domain: phys sdv

text:
Linda Ravazzano

Silvia Bonfanti

Roberto Guerra

Fabien Montel

Caterina A M La Porta

Stefano Zapperi

Spatial organization of nuclear pores in Xenopus laevis oocytes

Nuclear pores are protein assemblies inserted in the nuclear envelope of eukaryotic cells, acting as main gates for communication between nucleus and cytoplasm. So far, nuclear pores have been extensively studied to determine their structure and composition, yet their spatial organization and geometric arrangement on the nuclear surface are still poorly understood. Here, we analyze super-resolution images of the surface of Xenopus laevis oocyte nuclei during development, and characterize the arrangement of nuclear pores using tools commonly employed to study the atomic structural and topological features of soft matter. To interpret the experimental results, we hypothesize an effective interaction among nuclear pores and implemented it in extensive numerical simulations of octagonal clusters mimicking typical pore shapes. Thanks to our simple model, we find simulated spatial distributions of nuclear pores that are in excellent agreement with experiments, suggesting that an effective interaction among nuclear pores exists and could explain their geometrical arrangement. Furthermore, our results show that the statistical features of the geometric arrangement of nuclear pores do not depend on the type of pore-pore interaction, attractive or repulsive, but are mainly determined by the octagonal symmetry of each single pore. These results pave the way to further studies needed to determine the biological nature of pore-pore interactions.

Introduction

Genetic information in eukaryotic cells is well protected inside the cell nucleus that is divided from the outside cytoplasm by a membrane called nuclear envelope (NE). This segregation has the advantage of protecting the genome from sources of damage, but on the other hand communications based on exchange of macro-molecules, such as messenger RNAs (mRNA) or transcriptomic factors, are of vital importance during all the cell life cycle, to control protein synthesis and instruct gene expression 1,2 . The spatial architecture of the nucleus is crucial for the interaction between the genome and protein components of the nuclear complex and has a role in chromatin reorganization during cellular differentiation.

Nuclear pores, large protein assemblies inserted in the nuclear envelope, are responsible for selective nucleocytoplasmic transport, allowing the free diffusion of ions and small molecules and acting as selective gates for import and export of macromolecules, such as proteins and mRNAs [START_REF] Wente | Cold Spring Harbor perspectives in biology[END_REF] . Furthermore, nuclear pores are also involved in the organization of the genome and they contribute to gene regulation through physical interactions with chromatin [START_REF] Buchwalter | [END_REF] .

Since the discovery of nuclear pores in the 1950s, their structure has been the subject of extensive experimental investigations using Electron Microscopy (EM) and Cryo-Electron Tomography (cryo-ET) 5 . Great effort was spent on determining the composition of a single pore in terms of different protein complexes. Nuclear pores appear as modular assemblies of discrete constituents arranged with octagonal symmetry around a central axis 6 . Further investigations identified those discrete elements as multiple copies of about 34 protein subunits (nucleoporins). These peculiar proteins are remarkably conserved throughout eukaryotes, showing similar features in algae, yeast, vertebrates such as Xenopus Laevis, up to human.

The characteristic shape of a nuclear pore consists of two superimposed rings of nucleoporins, with eightfold symmetry, one on the outer face of the nuclear membrane and one on the inner face, with eight extended filaments departing from each ring. In contrast, the center of the pore, which forms the permeability barrier, is filled with disordered filaments of phenylanine-glycine (FG) repeats 1,5 . In this context, the experimental observations have already been accompanied by in silico studies involving all atoms and coarse-grained molecular dynamics simulations in order to to characterize the peculiar structure of nuclear pore complexes (NPCs). 7 , 8 , 9 .

Despite great progress in understanding the structure of a single nuclear pore, little is known on how nuclear pores are distributed across the surface of the nuclear membrane (in an average human cell there are approximately 2000-3000 nuclear pores) and whether and how they might interact among each other. Interactions are likely modulated by the nuclear lamina, a filamentous protein network underlying the nuclear envelope, but how the interaction occurs is still unclear.

Earlier studies, interestingly, revealed that highly proliferative cells such as embryos or tumors have an high density of nuclear pores on the nuclear membrane, while terminal differentiated cells have fewer, suggesting a link between number and distribution of pores and cell activity 10 . This link has been further explored in another early study focused on the changes in distribution of nuclear pores during spermatogenesis, following the evolution from spermatocytes to early spermatids. In particular, a clear change in nuclear pore spatial organization, from aggregation with hexagonal packing in pore rich areas coexisting with large pore-free areas in spermatocytes to a random distribution of pores in early spermatids, has been observed 11 . A further step in our understanding of the role of nuclear pore organization came from the observation of large pore-free islands in HeLa S3 human cells. These islands disperse with cell-cycle progression and reveal the importance of lamin A/C in regulating the pore distribution 12 .

In a recent paper, Sellés et al. performed super-resolution microscopy on Xenopus laevis oocytes observing the variation of nuclear pore distribution on the nuclear membrane during oocyte development 13 . In the present paper, we analyze those experimental data 13 to investigate the spatial distributions of nuclear pores across the nuclear membrane during the development of Xenopus laevis oocytes. To this end, we use tools typical of soft matter physics, such as the radial distribution function (RDF), local order parameters and Voronoi tessellation. To model the spatial distribution of nuclear pores observed experimentally, we introduce an effective interaction among them. We first define a potential with octagonal symmetry to properly model the shape of nuclear pores and then perform extensive numerical simulations of interacting nuclear pores, studying their behavior as the density varies. So far attempts of modeling NPCs geometrical arrangement on the nuclear surface and using computer simulations to deepen our understanding on it were missing. With our work we try to fill that gap proposing a first attempt to study in silico peculiarities and features of the spatial distribution of nuclear pores.

Materials and Methods

Experimental images

In this Section we analyze super-resolution experimental images of nuclear pores of Xenopus laevis oocyte by Sellés et al. 13 . According to the stage of development of the oocyte 14 we identify three groups of images: an early Stage II, an intermediate Stage IV, and a later Stage VI. A different number of samples were taken at each stage, specifically: 6 samples for Stage II, and 11 samples for both Stage IV and Stage VI. The images are 2560×2560 pixels (px) wide, with 1 px corresponding to 10 nm. Examples of nuclear pore experimental images are reported in Fig. 1a)-c).

