D Popov

A Buléon

M Burghammer

H Chanzy

N Montesanti

J-L Putaux

G Potocki-Véronèse

C Riekel

Crystal Structure of A-amylose: a Revisit from Synchrotron Microdiffraction Analysis of Single Crystals

The three-dimensional structure of A-amylose crystals, as a model of the crystal domains of A-starch granules, was revised using synchrotron radiation microdiffraction data collected from individual micron-sized single crystals. The resulting datasets allowed a determination of the structure with conventional X-ray structure determination techniques normally used for small molecules and not for polymers. Whereas the gross features of this improved structure do not differ extensively from previous structure determination, the high resolution of the diffraction diagrams, which is unusual for a crystalline polymer, allowed the resolution of important new fine details. These include a distortion of the amylose double helices resulting from the occurrence of two intracrystalline molecules of water and a tight network of hydrogen bonds involving each of the primary and secondary hydroxyl groups of the glucosyl moieties. Pairs of water molecules are located in discrete pockets that do not interfere with one another. In addition, the refinement of the new structure indicates a "parallel-down" organization of the amylose molecules within the unit cell as opposed to the previous "parallel-up" model. This new feature indicates that within the crystals, the nonreducing ends of the amylose molecules are oriented toward the c-axis direction of the unit cell. The description of this geometry is important to correlate the crystallography of the granules of A-starch with their ultrastructure and their mode of biosynthesis.

Here, we present for the first time the resolution of the structure of a polymer crystal from a full X-ray dataset collected on micron-sized polymer single crystals using synchrotron radiation microdiffraction. This achievement is a substantial advance, which opens the way to many more studies since the technique of growing polymer and biopolymer single crystals is well established.

Introduction

As opposed to the molecular and crystal structure determination of low molecular weight compounds, which is being done with great precision on a routine basis, the resolution of the structure of polymer crystals is still a major challenge since it requires a number of intuitive guesses together with trial-and-error methods. [START_REF] Tadokoro | Structure of Crystalline Polymers[END_REF] Indeed, many small molecules are susceptible to be crystallized as millimeter or sub-millimeter-sized crystals, yielding extensive diffraction datasets, with frequently more than 10 to 20 observed reflections per independent atom, the minimum ratio that is required for a proper structure refinement. [START_REF] Stout | In X-ray Structure Determination[END_REF] This is not the case for most polymers for which only crystals with dimensions ranging from sub-micron to the micron can be obtained. These crystals are far too small to yield substantial X-ray datasets when conventional X-ray diffractometers are used. In most cases, one relies on the study of crystalline fibers, in which a large number of extremely small polymer crystals are aligned with their chain axis lying along the fiber direction, but distributed in a more or less cylindrical organization perpendicular to the fiber axis. With a few exceptions, the resulting fiber diffraction diagrams are limited in resolution. Furthermore, their deconvolution into a three dimensional data set is frequently a major challenge that is not always easy to overcome unambiguously. Even simple crystallographic parameters such as those of the unit cell or the space group and symmetry elements are difficult to deduce from X-ray fiber diffraction diagrams of polymers.

In view of their small size, isolated polymer single crystals are usually amenable to electron diffraction analysis, [START_REF] Geil | Polymer Single Crystals[END_REF] provided that the electron beam intensity is kept low and that the vacuum of the transmission electron microscope does not damage the crystals. In the best cases, a series of meaningful spot electron diffraction diagrams can be recorded from individual polymer crystals. These electron diffraction diagrams, which frequently are of high resolution, have become quite useful to ascertain the unit cell parameters and symmetry elements of the crystal systems of many polymers. [START_REF] Perez | [END_REF] In particular, electron diffraction analysis can be of great help to deconvolute the fiber X-ray diagrams and in a number of cases, the input of electron diffraction has corrected erroneous unit cells and symmetry elements that had been proposed earlier from X-ray fiber diffraction analysis. Despite these advances, the exploitation of the electron diffraction intensities for crystal structure determination is not straightforward, since one needs to subtract dynamic contributions from electron diffraction intensities before any further analysis. At present, there is no satisfactory way to measure or calculate this dynamic contribution for polymer crystals.

The resolution of the crystal structure of A-amylose is the focus of this report.

Amylose, the linear constituent of starch can be recrystallized in vitro into the A or B allomorphs that are present in various native starches. The low crystallinity and complex ultrastructure of the starch granule do not allow a direct determination of the three-dimensional shape and distribution of the crystalline domains. Thus, structural models established from A-or B-amylose crystals can be transposed to the crystalline regions of native starch, which exhibit similar, but much less resolved diffraction diagrams. Getting an accurate molecular description of the A allomorph is important, since it is one of the main constituents of A-starch, [START_REF]Starch granules contain a high percentage of high molecular weight amylopectin, a highly branched macromolecule whose clusters of short linear (amylose-like) branches intertwine into parallel-stranded double helices, which pack together into crystalline domains, responsible for the crystallinity of native starch. Depending on the parent plant species and the packing of the double helices, one distinguishes two different allomorphs, namely A-and B-starch[END_REF] chiefly found in cereal starch, which not only constitutes an essential food product for humankind, but also serves as a base for a number of non-food industrial uses.

Whereas the first X-ray powder diagrams of A-amylose had already been recorded in 1930, [START_REF] Katz | [END_REF] it is only in the late seventies that a first three-dimensional model was proposed by the group of Sarko, using X-ray fiber diffraction diagrams showing 46 measurable diffraction spots. 7 At that time, it was proposed that A-amylose was crystallized as parallel stranded, right-handed double helices, organized in an orthorhombic unit cell, with unspecified space group. In addition, the model showed that the double helices were packed in an antiparallel fashion. Thanks to the successful preparation of micron-sized A-amylose single crystals 8 and their study by electron diffraction, 9 the X-ray fiber diffraction diagrams of Sarko could be re-indexed. A new unit cell was derived and the monoclinic space group B2, indicative of highly symmetrical structure, was proposed. Using the 34 strongest intensities of the dataset of Sarko's group, together with the electron diffraction information, an improved structure of Aamylose was proposed by Imberty et al., combining conformational analysis and X-ray refinement. 10 This new A-amylose model was still made of parallel-stranded double helices, but they were left-handed instead of the previous right-handed ones. In addition, these double helices were packed in a parallel fashion as opposed to Sarko's structure, where the packing was antiparallel. In view of the unit cell and symmetry elements, the structure proposed by Imberty et al. was described as resulting from an independent maltotriosyl residue, which was positioned in a parallel-up [START_REF]The "parallel-up" and "parallel-down[END_REF] mode within the unit cell and there was one water molecule per trisaccharide unit. 10 Despite its novelty, this model could not be refined, since the three dimensional coordinates of the 34 independent non-hydrogen atoms of this amylose hydrate could not be localized with precision, using only 34 diffraction intensities, each of them consisting of a number of overlapping reflections.

The recent development of microfocus beamlines at 3 rd generation synchrotron radiation sources, such as the ESRF in Grenoble, [START_REF] Riekel | [END_REF] has drastically changed the situation, as novel X-ray optical systems, allowing the production of focal spots with diameters in the micron and sub-micron sizes are now available. In addition, goniometers for keeping a single crystal in the center of micron-sized beams have also been developed. 13,14 Thus, when applied to very small crystalline objects such as a polymer single crystal, one can envisage the recording of X-ray datasets more complete than those recorded in electron diffraction experiments, and therefore susceptible to yield improved structural models. This paper follows a preliminary report, which showed that spot X-ray diagrams could be recorded on 5-10 µm needle-like single crystals of A-amylose, using a synchrotron X-ray beam of 10 m. 15 Here, we show how a full three-dimensional dataset was collected on such microcrystals and used to refine the structure of this amylose allomorph. To our knowledge, this is the first polymer structure determined using this technique on such small polymer single crystals. Getting an accurate atomic description of the A-amylose crystal is important since these crystals constitute part of the cereal starches, a food product of capital importance.

