Niki Baccile

Sophie Roelants

Eero Kontturi

Advanced Materials from Microbial Fermentation: the Case of Glycolipids and Nanocellulose

La chimie verte est une discipline relativement récente régie par douze principes fondateurs, incluant notamment l'économie d'atomes, la prévention de la pollution via des méthodes de synthèse chimique respectueuses de l'environnement, comme par exemple celles privilégiant le milieu aqueux aux solvants organiques, mais aussi le développement de produits chimiques et matériaux issus de la biomasse végétale. Dans ce contexte, la synthèse microbienne est un outil de choix pour supplanter, dans certains cas notables, les approches classiques basés sur la chimie organique de synthèse. Par exemple, les polyhydroxyalcanoates, des polyesters biodégradables, sont produits par voie bactérienne à partir de lipides et sucres et ils ont été identifiés comme des remplaçants potentiels au polypropylène dès les années '70.

Plus récemment, on commence à s'intéresser à la synthèse microbienne de composés sucrés polymériques, comme le dextrane ou la cellulose, ou lipidiques, comme les glycolipides amphiphiles. Bien que les pratiques de production microbienne de composés glycosylés datent de plusieurs décennies, l'essor de la chimie verte en tant que discipline au niveau international encourage des équipes de chercheurs pluridisciplinaires à se pencher sur la production, la diversification, l'étude des propriétés et les applications de cette classe de composés, dépassant ainsi la communauté des chercheurs en microbiologie, historiquement intéressés au développement des produits de fermentation de microorganismes.

Dans cet article nous souhaitons développer le thème mentionné ci-dessus en se focalisant sur la nanocellulose bactérienne, pour ce qui concerne les polymères glycosylés, et les biotensioactifs, pour ce qui concerne les systèmes lipidiques glycosylés. Le choix de ces deux systèmes est justifié par le fort développement des matériaux à base de nanocellulose mais aussi par le besoin de remplacer en partie les tensioactifs « classiques », sources non négligeable d'émission de CO2 au niveau mondial. Nous illustrerons les principales classes de molécules, les méthodes classiques de synthèse, les propriétés ainsi que quelques exemples d'application notoire. Nous terminerons par quelques perspectives sur le sujet.

Introduction

The present work has the goal of illustrating two notorious examples of a sustainable chemistry approach in the field of molecular and material science. For the former, we have chosen the field of glycolipids, while for the latter, we have chosen the field of nanocellulosic materials. Glycolipids are generally considered as standard "green" surfactants in their alkyl polyglycoside (APG) form, obtained by standard glycosylation reactions from sugars and fatty oils. 1 These products are commercially available in many detergent formulations. On the other side, nanocellulose has now became a standard "green" material derived from cellulose after a strong acid hydrolysis. To overcome these standard approaches, microbial fermemtation techniques have been developed since decades to prepare biobased glycolipids and nanocellulose. An overview of each domain, including standard synthesis, properties and up-to-date applications are presented below.

Bacterial nanocellulose

Nanocellulose: an introduction. Nanocellulose as a term originated only in the first decade of the 2000s 2 but many of the current nanocellulose species had been in existence at a much earlier date. 3 At present, one of the most researched aspects of nanocellulose concentrates on their use as a reinforcing phase in bio-based and/or biodegradable composites that may step in to replace plastics or appear as completely new materials in their own right. 4 In essence, nanocellulose is generally categorized under three discrete groups: cellulose nanocrystals (CNCs), cellulose nanofibers (CNFs) and bacterial cellulose (BC). 2 CNCs and CNFs are usually extracted from plant sources and the basis of their production involves disintegration of the plant cell wall. CNFs are in fact isolated cellulose microfibrils, the basic supramolecular units of native cellulose. Microfibrils are longitudinal threads of very high aspect ratio, just a few nanometers in width yet micrometers in length. 5 CNCs, in turn, are usually prepared by acid hydrolysis of native microfibrils. Controlled hydrolysis is able to degrade the disordered segments in the semi-crystalline cellulose microfibrils, leaving the crystallites intact. 6,7 The end result is CNCs: short, rigid rods of crystalline cellulose that spontaneously self-assemble into chiral nematic liquid crystals beyond a certain threshold concentration. In this short review, however, we will focus only on BC.

Bacterial cellulose: origin, preparation, morphology, general issues. BC is the odd one out of the nanocellulose family. It is usually treated as a separate category but essentially, BC could be considered a type of CNFs. BC consists of cellulose microfibrils synthesized by certain bacteria to an extracellular matrix. [START_REF] Iguchi | [END_REF] BC microfibrils are in a completely isotropic arrangement, lacking the tight hierarchical morphology of plant cellulose confined in the cell wall (Figure 1a). Therefore, one can say that BC is directly synthesized as a nanocellulose species without the need for specific isolation from a growth matrix. BC is also the only chemically pure form of nanocellulose as the other species are always embedded in a matrix of hemicellulose and -with land plants -lignin which are next to impossible to remove completely during nanocellulose preparation. 9 It is only certain types of bacteria that are able to synthesize BC from glucose. The most popular species for actual BC production is called Acetobacter xylinum or in modern nomenclature, Gluconacetobacter xylinus. The BC synthesis was first reported in 1886, 10 although the product was only much later identified as cellulose. 11 Culturing with G. xylinus is an aerobic process where BC grows at air/water surface in order to utilize the oxygen from air. [START_REF] Iguchi | [END_REF]12 The most used monosaccharide sources for BC production are glucose and sucrose. Glucose is converted via several steps into uridine diphosphoglucose (UDP-glucose) which is then polymerized into cellulose, i.e., a homopolymer consisting only of anhydroglucose units (Figure 1d). [START_REF] Iguchi | [END_REF]12 BC producing microbes are also able to utilize other monosaccharides to produce UDP-glucose and further to cellulose. A case in point is fructose -the other monosaccharide in the sucrose dimer -but several other monosaccharides have been trialed as a cellulose source as well. [START_REF] Iguchi | [END_REF] Commonly, the method of growing BC in static conditions at the air/water surface is relatively slow but nevertheless suitable for laboratory work. A few semi-continuous bioreactor designs for static culturing have been proposed throughout the past years. 13 Moreover, submerged fermentation has been investigated but the techniques face intrinsic problems, above all the supply of oxygen is usually too low for sufficient production. 14 BC consists chemically from exactly the same polysaccharide that makes up the cellulose microfibrils in plant cell walls. BC strands are also called microfibrils but their morphology is somewhat different to those in plants. Cellulose microfibrils in plants are monodisperse in width, generally 3-20 nm depending on the botanical source, and micrometers in length, that is, they are somewhat reminiscent of spaghetti in nanosized form. Instead of a more or less square cross sectional shape prevalent in plants, BC microfibrils possess a rectangular cross section with sides of 7 × 70-145 nm, thus rendering the appearance of the microfibrils in the shape of flat ribbons, somewhat reminiscent to tagliatelle in nanosized form. Fink et al. proposed that the flat microfibrils consist of elementary fibrils of 7 × 13 nm cross section. Indeed, when preparing CNCs from BC, the flat structure is broken also at the crystallite level and the cross sections of the CNCs bear roughly the dimensions of 7 × 7 nm. Applications of bacterial cellulose. The peculiar feature of BC is that it forms a hydrogel pellicle after the synthesis. The pellicle consists of isotropic BC microfibrils. Additionally, BC is exceptionally effortless to purify from other components that cellulose; a mild alkali washing is sufficient to remove the bacteria and additional sugars. In other words, BC is a nanocellulose hydrogel in a very pure form, which is something that many nanocellulose procedures are attempting to achieve from plant materials with much effort. It is, therefore, quite an obvious development that BC has been subjected to similar investigations as the rest of the nanocellulose family, composite materials being among the most popular targets for applications. Furthermore, biomedical applications, particularly wound dressing materials, have been fashionable with BC because of its chemical purity and non-toxicity. The rationale for using BC as a reinforcing phase in composites stems from its exceptionally high mechanical strength. Figure 1b shows the tensile strength and modulus of BC nanofibers in the context of a variety of other natural and synthetic fibers. 15 In general, the values reported for individual BC nanofibers as well as other nanocellulosic species vary somewhat, depending on the measurement technique. 15 It is obvious that BC works well as a composite reinforce: Figure 1c shows a collection of data from BC/polymer composites where the tensile strength has been plotted as a function of modulus. 15 The dashed lines denote the strength and modulus of poly(lactic acid) (PLA) because of its status as one of the highest performing non-petroleum derived polymers. The performance of BC and other nanocellulose grades has not lived up to their theoretical potential in polymer composite materials. 15 One of the major problems is the incompatibility between cellulose and the continuous composite matrix. Polymer matrices are often hydrophobic, resulting in aggregation and phase separation of the cellulose when mixed together. 16 Another seminal challenge is the susceptibility of BC to water and humidity: as with ordinary paper products, the strength of nanocellulose-containing products is usually impaired when in contact with water or vapor. 17 Multiple ways of surface modifying all kinds of nanocellulosic grades for better compatibility or water resistance exist 18 but the methods are usually not suited for large scale production required for composite preparation. For comprehensive reviews on polymer/nanocellulose composites, the reader is referred to articles by Siró and Plackett, 19 Siqueira et al. 20 and Lee et al. 15 Biomedical applications are another strong research trend with BC-containing materials. Wound dressing, in particular, has received a lot of attention with even commercial or pre-commercial products in the line. 21 Here, the high mechanical strength of BC, its hygroscopicity, non-toxicity, antimicrobial nature, and ability to geometrically conform in various shapes are seen as the principal assets. The similarity of BC nanofibers with collagen fibers of the skin improves the biocompatibility. Other biomedical applications of BC include tissue-engineering 22 and drug delivery. 23