Tracking of Nuclear Pores

-We analyze the trajectories of the nuclear pores with Trackpy v0.4.2, a Python package for particle tracking in 2D, 3D, and higher dimensions 15 . In particular, we first discriminate the nuclear pores using the function trackpy.locate, whose working principle is the following: i) preprocess the image by applying a bandpass filter (i.e. performing a convolution with a Gaussian to remove short-wavelength noise, and subtracting out long-wavelength variations by subtracting a running average, in order to retain intermediate scale features), ii) apply a threshold over the color channels, and iii) locate all the peaks of brightness, each referring to the position of a pore 15,16 . The parameters used for the tracking are: diameter = 9 px the diameter, and minmass, the minimum integrated brightness, working as a threshold value. The latter value is chosen based on the samples: minmass = 0 (no threshold) is used in the high-density samples of Stage II, while higher values of this parameter were necessary to correctly detect pores in the noisier experimental images of Stage IV and VI. The tracking procedure also allows us to determine the density of pores defined as the number of pores per unit area of the nuclear envelope from the experimental samples: 34.9 ± 2.3 NPC/µm 2 for Stage II, 25.6 ± 2.3 NPC/µm 2 for Stage IV, 20.5 ± 1.7 NPC/µm 2 for Stage VI. The errors here represent the standard deviation computed on the ensemble of samples for each developmental Stage. The above density values are slightly lower than those computed by Sellés et al. 13 . We attribute this discrepancy to the different tracking techniques employed and to an inherent uncertainty related to experimental measurements performed with super-resolution optical microscopy. In fact, with this technique, some fluorescent spots may be "fragmented" in the final image, due to small microscope movements. Thus it can happen that a single pore appears divided into several dots, introducing a certain arbitrariness in the counting of pores.

Numerical Simulations

To model the interaction among nuclear pores, we consider their peculiar octagonal shape, as observed in early experimental studies 6,17 (see e.g. Fig. 1a) and subsequently confirmed by structural studies on nucleoporins, and by recent advances in experimental techniques such as cross-linking mass spectrometry and cryo-electron tomography 5 . Coarsegrained models are of key importance for understanding the essential behaviors of biological phenomena without resorting to detailed modeling of the molecular structure 18 . In the following paragraph, we describe the coarse-grained model providing the details on the form of the potential for the pore-pore interaction, and the simulation protocol.

Model Potential for Nuclear

Pores -To account for the composite structure of each nuclear pore and its overall octagonal shape, we implement a coarse-grained model of the pore, which consists of a central particle surrounded by eight particles located at the vertices of a regular octagon, of circumradius R. For simplicity, the pore is treated as a rigid non-deformable object. Taking experimental data as reference 13,19 , in all simulations we set R = 67.5 nm. The overall interaction potential acting among the particles of two neighboring pores is composed of three terms, each of which consists of a Lennard-Jones (LJ) potential,

V (r i j ) = 4ε σ r i j 12 - σ r i j 6 , r i j < r cut (1)
with r i j = |r i -r j | is the distance between particle i and particle j, and ε, σ , and r cut parameters depend on the interaction term:

• center-center interaction -the central particle of a pore interacts repulsively with the central particle of a neighboring pore. For this term we set ε cc = 0.01 pg•µm 2 /µs 2 , σ cc = 0.12 µm, and the cutoff distance is set r cut cc = 2 1 /6 σ cc , to make the interaction purely repulsive. This term is necessary to avoid non-physical configurations, such as the case of overlapping pores that are otherwise rarely encountered.

• center-vertex interaction -the central particle of a pore interacts repulsively with the particle at the vertex of a neighboring pore. Again, this is introduced to avoid pore overlap and interpenetration. LJ parameters for this term are ε cv = 0.01 pg•µm 2 /µs 2 , σ cv = 0.08 µm, r cut cv = 2 1 /6 σ cv .

• vertex-vertex interaction -the particle at the vertex of a pore interacts with the particle at the vertex of a neighboring pore. We first considered the full LJ interaction (long-range attractive, short-range repulsive), but the purely repulsive case was also studied (see Supplementary Fig. S1). For the LJ interaction we set ε vv = 5•10 -4 pg•µm 2 /µs 2 , σ vv = 0.02 µm, and the cutoff distance is set to r cut vv = 2.5σ vv so to include the attractive part. For the purely repulsive case we set r cut vv = 2 1 /6 σ vv . By construction, in our model the overall pore-pore interaction is mainly driven by the present term, due to the fact that the central particles are surrounded by vertex particles.

In all cases, the LJ potentials are shifted to zero at the cutoff distance to avoid any energy discontinuity. Fig. 1d reports the total interaction energy of an octagonal pore (centered at the origin) with a corner particle from a second pore, as a function of the latter's position. The resultant potential energy surface (PES) shows a central strongly repulsive region (in blue) and trapping regions (in red) concentrated near the eight corner sites.

2.2.2

In silico nuclear pores configurations -The simulations of nuclear pore assemblies are performed using LAMMPS 20 with a timestep ∆t = 10 -5 µs. The starting configurations consist of 1000 randomly placed octagonal pores confined in a square periodic box of side L = 40 µm. To mimic the different experimental nuclear pore densities observed during oocyte development, the random configurations are alternately subjected to 10 5 steps of box compression at a constant temperature T = 2 3 ε vv k B followed by a 2•10 5 steps of annealing from high temperature T = ε vv k B down to T = 2 3 ε vv k B , in order to allow thermally-assisted rearrangements. Such procedure is iterated until the required density is obtained, and a final energy minimization at T = 0 K in 2×10 6 steps is performed. Following the above protocol we obtain configurations with a density of 20, 26, 36, 46, and 53 NPC/µm 2 . For each density value we obtain 10 different realizations starting from different random initial positions, in order to allow for proper statistical averaging. Examples of nuclear pore configurations obtained with numerical simulations are shown in Fig. 1e) and f).

Statistical analysis of nuclear pores structure

In this Section we describe three different quantities used to provide a statistical comparison of the simulations with the reference experimental data: the radial distribution function, the hexatic order parameter and voronoi diagram. where N is the number of particles in the system, L is the system size as specified above, and r i j is the distance between particles i and j and the average is over particles. The RDF is a key tool for the theory of monoatomic liquids, to characterize amorphous colloidal solids [START_REF] Jana | [END_REF] and to study glasses and the glass transition 23 .

g(r) = L 2 2πrN 2 N ∑ i=1 N ∑ j=1 j =i Experiments Simulations (a) (b) (c) (f) (e) (d)

Hexatic Order

Parameter -To characterize the geometrical properties of the structure formed by the nuclear pores, we compute for each particle the n-fold local orientational order parameter (hexatic order parameter):

ψ n (r i j ) = 1 nnn nnn ∑ j=1 e inθ (r i j ) ( 3
)
where nnn is the number of nearest neighbors of particle i, θ (r i j ) is the angle formed by the x axis and the vector r i j connecting particles i and j. Experimental nuclear pore complexes of Xenopus laevis at different developmental stages show ordered regions characterized by triangular and square lattice 13 , therefore we focus our analysis on order parameters i) with n = 6 for which |ψ 6 | = 1 for particles belonging to a perfect hexagonal structure, and ii) with n = 4 for which |ψ 4 | = 1 for particles belonging to a perfect square lattice. The determination of nearest neighbor particles is performed using a cutoff distance cut = 0.15 µm for the simulations and σ cut = 0.20 µm for the experimental images. Those values have been chosen considering the typical inter-particle distance in simulated and experimental samples. For all isolated particles (nnn < 2) we set ψ n =0 .

