Kris Wyckhuys

Francisco Sanchez-Bayo

Alexandre Aebi

Maarten Bijleveld Van Lexmond

Jean-Marc Bonmatin

Dave Goulson

Edward Mitchell

Stay true to integrated pest management

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

pest management has led to successes worldwide. Its Farmer Field Schools have transferred ecological concepts and nonchemical technologies to millions of small farmers, resulting in pesticide reductions of 92% in Bangladesh rice, 78% in India cotton, and 61% in Vietnamese tea (3). These FAO programs have attained pesticide cuts of 50 to 80% on millions of farms without yield loss (4), thus lifting farmers' profits, curbing energy use, and bolstering rural economies (5). Unfortunately, when some programs were dismantled in the mid-1990s, achievements were rapidly undone (6); Indonesia's pesticide use has since risen substantially.

Meanwhile, Croplife International's approach-i.e., pesticide-centered controlcontinues to proliferate across the globe (7,8). For example, insecticide-coated seeds are zealously backed by chemical enterprises (9), even though their on-farm use violates integrated pest management principles, triggers resistance development, exacerbates pollution, and degrades the resilience of farmland ecosystems (10). By thus deviating from established decision-making criteria and tolerating technology integration that occurs by chance instead of by design, this approach leads to ineffective pest control and negative social-ecological outcomes [START_REF] Pedigo | Entomology and Pest Management[END_REF]. Agro-ecology, digital tools, and innovative farming schemes can all remediate chemical

Stay true to integrated pest management

On 2 October 2020, the United Nations Food and Agriculture Organization (FAO) and CropLife International signed a letter of intent to establish a strategic partnership [START_REF]Letter of intent between the Food and Agriculture Organization of the United Nations and CropLife International[END_REF]. Moving forward, CropLife International's aspirations should be closely scrutinized. Checks and balances are essential to ensure that private interests do not take precedence over the common good.

For decades, both the FAO and CropLife International have advocated the uptake of integrated pest management-an approach that strategically integrates different crop protection methods to safeguard crop yields, secure farmer livelihoods, and preserve natural capital (2). However, their respective interpretations of the system's core constituents, underlying principles, and ultimate goals differ substantially. The FAO emphasizes how preventing pests through good agronomy, biological diversity, and ecological processes can lower the amount of pesticide required, whereas CropLife International focuses on the responsible use of agrochemicals, with far less desirable results.

The FAO's interpretation of integrated dependencies, but agro-industry meddling constitutes a prominent hurdle [START_REF] Goulson | [END_REF]. Ultimately, globe-spanning alliances that include agro-industry should only be built to scale up environmentally compatible practices and to pursue agri-food system transformations that are protective of human and environmental health. In the spirit of integrated pest management, as correctly interpreted and implemented by the FAO, biodiversity and ecological safeguards should constitute plant health programs' first line of defense, whereas pesticide use needs to be consciously downgraded to a measure of last resort.

Kris Wyckhuys 1,2,3 *, Francisco Sanchez-Bayo 4 , Alexandre Aebi 5 , Maarten Bijleveld van Lexmond 6 , Jean-Marc Bonmatin 7 , Dave Goulson 8 , Edward Mitchell 5,9

1 Institute of Plant Protection, China Academy of Agricultural Sciences, Beijing, China. 2 University of Queensland, Brisbane, QLD, Australia. 3 Chrysalis Consulting, Hanoi, Vietnam. 4 University of Sydney, Sydney, NSW, Australia. 5 University of Neuchatel, Neuchatel, Switzerland. 6 International Union for Conservation of Nature Task Force on Systemic Pesticides, Neuchatel, Switzerland. 7 Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique, Orléans, France. 8 University of Sussex, Brighton, UK.

9 Botanical

 9 Garden of Neuchatel, Neuchatel, Switzerland. *Corresponding author. Email: kagwyckhuys@gmail.com


Maria Genua

Laurie-Amandine Garçon

Yulia N Sergeeva

Els Saesen

Benjamin Musnier

Arnaud Buhot

Martial Billon

Evelyne Gout

Rabia Sadir

Hugues Lortat-Jacob

Christine Le Narvor

David Bonnaffé

Thierry Livache

Yanxia Hou

Discrimination of deletion to point cytokine mutants based on an array of cross-reactive receptors mimicking protein recognition by heparan sulfate

Keywords: Heparan sulfate, Interferon-γ, Cross-reactive, Pattern recognition, Surface plasmon resonance imaging

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

Introduction

Differential sensing of proteins based on cross-reactive arrays and pattern recognition is a promising alternative to the system based on 'lock-and-key' recognition principle such as biosensors for the detection and identification of proteins [START_REF] Garçon | A versatile electronic tongue based on surface plasmon resonance imaging and cross-reactive sensor arrays-a mini-review[END_REF] and for biomedical applications [START_REF] Geng | Array-based "chemical nose"sensing in diagnostics and drug discovery[END_REF]. This approach takes inspiration from the mammalian olfactory system, which uses cross-reactive receptors to create a pattern or a sample fingerprint. Thereby, it overcomes the difficulties associated with design and synthesis of highly selective receptors. In the last two decades, many different strategies have been developed to achieve high performance synthetic receptors or functionalized nanoparticles for differential sensing of proteins, [START_REF] Margulies | Combinatorial protein recognition as an alternative approach to antibody-mimetics[END_REF] such as tetra-carboxyphenylporphyrin coupled to different amino acids or dipeptides on their periphery developed by Hamilton and co-workers, [START_REF] Baldini | Pattern-based detection of different proteins using an array of fluorescent protein surface receptors[END_REF][START_REF] Zhou | Pattern recognition of proteins based on an array of functionalized porphyrins[END_REF] synthetic scaffolds consisting of an hexa-substituted benzene core combined with two tripeptide arms selected using combinatorial chemistry designed by Anslyn's group, [START_REF] Wright | Differential receptors create patterns that distinguish various proteins[END_REF] poly(p-phenyleneethynylene)s (PPEs) derivatives [START_REF] Miranda | Array-based sensing of proteins using conjugated polymers[END_REF] and later on gold nanoparticle conjugates developed by Rotello's group, [START_REF] You | Detection and identification of proteins using nanoparticle-fluorescent polymer 'chemical nose' sensors[END_REF][START_REF] De | Sensing of proteins in human serum using conjugates of nanoparticles and green fluorescent protein[END_REF][START_REF] Miranda | Enzymeamplified array sensing of proteins in solution and in biofluids[END_REF] ruthenium(II) tris(bipyridyl) protein surface mimetics by Hewitt and Wilson, [START_REF] Hewitt | Protein sensing and discrimination using highly functionalised ruthenium( II ) tris(bipyridyl) protein surface mimetics in an array format[END_REF] dual-ligand cofunctionalized fluorescent gold nanodots by Yuan et al., [START_REF] Yuan | Fluorescent gold nanodots based sensor array for proteins discrimination[END_REF] ionic liquids or their conjugates with quantum dots by Chen et al., [START_REF] Chen | Suspension array of ionic liquid or ionic liquid-quantum dots conjugates for the discrimination of proteins and bacteria[END_REF] blue-emitting fluorescent gold nanoclusters by Xu et al., [START_REF] Xu | A visual sensor array for pattern recognition analysis of proteins using novel blue-emitting fluorescent gold nanoclusters[END_REF] near infrared fluorescent dual ligand functionalized gold nanoclusters designed by Luo's group, [START_REF] Xu | Near infrared fluorescent dual ligand functionalized Au NCs based multidimensional sensor array for pattern recognition of multiple proteins and serum discrimination[END_REF][START_REF] Xu | A multicoloured Au NCs based cross-reactive sensor array for discrimination of multiple proteins[END_REF] aptamer-functionalized gold nanoparticles by Zhang's group [START_REF] Lu | Aptamer-based plasmonic sensor array for discrimination of proteins and cells with the naked eye[END_REF] and some other colorimetric sensor array based on gold nanoparticles summarized in the review [START_REF] Sun | Colorimetric sensor array based on gold nanoparticles: Design principles and recent advances[END_REF]. Based on these sensing materials, the obtained cross-reactive arrays were very efficient in detecting, identifying, and quantifying pure proteins at the low nanomolar concentration range [START_REF] You | Detection and identification of proteins using nanoparticle-fluorescent polymer 'chemical nose' sensors[END_REF] or to discriminate between a mixture of different proteins in human serum at physiological relevant concentrations. [START_REF] De | Sensing of proteins in human serum using conjugates of nanoparticles and green fluorescent protein[END_REF] Nevertheless, two major challenges remain: the production of sensing receptors with good selectivity for distinguishing between structurally similar analytes and the development of a single, label free, and reusable analytical device that allows monitoring of binding events in real-time.

In this study, we aim to develop a rational biomimetic methodology for preparing sensing receptors for the analysis of HSbps, inspired by the way HS polysaccharide recognizes its ligand, and to develop a reusable microarray that allows good discrimination between proteins featuring similar structures, such as deletion and point mutants.