Microbial glycolipid biosurfactants

The surfactant market. are performance molecules that intervene in nearly every product and aspect of human daily life and find applications in/as very broad array of markets and applications: chemical, pharmaceutical, cosmetic & personal care, packaging, carpets & textiles, detergents, paper, adhesives, (3D printing) inks, mining and leaching, healthcare, polymer, food and feed, paints, surface and industrial coating, etc. The global turnover of surfactants was worth USD 30.64 Billion in 2016 and is expected to reach USD 39.86 Billion by 2021, registering a CAGR of 5.4% between 2016 and 2021 24 which shows that they are bulk products with a significant economic and environmental impact. Despite the efforts to move towards a more environmental friendly economy, only 3 % of the surfactant market share consists out of molecules, which are 100 % biobased and even a smaller part < 0,5 % of entirely biologically produced biobased surfactants, such as microbial, plant based or enzymatically produced biosurfactants. There are myriad pathways to process crude oil or gas towards the hydrophobic building blocks of synthetic surfactants, e.g. Fischer-Tropsch synthesis, oxo process, olefin oligomerization or Friedel-Crafts alkylation. The constant consumer and hence market demand for sustainable, biobased and green solutions, has resulted in a substantial increase of the development and use of partly biobased and even wholly biobased surfactants (WBS) (100 % derived from renewable (non-fossil) biomass in such applications. 25 With respect to this terminology: the European Commission has issued a mandate to the European Commission, for Standardization (CEN) to generate general standards providing a common basis with respect to terminology, bio based content determination, sustainability aspects, life cycle analysis and declaration tools for Business to Business (B2B) and Business to Consumer (B2C), which was done within the CEN/TC-276 technical committee in cooperation with the ERASM Biosurfactant Taskforce (www.erasm.org). Shortly said, the four categories are shown in Figure 2a and range from > 95 % (wholly bio-based); > 50 % and < 95 % (majority bio-based); > 5 % and < 50 % (minority bio-based) and < 5% (non bio-based). The wholly biobased surfactants (WBS) according to ISO 16128 norm, represent about 3 % of the European surfactant market and have the best image. Subsequently, there is a strong drive of the industry to apply this type of surfactants in their products, of course without giving in performance or cost. Most WBS are based on sugars coupled to fatty acids and/or alcohols i.e. methylester sulphonates, alkylpolyglucosides, sorbitan esters, anionic APG derivatives, sucrose esters, methyl glucoside esters, fatty acid Nmethylglucamides, alkylpolypentosides, etc. However, some limitations/drawbacks are associated with the current WBS portfolio: -the largest part of the wholly biobased surfactants on the market are produced through chemical processes, which negatively impacts their environmental profile -functionality/variety is limited -they do not offer an easy possibility for further derivatization/functionalization -they show good performance, but do not boost performance by e.g. combining properties justifying their higher price -can only be produced from first generation substrates Several new technologies are currently being developed, that can alleviate these issues. Related to the first hurdle, full biological production processes are associated with an even better environmental profile and additionally offer better marketing opportunities to the companies, which is a big driver. Biosurfactants. The current state of the art for biologically produced surfactants (estimated by the authors to currently only account to a few thousand tonnes per annum thus representing only a very small amount of the surfactant market share) can be defined as: -plant derived molecules like e.g. saponins or cardolite commercialized by e.g. Foodchem, Dr. H. Schmittmann GmbH and Cardolite respectively -enzymatically produced molecules like e.g. enzymatic sugar esters, which are not on the market yet, but a lot of research is conducted in this field -microbially produced molecules like sophorolipids (SLs) launched on the B2B market by Soliance, Evonik and planned by Croda and applied by Ecover, Saraya, Henkel, Soliance, etc.; rhamnolipids (RLs) commercialized by Logos, Jeneil, Urumqi Unite Biotechnology, Biotensidon, Agae and Rhamnolipid.inc; Mannosylerythritol lipids (MELs) commercialized by Toyobo, Kanebo, Damy Chemicals, Biotopia co. and investigated by many more; Lipopeptides (LPs) commercialized by Lipofabrik, Kanebo and Kaneka and Xylolipids (XLs) to be commercialized by INS. Microbial surfactants already offer a solution to the first drawback mentioned above. They have been very extensively investigated as they offer some opportunities, e.g they can be produced from second generation (2G) substrates and waste streams like molasses, animal fats, dairy industry whey and other waste or side streams 26,27 . Recently, efficient sophorolipid production (wild type) was shown on 100 % 2G substrates (both hydrophilic as hydrophobic part) inside the framework of the BBI Carbosurf project, which will be published elsewhere. Derivatization efforts for sophorolipids -the best represented microbial biosurfactant on the market-have been investigated quite extensively 28 . In terms of chemical derivatization of wild type SLs, too many hurdles, like low yield and low uniformity related to the original complexity of the wild type SLs, are associated with these processes. Enzymatic derivatization strategies have also been investigated 28 and even commercialization efforts were done by the American company Synthezyme.

Three main reasons can be defined to explain low commercialization levels of biosurfactants. Cost considerations are to date the prime hindrance to market penetration of (microbial) biosurfactants. The maturity of the petrochemical industry makes purely cost-based competition unrealistic for most biosurfactants. Moreover, even the availability of biosurfactants with equivalent performance and at the same cost would not per se be sufficient to drive acceptance and utilization by consumers and brand owners. A better performance at an acceptable premium price would increase their marketability for mass consumption products as consumers anno 2017 demand environmentally friendly and benign products/processes, without wanting to give away functionality/performance. This situation requires the development of innovative and new solutions as the industry is currently increasingly driven by consumer awareness and opinion. A second hurdle is the lack of diversity. Formulation experts are like artists, demanding a diversified palette of molecules to shake and stir to get to a desired endpoint. Whereas the chemical industry has been very successful in developing an extraordinary wide variety of surfactant structures for a multitude of applications/functions, the biobased counterparts have not been able to offer the same diversity. To give an example, the chemically produced biosurfactants APG's (alkyl polyglucosides), mainly consist of mono-glucosylated (degree of polymerisation (DP) = 1) molecules and a small amount of diglucosylated (DP = 2) molecules. Consequently, the structural diversity of these biosurfactants is rather low with an average DP ranging between 1,2 and 1,6. Although APGs are one of the most prominent biosurfactants on the market, their narrow product spectrum limits further growth. Moreover, they do not offer a possibility for further derivatization/functionalization. Several new technologies are currently being developed. The large chemical company Evonik for example launched sophorolipids, a microbial biosurfactant, as an ingredient on the European B2B market in 2015, while they are currently taking actions to do the same for the microbial surfactant rhamnolipids. Also other big companies like Croda and Henkel are highly active in this field. Although these observations are already a step in the right direction, the biosurfactant diversity can still by far not compete with the portfolio of fossil derived surfactants. A third reason is the fact that (microbial) biosurfactants typically are defined by the occurrence of very similar congeners i.e. production of mixtures. For the microbial biosurfactants sophorolipids, these are mainly divided into a mixture of lactonic and acidic sophorolipids (Figure 2c). Additional variation within the congeners in the hydrophobic tail length, saturation degree and positioning of the sophorose head group (terminal (ɷ) and subterminal (ɷ-1) (Figure 2c). Easily over 30 congeners can as such be identified in 'sophorolipids', which are thus not defined by one single molecule. Mixtures might do the job and could thus be perfectly marketed, but these biological systems are sensitive to small variations in medium/cultures conditions, which will influence the composition of the mixture, which in turn gives rise to very different properties. This in-productvariation is one of the reasons of the very varying reports in the literature on their properties. The latter is simply unacceptable from an industrial point of view. Combined with the reasons mentioned above, this is one of the main reasons why microbial biosurfactants currently only represent a marginal part of the surfactant market.

A number of solutions can be defined for the mentioned bottlenecks. Variation can be increased by applying chemical and/or enzymatic derivatization possibly in combination with an expansion of the molecular variety through genetic engineering. The latter can also increase productivity (decrease of price) and uniformity (decrease of mixtures). The efforts spread over the past 15 years, 29 especially at the University of Ghent (Belgium), 30,31,32 resulted in the generation of a proprietary platform technology for the production of new types of glycolipid molecules (Figure 2b). This has been accomplished by the development of molecular techniques and constant expansion of the molecular toolbox for this non-conventional yeast and through the development and use of several -omic strategies. Although these engineering efforts have thus been quite successful, none of the abovementioned efforts have yet resulted in the commercialization of a new-to-nature compound. This is due to a few reasons: -new molecules translate/correspond with new processes, different properties and thus different applications.

-the non-optimized production processes and strains logically correspond with higher production costs -the absence of the molecules on the market works inhibitory for the economy of scale to kick in nor is regulatory approval granted (or data available for initiation of a regulatory dossier) although the related wild type SLs are.

