Ariane Peyret

Hang Zhao

Sébastien Lecommandoux
email: lecommandoux@enscbp.fr

Preparation and Properties of Asymmetric Synthetic Membranes based on Lipid and Polymer Self-Assembly

Keywords: membrane, asymmetry, biomimicry, lipid, polymer

Cell membrane asymmetry is a common structural feature of all biological cells.

Researchers have tried for decades to better study its formation and its function in membraneregulated phenomena. Especially, there has been increasing interest in developing synthetic asymmetric membrane models in the laboratory, with the aim of studying basic physicalchemistry properties that may be correlated to relevant biological function. The present article aims at summarizing the main presented approaches to prepare asymmetric membranes, which are most often made from lipids, polymers, or a combination of both.

INTRODUCTION

1972 was a milestone in research biology when the famous fluid mosaic model was proposed as an interpretation of biological membrane structure by Singer and Nicolson (Figure 1). [START_REF] Singer | The Fluid Mosaic Model of the Structure of Cell Membranes[END_REF] Biological membranes were defined as " […] analogous to a two-dimensional oriented solution of integral proteins (or lipoproteins) in the viscous phospholipid bilayer solvent." This model provided a solid base to start explaining membrane organization ("mosaic") and predicted many membrane phenomena regulated by lipid and protein dynamic rearrangement ("fluid"). Figure 1. Schematic three-dimensional representation of the fluid mosaic structure of the membrane, reproduced with permission from Ref. [1]. Copyright [1972] {Science]. Globular proteins inserted into a lipid matrix.

Around that same period of time, Bretscher introduced the concept of "asymmetry" to characterize membrane structure through the different repartition of lipids and proteins across the two leaflets of the bilayer. [START_REF] Bretscher | Membrane structure: some general principles[END_REF][START_REF] Bretscher | Asymmetrical Lipid Bilayer Structure for Biological Membranes[END_REF] One of his five guidelines for membrane structure was that the basis of a biological membrane is a bilayer enriched with choline lipid derivatives and glycolipids in the exoplasmic side and amino-phospholipids in the cytoplasmic side. [START_REF] Bretscher | Asymmetrical Lipid Bilayer Structure for Biological Membranes[END_REF] In 1974, a breakthrough in understanding of biomembrane, the bilayer couple hypothesis, was proposed by Sheetz and Singer. They hypothesized that the existed asymmetry feature within a bilayer would result in the two leaflets may respond independently and differently to a perturbation while maintaining coupled to one another, which underpin the mechanism of functional consequences, such as shape changes in a cell. [START_REF] Sheetz | Biological membranes as bilayer couples. A molecular mechanism of drug-erythrocyte interactions[END_REF] Evidence of asymmetry was already supported with several examples involving selective binding of substrates to one side or the other or phospholipase digestion. [START_REF] Verkleij | The asymmetric distribution of phospholipids in the human red cell membrane. A combined study using phospholipases and freeze-etch electron microscopy[END_REF][START_REF] Zwaal | Localization of red cell membrane constituents[END_REF][START_REF] Steck | Topographical Distribution of Complex Carbohydrates in the Erythrocyte Membrane[END_REF] For example, ferritin was shown to specifically bind to oligosaccharides with d-mannose or d-glucose residues on the outer surface of erythrocyte membranes and not on the inner cytoplasmic surface. [START_REF] Nicolson | Ferritin-Conjugated Plant Agglutinins as Specific Saccharide Stains for Electron Microscopy: Application to Saccharides Bound to Cell Membranes[END_REF] While proteins were also shown to be unevenly organized between both leaflets, asymmetry in biological membranes mostly comes from the distribution of lipids, which play a major structural and functional role. Indeed, their amphiphilicity allows them to spontaneously form membranes in the form of small compartments to segregate internal constituents from the external medium. They also act as messengers in signal transduction and molecular recognition processes and they are used for energy storage. [START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF] It is now well-established that the differences in constituents between both sides of the bilayer have a strong impact on membrane physical properties such as permeability, membrane curvature (linked to the intrinsic asymmetric nature of lipids), shape or surface charge (negatively-charged phospholipids on the cytoplasmic side). [START_REF] Rothman | Membrane asymmetry[END_REF][START_REF] Lodish | Biomembranes: Structural Organization and Basic Functions[END_REF][START_REF] Manno | Identification of a functional role for lipid asymmetry in biological membranes: Phosphatidylserine-skeletal protein interactions modulate membrane stability[END_REF][START_REF] Devaux | Static and dynamic lipid asymmetry in cell membranes[END_REF] Consequently, disruption of the normal membrane asymmetry often has significant physiological consequences such as blood coagulation or recognition and removal of apoptotic cells by macrophages. [START_REF] Fadeel | The ins and outs of phospholipid asymmetry in the plasma membrane: roles in health and disease[END_REF][START_REF] Lentz | Exposure of platelet membrane phosphatidylserine regulates blood coagulation[END_REF][START_REF] Zwaal | Pathophysiologic Implications of Membrane Phospholipid Asymmetry in Blood Cells[END_REF] For example, phosphatidylserine exposure on the outer leaflet is a sign for cell apoptosis. [START_REF] Fadok | Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages[END_REF][START_REF] Fadok | Apoptosis: Getting rid of the bodies[END_REF][START_REF] Verhoven | Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes[END_REF][START_REF] Schlegel | Phosphatidylserine, a death knell[END_REF] These processes are essential for cell development but if unregulated, they could be the source of pathologic conditions. [START_REF] Daleke | Regulation of transbilayer plasma membrane phospholipid asymmetry[END_REF] Asymmetry of lipid charge has shown impact on determination of transmembrane protein oridentation, [START_REF] Slusky | Charge asymmetry in the proteins of the outer membrane[END_REF] and a recent study proved that the segments of transmembrane contains smaller side chains prefer to exist in the outer monolayer of bilayer. [START_REF] Sharpe | A comprehensive comparison of transmembrane domains reveals organelle-specific properties[END_REF] Author manuscript of Langmuir 2018, 34, 3376-3385 DOI: 10.1021/acs.langmuir.7b04233 Thus, maintaining the repartition of constituents and mainly lipids between the two leaflets and hence maintaining asymmetry, is crucial for the proper functioning of membrane regulated cellular phenomena. [START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF][START_REF] Fadeel | The ins and outs of phospholipid asymmetry in the plasma membrane: roles in health and disease[END_REF][START_REF] Boon | Chemical control of phospholipid distribution across bilayer membranes[END_REF] As such, a lot of energy is invested by the cell to maintain the non-random distribution of species across the bilayer with the help of transporter enzymes such as flippases (ATP-dependent) or floppases (ATP independent) which are in charge of controlling the transmembrane distribution of lipids. [START_REF] Daleke | Regulation of transbilayer plasma membrane phospholipid asymmetry[END_REF][START_REF] Sharom | Flipping and flopping-lipids on the move[END_REF][START_REF] Pomorski | Lipid distribution and transport across cellular membranes[END_REF][START_REF] Devaux | Phospholipid flippases[END_REF][START_REF] Seigneuret | ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes[END_REF][START_REF] Bitbol | Measurement of outward translocation of phospholipids across human erythrocyte membrane[END_REF][START_REF] Meer | Dynamic Transbilayer Lipid Asymmetry[END_REF] The study of biological membranes has been facilitated by the preparation of synthetic symmetric lipid membranes either supported or in the form of vesicular systems. [START_REF] Iglic | Advances in Planar Lipid Bilayers and Liposomes[END_REF][START_REF] Huang | Emulsion templated vesicles with symmetric or asymmetric membranes[END_REF] Models with asymmetric membranes would however be more realistic in cell structural biomimicry studies but they have been difficult to establish experimentally mostly because of a lack of reliable methods to precisely control and quantify the asymmetry. Still, over the last few years, significant progress has been made in the preparation of such model systems using mostly lipid-only membranes. [START_REF] Marquardt | Asymmetric lipid membranes: towards more realistic model systems[END_REF] There are however many other possibilities to form asymmetric membranes with cell mimicking properties by using alternative synthetic pathways or constituents such as polymers or peptides. [START_REF] Huang | Emulsion templated vesicles with symmetric or asymmetric membranes[END_REF] The goal of this review is to gather and summarize the main cell mimicking asymmetric membrane models that have been proposed and studied over the last years. We aim at providing a non-exhaustive review of the different systems with respect to geometry and composition, focusing on the most relevant synthetic routes.

