Elisabetta Mileo

Marianne Ilbert

Alessandro Barducci

Patricia Bordes

Marie-Pierre Castanié-Cornet

Cyrille Garnier

Pierre Genevaux

Reynald Gillet

Pierre Goloubinoff

Françoise Ochsenbein

Gilbert Richarme

Chantal Iobbi-Nivol

Marie-Thérèse Giudici- Orticoni

Brigitte Gontero

Olivier Genest

Emerging fields in chaperone proteins: a French workshop

Keywords: Chaperone proteins, multifunctionality, plasticity, folding/misfolding, aggregation, structural dynamics

The "Bioénergétique et Ingénierie des Protéines (BIP)" laboratory, CNRS (France), organized its first French workshop on molecular chaperone proteins and protein folding in November 2017. The goal of this workshop was to gather scientists working in France on chaperone proteins and protein folding. This initiative was a great success with excellent talks and fruitful discussions. The highlights were on the description of unexpected functions and posttranslational regulation of known molecular chaperones (such as Hsp90, Hsp33, SecB, GroEL) and on state-of-the-art methods to tackle questions related to this theme, including Cryo-electron microscopy, Nuclear Magnetic Resonance (NMR), Electron Paramagnetic Resonance (EPR), simulation and modeling. We expect to organize a second workshop in two years that will include more scientists working in France in the chaperone field.

Introduction

The concept of molecular chaperones originates from the view that even though the structure (or absence of structure) of proteins is encoded by their primary amino acid sequence, sometimes they require "helper" proteins to reach their functional three-dimensional structure and avoid aggregation or degradation. The term of molecular chaperone was first coined to describe the function of a protein that assists the assembly of folded subunits into oligomeric protein complexes [START_REF] Laskey | Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA[END_REF], and later was extended to protein folding [START_REF] Ellis | Proteins as molecular chaperones[END_REF]. A consensus now emerges to define molecular chaperones as a diverse group of proteins that assist the folding/unfolding of other proteins and the formation/dissociation of macromolecular complexes without being a component of the final complex. Molecular chaperones are involved in a wide range of biological processes and play key roles in all living organisms.

They can be used or targeted for many medical, environmental or biotechnological applications.

At the end of November 2017, the Laboratory of "Bioénergétique et Ingénierie des Protéines" (BIP, Marseille) organized a workshop bringing together about fifty participants with guest speakers from France and one from Switzerland. Such an initiative had already been carried out in Europe, as for example in the United Kingdom (UK) and in the Netherlands. Meetings of this type are intended to improve communication within an emergent community and to increase collaborations and visibility at national and international levels.

In this report, we give an overview of the lectures (Figure 1). The first part focuses on the large panel of functions general chaperones may accomplish in various cellular processes under physiological as well as stressful conditions, showing an astonishing functional diversity. The two following sections concern chaperones that present particular features like intrinsic disorder and flexibility, and specific chaperones dedicated to a unique client. Here again, the diversity of the function of chaperones is illustrated. Finally, innovative techniques and tools to study or simulate chaperone behaviors that were exposed at the workshop are recorded in the last section of the report.

Breakthrough in known chaperones

General chaperones are proteins that maintain proteostasis in cells by preventing protein aggregation, helping in the native folding, activation and targeting of a large array of client proteins, in contrast to specific chaperones that are usually dedicated to a unique substrate.

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3 Some general chaperones require ATP for their function, whereas others are ATPindependent proteins. General chaperones have been extensively studied. Therefore, to continue making exciting breakthrough, original angles of research including unexplored model organisms and new chaperone discovery are needed.

2.1.

ATP and general chaperones

Protein homeostasis in cells depends on energy-consuming processes. For instance, de novo protein synthesis requires ATP hydrolysis for peptide bond formation, and controlled degradation by the chaperone-gated proteases requires ATP hydrolysis to unfold target proteins and render their peptide bonds accessible to hydrolysis. During and following translation, different classes of molecular chaperones require ATP hydrolysis to control the conformational state of proteins and favor their folding into their active conformation.

Furthermore, ATP-fueled unfolding chaperones, such as Hsp70, Hsp110 and Hsp104 can dynamically solubilize and reactivate stable protein aggregates that are formed under certain stress.

Pierre Goloubinoff (keynote lecture, Université de Lausanne, Switzerland) explained how he and his colleagues show, using various biochemical assays and physical modelling, that the highly conserved chaperones Hsp60 and Hsp70 by acting as ATP-powered unfolding enzymes, can inject the energy liberated by ATP-hydrolysis to destabilize misfolded polypeptides. By doing so iteratively at each ATP-cycle, these chaperones can artificially repopulate and transiently maintain the native state population of their substrate proteins, even under conditions that according to equilibrium thermodynamics, disfavor the native state. By acting as catalytic polypeptide unfolding nano-machines that specifically target misfolded and alternatively-folded protein substrates, chaperones may use the energy of ATP hydrolysis to reduce the cellular concentration of harmful non-functional protein aggregates and convert them into functional proteins [START_REF] Goloubinoff | Chaperones convert the energy from ATP into the nonequilibrium stabilization of native proteins[END_REF].

Recent quantitative proteomic studies show that in metazoans, the main ATPase chaperones Hsp90, Hsp70, Hsp60, correspond to 3-4 % of the total proteins of healthy terminally differentiated adult cells. Chaperones may be dramatically more abundant in healthy undifferentiated embryonic cells and in cancer cells, and much less abundant in aging and diseased tissues. For Pierre Goloubinoff, the future of chaperone research lies in the understanding of the fine-tune differences in chaperone and co-chaperone amounts and ratios in healthy embryonic cells and in cancer cells, both massively overexpressing chaperones, and

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diseased neuronal cells in degenerative diseases, like Parkinson and Alzheimer, that dramatically under-express chaperones and fail to repair or degrade toxic misfolded proteins.