-the lack of knowledge on the properties of these new molecules These hurdles are thus the next steps to take to valorized this technology and this is done by applying an integrative process design approach combining strain engineering with process development and optimization.

Applications. The discussion above mainly concerns the surface active properties, by far the most important from an industrial development point of view, of microbial glycolipid biosurfactants. However, a series of new perspectives have been opened by research groups working mainly in Japan, India, France and USA. The biocompatibility of most glycolipids and the presence of the free carboxylic acid group made them interesting candidates for the water stabilization of metal and metal oxide nanoparticles. The first work in this field was proposed by the group of Prasad at the NCL in Pune, India. Cobalt, 33 silver, 34 gold 35 but also iron oxide 36 nanoparticles (Figure 3a) have been used as systems to be coated with the acidic form of sophorolipids. In all cases, the resulting sophorolipid-coated system is highly dispersible in water and it was shown that, in the case of metal systems, sophorolipids can also act as a reducing agent, thus excluding the use of strong, classical, reducing agents like NaBH4. Cytotoxicity and genotoxicity tests performed on the gold and silver nanoparticles systems have shown no specific biological activity below 100 g/mL, 35 thus making these systems interesting candidates for biomedical applications. The impact of the sophorolipid coating on the colloidal stability was shown for the iron oxide nanoparticles, which were shown to be dispersible and stable in time up to 2 M KCl solution, thus showing the strong stability of the COOH-FeOx complex. 36 Antimicrobial activity of glycolipid biosurfactants in solution is a field of research since a long time. However, more recent works have shown their interest as surface antimicrobial and/or antiadhesive coatings. Dispersion of rhamnolipids on both hydrophobic (octadecyltrichlorosilane-modified glass) and hydrophilic (hydroxylrich glass) surfaces were shown to have an interesting antiadhesive effect on Gram-negative E. coli, P. putida and P. aeruginosa and on Gram-positive B. subtilis. In particular, adhesion of P. aeruginosa (number of attached cells per mm 2 ) was reduced by a factor 15 on the hydrophilic and by a factor 20, respectively, on the hydrophilic and hydrophobic glass substrates with respect to control. 37 (Figure 3d) This system, together with B. subtilis, were by far the most effective with respect to the other pathogens tested in the study. On the contrary, if the glycolipid (sophorolipid) is chemically grafted on the substrate via its pending COOH group (amidation reaction with a surface aminothiol primer deposited on gold), biocidal properties against both Gram-positive (L. ivanovii, E. faecalis, S. epidermidis, S. pyogenes) and Gram-negative (E. coli, P. aeruginosa, S. typhymurium) bacteria are observed instead. 38,39 If the efficiency is, in the best case, only of about 45% of the bacterial population present on the surface, these works show the first biocidal effect of a glucose derivative and join the debate concerning the interactions between carbohydrates and cell membranes. 40,[START_REF] Andersen | Proc. Natl. Acad. Sci[END_REF] Surfactants are known to spontaneously self-assemble in water, and glycolipid biosurfactants do not derogate this rule. Self-assembly properties have been reported for mannosylerythritol lipids, rhamnolipids, sophorolipids, glucolipids, cellobioselipids, just to cite the most important ones. [45][46][47][48][49][50] If the knowledge in this field is more or less advanced according to the effort that a specific research group has dedicated to a given family of molecules, one can summarize, without being exhaustive, the following morphologies: micelles, 45,50-52 vesicles, 45,48,49 fibers, 48,49 lamellar, 45,48 sponge 45 phases (Figure 3b-c). The nature of the molecule and concentration are the most obvious parameters that influence the self-assembled morphology, but pH 47,51,53 and temperature 45,47 have also been shown to have a strong influence on the self-assembly properties. This impressive load of work is still ongoing because the relationship between the structure of a given microbial glycolipid and the corresponding selfassembled phase is not obvious and cannot be based on the classical prediction based on packing parameter considerations, as recently shown for acidic oleic acid sophorolipids, which can form micelles or giant ribbons as a function of the purity but also, for a 100% pure compound, as a function of the preparation method. 54 One must here observe that microbial glycolipid systems are in fact never pure and in fact very hard to fully purify, because it is well known that microbial synthesis provide a system with a wide range of structurally-similar congeners where one or two are majoritarian.

Gene transfection, which consists in the delivery of genetic material across the cell membrane, is one of the applications in which control of self-assembly, and colloidal properties in general, is very important. In this field, mannosylerythritol lipids (MEL) have been employed as adjuvants in gene transfection of plasmid DNA using cationic liposomes as classical carriers and DNA binders. 45 It was shown that the presence of MEL increases the efficiency of gene transfection in NIH3, COS-7 and HeLa cells up to 50 to 70 times. The role of MEL seems to be the acceleration of membrane fusion between the cationic liposome and cell membrane, so that transfection efficiency is increased. More recently, quaternary ammonium-modified sophorolipids have been incorporated into negatively-charged DOPE liposomes and used as direct binders of negatively-charged plasmid DNA, to be transfected into A549, 16HBE and SKMEL28 cell lines. 55 Authors have found that two specific, long-chain, quaternary ammonium derivatives were highly efficient and with low cytotoxicity. The main difference between this work and the approach using MEL is the positively-charged sophorolipid obtained by chemical modification and its direct binding to plasmid DNA, thus giving it a direct role in terms of vectorization.

Conclusion

The present contribution shows two families of carbohydrate-rich compounds of microbial origin, one polymeric and the other molecular. Bacterial cellulose is an interesting polysaccharide challenging the use of plant cellulose for the easier approach in the purification and good waterdispersion properties. On the other side, microbial glycolipids are versatile compounds both in terms of lipid and carbohydrate structure that can self-assemble into a wide range of morphologies (micelles, vesicles, fibers…) and be used as antimicrobial and surface stabilizing agents, among others. Both families of compounds are biodegradable and non-toxic and have the goal of substituting, in the long run, petrochemical compounds. Nonetheless, in both cases, the industrial development of a fully microbial-based organic chemistry is far from being a present reality due to the high production costs and product variability.

Figure 1 -

 1 Figure 1 -a) Scanning electron micrograph of bacterial cellulose gel. The gel still contains the bacteria which are generally removed after culturing by a simple washing step. Reproduced from ref. 8. Copyright Springer 2000; b) Average tensile properties of BC in comparison to some commonly used synthetic and renewable fibers. c) Tensile strength versus tensile modulus of selected BC composites. The dashed lines denote tensile properties of neat PLA. Reproduced from ref. 15. Copyright Elsevier 2014. d) Simplified pathway from glucose to bacterial cellulose

Figure 2 -

 2 Figure 2 -a) Use of bio-based and non bio-based surfactants in the European Union, Norway, Switzerland and Iceland (2015). 25 b) Glycolipid portfolio developed by InBio.be based upon the yeast Starmerella bombicola. c) Representation of variation in wild type sophorolipids

Figure 3 -

 3 Figure 3 -a) Sophorolipid-capped silver nanoparticles (morphology, size, structure and organic content are respectively presented in panels A-B, C and D). Reproduced from ref. 42. Copyright Royal Society of Chemistry 2008. b) Self-assembled nanoscale twisted ribbons obtained from stearic acid sophorolipids. Reproduced from ref. 43. Copyright Wiley-VCH Verlag 2015. c) Glucosomes obtained from branched C22 sophorolipids. Reproduced from ref. 44. Copyright Wiley-VCH Verlag 2017. d) Antiadhesive effects of rhamnolipids-coated glass substrate. Reproduced from ref. 37. Copyright Wiley-VCH Verlag 2013


Jessica Cauchard
email: jcauchard@acm.org

Ehud Sharlin

"Come To Me Nice Butterfly" Drone Form in Collocated HDI

Keywords: collocated human-drone interaction, natural interfaces, intimate interaction with drones

Recent human-drone interaction (HDI) research is shifting away from the remote-control paradigm and increasingly exploring collocated interaction between people and drones. We are interested in intimate aspects of this new design space: in collocated interaction that affords tactile interaction and emotive touch. In this position paper, we briefly motivate emotive collocated HDI, present preliminary design ideation, and propose a research outline for further exploration using immersive collocated HDI simulations.

INTRODUCTION

Humans have an established set of interactions with volant (flying and gliding) animals, which has previously inspired human-drone interaction (HDI) design [START_REF] Ng | Collocated Interaction with Flying Robots[END_REF]. A small subset of these includes few exceptions, collocated interaction between people and flying animals is awkward, bothersome, and undesirable. On the other hand, cultural context can provide positive perspective, for example when interacting with pet birds [START_REF] Hines | A Kestrel for a Knave[END_REF], butterflies (Figure 2, [START_REF] Segal | What's Jewish about Butterflies?[END_REF]), or fairies. We expect collocated HDI to emerge in both domestic and work environments, for functional uses, such as delivery of goods, or search-and rescue, and for fun and leisure, such as in photo and videography. When considering this new design space we see the realistic negative context of interacting with volant animals as a design challenge, and turn to the much more positive, and unrealistic, cultural contexts for inspiration. We envision future drones that will share space with people, providing functionality and playing social, emotive and intimate roles, taking physical forms that convey positive relationship, inspired by cultural contexts (Figure 1). Such interactions are often seen in the popular culture, in movies and anime. For example, in Grave of the Fireflies [START_REF] Takahata | Grave of the Fireflies (火垂るの墓)[END_REF], we see the main character, within a cloud of fireflies, trying to catch and hold them, later using the fireflies for light when needed. We envision similar behaviors with swarms of drones, where people could catch one in their hand before releasing it to the swarm. Our vision is hindered by the current technical boundaries of flying machines. Drone technology, while advancing by leaps and bounds, still accounts for entities that become smaller in size but are still noisy, mechanical looking, and even dangerous. In order to explore the benefits and limitations of intimate collocated interaction with drones we propose the use of immersive simulations, and argue that the first challenge should be the exploration of drone form. In the following sections we provide a brief reflection on the state of the art of collocated HDI, describe our current low-fidelity ideation, present an exploration methodology and our proposal for an immersive simulation testbed.