We firstly describe the main different methodologies to prepare asymmetric membranes made exclusively from lipids. We then outline innovative approaches that have been presented with polymers and finally give an overview of a few systems that combine both lipids and polymers. These systems are schematically illustrated in Figure 2, which presents the main preparation routes, compositions and morphologies that have been reported for asymmetric lipid and polymer membranes. Figure 2. Schematic representation of some of the reported strategies to prepare synthetic asymmetric membranes with respect to composition and morphology, including pH gradient, [START_REF] Hope | Phospholipid asymmetry in large unilamellar vesicles induced by transmembrane pH gradients[END_REF] lipid insertion by proteins / enzymes or chemicals, [START_REF] Johnson | Use of phospholipid exchange protein to measure inside-outside transposition in phosphatidylcholine liposomes[END_REF][START_REF] Zilversmit | Lipid transfer proteins[END_REF][START_REF] Low | Phosphatidylinositol distribution and translocation in sonicated vesicles. A study with exchange protein and phospholipase C[END_REF] β-cyclodextrin, [START_REF] Cheng | Preparation and Properties of Asymmetric Large Unilamellar Vesicles: Interleaflet Coupling in Asymmetric Vesicles Is Dependent on Temperature but Not Curvature[END_REF][START_REF] Lin | Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry[END_REF][START_REF] Heberle | Subnanometer Structure of an Asymmetric Model Membrane: Interleaflet Coupling Influences Domain Properties[END_REF] droplet over an interface, [START_REF] Hamada | Construction of Asymmetric Cell-Sized Lipid Vesicles from Lipid-Coated Water-in-Oil Microdroplets[END_REF][START_REF] Richmond | Forming giant vesicles with controlled membrane composition, asymmetry, and contents[END_REF][START_REF] Hu | Microfluidic Fabrication of Asymmetric Giant Lipid Vesicles[END_REF][START_REF] Peyret | Asymmetric Hybrid Polymer-Lipid Giant Vesicles as Cell Membrane Mimics[END_REF] jetting, [START_REF] Kamiya | Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes[END_REF] supported bilayers, [START_REF] Visco | Asymmetric supported lipid bilayer formation via methyl-β-cyclodextrin mediated lipid exchange: influence of asymmetry on lipid dynamics and phase behavior[END_REF] nano-tubes, [START_REF] Li | Design of Asymmetric Peptide Bilayer Membranes[END_REF][START_REF] Lim | Spontaneous formation of nanometer scale tubular vesicles in aqueous mixtures of lipid and block copolymer amphiphiles[END_REF] triblock polymers, [START_REF] Mason | Polymersomes with asymmetric membranes based on readily accessible di-and triblock copolymers synthesized via SET-LRP[END_REF][START_REF] Konishcheva | PEO-b-PCL-b-PMOXA Triblock Copolymers: From Synthesis to Microscale Polymersomes with Asymmetric Membrane[END_REF][START_REF] Stoenescu | Vesicles with asymmetric membranes from amphiphilic ABC triblock copolymers[END_REF] and microfluidics. [START_REF] Matosevic | Layer-by-layer cell membrane assembly[END_REF][START_REF] Lu | Continuous microfluidic fabrication of synthetic asymmetric vesicles[END_REF][START_REF] Karamdad | Studying the effects of asymmetry on the bending rigidity of lipid membranes formed by microfluidics[END_REF][START_REF] Zhao | Synthetic membranes in microfluidic interfaces[END_REF]

DESIGN OF ASYMMETRIC MEMBRANES BASED ON LIPIDS

Lipids are the main constituents of all biological membranes. [START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF][START_REF] Pomorski | Lipid distribution and transport across cellular membranes[END_REF] Their amphiphilic character is responsible for their spontaneous organization into two leaflets that together form a bilayer.

Their polar headgroup is oriented towards the external aqueous environment while their hydrophobic tail (hydrocarbon chains) form the core of the membrane. Lipid-only asymmetric synthetic bilayer systems have been widely used and have proven to be highly successful models to study membrane properties. [START_REF] Marquardt | Asymmetric lipid membranes: towards more realistic model systems[END_REF][START_REF] Fernandez-Trillo | Vesicles in Nature and the Laboratory: Elucidation of Their Biological Properties and Synthesis of Increasingly Complex Synthetic Vesicles[END_REF] The wide available pool of lipids makes them suitable candidates to form different geometries through various processes detailed below.

Early examples of synthesized asymmetry in small and large vesicles relied on lipid transfer techniques involving lipid exchange proteins or enzymes. [START_REF] Johnson | Use of phospholipid exchange protein to measure inside-outside transposition in phosphatidylcholine liposomes[END_REF][START_REF] Zilversmit | Lipid transfer proteins[END_REF][START_REF] Low | Phosphatidylinositol distribution and translocation in sonicated vesicles. A study with exchange protein and phospholipase C[END_REF][START_REF] Markones | Engineering asymmetric lipid vesicles: accurate and convenient control of the outer leaflet lipid composition[END_REF] Lipid transfer proteins were discovered in 1975 in the group of Zilversmit after investigating how lipid components from plasma lipoproteins were taken up by liver cells. [START_REF] Johnson | Use of phospholipid exchange protein to measure inside-outside transposition in phosphatidylcholine liposomes[END_REF][START_REF] Zilversmit | Lipid transfer proteins[END_REF] Several groups studied the translocation rate of lipids from one monolayer to the other using the lipid exchange or conversion technique essentially by means of NMR after exposing small lipid vesicles to an exchange protein or often to a phospholipase enzyme that converts phosphatidylcholine (POPC) to phosphatidic acid (PA). [START_REF] Low | Phosphatidylinositol distribution and translocation in sonicated vesicles. A study with exchange protein and phospholipase C[END_REF][START_REF] De Kruijff | Rapid transbilayer movement of phospholipids induced by an asymmetrical perturbation of the bilayer[END_REF][START_REF] Pagano | Formation of asymmetric phospholipid membranes via spontaneous transfer of fluorescent lipid analogues between vesicle populations[END_REF] The lipid translocation (or flip-flop) rates varied from minutes to a few days depending on the system. [START_REF] Low | Phosphatidylinositol distribution and translocation in sonicated vesicles. A study with exchange protein and phospholipase C[END_REF][START_REF] De Kruijff | Rapid transbilayer movement of phospholipids induced by an asymmetrical perturbation of the bilayer[END_REF] Other examples of generating transmembrane bilayer asymmetry involve spontaneous transfer of a lipid from a donor to an acceptor lipid vesicle when the two populations are in close contact or a chemical control with a sodium ascorbate induced extinction of paramagnetism of spin-labelled lipids in the outer monolayer of lipid vesicles. [START_REF] Pagano | Formation of asymmetric phospholipid membranes via spontaneous transfer of fluorescent lipid analogues between vesicle populations[END_REF][START_REF] Mcconnell | Inside-outside transitions of phospholipids in vesicle membranes[END_REF] Later on, the pH gradient technique was proposed by Cullis and coworkers to induce transverse asymmetry in lipid bilayers. [START_REF] Hope | Phospholipid asymmetry in large unilamellar vesicles induced by transmembrane pH gradients[END_REF] They showed that they could influence the positioning of weak acid lipids such as phosphatidylglycerol (PG) by applying a transmembrane pH gradient in lipid vesicles (inside basic and outside acidic or the reverse). Charged lipids are known to move very slowly from one monolayer to the other but it was expected that using protonated (uncharged) PG lipids would facilitate the movement. It was found that when POPC vesicles with a fraction of PG were prepared with a transmembrane pH gradient (inside basic), almost 90 % of PG located in the inner monolayer. Interestingly, the reverse pH gradient led to the opposite asymmetry and the same vesicles formed with zwitterionic lipids only retained a symmetric distribution of lipids thus confirming the influence of pH on the movement of weak acid lipids. [START_REF] Hope | Phospholipid asymmetry in large unilamellar vesicles induced by transmembrane pH gradients[END_REF] Strategies based on the ability of cyclodextrin (CD) derivatives to bind and exchange phospholipids have been developed in the laboratory of E. London in the last decade to construct vesicles comprising an asymmetrical distribution of lipids between the lipidic monolayers. [START_REF] Cheng | Preparation and Properties of Asymmetric Vesicles That Mimic Cell Membranes[END_REF][START_REF] Chiantia | Asymmetric GUVs Prepared by MβCD-Mediated Lipid Exchange: An FCS Study[END_REF] Methyl-β-cyclodextrin (MβCD) molecules have the ability to bind hydrophobic species within their cavity and thus have attracted much attention as potential carriers for cholesterol, lipophilic drugs or other molecules with low solubility. [START_REF] Anderson | Calorimetric Measurement of Phospholipid Interaction with Methyl-β-Cyclodextrin[END_REF] In one of their initial studies, sphingomyelin (SM) multi-lamellar vesicles (MLVs) were obtained by rehydration of a dried lipid film. The assemblies were then incubated with highly concentrated MβCD to form SM-MβCD complexes which were purified and incubated with pre-formed small unilamellar lipid vesicles (SUVs) to allow insertion of SM in the outer lipid monolayer (Figure 3). [START_REF] Cheng | Preparation and Properties of Asymmetric Vesicles That Mimic Cell Membranes[END_REF] High performance thin layer chromatography confirmed a rapid (within minutes) exchange of about 80 % lipid with the outer monolayer of the SUVs whereas control samples with SUVs incubated with SM in the absence of MβCD only showed traces of exchanged SM. Fluorescence anisotropy measurements of fluorescent probes added to the vesicles were used to confirm asymmetry which was stable over a few days. The authors also showed that cholesterol could be inserted into these asymmetric SUVs by performing a second successive MβCD exchange step, confirming the versatility of the process. Following studies by the same group introduced the production of asymmetric large and giant unilamellar vesicles (respectively LUVs and GUVs) through the same MβCD-mediated lipid exchange with the aim of studying how asymmetry impacts ordered domain formation and inter-leaflet coupling in cell bilayer membranes. [START_REF] Cheng | Preparation and Properties of Asymmetric Large Unilamellar Vesicles: Interleaflet Coupling in Asymmetric Vesicles Is Dependent on Temperature but Not Curvature[END_REF][START_REF] Lin | Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry[END_REF][START_REF] Heberle | Subnanometer Structure of an Asymmetric Model Membrane: Interleaflet Coupling Influences Domain Properties[END_REF][START_REF] Chiantia | Asymmetric GUVs Prepared by MβCD-Mediated Lipid Exchange: An FCS Study[END_REF] The advantages of this method are essentially that it requires minimal equipment and that different lipids can be exchanged by this process, proving the flexibility of the method. The versatility in terms of vesicle size (SUVs, LUVs and GUVs) that can be used is also an important aspect as sometimes, methods to yield asymmetric vesicles are restricted/more efficient with a certain vesicle size (either giant or small/large). However, multiple steps (incubation, centrifugation, purification, etc.) are needed to yield the asymmetric vesicles making this method relatively tedious.

