Fabienne Dédaldéchamp
email: fabienne.dedaldechamp@univ-poitiers.fr

Christelle Moyen

Pierrette Fleurat-Lessard

Gabriel Roblin

Characteristic early membrane effects induced by tryptophan in pulvinar motor cells

Keywords: amino acids, Cassia fasciculata, membrane potential, Mimosa pudica, nasties, osmocontraction, pulvinus, tryptamine, tryptophan Ind, indole, OHTrp, 5-hydroxytryptophan, PMVs, plasma membrane vesicles, Ser, serine, Try, tryptamine, Trp, tryptophan

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

Introduction

The amino acid tryptophan (Trp) plays a primary role in all living organisms, from bacteria and fungi to plants and animals, by being an essential amino acid required for protein synthesis. In addition, Trp intervenes in the synthesis of many pivotal compounds. In animals which are incapable of synthesizing this amino acid, Trp after being ingested as residues within proteins is used for the synthesis of serotonin, melatonin and vitamin nicotinic acid [START_REF] Moffett | Tryptophan and the immune response[END_REF]. In plants, Trp plays a direct role in regulating plant development by providing precursors for the multiple pathways of synthesis of the hormone indole-3-acetic acid (IAA) that controls numerous physiological processes [START_REF] Woodward | A receptor for auxin[END_REF].

Furthermore, Trp has been shown to assist in plant tolerance of heavy metals. Thus, the Trp content increased in Noccaea caerulescens treated with increasing concentrations of Cd [START_REF] Zemanova | The significance of methionine, histidine and tryptophan in plant response and adaptation to cadmium stress[END_REF], resulting in reduced Cd availability to the plants, decreased Cd transport and reduced Cd accumulation [START_REF] Sanjaya | Overexpression of Arabidopsis thaliana tryptophan synthase beta 1 (AtTSB1) in Arabidopsis and tomato confers tolerance to cadmium stress[END_REF]. In addition, enzymes involved in Trp synthesis were induced under different kinds of stresses such as amino acid starvation, the oxidative stress-inducing herbicide acifluorfen and the abiotic elicitor α-amino butyric acid [START_REF] Zhao | Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor[END_REF].

Plants also utilize Trp as a precursor in the synthesis of a large variety of secondary metabolites, known to intervene in pathogen defense responses such as serotonin [START_REF] Ishihara | The tryptophan pathway is involved in the defense responses of rice against pathogenic infection via serotonin production[END_REF], phytoalexins (particularly camalexin), glucosinolates and indole-derived alkaloids [START_REF] Radwanski | Tryptophan biosynthesis and metabolism: biochemical and molecular genetics[END_REF][START_REF] Frerigmann | Regulation of pathogentriggered tryptophan metabolism in Arabidopsis thaliana by MYB transcription factors and indole glucosinolate conversion products[END_REF].

However, Trp has been shown to affect per se various long-term physiological processes in plants as shown by its effects observed on leaf growth and cell expansion in Arabidopsis [START_REF] Jing | Tryptophan deficiency affects organ growth by retarding cell expansion in Arabidopsis[END_REF].

In addition to its action on these types of processes involving gene regulation, Trp, similar to other amino acids, induces short-term cellular events, as evidenced by its triggering of rapid ionic fluxes during their uptake through the plasma membrane mediated by proton-coupled symports (Delrot et al., 2001 and references therein).

The aim of this work was to show whether, compared to other natural amino acids, Trp triggers characteristic early events at the membrane level, and in particular, noteworthy modifications of the bioelectrical transmembrane potential and disturbances of proton fluxes. For this purpose, the motor cells of pulvini of Mimosa pudica and Cassia fasciculata were chosen because the kinetics, energetics and some of the reaction steps of the stress-induced turgor driven leaf movements are now well documented [START_REF] Samejima | Changes in the extracellular ion concentration in the main pulvinus of Mimosa pudica during rapid movement and recovery[END_REF][START_REF] Moran | Osmoregulation of leaf motor cells[END_REF]. The reaction of the primary pulvini of M. pudica following a shock consists of a rapid spectacular drooping movement of the leaf in approximately 2 s, and the recovery takes 15-20 min [START_REF] Volkov | Mimosa pudica: electrical and mechanical stimulation of plant movements[END_REF]. Leaflet movements can be induced in M. pudica and C. fasciculata by transferring plants from light to darkness or from darkness to light. As the result of the coordinated contraction of the pulvinar cells, the motor reactions are driven by rapid turgor changes mainly associated with K + and Cl -fluxes. In plant cells, K + fluxes have been shown to be coupled to reverse H + fluxes driven by the regulation of the activity of a proton-pumping ATPase located at the plasma membrane [START_REF] Serrano | Structure and function of plasma membrane ATPase[END_REF]. Thus, attention has been paid on the way Trp may have a direct effect on the H + -ATPase using purified plasma membrane vesicles (PMVs) isolated from pulvinar tissues of M. pudica, which allow such an experimental approach.

The results obtained regarding Trp action on these different biological events are discussed in light of the comparative effects observed following treatments with 5-hydroxytryptophan (OHTrp) and by compounds linked either to Trp synthesis, namely, serine (Ser) and indole (Ind) [START_REF] Kriechbaumer | Characterisation of the tryptophan synthase alpha subunit in maize[END_REF] or to Trp catabolism, namely, tryptamine (Try) [START_REF] Kang | Enzymatic features of serotonin biosynthesis enzymes and serotonin biosynthesis in plants[END_REF]. It can be concluded that, among amino acids, Trp triggers specific actions on early ionic exchanges at the plasma membrane level and may serve as a signal modulating more long-lasting physiological processes triggered in plants by environmental changes.

Materials and methods

2.1.Plant growth conditions

Seeds of Mimosa pudica L. and Cassia fasciculata Michx. were germinated in an organic compost. Seedlings and older plants were grown in the compost watered daily and were kept in climate-controlled chambers at 27 ± 0.5 °C and 65 ± 5% relative humidity. Illumination was regulated to give 16 h of light (photophase 06:00-22:00 hours) provided by fluorescent tubes (mixing Osram fluora and Osram daylight types) giving a photon flux density (400-800 nm) of 80 µmol m -2 s -1 at the plant apex.

2.2.Electrophysiological measurements

The particular anatomy of the primary pulvinus of M. pudica characterized by many layers of parenchyma cells surrounding the central cylinder allows impalement of a microelectrode in a well-defined kind of cell. The transmembrane potential was measured by the classical electrophysiological method using glass microelectrodes (tip diameter < 1 µm, tip resistance from 5 to 30 MΩ) drawn from capillaries provided with an internal microfibre (GC 150F15; Clark Electromedical Instruments). Briefly, a leaf was excised from the stem of 2-month-old plants and the pulvinus fixed to the bottom of a 4 ml plexiglass chamber filled with a buffered medium (10 mM MES, pH 5.5) containing 1 mM NaCl, 0.1 mM KCl, and 0.1 mM CaCl2. The microelectrode was impaled into a motor cell of the abaxial ("extensor") half of the organ. Under these conditions, the calculated value of the resting transmembrane potential (Ψ0) from 33 assays was -118 ± 4 mV (SEM). For details, see [START_REF] Saeedi | Early events induced by salicylic acid in motor cells of Mimosa pudica pulvinus[END_REF] and references therein.

2.3.Measurement of pH variations

To record H + excretion, primary pulvini (400 mg) of 2-month-old M. pudica plants generally bearing 10 leaves were excised at 10:00 hours. The organs were divided in transverse sections and treated following the same procedure previously described [START_REF] Saeedi | Early events induced by salicylic acid in motor cells of Mimosa pudica pulvinus[END_REF]: an incubation medium composed of 0.50 mM CaCl2, 0.25 mM MgCl2, and variations of pH read on a pH-meter provided with combined electrodes (Futura micro-combination, Beckman Coulter) and linked to a potentiometric recorder. Compounds were added 3 hours after the beginning of the experiments when the pH decrease in the bathing medium reached a plateau. To quantify the amount of mobilized protons, titration was performed with NaOH or HCl at 5 x 10 -3 M onto a 2 ml incubation medium aliquot collected 3 h after application of the various tested compounds. The experiments were repeated at least 3 times.

2.4.Preparation and use of plasma membrane vesicles (PMVs)

Purified PMVs were prepared by phase partitioning of microsomal fractions from M. pudica primary pulvini. PMVs were frozen in liquid nitrogen and stored at -80 °C. The PMVs were put in the inside-out configuration by adding 0.05% Brij in the assay medium. In the 4 batches of vesicles used in this work, treatment with the plasma membrane H + -ATPase inhibitor sodium orthovanadate at 0.25 mM showed that 60% of the enzyme activity can be attributed to plasma membrane functioning.

Vanadate-sensitive ATPase activity of the PMVs was measured in a medium buffered with 50 mM MES/Tris at pH 6.5. Proton pumping was followed by the decrease of 9-aminoacridine absorbance at 495 nm in a medium buffered with 10 mM MES/Tris at pH 6.5. The reaction was started by the addition of 3 mM MgSO4 into the medium. For details, see [START_REF] Saeedi | Early events induced by salicylic acid in motor cells of Mimosa pudica pulvinus[END_REF].

2.5.Experimental procedure for observation of pulvinar movements

Plantlets of M. pudica bearing the first fully developed leaf were excised at 10:00 hours by cutting the hypocotyl 3 cm below the cotyledons and then were dipped in distilled water contained in small plastic tubes to allow for recovery from harvesting. Leaf position determined by the angle formed by the petiole and the line prolonging the hypocotyl was measured using a transparent protractor provided with a movable pointer, allowing angle measurement by homothety preventing therefore leaves from mechanical touch. At 14:00 hours (middle of the photophase), the distilled water was replaced by the tested solution buffered at pH 5.5 with 2.5 mM MES. After measurement of the initial angle (αi), the pulvini were stimulated by a shock at 1-h intervals for 4 h. Motor reactions were monitored by measuring the variation of the angle (∆α) within 10 s after the stimulation. The experiments were repeated 3 times on ten seedlings.

