Céline Douat

Mégane Bornerie

Stéphanie Antunes

Gilles Guichard

Antoine Kichler

Hybrid Cell-Penetrating Foldamer with Superior Intracellular Delivery Properties and Serum Stability

Sequence specific molecules with high folding ability (i.e. foldamers) can be used to precisely control the distribution and projection of side chains in space and have recently been introduced as tailored systems for delivering nucleic acids into cells. Designed oligourea sequences with an amphipathic distribution of Arg-and His-type residues were shown to form tight complexes with plasmid DNA, and to effectively promote the release of DNA from the endosomes. Herein, we report the synthesis of new cell-penetrating foldamer sequences in which the foldamer segment is conjugated via a reducible disulfide bond to a ligand that binds cell-surface expressed nucleoproteins with the idea that this system could facilitate both assemblies with nucleic acids and cell entry. This new system was evaluated for delivery of DNA in several cell lines and was found to compare favorably with all comparators tested (DOTAP and b-PEI as well as a number of known cell penetrating peptides) in various cell lines and particularly in culture medium containing up to 50% of serum. These results suggest that this dual molecular platform which is long lasting and non-cytotoxic could be of practical use for in vivo applications.

INTRODUCTION

In recent years, in a context of research seeking for modern pharmaceutical strategies, there has been a strong interest in the development of new and safe transport systems for the intracellular delivery of drugs and biomolecules such as nucleic acids (NA). A large fraction of engineered molecular transporters are inspired by cell-penetrating peptides (CPPs) [START_REF] Hoyer | Peptide Vectors for the Nonviral Delivery of Nucleic Acids[END_REF][START_REF] Bechara | Cell-Penetrating Peptides: 20 Years Later, Where Do We Stand[END_REF][START_REF] Boisguérin | Delivery of Therapeutic Oligonucleotides with Cell Penetrating Peptides[END_REF] and share a common chemical feature i.e. the presence of multiple guanidinium units in their structure (either part of the main chain or as side chain appendages). [START_REF] Rothbard | Arginine-Rich Molecular Transporters for Dr g Deli ery: ole of ackbone Spacing in Cell lar Uptake[END_REF] Guanidinium moieties facilitate cell intake by providing key interactions with anionic phosphate head groups of membrane lipids [START_REF] Futaki | Cell-Surface Interactions on Arginine-Rich Cell-Penetrating Peptides Allow for Multiplex Modes of Internalization[END_REF][START_REF] Takeuchi | Current Understanding of Direct Translocation of Arginine-Rich Cell-Penetrating Peptides and Its Internalization Mechanisms[END_REF][START_REF] Tung | Arginine Containing Peptides as Delivery Vectors[END_REF] and also mediate NA condensation and transfection. [START_REF] Emi | Gene Transfer Mediated by Polyarginine Requires a Formation of Big Carrier-Complex of DNA Aggregate[END_REF][START_REF] Alhakamy | Effect of Lipid Headgroup Charge and pH on the Stability and Membrane Insertion Potential of Calcium Condensed Gene Complexes[END_REF] Seminal works by Wender et al. have shown the possibility to efficiently mimic arginine-rich CPP with guanidinium-containing oligocarbamates, [START_REF] Wender | Oligocarbamate Molecular Transporters: Design, Synthesis, and Biological Evaluation of a New Class of Transporters for Drug Delivery[END_REF] a class of backbone that has been reinvestigated recently by Kumar et al. for NA delivery. [START_REF] Patil | Highly Efficient (R-X-R)-Type Carbamates as Molecular Transporters for Cellular Delivery[END_REF] Meanwhile, with the advent of foldamers, [START_REF] Gellman | Folda ers: A Manifesto[END_REF][START_REF] Guichard | Synthetic Foldamers[END_REF] various folded molecular architectures have been investigated for their capacity to spontaneously cross cellmembranes. Peptoids, [START_REF] Wender | The Design, Synthesis, and Evaluation of Molecules That Enable or Enhance Cellular Uptake: Peptoid Molecular Transporters[END_REF][START_REF] Unciti-Broceta | Synthesis, Penetrability and Intracellular Targeting of Fluorescein-Tagged Peptoids and Peptide-Peptoid Hybrids[END_REF][START_REF] Murphy | A Combinatorial Approach to the Discovery of Efficient Cationic Peptoid Reagents for Gene Delivery[END_REF] and γ-peptides [START_REF] Umezawa | Translocation of a β-Peptide Across Cell Membranes[END_REF][START_REF] Potocky | Effects of Conformational Stability and Geometry of Guanidinium Display on Cell Entry by Beta-Peptides[END_REF][START_REF] Demizu | A Preorganized β-Amino Acid Bearing a Guanidinium Side Chain and Its Use in Cell-Penetrating Peptides[END_REF][START_REF] Appelbaum | Arginine Topology Controls Escape of Minimally Cationic Proteins from Early Endosomes to the Cytoplasm[END_REF][START_REF] Daniels | Intrinsically Cell-Permeable Miniature Proteins Based on a Minimal Cationic PPII Motif[END_REF][START_REF] Farrera-Sinfreu | Cell-Penetrating Cis-γ-Amino-L-Proline-Derived Peptides[END_REF] and aromatic oligoamides [START_REF] Gillies | Development and Biological Assessment of Fully Water-Soluble Helical Aromatic Amide Foldamers[END_REF][START_REF] Iriondo-Alberdi | Cellular Internalization of Water-Soluble Helical Aromatic Amide Foldamers[END_REF] have shown promising results in term of cellular uptake. It has been postulated that the folded topology of these synthetic oligomers could play a key role in cell-membrane crossing. In most cases the defined helical geometry segregates the guanidinium moieties on one face of the structure thus conferring a facial amphiphilicity to these folded systems. [START_REF] Tang | Helical Poly(arginine) Mimics with Superior Cell-Penetrating and Molecular Transporting Properties[END_REF] This concept was also applied successfully to α-peptides, recent examples includ-ing the development of rigidified cationic polyproline II helix architectures, [START_REF] Nagel | Effect of Preorganized Charge-Display on the Cell-Penetrating Properties of Cationic Peptides[END_REF][START_REF] Fillon | Cell Penetrating Agents Based on a Polyproline Helix Scaffold[END_REF] and stabilized α-helices containing either aliphatic or cationic α,α-disubstituted amino acids [START_REF] Wada | Effect of Ala Replacement with Aib in Amphipathic Cell-Penetrating Peptide on Oligonucleotide Delivery into Cells[END_REF][START_REF] Yamashita | A Helix-Stabilized Cell-Penetrating Peptide as an Intracellular Delivery Tool[END_REF][START_REF] Kato | Plasmid DNA Delivery by Arginine-Rich Cell-Penetrating Peptides Containing Unnatural Amino Acids[END_REF] or hydrocarbon crosslinks between side chains. [START_REF] Tian | Achieving Enhanced Cell Penetration of Short Conformationally Constrained Peptides through Amphiphilicity Tuning[END_REF] Despite these significant advances, the interplay between folding, guanidinium distribution and cell penetration is often investigated in the absence of cargo and only few studies have reported cellular delivery of active molecules such as NAs. [START_REF] Tang | Helical Poly(arginine) Mimics with Superior Cell-Penetrating and Molecular Transporting Properties[END_REF] In this context, we have recently designed a first generation of cationic urea-based cell-penetrating foldamers (CPFs) as gene delivery system and we have shown that by introducing thiol moieties for effective foldamer dimerization (e.g. 1), we could 1) amplify the coating of 2.5-helical oligoureas on plasmid DNA (pDNA) and 2) facilitate the effective vector unpacking within the cell by a redox mechanism. [START_REF] Douat | A Cell-Penetrating Foldamer with a Bioreducible Linkage for Intracellular Delivery of DNA[END_REF] The resulting foldamer/pDNA assemblies, referred to as foldaplexes, exhibited high transfection efficiency with gene expression levels comparable to those obtained with branched polyethylenimine (b-PEI), N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) and LAH4 peptide, a potent 26residue long CPP developed for gene delivery. [START_REF] Kichler | Histidine-Rich Amphipathic Peptide Antibiotics Promote Efficient Delivery of DNA into Mammalian Cells[END_REF] CPF 1 which exhibits an amphipathic distribution of side chains along the 2.5-helix axis (Figure 1a), contains only one Argfor two His-type residues per oligourea segment, the Histype residues (His U ) being introduced to promote endosomal escape (i.e. so called proton sponge effect). [START_REF] Boussif | A Versatile Vector for Gene and Oligonucleotide Transfer into Cells in Culture and in Vivo: Polyethylenimine[END_REF][START_REF] Kichler | Polyethylenimine-Mediated Gene Delivery: A Mechanistic Study[END_REF] In this work, we have considered the possibility to increase further the transfection efficacy by combining the CPF sequence used in 1 with a multimeric cationic (Arg-rich) pseudopeptide (N5L) reported to bind nucleoproteins including cell-surface expressed nucleolin (NCL). [START_REF] Destouches | A Simple Approach to Cancer Therapy Afforded by Multivalent Pseudopeptides That Target Cell-Surface Nucleoproteins[END_REF][START_REF] Destouches | Suppression of Tumor Growth and Angiogenesis by a Specific Antagonist of the Cell-Surface Expressed Nucleolin[END_REF] NCL is known to play a key role in a variety of cellular processes such as transcription, replication and recombination of DNA and its expression although primarily known to be nuclear and cytoplasmic is enhanced at the surface of activated endothelial cells and of various types of cancer cells. [START_REF] Koutsioumpa | Cell Surface Nucleolin as a Target for Anti-Cancer Therapies[END_REF] Several recent studies have highlighted the potential of using NCL ligands (e.g. aptamer AS1411, [START_REF] Wu | Nucleolin Targeting AS1411 Modified Protein Nanoparticle for Antitumor Drugs Delivery[END_REF] the homing tumor peptide F3, [START_REF] Christian | Nucleolin Expressed at the Cell Surface Is a Marker of Endothelial Cells in Angiogenic Blood Vessels[END_REF] and multimeric pseudopeptides) [START_REF] Destouches | A Simple Approach to Cancer Therapy Afforded by Multivalent Pseudopeptides That Target Cell-Surface Nucleoproteins[END_REF][START_REF] Nisole | The Anti-HIV Pseudopeptide HB-19 Forms a Complex with the Cell-Surface-Expressed Nucleolin Independent of Heparan Sulfate Proteoglycans[END_REF] as anticancer agents but also to mediate receptor endocytosis and deliver active molecules into cells. Herein, we report the synthesis of the urea-based foldamer-peptide hybrid dimer (2) in which the CPF sequence of 1 was conjugated to a NCL pseudopeptide ligand via a reducible disulfide bond. This new system was evaluated for delivery of pDNA in several cell lines and was directly compared to known CPPs, oligourea 1 as well as cognate homodimers (Dim8 and Dim9, vide infra). We found that heterodimer 2 displays a remarkably high transfection efficacy, above that of the comparators in all cell lines tested and particularly in culture medium containing up to 50% of serum. These results suggest that this dual molecular platform could overcome some of the limitations of other NA delivery systems for in vivo applications.

