Thuy Minh Giang

Steven Le Feunteun
email: steven.le-feunteun@grignon.inra.fr

Sebastien Gaucel

Pierre Brestaz

Marc Anton

Anne Meynier

Ioan-Cristian C Trelea

Dynamic modeling

Keywords: Pancreatic lipase, Lipid digestion, Droplet size, Polyunsaturated Fatty Acids, Simulation

Introduction

Key parameters that govern food digestion in vivo are very hard to identify for practical reasons. In vitro methods, for which the conditions are strictly controlled, have therefore been, and are still, largely used. Concerning the digestion of emulsified lipids, both their compositional and structural properties can affect digestion [START_REF] Armand | Lipases and lipolysis in the human digestive tract: where do we stand?[END_REF]. It is known that short chain fatty acids are released faster and to greater extents than long-chain fatty acids [START_REF] Deckelbaum | Medium-chain versus long-chain triacylglycerol emulsion hydrolysis by lipoprotein-lipase and hepatic lipase -Implications for the mechanisms of lipase action[END_REF][START_REF] Armand | Effects of droplet size, triacylglycerol composition, and calcium on the hydrolysis of complex emulsions by pancreatic lipase -An in vitro study[END_REF][START_REF] Zhu | Free fatty acid profiles of emulsified lipids during in vitro digestion with pancreatic lipase[END_REF][START_REF] Li | New mathematical model for interpreting pH-stat digestion profiles: Impact of lipid droplet characteristics on in vitro digestibility[END_REF][START_REF] Li | Factors affecting lipase digestibility of emulsified lipids using an in vitro digestion model: Proposal for a standardised pHstat method[END_REF], and that polyunsaturated fatty acids such as docosahexanoic acid (DHA) and eicosapentaenoic acid (EPA) are highly resistant to lipolysis [START_REF] Bottino | Resistance of certain long-chain polyunsaturated fatty acids of marine oils to pancreatic lipase hydrolysis[END_REF][START_REF] Marze | In vitro digestion of fish oils rich in n-3 polyunsaturated fatty acids studied in emulsion and at the oil-water interface[END_REF]. The composition and the surface area of the interface surrounding lipids are also very important parameters because digestive lipases must adsorb on the droplet surface to reach their substrates. On the one hand, the kinetics of lipolysis increases with the interfacial area, and hence with decreasing droplet sizes [START_REF] Armand | Effects of droplet size, triacylglycerol composition, and calcium on the hydrolysis of complex emulsions by pancreatic lipase -An in vitro study[END_REF][START_REF] Li | New mathematical model for interpreting pH-stat digestion profiles: Impact of lipid droplet characteristics on in vitro digestibility[END_REF][START_REF] Li | Factors affecting lipase digestibility of emulsified lipids using an in vitro digestion model: Proposal for a standardised pHstat method[END_REF][START_REF] Armand | Emulsion and absorption of lipids: The importance of physicochemical properties[END_REF][START_REF] Borel | Hydrolysis of emulsions with different triglycerides and droplet sizes by gastric lipase in vitro -Effect on pancreatic lipase activity[END_REF]. On the other hand, the rate of the reaction depends on the lipase affinity for the interfacial layer and may therefore be modulated by the nature of the emulsifiers used to stabilize the emulsions [START_REF] Mun | Influence of emulsifier type on in vitro digestibility of lipid droplets by pancreatic lipase[END_REF][START_REF] Hur | Influence of initial emulsifier type on microstructural changes occurring in emulsified lipids during in vitro digestion[END_REF].

Moreover, if lipid digestion is initiated in the stomach by the gastric lipase, most of the reaction (70-90%) is performed in the upper part of the intestine by the combined action of the pancreatic lipase, its colipase, and bile salts [START_REF] Armand | Lipases and lipolysis in the human digestive tract: where do we stand?[END_REF]. Beyond the properties of the native emulsions, the structural modifications they may undergo within the stomach will affect the intestinal phase, and hence the overall kinetics of the digestion [START_REF] Singh | Structuring food emulsions in the gastrointestinal tract to modify lipid digestion[END_REF]. Emulsions can remain stable, flocculate or coalesce during the gastric phase depending on several parameters such as the initial droplet size, the type of emulsifiers, and the composition of the surrounding medium [START_REF] Golding | Impact of gastric structuring on the lipolysis of emulsified lipids[END_REF][START_REF] Marciani | Enhancement of intragastric acid stability of a fat emulsion meal delays gastric emptying and increases cholecystokinin release and gallbladder contraction[END_REF][START_REF] Wooster | Impact of different biopolymer networks on the digestion of gastric structured emulsions[END_REF][START_REF] Day | Tailoring the digestion of structured emulsions using mixed monoglyceride-caseinate interfaces[END_REF]. The rate of lipid hydrolysis during the intestinal phase can thus be impacted by changes in the droplet surface area induced by the gastric structuring of the emulsions: the lower the interfacial area when emptied into the duodenum, the slower the digestion. The stability of the oil droplets against flocculation and coalescence in the stomach is therefore essential to understand why two resembling emulsions may lead to different digestion kinetics.

Several studies have also highlighted that some emulsions can remain relatively stable during the gastric phase but undergo important modifications during the intestinal phase of digestion.

Whey protein stabilized submicron emulsions are good examples thereof. They have been reported to remain stable in size or flocculate in simulated gastric conditions [START_REF] Golding | Impact of gastric structuring on the lipolysis of emulsified lipids[END_REF][START_REF] Nik | Surface adsorption alters the susceptibility of whey proteins to pepsin-digestion[END_REF][START_REF] Li | Physicochemical behaviour of WPI-stabilized emulsions in in vitro gastric and intestinal conditions[END_REF]. In either case, the initial rate of lipolysis during the subsequent intestinal phase was fast and comparable to other gastric stable emulsions [START_REF] Golding | Impact of gastric structuring on the lipolysis of emulsified lipids[END_REF], suggesting that flocculation during the gastric step did not alter much the intestinal lipolysis kinetics. In parallel, other studies have shown that whey protein stabilized emulsions are highly prone to coalescence during the intestinal phase of in vitro digestion [START_REF] Mun | Influence of emulsifier type on in vitro digestibility of lipid droplets by pancreatic lipase[END_REF][START_REF] Singh | Structuring food emulsions in the gastrointestinal tract to modify lipid digestion[END_REF][START_REF] Li | Physicochemical behaviour of WPI-stabilized emulsions in in vitro gastric and intestinal conditions[END_REF][START_REF] Sarkar | Pancreatin-induced coalescence of oil-in-water emulsions in an in vitro duodenal model[END_REF]. The effect of such an intestinal coalescence of the oil droplets on the kinetics of pancreatic lipolysis have, however, not been investigated so far. Therefore, our study aimed at studying and modeling the influence of droplet coalescence on the kinetics of pancreatic lipolysis during an in vitro gastrointestinal digestion of a whey protein stabilized emulsion. The emulsion was prepared from a blend of medium-chain triacylglycerols and a microalgae oil rich in docosahexaenoic acid (DHA), a long-chain ω3 polyunsaturated fatty acid which daily recommended intake is 250 mg [START_REF] Efsa | Scientific opinion on dietary reference values for fats, including saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, trans fatty acids, and cholesterol[END_REF]. The evolution of both the oil droplet sizes and the lipolysis kinetics were monitored throughout gastrointestinal in vitro digestion experiments. A mathematical model, in line with previously published ones [START_REF] Li | New mathematical model for interpreting pH-stat digestion profiles: Impact of lipid droplet characteristics on in vitro digestibility[END_REF][START_REF] Verger | Interfacial enzyme kinetics of lipolysis[END_REF][START_REF] Jurado | Kinetics of the enzymatic hydrolysis of triglycerides in o/w emulsions: Study of the initial rates and the reaction time course[END_REF], was developed to quantitatively evaluate the relationships between the intestinal lipolysis kinetics and the evolution of the droplet surface area.

Material and methods

Materials

The oil containing medium-chain triacylglycerols (Miglyol 812S) (MCT): C8:0 (54%) and C10:0 (43%) was purchased from Sasol GmbH, Germany. The oil containing long-chain triacylglycerols (DHAsco) (LCT): docosahexahenoic acid (DHA, C22:6 n-3, 40%), C12:0 (4%), C14:0 (12%), C16:0 (12%) and C18:1 n-9 (24%) was obtained from Martek, via DSM Nutritional Products Ldt, Switzerland. Whey protein powder (Prolacta 95) was purchased from Lactalis Ingredients, France. Pepsin (P7012), mucin (M2378), pancreatin (P7545), pancreatic lipase (L3126) and bile extract (B8631) were from porcine origin and obtained from Sigma-Aldrich, France. Water was Milli-Q water. Solvents for liquid chromatography were chloroform for HPLC (Carlo Erba), methyl alcohol for HPLC (99.9%, Carlo Erba), ammonia solution (30%, Carlo Erba).

Emulsion preparation

An emulsion composed of 80% of aqueous phase and 20% of oil (w/w) was made. The aqueous phase was prepared by dissolving 4% (w/w) of whey protein powder, used as emulsifier, in a 0.1M sodium phosphate buffer at pH 7.0. The oil phase consisted of 62.5% LCT and 37.5% MCT (w/w) mixed together. A rotor-stator homogenizer (SilentCrusher M equipped with the 12F generator from Heidolph Instruments, Germany) was used in a preemulsification step (5 min, 20000 rpm). The coarse emulsion was successively homogenized for 5 min at 500 bars and for 10 min at 1000 bars under nitrogen flow with a high-pressure homogenizer (C3-EmulsiFlex, Sodexim SA, France) temperature-controlled at 4°C to produce the emulsion with droplet diameters below micron. A 50% (w/w) maltodextrin in water solution was then added as a 1:1 (v/v) ratio to the emulsion and the mixture was thereafter freeze-dried. The dried emulsion was then conditioned in oxygen hermetic bag under vacuum and kept at -20°C until uses. The dried emulsion was rehydrated in Milli-Q water to obtain a final oil concentration of 3.2% (w/w) on the day of the in vitro experiments.