HS, a large anionic polysaccharide, belongs to the family of glycosaminoglycan (GAG) that is widely present in the extracellular matrix and at the cell surface. It interacts with a multitude of HS binding proteins, including cytokines, chemokines, growth factors/morphogens and their receptors, etc, and thus has crucial regulatory roles in many biological processes. [START_REF] Bernfield | Functions of cell surface heparan sulfate proteoglycans[END_REF] It is crucially relevant to many disease processes, including inflammation, [START_REF] Parish | The role of heparan sulphate in inflammation[END_REF] angiogenesis, [START_REF] Chiodelli | Heparin/heparan sulfate proteoglycans glycomic interactome in angiogenesis: biological implications and therapeutical use[END_REF] neurodegeneration, [START_REF] Papy-Garcia | Glycosaminoglycans, protein aggregation and neurodegeneration[END_REF] and cancer. [START_REF] Sasisekharan | Roles of heparan-sulphate glycosaminoglycans in cancer[END_REF] This makes HS mimicry an attractive subject for the design of new efficient drugs or novel sensing methods targeting HSbps. Therefore, HS and their interaction with HSbps represents a very interesting model for our study.

HS is one of the most heterogeneous biopolymers with two levels of molecular diversity. [START_REF] Bonnaffé | Bioactive synthetic heparan sulfate and heparin derivatives: From long fragments mimetics to chimeras[END_REF][START_REF] Lubineau | Synthesis of tailor-made glycoconjugate mimetics of heparan sulfate that bind IFN-γ in the nanomolar range[END_REF] The first level corresponds to the micro-heterogeneities resulting from the variation in sulfation and epimerization of its constituting disaccharide building blocks (glucuronic/iduronic acidglucosamine).

In addition, the HS chain can be divided into three domains: A domains, which are mainly composed of D-glucuronic acid and N-acetylated D-glucosamine and thus have low charge density; S domains, which are rich in L-iduronic acid and N-sulfated D-glucosamine and highly charged; mixed A/S regions of variable length make the transition between A and S domains. Taken together, the various combinations of S, A and A/S domains generate multiple SAS charge topologies along the polymer chain and represent the second level of molecular diversity, as illustrated in Scheme 1a. Depending on cell type and activation state, HS chains with different negatively charged topologies are expressed, presumably promoting selective interactions with HSbps. [START_REF] Imberty | Structural view of glycosaminoglycan-protein interactions[END_REF][START_REF] Lortat-Jacob | Structural diversity of heparan sulfate binding domains in chemokines[END_REF] Recent studies reveal that many protein-HS interactions depend primarily on the overall charge density and distribution, rather than on the distinct saccharide sequence and the fine structure (specific sulfation and epimerization). [START_REF] Lindahl | Interactions between heparan sulfate and proteins-design and functional implications[END_REF][START_REF] Kreuger | Interactions between heparan sulfate and proteins: the concept of specificity[END_REF] Therefore, herein, we intended to mimic the biological properties of HS with a cross-reactive array, prepared by self-assembly of a small number of disaccharides used as building blocks (BBs) with different physicochemical properties on a surface of a chip. For this proof-of-concept study, two disaccharides neutral lactose (BB1) and negatively charged sulfated lactose (BB2) were used (Scheme 1b). We assumed that mixing these two molecules at different ratios should generate, following their self-assembly on the surface of a chip, various combinatorial surfaces with different charge densities and topologies. Each surface can be considered as a combinatorial cross-reactive receptor (CoCRR), which provides the micro-heterogeneities, just like the first level of diversity in HS. If such a biomimetic strategy works, more BBs with complementary physicochemical properties could be added in the CoCRR array to better mimic the biological properties of HS.

Herein, the efficiency of such a CoCRR array for protein sensing is evaluated using the cytokine interferon-γ (IFNγ) together with six mutants displaying a range of modification level. IFNγ strongly interacts with HS and plays an important role in modulating most phases of the inflammatory and immune response. [START_REF] Hu | Cross-regulation of signaling pathways by interferon-γ: implications for immune responses and autoimmune diseases[END_REF] For protein sensing, the CoCRR array was coupled to a SPRi optical detection system. SPRi measures modifications of the refractive index at the surface of the chip. The binding of the protein on the CoCRRs induces a shift of the plasmon curves and thus a variation of reflectivity. In contrast to commonly used colorimetric and fluorescent methods, [START_REF] Baldini | Pattern-based detection of different proteins using an array of fluorescent protein surface receptors[END_REF][START_REF] Zhou | Pattern recognition of proteins based on an array of functionalized porphyrins[END_REF][START_REF] Wright | Differential receptors create patterns that distinguish various proteins[END_REF][START_REF] Miranda | Array-based sensing of proteins using conjugated polymers[END_REF][START_REF] You | Detection and identification of proteins using nanoparticle-fluorescent polymer 'chemical nose' sensors[END_REF][START_REF] De | Sensing of proteins in human serum using conjugates of nanoparticles and green fluorescent protein[END_REF][START_REF] Miranda | Enzymeamplified array sensing of proteins in solution and in biofluids[END_REF] which require preparation of an array per sample, and high consumption of protein since the analysis needs to channel each protein sample to different spatially separated receptors, SPRi fits perfectly for protein sensing. Thanks to imaging mode, it allows for a label-free, parallel and synchronous observation of binding events over the whole array in a multiplex format. [START_REF] Wang | Recent advances in surface plasmon resonance imaging sensors[END_REF][START_REF] Wong | Surface plasmon resonance imaging sensors: a review[END_REF] The system is reusable after regeneration. More importantly, SPRi is able to monitor binding events in real-time, providing important information on the adsorption and desorption kinetics. [START_REF] Genua | SPR imaging based electronic tongue via landscape images for complex mixture analysis[END_REF][START_REF] Garçon | Landscapes of taste by a novel electronic tongue for the analysis of complex mixtures[END_REF] Therefore, it can provide supplementary discrimination parameters since similar affinities can result in different kinetics.

Materials and Methods

Materials

The two disaccharides (Scheme 1) lactose (BB1) and sulfated lactose (BB2) were prepared from a βlactoside, which was first converted to lactosylated disulfide BB1. Then, sulfatation was performed to obtain the fully sulfated disulfide BB2. The detailed synthesis of the disaccharides was reported previously. [START_REF] Hou | Continuous evolution profiles for electronic-tonguebased analysis[END_REF] Sodium chloride, sodium dihydrogen phosphate, HEPES, sodium dodecyl sulphate (SDS), Tween 20 and glycerol were purchased from Sigma-Aldrich. All of them were used as received. Protein lectin from Erythrina cristagalli (ECL), bovine serum albumin (BSA), Immunoglobulin G (IgG) from rabbit serum, and Cytochrome C (CytC) from horse heart were also purchased from Sigma-Aldrich and used as received. In this study, ultrapure water (18.2 MΩ.cm) used for preparation of buffer solutions was produced by a Purelab classic system (Elga).

IFNγ wild type and mutant expression and purification

Human IFNγ cDNA (a kind gift from Jean-Hervé Colle, Pasteur Institut) was cloned into pET11a expression vector (Novagen), and used to transform E. coli strain BL21 Star DE3 (Invitrogen). [START_REF] Saesen | Insights into the mechanism by which interferon-γ basic amino acid clusters mediate protein binding to heparan sulfate[END_REF][START_REF] Sarrazin | Heparan sulfate mimicry: a synthetic glycoconjugate that recognizes the heparin binding domain of interferon-γ inhibits the cytokine activity[END_REF] Cells were grown at 37 °C in Luria broth medium containing 100 µg/mL ampicillin, and induced with 0.5 mM IPTG for 5 h. Purification from inclusion bodies (IB) was then performed. Briefly, for IFNγ SD1, SD2, Δ136 and Δ124, IB were solubilized in 6 M guanidine HCl at 10 mg/mL, after which the protein was refolded by dilution to 0.36 mg/mL into 100 mM Tris-HCl buffer at pH 8 containing, 0.5 M L-Arg, 0.2 mM EDTA and protease inhibitors. Samples were then dialyzed against 20 mM Tris-HCl pH 8.0, containing 20 mM urea. For IFNγ S129 and S130, IB were also solubilized in 6 M Gdn/HCl but at 2.4 mg/mL and refolded by dilution to 0.2 mg/mL into 50 mM phosphate buffer and 0.5 M Gdn/HCl pH 7, supplemented with protease inhibitors.

The IFNγ samples were centrifuged for 20 min at 10 000 rpm, and the supernatants were subsequently brought to pH 6.8. Wild-type and mutant IFNγ were then purified by ion exchange chromatography (Source S HR 5/5 column in 50 mM phosphate buffer, pH 6.8) and gel filtration (Superdex 75 column equilibrated in 10 mM Tris and 10 mg/mL mannitol, pH 6.8).