RESULTS AND DISCUSSION

Hybrid Cell-Penetrating Foldamer Synthesis. The synthesis of the cysteamine-functionalized CPF sequence 8 used for the preparation of heterodimer 2 is shown in Scheme 1. The sequence of 8 differs from that in 1 by the position of the ureido cysteamine moiety. This modification was introduced to facilitate access of the corresponding oligourea using Boc chemistry by avoiding the need for the final HF cleavage of the resin. The solid-phase synthesis (SPS) using Boc chemistry is particularly robust, allowing the preparation of aliphatic oligoureas and related hybrids with good purities and yields. [START_REF] Teyssières | Proteolytically Stable Foldamer Mimics of Host-Defense Peptides with Protective Activities in a Murine Model of Bacterial Infection[END_REF][START_REF] Antunes | Solid-Phase Synthesis of Hybrid Urea Oligomers Containing Conservative Thiourea Mutations[END_REF][START_REF] Douat-Casassus | Microwave-Enhanced Solid-Phase Synthesis of , ′-Linked Aliphatic Oligoureas and Related Hybrids[END_REF][START_REF] Nelli | An Activated Building Block for the Introduction of the Histidine Side Chain in Aliphatic Oligourea Foldamers[END_REF] Here, the elongation of CPFs using Boc-protected activated monomers A was preferred over the use of azido building blocks [START_REF] Douat-Casassus | Microwave-Enhanced Solid-Phase Synthesis of , ′-Linked Aliphatic Oligoureas and Related Hybrids[END_REF] and TFAlabile resins, also because access to the activated His-type monomer required for such CPF sequences remains limited to its N-Boc protected version. [START_REF] Nelli | An Activated Building Block for the Introduction of the Histidine Side Chain in Aliphatic Oligourea Foldamers[END_REF] Our new strategy relies on the use of a resin equipped with a cystamine linker that can be cleaved under reductive conditions to release the CPF with a terminal thiol-function. It is worth mentioning that compared to standard oligourea synthesis on MBHA resin, there is no more requirement to introduce a -amino acid at the first position (see 1), [START_REF] Douat | A Cell-Penetrating Foldamer with a Bioreducible Linkage for Intracellular Delivery of DNA[END_REF][START_REF] Claudon | Consequences of Isostructural Main-Chain Modifications for the Design of Antimicrobial Foldamers: Helical Mimics of Host-Defense Peptides Based on a Heterogeneous Amide/Urea Backbone[END_REF] the cystamine-type linker and the first urea bond being stable to TFA treatment. Resins equipped with a cystamine linker have already been described in literature [START_REF] Quideau | Binding of Filamentous Actin and Winding into Fibrillar Aggregates by the Polyphenolic C-Glucosidic Ellagitannin Vescalagin[END_REF] and we hypothesized that besides its compatibility with the use of TFA needed for the successive, Boc deprotection steps, the disulfide bridge should also be resistant to microwave irradiation applied during the course of SPS. Resin 3 was prepared from MBHA resin as shown in scheme 1. To readily estimate the loading of resin 3 by colorimetric test (i.e. Kaiser test), the Boc-Lin-OH linker (4) was preassembled in solution [START_REF] Sun | A Collaborative Assembly Strategy for Tumor-Targeted siRNA Delivery[END_REF] prior to amide bond formation on the resin. Iterative coupling/deprotection cycles of activated succinimidyl carbamate monomers (A) bearing either Trp, Val or His side chains were next performed under classical coupling conditions and microwave irradiation. To note, an Orn-type monomer with an Alloc protecting group was incorporated in b position as a precursor of the Arg-type residue (Arg U ) because standard guanidine side-chain protecting group could not be used conveniently with our synthesis approach. After chain elongation was completed, resin-bound intermediate 5 was treated with palladiumtetrakis(triphenylphosphine) to remove the Alloc protection on the Orn U side chain and guanidinylation was subsequently performed using 1-amidinopyrazole hydrochloride (6) to furnish resin-bound oligourea 7. After final Tos deprotection of the two His U side chains of 7, oligourea 8 bearing a cysteamine moiety at first position [START_REF] Douat | A Cell-Penetrating Foldamer with a Bioreducible Linkage for Intracellular Delivery of DNA[END_REF] was successfully released from the support by reduction of the disulfide bridge in the presence of dithiothreitol (DTT) in basic medium and was recovered in 13% Scheme 1. New methodology relying on the use of a disulfide bridge resin (3) to assemble oligourea 8 on solid support. Abbreviations not defined in text: BOP, (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; DIEA, diisopropylethylamine; HOBt, hydroxybenzotriazole; Tos, tosyl; TFA, trifluoroacetic acid. yield after semi-preparative RP-HPLC purification. Heterodimer 2, was then obtained by disulfide bond formation between the C-terminal cysteine modified NCL multivalent ligand 9 [START_REF] Destouches | A Simple Approach to Cancer Therapy Afforded by Multivalent Pseudopeptides That Target Cell-Surface Nucleoproteins[END_REF] and the cysteamine spacer of oligourea 8 (Scheme 2). The formation of the disulfide bridge was carried out in two steps by first activating oligourea 8 in presence of 2,2′-dithiobis(5-nitropyridine) [START_REF] Douat-Casassus | Selective Catalysis with Peptide Dendrimers[END_REF] and then adding 9 to give 2 in 12% yield after semi-preparative RP-HPLC purification (see experimental section for details). For direct comparison with 1 and in order to evaluate the effect on cellular delivery of shifting the cysteamine position, 8 was homo-dimerized to provide Dim8 in 48% yield. To investigate the ability of the NCL ligand to transfect cells, we also carried out the homodimerization of 9 by DMSO oxidation procedure and the resulting Dim9 was recovered in 72% yield (see Supporting Information for details).