Fully expanded leaves of C. fasciculata were excised at 09:00 hours from 2month-old plants bearing 12 leaves. The cut petioles were dipped in distilled water to recover. Leaves were then transferred to test solutions buffered with 2.5 mM MES at pH 5.5 (except in particular assays dealing with pH action) for durations extending from 5 to 180 min and were exposed to darkness in the middle of the photophase of the photoperiodic cycle or in the light in the middle of the nyctophase (in this case, see additional data S2). At the end of the incubation period, the petiole tips were rinsed with buffer solution and the leaves were transferred to 2.5 mM MES adjusted at pH 5.5. The pulvinar movements induced by the variation in the light regime were monitored by measuring the distance between the leaflet tips with a calliper square which prevents disturbing of the leaf position by a mechanical touch. This linear measurement was then converted into angular values according to the formula d/2 = L/2 sin σ/2 in which σ was the calculated angle, d was the distance measured between the leaflet tips and L the maximum distance measured when the leaflets were fully opened (σ = 180 degrees). The curves represent the mean values obtained from the sum of at least three separate experiments on 8 individual leaves.

2.6.Uptake and metabolism of tryptophan

The experiments dealing with 1 mM labelled Trp uptake were carried out on the excised plantlets of M. pudica as described above. The uptake of [ 3 H] Trp (final specific activity of the solution 8 kBq ml -1 ) was measured by sectioning primary pulvini from the leaves with a razor blade at regular intervals. The excised organs were weighed and digested in a cocktail of 30% hydrogen peroxide, 70% perchloric acid and Triton X100 (2:1:1, v/v/v) for 24h at 55 °C. Radioactivity was then counted after addition of a scintillation medium (Ecolite, ICN) by liquid scintillation spectrometry (Packard Instruments). The experiments were repeated at least three times on 10 seedlings, so that each experimental point represents observations on 30 motor organs.

In a specific assay, a set of pulvini was harvested after 2 h of treatment in the labelled Trp, before being washed twice in 30 ml cold Trp-free buffered medium.

The samples were ground by mortar and pestle with 1 g quartz in 1 ml distilled water. After centrifugation, the residue was extracted again with medium and the combined extracts were vacuum evaporated. Thin-layer chromatography was performed to identify the Trp on Merck silica gel 60F254 plates (2 mm thick) developed with butanol / acetic acid / H2O (60:20:20, v/v/v). In these conditions, the RF value of the Trp visualized under UV light (254 nm) was 0.66. A 50-µl volume of the labelled extract was deposited onto a plate. After migration, the plate was dried and 0.5-cm-high silica gel areas were scraped off along the lane and placed into scintillation vials with 4 ml Ecolyte for counting.

Data analysis

Data were statistically analyzed according to Student-Fisher t test. Depending whether the number of sample (n) was above or below 20, data were respectively given as mean ± standard deviation (SD) and mean ± standard error (SE) in the figures and tables. A significant level of P < 0.05 was selected for determination of statistical significance.

2.8.Chemicals

All chemicals were purchased from Sigma-Aldrich Chimie (St-Quentin-Fallavier, France). Solutions of Trp, OHTrp, Try, and Ser were prepared at 10 mM final concentration and diluted in 2.5 mM HEPES (pKa 3.5 and 7.5) for experiments conducted at pH 4.5 and 7.5, and in 2.5 mM MES (pKa 6.1) for experiments conducted at pH 5.5 and 6.5. The pH of the solutions was adjusted with either 0.1 M KOH or 0.1 M HCl. Control assays have shown that buffering the media did not affect the pulvinar movements. Ind at 1 mM final concentration was dissolved in absolute ethanol, the final concentration of solvent being 0.3%. A stock solution of Fusicoccin (FC) prepared at 1 mM was dissolved in 7% final concentration of methanol. It has been verified that the respective solvent concentration assayed in controls did not affect the studied processes.

Results

3.1.Effect of tryptophan on the transmembrane potential of motor cells

The addition of Trp to the bathing medium of M. pudica motor cells at pH 5.5 induced a long-lasting hyperpolarization of the motor cell membrane in a concentration-dependent manner from 0.05 (threshold value) to 1 mM. Fig. 1A presents typical recordings of the time course of the bioelectrical modifications: the lag period lasted some minutes and the subsequent rise of potential reached a plateau within 20-30 min after application of the compound. Additional quantitative data are given in Table 1. The effect of Trp completely differed from that of OHTrp (Fig. 1A) and serine (Table 1) both of which induced a depolarization indicating that the effect of Trp is specific compared to that of the other tested amino acids. FC at 10 µM strongly hyperpolarized the cell membrane, ensuring the bioelectrical responsiveness of the impaled cell.

3.2.Effect of Tryptophan on the H + -ATPase activity

In a previous study, it was shown that slices of primary pulvini spontaneously acidified their incubation medium as a result of the FC-activated proton pump activity [START_REF] Saeedi | Early events induced by salicylic acid in motor cells of Mimosa pudica pulvinus[END_REF]. Fig. 1B shows the time course of the pH variation monitored in the bathing medium of the motor organ tissues after addition of the tested compounds. In the presence of Trp, the time course monitored in the controls was modified and displayed a biphasic pattern. First, Trp promoted a transient concentration-dependent acidification of the medium after a latency of approximately 5 min in the range 0.1 -1 mM, lasting from 0.5 h for 0.1 mM to 1.5 h for 1 mM. Then, in the next phase, it induced a dose-dependent rise in pH lasting for 2-3 h. By contrast, 1 mM OHTrp and 1 mM Ser both induced a sharp rise of pH observed some min after application which reached an optimum level after 2.5 h for OHTrp and 1 h for Ser, before decreasing back to the level of the control in about 5 h for OHTrp and 2 h for Ser. Another pattern was seen with 1 mM Try and 1 mM Ind as they both induced a continuous pH decrease in the bathing medium starting approximately 15 min after application of the products and lasting 3 h for Try and 5 h for Ind (Fig. 1B).

Additional data quantified the amount of mobilized proton following the various treatments (Table 1).

We further investigated whether a close relationship could be drawn between the H + fluxes monitored previously and a direct effect on the functioning of the plasma membrane H + -ATPase activity. In this goal, additional experiments were conducted on PMVs purified from pulvinar tissues in which proton pump assays and determination of ATPase catalytic activity were carried out. As seen in Table 2, Trp did not significantly modify both components of the proton motive force showing the same behavior as Ser and OHTrp. By contrast, Try strongly inhibited the proton pumping but did not modify the catalytic activity, presenting, therefore, the characteristics of a protonophore. Ind showed a vanadate-like behavior with particular characteristics. Indeed, Ind inhibited more strongly proton pumping (86% vs. 60%) but inhibited ATPase activity with a lower efficiency (37% vs. 66%).

3.3.Characteristics of the inhibition of the osmocontractile reaction by tryptophan

The seismonastic reaction of the M. pudica primary pulvinus was gradually inhibited in a dose-dependent manner for Trp concentrations higher than 1 mM (Fig. 1C). The effect was clearly seen one hour after the Trp addition and developed over the course of the experiment. After 2 h of Trp action, the inhibition was 6% at 1 mM, 18% at 5 mM and 35% at 10 mM. The time course of Trp effect can be closely paralleled with the amount of the labelled product measured in the motor organs (Fig. 1C). The Trp uptake inhibition (77%) obtained following a treatment with 10 µM of the protonophore CCCP argues for an active Trp uptake mechanism driven by a H + gradient-dependent process and supports the hypothesis of a Trp-H + symport in motor cells. TLC analysis using [ 3 H] labelled Trp indicated that after 2 h of treatment, 97% of the radioactivity was recovered at the Rf of the Trp migration spot, indicating that Trp metabolism occurred at a very low extent in these experimental conditions. Furthermore, the fact that the Trp-induced decrease of angle variation (∆α) resulted from an incomplete leaf sinking and not from an incomplete leaf recovery (since the pulvini returned to their initial position after the stimulation) argues for a restriction of the K + efflux, while the K + influx was not affected. OHTrp showed no significant effect whereas Try and Ind both strongly inhibited the reaction (25 and 64% after a 2 h treatment, respectively). Of note, Ind drastically hindered the recovery as measured by the increasing αi (inset in Fig. 1D).

As shown in Fig. 2A, Trp disturbed the dark-induced motor reactions of C. fasciculata leaflets. The handling of this model is simpler than that of M. pudica because of its low seismonastic sensitivity. The induced closure was inhibited in a dose-dependent manner at concentrations higher than 0.1 mM. After 2 h of Trp action, the inhibition was 6% at 0.1 mM, 26% at 0.5 mM, 50% at 1 mM and 78% at 5 mM. It should be stressed that the Cassia model is more sensitive (approximately 5 times) than the primary pulvini of M. pudica. To determine the influence of treatment duration, 1 mM Trp was applied for 5, 15, 30, 60, and 180 min before the onset of darkness. Fig. 2B shows that a treatment as short as 5 min induced a significant hindering of the reaction. This inhibitory effect increased with treatment duration being complete after 30 min. Applied at different pH in buffered media, 1 mM Trp inhibited the dark-induced reaction at all the pH values tested but the inhibition was well-marked towards alkaline pH values (Fig. 2C). Nevertheless, the choice of pH 5.5 in 2.5 mM MES was thought to be the most suitable in our experiments as it was near the physiological apoplastic pH. Data in Fig. 2D show that the L-or Dconfiguration of the molecule did not influence the reaction. By contrast, a modification of the structure by the introduction of an OH-group abolished the efficiency. Try and Ind both showed an inhibitory effect in the same range of concentrations (from 0.1 to 1 mM).

As shown in the histograms of Fig. 2E, the Trp inhibition induced on darkinduced reactions was noteworthy compared to other amino acids. While most of the amino acids tested did not influence the leaflet closure, it must be noted, however, that arginine, cysteine, phenylalanine and serine induced a low but significant inhibition when applied for 3h in this assay.