2.3.3

Voronoi Tessellation -We finally analyze the Voronoi diagram for the nuclear pore configurations, partitioning the image into regions of convex polygons around the center of each pore. This so-called Voronoi cells represent the area of space containing all points that are closer to one pore than to any other. For the experimental images, the co-ordinates of the pore centers were obtained from the tracking analysis and used as input for the Voronoi tesselation. For the simulations only the central particle of each octagon is considered in the Voronoi analysis. By construction, each Voronoi cell has polygonal shape, with a number of sides that corresponds to the number of neighbors. To compute the Voronoi tesselation we used the Python library Freud 24 , that allows to account for periodic boundary conditions. Using this method we extract for each pore the number of neighbors (pores are considered neighbors if they share an edge in the Voronoi diagram) and the size of each associated Voronoi cell.

Results

Global structure of NPC

From the RDF of the experimental samples, we note that at high density (early stage of development of the oocyte) g(r) shows a liquid-like shape, with two peaks clearly visible (see Fig. 2a). In fact in monoatomic liquids, at short range g(r) shows a pattern of peaks representing the nearest neighbour distances, and at large r it tends to unity due to total loss of order [START_REF] Hansen | Theory of simple liquids: with applications to soft matter[END_REF] . As the density decreases during oocyte development (Fig. 2b,c), the second peak of the experimental g(r) tends to disappear while the first peak tends to flatten out, thus converging toward a gas-like phase in which the order is lost. Previous analysis of the experimental images suggested a significant presence of square lattice domains of nuclear pores at low density (Stage II) 13 . For this reason, we have looked for a specific peak in the g(r) function. In the case of a regular square lattice, a second peak should be present at x sq = √ 2 x 1 (where x 1 represent the spatial coordinate of the first peak of the g(r) function), however we notice that a peak in this position is not visible. Nevertheless x sq falls on the tail of the first peak (which is quite flat), suggesting that some sparse regions with square lattice could be present inside the amorphous liquid. Similar considerations have been done for hexagonal structures at high densities: if a regular hexagonal packing of pores is present, the g(r) should display a peak at the position x hex = √ 3x 1 and again, this is not the case, with x hex falling only on the growing part of the second peak for the high density configurations. Some hexagonal structures at high density could be present but are not predominant, being the spatial distribution of NPCs mainly disordered. The obtained from simulations with the octagonal attractive potential (Fig. 2) reproduces the above described liquid-like profile presenting just a few peaks emerging over an otherwise flat profile. Furthermore, the positions of the peaks in the simulated g(r) match nicely with the experiments (especially for what concerns the first peak), suggesting that our model is able to capture the relevant features of the effective interaction among the nuclear pores on the nuclear membrane. This is a non-trivial result, as the position of the g(r) peaks closely reflects the interactions held between the constituents, and are a signature of each material and its peculiar properties as shown in previous studies of noble gases or water [25][26][27][28] . Ultimately, from this analysis, we can rule out the significant presence of extensive crystalline regions.

Orientational Order

In Fig. 3, we report the calculated local order parameter ψ 6 and ψ 4 , as defined in Section 4, for some experimental samples and for the configurations obtained from simulations. The cutoff to consider a pore as a neighbor was set to σ cut = 0.20 µm for the experiments and σ cut = 0.15 µm for the simulations. These values were chosen considering the typical pore-pore distances. From Fig. 3a and Fig. 3c it can be observed that only few pores are associated with square symmetry (ψ 4 ∼ 1). Instead, Fig. 3b and Fig. 3d show much broader regions associated with triangular lattice structures (ψ 6 ∼ 1). In those samples, in presence of higher pore densities, more nuclear pores belong to a regular structure and some regions with clear hexagonal order appears. To further investigate such behavior we computed the distribution of the local order parameters P(ψ 6 ) and P(ψ 4 ) averaged over all the samples for each value of the density (see Fig. 4). First, we can observe that the distributions of the experimental samples (Fig. 4a,b) are all unimodal, thus confuting the hypothesis of two different coexisting phases suggested previously 13 . Secondly, we notice that for both ψ 6 and ψ 4 the distributions are peaked at a value which increases with the density. The largest mode value is obtained for ψ 6 at the highest density (Stage II), suggesting a preference for hexagonal structures in the dense limit. The above trends of the distributions with the density are well reproduced by the simulated NPCs, as reported in Fig. 4c and Fig. 4d. For a more straightforward comparison we have reported in Fig. 4e It is worth to note that, in the explored density range, the |ψ 6 | value is far from approaching unity, corresponding to a crystalline structure, indicating that much larger densities would be required for such an ordered phase. Finally we note that in Fig. 4e the points associated to the experimental data do not fall exactly on the theoretical curves derived from the simulations. This can be partially explained with the uncertainties connected with the experimental observations of the nuclear pores, that affect the density evaluation. Despite that, the experimental points show a trend very close to that of the

Properties of Voronoi cells

Examples of Voronoi tesselation performed on high density NPC from the experimental images and from the simulated configurations are reported in Fig. 5a and Fig. 5d, respectively. The comparison in these high density samples shows similar tessellation patterns in experiments and simulations. A statistical analysis on the number of sides N of the Voronoi cells at different densities (Fig. 5b and Fig. 5e) clearly shows that the N = 6 occurrence increases with the density, and viceversa for the the N = 4 occurrence. Therefore, the Voronoi analysis enforces the idea, already anticipated above by the local order parameter, that the hexagonal configuration is favored at high densities at the expense of other kind of local order arrangements. Correspondingly, in agreement with experimental observations, the presence of some square structures at low density is also supported. However, we note at any density a significant number of cells with N = 5, about half between those with N = 4 and N = 6. We associate this with the particular sensitivity of the Voronoi tesselation method to "defect", i.e. deviations with respect to the ideal symmetric cases. To provide a further comparison, we report in Fig. 5c and Fig. 5f the distribution of the Voronoi cells area. Again, a good agreement between the experiments and our model is obtained, much narrower distributions at higher densities, shifting toward higher area values and broadening out as density decreases.

