf tube containing autoclaved NaCl/KCl saline solution. These solutions were subsequently mixed thoroughly using a Vortex. Serial dilutions were made in NaCl/KCl solution taking 100 µL aliquots. Then 100 µL aliquots were pipetted onto a nutrient agar plate, for the bacterial counting by the standard plate method. These agar plates were incubated, lid down, at 37°C for 24 h before colonies counting. The bacterial tests reported in this study were carried out in triplicates. To verify that no re-growth of E. coli occurs after the first bacterial inactivation cycle, the sputtered films were incubated on an agar Petri dish at 37°C for 24h. Statistical analysis are presented by mean of standard deviation (n=5%).

For the quantification of the ⦁ OH-radical, samples of 9 cm 2 were immersed in a solution of terephthalic acid (0.4 mmol L -1 ) dissolved in NaOH (4 mmol L -1 ). After preselected irradiation times, the solution was transferred in a quartz cell. The fluorescence of the photo-generated 2hydroxyterephthalic-acid was quantitatively monitored on a Perkin Elmer spectrometer. The spectra were recorded at a scan rate of 100 nm/min after excitation at 315 nm.

CuxO/TiO2 Characterization: Thickness, TEM, CA, spectral absorption and XPS

Thicknesses of the TiO2, CuxO and CuxO/TiO2 thin films were measured by profilometry (Alphastep-500, Tencor, USA) after deposition on Si-wafers. To be able to perform the transmission electron microscopy (TEM), the PES fabrics were embedded in epoxy resin (Embed 812) and cross-sectioned with an ultra-microtome (Ultracut E) up to thin layers of 80-100 nm thick. These thin layers were then placing on TEM holey carbon grid in order to image the samples. The Spot size was set to 5 µm, dwell time 50 µs and the real time of 600 s. The contact angles (CA) of TiO2, CuxO/TiO2 were determined by the sessile drop method on a Data Physics OCA 35 unit.

Diffuse reflectance spectroscopy (DRS) of TiO2, CuxO and CuxO/TiO2 was carried out a Perkin Elmer Lambda 900 UV-VIS-NIR spectrometer within the wavelength range of 250-750 nm. The background was subtracted using an unsputtered PES fabric. Optical spectra were analyzed by the Tauc's method.

An AXIS NOVA photoelectron spectrometer (Kratos Analytical, Manchester, UK) provided for with monochromatic AlKa (hʋ=1486.6 eV) anode. Binding energies (BE) were calibrated against the standard C1s binding energy at 284.6 eV [START_REF] Zeghioud | Photocatalytic performance of TiO2 impregnated polyester for the degradation of Reactive Green 12: implications of the surface pretreatment and the microstructure[END_REF][START_REF] Rtimi | TiON and TiON-Ag sputtered surfaces leading to bacterial inactivation under indoor actinic light[END_REF]. A Multipak (version 9 software) using 70:30 Gaussian: Lorentzian peak shape and a Shirley background function were used for Spectra deconvolution as previously reported [START_REF] Rtimi | TiON and TiON-Ag sputtered surfaces leading to bacterial inactivation under indoor actinic light[END_REF][START_REF] Rtimi | Growth of TiO2/Cu films by HiPIMS for accelerated bacterial loss of viability[END_REF][START_REF] Ochiai | Fabrication of a TiO2 nanoparticles impregnated titanium mesh filter and its application for environmental purification[END_REF][START_REF] Rtimi | Insight on the photocatalytic bacterial inactivation by co-sputtered TiO2-Cu in aerobic and anaerobic conditions[END_REF].

Results and discussion

Thickness calibration and TEM of sputtered CuxO/TiO2-PES

The thickness calibration of the CuxO deposed by HiPIMS at 20, 40 and 80 A on Si-wafer was investigated. Sputtering CuxO for 100 s at 100 A led to a layer of ~70 nm thick. This is equivalent to 350 atomic layers, each containing 10 15 Cu-atoms/cm 2 being deposited at a rate of ~5x10 15 atom/cm 2 s [START_REF] Mathews | Epitaxial Growth Part B[END_REF].

The PES fabrics were initially pretreated with UVC for 15 min as previously described. UVC pretreatment enhances the polarity, roughness and hydrophilicity of polyester improving the interfacial adhesion of TiO2 as previously reported by Kiwi et al. [START_REF] Mejia | Preparation, testing and performance of TiO2/Polyester for the degradation of gaseous methanol[END_REF] and R.A. Jelil [START_REF] Jelil | A review of low-temperature plasma treatment of textile materials[END_REF]. The UVC pre-treatment induced negatively charged functional groups, e.g., carboxylic, percarboxylic, epoxide and peroxide groups due to the atomic and excited O [START_REF] Elfalleh | Innovative And Stable TiO2 Supported Catalytic Surfaces Removing Aldehydes Under UV-Light Irradiation[END_REF]. The PES negative sites bind the positive ions generated in the plasma phase Ti 4+ /Ti 3+ /Cu + /Cu 2+ . This involves electrostatic attractions including chelation/complexation [START_REF] Ochiai | Fabrication of a TiO2 nanoparticles impregnated titanium mesh filter and its application for environmental purification[END_REF]. The TiO2 was deposited immediately after the UVC pretreatment due to the short lifetimes of the polar radical species.

Transmission electron microscopy (TEM) images of the sputtered CuxO/TiO2 on PES shows a continuous coating of TiO2 under layer on PES fabrics. TiO2 thickness of 10-15 nm is equivalent to 50 atomic layers [START_REF] Elfalleh | Innovative And Stable TiO2 Supported Catalytic Surfaces Removing Aldehydes Under UV-Light Irradiation[END_REF], taking an atomic layer thickness of 0.2 nm [START_REF] Mejia | Preparation, testing and performance of TiO2/Polyester for the degradation of gaseous methanol[END_REF]. During the deposition of the TiO2 under layer and the CuxO, the Ar/O2 ratio was fixed at 19. The oxygen atoms distribution shows the oxide nature of the Ti and the Cu upper layer. Uniform distribution of Cu-species on the TiO2 under layer with a high oxygen content. This reveals the oxidation of both under and upper layers during the deposition. Table 1 shows the Ti, O and Cu weight and atomic percentages in the sputtered films as determined by EDX. The CuxO layer uniformity and particulates shape/size are dependent on the number of deposited atomic layers. This controls the structure-reactivity relation of the Cu-catalyst clusters sputtered on the PES-substrate [START_REF] Sarakinos | High power pulsed magnetron sputtering: A review on scientific and engineering state of the art[END_REF][START_REF] Rtimi | Indoor light enhanced photocatalytic ultra-thin films on flexible non-heat resistant substrates reducing bacterial infection risks[END_REF]. Both layers were deposited in reactive mode in the presence of oxygen. In addition, the exposition of the sample to ambient environment after preparation can lead to further oxidation of the top-most layers as reported previously [START_REF] Rtimi | TiON and TiON-Ag sputtered surfaces leading to bacterial inactivation under indoor actinic light[END_REF][START_REF] Rtimi | Growth of TiO2/Cu films by HiPIMS for accelerated bacterial loss of viability[END_REF][START_REF] Rtimi | ZrNO-Ag co-sputtered surfaces leading to E. coli inactivation under actinic light: Evidence for the oligodynamic effect[END_REF][START_REF] Rtimi | Insight on the photocatalytic bacterial inactivation by co-sputtered TiO2-Cu in aerobic and anaerobic conditions[END_REF].

Photocatalytic experiments

Effect of the deposition Current of CuxO on TiO2-PES on the photocatalytic activity

under low intensity light: Chloroform removal.

Figure 2 shows the chloroform removal on CuxO/TiO2-PES sputtered for 20, 80 and 100 A. It is readily seen from Figure 2 that CuxO/TiO2-PES photocatalyst sputtered at 80 A led to the fastest chloroform removal compared to samples sputtered at 20 and 100 A. An increase in sputtering current from 20 to 80 A led to two times increase in the chloroform removal (from 40 to 72% of the initial 6 mg/m 3 of chloroform). This is due to the availability of the photocatalytic sites generated at the interface of the sample deposited at (80A). At low current values, the degradation will tend to a limit. It was recently reported that the deposition current in HiPIMS generates different ions/charged species [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]. This leads to differentiate charges at the thin films interface.

Effect of the deposition time of CuxO on TiO2-PES on their photocatalytic activities

The photocatalytic activities of CuxO deposed for different time (5, 20 and 100 s) on TiO2 under layer were investigated in details. For the copper-based photocatalyst (Figure 3) and when comparing different times of deposition under low intensity light, it is readily seen that the degradation of chloroform was significantly affected by the deposition time of CuxO and thus its amount on the surface. Indeed, the normalized concentrations of chloroform shown in Figure 3 decreased up to 71% within 15 h irradiation. This result is in agreement with other results reported for some other VOCs [START_REF] Mathews | Epitaxial Growth Part B[END_REF][START_REF] Elfalleh | Innovative And Stable TiO2 Supported Catalytic Surfaces Removing Aldehydes Under UV-Light Irradiation[END_REF]. It has been recently reported that the thickness/amount and microstructure of Cu are a function of the applied sputtering current and lead to the significant difference in the total-ions count [START_REF] Sarakinos | High power pulsed magnetron sputtering: A review on scientific and engineering state of the art[END_REF].