Experimental

Sample preparation. Amylose was enzymatically synthesized in vitro by amylosucrase 16 from sucrose as glucosyl donor and maltose as acceptor, using a molar ratio of 6. The reaction products were subsequently fractionated by preparative gel filtration, using a 7 L column of Biogel P-2 Fine (Biorad), and freeze-dried for further use. A fraction of amylose with an average degree of polymerization (DPn) of 17 and a polydispersity of 1.06 was used to prepare single crystals of A-amylose by recrystallizing dilute aqueous amylose solutions in presence of acetone vapors, as described elsewhere. 9 Typical needle-shaped crystals are shown in Figure 1a. Using a Kleindiek MM3A micromanipulator under an optical microscope, individual crystals were glued with Araldite to the tip of a tapered glass capillary (Figure 1b). The orientation of the needles with respect to the glass capillary varied from almost parallel to almost perpendicular.

Data collection.

Experiments were performed at the ID13 beamline of the European Synchrotron Radiation Facility (Grenoble, France), with a wavelength of =0.9465 Å defined by a Si-111 monochromator. The beam was focused with parabolic Be-refractive lenses 17 and collimated to either 10 or 30 m at the ID13 microgoniometer, 18 operating with a flux of 10 11 photons/s. The crystals were flash-frozen to 100 K using a N 2 cryostream system. Diffraction patterns were recorded on a MAR165 CCD with 2K2K pixels of 78.9478.94 µm 2 and 16 bit readout. Individual patterns, such as the one exemplified in Figure 2, were obtained on crystals that were rotated by 2 to 4° during data collection, with exposure times of 1 to 2 s.

The diffraction patterns were processed using the XDS package. [START_REF] Kabsch | Crystallography of Biological Macromolecules[END_REF] As was reported before, 15 severe radiation damage strongly reduced the number of patterns that could be collected on one crystal, because of structural decay and concomitant drop of the spot intensities. For this reason, and in order to increase redundancy and completeness of the diffraction information, datasets collected on 14 different crystals having 10-15 m length and 2-3 m thickness were merged using the XSCALE software. [START_REF] Kabsch | Crystallography of Biological Macromolecules[END_REF] The X-ray data sets were collected in 20-30 s of total exposure which corresponds to a change of unit cell parameters introduced by the radiation damage of about 0.1 Å. 15 A summary of the experimental parameters is given in Table 1. The compatibility of the collected data sets was checked by verifying that the correlation coefficients between the intensity values in each dataset were reasonably high. It was found that the dataset collected on a given crystal could be merged with at least 4 others with a correlation coefficients higher than 0.95. With a few other crystals, selected correlation coefficients deviated below this limit but were never smaller than 0.8. Typical I/σ value estimated at the stage of integration of the diffraction images were not higher than the final I/σ value obtained after the scaling of all the intensity data. The final value of the R mrgd-F =0.137 which reflects the quality of the intensity data in terms of statistics was significantly lower than R sym =0.193.

Data reduction and structure refinement. Previous results of data reduction 15 have confirmed the B2 space group already proposed by Imberty et al. 9 Data collected on four crystals showing the smallest structural change introduced by the radiation damage was used to refine the unit cell parameters: a=20.83(6) Å, b=11.45(4) Å, c=10.58(3) Å, =122.0(2)°.

The crystal structure was solved using a molecule replacement technique with the PATSEE software [START_REF] Egert | [END_REF] within the WinGX program complex. 21 A fragment of a regular left-handed 6-fold helix 10, 22 of 3 residues length without primary hydroxyl groups was used as a search fragment. A reasonable orientation was found for the search fragment with respect to 2-fold symmetry axis giving infinite double helix. The model was refined using SHELXL. 23 The positions of primary hydroxyl groups and two positions of water molecules were found on electron difference maps. At the beginning of the refinement, a rigid group approach was used and then bond length, angular and anti collision restraints were applied. Pyranose hydrogen atoms were inserted into calculated positions and included into refinement in the 'riding' model whereas the hydrogen atoms of the hydroxyl groups were not considered. During all the refinement, occupations of water molecules were fixed at 1 but after the final stage, they were refined starting from 0.5 and 1 with either fixed or unfixed thermal parameters of water molecules. In these four cases, the final value did not deviate from 1 by more than the estimated standard deviation (e.s.d.). The final refinement, which was done in the isotropic approach of thermal parameters, gave R1 and R1 free values of 0.17 and 0.22 respectively. The fact that these R-factors are relatively higher then those typically obtained with small molecules, is attributed to the unavoidable beam damage in our micron-sized crystals. Indeed, due to such irradiation, the exposure time had to be limited to 1 to 2 s/frame, with the result of a resolution of the X-ray data not better than 1.3 Å. Such resolution is lower than the one commonly obtained in the data collection on large crystals of small molecules.

The compatibility of the intensity values obtained on a given crystal was checked by re-refinement of the structure against the data collected on the other crystals. In order to do this, all 14 datasets collected on the selected A-amylose crystals were merged into two independent groups (7 original datasets per group) and the structure was re-refined against each of the two resulting datasets. The average discrepancy in the corresponding final atomic positions between these two groups was 0.03 Ǻ. The highest deviation of 0.156 Å was found for a primary hydroxyl group. These values reasonably match the e.s.d. of the atomic coordinates obtained after the refinement of the A-amylose structure against the whole diffraction dataset.

Results

Structure description

As outlined in Table 1, the structure was refined in the monoclinic space group B2, with unit cell parameters a = 20.83(6) Å, b = 11.45(4) Å, c (chain axis and monoclinic axis) = 10.58(3) Å and  = 122.0(2)°. In view of these parameters, the structure of A-amylose is based on the repeat of a maltotriosyl residue, and there are 4 such residues per unit cell. The labeling of the atoms along one of the amylose chain is shown in Figure 3, whereas the atomic final coordinates are presented in Table 2. The values of the bond lengths and angles are tabulated in the Supporting Information section. Each of these values falls in the range of those defined for polysaccharides containing pyranose rings. 25 The main conformational angles defining the chain geometry are listed in Table 3. For reference, we also have listed In Table 3, the conformational parameters of the earlier A-amylose structure derived from fiber and electron diffraction analysis. 10 A comparison of the present structure with the earlier one, indicates that the gross features of both structures are similar, but that they differ substantially in their finer details.

In agreement with the earlier structure, our A-amylose structure also consists of intertwined 6-fold left-handed parallel double helices, packed in a parallel fashion and tightly nested into one another. Each double helix is centered on the two-fold axis of the unit cell and the hydroxymethyl groups of each glucosyl residue adopt the gg conformation. [START_REF]The conformation of the hydroxymethyl group is defined by two letters, the first referring to the torsion angle  (O5-C5-C6-O6) and the second to the torsion angle ' (C4-C5-C6-O6). An ideal gg conformation would be defined as the set of two angles[END_REF] In both structures, the torsion angles  and  are not so different from one glucosyl residue to the next, with a range from 79 to 93° for , and from -145 to -153° for . In these two structures, the sets of  and  values that define the linkage of contiguous glucosyl residues indicate that the corresponding conformations fall in an area of minimum energy in the conformational energy  / map of maltodextrin. [START_REF] Pangborn | [END_REF]28 In addition, these values also indicate that such sets of conformational angles preclude the formation of intramolecular hydrogen bonds within contiguous glucosyl residues. 29 In contrast with the conformational torsion angles  and , which are in reasonable agreement between this structure and the earlier one, there is some difference in the bond angles in the present structure, ranges from 111 to 114°, whereas in the earlier structure, one of value was listed at 120.5°, a value that can be considered as rather high for -linked oligosaccharides. 29 Another main difference between the present structure and the previous one is the orientation of the amylose molecules within the unit cell.

