Bastien Fernandez

Beatriz Luna

Edgardo Ugalde

Spatial chaos of traveling waves has a given velocity

Keywords: numbers: 05, 45, Ra, 02, 30, -f

We study the complexity of stable waves in unidirectional bistable coupled map lattices as a test tube to spatial chaos of traveling patterns in open flows. Numerical calculations reveal that, grouping patterns into sets according to their velocity, at most one set of waves has positive topological entropy for fixed parameters. By using symbolic dynamics and shadowing, we analytically determine velocity-dependent parameter domains of existence of pattern families with positive entropy. These arguments provide a method to exhibit chaotic sets of stable waves with arbitrary velocity in extended systems.

Pattern formation in spatially extended systems can be regarded as the outcome of nonlinear dynamical processes [1]. This approach has proved successful to a vast phenomenology of various systems in Hydrodynamics, Optics, Solid-State Physics, etc. The most basic example concerns the spreading of simple coherent structures (e.g. fronts) in systems dominated by homogenization effects such as in reaction-diffusion [START_REF]Chemical Waves and Patterns[END_REF] or in some examples of population dynamics [START_REF] Murray | Mathematical Biology. I: an introduction[END_REF]. In other circumstances, when localization effects are predominant, spatial chaos can emerge along with the production of cohorts of stable heterogeneous profiles, as in bistable optical devices [START_REF] Firth | [END_REF] or in the Faraday instability of surface waves [5]. A breakthrough in the endeavor to elucidate this phenomenon has been to interpret solutions of stationary equations as the trajectories of a dynamical system where the original space variable plays the role of time. In this framework (which equally applies to continuous and discrete models) spatial chaos amounts to the existence of horseshoes and results from standard geometric constructions [6].

Beside standing patterns, extended systems also often generate a large variety of propagating structures especially when submitted to a global drift (e.g. open flows) [7]. In spite of this ubiquity, estimates on traveling pattern diversity are scarce, in particular as far as the velocity dependence is concerned. Indeed, stable waves with distinct velocities are known to possibly co-exist, but the occurrence of two or more chaotic sets of structures with distinct velocities remains unraveled. Appraising this issue from a theoretical viewpoint might have potential experimental applications in various fields, e.g. in fluid systems [7] or in secure communication [8]. In this context, the spatial dynamical system approach can also serve to construct horseshoes of traveling waves (TW) [9]. In principle, this tool also proves efficient to obtain conditions for chaos of waves with prescribed velocity (stationary solutions are regarded here as TW with vanishing velocity). However, since the velocity needs to be specified, it does not help to investigate the complexity dependence on velocity and parameters.

To gain intuition on these global features of spatial chaos in extended systems, we focus here on a minimal open flow model that can generate TW of arbitrary velocity, namely a unidirectional bistable Coupled Map Lattice (CML). Spatial chaos is quantified by the entropy of the sets of TW patterns with identical velocity. We numerically compute the entropy-velocity diagram in parameter space. The diagram unexpectedly reveals that spatial chaos occurs for at most a single velocity at once. To support this observation with analytical results, we rigorously infer, by means of symbolic dynamics, velocity-dependent parameter domains where specific TW pattern sets exist with positive entropy. These estimates show excellent agreement with numerical results and they extend to a larger class of models by structural stability. In a broader perspective, our shadowing arguments provide a new method to exhibit chaotic sets of stable waves with arbitrary velocity in extended systems.

The CML and its symbolic dynamics. We consider the unidirectional CML [7] on infinite real sequences,

x t+1 s = F (x t ) s = (1 -)f (x t s ) + f (x t s-1 ) (1)
where s ∈ Z indexes lattice sites, t represents time and the coupling constant lies in [0, 1]. The individual map f is piecewise linear, with two distinct branches separated by a jump at x = T (0 < T < 1), i.e. f (x) = ax + (1 -a)H(x -T ), H being the Heaviside function and 0 ≤ a < 1. This type of bistable CML is reminiscent of gradient flows modeling advection phenomena in atmospheric circulation systems [10]. The dynamics under iterations of f is elementary; any initial point located below the threshold T monotonically converges to the fixed point 0. Likewise, any point above T is attracted by 1.

The symbolic dynamics in this CML relies on the natural coding that consists in assigning a symbol 0 or 1 to each coordinate, according to its location with respect to T , namely let θ t s = H(x t s -T ). In that way, symbolic codes can be explicitly inserted in (1) via f (x t s-r ) = ax t s-r + (1 -a)θ t s-r (r = 0, 1). By iterating backward the resulting expression, one gains a formula for the coordinates of orbits in the attractor (i.e. of those sequences {x t } whose components exist and are bounded for all t ∈ Z) that exclusively depends on the code [11] x

t s = ∞ k=1 k n=0 n,k θ t-k s-n (2)
where the coefficients n,k = (1 -a)a k-1 k n (1 -) k-n n are positive and normalized. The expression ( 2) is useful for two reasons. Firstly, it establishes a continuous bijection between codes and CML orbits; especially that given a symbolic sequence {θ t s } s,t∈Z , there corresponds (if it exists) a single orbit {x t s } s,t∈Z in the attractor. This correspondence commutes with F and with space translations; hence all topological properties of the attractor express in symbolic codes. Secondly, (2) provides a substitute to the study of the attractor. An admissibility equation is obtained by requiring that the code computed based on (2) be indeed identical to the original symbolic sequence, i.e. a symbolic sequence {θ t s } codes for a genuine orbit iff the following relation holds

H ∞ k=1 k n=0 n,k θ t-k s-n -T = θ t s , ∀s, t ∈ Z (3)
Spatial complexity of traveling waves. Due to the advection term in (1), a characteristic feature of the CML is the prevalence of traveling patterns [7,10]. Accordingly, in the analysis of eq. ( 3), we focus on symbolic sequences corresponding to traveling waves, i.e. θ t s = θ s-vt where • stands for the integer part [START_REF]the corresponding orbit coordinates write x t s = φ(s -vt) where the profile is defined by φ(z) =[END_REF]. This consideration substantially reduces the variety of solutions to study as these spatio-temporal sequences are parametrized by a purely spatial sequence {θ s }, called a pattern, and by a velocity v. However, TW seem to capture the essential characteristics of the attractor in our case. Of note, for = 0 any TW sequence with v = 0 and arbitrary pattern in {0, 1} Z solves (3) (i.e. the uncoupled system F 0 has full diversity of fixed points), and similarly for = 1, any pattern is admissible with velocity v = 1.

Another simplification comes from symmetries in parameter space. Beside the trivial exchange (T, x t s ) ↔ (1 -T, 1 -x t s ) (for orbits off discontinuities) which implies reflection invariance with respect to T = 1/2, a more subtle symmetry exchanges any TW of velocity v for with a TW of velocity 1 -v for 1 -(reflection with respect to = 1/2). This property follows from the relation (the period L = 19 is chosen large enough for robustness). Gray levels (colors) correspond to velocities v, from 0 -left (light gray, yellow) to 1 -right (dark gray, brown) and including 1 7

F 1-• R = τ • R • F where (Rx) s = x -
, 1 6 , 1 5 , 1 4 , 2 7 , 1 3 , 2 5 , 3 7 , 1
2 and symmetric values. Intensity is proportional to entropy (maximal values along the axis T = 0.5). Curves delimit regions where TW symbolic sequences -with arbitrary pattern in a given chaotic set and given velocity -were analytically proved to solve the equation [START_REF] Murray | Mathematical Biology. I: an introduction[END_REF], see text. (Color online)

To get insights for arbitrary couplings, we have numerically estimated the TW entropy [13] depending on the velocity (Fig. 1). The figure reveals a surprisingly elaborated structure where the entropy appears to be positive for at most a single velocity when all parameters are fixed, i.e. no overlapping colored points were found. Hence, chaos of stable TW has a given velocity in our system. This property is rather unexpected because some TW with distinct speeds are known to coexist here. The selected velocity increases from 0 to 1 with coupling as expected from the definition of F , and remains locked on rational values according to structural stability [14]. In particular, a velocity plot vs. for T = 1/2 would show a Devil's staircase. Furthermore, the entropy itself remains constant on significant domains and surprisingly, besides reaching a maximum for symmetric maps f , this quantity for 0 < v < 1 may also reach high values far away from T = 1/2.

Fixed points analysis. To put numerical results on an analytic footing, we begin our investigations on eq. ( 3) by the spatial chaos of fixed points which materializes in the light gray (yellow) domain of Fig. 1. For a timeindependent code θ t s = θ s , the orbit is obviously a fixed point whose coordinates are given by x s = ∞ n=0 n θ s-n where

0 = (1 -a)(1 -) 1 -a + a and n = (1 -a) (a ) n-1 (1 -a + a ) n+1 , n > 0.
Eq. ( 3) stipulates that the coordinate x s at all sites s where θ s = 1 must exceed the threshold T (for simplicity, we do not consider the case of coordinates lying on T ). Likewise, all coordinates for which θ s = 0 must lie below T . Since all coefficients n are positive numbers, among all possible patterns and lattice sites, the smallest coordinate where θ s = 1 is x s = 0 . Using the normalization ∞ n=0 n = 1 and a similar argument for the sites where θ s = 0, one concludes that when the inequalities 1 -0 < T < 0 hold, all patterns solve (3) with v = 0. This condition is precisely the one that defines the central leftmost domain of maximal entropy log(2) in Fig. 1. In this case, no coordinate can cross the threshold T under F , independently of the initial point in [0, 1] Z , i.e. there cannot be any other admissible spatio-temporal sequence.

When T is tuned out of the central leftmost domain, large sets of patterns disappear through analogues of saddle-node bifurcations [15] and the entropy decreases [16] as evidenced by changes in color intensity in Fig. 1. The entropy however does not gradually fall off to zero. It remains locked to positive values on plateaus and suffers abrupt changes on short scales. This staircase behavior can be analytically established for a ≤ 3/4 (and under more general circumstances) as the fixed point bifurcation values belong to a Cantor set in this case. In addition, lower bounds on the entropy can be obtained by considering the sets, denoted by A L (L an arbitrary integer), that contain all patterns where any two consecutive 1's are separated by at least L consecutive 0's. Starting from A 0 (which incorporates all patterns) these sets form a nested sequence with decreasing and asymptotically vanishing entropy as L increases.