Over the last decade, there has been a growing interest in the development of new synthetic routes with higher control and precision to produce synthetic asymmetric lipid vesicles. Several groups reported on innovative protocols to generate membrane asymmetry with a special focus on giant vesicles (GUVs) that are thought to constitute more adequate cell mimics in terms of membrane physical properties, but they also have as a main strength facile manipulation and observation, as compared with small vesicles, which allows researchers to dig deeper into membrane physical properties. 41-43, 45, 64-65 Rather than through membrane exchange, giant asymmetric lipid vesicles can be assembled by emulsion methods where lipid-stabilized aqueous droplets are forced through a lipid-stabilized oil-water interface to generate the bilayer membrane. This process is referred as "droplet over an interface" or "emulsion-centrifugation" method (Figure 4). [START_REF] Pautot | Engineering asymmetric vesicles[END_REF][START_REF] Pautot | Production of Unilamellar Vesicles Using an Inverted Emulsion[END_REF] Weitz and coworkers proposed this method to prepare LUVs (about 500 nm in diameter) with an inner leaflet of POPC and outer leaflet of phosphatidylserine (POPS), or the reverse. Centrifugation was used to help the lipidstabilized aqueous droplets to cross the lipid-stabilized oil/water interface. One should note that the preparation of asymmetric SUVs was also previously demonstrated by a similar protocol. [START_REF] Xiao | Preparation of asymmetric bilayer-vesicles with inner and outer monolayers composed of different amphiphilic molecules[END_REF] The membrane asymmetry of the vesicles and its stability over time were demonstrated with a fluorescence quenching assay. This layer-by-layer assembly allowed insertion of a fluorescentlytagged lipid in one of the bilayer leaflets (POPC monolayer or POPS monolayer) and the fluorescence was measured before and after addition of a fluorescence quencher on the outer medium. For example, when the dye was inserted in the lipid-stabilized interface (outer leaflet of the vesicle membrane), about 95 % fluorescence was lost upon addition of the quencher to the vesicle solution, indicating an almost total asymmetry (no flip from the outer to the inner monolayer). Overall, authors demonstrated that highly asymmetric structures could be obtained using this method, with varying amphiphile molecules (they also performed a test with a lipidpolymer asymmetric membrane). A few years later, the group of Takagi followed a similar procedure to prepare cell-sized (GUVs) asymmetric vesicles with the aim of facilitating manipulation of "biological" vesicles under a controlled environment. [START_REF] Hamada | Construction of Asymmetric Cell-Sized Lipid Vesicles from Lipid-Coated Water-in-Oil Microdroplets[END_REF] Rather than through centrifugation, the authors used a sugar density gradient to induce spontaneous transfer of the denser lipid-stabilized sucrose droplets through an oil-glucose interface, a strategy that significantly increased vesicle yield. [START_REF] Hamada | Construction of Asymmetric Cell-Sized Lipid Vesicles from Lipid-Coated Water-in-Oil Microdroplets[END_REF] Other improvements of the "droplet over an interface" or "emulsion-centrifugation" methods involved microfluidics as an efficient way to produce sizecontrolled monodisperse emulsion droplets. [START_REF] Hu | Microfluidic Fabrication of Asymmetric Giant Lipid Vesicles[END_REF] In addition, the continuous droplet interface crossing encapsulation (cDICE) method has been described as an efficient way to produce giant vesicles with high throughput production of size-tunable monodisperse vesicles. [START_REF] Abkarian | Continuous droplet interface crossing encapsulation (cDICE) for high throughput monodisperse vesicle design[END_REF][START_REF] Claudet | Method to disperse lipids as aggregates in oil for bilayers production[END_REF] Vesicles are formed through continuous dripping of droplets off a capillary while forcing their passage through an interface using a centrifugal force. The authors presented the use of the cDICE method to form giant asymmetric vesicles. Advances in microfluidic strategies have led to generating monodisperse emulsion droplets at high-throughput. [START_REF] Shah | Designer emulsions using microfluidics[END_REF] Recent developments have enabled emulsions (w/o single emulsions or w/o/w double emulsions) to be used as templates to assemble vesicular structure via a bilayer selfassembly at interfaces, which provides opportunities to control over the composition in the membranes. [START_REF] Shum | Microfluidic fabrication of vesicles[END_REF] Asymmetric membranes constructed based on single emulsions by step-by-step coating with amphiphiles of interested were demonstrated in several group. [START_REF] Matosevic | Layer-by-layer cell membrane assembly[END_REF][START_REF] Lu | Continuous microfluidic fabrication of synthetic asymmetric vesicles[END_REF][START_REF] Karamdad | Studying the effects of asymmetry on the bending rigidity of lipid membranes formed by microfluidics[END_REF] A water in oil droplet was formed as precursor stabilizing with one kind of lipid molecules, and incubated in the channel of microfluidic device, and then coated with the second lipid phase. Recently Zhao et al. has reported that asymmetric bilayer vesicles (asymmetric lipid bilayer or lipid-polymer asymmetric bilayer) can be constructed by dewetting of thin-shell double emulsion templates which were fabricated in a single capillary microfluidic device. [START_REF] Zhao | Synthetic membranes in microfluidic interfaces[END_REF] The asymmetry property achieved by aforementioned microfluidic approaches was validated by both confocal laser scanning microscopy and fluorescence quenching assay indicating high degree of asymmetry can be readily produced by microfluidic technologies.