The Hsp90 chaperone

The 90-kDalton heat-shock chaperone protein (Hsp90) has been well-studied in eukaryotes [START_REF] Schopf | The HSP90 chaperone machinery[END_REF]. This three-domain protein is essential and allows in an ATP-dependent reaction the folding and activation of more than 300 client proteins with the assistance of about 20 cochaperones and the Hsp70 chaperone system. Among these clients, Hsp90 participates in the folding of oncoproteins in cancer cells. Therefore, many studies focused on Hsp90 inhibitor development to cure cancers. In addition, Hsp90 has been shown to regulate several amyloid proteins; it is thus considered as new target to treat amyloid diseases [START_REF] Blair | Targeting Hsp90 and its co-chaperones to treat Alzheimer's disease[END_REF]. For instance, Hsp90 interacts with the amyloid protein tau. The interaction of Hsp90 with tau has been suggested

to occur through tau aggregation-prone regions, including the VQIVYK motif. This hexapeptide Ac-VQIVYK-NH 2 is responsible of tau amyloid character and is widely used as a model to study in vitro amyloid formation mechanisms; it is known to self-aggregate and to form amyloid fibrils [START_REF] Bergen | Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure[END_REF]. Although it has been suggested that Hsp90 interacts with this hexapeptide, the role of the chaperone on the fibrillation process was still unknown. During his conference, Cyrille Garnier (MMDN Inserm, Montpellier) showed that Hsp90 (purified from pig brain) modulates in vitro the assembly and disassembly of amyloids structures formed from tau Ac-VQIVYK-NH 2 hexapeptide. Hsp90 inhibited polymerization of the hexapeptide maintaining it in its soluble form. At high hexapeptide concentration when Hsp90

was "overbooked", polymerization occurred; Hsp90 interacted with mature fibrils preventing their depolymerization. This important function could limit the spread of toxic amyloid species. A model explaining the dual effect of Hsp90 on the Ac-VQIVYK-NH 2 amyloid fibrillation process has been proposed [START_REF] Schirmer | Hsp90 directly interacts, in vitro, with amyloid structures and modulates their assembly and disassembly[END_REF]. Overall, this study gives insight into the roles of chaperone proteins in amyloid turnover.

In contrast to the great number of studies on eukaryotic Hsp90, Hsp90 from bacteria has been less investigated [START_REF] Buchner | Bacterial Hsp90--desperately seeking clients[END_REF]. In Escherichia coli, Hsp90 is dispensable, few client proteins are known, and no co-chaperone has been described so far. Therefore, studying new bacterial model is crucial to characterize better prokaryotic Hsp90. Olivier Genest (CNRS, Marseille)

presented experiments on the Hsp90 chaperone in Shewanella oneidensis (called Hsp90 So ), an aquatic gamma proteobacterium that adapts to stressful conditions. He showed that Hsp90 So is essential in S. oneidensis under heat stress, and that the ATPase activity of Hsp90 So is

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required for this function [START_REF] Honoré | Hsp90 Is Essential under Heat Stress in the Bacterium Shewanella oneidensis[END_REF]. To identify Hsp90 So clients, a genetic screen using the growth phenotype at high temperature of a strain lacking Hsp90 So was designed, and it allowed the identification of a protein involved in tRNA maturation, TilS. In the absence of Hsp90 So and at sub-lethal temperature, a significant decrease in TilS activity was measured and TilS was degraded. Therefore, this study links the major role of Hsp90 in S. oneidensis under stress conditions with an essential client protein, and opens up new avenues to understand better the role and mechanism of action of the Hsp90 chaperone in bacteria.

SecB-like chaperone

In Proteobacteria, the SecB chaperone assists protein export and participates in the folding of certain cytosolic proteins [START_REF] Castanié-Cornet | Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane[END_REF]. Pierre Genevaux (CNRS, Toulouse) talked about the specialization of a SecB like chaperone in Mycobacterium tuberculosis and its link with toxinantitoxin (TA) systems. Bacterial type II toxin-antitoxin systems are small genetic modules typically composed of a toxin and a more labile cognate antitoxin, which binds and inhibits the toxin. In response to certain stress the antitoxin is rapidly degraded by proteases and the free active toxin generally targets essential cellular processes, leading to a reversible growth inhibition known to facilitate bacterial persistence to drugs, virulence and survival in response to environmental insults. Tripartite TAC (Toxin-Antitoxin-Chaperone) modules are atypical TA systems composed of a conserved two-component TA that is specifically controlled by a molecular chaperone related to the canonical SecB chaperone. In this case, the SecB-like chaperone interacts with the antitoxin and protects it from both aggregation and degradation, and is thus strictly required for neutralization of the toxin by the antitoxin. In his talk, Pierre Genevaux presented some aspects of the molecular mechanism by which TA systems become chaperone-addicted [START_REF] Bordes | Chaperone addiction of toxin-antitoxin systems[END_REF], the specialization events that redirect a generic export chaperone towards the control of TA systems [START_REF] Sala | Directed evolution of SecB chaperones toward toxinantitoxin systems[END_REF] and some structural insights into the complex formed between the chaperone and the antitoxin. In addition, some part of the talk also concerned the direct evolution of the GroEL chaperone toward specific protein aggregates in E. coli

(Castanié-Cornet et al., in preparation).

Protein reparation

Beside their role in protein folding, some chaperones allow the reparation of other proteins.

Gilbert Richarme (keynote lecture, Université Paris Descartes-Sorbonne) reported that DJ-1/Park7, whose deficiency results in early onset parkinsonism, and previously described as a chaperone, repairs proteins from glycation. Glycation is a non-enzymatic covalent reaction

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discovered by Maillard in 1912, between reactive carbonyls (glucose and glyoxals (R-CO-CHO)), and amino acids (cysteine, arginine and lysine) or nucleotides (guanine), which results in the formation of Maillard adducts, followed by their maturation into the so-called advanced glycation end products (AGEs). Glycation is responsible for protein and nucleic acid inactivation, and is involved in aging, cancer, atherosclerosis, cataracts, neurovegetative, renal and autoimmune diseases. DJ-1 family proteins, named the "Maillard deglycases", are expressed in all organisms, and repair glycation by glyoxals, which represents about 70% of glycation damage [START_REF] Richarme | Parkinsonism-associated protein DJ-1/Park7 is a major protein deglycase that repairs methylglyoxal-and glyoxal-glycated cysteine, arginine, and lysine residues[END_REF]. Maillard deglycases also repair nucleic acids, especially the primary amino group of guanine, in a similar manner as they repair the lysine amino group [START_REF] Richarme | Guanine glycation repair by DJ-1/Park7 and its bacterial homologs[END_REF].

Deglycases from thermophilic organisms may also be used to prevent AGEs and acrylamide formation during food processing sterilization and storage [START_REF] Richarme | DJ-1 family Maillard deglycases prevent acrylamide formation[END_REF].