Recent reports investigated the ions distribution from the plasma phase (inside the magnetron sputtering chamber) until the resulting CuxO thin film (before and after exposition to light) [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]. S. Konstantinidis et al., reported on the diagnostics and understanding of the plasma-surface interaction in HiPIMS discharges [START_REF] Britun | Particle visualization in high-power impulse magnetron sputtering. II. Absolute density dynamics[END_REF][START_REF] Konstantinidis | Titanium oxide thin films deposited by high-power impulse magnetron sputtering[END_REF][START_REF] Britun | Plasma diagnostics for understanding the plasma-surface interaction in HiPIMS discharges: a review[END_REF]. The same authors followed the deposition rate of TiO2 films as a function of the applied current and showed that the deposition rate increases until a certain current intensity then it decreases [START_REF] Konstantinidis | Titanium oxide thin films deposited by high-power impulse magnetron sputtering[END_REF]. This can explain the lower photocatalytic activity observed for the sample sputtered at 100 A. Similar trends was observed by Kiwi et al., and was attributed to the inward charge diffusion in thick catalytic films [START_REF] Kiwi | Pulgarin Innovative self-cleaning and bactericide textiles[END_REF][START_REF] Kiwi | Self-Cleaning Textiles Modified by TiO2 and Bactericide Textiles Modified by Ag and Cu. Self-Cleaning Materials and Surfaces: A Nanotechnology Approach[END_REF][START_REF] Mejia | Innovative UVC light (185 nm) and radio-frequency-plasma pretreatment of nylon surfaces at atmospheric pressure and their implications in photocatalytic processes[END_REF][START_REF] Mejia | Selfcleaning modified TiO2-cotton pretreated by UVC-light (185 nm) and RF-plasma in vacuum and also under atmospheric pressure[END_REF]. The chloroform removal on CuxO/TiO2-PES with 100s of sputtering time was about twice time faster compared to the same catalyst with 5s of sputtering time; it reaches 80% removal in 12h with 20 s of sputtering as seen in Figure 3. On CuxO/TiO2-PES, only 40% of 6 g/m 3 of chloroform were removed within same period. This observation can be explained by the fact that 5 s is very low to form a layer of catalyst leading to chloroform degradation.

On other way, we see that increasing the time sputtering from 20 to 100 s has no effect of VOC removal. In fact, we note the same removal of chloroform. This observation is due to the fact that chloroform molecules adsorption is mainly a monolayer process and the increase in deposit time is considered could be associated to a multilayer coating thickness of the catalyst [START_REF] Rtimi | TiON and TiON-Ag sputtered surfaces leading to bacterial inactivation under indoor actinic light[END_REF]. Similar results were recently reported [START_REF] Rtimi | Effect of surface pretreatment of TiO2 films on interfacial processes leading to bacterial inactivation in the dark and under light irradiation[END_REF] when investigating bacteria degradation using TiO2 in water.

Analysis results were discussed according to the variation of deposition time and used galvanostatic current intensity during pulverization process.

Effects of indoor/outdoor pollutants on CuxO/TiO2-PES performance

Figure 4 shows that under 80 A and with visible light, the degradation of two kinds of pollutants, with equimolar inlet concertation, is quiet similar. However, when we decrease the intensity of light (data not shown), we observed that the degradation of chloroform is becoming slower than that of butyraldehyde. Therefore, we can conclude that the copper-based catalyst is more efficient for the degradation of butyraldehyde.

After 10 hours visible light illumination, the photocatalytic removal kinetics of chloroform was faster than butyraldehyde. This can be due to the chemical properties of pollutants, their affinities towards the catalyst and the accumulation of their intermediate by-products on the surface of catalyst leading to competition effect of VOC towards the catalytic active sites.

CuxO/TiO2-PES catalyst recyclability

The reusability of the tested samples is an important factor to investigate since it is a measure for its practical application potential degrading pollutants in the gas phase [START_REF] Mejia | Preparation, testing and performance of TiO2/Polyester for the degradation of gaseous methanol[END_REF][START_REF] Rtimi | Effect of surface pretreatment of TiO2 films on interfacial processes leading to bacterial inactivation in the dark and under light irradiation[END_REF]. To investigate the applicability of the developed copper sample to operate in four successive cycles, several visible photocatalytic oxidations were done. The copper catalyst was thoroughly washed after each photodegradation cycle and reused. The results of repetitive use of the CuxO/TiO2-PES photocatalyst up to the 4 th cycle l is shown below in Figure 5. We note no loss of sample photoactivity. The results reported in Figure 5 show the overall potential of the CuxO/TiO2-PES samples for application in air treatment. We note the same slope of the removal rate during each cycle.

Thus, this behavior can confirm the no deactivation of surface sites and an excellent catalytic stability of CuxO/TiO2-PES is confirmed. This trend can be attributed to the very low metals (copper and titanium) leaching during removal cycle. species and bacteria is very important for bacterial inactivation [START_REF] Baghriche | Innovative TiO2/Cu nano-surfaces inactivating bacteria in the minute range under low-intensity actinic light[END_REF]. In addition, Titanium dioxide exhibits antibacterial property under UV light illumination [START_REF] Li | Antibacterial activity of TiO2 nanotubes: influence of crystal phase, morphology and Ag deposition[END_REF]. After UV-visible light irradiation, photogenerated electron from the conduction band of cuprous oxide can be captured by Ti 4+ in TiO2 who is reduced to Ti 3+ ions. The valence band of CuxO forms a hole center, preventing the recombination of photogenerated electrons and holes [START_REF] Xiong | Bifunctional photocatalysis of TiO2/Cu2O composite under visible light: Ti 3+ in organic pollutant degradation and water splitting[END_REF]. The electron trapped in Ti 3+ can react with oxygen adsorbed on the TiO2 surface to form O2 -• , that could transfer to H + for the formation of HO2 • and • OH. The • OH (or/and HO2 • , O2 -• ) can kill the bacterial cells. The hole in the TiO2 valence band can migrate to the CuxO valence band, these photo-generated holes present in the VB of both semiconductors can be consumed by the oxidation of bacteria at the interface [START_REF] Xiong | Bifunctional photocatalysis of TiO2/Cu2O composite under visible light: Ti 3+ in organic pollutant degradation and water splitting[END_REF]. The optical properties of CuxO/TiO2-PES films were determined by diffuse reflectance spectroscopy (DRS) as shown in Figure 7. Normally a weak dependence is assumed for the scattering coefficient S on the wavelength [START_REF] Auer | Industrial Inorganic Pigments[END_REF][START_REF] Mamba | Synchronic coupling of Cu2O(p)/CuO(n) semiconductors leading to Norfloxacin degradation under visible light: Kinetics, mechanism and film surface properties[END_REF]. However, because of the relatively high scattering contribution of the PES, the reflectance spectra were treated to show the real optical absorption of the sputtered CuxO/TiO2-PES samples. A wide spectral range was seen for CuxO-PES up to almost 700 nm corresponding to a band-gap of ∼1.8 eV. When CuxO/TiO2 were sputtered on PES, a lower optical absorption (up to 600 nm) was observed corresponding to a bang gap of 2-2.1 eV. In general, the absorption in the spectral zone 500-600 nm reflects the interband transition of Cu2O-species [START_REF] Pachoalino | Gas phase photocatalytic decontamination using polymer supported TiO2[END_REF].The absorption between 600-800 nm is due to the intrinsic d-d transition induced in the Cu-oxide (especially CuO). In our case, this is observed only when we sputter CuxO on PES without TiO2 under layer. The low thickness of the upper layer of CuxO allow the applied light to reach the TiO2 under layer inducing photocatalytic electron/hole separation. It has been reported by Hashimoto et al., that very weak light intensities (1 µW/cm 2 ) can induce the photocatalytic activity of Cu-TiO2 thin films [START_REF] Sunada | Bactericidal activity of copper-deposited TiO2 thin film under weak UV light illumination[END_REF]. Furthermore, the same authors reported on low light intensities of 1 mW/cm 2 can induce bacterial inactivation at the interface of TiO2 thin films [START_REF] Sunada | Studies on photokilling of bacteria on TiO2 thin film[END_REF]. solar light [START_REF] Mamba | Synchronic coupling of Cu2O(p)/CuO(n) semiconductors leading to Norfloxacin degradation under visible light: Kinetics, mechanism and film surface properties[END_REF]. Figure 8a shows also the existence of two satellite peaks at 940 and 943.5 eV.