Circular dichroism (CD) was used to confirm the 2.5helical conformation of the urea-segment composing 2. The CD spectra, measured either in transfection conditions (150 mM NaCl) or in phosphate buffer (40 mM, pH 7.4), reveal a characteristic signature with a maximum of positive molar ellipticity at about 205 nm for 2 whereas at this wavelength no band is observed for 9, thus supporting the presence of a 2.5-helical secondary structure in 2. It is noteworthy that the CD band at 205 nm is enhanced for 2 compared to precursor 8 suggesting here strengthening of the helical folding in the hybrid foldamer (see Figure S1 in the Supporting Information).

Plasmid DNA/hybrid CPF assemblies for cell delivery. We next investigated the capacity of 2, Dim8 and Dim9 to associate with a plasmid DNA and transfect cells. Their association with a reporter luciferase expression plasmid (p-Luc) was first explored by performing a gel retardation assay (see Figure S2 in the supporting information). Heterodimer 2 was found to interact particularly strongly with NA as revealed by the complete loss of p-Luc migration at a 2/p-Luc mass ratio of only 1. The same experiment with Dim8 showed comparable results to those recently reported for 1.

The transfection efficiency of the new foldamers was then evaluated using luciferase as reporter gene and human embryonic kidney cells (HEK293) as target cells. CPF 1 and the cationic lipid DOTAP were first used as positive controls. Increasing amounts of CPFs were mixed with a fixed amount of p-Luc and the transfection capacity of the various compounds was determined by measuring the luminescence of cells (Figure 2a and Figure S3a). This assay showed that hybrid 2 exhibited promising transfection capacity with a level of luciferase expression upon transfection of HEK293 cells that compares well with that of DOTAP and exceeds that of 1. It is noteworthy that despite the increase of molecular weight, the charge ratio (N/P) of 2 (~3) remains slightly lower than that of 1 (~3.7) with an optimal 2/pDNA mass ratio (2.7) that is about 6 times less important to that of 1 (mass ratio = 16). Having a CPF active at low N/P and w/w ratios is indeed promising because that minimizes the risks of cytotoxicity and immune response potential (vide infra). Moreover, these low ratios ensure that no or only a few of CPF 2 remains uncomplexed in the transfection media. Moreover, these results also reveal that shifting the disulfide bridge from one ex-tremity in 1 to the other (Dim8) does not alter the transfection capacity of the CPF. In contrast, homodimer Dim9 displayed poor transfection properties demonstrating here the key role played by the oligourea 2.5-helix component in the DNA association and transfection processes.

With the aim to assess the transfection potential of 2, we conducted additional experiments in two other human cell lines: SV40-transformed MRC5 fibroblasts (MRC5-V2) and a breast cancer cell line (MDA-MB-231). Besides DOTAP, the 25 kDa cationic polymer b-PEIErreur ! Signet non défini. which is one of the best vectors to deliver genes and a "golden standard" in transfection assays was also introduced as comparator. The results obtained on these two cell lines unveiled that 2 is as good to transfect cells as these two commercially available reagents (Figure 2b-c and Figure S3) at a N/P ratio of solely 1.5 -3 and again superior luciferase expression was observed with foldaplexes composed of 2 compared to those formed either in the presence of 1 or Dim8 (Figure 2c). Furthermore, when this transfection experiment was repeated on MDA-MB-231 cells by this time including as controls three CPPs described as being amongst the most potent CPPs for gene delivery, CPF 2 was systematically more effective than LAH4, 33 LAH4-L1 [START_REF] Mason | The Antibiotic and DNA-Transfecting Peptide LAH4 Selectively Associates With, and Disorders, Anionic Lipids in Mixed Membranes[END_REF] and Vpr55-82 [START_REF] Kichler | Efficient DNA Transfection Mediated by the C-Terminal Domain of Human Immunodeficiency Virus Type 1 Viral Protein R[END_REF][START_REF] Coeytaux | The Cationic Amphipathic Alpha-Helix of HIV-1 Viral Protein R (Vpr) Binds to Nucleic Acids, Permeabilizes Membranes, and Efficiently Transfects Cells[END_REF] (Figure 2d). To gain insight into the cell uptake pathway of this second generation of foldaplexes, we next evaluated p-Luc expression after inhibition of the endosome acidification by concanamycin A. [START_REF] Dröse | Bafilomycins and Concanamycins as Inhibitors of V-ATPases and P-ATPases[END_REF] In the presence of this drug, the transfection efficiency of b-PEI and 2 was diminished by more than 100-and 1000-fold, respectively (Figure 2b), whilst that of DOTAP was almost unaltered (~3-fold less luciferase expression). At pH 7, the lipoplexes are indeed already protonated. Similar to what was previously established for homodimer 1, [START_REF] Douat | A Cell-Penetrating Foldamer with a Bioreducible Linkage for Intracellular Delivery of DNA[END_REF] these results emphasize that acidification of endocytic vesicles and therefore the protonation of His U residues are required to trigger the release of foldaplexes in the cytoplasm and consequently for an optimal activity of 2.

Additional information regarding the size and surface charge of the 2/pDNA foldaplex assemblies were gathered by performing dynamic light scattering experiments. In the absence of salt, we observed a majority of particles with a size of about 100 nm that compares well to the result previously reported for foldaplexes formed with 1. [START_REF] Douat | A Cell-Penetrating Foldamer with a Bioreducible Linkage for Intracellular Delivery of DNA[END_REF] In salt-rich conditions (150 mM NaCl), the foldaplexes were much larger with a mean diameter 10 times bigger ( 1000 nm). Thus, as earlier reported for 1 the presence of salts strongly influences the aggregation process. [START_REF] Douat | A Cell-Penetrating Foldamer with a Bioreducible Linkage for Intracellular Delivery of DNA[END_REF] Finally, the surface charge of the 2/pDNA foldaplexes (generated at the optimal mass ratio (2.7) for in vitro transfection) was investigated by zeta potential measurement and found to be positive (above +27 mV) thus rendering the particles capable to enter cells. NA transfection in serum-rich media. To further explore the potential of 2 as a gene carrier, we next determined the stability and activity of 2/pDNA foldaplexes in serum-rich media which is more realistic in terms of future in vivo use of this CPF-based delivery system. The recent finding that amphiphilic cationic oligourea sequences related to 8 display remarkable resistance to proteolysis and prolonged stability in biological fluids [START_REF] Teyssières | Proteolytically Stable Foldamer Mimics of Host-Defense Peptides with Protective Activities in a Murine Model of Bacterial Infection[END_REF] boded well for an improved behaviour of foldaplexes under such conditions. Up to 50% of foetal calf serum (FCS) was included in culture media of the three above mentioned cell lines and the level of luciferase expression was again monitored. DOTAP, b-PEI, LAH4 peptide and CPF 1 were included as references in this study (Figure 3a-b and Figure S4 for the MDA-MB-231 cell line). Remarkably, the 2/p-Luc foldaplexes were found to be extremely stable in serum-containing media and no loss of transfection capacity was observed in cell culture composed of up to 50% of FCS. When 25% FCS was included in the MRC5-V2 cell medium, 2 compared favourably with DOTAP, b-PEI and LAH4. It is likely that the dramatically reduced transfection properties observed for LAH4 reflects its reduced serum-stability (Figure 3a). The same trend was observed on HEK293 cell line with a culture medium composed with 50% FCS. Whilst 2 exhibits a transfection efficiency unaltered in the absence or presence of serum (Figure 2a), at the opposite 1 showed a transfection capacity that was diminished by ~ 700 fold. Another feature of 2, worthy to emphasize is that in contrast to DOTAP and b-PEI where an increase of the vector/pDNA mass ratio is needed to compensate serum destabilizing effects on lipo-and polyplexes, 54 in the case of 2 the same level of luciferase expression is observed in serum-free or enriched conditions illustrating here the remarkable capacity of this molecular transporter to deliver genes (Figure S5).