Discussion

4.1.Tryptophan as a multifunctional component in the plant cell life

Trp is an essential aromatic amino acid since it is one of the building blocks for protein synthesis and the source of many important compounds, particularly melatonin and nicotinamide adenine dinucleotide in animals [START_REF] Moffett | Tryptophan and the immune response[END_REF] and IAA and phytoalexins in plants [START_REF] Zhao | Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor[END_REF]. In plants, Trp has been shown to affect various long-term physiological processes such as organ growth [START_REF] Jing | Tryptophan deficiency affects organ growth by retarding cell expansion in Arabidopsis[END_REF], responses to stress and pathogen elicitors [START_REF] Zhao | Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor[END_REF][START_REF] Bednarek | A glucosinolate metabolism pathway in living plant cells mediates broad-spectrum antifungal defense[END_REF] and tolerance of heavy metals [START_REF] Sanjaya | Overexpression of Arabidopsis thaliana tryptophan synthase beta 1 (AtTSB1) in Arabidopsis and tomato confers tolerance to cadmium stress[END_REF].

Nevertheless, little has been reported about the mechanism by which Trp affects growth and development at the cellular and molecular level.

In this work, we focused our investigations on the early events triggered by Trp on plant cell functioning. Thus, we determined that Trp is rapidly recognized at the cell membrane level and induced characteristic and specific short-term cellular events by disturbing processes involved in rapid changes of membrane permeability to water and ions which are the early processes allowing osmocontractile pulvinar reactions.

4.2.Tryptophan as a putative signal molecule

The proposal that Trp is rapidly recognized at the plasma membrane level is greatly supported by the observation of the large concentration-dependent membrane hyperpolarization triggered a few minutes after application of the compound, indicating early modifications in ionic fluxes. Ion fluxes through the plasma membrane are common and early events reported as a prerequisite for subsequent cell reaction [START_REF] Colcombet | R-type anion channel activation is an essential step for ROS-dependent innate immune response in Arabidopsis suspension cells[END_REF]and references therein). The early recognition of Trp at the membrane level can be analyzed according to three phases.

(a) The occurrence of a lag period indicates that the recognition of the compound at the membrane triggers a chain reaction requiring a lapse of time to express the bioelectrical response and suggests therefore that Trp does not act directly on particular membrane effectors. A complementary study should now be carried out to identify the steps triggered before the onset of the hyperpolarization with a particular focus on the possible involvement of a Ca 2+ signaling process. Indeed, Ca 2+ is considered to be a ubiquitous internal second messenger that regulates various cellular processes acting through intracellular [Ca 2+ ] increase in response to various environmental stimuli [START_REF] Knight | Calcium signaling during abiotic stress in plants[END_REF] or following recognition of elicitors [START_REF] Lecourieux | Analysis and effects of cytosolic free calcium elevation in response to elicitors in Nicotiana plumbaginifolia cells[END_REF].

(b) The membrane hyperpolarization could result either from cation effluxes or anion influxes. However, close lag times in ∆Ψ and ∆pH variations (Table 1) and similar time courses of the bioelectrical and the pH variations noted in the bathing medium of pulvini strongly suggest that proton efflux may be the main process sustaining the hyperpolarization (Fig. 1). However, it can be concluded from experiments performed on PMVs that Trp did not increase the activity of the H + -ATPase by directly acting on the pump (Table 2) when compared to FC that also hyperpolarized the pulvinar cells and activated the H + -ATPase activity in PMVs [START_REF] Moyen | Membrane effect of 2,4dichlorophenoxyacetic acid in motor cells of Mimosa pudica L[END_REF]. This outcome clearly demonstrated different modes of action for these two compounds. Obviously, the membrane potential variations triggered by Trp strongly differed from those evoked by OHTrp (Fig. 1) and glycine [START_REF] Roblin | Cysteine: a multifaceted amino acid involved in signalling, plant resistance and antifungal development[END_REF]) on M. pudica motor cells and by various amino acids on Lemna gibba cells [START_REF] Fisher | Membrane potential changes related to glycine transport in Lemna gibba G1[END_REF] and on barley mesophyll cells [START_REF] Felle | Systemic signalling in barley through action potentials[END_REF]. In these latter cases, potential variations appear as depolarization spikes after a latency period reduced to seconds and followed by the return to the basal potential in approximately 30 min. [START_REF] Felle | Systemic signalling in barley through action potentials[END_REF] monitored the temporal sequence of the ionic migrations showing that Ca 2+ was the first ion to move and was required to trigger the subsequent Cl -and K + fluxes. As shown in Fig. 1B, the time course of the proton translocation appears complex possibly because of a dual role of Trp. In the first minutes following its application, Trp induced a continuous H + efflux from cells reminding of an auxin-like effect, as observed with IAA [START_REF] Bourbouloux | Effects of jasmonic acid on motor cell physiology in Mimosa pudica L. and Cassia fasciculata Michx[END_REF] and, in a second step, the observed H + influx may be related to its uptake (Fig. 1C) via a H + /substrate cotransport mechanism evidenced in amino acid transport by plant cells [START_REF] Delrot | Plasma membrane transporters: a machinery for uptake of organic solutes and stress resistance[END_REF]. The recorded curve represents the resultant of the bidirectional proton migration (See additional data S1).

(c) By comparing the time course of bioelectrical variations and proton fluxes, it should be stressed that both the plateau and the repolarization phase did not coincide with the beginning of the pH increase recorded in the bathing medium, suggesting that H + influx into the cells may not be the only driving component involved in this latter process. Considering the ionic species (Cl -and K + ) underlying the action potential [START_REF] Samejima | Changes in the extracellular ion concentration in the main pulvinus of Mimosa pudica during rapid movement and recovery[END_REF], it can be expected that these pivotal ions may also contribute to the early bioelectrical modifications triggered by Trp. The time course of pH variations also evidenced that H + fluxes were disturbed in a long-lasting manner (for hours) and, could therefore be considered to be involved in the regulation of long-term processes. In this direction, H + fluxes were suggested to be involved in elicitation processes [START_REF] Schaller | Modulation of plasma membrane H + -ATPase activity differentially activates wound and pathogen defense responses in tomato plants[END_REF] and cytosolic acidification to act as a second messenger, mediating MAP kinase activation in the response of plant cells to various stresses [START_REF] Tena | Cytosolic acidification but not auxin at physiological concentration is an activator of MAP kinases in tobacco cells[END_REF].

By considering its electrogenic properties shown here, Trp may constitute an intermediary in long distance signaling by inducing an electrical stimulus through its transport over long distance in plant as other amino acids. Thus, Trp may create a direct cell-to-cell electrical conduction following its accumulation and therefore may be implicated in plant resistance processes. Indeed, action potentials or woundinduced potential variations are able to transmit information over long distances [START_REF] Fromm | Electrical signals and their physiological significance in plants[END_REF] and to induce the expression of pathogen response genes [START_REF] Fisahn | Analysis of the transient increase in cytosolic Ca 2+ during the action potential of higher plants with high temporal resolution: requirement of Ca 2+ transients for induction of jasmonic acid biosynthesis and PINII gene expression[END_REF]. However, Trp is unlikely to be directly involved in the Hypersensitive Response as revealed by the assay of [START_REF] Ueno | Increase tryptophan decarboxylase and monoamine oxidase activities induce Sekiguchi lesion formation in rice infected with Magnaporthe grisea[END_REF] showing that following infection with Magnaporthe grisea, H2O2 formation and subsequent lesions on the leaves were due to an accumulation of Try resulting from increased Trp synthesis.

4.3.Tryptophan as an osmotic agent

The data obtained on the osmocontractile reaction of pulvinar cells (Fig. 1,2) indicated that the application of Trp modified bulk ionic migration and the associated water movement by considering the well documented mechanism driving motor cell shrinkage following the application of shock and darkness. Large K + and Cl -effluxes [START_REF] Samejima | Changes in the extracellular ion concentration in the main pulvinus of Mimosa pudica during rapid movement and recovery[END_REF][START_REF] Moran | Osmoregulation of leaf motor cells[END_REF] induced a rapid decrease in cell turgor.

The ionic migrations are likely regulated by the activity of K + and Cl -channels and the subsequent water movement allowed by the activity of aquaporins present in motor cell membranes [START_REF] Fleurat-Lessard | Distribution and activity of the plasma membrane H + -ATPase in Mimosa pudica L. in relation to ionic fluxes and leaf movements[END_REF][START_REF] Temmei | Water channel activities in Mimosa pudica plasma membrane intrinsic proteins are regulated by direct interaction and phosphorylation[END_REF][START_REF] Moran | Osmoregulation of leaf motor cells[END_REF]. As seen in Fig. 1C, there is a close relationship between the extent of inhibition of the motor reaction of M. pudica pulvini and the amount of Trp available in the vicinity of the motor cells. The fact that the sole drooping movement was affected but not the recovery argues for a process involving a vectorial water influx, resulting from the hindering of the K + efflux. This hypothesis is corroborated by the Trp-induced inhibitory effect on the dark-induced leaflet movement of C. fasciculata, (Fig. 2A) and by the Trp-promoting effect observed on the light-induced leaflet movement (See Supplementary data 2). However, it should be stressed that there is no direct link between motile reaction (and the underlying K + and water fluxes) and the process of Trp uptake, since the uptake of amino acids is stimulated at an acidic pH (Otsiogo-Oyabi and [START_REF] Otsiogo-Oyabi | Uptake of glycine by excised pulvini of Mimosa pudica in relation to proton pump activity[END_REF]; and reference therein) whereas leaflet movement was promoted at neutral/alkaline pH levels (Fig. 2C). These remarks concerning an increase in water availability in the cells are in accordance with some data showing that Trp increased the water content of wheat and maize cells and reduced the effect of drought in maize [START_REF] Rao | Role of foliar application of salicylic acid and L-tryptophan in drought tolerance of maize[END_REF]and references therein).

