Antoine Mallégol

Patrick Meyer

Sophie Loyer

A hydrographic risk assessment methodology integrating the user's preferences: application to a use-case in the English Channel

Keywords: risk assessment, hydrographic survey, MR-Sort, deSEAsion decision support tool

A hydrographic risk assessment methodology integrating the user's preferences: application to a use-case in the English Channel

Antoine Mallégol, Patrick Meyer IMT Atlantique, Lab-STICC, UMR CNRS 6285, F-29238 Brest, France {antoine.mallegol, patrick.meyer}@imt-atlantique.fr Sophie Loyer Shom, F-29200, Brest, France sophie.loyer@shom.fr

Abstract

The Shom (French hydrographic and oceanographic service) is in charge of describing the physical marine environment in its relationship with the atmosphere, the seafloor and coastal areas, and to predict its evolution. The main objective of its mission is the dissemination of all nautical information that guarantees safe navigation, mainly in the form of nautical charts. Given the extent of the area to be covered, a global assessment methodology for navigational risk appears to be essential to evaluate cartographic needs in an exhaustive and rational way, and to prioritize the hydrographic surveys to be carried out.

A risk is usually evaluated according to three main factors: a hazard, its probability of occurrence, and its severity of consequences. In the context of marine traffic, the hazard is represented by ship accidents. The probability of an accident depends on many criteria (seabed morphology, reliability of the hydrographic data, presence of dangerous zones, traffic, …). An accident can have several consequences (marine pollution, economic losses, …) which are again rated according to several criteria (proximity of a protected site, …).

To evaluate this navigational risk, the "Land Information New Zealand" (LINZ) has proposed the "New Zealand Hydrographic Risk Assessment" methodology, based on multiple aggregations through weighted sums, to estimate the probability of an accident and a score of the consequences of an accident. This risk score is then used to plan hydrographic survey campaigns, which should focus on high risk zones first. One of the drawbacks of this method is the somewhat arbitrary setting of the weights of these criteria, as well as the little information that is provided by the synthesized risk score.

We therefore propose in this work, to integrate a Multi-Criteria Decision Aiding technique (MR-Sort) into the "New Zealand Hydrographic Risk Assessment" methodology, in order to model the priorities of the end user more accurately, and to ensure greater explainability of the risk level. This should increase the user's confidence in the final recommendation, and allow the planning of hydrographic survey campaigns in a way that is more faithful to his / her priorities.


Radhe Shyam

Lionel Nauton

Gaetano Angelici

Olivier Roy

Claude Taillefumier

Sophie Faure
email: sophie.faure@uca.fr

NC-gem-dimethylated peptoid side chains: a novel approach for structural control and peptide sequence mimetics.

The design of linear peptoid oligomers adopting well-defined secondary structures while mimicking defined peptide primary sequences is a major challenge in the context of drug discovery. To this end, chemists have developed cis-inducing peptoid side chains to build robust PolyProline I-type helices. However, the number of efficient examples remains scarce and chemical diversity accessible through the use of these side chains remains limited. Herein, we introduce an array of NC-gem-dimethylated peptoid residues mimicking proteinogenic amino acids. Submonomer synthesis and block-coupling approaches were explored to access heterooligomers incorporating these novel types of side chains. NMR studies of monomer and trimer models showed that the NC-gem-dimethylated groups exert a complete cis control on the backbone amide conformation. Lastly, a preliminary molecular modelling study gave an insight into the preferred orientation of the substituents of the C-gem-dimethyl side chains relative to the peptoid backbone.

INTRODUCTION

Ever since their initial development in 1992, 1 N-substituted glycine oligomers or "peptoids", have been considered a highly promising class of protease resistant peptidomimetics due to their close similarity to peptides. Peptoids are sequence-defined oligomers that are generally synthesized by the robust "submonomer method" wherein each peptoid residue is created in two steps: an acylation step to create the peptoid carbon backbone followed by a substitution step using primary amines to install the side chains. 2 This methodology is particularly well-suited for the preparation of large combinatorial peptoid libraries. 3 Although chemically similar to peptides, peptoids do not retain their folding properties, primarily due to the absence of backbone intramolecular hydrogen bonding capacity and an achiral backbone. Furthermore, the cis/trans isomerism of the main chain N,Ndisubstituted amides also contributes to the conformational heterogeneity of the peptoid backbone. This may hamper the development of peptoid ligands with high and selective affinity for biological targets, including the development of folded peptoids as blockers of relevant protein-protein interactions. It is therefore essential to develop strategies for the control of peptoid folding while retaining the unique chemical diversity. Peptoid chemists have developed specific side chains capable of controlling the amide conformation by forming local interactions with the backbone. This has enabled the design of peptoid oligomers exhibiting various privileged secondary structures such as helices (Polyproline type I (PPI), 4 Polyproline type II (PPII) 5 and η-helix 6 ), ribbons, 7 loops 8 and Σstrands. 9 Despite a modest cis-directing control on the tertiary amide bond isomerism, the chiral aromatic phenylethyl side chain (spe) has been used extensively to promote the PPI peptoid helical conformation. Furthermore, its use as helix inducer imposes constraints in term of physicochemical diversity that are a serious brake on the SAR studies. 10 Other strong cis-inducing peptoid side chains include the chiral aromatic naphthylethyl (s1npe), 11 the triazolium-type side chain, 12 the chiral aliphatic tert-butylethyl (s1tbe), 13 and the achiral aliphatic tert-butyl (tBu) 14 and fluoro-alkyl groups. 15 In order to increase chemical diversity, functionalized chiral phenylethyl side chains carrying different substituents on the aromatic core have been developed. 16 However, neither these highly hydrophobic side chains nor the triazolium-type side chains represent optimal mimetics of amino acid side chains. 17 Among the cis-inducing peptoid side chains, the tert-butyl side chain is unique since it induces complete cis conformation via steric effects, regardless of the nature of the solvent (Figure 1A). 14,18 In addition, despite the absence of chirality, a network of weak interactions such as intramolecular CO(i) … HC(i-n) (n=1,2 or 3) hydrogen bonds and i/i+3 tBu … tBu London interactions help the folding of NtBu peptoid oligomers into the PPI-like helical conformation (Figure 1B). Through the design of NC-gem-dimethylated N-substituted glycine monomers mimicking natural amino acids (Figure 2), we herein address the challenge of controlling the amide bond isomerism while retaining the chemical diversity. These novel side chains may be regarded as "functionalized" tert-butyl side chains and we therefore speculated that they would likewise induce a high or even complete cis-amide control.

FIGURE 2 (a) Generic structure of a peptoid residue carrying a "substituted" tert-butyl side chain and its cis/trans isomerism; (b) Structures of a library of -gem-dimethylated side chains mimicking proteinogenic amino acids side chains and proposed peptoid residue nomenclature (the g letter refers to ,-gem-dimethyl and the h letter refers to homo)

The gem-dimethyl moiety is a special feature encountered in many natural products such as taxanes, epothilones or macrolides. 20 Furthermore, the gem-dimethyl group has been successfully introduced in biologically relevant compounds in order to enhance specific therapeutic effects (bioavailability, potency, selectivity, toxicity). In the field of peptides and peptidomimetics, the Thorpe-Ingold effect of the gem-dimethyl group has been exploited for the conformational restriction of peptide oligomers through the use of -aminoisobutyric acid (Aib). 21 As mentioned above, we speculated that the steric restriction generated by the NC-gem-dimethyl group should block the peptoid backbone amide in the cis conformation. Herein we thus show that a large array of cis inducing side chains closely mimicking natural amino acids side chains could be designed and introduced into peptoid oligomers.