In addition to these experiments, we analysed HEK293 cells by fluorescence microscopy following transfection with a green fluorescent protein expression plasmid (eGFP) in culture medium containing 50% FCS. The observed eGFP expression was compared to that produced when DOTAP was employed as transfection agent in a serumfree medium. As shown in Figure 3c, the number of GFP expressing cells 24 h after transfection with 2 is significant and surpasses the level of eGFP expression obtained with DOTAP. [START_REF] Douat | A Cell-Penetrating Foldamer with a Bioreducible Linkage for Intracellular Delivery of DNA[END_REF] The results show that 2 did not alter cell proliferation in culture medium containing or not 50% FCS (Figure 4a). Concurrently, the membrane integrity was assayed by measuring the extracellular activities of lactate dehydrogenase (LDH). The presence of this enzyme in the extracellular environment allows the detection of membrane alteration. We find that b-PEI and DOTAP slightly perturbed the cell membrane with around 1.5 and 2-fold more extracellular LDH respectively than in the control experiment, whilst in the presence of 2 no cell membrane alteration is observed whatever the medium (i.e. +/-serum in the culture medium) (Figure 4b). The last assay consisted in the determination of the total protein content after p-Luc transfection. [START_REF] Kichler | Histidine-Rich Amphipathic Peptide Antibiotics Promote Efficient Delivery of DNA into Mammalian Cells[END_REF] The data are in agreement with the MTT assay and support the conclusion that CPF 2 seems to be harmless to the cells (Figure 4c).

CONCLUSION

In summary, we have developed an SPS approach featuring a cystamine-type resin and Boc chemistry to facilitate access to new oligourea sequences equipped with a cysteamine termination for further functionalization using thiol reactivity. This synthesis approach was applied to the preparation of a new generation of CPFs combining a cationic oligourea sequence and a NCL peptide ligand. One such heterodimer (2) was found to be highly potent to deliver pDNA to various human cells and to mediate effective gene expression. It compared favorably with the "golden standard" transfection reagents DOTAP and b-PEI as well as a number of known CPPs. Remarkably, the efficacy of foldaplexes composed of 2 was not affected by the presence of up to 50% serum in the culture medium and cell cytotoxicity was low. For these reasons, the foldamer-peptide conjugates reported here may represent an alternative to overcome some of the current limitations of CPPs designed to deliver DNA. Future studies will aim at investigating the cell internalization process and at determining if it occurs through a cell-surface nucleolin internalization pathway and whether it is associated with some cell selectivity.

Figure 1 .

 1 Figure 1. Helical wheel representation of oligourea and formulae of a) the 1 st generation of homodimeric urea-based CPF (1) and b) 2 nd generation of urea-based foldamer-pseudopeptide hybrid (2).

Scheme 2 .

 2 Scheme 2. Synthetic routes to access Dim8 and 2 dimers through disulfide condensation of cysteamine-containing oligourea 8.

Figure 2 .

 2 Figure 2. Evaluation of the transfection efficiency of 2 and related dimers 1, Dim8 and Dim9. Increasing amounts of transfection reagent were mixed with a constant amount of reporter plasmid (1.5 g of p-Luc per duplicate). The complexes were incubated for 2-2.5 h in serum-free medium. Luciferase activity was measured 24-48 h post-transfection. The transfection efficiency is expressed as total light units per s per mg protein, and the average of duplicates is shown. Here, only are reported the conditions giving the highest luciferase expression. The complete assays are reported in the electronic supplementary information (see Figure S3, S5). a) Transfection of HEK293 cells with p-Luc in the presence of DOTAP (N/P = 2) and the four dimers 1, 2, Dim8 and Dim9. The mass (g) of compounds 1, 2, Dim8 and Dim9 used in the assay and the oligomer/pDNA charge ratio (N/P) are given in brackets; b) MRC5-V2 cells were transfected with DOTAP, PEI or 2 in the presence () or absence of concanamycin A (15 nM); c) Transfection levels obtained in MDA-MB-231 cells with DOTAP, b-PEI, 1, 2 and Dim8; d) Comparison of the transfection capacity of 2 vs. 3 CPPs namely Vpr55-82, LAH4-L1 and LAH4.

Figure 3 .

 3 Figure 3. Comparison of the transfection efficiency of 2 in serumfree or enriched media. The same conditions to those detailed in Figure 2 were applied. a) Transfection of MRC5-V2 cells with (•) or without the presence of 25% FCS. In this assay LAH4 peptide was included as control. The mass (g) of compound used in the assay and the charge ratio (N/P) are given in brackets; b) Transfection of HEK293 cells with culture medium containing 50% of FCS (•); c) eGFP expression 48 h after transfection of HEK293 cells with eGFP complexes without serum (photo on the left; with DOTAP as transfection agent) or in the presence of 50% FCS (right; with CPF 2) (n=2).

Figure 4 .

 4 Figure 4. The different transfection reagents were mixed with a constant amount of reporter plasmid (2.25 μg per triplicate (a, b) and 1.5 μg per duplicate (c)) and the complexes were incubated for 2h40 with MDA-MB-231 cells in the absence or in the presence (grid bar) of 50% serum. In graph c) the red dots represent the amount of protein in g per well (y axis on the right of the graph). Cytotoxicity determination of hybrid CPF 2. The cytotoxicity of 2/p-Luc foldaplexes was assayed on MDA-MB-231 cells by resorting to three different methods. First, cell

ACKNOWLEDGMENTS

This pro ect was partially f nded by d ordea C S Pro ets ploratoires Pre ier So tien Predoctoral fellowships fro D A and Conseil gional de o elle A itaine (to S.A.), and from the French Ministry of Research (to M. B.) are gratefully acknowledged. This work has benefited from the facilities and expertise of the Biophysical and Structural Chemistry platform at IECB, CNRS UMS3033, INSERM US001, Bordeaux University, France

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxxxxxx Detailed experimental protocols, materials and methods for cell transfection assays and additional figures S1-S5; synthesis and characterization of linker 4 and newly synthesized building block (A) equipped with Orn(Alloc) type side-chain, solid phase synthesis procedure of 8; syntheses of 2, Dim8 and Dim9; HPLC profiles and HRMS analyses of all foldamer hybrids related to this article; CD spectra of 2, 8 and 9.

AUTHOR INFORMATION Corresponding Author

* E-mail: g.guichard@iecb.u-bordeaux.fr * E-mail: kichler@unistra.fr

Notes

The authors declare no competing financial interest.


Ronan Fablet
email: rfablet@ifremer.fr

Françoise Daverat

Rançoise Daverat

Hélène De Pontual

Keywords: individual life histories, fish otoliths, Bayesian labeling, Gaussian mixture models, hidden Markov models, otolith microchemistry

published or not. The documents may come     Unsupervised Bayesian reconstruction of individual life histories from otolith signatures : case study of Sr : Ca transects of eel (Anguilla anguilla) otoliths

72% changed habitat once or of several times, mainly before age four. This method opens new avenues for the analysis of individual habitat strategies. In addition, it could be easily applied to any other measures (microchemistry or not) taken along an otolith growth axis to ories.