Gupta and Sen reported that if excited states were taken into account, the shake-up satellites should also be considered [START_REF] Gupta | Calculation of multiplet structure of core p-vacancy levels. II[END_REF]. They explained that the presence of the satellite is caused by as a shake-up result. The excitation of an unpaired 3d electron to a 4s energy level. This leads to the in the reduction of the kinetic energy of the 2p electron by the excitation energy of the unpaired 3d electron. Thus, two peaks occur, one for the 2p excitation and the other for the combined effect of the 2p and 3d excitations. The satellite occurs generally at higher binding energies (due to the reduction of the kinetic energy).

The O1s deconvolution (Figure 8b) shows the interdependence of CuxO (with its two oxidative states) and the TiO2 under layer. The existence of Cu-(OH) at the interface of the sputtered layers reflects a strong interaction of the thin film with water molecules. To quantify the water-surface relationship, wettability of the sputtered surface was carried out using by static water contact angle (CA) measurement.

The water CA on the CuxO/TiO2-PES surface was investigated. The CA at time zero was 101° and dropped to 52° after 2h irradiation. This can be attributed to the surface chemistry (especially OHgroups) and to the porous micro/nanostructured PES surface allowing the water droplet to vanish. Furthermore, this photo-switching behavior can be attributed to the physical adsorption of oxygen molecules on the topmost layer of the CuxO. The wettability of a flat surface can obey to the Wenzel model. This model is not sufficient when the surface composition is heterogeneous. The Cassie's law (based on the Cassie-Baxter equation, eq.1) can explain the wettability of these surfaces [START_REF] Marmur | Wetting of Hydrophobic Rough Surfaces: To be heterogeneous or not to be[END_REF]:

f Y cos( *) r cos( ) f 1 f θ θ = + - (1)
Where: rf is the roughness ratio of the wet surface area and f is the fraction of solid surface area wet by the liquid. When f = 1 and rf = r, the Cassie-Baxter equations is equivalent to the Wenzel equation. A wetting equilibrium on the rough PES surface is understood based on the competition between the hydrophobic surface and the photo-catalytically induced hydrophilicity (52° after 2h illumination). This involves oxidative states changes, as it was previously reported [START_REF] Rtimi | Innovative semi-transparent nanocomposite films presenting photo-switchable behavior and leading to a reduction of the risk of infection under sunlight[END_REF]. Table 2 shows the oxidative states changes of CuxO at the interface with VOCs and bacteria in ambient air. It is expected that Cu + can attain the d10 configuration by losing one electron from the s-subshell.

Nonetheless, because it has fully filled d-orbital, it is stable. However, in aqueous medium (in our case, bacterial suspension) or with moist air (in our case, indoor air), Cu + is unstable and tends to disproportionate leading to the formation of Cu 2+ , which presents incompletely filled d-orbital (d9 configuration). This lead to fast restoration of the surface reestablishing the initial oxidative states prior to the illumination.

Suggested mechanism for the CuxO/TiO2 pollutants degradation and bacterial inactivation

The XPS results suggest the reaction sequence on the sample surface of the sputtered catalysts, which are at the interface with the VOCs and the bacteria, as:

4Cu 0 +O2 → 2Cu2O E 0 0.41 V (2) Cu 2+ + Cu 0 + H2O → Cu2O + 2H + E 0 0.17 V (3) 2 Cu2O + O2 → 4CuO E 0 0.52 V (4)
The pattern of the Cu (and Ti) sputtered on the PES surface is complicated by the surface diffusion controlling the charge transport between Cu(II)/Cu(I) and Ti on the PES surface. In the topmost layers, the Cu 0 , Cu2O, CuO and TiO2 present motions leading to agglomerates presenting a different collective diffusion [START_REF] Venable | Epitaxial Growth Part[END_REF].

The distinct kinetics of bacterial inactivation and VOCs degradation times can be attributed to the intrinsic conduction band (cb) and valence band (vb) positions of CuO and Cu2O. On these bases, the vectorial charge displacement/flow is shown for both oxides in Figure 9a. The CuO present a band-gap (of 1.7eV), cb (-0.3 eV) and vb (+1.4 eV) while direct band-gap of Cu2O p-type presents a band-gap of 2.1 eV, cb (-1.4 eV) and vb (+0.7 eV). This can be summarized in the equations below:

CuO + hν → CuO(cbe-), CuO(vbh+) (5)
Under photon energies exceeding the CuO band-gap, the cbe-electron could either react directly with the O2 forming O2 -or reduce the Cu 2+ to Cu + as noted below and as shown in Figure 9 a:

CuO(cbe -) + O2 → CuO + O2 - (6)
CuO(cbe -) → CuO(Cu + ) (7) CuO(Cu + ) + O2 → CuO(Cu 2+ ) + O2 - Since the CuxO topmost layer is transparent allowing the light to reach TiO2, the photocatalytic reactions of CuxO/TiO2-PES under light can be summarized below by eq. [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] Guillerm | Removal of gas-phase ammonia and hydrogen sulfide using photocatalysis, non-thermal plasma, and combined plasma and photocatalysis at pilot scale[END_REF][START_REF] Palau | Effect of pre-treatments based on UV photocatalysis and photo-oxidation on toluene biofiltration performance[END_REF]. The highly mobile electrons transfer from Cu2Ocb to the TiO2cb as shown in Figure 9b. The vbh + will react leading to the observed bacterial inactivation and/or VOC degradation.

Cu2O + hν → Cu2O(cbe -), Cu2O(vbh + ) (10) Cu2O(cbe -) + TiO2 →TiO2 -or (Ti 3+ ) (11) TiO2 -+ O2 → TiO2 + O2 - (12) Cu2O(vbh + ) + bacteria → CO2, H2O, inorganic N,S (13) However, this mechanism presented in Figure 9b Sputtered materials were reported to degrade organic compounds [START_REF] Zeghioud | Photocatalytic Performance of CuxO/TiO2 Deposited by HiPIMS on Polyester under Visible Light LEDs: Oxidants, Ions Effect, and Reactive Oxygen Species Investigation[END_REF]. In the present study, the prepared material is tested for VOC removal and bacterial inactivation. This comprises ROS generation and their diffusion/migration in different media (liquid, suspension, air). This challenges systematic investigation of the possible photo-generated charges diffusion over different matrices.

Conclusion:

This report presents the testing of an innovative uniform and stable sputtered CuxO/TiO2-PES photocatalyst prepared at low temperature by high power impulse magnetron sputtering. This is the first report on uniform coatings of Cu/CuO on PES leading to the degradation of indoor air VOCs concomitant with an antibacterial activity. Two concentrate VOC models were removed at the interface of CuXO/TiO2-PES. Moreover, a stable removal performance of the chloroform by the CuxO/TiO2-PES samples was observed during repetitive recycling under visible irradiation. This catalyst feature shows an excellent catalytic stability and presents a promising potential for the application in air treatment. The CuxO/TiO2-PES photocatalyst exhibits a high antibacterial activity, which attained a total bacterial inactivation within 2 hours on CuxO/TiO2-PES sputtered at 80A. This makes the CuxO/TiO2 surfaces presented in the present study have promising applications in Lotus effect self-cleaning coatings for hospital textiles.

Figure 1 .

 1 Figure 1. Schematic of the batch photo-reactor with the central co-axial Sylvania CF-L 24W/840 light source (400-720 nm).

Figure 2 .

 2 Figure 2. Removal of chloroform with catalyst CuxO/TiO2-PES at different HiPIMS sputtering currents deposited for 100 s.

  Cu x O deposited by HiPIMS at 20 A Cu x O deposited by HiPIMS at 40 A Cu x O deposited by HiPIMS at 80 A

Figure 3 .

 3 Figure 3. Removal of chloroform on HiPIMS-sputtered CuxO/TiO2-PES (at 80 A) deposited at different times.

  x O deposited by HiPIMS (80 A) for 5 s Cu x O deposited by HiPIMS (80 A) for 20 s Cu x O deposited by HiPIMS (80 A) for 100 s

Figure 4 .

 4 Figure 4. Indoor/outdoor pollutants removal on CuxO/TiO2-PES HiPIMS-sputtered 80 A for 20s under a visible lamp irradiation.

Figure 5 . 3 . 4 .Figure 6 .

 5346 Figure 5. Degradation profile of chloroform with photocatalysis on a CuxO/TiO2-PES sample under visible light. Further experimental conditions: I=30W/m 2 , Chloroform concentration = 6 g/m 3 .

3. 3 .

 3 Diffuse reflectance spectroscopy (DRS), Surface oxidative states and wettability

Figure 7 .

 7 Figure 7. Tauc's plot for band-gaps estimation of: (1) TiO2, (2) CuxO and (3) CuxO/TiO2 deposited by HiPIMS on polyester. The oxidative states at the interface CuxO/TiO2-PES were investigated by XPS. The topmost layers are formed by Cu-species. The deconvolution of the CuxO (Cu2p3/2) peak in Figure 8a (time zero) identifies two peaks at 932 eV and 952 eV corresponding to Cu 2p3/2 and Cu 2p1/2 respectively. A mixture of Cu + and Cu 2+ forms the CuxO upper layer. The co-existence of CuO and Cu2O was recently reported by our laboratory and led to the fast Norfloxacin degradation under

Figure 8 .