Emulsion digestion

3 mL of the rehydrated emulsion, corresponding to an oil mass of about 96 mg, was placed into 22.4 mL headspace vials hermetically sealed with Teflon/silicon septa and aluminum caps. These vials were kept in a temperature controlled chamber at 37°C under magnetic stirring (400 rpm.min -1 ) throughout the duration of the experiments. The gastric phase duration was 60 min and was launched by adding 2.12 mL of simulated gastric fluid (SGF) and 40 µL of 1M HCl to reach a final pH of 2.5 in the reaction vials. The SGF solution contained 3.9 g.L -1 of pepsin, 2.4 g.L -1 of mucin, 120 mM of NaCl, 2 mM of KCl and 6 mM of CaCl 2 . The intestinal phase duration was then launched for 300 min maximum by adding 4.86 mL of simulated intestinal fluid (SIF) into the vials and 100 µL of 1M NaCO 3 to reach a final pH of 6.5. The simulated intestinal fluid (SIF) contained 30.8 g.L -1 of bile extract powder, 0.82 g.L -1 of pancreatin, 0.41 g.L -1 of pancreatic lipase, and the same electrolyte concentrations as the SGF.

The native emulsion (NE), and samples taken at the end of the gastric phase (G60), at t = 0 min of the intestinal phase (I0) using a modified SIF that contained all constituents except pancreatin and lipase, and at 15, 30, 60, 120 and 300 min of intestinal digestion (I15 to I300) were analyzed. One vial was used for one sampling time and one type of measurement (quantification of LCT and MCT by HPLC or droplet size by laser granulometry) so that the contents of 16 vials were analyzed in total (2 methods times 8 sampling times) for one digestion. Three independent digestion experiments, further denoted replicates, were performed. Samples devoted to droplet size measurements were analyzed immediately, whereas samples devoted to HPLC measurements were kept at -80°C until further analysis.

Quantification of LCT and MCT by HPLC

HPLC paired with an evaporative light scattering detector (ELSD) was used to quantify the decrease of both LCT and MCT masses during the time course of the in vitro digestions. Total lipids were extracted from 1.5 mL of the native emulsions or of the stomach media and from 3 mL of the intestinal media according to the Bligh and Dyer [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF] procedure with minor modifications in the ratio CHCl 3 /CH 3 OH/H 2 O 1/2/1. Before the HPLC analyses, the lipid extract was dissolved in CHCl 3 down to 0.3 mg.mL -1 for native emulsion and stomach media and to 0.7 mg.mL -1 for intestinal media. HPLC operating conditions were similar to those described in [START_REF] Kenmogne-Domguia | Gastric conditions control both the evolution of the organization of protein-stabilized emulsions and the kinetic of lipolysis during in vitro digestion[END_REF] using a Uptip-prep Strategy column (2.2 µm SI, 150×4.6 mm, Interchim, Montluçon, France) and 30 µL of injected lipid extract. As illustrated in Fig. 1, injected masses of pure MCT and LCT ranging from 0.5 to 9 µg led to a power law calibration curve with no distinction of the TAG chain-length.

The triacylglycerol region of an HPLC chromatogram stemming from an undigested sample is presented in Fig. 2. The retention times of LCT (1.21 min) and MCT (1.32 min) were different but their signals partly overlapped. As illustrated in Fig. 2, signal deconvolution was therefore undertaken using a specifically developed algorithm running with the Matlab™ software (The MathWorks Inc., Natick, MA) and the recovered LCT and MCT signals were converted into masses using the calibration curve (Fig. 1). Reliable results were obtained using this procedure as shown from the comparison of the mean LCT and MCT masses of 57.3 ± 2.7 and 35.0 ± 2.0 mg (estimated over 15 undigested samples) with the 59.7 and 35.7 mg targeted masses in each digestion vial, respectively. LCT and MCT masses were finally converted into lipolysis percentages using Eq. 1:

ሻݐሺݏ݅ݏݕ݈݈݅ ൌ ݉ ்ீ బ െ ݉ ்ீ ሺݐሻ ݉ ்ீ బ ൈ 100 ሺ1ሻ
where ݉ ்ீ బ and ݉ ்ீ ሺݐሻ are the masses (mg) of LCT or MCT initially present in the vials and measured by HPLC at time t, respectively.

Droplets size measurement

The volume-based distribution of oil droplet sizes was measured using a Mastersizer S (Malvern Instruments Ltd., Worcestershire, UK) equipped with a 2 mW He-Ne laser of λ = 633 nm and the 300RF lens with detection limits of 0.05 and 900 µm. The refractive index n 0 of the aqueous phase was 1.33 and the properties of the dispersed phase were 1.457 for the refractive index and 0.001 for the absorption. Samples were pre-diluted 100-fold with the desired solution (with or without sodium dodecyl sulfate, SDS, as deflocculating agent), and then diluted with distilled water to reach an oil volume concentration near 0.01% for the circulation in the measurement cell. All analyses were performed at room temperature as described previously [START_REF] Marze | In vitro digestion of fish oils rich in n-3 polyunsaturated fatty acids studied in emulsion and at the oil-water interface[END_REF][START_REF] Kenmogne-Domguia | Gastric conditions control both the evolution of the organization of protein-stabilized emulsions and the kinetic of lipolysis during in vitro digestion[END_REF]. The surface-weighted mean diameter, ݀ ଷଶ , corresponding to the droplet diameter having the same ratio of volume to surface area as the droplet distribution, was calculated according to:

݀ ଷଶ ൌ ∑ ݊ ݀ ଷ ∑ ݊ ݀ ଶ
where ݊ is the number of droplets of diameter ݀ .

Mathematical modeling

Model assumptions and equations

Lipolysis is mediated by the pancreatic colipase-lipase system which absorbs onto the droplet interface and splits the sn-1 and sn-3 ester bonds of triacylglycerols (TAG). The first step of the reaction generates one free fatty acid (FFA) and a diacylglycerol (DAG) which is further transforms into a second FFA and the sn-2-monoacylglycerol. Our model simulates the kinetics of lipolysis as inferred from the disappearance of the TAG molecules so that, in principle, it only characterizes the first step of the reaction. Nevertheless, the lack of detected DAG in the course of the experiments strongly suggests that the second reaction step was not rate-limiting in our experimental conditions, and hence that similar lipolysis kinetics would have been recovered by monitoring the appearance of the final products of the reaction. The main modeling assumptions were as follows:

A1: The rate of TAG hydrolysis was assumed proportional to the interfacial area of the droplets [START_REF] Singh | Structuring food emulsions in the gastrointestinal tract to modify lipid digestion[END_REF][START_REF] Golding | Impact of gastric structuring on the lipolysis of emulsified lipids[END_REF][START_REF] Verger | Interfacial enzyme kinetics of lipolysis[END_REF]. This means that the surface reaction rate was taken as constant in the considered experimental conditions. Thus:

݀݉ ்ீ ሺݐሻ ݐ݀ ൌ െ݇ . ܣሺݐሻ ሺ2ሻ
where ݇ is the hydrolysis rate constant (mg.m -2 .min -1 ) and ܣሺݐሻ is the interfacial area (m 2 ) at time t (min).

A2: The droplets were considered as spheres, and the interfacial area of the droplets was assumed to be adequately described by the size distributions measured by laser granulometry and their corresponding ݀ ଷଶ . Hence, one can write that:

ܣሺݐሻ ൌ 6 ܸ ்ீ ሺݐሻ ݀ ଷଶ ሺݐሻ ሺ3ሻ
where ݀ ଷଶ ሺݐሻ is the surface-weighted mean diameter (nm) at time t, and ܸ ்ீ ሺݐሻ is the volume of TAG in the sample (mm 3 ) at time t. Eq. 3 can be further transformed into:

ܣሺݐሻ ൌ 6 ݉ ்ீ ሺݐሻ ߩ. ݀ ଷଶ ሺݐሻ ሺ4ሻ
where ρ is the mass density of TAG (mg/mm 3 ) and ݉ ்ீ ሺݐሻ is the mass of TAG (mg) at time t.

Combining Eq. 1 and 4, the evolution of the TAG mass is given by:

݀݉ ்ீ ሺݐሻ ݐ݀ ൌ െ6݇ . ݉ ்ீ ሺݐሻ ߩ. ݀ ଷଶ ሺݐሻ ሺ5ሻ
To solve the differential equation Eq. 5, one also needs to know how the droplet sizes evolve as a function of time. In the present study, we resorted to two different assumptions on ݀ ଷଶ ሺݐሻ, leading to two different versions of the model:

A3a: In one version, it was assumed that all droplets had the same diameter and that the total number of droplets remained constant, hypotheses that lead to the following equation [START_REF] Li | New mathematical model for interpreting pH-stat digestion profiles: Impact of lipid droplet characteristics on in vitro digestibility[END_REF]:

݀ ଷଶ ሺݐሻ ൌ ݀ ඨ ݉ ்ீ ሺݐሻ ݉ ்ீ బ య ሺ6ሻ
where ݉ ்ீ బ is the mass of TAG initially introduced in the reaction vial and ݀ is the droplet diameter of the native emulsion.