Introduction

The recent bloom of ecology studies using otolith microchemistry emphasises the remarkable use of otolith between the environment e, sequential elemental measures acquired along an otolith growth axis are thought to record environmental information along the fish lifespan. River basin origin have been inferred from strontium hile salinity

). With more most popular water masses of different salinity for at least 20 fish species. To date, more than 28 published studies have exploited strontium calcium ratios for the analysis of life histories of eels species (Anguilla and costly rather than a easures were rmalised, the treatment of sequential Sr:Ca measures of eel otoliths data consisted in calculation of the mean values of each individual eel (Tsukamoto and Arai 2001[START_REF] Jessop | Migratory behavio[END_REF]. For assigned to a specific water mass according to the mean value of Sr:Ca measures. This is questionable as each Sr:Ca otolith measure is a specific indicator of a water mass so that the mean of two different water masses has no ecological meaning. Besides, the temporal dimension of the data was lost. Other studies interpreted directly individual Sr:Ca transects values plotted potential of the otolith accuracy for investigating fish life history traits. The chemistry to trace migration pathways is premised on a significant correlation elemental composition of otoliths and physicochemical properties of the ambient [START_REF] Campana | Chemistry an applications[END_REF]Martin and Thorrold 2005;Thorrold et al. 1997). Henc isotopes ratios (Milton and Chenery 2003;Thorrold and Shuttleworth 2000), w level has been inferred from ratios of strontium on calcium (Tzeng et al. 1994 than 200 research articles published, strontium calcium ratios became the microchemistry application to fish ecology, as a tool to track movements across (Troadec et al. 2000;[START_REF] Fablet | Semi-local extraction of ring structures in images of biological application to the Ba[END_REF] or other issues such as stock discrimination [START_REF] Campana | Stock Discrimination Using Can[END_REF], or fish individual status [START_REF] Cardinale | E stock, a Can[END_REF]). Within a signal processing framework, each Sr:Ca measure is associated with a hidden state variable standing for an environmental information (in our case, an habitat and the associated water mass), and the temporal nature of the sequence of Sr:Ca measures can be restored from the otolith growth pattern. Formally, the reconstruction of the individual patterns of habitat use is stated as the Bayesian reconstruction of the temporal sequence of the hidden state variables from the otolith growth pattern, the first years of the fish life have a greater spatial resolution last years of the fish life. As a consequence, evenly spaced Sr:Ca measures tran non equal number of measures for each year of the fish life. In addition, the cla individual migratory behaviours relied on a priori classes, which may not ac supervised method accounting for the mean and the variations of each ind (Daverat and Tomas in press). From classes of li of the eels resuled from a visual interpretation of the associated Sr:Ca signa consuming, such a scheme also appears rather subjective.

sequential measures and infer a relevant interpretation of the chemical signal (S terms of environmental information (here the habitat visited by the fish). Sign methods appeared as a promising tool to tackle this issue. Multidisciplinary already applied signal processing techniques to process fish otolith data for age observed sequence of Sr:Ca signatures. The proposed scheme mainly relies on Gaussian mixture models and hidden Markov models. All these developments were implemented under method was in terms of a method for the treatment of sequential measures taken on an otolith growth axis is further discussed. The interpretation of the macrostructures, so-called "rings", laid annually [START_REF] Berg | Age determination of eels, Anguilla anguilla (L.): comparison o otolith ring patterns[END_REF] observed on the otoliths along the longest growth axis, provided an estimation of individual growth patterns. The elver mark was set as the origin of the time axis and only the interval

Data

Material and Methods

An eel ecology study [START_REF] Daverat | Tracking conti shifts of eels using otolith Sr/Ca ratios: validation and application to the coastal, riverine eels of the Gironde-Garonne-Dordogne watershed[END_REF], led to the acquisition of 192 individual eel S series. The fish samples were collected in the Gironde river basin SW France habitats (water masses). 63 eels (33%) were collected in freshwater habitats, 11 movements across freshwater, brackish and marine habitats. Hence, only th otolith corresponding to the continental life of the eel was retained (from glass e edge). The acquisition method was described in [START_REF] Daverat | Tracking conti shifts of eels using otolith Sr/Ca ratios: validation and application to the coastal, riverine eels of the Gironde-Garonne-Dordogne watershed[END_REF], and electron microprobe measures of Sr and C was recorded as a distance from the glass eel mark.

between the elver mark and the edge was taken into account, as the ecological issue was the continental habitat use pattern of the eel after the glass eel stage until the time of capture.

ith respect to e series was

).

In the e will refer to this time axis as the age axis, since it refers to the time spent from the elver mark.

( )     - - = 2 ) ( exp 1 , H H m y m y g σ     2 2 2 2 H H H σ πσ
Considering a linear model, Θ H is explicitly defined by the mean value m H , the effect of the age λ A and the effect of the season λ S . For a Sr:Ca measure y at age a and hydrological season s (normalized average monthly flow), the associated likelihood g(y|a,s,Θ H ,σ H ) is given by:

     - = Θ 2 2 exp , , , H H H s a y g σ σ ( )    - - - 2 2 ) ( 2 1 S A H H s a m y λ λ πσ
The constant model is a particular case of the linear model with λ =λ =0. uld note that no labelled data is available to perform this estima a mixed set of Sr:Ca measures {y i } associated with unknown habitats (with considered ones). The estimation of the parameters of the hab

{ , Θ A S
Hence, in the subsequent, we will only detail the developments for the latter.

As a first step, we aim at determining for each habitat type H the associated model parameters (Θ H ,σ H ). One sho tion, but only in the three itat models

} { } M E R H H H H , , , ∈ σ π
is then stated as an unsupervised issue. To this end, given the Sr:Ca measures {y i } relative to explanatory variables {a i ,s i }, the whole distribution of {y i } is ussian habitat modelled as a Gaussian mixture issued from the superimposition of the three Ga models:

{ } { } ( ) { } ∑ ∈ ∈ Θ = Θ M E R H H H i i i h M E R H H H H i i i s a y g s a y p , , , , ) , , , ( , , , , σ π σ π
, where R, E and M stand for the labels relative to the three habitats: respectively, river (R), estuary (E) and marine area (M). π R , π E , π M are the prior probabilities for each habitat. Given {y i } and {a i ,s i }, we aim at estimating the parameters of the mixture model {π

H , Θ H , σ H } H ∈ {R,E,M} such that { } { } ( ) M E R H , , ∈
according to the maximum likelihood (ML). This model estimation is carried

EM (Expectation-Maximization) alg H H H s a y p , , , ,

Θ σ π

best fits to the distribution of the dataset {y i } out using the orithm [START_REF] Bishop | 200 patterns of American eels in an im microchemistry[END_REF]. The computations involved in this ing the mean values of the Gaussian modes. We rely on the statement that the lower the salinity of the habitat the lower the mean Sr:Ca measure (Fig. 1).

h mode was influence of between the contributions of each group of predictors permited to evaluate the relative importance of habitat, season and age (Silber et al. 1995). These statistical tests were performed with R ries of Sr:Ca measures as illustrated (Fig. 2). This issue resorts to the estimation of the temporal sequence of the habitat-related path and is formally stated as a Bayesian labelling issue, that is to say retrieving the temporal habitat sequence {x } corresponding to a given observed series of Sr:Ca measures {y t }, where, for each time t, x t is a label: R (river), E (estuary) or M (Marine area).

Within a Bayesian framework, this labelling issue comes to the determination of the best sequence according to the Maximum A Posteriori (MAP) criterion, that is to say iterative procedure are detailed in Annex I.

The estimated mixture parameters are finally assigned to each habitat by sort

The goodness of the fits of the constant model and the linear model for eac compared with AIC (Akaike Information Criterion) method [START_REF] Awad | Properties of the Akaike information criterion[END_REF] Further assumptions are required to solve for this minimization issue. Two investigated. First, assuming that Sr:Ca

{ } ) ( min ˆ, , t t M E R H t y H x p x = = ∈ arg Eq. 2 Using the estimated Gaussian mixture model { } { } M E R H H H H , , , , ∈ Θ σ π , the posterior likelihood function ) ( t t y H x p = is computed as: { } ( ) { } ) { } ∈ = Θ = M E R H H H H i i i H s a y H x p , , 2 2 21 1 1 1 , , , , , σ σ π ( ( ∑
∈ Θ Θ H i i i H M E R H H H H i i i i s a y p s a y p , , 1 2 , , , , , , π σ π
This first model is however rather simplistic and does not explictily model fish m among habitats. To account for these temporal dynamics, first-order Gaussian h models (Rabiner 1989) are used. These models were initially developed and speech analysis. As il ) ovements idden Markov exploited for lustrated (Fig. 3), two main components are involved: a prior on the temporal dynamics of the state variables {x t } which models fish movements from x t-1 to x t and a data-driven term characterizing the probabilistic distribution of the observed Sr:Ca measures y given the habitat type x .