Preparation of the combinatorial cross-reactive receptor array

For this, the pure and mixed solutions of BB1 and BB2 were deposited on gold surface of a prism, provided by Horiba Scientific (Orsay, France). Forty-eight hours prior to spotting, the prism was treated by plasma (0.6 mbar, 75% Oxygen, 25% Argon, power 40 W, 3 min) with a Femto plasma cleaner (Diener Electronic, Germany). Afterwards, eleven solutions with [BB1]/([BB1]+[BB2]) ratios (0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100%) were prepared in PBS (50 mM NaH 2 PO 4 , 150 mM NaCl, 10% glycerol, pH 6.8) with a total disaccharide concentration at 0.1 mM. Then these solutions were spotted at a volume of 8 nL using a non-contact spotter (sciFLEXARRAYER S3, Germany). For each ratio, four replicates were made and placed randomly on the prism. The prism was then left overnight at room temperature for the formation of CoCRRs via self-assembly. Notably, the addition of 10% glycerol in PBS minimized the evaporation of water in the spots during this self-assembly process and thus guaranteed a good reproducibility. Finally, the prism was cleaned thoroughly with ethanol and ultrapure water and dried under a flow of N 2 . It was kept at 4 °C in refrigerator before use.

Protein sensing by surface plasmon resonance imaging

Protein sensing was performed at 25 °C with a SPRi apparatus (Horiba Scientific), which was put in a temperature regulated incubator (Memmert, Germany). A collimated beam from a LED was polarized and sent towards the functionalized gold surface through the prism to illuminate the entire microarray.

The intensity of the reflected light was monitored by a CCD camera. In this study, SPRi measurements were performed at a fixed working angle, chosen at the highest slope of the plasmon curves.

The functionalized prism was placed in a PEEK flow cell with a volume of 10 µL and a hexagonal configuration. The flow cell was connected to a degassing system (Alltech, France), a 6-port medium pressure injection valve (Upchurch Scientific, USA) for sample injection, and a computer controlled syringe pump (Cavro XLP 6000, Cavro scientific instruments, USA).

In this study, freshly filtered and degassed HEPES solution (10 mM, pH 7.4) was used as running buffer solution for performing SPRi experiments. Protein samples were also prepared using this buffer and then injected onto the prism with a volume of 500 µL at a 100 µL/min flow rate (5 min infusion).

Once injected, molecular binding occurred. Consequently, all the spots of the cross-reactive receptors lighted up with different intensities according to their affinity with the protein. This binding event was registered using an 8-bit CCD video camera at a fixed interval of time (0.2 s). After each injection, the array was rinsed with running buffer at a 100 µL/min flow rate and then regenerated with 2% SDS (w/w) dissolved in ultrapure water for reuse. At least three replicated injections and measurements were performed in a random order for each protein sample on the same CoCRR array.

Moreover, a reference sample, protein ECL at 200 nM was used as a standard to evaluate the functionality of the eT in terms of stability and repeatability. [START_REF] Genua | SPR imaging based electronic tongue via landscape images for complex mixture analysis[END_REF] It was systematically injected onto the CoCRR array at the beginning before the injection of protein of interest, several times randomly in the middle and at the end of all protein sensing.

Data processing

SPRi monitors the changes of the refractive index taking place at the vicinity of the surface of the CoCRRs upon protein injection using the CCD video camera, which registers real-time images across the whole array. Using Imaging software (Horiba Scientific), all SPR images were converted to variations of reflectivity (expressed as R%) versus time and thus generated a series of kinetic binding curves, called sensorgrams. By plotting the response (R%) of each combinatorial surface for the protein sample at the end of injection versus BB1%, a 2D continuous evolution profile (CEP) was generated. By adding the BB1% in sensorgrams, 3D continuous evolution landscape (CEL) was generated for protein sample, which allows to integrating adsorption and desorption kinetics.

Results and Discussion

Scheme 1. Schematic representation of a) HS and b) the CoCRR array to demonstrate its rational design by mimicking HS, based on self-assembly of two disaccharides, lactose (BB1) and sulfated lactose (BB2),

at various ratios on the surface of a chip.

In practice, to construct the CoCRR array, BB1 and BB2 were prepared from β-allyl lactoside. [START_REF] Hou | Continuous evolution profiles for electronic-tonguebased analysis[END_REF] Using BB1 and BB2 at a constant total concentration, eleven solutions were prepared with

[BB1]/([BB1]+[BB2]) ratios of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100%. They were then spotted on the gold surface of a prism to generate, by formation of self-assembled monolayers (SAMs), an array composed of eleven CoCRRs. For each spot, the ratio of the two building blocks anchored on the gold surface was assumed to be equal to the molar ratio in the parent solution. [START_REF] Stranick | Nanometer-scale phase separation in mixed composition self-assembled monolayers[END_REF] Firstly, the protein IFNγ wild-type (IFNγ WT) at 50 nM prepared in 10 mM HEPES buffer solution (pH 7.4) was injected on the CoCRR array. Binding events were monitored in real-time by SPRi.

Herein, the variation of reflectivity (expressed as R%) for each combinatorial surface was recorded versus time, giving a series of kinetic binding curves, called sensorgrams as shown in Fig. 1a. Clearly, in coherence with our previous results, [START_REF] Hou | Continuous evolution profiles for electronic-tonguebased analysis[END_REF] the affinity of IFNγ WT to the combinatorial surfaces is dependent on BB2% in the BB mixtures: the higher the sulfated BB ratio, the higher the IFNγ WT affinity. Notably, the protein has the highest affinity to the combinatorial surfaces composed of 100 to 90% of sulfated BB2. Based on the sensorgrams, a pattern was generated for IFNγ WT by plotting the variation of reflectivity (R%) versus BB1% in the BB mixtures at equilibrium (13 minutes after protein injection).

Notably, according to the combinatorial approach, the composition of each sensing receptor is linked and close to that of its neighbors so that each receptor provides a signal that is coherent to that of its neighbor. Thus, the series of signals generated upon interaction between combinatorial surfaces and analytes can be considered as continuous. [START_REF] Hou | Continuous evolution profiles for electronic-tonguebased analysis[END_REF] Consequently, a 2D CEP can be generated, as shown in Fig. 1b. Moreover, thanks to SPRi, we can introduce adsorption and desorption kinetics as a new discrimination parameter by adding time as the third dimension in the CEP. In this way, a timedependent recognition pattern was obtained, called 3D CEL (see Fig. 1c). Both CEP and CEL are characteristic of the protein and thus can be used as "fingerprints" for its identification based on pattern recognition. However, CEL can provide supplementary information since two analytes presenting the same relative affinity for the CoCRR array may differ in their kinetics of interaction.

Furthermore, IFNγ WT was analyzed in a large range of concentrations: 25 nM, 50 nM, 100 nM, 200 nM, 400 nM, 800 nM, and 1600 nM. Its CEPs at various concentrations displayed Fig. 1d. For most combinatorial surfaces, the signal intensity is proportional to the protein concentration. Interestingly, the shape of all the CEPs are nearly the same. Therefore, the CoCRR array has a large dynamic range, capable of detecting IFNγ WT in the low nanomolar range before reaching saturation at high protein concentrations (> 800 nM).

Since the CoCRR array was designed to mimic biological properties of HS, it was expected that the system would show higher affinity to HSbps than to non-HSbps, even the ones having an isoelectric point (pI) above the pH of the running buffer (7.4). To challenge this hypothesis, the three non-HSbps Bovine Serum Albumin (BSA, pI = 5.2), Immunoglobulin G from rabbit serum (IgG, pI = 7.5), and Cytochrome C from horse heart (CytC, pI = 10.7) were injected on the CoCCR array under the same experimental conditions as used for IFNγ WT. For each protein, various concentrations were tested.

Gratifyingly, the CoCRR array has not only much lower affinity for BSA and IgG, but also for the positively charged CytC (at pH 7.4). It was found that in the low nanomolar range none of them interacts with the CoCRR array. Only at very high micromolar range concentrations is the signal intensity of their corresponding CEP (Fig. 2 a-c) comparable to that of IFNγ WT obtained at only 50 nM (Fig. 1b). In order to determine if the CoCRR array is able to differentiate between the protein IFNγ WT and some mutants with major modifications in D1 and D2, two mutants IFNγ Δ124 and IFNγ Δ136 were used. They were obtained by deleting the terminal sequences 125-143 (both D1 and D2) and 137-143 (only D2), respectively. The two proteins were injected onto the CoCRR array at 50 nM. Their corresponding CEPs are given in Fig. 3a and3b. For IFNγ Δ124, there was almost no interaction with the CoCRR array. This means that the deletion of the D1 and D2 led to the complete loss of affinity of the protein to the system (Fig. 3a). It is noteworthy that there are some other clusters of basic amino acids in IFNγ WT besides D1 and D2. However, obviously, they do not significantly contribute to the binding of the protein on the combinatorial surfaces at such a concentration. In contrast, for IFNγ Δ136, with only the removal of the D2, the shape of its CEP was similar to that of IFNγ WT but with lower signal intensity (Fig. 3b). Taken together, the CoCRR array is able to differentiate between the three proteins. Furthermore, we intend to determine the respective importance of D1 and D2 in the interaction between the CoCRR array and IFNγ WT. Based on these results, it is most likely that D1 is the predominant binding site since its removal led to the complete loss of affinity of the protein to the system.