 8 Figure 8. XPS deconvolution of (a) Cu2p and (b) O1s at the topmost layers of CuxO/TiO2-PES surface: species in direct contact with the pollutants and the bacteria.

Figure 9 .

 9 Figure 9. Proposed mechanisms of action of the prepared photocatalyst: (a) if we consider just CuxO interface, (b) if we consider a photocatalytic intervention of the TiO2 under layer.

  is low probable because of the very low amount of • OH-radical detected by fluorescence experiment.Many research groups reported the Fenton-like reaction allowing the generation of • OH-radical on copper surfaces[START_REF] Baghriche | Polystyrene CuO/Cu2O uniform films inducing MBdegradation under sunlight[END_REF] (see equation14). Furthermore, some other researchers went further by combining/associating the Fenton-like reaction to the Haber-Weiss cycle (equations 15 and 16) leading to the generation of • OH-radicals from H2O2, which in turn activates the copper Fentonlike reaction. Although Haber-Weiss reaction has long been studied and revived in different contexts, this association is still hypothetical and requires deep systematic investigation.Cu + + H2O2 → Cu 2+ + OH -+ • OH(14)H2O2 + • OH → H2O + O2 -+ H + (15)H2O2 + O2 -→ O2 + OH -+ • OH[START_REF] Zhong | Photocatalytic air cleaners and materials technologies -Abilities and limitations[END_REF]

Table 1 .

 1 EDX area and elemental mapping analysis of CuxO-TiO2-PES both sputtered for 100 s at 80 A.

	Element	Weight %	Atomic %
	O K	16.53	68.73
	Ti K	3.66	4.41
	Cu K	54.3	17.47

Table 2 .

 2 Oxidative states changes as determined by XPS peaks areas within indoor light

	irradiation for 2h.
		Cu +	Cu 2+
	CuxO/TiO2-PES Time zero	32.4	77.6
	CuxO/TiO2-PES After 2h irradiation	48	52

Acknowledgement:

S.R. would like to thank Prof. H. Hofmann for the support during the TEM and XPS experiment. Thanks Soheil Hasanzadeh for the images acquisition. A special thank goes to Prof. C. Pulgarin for the bio-lab facility.


Kevin Guedj

Yaël Abitbol

Dominique Cazals-Hatem

Marion Morvan

Léon Maggiori

Yves Panis

Yoram Bouhnik

Giuseppina Caligiuri

Olivier Corcos

Antonino Nicoletti
email: antonino.nicoletti@inserm.fr

Antonino Pr

Nicoletti

Adipocytes orchestrate the formation of tertiary lymphoid organs in the creeping fat of Crohn's disease affected mesentery

Keywords: Crohn's disease, adipocytes, mesentery, chemokines, tertiary lymphoid organs CD, "Crohn's Disease", LPS, "Lipopolysaccharide", LTo, "Lymphoid Tissue organizer", LTi, "Lymphoid Tissue inducer", SLO, "Secondary Lymphoid Organ", TLO, "Tertiary Lymphoid

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

ABSTRACT

The formation of tertiary lymphoid organs (TLOs) is orchestrated by the stromal cells of tissues chronically submitted to inflammatory stimuli, in order to uphold specific adaptive immune responses. We have recently shown that the smooth muscle cells of the arterial wall orchestrate the formation of the TLOs associated with atherosclerosis in response to the local release of TNF-α. Observational studies have recently documented the presence of structures resembling TLOs the creeping fat that develops in the mesentery of patients with Crohn's disease (CD), an inflammatory condition combining a complex and as yet not elucidated infectious and autoimmune responses.

We have performed a comprehensive analysis of the TLO structures in order to decipher the mechanism leading to their formation in the mesentery of CD patients, and assessed the effect of infectious and/or inflammatory inducers on the potential TLO-organizer functions of adipocytes.

Quantitative analysis showed that both T and B memory cells, as well as plasma cells, are enriched in the CD-affected mesentery, as compared with tissue from control subjects. Immunohistochemistry revealed that these cells are concentrated within the creeping fat of CD patients, in the vicinity of transmural lesions; that T and B cells are compartmentalized in clearly distinct areas; that they are supplied by post-capillary high endothelial venules and drained by lymphatic vessels indicating that these nodules are fully mature TLOs.

Organ culture showed that mesenteric tissue samples from CD patients contained greater amounts of adipocyte-derived chemokines and the use of the conditioned medium from these cultures in functional assays was able to actively recruit T and B lymphocytes. Finally, the production of chemokines involved in TLO formation by 3T3-L1 adipocytes was directly elicited by a combination of TNF-α and LPS in vitro.

We therefore propose a mechanism in which mesenteric adipocyte, through their production of key chemokines in response to inflammatory/bacterial stimuli, may orchestrate the formation of functional TLOs developing in CD-affected mesentery.

INTRODUCTION

Crohn's disease (CD) is a chronic inflammatory disease of the digestive tract, characterized by destructive lesions of variable topography, depth, and size.

Interestingly, CD-related intestinal lesions are characterized by a hypertrophy of the adjoining mesenteric adipose tissue, a process called "sclerolipomatosis" or "creeping fat" [START_REF] Kruis | Bacterial translocation -impact on the adipocyte compartment[END_REF]. This process is extremely specific to CD and is not observed in other chronic forms of enteritis. The extent of creeping fat correlates closely with transmural inflammation, fibrosis, muscularization, and stricture formation [START_REF] Sheehan | Fat-wrapping in Crohn's disease: pathological basis and relevance to surgical practice[END_REF] and is no longer considered a passive marker of intestinal inflammation, but rather a pathogenic process supporting the release of pro-adipokines [START_REF] Desreumaux | Inflammatory alterations in mesenteric adipose tissue in Crohn's disease[END_REF][START_REF] Siegmund | Mesenteric fat in Crohn's disease: the hot spot of inflammation?[END_REF] and local amplification of the inflammatory response in response to recurrent intestinal ulcerations ineluctably accompanied by bacterial translocation [START_REF] Peyrin-Biroulet | Mesenteric fat as a source of C reactive protein and as a target for bacterial translocation in Crohn's disease[END_REF].

Recent reports show that lymphoid nodules resembling tertiary lymphoid organs or TLOs are found impinged on lymphatic collecting vessels that enter and exit lymph nodes in the CD-affected mesentery [START_REF] Sura | Lymphatics, tertiary lymphoid organs and the granulomas of Crohn's disease: an immunohistochemical study[END_REF][START_REF] Randolph | Lymphoid Aggregates Remodel Lymphatic Collecting Vessels that Serve Mesenteric Lymph Nodes in Crohn Disease[END_REF]. We had previously shown that TLOs develop in the medial layer and adventitia of chronically inflamed arteries, likely due to the lymphatic drainage blockade [START_REF] Thaunat | Is defective lymphatic drainage a trigger for lymphoid neogenesis?[END_REF]. The formation of TLOs is orchestrated by the stromal cells of tissues chronically submitted to inflammatory stimuli and, in the case of atherosclerosis, we showed that the organizer role can be undertaken by arterial smooth muscle cells, in response to the local release of TNF-α. For triggering the formation of CD-associated TLOs, it is plausible that pathogen-derived stimuli might act in concert with TNF-α as blockade of the latter, alone, is not sufficient to suppress their formation in an experimental mouse model of CD.

In the present study, we demonstrate that the addition of a pathogen-derived stimulus, such as bacterial LPS to TNF-α, is able to confer adipocytes with a phenotype that resemble the one of "lymphoid tissue organizer cells", i.e. the stromal cells capable of orchestrating the formation of organized lymphoid structures. Our findings offer an explanation for the preferential development of striking fully mature TLOs within the hypertrophic, "creeping" adipose tissue that characterizes CD mesentery.

MATERIAL AND METHODS

2.1.

Selection of patients and tissue collection

This prospective and monocentric cohort study was performed on resection specimens of ileum from patients (n= 8) operated for CD or from controls (n= 6) operated for right colon adenocarcinoma. All patients provided informed written consent, and the protocol was approved by a French ethics committee (Comité d'Evaluation de l'Ethique des projets de Recherche Biomédicale Paris Nord: N° 2018-014).

2.2.

Cell and tissue analyses

Human tissues were characterized by immunohistochemistry. Cell suspensions were analyzed by flow cytometry. Please refer to the online-only Supplementary Data for the detailed methods.

Chemotaxis experiment

Chemotaxis assays were performed using polycarbonate filter with 5 µm pores (Neuro Probe). Please refer to the online-only Supplementary Data for the detailed methods.

Immunodetection of chemokines

Chemokine content was analyzed using the BioPlex® assay (Bio-Rad), as described in the online-only Supplementary Data.