As for the admissibility of A L , since there is no upper limitation on the 0-block length, the smallest coordinate for which θ s = 1 in this set is again x s = 0 . On the other hand, a straightforward argument based on positivity and decay of the coefficients n shows that the largest coordinate for which θ s = 0 is

x s = m L ≡ ∞ n=0 1+n(L+1) .
Hence under the condition m L < T < 0 all patterns in A L are admissible with velocity 0. The graphs of m L (L = 1, 2, 3) are shown in the leftmost domain in Fig. 1 and indicate that the sets A L indeed capture the main features of the fixed point entropy when T decreases. Moreover, the fine structure of the fixed point entropy can be explained by considering more elaborated families with intermediate restrictions on block lengths (results not shown). Finally, the bottom curve delimiting the leftmost domain represents the graph of m ∞ = 1 , below which the entropy turns out to cancel (see below).

Intermediate velocities. Unlike for extreme velocities, when v is fixed between 0 and 1, no parameter value exists where the TW entropy is maximal. Nonetheless, it is possible to exhibit domains where the pattern entropy is positive by shadowing orbits with fronts and anti-fronts of the same velocity. In a preliminary step, proceeding similarly as for CML with diffusive couplings [11], one shows that a TW front code (defined as θ t s = H(s-vt), v rational) solves the admissibility equation iff the param-eters satisfy the inequalities η(v -0) < T ≤ η(v) where η(v) = ∞ k=1 vk n=0 n,k increases with v. By symmetry, a front coexists with its symmetric anti-front under the condition

ω(v) ≡ max{η(v -0), 1 -η(v)} < T < 1 -ω(v).
This condition defines, for any rational velocity v = p q and any 0 ≤ a < 1, a (symmetric with respect to T = 1/2) rhombus-like domain S p/q in the ( , T )-square. Moreover, these domains are pairwise disjoint and contiguous when v changes as indicated in Fig. 1.

To prove spatial chaos of TW, we consider the pattern set B L of positive entropy where all finite 0-blocks and all finite 1-blocks have length L or larger. Any TW code with pattern in B L and velocity p q has the property to locally match, on any space-time window of size L, and modulo a time shift t 0 , either the corresponding front code (i.e. we have θ t s = H s -p q (t -t 0 ) for s -L < s ≤ s and t -L < t ≤ t) or the anti-front code. According to (2) and to normalization of the n,k , the coordinate x t s then turns out to be close to either the front coordinate, i.e.

x t s -φ front s - p q (t -t 0 ) < a L-1 (4)
or to the anti-front's coordinate. In addition, inside S p/q , all front and anti-front coordinates are bounded away from the threshold T uniformly in space-time, say at a distance δ > 0. By choosing L large enough so that a L-1 < δ (i.e. L ≥ L 0 ), the inequality (4) forces x t s and φ front s -p q (t -t 0 ) to be either both above or both below the discontinuity T (and similarly when x t s is close to the anti-front coordinate), i.e. eq. ( 3) holds at arbitrary point (s, t). We conclude that all patterns in B L0 are admissible with v = p q and a positive lower bound applies for the associated pattern entropy inside S p/q .

A similar shadowing argument can be applied to patterns in the sets A L above to gain further domains with positive entropy. The reference orbit in this case is unique; it is the soliton coded by θ t s = δ(s -vt) where δ stands for the Dirac distribution. In practice, we have determined soliton existence domains, analytically for the velocity 1/2 and numerically for v = 1/3 and 1/4. Their cross-sections for a = 0.8, called D p/q , are represented on Fig. 1 (bottom rhombus-like domains). As before, an excellent agreement is found with the corresponding regions of numerically evidenced positive entropy. Finally, we note that the shadowing argument can be repeated with further orbits to get additional predictions on the entropy. For instance, the numerically computed existence domains for the two-site solitons with v = 1/2, 1/3 and 1/4 are reported in Fig. 1. These are the rhombuslike structures intersecting their respective symmetric domain S p/q . Completing the diagram. In addition to domains of positive entropy, we determined regions where the entropy cancels. In particular we proved that, under the condition 0 < T < min{ 0 , 1 }, (i) any symbol 1 remaining steady long enough at s, eventually forces the neighboring symbol at s + 1 to become 1 and on the opposite (ii) the symbols 0 do not propagate (i.e. θ t s = 1 implies θ t+1 s = 1). In this case, any code asymptotically approaches a sequence composed of front profiles (i.e. θ t s = H(s -s(t))) and any TW entropy must vanish. By applying the symmetries T → 1 -T and → 1 -, one obtains large domains of low spatial complexity in the parameter space, that share common boundaries with positive entropy domains as shown on Fig. 2. Of note, in the limit a = 0 the CML becomes a cellular automaton. No TW with intermediate velocity exists in this case and there are four possible automaton rules according to the location of ( , T ) in the unit square.

Concluding remarks. Our analysis indicates that once the parameters are fixed in this system, various sets of TW co-exist with distinct velocities [17], but only a single ensemble can be chaotic. Numerical evidence has been supported by analytic arguments based on families of symbolic sequences. In a larger perspective, structural stability [14] implies that all chaotic sets exhibited here can be continued into suitable alterations of the piecewise affine model. Thus the existence of chaos of TW with arbitrary velocity generalizes to a larger class of bistable CML and beyond, to lattice systems with more general type of coupling (i.e. not fully asymmetric). Furthermore, based on uniqueness of the front velocity in bistable extended systems, we conjecture that the property of an at most one chaotic traveling patterns set holds in a broad context. Testing this property in further models provides a rewarding challenge for future studies.

Independently, our analytic methods proffer an alternative approach to the horseshoe construction, based on shadowing arguments. Indeed, the presence of a stable soliton (or the co-existence of stable fronts and anti-fronts of the same velocity) guarantees the existence of ensem-ble of traveling patterns with positive entropy. As it does not requires the explicit expression of localized solutions, this criterion is applicable to arbitrary spatially extended systems.

  s and (τ x) s = x s-1 are the spatial reflection and translation respectively.

7 FIG. 1 :

 71 FIG. 1: Entropy-velocity diagram of stable TW in ( , T )-space for a = 0.8. Painted points code for the numerical TW entropy log P 19,v 19

FIG. 2 :

 2 FIG. 2: Entropy-velocity diagram in the ( , T )-square and complementary domains where the entropy is analytically shown to cancel (outer light gray regions). (a) = 0.4 and (b) a = 0.8; the rectangle delimits the region displayed in Fig. 1. (Color online)

We are indebted to V. Afraimovich for constant stimulation and we thank C. Chandre, R. Coutinho and A. Torcini for a careful reading of the manuscript. We also acknowledge the host institutions for hospitality (i.e. BF to IF and BL and EU to CPT).Work supported by the ECOS-Nord programme M04M01.

∞ k=1 k n=0 n,k θ z-n+vk .

[13] For numerical purposes, the TW entropy is defined here as the exponential growth rate of the number PL,v of L-periodic patterns with a given velocity v, i.e. lim sup

. This quantity is related to the standard topological entropy based on the number of admissible words, see e.g. R. Badii and A. Politi, Complexity. Hierarchical structures and scaling in physics, Cambridge University Press (1997).

[14] Because of contraction, any orbit remaining at bounded distance of discontinuities (as for all orbits considered here) is Lyapunov stable and thus structurally stable. for L large enough. Thus, every fixed point pattern containing a (finite) 1-block of length L or larger cannot be admissible and the fixed point entropy must be smaller than log(2).

[17] For instance fronts with velocity v = 1/2 co-exist together with a chaotic set of waves with velocity 1/2 in the central bottom domain D 1/2 of Fig. 1.


Rinke Stienstra

Stéphane Mandard

Nguan Soon Tan

Walter Wahli

Christian Trautwein

Terrilyn A Richardson

Elgin Lichtenauer-Kaligis

Sander Kersten

PhD Michael Müller
email: michael.muller@wur.nl

PPARα controls the expression of the sIL-1ra in liver The Interleukin 1 receptor antagonist is a direct target gene of PPAR in liver

Keywords: PPAR, Liver, Inflammation, Microarray, Interleukin-1 receptor antagonist Stienstra et al., PPARα controls

Background/Aims: The Peroxisome Proliferator-Activated Receptor (PPAR)  belongs to the superfamily of Nuclear Receptors and plays an important role in numerous cellular processes, including lipid metabolism. It is known that PPAR also has an anti-inflammatory effect, which is mainly achieved by down-regulating pro-inflammatory genes. The objective of this study was to further characterize the role of PPAR in inflammatory gene regulation in liver.

Results: According to Affymetrix micro-array analysis, the expression of various inflammatory genes in liver was decreased by treatment of mice with the synthetic PPAR agonist Wy14643 in a PPAR-dependent manner. In contrast, expression of Interleukin-1 receptor antagonist (IL-1ra), which was acutely stimulated by LPS treatment, was induced by PPAR. Up-regulation of IL-1ra by LPS was lower in PPAR -/-mice compared to Wt mice. Transactivation and chromatin immunoprecipitation studies identified IL-1ra as a direct positive target gene of PPAR with a functional PPRE present in the promoter. Upregulation of IL-1ra by PPAR was conserved in human HepG2 hepatoma cells and the human monocyte/macrophage THP-1 cell line.

Conclusions:

In addition to down-regulating expression of pro-inflammatory genes, PPAR suppresses the inflammatory response by direct up-regulation of genes with antiinflammatory properties.

Introduction

Inflammation describes the comprehensive reaction of the host to various types of injury, which is generally protective and aimed at promoting tissue repair. The inflammatory response is mediated by a diverse group of cytokines and other signaling molecules that are able to profoundly influence cellular function. At the cellular level, numerous signaling pathways and transcription factors conspire in a complex network to produce the appropriate response. In recent years it has become clear that the Peroxisome Proliferator Activated Receptors (PPARs) modulate this response in a variety of organs. PPARs are members of the superfamily of Nuclear Receptors that play a pivotal role in mediating the effect of small lipophilic ligands on gene transcription [START_REF] Robinson-Rechavi | The nuclear receptor superfamily[END_REF]. The three isotypes of the PPAR-family, PPAR, PPAR/δ and PPAR, have been implicated in numerous processes, including lipid and glucose metabolism, and inflammation. Activation of the receptor occurs by binding of various ligands, ranging from natural compounds such as fatty acids to highly specific synthetic agonists. Upon ligand-activation, binding to so called PPAR Response Elements (PPRE) located in the promoter of target genes results in increased gene transcription. To accomplish activation of gene transcription it is essential that PPAR forms a heterodimer with the Retinoid X Receptor (RXR) [START_REF] Desvergne | Peroxisome proliferator-activated receptors: nuclear control of metabolism[END_REF].

