Aurélie Jourdain

Satish C Singh

Javier Escartin

Yann Klinger

K A Kamesh Raju

J Mcardle

Crustal accretion at a sedimented spreading center in the Andaman Sea

The combination of magmatic, tectonic, and hydrothermal processes that from the oceanic crust, relatively well understood along mid-ocean spreading centers, is strongly modified at sedimented spreading centers, which are still poorly understood owing to the lack of good crustal images. Here we present high-resolution deep seismic reflection images across the sedimented slow-spreading Andaman Sea spreading center that shows several sub-horizontal sills injected within the sedimentary strata beneath the axial graben. Below this sill-sediment sequence we observe reverse polarity reflections within the igneous crust, indicative of the existence of axial magma lenses (AMLs) at different depths. This results in a heterogeneous upper crust that incorporates metamorphosed sediments. The faults within the axial valley are steeply dipping (65-75º) and are in a stair-cased pattern. Their base coincides with a shallow dipping (30º) reflection, possibly defining the spreading axis morphology, controlled by the AMLs. We propose that as the sill-sediment sequences move away from the axial graben, they

are rotated and buried due to subsidence and faulting, forming the upper oceanic crust. The Moho reflections are observed along a significant part of the profiles, allowing the characterization of the whole crustal structure. We find that the upper oceanic crust is formed by a combination of sill-sediment sequence, followed by a thick gabbroic lower crust with some sediment along faults, with a mantle transition containing dunite lenses near the Moho.

INTRODUCTION AND SETTING

The oceanic crust forms at mid-ocean spreading centers by a combination of magmatic, tectonic, and hydrothermal processes that are relatively well studied along both fast-and slow-spreading mid-ocean ridges. However, sediments can drown spreading centers in the vicinity of continents or during the early stages of continental rifting and oceanization, as observed in the Guaymas Basin [START_REF] Lizarralde | Carbon release by offaxis magmatism in a young sedimented spreading centre[END_REF][START_REF] Kluesner | Late Pleistocene cyclicity of sedimentation and spreading-center structure in the Central Gulf of California[END_REF], northern Juan de Fuca Ridge [START_REF] Davis | Observations of natural-state fluid pressures and temperatures in young oceanic crust and inferences regarding hydrothermal circulation[END_REF], and Mohns Ridge [START_REF] Bruvoll | Burial of the Mohn-Knipovich seafloor spreading ridge by the Bear Island Fan: Time constraints on tectonic evolution from seismic stratigraphy[END_REF]. These sediments capping spreading centers can significantly modify the axial thermal regime and fluid circulation, and therefore the accretion processes (melt emplacement, faulting), and also the overall lithospheric structure and composition. To better understand the impact of sedimentation on crustal accretion, we present the first whole-crust, high-resolution multichannel seismic reflection images across the sedimented Andaman Sea Spreading Center (ASSC). The unprecedented resolution of these data allow us to image clearly the interactions of sedimentation and accretion, and to propose a general model of lithospheric formation and whole-crust structure at sedimented mid-ocean ridges.

The ASSC back-arc spreading center separates the Burmese sliver plate to the northwest from the Sunda plate (Eurasia) to the southwest (Fig. 1A) [START_REF] Curray | Tectonics of Andaman Sea and Burma[END_REF][START_REF] Kamesh Raju | New insights into the tectonic evolution of the Andaman basin, northeast Indian Ocean[END_REF]. It connects the northern Sagaing strike-slip fault with the southern Andaman-Nicobar and Sumatra faults [START_REF] Sieh | Neotectonics of the Sumatran fault, Indonesia[END_REF], which result from strain partitioning from the oblique Indian plate subduction beneath the Sunda plate [START_REF] Fitch | Plate convergence, Transcurrent Faults, and Internal Deformation Adjacent to Southeast Asia and the Western Pacific[END_REF]. Based on magnetic anomalies on the westernmost non-sedimented segment, ASSC spreading initiated 4 Ma ago at a full rate of 16 mm/yr, speeding to 38 mm/yr 2 Ma ago [START_REF] Kamesh Raju | New insights into the tectonic evolution of the Andaman basin, northeast Indian Ocean[END_REF].

Most of the Andaman Sea basin is sedimented and flat lying at 3000 m water depth. The ASSC, at the center of the basin, displays a ~12 km wide axial valley, and an also flat-lying rift valley floor at 3600 m water depth (Fig. 1B), infilled by sediments from the Irrawaddy river basin in Myanmar (Fig. 1A) [START_REF] Curray | Tectonics of Andaman Sea and Burma[END_REF]. This sedimented axial graben is bound by normal faults and tilted fault blocks, reminiscent of the structure found at slow spreading ridges (e.g., [START_REF] Needham | Some characteristics of the rift valley in the Atlantic ocean near 36°48[END_REF].

SEISMIC DATA

The seismic reflection data were acquired by Petroleum Geo-Services (PGS) in 2008 with a 6400 cubic inch air gun array and recorded by an 8-km long streamer with a group spacing of 12.5 m.

The air gun and the streamer were towed at 8 m and 10 m below the sea surface respectively, with a 25 m shot interval and a 9 s record length. Data were processed using a conventional time migration technique, which includes swell noise removal, trace editing to remove noisy traces, common midpoint binning, velocity analysis, upper mute, and stacking. Stacked data were migrated using a post-stack Kirchhoff migration technique. PGS provided the final migrated data and root mean square (RMS) velocities every 1 km (160 CMP), and no pre-stack data were available for our study. Here we present two N-S profiles crossing the sedimented segment of the ASSC and spaced ~40 km (Fig. 1B).

Figure 2 shows the interpreted seismic time sections along the two profiles that cross the rift valley; un-interpreted images are shown in Figure S1. Although the profiles are N-S, 25° NE off the orthogonal to the spreading axis, symmetry of structures on either side of the axial graben are clear, and show as well as the thick sediments filling the axial valley, and numerous faults penetrating down 2-2.5 s. The 7.5 s deep reflection is the Moho which appears intermittently along both profiles.

In the absence of seismic refraction studies, we converted RMS velocities into interval velocities, laterally smoothed to obtain a continuous velocity and seismic image. Although the RMSderived interval velocities are less accurate than those from refraction data, they provide insight into crustal structure. The P-wave velocity increases from 1.6 km/s near the seafloor to 3.0 km/s at the sedimentary sequence bottom/base? (Fig. S2), which combined with the linear seismic reflections suggest the presence of sedimentation layering. The alternating highly reflective and transparent layers within the sedimentary column could correspond to sediment deposits recording climate variability [START_REF] Kluesner | Late Pleistocene cyclicity of sedimentation and spreading-center structure in the Central Gulf of California[END_REF]. Further below velocity increases sharply to 4.5 km/s within a 0.6 s thick zone, associated with a layer displaying numerous bright reflections. Deeper and up to Moho, the reflective features are sparse but mostly steeply dipping.

Lacking seismic reflectors, the seismic velocity in this region is poorly constrained.

INTERPRETATION OF SEISMIC REFLECTORS AND RESULTS

In this section we first focus our interpretation on the interaction between magma sills emplacement sedimention, and the subsequent effects of faulting. We then address the nature of the crust-mantle boundary, to provide constraints on crustal accretion process at sedimented ridges.

Low velocity reflectors

Most of the reflections in the seismic profiles are of normal polarity as that of the seafloor reflection. However, both profiles display some reflections with reverse polarities (Figs.2A, 2B, 2C, 2D),, which suggest low velocity zones likely induced by fluids or by material with lower velocity than that of the surrounding medium.