A3b: In the other version, ݀ ଷଶ ሺݐሻ was estimated by linear interpolations of the experimental values recovered using laser granulometry.

For both model versions, the masses calculated by solving Eq. 5 were finally converted into percentages of lipolysis using Eq. 1 to enable the comparison of the model simulations with experimental data.

Model fitting and parameter estimation

The lipolysis of MCT was so fast that it was already finished at t = 15 min, i.e. the first sampling time. Only the lipolysis of LCT was therefore considered to confront the model to the experimental data. The differential equation Eq. 5 was numerically solved using a LCT mass density, ρ, of 0.92 g.cm -3 , and each of the previously described assumptions (A3a or b)

for the droplet size evolution as a function of time, ݀ ଷଶ ሺݐሻ. The unknown hydrolysis rate, ݇ , was then estimated by fitting model predictions to the LCT lipolysis results determined by HPLC. The recovered value, expressed in mg of TAG per minute and square meter of interfacial area (mg.m -2 .min -1 ), was then converted into µmol.m -2 .min -1 for comparison purposes with the literature using a LCT molar mass of 900 g.mol -1 . Numeric calculations were performed using Matlab™ software (The MathWorks Inc., Natick, MA).

Results and Discussion

Kinetics of lipolysis of LCT and MCT

The evolution of HPLC chromatograms during the intestinal phase stemming from one in vitro digestion experiment is shown in Fig. 3A, and the mean percentages of lipolysis calculated over the three replicates are presented in Fig. 3B. Initially, 2 peaks were clearly visible in the chromatograms. The signal of the native emulsion and at t = 0 min of the intestinal phase (modified SIF with no lipase) were similar since no gastric lipase was used in this study. At t = 15 min of intestinal digestion, the signal arising from MCT entirely disappeared, indicating that MCT were fully hydrolyzed in only few minutes. In contrast, about 20% of the initial LCT mass was still detected after 300 min (i.e. 5h) of intestinal digestion, showing that lipolysis was much slower for LCT than for MCT (Fig. 3B). In fact, lipolysis of LCT was relatively fast during the first 30 minutes of digestion but was greatly slowed down afterwards, resulting in a two-stage curve typical of most in vitro lipolysis studies on submicron emulsions made of long-chain triacylglycerols [START_REF] Zhu | Free fatty acid profiles of emulsified lipids during in vitro digestion with pancreatic lipase[END_REF][START_REF] Li | New mathematical model for interpreting pH-stat digestion profiles: Impact of lipid droplet characteristics on in vitro digestibility[END_REF][START_REF] Golding | Impact of gastric structuring on the lipolysis of emulsified lipids[END_REF].

Higher rates of lipolysis of MCT compared to LCT have been reported in many studies using pure MCT and LCT emulsions or MCT/LCT mixed emulsions as in the present study [START_REF] Deckelbaum | Medium-chain versus long-chain triacylglycerol emulsion hydrolysis by lipoprotein-lipase and hepatic lipase -Implications for the mechanisms of lipase action[END_REF][START_REF] Armand | Effects of droplet size, triacylglycerol composition, and calcium on the hydrolysis of complex emulsions by pancreatic lipase -An in vitro study[END_REF][START_REF] Zhu | Free fatty acid profiles of emulsified lipids during in vitro digestion with pancreatic lipase[END_REF][START_REF] Li | New mathematical model for interpreting pH-stat digestion profiles: Impact of lipid droplet characteristics on in vitro digestibility[END_REF].

This is generally attributed to the higher water solubility of medium-chain FFAs than longchain FFAs. Indeed, the low water solubility of long-chain FFAs would lead to their accumulation at the interface that would, in turn, inhibit the lipase activity by steric hindrance until they are removed by bile salts or by forming soap with calcium ions [START_REF] Zangenberg | A dynamic in vitro lipolysis model I. Controlling the rate of lipolysis by continuous addition of calcium[END_REF]. In contrast, the higher water solubility of medium-chain FFAs would facilitate their release from the interface, and hence promote further hydrolysis of triacylglycerols at the droplet surface. According to the composition of our emulsion, other factors may also have contributed to the higher rate of MCT hydrolysis. Indeed, it has been reported that triacylglycerols containing docosahexaenoic acid are more resistant to pancreatic lipase [START_REF] Bottino | Resistance of certain long-chain polyunsaturated fatty acids of marine oils to pancreatic lipase hydrolysis[END_REF], possibly because of an inhibitory effect induced by the presence of a double bond near the carboxyl group. For MCT/LCT mixed emulsions, it has also been shown that MCT hydrolysis can be promoted to the detriment of that of LCT [START_REF] Deckelbaum | Medium-chain versus long-chain triacylglycerol emulsion hydrolysis by lipoprotein-lipase and hepatic lipase -Implications for the mechanisms of lipase action[END_REF] because of a preferential location, or turnover, of MCT at the droplet interface [START_REF] Deckelbaum | Medium-chain versus long-chain triacylglycerol emulsion hydrolysis by lipoprotein-lipase and hepatic lipase -Implications for the mechanisms of lipase action[END_REF][START_REF] Armand | Effects of droplet size, triacylglycerol composition, and calcium on the hydrolysis of complex emulsions by pancreatic lipase -An in vitro study[END_REF]. It is therefore likely that different mechanisms have contributed to the marked difference we observed in the lipolysis kinetics of MCT and LCT.

Evolution of the droplet size

The evolution of the particle size distribution (measured without deflocculating agent) stemming from one in vitro digestion experiment is shown in Fig. 4A. The native emulsion (NE) presented a monomodal distribution with a mean ݀ ଷଶ of 0.26 µm. The mean size increased considerably during the gastric phase since the measured ݀ ଷଶ was 3.00 ± 0.46 µm after 60 min of contact with the SGF (G60). However, the size distribution and the measured ݀ ଷଶ returned close to their original values after dilution of the same sample in a 1% SDS solution (not shown) or after addition of a SIF with no pancreatin or lipase (I0). This demonstrates that the increase of the mean diameter during the gastric phase was caused by droplet flocculation, and that the subsequent addition of bile led to a deflocculation of these droplet aggregates. During the intestinal phase, the droplet size distribution remained similar during the first 15 min (I15). It was suddenly shifted toward considerably larger diameters at about t = 30 min (mean volume diameter of about 9 µm) and remained relatively stable until the end of the experiment (I30 to I300), with a good repeatability of the surface weighted diameters over the three replicates (Fig. 4B).

According to the demonstrated tendency of bile to deflocculate the droplet aggregates formed during the gastric phase, the increase of the particle size during the intestinal phase most assuredly resulted from a coalescence of the oil droplets. This conclusion is moreover consistent with previous studies showing that whey protein stabilized submicron emulsions undergo coalescence during in vitro intestinal digestion [START_REF] Mun | Influence of emulsifier type on in vitro digestibility of lipid droplets by pancreatic lipase[END_REF][START_REF] Singh | Structuring food emulsions in the gastrointestinal tract to modify lipid digestion[END_REF][START_REF] Li | Physicochemical behaviour of WPI-stabilized emulsions in in vitro gastric and intestinal conditions[END_REF][START_REF] Sarkar | Pancreatin-induced coalescence of oil-in-water emulsions in an in vitro duodenal model[END_REF]. We may even highlight that the evolution of the size distributions we measured is highly similar to previous results obtained during the digestion of a whey protein stabilized soya oil emulsion with an initial ݀ ଷଶ of 0.37 µm, and for which an intense coalescence was observed by confocal microscopy after 10 to 30 min of intestinal digestion [START_REF] Li | Physicochemical behaviour of WPI-stabilized emulsions in in vitro gastric and intestinal conditions[END_REF]. Hence, even if flocculation cannot be totally excluded from our own set of data, we will only refer to the term of coalescence in the rest of this article.

We may also highlight the remarkable simultaneity of the increase of the droplet size (Fig. 4B)

and of the decrease of the lipolysis rate (LCT in Fig. 3B). Although the authors did not point out this particular aspect, it seems that droplet coalescence during the intestinal phase was also concomitant with a decrease of the lipolysis rate in the recent study of Li and coworkers [START_REF] Li | Physicochemical behaviour of WPI-stabilized emulsions in in vitro gastric and intestinal conditions[END_REF]. It is indeed well known that the rate of lipolysis decreases with decreasing surface area, and hence with increasing droplet size [START_REF] Armand | Effects of droplet size, triacylglycerol composition, and calcium on the hydrolysis of complex emulsions by pancreatic lipase -An in vitro study[END_REF][START_REF] Golding | Impact of gastric structuring on the lipolysis of emulsified lipids[END_REF][START_REF] Wooster | Impact of different biopolymer networks on the digestion of gastric structured emulsions[END_REF][START_REF] Verger | Interfacial enzyme kinetics of lipolysis[END_REF][START_REF] Singh | Structural and biochemical factors affecting the digestion of protein-stabilized emulsions[END_REF]. We can therefore wonder how much of the decrease of the LCT lipolysis kinetics at about t = 30 min (Fig. 3B) was induced by droplet coalescence. This was further explored using a modeling approach.

Modeling results

We remind that MCT was fully hydrolyzed in less than 15 min so that only LCT lipolysis was considered for the modeling. The results obtained with three different mathematical models are presented in Fig 5A . First, the dotted line represents our model version that assumes a constant number of droplets of identical diameter (assumption A3a), and which decreases in size upon hydrolysis of the TAG they contain (Fig. 5B). The fit was very bad because the assumed mechanisms could not reproduce the strong decrease of the reaction kinetics at about 30 min.