In the earlier structure, a "parallel-up" situation [START_REF]The "parallel-up" and "parallel-down[END_REF] was proposed, indicating that the hemiacetal terminal moiety, the so-called reducing end, was oriented along the c-axis directionality of the unit cell. In the present case, a "parallel-down" packing is unambiguously observed, since the z coordinates of the C5 are higher than those of O5 in any given glucosyl moieties.

Water content

An electron density map of the independent maltotriosyl residue together with its hydration system is shown in Figure 4. This projection reveals without ambiguity the existence of two water molecules per maltotriosyl unit in the crystal structure of A-amylose, whereas, from density consideration, only one was proposed in the earlier structure. This new feature brings substantial difference, not only for the system of hydrogen bonds, which has to account for new possibilities, but also for a distortion of the helices and of their internal cavity. This feature is well illustrated in Figure 5, which presents the projections of two structures on the (a,b) plane: 5a for the present structure and 5b for the earlier one. In the latter, the projection of each double helix has nearly a hexagonal shape, with each projected glucosyl residue forming one side of the hexagon and also presenting a hexagonal central hydrophobic cavity, with a diameter of 3.5 Å. In such projection, which is that of a closepacked hexagonal arrangement, each double helix is in contact with six neighbors and the water molecules -seen as dots in Figure 5b -occupy the center of the interstitial spaces left open in such close packing. In contrast with this earlier structure, in the present structure, the projection of the double helices (Figure 5a) is no longer hexagonal, but somewhat deformed, as if they had been compressed along the b direction of the unit cell. This leaves an elliptical central cavity that presents a short dimension of 2.5 Å, and a longer one of 4 Å. This deformation is likely due to the presence of the pocket of the two water molecules that need to push their way in the close-packed arrangement of the amylose double helices. They do so by compressing the helices in one direction and by somewhat modifying the hexagonal closepacked arrangement and the connections between the double helices.

Hydrogen bonding

The hydrogen bonding parameters in the present structure of A-amylose crystal, together with those of the earlier structure are listed in Table 4. The values confirm that there is no intra-molecular hydrogen bond in these structures, but rather inter strands and interdouble-helical bonds. In the present structure, all the hydrogen bonds correspond to oxygenoxygen distances between 2.62 Å and 2.91 Å. In the inter-double helix category, two of the distances, namely the O3….O31 d and the O21….O21 f , that were too far in the earlier structure, fall now in the hydrogen bonding category and thus reinforced the crystal packing.

In the present structure, all the oxygen atoms of the primary and secondary hydroxyl groups appear to be involved into some type of hydrogen bonding, which was not the case in the earlier structure. As opposed to the earlier structure, where two intra-cyclic oxygen atoms were involved in hydrogen bonds with the water molecule, here only one of these, namely the O52 is at a close enough distance of the oxygen atom of OW2 to form a bond. The presence of two water molecules instead of one leads to a number of new hydrogen bonds, either between one of the amylose molecules and water or between the two molecules of water themselves. In Figures 6 and7, drawings represent two projections the network of hydrogen bonds that either connect the double helices together or the water molecules to the remaining of the structure. Figure 6, which is an a/c projection, highlights the nesting of the double helices and the presence of the pockets of two water molecules tightly hooked to one another through hydrogen bonds. The pockets themselves are hydrogen bonded to the adjacent double helices, but there is no interconnection between one pocket and the other. The a/b projections in Figure 7 show the details of the hydrogen bonds in a direction perpendicular to the double helix axes. In addition to the O21…O21 f , and the bifurcated O3…O31 d , which are new in this structure, one also notices the two strings of hydrogen bonds incorporating one or the other or both water molecules, namely O32…OW1…OW1 f …O32 f and O61…OW2…OW1….

OW1 f …OW2 f …O61 f .

Discussion

As aforementioned, the present resolution of the crystal structure of A-amylose modifies to some extent the existing knowledge on the ultrastructure of starch. Even if in their crystalline environment, the A-amylose helices appear less symmetrical than it was thought earlier. 9,10 They are nevertheless more tightly bound by a better established network of hydrogen bonds, which leaves no hydroxyl or hydroxymethyl group un-connected. In addition, the presence of two molecules of water per maltotriosyl residues indicates that close to 7% of the weight of the crystal structure consist of water molecules as opposed to only 3.5% proposed in the earlier structure. Such low water content differentiates the weakly hydrated crystals of A-amylose from those of B-amylose, where a water content of up to 27% resulted from the structural analysis. [30][31][32] This difference in hydration number explains why in A-amylose, the water molecules are located in discrete pockets that have no connection with one another, as opposed to B-amylose crystals where the water molecules are located between the double helices in wide channels occupying more than a quarter of the unit cell. When comparing the organization of the A-and B-amylose crystals, it is clear that the packing of the A-allomorph is significantly tighter than that of its B-counterpart, even if the structure of B-amylose, deduced only from fiber diffraction analysis is not known to high definition. [30][31][32] Indeed, in A-amylose, there is a number of hydrogen bonds between adjacent double helices whereas in B-amylose, the connections between the double helices are mainly through hydrate water bridges. [28][29][30] The crystal cohesion and therefore the thermal susceptibility of both allomorphs is thus expected to be substantially different. This is indeed the case since A-amylose crystals systematically melt by about 20°C above the B-specimens when subjected to the same hydrothermal treatment. 33 In addition, the lower stability of B-versus A-sample toward heating is further enlightened when starch granules, such as those of potato, which present a B-crystal X-ray diagram, get irreversibly converted into the A-type after heating at low moisture for several hours. 34 A similar conversion, but much faster, occurs when the hydrothermal treatment is directly applied to the crystalline fraction of potato starch recovered after partial acid hydrolysis of the granules. 35 Quite interestingly, the preparation of A-amylose crystals requires conditions where the crystallization takes place in aqueous mixtures containing a small percentage -typically of the order of 10 to 15% -of a low polarity precipitant such as ethanol or acetone. 8 Whereas the absence of precipitant leads to the formation of B-amylose crystals, 8 its use at higher concentration, which intuitively should yield A-amylose crystals with a lower water content, invariably leads to V-amylose crystals, which are single-helical. 8,36 Thus, the growth of the A-amylose crystals results from a tight balance between hydrophobic and hydrophilic interactions. Obviously, the dominant hydrophilic character is required to stabilize the amylose chains in double helices and not in single helices. On the other hand, the amount of hydrophobic precipitant required to obtain A-amylose crystals confines the size of the hydration domains to small water pockets, and not to large helical channels reported for Bamylose, when no hydrophobic precipitant is used to trigger the crystallization.