To further confirm this point, the mutants IFNγ SD1 (amino acids 124-143, ASTGSSSSSQMLFRGRRASQ) and IFNγ SD2 (amino acids 124-143, AKTGKRKRSQMLFSGSSASQ) were designed by substituting all basic amino acid residues with the neutral serine in D1 and D2, respectively. For IFNγ SD1 , the CEP (Fig. 3c) showed that, just like IFNγ Δ124, there was almost no interaction with the CoCRR array despite the presence of D2. These results

show that similarly to HS, the CoCRR array is extremely sensitive to major changes in D1 cluster.

While for IFNγ SD2 with major modification in D2 (Fig. 3d) a significant decrease of CEP signal intensity was observed but this mutant did not completely lose affinity to the CoCRR array. Therefore, these data confirm that D1 is the predominant binding site for the interaction between the CoCRR array and IFNγ WT. mutants are somehow different to that of IFNγ WT. In particular, the differences are evident for their CELs, in which the kinetics of interaction were taken into account.

It is important to mention that the three dimensional structure of IFNγ has been extensively studied. It is known that the C-terminal domain, including the two HS binding sites, is fully solvent exposed and does not adopt a specific folding. Consequently, it is not engaged in intramolecular contacts. [START_REF] Walter | Crystal structure of a complex between interferon-γ and its soluble high-affinity receptor[END_REF] In addition, in our previous work, IFNγ lacking parts of the (or the entire) C-terminal domain had been expressed and was shown to be correctly folded. [START_REF] Saesen | Insights into the mechanism by which interferon-γ basic amino acid clusters mediate protein binding to heparan sulfate[END_REF] Therefore, it is known that the mutations we generated do not disturb the protein fold. We could thus conclude that the changes in binding are linked to the charge in basic amino acid residue's content in the protein C-terminal domain.

Therefore, these findings indicate that the CoCRR array is also capable of perceiving such subtle changes made in the C-terminal region of the protein. To the best of our knowledge, to date there is no multisensor system that has achieved such a good selectivity with the capability to differentiate between proteins with only a single amino acid difference, based on the cross-reactivity principle and pattern recognition.

Figure 1

 1 Figure 1. a) Sensorgrams showing the response of each combinatorial surface for IFNγ WT at 50 nM. b) The pattern generated by the CoCRR array for IFNγ WT (50 nM), called continuous evolution profile (CEP) showing the response of each combinatorial surface for IFNγ WT at 13 th minute after protein injection (Error bars were obtained with the four replicates of each combinatorial surface on the chip.). c) The second pattern generated for IFNγ WT (50 nM) by integrating adsorption and desorption kinetics thanks to SPRi, called 3D continuous evolution landscape (CEL). Note: R% increases from red yellowgreen blue. d) CEPs of IFNγ WT at different concentrations.

Figure 2 .Figure 3 .

 23 Figure 2. Sensing of non-heparan sulfate binding proteins with the CoCRR array for comparison with IFNγ WT. CEPs of a) BSA at 20 μM, b) IgG at 1 μM, and c) CytC at 1 μM. Error bars were obtained with the four replicates of each combinatorial surface on the chip.For example, Fig.2shows the CEPs of the three proteins generated at selected concentrations: BSA (20 μM), IgG (1 μM) and CytC (1 μM). For negatively charged BSA and neutral IgG, the proteins bound to all the combinatorial surfaces almost in the same way due to simple non-specific adsorption.

Figure 4 .

 4 Figure 4. Sensing of the IFNγ WT's single-point mutants with the CoCRR array. a) Sensorgrams, b) CEP and c) CEL of IFNγ S129, d) Sensorgrams, e) CEP and f) CEL of IFNγ S130. Both proteins were injected on the CoCRR array at 50 nM. Error bars for b and e were obtained with the four replicates of each combinatorial surface on the chip. Note: for c) and f), R% increases from red yellowgreen. Finally, we have evaluated the capability of the CoCRR array to discriminate between IFNγ WT and its single-point mutants. For this, two single amino acid mutants in D1 were prepared, IFNγ S129 and IFNγ S130 in which the residues R 129 and K 130 were substituted with a S residue respectively (amino acids 124-143, AKTGKSKRSQMLFRGRRASQ and AKTGKRSRSQMLFRGRRASQ). They were subsequently injected onto the CoCRR array at 50 nM. The obtained sensorgrams, CEPs and CELs for the two mutants are shown in Fig. 4. Compared to the protein IFNγ WT, the two mutants both have reduced affinity for the CoCRR array. Moreover, the CEP and CEL of these two single amino acid

Acknowledgment

The authors thank greatly Labex LANEF at Grenoble (ANR-10-LABX-51-01) for supporting the PhD scholarship of Laurie-Amandine Garçon and the CEA's "Programme Transversal Nanosciences" for supporting the postdoc of Yulia Sergeeva. IRIG-SyMMES is a part of Labex ARCANE and CBH-EUR-GS (ANR-17-EURE-0003) and GRAL (ANR-10-LABX-49-01).

For the positively charged CytC (at pH 7.4), higher interaction with the combinatorial surfaces rich in negatively charged BB2 was observed, probably due to non-specific electrostatic interaction. However, a 20-fold higher concentration was required to obtain a signal intensity comparable to that of IFNγ WT (50 nM), whose pI (10.1) is similar to that of CytC. Moreover, in our previous work, [START_REF] Hou | Continuous evolution profiles for electronic-tonguebased analysis[END_REF] the CoCRR array was used to analyse two other HSbps: the α and γ isoforms of the chemokine CXCL12. These two proteins can be easily discriminated from each other based on their CEPs. When compared with IFNγ, the CEP of IFNγ was distinct from that of CXCL12α but barely distinguishable from the one of CXCL12γ. This was explained by their structures. In fact, both CXCL12γ and IFNγ possess two distant HS binding domains. At least one of them is located in an unfolded part of the protein, whose flexibility could thus maximize contact points with their ligands or with low charged CoCRRs through conformational fluctuations. For a better discrimination between IFNγ and CXCL12γ, more BB1/BB2 mixtures and/or their mixtures with some other BBs with complementary physicochemical properties should be integrated into the CoCRR array. All these results confirm that the CoCRR array, designed to mimic the biological properties of HS, is able to discriminate not only HSbps from non-HSBPs, but also between different HSbps .

In the following study, to better understand the high affinity between the CoCRR array and IFNγ WT and in particular to investigate if the CoCRR array is able to discriminate between proteins with very similar structures, different IFNγ mutants were designed. The carboxyl-terminal domain of IFNγ (amino acids 124-143, AKTGKRKRSQMLFRGRRASQ) consists of two adjacent clusters of basic amino acids including domain 1 (D1) with residues 125-131 (KTGKRKR) and domain 2 (D2) with residues 137-140 (RGRR). [START_REF] Vanhaverbeke | NMR characterization of the interaction between the C-terminal domain of interferon-γ and heparin-derived oligosaccharides[END_REF] According to our previous findings both D1 and D2 are implicated in HS recognition but D1 is the predominant binding site. [START_REF] Saesen | Insights into the mechanism by which interferon-γ basic amino acid clusters mediate protein binding to heparan sulfate[END_REF]

Conclusions

In conclusion, based on our rational biomimetic methodology, the CoCRR array, prepared by using simple and easily accessible disaccharides, can mimic the biological properties of HS. Coupled with SPRi, such an array is very efficient for the analysis of HSbps with high sensitivity and selectivity. In addition, and more importantly, it is even capable of discriminating subtle differences between proteins, such as shown for IFNγ and its single point mutants, used here as a model system. This is the first time that such a good selectivity is obtained based on cross-reactive arrays. The CoCRR array-SPRi system is reusable after regeneration. Furthermore, by introducing novel BBs with complementary physicochemical properties, the CoCRR array with improved protein binding properties could be developed. Thus, it represents a simple and promising tool for protein sensing as well as for better understanding of HS-protein interaction mechanisms. Furthermore, it could be highly useful for research on novel GAG mimetic drugs.

Declarations

The authors declare no conflicts of interest.


Edith Grosbellet

Stephanie Dumont

Carole Schuster-Klein

Beatrice Guardiola-Lemaitre

Paul Pevet

François Criscuolo

Etienne Challet
email: challet@inci-cnrs.unistra.fr

Circadian phenotyping of obese and diabetic db/db mice

Keywords: Diabetes, Obesity, Circadian rhythm, Hypothalamus ANOVA, analysis of variance, ARC, arcuate nucleus, CRY, cryptochrome, DMH, dorso-medial hypothalamic nucleus, NS, non-significant, PB, phosphate buffer, PBS, phosphate buffer saline, PER2,

Introduction

In mammals, most metabolic parameters are under the control of the circadian timing system.