Second, the dashed line represents the model proposed by Li and McClements [5]. This model is similar to the previous one but further assumes that a fraction of the TAG can remain undigested. It provided a very good fit of our experimental data (R 2 = 0.9888) and led to a lipolysis extent of 77.3 ± 2.6 % and a surface rate constant (݇ ) of 9.4 ± 1.5 µmol.m -2 .min -1 , which is rather close to the 13.8 µmol.m -2 .min -1 reported for a corn oil emulsion in [START_REF] Li | New mathematical model for interpreting pH-stat digestion profiles: Impact of lipid droplet characteristics on in vitro digestibility[END_REF]. As noticed by the authors, these rate constants should nevertheless be considered as upper values whenever droplet flocculation or coalescence takes place because, in such cases, the model would not adequately simulate the evolution of the available interfacial area. This is also why the mean diameter simulated with this model decreased from 350 to 213 nm (Fig. 5B), a trend that is not consistent with our experimental results (Fig. 4).

Finally, the solid line represents our model version that accounts for the experimentally measured ݀ ଷଶ (Fig. 5A andB). This model also reproduced the experimental data very well (R 2 = 0.9882). The underlying interpretation is however entirely different since the marked slowdown of the reaction kinetics around 30 min is here fully explained by the decrease of the interfacial area caused by droplet coalescence (i.e. no upper fraction of the digested lipids is assumed here). The only unknown parameter in this model is the surface reaction rate (݇ ) that was estimated to be 2.4 ± 0.1 µmol.m -2 .min -1 .

To support the above considerations, the interfacial surface area was calculated for both our model accounting for coalescence and the model of Li and McClements (Fig. 6). According to the model of Li and McClements, the droplet size reduces upon TAG hydrolysis, leading to a corresponding decrease of the surface area. The lipolysis nevertheless plateaued (Fig. 5A) despite a large remaining droplet surface area. This suggests that the interface was no more available for the enzymatic action, or in other words, that the reaction was inhibited by a mechanism such as an accumulation of the reaction products at the droplet surface. According to our model however, the slowdown of lipolysis was clearly, and solely, related to the sharp decrease of the interfacial area induced by droplet coalescence. At 300 min, the available interfacial area was low but nonzero, in agreement with the fact that lipolysis still proceeded slowly. Note also that the reaction rate constants estimated with both models reflect these differences of simulated surface area since they are expressed per unit of interfacial area. This is indeed why the value recovered using our model (assumption A3b) is about 4 times smaller than the value estimated with the model of Li and McClements (2.4 and 9.4 µmol.m -2 .min -1 , respectively).

Taking into account the evolution of the experimentally measured droplet sizes, the mathematical model allowed a good prediction of the intestinal lipolysis kinetics. The decrease of the interfacial area was thus the major reason for the slowdown of the reaction rate after about 30 min. We may even highlight that our model slightly overestimates the last experimental point of the kinetics at t = 300 min (Fig. 5A). Thus, our results are still compatible with an inhibition of the enzymatic reaction at the interface, but as a second order factor.

On the one hand, our results confirm that the kinetics of lipolysis are essentially proportional to the interfacial area [START_REF] Verger | Interfacial enzyme kinetics of lipolysis[END_REF]. On the other hand, they may also offer a complementary explanation for the strong decreases of the lipolysis kinetics that are frequently observed after few minutes of intestinal in vitro digestions. Indeed, such slowdowns are often attributed to an inhibition induced by the reaction products that accumulate at the interface, with no or little attention given to a possible coalescence or flocculation of the emulsion droplets. This is most probably because such phenomena have not been expected to occur in the conditions encountered in the intestinal phase because of high concentrations of bile salts and no macroscopic visual evidences. Nevertheless, several recent studies have shown that droplet flocculation and coalescence might in fact be encountered during the intestinal phase of in vitro experiments [START_REF] Mun | Influence of emulsifier type on in vitro digestibility of lipid droplets by pancreatic lipase[END_REF][START_REF] Singh | Structuring food emulsions in the gastrointestinal tract to modify lipid digestion[END_REF][START_REF] Day | Tailoring the digestion of structured emulsions using mixed monoglyceride-caseinate interfaces[END_REF][START_REF] Li | Physicochemical behaviour of WPI-stabilized emulsions in in vitro gastric and intestinal conditions[END_REF][START_REF] Sarkar | Pancreatin-induced coalescence of oil-in-water emulsions in an in vitro duodenal model[END_REF], similarly to what has now been established for the gastric phase [START_REF] Golding | Impact of gastric structuring on the lipolysis of emulsified lipids[END_REF][START_REF] Wooster | Impact of different biopolymer networks on the digestion of gastric structured emulsions[END_REF][START_REF] Day | Tailoring the digestion of structured emulsions using mixed monoglyceride-caseinate interfaces[END_REF].

The exact cause for the occurrence of coalescence still remains to be studied because the nature of the interfacial layer is continuously evolving, especially in the case of proteinstabilized emulsion. The TAG composition of the droplets is evolving in the course of the reaction [START_REF] Ye | Effect of calcium on the kinetics of free fatty acid release during in vitro lipid digestion in model emulsions[END_REF] and a competitive adsorption process takes place at the interface between emulsifier molecules, enzymes, bile salts and the products of the lipolysis reactions. It has been reported that hydrolysis of the proteins adsorbed at the interface can weaken the droplet repulsion forces and favor droplet flocculation or coalescence [START_REF] Li | Physicochemical behaviour of WPI-stabilized emulsions in in vitro gastric and intestinal conditions[END_REF][START_REF] Kenmogne-Domguia | Gastric conditions control both the evolution of the organization of protein-stabilized emulsions and the kinetic of lipolysis during in vitro digestion[END_REF], possibly explaining why protein stabilized emulsions seem more sensitive to these phenomena [START_REF] Singh | Structural and biochemical factors affecting the digestion of protein-stabilized emulsions[END_REF]. Droplet coalescence during digestion has also been reported to be promoted by the accumulation of monoacylglycerols and fatty acids at the interface [START_REF] Wooster | Impact of different biopolymer networks on the digestion of gastric structured emulsions[END_REF][START_REF] Ye | Effect of calcium on the kinetics of free fatty acid release during in vitro lipid digestion in model emulsions[END_REF], that is, by the same mechanism as that usually put forward to support an inhibition of the enzymatic reaction. More studies are therefore needed to determine the frequency of droplet coalescence during in vitro intestinal digestions and to better understand its consequences on the lipolysis kinetics.

Conclusion

Our study confirms previously reported results showing that the kinetics of lipolysis is much faster for MCT than for LCT when they are mixed together in the same emulsion. In agreement with recent studies, it also confirms that whey protein stabilized submicron emulsions are prone to coalescence during the intestinal phase of in vitro digestions.

Moreover, by accounting for the experimentally measured droplet distributions in a modeling approach, we were able to adequately reproduce the two-stage lipolysis curve recovered for LCT with an initial fast reaction rate that markedly slowed down after about 30 min. These modeling results demonstrate that droplet coalescence had a considerable impact on the lipolysis kinetics of the remaining LCT by causing a sharp reduction of the interfacial area available for the adsorption of pancreatic lipase-colipase. Contrarily to what is generally postulated for intestinal lipid digestion, our findings suggest that inhibition of the enzymatic reaction might not always be the key mechanism explaining why in vitro lipolysis kinetics of emulsified lipids often plateaus before the reaction is completed.

Fig 1 .

 1 Fig 1. Calibration curve relating the HPLC signal to the injected mass of TAG. The light

Fig 2 .

 2 Fig 2. HPLC chromatogram stemming from an undigested sample (circles) superimposed

Fig 3 .

 3 Fig 3. (A) Typical evolution of HPLC chromatograms during the intestinal phase of in vitro

Fig 4 .

 4 Fig 4. (A) Typical evolution of the droplet size distributions measured without deflocculating

Fig 5 .

 5 Fig 5. (A) Extent of LCT lipolysis measured by HPLC (symbols) and the fits obtained with i)

Fig 6 .

 6 Fig 6. Evolution of the interfacial area during the intestinal phase according to our model that

  Figure4

Acknowledgements

The authors thank Valérie Beaumal (UR1268 BIA) for the preparation of the freeze-dried emulsion and Claude Genot and Sebastien Marze for helpful remarks and discussions. This work was supported by the Institut National de la Recherche Agronomique and the Institut Carnot QUALIMENT (France). The authors are involved in the Food and Agriculture COST (European Cooperation in Science and Technology) Action FA1005 'Improving health properties of food by sharing our knowledge on the digestive process (INFOGEST)', http://www.cost-infogest.eu


Thibaut Boibessot

Christopher Zschiedrich

Alexandre Lebeau

David Bénimélis

Catherine Dunyach-Remy

Jean-Philippe Lavigne

Hendrik Szurmant

Zohra Benfodda

Patrick Meffre

The Rational Design, Synthesis, and Antimicrobial Properties of Thiophene Derivatives That Inhibit Bacterial Histidine Kinases

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


Guillaume Drutel

Markus Kathmann

Anne Heron

Jean-Charles Schwartz

Jean-Michel Arrang

Cloning and Selective Expression in Brain and Kidney of ARNT2 Homologous to the Ah Receptor Nuclear Translocator (ARNT)

Arnt2, a new member of the basic-helix-loop-helix transcription factor family, was cloned from rat brain cDNAs. Its deduced 712 amino acid sequence displays 63% identity with that of the aryl hydrocarbon receptor nuclear translocator (Arnt1) that was completely established. Whereas Arnt2 gene expression, established by Northern blotting and in situ hybridization histochemistry, occurred selectively in brain and kidney, that of Arnt1 was ubiquitous, suggesting that the two proteins play distinct roles, presumably via dimerization and DNA binding with different partners.