4.4.The effects induced by tryptophan are specific in several aspects

Many previous studies have claimed that the physiological responses seen after an exogenous application of Trp may result from the conversion of Trp into IAA [START_REF] Abdel-Monem | The role of tryptophan and Prozac (5-hydroxytryptophan) on the growth, some biochemical aspects and yield of two sunflower cultivars grown in saline soil[END_REF]and references therein). However, other works have also shown that Trp exerts a direct effect on plant cell physiology in processes as different as embryo germination in Triticum aestivum [START_REF] Morris | Identification of Ltryptophan as an endogenous inhibitor of embryo germination in white wheat[END_REF] and on cell expansion in Arabidopsis mutants [START_REF] Jing | Tryptophan deficiency affects organ growth by retarding cell expansion in Arabidopsis[END_REF]. A major point to ascertain in this study was that Trp acted itself and that the effects may not be attributed to products of its metabolism, in particular to the possible synthesis of IAA. On the motor cell model, Trp induced similar physiological effects as IAA molecules regarding the induction of membrane hyperpolarization, the inhibitory effect on the shock-induced reaction (unpublished result), the inhibitory effect on the dark-induced reaction and the promoting effect on the light-induced reaction (Everat- [START_REF] Everat-Bourbouloux | Comparative effects of indole-3-acetic acid, gibberellic acid and benzylaminopurine on the dark-and light-induced pulvinar movements in Cassia fasciculata[END_REF]. It should be emphasized that Trp is less effective than IAA, since 1 mM of the amino acid was required to trigger reactions of similar amplitude as those obtained with 10 µM IAA. However, Trp triggers a complex biphasic change in H + fluxes as previously described (Fig. 1B) in contrast with IAA and FC which continuously acidified the bathing medium of motor organs [START_REF] Bourbouloux | Effects of jasmonic acid on motor cell physiology in Mimosa pudica L. and Cassia fasciculata Michx[END_REF]. This pattern of Trp responses looks similar to those obtained with 2,4-D. Indeed, the herbicide also induced at first a pH acidification followed by a pH rise in relation to its phenolic structure [START_REF] Moyen | Membrane effect of 2,4dichlorophenoxyacetic acid in motor cells of Mimosa pudica L[END_REF], indicating that the proton exchanges are modulated according to the effect triggered by each moiety of the molecular complex. The fact that Trp is the active component was first supported by the short latency (within min) occurring between the Trp application and the observation of the cell responses which tended to rule out a putative effect of a compound resulting from the metabolism of Trp by the motor cells. Such eventuality was disproven with the use of [ 3 H] labelled Trp combined with TLC analysis, which evidenced a very low rate of metabolism during the first 2 h of Trp uptake by the tissues.

We have also characterized the specific effect of Trp by including in our study the effects of compounds involved in its synthesis (Ind and Ser) and its metabolism (Try). Clearly, we have shown that these compounds acted through mechanisms which are fundamentally different from those underlying Trp effects, although they can result in the same final osmoregulated reaction of the pulvinar cells. Thus, we showed that Try acted on membrane functioning through a protonophore effect and that Ind acted directly on both processes of the H + -ATPase activity as observed on PMVs. In previous reports, we showed that disturbing the H + -ATPase functioning resulted in modification of the motile reactions [START_REF] Saeedi | Early events induced by salicylic acid in motor cells of Mimosa pudica pulvinus[END_REF]. However, an additional effect has to be considered, since a sole protonophore action would induce a pH alkalinisation in the bathing medium of motor tissues as observed with dinitrophenol [START_REF] Saeedi | Early events induced by salicylic acid in motor cells of Mimosa pudica pulvinus[END_REF]. In contrast, Try induced a continuous acidification of the medium, thus possibly meaning that Try induced a modification of the membrane permeability leading to a leakage of protons. This continuous H + efflux may be one step in a chain reaction leading to a necrotic effect linked to H2O2 formation as observed in rice infected with the fungus Magnoporthe grisea [START_REF] Ueno | Increase tryptophan decarboxylase and monoamine oxidase activities induce Sekiguchi lesion formation in rice infected with Magnaporthe grisea[END_REF]. In the case of Ind, stronger ionic disorders have to be considered as indicated by the also unexpected medium acidification and by the observation of the large gradual increase in initial angle in course of the assay (see inset in Fig. 1D).

These last data may indicate that Ind drastically disturbed cell functioning and led to a necrosis of the pulvinar cells upon increased treatment duration that requires further microscopic observations to identify the wounded cell compartments.

It can further be highlighted that, compared to many other amino acids, Trp affects in a specific way the electrophysiological events, the proton exchange status and more generally the ionic migrations as indicated by the data on the stimulusinduced motile reactions. Of note, the Trp-induced membrane potential changes strongly differed from those induced by the structurally related compound OHTrp which triggered a spiky depolarization of the motor cell membrane identical to that evoked by glycine application [START_REF] Roblin | Cysteine: a multifaceted amino acid involved in signalling, plant resistance and antifungal development[END_REF]2018). Furthermore, differences were also apparent in the shock-and dark-induced reactions: Trp strongly inhibited these reactions while glycine promoted them [START_REF] Otsiogo-Oyabi | Effects of glycine on dark-and light-induced pulvinar movements and modification of proton fluxes in the pulvinus of Mimosa pudica during glycine uptake[END_REF].

Interestingly, arginine, cysteine, phenylalanine and Ser also inhibited the darkinduced reactions in C. fasciculata leaflets when observed after 3 hours of action (Fig. 2E). The case of Ser is interesting since this amino acid combines with Ind to form Trp [START_REF] Kriechbaumer | Characterisation of the tryptophan synthase alpha subunit in maize[END_REF], so that we can suppose that the effect observed 4 h after the application on the leaves could result from Trp synthesis. Altogether, these data prompted us to investigate further whether these particular amino acids may play particular roles in the plant cell functioning. In this way, a recent study concerning cysteine has confirmed that this amino acid also induced characteristic effects on the processes studied here [START_REF] Roblin | Cysteine: a multifaceted amino acid involved in signalling, plant resistance and antifungal development[END_REF].

Conclusions

The data highlight that some amino acids may have specific roles outside of a simply metabolic role as demonstrated by the characteristic properties evoked by Trp in plant cell physiology. Further experiments should be devoted to the underlying mechanisms of these non-metabolic effects, since some actions of Trp did not correspond to a classical mode of action as compared to other amino acids. In particular, the mechanism sustaining the variation of membrane permeability to water and ions should be investigated further, as it does not result from an activation of the proton pump activity, raising the hypothesis of a direct action on ionic Table 1. Effect of tryptophan (Trp) at various concentrations, 5-hydroxytryptophan (OHTrp) at 1 mM and serine (Ser) at 1 mM on the characteristics of the transmembrane potential of pulvinar motor cells and on the characteristics of the proton fluxes measured in the bathing medium of excised pulvini of Mimosa pudica. Calculations of ∆Ψ and of the amount of mobilized protons were respectively made 10 min and 90 min after application of the compounds. n: number of assays. Mean ± SD. Superscript letters indicate significant differences at the 5% probability by Student-Fisher t test (b-f) or values not significantly different from the control (a). +, Hyperpolarization; -, depolarization.

Membrane potential changes

Proton fluxes

Fig. 1 .

 1 Fig. 1. Characteristics of the early events induced by tryptophan (Trp) in primary pulvini of Mimosa pudica. (A) Typical recordings of transmembrane potential variations in pulvinar parenchyma motor cells induced by tryptophan at various concentrations (0.05, 0.1, 0.5, 1 mM) and by 1 mM 5OH-tryptophan (OHTrp). Compounds were added at time 0 (black arrow) and 10 µM fusicoccin (FC) was added at 60 min (black arrowheads). The number of assays is indicated in Table 1. (B) Representative time courses of pH variations monitored in the bathing medium of pulvinar tissues induced by various compounds. In the upper part, effect of tryptophan at various concentrations (0.1, 0.5, 1 mM) and of 1 mM 5OH-tryptophan (OHTrp). In the lower part, effect of serine (Ser), tryptamine (Try) and indole (Ind) applied at 1 mM. The experiments were conducted at least three times with the same general result. (C) Time courses of the inhibition of the seismonastic pulvinar osmocontractile reaction following treatment with tryptophan at various concentrations expressed in mM (mean ± SE, n = 30) and time course of the amount of 1 mM labelled tryptophan absorbed by the pulvinar tissues from excised leaves (white symbols, dotted line) and effect of CCCP ( ) at 10 µM on the uptake (mean ± SD, n = 4). (D) Comparative time courses of the inhibition of the seismonastic pulvinar osmocontractile reaction observed after application of serine, 5OHtryptophan, tryptamine and indole (at 1 mM) (mean ± SE, n = 30). In inset, initial angle in degrees measured at the beginning of the treatment (αio) and after 4 h (αi4) (mean ± SE, n = 30). Co: control.

Fig. 2 .

 2 Fig. 2. Characteristics of the inhibitory effect induced by tryptophan on the dark-induced closure of Cassia fasciculata leaflets. (A) Modification in the time course of the closure as a function of concentration applied (from 0.1 to 5 mM) for 3 h before the darkness (black arrow). (B) Effect of 1 mM tryptophan treatment duration. (C) Effect of the pH of the incubation medium as indicated in controls (black columns) and in 1 mM tryptophan-treated leaves (white columns). (D) Effects of 1 mM, tryptophan in the L-and D-form, 1 mM 5OH-tryptophan (OH), and of indole (Ind) and tryptamine (Try) at various concentrations (a, b, c respectively 0.1, 0.5 and 1 mM). (E) Comparative effect of tryptophan with other natural amino acids (Co: control; a: tryptophan, b: alanine; c: arginine; d: asparagine; e: aspartic acid; f: cysteine; g: glutamine; h: glutamic acid; i: glycine; j: histidine; k: hydroxyproline, l: isoleucine; m: leucine; n: lysine; o:methionine; p: phenylalanine; q: proline; r: serine; s: threonine; t: tyrosine; u: valine).Otherwise indicated, the calculations were made 1 h after the start of darkness on sets of leaflets treated for 3 h with the compounds prepared in buffer at pH 5.5. Mean ± SE; n = 24. Superscript letters and asterisks in (E) indicate significant differences at 5% probability level by Student-Fisher t test (b-d) or values not significantly different from the control (a).

Table 2 .

 2 Modifications of net proton movement (measured by absorbance variation from the control absorbance at 495 nm) and vanadate-sensitive ATPase activity in plasma membrane vesicles purified from Mimosa pudica pulvini treated with tryptophan, 5-hydroxytryptophan, tryptamine, indole, serine and vanadate. Values are mean ± SD. Into brackets, percentage of inhibition (-) or activation (+). Letters following bracket indicate significant differences at the 5% probability by Student-Fisher t test (b, c) or values not significantly different from the control (a).