Another significant feature revealed in the present study is that the A-amylose crystal structure adopts a "parallel-down" organization, [START_REF]The "parallel-up" and "parallel-down[END_REF] indicating that the non-reducing end of the amylose molecules is oriented toward the positive direction of the unit cell c-axis. This result is important since it allows correlating the unit cell matrix of A-amylose crystals with the biogenesis of these crystals within their starch environment. Indeed, it is known that starch synthases elongate starch molecules by adding D-glucopyranosyl units at the non-reducing ends of amylopectin, [START_REF] French | Starch, Chemistry and Technology[END_REF][START_REF] Buléon | [END_REF][39] therefore also toward the positive direction of the c-axis of the Aamylose lattice. In a given amylopectin molecule, there is only one reducing end but multiple non-reducing ends are located at each dangling end of the short amylose fragments that branch out of amylopectin and form the A-amylose crystals. [START_REF] Buléon | [END_REF]39 The fact that starch granules grow by apposition, since newly formed starch is deposited at or near the granule surface, [START_REF] French | Starch, Chemistry and Technology[END_REF] would indicate that in the radial organization of the amylopectin chains, the reducing ends of these molecules should point toward the center of the granule, with the consequence of a radial organization of the positive direction of the c-axis, oriented from the granule center toward its periphery. So far, such geometry has not been proven due to the complex ultrastructure of starch granules that seem to consist of oblong crystalline blocklets having of the order of 100 nm in size and encompassing several amylopectin chains. [START_REF] Buléon | [END_REF][39][40] At present, the directionality of the amylose crystals and therefore that of the amylopectin molecules within these blocklets are not known with certainty. Furthermore, the orientation of the individual blocklets with respect to one another is also a subject of debate. To answer such fundamental questions, there is thus a need to correlate the crystallographic geometry with the ultrastructure of native starch, the goal being to map out the directionality of the c-axis throughout the starch granules at the 100 nm level. At present, only X-ray microdiffraction maps at a 5 µm scale have been recorded on individual starch granules. [41][42][43] We believe that it is the first time that a recording of full X-ray datasets on micron-sized polymer crystals has been made. This study thus opens the way to determine with precision the three-dimensional structure of polymers. So far, the only accessible diffraction data from polymers crystals resulted from either X-ray fiber diagrams or single crystal electron diffraction diagrams. Whereas the data from the former were limited and difficult to deconvolute, those of the latter were important to yield lattice parameters and symmetry elements, but frequently failed to provide processible diffraction intensities, due to overlapping dynamic contributions that were proven hard to evaluate. With the current development of synchrotron microfocus beamlines, it now becomes possible to envisage the recording of reliable X-ray datasets on one polymer single crystal. This study has shown the benefit of this recent development for the resolution of a polymer crystal structure, using a conventional crystallography structure determination protocol. The present structure of Aamylose describes only one of the numerous allomorphs in which amylose is susceptible to crystallize as micron-sized single crystals. 44,45 Many of these are in fact crystallosolvates, where amylose single helices are co-crystallized with guest molecules that are located either within the helical cavities and/or in between the helices. These easy-to-prepare crystalline complexes are quite important for the science of starch, since they allow not only the fractionation of starch, but also the slow release of the guests over a number of days or even months. Despite extremely rich electron diffraction diagrams, indicative of a high crystal perfection, these complexes have so far eluded any structural determination. The resolution of their structures, using the methods that have been presented here, would represent a substantial advance to understand and predict the numerous structural possibilities offered by amylose in vitro and therefore by starch inside native granules.

The present dataset was collected with an X-ray microbeam that had a diameter of 10-30 m. X-ray beams that are at least 10 times narrower are already available today on a routine basis for single crystal structure analysis. 13,14 As already shown in protein crystallography, this will allow structure determination on even smaller crystal volumes of polymers. 14 A potential target might be the structure of B-amylose. We also note that 100 nm and smaller beams start to become available. 46 With such nanobeams, it will be possible to probe the local structure of starch blocklets in thin sections, to test their crystallinity and orientation and thus to transpose the data obtained with the in vitro model system investigated here to the starch granule itself and understand the relation existing between its biogenesis and its ultrastructure.

Figure captions

Figure 1 .

 1 Figure 1. a: SEM image of A-amylose crystals prepared by recrystallization of synthetic amylose. b: One crystal glued to a borosilicate glass capillary tip.

Figure 2 .

 2 Figure 2. One quadrant of a typical X-ray diffraction diagram of a crystal, as in Figure 1b, obtained in 1 s during a 2° rotation. Insert: example of the intensity distribution across one of the diffraction spots (circled).

Figure 3 .

 3 Figure 3. Labeling of the carbon and oxygen atoms together with the torsion angles  and  and the bond angle along one of the strands of an A-amylose double helix.

Figure 4 .

 4 Figure 4. Independent maltotriosyl residue and two hydration water molecules in 2Fo-Fc 1

Figure 5 .

 5 Figure 5. Projection on the (a, b) plane of a: the present structure; b: earlier structure 10 derived from a fiber and electron diffraction data. The dots represent the oxygen atoms of the water molecules.

Figure 6 .

 6 Figure 6. Projection on the (a, c) plane of the structure of A-amylose. The dots correspond to the oxygen atoms of the water molecules. Details of the hydrogen bonding are represented by dashed lines.

Figure 7 .

 7 Figure 7. Hydrogen bonds of structure of A-amylose in the (a, b) plane. a: general view; b: details of a.

 1

 1

Figure 4 29

 4 Figure 4

Figure 5

 5 Figure 5

Table 1 .

 1 Experimental details

	Crystal data
	Chemical formula Cell setting, space group	C 18 0 17 H 34 monoclinic, B2
	a (Å)	20.83 (6)
	b (Å)	11.45 (4)
	c (Å)  ° V (Å 3 )	10.58 (3) 122.0 (2) 2140(11)
	Z	4
	Calculated density	1.622
	Radiation type	synchrotron X-ray
	 (Å)	0.9465
	Data collection
	Diffractometer	ESRF ID13
	Highest resolution (Å) Number of unique reflections	1.3 987 (155) 1
	Number of free reflections Total number of reflections measured	98 5786 (378) 1
	Reflections > 2(I)	676
	Temperature ( K)	100
	Range of h	-14 =< h =< 16
	Range of k	-8 =< k =< 8
	Range of l Completeness % R sym 2 R sym (expected) 3, R mrgd-F 2 R meas 2 I/ (I)	-8 =< l =< 7 98.6 (91.8) 1 0.193 (0.712) 1 0.191 (0.597) 1 0.137 (0.677) 1 0.202 (0.813) 1 9.37(2.62) 1
	Refinement
	Total number of the parameters refined	143
	Total number of restraints R1 4 R1 free 4	89 0.1749 0.2216
	1 The numbers in parentheses correspond to the outer resolution shell 1.4/1.3 Å 2 Ref. 24 3 Expected R-factor derived from (I) 4 R1=Σ||F o |-|F c ||/Σ|F o |

Table 2 .