Figure 1: Overview of the topics discussed during the meeting. The scheme showing the disordered and ordered protein structures has been reprinted from reference [START_REF] Bardwell | Conditional disorder in chaperone action[END_REF] (Figure 1, Copyright (2018), with permission from Elsevier). In the panel "Tools to study chaperone structural dynamics", the NMR spectrum has been modified from reference [START_REF] Chang | Identification of the Calmodulin-Binding Domains of Fas Death Receptor[END_REF], the Molecular Dynamics Simulations image has been reprinted from reference [START_REF] Malinverni | Modeling Hsp70/Hsp40 interaction by multi-scale molecular simulations and coevolutionary sequence analysis[END_REF], the Cryo-EM maps have been reprinted from reference [START_REF] Macé | The structure of an elongation factor G-ribosome complex captured in the absence of inhibitors[END_REF].

Plasticity in chaperone proteins

Some proteins challenge the classical structure-function paradigm because they do not have a well-defined three-dimensional structure. These proteins were originally termed intrinsically unstructured proteins (IUPs) and nowadays are called intrinsically disordered proteins (IDPs).

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8

Despite their lack of a well-defined three-dimensional structure, many proteins containing disordered regions/domains (IDRs) or completely disordered proteins are functional. These proteins carry out basic functions such as regulation of metabolic pathways, transcription, translation, cellular signal transduction; they can act as scavengers of toxic molecules and they play a key role in the assembly of multi-protein complexes. Moreover, recently they were shown to have other functional capacity as they can act as chaperones assisting the folding of other proteins and preventing their aggregation. For some proteins, a conformational switch between disordered and folded states or reciprocally, is redoxcontrolled by the formation/breakage of disulfide bonds (oxidation/reduction). These proteins belong to the conditionally disordered protein (CDP) family. Hsp33 and CP12 are two CDPs that both have chaperone activity [START_REF] Reichmann | The roles of conditional disorder in redox proteins[END_REF].

Hsp33 chaperone

Marianne Ilbert (CNRS, Marseille) described the molecular mechanism of Hsp33, a molecular chaperone specifically activated by oxidative stress conditions that lead to protein unfolding. Exposure to peroxide stress at elevated temperature activates Hsp33. To respond to this dual stress, Hsp33 has developed a very ingenious system with (i) a C-terminal redox switch domain which detects oxidizing conditions by disulfide bond formation and (ii) an adjacent metastable linker region which responds to unfolding conditions [START_REF] Ilbert | The redox-switch domain of Hsp33 functions as dual stress sensor[END_REF]. Hsp33 is then turned into an active chaperone that protects the cells against oxidative protein aggregation.

Use of a faster oxidative agent, hypochlorous acid (HOCl) produced by the immune system in response to bacterial attacks, can activate the chaperone function of Hsp33 without the requirement of elevated temperature, underlying an even more interesting mode of regulation [START_REF] Winter | Bleach activates a redox-regulated chaperone by oxidative protein unfolding[END_REF].

CP12 protein

Brigitte Gontero (CNRS, Marseille) presented a small chloroplast protein called CP12 found in most photosynthetic organisms [START_REF] Gontero | An intrinsically disordered protein, CP12: jack of all trades and master of the Calvin cycle[END_REF]. In the green alga, Chlamydomonas reinhardtii, CP12

is constituted of about 80 amino acids and has two pairs of cysteine residues that under oxidizing conditions can form two disulfide bridges, one at the N-terminus and one at the Cterminus. Oxidized CP12 is present under dark conditions, and plays a vital role in the assembly of a complex involving phosphoribulokinase (PRK) and glyceraldehyde-3phosphate dehydrogenase (GAPDH), two enzymes belonging to the Calvin cycle responsible for CO 2 assimilation. CP12 is required for the ternary complex assembly [START_REF] Graciet | The small protein CP12: a protein linker for supramolecular complex assembly[END_REF]. Its release from

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9 the complex is triggered by reducing conditions corresponding to light. Under these conditions, the ternary complex is dissociated, the released enzymes are fully active and reduced CP12 is fully disordered and highly flexible. CP12, besides its role in the assembly of the supra-molecular complex, can prevent the in vitro thermal inactivation and aggregation of GAPDH and as such has a chaperone activity [START_REF] Erales | CP12 from Chlamydomonas reinhardtii, a permanent specific "chaperone-like" protein of glyceraldehyde-3-phosphate dehydrogenase[END_REF].

Specific chaperones

During the workshop, a session was dedicated to specific chaperones. In contrast to a general chaperone that acts on a broad panel of client proteins, a specific chaperone is dedicated to a targeted client. During this workshop, two families of specific chaperones have been highlighted. One is dedicated to histones during the assembly process of chromatin, and the second is involved in the maturation of bacterial respiratory molybdoenzymes. These two examples show the large impact that specific chaperones possess in various cellular processes just as do general chaperones.

Histone chaperones

Chromatin assembly and disassembly processes involve a number of chaperoning factors playing crucial roles in the maintenance of the genome. Factors that assemble and remodel chromatin are fascinating machineries that not only act in the mechanical packaging of DNA but are also involved in the tight regulation of DNA accessibility in processes such as transcription, replication and repair. Francoise Ochsenbein (CEA, Gif-sur-Yvette) focused her talk on Asf1 (Anti-silencing function 1) a highly conserved histone chaperone that binds and assembles histones H3-H4 onto the DNA, and is required for cell proliferation, cell cycle progression, resistance to genotoxic stresses and the establishment of specific histone epigenetic marks [START_REF] Mousson | The histone chaperone Asf1 at the crossroads of chromatin and DNA checkpoint pathways[END_REF]. During her talk, Francoise Ochsenbein exposed an innovative peptidic development on the interaction between Asf1 chaperone and H3-H4. This high-affinity peptide competes with Asf1 binding in vitro, and binds Asf1 in human cells. On cell cultures, the inhibitory peptide was found to be toxic for U2OS cancer cells impeding proliferation by slowing down the cell cycle progression but remained sensitive to proteolysis. Since a recent study showed a striking correlation between the severity of breast cancers and the level of Asf1 expression, this phenotype highlights the specificity of the peptide action and opens major perspectives in chaperone targeted therapies.