RELATED WORK

Prior work on interaction with small-sized drones explored control mechanisms for collocated interactions including voice [START_REF] Draper | Manual Versus Speech Input for Unmanned Aerial Vehicle Control Station Operations[END_REF][START_REF] Pourmehr | You are green: a touch-to-name interaction in an integrated multi-modal multi-robot hri system[END_REF], gestures [START_REF] Landay | Drone & Wo: Cultural Influences on Human-Drone Interaction Techniques[END_REF][START_REF] Ng | Collocated Interaction with Flying Robots[END_REF][START_REF] Cauchard | Drone & me: an exploration into natural human-drone interaction[END_REF][START_REF] Jensen | Knowing You, Seeing Me[END_REF][START_REF] Pfeil | Exploring 3d gesture metaphors for interaction with unmanned aerial vehicles[END_REF], gaze [START_REF] Khamis | GazeDrone: Mobile Eye-Based Interaction in Public Space Without Augmenting the User[END_REF], and touch [START_REF] Abtahi | Drone Near Me: Exploring Touch-Based Human-Drone Interaction[END_REF][START_REF] Braley | GridDrones: A Self-Levitating Physical Voxel Lattice for Interactive 3D Surface Deformations[END_REF]. Several feedback mechanisms have been proposed such as using a screen, a projector, or using the flight path to convey affect and emotions [START_REF] Cauchard | Emotion encoding in human-drone interaction[END_REF][START_REF] Arroyo | Daedalus: a sUAV for human-robot interaction[END_REF]. Affect and emotion have been specifically investigated as a first step towards integrating drones into humans' social environments. In their preliminary results, Arroyo et al. [START_REF] Arroyo | Daedalus: a sUAV for human-robot interaction[END_REF] show that different emotional states can be recognized and suggest that HDI can be improved if the drone conveys different emotional states. Cauchard, et al. [START_REF] Cauchard | Emotion encoding in human-drone interaction[END_REF] explored how a drone's emotions could be conveyed through different behaviors and flying paths. They showed that people can accurately associate emotional states to a drone. Recently, researchers have been working towards design guidelines for social drones suitable for interaction and companionship. Kim et al.'s ideal companion drone [START_REF] Young | The Naughty Drone[END_REF] presents "adorability" features. Yeh et al. [START_REF] Yeh | Exploring proxemics for human-drone interaction[END_REF] proposed a blue oval shaped drone and discussed how a tablet can be used to display a "friendly face". Karjalainen et. al. [START_REF] Daniel Karjalainen | Social Drone Companion for the Home Environment[END_REF] investigated several features and found that emotional characteristics were desirable, and they also suggest that the drone appearance should be a round shape with a face. The above literature shows that appearance and behavior is a central aspect of designing social drones intended to interact with people. We propose an investigation of this new HDI design space by liberally exploring unrealistic cultural contexts of interaction with collocated flying entities. We further suggest that this early design exploration should be unhindered by current drone technology, and instead conducted first within immersive simulations.

EXPLORING FORM IN IMMERSIVE COLLOCATED HDI

We are planning to design and implement a collocated HDI immersive simulator that would allow users to perform various simple tasks while interacting with a collocated drone. The simulator will be implemented in VR, though an AR iteration is also possible. The simulator will include 3D visual features relating to the drone, the setting (e.g. domestic environment, or a workplace) and the task. The testbed will include haptic feedback supporting intimate interactions: touching the drone, sensing its landing on a palm or taking off (note that others proposed the use of physical drones to provide such haptic feedback [START_REF] Knierim | Tactile drones-providing immersive tactile feedback in virtual reality through quadcopters[END_REF][START_REF] Hoppe | VRHapticDrones: Providing Haptics in Virtual Reality through Quadcopters[END_REF]), and possibly synchronized air flow device (e.g. fan [START_REF] Somniacs | Birdly® -The Ultimate Dream of Flying[END_REF]), enabling sensation of the drone rotors in proximity. The simulator will allow iteration of various collocated HDI form and behavior approaches, ranging from interaction with drones that look, for example, like butterflies, fairies, and birds (see Figure 1 for artist renditions of some of our current design ideas). The drones rendered in the simulator can take a range of forms, rendered as naïve entities, or as mechanical looking and moving flying machines. The simulator will support different drone behavior and varying flight characteristics (speed, acceleration and jerk) and proxemics behaviors. The simulator will support single drone as well as a group of drones moving individually or as a flock or swarm. The simulated drones will be sensitive to the user's pose and gestures, as well as their voice and gaze. The testbed will also allow the simulated drones to express themselves, using body and wings gestures, gaze, "facial" expression, chirping, and voice.

LIMITATIONS

Our proposed collocated HDI simulator stops short of practical reflections on the many potential technical limitations and bottlenecks derived from some of our design ideas. Implementing drones for collocated interaction is a hard technical challenge, and it is questionable how many of our simulated design ideas could be scaled to the physical realm and implemented as collocated drones, in the short-term.

CONCLUSION

Realistic interaction between people and collocated volant animals is very limited at best, and undesirable in most practical cases. However, collocated interaction with flying entities in cultural contexts, in literature, film, media and the arts, while often unrealistic, can be quite positive. We suggest an exploration of the realm of purposely unrealistic collocated HDI metaphors, starting from drone form and behavior. We propose an immersive simulation testbed that would allow people to interact with different forms of collocated drones in various settings and tasks. Lessons drawn from the testbed could inform the design of the form and behavior of future drones, and be used in the design of more natural HDI.

  collocated interactions, for examples, dealing with flying insects, petting bats, or falconeering. With This paper is published under the Creative Commons Attribution 4.0 International (CC-BY 4.0) license. Authors reserve their rights to disseminate the work on their personal and corporate Web sites with the appropriate attribution. iHDI '19 -International workshop on Human-Drone Interaction, CHI '19 Extended Abstracts, May 5, 2019, Glasgow, Scotland, UK http://hdi.famnit.upr.si © 2019 Creative Commons CC-BY 4.0 License.

Figure 1 :

 1 Figure 1: artist's rendition of drone form in collocated HDI (sketches courtesy of Norm)

Figure 2 :

 2 Figure 2: From the cover of Fania Bergstein's "Come to Me Nice Butterfly" [6]

"Come to me, nice butterfly. Sit with me, in the palm of my hand. Sit, rest, don't fear. and fly away again." From: "Come to Me Nice Butterfly", by Fania Bergstein (translated from Hebrew [6])


Susanne Boll
email: susanne.boll@uol.de

Marion Koelle
email: marion.koelle@uol.de

Understanding the Socio-Technical Impact of Automated (Aerial) Vehicles on Casual Bystanders

Keywords:

Automated vehicles are coming to our streets and into our air. These automated vehicles are not acting as fully independent entities but are embedded into our social space and are affecting humans with which they interact. Recent advances are looking at the direct cooperation of human and machine in concrete interaction scenes such as steering a semi-automated drone or interacting with an automated car as a pedestrian. What we do not understand yet, is the reaction of automated systems on individuals that are casual bystanders of the automated systems. Cooperation and social acceptance of the casual bystanders are crucial in many situations. Affects such as irritation, anxiety or frustration may be easily invoked by the automated object. We need to anticipate effects on bystanders and include this into the interaction design space.

INTRODUCTION

Understanding an automated vehicle is challenging and can cause negative side effects, such as anxiety or frustration. Because today's automated vehicles omit the human side of person-to-person communication: when communicating with an automated vehicle, users and bystanders cannot rely on established practices as in communicating with other individuals. While the automated vehicles of today are being designed to work properly, robustly and safely, they do not yet act socially. Two humans that can see each other, such as a pedestrian and a driver, still use many subtle cues to cooperate and indicate each others intention. This may not be true for a full automated vehicle in cooperation with a pedestrian, who may not understand it, not trust it, or just be surprised by the automated vehicles' behavior. Similarly, drones might distract or surprise bystanders and even cause anxious behavior, as when a drone approaches a person that is not expecting it and cannot understand the intention of the drone.

We propose that a systematic exploration of the design space is needed for automated vehicles. This includes their appearance and actions to communicate intent to casual bystanders in everyday situations and increase cooperative, prosocial behavior. We hypothesize that bystanders and affected persons not only need to recognize the automated vehicle but clearly understand its intentions and upcoming actions to increase social acceptability and successful cooperation.