The temporal prior is stated as a first-order Markov chain. This resorts to the assumption that, given the sequence of state variables (x 0 , x 1 , ..., x t-1 ) from time 0 to time t-1, the state variable at time t only depends on x t-1 . It means that this model only keeps the memory of its last state to jump to the next one. Formally, this leads to the property that t t ) ( ) , ,..., (

1 0 1 1 - - = t t t t x x p x x x x p
. Consequently, a first-order Markov chain is fully characterized by its transition matrix Γ:

) , ( ) ( H H H x H x p Γ = = = 2 1 2 1 1 t t -
which specifies the likelihood that the fish is in habitat type H (Fig. 4), whereas direct transitions from A to C are impossible, paths from A through B are possible.

( )

s a y g H x y p σ , , , ) , ( Θ = Φ = H H t t t1
where denotes the set of habitat-related Sr:Ca models ,

{ } { } M E R H H H t t , , , ∈ Θ = Φ σ .
To exploit this Gaussian hidden Markov model, we first need to estimate the parameters of the prior term. Similarly to the estimation of the parameters of the Gaussian mixture model, etermine the transition matrix associated with the maximum likelihood for the whole set of samples: this estimation is performed according to the ML criterion which resorts to d

∏ Γ = Γ Γ i i T i i T i y y x x p ) , ,..., ,..., ( max arg ˆ0 0
This maximization issue is solved for using the EM algorithm. We let the reader refer to Rabiner (1989) for a detailed description of this estimation procedure. We only review its main characteristics. This procedure iterates two steps until convergence. Given the current estimate Γ k of the transition matrix, the Expectation step resorts to the computation of the posterior likelihoods:

) , ,..., ,. ( ) ( 0 1 1 2 2 1 k i T i i t i t i H H y y H x H x p t Γ = = = - ξ , 1 i ) , ,..., . ( ) ( 0 1 k i T i i t H y y H x p t Γ = = γ .
The M-step follows to update the transition matrix Γ k+1 from these posterior likelihoods as their average over the whole dataset:

∑∑ ∑∑ = = i i N T i H H i i t t ) ( ) ( γ ξ = = = Γ N T t H i t H H 1 0 1 0 2 1 ) , ( 1 2 1 .
Given the estimate of the parameters of the hidden Markov model, the determ optimal MAP sequence ( ) Since the habitat sequences are calibrated over time, a variety of measures can also be defined to characterize individual life traits. We focus on the analysis of the time at which the transitions from one habitat to another occur, and of the time spent in a given habitat between two transitions. For a given type of transition from habitat type H 1 to habitat type H 2 (i.e., within the set of transitions {R to E, R to M, E to M, E to R, M to R, M to E}), the whole set of habitat sequences

Analysis of habitat sequences

Given the set of the individual habitat sequence, a quantitative analysis of fish m carried out. First the global analysis of the habitat sequences delivers a qua by the quality and the order of the visited habitats: for instance, the movement p from habitat sequence RRRREEEERRRRR is RER. Given the whol automated and unsupervised classification of individual movement behav determined, as well as the relative frequencies of these categories of movement p

( ) { } { } { } N i T t i ,..., 1 ,..., 0 ∈ H 1 to H 2.
{ } n H H t 2 1
is performed in terms of age, month and age group at which the transitions occur. Scale parameter µ is set to 1 for quantities given as monthly values and age groups and to 1/10 2 for quantities given as ages. 45167) has a ue of 46465).

Habitat, age and hydrological season (river flow) factors all had a significant influence on Sr/Ca values for each habitat (p<0.001) (Table 1).

The comparison of the relative contribution of the habitat factor with the contribution of both f SrCa values I 4.38-4.79], l. 1995). Since the effect of age and season is significant in terms p-statistics, we report the results of the analysis of the individual chronologies of habitat with respect to the linear model. odel and the ig. 5). As the e associated habitat sequences may be chaotic and may involve numerous short and unlikely transitions. On the contrary, the explicit modelling of fish movements between habitats thanks to the estimated transition odel.

Consequently, this model was chosen to further characterize life traits from the estimated habitat sequences. Eels sampled in the Gironde watershed displayed a wide repertoire of habitat use patterns, such as residences all life long in the same habitat as defined in the model (river, estuary, marine area) or as single or multiple shifts among habitats (Fig. 6).

Results

Comparison between constant and linear models.

The linear model accounting for habitat, age and season effects (AIC value of better performance than the model only accounting for habitat effect (AIC val age and season factors revealed that habitat contributes more to the variation o than age and season with a ratio of effect standard deviations of 4.58 [95%C (Silber et a

Individual chronologies of habitat use.

The comparison of the habitat sequences issued from the Gaussian mixture m hidden Markov models showed the improvements brought by the latter one (F Gaussian mixture models do not account for time coherence, th matrix leads to the smoother results reported for the hidden Markov m of movement ur analysis is rns up to age 6. As illustrated, 37 patterns are represented (Fig. 7). While only the first 20 patterns account for more than 80% of the samples, the first five patterns occur with a frequency greater than This resident ment patterns 6.5% of the a. While 33% of the sample was collected in the river, only 14.3% of the samples are labeled as residents in the river habitat. Similarly, the reported categorization leads to only 7.8% of residents in the the estuary. ll as patterns nly between eably, up to 5 successive migrations between the river and the estuarine area can be observed for the same individual before age 6. Fewer samples involve migrations among the three habitats.

However, about 9% of the samples are associated to patterns including a first migration from the river to the estuarine area, and then one or several movements between the estuarine and the marine areas. Conversely, very few individuals (below 2%) move from the marine area to the river after a stay in the estuarine area.

Analysis and classification of movement patterns

From the overall analysis of the reduced habitat sequences, the different classes patterns associated with the considered dataset were automatically determined. O restricted to fish older than four years and take into account the movement patte 5%. Among the first four patterns, three correspond to resident behaviour. behaviour however account for only 28% of the samples and 72% of the move involve at least one movement. Residents in the marine habitat account for sample which is quite consistent with the 7% of fish collected in the marine are estuary area while our sample is composed of 60% of fish collected in Concerning migration behaviours, patterns involving only one migration as we involving several migrations between two types of habitats are encountered, but o the river and the estuarine area or between the estuarine and marine areas. Notic Transitions between the river and the marine area did not occur. Besides, the reported results n-dependent.

Analysis of transitions schedules and duration of habitat use

at use before their absence, ary did not seem age specific (Fig. 9), but their frequency decreased with age with a maximum of transitions occurring at age 1. The duration of the stay in the river was less than two years with a maximum of fish spending less than one year in the river before moving to the estuary (Fig. 9).

Transitions from the estuary to the river and transitions from the estuary to the marine area did not occur for a specific age of the eel, as shown (Fig. 10) but decreased as the age of the fish increased. Most eels spent less than one year in the estuary before moving either to the river or to the marine area (Fig. 10).

Analysis of the transitions between habitats.

In order to keep a consistent temporal resolution through the fish life, only the f life of the fish were further considered for the rest of the analysis. The propo transitions from one habitat to another one were evaluated first as a function of habitat as presented for instance for the estuary (Fig. 8) at the individual level.

that movements among habiats are seldom compared also shows that the occurrence of the transition is seaso Age at transition between the river and the estuary and duration of the habit changing habitat were investigated for eels of more than four years old. Due to transitions between the marine habitat and the river were not analysed. As expected, the contribution of habitat has been shown to be much greater than that of age and season. Outside the metamorphosis from leptocephalus larvae into glass eel, no significant effect of ontogeny due to growth or age was observed so far on Sr:Ca incorporation into eel otolith [START_REF] Daverat | Tracking conti shifts of eels using otolith Sr/Ca ratios: validation and application to the coastal, riverine eels of the Gironde-Garonne-Dordogne watershed[END_REF][START_REF] Kawakami | Factors influencing otolith strontium/calcium ratios in Anguilla japonica elvers[END_REF]Kraus and Secor 2003;Tzeng 1996). A validation using another fish species reared for two years in constant salinity failed in detecting any age effect on Sr:Ca incorporation into otoliths (Elsdon and

Spatial and temporal resolution of the analysis

The integration of the temporal dimension in Sr:Ca series was an important issue Time series were reconstructed using resampling techniques. The spatial reso analysis on the otolith was constrained by analytic requirements. In this study, analyse only the first 6 years of life in order to keep a consistent temporal re method was based on a constant growth of the otolith through the year. It would by accounting for the sea

Habitat-related modelling of otolith signatures

In this study, a mono-proxy approach was used to model habitat-related otolith c

The proposed unsupervised scheme based on Gaussian mixture models permits to estim model parameters for each habitat zone from unlabelled data. A linear model Gillanders 2005). Age may affect Sr incorporation at a greater time scale than a few years, especially for some eels that can spend up to 20 years in their feeding habitats. As the mean ered, the age ry weak, was asses, of the variations of the river flow were introduced in the model developed here. In this study site, measures of Sr:Ca ratio in the water, collected at different seasons showed that values were slightly estuarine and and Sr:Ca value erimental and or 2004).