MATERIALS AND METHODS

Materials

Chemicals obtained from commercial sources were used without further purification unless stated otherwise. tert-Butylamine (98% purity), tert-amylamine (98% purity), 2-amino-2-methyl-1-propanol (99% purity) and diphenylphosphinic acid pentafluorophenyl ester were purchased from Aldrich. Benzyl bromoacetate (97% purity), ethyl bromoacetate (98% purity) and bromo acetylbromide (98% purity) were purchased from Alfa aesar. tert-Butylbromoacetate (98% purity) was purchased from TCI. Piperidine (99% purity) was purchased from Avocado. THF, CH 2 Cl 2 and MeOH were dried over aluminum oxide via a solvent purification system and stored over 4Å molecular sieves. EtOAc, CH 2 Cl 2 , cyclohexane, and MeOH for column chromatography were obtained from commercial sources and were used as received. Et 3 N was dried over KOH, distilled and stored over 4Å molecular sieves.

Preparation of -gem-dimethylated amines

The required NC-gem-dimethylated amines a-g (Figure 3) were purchased or prepared in gram scale (Supporting Information Scheme S1, S2 and S3).

Preparation of peptoid monomer models carrying NC-gem-dimethylated side chains

To a solution of piperidine (1.0 equiv, 0.2 M) in THF at 0 °C under argon was added Et 3 N (1.2 equiv) and then bromoacetyl bromide (1.2 equiv). After stirring for 1 h at 0°C, the resulting mixture was diluted with EtOAc (10 mL per mmol of starting material) and filtered, washing the solids with EtOAc. The filtrate was then concentrated and dried in vacuo, yielding N-(2-bromo-1oxoethyl)piperidine which was purified by flash chromatography on silica gel using EtOAc/cyclohexane 50:50 as eluent. To a solution of the bromoacetyl amide (1.0 equiv, 0.2 M) in THF at 0 °C under argon was added Et 3 N (2.0 equiv) followed by the chosen -gem-dimethylated amine (4.0 equiv). After stirring overnight at room temperature, the resulting mixture was diluted with EtOAc (10 mL per mmol starting material) and filtered, washing the solids with EtOAc. The filtrate was then concentrated under reduced pressure. When the primary amine used could not be removed by simple evaporation, the product was purified by flash chromatography on silica gel. The secondary amine was then acetylated with acetic anhydride (4.0 equiv) and Et 3 N (2.0 equiv) in EtOAc at 0 °C overnight. The resulting mixture was diluted with EtOAc (10 mL per mmol starting material) and filtered, washing the solids with EtOAc. The filtrate was then concentrated and dried in vacuo, and the crude monomeric peptoid model was purified by flash chromatography on silica gel.

Submonomer synthesis of peptoid oligomers

Substitution step

To a solution of tert-butyl bromoacetate, benzyl bromoacetate, ethyl bromoacetate or bromoacetyl amide (1.0 equiv, 0.2 M) in THF at 0 °C under argon was added Et 3 N (2.0 equiv) followed by the chosen primary amine (4.0 equiv). After stirring overnight at room temperature, the resulting mixture was diluted with EtOAc (10 mL per mmol starting material) and filtered, washing the solids with EtOAc. The filtrate was then concentrated under reduced pressure. EtOAc was added to the residue which was then concentrated under reduced pressure. When the primary amine used could not be removed by simple evaporation under reduced pressure, the product was purified by flash chromatography on silica gel.

Acylation step

To a solution of the crude secondary amine (1.0 equiv, 0.2 M) in EtOAc at 0°C under argon, was added Et 3 N (1.2 equiv) and then a solution of freshly prepared bromoacetic anhydride (1.2 equiv) in EtOAc. After stirring for 2 h at room temperature the resulting mixture was filtered, washing the solids with EtOAc. The filtrate was then concentrated and dried in vacuo. Flash chromatography on silica gel yielded the desired bromoacetyl amide.

N-terminal acetylation

To a solution of the peptoid (1.0 equiv) and Et 3 N (4.0 equiv) in EtOAc (0.2 M) at room temperature was added acetic anhydride (8.0 equiv). After stirring overnight at room temperature, the mixture was filtered, washing the solids with EtOAc. The filtrate was then concentrated and dried in vacuo, yielding the crude N-acylated compound. The final oligomer was purified by flash chromatography on silica gel.

2.5 Access to peptoid oligomers by blockwise coupling approach 2.5.1General procedure of coupling with pentafluorophenyl diphenyl phosphinate (FDPP)

The required free-acid block peptoid oligomer was obtained by hydrogenolysis of the benzyl ester precursor using Pd/C 10% in methanol. To a solution of the free-acid block peptoid oligomer (1.0 equiv., 0.4M)in dry CH 2 Cl 2 at room temperature under argon was added FDPP (1.2 equiv). The mixture was stirred for 5 min and a solution of the free-amine block peptoid oligomer (1.0 equiv., 0.4M) and DBU (3.0 equiv.) in CH 2 Cl 2 was slowly added dropwise. The resulting mixture was stirredat room temperature for 48h and was then diluted with CH 2 Cl 2 and subsequently washed with 5% aq. citric acid solution and satd. aq.NaHCO 3 . The organic fraction was dried over Na 2 SO 4 , filtered and concentrated in vacuo, yielding the crude product. The products were purified by flash chromatography on silica gel column.

General procedure of coupling with pentafluorophenyl trifluoroacetate (TFAPfp)

The required free-acid block peptoid oligomer was obtained by hydrogenolysis of the benzyl ester precursor using Pd/C 10% in methanol. To a solution of the free-acid block peptoid oligomer (1.0 equiv., 0.3M) in dry CH 2 Cl 2 at room temperature under argon was added pentafluoropheny trifluoroacetate (1.5 equiv.) and pyridine (1.5 equiv.). The mixture was stirred for 1 hand was then diluted with CH 2 Cl 2 and washed with 1M HCl and satd.aq.NaHCO 3 . The organic fraction was dried over Na 2 SO 4 , filtered and concentrated in vacuo, yielding the crude activated ester which was immediately used in the next step. To the solution of the activated ester (1.0 equiv., 0.2 M) in dry CH 2 Cl 2 at room temperature under argon was added slowly a solution of the free-amine block peptoid oligomer (1.0 equiv., 0.2M) and DBU (3.0 equiv.) in CH 2 Cl 2 . The mixture was stirred for 48h and was then diluted with CH 2 Cl 2 and subsequently washed with 5% aq. citric acid solution and satd. aq. NaHCO 3 . The organic fraction was dried over Na 2 SO 4 , filtered and concentrated in vacuo, yielding the crude product. The products were purified by flash chromatography on silica gel column.

Mass spectrometry

LC-MS were recorded on a Micromass Q-Tof Micro (3000 V) apparatus or a Q Exactive Quadrupole-Orbitrap Mass Spectrometer coupled to a UPLC Ultimate 3000 (Kinetex EVO C18; 1.7µm; 100mm x 2.1mm column with a flow rate of 0.45 mL•min -1 with a linear gradient of solvent B from 5% to 95% over 7.5 min (solvent A = H 2 O + 0.1% formic acid, solvent B = acetonitrile + 0.1% formic acid); equipped with a DAD UV/VIS 3000 RS detector).