	Product	Concentration	Absorbance variation n Vanadate-sensitive activity n
		(mM)	(unit mg.prot -1 .min -1 )		(nmol Pi.mg prot -1 .min -1 )
	Control	0	0.35 ± 0.03 a	8	433 ± 17 a	8
	Tryptophan	1	0.34 ± 0.01 [-3] a	8	452 ± 28 [+4] a	8
	Tryptamine	1	0.05 ± 0.03 [-86] b	6	444 ± 23 [+3] a	8
	Indole	1	0.05 ± 0.02 [-86] b	8	272 ± 43 [-37] b	8
	Vanadate	0.25	0.14 ± 0.06 [-60] c	8	147 ± 25 [-66] c	8
	5OH Tryptophan 1	0.31 ± 0.02 [-11] a	4	421 ± 15 [-3] a	4
	Serine	1	0.36 ± 0.05 [+3] a	3	441 ± 13 [+2] a	4

Acknowlegments This work was supported by UMR CNRS 7267 Laboratoire EBI Ecologie et Biologie des Interactions, Equipe SEVE Sucres & Echanges Végétaux Environnement,

channels (K + channels in particular). Another pivotal line of research would be focused on the processes linked to the membrane sensing of Trp. The perception at the membrane level may be achieved through a specific receptor as shown for IAA [START_REF] Woodward | A receptor for auxin[END_REF] or may be induced through a transceptor [START_REF] Dinkeloo | Update on amino acid transporter functions and on possible amino acid sensing mechanisms in plants[END_REF] linking transport function and signaling in the cells.

Supplementary material

Supplementary data S1. Putative explanation of the complex time course of pH variations monitored in the bathing medium of pulvinar tissues of M. pudica following application of tryptophan. Supplementary data S2. Effect of 1 mM tryptophan and 1 mM serine on the lightinduced opening of Cassia fasciculata leaflets.

Author contributions

Christelle Moyen performed the electrophysiological studies, Fabienne

Dédaldéchamp participated to the assays on the metabolism experiments, Pierrette Fleurat-Lessard and Gabriel Roblin carried out the observations on leaf reactions and the analysis carried out on PMVs. All the authors participated in the writing of the manuscript and approved the final version.


Jérémie Jayet

Aurélien Vallée

Delayed Femoral Embolism of Biological Glue Following the Repair of Type a Aortic Dissection

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


MD Quentin Hennocq
email: quentin.hennocq@aphp.fr

MD Amin Bennedjaï

MD François Simon

MD PhD Sylvie Testelin

MD PhD Bernard Devauchelle

MD Jean-François Tulasne

MD Stéphanie Dakpé

Roman Hossein Khonsari

MD PhD Hossein Khonsari

Maxillofacial surgery in wartime Middle-East: Paul Tessier's missions to Iran

Keywords: war injuries, ballistic trauma, Iraq-Iran, facial reconstruction, war surgery

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

INTRODUCTION

In addition to the 200,000-600,000 casualties, more than 400,000 people -both soldiers and civilians -were injured on the Iranian side between 1980 and 1988 during the Iraq-Iran conflict [START_REF] Khosravi | Facial injuries in Iranian veterans during the Iraq-Iran war (1980-88): differences from recent conflicts[END_REF]. Figures were of the same order of magnitude on the Iraqi side [START_REF] Khosravi | Facial injuries in Iranian veterans during the Iraq-Iran war (1980-88): differences from recent conflicts[END_REF]. According to Carey (Carey, 1991), among the American armed forces in Iraq, 17.3% received head wounds, 4.3% neck wounds, 5.8% chest wounds, 9.3% abdominal wounds, and 90% extremity wounds.

Maxillo-facial injuries were frequent, as in most 20th-century conflicts. Several public health studies have noted an increase in the proportion of maxillofacial injuries in the 20th and 21st centuries in American and British armies, probably due to an improvement in chest and abdomen protection devices and the more common use of explosive weapons [START_REF] Khosravi | Facial injuries in Iranian veterans during the Iraq-Iran war (1980-88): differences from recent conflicts[END_REF].

Paul Tessier (1917Tessier ( -2008) ) was a French plastic surgeon and a pioneer of craniofacial surgery [START_REF] Wolfe | The influence of Paul Tessier on our current treatment of facial trauma, both in primary care and in the management of late sequelae[END_REF]. Tessier took part in several missions to Iran, supported by the JAO (Janbazan Affairs Organization). Jean-François Tulasne (Paris, France), David Matthews (Charlotte, USA), Joe Murray (Boston, USA), Fernando Ortiz Monasterio (Mexico City, Mexico), Yvon Raulo (Paris, France), and Luigi Clauser (Ferraro, Italy) were part of the international team of surgeons that travelled to post-war Iran in order to operate on facial injuries with Tessier.

The purpose of Tessier's missions to Iran was to provide advanced reconstructive surgery to patients with facial war injuries. Eiseman [START_REF] Eiseman | Casualty care planning[END_REF]) defined four types of war according to their spatial extent: (1) terrorist attacks, (2) rural attacks, (3) minor conventional wars, and (4) major conventional wars. The Iraq-Iran conflict was classed as a minor conventional war (type 3). Maxillofacial trauma is more common in conflicts with small spatial extent: 21% of injuries in terrorist attacks are maxillo-facial, as opposed to 15% in major conventional wars [START_REF] Dobson | Trends in maxillofacial injuries in war-time (1914-1986)[END_REF]. The face is also easily targeted by small arms and light weapons, and facial protective devices are often lacking in small conflicts.

We collected 322 files on patients with facial war injuries who were operated on by Paul Tessier in Iran from 1990 to 1993, as part of international surgical missions involving a team of prominent plastic surgeons. These files were the property of the Association Française des Chirurgiens de la Face and are part of the Tessier Collection, stored in the University Hospital of Amiens, France. We collected epidemiological parameters and details on surgical procedures.

Descriptive statistics were used to characterize the cohort, and a multivariate logistic model to assess the factors associated with severe eye injuries within all facial injuries.

METHOD

The files of 322 patients managed by Paul Tessier in Tehran, Iran, between 1990 and 1993, were collected for this study. The variables of interest were: date of assessment, age, date of trauma, gender, injury mechanism (shell fragment, bullet, missile, blast, burn/chemical burn, car accident). The facial lesions were divided into: (1) upper third skeleton -including frontal bone and orbits (uni-or bilateral enucleation); (2) middle third skeleton -including nasal bones, zygoma, maxilla and alveolar bone, maxillary sinuses, and hard palate; (3) lower third skeleton -including the mandible, the temporo-mandibular joint (TMJ); (4) occlusal disorders; and (5) soft tissue damage -including tear ducts, eyelids, nose, lips, ears, and burn scars. The number of reconstructive surgery procedures and the country in which they were performed were recorded. The number of procedures involving a bone graft and/or a flap was noted. The types of procedure were as follows: (1) bone and/or costal grafts; (2) foreign body removal; (3) dental implant placement; (4) orthognathic surgery; (5) TMJ ankylosis surgery; [START_REF] Dobson | Trends in maxillofacial injuries in war-time (1914-1986)[END_REF] uni-or bilateral canthopexy; (7) lip surgery -modeling cheiloplasty, vestibuloplasty, Abbé flap;

(8) orbital cavity refection -prosthetic rehabilitation after enucleation; and (9) local plastic procedures -local advancement or rotation flaps, Z/W/V-Y plasties, excision of hypertrophic scars, rhinoplasty, expander placement, tear duct surgery, otoplasty, and regional flapstemporal, Abbé, Baron-Tessier, scalp, other regional flaps, free flap. Among the bone and/or costal grafts, the harvest site was noted when available (iliac, frontal and/or parietal, temporal osteo-muscular flap, or costal), as well as the receiver area (frontal, including the anterior wall of the frontal sinus and the upper rim of the orbit; orbits, including the lateral and medial walls and the floor of the orbit; the nasal dorsum; the zygoma, including the lower edge of the orbit but not the orbital floor; the maxilla, including the alveolar bone, the anterior wall of the maxillary sinus and the hard palate; and, finally, the mandible. The orthognathic procedures were: Le Fort I osteostomy, sagittal split of the mandible, and genioplasty. Epithelial inlays or total skin grafts for vestibuloplasties and orbital cavity reconstructions were noted, as well as conformer placements and pseudorraphy for orbital cavity repair. Bone grafts, septoplasties, and conchal cartilage grafts associated with rhinoplasties were recorded. Complications were also considered: infection, graft pseudarthrosis, graft necrosis, bleeding, and other complications.

A multivariate logistic model was used to assess the statistical relationship between enucleation and injury mechanism in victims of shell fragment and bullet trauma. The age and gender variables were integrated into the model in order to eliminate potential confusion factors. The hypotheses of normality and homoscedasticity of the residues were tested. The parameters were compared with 0 using a Student test, and a value of p < 0.005 was defined as a statistically significant relationship. Odds ratios, with their confidence intervals, were computed using these parameters. The statistical analyses were performed using R v. 3.3.2 (R Core Team, 2016).

RESULTS

Records for 322 patients assessed by Paul Tessier between 1990 and 1993 in Tehran, Iran were collected. 305 (94.72 %) were male, 17 (5.28 %) were female (Table 1). The ages at assessment ranged from 5-67 years (average: 27.15 ± 6.97) (Supp. Fig. 1). The ages at trauma ranged from 2-62 years of age (average: 20.65 ± 7.04). Sixty-eight patients (21.12%) were under 18 years of age at the time of trauma. Trauma occurred between 1971 and 1992 (Figure 2). CT-scans were performed on 54 patients, of which 10 had 3D reconstructions.

Information on injury mechanism was available for 240 patients (74.53%): (1) shell fragments, 161 patients (50%); (2) bullets, 27 patients (8.38%); (3) burns, 21 patients (6.56%) -including three chemical burns (0.93%); (4) blasts, 15 patients (4.66%); (5) missiles, 12 patients (3.73%); and (6) road accidents, 4 patients (1.24%) (Figure 3).