Besides their ability to enhance gene transcription [START_REF] Mandard | Peroxisome proliferator-activated receptor alpha target genes[END_REF], PPARs are also able to suppress gene expression. For example, it has been shown that activated PPAR lowers the expression of several enzymes connected with amino acid metabolism (4), although the mechanism behind the observed down-regulation remains unknown. In addition, the effects of PPAR on inflammation are mainly achieved by suppressing gene expression. Since the initial observation that PPAR-/-mice have a prolonged inflammatory response [START_REF] Devchand | The PPARalpha-leukotriene B4 pathway to inflammation control[END_REF], an important role for PPARs in regulating inflammatory responses has clearly emerged. Although numerous studies have demonstrated the protective and anti-inflammatory effects of PPAR-Stienstra et al., PPARα controls the expression of the sIL-1ra in liver activation in liver [START_REF] Delerive | Peroxisome proliferator-activated receptors in inflammation control[END_REF] [START_REF] Verme | The nuclear receptor peroxisome proliferator-activated receptor-alpha mediates the antiinflammatory actions of palmitoylethanolamide[END_REF], information about the precise molecular mechanisms involved is somewhat limited. One of the mechanisms by which this nuclear receptor exerts its antiinflammatory action is through modulation of the NF-B pathway. Physical interaction of PPAR with NF-B prevents its activation and downstream pro-inflammatory effects [START_REF] Delerive | Peroxisome proliferator-activated receptor alpha negatively regulates the Stienstra et al., PPARα controls the expression of the sIL-1ra in liver vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappaB and AP-1[END_REF].

Moreover, PPAR has been shown to up-regulate the expression of IB, the natural NF-B inhibitor that prevents the nuclear translocation and activation of the pro-inflammatory transcription factor [START_REF] Delerive | DNA binding-independent induction of IkappaBalpha gene transcription by PPARalpha[END_REF].

Anti-inflammatory properties have also been assigned to the other two PPAR isotypes.

Activation of PPAR controls the inflammatory status of the intestinal tract [START_REF] Dubuquoy | Role of peroxisome proliferator-activated receptor gamma and retinoid X receptor heterodimer in hepatogastroenterological diseases[END_REF] and is responsible for the down-regulation of a specific subset of pro-inflammatory genes in macrophages [START_REF] Welch | PPARgamma and PPARdelta negatively regulate specific subsets of lipopolysaccharide and IFN-gamma target genes in macrophages[END_REF]. The recent generation of macrophages lacking PPAR/δ has also revealed a specific role for PPAR/δ in regulating inflammatory processes [START_REF] Lee | Transcriptional repression of atherogenic inflammation: modulation by PPARdelta[END_REF].

To better understand the regulatory role of PPAR in liver and to identify possible new target genes under the control of PPAR, we studied PPAR-dependent gene expression levels in mouse liver by means of Affymetrix microarray analysis. Activation of PPAR was achieved by treating Wildtype (Wt) and PPAR -/-mice with the synthetic PPAR agonist Wy-14643.

While numerous inflammatory genes were found to be down regulated by PPAR activation, the IL-1 receptor antagonist, however, was highly up-regulated by PPAR Additional experiments indicated that the IL-1 receptor antagonist is a direct target gene of PPAR. Our data suggest that PPAR may modulate inflammation by direct up-regulation of target genes.

Stienstra et al., PPARα controls the expression of the sIL-1ra in liver

Material and Methods

Chemicals

Wy-14643 was obtained from ChemSyn Labarotories. Cell culture medium, fetal calf serum and penicillin/streptomycin/fungizone were from BioWhittaker Europe (Cambrex Bioscience). SYBR green was from Eurogentec (Seraing, Belgium).

The human and mouse antibody against IL-1ra and the recombinant hIL-1β were from R&D systems (R&D Systems Europe Ltd, Abingdon, UK). Otherwise, chemicals were from Sigma-Aldrich (Zwijndrecht, The Netherlands).

Animal experiments

Sv129 PPAR-/-mice and corresponding Wt mice were purchased at the Jackson Laboratory (Bar Harbor, Maine, USA). For the fasting experiment, male mice were fasted for 24 hours starting at the onset of the light cycle. For the feeding experiments with Wy-14643 (0.1%), L165041 (0.025%) and Rosiglitazone (0.01%), ligands were mixed in the food and given to female mice for 5 days. Liver was dissected and directly frozen into liquid nitrogen.

Lipopolysaccharide (LPS) (E. coli; Sigma-Aldrich, St-Louis, MO) was administered at a dose of 4 mg/kg IP. After 3 hours of treatment, liver was dissected and frozen into liquid nitrogen.

The animal experiments were approved by the animal experimentation committee of the Wageningen University, the Netherlands and the district government of Lower Saxony, Germany.

Oligonucleotide microarray

Total RNA was isolated from mouse liver using Trizol reagent (Invitrogen, Breda, The Netherlands) following the supplier's protocol. For the microarray experiment, 10 μg of total Stienstra et al., PPARα controls the expression of the sIL-1ra in liver liver RNA pooled from 5 or 3 mice was used for cRNA synthesis. To confirm integrity of the RNA, bioanalyzer (Agilent) analysis was done before the hybridization process was started.

The Affymetrix Mouse Expression array 430A was used and results were analyzed using Microarray Suite and Data Mining Tool (DMT) software following instructions of the manufacturer. Heat Map analysis was done using Spotfire DecisionSite software (Spotfire Inc, Sommerville, MA).

RNA Isolation and RT-PCR

RNA from animal tissue or cells was extracted with Trizol reagent using the supplier's instructions. After treatment with Dnase I amplification grade (Invitrogen), 4 or 5 μg of RNA was used for reverse transcription with Superscript II RT Rnase H (Invitrogen) using oligo (dT) primers following manufacturer's instructions.

Real Time Quantitative PCR

PCR was performed with platinum Taq polymerase (Invitrogen) and SYBR green using an iCycler PCR machine (Bio-Rad Laboratories BV, Veenendaal, The Netherlands). TTTCTCTAGGGCTGAGGACAGCAAACTTCT 3' combined with primer sIL-1ra-Rprom and the reverse primer sIL-1ra-mutR 5' AGAAGTTTGCTGTCCTCAGCCCTAGAGAAA 3' combined with primer sIL-1ra-Fprom were used to generate the two partially overlapping PCR fragments. In a final PCR, the two fragments with overlapping ends were used to amplify the mutated sIL-1ra promoter with the forward primer sIL-1ra-Fprom and the reverse primer sIL-1ra-Rprom.The final product was cloned into the XhoI and HindIII sites of the pGL-3 basic vector. cDNA corresponding to mPPAR was cloned into pSG5 (Stratagene).

After cloning, fragments were sequenced to confirm the integrity of the constructs. The RXR expression plasmid was a generous gift of Dr. S.A. Kliewer.

Primary mouse hepatocyte isolation

Primary mouse and rat hepatocytes were isolated as described previously [START_REF] Kuipers | Impaired secretion of very low density lipoprotein-triglycerides by apolipoprotein E-deficient mouse hepatocytes[END_REF]. Briefly, after cannulation of the portal vein, the liver was perfused with calcium free HBSS which was pregassed with 95% O2/5% CO2. Next, the liver was perfused with a collagenase solution until

Stienstra et al., PPARα controls the expression of the sIL-1ra in liver swelling and degradation of the internal liver structure was observed. The hepatocytes were released, filtered and washed several times using Krebs buffer. The viability was assessed by Trypan blue staining and was at least 80%. Cells were cultured in William's Medium E supplemented with 10% FCS, penicillin/ streptomycin/fungizone, insulin and dexamethasone.

Cells were plated in collagen (Serva Feinbiochemica, Heidelberg, Germany) coated wells with a density of 0.5 x 10 6 cells/ml. After 4 h of incubation, the medium was removed and replaced with fresh medium. The next day, hepatocytes were used for experiments and treated with IL-1 (5 ng/ml) for 24 h.

Cell culture and transfection

Human hepatoma HepG2 cells were obtained from the ATCC (Manassas, VA, USA) and grown in DMEM containing 10% FCS and PSF. THP-1 cells were from ATCC and grown in RPMI-1640 containing 10% FCS and PSF. HepG2 cells were transfected using calcium phosphate precipitation. A -galactosidase reporter was co-transfected to normalize for differences in transfection efficiency. After transfection, cells were treated with the PPAR ligand Wy14643 at 50 M or vehicle (DMSO) for 24 h prior to lysis. Promega luciferase assay (Promega) and standard -galactosidase assay with 2-nitrophenyl-BD galactopyranoside were used to measure the relative activity of the promoter. For expression experiments in HepG2 cells, FCS was removed from the medium when ligand or cytokines were added. THP-1 cells were differentiated towards macrophages using phorbol myristic acid (Sigma-Aldrich) at a concentration of 100 M.

Chromatin Immunoprecipitation (ChIP)

Wt or PPAR -/-mice were used and fed by gavage with either Wy-14643 (50 mg/kg/day) or vehicle (0.5 % carboxymethyl cellulose) for 5 days or fasted for 24h. After treatment, mice Stienstra et al., PPARα controls the expression of the sIL-1ra in liver were sacrificed by cervical dislocation and the liver was perfused with prewarmed (37 C) phosphate-buffered saline for 5 minutes followed 0.2% collagenase for 10 min. The liver was diced and forced through a stainless steel sieve and the hepatocytes were collected into DMEM containing 1% formaldehyde. After incubation at 37 C for 15 min, the hepatocytes were pelleted and ChIP was performed using a mouse PPAR-specific antibody as previously described [START_REF] Poi | Antiapoptotic role of PPARbeta in keratinocytes via transcriptional control of the Akt1 signaling pathway[END_REF]. The sequences of the primers used for PCR were 5'-CAGATGCAGAATTGGGAAAAGATG-3' for the forward primer and 5'-GCAAGCAATAGGGCCTGGTGAAC-3' for the reverse primer. Control primers used were 5'-CTCCCTTTCCCCTTCTGTCCCTCTCATT-3' for the forward primer and 5'-TTCCCAAACTCCCCACCCCATCC-3' for the reverse primer.