Nuclear magnetic resonance spectroscopy

NMR spectra ( 1 H, 13 C, Jmod, COSY, HSQC, HMBC) were recorded on a Bruker AC 400 spectrometer or a Bruker AC-500 spectrometer. Chemical shifts were referenced to the residual solvent peak and coupling values J were given in Hz. The following multiplicity abbreviations were used: s for singlet, bs for broad singlet, d for doublet, t for triplet, q for quartet and m for multiplet. Assignments were based on the following 1D and 2D experiments: 1 H, COSY, HMBC, HSQC and Jmod-experiments.

Molecular modelling

Gas-phase full geometry optimizations and scans were performed at the density functional theory (DFT) level using B3LYP functional with 6-31+G(d,p) basis sets. All the DFT calculations were carried out with Gaussian09 software. 22 Structure preparations and visualizations were performed using chimera software. 23 On peptoid monomer models, a 360° scan of the dihedral angle χ1 was performed by intervals of 5° at the B3LYP/6-31+G(d,p) level. Molecular dynamics was performed using NAMD 24 and the charmm22 25 force field parameters. A hexamer model was constructed after parametrization of each side chain with Gaussian 09 and using the paratool module from VMD (visual Molecular Dynamic). 26 For the initial construction, the ω dihedral angles were considered to be 0º (cis conformation), the φ dihedral angles were considered to be -90º and the ψ dihedral angles were considered to be ±180º. The χ1 angle values were chosen to correspond one of the most stable positions identified during the χ1 scan of monomer models; for the tert-butyl side chain (χ1= +60º), for the NgIle side chain (χ1= -60°) and for the NgPhe side chain (χ1= ±180º).The hexamer was capped with an N-terminal acetyl group and a C-terminal benzyl ester. The molecular dynamics (MD) simulation included an initial phase of minimization followed by 40 ns of simulation. MD simulation was performed in gas with a dielectric constant of 37.5 corresponding to acetonitrile as solvent, at a temperature of 298K.

A large number of -gem-dimethyl primary amines are commercially available, including those that are necessary for mimicking the side chain of naturally-occurring amino acids (Figure 2). In the present study, six different NC-gem-dimethyl side chains were installed and their capacity to control the backbone amide conformation was studied. These six side chains originated from the use of tertbutylamine a, 1,1-dimethylpropylamine b, 2-amino-2-methyl-1-propanol c, -dimethylbenzylamine d, -dimethyl-1H-indole-3-ethanamine f and tert-butyl N-(4-amino-4-methylpentyl) carbamate g (Figure 3). Although commercially available, amines d, f and g were prepared on gram-scale according to known procedures (Supporting Information).

We first synthesized a series of peptoid monomer models carrying these novel side chains in order to study their capacity to control the peptoid amide geometry. The cis-directing effect of these new side chains was thus assessed using a C-piperidinyl amide monomer model capped with an acetyl group at the N-terminus (Scheme 4). This peptoid model has previously proven to be well suited for the determination of amide cis/trans ratios. [11][12][13][14][15] The cis/trans ratio of the acetamide may indeed be investigated by NMR spectroscopy since the cis and trans rotamers display distinct 1 H NMR resonances which allows for determining K cis/trans ratios. 11,12 Starting from N-(2-bromo-1-oxoethyl) piperidine, bromine substitution with an excess of primary amine in the presence of triethylamine followed by acetylation provided the monomeric models carrying a selection of "functionalized" tertbutyl side chains in moderate to good yields (Scheme 1).

SCHEME 1 Synthesis of peptoid monomer models 1

As previously described, the steric effect of the tert-butyl group results in the presence of only one amide rotamer in the 1 H NMR spectra of monomer 1a in a variety of solvents (CDCl 3 , CD 3 CN, CD 3 OD, acetone-d 6 , and D 2 O, Supporting Information). This conformation was shown to be cis by NOESY experiments. 14 The absence of the trans rotamer signals was therefore interpreted as the tertbutyl group resulting in a cis/trans ratio of > 95:5 (K cis/trans ratio>19). Importantly, the 1 H NMR spectra of the new peptoid models 1b ,1e and 1g likewise only showed a single set of signals, corresponding to the cis conformation of the acetamide (K cis/trans ratio>19).

The cis inducing effect of the C-gem-dimethyl side chains was further studied on trimeric peptoids. A series of trimers with two consecutive tert-butyl side chains and a C-gem-dimethyl side chain on the terminal acetamide was therefore designed. The trimer models 4-8 (Scheme 2) were prepared following the submonomer solution-phase synthesis methodology using a bromoacetic anhydridebased protocol developed for sterically hindered and deactivated primary amines. 14 When using these optimized conditions and when employing volatile primary amines, chromatography is only required after the acylation step. 27 Indeed, only filtration of salts and evaporation are needed after the substitution step. However, when a non-volatile primary amine is used in the substitution reaction, purification by flash chromatography is also required after this step. The use of solid-phase synthesis was avoided since it is our experience that oligomers of different lengths that are difficult to separate are obtained when synthesizing homooligomeric peptoids with tBu side chains. The common bromo intermediates 2 and 3 were prepared in four steps starting from ethyl bromoacetate or benzyl bromoacetate with yields of 42% and 41%, respectively (Scheme 2). Nucleophilic substitution using the-gem-dimethyl amines c, d, f or g followed by acetylation provided trimers 4-8 (Table 1). Displacement of the bromide was also achieved with the non-gem-dimethylated amine h (N-Boc-1,4diaminobutane) to provide peptoid 9 for comparative studies. The cis/trans ratio of the terminal acetamide was determined for all the trimer peptoids 4-9 by 1 H NMR integration of singlets corresponding to the acetyl CH 3 protons (Figure 4). According to previous studies, 19,28 the protons of trans peptoid acetamides are deshielded which results in a chemical shift of ˃ 2 ppm. On the contrary, it was observed that the protons of cis acetamides display a chemical shift of < 2 ppm. The 1 H NMR spectrum of trimer 9 in CD 3 OD showed two singlets at 1.93 and 2.15 ppm in 48:52 proportion, indicating the presence of two conformational isomers in a 48:52 cis/trans ratio. By contrast, the 1 H NMR spectrum in CD 3 OD of the corresponding trimer 8 with a NC-gemdimethylated-functionalized lysine side chain showed a major singlet at 1.91 ppm indicating a strong preference for the cis conformation of the terminal acetamide. A very small singlet detected at 2.15 ppm may correspond to the trans rotamer (< 3%). The same trend was observed for trimers 4, 5, 6, and 7 carrying the NC-gem-dimethylated-functionalized phenylalanine, homo-serine, homo-tryptophan and lysine side chains respectively (Figure 4). This NMR study on model trimers clearly indicates that the C-gem-dimethylated side chains have the same highly selective cis-directing effect as the tert-butyl side chain. This novel family of side chains thus locks the backbone amides in the cis conformation while maintaining access to a high degree of diversity. To further demonstrate the value of C-gem-dimethylated side chains in a drug discovery context we therefore synthesized longer oligomers.