The master circadian clock, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, is reset by the environmental light-dark cycle and synchronizes secondary oscillators present in many regions of the brain (e.g., nuclei of the medial hypothalamus, involved in feeding-fasting cycle) and in most peripheral organs (e.g., liver and adipose tissue) [START_REF] Froy | Metabolism and circadian rhythms--implications for obesity[END_REF][START_REF] Bechtold | Hypothalamic clocks and rhythms in feeding behaviour[END_REF]. The circadian control of metabolism is reflected, for instance, by daily variations of metabolic hormones, such as leptin, and plasma glucose [START_REF] Kalsbeek | The suprachiasmatic nucleus generates the diurnal changes in plasma leptin levels[END_REF][START_REF] Gimble | Prospective influences of circadian clocks in adipose tissue and metabolism[END_REF]. The molecular clockwork relies on transcriptional and translational feedback loops involving clock genes and proteins and generating a rhythmic transcriptional activity with a ~24-h period. The main feedback loop involves CLOCK-BMAL1 heterodimer, which stimulates the transcription of Period (e.g., Per1-2) and Cryptochrome (Cry 1-2) genes. PER-CRY heterodimers inhibit in turn the transcriptional activity of CLOCK-BMAL1. CLOCK-BMAL1 also stimulates the transcription of clock-controlled genes, many of them being also involved in metabolism control, thus providing a molecular mechanistic basis for a circadian control of metabolism [START_REF] Froy | Metabolism and circadian rhythms--implications for obesity[END_REF].

In turn, metabolic cues can affect circadian rhythmicity. Feeding time and concomitant changes in nutritional and hormonal signals are potent synchronizers of peripheral oscillators, such as the liver and white adipose tissue [START_REF] Schibler | Peripheral circadian oscillators in mammals: time and food[END_REF][START_REF] Damiola | Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus[END_REF][START_REF] Stokkan | Entrainment of the circadian clock in the liver by feeding[END_REF][START_REF] Zvonic | Characterization of peripheral circadian clocks in adipose tissues[END_REF]. Under certain conditions of feeding (hypo-or hypercaloric diets), metabolic cues affect the master clock and modify its circadian responses to light [START_REF] Challet | Fos-like immunoreactivity in the circadian timing system of calorie-restricted rats fed at dawn: daily rhythms and light pulseinduced changes[END_REF][START_REF] Resuehr | Caloric restriction and melatonin substitution: effects on murine circadian parameters[END_REF][START_REF] Kohsaka | High-fat diet disrupts behavioral and molecular circadian rhythms in mice[END_REF][START_REF] Mendoza | High-fat feeding alters the clock synchronization to light[END_REF][START_REF] Mendoza | Feeding cues alter clock gene oscillations and photic responses in the suprachiasmatic nuclei of mice exposed to a light/dark cycle[END_REF]. This reciprocal relationship is potentially of great importance for human health since metabolic disorders like obesity or diabetes are concomitant to circadian disturbances [START_REF] Froy | Metabolism and circadian rhythms--implications for obesity[END_REF][START_REF] Gimble | Prospective influences of circadian clocks in adipose tissue and metabolism[END_REF].

Various experimental models of obesity or diabetes were previously used to study the impact of metabolic disorders on circadian rhythmicity. For example, diet-induced obesity in rodents lengthens the endogenous period of wheel-running behavior and body temperature under constant darkness, and reduces photic resetting of the master clock, as shown by smaller lightinduced phase-advances and slower rate of re-entrainment after a jet-lag (phase-advance) [START_REF] Kohsaka | High-fat diet disrupts behavioral and molecular circadian rhythms in mice[END_REF][START_REF] Mendoza | High-fat feeding alters the clock synchronization to light[END_REF]. Genetically obese ob/ob mice, lacking functional leptin, display no change in the endogenous period, but display altered photic resetting, as shown by larger light-induced phasedelays and faster rate of re-entrainment after a delayed light-dark cycle [START_REF] Sans-Fuentes | Light responses of the circadian system in leptin deficient mice[END_REF][START_REF] Grosbellet | Leptin normalizes photic synchronization in male ob/ob mice, via indirect effects on the suprachiasmatic nucleus[END_REF], and disturbances of peripheral clocks [START_REF] Ando | Impairment of peripheral circadian clocks precedes metabolic abnormalities in ob/ob mice[END_REF]. Moreover, insulino-dependent diabetes, induced in rodents by the chemical destruction of β-pancreatic cells, leads to circadian disturbances in peripheral clocks [START_REF] Kuriyama | Daily injection of insulin attenuated impairment of liver circadian clock oscillation in the streptozotocin-treated diabetic mouse[END_REF][START_REF] Oishi | Gene-and tissue-specific alterations of circadian clock gene expression in streptozotocin-induced diabetic mice under restricted feeding[END_REF] and alterations of photic resetting of the master clock [START_REF] Lahouaoui | Clock genes and behavioral responses to light are altered in a mouse model of diabetic retinopathy[END_REF][START_REF] Challet | Altered circadian responses to light in streptozotocin-induced diabetic mice[END_REF].

Db/db mice, lacking functional leptin receptor, are a recognized model of obesity and type 2 diabetes [START_REF] Herberg | Laboratory animals exhibiting obesity and diabetes syndromes[END_REF]. Therefore, db/db mice offer a model of choice to study the circadian alterations resulting from obesity combined with severe diabetes. Slight alterations in transcriptional levels of Per2 in SCN, as well as impairments of hepatic molecular clockwork have previously been shown in db/db mice [START_REF] Kudo | Night-time restricted feeding normalises clock genes and Pai-1 gene expression in the db/db mouse liver[END_REF]. The aim of this study was first to further investigate circadian alterations in the master clock of db/db mice, by studying free-running rhythms, circadian oscillations of clock proteins (PER1, PER2 and BMAL1), as well as behavioral and molecular responses to light. Wild-type mice fed with high fat and ob/ob mice display slower rate of reentrainment after an advanced light-dark cycle, suggesting that obesity in both cases alters lightinduced phase-advances [START_REF] Mendoza | High-fat feeding alters the clock synchronization to light[END_REF][START_REF] Grosbellet | Leptin normalizes photic synchronization in male ob/ob mice, via indirect effects on the suprachiasmatic nucleus[END_REF]. The study of circadian responses to light in late night was expected to highlight the additional effect of severe diabetes in db/db mice. Second, because db/db mice display alterations in feeding behavior, including daytime hyperphagia [START_REF] Grosbellet | Leptin modulates the daily rhythmicity of blood glucose[END_REF], we wondered whether alterations in feeding rhythm could be related to alterations of molecular clockwork in the SCN or in downstream structures, such as arcuate (ARC), ventro-(VMH) and dorso-medial hypothalamic (DMH) nuclei, possibly involved in feeding rhythm.

Material and Methods

Ethics statement

All experiments were performed in accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996), the French National Law (implementing the European Union Directive 2010/63/EU) and approved by the Regional Ethical Committee of Strasbourg for Animal Experimentation (CREMEAS AL/01/06/03/12).

Animals, housing and diet

Eight-week-old male BKS(D)-Lepr db /JOrlRj (db/db) mice and control littermates (db/+) were purchased from Janvier Labs breeding centre (Le Genest-Saint-Isle, France). Unless otherwise stated, they were housed in individual cages, kept at 23 ± 1°C under a 12:12 h light-dark cycle with lights on at 07:00 AM and lights off at 07:00 PM, defining Zeitgeber time (ZT) 0 and ZT12, respectively. Food (standard chow pellets, 105, SAFE, Augy, France) and tap water were available ad libitum.

Circadian phenotyping of db/+ and db/db mice

In a first series, 32 db/+ mice and 32 db/db mice were studied. Among them, 15 db/+ and 16 db/db mice were implanted intraperitoneally under isoflurane anesthesia, with Vitalview telemetry devices (Mini-Mitter Co., Sunriver, OR, USA) to record general activity and body temperature in 5 min bins during all the experimental procedure. After surgery, animals were maintained at 23°C under a 12 h light/12 h dark cycle (lights on at 7:00 AM for 2 weeks before experiments began. Other mice were exposed to the same conditions, without telemetry. Mice were first exposed to a light-dark cycle (lights on, 7 AM; lights off, 7 PM) for two weeks. The quantity of water consumed in 24 h was estimated by weighing water bottles. Then, mice were transferred under constant darkness (DD) for 10 days to determine their endogenous period.

Afterwards, mice were resynchronized to a light-dark cycle (lights on 7:00 AM; lights off 7:00 PM) during 3 weeks. On the day of sacrifice, mice were transferred in constant darkness and sacrificed every 6 h at projected ZT2, 8, 14 and 20 (projected ZT12 corresponding to the time of light offset the day before). A dim red light (TL-D 18W Red SLV, Philips, <3 lux at the level of animals) was used to euthanize mice. Brains were collected to determine oscillations of clock proteins by immunohistochemistry in suprachiasmatic nucleus (SCN) and medial hypothalamus.