Finally, uncommon original techniques have been presented to construct lipid vesicles comprising an asymmetric membrane. Especially, microfluidic jetting has been introduced. [START_REF] Richmond | Forming giant vesicles with controlled membrane composition, asymmetry, and contents[END_REF][START_REF] Kamiya | Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes[END_REF][START_REF] Stachowiak | Inkjet formation of unilamellar lipid vesicles for cell-like encapsulation[END_REF][START_REF] Stachowiak | Unilamellar vesicle formation and encapsulation by microfluidic jetting[END_REF] The main concept is that a jet flow is used to deform a lipid bilayer and induce formation of vesicles (Figure 5). Kamiya and coworkers formed cell-sized asymmetric 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) / 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) vesicles using this technique by induced break up of a phospholipid microtube formed by applying a jet flow on a lipid planar bilayer. [START_REF] Kamiya | Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes[END_REF] They demonstrated that upon deformation of the lipid bilayer with an applied jet flow, the microtube breakdowns in two distinct vesicle populations of respectively 100-200 μm and 3-20 μm in diameter. With this method, authors were able to produce monodisperse asymmetric vesicles with little residual solvent in the membrane to investigate lipid flip-flop. They also examined the amount of residual organic solvent using confocal Raman scattering microscopy, which showed that the cell-sized vesicles accommodated tiny amount of the solvent that hardly produced a layer between the two leaflets.

In addition, in vitro membrane protein synthesis was demonstrated, proving the biological relevance of the system.

Another approach resides in focusing on the membrane alone in the form of a supported lipid bilayer, instead of designing objects which require a precise control over the final shape and which might complicate membrane study. In particular, supported lipid bilayers have been broadly studied and used as membrane models. [START_REF] Sackmann | Supported Membranes: Scientific and Practical Applications[END_REF] They consist of a lipid bilayer either fixed or freely deposited on a solid surface (e.g. gold or mica) and are classically formed by the Langmuir-Blodgett technique (transfer of successive lipid monolayers) or by vesicle fusion (liposomes deposited and allowed to fuse on the surface). [START_REF] Sanderson | Resolving the kinetics of lipid, protein and peptide diffusion in membranes[END_REF] In a recent study, dioleoylphosphatidylcholine (DOPC) supported bilayers were formed by vesicle fusion and MβCD was used to enrich the upper DOPC leaflet with sphingomyelin (SM) thus generating an asymmetric bilayer which stayed stable for up to a few hours, before SM started flipping to the lower leaflet. [START_REF] Visco | Asymmetric supported lipid bilayer formation via methyl-β-cyclodextrin mediated lipid exchange: influence of asymmetry on lipid dynamics and phase behavior[END_REF] While this approach does not allow a precise control over the degree of asymmetry it is however tunable, easy to handle and solvent-free, making it suitable to reconstruct synthetic asymmetric membranes. Similar studies showed the formation of asymmetric domains in supported bilayers. [START_REF] Lin | Lipid Asymmetry in DLPC/DSPC-Supported Lipid Bilayers: A Combined AFM and Fluorescence Microscopy Study[END_REF][START_REF] Wacklin | Composition and Asymmetry in Supported Membranes Formed by Vesicle Fusion[END_REF] Overall, important advances have been made in the field of asymmetric membrane models, especially in the last decade, using lipidic systems. The different proposed technologies to construct lipid-based membrane asymmetry allowed gaining more control into the design of synthetic membranes. In addition, these approaches have considerably helped the investigation and understanding of lipid dynamics in biological membranes.

DESIGN OF ASYMMETRIC MEMBRANES BASED ON BLOCK COPOLYMERS

When considering the design of biological membrane mimics, lipids come as an obvious choice for a starting material. However, cellular membranes are only made with about 60 % of lipids and are actually much more complex than pure lipidic bilayers. [START_REF] Cooper | Structure of the Plasma Membrane[END_REF] Indeed, proteins, glycoproteins, glycolipids, glycans or cholesterol that are incorporated in cell membranes convey precise mechanical properties that cannot be effectively represented by lipid-only membrane models. [START_REF] Marquardt | Asymmetric lipid membranes: towards more realistic model systems[END_REF] More recently, new synthetic asymmetric membranes have been proposed, with other constituting materials and morphologies, with the shared goal of determining to what extent asymmetry influences membrane properties.

A promising approach in the field of cellular membrane mimics resides in the use of polymers.

Because of their synthetic nature which makes them chemically stable and tunable, polymers have attracted a lot of attention for the bottom-up assembly of cell mimics, especially in the form of polymer vesicles, or polymersomes, which possess tougher and thicker membranes than their lipid analogues. [START_REF] Kamat | Engineering Polymersome Protocells[END_REF][START_REF] Marguet | Multicompartmentalized polymeric systems: towards biomimetic cellular structure and function[END_REF][START_REF] Städler | Polymer hydrogel capsules: en route toward synthetic cellular systems[END_REF][START_REF] Discher | Polymersomes: Tough Vesicles Made from Diblock Copolymers[END_REF][START_REF] Discher | Polymer Vesicles[END_REF][START_REF] Meins | Recent trends in the tuning of polymersomes' membrane properties[END_REF][START_REF] Mason | Polymersomes as protocellular constructs[END_REF] Such unique properties have been advantageously used to design complex systems, ranging from compartmentalized vesicles to mixed systems. [START_REF] Marguet | Multicompartmentalized polymeric systems: towards biomimetic cellular structure and function[END_REF][START_REF] Peyret | Liposomes in polymersomes: Multicompartment system with temperature-triggered release[END_REF][START_REF] Perro | Mastering a Double Emulsion in a Simple Co-Flow Microfluidic to Generate Complex Polymersomes[END_REF][START_REF] Shum | Multicompartment Polymersomes from Double Emulsions[END_REF][START_REF] Kim | Multiple Polymersomes for Programmed Release of Multiple Components[END_REF][START_REF] Peters | Cascade Reactions in Multicompartmentalized Polymersomes[END_REF][START_REF] Chiu | Polymer Vesicles Containing Small Vesicles within Interior Aqueous Compartments and pH-Responsive Transmembrane Channels[END_REF][START_REF] Fu | Multicompartmentalized polymersomes for selective encapsulation of biomacromolecules[END_REF][START_REF] Städler | A Microreactor with Thousands of Subcompartments: Enzyme-Loaded Liposomes within Polymer Capsules[END_REF][START_REF] Trantidou | Engineering Compartmentalized Biomimetic Micro-and Nanocontainers[END_REF] As such, Zhang and coworkers presented a phase-guided assembly technique to form giant polymersomes from two amphiphilic diblock copolymers: poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL) and dextran-b-poly(ε-caprolactone) (DEX-b-PCL). [START_REF] Zhang | Polymersomes of asymmetric bilayer membrane formed by phase-guided assembly[END_REF] The vesicles self-assembled from an aqueous two-phase system, where PEO formed the continuous phase and dextran the dispersed phase.