The deep structures of the upper third of the face were affected in 124 patients (38.50%), compared with 72 (22.36%) for the middle third and 86 (26.71%) for the lower third (Figure 4, Supp. Fig. 2). Injuries of the upper third of the face included 13 frontal fractures (4.04%), 60 orbital fractures (18.63%), and 95 uni-or bilateral enucleations (29.50%). Injuries of the middle third of the face included 18 nasal bone fractures (5.59%), 29 zygomatic fractures (9.01%), and 35 maxillary fractures (10.87%). Injuries of the lower third of the face included 36 mandibular body fractures with pseudarthroses (11.18%), 15 TMJ ankyloses (4.66%), and 12 occlusal abnormalities (3.73%). Other more superficial lesions affected 136 patients (42.24%): 39 uni-or bilateral eyelid injuries (12.11%), 32 lip injuries (9.94%), 18 nasal mutilations (5.59%), 18 burn injuries (5.59%), eight tear duct lesions (2.48%), and three ear mutilations (0.93%).

Patients seen by Tessier and his team had previously benefited from 0-40 surgeries (average: 6.22 ± 7.25). This information was available for 211 patients (Supp. Fig. 3). Of these surgeries performed before Tessier's missions, 171 procedures had been performed in Iran, 34 in Germany, five in the UK, two in Spain and one in each of the following countries: France, Jordan, Turkey, Australia, Iraq, and Austria. During these previous procedures, 46 bone grafts had been performed, and 28 flaps (local or free flaps).

Paul Tessier himself used bone grafts extensively for facial reconstruction. A total of 175 grafts were harvested, of which 72 (41.14%) were iliac grafts, 94 (53.71%) frontal and/or parietal grafts, seven (4.00%) temporal osteomuscular flaps, and two (1.14%) rib grafts (Figure 6). A total of 198 grafts were performed (one harvested graft could be used in several sites): 13 (6.57%) grafts in the frontal area, 58 (29.29%) periorbital grafts, 26 (13.13%) nasal grafts, 28 (14.14%) zygomatic grafts, 36 (18.18%) maxillary grafts, and 37 (18.69%) mandibular grafts (Figure 6). The other procedures performed by Tessier were: 65 uni-or bilateral canthopexies and 61 orbital cavity reconstructions; 58 local flaps (advancement flaps, rotation flaps or V, V-Y, W, or Z plasty) and 53 regional or free flaps; 34 lip surgeries (commissuroplasties and/or vestibuloplasties); 28 rhinoplasties; 24 removals of foreign bodies; 22 expander placements; 19 dental implant placements; 15 orthognathic procedures, including four Le Fort I osteotomies, two sagittal splits of the mandible, and nine genioplasties; 11 TMJ ankylosis surgeries (temporal aponeurosis flap after ankylosis block resection); nine tear duct surgeries; and two otoplasties (Figure 5).

The vestibuloplasties (28) involved an epithelial inlay in six cases (21.43%) and a total skin graft in 10 cases (35.71%). Orbital cavity reconstructions used an epithelial inlay in 20 cases (32.79%) and a total skin graft in 27 cases (44.26%). An intra-orbital conformer with pseudorraphy was used in 31 patients (50.82%) and pseudorraphy alone in 47 patients (77.05%). Rhinoplasty techniques used by Tessier included 26 bone grafts on the nasal dorsum (92.86%), 14 septoplasties (50.00%), and 11 conchal grafts (39.29%) (Supp. Fig. 4). Tessier performed 53 flaps, including 22 temporal flaps (41.51%), five Abbé flaps (9.43%), seven Baron-Tessier flaps (13.21%), 14 scalp rotation flaps (26.42%), and three free flaps, including one fibula flap, one radial forearm flap, and one tubed abdominal flap (5.66%) (Supp. Fig. 5).

Complications were reported in 25 patients (7.76%): 21 infections (6.52%), four graft mobilities (1.24%), five graft necroses (1.55%), and one case of severe nasal bleeding (0.31%). A peroperative death due to an anesthetic accident was also reported (Supp. Fig. 6).

A multivariate logistic model showed that patients injured by shells were 4.039 (1.325; 17.599) more likely (p = 0.029) than patients with gunshot wounds to have uni-or bilateral enucleation, at equal age and sex (Table 2, Supp. Fig. 7). The odds ratios for age and sex were not significant, and their confidence interval included 1.

DISCUSSION

Paul Tessier has contributed to the systematization of the management of severe maxillofacial and craniofacial trauma, and his work on complex fractures and ballistic trauma is especially relevant in the context of war-time facial injuries [START_REF] Wolfe | The influence of Paul Tessier on our current treatment of facial trauma, both in primary care and in the management of late sequelae[END_REF][START_REF] Wolfe | A man from Heric. The life and work of Paul Tessier, MD, father of craniofacial surgery[END_REF]. The epidemiology of wartime maxillofacial injuries has been described by Dobson et al. [START_REF] Dobson | Trends in maxillofacial injuries in war-time (1914-1986)[END_REF]) and Eiseman [START_REF] Eiseman | Casualty care planning[END_REF]: their overall mean incidence was 16%, which is greater than values based on the ratio of the surfaces of the face to the whole body (12%). The incidence of head and neck injuries was 12% for World War I (1914-18), 9% for the Spanish War (1936-39), 4% for World War II (1939-45), 19% for the war in Northern Ireland (1968-98), and 29% for the Falklands War in 1982 [START_REF] Dobson | Trends in maxillofacial injuries in war-time (1914-1986)[END_REF]). In addition, Breeze et al. [START_REF] Breeze | Maxillofacial injuries in military personnel treated at the Royal Centre for Defence Medicine, June 2001 to December 2007[END_REF] found an increasing trend in the proportion of maxillofacial injuries from 2005 to 2007 in British military personnel. We therefore observe an increase in the incidence of maxillofacial lesions, both with time and related to a smaller spatial extent of the conflicts.

Several clinical series reporting the injury patterns of military victims of recent conflicts

are available in the literature. Sadda [START_REF] Sadda | Maxillofacial war injuries during the Iraq-Iran war: an analysis of 300 cases[END_REF] published a series of 300 Iraqi soldiers operated on for craniofacial lesions: 80.7% were shell injuries, i.e. low-velocity trauma, and 19.3% bullet injuries, i.e. high-velocity trauma. Among all craniofacial lesions, [START_REF] Sadda | Maxillofacial war injuries during the Iraq-Iran war: an analysis of 300 cases[END_REF] found 68.3 % facial fractures, including 24.3 % lesions of the middle third of the face and 40.3% mandibular fractures. These authors hypothesized that high-velocity projectiles caused limited entry point lesions and wide exit wounds, with temporary cavitation and a shock-wave effect (Al-Shawl, 1986); low-velocity projectiles caused small entry lesions with no exit wound, thus causing much less damage. These results were not in line with our findings, which showed a higher risk of enucleation after low-velocity shell trauma when compared with high-velocity gunshot wounds. Nevertheless, [START_REF] Sadda | Maxillofacial war injuries during the Iraq-Iran war: an analysis of 300 cases[END_REF] had not assessed the specific risk for enucleation and our results were not directly comparable to his. Motamedi and Behnia (Motamedi and Behnia, 1999) reported 33 cases of war injury from the Iraq-Iran war, including 70% bullet wounds and 21% shrapnel (shell) wounds, causing 15% periorbital lesions, 39% middle-third lesions, and 45% perioral damage. Reconstructions only used iliac bone grafts. Taher [START_REF] Taher | Management of weapon injuries to the craniofacial skeleton[END_REF] reported 1135 war-injured patients treated in Tehran, Iran between 1984 and 1990. In their series, 72.6% of patients had mandibular bone injuries, 36.30% of patients had injuries of the middle third of the face and 20% of patients had injuries of the upper third of the face; 29.4% of all patients had associated eye injuries. The authors reported three main types of ballistic injury:

(1) avulsive, (2) penetrating, and (3) perforating. We were able to sort the lesions in our series according to the same three categories: (1) avulsive lesions included soft tissue losses, bone defects, dental losses, and eye enucleation; (2) penetrating lesions encompassed foreign bodies and lesions without an exit orifice; (3) perforating lesions included hard and soft tissue lesions with an exit orifice. Akhlaghi et al. [START_REF] Akhlaghi | Management of maxillofacial injuries in the Iran-Iraq war[END_REF] reported the surgical management of 210 war-injured patients in Tehran, Iran between 1981 and 1986. In their series, 20.63% of patients benefited from mandibular bone grafts and 1.59% of patients required orbital reconstruction, with a complication rate of 11.43%, which was higher than the rates reported by Tessier. However, these complication rates were not comparable: Akhlaghi et al. [START_REF] Akhlaghi | Management of maxillofacial injuries in the Iran-Iraq war[END_REF] reported acute ballistic trauma cases with a high risk of postoperative infection (foreign bodies, local battlefield conditions, associated burns and blasts), whereas Tessier only practiced secondary surgery distant from the initial trauma.

A series of 43 war-injured patients operated on in Iran by Tessier had previously been reported by Simon et al. [START_REF] Simon | Lower jaw reconstruction and dental rehabilitation after war injuries: the experience of Paul Tessier in Iran in the late 1980s[END_REF], with a focus on mandibular reconstruction. In this series, Tessier had performed 24 bone grafts on 19 patients (44%), including 15 iliac grafts and four frontal-parietal grafts; 40% of the injuries were caused by shells and 17% were bullet wounds. The authors described some surgical techniques for which Paul Tessier and his team had developed a specific expertise, such as the Baron-Tessier flap [START_REF] Talmant | Use of neck muscle in the correction of facial asymmetry. The Barron-Tessier flap[END_REF][START_REF] Tessier | Platysmabased myocutaneous clavicular island flap for intraoral reconstruction[END_REF] for lower lip and vestibular reconstructions, or the scalp flap for skin loss in zones with facial hair. Tessier systematically applied his principles of craniofacial 'autarchy': he considered the head and neck region to be self-sufficient for reconstruction and preferred, for instance, using parietal bone grafts and local/regional flaps, even for complex hard and soft tissue defects [START_REF] Wolfe | The influence of Paul Tessier on our current treatment of facial trauma, both in primary care and in the management of late sequelae[END_REF][START_REF] Wolfe | A man from Heric. The life and work of Paul Tessier, MD, father of craniofacial surgery[END_REF]. These specificities in the management of war injuries were also found in the series we report on.

Single or bilateral enucleation affected 29.50% of patients managed by Tessier in this series. A multivariate logistic model based on this dataset showed that enucleation was not related to age or gender, but only to the type of projectile: shell wounds were about four times more likely to be associated with enucleation than bullet wounds. Shells are in fact low-velocity weapons acting by fragmentation, and are thus associated with a wider range of tissue damage (Ansell et

Figure 1 .