STATE OF THE ART

The interaction between humans and drones is a field in its beginning. Nevertheless, drones are more and more becoming automated (aerial) vehicles. We are convinced that the work on interaction between other types of automated vehicles and pedestrians and other traffic participants will be inspiring and supporting for the work on human-drone interaction. Research and industry are already making first proposals and showcase concept studies of communicating with bystanders to inform them and to keep them in the loop. In consequence it is timely and topical to conduct research on the effect of (automated) drones on casual (human) bystanders.

Rothenbücher et al. [START_REF] Rothenbücher | Ghost Driver: A Platform for Investigating Interactions Between Pedestrians and Driverless Vehicles[END_REF] developed a Wizard-of-Oz technique for investigating the interaction between automated vehicle and traffic participants (or bystanders) by hiding the actual driver (the "Wizard") of the car. They considered how pedestrians interacted with the car at crossings in the Stanford University Campus and found that most pedestrians were able to decide whether to cross the road or not without explicit communication, but also that it remains relevant to acknowledge that a pedestrian was noticed. However, this was a first experiment in a confined campus setting where people are expectedly open to technological innovations and with less of a surprising effect than in a narrow urban road scenario in Europe. Similarly, Petterson et al. [START_REF] Pettersson | Design Techniques for Exploring Automotive Interaction in the Drive Towards Automation[END_REF][START_REF] Pettersson | Living Room on the Move: Autonomous Vehicles and Social Experiences[END_REF] report on explorations of social situations between (seemingly) autonomous cars and pedestrians using similar techniques where the actual driver was hidden behind a cover resembling a car seat (c.f., Habibovic et al. [START_REF] Habibovic | Evaluating interactions with non-existing automated vehicles: three Wizard of Oz approaches[END_REF] ). They uncovered two-fold, specific information needs: information the pedestrian may need from the autonomous vehicle in certain situations, and vice versa. Chang et al. [START_REF] Chang | Eyes on a Car: An Interface Design for Communication Between an Autonomous Car and a Pedestrian[END_REF] equipped cars in Virtual Reality (VR) with eyes to help pedestrians assessing if they were acknowledged. Their experiment showed that pedestrians made quicker decisions and felt safer when they could see where the car is "looking". Mahadevan et al. [START_REF] Karthik Mahadevan | Communicating Awareness and Intent in Autonomous Vehicle-Pedestrian Interaction[END_REF] explored possible interfaces and designs for explicit vehiclepedestrian communication and tested them with an equipped car and a Segway. They could show that explicit signals help pedestrians to make faster decisions. While reviewing, relevant literature shows that research towards cues and signaling of automated vehicles exists, there are still no common communication strategies or even standardized designs. Also, current research focuses on functional traffic interaction such as crossing a road but does not necessarily investigate how cues need to be designed for prosocial behavior and inclusion of effects on casual bystanders.

The social acceptability of drones as guidance vehicles for navigating persons with visual impairments has been explored by Avila Soto et al. [START_REF] Avila | Look, a Guidance Drone! Assessing the Social Acceptability of Companion Drones for Blind Travelers in Public Spaces[END_REF]. They investigated both, the (visually impaired) user's perspective, and the perspective of (sighted) casual bystanders, and note that knowledge about the purpose, functioning, and benefits of the guidance drone are relevant for social acceptability. However, they did not elaborate on autonomous behavior of the drone and on how the drone interface might communicate its intent. Communication of usage purpose and intention has been explored in the area of body-worn cameras [START_REF] Koelle | Beyond LED Status Lights -Design Requirements of Privacy Notices for Body-worn Cameras[END_REF]. In this context, design strategies including physicality, signaling, as well as transfer of control have been explored to create a sense of situational awareness for the bystander, and justification on the device user's side. The authors also explored how bystanders can take over control by using gestures to explicitly express consent (Opt-in) or disapproval (Opt-out) with being recorded by a body-worn camera [START_REF] Koelle | Your Smart Glasses' Camera Bothers Me!: Exploring Opt-in and Opt-out Gestures for Privacy Mediation[END_REF]. Similar technologies could be used for drones when people might want to use explicit consent to be recorded or not.

In our own previous work, we have been designing and evaluating multimodal interfaces that form the foundation for communicating information about upcoming tasks and objects in pervasive spaces by multimodal cues. We systematically explored different sensor modalities in pervasive user interfaces: light-based interfaces [START_REF] Matviienko | Deriving Design Guidelines for Ambient Light Systems[END_REF], projection-based interfaces [START_REF] Anke | FlyMap: Interacting with Maps Projected from a Drone[END_REF], auditory interfaces [START_REF] Wilko Heuten | Interactive 3D Sonification for the Exploration of City Maps[END_REF], and haptics and vibro-tactile interfaces [START_REF] Cauchard | ActiVibe: Design and Evaluation of Vibrations for Progress Monitoring[END_REF][START_REF] Pielot | Where is My Team: Supporting Situation Awareness with Tactile Displays[END_REF][START_REF] Strasnick | BrushTouch: Exploring an Alternative Tactile Method for Wearable Haptics[END_REF]. For many years now, we explored assistance systems with increasing levels of automation. We studied the effect of multimodal cueing for situation awareness and spatial awareness in supervisory tasks [START_REF] Fortmann | Supporting Situation Awareness with Peripheral Feedback on Monitoring Behavior[END_REF] and attention shifting to other work tasks [START_REF] Müller | Ambient Timer -Unobtrusively Reminding Users of Upcoming Tasks with Ambient Light[END_REF]. We studied how wearable technology can give feedback on biosignals using visual, audio, and haptic modalities [START_REF] Frey | Breeze: Sharing Biofeedback Through Wearable Technologies[END_REF]. We have been designing multimodal cues for take over in highly automated driving [START_REF] Shadan Sadeghian Borojeni | Assisting Drivers with Ambient Take-Over Requests in Highly Automated Driving[END_REF] and priming individual with information about the upcoming task after taking over [START_REF] Shadan Sadeghian Borojeni | From Reading to Driving: Priming Mobile Users for Take-over Situations in Highly Automated Driving[END_REF]. In recent work, we investigated how we can shift attention in larger cyber-physical system and environments to (automated) objects that are currently out of view [START_REF] Gruenefeld | EyeSee360: Designing a Visualization Technique for Out-of-view Objects in Head-mounted Augmented Reality[END_REF][START_REF] Claudius Stratmann | Exploring Vibrotactile and Peripheral Cues for Spatial Attention Guidance[END_REF].

To date, research on human-drone interaction has aimed to communicate the intent of a drone, such as using LEDs around a quadcopter to communicate direction [START_REF] Szafir | Communicating directionality in flying robots[END_REF] or modifying the drone's flight path, using techniques such as arcing, to communicate directional intent [START_REF] Szafir | Communication of intent in assistive free flyers[END_REF]. We find that several works that looked at the adequacy of multimodal interaction with drones [START_REF] Cauchard | Drone & Me: An Exploration into Natural Human-drone Interaction[END_REF], or how drones might convey information about themselves. As one example, Cauchard et al. [START_REF] Jessica R Cauchard | Emotion encoding in human-drone interaction[END_REF] modified the flight path of the drone to communicate the drone's emotions. However, none of this work investigated the automation level of the drone and how it could handover control to passersby or provide the option to listen to their command. As with autonomous cars, the technology's current level of automation for a task is not conveyed to passersby. It is crucial for the autonomous vehicles to become more transparent so that they can be accepted into our environments. In order to explore concepts and interaction designs, this research can be inspired by different levels of simulation of automated situations which enables us to obtain higher levels of mundane realism [START_REF] Pettersson | Design Techniques for Exploring Automotive Interaction in the Drive Towards Automation[END_REF], to evaluate designs for interaction with automated vehicles in different traffic scenarios.

PROSOCIAL BEHAVIORS IN HUMAN-AUTONOMOUS VEHICLE COMMUNICATION

So far, research and industry has shown interesting prototypes and design concepts of such displays outside the vehicle. However, commercial products coming with a standard set of communication patterns have not been developed. Here, the industry has not yet demonstrated a clear understanding of a cooperative and understanding interaction between automated vehicles and individual bystanders. To better understand the impact of the role of bystanders for the design of automated (aerial) vehicles for acceptance and cooperation of automated vehicles with casual bystanders we need to understand how an automated (aerial) vehicle and its goals can be recognized and understood. We need to design for prosocial behavior with the automated vehicle and how cues and signals of the automated vehicles can support cooperative behavior and communication with bystanders. We identify three core aspects of that form the basis for a successful interaction between an automated vehicle and bystander(s):

Situational Awareness (SA) One aspect is the Situation Awareness of the automated vehicle by the bystander. We are following Endsley's well established notion of Situation Awareness [START_REF] Endsley | Measurement of situation awareness in dynamic systems[END_REF], in which SA comprises the perception of the objects in the environment, understanding their behaviour and the individuals' projection of future

Key Factors

We hypothesize that are three key factors that are core to such communication aspects for moving automated vehicles, such as cars and drones, as described above. These core factors, that we envision to be addressed by future research are:

(1) Physicality. The device itself may have a form, shape and (aesthetic) appearance that is selfexplaining its functions and potential actions. What role does the shape of the device play for the acceptance of the automated devices' actions? Can the intended function already be encoded in the appearance? (2) Signaling. Beyond the actual appearance, the device can use integrated and added displays to signal its behavior to the bystander. It needs to be further investigated what and how should it signal with visual and auditory displays? (3) Movements. As the automated vehicle is moving, it is in demand to better understand how to express cooperative behavior with movements. How should such vehicles approach a person to express intent of communication?