Phase separation led DEX-b-PCL to align at the surface of the dextran droplets (with dextran facing the interior) and PEG-b-PCL to form the outer leaflet of the bilayer, with PEG facing the continuous phase. The asymmetry of the GUVs was confirmed by means of NMR and fluorescence experiments. The authors also demonstrated the possibility to encapsulate erythropoietin with high loading efficiency (ascribed to the thermodynamically favored partition) and a well-preserved bioactivity, thus demonstrating the biological relevance of their system. A majority of investigated copolymers have an AB structure, where A is hydrophilic and B is hydrophobic, which self-assemble into symmetric membranes. However, ABC triblock copolymers (A and C water soluble but chemically different blocks and B hydrophobic middle block) were recently introduced to form polymersomes bearing an asymmetric membrane upon film rehydration. [START_REF] Mason | Polymersomes with asymmetric membranes based on readily accessible di-and triblock copolymers synthesized via SET-LRP[END_REF][START_REF] Konishcheva | PEO-b-PCL-b-PMOXA Triblock Copolymers: From Synthesis to Microscale Polymersomes with Asymmetric Membrane[END_REF][START_REF] Stoenescu | Vesicles with asymmetric membranes from amphiphilic ABC triblock copolymers[END_REF][START_REF] Blanazs | Tailoring Macromolecular Expression at Polymersome Surfaces[END_REF][START_REF] Liu | The highly efficient delivery of exogenous proteins into cells mediated by biodegradable chimaeric polymersomes[END_REF] When using such polymers, three different conformations can be obtained following vesicle formation: A block on the inside and C on the outside, the reverse, or a mixture of A and C on either side (symmetric). Due to their intrinsic asymmetric nature, if the orientation of the triblocks can be controlled or influenced inside the membrane, it is thus possible to present total asymmetry. For example, designing triblocks with different molecular weights for the A and C blocks should result in the longer one segregating on the outside of the vesicle due to a larger radius of curvature (differing volume fraction) and the smaller one segregating on the inside. [START_REF] Stoenescu | Vesicles with asymmetric membranes from amphiphilic ABC triblock copolymers[END_REF][START_REF] Luo | Thermodynamic Stabilization Mechanism of Block Copolymer Vesicles[END_REF] Furthermore, by adopting in the same fashion, Eisenberg et al. has expanded chemical localization of distinct groups facing to the interior or exterior aqueous environment, which was utilized to form polymeric vesicles with compositional asymmetric bilayers built by preferentially segregation of diblock copolymer molecules. [START_REF] Luo | One -Step Preparation of Block Copolymer Vesicles with Preferentially Segregated Acidic and Basic Corona Chains[END_REF] Recently, Tordarson has demonstrated that a family of synthesized diblock copolymers featured with different hydrophilic moieties can be used to generate polymersomes with asymmetric membrane to mimic the asymmetry across cell membranes. Interestingly, triblock copolymers have the same block ratios as the mixture of diblock copolymers extensively produced micelles rather than vesicular archetectures might due to the short hydrophobic block, which was evidenced by cryo-TEM. [START_REF] Mason | Polymersomes with asymmetric membranes based on readily accessible di-and triblock copolymers synthesized via SET-LRP[END_REF] Using charged blocks is another technique to help block segregation on either side of the membrane as has been shown by Zhong and coworkers who formed biodegradable chimaeric polymersomes from poly(ethylene oxide)-b-poly(caprolactone)-bpoly(2-(diethylamino) ethyl metacrylate) (PEO-b-PCL-b-PDEA) triblock copolymers with an outer PEO block and inner PDEA charged block assumed to facilitate efficient protein encapsulation and stabilization. [START_REF] Liu | The highly efficient delivery of exogenous proteins into cells mediated by biodegradable chimaeric polymersomes[END_REF] Others have used cleavable peptide-linked triblock copolymers to form vesicles with asymmetric membranes that can undergo shape transformation into multicavity vesicles upon cleavage. 100 While vesicles with asymmetric membranes formed from polymers are still in an early research stage, they hold promise in the design of new synthetic cellular analogues and have great potential for biomedical applications.

MEMBRANE ASYMMETRY IN HYBRID LIPID-POLYMER SYSTEMS

Mixing lipids and polymers to form synthetic asymmetric membranes has been rarely exploited but has a lot of potential interest. Indeed, combining the advantages of lipids as an intrinsic cell component and the chemical versatility of polymers may provide a powerful and versatile way to prepare original membranes with the ability to mimic the physical properties of cells. To date, such combination has only been used to develop so-called hybrid membranes that can be engineered to be homogeneous or heterogeneous (phase separation) in composition. [101][102][103][104][105][106][107][108][109] In this context, our group has recently reported the preparation of giant vesicles comprising an asymmetric polymer/lipid membrane using the "droplet over an interface" protocol (emulsioncentrifugation process that has been optimized for both polymers and lipids). [START_REF] Peyret | Asymmetric Hybrid Polymer-Lipid Giant Vesicles as Cell Membrane Mimics[END_REF][110][111] As schematically represented in Figure 6, a polymer stabilized emulsion droplet is poured over an oil-water lipid stabilized interface and, with help of centrifugation, the resulting vesicles are recovered in the lower water phase. As a result, the generated giant vesicles possess an asymmetric membrane composed of an outer lipid (POPC) monolayer and inner polymer (PButb-PEO) layer. We also showed that the reverse asymmetric vesicles could be formed, with the lipid facing the inside of the vesicle and polymer facing the outer medium. Complete membrane asymmetry was demonstrated with fluorescence quenching assays and membrane properties were determined by evaluating lipid lateral and transverse diffusion coefficients. Figure 6. Schematic representation of the "droplet over an interface" method to generate asymmetric lipid-polymer vesicles. [START_REF] Peyret | Asymmetric Hybrid Polymer-Lipid Giant Vesicles as Cell Membrane Mimics[END_REF] An aqueous droplet stabilized by a polymer is poured over a lipid-stabilized oil-water interface. 110 Although vesicles appear as an optimal choice in the field of cell biomimicry, alternative morphologies comprising asymmetric membranes have also been investigated. In particular, tubular structures have been presented. [START_REF] Li | Design of Asymmetric Peptide Bilayer Membranes[END_REF][START_REF] Lim | Spontaneous formation of nanometer scale tubular vesicles in aqueous mixtures of lipid and block copolymer amphiphiles[END_REF]112 As illustrated by Liedberg and co-authors, an equimolar proportion of poly(butadiene)-b-poly(ethylene oxide) and POPC led to the formation of nano-tubular vesicles. They also demonstrated that asymmetry generates a net spontaneous curvature, with the lipid segregating into the inner compressed leaflet, that is required for the stabilization of the tubular morphology. [START_REF] Lim | Spontaneous formation of nanometer scale tubular vesicles in aqueous mixtures of lipid and block copolymer amphiphiles[END_REF] Finally, amphiphilic peptides were also interestingly considered in generating spontaneous membrane asymmetry in nanotubes or nanoribbons. [START_REF] Li | Design of Asymmetric Peptide Bilayer Membranes[END_REF]112 Amphiphilic peptides based constructs show promising potential as building blocks for the assembly of membrane with compositional asymmetric arrangement. Taking their advantages of being rationally designed for specific bio-macromolecular targets and being highly biocompatible as well as easy degradation in the in vivo environment enable new class of peptide-based biomembranes to open the new avenues to assemble the asymmetric membranes in the form of intracellular delivery vehicles, personalized medicine carriers and ultra-sensitive biosensors.

EFFECTS AND APPLICATIONS OF ASYMMETRIC BILAYER SYSTEMS

Biological membrane systems possess some degree of lipid asymmetry with respect to bilayer composition prepared by methods as reviewed above have been studied on physical properties of asymmetric biomembranes and membrane-mediated cellular process. Microfluidic-assisted approaches enable well-controlled construction of asymmetric lipid bilayer vesicles at high throughput fashion. [START_REF] Karamdad | Studying the effects of asymmetry on the bending rigidity of lipid membranes formed by microfluidics[END_REF]113 The effects of tailored lipid bilayer on the mechanical property has been determined by the fluctuation analysis technique and micropipette aspiration system. In both works, the bending modulus of synthetic asymmetric vesicles were at 30 % higher than the values of acquired for symmetric bilayer vesicles. The implication of the findings on the effects of transbilayer asymmetry on mechanical property could pave the way for investigating proteinlipid interaction, especially the mechanosensitive channel whose integration into membrane is controlled by membrane asymmetry. 114 Asymmetric biomembranes were utilized as a promising models to govern the orientation of membrane proteins during incorporating into synthetic biomembranes. [115][116] Meier group reported that triblock copolymer built asymmetric membranes can induce a favor insertion orientation of Aquaporin 0 which remain functional inside polymeric membranes. Oiki et al. successfully demonstrated that oriented reconstitution of a potassium channel (KcsA) driven by where the preferred lipid deposits within the asymmetric bilayer. A recent study was undertaken by Chen revealing a transbilayer asymmetry induced translocation of β-galactosidase. 117 To address issue of random incorporation of membrane proteins using asymmetric biological membranes would not to broaden knowledge of interactions between membrane proteins and biomembranes, but also propose a versatile way to produce highly functional bio-device.

CONCLUSION

Over the last years, reconstitution of model membrane systems has helped researchers gain better understanding into the complex machinery of cellular processes, from simple diffusion through membranes to incorporation of transmenbrane proteins or lipid dynamics and clustering.

Asymmetric biomembranes are more accurate mimicking systems to resemble natural cell membranes than the unsymmetrical membrane systems. The construction of asymmetric synthetic membranes through a variety of different methods and components open new opportunities to dig deeper into our understanding on the cell machinery and could lead to the development of new therapeutic approaches where lipids should play a major role. Besides as to artificially build a cell upon lifeless components, bioinspired vesicles featured with asymmetric bilayer are a step forward to the resemblance of cells in terms of function and structure of biological membranes. The proposed systems so far are very simple and mimic in a very simplistic manner some of the basic properties of cell asymmetry and not at all the biological phenomenon beyond this. Future work will focus on designing new strategies are desirable to reach stable artificial asymmetric membranes with high control, precision and biological resemblance, from both structural and functional view-points. Eventually, these systems might find interest to better elucidate the significance of lipid asymmetric distribution in cell membranes and uncover how asymmetry is initially formed and maintained as well as assist to gain insight in cellular functions associated with membrane asymmetry.