A second series of 8 mice (4 db/+ and 4 db/db) was implanted with telemetry devices, as reported above. Mice were kept under a 12:12 h light-dark cycle. During baseline, water was available ad libitum. Then, daily access to water was restricted to 8 h per day (from ZT12 to ZT20) during two weeks. Thereafter, water was again available ad libitum for two weeks.

Photic resetting of SCN clock

In a third series, 28 mice (n=14 per genotype) were transferred under dark conditions after two weeks of habituation under a light-dark cycle (12 h light; 12 h dark) cycle, mice were exposed to a 6-h phase advance of the light-dark cycle (jet-lag test, advance shift). After two weeks under the new light-dark cycle, mice were transferred to constant darkness. On the first night, half animals (7 db/+ and 7 db/db mice) were exposed to a 30-min white light pulse (200 lux at the level of the animals) at projected ZT22 (i.e. the late period of the subjective night). Mice were euthanized under dim red light (see above) one hour after the beginning of the light pulse.

Dark control animals (n=7 per genotype) did not receive the light pulse and were sacrificed at the same time and in the same conditions as light-exposed mice.

Immunohistochemistry

Animals were deeply anesthetized with sodium pentobarbital (i.p. 150 mg/kg) and intracardially perfused with saline (NaCl 0.9%) followed by 4% paraformaldehyde in 0.1M phosphate buffer (PB). Each brain was rapidly removed and post-fixed in same fixative for 24 h, then cryoprotected in 30% sucrose for 48-72 h. Brains were quickly frozen in isopentane cooled at -40°C and stored at -80°C. Coronal cryosections of 30 µm through the SCN and the medial hypothalamus (ARC, VMH and DMH) were prepared on a cryostat at -20°C.

Free-floating sections were washed in 0.01M Phosphate buffer saline (PBS) and incubated in a solution of 3% of H2O2 (Sigma-Aldrich, St Louis, MO, USA) in PBS for 30 min. Brain sections were then rinsed in PBS for 10 min three times. For PER2, BMAL1, c-FOS and P-ERK1/2 staining, sections were incubated for 2 h in a blocking solution in 10% normal goat serum in PBS with 0.3% Tween-20, followed by an incubation with a rabbit polyclonal anti-PER2

(1:3000, Cat # PER-21-A, Alpha Diagnostic International, San Antonio, TX, USA), anti-BMAL1 (1:3000, ab3350, Abcam, Cambridge, MA, USA), anti-c-FOS antibody (1:10,000, SC-52, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or a rabbit monoclonal anti-Phosphorylated extracellular signal-regulated kinase1/2 (P-ERK1/2) antibody (1:40,000; #4370, Cell Signaling Technologies, Danvers, MA, USA) for 18 h at 4°C. For PER1 staining, sections were blocked in 10% normal horse serum in PBS with 0.3% Tween-20, followed by incubation with a goat polyclonal anti-PER1 (1:3000; SC-7724; Santa Cruz Biotechnology).

Sections were then rinsed three times for 10 min in PBS-T20 and, for PER2, BMAL1, c-FOS and P-ERK1/2, incubated for 2 h at 4°C with a biotinylated anti-rabbit IgG made in goat (Vectastain Standard Elite ABC Kit PK6101, Vector Laboratories, Inc., Burlingame, CA, USA) diluted 1:500 in in PBS with 0.3% Tween-20. Secondary antibody used for PER1 was a biotinylated anti-goat IgG, made in horse (BA-9500, Vector Laboratories Inc.), diluted 1:500 in in PBS with 0.3% Tween-20. Sections were rinsed in in PBS with 0.3% Tween-20 and incubated for 1 h at room temperature with an avidin-biotin-peroxidase complex (Vectastain Standard Elite ABC Kit; Vector Laboratories Inc.). Sections were rinsed three times for 10 min in PBS, and incubated with 0.05% 3,3'-diaminobenzidine (Sigma-Aldrich) with 0.015% H2O2 in tap water. Then, sections were rinsed with PBS, wet-mounted onto gel-coated slides, dehydrated through a series of alcohols, soaked in toluene and coverslipped with Eukitt (Chem Lab, Zedelgem, Belgium).

Quantification of immunostaining

Images were taken on a Leica DMRB microscope (Leica Microsystems, Rueil-Malmaison, France) connected to an Olympus DP50 digital camera (Olympus France, Rungis, France). For staining intensity, all lighting parameters on the microscope and the camera software (Viewfinder Lite; Olympus) were standardized to ensure stable lighting throughout the image capture procedure. Using the atlas of Paxinos and Franklin [START_REF] Paxinos | The Mouse Brain Stereotaxic Coordinates[END_REF]), one brain section per structure per animal was carefully selected to compare the same level of structures between animals, corresponding to bregma levels -0.46mm for SCN (Coronal plate 35 of the atlas), -1.58mm for ARC and VMH (Plate 44), -1.82mm for DMH (Plate 46). Boundaries of structures were chosen by comparison with sections stained with Cresyl Violet and by using anatomical landmarks (e.g., third ventricle, fornix and mammillothalamic tract for DMH). Quantifications were performed with ImageJ software (W. S. Rasband, U.S. National Institutes of Health, Bethesda, MD, USA) by delimiting so-called "Regions of Interest" covering each side of the nuclei. For each animal, 3 pictures were taken: one of the target structure, one part of the section without specific staining (internal capsule) and one of the slide without section. The latter was substracted from the two other images to compensate for inhomogeneities in the illumination of the image field. Then, the mean intensity of gray pixel value was calculated and averaged for both sides of target structures, and for unstained tissue, which corresponds to tissue background, varying between animals. By substracting the mean intensity of background tissue to the mean intensity of selected structure, we obtain the final value of intensity, proportional to the quantity of targeted protein.

Statistical analysis

Data are presented as mean ± SEM. Normality was assessed with a Shapiro-Wilk test. Student's t test was used to compare two groups. Two-way analysis of variance (ANOVA) was performed to assess the effects of [genotype and light conditions] or [genotypes and time] and the interaction between these factors (Statistica 10.0, StatSoft, Tulsa, OK, USA). p values > 0.05 were considered non-significant (NS).

The period of entrainment (T) under a light-dark cycle and the endogenous period (t) under constant darkness were measured for general cage activity and body temperature by using the χ 2 Periodogram function of Clocklab software (Actimetrics, Evanston, IL, USA). For assessing daily rhythmicity, we also used a cosinor analysis to determine mean level, amplitude and acrophase of the considered parameter with SigmaPlot software (Systat software Inc., San Jose, CA, USA). Data for body temperature under a light-dark cycle were fitted to the following regression: [y=A+B•cos(2•π•(x-C)/24)] where A is the mean level, B the amplitude, and C the acrophase of the rhythm. For assessing circadian rhythmicity of body temperature in constant darkness, we used a cosinor analysis to determine mean level, amplitude, acrophase, and endogenous period of the considered parameter with SigmaPlot. Data were fitted to the following regression: [y=A+B•cos(2•π•(x-C)/D)] where A, B and C were defined as above, while D is the endogenous period of the free-running rhythm. Cosinor analyses of clock proteins (PER1, PER2 and BMAL1) were performed on data at points ZT2, 8, 14 and 20. To clarify the graphic representations, ZT2 point was double-plotted in Figures 2 and4, but the repeated point was excluded for statistical analysis.

Results

Locomotor activity and body temperature rhythms

General activity and body temperature rhythms of db/+ mice were synchronized on the lightdark cycle (Figure 1A andB) and free-ran under constant darkness conditions with an endogenous period close to 23.7 h for both parameters (Table 1). By contrast, under both lightdark cycle and DD conditions, most db/db mice (12/16 and 10/16, respectively) displayed arrhythmicity of general activity (Figure 1C andD; Table 1). Despite much disruption of body temperature rhythm in db/db compared to db/+ mice, significant rhythmicity can be detected under both light-dark cycle and constant darkness conditions, albeit with lower amplitude (Table 1). When measurements were possible, the endogenous period in constant darkness was significantly longer in db/db mice for both activity and temperature rhythms (+30 and +18 min, respectively; Table 1). Moreover, the mean temperature was significantly lower in db/db mice (36.3 ± 0.05 vs 34.6 ± 0.1°C in db/+ and db/db mice, respectively; p<0.01). We also observed that db/db mice consumed six-fold more water than db/+ mice (6.3 ± 0.2 vs 39.1 ± 3.0 ml/day, for db/+ and db/db mice, respectively; p<0.01), revealing a polydipsia characteristic of severe diabetes.