Discussion and Conclusions

A first attempt to investigate the spatial distribution of nuclear pores goes back to the '70s, when the positions of NPCs on the surface of rat kidney nuclei was observed and distances among them measured 29 . Already in this study, some regularities were found in the distribution of pore-pore distances measured in the samples, suggesting a non random spatial distribution and some peaks corresponding to hexagonal structures, even if the statistics was too poor to reach further conclusions. More recently, Sellés et al. investigated the angular distribution between first neighbors of nuclear pores, revealing no preferential angles for Stage II and IV Xenopus laevis oocytes (high density) and two distinct peaks at 90 • and 180 • for later Stage VI, suggesting the presence of square lattice regions at low density 13 . In our study, from the analysis of the radial distribution function g(r) of the nuclear pores on the nuclear membrane of Xenopus laevis oocytes, we could not observe peaks in correspondence of peculiar geometrical structures, meaning that even though some crystalline regions are present, they are quite rare and do not statistically influence the overall NPC spatial distribution. Interestingly by analysing the g(r) of the nuclear pores, we were able to identify an amorphous, liquid-like structure in which, in the early phase of oocyte development (when NPC density is high), long-range order is soon lost. On the other hand, as the oocyte develops, the nuclear pore density decreases and shows a behaviour compatible with a more dilute, gas-like system. From a biological point of view, the early stages of oocyte development are associated with intense transcriptional activity, as the oocyte needs to build up a huge reserve of gene products such as mRNAs, tRNAs and proteins in order to correctly fulfil its future role after fertilisation. Once the necessary maternal mRNAs have been copied, transcriptional activity in the later stages of oocyte development becomes lower 30 . These changes in transcriptional activity could be linked to changes in the spatial distribution of NPCs during oocyte development, particularly changes in density. It would be extremely interesting to further explore this connection from a biophysical point of view, e.g. by trying to quantify the flow of matter through the pores, (as has already been done in some previous kinetic studies, which showed that a single NPC can a mass flow of nearly 100 MDa/s 31 ), at different stages of the cell's The positions of the peaks in the g(r) we computed for nuclear pores are another key point of our results. it is known from the physics of matter that the positions of the peaks of the radial distribution function and their relative distances give actual information about the geometrical arrangement of the particles within a material, and are a signature of the material itself [START_REF] Chandler | Mechanics[END_REF] . In particular, the peak positions allow to indirectly infer the type of interactions among the constituents of a specific material. Here, we have shown that the eight-fold potential used to model the NPC in our simulations is able to nicely reproduce the experimental g(r) peak locations. In particular, the position of the first peak obtained from the simulations is in excellent agreement with Sellés et al. 13 and with previous observations 11,29 . We also checked that assuming a simple LJ potential acting among the pores (i.e. with spherical symmetry) with parameters compatible with experimental pore sizes, the prediction of the g(r) peaks is not in agreement with experimental results (see Supplementary information). Even if the hypothesis of a pore-pore potential with spherical symmetry (perhaps with an effective interaction size for the pore that does not coincide with its physical size) can not be excluded, our work suggests that the octagonal shape of the pore and the associated eight-fold symmetry of its interaction potential plays a crucial role in determining the correct spatial distribution of the pores. These facts are worth to notice, since our simplified model based on the assumption of an effective eight-fold pore-pore interaction is able to catch a crucial signature of the spatial distributions of nuclear pores, the radial distribution function peaks positions, even if the interaction details (e.g. if the pore-pore potential is attractive or repulsive) are not known (see Supplementary). Hopefully this could help to deepen the investigation of the nature of the pore-pore interplay, allowing to study also in silico an inter- action that in reality is not fully understood under a biological point of view. Pore-pore interactions are unlikely to be direct, but rather mediated by the lamin scaffold through complex interactions are hard to model explicitly. In this sense, our assumption that nuclear pore arrangement can be modeled as assembly of interacting octagons is possibly oversimplified, since after development pores are stuck within the lamina and are unlikely to diffuse. We can, however, imagine that as the nuclear envelope is formed the nuclear pores are arranged in way that is dictated by their geometry and which could then be by our model. Since extensive MD simulations of lamina filaments forming a three-dimensional network beneath the nuclear envelope have recently been performed [START_REF] Sapra | [END_REF] , it be interesting to try to go further in modelling the outer regions of the cell nucleus, linking the lamina network and the spatial distribution of the nuclear pores.

Coming back to our analysis, the spatial distribution of NPCs, investigated through the local order parameters shows that at high density the pores tend to arrange following the tri-angular lattice. Even though the g(r) does not show explicit peaks in correspondence to a triangular lattice, the study of Ψ 6 (Fig. 3) and the Voronoi tessellation method (Fig. 5) prove that at high density islands of six-fold symmetry packed pores appear. Noticeably, such behavior has been already reported in previous experimental observations. During apoptosis the distribution of nuclear pores on the cell nucleus strongly changes, bringing the NPCs to be highly concentrated in small regions of the nuclear envelope (on mouse cell nuclei) and leaving the rest of the surface pore-free. Those clusters of pores showed a hexagonal packing and were supposed to be correlated with diffuse chromatine areas 34 . Occasional areas of very regular hexagonal packing of nuclear pores have been also observed to emerge during the development of male germ cells, in rodent spermatocytes 11 . Those facts open interesting questions on how the geometrical disposition of the pores in some areas, or even more simply, their density, are influenced by the underlying nuclear activity and on what are the biological causes responsible for the effective interaction among NPCs. Consid- ering the pores under a geometrical and topological point of view, underlying the importance of their octagonal shape, like our simple model does, could be extremely interesting also in the contest of membranes studies. In fact in a recent paper by Torbati et al. 35 , the authors studied the mechanical stability of the lipid bilayer membrane of the nuclear envelope, considered as two concentric membrane shells fused at numerous sites with toroid-shaped nuclear pores (here simply modeled as circular holes). Using mechanistic arguments based on elasticity, they showed that in-and out-of-plane stresses can give rise to the pore geometry and the geometric topology observed in cell nuclei, finding simulated interpore distances in good accord with the ones observed in mammalian cells nuclei. How octagons can contribute to stabilize the curvature of a spherical membrane 1 and how they tend to be spatially arranged on such a geometry could be an issue to consider to better clarify the process of nuclear pores formation.

2. 3 . 1

 31 Radial Distribution Function -To gain insight on the local structure of the nuclear pore complex on the nuclear membrane, we analyze the radial distribution function (RDF) 21 :

Fig. 1

 1 Fig. 1 Comparison of experimental and simulated nuclear pore images. -(a) Detached pore complexes released onto the microscope grid from a nuclear envelope immersed in a low-salt medium containing 0.1% Triton X -100, from which the octagonal shape is clearly visible. Scalebar is 0.1µm. (b),(c) Portions of experimental images of nuclear pores in Xenopus laevis oocyte at different developmental stages, respectively Stage II (b) and Stage VI (c), obtained using super-resolution microscopy. Scalebars are 1µm. Panel (a) is an adaptations from Unwin et al.6 , panels (b) and (c) are adaptations from Sellés et al.13 . Scalebars are 1 µm. (d) Potential energy surface obtained from the modeled interaction between a pore and a corner of a neighboring pore: the blue area marks a strongly repulsive region, while the red areas mark the trapping centers. (e),(f) Example of two configurations of nuclear pores obtained from numerical simulations for comparison with experimental data. The density is 36 NPC/µm 2 for (e) and 20 NPC/µm 2 for (f). Scalebars are 1µm.

  the average value of |ψ 6 | and |ψ 4 | as a function of the pores density for both simulations and experiments. For the former, we observe that both |ψ 6 | and |ψ 4 | values slowly increase with the density, showing the same values until a density of 36 NPC/µm 2 where a bifurcation occurs. Beyond that density value, |ψ 4 | seems to saturate while |ψ 6 | keeps increasing, thus favoring the hexagonal order at high density.

Fig. 2

 2 Fig. 2 Radial Distribution Function of nuclear pores -The g(r) is reported for three different densities of the nuclear pores. Blue curves are from averages over ten simulations with attractive octagon potential. Dashed red curves are obtained from the experimental images for Stage II -(a), Stage IV -(b), Stage VI -(c).

SimulationsFig. 3

 3 Fig. 3 Color maps of local order parameters -Snapshots of pores colored as a function of the local order parameter: (a) a zoomed region of an experimental sample at Stage VI, for which we estimate a density of 20.5 ± 1.7 NPC/µm 2 ; (b) a zoomed region of an experimental sample at Stage II, for which we estimate a density of 34.9 ± 2.3 NPC/µm 2 ; (c) and (d) a simulation box with pores density 20 NPC/µm 2 and 36 NPC/µm 2 respectively.