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ABBREVIATIONS

Figure 3 .

 3 Figure 3. Schematic representation of methyl-β-cyclodextrin (MβCD) mediated lipid exchange to

Figure 4 .

 4 Figure 4. Schematic representation of the "droplet over an interface" method to generate

Figure 5 .

 5 Figure 5. Schematic representation of « jetting ». A jet flow is applied on a lipid asymmetric

  cDICE, continuous droplet interface crossing encapsulation; CD, cyclodextrin; DEX-b-PCL, dextran-b-poly(ε-caprolactone); DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPS, 1,2dioleoyl-sn-glycero-3-phospho-L-serine; GUVs, giant unilamellar vesicles; LUVs, large unilamellar vesicles; MβCD, methyl-β-cyclodextrin; MLVs, multi-lamellar vesicles; PA, phosphatidic acid; POPC, phosphatidylcholine; PG, phosphatidylglycerol; POPS, phosphatidylserine; PEO-b-PCL, poly(ethylene oxide)-b-poly(ε-caprolactone); PEO-b-PCL-b-PDEA, poly(ethylene oxide)-b-poly(caprolactone)-b-poly(2-(diethylamino) ethyl metacrylate); SM, sphingomyelin; SUVs, small unilamellar vesicles.

ACKNOWLEDGEMENT

This work was funded by the French Ministry of Research and Education (fellow A.P.) and the Agence Nationale de la Recherche (ANR program NO-SynthCell N8 ANR-14-CE16-0015).

CPER CampusB project funded by the French state and the Région Nouvelle Aquitaine is also acknowledged.

AUTHOR INFORMATION


Thomas A Darde

Frédéric Chalmel

Terje Svingen

Exploiting advances in transcriptomics to improve on humanrelevant toxicology

Keywords: toxicogenomics, risk assessment, mechanisms, RNA-seq, toxicology

Modern toxicology is challenged in its aspiration to test and risk-assess the myriad of chemicals humans potentially can be exposed to. We still lack sufficient data to perform proper human risk assessments for a large proportion of environmental chemicals and we still know very little about many of the molecular mechanisms that cause adverse effects. In turn, lack of mechanistic insight stalls the development of more cost-efficient and highthroughput alternative assays, which is a prerequisite if we are to deal with the large number of untested compounds. One way to help speed up the effort is to take advantage of the many advances in genomics technologies and apply them to toxicity testing strategies, or at the very least use them to better characterize the causative molecular mechanisms. For instance, single-cell Digital Gene Expression-(DGE-) and single-cell RNA (scRNA-) sequencing techniques hold great promise for not only enabling the analyses of large sample sizes at low cost, but also capturing toxico-molecular events and genomic susceptibilities at the cellular level. In this paper, we discuss some of the advances in transcriptomics and how they can be applied to toxicology to advance human-relevant risk assessment.

Introduction

Contemporary toxicology is grappling with increasing demands for faster, cheaper and more reliable test strategies. There is also an increasing demand to develop and incorporate more human-relevant models and assays when assessing the risk to human health; and ideally, real-life exposure scenarios and sub-group susceptibilities should also be accounted for [START_REF] Collins | Transforming environmental health protection[END_REF].

These are all individually big challenges, but combined they represent an enormous challenge that cannot be solved using traditional toxicology approaches of assessing single chemicals one at a time by standard testing regimens. This is especially true if we considering the tens-of-thousands of chemicals that are continuously being manufactured and used across the world, chemicals for which we still lack sufficient data to perform proper risk assessments. In fact, global chemical pollution has been considered so immense and complex that its impact on the planet and its inhabitants is beyond quantifiable [START_REF] Rockström | A safe operating space for humanity[END_REF]. To help solve some of these challenges, modern genomics methods can be combined with toxicology approaches to assess toxicity in new ways -a modern field coined toxicogenomics.

The term toxicogenomics first appeared in the late 1990s. It heralded a new era for chemical toxicology by advancing our understanding of mechanisms of toxicity and thereby predictive capabilities [START_REF] Nuwaysir | Microarrays and toxicology: the advent of toxicogenomics[END_REF]. In 2007, the American National Research Council defined toxicogenomics as "combin[ing] toxicology with information-dense genomic technologies to integrate toxicantspecific alterations in gene, protein and metabolite expression patterns with phenotypic responses of cells, tissues and organisms" [START_REF]Applications of Toxicogenomic Technologies to Predictive Toxicology and Risk Assessment[END_REF]. The main hypothesis was that an organismal response to a toxicant leading to pathological or phenotypical changes in certain organs at a low dose could be detected as changes to gene, protein, or metabolite expression. The field

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4 has yet to deliver on all its promises, but has come a long way. Currently gene expression is the most sensitive of the above-mentioned parameters, also with the highest cost-benefit ratio. Consequently, toxicogenomics has by some been defined more narrowly to only include ''the study of altered gene expression after toxicant exposure'' [START_REF] Haschek | Haschek and Rousseaux's handbook of toxicological pathology[END_REF].

Irrespective of current state of the science, toxicogenomics has a broad application potential and can be used for mechanistic analyses, biomarker discovery, risk assessments and drug development, and so forth. And perhaps more importantly, toxicogenomics holds the potential to deliver more discriminate, sensitive and predictive information with regard to pharmaceuticals and environmental chemicals and their potential effects on human health. A looming challenge, however, comes from the successes achieved with genomics technologies that now are capable of retrieving mass amount of data all the way down to the scale of single cells. Unless handled appropriately, such terabytes of information can serve to obscure rather than illuminate the picture; or to quote Cervante's immortal character Don Quixote, "facts [can become] the enemy of truth".

Transcriptomics for human-relevant toxicity testing

Although genomics can be applied to toxicology in numerous ways, here we will focus on where transcriptomics can, and already do, make an impact on human-relevant chemical toxicity. Three important areas are mechanistic insight, biomarker identification, and mixture risk assessment. One: A recent comparative case study on human risk to benzo(a)pyrene in drinking water found that genomics-informed assessments yielded comparative points-of departure (POD) values as did traditional assessment approaches; however, toxicogenomics could also be used to delineate more detailed modes-of-action in

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5 the mouse toxicity model and asserting human relevance by showing consistency in perturbed pathways between the mouse tissue and expression data from human cells [START_REF] Moffat | Comparison of toxicogenomics and traditional approaches to inform mode of action and points of departure in human health risk assessment of benzo[a]pyrene in drinking water[END_REF].

Two: A recent report by Makarov and colleagues presented an efficient method for identifying reliable biomarkers using toxicogenomics datasets. Using two different algorithms ('search marker' and 'recognition algorithm') on the well-known toxicogenomics dataset DrugMatrix [START_REF] Makarov | Computational method for discovery of biomarker signatures from large, complex data sets[END_REF], they were able to identify genomic patterns specific to chemical compounds regardless of dose or time. Three: Several studies have attempted to implement mixture modelling in transcriptomics analysis to better understand the interactions of combined chemicals [START_REF] Tilton | Transcriptional impact of organophosphate and metal mixtures on olfaction: copper dominates the chlorpyrifos-induced response in adult zebrafish[END_REF][START_REF] Hutchins | Transcriptomic signatures in Chlamydomonas reinhardtii as Cd biomarkers in metal mixtures[END_REF][START_REF] Altenburger | Mixture toxicity revisited from a toxicogenomic perspective[END_REF], a topic considered of utmost relevance if we are to appreciate the true impact environmental chemicals can have on human health [START_REF] Svingen | The risk of chemical cocktail effects and how to deal with the issue[END_REF][START_REF] Kortenkamp | Regulate to reduce chemical mixture risk[END_REF]. Although our understanding of the molecular and cellular responses to complex mixtures remains lacking -and the use of classical mixture toxicity models to predict mixture effects from toxicogenomics data is still in its infancy [START_REF] Song | Deciphering the Combined Effects of Environmental Stressors on Gene Transcription: A Conceptual Approach[END_REF] -we predict great advances in this area within the near future.