Access to longer oligomers was first explored using the submonomer method. This method was thus used to synthesize peptoid hexamer 14 carrying three different types of NC-gem-dimethylated residues: NgPhe, NgIle and NghSer (Scheme 3). The monomer 10 was first synthesized from tertbutyl bromoacetate and amine b. Five submonomer cycles then furnished the hexamer 13 in a nonoptimized overall yield of 10%. Subsequent acetylation using acetic anhydride and tertbutyldimethylsilyl deprotection with tetrabutylammonium fluoride furnished the hexapeptoid Ac-(NghSer-NgPhe-NgIle) 2 -OtBu 14 as a white powder.

SCHEME 3 Submonomer synthesis of peptoid hexamer Ac-(NghSer-NgPhe-NgIle) 2 -OtBu 14 (conditions (i) for the substitution step and (ii) for the acylation step; all oligomers present amide bonds in a dominant cis conformation, but are drawn trans for convenience)

A convergent fragment-coupling approach which is more appropriate to access longer oligomers was then explored. Using this method, hexamer 18 was synthesized (Scheme 4). First, the trimer block amine 15 and trimer block acid 17 were prepared by submonomer synthesis, incorporating successively the amines b (tert-amylamine), d -dimethylbenzylamine) and a (tert-butylamine). The coupling of these two trimers using FDPP as coupling reagent in the presence of 1,8diazabicyclo[5.4.0]undec-7-ene (DBU) as base, yielded the hexamer 18 in 44% yield. The use of a two-step procedure involving the corresponding activated pentafluorophenyl ester intermediate 19 instead proved less efficient. Indeed, only 12 % of hexamer 18 was formed and nearly 50% of the trimer block amine 15 was recovered after purification of the crude product.

SCHEME 4

Fragment-coupling approach to access peptoid hexamer Ac-(NghAla-NgPhe-NgIle) 2 -OBn 18 (conditions (i) for the substitution step and (ii) for the acylation step; all oligomers present amide bonds in a dominant cis conformation, but are drawn trans for convenience)

In order to provide insight into the preferred orientation of the substituents of the C-gem-dimethyl side chains with respect to the peptoid backbone, the total energy of peptoid models with the acetamide in the cis conformation was determined by a systematic variation of the χ1 torsion angle by intervals of 5° at the B3LYP/6-31+G(d,p) level (Figure 5). The calculations were performed on C- As recently reported, the side chain 1 dihedral angle of NtBu peptoid units differs significantly from that of peptoid monomers bearing a disubstituted side chain such at the NC  carbon as for example an isopropyl side chain. 19 For the Ac-NtBu-OMe model, three minima of equal energies were observed on the energy diagram for  1 values around +60°, ±180° and -60°(300°) (Figure 5A). These staggered conformations prevent steric clashes between the Me groups and the amide carbonyl group of residue (i-1). By contrast, for the Ac-NiPr-OMe model, two minima were observed for  1 values around 0° and ±180°. The lowest energy minimum corresponds to the eclipsed conformation with the NC  -H bond facing the C=O bond. For the Ac-NgIle-OMe model, three minima were observed corresponding to those observed for the Ac-NtBu-OMe model. The two lowest energy minima correspond to gauche conformations (+60° and -60°(300°)) and the third minimum corresponds to the anti-conformation (180°) (Figure 5B). The anti-conformation is 1 kcal/mol higher in energy than the gauche conformation due to a small steric clash between the ethyl CH 3 group and the backbone C  protons.

methyl
For the Ac-NgPhe-OMe model, three minima centered at about +60°, ±180 and -60°(300°) were also observed, but the profile of the energy diagram between these minima is unique. In this case, the  2 dihedral angle corresponding to the phenyl group rotation is also an important factor. However, the three lowest energy minima correspond to those observed for the Ac-NtBu-OMe model.

Peptoid oligomers with cis-amide bonds adopt a structure similar to the polyproline type I helix and the helical screw sense is induced by the stereochemistry of -chiral side chains. Recently, we reported that achiral NtBu-based peptoids may also adopt a PPI-like structure stabilized by weak backbone CH … O=C interactions and side chain tBu … tBu London interactions. 19 The same should be observed in peptoid oligomers comprised of C-gem-dimethylated units. We have therefore performed a 40 ns molecular dynamic (MD) simulation of peptoid hexamer 18. The initial structure was generated with backbone dihedral angles (,)=(0°,-90°, ±180°) near the polyproline type I helix and the side chain  1 dihedral angles were chosen according to the energy minima observed during the scan of the  1 dihedral angle. The  1 values were therefore initially fixed at +60° for the NtBu units, at ±180° for the NgPhe units and at -60° for the NgIle units. In the course of the MD simulation, the helical conformation was maintained revealing the stability of this structuration that closely resembles the PPI helix (Figure 6 and Supporting Information Figure S3). According to this model, the formation of a PPI-like helical structure with outward distribution of the R groups is thus favored for peptoid oligomers incorporating C-gem-dimethylated side chains.

CONCLUSION

We have shown the high cis-directing effect of C-gem-dimethylated side chains by NMR spectroscopy studies of monomeric and trimeric peptoid models. Hexamers carrying C-gemdimethylated side chains were furthermore obtained in solution by the submonomer method or a convergent fragment-coupling approach. The privileged positioning of the R group of the C-gemdimethylated side chains was scrutinized according to the χ1 torsion angle value by determining the total energy of peptoid models with the acetamide in the cis conformation. As observed for the tertbutyl side chain, staggered conformations preventing steric clashes between the methyl groups and the amide carbonyl group of residue (i-1) were preferred. A model of the PPI-type helix was constructed and studied by molecular dynamics simulations. This helical conformation appeared to be highly favorable although the achiral side chains do not allow controlling the helix direction i.e. the sign of  dihedral angle (≈-90°or +90°). 19 Indeed in peptoid folding, the chirality of the side chains is critical to get well-folded secondary structures of defined handedness. This aspect has to be taken into account in the future design of NC-gem-dimethylated N-substituted glycine oligomers mimicking specific peptide sequences.

Overall, these novel C-gem-dimethylated side chains represent a valuable addition to the "toolbox"

for the construction of folded functionalized peptoid oligomers. This family of side chains retains structural diversity while maintaining a high degree of conformational control. The close mimic of peptide sequence is a crucial subject in a drug discovery context and these novel side chains will, for example, enable careful structure-activity relationship studies in the domain of peptoid-protein recognition.

19

 19

FIGURE 1

 1 FIGURE 1 (a) Peptoid oligomer carrying the tert-butyl side chain; (b) X-Ray structure of the pentamer Ac-(NtBu) 5 -OBn 19

FIGURE 3

 3 FIGURE 3 List of the  -gem-dimethylated amines used in the present study: (A) commercially available and (B) amines prepared by synthesis

FIGURE 4

 4 FIGURE 4 Acetyl proton region of the 1 H NMR spectra of trimers 4, 5, 6 (on the left) and 7, 8, 9 (on the right) in CD 3 OD

  ester peptoid monomers capped with an acetyl group at the N-terminus with different Cgem-dimethyl side chains (models Ac-NtBu-OMe, Ac-NgIle-OMe and Ac-NgPhe-OMe) or the isopropyl group (model Ac-NiPr-OMe).