On-axis we observe two low velocity, revese polarity reflectors. We suggest correspond to a magma bearing axial melt lens (AML), rather than other fluids such as water, as they are located below the ASSC axis; such reserve polarity reflections are commonly interpreted as the present of AMLs beneath fast spreading ridges [START_REF] Detrick | Multi-channel seismic imaging of a crustal magma chamber along the East Pacific Rise[END_REF][START_REF] Kent | Distribution of Magma Beneath the East Pacific Rise Between the Clipperton Transform and the 9°17'N Deval From Forward Modeling of Common Depth Point Data[END_REF] and more recently at slow-spreading (Singh et al., 2006b) ridges. The shallowest one, which is 0.8 km wide at 6 s two-way travel time (TWTT) present on both profiles and we name it upper axial melt lens (U-AML). The second deeper on, which is 2-km wide at 7.2 s, is present only on profile PGS08-23 and we name it lower axial melt lens (L-AML).

We also observe some reverse polarity reflections off-axis, but owing to their position and relatively short lateral extension, they probably correspond to fluids other than magma such as water or gas circulating near faults or along sediment-sill interfaces (Fig. 2).

High velocity reflectors

Within the sedimentary layers and underneath them, we observe two sets of strong and discontinuous normal polarity reflections (Figs. 2A,2B) that should correspond to high, positive velocity contrasts. We interpret those that are sub-horizontal (sloping < 20°) as frozen igneous bodies or sills (Figs. 2A, 2B), following similar criteria used in other studies in similar environments (Trude et al. 2003;[START_REF] Lizarralde | Carbon release by offaxis magmatism in a young sedimented spreading centre[END_REF]. These reflectors tilt primarily toward the axis, where they can show a saucer-shape [START_REF] Hansen | The three-dimensional geometry and growth of forced folds above saucer-shaped igneous sills[END_REF].

Steeper reflections (>20°) can be interpreted either as highly rotated sills or as fault reflectors owing to their orientation and position in the proximity of prominent and continuous faults (Figs.

2A, 2B). We prefer to interpret them as sills because, in the sedimentary strata, faults offset sediment layers and lack any reflectivity contrast (see below). Furthermore, the high reflectivity of the frozen magma sills below the sedimentary sequence requires a surrounding medium that is not of magmatic origin.

Tectonic structures

The 600 m deep and 12-km wide axial graben is flanked by a series of high-angle (65-75º) normal faults. They have a ~1 km spacing (Figs. 2A,2B,3A) with a staircase pattern, dipping towards the center of the basin, and are responsible for the local subsidence. These faults are recent as they cut through the most recent sediments. The seismic profiles and the seafloor bathymetry suggest that the nature of the graben deformation and associated faulting is similar along the whole segment. A clear bounding fault buried under more recent sediments is imaged around CMP 26000-27000 (Fig. 2A). This fault penetrates down to 700 ms above the Moho, with 500 ms thick reflective zone above the fault's lower extent (Fig. 3B). A similar, but smaller, structure is present on the conjugate side to the south. On PGS08-23, these OBFs are also visible, and have some high angle faults in a staircase pattern below them. These low-angle reflections may thus be akin to the present-day seafloor surface of the axial graben flank. The identification of high-angle, staircase patterns is increasingly difficult nearing the transform fault in the east near profile PGS08-21, where the block rotation and the deformation are the highest. Locally, we observe a sediment infill that has been tilted away from the axis, consistent with flexural rotation associated with ridgeward normal faulting. Deep-rooted low-angle faults close to the axis rupture at the seafloor. Other faults are sealed by recent sediments, forming an unconformity with the tilted blocks. Hence, the faults responsible for the tilt of these blocks are not active at the present time. This observation, together with the lack of a tectonic signal on the most recent sediment cover, lack of fault scaprs at the seafloor away from the axis, and the concentration of seismicity along the axial graben [START_REF] Diehl | Back-arc extension in the Andaman Sea: Tectonic and magmatic processes imaged by high-precision teleseismic double-difference earthquake relocation[END_REF], clearly demonstrate that tectonic deformation concentrates along the graben. Little or no deformation occurs off-axis, and all the tectonic structures observed off-axis have been passively rafted due to oceanic accretion on-axis, where we focus our analysis.

Crust-Mantle boundary

The Moho reflection is generally 1-5-2.5 s into the oceanic crust [START_REF] Kent | Uniform accretion of oceanic-crust south of the Garrett transform at 14-degrees-15's on the East Pacific Rise[END_REF]. On our data it is imaged at ~7.6 s on profile PGS08-21 and around 7.5-7.8 s on profile PGS08-23. It is slightly shallower beneath the axial valley (Figs. 2A,2B), and can be followed along a significant part of the profile. If the base of the sedimentary strata is the top of the oceanic crust and the Moho reflection its base, then crustal thickness would vary from 1.6 s to 2.4 s. On Profile PGS08-23, between CMP 77000 and 79000, the Moho displays a complex pattern, with lenstype structures (Figs. 2B, 3C); similar reflectors are observed near CMP 16000 and 25000 on profile PGS08-21, and around CMP 69000 and 80000 on profile PGS08-23. Beneath the spreading axis we observed a weak reflection ~ 250 ms below the L-AML, which we also interpret as the Moho.

DISCUSSION AND CONCLUSIONS

A combination of the interval P-wave velocity data and seismic reflection images of the full crustal thickness allows us to characterize for the first time with sufficient detail the nature of the whole crust formed along the axis of a sedimented spreading center.

Axial melt supply

Multiple melt lenses have been recently reported beneath fast spreading centers [START_REF] Arnulf | Seismic evidence of a complex multi-lens melt reservoir beneath the 9° N Overlapping Spreading Center at the East Pacific Rise[END_REF]Marjanovic et al., 2014), but the presence of two melt sills (U-AML and L-AML) beneath a slow spreading ridge is novel as it has been difficult to image melt lens below them (Singh et al., 2006b). Furthermore, the separation between the two melt sills is much larger (~1.2 s) than that observed at fast spreading ridge (0.2-0.3 s), and therefore, their roles should be different.

The U-AML is relatively shallow when compared to the predicted depth for AMLs for the spreading rate of 3.8 cm/yr [START_REF] Chen | A Nonlinear Rheology Model for Mid-Ocean Ridge Axis Topography[END_REF]). In the absence of any enhanced melt supply to the axis, this prediction would suggest that the apparently shallow U-AML is likely a transient feature at this spreading rate, but instead the presence of the reflector on the two profiles 40-km apart indicates that the U-AML is instead a prevalent feature. This shallow melt lens position could also be facilitated by the thick sediment layer blanketing the axial valley, that likely acts as an insulating layer resulting a warmer thermal regime that could help retain a melt for longer time.

The U-AML, which lies at the base of the sedimentary cover,is also at the intersection of the median valley bounding faults, suggesting that the shallow dipping bounding faults may root at the U-AML. The U-AML is also shifted ~0.5 km northward from the axial graben on PGS08-21 and ~1.2 km on PGS08-23, inducing the southern boundary fault to dip more shallowly than the northern one. Such an asymmetry in faulting caused by the AML position has also been suggested at the Afar rift [START_REF] Grandin | Manda Hararo-Dabbahu rifting event Afar (Ethiopia) : Constraints provided by geodetic data[END_REF].

The depth of the L-AML, at ~2.3 s below the seafloor or ~4.5 km within the igneous crust, is consistent with the prediction from lithospheric cooling models at this rpreading rate [START_REF] Chen | A Nonlinear Rheology Model for Mid-Ocean Ridge Axis Topography[END_REF]. The L-AML likely feeds the U-AML on axis, and this magmatic system bay be the source for other crustal melt sills near and off the spreading axis now frozen, although the the dynamics and interactions of multiple melt lenses are not well-known.