Genomics in the twenty-first Century

Modern genomics has seen enormous advances within a relatively short space of time.

What seemed like impossible less than two decades ago is now possible even for a small research laboratory. Today one can sequence a person's genome in two days for less than $4,000, as well as analyse the transcriptome of single cells from complex tissues. In comparison, the first human genome took 13 years to complete at a cost of upwards of $1 billion [START_REF]The cost of sequencing a human genome[END_REF]. Hence, the main challenge seems no longer to be what data we can extract, but what to do with the information once it has been collected.

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6 Advances in ultra-high-throughput sequencing technologies have greatly reduced the overall cost of running these assays, and thus also greatly boosted their use [START_REF] Sims | Sequencing depth and coverage: key considerations in genomic analyses[END_REF]. Today, RNA sequencing (RNA-seq) is the dominant technique in transcriptomics and has several advantages over other approaches such as the early Sanger sequencing or microarray-based methods. Indeed, RNA-seq has quickly proven to produce accurate quantification of gene expression and to correlate well with other technologies to eventually outperform the alternative methods in terms of accurately quantifying low-abundance transcripts [START_REF] Izadi | A comparative analytical assay of gene regulatory networks inferred using microarray and RNA-seq datasets[END_REF][START_REF] Chen | Correlation between RNA-Seq and microarrays results using TCGA data[END_REF][START_REF] Wang | The concordance between RNA-seq and microarray data depends on chemical treatment and transcript abundance[END_REF].

Because of the many advantages coinciding with parallel development of more sophisticated software and algorithms to handle ever larger data sets, RNA-seq has opened up possibilities for comprehensive characterization of the genomic landscapes to the point of transcriptome data at single-cell resolution by Single-cell RNA-sequencing (scRNA-seq).

This has made it possible to catalogue the expression of individual genes in individual cells from a given tissue with reasonable accuracy. This powerful tool can ultimately reveal the inherent variability between cells, both in normal and disease states [START_REF] Linnarsson | Single-cell genomics: coming of age[END_REF]. These advances can give access to a vast amount of information that will allow us to address a myriad of scientific questions regarding cell differentiation and lineage specification, cellular heterogeneity, and development of disease from earliest of stages, all within one tissue from one individual. The potential applications to biology, toxicology and medicine are enormous.

Another recent development in genomics is Digital Gene Expression (DGE) profiling, a variant of the SAGE method using ultra-high-throughput sequencing techniques [START_REF] Audic | The significance of digital gene expression profiles[END_REF]. Simply put, DGE can be used to quantify levels of expression by directly counting reads from raw data obtained from sequencing the short fragments located at the 3' poly(A) of RNA

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messenger, as opposed to RNA sequencing which covers the entire genome. This technique, originally developed to study scRNA-seq or small cell populations, provides a reliable estimate of the gene expression level at extremely low costs -€30-50 per samplecompared with conventional "bulk" RNA sequencing [START_REF] Soumillon | Characterization of directed differentiation by high-throughput single-cell RNA-Seq[END_REF].

Together these emerging genomics technologies can be applied across many biomedical research fields, including diagnostics [START_REF] Nikitina | Primary candidate rna biomarker screening by RNA-seq for prostate cancer diagnostics[END_REF], disease profiling [START_REF] Costa | RNA-Seq and human complex diseases: recent accomplishments and future perspectives[END_REF], gene function and annotation [START_REF] Bischler | Differential RNA-seq (dRNA-seq) for annotation of transcriptional start sites and small RNAs in Helicobacter pylori[END_REF], as well as response to environment and toxicants [START_REF] Yin | Gene expression profiling analysis of bisphenol A-induced perturbation in biological processes in ER-negative HEK293 cells[END_REF]. Below, we will discuss more how transcriptomics can be used in toxicology to both advance our knowledge of modes of action and perform better risk assessments at lower costs than previously possible.

Toxicogenomics in the twenty-first Century

Depending on endpoint to be evaluated, many toxicity assessments use intact animals or cell cultures to measure several parameters, including morphometric and histopathological evaluations, cell viability, cytotoxicity, hepatotoxicity, or nephrotoxicity. These classical approaches can detect toxicity and be used to assess points-of-departure levels (POD) for specific chemicals. Data generated by omics technologies on the other hand, are envisioned to detect toxicities that may not be observable by conventional assessment strategies, thereby facilitating more accurate and predictive decision-making based on toxicity mechanisms [START_REF] Urushidani | Prediction of hepatotoxicity based on the toxicogenomics database[END_REF]. It comes with certain challenges, however, for example lack of uniform study designs, multiplicity of normalization and analysis strategies [START_REF] Quackenbush | Microarray data normalization and transformation[END_REF], reproducibility of microarray data across platforms [START_REF] Mah | A comparison of oligonucleotide and cDNA-based microarray systems[END_REF], absence of data quality control measures and standards, and lack of effective data sharing and reporting standards.

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The ability to profile global transcriptional responses has become a valuable resource in toxicology. One challenge, however, is that in vivo toxicity studies that include transcriptomics rarely exceed 10-12 samples. It is close to impossible to detect robust toxicogenomic signatures in humans with such small samples sizes [START_REF] Miller | Improving reproducibility in toxicology[END_REF][START_REF] Poland | The elephant in the room: reproducibility in toxicology[END_REF]. To overcome this problem, a viable alternative is to use low-cost sequencing technologies such as DGE to allow for the study of larger collection of samples in order to detect stronger toxicogenomic signatures with greater statistical power. This would also help discriminate sub-groups of individuals based on their susceptibility to disease, making it possible to identify common constitutional factors.

Susceptibility to an adverse effect may also exist within cellular subpopulations within a defined tissue. Classical "bulk" transcriptomic technologies cannot detect this phenomenon at the organ level. Recent advances in scRNA-seq approaches make it possible to study the impact of chemical exposure at the level of cellular subpopulation, as well as to identify specific biomarkers [START_REF] Niu | Potentials of single-cell biology in identification and validation of disease biomarkers[END_REF] to enhance the toxicological characterization and underlying mechanisms at the cellular level. Single-cell technologies have already been used successfully in studies on tumor heterogeneity. In a landmark study by Dalerba and colleagues [START_REF] Dalerba | Single-cell dissection of transcriptional heterogeneity in human colon tumors[END_REF], scRNA-seq revealed that multi-lineage cell differentiation determines the transcriptional diversity of cancer tissues. With regard to toxicology, scRNA-seq has been highlighted as a potential powerful approach to discover new biomarkers that reflect the efficacy, metabolism, pharmacodynamics, absorption and toxicology of chemicals [START_REF] Niu | Potentials of single-cell biology in identification and validation of disease biomarkers[END_REF].

Using toxicogenomics to bridge the gaps between rodent and human data

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For more than a century, rodents have been indispensable for experimental medicine, drug development and toxicology. Rats and mice continue to serve as model organisms in drug design, biomarker discovery and chemical risk assessment, but the use of animal models is also under increasing pressure, both from an ethical point of view and for not necessarily providing human-relevant data. Several studies have shown that rats and humans share a high degree of genomic and physiologic similarities [START_REF] Gibbs | Genome sequence of the Brown Norway rat yields insights into mammalian evolution[END_REF][START_REF] Mattes | Detection of hepatotoxicity potential with metabolite profiling (metabolomics) of rat plasma[END_REF], but simultaneously, important differences have been reported, including functional differences within nonpharmacokinetic metabolism. Such differences could influence how and if a compound induce toxicity or elevate a biomarker [START_REF] Luo | Evaluation of serum bile acid profiles as biomarkers of liver injury in rodents[END_REF][START_REF] Shannahan | Pulmonary oxidative stress, inflammation, and dysregulated iron homeostasis in rat models of cardiovascular disease[END_REF]. It is thus paramount that we characterize any differences between rodents and humans if we are to accurately interpret animal studies used for biomarker discovery, toxicity testing or comparative toxicogenomics analyses [START_REF] Clayton | Pharmaco-metabonomic phenotyping and personalized drug treatment[END_REF][START_REF] Uehara | The Japanese toxicogenomics project: application of toxicogenomics[END_REF]. To this, transcriptomic analyses can help reveal inter-species similarities and differences [START_REF] Rye | Unraveling the genetics of otitis media: from mouse to human and back again[END_REF].