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Chaperones of metalloenzymes

Bacterial metalloenzymes and especially molybdoenzymes are fully involved in environmental purposes since they have crucial functions in respiration, detoxification processes and are part of various geochemical cycles. In bacteria, most of the molybdoenzymes require specific chaperones for their stability and maturation (i.e. insertion of a complex molybdenum cofactor) [START_REF] Lemaire | Chaperones in maturation of molybdoenzymes: Why specific is better than general?[END_REF]. The knowledge acquired on both the function and the mechanism of these dedicated auxiliary proteins is of great applied interest. The molybdoenzyme TorA and its mate chaperone TorD have been studied in E. coli and S.

oneidensis in particular by Marianne Ilbert, Olivier Genest, and Chantal Iobbi-Nivol (CNRS, Marseille). These studies allowed: (i) to define a family of specific chaperones for molybdoenzymes, the TorD like family; (ii) to demonstrate that the specific chaperone TorD prevents the degradation of the non-mature form of TorA by the Lon protease [START_REF] Redelberger | Quality control of a molybdoenzyme by the Lon protease[END_REF]; (iii) to understand the mechanism of insertion of the molybdenum cofactor into TorA [START_REF] Leimkühler | Bacterial molybdoenzymes: old enzymes for new purposes[END_REF].

Other studies performed on the membranous nitrate reductase NarGHI showed that its specific chaperone NarJ is also involved in the assembly of the three subunits and the anchoring of the enzymatic complex to the membrane. This chaperone protein has been the focus of another talk (see part 5). Altogether, studies performed on the various members of the TorD-like family highlight the wide panel of tasks undertaken by these specific chaperones and that they share a highly conserved 3D-structure although they present a low level of amino acid sequence homology [START_REF] Leimkühler | Bacterial molybdoenzymes: old enzymes for new purposes[END_REF].

Experimental challenges and biophysical tools for studying chaperone structural dynamics and their interactions

Protein structural flexibility and dynamics play a central role in biological functions. Indeed, there is an intimate relation between dynamics and molecular function. Structural transitions are involved in many key biological processes and play an important role in molecular recognition processes, catalysis, ligand binding, assembly of complex structures and disorderto-order transitions, as already mentioned for CP12 and Hsp33 (see part 3).

Chaperones are a fascinating group of proteins that exhibit different degrees of structural flexibility and are able to interact with different partners or different regions of the same partner thereby modulating their properties. Understanding chaperone dynamics and their interaction with other biomacromolecules is a key objective to decipher how they function

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and how their dynamics is related to their biological activity. However, due to the intrinsic dynamics and to the high structural fluctuations of chaperones, this kind of studies is currently difficult. During the workshop, several talks focused on the importance of biophysical approaches and on emerging experimental techniques in studying chaperone dynamics.

Modeling

Alessandro Barducci (INSERM, Montpellier) showed how the combination of analytical models, multi-scale molecular simulations and statistical analysis of protein sequences is a powerful approach to shed light on the complex functional cycle of Hsp70 chaperones. The ubiquitous Hsp70s are highly versatile, ATP-dependent molecular machines in the chaperone toolbox that assist a plethora of cellular processes including co-translational folding, refolding of misfolded and aggregated proteins, protein translocation, and protein degradation.

Particularly, A. Barducci explained how rate models based on biochemical rates can be used to quantitatively investigate the non-equilibrium cycling of Hsp70 chaperones, which take advantage of the energy released by ATP-hydrolysis to achieve ultra-affinity for the client proteins [START_REF] De Los Rios | Hsp70 chaperones are non-equilibrium machines that achieve ultraaffinity by energy consumption[END_REF]. Furthermore, it was discussed how multi-scale molecular simulations can be profitably combined with co-evolutionary sequence analysis to characterize large-scale conformational dynamics and homo-dimerization in Hsp70 [START_REF] Malinverni | Large-Scale Conformational Transitions and Dimerization Are Encoded in the Amino-Acid Sequences of Hsp70 Chaperones[END_REF], as well as to elucidate key structural features of the transient complex formed by this chaperone with essential cochaperones, such as J-domain proteins [START_REF] Malinverni | Modeling Hsp70/Hsp40 interaction by multi-scale molecular simulations and coevolutionary sequence analysis[END_REF].

Cryo-electron microscopy

In bacteria, trans-translation is the primary quality control mechanism for rescuing bacterial ribosomes arrested during translation. This key process is universally conserved and plays a critical role in the viability and virulence of many pathogens. Taking into account the necessity for bacterial survival to avoid ribosome stalling and the absence of trans-translation in eukaryotes, this process is a promising target for novel antibiotics or for increasing the activity of currently used protein synthesis inhibitors. Reynald Gillet (Université de Rennes) presented recent results obtained on structural aspects of trans-translation using cryo-electron microscopy, addressing the movements of the complex between transfer-messenger RNA (tmRNA) and SmpB protein (tmRNA-SmpB) through a stalled ribosome, and more particularly the conformational changes needed to transit from one ribosomal site to another [START_REF] Giudice | Trans-translation exposed: understanding the structures and functions of tmRNA-SmpB[END_REF]. The data obtained from this study were used to develop an in vivo double fluorescent

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reporter system for simultaneous quantification of trans-translation and associated proteolysis activities in bacteria [START_REF] Macé | A Genetic Tool to Quantify trans-Translation Activity in Vivo[END_REF]. This assay can be used to discover potent inhibitors of transtranslation in vivo.

Magnetic Resonance Studies

Françoise Ochsenbein (CEA, Gif-sur-Yvette), whose work has been already introduced (see part 4), showed the importance of integrating NMR spectroscopy, X-ray crystallography and modeling methods to characterize the structure of protein complexes. In particular, she highlighted how the characterization and the understanding of the interaction network between Asf1 histone chaperone and histones H3-H4 was possible thanks to this integrative biophysical approach [START_REF] Richet | Structural insight into how the human helicase subunit MCM2 may act as a histone chaperone together with ASF1 at the replication fork[END_REF][START_REF] Jiao | Surprising complexity of the Asf1 histone chaperone-Rad53 kinase interaction[END_REF].