Western Blot

Western blotting was carried out using an ECL system (Amersham Biosciences) according to the manufacturer's instructions. Acetone precipitated protein from equal amounts of medium from HepG2 cells or equal amounts of mouse total liver cell lysates as determined by Bio-Rad Protein Assay reagent (Bio-Rad Laboratories BV) were used and resolved by SDS/PAGE on a 12% polyacrylamide gel. A protein marker (Invitrogen) was used to determine the sizes of the separated proteins. Separated proteins were transferred to Immobilon-P transfer membranes (Millipore).The primary antibody was used at a dilution 1:1000 and the membranes were incubated overnight at 4 C. The secondary antibody was used at a dilution of 1:5000. All incubations were performed in 1X Tris-buffered saline, pH 7.5, with 0.1 % Tween 20 and 5% dry milk. In the final washings, dry milk was removed from the solution.

Statistical analysis

The Student's T-test was used to calculate statistical significant differences.

Stienstra et al., PPARα controls the expression of the sIL-1ra in liver

Results

PPAR, but not PPAR/δ and PPAR, is involved in the negative regulation of inflammatory related-genes in liver

To screen for potential novel target genes of PPAR involved in inflammation in liver we performed micro-array analysis. A comparison was made between liver RNA from Wt and PPAR-/-mice treated or not with the synthetic PPAR ligand Wy14643. The expression of a large number of genes involved in inflammation was decreased by Wy14643 in Wt but not PPAR-/-mice (Fig. 1A). As shown in the Fig. 1B, the expression of all of these genes was up-regulated during Lipopolysaccharide (LPS)-induced acute inflammation. These included serum amyloid A, orosumucoid, metallothionein, Signal Transducer and Activator of Transcription 3, IL-18, and IL-1RAcP. An exception to this pattern of regulation was IL-1Ra, which in contrast to the numerous other genes induced by LPS, was up-regulated by PPAR activation. Real-time quantitative PCR (qPCR) confirmed the marked suppression of the selected genes involved in inflammation by Wy14643 in Wt but not PPAR-/-mice (Fig. 2).

To evaluate whether the down-regulation of inflammatory genes in liver is specific to PPAR activation, we studied the effect of synthetic PPAR/δ and PPAR agonists. Whereas

Wy14643 markedly reduced expression of the selected set of genes, no changes or an opposite response was observed after administration of L165041 and rosiglitazone (Fig. 3).

Stienstra et al., PPARα controls the expression of the sIL-1ra in liver

PPAR activation enhances gene expression of the anti-inflammatory IL-1 receptor antagonist

While many genes involved in inflammation were down-regulated by Wy14643, hepatic expression of Interleukin 1 receptor antagonist (IL-1ra) was highly increased by PPAR activation (Fig. 1). IL-1ra is believed to have mainly anti-inflammatory properties [START_REF] Lennard | Interleukin-1 receptor antagonist[END_REF]. The IL-1ra gene is expressed in multiple forms using two different promoters, resulting in three intracellular forms transcribed via the 5' promoter and one soluble form transcribed via a different promoter [START_REF] Butcher | Comparison of two promoters controlling expression of secreted or intracellular IL-1 receptor antagonist[END_REF]. To more closely examine the regulation of IL-1ra by PPAR in liver, specific primers were developed to distinguish between the intracellular (icIL-1ra) and soluble (sIL-1ra) form of IL-1ra. QPCR showed that the soluble form but not the intracellular form is regulated by Wy14643 and fasting in mouse liver in a PPAR-dependent manner (Fig. 4A,B).

It should be mentioned that icIL-1ra mRNA was difficult to detect compared to sIL-1ra and to our knowledge no function has been assigned to icIL-1ra in liver. Further analysis showed that only the PPAR isotype is involved in the upregulation of sIL-1ra expression in liver (Fig. 4C). Protein levels of IL-1ra analyzed in liver cell lysates matched the gene expression pattern (Fig. 5).

PPAR is essential to induce expression of sIL-1ra during inflammatory conditions in liver

To investigate whether PPAR is involved in the regulation of sIL-1ra during inflammatory conditions, Wt and PPAR -/-mice were exposed to LPS. As expected, LPS treatment increased sIL-1ra gene expression, however the increase was significantly higher in Wt mice compared to PPAR -/-mice (Fig. 6A). Induction of IL-1expression was similar between

Wt and PPAR -/-mice after exposure to LPS (data not shown). Subsequently, the ratio of IL-1ra to IL-1, which is often used to determine the activation of IL-1 signaling pathways, Stienstra et al., PPARα controls the expression of the sIL-1ra in liver was drastically improved in Wt mice compared to PPAR mice after exposure to LPS, suggesting a shift towards a more anti inflammatory phenotype (Fig. 6A). Treatment of Wt and PPAR -/-primary mouse hepatocytes with IL-1 to induce sIL-1ra gene expression gave comparable results, as shown by a more pronounced increase in sIL-1ra gene expression in hepatocytes expressing PPAR compared to PPAR -/-hepatocytes (Fig. 6B).

sIL-1ra is a true target gene of PPARα in mouse liver

To determine whether the sIL-1ra promoter is directly regulated by PPAR, 1.9 kb of promoter region of the soluble isoform was cloned into a luciferase reporter vector and used in transactivation assays. Co-transfection with PPAR and RXR expression plasmids revealed that while RXR alone did not have any effect on the promoter activity (data not shown), transfection of both PPAR and RXR expression plasmids increased the activity of the sIL1-ra promoter (Fig. 6A). This effect was further enhanced by treatment of cells with Wy14643. In silico analysis of the sIL-1ra promoter using Nubiscan V2.0 software identified a possible PPRE located at around 700 bp upstream of the transcription start site. To investigate whether this PPRE may be responsible for PPAR-mediated transactivation of the sIL-1ra gene, the response element was disabled by site-directed mutagenesis (Fig. 6B). After mutating the PPRE, the response to PPAR and Wy14643 was completely abolished, suggesting that the effects of PPAR are mediated by the PPRE identified (Fig. 6C).

Finally, to investigate if PPAR is bound to the PPRE identified in mouse liver, in vivo chromatin immunoprecipitation (ChIP) was performed using an anti-PPAR antibody. In mice, treatment with Wy-14643 and fasting increased the binding of PPAR to this PPRE in Wt but not PPAR -/-mice. No binding was observed to a control sequence located 1800

Stienstra et al., PPARα controls the expression of the sIL-1ra in liver base pairs upstream from the PPRE (Fig. 6D). Taken together, these data indicate that the PPRE identified mediates the effects of PPAR on sIL-1ra gene expression.

sIL-1ra is also regulated by PPAR in human cells

To evaluate whether the effects seen in mouse liver can also be reproduced in human liver cells, HepG2 cells were used. HepG2 cells only express the soluble form of the IL-1ra gene (17) and treatment of the cells with IL-1β, an inducer of sIL-1ra expression, resulted in the expected increase in gene expression levels (Fig. 7A). Incubation with RXR and PPAR agonists alone did not result in major changes in gene transcription. However, combined treatment with both IL-1β and PPAR/RXR agonists did synergistically increase sIL-1ra expression. Since soluble IL-1ra is secreted, medium from treated HepG2 cells was analyzed for IL-1ra protein concentration. The protein levels followed the sIL-1ra mRNA induction in these cells (Fig 7B). The upper band represents the glycosylated form of the sIL-1ra protein and the lower band the unglycosylated protein [START_REF] Gabay | Interleukin 1 receptor antagonist (IL-1Ra) is an acute-phase protein[END_REF]. Next, the expression of the sIL-1ra was examined in the human derived monocyte/macrophage THP-1 cell line (Fig 7C). Since it is known that PPAR localization shifts from hepatocytes towards Kupffer cells during hepatic inflammation [START_REF] Orfila | Immunohistochemical distribution of activated nuclear factor kappaB and peroxisome proliferator-activated receptors in carbon tetrachloride-induced chronic liver injury in rats[END_REF], it was of interest to test the ability of PPAR to regulate the expression of sIL-1ra in this cell type. The expression of sIL-1ra was increased by differentiation of the cells towards macrophages using phorbol myristic acid (PMA). In contrast to the expression levels in monocytes, the expression level of sIL-1ra in macrophages was increased by Wy14643. These data indicate that sIL-1ra is a target of PPAR in human hepatocytes and macrophages.

Stienstra et al., PPARα controls the expression of the sIL-1ra in liver

Discussion

Since the initial discovery of a possible role for PPAR in controlling the inflammatory response (5), considerable progress has been made towards resolving the function of PPAR during inflammation [START_REF] Delerive | Peroxisome proliferator-activated receptor alpha negatively regulates the Stienstra et al., PPARα controls the expression of the sIL-1ra in liver vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappaB and AP-1[END_REF][START_REF] Delerive | DNA binding-independent induction of IkappaBalpha gene transcription by PPARalpha[END_REF][START_REF] Kleemann | Fibrates down-regulate IL-1-stimulated C-reactive protein gene expression in hepatocytes by reducing nuclear p50-NFkappa B-C/EBP-beta complex formation[END_REF]. It is now clear that PPAR mainly governs inflammation by down-regulating expression of genes, including several acute phase genes.

Here, we show that soluble Interleukin 1 Receptor antagonist is a direct target gene of PPAR with a functional PPRE in its promoter, suggesting that PPAR may also govern inflammation by direct up-regulation of target genes. IL-1ra is an inhibitor of cytokine signaling and is produced by many cells including hepatocytes. During the acute phase response, the expression of IL-1ra is strongly induced and IL-1ra is therefore referred to as a positive acute phase protein. In our hands, hepatic sIL-1ra expression was highly up-regulated after LPS treatment. By binding to the IL-1 receptor with almost equal affinity as IL-1, it prevents activation of the pro-inflammatory IL-1 pathway and downstream NF-B activation [START_REF] Gabay | Production of IL-1 receptor antagonist by hepatocytes is regulated as an acute-phase protein in vivo[END_REF]. Under non-inflammatory conditions, PPAR activation by Wy14643 or fasting markedly increased sIL-1ra gene expression in liver, which was not observed in PPAR -/mice. Thus, PPAR seems to be able to increase the expression of sIL-1ra in liver independently of any inflammatory stimulus. Transactivation and chromatin immunoprecipiation experiments indicated that PPAR regulates sIL-1ra expression via a PPRE located around 700 bp upstream of the transcription start site.