Fig. 4

 4 Fig. 4 Distribution of the local order parameters -The distribution of the local order parameters at different pores densities is reported for (a),(b) experimental samples and (c),(d) for simulated configurations; (e) the average |ψ 6 | and |ψ 4 | values as a function of the density. The dashed lines report the values obtained from the simulations, with shadows highlighting the respective standard deviation. The points represent the values computed from the experimental samples, with vertical errorbars for the standard deviation, and horizontal errorbars reporting the error on the density, as described in Section 2.1.1.

Fig. 5

 5 Fig. 5 Voronoi tesselation applied to nuclear pores -Examples of Voronoi tesselation are provided for (a) an experimental samples at Stage II and (d) for a simulation with density NPC/µm 2 ; (b) and (e) histogram of the number of Voronoi cell facets, for different densities; (c) and (d) the corresponding distribution of the Voronoi cells area.

| 1-10

Acknowledgements

We thank Zoe Budrikis for preliminary developments of the simulation code.


How should we distantiate our perception of the colours of the past ?

1) Colours have changed and cannot be restored a. An exhibition took place in 2012 at the Musée Cognacq-Jay 'La Patine du temps,' arguing that it is impossible to restore paintings to their original state. One of the most telling examples was a comparison of two portraits of a child by Greuze (1775), where the unrestored painting, though crackled, had depth, whereas the relined painting, though it had a faultless surface in an attempt to restore it to its original state, gave a very flat effect and had lost its depth. You will note that the exhibition was contemporary with the controversial restoration of Leonardo's Saint Anne in the Louvre, and remember that Cognacq-Jay is a museum of the City of Paris whereas the Louvre is a national museum; they may have different approaches. b. 17th and 18th-century writers knew that colours change; they knew that in addition, varnish darkens, and so a dark picture was an asset -a proof that it was an Old Master painting, so much so that forgers sold deliberately darkened pictures. Hogarth makes fun of the taste for dark paintings in his Time Smoking a Picture (1761).

Experimenting on pigments sometimes had a disastrous result, as some of Reynolds's paintings show. He experimented on various mixtures, which in the course of time proved to be unstable and darken.

As part of the debate on the relative merits of drawing and colour initiated in the academies of the 17th century, and on the relative merits of painting and of literature, colour was an image of transience, a view put forward by Pope in An Essay on Criticism (1709)

The treach'rous colours the fair art betray, And all the bright creation fades away ! (494-95) So perhaps we are closest to our 17th and 18th-century forbears when we recognise that we cannot see the colours of their paintings?

2) We should historicise colours and our perception of them a. Now to look at material culture, the period we study is characterised by the coexistence of traditional natural colours and the first synthetic chemical colours. The first of them, Prussian blue, a cyanide, was invented in the first decade of the 18th century by a Prussian colourman. It was soon adopted by painters, frequently in a light form to paint the skies, as it opened new possibilities to them. Among those who most widely used it for their skies was Canaletto, and we should bear in mind that our mental image of Venice, largely influenced by the vedutisti, with its light bluish-greenish sky, is the result of a then recent chemical discovery. b. But can we see Canaletto's paintings as British collectors of the 18th c. saw them ? A major collection is to be found in the Canaletto room at Woburn Abbey (see 'Woburn Abbey'), where they were placed in 1800, coming from Bedford House in London for which they had been commissioned in the 1730s. As we can see, the room has windows opening into the grounds and is very bright. This is not the original lighting; when the paintings arrived, the Duke of Beford had the windows blocked so that all the paintings could be hung in one room, and the windows were reopened only in 1955, with three paintings removed to other rooms. The guests in 1800 would see them in a dark room, and in addition with the flickering light of candles, not with our stable lighting. Can we really reconstruct their experience or recreate their perception? we can attempt to do so in an animation with modern media, as long as we are aware that it is only an approximation.

3) How far does 19th-and 20th-century research on colours separate us from earlier centuries?

Just a few examples of approaches to colour which have emerged since then: a. Among the discoveries on colours in the 19th century was the discovery of the reason why the sky seems to be blue, an experiment (1868) that you may see at the Royal Institution in London in the section on Tyndall -the reason being that the sky (like so-called blue eyes) is transparent, but blue light having a shorter wavelength is more easily scattered (see 'Interactive Timeline'). b. Digital colours are a late 20th century feature. They are based on arithmetics: 256 colours in the basic palette because 256=2 8 -eight bits each having two options, so 256 combinations. An interesting point is that, in geometrical constructions of volumes with 3D programmes, colours and textures can create volumes -e.g. the Voronoi texture (Voronoi being the name of a mathematicianaround 1900) which can be used to make surfaces rise so as to produce hills or waves on a river where changes of colour and texture occur. So colours are not always subordinate to form, they can create them -an old debate. other things that in a competition all the competitors much operate in similar lighting (see Lego). Contrasts of dark/light colour guide motion, depending on light. The issues of the relationship between colours, darkness and shadow, light, which have been discussed for centuries ever since Leonardo, take on a new dimension: then and now, colours need to be studied in context. We have to know the past in order to understand the present, but we also need to be aware of present-day research if we are to perceive the significance of the past.

4) Present

Marie-Madeleine Martinet

Sorbonne Université

  -day research : arts and sciences -returning to Early-Modern universality ? a. Recent research in museums and galleries combines artistic and scientific skills : the National Gallery exhibition 'Making Colour' (2014) studied colours in an 'artistic and scientific voyage of discovery' through the chemical nature of the pigments used for centuries, complemented by an interactive display experimenting on the visitors' perception of colour. The 'Regency Colour' exhibition at the Royal Pavilion in Brighton (2013) showed the scientific basis of Regency approaches to colour. Numerous projects such as 'Polyre' (2016) combine arts and sciences and rest on partnerships between laboratories in several fields, in the same way as medical tools of investigation are now used in archaelogy. It is therefore essential that we give our students both an artistic and a scientific grounding if they are to make a significant contribution in present-day society by establishing partnerships with researchers or creative workers from several fields of study. An age-old question is the relationship between colours on the one hand, and light and dark effects on the other hand, ranging from Renaissance studies of shadows to definitions of colour 'tone' or dark/light 'value' by painters and their use of Claude Lorraine glasses to correct their paintings. An 18th-century text, Johann Heinrich Lambert's Photometria, sive De mensura et gradibus luminis, colorum et umbrae (1760) is the basis for present-day studies and practice in information visualisation, including the algebraic and trigonometric formula used to create reflection on colours in computer graphics, taking into account the Lambertian amount of light penetrating the pigment : (see Ware 43). It would be interesting to study, among the mathematics and physics behind the new media, which ones belong to classical physics dating back to the period we are studying, and which ones are based on more recent scientific revolutions. b. Light/dark contrasts have been given a new lease of life by recent technologies in robotics: certain robots (2017) are programmed to follow paths indicated by dark lines on a white ground, which they can trace as they are equipped with sensors; this means among