Concerning the "emerging experimental techniques", the study of biomolecules in living cells appears as the most reliable approach to investigate their structural properties and dynamic features. The macromolecular crowding and the stickiness of the cytoplasm, non-specific weak protein-protein interactions, as well as a myriad of potential interactors constitute parameters that can perturb protein structure and dynamics. Over the last years, several studies have highlighted the importance of exploring biomolecules directly in their intracellular environment [START_REF] Theillet | Physicochemical properties of cells and their effects on intrinsically disordered proteins (IDPs)[END_REF]. However, a number of methodological and spectroscopic challenges still need to be addressed to make this approach broadly applicable to NMR, EPR and fluorescence, for instance. Elisabetta Mileo (CNRS, Marseille) presented how EPR spectroscopy can be used to investigate chaperones in actions directly inside cells [START_REF] Karthikeyan | A Bioresistant Nitroxide Spin Label for In-Cell EPR Spectroscopy: In Vitro and In Oocytes Protein Structural Dynamics Studies[END_REF]. In

Outlook

This workshop demonstrated the different areas of biology and the growing number of technologies that the chaperone field now involves. The different talks presented at this conference highlighted the complexity hidden within these proteins and their variable functions. It is clear that emerging tools applied to different model organisms are necessary to understand better the ubiquitous role of chaperones.

There are still many outstanding questions in this field and we still have lots of work to do before solving all the mysteries of chaperones. Since this event was highly successful (Figure 2), we are looking forward to organize a second event. We are aware that there are many other scientists working in France in the field of chaperone proteins who were not present at this first workshop. Therefore, the next challenge for the second edition will be to gather new people to increase the French chaperone community.

  particular, Site-Directed Spin Labeling (SDSL)-EPR including Double Electron-Electron Resonance (DEER) techniques were used to get information on the structure and conformational dynamics of the NarJ chaperone[START_REF] Lorenzi | Conformational selection underlies recognition of a molybdoenzyme by its dedicated chaperone[END_REF], dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein in E. coli. Thanks to the development of an efficient method to deliver the paramagnetic-labeled-NarJ inside E. coli cells, E. Mileo and coworkers succeeded in performing intracellular EPR experiments at room temperature and at endogenous concentration (30-40 µM), and they were able to follow the EPR signal in cells for more than 2 hours. These exciting results open up the perspective of achieving high resolution EPR studies of other chaperone proteins and their complexes in their native environment. M A N U S C R I P T A C C E P T E D

Figure 2 :

 2 Figure 2: Group picture of the first French workshop on chaperone proteins and protein folding.

Acknowledgements

We would like to thank the BIP laboratory for funding and support, Laure Azzopardi for her precious assistance in the organization of the workshop, all participants, and of course the members of our respective groups.

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Highlights

• Meeting report for the First French Workshop on Chaperone Proteins, France, 2017.

• The meeting gathered scientists working in France on chaperone proteins.

• Themes were: breakthrough in chaperones, plasticity, specific chaperones, biophysical tools.


Fan Zhang

Arnaud Erriguible
email: erriguible@enscbp.fr

Théo Gavoille

Mike T Timko

Samuel Marre
email: samuel.marre@icmcb.cnrs.fr

Inertia-Driven Jetting Regimes in Microfluidic Coflows

Microfluidics have been used extensively for study of flows of immiscible fluids, with specific focus on the effects of interfacial forces on flow behavior. In comparison, inertia-driven flow of confined coflowing fluids has received scant attention at the microscale, despite the fact that the effects of microscale confinement are expected to influence inertia-driven flow behavior as observed in free jets. Herein, we report three distinct modes for break-up of co-flowing, confined, microscale jets: the conventional Rayleigh mode and two additional inertia-driven modes occurring at higher Reynolds numbers flows, namely a sinuous wave breakup and an atomization-like mode. Each of the three modes is differentiated by a characteristic droplet size, size distribution, and dependence of the jet length as a function of the external fluid velocity (vext). A unified phase diagram is proposed to categorize the jet breakup mechanisms and their transitions using as a scaling factor the ratio of the jet inertial forces to the sum of the viscous and interfacial forces for both the inner and outer fluids. These results provide fundamental new insight into the flow behavior of microscale-confined co-flowing jets.

Droplets and jets are of primary importance for applications such as generation of emulsions with at submicron scale objects, sprays, and other multiphase flows, all of which are involved in a wide variety of chemical and industrial processes. Multiphase flows have been especially useful to the microfluidics community since dripping and jetting can be used to generate nearly monodisperse droplets that act as nanoliter reactors in series to ensure precise control of residence time and residence time distributions, with enhancing mixing [START_REF] Seemann | Droplet based microfluidics[END_REF]. Such approaches have been extensively used for microfluidics applications including foams generation [START_REF] Shah | Designer emulsions using microfluidics[END_REF][START_REF] Chokkalingam | Selfsynchronizing pairwise production of monodisperse droplets by microfluidic step emulsification[END_REF][START_REF] Hashimoto | Synthesis of Composite Emulsions and Complex Foams with the use of Microfluidic Flow-Focusing Devices[END_REF], dropletsbased microfluidic [START_REF] The | Droplet microfluidics[END_REF], jet stabilization [START_REF] Utada | Dripping, Jetting, Drops, and Wetting: The Magic of Microfluidics[END_REF][START_REF] Keenan | Microfluidic "jets" for generating steady-state gradients of soluble molecules on open surfaces[END_REF], organic and inorganic micro and nanostructures synthesis [START_REF] Marre | Synthesis of micro and nanostructures in microfluidic systems[END_REF], and chemical reactions [START_REF] Song | Reactions in droplets in microfluidic channels[END_REF][START_REF] Elvira | The past, present and potential for microfluidic reactor technology in chemical synthesis[END_REF].

Jets are metastable hydrodynamic structures, which eventually break into droplets. Jet breakup is a wellknown behavior, which occurs via various mechanisms, depending on the properties (velocity, density, viscosity, surface tension, etc.) of the fluid forming the jets and of the outer fluid [START_REF] Rayleigh | On the instability of jets[END_REF]. Coflow geometries have been considered extensively to study the dripping-to-jetting transition in confined geometries, both for liquid-liquid and liquid-gas coflows [START_REF] Guillot | Stability of a Jet in Confined Pressure-Driven Biphasic Flows at Low Reynolds Numbers[END_REF][START_REF] Utada | Dripping to Jetting Transitions in Coflowing Liquid Streams[END_REF][START_REF] Mak | The dripping-to-jetting transition in a co-axial flow of aqueous two-phase systems with low interfacial tension[END_REF][START_REF] Zhao | Two-phase microfluidic flows[END_REF]. This transition depends on the propagation of an absolute instability originating from a growing disturbance downstream, itself arising from the Rayleigh-Plateau instability [START_REF] Utada | Absolute Instability of a Liquid Jet in a Coflowing Stream[END_REF].