FIGURE 5

 5 FIGURE 5 Energy diagram of the total scan of the  1 dihedral angles of model peptoid monomers: (A) Models carrying the tert-butyl side chain (R=Me) in black and the isopropyl side chain (R=H) in blue; (B) Models carrying the tert-butyl side chain (R=Me) in black, the C-gem-dimethyl propyl side chain (R=Et) in green and the C-gem-dimethyl benzyl side chain (R=Ph) in red; (C) Chemical structure of the models and definition of the dihedral angles :  [C  (i-1); C(i-1); N; C  ],  [C(i-1); N; C  ; C],  [N; C  ; C; O],  1 [C(i-1)  N; NC  ; NC  R)] or [C(i-1)  N; NC  ; H)] for the Ac-NiPr-OMe model

FIGURE 6

 6 FIGURE 6 Model of hexamer 18 obtained by molecular dynamic (A) view perpendicular to the helical axis and (B) view parallel to the helical axisMonitoring of the  1 dihedral angle variation over the course of the simulation as expected showed that rotation of the tert-butyl group occurred with three privileged positions ( 1 = +60°, -60° or 180°) (Supporting Information FigureS2). By contrast, in the case of the C-gem-dimethylated side chains no rotation was observed during the simulation, apart from in the very beginning. Indeed, the  1 dihedral angle for the NgPhe units rapidly reached approximately -60°, which is identical to those of the NgIle units. This  1 dihedral angle of -60° corresponds to a spatial arrangement where the R substituents of the C-gem-dimethylated side chains are pointing away from the helical axis.

SCHEME 2

 2 Synthesis of peptoid trimers 4-8 (conditions (i) for the substitution step and (ii) for the acylation step)

	Peptoid trimers	Amines Yield a Exact mass HRMS (TOF MS ES+)
	Ac-NgPhe-NghAla-NghAla-OEt 4	a, a, d	54%	C 27 H 44 N 5 O 3 [M+H + ]: calc. 490.3270, found 490.3275
	Ac-NghSer-NghAla-NghAla-OBn 5	a, a, c	72%	C 27 H 44 N 3 O 6 [M+H + ]: calc. 506.3214, found 506.3224
	Ac-NghTrp-NghAla-NghAla-OBn 6	a, a, f	74%	C 35 H 49 N 4 O 5 [M+H + ]: calc. 605.3703, found 605.3703
	Ac-NgLys(Boc)-NghAla-NghAla-OEt 7	a, a, g	28%	C 29 H 55 N 4 O 7 [M+H + ]: calc. 571.4065, found 571.4053
	Ac-NgLys(Boc)-NghAla-NghAla-OBn 8 a, a, g	54%	C 34 H 57 N 4 O 7 [M+H + ]:calc. 633.4227, found 633.4224
	Ac-NLys(Boc)-NghAla-NghAla-OBn 9	a, a, h b 57%	C 32 H 53 N 4 O 7 [M+H + ]:calc. 605.3909, found 605.3892

a Overall yields calculated from the bromo derivatives 2 or 3; b The primary amine h is N-Boc-1,4-diaminobutane.

TABLE 1

 1 Peptoid sequences and yields of trimers 4-9

ACKNOWLEDGMENT

We gratefully thank the French Ministry of Higher Education and Research for the grant to RS. We are grateful to M. Leremboure for mass spectrometry analysis (UCA PARTNER).Computations have been performed on the supercomputer facilities of the Mésocentre Clermont Auvergne. This work was supported by the ANR project ARCHIPEP.


Stéphane Potteaux

Virginie Deleuze

Régine Merval

Michel F Bureau

Bruno Esposito

Daniel Scherman

Alain Tedgui

Ziad Mallat
email: <mallat@larib.inserm.fr>

In vivo electrotransfer of interleukin-10 cDNA prevents endothelial upregulation of activated NF-jB and adhesion molecules following an atherogenic diet

Keywords: atherosclerosis, endothelial factors, gene therapy, inflammation

Objectives. Interleukin (IL)-10 has anti-atherogenic properties. However, the molecular mechanisms involved in IL-10 protection against atherosclerosis in vivo remain poorly understood. In this study, we examined the effect of IL-10 cDNA in vivo electrotransfer on diet-induced, endothelial activation. Methods. C57BL/6J mice were fed an atherogenic diet for 10 days. Expression of VCAM-1 and ICAM-1 was examined in the aortic sinus, a region predisposed to atherogenesis in mice, using immunohistochemistry. NF-jB activation was examined using a monoclonal antibody that selectively reacts with the activated form of the p65 subunit. Results. We detected a low basal expression of activated NF-jB, VCAM-1 and ICAM-1 in the endothelium of the aortic sinus. Endothelial expression of activated NF-jB, VCAM-1 and ICAM-1 was markedly increased after 10 days on the atherogenic diet (p < 0.001). In vivo electrotransfer of a murine IL-10-encoding plasmid completely prevented diet-induced endothelial upregulation of activated NF-jB, VCAM-1 and ICAM-1 (p < 0.01). Conclusion. In vivo electrotransfer of IL-10 cDNA prevents diet-induced endothelial activation. These results suggest that the protective effects of IL-10 may already occur in the very early stages of atherogenesis.