Arnt, the Ah receptor nuclear translocator, is a member of the basic helix-loop-helix (bHLH) superfamily of DNA binding proteins, originally found to form a heterodimer with the aryl hydrocarbon receptor (Ahr) in response to environmental pollutants such as dioxins. The Ahr/Arnt heteromer translocates to the nucleus and binds specific recognition sequences localized upstream of various dioxin-inducible genes such as cytochromes P450 [reviewed in refs. [1][2][3].

Arnt sequence contains a conserved region termed the PAS domain and shared by other transcription factors [4]. Recent observations suggest that Arnt might have regulatory roles via heterodimerization not only with Ahr but also with other bHLH-containing PAS partners such as the single-minded protein (Sim), involved in central nervous system development [5,6] and the hypoxia-inducible factor 1a (HIF-1a) involved in adaptive responses of various cells and tissues to hypoxia [START_REF] Wang | Proc. Natl. Acad. Sci[END_REF].

Following RTPCR amplification of cerebral mRNAs using degenerate amplimers of heptahelical receptors, we unexpectedly cloned a cDNA of unknown sequence displaying however distinct homology with that of human or mouse Arnt (called Arnt1 here) [4,[START_REF] Li | [END_REF]. Here we report the full length sequence of this novel protein (designated Arnt2) as well as that of rat Arnt1, of which only a fraction was previously reported [9]. The latter was established for purpose of comparison and in order to design selective hybridization probes. Northern blot and in situ hybridization studies indicate that the two homologous gene transcripts are differently distributed among rat tissues. Recently, while this manuscript was in preparation, the sequence and properties of a mouse Arnt2 were disclosed [10].

MATERIALS AND METHODS

Cloning and sequencing of a rat Arnt2 cDNA. A single strand cDNA was synthesized using AMV-reverse transcriptase (20 units, Boehringer) and rat frontal cortex poly(A) / mRNAs (1 mg). This template was amplified for 35 cycles (947C, 507C and 727C for 1, 1 and 3 min, respectively) using Taq polymerase (2,5 units, Cetus) and two degenerate oligonucleotides based on the sequence of the third and sixth transmembrane domains of heptahelical receptors [11]. Sense (5*-CCCAAGCTTAAGTAGGG(CA)A(GA)CCAGCA(GTC)AI(GA)A(GT)GAA-3*) and anti-sense (5*-CCCAAGCTTCT(CG)TT(CT)(AG)T(CG)AT(CT)AGIIT(GT)GA(CT)(CA)GCG-3pr) primers were synthesized with HindIII restriction sites at their 5* ends. PCR products were cloned into pGEM 4Z (Promega) and sequenced by the dideoxy nucleotide chain termination method [START_REF] Sanger | Proc. Natl. Acad. Sci. USA[END_REF] with Sequenase version 2.0 (United States, Biochemical Inc.). Unexpectedly, one clone (A31) contained a 0.75 kb fragment which displayed significant homology with the PAS domain of the human and mouse Arnt1 [4,[START_REF] Li | [END_REF]. Therefore, the corresponding protein was designated Arnt2.

To obtain a full length Arnt2 sequence, a rat hypothalamus cDNA library constructed in l ZapII was screened (10 6 phages) at high stringency with a 32 P-labelled DNA fragment corresponding to A31. Among 24 positive clones, one exhibited an open reading frame encoding a protein of 712 amino acids.

Cloning and sequencing of a full-length rat Arnt1 cDNA. A single strand cDNA was synthesized using AMVreverse transcriptase (20 units, Boehringer) and rat stomach poly(A) / mRNAs (1 mg). This template was amplified for 35 cycles (947C, 567C and 727C for 1, 1 and 2 min, respectively) using the Expand high fidelity PCR system (Boehringer) and two primers derived from the 5* and 3* non coding sequences of the human Arnt1 [4]. The sense (5*-GGTGGCGCGGCGGCGGTGGCATCA-3*) and antisense (5*-CCTTATCCTCACCCCAATAGTTCT-3*) primers corresponded to nucleotides 27 to 50 and 2426 to 2448 respectively. The amplified DNA was subcloned into pGEM 4Z and sequenced.

Northern blotting. Poly(A) / mRNAs (8 mg) from various tissues of adult male Wistar rats were denaturated, electrophoresed and transferred to nitrocellulose membranes as described [START_REF] Traiffort | Proc. Natl. Acad. Sci[END_REF]. Hybridization was carried out overnight at 427C with probes 32 P-labelled by random priming and corresponding to fragments of the rat Arnt1 or Arnt2 sequence (amino acids (a.a) 153 to 374 and 195 to 433 respectively) (Fig. 1). Blots were washed twice in 2 1 SSC /0.1% SDS at 427C for 30 min and once in 0.2 1 SSC /0.1% SDS at 427C for 30 min. They were then exposed to X-ray films at 0807C for 21 days.

In situ hybridization. Cryostat sections (12 mm) of 4-day old rats were treated essentially as described [START_REF] Bouthenet | [END_REF]. The sections were hybridized in the presence of 50% formamide and 6 1 10 6 dpm antisense or sense RNA probes at 507C (Arnt1) or 557C (Arnt1). The highly variable C-terminal coding and non coding nucleotide sequences of rat Arnt1 or Arnt2 were used as probes for hybridization experiments. The probe corresponded to a.a. 683 to 796 for Arnt1. A 0.39 kb probe corresponding to the nucleotide sequence encoding from a.a. 608 and encompassing a part of the 3* non coding sequence was used for Arnt2. After subcloning into pGEM 4Z, antisense and sensestrand 33 P-labeled probes were synthesized in vitro using SP6 or T 7 RNA polymerase of Riboprobe kit (Promega).

RESULTS

Deduced amino acid sequences of rat Arnt1 and Arnt2. The full length cDNA sequence of rat Arnt2 derived from degenerate PCR and brain cDNA library screening presented an open reading frame (ORF) encoding a protein of 712 aminoacids (Fig. 1). This ORF starts by an ATG codon, the most upstream of a series of ATG codons and surrounded by 7 out of 10 nucleotides belonging to a consensus initiation sequence [15]. This sequence displayed high degrees of homology with those of full length human Arnt1 [4] and the partial sequence of rat Arnt1 [9]. We also cloned and sequenced a full length rat Arnt1 using PCR with primers designed in highly conserved regions of the human Arnt1. Two kinds of PCR products were obtained with ORFs encoding proteins of either 785 or 800 amino acids, presumably depending on the presence or absence of an alternative exon (sequence underlined in Fig. 1). Arnt1 and Arnt2 sequences display a 63% identity overall, a value increased to 75% and 95% considering the conserved PAS bHLH domains respectively. The C-terminal end of sequences, following the PAS domain, displayed only 45% identity and a glutamine-rich region was present in both, although much longer in rArnt1 than in rArnt2 (Fig. 1), mArnt1 or hArnt1 [4,[START_REF] Li | [END_REF].

Analysis of the sequences by the PROSITE program [16] showed 1, 9 and 8 consensus phosphorylation sites for protein kinases A and C and casein kinase II respectively in Arnt2 whereas corresponding values for Arnt1 were 1, 10 and 16 respectively. Protein kinase C sites were absent from the hHLH domains of rArnt1 and rArnt2 whereas they occur in this domain of human AhR [17].

Analysis of Arnt1 and Arnt2 gene expression in rat tissues. Northern blot analysis of Poly(A) / mRNAs from rat tissues indicated the presence of a major 4.8-kb Arnt1 mRNA, highly expressed in adrenals, kidney, stomach and lung; lower signals were also detected in cerebral cortex and heart (Fig. 2).

Analysis of the same blot using a probe for rat Arnt2 mRNAs indicated the presence of a 7-kb transcript, highly expressed in cerebral cortex and kidney. A lower signal of the 7-kb transcript was detected in the stomach, a 6.7-kb Arnt2 mRNA in the lung and no signals in adrenals and heart (Fig. 2).

The distribution of Arnt1 and Arnt2 gene transcripts was studied further by in situ hybridization analysis performed on sagittal sections of newborn rats with an antisense RNA probe prepared in a specific (C-terminal) part of the Arnt1 or Arnt2 sequences. When compared with the corresponding sense probes for which no background signals could be seen (data not shown), the Arnt1-and Arnt2-antisense riboprobes generated clear signals (Fig. 3). The Arnt1 gene was expressed in many (if not all) tissues. The highest expression level occured in the kidney, adrenals, intestine, spleen, salivary glands and retina. Signals could also be easily detected in the thymus, stomach, lung, brain and skin. Lower signals were found in many other tissues such as the heart and liver. The expression pattern of Arnt2 was very different from that of Arnt1, with a highly discrete distribution of transcripts. High signals were found only in the brain, spinal cord (data not shown), sympathetic and spinal ganglia and kidney. In the latter, Arnt2 was highly expressed both in the external cortex and medulla. Lower signals were also found in the retina (Fig. 3).

FIG. 2.

Northern blot analysis. Poly(A) / mRNAs (8 mg) from adult rat tissues were used. The blot was hybridized first with the 0.72-kb Arnt2 cDNA probe, and then dehybridized before hybridization with the 0.71-kb Arnt1 probe. As a control, the blot was finally rehybridized with a b-actin cDNA which showed similar signals and minimal degradation in all samples (not shown). Blots were exposed for 21 days at 0807C with intensifying screens. The 0.24 to 9.5 kb RNA ladder was used for size markers, on the left. Cer. Cortex, cerebral cortex.