Further applications of this model could take into account other types of effects such as physiology parameters, fooding conditions or temperature [START_REF] Campana | Chemistry an applications[END_REF]Campana et al. ponential, ...) is framework vironments or states experienced of Sr:Ca ratio with oxygen isotopes ratios as a proxy of estimation of

Reconstruction of habitats use chronologies

The proposed method turned out to be particularly adapted to the analysis of large data sets.

Our original data consisted in 192 individual Sr:Ca series containing 70 points of Sr:Ca measures on average. Hence a total of 14649 Sr:Ca measures, were analysed as 14649 events age of our sample was 7 years, and only the six first years of life were consid effect was weak compared to the habitat effect. The season effect, although ve explained by the seasonal variations of freshwater flows into marine water m Gironde watershed and by the variations of water temperature. Hence, seasonal fluctuating over the seasons without affecting the discrimination of marine, river habitats [START_REF] Daverat | Tracking conti shifts of eels using otolith Sr/Ca ratios: validation and application to the coastal, riverine eels of the Gironde-Garonne-Dordogne watershed[END_REF]. As expected, the relation between habitat was very strong, a result validated for eels and other species using coupled exp field validations [START_REF] Daverat | Tracking conti shifts of eels using otolith Sr/Ca ratios: validation and application to the coastal, riverine eels of the Gironde-Garonne-Dordogne watershed[END_REF][START_REF] Elsdon | Consistency of patterns betwee experiments and field collected fish in otolith chemistry : an example and ap salinity reconstructions[END_REF]Kraus and Sec 2000). In addition, other kinds of parametric models (polynomial, log-normal, ex could also be straightforwardly used. Besides, multi proxy approaches using multidimensional structural and/or chemistry otolith signatures may also investigated within th with a view to retrieving a more precise estimation of the en thought the fish life. The combination water temperature (Nelson et al. 1989) may for instance resort to a more precise the temporal resolution of the measures in the example developed here.

representing an habitat use. The proposed approach is computationally efficient, since only a few minutes are required to process the whole sample set, including both the estimation of the al patterns of ework is that ation of individual Sr:Ca series in terms of habitat use is provided From a methodological point of view, hidden Markov models have been shown to be much more efficient than Gaussian mixture models to reconstruct individual state sequences. These physiological for instance, ture). Besides, recent developments in the field of conditional random fields might also be investigated to take into account more complex time dynamics or continuous state sequence. ubjective and . 37 different sample, were identified within the processed sample set. The treatment of the same data set was performed according to a supervised classification in a previous study (Daverat and Tomas in press).

This categorization exploited only six classes and failed in describing with precision the repertoire of behaviour of the eels. Those six classes had been defined a priori from the visual inspection of all the plots of the individual Sr:Ca series and from results for other population found in the literature which was not very robust. Previous work indeed mainly relies on such supervised classification with a view to testing a priori hypotheses on patterns of habitat use. In most cases, some individual patterns did not fit to these a priori hypotheses [START_REF] Kotake | Seasonal variation in the migratory history of the japanese eel Anguilla japonica in Mikawa Bay, Japan[END_REF]Tsukamoto and Arai 2001;[START_REF] Jessop | Migratory behavio[END_REF] and were withdrawn from the analysis. tterns greatly the direct determination of the t use patterns found for A. anguilla in the Baltic sea (Limburg et al. 2003;Tzeng et al. 1997) As a by-product of the proposed approach, statistical descriptors of the fish movements between habitats, such as the distribution of the transition time from one habitat to another or the distributions of the time spent in a given habitat after a transition, were computed over the whole sample set. This resulted in a huge gain in analysis time and power, compared to previous methods that required to retrieve the information individually from each Sr:Ca On the contrary, the robust and unsupervised categorization of movement pa improves the investigation of unknown populations thanks to diversity of the patterns of habitat use and of the associated proportions.

The diversity of habitat use chronologies reported here is consistent with habita [START_REF] Jessop | Migratory behavio[END_REF]), A. rostrata (Cairns et al. 2004;[START_REF] Jessop | Migratory behavio[END_REF]; Morrison e well as A. australis and A. dieffenbachii [START_REF] Arai | different habitat use by New Zealand freshwater eels Anguilla australis and A. as revealed by otolith microchemistry[END_REF]). The present work existence of eels resident of their capture site (about 28%). Besides, as sugge named "transients" (Tsukamoto & Arai 2001) or "nomads" in (Daverat et al. 200 collected in the estuary might be interpreted as an absence of movement under fluctuations of river flows into the estuary. However, the estimated habitat-re account fo whatever the season. This makes unlikely the reconstruction of such mislabelle series. The analysis of transitions revealed that movements between two different habitats were not as frequent as the residence in the same habitat along the fish life. The same result was obtained for studies using mark recapture techniques [START_REF] Jellyman | Movements of shortfinned e australis, in Lake Ellesmer tracking[END_REF]Secor 2003) and telemetry (Parker 1995) that found that most yellow eels ten resident behaviour. Transitions are rare temporal events along the fish life whic difficult to observe directly. In this study, transitions between two different habi press). Analysis of transitions also revealed that most eels spend less than on habitat before changing again.

The unsupervised categorization framework enlarges the scope of possible ana of fish ecology. Further developments of the analysis of eel habitat use cou More generally it provides a powerful tool to assess the relative efficiency tactics in terms of fitness. At a broader scale, the proposed approach demonstra interest in exploiting advanced processing techniques to fully exploit the rich individual biolog Annex I: EM parameter estimation for Gaussian mixture models ied out according to the maximum likelihood (ML) criterion. It resorts to the following maximization issue:

The estimation of the parameters of the Gaussian mixture models is carr

{ } { } ( ) Θ = Θ M E R , , ˆσ π σ π { } { } { } ∏ ∈ Θ ∈ ∈ i H H H H i i i M E R H H H H s a y p M E R H H H H , , , , , , , , max arg , , , , σ π
{ } { } ( ) M E R H k H k H k H i i i s a y x p
{ } { } M E R H , , ∈ : k H k H k H , , Θ σ π { }
To solve for this maximization issue, we use the EM (Expectation-Maximization) algorithm [START_REF] Bishop | 200 patterns of American eels in an im microchemistry[END_REF]. Let us denote by x i the variable stating that the i th sample is issued from habitat x i . The EM algorithm iterates until convergence two steps. At iteration k, the E-step computes the posterior likelihood

{ } ( ) { } { } ∈ = Θ = M E R H k H H H i i i H k k k i s a y p s a y H x p , , 1 2 2 2 21 1 1 1 , , , , , , , , σ π ( ) ( ∑
∈ Θ Θ k H i i i k H k k k M E R H H H H i i i s a y p , , , , , σ π σ π
Given the posterior probabilities, the M-step aims at updating model parameters

{ } { } M E R H k H k H k H , , 1 1 1 , , ∈ + °+ Θ σ π
. To simplify the notations, let us denote by τ iH the posterior

} { } ) M E R H H , , ∈ . H H i i i i { } M E R H H , , ∈ ) { ( k k k s a y x p , , , , , Θ σ π ew priors { } k 1 + π are updated a ( ) H H H k H m µ λ , , 1 = Θ +
The n s: Figure 4: Illustration of the characteristics of the transition matrix which temporal dynamics of the state variable for a model involving four states A, graphical representation of this transition matrix (the arrows illustrate the dependencies between these states with associated likelihood with the associa probabilities). As illustrated, the particular case of transitions is the one corresponding to staying in the curr to one of the three states (namely, river (dots), estuary (plain), marine (dash)) as age or month.