Gene expression analysis

Total RNA was analyzed by real-time polymerase chain reaction, as described in the online-only Supplementary Data.

Statistical analysis

The means, SEMs and p values were calculated using the software JMP®. The differences between conditions were considered statistically significant for the probability value <0.05 using Student t-tests and Mann-Whitney non-parametric tests, as appropriate.

RESULTS

Fully mature TLOs develop in CD mesenteric tissues

Lymphoid nodules were detected in resected mesenteric tissues from CD patients, but not in control tissues (Supplementary Figure 1). These nodules were mostly located under the subsereous mesentery, adjacent to the deep muscle layer, sometimes they were found deeper in the mesentery, in which case they formed along the vascular peduncle. The nodule density was particularly high in the regions of the intestine where transmuscular damages were manifest. These nodules were composed of CD3 + T cells surrounding CD20 + B cell clusters, with a structural organization reminiscent of that of B cell follicle and T cell areas in SLOs (Figure 1). Additionally, the mesenteric lymphoid structures were irrigated by CD31 + blood vessels and were drained by a gp38 + lymphatic network (Figure 1). Of note, the lymphoid nodules were not surrounded by continuous gp38+ lymphatic capsules, and therefore, are not SLOs. Some of the blood vessels feeding the lymphoid structures were layered with PNAd + endothelial cells displaying a cuboidal shape, demonstrating the presence of high endothelial venules, which are known to be mandatory for the recruitment of naïve and memory cells within SLOs [START_REF] Ruddle | High Endothelial Venules and Lymphatic Vessels in Tertiary Lymphoid Organs: Characteristics, Functions, and Regulation[END_REF].

These observations were supported by flow cytometry analysis performed on cell suspensions obtained after enzymatic digestion of the human mesenteric tissues where we found that the mesentery from CD patients was enriched in leukocytes, especially T CD3 + and B CD19 + lymphocytes, and neutrophils, compared with mesentery from control subjects (Figure 2). These data strongly suggest that the lymphoid structures present in the mesentery of CD patients display key features of TLOs, and their spatial organization indicates a high degree of maturation.

Functional germinal centers are present in CD mesenteric tissues

To further analyze the composition of these lymphoid structures, we searched for the presence of cells known to support the function of germinal centers. Located within the B cell compartment of mesenteric TLOs, we found dense networks of follicular dendritic cells (FDCs) identified by their expression of CD21 (Figure 3), CD23, CD35, SSTR2A and Podoplanin (Supplementary Figure 2). In SLOs, FDCs are keys cells of the germinal center reaction since they can capture and present antigens in their native forms to B cells during the selection of high affinity clones. The presence of such cells therefore suggested that mesenteric TLOs are highly mature and that they can support the local maturation, proliferation, and selection of B lymphocytes (Figure 3). This hypothesis was confirmed by the immunodetection of CD20 + AID + , of CD20 + Bcl6 + , and of CD20 + PCNA + B cells (Figure 4). Indeed, the fact that B cells expressed AID (Activationinduced cytidine deaminase, the enzyme mediating) is the demonstration that these cells are ongoing class switch recombination and somatic hypermutation of immunoglobulin genes [START_REF] Muramatsu | Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme[END_REF] BCL6 gene expression is tightly regulated during mature B cell differentiation. The presence of BCL6 protein in B cells indicate that they are at the germinal center stage of their differentiation (ref: [START_REF] Basso | Roles of BCL6 in normal and transformed germinal center B cells[END_REF]). The detection of CD20+

expressing PCNA (Proliferating cell nuclear antigen) in the TLOs is the demonstration that these B cells are undergoing proliferation. An extended flow cytometry analysis revealed an enrichment of CD19 + IgD -CD27 + memory B cells, CD19 + IgD -CD38 + plasma cells, as well as CD4 + CD45RO + memory and CD3 + CD4 + CD127 + CXCR5 + follicular helper T cells in the mesentery of CD patients compared with the mesentery of the controls (Figure 5; please see Supplementary Figure 3 for the full gating strategy), thereby further showing that TLOs sustain local germinal center reactions. CD4 + CD127 -CD25 + T regulatory cells were also detected, indicating that immune responses taking place in CD's mesenteric TLOs might be, at least partially, controlled.

The mesenteric adipose tissue as the organizer of TLOs

As shown in Figure 6A, the mesenteric adipose tissue is dramatically hypertrophied in CD patients. Importantly, TLOs were located within this tissue, in close proximity with the muscular layer of the intestinal wall, with a high prevalence where transmuscular damages were observed (Figure 6B). They were also observed around the vascular peduncles spanning the mesentery. The almost systematic embedding of TLOs into the adipose tissue suggested that chemokines able to attract T and B cells were produced within this tissue. Indeed, the expression of CCL19 (P<0.05), CCL20 (trend), CCL21

(trend), CXCL13 (trend), and CXCL16 (P<0.05) chemokines in mesenteric tissues adipocytes was higher in tissue from CD patients as compared to control intestinal samples (Figure 7A). In functional chemotaxis assays, we found that soluble mediators released by CD mesenteric pieces (conditioned-media) could specifically trigger the recruitment of human CD19 + B cells, compared with that of conditioned-media prepared from control tissues (Figure 7B).

Interestingly, the chemokine-producing cells were distributed according to specific patterns, as detected by immunohistochemistry (Supplementary Figure 4). CXCL16 and CCL20 chemokines were preferentially found in the T lymphocyte zone, while CCL21 and CXCL13 chemokines were restricted to the B cell area. CCL19 chemokine was found to be equally distributed in the two zones. These observations suggest that CXCL16, CCL20, CCL21, and CXCL13 play a central role in the establishment/maintenance of the B cell/T cell compartmentalization.

Strikingly, we also found that adipose cells proximal to the TLOs were strongly positive for the CCL20 and CXCL13 chemokine (Figure 8A), which also colocalized with intracellular actin staining (Supplementary Figure 5), thereby indicating that these stromal cells participate to the chemoattraction of B and T cells. This could explain why the mesenteric adipose tissue in CD is favorable for the development of TLOs.

Additionally, this suggested that adipose cells could participate in the process of mesenteric TLO formation.

TNF-α and LPS trigger the recruitment of immune cells by mesenteric adipocytes

As mentioned above, the mesenteric territory wherein TLOs develop is anatomically close to deep ulcerations and fistulae in the inflamed intestine of CD patients. Therefore, the mesenteric tissue of CD patients is in contact both with inflammatory molecules such as TNF-α (which is increased in the serum of patients [START_REF] Maeda | Serum tumor necrosis factor activity in inflammatory bowel disease[END_REF]), and with bacterial components such as LPS. Of note, during lymphoid (neo)genesis, TNF-α confers stromal cells with a LTo potential [START_REF] Guedj | M1 macrophages act as LTbetaR-independent lymphoid tissue inducer cells during atherosclerosis-related lymphoid neogenesis[END_REF][START_REF] Guedj | Inflammatory micro-environmental cues of human atherothrombotic arteries confer to vascular smooth muscle cells the capacity to trigger lymphoid neogenesis[END_REF].

We therefore tested whether TNF-α and/or LPS could trigger the expression of chemokines in adipocytes. The expression of CCL19, CCL21, CXCL13, CCL20 and CXCL16 was analyzed by RT-qPCR in murine 3T3-L1-adipocytes, stimulated or not by TNF-α or LPS. We found that TNF-α strongly induced the expression of all but one (CCL21) chemokine (Figure 8B). Besides, LPS induced a strong expression of CCL21 and CCL19.

These results were corroborated by the measurement of the chemokines in the cell culture supernatants, except for CCL21 for which we failed to detect increased protein levels, regardless of the culture condition (Figure 8C). The combination of LPS and TNFα is therefore expected to be optimal to confer adipocytes with a full LTo cell potential.

Finally, we found that the supernatant of 3T3-L1 cells stimulated with TNF-α and LPS induced the recruitment of several subpopulations of T and B cells, while the supernatant of unstimulated cells did not have such an effect (Figure 8D).

DISCUSSION

Herein, we report that the mesenteric tissue of CD patients is enriched in memory lymphocytes and plasma cells, and hosts TLOs. CD mesenteric TLOs displayed compartmentalized B and T cell areas that were irrigated by blood vessels layered by PNAd + endothelial cells displaying a cuboidal shape. These vessels are likely high endothelial venules. PNAd + high endothelial venules are specialized for the recruitment of CD62L+ lymphocytes in SLOs (besides spleen) and in the thymus. They likely play the same role in TLOs for the regulation of the entry of naïve and memory lymphocytes.

Mesenteric TLOs in CD patients also displayed a network of CD21 + FDCs, indicating ongoing local B cell responses. Indeed, FDCs have the capacity to capture and to coat their surface with antigens on which B cells can test their B cell receptors during the germinal center reaction through which the selection of high affinity B cell clones occurs [START_REF] De Silva | Dynamics of B cells in germinal centres[END_REF].