CONCLUSION HCI research with autonomous devices is still in its early stages. Autonomous cars are increasingly being studied and have an obvious widespread reach as we saw companies like Uber using autonomous taxis driving passengers from 2016 to 2018. On the other hand, drones are just reaching the point of technological maturity for them to interact with people. We posit that the research done in autonomous devices can be designed with similar methodologies for cars and drones. In particular, we show the importance of including methodologies taking in consideration passerby and not only designated users. This will be crucial for the technology to become acceptable to all.

Figure 1 :

 1 Figure 1: Person interacting with a drone showing it to move sideways.

Figure 2 :

 2 Figure 2: Person interacting with automated vehicles in a VR Simulation -interacting with an automated vehicle to indicate it should give way.

states and events. We propose that the following questions need to be answered to understand which cues and signals issued by the automated vehicle are good prerequisites to increase the situation awareness of the vehicle:

• How to make the bystander aware of the automated (aerial) vehicle?

• How to allow the bystander to identify whether a present automated (aerial) vehicle is affecting her/him?

Affordance

Based on the longstanding knowledge from the field of human computer interaction, we know that interaction is more successful and satisfying if the object of interaction offers affordance on how to interact with it [START_REF] Donald A Norman | Affordance, conventions, and design[END_REF]. As an attribute of interaction design, an affordance is a feature that is offered to the user, what the interaction design provides or furnishes [START_REF] James | The ecological approach to visual perception: classic edition[END_REF]. We want to understand what kind of physical (real) affordance, cognitive (perceived) affordance, and functional affordance [START_REF] Hartson | Cognitive, physical, sensory, and functional affordances in interaction design[END_REF] the automated vehicle can offer to the bystander, such that it easily reveals its functions and actions. With such an affordance concept, the bystander can understand if and how they may be able to interact with the automated (aerial) vehicle such as allowing it to come closer or to stop.

• Do the automated (aerial) vehicle's features help the bystander to understand what it does/and provides? • Does the automated (aerial) vehicle reveal if it is ready to receive inputs and if a bystander may interact with it? • Does the automated (aerial) vehicle offer functions to interact with it?

Conversation

The interaction with an automated vehicle might not be a one-shot or uni-directional interaction, but rather an actual negotiation between an individual and an automated (aerial) vehicle. For example, in the case of a car, the determination of who will go first and decide that the individual may interfere and signal a decision (c.f., Figure 2). In the case of a drone, the person may want to tell the drone that they do not want to appear in recorded footage, provide instructions for guidance and navigation aids, or acknowledge receipt of a delivery.

• How does the automated (aerial) vehicle get into a conversation with a bystander? • How can the bystander ask and get explanations on the intentions and actions of an automated (aerial) vehicle? • How to interact with the automated (aerial) vehicle such as asking it to come closer?


Damien Ulveling

Claire Francastel

Dr Florent Hubé
email: florent.hube@univ-paris-diderot.fr

Identification of potentially new bifunctional RNA based on genome-wide datamining of alternative splicing events

Keywords: Intron retention, alternative splicing, non-coding RNA, bifunctional RNA, Steroid Receptor RNA Activator SRA

Identification of potentially new bifunctional RNA based on genome-wide data-mining of alternative splicing events

Damien Ulveling, Claire Francastel, Florent Hubé

Completion of the Human Genome Project and, later on, advances in whole genome tiling arrays and high-throughput cDNA/EST sequencing have led to at least two unexpected outcome which have advanced our somewhat dichotomous vision of genome expression which placed genes and their messenger RNA products in the limelight for decades, whereas the remainder was relegated as junk DNA. The predicted 50-100,000 protein-coding gene units, that would reflect the great complexity of the human organism, were first revised downwards to about 20,000 coding genes [START_REF] Ast | The alternative genome[END_REF]. In parallel, it strengthened the view that genomes are widely transcribed while mRNA represents only 1-2% of this transcriptional output. The vast majority of these RNA was classified as non-coding RNA (ncRNA) [2; 3] primarily owing to the fact that, by definition, no open reading frame (ORF) long enough to be considered, nor protein, have been associated with them. Generally speaking, this classification incorporates transcripts generated by pervasive transcription outside of annotated gene loci for which no function has been identified yet, although many studies are continuing to add to our knowledge of the fundamental role of these RNA in most biological processes (for a review see [START_REF] Mercer | Long non-coding RNAs: insights into functions[END_REF]). Most non-coding RNA, and especially long non-coding RNA, are transcribed by RNA polymerase type II, capped and polyadenylated, undergo splicing, and can not be discriminated from mRNA [START_REF] Carninci | Noncoding RNA transcription beyond annotated genes[END_REF][START_REF] Dinger | Differentiating proteincoding and noncoding RNA: challenges and ambiguities[END_REF][START_REF] Griffiths-Jones | Annotating noncoding RNA genes[END_REF][START_REF] Ulveling | When one is better than two: RNA with dual functions[END_REF], except by their protein-coding potential. This view has been challenged recently, with the concept of bifunctional RNA (for a review, see [START_REF] Ulveling | When one is better than two: RNA with dual functions[END_REF] and Figure 1). In essence, bifunctional RNA hold the dual capacity of serving as both intermediate molecules translated into protein and functional RNA. Several examples have been described recently in the animal kingdom, with p53 being a prime example (nicely reviewed by M. Candeias in this issue). Both p53 mRNA and protein can interact with the Mdm2 protein with opposing effects on p53 synthesis and proteasome-mediated degradation [10; 11]. Historically, the founding member of this new class of ncRNA exhibiting the ability to encode for proteins was SRA (Steroid Receptor RNA Activator), first identified as a structural ncRNA molecule in hormone receptor complexes, characterized by discrete stem-loop structures required for the coactivator function of SRA [START_REF] Lanz | Distinct RNA motifs are important for coactivation of steroid hormone receptors by steroid receptor RNA activator (SRA)[END_REF] extensively reviewed by Cooper et al. in this issue. A few years later, new SRA isoforms were identified, exhibiting an additional exon upstream of the core exons, containing two initiating methionines and a predicted open reading frame (ORF) of 236/224 amino acids [13; 14], for which two associated SRAP proteins were detected shortly afterwards [13; 15; 16]. Interestingly, the existence of both coding and non-coding SRA transcripts seems to be regulated, at least in part, by the differential splicing of the first intron of SRA. Therefore, the regulation of alternative splicing on this type of molecule might regulate the balance between RNA and protein-coding functions and influence the overall effect of SRA expression on the regulation of gene expression and cell differentiation [6; 13-19]. In this respect, we recently established that while SRA ncRNA was an enhancer of MyoD transcriptional activity and myogenic differentiation, SRAP prevented this SRA RNA-dependant co-activation through interaction of SRAP with its RNA counterpart.

Such an example of a genetic locus producing both coding and non-coding RNA, depending primarily on an event of alternative splicing, is to our knowledge an isolated case. But it can be predicted that bifunctional RNA could be more widespread than expected and could apply to hundreds of mRNA that could function as functional RNA or, conversely as non-coding RNA that could hide a capacity to encode peptides, simply because these aspects have rarely been formally addressed [20; 21]. We propose here to take advantage of the massive amount and diversity of data generated by sequencing projects to perform a bioinformatical identification of mRNA with ORF disrupted by alternative splicing, that could potentially represent new bifunctional RNA for which experimental approaches may uncover a functional implication (Figure 2).

The number of introns in a genome seems to increase with organismal complexity since it greatly increased during evolution [START_REF] Koralewski | Evolution of exon-intron structure and alternative splicing[END_REF], from 4 introns in the whole genome of Giardia intestinalis to about 7.8 per gene in humans. With the number of coding genes estimated between 19,000 and 25,000, the human genome contains about 150,000 to 200,000 introns [1; 20]. A particular case in the general process of alternative splicing of introns leads to maintenance of certain introns. In vertebrates, this retention of introns affects predominantly short introns, i.e. shorter than 200 nucleotides in length [22]. Although this only accounts for about 3.1% of all reported alternative splicing events, hundreds of genes may be affected [START_REF] Koralewski | Evolution of exon-intron structure and alternative splicing[END_REF]. Whether this retention of introns produces functional and stable molecules, as reported in the case of SRA, remains to be explored.