Formation of the upper crust

We identify a steep gradient in P-wave velocity ranging from 3.0 to 4.5 km/s att he base of the recent sediments, clearly associated with the numerous sills observed at this depth (Fig. S2). As for the Guyamas basin [START_REF] Kluesner | Late Pleistocene cyclicity of sedimentation and spreading-center structure in the Central Gulf of California[END_REF], the reflectors in this area show amplitude whitening between sills, which could be due to fluid-expulsion and metamorphism or the presence of gas (steam). The P-wave velocity in the sediments is likely to increase due to compaction and fluid expulsion, and to metamorphism around sill intrusions [START_REF] Aarnes | The impact of host-rock composition on devolatilization of sedimentary rocks during contact metamorphism around mafic sheet intrusions[END_REF]. Based on the P-wave velocity, we suggest that the upper crust is a 1.2 km thick velocity gradient zone composed of metamorphosed sediments and sill intrusions (Fig. 4).

Deflation due to melt extraction in the U-AML, in addition to normal faulting along the axial graben, can contribute to axial subsidence and promote the inward tilt of the sill/sediment sequences. Extension concentrated at the rift valley and its flanks would thus induce the development of steeply dipping faults (stair-case normal faults), possibly facilitating further injection of sills in the upper sedimentary strata. High-amplitude reflections below the highvelocity gradient zone are sparse, sometimes concentrating as lenses along low-angle faults. The relatively higher velocity in this crustal section is indicative of magmatic accretion, but lacking adequate velocity constrains we do not exclude it to be metamorphosed sill-sediment lenses similar in composition to the upper crust. The former interpretation would be induced by the proximity of these faults with the magma supply at the axis. The latter one would suggest high variability of the metamorphic upper crustal thickness produced by locally high subsidence, and is more consistent with the presence of high velocity contrasts required to image these reflections.

The U-AML can define the effective brittle-ductile boundary locally while it is active, controlling the location of the inner bounding faults. After a certain amount of extension and magmatism, a new inner bounding fault would develop inward from the existing bounding faults, the older faults becoming inactive, rafted off-axis, and buried by incoming sediments. With such a process, all sill-sediment sequence now present in the graben likely forms the bulk of the upper part of the oceanic crust here. These results also suggest that the magmatic crustal accretion at the ASSC is taken up solely by intrusive processes, consistent with the absence of clear magnetic anomalies that can be attributed to eruptive units [START_REF] Kamesh Raju | New insights into the tectonic evolution of the Andaman basin, northeast Indian Ocean[END_REF], and to the lack of volcanic features along the axial graben floor. Variation in magmatic supply to the AML could be responsible for the two modes of faulting of the ASSC and would exclude the hypothesis of a halt in the oceanic crustal accretion as suggested by [START_REF] Morley | Is spreading prolonged, episodic or incipient in the Andaman Sea? Evidence from deepwater sedimentation[END_REF]. On the other hand, the L-AML may locally control the rheology of the lithosphere, pinnin the root of outer bounding fault, while the two melt lenses regulate activity on the outer and inner bounding faults, and hence the nature of the median valley (Fig. 4).

Crustal thickness and Moho transition

Widespread Moho reflections allows us to constrain the nature of the lower crust and its base.

Lacking accurate seismic refraction velocities, we assume a P-wave velocity of 6.8 km/s in the lower crust (e.g., [START_REF] Lizarralde | Variation in styles of rifting in the Gulf of California[END_REF]. This yields a lower crustal thickness of <5.5 km, and a total crustal thickness of <6.7 km, overlain by 1.5 km of sediments, consistent with gravityderived estimations. [START_REF] Morley | Is spreading prolonged, episodic or incipient in the Andaman Sea? Evidence from deepwater sedimentation[END_REF].

A complex Moho with lenses just above it could result from either dunite lenses (melt residue) or gabbro lenses in the mantle-crust transition zone. Frozen melt sills below the Moho have been observed beneath the Juan De Fuca Ridge (Nedimovic et al., 2005), but ASSC lenses seem to lie within the Moho transition zone, and could be the residue of former L-AMLs. The complex Moho transition zone with dunite-gabbro lenses observed in the Oman ophiolite [START_REF] Braun | Dunite distribution in the Oman ophiolite: Implications for melt flux through porous dunite conduits: Geochemistry[END_REF] may be a suitable analog to the complex Moho we image here. The proximity of the Moho with lower melt lens creates a complex mantle transition zone, which combined with the presence of multiple sills, makes the sedimented ridge a special class of ocean spreading center forming unique marginal crust, but accurate seismic velocities are required to estimate the magmatic supply to the axis from the relative proportion of sediment and magma and its variation in depth.

Ridge hydrothermal field: Nature, v. 442, p. 1029-1032. Smith, W. H. F., and Sandwell, D. T., 1997, Global sea floor topography from satellite altimetry and ship depth sounding: Science, v. 227, p. 1956-1962. Trude, J., Cartwright, J., Davies, R., J., and Smallwood, J., 2003, New technique for dating igneous sills: Geology, v. 31, no. 9, p. 813-816.

FIGURE CAPTIONS

  Further away from the axis and central graben,shallow-dipping (20-40º) reflections at both flanks of the median valley are shown in PGS08-21, and only on one flank for PGS08-23. In some cases, these low-angle reflectors reach deep crustal levels and show a clear tilt of the adjacent crust block. This indeed suggests a fault-related flexural rotation resulting in a shallow dip of sedimentary reflectors away from the axis. This is best shown by two of the major inwarddipping low angle Outer Bounding Fault North and South faults (OBF-N and OBF-S in Figs. 2A, 2B).

Figure 1 :

 1 Figure 1: Andaman Sea Spreading Centre: A: General map of the study area (Multibeam

Figure 2 :

 2 Figure 2: Seismic profiles: Interpreted seismic images A: PGS08-21 and B: PGS08-23. Time

Figure 3 :

 3 Figure 3: Expanded view of seismic data: Interpretation uses the same colors as figure 2. A:

Figure 4 :

 4 Figure 4: Crustal model of sedimented ASSC: Schematic 3D block diagram synthesizing

Figure S1 :

 S1 Figure S1: Un-interpreted seismic images: A: Profile PGS08-21. B: Profile PGS08-23.

Figure S2 :

 S2 Figure S2: Interval Velocity: Interval velocity as a function of time derived from the root mean

ACKNOWLEDGMENTS

We would like to thank Petroleum Geo-Services (PGS) for providing the data, and Adrien Arnulf and Wayne Crawford for discussions.


Delphine Halie

Joëlle Pérard-Viret

Jacques Royer

SYNTHESIS OF CHIRAL NON RACEMIC INDOLIZIDIN-3-ONES AS PEPTIDOMIMETIC SCAFFOLD

A new asymmetric strategy has been devised to attain indolizidin-3-ones wich can be used as peptidomimetic scaffolds. The sequence is a two-step synthesis.

Local constraint of peptide geometry has been widely considered as a useful strategy for attaining peptide secondary structure or modifying the specificity for a receptor and could be achieved through the properties of cyclic dipeptide mimetics. 1 Further possibilities to functionalize these mimics is also of great interest as this would allow the introduction of lipophilic moieties or known drugs (for vectorisation) or others. [START_REF] Temming | [END_REF] In this context, we developped the preparation of the indolizidone structure (1) (Figure 1), which could be further derivatized to (2) countaining all the required structural features: a constrained bicyclic dipeptide bearing an extra functional group for the attachment of other entities. The indolizidinone skeleton has been largely used as a scaffold for the design and elaboration of constrained peptidomimetics 3 but mainly as indolizidin-5-one and rather scarcely as indolizidine-3-one 2 (or C-terminal pipecolic acid). 4 We now present herein a new approach toward a total asymmetric synthesis of this later type of compound.