DISCUSSION

The sequence of the rat Arnt2 protein that we disclose here indicates that Arnt2 represents a new member of the bHLH-PAS family of transcription factors. Analysis of the bHLH and PAS domains revealed that Arnt2 is most similar to rat Arnt 1 than to any other member of this family (Fig. 1) suggesting that Arnt2 functions, like Arnt1, as a nuclear translocator. The high homology (95% and 75% identity, respectively) found within the two domains suggests that Arnt1 and Arnt2 interact with the same partners (through the HLH and PAS motifs) and DNA recognition sequences (through the DNA binding (b) motif). The 15 a.a. sequence encoded by an alternative exon within the N terminal part of Arnt1 ( [4] and Fig. 1) and identifying two isoforms of this protein was not found in the corresponding sequence of Arnt2. However the putative existence of alternative exons in Arnt2 remains to be assessed by additional studies. The C-terminal sequence of both proteins beyond the PAS region is quite variable, with a remarkable Gln repeat (16 residues) found in the Q-rich region of Arnt1 but a shorter one in Arnt2, possibly indicating a different transcriptional activity of this region of the two (putative) translocators. In addition, Arnt1 and Arnt2 sequences contain multiple but different consensus sites for protein kinases. Whether the respective phosphorylation of Arnt1 and Arnt2 proteins differentially affects the heterodimerization with their respective bHLHPAS partners remains to be elucidated [1].

FIG. 3.

In situ hybridization analysis. Sagittal sections of newborn (postnatal day 4) rats were hybridized with 33 Plabelled antisense or sense riboprobes for Arnt1 or Arnt2 and exposed for 21 days. Note the restricted localization of Arnt2 mRNAs in brain and kidney and the ubiquitous distribution of Arnt1 mRNAs. Adjacent sections hybridized with a sense probe did not show any signal (not shown). The panels are dark-field photographs of autoradiograms. Saliv. Gl., Salivary gland; gg, ganglion.

Northern blot analysis showed different sizes for the transcripts of the two genes in rat tissues, i.e. 4.8 kb for Arnt1 and 7.0 kb for Arnt2. In lung there was an additional Arnt2 mRNA of 6.7-kb which may correspond to the use of distinct initiation or polyadenylation sites or to a splice variant.

In situ hybridization data of whole body sections of young rats, in agreement with data from Northern blot analysis, showed that the two genes are expressed in a markedly contrasted manner among rat tissues. Thus, Arnt1 gene is expressed in a highly ubiquitous manner in many peripheral organs as well as in selected regions of the central nervous system (hippocampus, cerebral and cerebellar cortices). This observation is consistent with data from Northern blot analysis and ribonuclease protection assays in selected rat or mice tissues [5,9] and with in situ hybridization histochemistry of rat brain [18]. Our data also point out to additional, previously non disclosed localisations of Arnt1 mRNAs such as in retina, adrenals or salivary glands.

In contrast, high expression of the Arnt2 gene was mainly restricted to the central and peripheral nervous system (brain, spinal cord, autonomic ganglia) and to the kidney (outer cortex and medulla).

All these data taken together strongly suggest that Arnt1 and Arnt2 play different physiological roles probably involving different partners.

While our work was progressing, Hirose et al. [10] reported the cloning of a mouse Arnt2, the amino acid sequence of which displays 99% identity with that of rat Arnt2. In agreement with our data, mouse Arnt2 was only expressed in the brain and kidney of adult mice when analyzed in northern blots, whereas the expression of Arnt1 was ubiquitous. In situ hybridization experiments with early mouse embryos in which AhR and Sim were previously shown to be expressed, also revealed a distinct expression pattern of Arnt1 and Arnt2.

FIG. 1 .

 1 FIG. 1. Amino acid sequences of rat Arnt1 and Arnt2. Alignment was done by the program TREEALIGN of Hein et al. [19]. Conserved amino acids are indicated (*). The sequence corresponding to the alternative exon of Arnt1 is underlined. Dashed lines indicate gaps inserted to improve alignment. The nucleotide sequence of the Arnt1 and Arnt2 clones has been submitted to the GenBank data base with accession numbers U61184 and U61405, respectively.

ACKNOWLEDGMENTS

The authors thank Véronique Cochois for skilled technical assistance and Annie Galtier for typing the manuscript. Guillaume Drutel is a recipient of a fellowship from Association pour la Recherche sur le Cancer.


A Mutschler

A Lemasson

O Sorlin

D Bazin

C Borcea

R Borcea

Z Dombrádi

J.-P Ebran

A Gade

H Iwasaki

E Khan

A Lepailleur

F Recchia

T Roger

F Rotaru

D Sohler

M Stanoiu

S R Stroberg

J A Tostevin

M Vandebrouck

D Weisshaar

K Wimmer

A proton density bubble in the doubly-magic 34 Si nucleus

Keywords:

Many properties of the atomic nucleus, such as vibrations, rotations and incompressibility can be interpreted as due to a two-component quantum liquid of protons and neutrons. Electron scattering measurements on stable nuclei demonstrate that their central densities are saturated, as for liquid drops. In exotic nuclei near the limits of mass and charge, with large imbalances in their proton and neutron numbers, the possibility of a depleted central density, or a "bubble" structure, was discussed in a recurrent manner since the seventies. Here we report first experimental evidence that points to a depletion of the central density of protons in the short-lived nucleus 34 Si. The proton-to-neutron density asymmetry in 34 Si offers the possibility to place constraints on the density and isospin dependence of the spin-orbit force -on which nuclear models have disagreed for decades-and on its stabilizing effect towards limits of nuclear existence.

Microscopic systems composed of atoms or clusters can exhibit intrinsic structures that are bubble-like, with small or depleted central densities. For example, the fullerene molecules, composed of C atoms, are structures with extreme central depletion [START_REF] Schultz | Topological Organic Chemistry. Polyhedranes and Prismanes[END_REF]. In nuclear physics, depletions also arise in nuclei with well-developed cluster structures when clusters are arranged in a triangle or ring-like structure -such as in the triple-α Hoyle state [START_REF] Hoyle | On Nuclear Reactions Occurring in Very Hot Stars. I. the Synthesis of Elements from Carbon to Nickel[END_REF][START_REF] Freer | The Hoyle state in 12 C[END_REF]. Unlike such a non-homogeneous, clustered system, central density depletions or bubble-like structures would be much more surprising in homogeneous systems, such as typical atomic nuclei with properties characteristic of a quantum liquid [START_REF] Ebran | How atomic nuclei cluster?[END_REF].

This hindrance of bubble formation in atomic nuclei is inherent in the nature of the strong force between nucleons, which is strongly repulsive at short distances (below 0.7 fm), attractive at medium range (≈1.0 fm) and vanishes at distances beyond 2 fm. In a classical picture, the medium-ranged attraction of nuclear forces implies that nucleons interact strongly and attractively only with immediate neighbors, leading to a saturation of the nuclear central density, ρ 0 . Quantum mechanically, the delocalization of nucleons [START_REF] Mottelson | Nuclear Structure, Trends in Nuclear Physics, 100 years later[END_REF] leads to a further homogeneity of the density. Extensive precision electron scattering studies from stable nuclei [START_REF] Hofstadter | Electron Scattering and Nuclear Structure[END_REF] confirm that their central densities are essentially constant, with ρ 0 ≈ 0.16 fm -3 , independent of the number of nucleons A. As a consequence, like a liquid drop, the nuclear radii and volumes increase as A 1/3 and as A, respectively. Thus, a priori, bubble-like nuclei with depleted central densities are unexpected.

Historically, the possibility of forming bubble nuclei was investigated theoretically in intermediate-mass [START_REF] Campi | Possible bubble nuclei 36 Ar and 200 Hg[END_REF][START_REF] Davis | Generalized shells in nuclei: Hartree-Fock calculations for bubble nuclei[END_REF][START_REF] Khan | Detecting bubbles in exotic nuclei[END_REF][START_REF] Grasso | Nuclear 'bubble' structure in 34 Si[END_REF], superheavy [START_REF] Bender | Shell structure of superheavy nuclei in self-consistent mean-field models[END_REF] and hyperheavy systems [START_REF] Dechargé | Bubbles and semi-bubbles as a new kind of superheavy nuclei[END_REF]. In general, central depletions will arise from a reduced occupation of single particle orbits with low angular momentum . These wave functions extend throughout the nuclear interior whereas those with high-are more excluded by centrifugal forces. For example, in a comparison of the charge densities of 206 Pb and 205 Tl, the contribution from =0 orbits (there 3s) is peaked at the nuclear center [START_REF] Cavedon | Is the Shell-Model Concept Relevant for the Nuclear Interior?[END_REF]. The amplitude of this central depletion in 205 Tl is of order 11%. A much larger central depletion of protons, of about 40% compared to stable 36 S, was proposed in 34 Si [START_REF] Grasso | Nuclear 'bubble' structure in 34 Si[END_REF][START_REF] Li | Pseudospinorbit splitting and its consequences for the central depression in nuclear density[END_REF] using various mean field approaches, arising from the proton occupancy of the 2s 1/2 orbital. However, recent theoretical calculations suggest that nuclear correlations act to smoothen these orbital occupancies in both the heavy and superheavy nuclei [START_REF] Wang | Pseudospin-orbit splitting and its consequences for the central depression in nuclear density[END_REF][START_REF] Afanasjev | Central depression in nuclear density and its consequences for the shell structure of superheavy nuclei[END_REF] and in 34 Si [START_REF] Yao | Beyond-mean-field study of the possi-ble 'bubble' structure of 34 Si[END_REF]. Here, we use the one-proton removal (-1p) reaction technique to show that the 2s 1/2 proton orbit in 34 Si is in fact essentially empty, in contrast to 36 S where this 2s 1/2 orbit is almost fully occupied by 1.7(4) protons compared to the maximum occupancy of 2 [START_REF] Khan | The mass of 35 P and spin-parity assignments for excited 35 P states[END_REF][START_REF] Mutschler | Spectroscopy of 35 P using the oneproton knockout reaction[END_REF].