Efforts have been put towards setting up large, comprehensive collections of comparable rodent and human transcriptomic data. New computational methods for integrating gene expression measurements into genome-scale network reconstruction have also been developed. 'Systems toxicology' approaches rely on the integration of multiple toxicology data (e.g. transcriptomic, metabolomic, epigenomic) with quantitative analysis of large networks of molecular and functional changes occurring across multiple levels of biological organization. It can be used to study the full spectrum of toxicological responses from xenobiotic insult, as well as enable comparative predictions across individual patients, treatment conditions, tissue types and species [START_REF] Blais | Reconciled rat and human metabolic networks for comparative toxicogenomics and biomarker predictions[END_REF][START_REF] Davis | The Comparative Toxicogenomics Database: update 2017[END_REF]. These efforts have been made possible by the implementation of services for toxicogenomics data management.

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Potential impact of toxicogenomics on 'uncertainty factors' in chemical risk assessment

In order to account for uncertainties when using effect assessment data for human chemical risk assessment, so-called assessment factors (AF; also referred to as uncertainty factors) are used to take into account inter-and intra-species variabilities and uncertainties [START_REF]Chapter R.8: Characterisation of dose [concentration]-response for human health[END_REF].

Although the 'default' AF of 100 is often referred to (10x to account for differences in experimental animal to humans; 10x to account for individual differences in human population), there are in fact many different AFs employed within regulatory frameworks depending on experimental design and substances that are tested [START_REF]Chapter R.8: Characterisation of dose [concentration]-response for human health[END_REF][START_REF] De Raat | ECHA Science: Understanding the importance of assessment factors in finding safe human exposure levels[END_REF]. AF values must be adjusted in cases where information is available that allows for greater certainty. Such amended AFs can be contentious however, and a source of debate [START_REF] De Raat | ECHA Science: Understanding the importance of assessment factors in finding safe human exposure levels[END_REF]. In any case, it is likely that toxicogenomics can impact how AFs are determined in the future. For instance, by mapping the genetic differences between populations associated with susceptibility to a given disease, concomitant with elucidation of the detailed regulatory pathways that are affected by specific chemicals, there will be much higher certainty in the use of effect data for calculating health-based guidance values. Hence, the AF for intra-species (i.e. human) differences could be set much lower than the default 10x. This line of though can also be extended to default AFs for inter-species variations. By gaining more knowledge about the genomic landscape and how different species respond to the exposome, it is likely that more precise AFs can be set for individual species (e.g. rat), or even strains of the same species, depending on how well they relate to humans for any given effect endpoint.

Toxicogenomics data management and online services

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The sheer volume of available transcriptomic data has now grown so large that it starts to impose additional challenges to research communities, including toxicology; namely the complex tasks of processing, hosting and interpreting data [START_REF] Raja | A review of recent advancement in integrating omics data with literature mining towards biomedical discoveries[END_REF]. Among the many practical points that should be considered when funding, planning and reporting toxicology studies in the future is improved data transparency [START_REF] Poland | The elephant in the room: reproducibility in toxicology[END_REF][START_REF] Wilkinson | The FAIR Guiding Principles for scientific data management and stewardship[END_REF]. This should also include negative (read: no effect) data or contradictory results. The European Commission has started to address this issue by attempting to establish a model service for systems biology data management through the European Research Infrastructure Consortium (ERIC). Its objective is to make biological data FAIR: Findable, Accessible, Interoperable and Reusable. This demands that investigators make raw data publicly available. Although great efforts have been made to store raw data in public repositories [START_REF] Barrett | NCBI GEO: archive for functional genomics data sets--update[END_REF][START_REF] Kolesnikov | ArrayExpress update--simplifying data submissions[END_REF], the all-important processed data (e.g. list of regulated genes) remains inaccessible to the scientists (i.e. supplementary files provided by authors).

To meet a growing demand of accessing pre-processed omics data without having to download and re-process raw data, several on-line resources have been developed (Table 1). For example, the CTD [START_REF] Davis | The Comparative Toxicogenomics Database's 10th year anniversary: update 2015[END_REF], diXa [START_REF] Hendrickx | diXa: a data infrastructure for chemical safety assessment[END_REF], ToxDB [START_REF] Hardt | ToxDB: pathway-level interpretation of drug-treatment data[END_REF], CEBS [START_REF] Lea | CEBS: a comprehensive annotated database of toxicological data[END_REF], Drug2Gene [START_REF] Roider | Drug2Gene: an exhaustive resource to explore effectively the drug-target relation network[END_REF] and ToxCast [START_REF] Richard | ToxCast Chemical Landscape: Paving the Road to 21st Century Toxicology[END_REF], which have paved the way for improved storage, exchange, and analysis of toxicological data [START_REF] Miller | Data sharing in toxicology: beyond show and tell[END_REF]. More recently, we launched TOXsIgN [START_REF] Darde | TOXsIgN: a cross-species repository for toxicogenomic signatures[END_REF] as an innovative platform designed to share standardized processed data that also provide tools for predictive toxicology. The success of this and other repositories, however, ultimately depends on researchers' compliance to the FAIR principles. But conversely, the success of FAIR depends on the availability of suitable repositories such as TOXsIgN. In essence, we encourage the uploading of toxicogenomic and toxicological signatures in dedicated repositories such as Less than two decades ago, proposing to sequence the transcriptome at single cellresolution in a complex organ and use this data to extract a personalized toxicogenomic profile to predict future effects from exposure to a real-life mixture of environmental chemicals, would likely have been considered quixotic. With recent advances in genomics and computer technologies, however, this proposition does no longer seem exceedingly idealistic or delusional. Massive amounts of gene expression data are now available in the public domain, enabling new biological questions to be addressed through data re-use without the need for further experimentation [START_REF] Igarashi | Open TG-GATEs: a large-scale toxicogenomics database[END_REF][START_REF] Ganter | Toxicogenomics in drug discovery and development: mechanistic analysis of compound/class-dependent effects using the DrugMatrix database[END_REF][START_REF] Lamb | The Connectivity Map: using geneexpression signatures to connect small molecules, genes, and disease[END_REF][START_REF] Liu | Compound signature detection on LINCS L1000 big data[END_REF]. And we are optimistic that the future of toxicogenomics will deliver on many of its promises and thereby also aid contemporary toxicology to deliver on increasing demands from governing bodies and the public to safeguard human health against xenobiotic insults at ever lower costs and better predictability. To end, we like to highlight five points where we believe toxicogenomics will have significant impact in the coming years.

• Enhance our knowledge about mechanism of action at the molecular and cellular levels induced by exposure to xenobiotics.

• Enable the progressive implementation of the 3R-principles (replace, reduce, refine) for animal toxicity studies, not least by helping to verify the suitability of emerging technologies such as human-based organoids and organs-on-chip. Genomics technologies will likely play a major role in future chemical risk assessment.

• Facilitate comparative studies between species, in particular with regard to human relevance and thereby enable more accurate extrapolations from model organisms to human risk assessment. • Allow for the identification of susceptible sub-populations or individuals with regard to effects caused by chemical exposures. This could also include refinements to the arbitrary '10x uncertainty factor' used in risk assessments. Using DGE, many analyses could be performed, for instance signature matching methods assuming that compounds which induce similar gene expression signatures will also have similar effects in a biological system; or the biological networking (i.e. co-expression network, protein-protein interaction), especially directed signalling networks, which allow scientists to follow the cellular response of a compound treatment from the compound's target to the differentially expressed genes. From these methods, and using a sufficient dataset, there is an obvious opportunity to estimate the toxicity of a compound, its mechanism of toxicity, and a related Adverse Outcome Pathway. With increasing data becoming available, and increasing sophistication of methods, the aim is that this will, over time, result in decreased animal testing and decreased the number of failures during drug development.

14 •

 14 Support the development of integrated approaches needed to advance the area of mixture risk assessment by offering more integrated knowledge about molecular and cellular responses to exogenous compounds.

Figure 1 -

 1 Figure 1 -Typical toxicogenomics analysis pipeline based on gene expression analysis.

Acknowledgements

We thank Dr Julie Boberg for helpful comments on the manuscript. This work was partially funded by the French agency for food and safety [ANSES n °EST-13-081 to F.C], the French National Research Agency [ANR n° 16-CE14-0017-02 to F.C], and the Fondation pour la recherche médicale [FRM n °DBI20131228558 to FC].

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12 TOXsIgN before submission of manuscripts for publication. This would increase both their visibility and accessibility.