In human HepG2 cells PPAR activation was able to increase sIL-1ra gene expression when cells were co-treated with IL-1 which is known to induce expression of sIL-ra by NF-κB and C/EBP binding to the sIL-1ra promoter [START_REF] Gabay | Mouse IL-1 receptor antagonist isoforms: complementary DNA cloning and protein expression of intracellular isoform and tissue distribution of secreted and intracellular IL-1 receptor antagonist in vivo[END_REF]. In THP-1 cells differentiated towards macrophages, PPAR was also capable of inducing sIL-1ra expression. Since macrophage/monocyte type cells including Kupffer cells have an important role in controlling Stienstra et al., PPARα controls the expression of the sIL-1ra in liver hepatic inflammation [START_REF] Gregory | Neutrophil-Kupffer cell interaction: a critical component of host defenses to systemic bacterial infections[END_REF], the PPAR-dependent regulation of the IL-1ra gene in these cells might contribute to the immune-modulating functions of PPAR in liver. Together, these data indicate that sIL-1ra is a direct target gene in mouse and human hepatocytes, as well as in human macrophages. By inhibiting binding of IL-1β to its receptor via increased expression of its natural antagonist, PPAR might prevent or counteract the activation of the IL-1βsignalling cascade including the pro-inflammatory NF-κB pathway.

The availability of microarray techniques offers the opportunity to screen for new PPAR controlled genes and pathways with the aim of elucidating the effects of PPAR on inflammation. Using this approach, our initially analysis focused on inflammation-related genes that are regulated by PPARactivation in liver under normal conditions. Many of the genes that were decreased in liver of mice treated with Wy14643 were increased during acute inflammation mimicked by LPS administration. Several acute phase response genes, cytokine receptor components, and intracellular signaling transducers were affected. The results for several of these genes corresponds well with earlier studies [START_REF] Gervois | PPARα controls the expression of the sIL-1ra in liver vivo treatment with the peroxisome proliferator-activated receptor-alpha activator fenofibrate[END_REF][START_REF] Gervois | Negative regulation of human fibrinogen gene expression by peroxisome proliferatoractivated receptor alpha agonists via inhibition of CCAAT box/enhancer-binding protein beta[END_REF], whereas for other genes the linkage to PPAR is entirely novel.

IL-18, a pro-inflammatory cytokine with an established role in liver injury [START_REF] Chikano | IL-18 and IL-12 induce intestinal inflammation and fatty liver in mice in an IFN-gamma dependent manner[END_REF], is one of the genes that was decreased after PPAR activation. Furthermore, the LIF-receptor, which belongs to the IL-6 receptor family, was strongly decreased after PPAR-agonist treatment in mouse liver. Downstream of this pathway, PPAR is also ably to suppress the expression of STAT3, a transcription factor implicated in the regulation of inflammatory signaling in liver [START_REF] Desiderio | A genome-wide analysis of the acute-phase response and its regulation by Stat3beta[END_REF]. Finally, PPAR seems to have a role in regulating IFNγ-activated genes which is illustrated by the suppression of IFNγ-inducible genes by PPAR.

Stienstra et al., PPARα controls the expression of the sIL-1ra in liver Our analysis clearly demonstrates an important immunosuppressive effect by PPAR in liver.

One limitation of the present study is that we did not address the effect of PPAR activation during LPS-induced inflammation. However, a previous study showed that when the expression of specific components of the inflammatory signaling cascade was suppressed by PPAR agonists prior to IL-6 treatment, the subsequent acute phase responses in liver were diminished [START_REF] Gervois | PPARα controls the expression of the sIL-1ra in liver vivo treatment with the peroxisome proliferator-activated receptor-alpha activator fenofibrate[END_REF]. Although future experiments should aim at combining both PPAR activation and inflammation in liver to appraise the importance of PPAR in regulating inflammation, our data reveal several candidate genes by which PPAR might exert its potent anti-inflammatory effects in liver.

In conclusion, by negatively regulating multiple components of inflammatory signalling pathways ranging from cytokines to receptor complexes and downstream target genes, PPAR activation may render the liver less susceptible to the effects of inflammation. Upregulation of soluble IL-1ra by PPAR may contribute to its immune-modulating effects. Figure 6 PPAR is essential for the induction of sIL-1ra in liver during LPS-induced inflammation.

(A) Wildtype and PPAR -/-animals were exposed to LPS or saline for 16 h. Liver RNA was isolated and expression of sIL-1ra was measured by qPCR. Significant differences for sIL-1ra

Figure 1 Figure 2

 12 Figure 1

Figure 3 PPARFigure 4 PPAR

 34 Figure 3

Figure 5

 5 Figure 5

Figure 7

 7 Figure 7

Figure 8

 8 Figure 8

StienstraFigure 6

 6 Figure 6

Figure 7 Figure 7

 77 Figure 7 Figure 7

Figure 8 Figure 8

 88 Figure 8 Figure 8

  PPARα controls the expression of the sIL-1ra in liver PPAR -/-mice were treated with IL-1 (5 ng/ml) or vehicle. After 24 h, expression levels of sIL-1ra were determined via qPCR.

	expression between Wt and PPAR -/-mice treated with LPS were observed (Student's t-test,
	P < 0.01). The sIL-1ra to IL-1b ratio after exposure to LPS was calculated in Wildtype and
	PPAR -/-mice after LPS treatment. IL-1b expression was determined via qPCR. sIL-1ra to

IL-1ratios of Wildtype and PPAR -/-mice were significantly different (Student's t-test, P < 0.002). Error bars represent SEM (n = 4). (B) Primary mouse hepatocytes from Wt and

Stienstra et al.,

Table 1

 1 Primer sequences used for Real Time PCR

	Gene	Forward primer	Reverse primer	Product size
	Mouse sIL-1ra	AAATCTGCTGGGGACCCTAC	TGAGCTGGTTGTTTCTCAGG	164 bp
	Mouse icIL-1ra	CAGTTCCACCCTGGGAAGGT	AGCCATGGGTGAGCTAAACAGGACA	128 bp
	Mouse STAT3	GACCCGCCAACAAATTAAGA	TCGTGGTAAACTGGACACCA	214 bp
	Mouse IL-18	ACAACTTTGGCCGACTTCAC	GGGTTCACTGGCACTTTGAT	127 bp
	Mouse IL-1RAcP	TTGCCACCCCAGATCTATTC	CCAGACCTCATTGTGGGAGT	122 bp
	Mouse SAA	GCGAGCCTACACTGACATGA	TTTTCTCAGCAGCCCAGACT	121 bp
	Mouse LIF-receptor	AGGAATGCCACAATCAGAGG	AACCCGGAAAGTGTATGCAG	90 bp
	Mouse IL-6 receptor	CAGCGACACTGGGGACTATT	AGTCACTCTTCCCGTTGGTG	220 bp
	Mouse -actin	GATCTGGCACCACACCTTCT	GGGGTGTTGAAGGTCTCAAA	139 bp
	Human	GCCTCCGCAGTCACCTAAT	TCCCAGATTCTGAAGGCTTG	108 bp
	sIL-1ra
	Human	AACACCCCAGCCATGTACG	ATGTCACGCACGATTTCCC	254 bp
	-actin

Acknowledgements

We thank Jolanda van der Meijde for performing the microarray experiment and René Bakker for assisting with the animal experiments. We thank Dr. Mark Leibowitz (Ligand Pharmaceuticals) for the kind gift of Lg100268. This study was supported by the Centre for Human Nutrigenomics, with additional support by the Netherlands Organization for Scientific Research (NWO), the Wageningen Center for Food Sciences, the Dutch Nutrigenomics Consortium, the Swiss National Science Foundation and Etat de Vaud and the National Institutes of Health grant GM46897. Stienstra et al., PPARα controls the expression of the sIL-1ra in liver


Eric Lambaudie
email: lambaudie@marseille.fnclcc.fr

Fabrice Narducci

Marie Bannier

Camille Jauffret

Nicolas Pouget

Eric Leblanc

Gilles Houvenaeghel

Role of robot-assisted laparoscopy in adjuvant surgery for locally advanced cervical cancer

Keywords: Locally advanced cervical cancer, Adjuvant surgery, Robot-assisted laparoscopy

Objective. The aim of this study was to compare the feasibility and efficacy of robot-assisted laparoscopy with traditional laparotomy and conventional laparoscopy in a series of patients with locally advanced cervical cancer managed in our two institutions.

Methods. Twenty-two patients who underwent robot-assisted laparoscopy were compared with 20 patients who underwent adjuvant surgery by laparotomy and 16 who underwent conventional laparoscopy, before the arrival of the Da Vinci surgical system.

Results. There was no significant difference between the three groups in terms of body mass index, FIGO stage, or tumor histology. The complication rate was similar in the three groups of patients, although there was a trend towards more lymphatic complications in the robotassisted subgroup managed medically. There was no significant difference in the recurrence rate between the robot-assisted laparoscopy, conventional laparoscopy and laparotomy groups (27.3%, 29.4% and 30%, respectively).

Conclusion.

Robot-assisted laparoscopy is feasible after concurrent chemoradiation and brachytherapy in cases of locally advanced cervical cancer. This new surgical approach reduces hospital stay, and seems to result in less severe complications than conventional laparotomy without modifying the oncological outcome.

Introduction

Chemoradiation is recognized as the standard treatment for locally advanced cervical cancer [START_REF] Green | Concomitant chemotherapy and radiation therapy for cancer of the uterine cervix[END_REF]. Whenever technically possible, chemoradiation should be complemented by adjuvant brachytherapy. If residual cervical tumors or lymph node involvement are observed, adjuvant surgery (or "end" surgery) is favored to optimize local control [START_REF] Keys | Cisplatin, radiation, and adjuvant hysterectomy compared with radiation and adjuvant hysterectomy for bulky stage IB cervical carcinoma[END_REF][START_REF] Classe | Surgery after concurrent chemoradiotherapy and brachytherapy for the treatment of advanced cervical cancer: morbidity and outcome: results of a multicentric study of the GCCLCC[END_REF].