Endothelial cell activation with the consequent expression of cell adhesion molecules on the endothelial cell surface, is the primary event in the cascade leading to the development of atherosclerotic lesions [START_REF] Ross | Atherosclerosis--an inflammatory disease[END_REF][START_REF] Hansson | Inflammation, atherosclerosis, and coronary artery disease[END_REF]. Induction of endothelial cell adhesion molecules, particularly vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1, is triggered by a variety of pathophysiologically relevant stimuli, including abnormal blood flow hemodynamics [START_REF] Walpola | Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries exposed to altered shear stress[END_REF][START_REF] Chappell | Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium[END_REF][START_REF] Gonzales | Hemodynamic modulation of monocytic cell adherence to vascular endothelium[END_REF][START_REF] Nagel | Shear stress selectively upregulatesintercellular adhesion molecule-1 expression in cultured human vascular endothelial cells[END_REF], oxidized LDL (oxLDL) [START_REF] Jeng | Oxidized low-density lipoprotein enhances monocyte-endothelial cell binding against shear-stress-induced detachment[END_REF][START_REF] Frostegard | Mononuclear leukocytes exposed to oxidized low density lipoprotein secrete a factor that stimulates endothelial cells to express adhesion molecules[END_REF][START_REF] Khan | Modified low density lipoprotein and its constituents augment cytokineactivated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells[END_REF] and atherogenic diets [START_REF] Li | An atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium[END_REF], as well as proinflammatory cytokines [START_REF] Caterina | Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines[END_REF]. This induction is highly modulated by the activation of the proinflammatory nuclear transcription factor NF-jB [START_REF] Iademarco | Characterization of the promoter for vascular cell adhesion molecule-1 (VCAM-1)[END_REF][START_REF] Neish | Functional analysis of the human vascular cell adhesion molecule 1 promoter[END_REF][START_REF] Marui | Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells[END_REF][START_REF] Voraberger | Cloning of the human gene for intercellular adhesion molecule 1 and analysis of its 5'regulatory region. Induction by cytokines and phorbol ester[END_REF]. Interleukin (IL)-10 is a potent antiinflammatory cytokine expressed in the atherosclerotic lesion [START_REF] Uyemura | Cross-regulatory roles of interleukin (IL)-12 and IL-10 in atherosclerosis[END_REF][START_REF] Mallat | Expression of interleukin-10 in advanced human atherosclerotic plaques : relation to inducible nitric oxide synthase expression and cell death[END_REF], and has been shown to exert a major protective role against the development of atherosclerosis in C57BL/6 mice [START_REF] Mallat | Protective role of interleukin-10 in atherosclerosis[END_REF][START_REF] Oslund | Interleukin-10 blocks atherosclerotic events in vitro and in vivo[END_REF][START_REF] Namiki | Intramuscular gene transfer of interleukin-10 cDNA reduces atherosclerosis in apolipoprotein E-knockout mice[END_REF]. Different studies have demonstrated the beneficial role of IL-10 through various experimental models of atherosclerosis. IL-10 deficiency in C57Bl/6 mice fed an atherogenic diet has been shown to enhance early lesion formation [START_REF] Mallat | Protective role of interleukin-10 in atherosclerosis[END_REF][START_REF] Oslund | Interleukin-10 blocks atherosclerotic events in vitro and in vivo[END_REF]. More recently, similar observations were obtained in apolipoprotein E (apoE)/IL-10 double-knockout (KO) mice fed a standard diet [START_REF] Caligiuri | Interleukin-10 deficiency increases atherosclerosis, thrombosis, and low-density lipoproteins in apolipoprotein E knockout mice[END_REF], and in LDL receptor-KO mice with bone marrow deficiency in IL-10 [START_REF] Potteaux | Leukocyte-derived interleukin 10 is required for protection against atherosclerosis in low-density lipoprotein receptor knockout mice[END_REF]. In parallel, other studies confirmed the anti-atherogenic effect of IL-10 by overexpressing it in LDLr-or apoE-deficient mice [START_REF] Namiki | Intramuscular gene transfer of interleukin-10 cDNA reduces atherosclerosis in apolipoprotein E-knockout mice[END_REF][START_REF] Pinderski | Overexpression of interleukin-10 by activated T lymphocytes inhibits atherosclerosis in LDL receptor-deficient mice by altering lymphocyte and macrophage phenotypes[END_REF]. Although these consistent data emphasize the anti-atherogenic properties of IL-10, the molecular mechanisms involved in IL-10-mediated protection against atherosclerosis in vivo remain poorly understood. In vivo studies have demonstrated that atherosclerotic lesions of IL-10-deficient mice showed increased infiltration of inflammatory cells [START_REF] Mallat | Protective role of interleukin-10 in atherosclerosis[END_REF][START_REF] Potteaux | Leukocyte-derived interleukin 10 is required for protection against atherosclerosis in low-density lipoprotein receptor knockout mice[END_REF]. In vitro studies have revealed that IL-10 inhibited the interaction between activated endothelial cells and monocytes in an atherogenic context [START_REF] Oslund | Interleukin-10 blocks atherosclerotic events in vitro and in vivo[END_REF]. Based on these data, we hypothesized that at least some of the beneficial effects of IL-10 in atherosclerosis may involve a protective role against the up-regulation of cell adhesion molecules and the recruitment of inflammatory cells into the vessel wall. IL-10 has been shown to inhibit NF-jB activation in vitro and in vivo [START_REF] Wang | Interleukin (IL)-10 inhibits nuclear factor kappa B (NFkappa B) activation in human monocytes. IL-10 and IL-4 suppress cytokine synthesis by different mechanisms[END_REF][START_REF] Lentsch | In vivo suppression of NF-kappa B and preservation of Ikappa B alpha by interleukin-10 and interleukin-13[END_REF] although no study has reported an inhibitory effect in endothelial cells. With regard to expression of cell adhesion molecules, contradictory results have been reported in vitro [START_REF] Krakauer | IL-10 inhibits the adhesion of leukocytic cells to IL-1-activated human endothelial cells[END_REF][START_REF] Noble | Interleukin 10 regulates cellular responses in monocyte/endothelial cell co-cultures[END_REF], whereas lack of IL-10 was associated with increased VCAM-1 and ICAM-1 expression in a model of colitis in vivo [START_REF] Kawachi | Cytokine and endothelial cell adhesion molecule expression in interleukin-10-deficient mice[END_REF]. In vivo electrotransfer of plasmid DNA has emerged as a highly efficient method of ensuring a sustained and significant delivery of specific molecules with no or minimal side effects [START_REF] Aihara | Gene transfer into muscle by electroporation in vivo[END_REF][START_REF] Mir | High-efficiency gene transfer into skeletal muscle mediated by electric pulses[END_REF][START_REF] Nakano | Cytokine gene therapy for myocarditis by in vivo electroporation[END_REF]. Therefore, we designed this study to examine the effects of a single in vivo, intramuscular IL-10 cDNA electrotransfer on endothelial NF-jB activation and expression of VCAM-1 and ICAM-1 in the aortic sinus of C57BL/6 mice fed an atherogenic diet. As previous studies showed that induction of endothelial cell adhesion molecules following an atherogenic diet is rapid, and precedes the recruitment of circulating mononuclear cell into the vessel wall [START_REF] Nakashima | Upregulation of VCAM-1 and ICAM-1 at atherosclerosis-prone sites on the endothelium in the ApoE-deficient mouse[END_REF][START_REF] Iiyama | Patterns of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression in rabbit and mouse atherosclerotic lesions and at sites predisposed to lesion formation[END_REF], the time course of this study was limited to 10 days.

METHODS

Mice

Female C57BL/6J (5 to 9 mice per group), 7-8 weeks old were fed either a standard "A04" diet, which contained 3 % fat (UAR, France) or were placed on an atherogenic diet for 10 days. The atherogenic diet was generated by addition of 15 % cacao butter, 1.25 % cholesterol and 0.5 % sodium cholate to the standard diet. Mice were kept in accordance with standard animal care requirements, housed 4-5 per cage and maintained on a 12-hour lightdark cycle. Water and food were given ad libitum. We chose the C57BL/6J model in order to control the induction of high blood cholesterol levels starting from normal baseline values. The 10-day period was chosen in order to study the precise role of IL-10 on endothelial activation, before any invasion of the arterial wall by inflammatory cells.

Immunohistochemical studies

After 10 days on the atherogenic diet, mice were killed by CO 2 overdose. The basal half of the ventricles and the ascending aorta were removed, embedded in OCT compound (tissue Tek), frozen in isopentane, and stored at -70 °C until processing. Serial 10 lm sections of the aortic sinus with valves (80-90 per mouse) were cut on a cryostat. Each slide contained 6 to 10 sections that were representative of the whole aortic sinus. Frozen sections were fixed in acetone before immunostaining. They were incubated with either a primary goat polyclonal antibody against mouse VCAM-1 (Santa Cruz), a primary goat polyclonal antibody against mouse ICAM-1 (Santa Cruz), or a primary mouse monoclonal antibody against NF-jB, p65 subunit (Boehringer Mannheim). This latter antibody recognizes the IjB binding region on the p65 DNA binding subunit and therefore selectively reacts with p65 in the activated form of NF-jB. This antibody has been useful in numerous studies for immunohistochemical detection of activated NF-jB in specific cell types [START_REF] Brand | Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion[END_REF]. Immunostains were visualized after incubation with the corresponding pre-adsorbed, secondary biotinylated antibodies (Vector Laboratories) and the use of avidin-biotin horseradish peroxidase visualization systems (Vectastain ABC Kit) (Vector Laboratories). We took advantage of the InnoGenex Mouse-to-Mouse Iso-IHC Kit to abrogate any background staining that may have resulted from the use of the mouse primary anti-NF-jB antibody on mouse tissues. Irrelevant immunoglobulins were used for negative controls. Six to ten sections per animal (representative of the whole aortic sinus) were analyzed for each immunostaining. A quantitative analysis of the percentage of positive cells for each immunostaining was performed at a magnification of x 400 in a strictly blinded manner.