Several works report detailed comprehensive modeling of the dripping-to-jetting transition observed in liquidliquid and liquid-gas microsystems [START_REF] Utada | Dripping to Jetting Transitions in Coflowing Liquid Streams[END_REF][START_REF] Herrada | Liquid flow focused by a gas: Jetting, dripping, and recirculation[END_REF][START_REF] Cordero | Quantitative analysis of the dripping and jetting regimes in co-flowing capillary jets[END_REF][START_REF] Haase | Characterisation of gas-liquid two-phase flow in minichannels with co-flowing fluid injection inside the channel, part I: unified mapping of flow regimes[END_REF] for low Reynolds numbers flows (Re ≡ (vρd) /η), typically Re < 10,where ρ is the fluid density, v is the fluid velocity, d is the flow characteristic length and η is the dynamic viscosity. In such cases, the dripping and Rayleigh jetting regimes and the transitional boundaries between them can be organized using a map of the inner fluid Weber number W e in ≡ v 2 int ρ int d /σ , where σ is the interfacial tension between the two immiscible fluids, versus outer fluid Capillary number (Ca ext ≡ (v ext η ext ) /σ), as previously described [START_REF] Utada | Dripping to Jetting Transitions in Coflowing Liquid Streams[END_REF][START_REF] Mak | The dripping-to-jetting transition in a co-axial flow of aqueous two-phase systems with low interfacial tension[END_REF].

Aside from the Rayleigh regime observed for confined flows, jets exhibit other breakup modes that have been investigated for jetting into quiescent outer fluids [START_REF] Birouk | Liquid jet breakup in a quiescent atmosphere: a review[END_REF]. Four distinct jetting regimes have been identified and reported so far [START_REF] Reitz | Mechanisms of breakup of round liquid jets[END_REF] including the Rayleigh mode, the first and second wind induced breakup modes, and the atomization regime [START_REF] Lin | Drop and spray formation from a liquid jet[END_REF][START_REF] Erriguible | Numerical investigations of liquid jet breakup in pressurized carbon dioxide: Conditions of two-phase flow in Supercritical Antisolvent Process[END_REF]. These regimes and the transitions between them depend on several factors such as inertia, interfacial tension, and viscosity ratio of the fluids [START_REF] Lefebvre | Atomization and Sprays[END_REF].

Even though inertia-driven jet breakup regimes occurring at larger Reynolds numbers (Re > 100) have been identified and detailed in several experimental works of unconfined jets, the case of microscale confined jetting has received scant attention. This is primarily due to the technical difficulties encountered when studying the high flowrates required to reach high Re regimes and the resulting high pressure drops, which complicate experimental investigation.

Studies using high pressure microsystems can access high Re regimes consisting of gas-liquid, liquid-liquid, and even supercritical fluid (SCF)-liquid components. Recent publications have demonstrated the use of microfluidic systems at high pressures [START_REF] Marre | Supercritical microfluidics: Opportunities in flow-through chemistry and materials science[END_REF][START_REF] Ogden | Fluid behavior of supercritical carbon dioxide with water in a double-Y-channel microfluidic chip[END_REF][START_REF] Qin | Highly pressurized partially miscible liquid-liquid flow in a micro-T-junction. I. Experimental observations[END_REF]. SCFs are of particular interest as working fluids, as they combine liquid-like densities and gas-like viscosities [START_REF] Loppinet-Serani | Current and foreseeable applications of supercritical water for energy and the environment[END_REF]. Moreover, SCFs properties can be adjusted with minor changes of pressure and/or temperature, permitting study of inertial-viscous regimes that might otherwise be difficult to access experimentally. Accordingly, the use of highpressure microsystems and SCFs permits the study of new conditions of flow fragmentation that have not been investigated previously. Of particular interest are inertiadriven regimes, where inertial forces F iner = ρv 2 d 2 can overcome viscous (F v = ηvd) and interfacial (F IT F = σd) forces that typically dominate at the microscale. Previous work has exploited the experimental flexibility afforded by SCFs, and dripping to jetting transition has already been reported for a micro-confined liquid/SCFs co-current flow [START_REF] Marre | Dripping to jetting transitions observed from supercritical fluid in liquid microcoflows[END_REF][START_REF] Guillaument | Numerical simulation of dripping and jetting in supercritical fluids/liquid micro coflows[END_REF]. In this letter, we identify three jetting modes observed for co-flows of either dense CO 2 (liquid or supercritical) or liquid pentane and water in a capillary microreactor. Each mode was identified by distinctive droplet sizes, size distributions, and the evolution of the jet length and shape. These are the first reported observations of such modes under microscale confined conditions. were used to feed CO 2 or pentane and water at constant flow rates, while a third pump was used in constant pressure mode as a back pressure regulator to maintain constant the outlet pressure at p = 10 M P a for the CO 2water system, i.e. above the critical pressure of CO 2 (p c(CO2) = 7.38 M P a) or at p = 0.1 M P a for the pentane -water system. The capillary assembly was placed in a temperature-controlled bath (20 < T (°C) < 50) and the coflow was monitored using a high speed CCD camera (Phantom Miro 340, Vision Research, Inc.) mounted on a binocular microscope. Image resolution was ∼ 1.5 µm /pixel, which allowed resolution of features larger than about 5 µm. Jet lengths, droplets sizes, and size distributions were extracted from still images using the ImageJ software.

Actual fluid velocities inside the microchannel (v int , v ext ) were estimated from the pump flow rates and accounting for the temperature dependence of density:

v i = Q i(pump) Si × ρ i(pump) ρ i(bath)
, where S i is the internal cross section area out of which the fluid i is passing, Q i(pump) is the pump volumetric flowrate, while ρ i(pump) and ρ i(bath) Table I summarizes relevant physical properties and dimensionless numbers used in this work.

The conventional strategy to classify the jet break up regimes is to monitor the evolution of the length of the coherent portion of the jet, the mean droplet size and the droplet size distribution as functions of the external fluid velocity (v ext ). In a typical case, four flow regimes can be distinguished (Fig. 2).

Dripping. At low flow velocities, the classical dripping regime is observed, displaying an absence of jets with generation of large, monodisperse droplets at the tip of the inner capillary (Fig. 2a).