  processing issue embedded in a Bayesian framework. The proposed computational methodology was applied to a set of 192 eel (Anguilla anguilla) another were modelled. Major movement characteristics such as age at transition between habitats and time spent in one habitat were estimated. As a straightforward output, an unsupervised classification of habitat use patterns was determined and showed

  spp). Data acquisition of otolith microchemistry remains technically, timely demanding. Hence, the challenge has first been a matter of data acquisition matter of data interpretation. So far, most otolith microchemistry transects of m interpreted by a visual evaluation of each individual signal. Whenever fo instance, Tzeng et al. 2002 classified eel life histories from the number of years against age class graphs, with Sr:Ca values assigned to a water mass according to their level(Morrison et al. 2003). This was not either satisfactory, as, due to the non-linearity of in the processed dataset( Tzeng et al. 2002, Daverat and Tomas in press). For instance, a large data set of 270 eel Sr:Ca transects was classified with a ividual curve fe histories defined a priori, the classification l. While time It emerged from this overview of previous work a need for an unsupervised and well-founded computational method that could at the same time account for the temporal nature of the r

  habitats and 14 eels (7%) in the coastal habitats. The aim of this study was to characterize the habitat use patterns of eels from the Gironde river basin, during their continental growing phase as a yellow eel. Sr:Ca ratios transects were used to track the eels e part of the el mark to the consisted in a concentrations in 8 µm diameter spots, evenly spaced every 20 µm along the otolith longest growth axis from the glass eel mark to the edge of the otolith. Along this transect of Sr:Ca measures, the position of each annual age mark Calibration over time of Sr :Ca series .

  Matlab 7 using Netlab (Nabney 2001) and CRF (Murphy 2004) toolboxes. This applied to the interpretation of 192 eel otolith Sr:Ca transects of measures individual habitat use histories. The generalisation of such

  and the age and season on Sr:Ca value was tested according to correlation statistics model prediction and the data (McCullagh and Nelder 1989). The comparison of the software (RDevelopmentCoreTeam 2005).Estimation of individual habitat use from Sr:Ca seriesOur goal was to analyze the individual patterns of habitat use from the se t sequence of habitat categories corresponding to the maximum posterior likelihood given the acquired series of Sr:Ca measures: {y t } and labels {x t } are statistically independent. Equation (2) reduces for each time t to:

  the observed movement patterns. The movement pattern is defined as the sequence of the successive habitats visited by the fish. This sequence is defined attern issued e otolith set, the iours can be atterns.

  , where Z is the normalization factor. putation of the The com

  of season. In the overall sample (192 eels), transitions as a function of age or as a function of season, between two different habitats, were less frequent than stays in the same This indicates to the residence in one habitat.

  20 µm provided an approximate temporal resolution of one month for the first years of life up three months and more later than the 6 th year. This constraint leads us to solution. Our be improved sonal variations of the eel otolith growth. Despite the general variations of the eel otolith are known (Mounaix and Fontenelle 1994), a formalised model is, as far as we know, not available. haracteristics. ate was used to account for the significant influence on Sr:Ca values of age and season in addition to habitat.

  habitat Sr:Ca Gaussian mixture model and the reconstruction of all the individu habitat use. Compared to previous work, the key feature of this quantitative fram a non-subjective interpret from an unsupervised analysis. models could obvisouly take into account other types of discrete such as parameters, but they might be extended to continuous state variables ( tempera Unsupervised extraction and analysis of movement patterns A major contribution of the proposed Bayesian framework lies in the non-s unsupervised thus exhaustive categorization of individual movement patterns patterns of habitat use, with 20 patterns accounting for more than 80 % of the

  as well as those of other temperate eel species such as Anguilla japonica (Tsukamoto and Arai 2001; t al. 2003), as confirmed the sted by eels 4; Daverat et al. 2005), a significant number of eels (about 72%) changed habitats once or more. Chronologies with multiple transitions between the river and the estuary found for some eels the seasonal lated models r these seasonal fluctuations and Sr:Ca distributions are well discriminated d sequences, so that the hypothesis of patterns with multiple or seasonal movements was confirmed in the present study.

  of the eels increased. Similar results were obtained for Anguilla japonica (Tzeng et al. 2002) and A. rostrata (Morrison et al. 2003) as well as A. anguilla (Daverat and Tomas in e year in one lysis in terms ld consist in comparing individual parameters (size at age, age at maturity), of the different habitat use patterns. Such analysis could also focus on a specific stage of the fish (age or size class). of individual tes the actual potential of ical archive, such as fish otoliths, to characterize individual life traits. A wide range of applications for the analysis of individual life traits might be stated as such a Bayesian reconstruction of the time series of a state sequence from a set of chemical and structural measures.

  is the vector defined by [1 a i s i ]. This weighted linear regression leads to:

Fig

  Fig. 1.

Fig

  Fig. 2.

Fig

  Fig. 3.

  x

  Fig.4

Fig. 8

 8 Fig. 8

Fig. 9

 9 Fig. 9

Fig

  Fig. 10.

Figure 1 :

 1 Figure 1: Distribution of Sr:Ca values and associated fitted Gaussian modes.

Figure 8 :

 8 Figure 8: Proportions (Y axis, log scale) of instantaneous transitions from the estuarial states a function of

Figure 10 :

 10 Figure 10: Distribution of ages at transition from the estuary to the river (plain line) or to the marine habitat (dots) (left panel) and distribution of the estuary habitat use duration anterior to the transition (right panel).

Figure 2 :

 2 Figure 2: Principle of the reconstruction of the time mesures spatially sampled along a growth axis of the otolith.

Figure 3 :

 3 Figure 3: Graphical representation of the Gaussian hidden

Figure 5 :Figure 6 :

 56 Figure 5: Habitat use pattern issued from a given Sr:Ca series as obtained with mixture model (GMM) (left panel) or the hidden Markov model (HMM) (right p

Figure

  Figure 7: A from the estimated habitat sequences.

Determination of habitat-related Sr:Ca model

	explanatory variables (fish age and month) was also considered. Formally, let us denote by
	g(.|Θ H ,σ H ) the Gaussian distribution of Sr:Ca measures for habitat H, parameterized by the
	Annual rings were used as time references to transform Sr :Ca series acquired w ard deviation σ H . Using a constant model Θ H =m H , g(.|m H ,σ H ) is mean model Θ H and the stand
	the distance to the elver mark to time series using a linear interpolation. The tim computed for a Sr:Ca measure y as:
	interpolated at a monthly precision (that is to say a time sampling rate of 1/12
	following, w
	The actual temporal resolution of the Sr:Ca series depends both on the sampling resolution of
	the electron microprobe and obviously of the otolith growth rate. In a previou	s experiment
	nd for young (Daverat et al. 2005), a mean otolith growth rate of 20 µm per month was fou
	individuals, so that about 11 Sr:Ca measures are usually sampled for the first	growth years,
	whereas from the 6 th year fewer measures (down to 3 or 4) may be available	due to slower
	growth. Therefore, the results issued from the analysis of interpolated Sr:Ca time series need
	to be cautiously analyzed in terms of temporal precision, especially for the last years of the
	life of the older individuals.
	Following Daverat et al. (2005), Sr :Ca measures can be regarded as a proxy	of the habitat
	type for eels of the Gironde watershed. Three habitat categories were considered according to
	salinity compartment: river, estuary and marine habitats. We further modelled the distribution
	of Sr:Ca signatures for each habitat as a Gaussian distribution parameterized by a mean model
	and a standard deviation.
	Different models could be chosen. In this study, two different cases were investigated. The
	first one was a constant model parameterized by a mean value. In order to test for the
	influence of seasonality and age on the incorporation of strontium, a linear model with two

  These transitions are characterized by their transition times

	i t x ∈	is analyzed to extract the set of all the transitions from
		{ } n H H t 2 1	and the times
	spent in H 2 { }

Table 1 : Anova table for the linear model.

 1