Next, we challenged db/db and db/+ mice with a daily restricted access to water to investigate its impact on daily rhythmicity. Scheduled access to water restores significant behavioral rhythmicity in non-rhythmic db/db mice, while not altering rest/activity rhythm in db/+ mice (Figure 2; Table 2).

Circadian oscillations of clock proteins in SCN

PER1, PER2 and BMAL1 oscillations were not affected in the SCN of db/db mice compared with db/+ mice (2-way ANOVA, no effect of genotype, p>0.05 for each protein) (Figure 3). An effect of time was found for the three proteins (p ≤ 0.01). A cosinor analysis revealed a significant circadian rhythm for PER2 in both genotypes, with a peak close to ZT 15-16 (Table 3). The acrophase of PER1 oscillation appeared to be close to ZT12.5 in both genotypes. The circadian rhythmicity, however, was not significant in db/db mice since the amplitude did not reach the threshold of significance (p= 0.077). BMAL1 peaked around ZT18-19 in both genotypes, while the amplitude of the rhythm did not reach the threshold of significance for db/+ mice (p=0.068).

Photic resetting in SCN of db/db mice

Given that rhythms of body temperature of db/db mice were less disrupted than general activity, we tried to quantify the rate of reentrainment of db/db mice after a 6-h advance of the lightdark cycle (i.e. the numbers of days necessary for resynchronization). Unfortunately, the weaker rhythmicity of body temperature rhythm in db/db mice as compared to db/+ controls, precluded a precise quantification of rate of reentrainment (Figure 4).

As expected, a light pulse at the end of the subjective night increased the levels of the markers of cellular activation c-FOS and P-ERK1/2 in the SCN of control mice [START_REF] Kornhauser | Photic and circadian regulation of c-fos gene expression in the hamster suprachiasmatic nucleus[END_REF][START_REF] Obrietan | Light and circadian rhythmicity regulate MAP kinase activation in the suprachiasmatic nuclei[END_REF] (Figure 5).

Interestingly, the light-induction of c-FOS and P-ERK1/2 was respectively twice and 1.5 times higher in db/db mice, suggesting a higher responsiveness to light-induced phase-advances in the diabetic and obese mice.

Clock protein oscillations in medial hypothalamus

Since db/db mice show an altered pattern of feeding, with increased food intake during the light period [START_REF] Grosbellet | Leptin modulates the daily rhythmicity of blood glucose[END_REF], we wondered whether molecular clockwork was altered in oscillators of medial hypothalamus involved in feeding rhythm, like arcuate nucleus (ARC), ventro-and dorsomedial hypothalamic nuclei (VMH and DMH, respectively). In VMH and DMH, the levels of PER1 and BMAL1 were similar between db/+ and db/db mice and no circadian oscillation was detected (Figure 6). Similarly, BMAL1 levels did not oscillate and nor they differ between db/+ and db/db mice in ARC. We found a significant rhythmicity of PER1 in ARC of db/+ mice (p=0.0076), which was lost in db/db mice (NS). However, no effect of genotype was found (effect of genotype: NS), although the interaction [genotype x time] was very close to significance threshold (p=0.055).

Discussion

This study provides new information on the circadian alterations of obese and diabetic db/db mice. General activity of db/db mice is arrhythmic under both light-dark cycle and constant dark conditions, while body temperature, although disrupted, is still rhythmic under both conditions of lighting. The free-running period of body temperature is lengthened in db/db compared to db/+ mice. After a light pulse in late night, the photic induction of c-FOS and P-ERK1/2 is increased in SCN of db/db compared with db/+ mice while their rate of resynchronization is quicker, suggesting alterations in photic responses of the master clock.

Almost no alteration is detected in the daily oscillations of clock proteins in the SCN, VMH, DMH and ARC. Therefore, our results do not support a direct relationship between altered patterns of activity and feeding and alterations in SCN and medial hypothalamic oscillators.

Activity and temperature rhythms in db/db mice

Given that our db/db mice were 8 weeks of age at the beginning of the experiment, they exhibit severe diabetes [START_REF] Herberg | Laboratory animals exhibiting obesity and diabetes syndromes[END_REF]. The observed behavioral arrhythmicity in most db/db mice (Figure 1, Table 1) is consistent with a previous study [START_REF] Kudo | Night-time restricted feeding normalises clock genes and Pai-1 gene expression in the db/db mouse liver[END_REF], while another work showed only a decreased amplitude of rest/activity rhythm [START_REF] Laposky | Sleep-wake regulation is altered in leptin-resistant (db/db) genetically obese and diabetic mice[END_REF]. Because ob/ob mice do not show similar alterations of rest/activity rhythm [START_REF] Sans-Fuentes | Light responses of the circadian system in leptin deficient mice[END_REF][START_REF] Grosbellet | Leptin normalizes photic synchronization in male ob/ob mice, via indirect effects on the suprachiasmatic nucleus[END_REF], the prominence of arrhythmicity of motor behavior, but not body temperature, in db/db mice suggests functional links with diabetes. Here we report an important polydipsia in db/db mice, characteristic of severe diabetic state [START_REF] Herberg | Laboratory animals exhibiting obesity and diabetes syndromes[END_REF]. Restriction of water during the dark phase was able to restore daily variations in rest/activity in behaviorally arrhythmic animals. Therefore, we propose that behavioral arrhythmicity of db/db mice is due in most part to a masking effect of polydipsia. In db/db mice under a light-dark cycle, there may be also a decreased masking effect of light that normally inhibits motor activity of nocturnal mice during the light phase, including in mice with defective circadian clocks [START_REF] Van Der Horst | Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms[END_REF][START_REF] Bunger | Mop3 is an essential component of the master circadian pacemaker in mammals[END_REF]. This hypothesis of masking effects is in accordance with the fact that rhythms of general activity and temperature are not correlated in db/db mice, while both rhythms totally fit in db/+ mice. Db/db mice also display hypothermia, which is consistent with defects in thermogenesis and cold tolerance previously reported [START_REF] Leibel | The molecular genetics of rodent single gene obesities[END_REF]. Of interest, a previous study showed that feeding restricted to nighttime also increases the robustness of behavioral rhythmicity in db/db mice [START_REF] Kudo | Night-time restricted feeding normalises clock genes and Pai-1 gene expression in the db/db mouse liver[END_REF].

Free-running period and oscillations of clock proteins in the SCN of db/db mice

Furthermore, the endogenous period is increased in db/db mice, which highlights a faster master clock. Of note, diet-induced obese mice also display lengthened free-running period [START_REF] Kohsaka | High-fat diet disrupts behavioral and molecular circadian rhythms in mice[END_REF][START_REF] Mendoza | High-fat feeding alters the clock synchronization to light[END_REF], whereas the endogenous period is not changed in obese ob/ob mice [START_REF] Sans-Fuentes | Light responses of the circadian system in leptin deficient mice[END_REF][START_REF] Grosbellet | Leptin normalizes photic synchronization in male ob/ob mice, via indirect effects on the suprachiasmatic nucleus[END_REF]. These differential effects between ob/ob and db/db mice suggest that the lengthening of the endogenous period in db/db mice is not due to obesity or defective leptin signaling, but could result from severe diabetes. In that view, lengthening of the endogenous period in high-fat fed mice would be due to processes not directly related to diabetic symptoms. To try to explain the functional defects of the SCN clock in db/db mice, we investigated clock protein oscillations in their SCN (Figure 2). We did not detect any significant difference between db/+ and db/db mice for the three proteins studied, PER1, PER2 and BMAL1. The circadian rhythmicity did not reach, but was close to, the threshold of significance for BMAL1 in db/+ mice and PER1 in db/db mice. The difference between db/+ and db/db mice is thus very subtle. These results are consistent with those of Kudo and colleagues, who reported no difference in transcriptional levels of Per1 and found a slight increase of Per2 mRNA in the SCN of db/db mice [START_REF] Kudo | Night-time restricted feeding normalises clock genes and Pai-1 gene expression in the db/db mouse liver[END_REF]. Here PER1 and PER2 levels also tend to be slightly increased in db/db mice. Further investigations are needed to assess whether this modest increased amplitude could strengthen the negative feedback loop and somehow lengthen the endogenous period of the SCN clockwork in db/db mice.

Photic responses of SCN in db/db mice

Classical light-induced phase-shifts of rest/activity rhythm cannot be determined in db/db mice because of behavioral arrhythmicity or noisy circadian rhythmicity. At least at molecular levels, a light pulse in late night induces a higher increase of c-FOS and P-ERK1/2 in the SCN of db/db mice compared with db/+ mice (Figure 3). Of note, db/db mice are known to develop retinopathy due to hyperglycemia-induced microvascular damage, and even loss of ganglion cells [START_REF] Bogdanov | The db/db mouse: a useful model for the study of diabetic retinal neurodegeneration[END_REF]. In view of the higher sensitivity to light of the SCN cells in db/db mice, it is unlikely this effect is due to altered photo-reception or transduction in their retina and favors the idea of intra-SCN effects. The higher responsiveness of the db/db SCN to light may partly be due to their longer free-running period (i.e., close to 24 h) that would increase light-induced phaseadvances in late night. This possibility, however, needs to be tested further because high-fat fed mice that also have a longer period (also close to 24 h) display a reduction in light-induced phase-advances [START_REF] Mendoza | High-fat feeding alters the clock synchronization to light[END_REF], suggesting additional metabolic modulation of photic resetting (see below).