N O OH O N O OR O 12 NH 2 X X X = OH, OR' Figure 1
The synthesis of racemic 1 was already reported in the literature 5 but is difficult to adapt in an asymmetric manner. We preferred a different strategy also based on the acyliminium ion cyclization 6 and using the chemistry developed in our laboratory for some years. The key step is the cyclization to an acyliminium derived from chiral α,β-unsaturated γ-lactam. 7 Some years ago, we reported the condensation of primary amines with dimethoxydihydrofuran to give α,β-unsaturated γ-lactam. The later was the precursor of an acyliminium species (through acidic treatment) and then, if the starting amine bears a nucleophilic function, cyclization can occur quite readily according to the following scheme (Scheme 1)

N O R NH 2 O MeO OMe + Nu R Nu N O R Nu Scheme 1
By the use of this strategy, we also already reported that electron-enriched aromatics led in a one-pot sequence to bicyclic lactams. 8 We now want to extend this reaction to olefin cyclization: in this case a nucleophilic terminator is necessary to stabilize the so-formed carbocation.

We started with the condensation of rac-methyl allylglycinate and dimethoxydihydrofuran which gave the γ-lactam (3) as a mixture of α,β/β,γ-unsaturated regioisomers (3a+3b) in a 55% overall yield after purification. The treatment of this regioisomeric mixture with various nucleophiles XH and under acidic conditions allowed the attempt cyclization to occur. Nevertheless, depending upon the nucleophile and the experimental conditions, cyclization proceeded with various yields and was, in some cases, accompanied by transesterification products. The table shows the different attempts. Water was first tried as a nucleophile in limited amount and in various solvents to avoid ester hydrolysis (Entries 1-3). While no cyclization was observed in dioxan, 1a was isolated in DME (in low yield) and in CF 3 CH 2 OH (Entry 3).

Two experiments were conducted in benzyl alcohol (Entries 4,5) with the hope to directly introduce a benzyl ether group. Indeed, while there was no cyclization at rt, the two cyclization products (1a and 1b)

obtained in dichloroethane at 90°C bear a free OH group showing a poor nucleophilicity of this alcohol.

When the cyclizations were conducted with H 2 SO 4 in methanol, free alcohol (1a) was still present but accompanied (1/1 mixture) with the methoxy derivative (1c) (Entry 7). The use of acetonitrile (Entry 9) was of great interest since a Ritter reaction took place to give an interesting amide (compound 1f) but unfortunately, we were not able to rise the yield to satisfactory values. At that stage, very simple processes were established to attain the target compounds, with fixed configuration. Reflux in formic acid (Entry 8) gave access to a diastereomeric mixture but in very high yield. Conditions were found to stereoselectively access the cis alcohol in acceptable combined yields (Entries 3 and5).

N COOMe O nucleophile H + N O H COOR X 1 X 2 N COOR O X + 1 4 3 a X 1 =OH, X 2 =H, R=Me b X 1 =OH, X 2 =H, R=Bn c X 1 = H, X 2 =OMe, R=Me d X 1 =OCHO, X 2 =H, R=Me e X 1 =H, X 2 = OCHO, R=Me f X 1 =NHCOCH 3 , X 2 =H, R=Me a X=OH, R=H
An important aspect should now be examined and is concerned with the possible application of this scheme in the chiral non-racemic series. The question is to be addressed since it was early reported that the iminium ion cyclization may be accompanied by a partial, or total racemization through a possible aza-Cope rearrangement. 9 Indeed, a brief review of the literature revealed that the presence of the ester group was not a favorable structural feature for such a rearrangement. 10,11 The reaction sequence was then conducted with chiral non-racemic methyl allylglycinate and the cyclization carefully checked. gave the expected bicyclic lactam (1d + 1e) in 47% overall yield from (S)-allylglycine. These compounds were the only products of the cyclization process. Diastereomers (1d and 1e) were separated by column chromatography on silica gel and the integrity of the chiral center of the starting material was checked on each compound. Cis-isomer (5S, 7S, 8aR)-(1d) [START_REF]5S, 7S, 8aR)-1d : [α] D 33 -19.6 (c 1. 0, CHCl 3 ), HRMS (ES +[END_REF] as well as trans-compound (5S, 7R, 8aR)-(1e) [START_REF]5S, 7R, 8aR)-1e : [αD 33 -29.7 (c 1. 0, CHCl 3 ), 1 H NMR (400MHz, CDCl 3 ) δ[END_REF] were both found as single isomer by HPLC analysis on chiral column [START_REF]Chiral pack AD column, 250mm, n-hexane/EtOH 85/15, ∆Rt = 2 min[END_REF] (the racemic compound (1d) nicely exhibited a two peaks pattern of equal intensities with a ∆Rt = 8 min. and the racemic 1e two peaks of equal intensities and a ∆Rf= 1min.).

The presented sequence is thus applicable to the preparation of the enantiomerically pure target compound.

It appears as a rapid and efficient synthesis of such intermediates. (MS Word Style "05 Het-Text")

  Scheme 2

  Scheme 4

  MeOH)) subjected to condensation to give 3a,b. The cyclization of 3a,b in formic acid

Table Eventually

 Eventually

when the reaction was conducted in formic acid the formation of the two epimeric formates (1d) and (1e) (1:1.4) was observed in 91% yield.


Maria Kitsara
email: kitsara.m@gmail.com

George Tassis

Aristeidis Papagiannopoulos
email: apapagiannopoulos@eie.gr

Alexandre Simon

Onnik Agbulut

Stergios Pispas

Dr M Kitsara

Mr A Simon

Prof O Agbulut

Dr Mr G A Tassis

Dr S Papagiannopoulos

Polysaccharide-protein multilayers based on chitosan-fibrinogen assemblies for cardiac cell engineering

Keywords: polysaccharide-protein multilayers, chitosan-fibrinogen assemblies, tunable hydrophilicity/hydrophobicity, cell-material interaction, cardiomyocytes culture

The cell and tissue culture substrates play a pivotal role in the regulation of cell-matrix and cellcell interactions. The surface properties of the materials control a wide variety of cell functions.

Amongst various methods, layer-by-layer (LbL) assembly is a versatile surface coating technique for creating controllable bio-coatings. Here, polysaccharide/protein multilayers are proposed, which are fabricated by immersive LbL assembly and based on the chitosan /fibrinogen pair for improving the adhesion and spreading of cardiomyocytes. Two approaches in LbL assembly are employed for clarifying the effect of the bilayers order and their concentration on cardiomyocytes viability and morphology. Fourier transform infrared spectroscopy (FTIR) measurements show that the adsorption of the biopolymers is enhanced during the LbL deposition in a synergistic manner. Contact angle measurements indicate that the multilayers are alternating from less to more hydrophilic behavior depending on the biopolymer that is added last. Confocal microscopy with immunostained fibrinogen reveals that the amount of the protein is higher when the concentration of the immersion solution is increased, however, for low solution concentration it is speculated that interdigitation between the separate biopolymer layers takes place. This work motivates the use of fibrinogen in polysaccharide/protein multilayers for enhanced cytocompatibility in cardiac tissue engineering.