 2 Fractional coordinates and thermal parameters of independent atoms (e.s.d. values are given in parentheses).

	Atom	x	y	z	U iso , Å 2
	O1	0.0995(14)	0.272(3)	0.673(3)	0.124(10)
	O2	0.2542(16)	0.413(3)	0.626(3)	0.129(10)
	O3	0.2940(13)	0.304(3)	0.838(3)	0.108(9)
	O5	0.1216(15)	0.381(3)	0.867(3)	0.131(10)
	O6	0.114(2)	0.357(4)	1.136(4)	0.179(15)
	C1	0.1498(14)	0.391(3)	0.743(3)	0.082(12)
	C2	0.2259(15)	0.406(3)	0.750(3)	0.116(17)
	C3	0.2238(14)	0.292(3)	0.822(3)	0.117(14)
	C4	0.1853(15)	0.270(3)	0.951(3)	0.100(13)
	C5	0.1112(14)	0.264(3)	0.929(3)	0.094(13)
	C6	0.069(2)	0.247(4)	1.053(4)	0.17(2)
	O11	-0.0710(14)	0.122(3)	0.343(3)	0.142(10)
	O21	0.0220(16)	0.401(3)	0.299(3)	0.154(11)
	O31	0.1355(18)	0.473(4)	0.489(4)	0.25(2)
	O51	-0.0884(13)	0.168(2)	0.553(3)	0.111(10)
	O61	-0.070(2)	0.183(4)	0.811(4)	0.184(14)
	C11	-0.0718(15)	0.218(2)	0.427(3)	0.113(14)
	C21	0.0060(15)	0.3470(17)	0.421(4)	0.20(3)
	C31	0.0692(16)	0.338(4)	0.480(4)	0.159(18)
	C41	0.0437(14)	0.285(4)	0.616(4)	0.14(2)
	C51	-0.0328(14)	0.148(3)	0.608(4)	0.135(17)
	C61	-0.0625(18)	0.086(3)	0.739(4)	0.25(4)
	O12	-0.1670(15)	-0.142(2)	0.0000	0.126(10)
	O22	-0.2071(16)	0.047(3)	-0.041(3)	0.137(11)
	O32	-0.1354(13)	0.223(2)	0.167(3)	0.099(9)
	O52	-0.2224(14)	-0.195(2)	0.198(3)	0.112(9)
	O62	-0.2232(19)	-0.183(3)	0.464(4)	0.149(12)
	C12	-0.224(2)	-0.147(3)	0.077(3)	0.18(2)
	C22	-0.2154(17)	-0.008(3)	0.081(3)	0.103(13)
	C32	-0.1441(17)	0.095(2)	0.156(3)	0.092(12)
	C42	-0.1434(18)	0.034(2)	0.284(3)	0.113(14)
	C52	-0.1557(18)	-0.107(2)	0.267(3)	0.118(17)
	C62	-0.164(2)	-0.176(4)	0.396(4)	0.15(2)
	OW1	-0.074(2)	0.429(4)	0.992(4)	0.203(18)
	OW2	-0.121(4)	0.350(6)	0.750(6)	0.25(2)

Table 3 .

 3 Conformational parameters. The e.s.d. values for the present results are given in parentheses while the e.s.d. values for the results from Ref. 10 are unknown.

	Glucosidic bond parameters (°)	Present work	Previous fiber and powder data 8

Table 4 .

 4 Hydrogen bonding in A-amylose. The e.s.d. values for the present results are given in parentheses while the e.s.d. values for the results from Ref. 10 are unknown.

	Bond	Present work	Previous fiber and powder data 8
		d (Å)	d (Å)
		Inter-strand hydrogen bonds
	O2…O62 a	2.91(4)	2.93
	O6…O21 b	2.81(5)	2.75
	O61…O22 b	2.89(5)	2.66
		Inter-double-helix hydrogen bonds
	O2…O6 c	2.62(5)	2.63
	O3…O31 d	2.70(4)	3.08
	O3…O32 e	2.78(3)	2.78
	O21…O21 f	2.86(6)	3.53
	O22…O62 g	2.64(4)	2.68
		Hydrogen bonds with water molecules
	OW1…O32 b	2.73(5)
	OW1…OW1 f	2.61(9)
	OW1…OW2	2.72(7)
	OW2…O61	2.72(7)
	OW2…O52 h	2.82(7)
	a -x, -y, z b x, y, z+1 c -x+1/2, -y+1, z-1/2 d -x+1/2, -y+1, z+1/2 e x+1/2, y, z+1/2 f -x, -y+1, z

g -x-1/2, -y, z-1/2 h -x-1/2, -y, z+1/2

Acknowledgments. The authors thank C. Albenne and P-C. Escalier (LISBP) for preparing the fraction of synthetic amylose used in this study, as well as K. Mazeau (CERMAV) for his help with molecular graphics and Y. Nishiyama (CERMAV) for valuable discussions.

Supporting Information Available: List of bond lengths and angles. This information is available free of charge via the Internet at http://pub.acs.org.


David Bellocq

Jean-François Korobelnik

Carole Burillon

Nicolas Voirin

Corinne Dot

Eric Souied

John Conrath

Solange Milazzo

Pascale Massin

Stéphanie Baillif

Laurent Kodjikian

Effectiveness and safety of dexamethasone implants for post-surgical macular oedema including Irvine-Gass syndrome: The EPISODIC study

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

Effectiveness and safety of dexamethasone implants for post-surgical macular oedema including Irvine-Gass syndrome: the EPISODIC study INTRODUCTION Irvine-Gass (IG) syndrome describes a macular oedema that develops following ophthalmic surgery. It was first clinically described in 1953 by Irvine [START_REF] Irvine | A newly defined vitreous syndrome following cataract surgery[END_REF] in his Proctor lecture. Gass and Norton subsequently published an angiographic study of its characteristics in 1966. [START_REF] Gass | Cystoid macular edema and papilledema following cataract extraction. A fluorescein fundoscopic and angiographic study[END_REF] Maumenee was the first to name it the IG syndrome. The most likely physiopathological hypothesis is that there is an inflammatory response instigated by inflammatory mediators released during and after surgical procedures, causing alterations to the blood-retinal barrier.

With improvements in cataract surgery, notably the considerable reduction in the size of the incisions required during phacoemulsification, clinically significant cystoid macular oedema (CME) with reduced visual acuity and metamorphopsia only occurs in 1-2% [START_REF] Packer | Incidence of acute postoperative cystoid macular edema in clinical practice[END_REF] of patients, with peak incidence occurring on average 6 weeks after surgery. However, subclinical CME-that is, with no visual impairment-is detected in almost 30% of patients with post-surgical angiography and a further 11-41% with optical coherence tomography (OCT), despite the administration of preventive treatment. [START_REF] Perente | Evaluation of macular changes after uncomplicated phacoemulsification surgery by optical coherence tomography[END_REF] Incidence increases in the event of complications during surgery. The identified risk factors [START_REF] Frost | Vitreous loss in planned extracapsular cataract extraction does lead to a poorer visual outcome[END_REF] are posterior capsule rupture and vitreous loss, as well as the use of iris retractors. The presence of an epiretinal membrane, a vein occlusion, a history of uveitis or diabetes, and the use of prostaglandin eye drops also increase the incidence of pseudophakic CME.

Post-surgical macular oedema is the main cause of reduced visual acuity following ophthalmic surgery. Managing the disease has proven to be complicated and no randomised studies have been conducted to establish the best therapeutic options in the last 20 years. The most common first-line treatment is a combination of oral acetazolamide, used off label, and the topical administration of non-steroidal antiinflammatory drugs (NSAIDs), used in compliance with the approved indications.

Second-line treatments include a range of therapies used off label: intravitreal injection of steroid such as triamcinolone or dexamethasone implants, [START_REF] Jonas | Intraocular injection of crystalline cortisone as adjunctive treatment of diabetic macular edema[END_REF][START_REF] Benhamou | Intravitreal triamcinolone for refractory pseudophakic macular edema[END_REF][START_REF] Thach | A comparison of retrobulbar versus sub-Tenon's corticosteroid therapy for cystoid macular edema refractory to topical medications[END_REF][START_REF] Randazzo | Chronic macular edema medical treatment in Irvine-Gass syndrome: case report[END_REF][START_REF] Koutsandrea | Intraocular triamcinolone acetonide for pseudophakic cystoid macular edema: optical coherence tomography and multifocal electroretinography study[END_REF][START_REF] Williams | Dexamethasone posterior-segment drug delivery system in the treatment of macular edema resulting from uveitis or Irvine-Gass syndrome[END_REF] or intravitreal injection of antivascular endothelial growth factor (VEGF), 12 13 or subcutaneous injections of interferon α2a. [START_REF] Deuter | Successful treatment of chronic pseudophakic macular edema (Irvine-Gass syndrome) with interferon alpha: a report of three cases[END_REF] Ozurdex is a biodegradable intravitreal implant which delivers 0.7 mg of the corticosteroid dexamethasone into the vitreous and the retina. The dexamethasone acts on all inflammatory mediators. The implant has been approved for the treatment of macular oedema (ME) secondary to retinal vein occlusion [START_REF] Kodjikian | Ranibizumab versus bevacizumab for neovascular age-related macular degeneration: results from the GEFAL Noninferiority Randomized Trial[END_REF] and for non-infectious posterior uveitis. [START_REF] Lowder | for the Ozurdex® HURON Study Group. Dexamethasone intravitreal implant for noninfectious intermediate or posterior uveitis[END_REF] As IG is primarily an inflammatory response and O z u r d e xh a sa l r e a d yb e e nu s e dt ot r e a t2 7p a t i e n t s with IG in a phase 2 study on uveitis, [START_REF] Williams | Dexamethasone posterior-segment drug delivery system in the treatment of macular edema resulting from uveitis or Irvine-Gass syndrome[END_REF] we believed it would be of interest to collect data on a larger number of patients. Moreover, in the study by Williams et al, [START_REF] Williams | Dexamethasone posterior-segment drug delivery system in the treatment of macular edema resulting from uveitis or Irvine-Gass syndrome[END_REF] patients with IG syndrome were unfortunately mixed in with uveitis patients.