Since the late 1990s, robotic assistance (Da Vinci; Intuitive Surgical) has found an important place in mini-invasive surgical procedures. Recently, various applications have been described in gynecological malignancies: endometrial cancer staging, radical hysterectomy [START_REF] Boggess | A case-control study of robot-assisted type III radical hysterectomy with pelvic lymph node dissection compare with open radical hysterectomy[END_REF], trachelectomy [START_REF] Persson | Robot-assisted abdominal laparoscopic radical trachelectomy[END_REF], and lymph node staging via the trans-peritoneal or extraperitoneal route [START_REF] Vergote | Robotic retroperitoneal lower para-aortic lymphadenectomy in cervical carcinoma: first report on the technique used in 5 patients[END_REF][START_REF] Magrina | Robotic radical hysterectomy: comparison with laparoscopy and laparotomy[END_REF].

We report our experience of this procedure in a subgroup of patients managed for advanced cervical cancer, after carrying out 100 robotic surgical gynecological procedures in our two cancer centers. To our knowledge, this is the first report to describe the use of this procedure in oncology patients, because "end surgery" is an option in advanced cervical cancer management and because robotic assistance is a new surgical technique. All of our patients were managed by experienced oncological surgical teams, especially with regard to conventional laparoscopy.

The aim of this study was to demonstrate the feasibility of robot-assisted laparoscopy in comparison to traditional laparotomy and conventional laparoscopy in a series of patients with locally advanced cervical cancer.

Patients and Methods

All the procedures has been performed by 4 surgeons, in our 2 institutions

Robot-assisted laparoscopy patients

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 4
Twenty-two patients underwent robot-assisted laparoscopy. Peri-and post-operative data were recorded prospectively, including operation time from skin incision to skin closure, and installation and docking time for the Da Vinci surgical system. Blood loss, peri-and postoperative morbidity, length of hospitalization (from the day of surgery), and histological results were also recorded.

Data concerning the characteristics of the patients, the surgical procedure, histological findings and follow-up were also collected.

Surgical procedure

Since the arrival of Da Vinci S in our oncological surgical departments (Marseille and Lille, France) in February 2007, adjuvant surgery in patients referred for advanced cervical cancer has been carried out laparoscopically with robot assistance.

All patients were placed in the low lithotomy position with their arms padded and tucked to the side. The Da Vinci unit was positioned between the legs for all pelvic procedures, with or without lombo-aortic exploration. In all cases, five ports were placed: four for the Da Vinci surgical system's arms (one camera port, three instrument ports) and the fifth as a classical laparoscopic port for the assistant (suction, specimen removal, needle application, etc).

The first port was placed after opening the abdominal cavity with a small abdominal incision to introduce the camera. The position of the camera depended on the anatomical site of the intended procedure. For pelvic surgery, the camera port was placed 1-2 cm above the umbilicus and the four additional ports were positioned in a curved line, keeping a 7-8 cm distance between the ports. After routine exploration of the peritoneal cavity, the Da Vinci unit was docked.

In the case of locally advanced cervical cancer (IB2, II, III), treatment included lomboaortic lymph node staging prior to concurrent chemoradiation to define the radiation field,

Traditional laparotomy and laparoscopy patients

Two groups of patients underwent the same treatment with adjuvant surgery performed by laparotomy (n=20) or conventional laparoscopy (n=16). Before the arrival of the Da Vinci robotic system, the choice of surgical approach depended on the surgeon's previous laparoscopy experience. However, if a bulky residual tumor was suspected on MRI after chemoradiation, laparotomy was the procedure of choice. In the conventional laparoscopic subgroup, radical hysterectomy was performed systematically.

Statistical analysis

Statistical analysis was performed using the Kruskall Wallis test and Chi-square test. A pvalue of 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 12 software for Windows. Differences in the survival curves or in the disease-free survival curves were estimated by log-rank test.

Results

Demographic and clinical characteristics of the patients

The characteristics of the 58 patients included in the study are shown in Table 1. There was no significant difference between the three groups in terms of body mass index, FIGO stage, or tumor histology. Two cases of FIGO stage IB1 and one case of FIGO stage IA2 were included as advanced cervical cancer because of pelvic lymph node involvement diagnosed surgically (laparoscopic lymph node staging before brachytherapy).

Comparison of surgical procedures

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 6
In terms of per-operative criteria, a significant difference was observed between robotassisted laparoscopy, conventional laparoscopy and laparotomy in terms of operative time; conventional laparoscopy took longer than robot-assisted laparoscopy and laparotomy in this indication (Table 2). This can be explained by the surgical approach used, which consisted of systematic radical hysterectomy in the subgroup treated by conventional laparoscopy.

On the other hand, a significant difference was observed in hospital stay between laparoscopy (robot-assisted and conventional) and laparotomy patients, while there was no difference between robot-assisted laparoscopy and conventional laparoscopy. No conversion was necessary in the two laparoscopic groups and blood transfusions were necessary in only two cases (one in the robotic group, one in the laparotomy group).

There was no significant different in complication rate between the three groups of patients (Table 2). However, there was a trend towards more lymphatic complications in the robot-assisted subgroup managed medically (painkillers) or with drains (two cases).

Histological findings and follow-up

The histological findings in the 56 patients are summarized in Table 3; there was no significant difference between the three groups, although residual cervical tumor was more frequent in the laparotomy group (patient selection after chemoradiation, with suspected bulky residual tumor). There was also no difference in median number of lymph nodes removed between the three groups. Follow-up was significantly higher with conventional laparoscopy and laparotomy.

Since the arrival of Da Vinci, all surgical indications previously managed by the traditional route are now managed robotically. There was no difference in recurrence rate between robotassisted laparoscopy, conventional laparoscopy and laparotomy (27.3%, 29.4% and 30%, respectively. The time interval between surgery and recurrence was also similar in the three

Discussion

The benefit of adjuvant surgery in the management of locally advanced cervical cancer is debatable. Only one randomized GOG study reported lower relapse-and higher progression-free survival in patients treated with radiation and surgery. Overall survival was similar in the two groups and all patients were treated with concurrent chemoradiation.

Surgery-associated morbidity is high: about 10% Grade 3-4 morbidity described in the randomized trial of Keys et al. [START_REF] Keys | Radiation therapy with and without extrafascial hysterectomy for bulky stage IB cervical carcinoma: a randomized trial of the Gynecologic Oncology Group[END_REF].

In a previous study, residual cervical tumors were observed in 40-50% of patients and pelvic positive lymph nodes in 16% [START_REF] Houvenaeghel | Contribution of surgery in patients with bulky residual disease after chemoradiation for advanced cervical carcinoma[END_REF]. In the current series, these data were not recorded since only two stage IIIs and one stage IV were observed in the three groups. It is known that that the rate of residual disease increases with FIGO stage: 43-52% in stage IB2, 41-56% in stages IIA and IIB, 51.6%-68% in stage III, and 72.7-73.7% in stage IVA [START_REF] Keys | Cisplatin, radiation, and adjuvant hysterectomy compared with radiation and adjuvant hysterectomy for bulky stage IB cervical carcinoma[END_REF][START_REF] Houvenaeghel | Recurrences of pelvic cancers. Pelvic reconstruction: when, how, why?[END_REF][START_REF] Classe | Pathological evaluation of pelvic radiotherapy with concurrent cisplatin-based chemotherapy followed by surgery in treatment of locally advanced cervical cancer: a retrospective multicentric experience of French Federation of Cancer Centers[END_REF].

In our experience, and as reported previously, stages III and IV seem to have an impact in terms of mortality but with no significant difference concerning overall survival compared to stages I and II [START_REF] Carcopino | Equivalent survival in patients with advanced stage IB-II and III-IVA cervical cancer treated by adjuvant surgery following chemoradiotherapy[END_REF]. In patients who do not respond to chemoradiation, in the absence of para-aortic involvement, 80% of patients survive to 5 years after curative surgery [START_REF] Houvenaeghel | Contribution of surgery in patients with bulky residual disease after chemoradiation for advanced cervical carcinoma[END_REF]. This argument justifies our therapeutic strategy and the absolute necessity to reduce the complication rate or grade. Mini-invasive surgery seems to be an attractive option and constitutes a new surgical field for robotically-assisted laparoscopy.

Several publications have reported on the feasibility of robot-assisted laparoscopy in cervical cancer: lymph node staging, radical hysterectomy, trachelectomy [START_REF] Boggess | A case-control study of robot-assisted type III radical hysterectomy with pelvic lymph node dissection compare with open radical hysterectomy[END_REF][START_REF] Persson | Robot-assisted abdominal laparoscopic radical trachelectomy[END_REF][START_REF] Lambaudie | Robot-assisted laparoscopy in gynaecologic oncology[END_REF], and the

M A N U S C R I P T A C C E P T E D

ACCEPTED MANUSCRIPT 8 first case of anterior pelvic exenteration has also been reported (Lambaudie et al., Gynecol Oncol 2009 in Press). Thus, there is no doubt about the feasibility of extrafascial or radical hysterectomy after chemoradiation, as described in this study. Indeed, 3-dimensional vision, tremor filtration and the precision and maneuverability of robotic instruments are advantages that assist in easier dissection, particularly in the case of fibrosis or tissue retraction after chemoradiation.

Boggess et al. [START_REF] Boggess | A case-control study of robot-assisted type III radical hysterectomy with pelvic lymph node dissection compare with open radical hysterectomy[END_REF], Magrina et al. [START_REF] Magrina | Robotic radical hysterectomy: comparison with laparoscopy and laparotomy[END_REF] and Sert et al. [START_REF] Sert | Robotic radical hysterectomy in early-stage cervical carcinoma patients, comparing results with total laparoscopic radical hysterectomy cases. The future is now?[END_REF] in their respective series of radical hysterectomy observed significant difference between robot-assisted laparoscopy, conventional laparoscopy and laparotomy in terms of operation time, mean blood loss, hospital stay and histological findings (particularly quality of lymph node dissection).

As in the current series, Nezhat [START_REF] Nezhat | Minimally invasive surgery in gynaecologic oncology: laparoscopy versus robotics[END_REF] did not observe any significant differences in peroperative criteria or histological findings between two groups of patients who underwent laparoscopic robot-assisted radical hysterectomy (n=13) or conventional laparoscopic radical hysterectomy (n=30). The conclusion of this prospective experience was that despite the multiple advantages of robotic assistance published in the literature, robot-assisted procedures have no advantages compared to traditional total laparoscopic radical hysterectomy when performed by an experienced laparoscopic surgeon.