A beam of 4 × 10 5 34 Si nuclei per second was produced by the fragmentation of a 140 MeV/u 48 Ca primary beam on a 846 mg•cm -2 thick 9 Be target at the Coupled Cyclotron Facility at the National Superconducting Cyclotron Laboratory. The 34 Si then impinged on a 9 Be secondary target (100 mg•cm -2 ) producing 33 Al nuclei through the (-1p) reaction. These 33 Al residues were identified through their measured energy-loss in an ionization chamber located at the focal plane of the S800 spectrograph, and their time-of-flight between two scintillators placed at the object and image focal planes of the device. Their trajectories were obtained from their positions measured at two cathode-readout drift chambers.

Prompt γ-rays, originating from the in-flight decay of excited 33 Al produced during the reaction, were detected in coincidence with the 33 Al residues in the seven modules of the GRETINA array [START_REF] Paschalis | The performance of the Gamma-Ray Energy Tracking In-beam Nuclear Array GRETINA[END_REF] that surrounded the target at angles near 90 • and 58 • . Event-by-event Doppler reconstruction was performed using the deduced 33 Al velocity at the mid-target position, the position reconstruction on the target, and the γ-ray detection angle -determined from the position of the greatest energy deposition in the GRETINA array. An absolute in-flight efficiency of 6.5% and an energy resolution of σ 2 keV, respectively, were obtained at 1 MeV, based on the use of calibrated sources and GEANT4 simulations [START_REF] Agostinelli | Geant4: a simulation toolkit[END_REF] to account for the Lorentz boost. A systematic uncertainty of 0.25% is estimated on the γ energy centroid.

The γ singles (first row of Fig. 1), γ -γ coincidences (second to third rows of Fig. 1), and the relative γ intensities were used to establish the level scheme of excited states in 33 Al, shown in the left part of Fig. 2. Energies and branching ratios are given for each populated 33 Al final state. The energy of seven γ-rays match, within uncertainties, those observed in the β-decay of 33 Mg [START_REF] Tripathi | Intruder Configurations in the A=33 Isobars: 33 Mg and 33 Al[END_REF]. However, the level scheme proposed in Fig. 2 differs significantly from that of Ref. [START_REF] Tripathi | Intruder Configurations in the A=33 Isobars: 33 Mg and 33 Al[END_REF] where, unlike in the present work, γ -γ coincidences were rarely exploited.

The orbital angular momenta of the protons removed from 34 Si are determined by comparing, in the right panel of Fig. 2, the experimental (γ-gated) and theoretical longitudinal momentum distributions (p ) of the 33 Al. The latter are described in the Section 'Methods'. The ground state momentum distribution in Fig. 2 is obtained by subtracting contributions from excited states. The p distributions of the 0-, 1621-, and 1651-keV states are characteristic of =2 proton removal. The much narrower p of the 210-and 3704-keV states suggest =0 assignments.

Normalized spectroscopic factors, C 2 S exp norm , and proton occupancies of the orbits are derived from the experimental cross sections, as described in Section 'Methods'. The summed spectroscopic factors for the first three =2 states is 5.5 ± 1.0, consistent, within uncertainties, with a full occupancy (2J + 1=6) of the 1d 5/2 orbit. The weak FIG. 1. Gamma-ray spectra of 33 Al. Singles (first row) and γ-gated (second to third rows) Doppler-corrected γ-ray spectra of 33 Al. The γ-rays labeled with an empty triangle, accounting for 5.8 % of the cross section, are not produced by the one-proton removal mechanism and are not relevant for determining the proton occupancies. Their origin is discussed in Ref. [START_REF] Mutschler | Spectroscopy of 35 P using the oneproton knockout reaction[END_REF].

population of the 3926 keV state, with a spectroscopic factor of 0.11 [START_REF] Freer | The Hoyle state in 12 C[END_REF], is tentatively attributed to an =2 proton knockout from the 1d 3/2 orbit. A proton occupancy of the 2s 1/2 orbital of 0.17(3) is deduced from the two observed =0 excited states at 2101 and 3704 keV. Assuming, though very unlikely, that the four (unassigned) weakly populated states at 2666, 2696, 2816 and 3193 keV were also =0, this occupancy would be increased by only 0.07 (3). This very small proton occupancy of the 2s 1/2 orbital, 10% of that in 36 S, results in a large depletion of the central density of protons in 34 Si.

On the other hand, the neutron 2s 1/2 orbit is essentially fully occupied in 34 Si, with a summed spectroscopic factor value of 2.0(3) being deduced from the corresponding neutron removal reaction from 34 Si [START_REF] Enders | Single-neutron knockout from 34,35 Si and 37 S[END_REF]. Thus, 34 Si exhibits a large proton-to-neutron density asymmetry that, to our knowledge, has not been revealed in any other nucleus. It is favored because 34 Si can be viewed as a doubly-magic nucleus in which mixing between normally occupied and valence orbits is very limited [START_REF] Enders | Single-neutron knockout from 34,35 Si and 37 S[END_REF][START_REF] Burgunder | Experimental Study of the Two-Body Spin-Orbit Force in Nuclei[END_REF]. The high energy of its first excited state (3.3 MeV), its low reduced transition probability B(E2; 0 + → 2 + ) [START_REF] Ibbotson | Quadrupole Collectivity in 32,34,36,38 Si and the N=20 Shell Closure[END_REF] and the small electric monopole strength ρ(E0; 0 + 1 → 0 + 2 ) [START_REF] Rotaru | Unveiling the Intruder Deformed 0 + 2 State in 34 Si[END_REF] complete this picture of double magicity. Fig. 3 visualizes the changed proton densities and almost unchanged neutron densities between 34 Si and 36 S from Relativistic values result from the uncertainty in extracting the intensity of the γ transitions decaying from the corresponding levels. When these levels are fed from higher lying states, the corresponding feeding contribution was subtracted, inducing larger error bars. As fed by many transitions, the branching ratio to the ground state therefore has the largest error bar. The J π assignments and experimental spectroscopic factors Hartree-Fock Bogoliubov (RHFB) calculations that use the PKO2 energy density functional [START_REF] Ebran | Relativistic Hartree-Fock-Bogoliubov model for deformed nuclei[END_REF] and which predict very similar proton and neutron occupancies to those deduced here. It should however be noted that mean field calculations do not all predict similar neutron and proton density profiles in 34 Si. Indeed they are very sensitive to the size of the proton and neutron gaps derived from the choice of functionals, as well as to the treatment of pairing and quadrupole correlations that act to reduce the central density depletion, as found in Ref. [START_REF] Yao | Beyond-mean-field study of the possi-ble 'bubble' structure of 34 Si[END_REF]. Indeed, this model-dependence of the predictions of the existence of a central depletion was a major motivation to perform the present experiment.

With this differential two-fluid behavior, 34 Si offers unique possibilities to test the density and proton-toneutron (isospin) dependence of the nuclear spin orbit (SO) potential -which generates most of the shell gaps that stabilize magic nuclei in the chart of nuclides [START_REF] Sorlin | Evolution of the N=28 shell closure: a test bench for nuclear forces[END_REF][START_REF] Sorlin | Nuclear magic numbers: New features far from stability[END_REF]. In most theoretical models, the SO potential can be expressed in terms of the derivative of the proton and neutron densities, with coefficients that differ by a factor of as much as two between various relativistic or non-relativistic approaches (see e.g. the discussions in [START_REF] Bender | Shell structure of superheavy nuclei in self-consistent mean-field models[END_REF][START_REF] Sorlin | Nuclear magic numbers: New features far from stability[END_REF][START_REF] Sharma | Shell effects in nuclei near the neutron-drip line[END_REF][START_REF] Sharma | Isospin Dependence of the Spin-Orbit Force and Effective Nuclear Potentials[END_REF][START_REF] Lalazissis | Reduction of the spin-orbit potential in light drip-line nuclei[END_REF][START_REF] Holt | Nuclear energy density functional from chiral two-nucleon and three-nucleon interactions[END_REF]). These as yet unknown density and isospin dependences of the SO interaction strongly impact (i) the evolution of the spin-orbit interaction and magic numbers as one approaches the drip lines [START_REF] Sharma | Shell effects in nuclei near the neutron-drip line[END_REF][START_REF] Lalazissis | Reduction of the spin-orbit potential in light drip-line nuclei[END_REF], where the surface diffuseness is increased and consequently the SO interaction is expected to be reduced. This influences the binding energies, the lifetimes, and nuclear capture rates of the nuclei close to the neutron drip line that are involved in the synthesis of elements in the Universe beyond Fe through the rapid neutron capture process. This also impacts (ii) the location of a possible island of stability for superheavy nuclei [START_REF] Bender | Shell structure of superheavy nuclei in self-consistent mean-field models[END_REF] that differ strongly depending on the theoretical models used, and (iii) the puzzling discontinuity in the isotope shifts observed for the Pb isotopes [START_REF] Reinhard | Nuclear effective forces and isotope shifts[END_REF][START_REF] Sharma | Isospin Dependence of the Spin-Orbit Force and Effective Nuclear Potentials[END_REF], a phenomenon that seems to be accounted for only by a certain category of models. These aspects of the SO force have not previously been accessible to experimental scrutiny as, in the vast majority of nuclei, the saturation of the nuclear forces implies (a) a near-constant central density for protons and neutrons, and (b) an almost universal surface diffuseness. The result is a SO force peaked at the proton and neutron surfaces having a similar strength for all models. The central proton density depletion in 34 Si drives an additional (interior) component of the SO force, with a sign opposite to that at the surface. Therefore, low-nucleons, that can probe the interior of the bubble, should encounter a much weaker overall SO force (e.g. [START_REF] Bender | Shell structure of superheavy nuclei in self-consistent mean-field models[END_REF][START_REF] Todd-Rutel | Spinorbit splitting in low-j neutron orbits and proton densities in the nuclear interior[END_REF]) and display a significantly reduced SO splitting. This prediction is in line with the observed reduction of the neutron 2p 3/2 -2p 1/2 splitting in 35 Si [START_REF] Burgunder | Experimental Study of the Two-Body Spin-Orbit Force in Nuclei[END_REF], when compared to neighbouring N = 21 isotones. Such a sudden change by a factor of two in amplitude is unique on the chart of nuclides and seems clearly connected to the change in central nuclear density observed here. Moreover, having different proton and neutron central densities, 34 Si can be used to constrain the isospin dependence of the SO interaction in an unprecedented manner, for example, by identifying models that predict the correct amplitude of the SO reduction.