Rayleigh Jetting. With increasing external flow velocity, classical dripping transitions to jetting occur. Jet length grows almost linearly with the external fluid velocity, as shown in Fig. 3a. Downstream, the jet eventually breaks into monodisperse droplets due to the Rayleigh-Plateau instability (Fig. 2b and2c and movie [30]). Breakup results from the growth of long wavelength perturbations when the inertial forces (F iner = ρv 2 d 2 ) become equal to or greater than the interfacial forces (F IT F = σd), i.e. W e ∼ 1. These two first regimes, mostly driven by interfacial forces, have been observed and studied several previous times at the microscale for liquid-liquid [START_REF] Utada | Dripping to Jetting Transitions in Coflowing Liquid Streams[END_REF][START_REF] Mak | The dripping-to-jetting transition in a co-axial flow of aqueous two-phase systems with low interfacial tension[END_REF], liquid-gas [START_REF] Haase | Characterisation of gas-liquid two-phase flow in minichannels with co-flowing fluid injection inside the channel, part I: unified mapping of flow regimes[END_REF], and supercritical fluid-liquid [START_REF] Marre | Dripping to jetting transitions observed from supercritical fluid in liquid microcoflows[END_REF] coflows. Inertia-driven jetting-sinuous wave breakup. With increasing velocity, the jet undergoes destabilization due to the effects of external inertial forces, in contrast with the behavior observed in the Rayleigh jetting mode. The viscous forces (F v = ηvd), which tend to stabilize the jet, are largely overcome by the inertial forces for Re > 500, resulting in the formation of sinuous waves, as previously observed for unconfined flows [START_REF] Lin | Drop and spray formation from a liquid jet[END_REF][START_REF] Lefebvre | Atomization and Sprays[END_REF]. In the sinuous wave breakup regime, breakup stems primarily from the unstable growth of short wavelength perturbations, probably arising from Kelvin-Helmholtz instabilities. The Kelvin-Helmholtz instabilities form due to the localized high velocity ratio ( vext /vint) present on the surface of the jet (Fig. 2d and 2e and movie [31]). Two important characteristics can be highlighted: (i) the jet length continuously decreases with increasing outer fluid velocity and, (ii) the polydispersity of the droplet size increases, relative to the distributions observed in the Rayleigh jetting mode, since the jet breaks into fluid ligaments through a pullout mechanism, with the fragments later forming droplets due to Rayleigh mechanism or minimization of surface energy. Filament breaking, which can be described as secondary atomization [START_REF] Zandian | Planar liquid jet: Early deformation and atomization cascades[END_REF] produces a droplet distribution with much greater polydispersity than is observed in the dripping or Rayleigh jetting regimes (Fig. 3b). Inertia-driven jetting-Atomization-like. At high values of external fluid inertial force (F iner,ext ), the jet length stabilizes with increasing fluid velocity (Fig. 2f, Fig. 2g andmovies [? ]). Smaller ligaments form, which transform nearly instantaneously into droplets. In this regime, droplet size can reach values as small as 0.4d (Fig. 3). One of the main distinguishing features of this regime is characterized by an enlargement of the jet diameter, relative to the nozzle diameter. To confirm this unexpected behavior, we have performed numerical simulations using a 3D incompressible one-fluid model [START_REF] Kataoka | Local instant formulation of two-phase flow[END_REF], already validated for liquid jet breakup in pressurized CO 2 [START_REF] Guillaument | Numerical simulation of dripping and jetting in supercritical fluids/liquid micro coflows[END_REF]. This model is selected since the flow behavior is clearly non axisymmetrical for inertial modes (sinuous wave breakup and atomization-like modes). Compressed CO 2 and water used in this work are considered as immiscible fluids in the investigated conditions, so the computational study must account for two-phase flow. Accordingly, the Brackbill model [START_REF] Brackbill | A continuum method for modeling surface tension[END_REF] (see ESI) was employed to compute the interfacial forces, while the VOF approach was used with a piecewise linear interface construction (PLIC) for the interface tracking [START_REF] Youngs | Time-dependent multi-material flow with large fluid distortion[END_REF]. As observed in numerical simulations (Fig. 2h and movies [37]), the high velocity ratio ( vext /vint > 10) generates hydrodynamic recirculating vortices within the jet, which confine the inner fluid at the tip and lead to noticeable jet enlargement.

Next, we sought to generalize our observations, by studying the effects of the experimental parameters (inner and outer fluid velocities, p, T ) on the interfacial and viscous forces which govern jet behavior. All the three systems (i.e. scCO 2 -water, liquid CO 2 -water and pentane -water) were studied so that the widest possible range of experimental parameters could be included in this work and to arrive at the most general possible conclusions. We have varied the experimental parameters to investigate the effects of inertia compared to interfacial and viscous forces. These conditions result in high Reynolds number flows for both the inner and outer fluid (up to Re = 2200, Table I), which have not been previously investigated due to the practical restrictions associated with working with high flowrates and the resultant high pressure drops. Investigating these effects provides data to unify in a single picture the four distinct coflow regimes. Utada et al. [START_REF] Utada | Dripping, Jetting, Drops, and Wetting: The Magic of Microfluidics[END_REF] proposed categorizing the transition between the dripping and the jetting regime in micro-coflows using the [W eint , C aext ] diagram, which takes into account the inner flow inertial and interfacial forces, and the outer flow viscous and interfacial forces. These previous studies considered only inner and outer flows with very low inertial forces ∼ 10 -3 µN , possibly preventing observations of the sinuous wave and atomization break up regimes shown here. In contrast, we explored conditions where the inertial forces are in the range 10 -2 -10 1 µN.

We categorize the jetting regimes and their domains of existence by using the ratio of the inertial forces over the sum of the other forces (viscous and interfacial) for both the inner and outer fluid. This approach makes possible the representation of the jet phase diagram, Y = f (X) consisting of a log-log plot non-dimensional scaling parameters: X = Finer,ext /(F IT F,ext +Fv,ext) and Y = Finer,int /(F IT F,int +Fv,int).

This non-dimensionalization emphasizes the effects of inertial forces over viscous and interfacial ones. In Fig. 4, we have represented all available experimental data, and categorized their flow behavior into one of the 4 regimes. Fig. 4 provides data for both the sc-CO 2 /water (48 °C, 10 MPa) , the liquid CO 2 /water (20 °C, 10 MPa) and the liquid pentane /water (20 °C, 0.1 MPa) systems. As a first observation, Fig. 4 shows that the transition from dripping to jetting occurs when X > 1 and/or Y > 1, (Fig. 4), as expected. Indeed, the inertial forces at these conditions are greater than the interfacial and viscous forces, leading to jetting.