M.D. Aymeric Becq
email: aymeric.becq@aphp.fr

Charles Houdeville

My-Linh Tran

MD Nils Steuer

MD David Danan

Einas Marie Anne Guillaumot

Abou Ali

MD Maximilien Barret

PhD Aurélien Amiot

MD, PhD Nicolas Carbonell

MD My-Linh Charles Houdeville

MD Minh Tran

MD Einas Abou Ali

MD Philippe Marteau

PhD Ulriikka Chaput

MD Xavier Dray

MD, PhD Marine Camus

MD Samuel Giday
email: samgiday@gmail.com

My-Linh Tran

Einas Abou

Team (POET Parisian On-Call Endoscopy

Cyriaque Bon

Benoît Bordacahar

Bertrand Brieau

Charlotte Delattre

Johann Dreanic

Julien Kirchgesner

Cécilia Landman

Anne Laurain

Guillaume Le Gall

Romain Leenhardt

Sara Lemoinne

Marie Lequoy

Nelson Lourenco

Chloé Martineau

Frédérick Moryoussef

Jean-Charles Nault

Jeanne Netter

Violaine Ozenne

Anna Pellat

Olivia Pietri

Pierrre Rompteaux

Adrien Sportes

Aziz Zaanan

Aymeric Drs

Charles Becq

My-Linh Houdeville

Minh Tran

Experience with the use of a hemostatic powder in 152 patients undergoing urgent endoscopy for gastrointestinal bleeding

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

ABSTRACT

Background and Study Aims

In the recent years, topical hemostatic powders have been used for the management of upper gastrointestinal bleeding. The aim of this study was to report on the use of an hemostatic powder (Hemospray ® ), outside regular hours, by on-call endoscopists during urgent endoscopic procedures.

Material and methods

In this retrospective multicenter cohort study, consecutive patients having undergone an urgent endoscopy with the use of Hemospray ® from November 2015 to December 2018 in the Paris and suburbs area were included. We collected clinical, biological and endoscopic variables. The outcomes such as the recurrence, repeat endoscopy and hemostatic treatment need, complications and survival were also collected.

Results

A total of 152 patients (mean 65 years old, 70.4% male) were included. Amongst the 31 endoscopists, 11 were "more experienced", and performed 48% of the endoscopies. The most common causes of bleeding were peptic ulcer (47.7%), malignancy (22.2%) and esophagitis (12.4%). Most bleedings originated from the upper GI tract (95.0%). Hemospray ® was used as a salvage therapy in 60.8% of cases. Other hemostatic techniques were used in 52.9% of cases.

Immediate bleeding cessation was noted in 79.0% of cases, recurrence in 39.9% of cases, and 26.4% of patients benefited from a repeat endoscopic hemostasis. 34 (23.0%) patients required a non-endoscopic treatment. At day 30, the survival rate was 71.6%. One complication was reported (perforation).

Conclusions

Hemostatic powder application by on-call endoscopists outside regular hours is technically feasible, but comes with a high risk of rebleeding in severely ill patients. Keywords: Hemospray ® , hemostatic powder, digestive bleeding, gastrointestinal hemorrhage ABBREVIATIONS -GIB: Gastrointestinal bleeding -UGIB: Upper gastrointestinal bleeding -PUD: Peptic ulcer disease -POET: Parisian On-call Endoscopy Team

INTRODUCTION

Acute gastrointestinal bleeding (GIB) is a common condition, associated with a significant morbidity and mortality [START_REF] Van Leerdam | Epidemiology of acute upper gastrointestinal bleeding[END_REF][START_REF] Hearnshaw | Acute upper gastrointestinal bleeding in the UK: patient characteristics, diagnoses and outcomes in the 2007 UK audit[END_REF]. Mortality rates have been reported ranging from 3 to 14%, depending on the age, co-morbidities, clinical status, cause of bleeding [START_REF] Van Leerdam | Epidemiology of acute upper gastrointestinal bleeding[END_REF][START_REF] Hearnshaw | Acute upper gastrointestinal bleeding in the UK: patient characteristics, diagnoses and outcomes in the 2007 UK audit[END_REF]. Upper gastrointestinal bleeding (UGIB) accounts for over 80% of all GIB. Guidelines on the management of variceal and non-variceal UGIB have been published [START_REF] Barkun | International consensus recommendations on the management of patients with nonvariceal upper gastrointestinal bleeding[END_REF][START_REF] De Franchis | Expanding consensus in portal hypertension: Report of the Baveno VI Consensus Workshop: Stratifying risk and individualizing care for portal hypertension[END_REF]. Endoscopic hemostasis is the cornerstone of the management of GIB and relies on injection, thermal and mechanical therapies [START_REF] Gralnek | Diagnosis and management of nonvariceal upper gastrointestinal hemorrhage: European Society of Gastrointestinal Endoscopy (ESGE) Guideline[END_REF]. Injection therapy (epinephrine, sclerosing agents, and glue), thermocoagulation (including plasma argon coagulation and bipolar electrocautery), as well as hemostatic clips and band ligation have been shown to be effective methods in this setting [START_REF] Laine | Endoscopic ligation compared with sclerotherapy for treatment of esophageal variceal bleeding. A meta-analysis[END_REF][START_REF] Laine | Endoscopic therapy for bleeding ulcers: an evidence-based approach based on meta-analyses of randomized controlled trials[END_REF][START_REF] Sung | Endoscopic clipping versus injection and thermocoagulation in the treatment of non-variceal upper gastrointestinal bleeding: a meta-analysis[END_REF][START_REF] Hwang | The role of endoscopy in the management of acute non-variceal upper GI bleeding[END_REF]. However, they are not always efficient and offer limited success in cases of large bleeding areas (i.e. malignancy) [START_REF] Chen | Hemostatic Powders in Gastrointestinal Bleeding: A Systematic Review[END_REF].

In the more recent years, topical hemostatic powders such as Hemospray ® (TC-325; Cook Medical, Winston-Salem, NC, USA) have been tested on porcine models, then used for the management of UGIB in humans [START_REF] Chen | Hemostatic Powders in Gastrointestinal Bleeding: A Systematic Review[END_REF][START_REF] Hagel | The Application of Hemospray in Gastrointestinal Bleeding during Emergency Endoscopy[END_REF][START_REF] Giday | Safety analysis of a hemostatic powder in a porcine model of acute severe gastric bleeding[END_REF][START_REF] Giday | Long-term randomized controlled trial of a novel nanopowder hemostatic agent (TC-325) for control of severe arterial upper gastrointestinal bleeding in a porcine model[END_REF][START_REF] Chen | TC-325 hemostatic powder versus current standard of care in managing malignant GI bleeding: a pilot randomized clinical trial[END_REF]. Hemospray ® is an inorganic, absorbent powder, which upon contact with the moisture of an active bleeding, becomes coherent, creating a mechanical barrier. It also activates platelet aggregation and possibly concentrates coagulation factors [START_REF] Chen | Hemostatic Powders in Gastrointestinal Bleeding: A Systematic Review[END_REF]. The resulting coagulum typically sloughs off within 24 hours [START_REF] Chen | Hemostatic powder TC-325 in the management of upper and lower gastrointestinal bleeding: a two-year experience at a single institution[END_REF]. Hemospray ® has received clearance in multiple countries, specifically in Europe and has recently been FDA approved [START_REF] Hagel | The Application of Hemospray in Gastrointestinal Bleeding during Emergency Endoscopy[END_REF][START_REF] Commissioner | Press Announcements -FDA permits marketing of new endoscopic device for treating gastrointestinal bleeding[END_REF]. Only a few number of studies have been published on Hemospray ® , showing it to be technically feasible and a viable option as primary or salvage hemostatic treatment of UGIB [START_REF] Sung | Early clinical experience of the safety and effectiveness of Hemospray in achieving hemostasis in patients with acute peptic ulcer bleeding[END_REF][START_REF] Ibrahim | Endoscopic treatment of acute variceal hemorrhage by using hemostatic powder TC-325: a prospective pilot study[END_REF][START_REF] Haddara | A novel hemostatic powder for upper gastrointestinal bleeding: a multicenter study (the "GRAPHE" registry)[END_REF].