Finally, atomic nuclei are usually highly incompressible, the corresponding monopole modes involving very high excitation energies [START_REF] Harakeh | Direct Evidence for a New Giant Resonance at 80A -1/3 MeV in the Lead Region[END_REF][START_REF] Youngblood | Isoscalar Breathing-Mode State in 144 Sm and 208 Pb[END_REF]. Exhibiting a central density that is significantly lower than the saturation density, 34 Si may present new (soft) compression modes at low energy with the potential to shed light on the recently observed fragmentation of the giant monopole at low energy in the neutron-rich 68 Ni nucleus [START_REF] Vandebrouck | Measurement of the Isoscalar Monopole Response in the Neutron-Rich Nucleus 68 Ni[END_REF]. This information would in turn be useful for testing different models of the nuclear equation of state at a density below the saturation density, important for instance in the modeling of the neutron star crust.

METHODS

The eikonal model and choice of parameters used to calculate the proton removal single-particle cross sections, σ sp α , and the parallel momentum, p , distributions of the residues are detailed in Ref. [START_REF] Gade | Reduction of spectroscopic strength: Weakly-bound and strongly-bound single-particle states studied using one-nucleon knockout reactions[END_REF]. The shapes of the high momentum parts of these distributions are used in the comparisons with experiment in Fig. 2 as more dissipative collisions, treated only approximately in the eikonal model, affect measured distributions at the lower momenta [START_REF] Stroberg | Single-particle structure of silicon isotopes approaching 42 Si[END_REF]. In comparing to experiment, the theoretical p distributions are convoluted with (i) the momentum dispersion of the secondary beam, (ii) the beam straggling in the target, and (iii) the momentum broadening due the reaction's position within in the target.

The experimental partial cross sections, b KO f σ exp inc,KO , correspond to the removal of a proton with quantum numbers n J from the ground state of 34 Si. Here σ exp inc,KO is the experimental inclusive removal cross section, that amounts to 27.7(1.0) mb, and b KO f is the experimental branching ratio (in %) for populating final state f . Following Ref. [START_REF] Mutschler | Spectroscopy of 35 P using the oneproton knockout reaction[END_REF], the normalized knockout spectroscopic factors are expressed as:

C 2 S exp norm = b KO f σ exp inc,KO Rsσ sp f , (1)
where σ sp f is the theoretical single-particle knockout cross section [START_REF] Gade | Reduction of spectroscopic strength: Weakly-bound and strongly-bound single-particle states studied using one-nucleon knockout reactions[END_REF]. Rs accounts for the systematic quenching of measured nucleon knockout cross sections when compared to those calculated when combining these eikonal model σ sp f with shell model spectroscopy and C 2 S [START_REF] Tostevin | Systematics of intermediateenergy single-nucleon removal cross sections[END_REF]. With this normalization, the C 2 S exp norm sum rule (or orbit occupancies) to states in 33 Al are normalized to the maximal occupancy of a given sub-shell, that is 2J + 1.

We are aware that short-range correlations [START_REF] Pandharipande | Independent particle motion and correlations in fermion systems[END_REF] and coupling to collective degrees of freedom [START_REF] Barbieri | Role of Long-Range Correlations in the Quenching of Spectroscopic Factors[END_REF] usually complicate the determination of spectroscopic factors (or their related shell occupancies and vacancies), which are not directly observable [START_REF] Dickhoff | Self-consistent Green's function method for nuclei and nuclear matter[END_REF][START_REF] Th | Nonobservable nature of the nuclear shell structure: Meaning, illustrations, and consequences[END_REF][START_REF] Furnstahl | Are occupation numbers observable?[END_REF]. Moreover, present reaction models use effective potentials that do not capture the full microscopic complexity of the nucleus and often induce uncertainties in the deduced results. However, under the reasonable assumption that these effects are similar between neighboring nuclei, here between the closed-shell nuclei 36 S and 34 Si, the consideration of a differential evolution of spectroscopic strengths and occupancies is sensible, a view supported by tests of sum rules for occupancy and vacancy of orbitals derived from experimental cross sections [START_REF] Schiffer | Test of Sum Rules in Nucleon Transfer Reactions[END_REF]. Error bars on the occupancy values quoted in the main document include statistical and systematical errors, the latter being derived from Ref. [START_REF] Tostevin | Systematics of intermediateenergy single-nucleon removal cross sections[END_REF].

We note that the measured inclusive cross section for the removal of an = 0 proton is about 12 times larger in 36 S than in 34 Si. This directly measured cross-section ratio, attributed to the almost complete depletion of the 2s 1/2 proton orbit between the two nuclei, has a value that is very similar to the ratio of occupancies derived from the reaction model calculations presented.

It is hoped that the present study will stimulate new developments in the modeling of nuclear reactions and their application to nuclear spectroscopy and further motivate the construction of a high-luminosity electron -radioactive nuclei collider facility that would enable a more direct experimental determination of the proton density distribution in 34 Si.

Data availability: Raw data were obtained at the Coupled Cyclotron Facility at the National Superconducting Cyclotron Labo-ratory, Michigan State University, USA. All other derived data used to support the findings of this study are available from the authors upon request and a thorough explanation of the analysis method can be found in Ref. [START_REF] Mutschler | Le noyau-bulle de 34 Si : Un outil exprimental pour étudier l'interaction spin-orbite ?[END_REF].

FIG. 2 .

 2 FIG. 2. Level scheme of33 Al with parallel momentum distributions of the strongest populated states. Left: Level scheme of33 Al, obtained from the γ-γ coincidence spectra of Fig.1, with energies (in keV) and branching ratios b KO f

  C 2 S exp norm of the strongest populated states are shown. Uncertainties on the C 2 S exp norm values are derived from Equation . They include those on b KO f discussed above and on the empirical quenching factor RS, that amounts to about 20%. Right (a-f): Experimental parallel momentum, p , distributions for the strongest populated states in 33 Al (black crosses) are compared to theory, assuming removal of an =0 (red curves) or =2 (blue curves) proton from the 34 Si ground state. As explained in Section 'Methods', the high momentum part of p is considered in this comparison. Momentum distributions for weakly populated states (b KO f < 1%) have insufficient statistics to be exploited. Horizontal bars correspond to the binning on the p value. Vertical error bars are deduced from uncertainties on b KO f per bin of p value.

FIG. 3 .

 3 FIG.3. Neutron and proton density distributions of the34 Si and 36 S nuclei. Neutron (a) and proton (b) density distributions of34 Si computed using RHFB calculations with the PKO2 interaction. (c) and (d) are those for 36 S. While the proton and neutron density distributions are similar in 36 S, they are significantly different in34 Si, with a sizeable central depletion of protons. Orbital occupancies obtained from these calculations are very similar to those deduced experimentally, providing a visualization of the proton and neutron density distributions in the two nuclei.

Acknowledgments This work is supported by the National Science Foundation (NSF) under Grant Nos.

PHY-1102511 and PHY-1306297, the OTKA Contract No. K100835, as well as by the Institut Universitaire de France. GRETINA was funded by the US DOE -Office of Science. Operation of the array at NSCL is supported by NSF under Cooperative Agreement PHY-1102511 (NSCL) and DOE under grant DE-AC02-05CH11231 (LBNL). J.A.T acknowledges support of the Science and Technology Facility Council (UK) grant ST/L005743.

Author Contributions A.M performed the off-line data analysis, A.Lem, D.W, K.W performed on-line data analysis and checked the integrity of data taking. K.W, A. Lem performed GEANT4 simulations and wrote parts of the offline sorting code. A.G and J.T performed reaction theory calculations. D.B operated the S800 spectrometer. D.W and F.R were responsible for the setting up, calibration and operation of the Gretina array. H.I,K.W helped to setup the Gretina array. The manuscript was prepared by O.S, A.M, J.T, A.G, A.Lem and E.K. J-P.E performed relativistic mean field calculations. Z.D and D.S contributed to the off-line data analysis of the γ-ray spectra and C.B, R.B, E.K, A.Lem, A.Lep, H.I, T.R, M.S, M.V, R.S checked data accumulation on-line. O.S. proposed the experiment and supervised the analysis.

Author Information The authors declare no competing financial interests. Readers are welcome to comment the online version of the paper. Correspondence and requests for material should be addressed to O.S. (sorlin@ganil.fr).