We next tried to decipher why TLOs preferentially developed in the creeping fat of the mesentery [START_REF] Randolph | Lymphoid Aggregates Remodel Lymphatic Collecting Vessels that Serve Mesenteric Lymph Nodes in Crohn Disease[END_REF], a tissue that is a deeply remodeled with a massive adipose tissue expansion in CD patients. The extent of this creeping fat (or "sclerolipomatosis") is such that it guides the surgeon in delineating the intestinal segment that requires resection.

Our data suggest that the localization of the TLOs in this tissue is due to the local high concentration of CCL19, CCL20, CCL21, CXCL13, and CXCL16 chemokines, likley produced by the adipocytes in the vicinity of TLOs, as well as by the cells comprised within the TLOs themselves, further sustaining their implantation.

Thus, similar to what occurs in chronically inflamed arteries [START_REF] Guedj | M1 macrophages act as LTbetaR-independent lymphoid tissue inducer cells during atherosclerosis-related lymphoid neogenesis[END_REF][START_REF] Guedj | Inflammatory micro-environmental cues of human atherothrombotic arteries confer to vascular smooth muscle cells the capacity to trigger lymphoid neogenesis[END_REF][START_REF] Grabner | Lymphotoxin beta receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE-/-mice[END_REF][START_REF] Lotzer | Mouse aorta smooth muscle cells differentiate into lymphoid tissue organizer-like cells on combined tumor necrosis factor receptor-1/lymphotoxin beta-receptor NF-kappaB signaling[END_REF], where the most abundant stromal cells, the vascular smooth muscle cell, adopt the function of TLO organizer cells, in the sclerolipomatous tissue of CD patients where TLOs are embedded, the most abundant stromal cell of the mesenteric fat, adipocytes appear able to orchestrate the development of fully mature TLOs. The highly plastic adipose cells were already known to be able to produce vast amounts of a large panel of cytokines [START_REF] Fantuzzi | Adipose tissue, adipokines, and inflammation[END_REF]. It is not surprising that they can also produce chemokines.

While our vitro experiments demonstrate that adipocytes can produce these chemokines, we recognize that the immunodetection of chemokines in adipocytes from human CD mesenteric tissue is not a definitive demonstration that they do so since these cells could have taken up these chemokines. Further investigations are required in order to rule out whether different stromal cells, other that adipocytes, could also be endowed with lymphoid tissue-organizer capacities in CD mesenteric tissue.

Stimulation with TNF-α alone however does not seem to be sufficient to trigger the organizer cell function of adipocytes, the addition of LPS was necessary in order to produce the full panel of chemokines required to recruit the different cell populations that form TLOs. This finding may explain why CD-associated TLO develop close to the sites of lesions of the intestinal barrier, where bacterial-derived inflammatory stimuli can act in a synergistic manner with the TNF released by the damaged tissue to trigger the production of homeostatic chemokines by the local adipocytes.

CD is not the sole intestinal pathological condition associated with an alteration of the intestinal barrier. Indeed, the translocation of bacterial components can be observed in other enteritis such as radiation enteritis, tuberculosis enteritis and ischemic enteritis.

Thus, the "mesenteric inflammatory environment" is not specific to CD. At variance, the sclerolipomatosis is pathognomonic of CD. The presence of the wrapping fat subjected in an inflammatory environment triggered by an altered epithelial barrier would therefore constitute the key combination for the development of TLOs specifically in the mesentery of CD patients.

Concerning the impact of mesenteric TLOs, whether they are solely protective structures restraining the bacterial entry across the damaged intestinal epithelium or instead play a role in the perpetuation of the intestinal damage will require further studies. It is worth mentioning that immune responses mounted in TLOs develop in the inflamed tissue and outside of controlled lymphoid organs. As a consequence, stochastic and potentially harmful immune responses emerge from TLOs [START_REF] Thaunat | Immune responses elicited in tertiary lymphoid tissues display distinctive features[END_REF]. We however found Tregs cells the mesentery of CD patients indicating that the local immune responses may be regulated to some extent. This regulation might be insufficient and TLOs could play deleterious role in CD since we found that the mesenteric lymphoid structures were associated with a more severe phenotype of CD (a study that is currently submitted).

As a perspective to our findings, monitoring the formation and/or the expansion of TLOs in longitudinal studies (by cellular/molecular imaging of B cells or other TLO-specific molecules) should be considered for clinical staging of CD. Finally, targeting the formation of TLOs and/or the function of the immune effectors generated locally appears as a new promising strategy to impact on the pathologic process of CD. An additional way to impact on mesenteric TLOs is the resection of the mesentery support tissue. Indeed, it was recently shown that removing the mesentery during ileocolic resection in CD patients reduced post-operative recurrence [START_REF] Coffey | Inclusion of the Mesentery in Ileocolic Resection for Crohn's Disease is Associated With Reduced Surgical Recurrence[END_REF]. This lends support to our idea that the mesentery and the immune response that it hosts play a crucial role in the pathophysiology of CD.

Figure 1 :

 1 Figure 1: B cell follicles develop in the mesentery of human CD

Figure 2 :

 2 Figure 2: Increased inflammatory infiltrate in the mesentery of CD

Figure 3 :

 3 Figure 3: CD-associated lymphoid structures contain germinal centers

Figure 4 :

 4 Figure 4: CD-associated GCs present key features of selection, maturation

Figure 5 :

 5 Figure 5: Specific leukocyte populations in the mesentery of CD patients

Figure 6 :

 6 Figure 6: Mesenteric TLOs are systematically embedded in hypertrophic

Figure 7 :

 7 Figure 7: The mesentery in CD produces specific chemokines capable of

Figure 8 :

 8 Figure 8: Adipocytes play a central role in mesenteric lymphoid neogenesis

ACKNOWLEDGMENT

This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Assistance Publique-Hôpitaux de Paris (AP-HP) and by the MSD Avenir fondation.


Nicolas Fabresse

Islam Amine Larabi

Isabelle Etting

Charlotte Mayer

Philippe Charlier

Geoffroy Lorin De

La A Grandmaison

Jean-Claude Alvarez
email: jean-claude.alvarez@aphp.fr

Geoffroy Lorin De

Jean-Claude Pr

Alvarez

Identification of toxics in adipocere: Two case reports

Keywords: adipocere, postmortem changes, toxicological analysis

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

Introduction

Adipocere (or "grave wax") is a grayish to white crumbly wax-like and water-insoluble organic material formed by the anaerobic bacterial hydrolysis of fat (i.e. saponification) in human (especially women, infants and overweighed individuals) and some animal tissues [START_REF] Rabl | Postmortem changes after 50 years immersion (1939 reconstructed suicide)[END_REF][START_REF] Manulik | Modern concepts of adipocere formation mechanisms[END_REF][START_REF] Fiedler | Decomposition of buried corpses, with special reference to the formation of adipocere[END_REF][START_REF] Forbes | The identification of adipocere in grave soils[END_REF][START_REF] Forbes | A preliminary investigation of the stages of adipocere formation[END_REF]. When appearing, putrefaction is replaced by a permanent and stable firm cast of fatty tissues and face, and, in some cases, of internal organs. It is mostly composed of saturated fatty acids (palmitic, myristic and stearic acids) over unsaturated ones, with variation of fatty acid composition between different samples attributed to their age and the burial conditions [START_REF] Jaggers | The effects of soil environment on postmortem interval: a macroscopic analysis[END_REF][START_REF] Nushida | Adipocere formation via hydrogenation of linoleic acid in a victim kept under dry concealment[END_REF]. Some environmental conditions help the formation of such a substance: absence of oxygen, old and humid place [START_REF] Fründ | Quantification of adipocere degradation with and without access to oxygen and to the living soil[END_REF]. A role of bacteria such as Clostridium perfringens, or Micrococcus luteus has been suspected [START_REF] Takatori | Investigations on the mechanism of adipocere formation and its relation to other biochemical reactions[END_REF][START_REF] O'brien | Waxing grave about adipocere: soft tissue change in an aquatic context[END_REF]. Various examples have been described in bodies deposed in shallow, moist, clay, soiled grave or thrown into pools or submerged in water [START_REF] Kahana | Marine taphonomy: adipocere formation in a series of bodies recovered from a single shipwreck[END_REF][START_REF] Fiedler | Characterization of soils containing adipocere[END_REF][START_REF] Pakosh | Soft tissue decomposition of submerged, dismembered pig limbs enclosed in plastic bags[END_REF]. Coffins retard the rate at which adipocere forms and clothes enhance its formation [START_REF] Forbes | The effect of the method of burial on adipocere formation[END_REF]. Variations in the amount of sodium, potassium, calcium, and magnesium between individuals may also explain differences of formation delay and composition [START_REF] Notter | The initial changes of fat deposits during the decomposition of human and pig remains[END_REF].