Systemic delivery of murine IL-10 by intramuscular injection of expression plasmid DNA

To assess the effects of IL-10 supplementation on in vivo endothelial NF-jB activation and expression of VCAM-1 and ICAM-1, mice placed on the atherogenic diet were injected at day 0 with either murine IL-10 expression plasmid, pCor-IL10, or an empty plasmid as control, as previously described [START_REF] Mallat | Protective role of interleukin-10 in atherosclerosis[END_REF]. IL-10 or control expression plasmid (15 lg) was injected into both tibial cranial muscles of anesthetized mice. Briefly, transcutaneous electric pulses (8 square-wave electric pulses of 200 V/cm, 20 ms each, at 2 Hz) were delivered by a PS-15 electropulsator (Genetronics) using two stainless steel plate electrodes placed 4.2 to 5.3 mm apart, each side of the leg. Blood samples were obtained by vein puncture at day 2 and day 10 after transfection. Circulating IL-10 levels were determined in the serum samples using of a highly sensitive ELISA kit specific for murine IL-10 (BioSource International).

Statistical analysis

Data are expressed as mean ± SEM. Simple regression analysis was performed to analyze the relation between expression of activated NF-jB and that of VCAM-1 and ICAM-1. The effects of the atherogenic diet or in vivo IL-10 cDNA electrotransfer on endothelial expression of activated NF-jB, VCAM-1 and ICAM-1 were determined using a t-test. A value of p < 0.05 was considered statistically significant.

RESULTS

Basal expression of activated p65, VCAM-1 and ICAM-1 in lesion-prone areas

A careful study of the aortic sinus revealed constitutive immunoreactivity for the activated form of NF-jB in occasional endothelial cells, exclusively located at atherosclerosis-prone areas (figure 1A). Otherwise, the staining was negative indicating that the antibody did not recognize the non-activated form of NF-jB. Negative staining using an irrelevant IgG3 further confirmed the specificity of the reaction (data not shown). As expected, we also observed constitutive VCAM-1 (figure 1B) and ICAM-1 expression in the endothelial cells of lesion-prone areas (table 1).

Diet-induced up-regulation of activated p65, VCAM-1 and ICAM-1

We found a clear increase in immunoreactivity for activated NF-jB in the endothelial cells of the aortic sinus after 10 days on the atherogenic diet (figure 2A). The increase in endothelial NF-jB activity was associated with a substantial up-regulation of endothelial VCAM-1 and ICAM-1 (figure 2B and2C, table 1). Quantitative analysis of the percentage of positive cells showed a highly significant difference between the mice on the atherogenic diet and those on the standard diet (table 1).

Prevention of diet-induced endothelial activation by in vivo electrotransfer of IL-10-encoding plasmid

The in vivo eletrotransfer technique ensured the production of high levels of circulating IL-10 in the mice transfected with pCor-IL-10 plasmid (2015.25 ± 121.16 pg/mL at day 2, n = 3, and 319.75 ± 35.40 pg/mL at day 10, n = 3). Circulating levels of IL-10 following injection with the empty plasmid were below the level of detection. In vivo electrotransfer of pCor-IL-10 plasmid completely prevented diet-induced expression of activated p65 and inhibited VCAM-1 and ICAM-1 up-regulation (figure 2D-2F, table 1). Overall, there was a very significant positive correlation between the presence of activated p65 and the expression of both adhesion molecules on the endothelial lining (r = 0.82, p < 0.0001 for VCAM-1; r = 0.68, p < 0.001 for ICAM-1).

DISCUSSION

Endothelial expression of VCAM-1 and ICAM-1 is a critical event in the initiation of the atherosclerotic process [START_REF] Ross | Atherosclerosis--an inflammatory disease[END_REF][START_REF] Hansson | Inflammation, atherosclerosis, and coronary artery disease[END_REF]. These adhesion molecules are up-regulated in atherosclerotic-prone areas and their expression precedes the recruitment of inflammatory cells within the arterial wall [START_REF] Nakashima | Upregulation of VCAM-1 and ICAM-1 at atherosclerosis-prone sites on the endothelium in the ApoE-deficient mouse[END_REF][START_REF] Iiyama | Patterns of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression in rabbit and mouse atherosclerotic lesions and at sites predisposed to lesion formation[END_REF]. Moreover, the use of neutralizing antibodies that block the ligation of these molecules was shown to inhibit leukocyte adhesion on the endothelial cell surface and to reduce the development of atherosclerotic lesions [START_REF] Shih | Blocking very late antigen-4 integrin decreases leukocyte entry and fatty streak formation in mice fed an atherogenic diet[END_REF][START_REF] Patel | Inhibition of alpha4 integrin and ICAM-1 markedly attenuate macrophage homing to atherosclerotic plaques in ApoE-deficient mice[END_REF][START_REF] Schapira | Genetic deletion or antibody blockade of alpha1beta1 integrin induces a stable plaque phenotype in ApoE-/-mice[END_REF]. ICAM-1 is constitutively expressed on the endothelial cell surface. In this study, we also observed a constitutive expression of VCAM-1 on the endothelial lining of lesion-prone areas, a finding consistent with the results of Iiyama et al. [START_REF] Iiyama | Patterns of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression in rabbit and mouse atherosclerotic lesions and at sites predisposed to lesion formation[END_REF]. We also showed constitutive expression of the activated form of p65 in occasional endothelial cells exclusively located in lesion-prone areas, as shown by Hajra et al. [START_REF] Hajra | The NF-kappa B signal transduction pathway in aortic endothelial cells is primed for activation in regions predisposed to atherosclerotic lesion formation[END_REF]. It is likely that this represents the in vivo effects of local changes in shear stress on NF-jB activation, a phenomenon that has been established in vitro [START_REF] Chappell | Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium[END_REF][START_REF] Gonzales | Hemodynamic modulation of monocytic cell adherence to vascular endothelium[END_REF]. We also found a significant increase in endothelial expression of activated NF-jB shortly after the administration of the atherogenic diet and showed a close relationship between this activation and the up-regulation of VCAM-1 and ICAM-1. Our in vivo findings are in agreement with in vitro studies showing that upregulation of VCAM-1 and ICAM-1 on endothelial cells is under inflammatory control and is highly dependent on NF-jB activation [START_REF] Weber | Monocyte arrest and transmigration on inflamed endothelium in shear flow is inhibited by adenovirus-mediated gene transfer of IkappaB-alpha[END_REF][START_REF] Lockyer | Inhibition of nuclear factor-kappaBmediated adhesion molecule expression in human endothelial cells[END_REF]. We and others have shown that both the development and the stability of atherosclerotic lesions depend on the balance between pro-and anti-inflammatory cytokines and that IL-10 plays a major role in this balance [START_REF] Mallat | Expression of interleukin-10 in advanced human atherosclerotic plaques : relation to inducible nitric oxide synthase expression and cell death[END_REF][START_REF] Oslund | Interleukin-10 blocks atherosclerotic events in vitro and in vivo[END_REF]. Pinderski et al. reported

an in vitro decrease in monocyte adhesion to endothelial cells transfected with an

Representative photomicrographs of tissue sections from the aortic sinus of C57BL/6 female mice fed a standard diet and injected at day 0 with a control empty pCor plasmid. The sections were stained for activated p65 NF-jB (A) and VCAM-1 (B). Original magnifications: x 400.