Introduction

One of the critical aspects of biomaterials for tissue engineering and regenerative medicine is their ability to control cell functions such as adhesion, morphology, migration, differentiation and proliferation. The surface properties of biomaterials and generally tissue culture substrates play a pivotal role in the regulation of cell-matrix and cell-cell interactions [1] . Therefore, surface functionalization with biomimetic coatings is of paramount importance towards the direction of resembling the extracellular matrix microenvironment. In specific, coatings with natural biopolymers have the advantage of possessing the necessary biochemical cues for cells attachment and at the same time their degradation products are non-toxic and have a low immune response [2] .

Various methods for surface functionalization with biomimetic coatings have been employed, but layer-by-layer (LbL) assembly consists of a versatile and low-cost coating technique leading to controllable bio-coatings. [3] . In this technique, polyelectrolyte multilayers (PEMLs) are fabricated that consist of alternating anionic and cationic polymers in aqueous media that self-assemble on an interface driven by electrostatic interactions [4], [5] . This way, stable films with high adsorbed amounts can be obtained whereas in simple adsorption of a single polyelectrolyte the effect would be weak [6] . Protein-polyelectrolyte (or polysaccharide) complexation can be used to introduce the versatility of proteins regarding their pH-dependent charge distribution and patchy hydrophobic surface [7,8] . Recently the multilayer formation between polyelectrolytes and protein/polyelectrolyte complexes was proposed as a method for stable multilayers [9,10] . PEMLs are proposed for applications related to tissue engineering [11,12] , drug delivery [13] and cell culture in general [14][15][16][17] . LbL self-assembled PEMLs have effectively maintained the concentration of a growth factor for cell cultures to avoid repeated growth factor addition and intervention [14] . Interdigitation between the polyelectrolytes in PEMLs has been suggested as a modulating factor for cell adhesion [15] and cell morphology and growth [18] .

Even though LbL assembly is a well-known technique, the combination of materials for assembling functional coatings for cardiac tissue engineering has not yet been explored. In this work we propose polysaccharide/protein multilayers (PPMLs) based on the chitosan (Chit)/fibrinogen (Fbg) pair in order to improve the adhesion and spreading of cardiomyocytes in culture. Chit as a polysaccharide is biodegradable, biocompatible and non-toxic [19] , and has been used in tissue engineering and cell growth studies in the form of hydrogels [20] , films [21,22] and nanoparticles [23] . Fbg is a blood plasma protein and has the ability to polymerize into fibrin and in this way plays a major role in blood clotting and platelet aggregation [24] . Its active sites allow for binding fibroblasts, endothelial cells and their growth factors [25,26] . Micro-and nanostructured Fbg-based biomaterials have shown potential for the delivery of drugs, biomolecules and genes in tissue engineering applications [27] . In this work, we employed LbL deposition of five bilayers of Chit/Fbg in order to create stable films that can support the culture of cardiomyocytes. We studied the effect of the layers' order as well as of their concentration on the cardiomyocytes attachment and spreading. Therefore, we deployed PPMLs where the top layer, which was in contact with the cells, in one case was Fbg and in the other case Chit.

Then the effect of their concentration on cell adhesion and spreading was evaluated by using two different concentrations for both Chit and Fbg.

Results and Discussion

PPMLs physicochemical characterization

Fbg has a well-documented trinodular structure. It consists of three nodules (D-E-D domain), which are connected by two triple-stranded amino-acid helices [28] . The two-end αC domains interact electrostatically with the central E domain in a manner that is pH-dependent [29] . E and D domains are negatively charged at neutral pH and positively charged at acidic pH (below 3.5). αC domains are positively charged at both neutral and acidic pH as they contain arginine and lysine residues. Therefore, near Fbg's isoelectric point (pI=5.5-5.8) Fbg contains domains of both negative (E and D) and positive (αC) charge [30,31] . The αC domains of Fbg are hydrophilic while E and D domains are hydrophobic [32,33] . Adsorption of Fbg and plasma proteins on solid surfaces has been thoroughly investigated because it is very crucial for the interaction of blood plasma with biomaterials [34,35] . Fbg can adsorb on hydrophilic surfaces by electrostatic interactions and to hydrophobic surfaces by anchoring its hydrophobic groups.

FTIR experiments were performed on PPMLs deposited on gold-coated surfaces. This was done because glass lamellae would not support the mechanical stresses from the ATR tool press and more importantly did not provide adequate reflection of light for ATR detection. Although the substrate used was not the actual substrate where the ML films for cell culture were formed, the synergistic accumulation of the two biopolymers and the interactions with each other in the formed films, can be characterized. In addition, it can be assumed that the top layers of both the MLs either on glass or on gold, which are away from the solid/water interface, have similar properties. Adsorption from separate pure solutions on gold surfaces is expected due to hydrophobic moieties on Chit [36,37] (hydrophobic content of Chit affects its interactions even at high degrees of deacetylation) and Fbg [32] . Experiments with pure Fbg solutions have shown that 5 min of adsorption is enough to reach adsorbed amounts higher than 10 mgm -2 on gold even for 0.1 mg.ml -1 solution concentration [38] . More importantly, at times longer than 5 min adsorption kinetics is greatly slowed down as adsorption proceeds towards plateau coverage.

Therefore, the multilayer deposition approach is expected to effectively increase the immobilized amount of Fbg as it exploits the Chit/Fbg interactions. The characteristic bands of adsorbed Fbg and Chit are found in Figure 1a andc and Figure 1b and d respectively as first layers. There is a distinctive region at about 1100 cm -1 where Chit's characteristic groups absorb [39][40][41] . Fbg characteristic bands amide I and II are found at 1652 and 1541 cm -1 respectively [42,43] . Chit's amide II bending (1647-1650 cm -1 ) and N-H bending (1535 cm -1 ) are compatible with the FTIR spectra although they are near Fbg' amide I and II bands [44,45] . The characteristic bands generally increase in intensity upon successive depositions, which shows the multilayer build-up. It has to be noted that this was not an in-situ experiment but nevertheless measurements were taken on at least 3 different spots of the gold surface to confirm homogeneity and reproducibility. In any case, it is informative to discuss the evolution of separate regimes of the spectra. The increase in absorbance at e.g. 1100 cm -1 is possibly related to adsorbed Chit, because Fbg does not show any strong characteristic absorption in the region 1200-1000 cm -1 (Figure 1). Sequential adsorption on preformed layers of Fbg is especially evident in Fig. 1c. Enhancement at 1100 cm -1 is observed from the 1 st to the 2 nd layer and from the 9 th to the 10 th layer (corresponding to Chit addition). This effect is not found in Figure 1 b andd where Fbg is the added 2 nd and 10 th layer. At the same time the increase in the amide I and II bands of Fbg is not so strong when Chit is added (2 nd and 10 th stage in Figure 1a andc) in comparison to the case when Fbg is added (2 nd and 10 th stage in Figure 1b andd). This is because these peaks appear relatively weaker in Chit (Figure 1b and d, 1 st layer) in comparison to Fbg (Figure 1c). A band at 1265 cm -1 that appears only when Chit is present in the layers can be attributed to characteristic CH2 wagging vibrations and combined N-H deformation and C-N stretching [45,46] . This band increases as more Chit is added on the layers. Therefore, ATR-FTIR experiments show that sequential adsorption of the two biopolymers effectively increases their adsorption on a surface in a synergistic manner and can be used to create multilayers on other surfaces too. Fbg is expected to adsorb strongly on glass surfaces at pH values both below and above its pI because it contains positively charged groups that may anchor to the negative charges on the glass/water interface [31,47] . Adsorption times shorter than 1 min are enough for Fbg adsorption on hydrophilic SiO2 surfaces at neutral pH [48] . Glass surfaces maintain their negative surface charge at pH values down to 2 [31] . Adsorption of cationic polymers on glass surfaces is driven by the negative surface charge of glass when in contact with water [49] , while Chit remains positively charged up to pH 6.5 [50] .