The goal of our study was to report on the real-life use of Hemospray ® , in the setting of urgent endoscopies, performed by mobile on-call endoscopists, outside regular hours, in multiple Parisian teaching hospitals.

PATIENTS AND METHODS

We performed a retrospective descriptive multicenter cohort study to investigate the use of Hemospray ® during emergencies, by on-call endoscopists. This study was approved by the local ethics committee. IRB approval was not required for this study. Patients' data was de-identified.

Outside regular hours (nights and week-ends), urgent endoscopy procedures are performed by an on-call endoscopist in over 20 Parisian teaching hospitals, covering a 5 million inhabitant area. All endoscopists are senior attending physicians, fully trained in all the hemostatic techniques. All have received hands-on training sessions on the use of Hemospray ® . A taxicab is used to go from one hospital to another. The endoscopist is based at Saint-Antoine Hospital where all the equipment is stored. The on call team and equipment is transported by taxicab to perform hemostatic technics such as clipping, epinephrine injection, band ligation, thermocoagulation and hemostatic powder application (figure 1).

We included consecutive patients having undergone an urgent endoscopy with the use of Hemospray ® from November 2015 to December 2018. The indication and treatment decision making was at the discretion of the endoscopist. Hemospray ® was applied using the standard seven-French catheter with its tip a few centimeters from the bleeding source. The endoscopist was assisted by a nurse and performed bedside endoscopies in intensive care units with a portable endoscopy processor (Tele Pack X GI, Karl Storz SE & Co. KG, Tuttlingen, Germany), nontherapeutic gastroscopes (Karl Storz Silver Scope -13821 PKSK/NKSK, Karl Storz SE & Co. KG, Tuttlingen, Germany), and a portable electrosurgical unit (ERBE -VIO100C, Erbe Elektromedizin GmbH, Tuebingen, Germany). The procedures were all performed with general anesthesia, after patient intubation, given the high risk of aspiration in UGIB secondary to bloodfilled esophagus and/or stomach, or because in these urgent settings patients are not always with empty stomach as they may have recently eaten or drank. Salvage therapy was defined as the application of Hemospray ® for persistent bleeding after failure of other hemostatic modalities either during the same endoscopic procedure or after failure of previous endoscopic attempts.

Immediate hemostasis was defined as the absence of ongoing visible active bleeding after Hemospray ® application. Recurrence was defined as the requirement of a subsequent hemostatic treatment, meaning either a repeat endoscopic treatment and/or a non-endoscopic treatment (arterial embolization, surgery, radiation therapy) within 30 days.

We collected clinical data. Data regarding the hemorrhagic event was also collected. History of recent endoscopy in the 30 days prior to the one performed by the on-call endoscopist with use of Hemospray ® , and hemostatic treatment requirement was collected. Endoscopic findings were collected as well: cause, origin, type of bleeding, other hemostatic techniques used, primary or salvage Hemospray ® , number of kits used, technical issues, and immediate efficacy. Outcomes were collected, including the need for repeat endoscopy at day 3 and 30, subsequent endoscopic or non-endoscopic hemostatic treatment, complications, and survival at day 30. The level of experience of the on-call endoscopist was collected. We considered an endoscopist as "less experienced" if he had been participating in on call shifts for less than four years at the start date of data collection (i.e. November 2015). We considered an endoscopist as "more experienced" if he had been participating in on-call shifts for more than four years at the start date of data collection.

The primary outcome of the study was the rate of bleeding recurrence which was calculated based on the number of subsequent hemostatic interventions needed. The secondary outcomes were the technical feasibility, the use of Hemospray ® as a primary treatment, and the survival at 30 days.

RESULTS

In the span of the 3 years of the study period, 2840 endoscopies were performed during nonworking hours for suspected bleeding, with hemostatic treatment in 1143 of cases (40.2%).

Hemospray ® was used in 13.4% of all procedures with hemostatic treatment, accounting for 11% of endoscopies performed for hemostatic purposes in 2016, 8% in 2017 and 11% in 2018. During the study period, 152 patients underwent 153 endoscopic procedures in the setting of acute GIB with Hemospray ® application. Four patients were excluded due to missing follow-up data (4) (flow chart). These endoscopies were performed by 31 different endoscopists. Amongst these endoscopists, 11 were 'less experienced' and performed 73 of the 153 endoscopies (48%).

The mean age of the patients was 65.0 ± 12.2 years. 70.4% of patients were male and 61 patients (40.1%) were on an antiplatelet and/or anticoagulant medication. Sixty-four patients (42.1%) required a vasopressive drug and 142 (93.4%) blood transfusion (table 1).

Forty-six (30.1%) had endoscopic procedures within the 30 days prior, 26 (56.5%) of which received hemostatic treatment. The most common causes of bleeding were peptic ulcer disease (PUD) (47.7%), bleeding tumors (22.2%) and esophagitis (12.4%). Amongst the nine patients with post-endoscopy bleeding, five were secondary to a sphincterotomy. One patient had an aortoenteric fistula. Most bleedings were localized in the upper GI tract (95.0%) and were active (95.4%). Regarding the four patients with non-active bleeding, two had a post-sphincterotomy bleed with a clot seen at the level of the papilla. Hemospray ® was used as a primary therapy in 60 cases (39.2%) and as a salvage therapy in 93 cases (60.8%). The use of Hemospray ® as a primary or salvage therapy was similar between "less" and "more" experienced endoscopists (38% and 40% as a primary therapy, and 62 and 60% as a salvage therapy, respectively) (figure 2). Other hemostatic techniques were used in 81 cases (52.9%). Immediate bleeding cessation was noted in 121 cases (79.0%). Use of 2 Hemospray ® kits was required in one patient (0.7%). Hemospray ® application was challenging in five cases (3.3%) (table 2). Among the patients with persistent bleeding, 7 (43.7%) had received Hemospray ® as a primary therapy and 9 (56.3%) as a salvage therapy. Among the patients with bleeding, 41 (33.9%) had received Hemospray ® as a primary therapy and 80 (66.1%) as a salvage therapy (figure 3).