Robot-assisted laparoscopy a major evolution of laparoscopy. Robot-assisted laparoscopy and conventional laparoscopy are similar in terms of operation time (without docking time), hemorrhagic complications, median number of resected lymph nodes and hospital stay. Difference concerning lymphatic complications has to be confirmed. We think that it is due to the greater interest of this type of complications since almost 5 years (but not to surgical technique which is not different in our 3 groups concerning lymph node dissection), which has an impact on quality of life.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

9 However, in this study, surgical teams were experienced in conventional laparoscopy.

In an experienced oncological surgeon's hands, robot-assisted laparoscopy, with a shorter learning curve, should develop for the patients' benefit (little scarring, lower hemorrhagic complications, and shorter hospital stay). The real advantage of robot-assistance is its application to advanced laparoscopic surgical procedures with less training than for conventional laparoscopy.

Reynolds and Advincula [START_REF] Reynolds | Robot assisted laparoscopic hysterectomy: technique and initial experience[END_REF] suggested a cutoff at 30 patients to obtain a similar complication rate and operation time for robot-assisted hysterectomy compared to abdominal hysterectomy. We think that previous laparoscopic experience and the possibility of performing the first robotic procedure by a team of two surgeons should contribute to a reduced learning curve.

In conclusion, robot-assisted laparoscopy is feasible after concurrent chemoradiation and brachytherapy in cases of locally advanced cervical cancer. This new surgical approach reduces hospital stay, and is associated with a lower rate of serious complications than laparotomy without modifying the oncological outcome. In the hands of experienced oncological surgeons, robot-assisted laparoscopy with a shorter learning curve should develop for the patients' benefit (laparoscopic advantages: little scarring, lower hemorrhagic complications and shorter hospital stay).

Conflict of Interest Statement

  surgery (simple colpohysterectomy + lymphadenectomy). Radical hysterectomy was performed only if difficulties were encountered during the surgical procedure in exposing surgical landmarks (ureter, uterine pedicle) due to fibrosis and retraction after concomitant chemoradiation and brachytherapy.

  of the short follow up, we did not observe significative difference in term of overall survival and disease-free survival.

EricTable 1 :Table 2 :Table 3 :

 123 FIGO stage

Table 4 :

 4 Follow-up, recurrences and mortality of patients undergoing robot-assisted laparoscopy, conventional laparoscopy and laparotomy

		Robot-assisted	Conventional	Laparotomy	p
		laparoscopy	laparoscopy	(n=20)
		(n=22)	(n=16)
	Median follow-up (months)	11.55	19.45	34.6	<0.001
	(range)	(3.5-22.5)	(2.4-50)	(15.8-84.4)
	Median time interval between
	surgery-recurrence (months)	7.05	11.9	7.9	NS
	(range)	(4.1-7.6)	(7.9-29.1)	(7.5-43.3)
	Recurrences, n (%)	6 (27.3%)	4 (25%)	6 (30%)	NS
	Local	2	2	2
	Nodes	2		3
	Lung metastasis	2
	Liver metastasis			1
	Brain metastasis		1
	Trocart port metastasis		1
	Mortality, n (%)	2 (9.1%)	2 (11.8%)	4(20%)	NS


Jaroslav Pec

ˇ • Isvett

Josefina Flores-Sanchez

Young Hae Choi

Robert Verpoorte
email: verpoort@chem.leidenuniv.nl

I J Flores-Sanchez
email: i.flores.sanchez@chem.leidenuniv.nl

Á R Verpoorte

Metabolic analysis of elicited cell suspension cultures of Cannabis sativa L. by 1 H-NMR spectroscopy

Keywords: Cannabis sativa, Cell suspension cultures, Elicitation, Metabolomics, Nuclear magnetic resonance spectroscopy, Tyrosol

Cannabis sativa L. plants produce a diverse array of secondary metabolites. Cannabis cell cultures were treated with jasmonic acid (JA) and pectin as elicitors to evaluate their effect on metabolism from two cell lines using NMR spectroscopy and multivariate data analysis. According to principal component analysis (PCA) and partial least squarediscriminant analysis (PLS-DA), the chloroform extract of the pectin-treated cultures were more different than control and JA-treated cultures; but in the methanol/ water extract the metabolome of the JA-treated cells showed clear differences with control and pectin-treated cultures. Tyrosol, an antioxidant metabolite, was detected in cannabis cell cultures. The tyrosol content increased after eliciting with JA.

Introduction

Cannabis sativa L. is an annual dioecious plant from Central Asia. A number of secondary metabolites have been described to occur in this plant [START_REF] Flores-Sanchez | Secondary metabolism in cannabis[END_REF]. Cannabinoids are a well known group of natural products in this plant and more than 70 different cannabinoids have been found so far [START_REF] Elsohly | Chemical constituents of marijuana: the complex mixture of natural cannabinoids[END_REF]. Several therapeutic effects of cannabinoids have been described [START_REF] Williamson | Cannabinoids in clinical practice[END_REF] and the discovery of the endocannabinoid system in mammals marked a renewed interest in these compounds [START_REF] Heifets | Endocannabinoid signaling and long-term synaptic plasticity[END_REF].

Metabolomics is a rapidly developing discipline in plant biology and has become an integral part of many functional genomics programs [START_REF] Hagel | Plant metabolomics: analytical platforms and integration with functional genomics[END_REF]. In recent years metabolite profiling has become routinely applicable across biology, being useful for phenotyping and diagnostic analyses. Several analytical platforms have been proposed for characterizing and quantifying the metabolites in plants in a systematic manner [START_REF] Hagel | Plant metabolomics: analytical platforms and integration with functional genomics[END_REF]. 1 H-NMR spectroscopy is one of these platforms which is currently being explored together with principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA), the most common methods to analyze the variability in a group of samples.

Metabolic pathway fluxes depend on the environment in which the cell is grown. For example, in plant cell cultures and plants treated with biotic elicitors clear qualitative and quantitative variations in the metabolic pool were shown [START_REF] Sanchez-Sampedro | Metabolomic alterations in elicitor treated Silybum marianum suspension cultures monitored by nuclear magnetic resonance spectroscopy[END_REF][START_REF] Jahangir | Metabolomic response of Brassica rapa submitted to pre-harvest bacterial contamination[END_REF]. Although, cannabis cell cultures do not produce cannabinoids, even after elicitation treatments [START_REF] Flores-Sanchez | Elicitation studies in cell suspension cultures of Cannabis sativa L[END_REF], analyses in the content of the metabolite pool from two cell lines were made in order to study the effect of elicitation on the Cannabis sativa cell suspension culture metabolome. Here, we report the results from these studies.

Materials and methods

Chemicals

Chloroform-d (99.80%, v/v) and methanol-d 4 (99.80%, v/v) were obtained from Euriso-top (Paris, France). Water-d 2 (99%, v/v) was acquired from Spectra Stable Isotopes (Columbia, MD, USA). NaOH-d was purchased from Cortec (Paris, France). All chemical products and mineral salts were of analytical grade.

Plant material and cell culture methods

Cell suspension cultures were induced from leaf explants of Cannabis sativa L. (drug type variety Fourway) and were maintained in MS basal medium [START_REF] Murashige | A revised medium for rapid growth and bioassays with tobacco cultures[END_REF] supplied with B5 vitamins [START_REF] Gamborg | Nutrient requirements of suspension cultures of soybean root cells[END_REF]), 2 mg 2,4-D/l, 1 mg kinetin /l and 30 sucrose g/l. Cells were subcultured with a 3-fold dilution every 2 weeks. Cultures were grown at 25°C with shaking at 110 rpm and under a light intensity of 14-23.8 lmol/m 2 s. Cellular viability measurement was according to [START_REF] Widholm | The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells[END_REF].

Elicitation

Jasmonic acid (JA, Sigma) was dissolved in 30% (v/v) ethanol. Pectin suspensions from Citrus fruits (87% galacturonic acid and 8.7% methoxy groups, Sigma) were prepared according to the method of [START_REF] Flores-Sanchez | Biosynthesis of sterols and triterpenes in cell suspension cultures of Uncaria tomentosa[END_REF].

Erlenmeyer flasks (250 ml) containing 50 ml medium were inoculated with 5 g fresh cells. Five days after inoculation 100 lM JA or 50 mg pectin was added to the suspension. The cultures were harvested every 2 days.

Extraction of compounds for the metabolic profiling

Metabolite extraction was carried out as described by [START_REF] Choi | Metabolomic differentiation of Cannabis sativa cultivars using 1 H-NMR spectroscopy and principal component analyses[END_REF] with slight modifications. To 0.1 g of lyophilized plant material either 4 ml methanol/ water (1:1, v/v) or 4 ml chloroform was added, mixed for 30 s and sonicated for 10 min. The mixtures were centrifuged at 4°C and 12009g for 20 min. Each extraction was performed twice. The methanol/water and chloroform fractions were separated and evaporated. Extractions with ethyl acetate were also performed. The organic phase was dried over anhydrous Na 2 SO 4 and evaporated. Extracts were stored at 4°C.