 1 Figure 1. Preoperative and postoperative images of a 28-year-old man assessed by Tessier in

Figure 2 .

 2 Figure 2. (left) Age distribution density curve at the time of trauma -dotted line; 18 years of

Figure 3 .

 3 Figure 3. Distribution diagram for injury mechanism (percentages); NA = not available.

Figure 4 .

 4 Figure 4. Anatomical distribution of craniofacial bone lesions: upper third, middle third, and

Figure 5 :

 5 Figure 5: Distribution of the different procedures performed by Tessier and his team; TMJ =

Figure 6 :Fig. 1 .Fig. 2 .

 612 Figure 6: (Upper row) Bone or rib graft site distribution (percentages); OM temporal graft =

Table 1 .

 1 al., 2014 and Mader et al, 2006). Description of the cohort of patients operated on by Paul Tessier in Iran from 1990 to 1993.

	Legends

Table 2 .

 2 Results of the multivariate logistic model; OR = odds ratio

Table 2 .

 2 Results of the multivariate logistic model; OR = odds ratio.

		OR	2.5%	97.5%	p
	Female	0.781	0.045	2.401	0.789
	Age	0.964	0.905	1.017	0.211
	Shell	4.039	1.325	17.599	0.029

Acknowledgment

The authors would like to express their sincere gratitude to the Association Française des Chirurgiens de la Face (AFCF), in particular Prof. Testelin and Prof. Devauchelle, for granting access to the documents and photos of the Tessier Collection (Centre Hospitalo-Universitaire d'Amiens, Université de Picardie). This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CONCLUSION

Paul Tessier was a pioneering surgeon who applied the same systematic approach to each step of his surgical work (planning, procedure, follow-up). Notably, his conception of craniofacial 'autarchy' deserves further attention and investigations: free flaps are nowadays a standard procedure for the reconstruction of complex craniofacial defects, but iliac and fronto-parietal grafts associated with diverse soft tissue reconstruction techniques can still provide a wide range of solutions [START_REF] Mertens | Early bone resorption of free microvascular reanastomized bone grafts for mandibular reconstruction -a comparison of iliac crest and fibula grafts[END_REF], especially in precarious situations during or in the aftermath of war. The 322 patients we have presented were all operated on within a short timeframe by the same surgeon; the series is unique in its representation of 20th-century warfare facial injuries and shows the predominance of shrapnel as a cause of extensive facial trauma.


Miguel Albuquerque

Dominique Luton

Julie Le Faouder

Pierre Bedossa

Jean Guibourdenche

Dr Pierre-François Ceccaldi
email: pf.ceccaldi@aphp.fr

Variation of fetuin-A in maternal and fetal serum during human parturition

Keywords: Fetuin-A, parturition, calcium, Fetuin-A in human parturition

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

Introduction

Human parturition involves multiple interactions and signaling pathways for cervical ripening and uterine contractions. Traditionally, three general contemporaneous theories describe labor initiation: functional loss of pregnancy maintenance factor, synthesis of factors that induce parturition and also signal for parturition beginning [START_REF] Williams | Williams obstetrics[END_REF]. Most advances in research have come from animal models (pregnant sheep, rodents, rhesus monkeys) and underline the importance of fetal development and maturation in the timing of labor. One of the pathways most studied is the fetal pituitary-adrenal (FPA) axis. Maternal levels of free corticotropin-releasing hormone rise with placental mass during gestation and this increase correlates positively with the onset of parturition, probably stimulating fetal adrenal production of dehydroepiandrosterone sulfate and cortisol [START_REF] Ceccaldi | Modulation of free corticotrophin-releasing hormone, adrenal and placental steroid hormone levels induced by mifepristone during pregnancy[END_REF]. The role of the maturation of the fetal hypothalamic-pituitary-adrenal axis has recently been completed by the description of the central action of the nuclear factor kappa B (NF-kB), an important transcription factor, especially in inducing uterine contractions and cervical modifications [START_REF] Mitchell | Are animal models relevant to key aspects of human parturition?[END_REF][START_REF] Golightly | Endocrine immune interactions in human parturition[END_REF] . As mentioned Menon R et al, human parturition seems to be the end of a counting device that measures maturation of the fetal organ systems and the production of hormones and other soluble mediators and that promotes inflammation and orchestrates an immune cascade to propagate signals across different uterine compartments [START_REF] Menon | Novel concepts on pregnancy clocks and alarms: redundancy and synergy in human parturition[END_REF].

New techniques, such as proteomic analysis, applied to maternal blood allow the identification of circulating molecules of potential interest, particularly serum proteins involved or reflecting parturition. Yuan et al used proteomic techniques to look at variations in small proteins (MW < 10 000 Da) in the placental blood of pregnant women undergoing different modes of delivery [START_REF] Yuan | Low abundance plasma proteins in labour[END_REF]. Most changes during labor occurred in proteins related to inflammation and immunomodulatory processes.

The main objectives of our prospective study were first to analyze longitudinally maternal serum peptides in low-risk pregnant women, from 39 weeks of gestation (WG) to delivery in physiological conditions, and second to investigate whether one or more of those peptides could be of interest in the days preceding delivery in correlation with observed maternal and fetal clinical parameters.

Materials and methods

Clinical survey and collection of sera

Sixty low-risk pregnant women enrolled in a prospective cohort after giving written consent (Institutional Review Board (IRB) 00003835) were seen at regular intervals, weekly from 39 WG and then every 48 hours at 41 WG in the Department of Obstetrics and Gynecology at the Beaujon teaching hospital (Assistance Publique -Hôpitaux de Paris). Maternal blood was sampled during consultations, at delivery and during a postnatal consultation, 6 to 8 weeks after childbirth. All patients had given birth at the latest by 42 WG. For patients at 41 WG and 5 days, we proposed induction of delivery using prostaglandins according to our usual clinical protocol. To compare fetus, we use a statistical tool based on Z-scores, also known as standardized scores [START_REF] Salomon | Estimation of fetal weight: reference range at 20-36 weeks' gestation and comparison with actual birth-weight reference range[END_REF]. The Z-score describes the deviation in standard deviation (SD) from the mean. It is recommended by the WHO (World Health Organization) and allows percentiles to be calculated with precision, especially for values close to extremes (± 2 SD) [START_REF] Kiserud | The World Health Organization fetal growth charts: concept, findings, interpretation, and application[END_REF].

In a two-step analysis, we first compared longitudinally maternal sera at different terms of gestation and identified the most significant peaks of peptides, and second we analyzed variations of those significant identified peaks and clinical events. Cord blood was also collected at delivery.

Proteomic analysis of maternal serum and peak identification (Graphic 1)

Maternal serum proteomic profiles were established at the INSERM U773 laboratory using a serial analyzer SELDI 4000 (Surface-Enhanced Laser Desorption Ionization) followed by analysis by mass spectrometry (PCS4000; Bio-Rad). The analysis generated a molecular profile characterized by an inventory of the molecular masses of proteins. Each peak was characteristic of one protein, the relative abundance of which was reflected by the intensity of the peak. A longitudinal analysis of the proteomic profiles of 33 patient sera using Protein Chip Data Manager (Bio-Rad; array tool Biometric Research Branch, NCI) identified discriminating peaks. Molecular species were identified according to the following stages: selection of the samples where peaks were the most represented, enrichment and partial purification by four different strategies, analysis of the peptide imprint by means of dedicated programs (Profound, Mascot) and databases (Swiss-Prot, NCBI) after sequencing by mass spectrometry MS-MS to achieve identification. The different strategies were:

-Purification by depletion of 14 proteins (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin fibrinogen, alpha-2-macroglobulin, alpha-1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, complement C3 and transthyretin) using an Agilent Human 14 Multiple Affinity Removal System Column (Agilent Technologies, USA), followed by desalting and concentration by means of a Millipore ZipTip C18 (Millipore, Sigma Aldrich, USA) and fractionation by microLC. Fractions on MALDI-TOF were read and checked to detect peaks of interest.

-Depletion with the Proteoprep® Protein Precipitation Kit (Sigma Aldrich, USA), sample fractionation by microLC and then reading the fractions by MALDI-TOF.

-Depletion with the Proteoprep® Protein Precipitation Kit (Sigma Aldrich, USA) and Profinity IMAC Resin, uncharged (Bio-Rad, USA) to affine to divalent ions, followed by fractionation of the sample by microLC and reading of fractions with MALDI-TOF.

-Deposit samples on Weak Cation Exchange (WCX, Bio-Rad, USA) with different types of eluting solvents, followed by fractionation of the sample by microLC and reading of fractions.

Lower masses (2000 Da) were not taken into account because in this part of the spectrum the matrix interferes with detection. The usual protocol was a first detection with a signal-to-noise ratio of more than 5 and the second with a signal-to-noise ratio of more than 2. Spectral peaks were aligned to fit peaks to the same molecular weights in all profiles. Peaks within a mass window of ± 0.3% (given by the manufacturer) were considered identical.

Peaks detected with the software ProteinChip Data Manager Client 3.5 were included in the analysis if the coefficient of standardization was between 0.5 and 2.5. Mann-Whitney tests were performed using the BRB-ArrayTools software. Standard analyses were done with GraphPad Prism 7.0 (Windows version GraphPad Software, California, USA). A 2-day period was taken into account, because for the initial inclusions from 39 WG the terms were calculated from ultrasound exams done early in pregnancy.

A 1µL sample was removed from every fraction, mixed with 5-mg/mL cyano-4hydroxycinnamic acid in ACN/water/TFA (50/50/0.1) and analyzed by MALDI-MS on a MALDI-TOF/TOF Autoflex III in positive linear mode. Fractions containing an m/z relationship of interest lower than 4000 Da were analyzed in reflectron mode. The MS/MS spectrum obtained was submitted to the local search engine Mascot 2.3.0 (Matrix Science), using a tolerance for the precursor of 0.6 Da, an MS/MS tolerance of 0.5 Da, with the oxidation of methionine specified as a variable modification and no indicated enzyme.

Immunoassay of maternal serum

Sera were stored in PerinatCollection (ANR-10-EQPX-0010) until assay. Fetuin-A was quantified using an immunoassay (ab119594 Abcam ®).