The objective of this study was to assess the anatomic and functional EffectIveness, as well as the Safety Of Dexamethasone implants (Ozurdex) in treating postsurgical macular oedema including Irvine-Gass syndrome (EPISODIC study).

PATIENTS AND METHODS

A descriptive, observational, retrospective, consecutive, uncontrolled multicentre pilot case series was conducted in France from March 2011 to June 2013.

All patients received clear, detailed prior information on the treatment and expected risks and benefits. As the data were collected retrospectively and the patients' management was not modified, according to French law (n°2004-806, 9 August 2004), this study did not require research ethics committee approval. It was conducted in accordance with the law on data protection (n°2004-801, 6 August 2004).

All consecutive adult patients presenting with clinical or subclinical post-surgical macular oedema, including IG syndrome, treated with intravitreal injections of dexamethasone implant 0.7 mg (Ozurdex) and followed up for at least 6 months, were included in the study.

The patients were treatment experienced and had received at least one previous treatment of NSAID eye drops combined with oral acetazolamide. All the previous treatments administered to each patient were identified.

Patients treated with oral corticosteroids, patients with any of the contraindications for Ozurdex set out in the June 2013 marketing approval, and patients with uncontrolled diabetes with glycosylated haemoglobin >13% were excluded. [START_REF] Williams | Dexamethasone posterior-segment drug delivery system in the treatment of macular edema resulting from uveitis or Irvine-Gass syndrome[END_REF] Low baseline visual acuity, anatomic OCT macular changes due to oedema persistence, and a long duration of macular oedema did not constitute exclusion criteria.

Each patient underwent a standardised examination at the initial visit, and at each monthly follow-up visit, with measurement of best-corrected visual acuity (BCVA) in ETDRS letters (Early Treatment Diabetic Retinopathy Study), air-puff or applanation tonometry to measure intraocular pressure (IOP), ophthalmoscopy, and spectral-domain OCT (Cirrus HD-OCT Zeiss) to measure central subfield macular thickness (CSMT) (thickness of a circular area of 1 mm, concentric to the foveal centre). Patients who received more than one injection received the same follow-up, with a monthly examination and recording of the same clinical and OCT data.

The main objective of our study was to assess the benefito f Ozurdex in treating post-surgical macular oedema including IG syndrome, with monthly visits over a 6-month follow-up period, by measuring BCVA in ETDRS letters. Several secondary objectives were also analysed. First and foremost, we wanted to assess the anatomic effectiveness of Ozurdex by measuring CSMT each month. We then assessed the implant's tolerance, measuring local tolerance in terms of IOP, iatrogenic retinal detachment or exogenous endophthalmitis, as well as overall tolerance. We also analysed the functional effectiveness according to initial visual acuity, and searched for a significant difference in gain between patients with an initial visual acuity ≤50 letters and patients with an initial acuity >50 letters. Finally, we assessed the anatomic and functional effectiveness of Ozurdex in cases of repeat intravitreal injections.

Statistical methods

BCVA, CSMT, and IOP were described over time using mean ±SD at each time point. Baseline values were at the day of Ozurdex injection and measures were repeated for each patient at months 1, 2, 3, 4, 5, and 6. A subgroup of patients underwent a second injection, and measures were also repeated for each patient at months 1, 2, 3, 4, 5, and 6 after this second injection.

Differences in BCVA, CSMT, and IOP at months 1, 2, 3, 4, 5, and 6 compared to baseline values were assessed using univariate analysis of variance (ANOVA), taking into account the dependence of repeated measures over time for each patient. For this, the lme function of the package nlme [START_REF] Pinheiro | _nlme: Linear and Nonlinear Mixed Effects Models[END_REF] in the software R [START_REF] Core | R: a language and environment for statistical computing[END_REF] was used. A value of p<0.05 was considered statistically significant.

RESULTS

This retrospective study was conducted in nine centres located in mainland France (see online supplementary appendix 1). A case series was constituted, with 50 consecutive patients included between March 2011 and June 2013, with a minimum follow-up period of 6 months. The population characteristics are shown in table 1.

The mean age was 72.1 years. The population was made up of more women than men (58% women). There was no difference in terms of laterality.

In regard to the cause of macular oedema, 82% of the cases were secondary to phacoemulsification cataract surgery (IG syndrome), 6% were secondary to epiretinal membrane peeling, 10% were secondary to combined cataract surgery and membrane peeling, and 2% were secondary to vitrectomy for retinal detachment.

The mean time between surgery and the diagnosis of postsurgical macular oedema was 7.8 weeks (range 4.1-14.4). All patients were treatment experienced: 62% of patients had been treated with topical NSAIDs and oral acetazolamide, 16% had received the same treatment associated with intravitreal injection of triamcinolone, 7% had the same treatment associated with intravitreal injection of anti-VEGF, and 8% had received at least three different treatments before their first intravitreal injection of Ozurdex. Functional effectiveness was assessed on a monthly basis by measuring mean±SD BCVA using the ETDRS scale. Baseline BCVA, measured on the day of the first injection of Ozurdex , was 55.7±15.4 letters (L). At month 1, mean BCVA was 68.3 ±13.0 L, representing a gain of 12.6 L ( p<0.001). At month 2, BCVA was 71.8±10.5 L, a gain of 16.1 L (more than three lines on the ETDRS scale) ( p<0.001). From month 3 onwards, functional recurrence was observed with a reduction in BCVA to 67.7±13.3 L (a gain of 12.0 L from baseline) ( p<0.001). The drop in visual acuity continued throughout the follow-up period with BCVA of 63.1±15.5 L at month 4 ( p<0.001), then 61.9±14.0 L in month 5 ( p<0.001), and 59.6±13.5 L in month 6 ( p=0.002) (figure 1), meaning that BCVA at 6 months was still better than baseline. The percentage of patients with an increase >15 letters was also assessed over the course of the follow-up period. At month 2, 61.2% of patients had benefited from an increase of this magnitude and 43.8% at month 4 ( p<0.001). Anatomic effectiveness was assessed by measuring mean CSMT using spectral-domain OCT. Initial mean±SD CSMT was 544±117.2 μm. At month 2, mean CSMT was 302±76.3 μm, a reduction of 242 μm ( p<0.001). From month 3 onwards, anatomic recurrence of CME was observed with CSMT of 350±68.2 μm. Anatomic and functional recurrences were both first detected from month 3 and continued throughout the follow-up period, with values consistently above the baseline (figure 2).