A remarkable result arises at the transition from Rayleigh jetting to sinuous wave breakup regime. Indeed, the experimental data indicate that, independent of the inner fluid force ratio, the "inertial regime" (i.e. sinuous wave and/or atomization-like break up) is never attained provided that X <5 (Fig. 4). For X < 5, the outer fluid viscous and interfacial forces are sufficient (although smaller than the inertial forces) to stabilize the jet and prevent unstable growth of short wavelength, Kelvin-Helmhotz perturbations that would otherwise lead to the sinuous wave breakup regime. With increasing X, the jet enters the atomization regime. Reaching atomization occurs when the inner fluid viscous and interfacial forces overcome the inertial forces (low Y values, Fig. 4). In contrast, when the inertial forces of the inner fluid are sufficient (higher Y values), they tend to extend the length of the jet, stabilizing the sinuous wave break up mode (Fig. 4).

The observations and diagram presented in here are not unique to [dense CO 2 (liquid or supercritical) -water] micro coflows. Indeed, the pentane-water system (Fig. 4, triangles), obeys the same jetting behavior as the sc-CO 2 -water system (Fig. 4, squares) and the liquid CO 2 -water (Fig. 4, circles), when non dimensional scaling parameters X and Y are used for data interpretation.

In conclusion, we have studied the jetting behavior of micro-confined co-flows using a high pressure microchannel device. Several fluid-fluid systems were studied, including scCO 2 -water, liquid CO 2 -water, and pentane -water. In addition to the well know dripping and Rayleigh jetting modes, two new inertia-driven modes were observed, namely the so-called sinuous wave breakup and the atomization-like regime. We find that all our data can be captured by a log-log plot using dimensionless scaling parameters. This study extends previous work on microscale-confined coflowing liquids to permit observation of qualitatively new behavior and unifies previous studies by mapping jet behavior onto a single diagram.

This work provides the basis for many new microfluidics experiments, including those involving production of sub-micron droplets, as reported by other approaches [START_REF] Jeong | Controlled generation of submicron emulsion droplets via highly stable tip-streaming mode in microfluidic devices[END_REF]. Future fundamental studies should investigate areas of the phase diagram which remain to be explored, especially those for Y < 10 -2 and Y > 10 1 . Data are required at these conditions to provide a full understanding of the transition between between viscous/interfacialdriven flows and inertia driven flows.

This study was supported by the French National Research Agency (ANR SUPERFON), the University of Bordeaux (PhD funding of FZ), The IFPEN (PhD funding of TG), the CNES and the Région Nouvelle Aquitaine. This work has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement n°725100, project: Big Mac). MTT thanks the U.S. National Science Foundation for support (CBET 1554283). SM and AE thank Fabien Palencia for technical support.

Figure 1 .

 1 Figure 1. Coflowing set-up developed for this study. D and d are the inner diameters of the outer and inner tubing, respectively. L is the jet length, while a is the droplets diameter.

a

  × 10 -4 -1.5 × 10 -3 1.6 × 10 -3 -0.15 1.3 × 10 -4 -1.6 × 10 -3 1.3 × 10 -3 -0.11 2.3 × 10 -4 -9.4 × 10 -3 Re b Versus water. b Depending on flow rates. For the calculation, we have used: d = 100 µm, D = 250 µm 25 < QCO 2 µl.min -1 < 1000 50 < QH 2 O µl.min -1 < 10000 25 < QP entane µl.min -1 < 1000 are the fluid density in the pump (at p = p exp and room temperature -RT ) and in the capillary (p = p exp and T = T exp ), respectively. The physical properties of CO 2 and H 2 O at experimental conditions were obtained from the REFPROP software [38] or from the literature [39].

Figure 2 .

 2 Figure 2. (a) Optical images of the evolution of the jetting as of function of the outer fluid velocity: (a) Dripping, (b, c) Rayleigh jetting, (d, e) Sinuous wave breakup mode, (f, g) atomization-like mode. The pictures were obtained for p = 10 MPa, T = 20 °C, inner fluid: liquid CO2, outer fluid: Water, v int(CO 2 ) = 0.25 m.s -1 , (h) Median plane visualization of the 3D numerical modelling of the atomization-like jetting mechanism displaying the velocity vectors inside the core of the jet (p = 10 MPa, T = 20 °C, vint(CO2) = 0.25 m.s -1 ; vext (H2O) = 4 m.s -1 ). The arrows represent the velocity vectors, which magnitude are proportional to their length, while the color represents the relative pressure (Pr) spatial variations (the total pressure can be calculated as: P = Pr + 10 M P a).

Figure 3 .

 3 Figure 3. Example of the regimes characteristics used to categorize the breakup mechanisms. (a) Evolution of the jet length and the droplets mean size ā/d as a function of vext and (b) the droplets size distributions for different values of vext, corresponding to yellow circled point in (a). The data were obtained for p = 10 MPa, T = 20 °C, inner fluid: liquid CO2, outer fluid: Water, v int(CO 2 ) = 0.25 m.s -1 .

Figure 4 .

 4 Figure 4. Log-log plot of the ratio (X ; Y) of the inertial forces over the sum of the viscous and interfacial forces for the sc-CO2-water (p = 10 MPa, T = 48 °C: P), pentanewater (p = 0.1 MPa, T = 20 °C : a) and liquid CO2 -water (p = 10 MPa, T = 20 °C: ) systems. The dripping regime is indicated in black, the Rayleigh type jets in green, the sinuous wave breakup mode is in blue and the atomization-like mode points are in red.

Table I .

 I Physical properties and corresponding values for the dimensionless numbers (W e, Ca and Re) of CO2 or pentane and water flows at p = 10 MPa (or p = 0.1 MPa) for T = 20 °C (liquid CO2 and liquid pentane) and T = 48 °C (sc-CO2),

	respectively.
	T	20 °C		48 °C		20	°C
	P	10 MPa	10 MPa	0.1 MPa
	Fluid	Liquid CO2	H2O	sc-CO2	H2O	Pentane
	σ a mN m -1	37.1	/	28.8	/	51.2
	η (µPa s)	81.5	998.8	30.6	567.3	227.5
	ρ kg m -3	856.3	1002.7	421.6	993.2	625.8