Even if some exceptional cases have been described (minimal delay of 3 days only), a minimal delay of a month seems necessary between death and the early beginning of adipocere transformation [START_REF] Kumar | Early adipocere formation: a case report and review of literature[END_REF]. Determination of postmortem interval (PMI) has been proposed by some authors using the rhythm of adipocere formation [START_REF] Rothschild | Adipocere--problems in estimating the length of time since death[END_REF][START_REF] Yan | Preliminary quantitative investigation of postmortem adipocere formation[END_REF]. Sections of organs or tissues may be conserved within these adipocere formations, such as vascular sections; indeed a case of atherosclerosis diagnosis was carried out on an exhumed body of a 40-year-old man from the macroscopic and microscopic analysis of coronaries and aorta [START_REF] Ambade | Atherosclerosis: a medicolegal tool in exhumed decomposed bodies[END_REF].

Gunshot or strangulation wounds may be long-term conserved with bodies completely converted into adipocere, making the retrospective diagnosis possible [START_REF] Kobayashi | A « keyhole lesion » gunshot wound in an adipocere case[END_REF][START_REF] Singh | Fact Speaking Blood Vessel A Case Report on Ligature Strangulation[END_REF].

To the best of our knowledge, very few examples of forensic toxicological analysis carried out on adipocere samples have been published. We report two forensic cases of death with adipocere formation where toxicological analyzes were performed on adipose material.

Material and method

a. Adipocere treatment A 10 mg/L of lipase solution (LS) was prepared in phosphate buffer 0.1 M (NaH2PO4, pH 7.4) with 4 mg/mL of lauryl sulfate. Ten mg of adipocere sample were weighed and 500 µL of LS were then added, the mixture was thoroughly mixed and incubated 16 h at 50°C. b. Liver treatment Liver samples (5 g) were homogenized in deionized water (5 mL). Final quantification of each sample was based on an appropriate dilution in deionized water if necessary.

c. GC-MS and LC-DAD screening

Adipocere samples were analyzed with our routine methods. Briefly, 500 µL of adipocere lysate were extracted two times, once after addition of 1 mL of hydrochloric acid 0.2 M and once after addition of 1 mL of 0.2 M sodium hydroxide. Samples were then extracted by liquid-liquid extraction (LLE) with 4 mL of diethylether/dichloromethane/hexane/isoamylic alcohol (250/150/100/2.5; v/v/v/v). Extracts were then pooled and evaporated. The dry residue was reconstituted and injected into the GC-MS system or LC-DAD.

d. LC-MS/MS

Samples were analyzed with a sensitive method devoted to drug facilitated sexual assault cases, allowing the detection of psychotropic substances. Two hundred µL of carbonate buffer (1 M, pH 9.7) were added to 200 µL of adipocere lysate. Liquid-liquid extraction (LLE) was realized with 2 mL of hexane/ethyl acetate (1:1/v:v), mixed during 15 min and centrifuged at 3500 rpm for 10 min. The organic layer was then transferred into a glass tube and evaporated.

The dry residue was solved in 100 µL of 2 mM ammonium formate in 0.1% formic acid and 10 µL were injected into the system. Chromatography was performed on a Thermo-Finnigan Surveyor high-performance liquid chromatography (HPLC) system (Thermo Fisher, Les Ulis, France), with an autosampler injector using a 1.9 µm HypersilGold PFP column (100 mm x 2.1 mm i.d.; Thermofisher, Les Ulis, France). The device was completed with a pre-column (HypersilGold PFP, 1.9 µm, 10 mm x 2.1 mm i.d., Thermofisher, Les Ulis, France). The elution gradient was achieved as follow: 80% A (ammonium formate 2 mM, 0.1% formic acid) and 20% B (acetonitrile, 0.1% formic acid), linear gradient to 90% of B in 10 min, held for 2 min. The column re-equilibration was performed with linear gradient to 20% A in 0.5 min, held for 2.5 min. The flow rate was set at 300 µL.min e. Barbiturates assay Two hundred and fifty µL of sodium acetate 0.2 M (pH 6) were added to 100 µL of adipocere lysate or liver homogenate. LLE was performed with 150 µL of hexane, mixed 30s and centrifuged 5 min. Fifty µL of the supernatant were transferred into a vial and 1 µL was injected into the GC-MS system. Chromatography was performed on a Focus Gas Chromatography (Thermofisher, Les Ulis, France) with an auto sampler TriPlus (Thermofisher, Les Ulis, France). The device was coupled with a column UB5P (30 m x 0.25 mm x 0.25 µm, Uptibond, Interchim, Montluçon, France). The temperature gradient was achieved as follow: 80°C for 0.5 min, linear gradient to 160°C at 30°C/min, to 220°C at 5°C/min and to 290°C at 40°C/min held for 1 min. The gas flow rate (helium) was set at 16 mL.min -1 . Ions were detected with a DSQ II (Thermofisher, Les Ulis, France) mass spectrometer in selective ion monitoring (SIM) mode. Phenobarbital quantification m/z was 232 and confirmation 260 and 175. Heptabarbital was used as internal standard. The linearity range was 0.5-50 µg/g.

Cases report a. Case report 1

The adipocerous body of a 39-year-old woman was accidentally found in a river 12 years after she committed suicide by drowning in her car. Adipocere was present in the following anatomic sites: foot, legs, thighs, buttocks and inferior part of the trunk (Figure 1). All other parts of the body were skeletonized or absent. The identification was possible according to clothes, identity documents and personal items accompanying the rests. No traumatic lesion or arguments against the hypothesis of a suicide were found.

Toxicological analyses were carried out on adipocere. Diazepam, nordazepam, and flunitrazepam were detected at 2.1 ng/g, 1.8 ng/g and 1.2 ng/g, respectively.

b. Case report 2

The body of a 50-year-old man was accidentally discovered by a farmer harvesting a field plot 24 days after he disappeared. A box of Gardenal® (phenobarbital) was found on the corpse. The identification of the body was made possible by a scar at the forehead and a femoral prosthesis. The individual was diagnosed with an end-stage throat cancer. Given the state of advanced decomposition of the body and the absence of blood, toxicological analyzes were performed on liver and adipocere present on the abdominal part of the corpse. It revealed the presence of phenobarbital at 26 µg/g and 59 µg/g, respectively. The phenobarbital ion chromatogram corresponding to the adipocere sample is presented in figure 2.

Discussion

Forensic toxicologists can be faced to special cases, where the advanced stage of decomposition of corpses leave very few matrices of interest to explore the pre-mortem exposure of the deceased. In particular conditions of temperature, humidity and pH, the transformation of soft tissue into adipocere makes it possible to preserve a tissue for very long periods. The record for adipocere persistence is probably the Tyrolean Iceman (3350 -3100 BC) [START_REF] Hess | Histological investigations on the Tyrolean Ice Man[END_REF]. The analysis of adipocere is made difficult by the lipidic composition of the matrix, which is similar to soap. The method applied in our cases is derived from a previously published method for amiodarone quantification in adipose tissue [START_REF] Lafuente-Lafuente | Amiodarone concentrations in plasma and fat tissue during chronic treatment and related toxicity[END_REF]. Lipase allows a complete degradation of fat before LLE. In case 1, adipocere analysis highlighted the presence of three benzodiazepines (diazepam, nordazepam and flunitrazepam) with low concentrations. Despite detection and quantification of these substances, interpretation remains difficult due to the lack of data regarding the evolution of drug concentration in adipocere, concentrations should be very different from blood or serum at the moment of death.

In case 2, we found phenobarbital in the liver and adipocere; liver concentration was in the therapeutic values range [START_REF] Sakata | Distribution of antiepileptic drugs in human blood and tissue[END_REF]. Levine et al. evaluated the stability of phenobarbital in blood and liver at 25°C and at 4°C during 3 months and greater than 75% of the drug was recovered [START_REF] Levine | Postmortem stability of barbiturates in blood and tissues[END_REF]. In our case, the concentration found 24 days after death should be close to the initial amount, and probably not the cause of death. However, it is interesting to see that liver and adipocere concentrations are of the same order of magnitude.

Physicochemical properties (stability and lipophilicity) probably condition the ability of some molecules to bind to adipose tissue and persist for a long period; the compounds found were either acidic or lipophilic.

We find very few studies in the current literature where a toxicological analysis was done on adipocere. Inoue et al. reported the identification of toluene in the adipoceratous body of a 24year-old man found dead in a car drown in a river for about 3 months (the presence of diatom in lung, liver and kidney suggested that death was caused by drowning) [START_REF] Inoue | Detection of toluene in an adipoceratous body[END_REF]. The cadaver was almost completely adipoceratous and autopsy findings revealed that there were neither remarkable injuries nor lethal diseases. Toluene, ethanol, 1-propanol, 2-propanol, 1-butanol, dimethyl sulfide, dimethyl disulfide, isovaleraldehyde and n-butyl n-butyrate were detected in the specimens collected at the autopsy by head-space gas chromatography/mass spectrometry (GC/MS). The toluene concentrations were 31.0 µg/g in brain, 10.6 µg/g in liver, 5.4 µg/g in kidney, 15.0 µg/g in skeletal muscle and 187.1 µg/g in adipose tissue. The individual was a painter; these results suggested that he had been sniffing paint thinner.