As shown in Figure 3, the vast majority of the observed retention of introns (1962 out of 2241 reported events) fall within coding sequences (CDS), which only represent about half of the full mRNA length [START_REF] Galante | Detection and evaluation of intron retention events in the human transcriptome[END_REF], suggesting a non-random distribution of retained introns. Those retained at the extremities of untranslated regions (UTR), 140 events in 5'UTR and 139 and 3'UTR, might contribute to the stabilization (or destabilization) of the associated transcript or might offer target sites for miRNA and therefore participate in the regulation of mRNA degradation. The appearance of stop codon(s) in such regions would not interfere with the protein coding capacity of the transcripts. Indeed, in 620 cases, retention of an intron did not change the reading frame, but could potentially lead to the formation of additional protein domains of up to 66 amino acids since we selected introns shorter than 200 nucleotides. This type of alternative splicing has been known for decades to participate in the enrichment of the diversity of proteins produced from a single gene and potentially diversification of protein function. However, when introns are retained within the CDS, the appearance of a stop codon would contribute to the production of a truncated peptide (if inserted close to the 3'-end) or the absence of a protein product (if inserted downstream of the start codon). We found here that the translatability of about 318 genes might indeed be interrupted by the retention of an intron in the first third of the CDS. This type of event was typically disregarded owing to the absence of protein products associated with them, but in the light of recent data, they might also contribute to the diversification of the information carried by genes, by producing functional RNA.

During our data-mining for alternative splicing events described above and in the legend of Figure 2, we identified 318 potentially new bifunctional RNA. SRA, the first reported case of retention of an intron that influences the production of two distinct molecular entities with opposing functions in the same pathways, which served as the base for this study, was indeed found in this cluster of 318 transcripts, validating the potential and accuracy of the approach employed. One could argue that these alternatively spliced and untranslated mRNA are usually considered as defective RNA and thought to be directed to nonsense mediated decay (NMD) pathways to be degraded. However, the database we used contained entries exhibiting various types of alternative splicing events and the denoted 'retained intron' was based on an analysis of splicing graphs, conserved between human and mouse, and further confirmed by their presence in EST or cDNA database, indicating that they may well exist under physiological conditions [START_REF] Sugnet | Transcriptome and genome conservation of alternative splicing events in humans and mice[END_REF]. Of course, these potentially new bifunctional RNA await experimental validation to test their functionality at the RNA level.

Figure 1.

Influence of alternative splicing events on the transcriptional output of a gene. In addition to the classical enrichment of protein diversity, alternative splicing might also contribute to the diversification of the information carried by genes, by producing functional RNA instead of a protein product. For example, SRA RNA exists as coding and non-coding isoforms, through alternative splicing of the first intron, leading to the production of a protein-coding mRNA whereas its retention leads to the production of a non-protein-coding functional RNA (ncRNA), forming the basis for the concept of "bifunctional RNA". (ORF, Open Reading Frame).

Figure 2. Workflow for data mining of alternative splicing events. Datasets of 'Human

Introns', 'Alternative Events' and 'Genes' were retrieved from the UCSC table browser (http://genome.ucsc.edu), using "Genes and gene prediction", "Alt events" or "RefSeq Genes" tracks, respectively, from the hg19 assembly covering the whole human genome. Using Galaxy (http://main.g2.bx.psu.edu) and 'Operate on Genomic Intervals' tool, we have selected only introns shorter than 200 nucleotides and 100% covered in 'Alternative Events' datasets. Location of intron retention relative to the start and stop codons was obtained by genomic coordinate comparison (from 'Genes' dataset). ORF disruption was then assessed using a custom Perl script.

Figure 3 .

 3 Figure 3. Number of entries identified throughout the workflow described in Figure 2,

Acknowledgements F.H. was supported by "Association Française contre les Myopathies" and the "Association le Cancer du Sein, Parlons-en!". Work in C.F. lab is supported by research founding from grants from LNCC (Ligue Nationale Contre le Cancer).


Baptiste Bogard

Claire Francastel
email: claire.francastel@univ-paris-diderot.fr

Florent Hubé
email: florent.hube@univ-paris-diderot.fr

Dr Jin Li

Dr Junjie Xiao

A new method for the identification of thousands of circular RNAs

Keywords:

Identified in the early 1990s, mammalian circular RNAs have recently generated a lot of interest as a novel type of regulatory molecules involved in regulation of gene expression and unconventional therapeutic targets. In line with the need to develop and improve new methods for their identification and to study their role in molecular circuitries, the study by Panda et al. published in Nucleic Acids Research in July 2017 offers a new strategy for the systematic identification of circRNAs and revealed a special class of intron-derived circRNAs.

Circular RNAs (CircRNAs) have recently emerged as a novel class of abundant endogenous non-coding RNA (ncRNAs) with regulatory potential in mammalian cells. Owing to the difficulty to identify these RNAs through traditional methods dedicated to the analysis of linear RNAs, our knowledge of these intriguing RNA species has remained limited. Recent efforts to develop novel biochemical enrichment strategies coupled to deep sequencing allowed their systematic identification for comprehensive studies of their biogenesis and function. The manuscript by Panda et al. discussed herein provides an improvement to existing methods usually based on depletion in polyadenylated RNAs and resistance to linear-RNA exonucleases [START_REF] Panda | High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs[END_REF].

Discovery of circular RNAs

The existence of circular RNAs (circRNAs) was first reported in the 1970s in plant viroids and yeast mitochondria (2;3), and later on in higher eukaryotes [START_REF] Nigro | Scrambled exons[END_REF]. Very few new circRNAs were identified over the years including circRNAs from the Sry gene in mouse testis [START_REF] Capel | Circular transcripts of the testis-determining gene Sry in adult mouse testis[END_REF] or from the cytochrome P450 gene in rats [START_REF] Zaphiropoulos | Circular RNAs from transcripts of the rat cytochrome P450 2C24 gene: correlation with exon skipping[END_REF] where their levels are correlated with exon skipping. Despite the fact that circRNAs have been detected in human nuclear extracts [START_REF] Pasman | Exon circularization in mammalian nuclear extracts[END_REF], they have long been considered as byproducts of splicing or debranching of the lariat produced by the Debranching enzyme 1 (DBR1), at least over the last two decades [START_REF] Jeck | Detecting and characterizing circular RNAs[END_REF].

Circular RNAs: origin, biogenesis and nomenclature

In addition to being considered as "junk" RNA, circRNAs were not identified in traditional sequencing techniques because they cannot be separated on the criterion of size and they do not have, by definition, 5'-and 3'-ends nor poly(A) tails [START_REF] Jeck | Detecting and characterizing circular RNAs[END_REF]. With the advent of high throughput sequencing techniques and the development of dedicated bioinformatic analysis (for review see ( 9)), many new circRNAs have now been identified and further classified according to their origin and biogenesis. Since the nomenclature of circular RNAs can lead to some confusion, we first review the main referenced classes that are depicted on Figure 1. The first class is referred to as circular intronic RNAs (ciRNAs) that, as their name implies, originate from the circularization of intronic sequences. During the splicing reaction, the lariat that is formed may escape debranching by DBR1. The 3'-linear extremity is then trimmed by an exonuclease leading to the formation of a perfect circular RNA. A different class is designated as circular RNAs (circRNAs) that are covalently closed by a mechanism called backsplicing. In canonical linear splicing, the 5' donor splice site joins the 3' acceptor splice site to form a 5'-3' splice junction (consensus sequence AG/GT), combining exons in a sequential order. In contrast, backsplicing goes in the reverse direction. Thus, a donor splice site will react with an acceptor splice site located upstream, allowing the formation of a 3'-5' backsplice junction with the particular consensus sequence GT/AG. CircRNAs that retain introns between two or more exons are called Exon-Intron circRNAs (EIcircRNAs) as opposed to Intronic circRNAs (IcircRNAs) exclusively composed of intronic sequences or Exonic circRNAs (EcircRNAs) that contain only exons (1;4).

CircRNAs are between 100 nt and several kb in size, they originate from both coding and non-coding genes, and their levels are not correlated with expression of the host gene [START_REF] Cheng | A lariat-derived circular RNA is required for plant development in Arabidopsis[END_REF]. CircRNAs are also very stable in the cells with a half-life of more than 48 hours, although not in blood serum (< 15 sec) probably due to the presence of endonucleases.

EcircRNAs have been shown to adopt a cytoplasmic localization (4;8) although the export system remains unknown, in contrast to EIcircRNAs that are preferentially located in the nucleus [START_REF] Li | Exon-intron circular RNAs regulate transcription in the nucleus[END_REF]. The combination of these features, especially the lack of correlation between the expression of the host genes and the levels of circRNAs, strongly suggests that their release is actively regulated and operates physiological functions in the cells [START_REF] Jeck | Detecting and characterizing circular RNAs[END_REF].

Biological functions of circRNAs

Many studies have suggested that the exonic sequences carried by circRNAs contain target sequences for microRNAs (miRNAs) and could act as sponges or endogenous competitors for miRNAs [START_REF] Jeck | Detecting and characterizing circular RNAs[END_REF]. This is the case for the circRNA identified in Sry, which contains several binding sites for the mmu-miRNA-138. Overexpression of ectopic circRNA Sry leads to the reduction of the mmu-miRNA-138-mediated knockdown of a luciferase contruct containing miRNA-138 binding sites [START_REF] Hansen | Natural RNA circles function as efficient microRNA sponges[END_REF]. Similarly, the circWDR77, circular RNA produced from linear WDR77 transcripts, acts as a sponge for the hsa-miRNA-124 and prevents the knockdown of FGF-2 [START_REF] Cheng | A lariat-derived circular RNA is required for plant development in Arabidopsis[END_REF]. As previously mentioned, EIcirRNAs represent a particular class of circRNAs localized in the nucleus. They have been shown to regulate transcription in cis through their interaction with the RNA polymerase II, U1 nuclear RNA and the promoter of their host gene. In addition, knockdown of EIcircRNAs can cause a decrease in the mRNA levels of their host genes [START_REF] Li | Exon-intron circular RNAs regulate transcription in the nucleus[END_REF]. As it has already been shown that almost all RNAs interact with RNA binding proteins, it is very likely that circRNAs belong to large complexes called circRNPs [START_REF] Lee | Emerging roles for ribonucleoprotein modification and remodeling in controlling RNA fate[END_REF]. Indeed, circRNAs can exist in complex with argonaute (AGO) proteins [START_REF] Jeck | Detecting and characterizing circular RNAs[END_REF] or with the IGF2BP3 or Insulin-like Growth Factor 2 Binding Protein 3 [START_REF] Schneider | CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs[END_REF]. It has also been proposed that circRNAs could act as sponges for RBPs or as scaffolds to facilitate the interaction between several RBPs [START_REF] Jeck | Detecting and characterizing circular RNAs[END_REF].