We then analysed the tolerance of the dexamethasone implant, primarily local tolerance, by measuring mean±SD IOP. Before the first injection, mean IOP was 12.3±2.6 mm Hg. At month 1, mean IOP was 15.3±4.6 mm Hg, the maximum value recorded ( p<0.001). After this first month, mean IOP decreased consistently with a mean value of 13 mm Hg (figure 3). We also analysed the number of patients with IOP >25 mm Hg, which concerned three patients at the first month visit (6% of patients) and four patients at the second month visit (8% of patients). The number of patients affected then decreased; at month 3, two patients had IOP >25 mm Hg, and at month 4 just one patient. In total, 10 patients presented ocular hypertension (IOP >21 mm Hg) with mean IOP of 26 mm Hg (range 22-33). Seven patients received one single anti-glaucoma eye drop treatment and three were treated with two eye drop treatments. No filtering surgery was required.

We then analysed the functional effectiveness according to initial visual acuity. Twenty patients had an initial visual acuity ≤50 letters (mean 40.7 L) and 30 patients had initial visual acuity >50 letters (mean 65.8 L). The mean change in visual acuity at month 2 was statistically different between the two groups, with a mean gain of 22.7±8.5 L at month 2 for the group '≤50 letters' and a mean gain of 11.3±8.1 L at month 2 for the group '>50 letters' (p<0.001), with BCVA at 63.4 and 77.1 L, respectively. At month 4, there was no statistically significant difference, with a mean gain of 9.1±11.8 L for the group '≤50 letters' and 6.2±8.7 L for the group '>50 letters' (p=0. 19).

Finally, we analysed the changes following the first intravitreal injection. Out of the 50 patients included in the study, 39 were followed up for at least a 12-month period. Within this cohort, 19/39 patients (48.7%) developed functional and/or anatomic recurrence of macular oedema, meaning that more than half of the patients, followed up for at least 1 year (39 patients), only needed one injection of Ozurdex. Each of these patients was re-treated with Ozurdex. The changes in BCVA and mean CSMT were measured. The average time to re-treatment was 5.08 months (range 4.01-11.2). Reproducibility was confirmed in terms of both functional and anatomic effectiveness. The day of the second intravitreal injection, mean BCVA was 61.1±9.8 L. At the first month visit after the second intravitreal injection, mean BCVA was 65.8±9.2 L. At month 2, BCVA was 71.9±6.7 L, representing a mean increase of 16.2 L compared to initial baseline (p<0.001). The pattern of recurrence was similar to the pattern following the first intravitreal injection with a progressive decrease in mean BCVA throughout the follow-up period, with BCVA at 62.7±8.9 L at month 4 (p<0.001) (figure 1). In terms of anatomic effectiveness, CSMT on the day of the second intravitreal injection was 421±60 μm. At the first monthly visit after the second injection, mean CSMTwas 296±26.6 μm, representing a decrease of 248 μmcompared to the mean baseline macular thickness (p<0.001). At month 2, mean CSMT was 298±24.1 μm, a reduction of 246 μm (p<0.001). The pattern of recurrence was also identical, with an increase in mean CSMT from month 2 onwards, measured at 435 ±74.3 μm at month 4 (p<0.001) (109 μm below baseline), a figure that remained consistent over the final 2 months (figure 2) (p<0.001).

The final parameter analysed was the tolerance after a second intravitreal injection of Ozurdex. In terms of IOP, mean pressure was 16.8±3.2 mm Hg at the first monthly visit compared to the mean IOP before the second intravitreal injection of 13 mm Hg. IOP then progressively decreased as shown in figure 3. Three further patients presented ocular hypertension after the second intravitreal injection, with mean IOP of 22 mm Hg (mean IOP 19 mm Hg before the second injection). Two patients received one single pressure-lowering drug treatment and one patient received two drug treatments. No filtering surgery was required. Furthermore, there were no reported cases of retinal detachment or endophthalmitis during the study. No systemic adverse effects occurred over the course of the study.

DISCUSSION

Nowadays, patients expect an immediate benefit following cataract surgery. Post-surgical macular oedema is rare. It can heal spontaneously, but it can also persist and cause permanent damage to the macular and decrease visual acuity. IG syndrome and others kinds of post-surgical macular oedema can become a genuine therapeutic challenge, as some cases are refractory to therapy and there is no consensus on the best care management to offer patients.

As previously stated, first-line treatments often include oral acetazolamide associated with NSAID eye drops. 19 20 Acetazolamide enhances the pump action of the retinal pigment epithelium by acting on carbonic anhydrase. [START_REF] Marmor | Enhancement of retinal adhesion and subretinal fluid resorption by acetazolamide[END_REF] The disadvantage of this treatment is the numerous adverse effects linked to acetazolamide, including renal colic, cramps, formication, and asthenia.

In the event of capsular rupture with vitreous loss and presence of vitreous in the anterior segment, the safe and thorough evacuation of vitreous from the anterior chamber, sometimes associated with a posterior vitrectomy, is the recommended firstline response.

Second-line treatments include a range of therapies used off label. Intravitreal injection of anti-VEGF can be used to inhibit VEGF released during surgical procedures. Various publications have assessed the effect of using bevacizumab or ranibizumab to treat IG syndrome, and have reported functional and anatomic benefits after several intravitreal injections. The case series conducted by Barone et al, [START_REF] Barone | Short-term safety and efficacy of intravitreal bevacizumab for pseudophakic cystoid macular edema[END_REF] composed of 10 eyes with IG syndrome, reported an increase of two lines on the ETDRS scale, after one or more intravitreal injections of bevacizumab. However, other case series have found intravitreal injection of anti-VEGF to be ineffective. The case series conducted by Spitzer et al, [START_REF] Spitzer | Efficacy of intravitreal bevacizumab in treating postoperative pseudophakic cystoid macular edema[END_REF] composed of 16 eyes, showed no benefit in the treatment of CME refractory to first-line treatments.

Infliximab is an anti-TNFα agent with immunosuppressive properties. A case series has suggested its effectiveness when administered by intravitreal injection to seven eyes with IG syndrome after the failure of intravitreal injection of anti-VEGF. [START_REF] Wu | Intravitreal infliximab for refractory pseudophakic cystoid macular edema: results of the Pan-American Collaborative Retina Study Group[END_REF] Indeed, after an average of 2.7 intravitreal injections, mean BCVA increased from 1.14±0.59 logMAR to 0.51±0.35 logMAR ( p=0.0156) after 6 months' follow-up. However, adverse effects were also reported, in particular retinotoxicity. [START_REF] Giganti | Adverse events after intravitreal infliximab (Remicade)[END_REF] Another case series suggested the effectiveness of subcutaneous injections of interferon α2a with an improvement in BCVA in three out of the four patients initially treated. [START_REF] Deuter | Successful treatment of chronic pseudophakic macular edema (Irvine-Gass syndrome) with interferon alpha: a report of three cases[END_REF] The use of corticosteroids to treat post-surgical macular oedema including IG syndrome is also commonplace, often in the form of intravitreal injections of triamcinolone. Several publications have reported the effectiveness of triamcinolone in treating macular oedema secondary not only to diabetes or uveitis but also to ophthalmic surgery. [START_REF] Jonas | Intraocular injection of crystalline cortisone as adjunctive treatment of diabetic macular edema[END_REF][START_REF] Benhamou | Intravitreal triamcinolone for refractory pseudophakic macular edema[END_REF][START_REF] Thach | A comparison of retrobulbar versus sub-Tenon's corticosteroid therapy for cystoid macular edema refractory to topical medications[END_REF][START_REF] Randazzo | Chronic macular edema medical treatment in Irvine-Gass syndrome: case report[END_REF][START_REF] Koutsandrea | Intraocular triamcinolone acetonide for pseudophakic cystoid macular edema: optical coherence tomography and multifocal electroretinography study[END_REF] The doses of triamcinolone used in the different studies vary from 2-4 mg. However, recurrence of CME is common after 6 weeks to 3 months post-injection, and the effectiveness of re-treatment varies. The case series conducted by Koutsandrea et al, [START_REF] Koutsandrea | Intraocular triamcinolone acetonide for pseudophakic cystoid macular edema: optical coherence tomography and multifocal electroretinography study[END_REF] composed of 14 eyes, demonstrated the benefit of intravitreal injections of triamcinolone for pseudophakic CME, with an improvement in visual acuity and macular thickness. Periocular corticosteroids have also been shown to be effective for pseudophakic CME refractory to topical treatments. Thach et al, [START_REF] Thach | A comparison of retrobulbar versus sub-Tenon's corticosteroid therapy for cystoid macular edema refractory to topical medications[END_REF] in a series involving 48 patients with refractory CME, demonstrated that sub-Tenon injections improved visual acuity from 20/92 to 20/50 ( p=0.0001), and retrobulbar injections improved visual acuity from 20/97 to 20/58 ( p=0.035), with no difference between the two techniques. Randazzo and Vinciguerra 9 also reported improved anatomic and visual outcomes in a patient with chronic CME treated with sub-Tenon betamethasone 4 mg.