Contact angle (CA) experiments provide straight-forward observation of the wettability properties of surfaces. In Figure 2 the LbL formation of Chit/Fbg MLs is followed by CA measurements. The CA on clean glass lamella was CA= 53±2° which is compatible with literature results [51,52] . At low solution concentrations (0.1 mg.ml -1 ) when Fbg is the first component, adsorption and layer formation on the glass/water interface changes CA significantly (Figure 2a). CA drops towards lower values pointing to a more hydrophilic interface. The hydrated layer of adsorbed Fbg is hydrophilic, apparently due to the dissociated Fbg groups. The CA value of about 40° is compatible with the value measured for hydrated Fbg films on glass i.e. 46.5° [53] . Applying the Chit solution leads to an increase in CA (Figure 2a). This tendency towards hydrophobic behavior comes from the fact that Chit interacts electrostatically with the pre-adsorbed Fbg. Upon neutralization of the negative charges of Fbg's E and D domains their hydrophobic nature is more pronounced. In addition, neutralization of Chit monomers makes its chain more hydrophobic due to the presence of methyl groups [54][55][56] . Alternating Chit and Fbg layers lead to a saw-tooth pattern with successive changes from less to more hydrophilic behavior respectively.

The situation is very similar when Chit is the first layer (Figure 2b). Nevertheless, application of the Chit 0.1 mg.ml -1 solution on bare glass lamella has no significant change on the CA, indicating its low initial adsorption. However, the saw-tooth pattern follows similarly to Fig. 2a after the application of Fbg solution. Consequently, alternating Chit with Fbg leads successively to more hydrophobic or more hydrophilic layers (respectively) either if Chit or Fbg is the first solution applied on the glass surface. In more detail, when Fbg is the first layer the mean CAs for Chit and Fbg as top layers are 72°±12° and 39°±7° respectively whereas for Chit as a first layer the values are 68°±7° and 53°±17° (Figure 2a andb).

For solutions of high concentration (1 mg.ml -1 ) (Figure 2c andd) a saw-tooth pattern is also followed, at least for 6 layers when Fbg is the first layer (Figure 2c), and for all 10 layers when Chit is the first layer although with a weaker intensity (Figure 2d). When Fbg is the first layer the mean CAs for Chit and Fbg as top layers are 61°±10° and 48°±11° respectively whereas for Chit as a first layer the values are 61°±8° and 51°±3° (Figure 2c andd). In conclusion, the hydrophilicity/hydrophobicity of the mixed layers can be tuned by choosing one of the two components as the outermost component (especially by adsorption from solutions of 0.1 mg.ml - 1 ).

In addition, the various PPMLs were immunostained with an antibody against Fbg for identifying the differences in the amount of the attached Fbg on the glass. Confocal microscopy analysis showed that all the combinations of PPMLs contain a high amount of Fbg (Figure 3).

The fluorescent intensity of Fbg is higher when it corresponds to the last deposited layer, which indicates a degree of separation between the sequentially deposited layers (Figure 3b,d). In addition, the fluorescent intensity of Fbg is higher when the solution concentration is higher (Figure 3d). The Fbg layer appears to be less uniform in the case of PPMLs with Chit as last layer obtained using the lower concentration, leading to the speculation that interdiffusion of Fbg and Chit layers took place (Figure 3a). These interpenetrated layered structures are in accordance to the literature, indicating that immersive LbL assembly leads to films that grow in a nonlinear manner [57,58] . This strong interpenetration possibly allows the formation of network structures consisting of Fbg molecules which could explain the morphology observed in Figure 3a. In any case confocal microscopy with immunostained Fbg proves the successful deposition of PPMLs on the glass substrate and illustrates the tunability of the adsorbed amount (see movies, SI).

The information derived from fluorescence microscopy can be useful for identifying the optimized coating amount for efficient cell culture. Combining the results of FT-IR and contact angles with the confocal images, it seems that the PPMLs with higher concentration can very possibly lead to the same amount of deposited Chit and Fbg. Aiming to scrutinize the synergistic effect of Chit and Fbg, we proceeded with the cell culture studies using the PPMLs having Chit as last layer (Figure 3a,c). In this case, there are most certainly Chit segments exposed to the exterior. As the layers appear mixed, Fbg units are possibly also exposed to the external medium. Therefore, in the particular PPMLs (with Chit as the last added layer) both biopolymers certainly affect cell-PPML interactions.

Cardiomyocytes cytocompatibility on PPMLs

The cytocompatibility of both Chit and Fbg has been reported in various studies using different cell types [25,26,59] . Nevertheless, the combination of both biomaterials as coating for cell culture

has not yet been explored. In this work, we aimed at scrutinizing the interaction of these PPMLs with cardiomyocytes. It has to be pointed out that cardiomyocytes do not adhere, consequently do not survive, in pure glass without a coating. In addition, this type of cells does not proliferate, rendering their successful culture challenging. Herein, we cultured primary cardiomyocytes (NRCMs) for 3 days on the surface of all samples and then we assessed their viability and morphology. Pure glass lamella served as control in addition to laminin coating, which is the most common used for NRCMs culture.

Before comparing the effect of multiple layers of Chit/Fbg on cell viability, we cultured NRCMs on single layers of Chit and Fbg with concentrations 0.1 and 1 mg.ml -1 . NRCMs survival was extremely low on the surfaces with one layer of Chit (Figure 4a-b). This result is in accordance with the literature, where poor NRCMs adhesion with rounded morphology was observed when cultured on Chit surfaces [60] . On the contrary, a single layer of Fbg was sufficient for the attachment and spreading of NRCMs, even at the lower concentration, indicating its strong adsorption to the glass substrate (Figure 4c-d, 5). same levels of single layers of Fbg and PPMLs. The PPML with high concentration exhibited higher viability, even though this difference is not significant when compared with laminin. We further assessed the cytocompatibility of PPMLs in terms of cell morphology employing immunofluorescence. Confocal microscopy analysis of cultured NRMCs on coated glass showed that they have an elongated shape, close to the in vivo one (Figure 6). While the small number of cells that attached and survived on glass lamella presented round shape with short bundles of α-actinin (Figure 6a). Among the various coatings, the PPML with Fbg as first layer with high concentration showed better-defined striations of sarcomeres as indicated by αactinin immunostaining (Figure 6f,h). For better visualization of the sarcomeres, the Figure 6g,h shows magnified confocal images of cells stained with phalloidin together with α-actinin immunolabelling on laminin-coated and PPMLs of 1mg.ml -1 samples. Phalloidin stains the actin filaments, which consist part of the sarcomere together with myosin II filaments [61] . It is obvious that laminin coating did not lead to the creation of well-structured striations of the sarcomeres as in the case of PPMLs of 1mg.ml -1 . A well-constructed sarcomere is key element for the function of cardiomyocytes as it consists their contractile unit [62] , and is of paramount importance when developing in vitro cardiac models. As cardiac contractile performance is highly dependent on sarcomere length (SL), we calculated the latter and compared it to the literature [63] . The SL of NRCMs cultured on PPML with high concentration is 1.72 ± 0.2 μm, which is within the range of the reported values (Figure S1) [64][65][66] . The obtained SL (1.61 ± 0.54 μm) upon culture on laminin coating is not only lower but also exhibits high variation, thus further validating the results of immunofluorescence. Combining these observations with the viability results, we can conclude that the PPML with Fbg as first layer and concentration of 1mg.ml -1 is the most suitable coating for the culture of NRCMs.