Table 1

Quantitative analysis of the percentage of positive staining for activated p65 NF-jB, VCAM-1 and ICAM-1 in endothelial cells of the aortic sinus of C57BL/6 mice (n = 5 to 9 for each staining) C,F). We detected a significant activation of p65 NF-jB in the endothelium after 10 days on the atherogenic diet (A). In vivo transfer of IL-10 resulted in nearly complete inhibition of endothelial p65 NF-jB activation (D). We also detected a significant up-regulation of endothelial VCAM-1 (B) and ICAM-1 (C) after 10 days on the atherogenic diet. In vivo transfer of IL-10 significantly prevented the induction of both VCAM-1 (E) and ICAM-1 (F). Original magnifications: x 400.

2

 2 Figure 2Representative photomicrographs of tissue sections from the aortic sinus of C57BL/6 female mice fed an atherogenic diet for 10 days, and injected at day 0 with either a control (A-C) or an IL-10-encoding plasmid (D-F). The sections were stained (in red) for activated p65 NF-jB (A, D), VCAM-1 (B, E) or ICAM-1 (C, F). We detected a significant activation of p65 NF-jB in the endothelium after 10 days on the atherogenic diet (A). In vivo transfer of IL-10 resulted in nearly complete inhibition of endothelial p65 NF-jB activation (D). We also detected a significant up-regulation of endothelial VCAM-1 (B) and ICAM-1 (C) after 10 days on the atherogenic diet. In vivo transfer of IL-10 significantly prevented the induction of both VCAM-1 (E) and ICAM-1 (F). Original magnifications: x 400.

Eur. Cytokine Netw., Vol. 17 n°1, March 2006, 1-6


Reuse agreement template between Cultural Heritage Institutions and researchers

Document initiated by: Laurent Romary (INRIA), Dorian Seillier (INRIA) and Erzsébet Tóth-Czifra (DARIAH-EU) in 2019.

A defining feature of data and data workflows in the arts and humanities domain is their dependence on cultural heritage sources hosted and curated in museums, libraries, galleries and archives. A major difficulty when scholars interact with heritage data is that the nature of the cooperation between researchers and Cultural Heritage Institutions and the researchers working in CHIs (henceforth CHIs) is often constrained by structural and legal challenges but even more by uncertainties as to the expectations of both parties.

This recognition led several European organizations such as APEF, CLARIN, Europeana, E-RIHS to come together and join forces under the governance of DARIAH to set up principles and mechanisms for improving the conditions for the use and re-use of cultural heritage data issued by cultural heritage institutions and studied and enriched by researchers. As a first step of this joint effort is the Heritage Data Reuse Charter (https://datacharter.hypotheses.org/) establishes 6 basic principles for improving the use and re-use of cultural heritage resources by researchers and , to help all the relevant actors to work together to connect and improve access to heritage data. These are: Reciprocity, Interoperability, Citability, Openness, Stewardship and Trustworthiness.

As a further step in translating these principles to actual data workflows the survey below serves as a template to frame exchanges around cultural heritage data by enabling both Cultural Heritage Institutions, infrastructure providers and researchers and to clarify their goals at the beginning and the project, to specify access to data, provenance information, preferred citation standards, hosting responsibilities etc. on the basis of which the parties can arrive at mutual reuse agreements that could serve as a starting point for a FAIR-by-construction data management, right from the project planning/application phase.

In practice, the survey below can be flexibly applied in platform-independent ways in exchange protocols between Cultural Heritage Institutions, researchers, and all other parties involved in cultural heritage data exchange.

Institutions who sign the Charter could use it (and expect to use such surveys) in their own exchange protocols. Another direction of future developments is to set up a platform dedicated to such exchanges.

On the other hand, researchers are encouraged to contact the CHIs during the initial stages of their project in order to explain their plans and figure details of transaction together. This mutual declaration can later be a powerful component in their Data Management Plans as it shows evidence for responsible and fair conduct of cultural heritage data, and fair (but also FAIR) research data management practices that are based on partnership with the holding institution.

As enclosing a Research Data Management Plan to grant applications is becoming a more and more common requirement among research funders, we need to raise the funders' awareness to the fact that such bi-or trilateral agreements and data reuse declarations among researchers, CHIs and infrastructure providers are crucial domain-specific components of FAIR data management.

A richer context to the adoption of the template below can be found at: https://datacharter.hypotheses.org/ For further information and feedback (which is more than welcome), please contact: laurent.romary@inria.fr and erzsebet.toth-czifra@dariah.eu.

Reciprocity

« Both Cultural Heritage Institutions and Researchers agree to share content and knowledge equally with each other, making use of data centres and research infrastructure »

• Researcher

Are you planning to communicate your research results to the institution that gave you access to the primary sources as well as the equipment providers, enabling them to enrich their original resources?

If so, could you please provide us with contact information to the corresponding person?

• Heritage Institution

Under what conditions would you allow researchers to access the collection in question?

Please specify any potential legal and/or technical issues involved in allowing access to your resources.

How would you like to benefit from the research results obtained using your sources, including potential corrections or enrichments regarding your initial data? Are there any specific enrichments you are especially interested in? Are there any mechanisms at your disposal to facilitate these exchanges (mail, online form, an open API etc.)?

• Research Facility / Equipment (if applicable)

Would you like to be kept informed about the research results? In which form (specific message, publication, etc.)?

Are there any specific enrichments you are especially interested in? Are there any mechanisms at your disposal to facilitate these exchanges (mail, online form, etc.)? Interoperability « Cultural Heritage Data will be made accessible in a form that facilitates reuse of the data for research. Formats should work and be interoperable for both scholars and CHIs. »

• Researcher

Are you planning to share your data and/or metadata in standardised formats, processes and protocols ? If so, in which ones?

• Heritage Institution Are you planning to share your data and/or metadata in standardised formats, processes, and protocols? If so, in which ones?

• Research Facility Do the data you provided rely on a standardised (and open if possible) and/or documented format? If not, is there a reliable documentation on the format(s) being used? Citability « Cultural Heritage data and any resulting research need to be fully citable to increase their visibility and impact. Relevant data citation standards should be applied » • Heritage Institution Which licenses do you use to disseminate your data? Are there any particular constraints regarding the dissemination of the research data obtained using your material (sensitive data, intellectual property, etc.)? Would you recommend using specific licenses to disseminate these data?

• Research Facility

What is your policy regarding data obtained using your equipment? Would Do you recommend using specific licenses? Stewardship « Long-time preservation, persistence, accessibility and legibility of cultural heritage data should be a priority »

• Researcher

Where are you planning to store your data to ensure their long-term preservation? If so, what infrastructure will you use? (This might be a repository or other appropriate solution.)

• Heritage Institution Do you provide a data storage service for the research results or are you associated with any reliable and public host? documenting the provenance of the data that they share, including records of any relevant materials, equipment, techniques, procedures and protocols used. They will attribute any resources used and flag any issues of authenticity or missing data and will also alert to any defamatory use of Cultural Heritage data. »

• Researcher

Are you planning to document all those who participated in the data production process in order to ensure its traceability?

• Heritage Institution Will you consent to be all your materials used for a research project listed and cited under its provenance metadata?

• Research Facility Will you consent to be all your materials used for a research project listed and cited under its provenance metadata?