CircRNAs were long considered as ncRNAs because they were not detected in polysomes (5;8;14). However, an unexpected function emerged along which some circRNAs have the ability to code for proteins, or at least peptides. Three publications, summarized by Schneider and Bindereif [START_REF] Schneider | Circular RNAs: Coding or noncoding?[END_REF], revealed the translation of circRNAs into small proteins through a cap-independent translation initiation, further confirmed by polysome fractionation and mass spectrometry experiments [START_REF] Legnini | Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis[END_REF][START_REF] Pamudurti | Translation of CircRNAs[END_REF][START_REF] Yang | Extensive translation of circular RNAs driven by N(6)-methyladenosine[END_REF]. Therefore circRNAs may possess the ability to act both as coding and non-coding RNAs, as it has already been reported in the case of the socalled bifunctional RNAs [START_REF] Ulveling | When one is better than two: RNA with dual functions[END_REF].

More generally, several circRNAs have been implicated in development, pluripotency, proliferation, differentiation or migration of normal and tumor cells [START_REF] Legnini | Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis[END_REF]. For example, overexpression of circPTK2 promotes proliferation and migration of bladder cancer cells consistent with its high levels found in these cancer cells [START_REF] Xu | Circular RNA hsa_circ_0003221 (circPTK2) promotes the proliferation and migration of bladder cancer cells[END_REF]. CircBIRC6 contributes to maintenance of pluripotency in human embryonic stem cells by "sponging" hsa-miRNA-34a and -145, which otherwise target pluripotency genes [START_REF] Yu | The circular RNA circBIRC6 participates in the molecular circuitry controlling human pluripotency[END_REF]. CircRNAWDR77, described above, facilitates proliferation and migration of vascular smooth muscle cells [START_REF] Cheng | A lariat-derived circular RNA is required for plant development in Arabidopsis[END_REF].

Given the many physiological functions that circRNAs may operate, and in order to understand their molecular functions, there is a burning need to proceed with their exhaustive identification. This will ultimately allow circRNAs to be used as innovative biomarkers and open up new therapeutic approaches in the treatment of cancer or other human diseases where RNA splicing, and hence the production of circRNAs, is defective.

A new method allows the identification of many new circRNAs

Since most of circular RNAs reported to date are the result of backsplice reactions ((22); circBase, http://www.circbase.org), biocomputational strategies developed for their identification were based on the detection of the backsplice signature GT/AG [START_REF] Jeck | Detecting and characterizing circular RNAs[END_REF]. In order to improve their identification experimentally, several studies have used the property of circRNAs to be resistant to RNase R, an exonuclease that specifically degrades linear RNAs.

Therefore, the use of libraries depleted in the majority RNAs (rRNAs and mRNAs) and treated with RNase R allowed the enrichment in circRNAs and thus eased their identification.

As a result of these strategies, hundreds of circRNAs were identified and characterized ((22); circBase, http://www.circbase.org).

However, and even if these approaches indeed allowed significant enrichment in circRNAs, a large proportion of linear RNAs escapes the digestion by RNase R. In the recent study by Panda et al. that we highlight here (Figure 2), an additional step has been added to overcome this issue and to remove the remaining RNase R-resistant linear RNAs [START_REF] Panda | High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs[END_REF]. The strategy was to perform a poly(A) tailing reaction on the remaining linear RNAs, i.e. with a free 3'-OH end, such as those that are processed in a posttranscriptional manner and inherently lack a poly(A) tail (miRNAs, snoRNAs, snRNAs...), or that escape digestion because of their highly structured folding. A poly(A) depletion is then perform on the modified RNA samples. This strategy, called "RNase R treatment followed by polyadenylation and poly(A) + RNA depletion" (RPAD, Figure 2), was used to isolate highly enriched circRNAs from total RNA (1). However, it should be noted that this approach also removes ciRNAs since they still have a 3'-OH extremity, until trimming of the lariat is complete, that is free for the re-polyadenylation procedure (Figure 1).

With this new strategy, the authors were able to identify a large number of circRNAs in human HeLa and murine C2C12 cells (38,651 and 17,341 respectively) and release their full-length sequences, Of these, 1,374 were identified as EcircRNAs generated from backsplicing of exons, including 783 already known EcircRNAs (57%), which validated their method. In addition, they also identified 591 new EcircRNAs (43%). Maybe more striking, they uncovered high numbers of yet unidentified IcircRNAs (Figure 1) (37,277 in human and 16,768 in mouse). The authors further experimentally validated some EcircRNAs and IcircRNA candidates by RT-PCR using divergent primers and confirmed the backsplice consensus signature by sequencing. In agreement with the idea that circRNAs may function as sponges for RBPs, the authors also identified several binding sites for RBPs in silico (8;14), supporting the implication of circRNAs in molecular circuiteries. Against all odds, because circRNAs are defined to originate from splicing reactions, some of the newly identified circRNAs mapped to intergenic regions. However, this is an interesting finding that may simply imply that the host transcript of these circRNAs has not yet been identified and annotated in the chosen cellular contexts.

As mentioned above, Panda et al. identified for the first time intronic circular RNAs whose biogenesis still remains unclear [START_REF] Panda | High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs[END_REF]. The biogenesis of IcircRNAs might be different form that of EcircRNAs, i.e. through backsplicing. However, they are not produced by canonical splicing like ciRNAs, which were eliminated from the analysis (see above). Despite their abundance revealed by this study, IcircRNAs may originate from poorly preserved splice junctions that cannot be used as a predictive signature to discover additional IcircRNAs.

Again, it is worth noting that IcircRNAs were 20-fold more abundant than EcircRNAs.

The most reasonable explanation comes from the genome composition; indeed, introns represent about half the human genome, whereas exons account for only 2% of it [START_REF] Hube | Mammalian introns: when the junk generates molecular diversity[END_REF].

Despite the difficulty of studying IcircRNAs as suggested by the authors -e.g. IcircRNAs do not share a well-defined consensus backsplice junction -and given their abundance in the cell, their characterization and mechanisms of action remains to be quickly clarified.

Conclusion

Panda et al. have developed a new method, called RPAD, to enrich circRNAs. With this strategy, they identified thousands of known and unknown circular RNAs. More importantly, they discovered a new class of abundant intronic circular RNAs. We are convinced that this new method is an important first step towards the characterization and the comprehension of the involvement of circRNAs in cells. Figure 1. Classification and nomenclature of circular RNAs according to their biogenesis pathways. (A) ciRNAs originate from splicing. Once the lariat is formed, an exonuclease trims the 3'-end to form a circular RNA. Since the branch point, through the 2'-OH of its adenosine, attacks the 5'-end donor site, the adenosine of the branch point will still conserve its 3'-OH extremity free after the trimming reaction. (B) circRNAs are generated by backsplicing. Backsplicing means that the 5' donor splice site of an exon can react with the 3' acceptor splice site of an upstream exon. EIcircRNAs are the result of the circularization of 2 or more exons with their corresponding introns (note that it is possible to have the circularization of an exon on itself). Thereafter the EIcircRNAs can remain intact or can be spliced to form EcircRNAs and an intronic lariat that can be further processed into a ciRNA.

Figures legend

IcircRNAs are generated by the circularization of an intron on itself through a mechanism that is still unclear. ciRNAs, circular intronic RNAs; circRNAs, circular RNAs; EcircRNAs, exonic circRNAs; IcircRNAs, Intronic circRNAs; EIcircRNAs, Exon-Intron circRNAs.

Figure 2 .

 2 Figure 2. Schematic representation of the RPAD method. RNA samples are first digested by RNase R (3'-exonuclease) in order to remove all linear RNAs. After the digestion, some linear RNAs remain intact because of their strong structured folding. A second step is therefore added which consists in the polyadenylation of the remaining linear RNAs, which are then depleted. In the last step, an rRNA depletion is performed to ensure a collection of circRNAs with high-purity. The RPAD sample is finally subjected to sequencing. The RPAD method reveals that all the identified Intronic circRNAs are a new class of circRNAs. circRNAs, circular RNAs; EcircRNAs, exonic circRNAs; IcircRNAs, Intronic circRNAs.

Acknowledgements

Funding: AFM, Association Française contre les Myopathies, Research Grant #20534; AFM PhD Fellowships #21363.

Conflicts of Interest

The authors have no conflicts of interest to declare.