Therefore, these findings can be used for the functionalization of scaffolds based on polycations with Fbg via LbL assembly. In this way a composite material will be created combining favourable properties of both biomaterials, which is a key parameter in cardiac tissue engineering [67] . The multilayer structure and internally modulated hydrophilicity of the composite coatings allows for the entrapment of various active components that could be beneficial for the differentiation of cells. In addition, LbL construction including Chit provides a means to tune the adsorbed amount of Fbg and a quasi-3D structure to the adsorbed films.

Conclusion

We investigated two approaches in LbL assembly for unmasking the optimal PPMLs combination that leads to improved cardiomyocytes viability and morphology: the bilayers order namely the coating that is in contact with the cell surface, and the biopolymers' concentration. Concentration increase was found to enhance the amount of adsorbed protein while it reduced the degree of mixing between the successive layers. The choice of the biopolymer that was added on top was found to influence the hydrophilicity of the Chit/Fbg coating as Chit led to more hydrophilic layers in comparison to Fbg. Pure Fbg coating was sufficient for good NRCMs adhesion and spreading, but NRCMs morphology is superior when cultured on 5 bilayers of PPMLs of 1 mg.ml -1 concentration. This result was further confirmed by the value of sarcomere length (SL), which is a key determinant of cardiac mechanical function. Additionally, the presence of Chit facilitates the tuning of Fbg adsorbed amount and the incorporation of other bioactive compounds that could bind to Fbg. These findings are useful for the optimization of the surface properties of biomaterials that can be applied to cardiac tissue engineering. For example, synthetic scaffolds can be functionalized by the proposed LbL assembly for enhancing their cytocompatibility. In addition, these PPMLs exhibited long shelf-life, which increases the versatility of their use in cell cultures.

Experimental Section

Materials: Chit (low molecular weight, 75-85% deacetylated) and Fbg from bovine plasma (type I-S, 65-85% protein) were purchased from Sigma and were used without further treatment. Stock solutions of Chit (1 mg.ml -1 ) or Fbg (10 mg.ml -1 ) were prepared by dissolving the Chit powder in aqueous solution of acetic acid (pH 5) and Fbg in a 0.15 M NaCl solution (pH 7) and were left overnight at 4 °C to equilibrate. Solutions at lower concentrations were prepared by diluting the stock solutions with their aqueous solvents. Laminin (L2020, 1-2 mg.ml -1 in Tris buffered NaCl) was purchased from Sigma-Aldrich and was stored at -20°C.

Before use, laminin was diluted in Dulbecco's phosphate buffer saline (DPBS) (Thermo Fisher Scientific, Saint-Herblain, France), in order to obtain a final concentration of 0.02-0.04 mg.ml - 1 .

Figure 1 .

 1 Figure 1. ATR-FTIR spectra at 1 st (black), 2 nd (red), 9 th (green) and 10 th (blue) stage of the sequential adsorption of 0.1 mg.ml -1 : Fbg (a), Chit (b) as the first layer and 1.0 mg.ml -1 : Fbg (c), Chit (d) as the first layer of polysaccharide/protein multilayers (PPMLs).

Figure 2 .

 2 Figure 2. Contact angle from Chit (red)/Fbg (blue) MLs on glass substrate (black) from solutions of 0.1 mg.ml -1 : Fbg (a), Chit (b) as the first layer and 1 mg.ml -1 : Fbg (c), Chit (d) as the first layer of polysaccharide/protein multilayers (PPMLs).

Figure 3 .

 3 Figure 3. Fluorescent images of Fbg on the different PPMLs of five alternating bilayers from solutions of 0.1 mg.ml -1 : Fbg (a), Chit (b) as the first layer and 1 mg.ml -1 : Fbg (c), Chit (d) as the first layer (the sample was scratched, for this reason the dark area appears).

Figure 4 .

 4 Figure 4. Optical live images of NRCMs seeded on single layers of Chit (a, b) andFbg (c, d) with concentrations of 0.1 (a, c) and 1 (b, d) mg.ml -1 respectively.

Figure 5 .

 5 Figure 5. Viability results of NRCMs cultured on single layers (1L) of Chit and Fbg with concentrations of 0.1 (low) and 1 (high) mg.ml -1 and on PPMLs with Fbg as first layer and concentrations of 0.1 (low) and 1 (high) mg.ml -1 , compared with laminin coating and bare glass lamellae.

Figure 6 .

 6 Figure 6. Confocal images of NRCMs seeded on: glass lamella (a) and laminin coating (b), single layers of Fbg with concentrations of 0.1 (c) and 1 (d) mg.ml -1 , PPMLs with Fbg as first layer and concentrations of 0.1 (e) and 1 (f) mg.ml -1 . (g)-(h): zoomed images of (b) and (f), respectively. Scale bar a-f: 30μm, g-h: 20μm. Green: α-actinin, red: phalloidin, blue: nuclei.

Acknowledgements

The authors thank Dr. Georgios D. Chryssikos and Dr. Eirini Siranidi for their help in ATR-FTIR measurements. The authors also thank the personnel of the Photon Microscopy Facility of IBPS for helpful advice and technical assistance during microscopy image acquisition and analysis. This work was supported by the LabEx REVIVE (ANR-10-LABX-73). Also, this work has been sponsored by the Ile-de-France Region in the framework of Respore, the Île-de-France network of Excellence in Porous Solids". MK acknowledges personal funding from both LabEx REVIVE and DIM Respore too.

Formation of polysaccharide/protein multilayers (PPMLs) based on the Chit/Fbg pairs:

Films of alternating Chit/Fbg were deposited on glass lamellae by the LbL deposition method.

Briefly, fresh glass lamellae were rinsed with distilled water and dried under nitrogen stream to remove any dust particles. A glass lamella of 12mm-diameter was immersed vertically in the solution of Chit or Fbg for 5 min before it was gently rinsed with water in order to remove material that was not adsorbed on the surface. The procedure was repeated for the other macromolecule i.e. Fbg or Chit respectively, until five pairs (bilayers) were deposited. Two different solution concentrations were used for Chit and Fbg: 0.1 and 1.0 mg/ml to prepare Chit/Fbg multilayers. The samples were dried at room temperature and then were stored at 4 0 C until use. The coatings were stable even after six months of storage indicating their long shelflife. For the control experiments with laminin, the coatings were prepared just before the cell culture. The diluted laminin solution was deposited on top of the glass lamellae for 2 h at room temperature, followed by a wash with DPBS.

FTIR experiments: FTIR measurements were realized on gold-coated microscope slides.

The LbL deposition was performed on a commercial gold-coated (100 nm) microscopy slide.

An attenuated total reflectance (ATR) diamond accessory, from SENS-IR, and a press were used to record Infrared spectra on a Bruker Equinox 55 Fourier Transform Instrument. Two measurements were taken from different spots of the glass slides placed at the center of the sample holding device and 64 scans were performed in the range 500-5,000 cm -1 , at a resolution of 2 cm -1 .