The time to recurrence of macular oedema after the first intravitreal injection of triamcinolone for IG syndrome varies between case series and occurs from the second month after the intravitreal injection onwards. [START_REF] Benhamou | Intravitreal triamcinolone for refractory pseudophakic macular edema[END_REF] The time to re-treatment also varies widely (2.5-24 weeks), giving a mean figure of around 8 weeks following the first intravitreal injection. [START_REF] Benhamou | Intravitreal triamcinolone for refractory pseudophakic macular edema[END_REF] In comparison, the mean time to recurrence after the first intravitreal injection in our case series is around 3 months and the mean time to re-treatment is much longer at 22.9 weeks. However, more than half of the patients followed-up for at least 1 year (20/39) presented neither functional nor anatomical recurrence.

Dexamethasone implants have not received specific marketing approval for the treatment of post-surgical macular oedema, including IG syndrome, although IG cases were included in the phase 2 trial. [START_REF] Williams | Dexamethasone posterior-segment drug delivery system in the treatment of macular edema resulting from uveitis or Irvine-Gass syndrome[END_REF] Ozurdex marketing approval specifies that this implant is indicated for the treatment of adult patients with inflammation of the posterior segment of the eye presenting as non-infectious uveitis. Several physiopathological models have been put forward to explain post-surgical CME and to date the most likely cause is inflammation. [START_REF] Xu | Cataract surgery induces retinal proinflammatory gene expression and protein secretion[END_REF] Indeed, surgery releases large quantities of inflammatory mediators such as arachidonic acid responsible for inflammatory cascade, pro-inflammatory cytokines, lysozyme or anti-VEGF. This alters the blood-retina barrier and increases vascular permeability. [START_REF] Xu | Cataract surgery induces retinal proinflammatory gene expression and protein secretion[END_REF] Liquid then accumulates in the external plexiform layer and inner nuclear layer, forming cystic cavities. It could therefore be suggested that postsurgical macular oedema including IG constitutes a physiopathogenic inflammation mechanism similar to posterior segment inflammation, and that using Ozurdex to treat IG could therefore be considered to constitute an approved use of the implant. Ozurdex has already been used to treat 27 patients with IG in the phase 2 case series on uveitis conducted by Williams et al. [START_REF] Williams | Dexamethasone posterior-segment drug delivery system in the treatment of macular edema resulting from uveitis or Irvine-Gass syndrome[END_REF] But patients with IG syndrome were mixed in with uveitis patients, so that no firm conclusions can be drawn regarding the IG patients. The results from this series showed that 54% patients gained 15 letters at the third month of follow-up. The case series conducted by Dutra Meideros et al [START_REF] Medeiros | Dexamethasone intravitreal implant for treatment of patients with recalcitrant macular edema resulting from Irvine-Gass syndrome[END_REF] assessed the therapeutic response to Ozurdex on nine eyes with treatment-refractory IG syndrome, and showed an improvement in BCVA and CSMT over a 6-month follow-up period.

Our retrospective non-comparative case series included 50 consecutive patients with post-surgical macular oedema including IG syndrome in nine centres in France. The minimal follow-up was 6 months but 78% of patients were followed for 12 months. This study shows significant functional and anatomic improvements as compared to baseline, with a functional gain of more than three ETDRS lines over the follow-up period. Although some patients experienced a recurrence in CME, BCVA and/or mean CSMT at the end of follow-up were still better than at baseline. It should also be noted that very few adverse effects were reported during follow-up. Cases of ocular hypertension were reported but were controlled with pressure-lowering medication. No filtering surgery was required. Furthermore, no cases of endophthalmitis or iatrogenic retinal detachment were reported. No systemic adverse events were observed.

In conclusion, the dexamethasone implant Ozurdex seems to be a safe and effective therapeutic option for post-surgical macular oedema including IG syndrome refractory to first-line treatments. Only half of the patients needed a second injection during the 1-year follow-up. In patients who received two injections, the effectiveness and safety of the two implants were similar. An interventional, prospective, comparative, randomised study to compare the effectiveness and safety of different second-line treatments would be helpful in confirming the results of this pilot study.

Figure 1

 1 Figure 1 Functional effectiveness: changes in mean best-corrected visual acuity (BCVA) after the first two intravitreal injections of dexamethasone implant 0.7 mg (Ozurdex). M, month.

Figure 2

 2 Figure 2 Anatomic effectiveness: changes in mean central subfield macular thickness (CSMT, in μm) after the first two intravitreal injections of dexamethasone implant 0.7 mg (Ozurdex). M, month.

Figure 3

 3 Figure 3 Tolerance: changes in mean intraocular pressure (IOP, mm Hg) after the two first intravitreal injections of dexamethasone implant 0.7 mg (Ozurdex). M, month.

Table 1

 1 Baseline patient characteristics

	Number	%

BCVA, best-corrected visual acuity; CSMT, central subfield macular thickness; ERM, epi-retinal membrane; NSAIDs, non-steroidal anti-inflammatory drugs; VEGF, vascular endothelial growth factor.

Competing interests J-FK: Consultant for Alcon, Allergan, Bayer, Novartis, Thea, Roche, Zeiss. CD has sat on advisory boards for Alcon, Allergan, Bayer; lecture fees from Alcon, Alimera, Allergan, Bayer, Essilor, Novartis. ES expert for Novartis, Allergan; advisory boards for Allergan, Bayer, Novartis; lecture fees from Allergan, Heidelberg, Novartis. JC has sat on advisory boards for Alcon, Alimera, Allergan, Bayer, Novartis; lecture fees from Allergan, Bayer, Novartis. SM Investigator for trials sponsored by Alimera, Allergan, Bayer, Novartis, Leurquin and lecture fees from Alcon, Allergan, Bayer, Essilor, Novartis. PM advisory boards for Alimera, Allergan, Bayer, Novartis, Sanofi; lecture fees from Allergan, Novartis. SB: board for Novartis, Bayer, Allergan. LK Principal Investigator for trials sponsored by Novartis, Bausch&Lomb, Théa, Alcon; has sat on advisory boards for Alcon, Alimera, Allergan, Bayer, Bausch&Lomb, Novartis, Théa; lecture fees from Alcon, Alimera, Allergan, Bayer, Bausch&Lomb, Novartis, Théa.

Ethics approval As the data were collected retrospectively and the patients' management was not modified, according to French law (n°2004-806, 9 August 2004) research ethics committee approval was not required.

Provenance and peer review Not commissioned; externally peer reviewed.