Db/db mice are hyperglycemic and their rhythm of glucose is reversed (peak during the night).

Their insulinemia is also elevated, with higher values during the day, opposite to the rhythm of insulin in db/+ mice [START_REF] Grosbellet | Leptin modulates the daily rhythmicity of blood glucose[END_REF]. Glucose availability has been shown to modulate SCN responses to light. Blocking glucose utilization with 2-deoxy-D-glucose reduces light-induced phase-shifts in mice [START_REF] Challet | Reduced glucose availability attenuates circadian responses to light in mice[END_REF]. By contrast, streptozotocin-induced hyperglycemia in mice increase the shifting effect of light during early night [START_REF] Challet | Altered circadian responses to light in streptozotocin-induced diabetic mice[END_REF]. Furthermore, glucose has been shown to alter the circadian firing rate of SCN slice in vitro [START_REF] Hall | Suprachiasmatic nucleus neurons are glucose sensitive[END_REF]. Glucose uptake of neurons being independent of insulin [START_REF] Heidenrich | Glucose transport in primary cultured neurons[END_REF], the increase of glucose availability in db/db mice may affect the metabolic activity of SCN cells. Molecular metabolic actors being closely connected with molecular clockwork, this could impact the circadian responses to light of SCN cells.

We investigated the effects of light exposure in early night (i.e., inducing phase-delays) in db/db mice and found no difference in light-induced expression of c-FOS and P-ERK1/2 between db/db and db/+ mice (see Supplemental Fig. S1). This result is reminiscent of the lack of molecular changes observed in the SCN of ob/ob mice exposed to light in early night [START_REF] Grosbellet | Leptin normalizes photic synchronization in male ob/ob mice, via indirect effects on the suprachiasmatic nucleus[END_REF] and different from the decrease in c-FOS observed in streptozotocin-induced diabetic rats [START_REF] Yamanouchi | Decreased level of light-induced Fos expression in the suprachiasmatic nucleus of diabetic rats[END_REF]. This indicates that in db/db mice, molecular responses to light are not changed in early night, and are specifically altered in late night. The increased molecular responses in the SCN of diabetic db/db mice after a light pulse in late night are actually opposed to those induced by similar light in high-fat fed mice which display weaker induction of c-FOS and P-ERK1/2 in the SCN, correlated with smaller phase-advances of behavioral rhythm, in spite of longer free-running period as compared to chow-fed control mice [START_REF] Mendoza | High-fat feeding alters the clock synchronization to light[END_REF]. This suggests that alterations in photic resetting of db/db mice are not a passive consequence of obesity, but could be due to two causes, namely the lack of leptinergic activity and/or severe diabetes. The lack of leptin has already been associated with altered circadian phenotype in ob/ob mice [START_REF] Grosbellet | Leptin normalizes photic synchronization in male ob/ob mice, via indirect effects on the suprachiasmatic nucleus[END_REF][START_REF] Ando | Impairment of peripheral circadian clocks precedes metabolic abnormalities in ob/ob mice[END_REF]. Photic resetting associated with phase-advances is decreased in ob/ob mice, because a slower resynchronization was found after a phase-advance of the light-dark cycle [START_REF] Grosbellet | Leptin normalizes photic synchronization in male ob/ob mice, via indirect effects on the suprachiasmatic nucleus[END_REF]. Together, these comparative data suggest that severe diabetes, rather than obesity, could explain the potentiated photic responses during late night in db/db mice.

Alterations in medial hypothalamic oscillators of db/db mice

Db/db mice also show altered feeding behavior, with higher food consumption during the light phase [START_REF] Grosbellet | Leptin modulates the daily rhythmicity of blood glucose[END_REF]. Phase-advance of feeding rhythm has also been observed in diabetic Zucker (fa/fa) rats, bearing a missense mutation in long-form leptin receptor [START_REF] Mistlberger | Circadian rhythms in the Zucker obese rat: assessment and intervention[END_REF]. Since nuclei of the medial hypothalamus have been involved in feeding rhythms [START_REF] Bechtold | Hypothalamic clocks and rhythms in feeding behaviour[END_REF], we investigated the clock protein oscillations in ARC, VMH and DMH. Contrary to our previous study [START_REF] Feillet | Forebrain oscillators ticking with different clock hands[END_REF], here we did not detect oscillations of PER1 in DMH of control mice. Due to the sharp and transient increase of PER1 in the mouse DMH, the chosen time of sampling in the present study might not be adequate to detect this rhythm. As previously shown [START_REF] Feillet | Forebrain oscillators ticking with different clock hands[END_REF], we found a significant rhythmicity of PER1 in ARC. ARC being a crucial structure involved in the generation of feeding rhythm [START_REF] Li | Leptin-sensitive neurons in the arcuate nuclei contribute to endogenous feeding rhythms[END_REF], it would be tempting to think that alteration of PER1 rhythmicity in ARC could be involved in altered feeding behavior of db/db mice. However, our data do not support such a hypothesis. Supplemental Fig. S1

Conclusions

Legends to figures

Fig. 1 .

 1 Fig. 1. Representative double-plotted actograms of general activity (A, B) and thermograms of body temperature (C D) of db/+ (left) and db/db mice (right). After 2 weeks in 12:12 h lightdark (LD) cycle, mice were transferred under constant darkness (DD) for 10 days (indicated with arrows), before being resynchronized to new a regular light-dark cycle for 3 weeks (LD pZT, projected Zeitgeber time. Projected nighttime is indicated with a shaded area.

Fig. 2 .

 2 Fig. 2. Double-plotted actograms of general activity of db/+ (left) and db/db mice (right) challenged with daily restricted access to water. After 2 weeks in 12:12 h light-dark cycle with water ad libitum, water access was limited to 8-h per day, from ZT (Zeitgeber time) 12 to ZT20 (shaded rectangle) during two weeks (indicated with arrows). ZT, Zeitgeber time.

Fig. 3 .

 3 Fig. 3. Circadian profiles of PER1, PER2 and BMAL1 expression (means ± SEM) in the suprachiasmatic nucleus (SCN) of db/+ (black circles) and db/db (white circles) mice. Data for ZT2 are double-plotted. pZT, projected Zeitgeber time. Projected nighttime is indicated with a shaded area. Scale bar: 100 m. Significant and non-significant cosinor regressions are shown with solid and dashed curves, respectively.

Fig. 4 .

 4 Fig. 4. Double-plotted thermograms of db/+ (left) and db/db mice (right) before and after a 6h phase advance of the light-dark cycle. Arrows on day 11 indicate the first day of the 6-h advance.

Fig. 5 .

 5 Fig. 5. c-FOS and P-ERK1/2 responses in the suprachiasmatic nucleus (SCN) of db/+ and db/db mice exposed (Light pulse) or not (Dark control) to a 30-min light pulse at projected ZT 22 (means ± SEM). Groups with different letters are significantly different (p<0.05). Representative photomicrographs of c-FOS and P-ERK1/2 staining in SCN of db/+ and db/db mice. Scale bar: 100µm.

Fig. 6 .

 6 Fig. 6. Circadian profiles of PER1 and BMAL1 expression (means ± SEM) in the arcuate nucleus (ARC), ventromedial and dorsomedial hypothalamus (VMH and DMH, respectively) of db/+ (black circles) and db/db (white circles) mice. Data for ZT2 are double-plotted. pZT, projected Zeitgeber time. Projected nighttime is indicated with a shaded area.

Figure 6

 6 Figure 3

Acknowledgements:

We thank S. Gourmelen and Dr D. Sage-Ciocca for their expert assistance with animal care and actimetry, respectively. This work was supported by the Centre National de la Recherche Scientifique, University of Strasbourg (E.C., F.C. and P.P.), ProjEx H2E "Contributions of exotic animal models in the discovery of new therapeutic approaches in human pathophysiology", University of Strasbourg (to F.C. and E.C.), the Institut de Recherches Internationales Servier (C.S.K. and B.G.L.), and a doctoral fellowship from the French Ministry of National Education and Research (E.G.).

These results show that db/db mice display circadian alterations. At behavioral level, db/db mice are arrhythmic, most likely due to a masking effect of polydipsia. The rhythm of body temperature is less disrupted and free-ran under constant darkness with a longer endogenous period compared with db/+ mice. Moreover, altered photic response of the SCN clock in db/db mice, characterized by higher induction of c-FOS and P-ERK1/2 after a light pulse in late night, could be due to severe diabetes. Therefore, the present study provides new experimental information showing that genetic obesity and type 2 diabetes impacts the circadian timing system by affecting the master clock.