Contact angle (CA) experiments:

CA measurements of water sessile drops were performed, using a custom-made experimental set-up, on the glass lamellae that were utilized for the cell studies. Images of 5 μL water drops on the surface of each substrate after rinsing with water were obtained by a 2.0MP 500x USB Digital Microscope within 30 s from the droplet deposition. The drop profiles were analyzed by standard MATLAB functions. In brief, the drop edge profile was determined after transforming the images to grayscale. For numerical calculations the origin of the coordinates was chosen on the intersection between the apparent perpendicular axis of symmetry of the drop and the solid/liquid interface. Edge profiles were treated in polar coordinates (𝑟, 𝜃) because 𝑟(𝜃) varies smoothly with 𝜃 and is hence effectively fitted by polynomial functions. The values of CA were extracted from the derivatives on the left and right-side contacts of the drop with the surface and transformed back to Cartesian coordinates. The average of the two values from different spots on the glass lamella was reported as the measured CA. Typically, the deviation between left and right contact angles was less than 2° and their average was taken as the CA for the particular sessile drop. The CAs were measured at room temperature.

Isolation of neonatal rat cardiomyocytes: Newborn rat cardiomyocytes (NRCMs) were obtained from 1-day-old Wistar RjHan rat (Janvier Labs, Saint-Berthevin, France). The hearts were cut in small pieces and the cardiac cells were isolated, using a neonatal heart dissociation kit (Miltenyi Biotec, Paris, France) according to the manufacturer's instructions.

Cardiomyocytes were purified by depletion of non-target cells (such as fibroblasts, endothelial cells), using a neonatal cardiomyocyte isolation kit (Miltenyi Biotec, Paris, France). 5 x 10 5 cells were seeded on glass lamellae of 12mm-diameter coated with the PPMLs and cultured with DMEM medium (with 4.5g/L D-Glucose and without Na Pyruvate) supplemented with Lglutamine, penicillin-streptomycin, 10% horse serum and 5% fetal bovine serum (FBS) at 37 °C and 5% CO2 for 3 days. Immunostaining and cell morphology analysis: For the Fbg immunostaining, PPMLs samples without cells were immunostained using an antibody against Fbg (rabbit polyclonal antibody, dilution 1:200, Bioss Antibodies). After 3 washes in PBS, the samples were mounted with mowiol.

The NRCMs, cultured on PPMLs and controls, were washed with PBS and fixed with 4% paraformaldehyde solution. After 3 washing steps, they were incubated with 5% bovine serum albumin (BSA) containing 0,1 % Triton X-100 for 1 hour. The cells were incubated for 90 minutes at room temperature with an antibody against α-actinin (mouse IgG, dilution 1:300, Sigma-Aldrich, Saint-Quentin-Fallavier, France). After 3 washes in PBS, cells were incubated in the presence of the secondary antibody (Alexa 488 goat-anti-mouse) and Alexa Fluor® 647 phalloidin (Life technologies). Then samples were mounted with mowiol containing 5 µg/ml Hoescht 33342 (Life Technologies).

All images were captured using a motorized confocal laser scanning microscope (Leica TCS SP5). Sarcomere length (SL) was measured by α-actinin immunostaining using ImageJ.

Viability assay: The viability of NRCMs was assessed by using the Muse® Count & Viability Kit. The kit differentially stains viable and non-viable cells based on their permeability to the two DNA binding dyes present in the reagent. The measurements were repeated at least 3 times, using 3 samples per time.

Statistics: Analyses were conducted using GraphPad Prism 8.00 (GraphPad Software Inc., SD, USA). For one-way analysis of variance, the normality was checked using the Shapiro-Wilk normality test. If the normality of distribution assumption was not met, the nonparametric Kruskal-Wallis test was used instead of ANOVA. If a significant difference was found, then multiple comparison tests were performed to compare the different groups analyzed by controlling the False Discovery Rate. A p-value < 0.05 was considered significant. Values are given as the means ± standard error of the mean.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Conflicts of interest

There are no conflicts to declare.


Jeanne Kergomard

Mélina Robert

Nathalie Barouh

Gilles Paboeuf

Bruno Barea

Pierre Villeneuve

Olivier Schafer

Tim J Wooster

V Vié

Effects of processing and concentration on the oxidative stability and interfacial behavior of tree nuts

Jeanne Kergomard 1,2* , Mélina Robert 3 , Nathalie Barouh 3 , Gilles Paboeuf 2 ,Bruno Barea 3 , Pierre Villeneuve 3 , Olivier Schafer 4 , Tim J. Wooster 4 , Véronique Vié 1 and Claire Bourlieu 2

Conclusion:

Overall, the results indicate that the fat content tends to stabilize the cashew juices and pastes, in particular thanks to the presence of free phenolic compounds in the matrix, which plays the role of sacrificial agents against oxidation. The FA profile, rich in saturated and monounsaturated fatty acids, also helps to limit the phenomenon of lipid oxidation. The processing tends to destabilize the systems and the larger scale of homogenization has induced an alteration of the OB interface. As previously described, the native OB assemblies are remarkably stable against oxidation, which raises the interest of their use in food applications.

Results: Impact of ultra-structure -Does processing induced an increase in the reactivity of the systems?

Material and methods

In this aim, we need to understand i) the impact of fat content in the matrix, and ii) the modification of ultrastructure induced by usual industrial processes (High pressure homogenization, heat treatment).

Introduction:

The demand for plant-based beverages as an alternative to dairy products has considerably increased over the recent years.

To preserve the nutritional qualities of tree nut oil bodies (OB) in these products, we need to control their physico-chemical stability.

Results: Impact of fat content -Does increasing fat concentration tend to stabilize the systems?

What are OB?

VS OB dispersion Cashew juice/paste

Characterization

Oxidative challenge tests (PV, TBARS) Confocal microscopy Tensiometry/Ellipsometry Atomic Force Microscopy

Conclusion:

The results of the storage test on 4% wt. milks indicate that HHP* tends to destabilize the systems, however no significant difference was obtained between homogenization strength (around 2.0 meq0 2 /kg of fat for HHPH and HHPL after 30 days of storage test). Nevertheless, ellipsometric studies indicate that HHPH caused greater deterioration of compounds in the nut-juice system than HHPL. Indeed, the lag phase observed before the increase of the surface pressure indicates that the particles cannot properly stabilize at the interface, probably due to a reduction in hydrophobic interactions. AFM images confirm the hypothesis, with more aggregation at the interface of the HHPH sample. OB also show a very good stability to oxidation, which confirms the relevance of keeping the native assembly, as shown in a previous study on walnuts [Kergomard et al., Food Chem, 2021].

*

  Corresponding author: Jeanne.kergomard@univ-rennes1.fr EFFECTS OF PROCESSING AND CONCENTRATION ON THE OXIDATIVE STABILITY AND INTERFACIAL BEHAVIOR OF TREE NUTS OIL BODIES (OB)

  tree nuts, OB are remarkably stable lipoprotein assemblies that are rich in essential nutrients (polyunsaturated fatty acids, tocopherols, antioxidant phenolic compounds…). Level of processing HHPH* vs HHPL** Fat content 4 vs 25 vs 38% wt.

  pressure)  molecular interfacial interactions Δ (ellipsometric angle)  amount of matter at the a/w interface The results show that increasing fat content tends to stabilize the systems over time. Values of 25.2 ± 2.6 and 68.4 ± 5.2 meqO 2 /kg of fat were reported for 4% wt and 25% wt. respectively after 75 days, supporting this hypothesis. The stability of the three matrices over 30 days of storage can be explained by their high content of free phenolic compounds, which can act as sacrificial agents and protect lipids from oxidation.The FA composition may also contribute to the chemical stability of the systems, as the samples are composed of 20% saturated fatty acids and 60% oleic acid, less prone to oxidation than PUFA.Milk HHPL vs Milk HHPH vs OB (4% wt.) Roasted milk, RP: Roasted paste

  pf= 12,66 ± 0,17 mN/m *HHPH: High pressure homogenization High strength (300/30 bars), **HHPL: High pressure homogenization Low strength (90/10 bars)