NMR measurements and data analyses

The dried fractions were dissolved in chloroform-d and methanol-d 4 :water-d 2 (1:1, v/v, pH 6), respectively. KH 2 PO 4 was used as a buffering agent for methanol-d 4 :water-d 2 . Sodium trimethylsilyl propionate (TMSP) was used as internal standard for methanol-d 4 /water-d 2 . Dried medium extracts were dissolved in methanol-d 4 . Measurements were carried out at 25°C using a Bruker AV-500 NMR instrument operating at a proton NMR frequency of 500.13 MHz. For each sample, 128 scans were recorded using the same parameters as previously reported by [START_REF] Choi | Metabolomic differentiation of Cannabis sativa cultivars using 1 H-NMR spectroscopy and principal component analyses[END_REF]. The 1 H-NMR spectra were automatically reduced to ASCII files using AMIX (v. 3.7,Bruker Biospin). Spectral intensities were scaled to total intensity for the chloroform and methanol extracts and to TMSP for the methanol/water extract and reduced to integrated regions of equal width (0.04 ppm) corresponding to the region d 0.3-10. The regions of d 3. 30-3.34, d 4.74-4.98 and d 7.18-7.3 were excluded from the analyses because of the residual signal of methanol, water and chloroform, respectively. Principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) were performed with the SIMCA-P software (v. 11.0, Umetrics Umea ˚, Sweden). Compounds were quantified by the relative ratio of the intensities of their peak-integrals and the ones of internal standard according to [START_REF] Flores-Sanchez | Elicitation studies in cell suspension cultures of Cannabis sativa L[END_REF]. The characteristic 1 H-chemical shifts (d) and coupling constant (Hz) of identified metabolites were previously reported [START_REF] Flores-Sanchez | Elicitation studies in cell suspension cultures of Cannabis sativa L[END_REF]. For structure elucidation of aromatic metabolites, 1 H-NMR and 2D-NMR (HMBC, HMQC, J-Resolved and 1 H-1 H-COSY) was used as described by [START_REF] Choi | Metabolomic differentiation of Cannabis sativa cultivars using 1 H-NMR spectroscopy and principal component analyses[END_REF] and for aromatic compound isolation Sephadex LH20 column chromatography with methanol as eluent was used. Eighteen fractions were collected and the profiles were analyzed by TLC with silica gel 60F 254 plates, developed in methanol/ chloroform (9:1,v/v) and revealed with anisaldehyde/sulfuric acid reagent. Fractions 4 and 5 were mixed and purified by solid phase extraction (SPE) using SEP-Pak Cartridge Plus C18 (Waters, Milford, MA, USA). From the first elution using water, phenylalanine together with traces of tyrosine and tyramine were identified. From the second elution using methanol/water (1:1, v/v), tyrosine, tyrosol, tyramine and tyramine glycoside were identified (Fig. 7).

Statistics

Data were analyzed by MultiExperiment Viewer MEV 4.0 software (TM4 Software Suite, J. Craig Venter Institute, Rockville, MD, USA). For analyses involving two and three or more groups paired t-test, ANOVA, PCA and PLS-DA were used, respectively with a = 0.05 for significance. Values are expressed as means of three replicates with standard deviations.

Results and discussion

Two cell lines were generated from leaf explants which were either green (GC cells) or greenish brown (GB cells). According to the principal component analysis (PCA) from the chloroform fraction, the two cell lines were separated by cell line type (second principal component, PCA2) and developmental stage (first principal component, PCA1) (Fig. 1a). The loading plot suggests that in the younger samples unsaturated fatty acids are predominant; while in older samples saturated fatty acids are predominant (Fig. 1b). The 1 H-NMR spectral region of 5.08-5.4 ppm (d) represents signals of double bonds from unsaturated fatty acids and the region of d 0.8-1.9 represents signals from saturated fatty acids. Both cell lines have a different profile of saturated fatty acids and unsaturated fatty acids could be a marker for the cell line GB as they are the predominant fatty acids in this cell line (Fig. 1c).

Both cell lines were elicited with JA and pectin. According to partial least square-discriminant analysis (PLS-DA) from the chloroform fraction the pectin treatment had more effect on the metabolism of saturated fatty acids in both cell lines than the JA treatment (Fig. 2). The separation between control and JA-treated cultures is due to a difference in the unsaturated fatty acid contents. No cannabinoid signals were detected, as it was reported previously [START_REF] Flores-Sanchez | Elicitation studies in cell suspension cultures of Cannabis sativa L[END_REF].

When the methanol/water fraction from control samples was analyzed, a separation was found between the two cell lines based on cell line type (PC4) and developmental stage or culture age (PCA1) (Fig. 3). According to the loading plot of PC1 (Fig. 3b), aromatic compounds tend to predominate in older cultures. Based on PC4, in GB cultures alanine (d 1.48), GABA (c-aminobutyric acid, d 1.90, d 2.31 and d 3.0) and sucrose (d 5.4) were dominant metabolites. In GC cultures leucine (d 1.71), glutamine (d 2.13), glutamic acid (d 2.45), aspartic acid (d 3.95), tryptophan (d 7.72) and tyrosine (d 3.86) were the dominant metabolites (Fig. 3c). The methanol/water fractions from elicited samples show a different metabolic profiling compared to the control samples (Fig. 4). JA elicitation had a stronger effect on the metabolome of both cell lines than pectin elicitation. Based on PLS-DA analysis sucrose, glucose, choline, valine, isoleucine, glutamic acid and GABA were the predominant metabolites in pectin-treated cultures from both cell lines (Fig. 4b).

Phenylalanine, tyrosine, tyrosol, tyramine and tyramine glucoside (Fig. 7) were the aromatic compounds identified in the aromatic region (d 6.0-8.0) of the NMR spectra of JA-treated cultures (Supplementary Table 1; Fig. 4b). Induction of phenylalanine, tyramine and tyrosine in C. sativa cell suspension cultures by JA elicitation has been reported [START_REF] Flores-Sanchez | Elicitation studies in cell suspension cultures of Cannabis sativa L[END_REF]. A time-course shows that the elicitation with JA increased the tyrosol concentration twofold in GB cell cultures if compared to GC cell cultures at day 16 (Fig. 5). The content of tyrosol from Numbering is developmental stage or culture age (from day 0-28). b Control region in loading plot contains leucine (1), aspartic acid (2), tryptophan (3), trigonelline (4), glutamine (5), threonine (6), adenosine (7) and cytidine (8); pectin-treated region contains sucrose (9), glucose (10), choline (11), valine (12), isoleucine (13), c-amino-butyric acid (GABA, 14) and glutamic acid (15); and JA-treated region contains phenylalanine (16), tyrosine (17), tyramine (18), tyramine glycoside (19), tyrosol (20) [START_REF] Aturki | Analysis of phenolic compounds in extra virgin olive oil by using reversed-phase capillary electrochromatography[END_REF]Sotiroudis and Kyrtopoulos 2008). It has an antioxidant activity [START_REF] Giovannini | Tyrosol, the major olive oil biophenol, protects against oxidized-LDLindiced injury in Caco-2 cell[END_REF]. Tyrosol occurs in plants of the Rhodiola species [START_REF] Li | Identification and comparative determination of rhodionin in traditional Tibetan medicinal plants of fourteen Rhodiola species by high-performance liquid chromatography-photodiode array detection and electrospray ionization-mass spectrometry[END_REF] and in foliar fungal endophytes of Picea glauca [START_REF] Sumarah | Characterization of polyketide metabolites from foliar endophytes of Picea glauca[END_REF]. There are no previous reports about the presence of tyrosol in C. sativa. Analysis of the tyrosol content by 1 H-NMR spectroscopy between virgin olive oil (0.06 lmol/100 mg; Christophoridou and Dais 2009) and the elicited cannabis cell lines (2.7 ± 0.1 lmol/100 mg in GB line and 1.2 ± 0.01 lmol/100 mg in GC line) suggests that cannabis cell suspension culture could be used for the production of this antioxidant metabolite.

Principal component analysis (PCA) analysis of the methanol fraction from medium extracts of the control and elicited cell cultures shows that the cell line GC secretes tyrosine, tyrosol and tyramine into the culture medium (Figs. 6,7; Supplementary Table 2). Apparently, the excretion of these aromatic compounds was not affected by JA or pectin treatment in the GC cell line and it could be an inherent part of the metabolism of this cell line. 10.00000" 9.64000" " 9.24000" " 8.84000" " 8.44000" " 8.04000" " 7.64000" " 7.24000" " 6.84000" " 6.44000" " 6.04000" " 5.64000" " 5.24000" " 4.32000" " 3.84000" " 3.44000" " 3.00000" " 2.60000" " 2.20000" " 1.76000" " 1.36000" " 0.88000" " 0.48000" " 10.00000" 9.64000" " 9.24000" " 8.84000" " 8.44000" " 8.04000" " 7.64000" " 7.24000" " 6.84000" " 6.44000" " 6.04000" " 5.64000" " 5.24000" " 4.32000" " 3.84000" " 3.44000" " 3.00000" " 2.60000" " 2.20000" " 1.76000" " 1.36000" " 0.88000" " 0.48000" "

Fig. 1

 1 Fig. 1 Principal component analysis (PCA) from 1 H-NMR data of CHCl 3 fractions from the control cell lines green (GC, open circle) and greenish-brown (GB, filled circle). a Score plot, b PC1 loading plot, c PC2 loading plot. Numbering represents developmental stage or culture age (from day 0-28)

Fig. 2 Fig. 4

 24 Fig. 2 Score (a) and loading (b) plots of PLS-DA analysis from 1 H-NMR data of chloroform fractions. open circle control, asterisk JA-treated, filled circle Pectin-treated. a Numbering represents developmental stage or culture age (from day 0-28). b Control region in loading plot contains unsaturated fatty acids (1); pectin-treated region contains saturated fatty acids (2) and JA-treated region contains unsaturated fatty acids (3)

C

  Effect of cell line type compounds present in olive oil

Fig. 5 Fig. 6

 56 Fig. 5 Time course of tyrosol content in the control, JA and pectin-treated cannabis cell cultures. GC green cell line, GB greenish-brown cell line

Fig. 7

 7 Fig.7Structures of tyrosine and its metabolites identified from methanol/water and methanol fractions

  and ethanol glucoside (21)

		0.40
		0.30
	PC1	0.20
		0.10
		0.00
		-0.10
		10	9.64	9.24	8.84	8.44	8.04	7.64	7.24	6.84	6.44	6.04	5.64	5.24	4.6	4.2	3.8	3.4	2.96	2.56	2.16	1.76	1.36	0.96	0.56
										Chemical shift (ppm)
											SIMCA-P+ 12 -2009-11-04 06:52:08 (UTC+1)
		0.30
		0.20
		0.10
	PC4	-0.00
		-0.10
		-0.20
		-0.30
		-0.40
		10	9.64	9.24	8.84	8.44	8.04	7.64	7.24	6.84	6.44	6.04	5.64	5.24	4.6	4.2	3.8	3.4	2.96	2.56	2.16	1.76	1.36	0.96	0.56
										Chemical shift (ppm)
											SIMCA-P+ 12 -2009-11-04 06:54:10 (UTC+1)

BiotechnolLett (2010) 32:935-941

Acknowledgements I.J. Flores Sanchez received a partial grant from Consejo Nacional de Ciencia y Tecnologia (Mexico) and J. Pec ˇreceived a grant from Ministry of Education of the Czech Republic (MSM 0021620822).