Grellner W. reported the case of a 31-year-old man found dead in a forest nearly 3 years after death [START_REF] Grellner | Possibilities for toxicologic evidence in adipocere formation and postmortem interval of several years[END_REF]. The residual soft tissue was completely transformed into adipocere. Toxicological analysis was performed on adipoceratous samples and revealed the presence of two heavy metal (cadmium: 0.30 µg/g; thallium: 0.91 µg/g) and three drugs with minor levels (phenobarbital: 0.32 µg/g; nordazepam: 140 ng/g; salicylic acid: 0.04 µg/g). Few details about the analytical method are given. The cause and manner of death were supposed to be a suicidal intoxication to cadmium sulfate and thallium sulfate found on the cadaver. Nishimura et al. reported a case of an adipocere body found in the sea after suicide by drowning 5 months ago. The brain was almost completely converted into adipocere.

Toxicological analyses were performed on femoral muscle, brain, liver and kidney by gas chromatography. Nicotine and cotinine were detected in all tissues and brain concentrations were 54.3 and 414 ng/g, respectively [START_REF] Nishimura | Forensic implication of muscle level of nicotine in an adipocere body found in the sea[END_REF]. The authors compared their results with previous cases and conclude that the concentrations found were high, close to those found in suicidal smokers suggesting a suicidal death.

Conclusion

The adipocere is formed under very particular environmental conditions. Once formed, adipocere can persist for very long period. A toxicological analysis carried out on adipocere can make it possible to explore the pre-mortem exposure of the deceased even if advanced adipocere formation with a longer post-mortem interval is present. The interpretation of quantitative results remains difficult given the lack of data in the literature.

Conflict of interest : none

  -1 . The compounds were detected by a Thermo-Finnigan LCQ Advantage ion trap MS (Thermofisher, Les Ulis, France) equipped with an ESI source set in the positive ionization mode. Data were collected either in full-scan mode at m/z 100-400 or in full scan MS-MS mode. Parent and confirmation m/z were respectively: 271.5 and 243.1 for nordazepam (collision energy CE = 42); 285.2 and 257.1 for diazepam (CE = 40), 314.2 and 267.1 for flunitrazepam (CE = 41). The linearity range was 1-500 ng/g. Nordazepam-D5 was used as internal standard.

Figure 1 .

 1 Figure 1. (A) Bones of the legs and pelvis with the remains of clothes; (B) Adipocere

Figure 2 .

 2 Figure 2. Phenobarbital ion chromatogram of a blank sample (A) and an adipocere sample


Pauline Infantes

MD Matthieu Mezzadri

MD Rea Lo Dico
email: rea.lo-dico@aphp.fr

MD Rea Lo Dico

Surgical technique: external iliac (or ilio-obturator) lymph node dissection via laparotomy

INTRODUCTION:

The external iliac lymphatic chain is the initial lymph node pathway station for cancer cell metastases from gynecological cancer.

Consequently, external iliac lymph node dissection (EILND) is indicated in several carcinologic settings.

For cervical cancer, pelvic lymph node staging requires EILND or sentinel lymph node (SLN) biopsy via laparoscopy, usually associated with frozen section pathology examination, for early stage disease: I-A (with emboli) to I-B1). If the SLN biopsy is negative, an extended hysterectomy is indicated. If the SLN biopsy is positive, lumbo-aortic lymph node dissection (LALND) is performed, usually via laparoscopy without hysterectomy. Thus, management is akin to that for locally advanced cancer. For locally advanced stages: I-B2 to IV-A), laparoscopic lumbo-aortic staging without EILND is indicated through a trans-or retroperitoneal approach, before concomitant radiochemotherapy. EILND is performed only if there is bulky lymphadenopathy, with the goal of improving the outcome of subsequent radiation therapy.

For endometrial cancer [START_REF] Colombo | ESMO-ESGO-ESTRO Consensus Conference on Endometrial Cancer: diagnosis, treatment and follow-up[END_REF], three prognostic groups have been defined based on pre-operative findings. For "low risk" cancers (endometroid grades 1-2, stage I-A), EILND is not recommended. For "intermediate risk" cancers (endometroid grade 3, stage I-A, grades 1-2, and stage I-B), experienced groups perform SLN biopsy. LALND without EILND is performed if the SLN is positive. Lastly, for "high risk" cancers (endometroid grade 3, stage I-B and stage ≥ II and non-endometroid cancer), LND is always indicated and associates SLN biopsy and EILND via laparoscopy or laparotomy.

In vulvar cancer [START_REF] Le | The definition of optimal inguinal femoral nodal dissection in the management of vulva squamous cell carcinoma[END_REF], EILND is only rarely indicated.

Lastly, for ovarian carcinoma [START_REF] Querleu | European Society of Gynaecological Oncology (ESGO) Guidelines for Ovarian Cancer Surgery[END_REF], EILND is theoretically indicated for all stages, and is performed according to the degree of resectability and operability of the patient. Laparoscopy is possible in early stage cancer.

In case of advanced stage (70% of ovarian cancers are stage IIIC at diagnosis), EILND and LALND are performed via laparotomy in association with cytoreduction of peritoneal tumor.

FIG 1)

Regional anatomy

There are three external iliac lymph node groups:

FIG 2) PATIENT POSITION AND INCISION

The patient is positioned supine, in slight Tredelenburg, with both arms extended and legs apart (double team approach). A bladder catheter is inserted.

A low midline (infra-umbilical) incision is made in the rare circumstances where EILND alone is performed. Otherwise, the midline incision is supra and infra-umbilical (cytoreductive surgery or associated LALND).

FIG 3) PERITONEAL APPROACH

The umbilical artery is identified first (Fig. 3a)

The peritoneum is incised laterally between the lumbo-ovarian pedicle and the external iliac vessels (Fig. 3b), extending from the paracolic gutter (Fig. 3c) to the round ligament of the uterus, which is then divided (Fig. 3d).

FIG 4) URETEROLYSIS

The ureter is identified at the level of the iliac artery bifurcation. The left ureter (Fig. 4a) usually crosses the external iliac artery at the level of the bifurcation whereas the right ureter (Fig. 4b) crosses the iliac artery slightly distal to the bifurcation. The ureter is then dissected free all along its pelvic course (Fig. 4c). A vascular tape placed around the ureter can help, but is by no means routinely necessary.

The umbilical artery is then grasped and dissected up to its origin (Fig. 4d).

Lymph node dissection is pursued by opening the paravesical space down to the levator ani muscles (Fig. 4e).

FIG 5) EXCISION OF THE SUPRA-ARTERIAL AND PRE-VENOUS

CHAIN

To accomplish this, the sheath of the external iliac is grasped on its anterior aspect in order to delicately pull on the supra-arterial lymph node package (Fig. 5a). Dissection is pursued by skeletonization of the artery from its bifurcation to the origin of the previously divided round ligament (Fig. 5b).

The dissection is continued along the lateral aspect of the external iliac artery posteriorly to the psoas muscle and also pursued in the space between the external iliac artery and vein, to extract the prevenous lymphatic chain (Fig. 5c).

FIG 6) DISSECTION AND EXCISION OF THE INFRA-VENOUS CHAIN

Dissection is pursued along the medial aspect of the external iliac vein and then beneath the vein down to the internal obturator muscle laterally and the inguinal ligament inferiorly (Fig. 6a). A vein retractor is placed under the external iliac vein (Fig. 6b) to expose the subvenous tissues and reach the lateral limit of the dissection at the level of the obturator muscle. The obturator nerve marks the deep extent of the dissection (Fig. 6c). Once all the elements have been identified, the lymphatic tissue is separated from it paravesical attachments and mobilized superiorly and anteriorly along the posteriorly-located obturator nerve up to the iliac bifurcation. This is performed with Dubost type scissors, opened just slightly, pushing the dissected lymph node package over the obturator nerve, by a bottom-to-top sweeping movement (likened to barber-style hair cutting).

External iliac lymph node dissection is then finished. Routine pelvic drainage is not recommended because this can enhance the onset or persistence of lymphoceles.

  -lateral: supra-arterial external iliac lymph node chain located between the psoas muscle and the external iliac artery (1) -middle (inconstant): pre-venous external iliac lymph node chain between the external iliac artery and vein (2) -medial: sub-venous external iliac lymph node chain located between the external iliac vein and obturator muscle (3) The anatomic limits of lymph node dissection are: -superior: iliac bifurcation (1a) -inferior: inguinal ligament or Cooper's ligament (1b) -lateral: internal obturator muscle (1c) -medial: umbilical artery (1d) -deep: obturator nerve (1e)