Results

After standardization of mass spectrometry analysis, 8 of 270 profiles were not retained. To check reproducibility and estimate variability in the tests, a serum control was randomly deposited both pins. The inter-chip coefficient of variability of the intensities of the peaks present in 100% of spectra was calculated from the controls. We had a 25% relative standard deviation for the small masses, which complies the manufacturer's standards. Fifty-six peaks were detected for the profiles of masses under 10 000 Da (Graphic 2).

For identification of the peaks of interest according to term, we defined a cohort for which the time between delivery and inclusion (39 WG) was the longest, so as to have a longitudinal follow-up of these patients. Clinical data of our initial cohort are described in Table 1.

Twenty-two patients (36.7 %) were kept for at least ten days between inclusion and parturition, corresponding to the median in the initial cohort. other fragments correspond to a carrier of crystals of apatite and a TGF-β binding site [START_REF] Tajirian | Regulation of human monocyte proMMP-9 production by fetuin, an endogenous TGF-beta antagonist[END_REF].

The second peak, CM10-3884.6, was highlighted in the linear analysis by MALDI, but unfortunately we were unable to fractionate it. Fiedler et al reported that this peak corresponds to a doubly charged fragment of platelet factor 4 (PF4, CXCL4), which is found in granules of platelets and could be released during centrifugation of maternal serum samples as a bias [START_REF] Fiedler | Serum peptidome profiling revealed platelet factor 4 as a potential discriminating Peptide associated with pancreatic cancer[END_REF] .

The peaks at 6819.3 Da and 6614.2 Da were two components of apolipoprotein C1, a lipoprotein produced primarily in the liver and found in plasma. It has an important role in the exchange of esterified cholesterol between lipoproteins [START_REF] Flood-Nichols | Longitudinal Analysis of Maternal Plasma Apolipoproteins in Pregnancy: A Targeted Proteomics Approach[END_REF].

To investigate the peak CM10-2737.8, identified as a fragment of fetuin-A, we measured maternal serum fetuin-A (mSF-A) using an ELISA in our entire cohort, even in cord blood.

The kinetics of this fragment in the days before the delivery made us continues the investigations. Levels ranged from 15.3 ± 6.46 ng/mL to 10.6 ± 4.24 ng/mL at delivery. We established that there was a significant decrease in the days preceding delivery (Graphic 3). At delivery, there was no observed difference between induced or natural parturition and vaginal delivery or cesarean section.

At delivery, despite we observed in our cohort significant higher Z-scores for males than females (Table 2, p= 0.026), there was not difference for concentrations of fetuin-A in blood cord between males and females. Serum concentrations of fetuin-A were distributed according to fetal trophicity. Despite this, there is no significant difference in serum concentrations of Fetuin-A on either side of the placenta at birth. The mean level of fetuin-A in fetal cord blood was only 1.15 times that in maternal blood, with no significant correlation (r2=0.0053, p=0.674) (7.3-18.1 ng/mL).

At postpartum, more than six weeks after delivery mean maternal fetuin-A level was higher than at delivery (13.7 ±3.23 ng/mL vs 10.6 ±4.24 ng/mL, p=0.003).

Discussion

Our study by SELDI confirms that several maternal serum proteins fluctuate three weeks before delivery, especially proteins involved in inflammatory and immunotolerance processes.

Using MALDI-TOF technology, we identified the N-terminal fragment of fetuin-A. Fetuin-A is a complex multifunctional protein expressed in numerous tissues such as bone before and after birth and is essentially secreted by hepatocytes in adult life and placenta during pregnancy [START_REF] Dziegielewska | alpha 2HS-glycoprotein is expressed at high concentration in human fetal plasma and cerebrospinal fluid[END_REF] . Fetuin-A is a natural antagonist of TGF-beta and is able to bind calcium [START_REF] Mori | Fetuin-A: a multifunctional protein[END_REF] .

It circulates at high levels in fetal blood and then declines in childhood and in adulthood, when it fluctuates widely under the influence of many factors (genetic, diet, exercise, age) [START_REF] Robinson | From infancy to aging: Biological and behavioral modifiers of Fetuin-A[END_REF].

Fetuin-A circulates in the blood and contributes to different physiological and pathological processes: calcification in many tissues such as in bone and tooth mineralization, insulin resistance and dyslipidemia leading to obesity and cardiovascular diseases, acute and subchronic inflammation. Also, low levels of fetuin-A are associated with vascular calcification and inflammation [START_REF] Laughlin | The association of fetuin-A with cardiovascular disease mortality in older community-dwelling adults: the Rancho Bernardo study[END_REF][START_REF] Reinehr | Fetuin-A and its relation to metabolic syndrome and fatty liver disease in obese children before and after weight loss[END_REF][START_REF] Xu | Serum fetuin-A is correlated with metabolic syndrome in middle-aged and elderly Chinese[END_REF]. Siegel-Axel et al found that fetuin-A influences the expression of proinflammatory and angiogenic proteins via toll-like receptor 4 (TLR4) pathway in perivascular fat cells cultured in vitro [START_REF] Siegel-Axel | Fetuin-A influences vascular cell growth and production of proinflammatory and angiogenic proteins by human perivascular fat cells[END_REF] . The TLR4 pathway culminates in activation of the transcription factor NF-kB, which controls the expression of an array of inflammatory cytokine genes [START_REF] Pal | Fetuin-A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance[END_REF] . NF-kB leads numerous proinflammatory cytokines that during parturition and the withdrawal of progesterone allow the contraction of the myometrium via estrogens and prostaglandins. transduction of parturition has pathways that vary among modes of delivery and that the hepatocyte nuclear factor 1 homeobox A (HNF1A) is likely to play a key role [START_REF] Yuan | Low abundance plasma proteins in labour[END_REF] . Induction of these pathways during labor was characterized by the appearance of several inflammationrelated proteins, as fetuin-A. In our study, we did not observe significant variation in fetuin-A induced by the mode of delivery but spontaneously few days before delivery.

On the fetal side, Iliodromiti et al studied fetuin-A in amniotic fluid in Down Syndrom fetus in the second trimester and founded reduced levels than euploid fetus (5,3 ng/mL vs 6,8 ng/mL, p= 0,008) [START_REF] Iliodromiti | Fetuin A concentration in the second trimester amniotic fluid of fetuses with trisomy 21 appears to be lower: phenotypic considerations[END_REF]. They suggested an association with altered metabolic pathways and could be potentially associated with features like growth restriction or impaired osteogenesis.

In our study there is no difference between macrosomic and fetal growth restriction fetus, or male and female fetus.

Fetuin-A has an important role in mineralization biology. Schafer et al. described its affinity for calcium phosphate minerals like hydroxyapatite and in fetuin-A knockout mice [START_REF] Schafer | The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification[END_REF][START_REF] Jahnen-Dechent | Fetuin-A regulation of calcified matrix metabolism[END_REF]. In fetal side, concentrations of fetuin-A were decreased in amniotic fluid of idiopathic preterm premature rupture of the membranes (PPROM) [START_REF] Shook | Calciprotein particles as potential etiologic agents of idiopathic preterm birth[END_REF]. Taken together, the serum variation of fetuin-A on both the fetal and maternal side and the proteins involved in the calciprotein particles mechanism would be an interesting avenue to explore in parturition or in some diseases during pregnancy like diabetes or preeclampsia.

Graphic 1: Summary of the proteomic analysis of maternal serums.

Graphic 2: Results of the proteomic analysis

Table 1: Maternal and fetal characteristics (N=60) Graphic 3: maternal fetuin-A concentration few days before and at delivery (r2= 0,142; p< 0,0001)

Under 10 000

 10 Da, four peaks were observed: 3884.6 Da, 2737.8 Da, 6819.3 Da and 6614.the Mascot search pointed to the N-terminal fragment of α-2-HS glycoprotein (AHSG, fetuin-A). Fetuin-A is a 55-59 kDa phosphorylated glycoprotein of two polypeptide chains. This N-terminal fragment has no known function;

Kalabay et al described

  an increase of mSF-A determined by radial immunodiffusion in the second part of a normal pregnancy[START_REF] Kalabay | Correlation of maternal serum fetuin/alpha2-HS-glycoprotein concentration with maternal insulin resistance and anthropometric parameters of neonates in normal pregnancy and gestational diabetes[END_REF] . Gomez et al observed in vitro that fetuin-A reduced extravillous cytotrophoblast viability and invasion in the first trimester and particularly reduced expression of insulin receptor substrate-1 and of tyrosine phosphorylated insulin receptor substrate-1[START_REF] Gomez | Effects of increased fetuin-A in human trophoblast cells and associated pregnancy outcomes[END_REF]. In a prospective cohort of 309 women in preterm labor with suspected intraamniotic infection, Pereira et al identified 23 peptide masses that discriminated preterm labor from spontaneous preterm birth[START_REF] Pereira | Insights into the multifactorial nature of preterm birth : proteomic profiling of the maternal serum glycoproteome and maternal serum peptidome among women in preterm labor[END_REF]. Also, 48 of 52 proteins were classified into different functional pathways involved in cases of preterm labor: complement/coagulation cascade, inflammation/immune response, fetal or placental development, extracellular matrix proteins. Particularly, fetuin-A, classified in the inflammation response or in fetoplacental development, showed a negative fold change (-1.22, p<0.0001) and was associated with preterm birth. Yuan et al reported that the signal

Table 2 : Fetal Z-scores and fetuin-A at delivery, both side of the placenta

 2

Conclusion

At the beginning of parturition, there is mobilization of proteins, such as fetuin-A, that are likely to be involved in inflammatory and immune tolerance processes and maternal adaptation to pregnancy. Fetuin-A may play a role between the pro-inflammatory pathway NF-kB and the transcription factor HNF1A, which is involved in the expression of several liver-specific genes. The literature describes fetuin-A as a potential biomarker for organ dysfunction and metabolic syndrome disorders. The protein mineral homeostasis would be an interesting pathway to explore during human parturition.

The authors report no conflict of interest.

Maternal Characteristics

-Age (years, Median, min -max) -Parity -BMI at end of pregnancy (Median, min -max) -GA at delivery (WG, median, min-max) -Vaginal delivery (%) -Induction of parturition (%)