Four patients were lost to follow-up. Seventy-one patients (48.0%) required 88 repeat endoscopic procedures for suspected recurrence. A repeat endoscopy was required in 37 patients before day 3, in 17 patients before day 30, and in 17 patients before day 3 and a second time before day 30.

Thirty-nine patients (26.4%) required a repeat hemostatic treatment (48 endoscopies) (table 3).

Thirty-four (23.0%) patients required a non-endoscopic hemostatic treatment such as arterial embolization (9.5%), surgery (8.8%), arterial embolization followed by surgery (2.7%) and radiation therapy (2.0%). Overall, 59 (39.9%) patients had a recurrence requiring endoscopic and/or non-endoscopic hemostatic treatment. Fourteen (9.5%) patients required both endoscopic and non-endoscopic hemostatic treatment. One perforation was reported. Verification of the patients' records showed the diagnosis was made by Computed Tomography, 48 hours after the endoscopy, because of worsening clinical condition. Based on the endoscopy report, the patient had a deep peptic ulcer and there was no suspicion of perforation prior or during the endoscopy. At day 30, 106 patients (71.6%) had survived (table 3). Finally, 23% and 33% of patients with and without bleeding cessation respectively did not survive after 30 days of follow-up (Odds Ratio 0.69, 95% Confidence interval [0.21; 2.63], Fisher's exact test, p = 0.55) (Figure 4).

Immediate hemostasis was achieved in 121 (79%) patients, amongst which 40.9% (49 patients) did not require further treatment (endoscopic or non-endoscopic) and survived at 30 days. Overall, 57 (37.5%) of the 152 patients did not require further treatment (endoscopic or non-endoscopic) and survived at 30 days, regardless of initial bleeding cessation.

DISCUSSION

In this study, we report on the real life use of the hemostatic powder Hemospray ® , in the setting of urgent endoscopic procedures performed outside working hours, by mobile on-call endoscopists. Hemospray ® was mostly used for upper GIB (95.0%), nearly half of which were due to PUD (47.7%). Hemospray ® was used as salvage therapy in 60.8% of cases. During follow up, 39.9% had a recurrence. At 30 days of follow-up, the mortality rate was 28.4%.

The use of Hemospray ® by our group was stable after becoming available and did not increase with the endoscopists experience. It's use accounts for 10% of all our cases. It should however be noted that one third of our patients had a prior procedure, nearly two thirds benefited from Hemospray ® as a salvage therapy, and that these endoscopies were performed in an urgent setting, in non-dedicated units.

Our study adds to the existing body of literature regarding the use of Hemospray ® . First, our study serves to show that Hemospray ® application is user friendly, consistent with previously published data. The application was challenging in five cases (3.3%), due to difficult anatomy, scope position and/or obstruction of the catheter. Second, previous studies have reported on the efficacy of Hemospray ® application in acute GIB [START_REF] Sung | Early clinical experience of the safety and effectiveness of Hemospray in achieving hemostasis in patients with acute peptic ulcer bleeding[END_REF][START_REF] Ibrahim | Endoscopic treatment of acute variceal hemorrhage by using hemostatic powder TC-325: a prospective pilot study[END_REF][START_REF] Haddara | A novel hemostatic powder for upper gastrointestinal bleeding: a multicenter study (the "GRAPHE" registry)[END_REF][START_REF] Alzoubaidi | Outcomes from an International Multicentre Registry of patients with acute gastrointestinal bleeding undergoing endoscopic treatment with Hemospray[END_REF][START_REF] De Santiago | Hemostatic spray powder TC-325 for GI bleeding in a nationwide study: survival and predictors of failure via competing risks analysis[END_REF]. The largest study to this day, published in 2019 by Alzoubaidi et al., included 314 patients. The immediate efficacy rate was 89.5%. The recurrence rate amongst these patients was 10.3%. The 30-day all-cause mortality was 20.0% (63 of 314 patients) [START_REF] Alzoubaidi | Outcomes from an International Multicentre Registry of patients with acute gastrointestinal bleeding undergoing endoscopic treatment with Hemospray[END_REF]. A meta-analysis by Faccuirusso et al. including 1063 patients showed an immediate hemostasis rate of 93.5%, a 30 day recurrence rate of 16.9%, and an all-cause mortality rate of 7.6% [START_REF] Facciorusso | Efficacy of hemostatic powders in upper gastrointestinal bleeding: A systematic review and meta-analysis[END_REF]. In our study, the immediate efficacy rate was 79%, the recurrence rate was 39.9%, and 28.4% of patients did not survive at day 30. The plausible reasons for these differences are many.

In our study, 92% of patients had comorbidities, and 42.1% required vasopressive support. The mean hemoglobin level was very low (7 g/dL), 93.4% were transfused, 40.1% were on an antiplatelet and/or anticoagulant medication, and 17% had prior endoscopic hemostatic treatment in the last 30 days. Furthermore, endoscopies were performed in difficult conditions: nondedicated units, overnight or during weekends.

A majority of our patients with bleeding cessation had Hemospray ® as salvage therapy (66.1%).

Amongst the 16 patients with ongoing bleeding, Hemospray ® as primary or salvage therapy did not seem to differ (7 of the 60 patients with Hemospray ® as primary therapy vs. 9 of the 93 patients with Hemospray ® as salvage therapy). We do not believe, based on these results, that Hemospray ® should be specifically used as salvage therapy. In some cases, Hemospray ® would naturally appear as the first and only choice of treatment (i.e. extensive esophagitis, large tumors), and it seems the rate of ongoing bleeding would not be higher. However, given the cost of this treatment, future studies should specifically assess whether or not it is a cost-effective treatment or not, especially given the likely lower efficiency as a primary therapy.

There was no statistical difference between patients with or without bleeding cessation in terms of mortality rate at day 30 (23 vs 33%, p-value: 0.55). This was unsurprising as severely ill patients likely die of other causes, especially those with bleeding cessation, as shown in a previously published study [START_REF] Facciorusso | Efficacy of hemostatic powders in upper gastrointestinal bleeding: A systematic review and meta-analysis[END_REF].

Finally, after other hemostatic techniques had failed, or were judged inappropriate, Hemospray ® yielded an immediate bleeding cessation in 121 patients, 40.5% of which did not require further intervention and had survived at 30 days of follow-up. Given the temporary hemostatic effect of Hemospray ® , these results suggest that this technique can be a positive step in stopping an acute bleeding. However, the rebleeding (41.3%, 50/121) and mortality (22.3%, 27/121) rates were significant in these patients. We believe that when Hemospray ® has been beneficial at first, close monitoring should be implemented to avoid delay of further non-endoscopic hemostatic treatment if needed.

All in all, Hemospray is mostly used as salvage therapy in UGIB, when other techniques have failed, but can also be used as a first line hemostatic treatment when appropriate. Hemospray application should be considered as a temporary treatment, and as being a potential bridge to a non-endoscopic treatment in some cases.

Guidelines recommend the use of Hemospray ® in active non-variceal upper GIB. In this study, Hemospray ® was mainly performed in patients with upper GIB (95.0%). In addition, it was used on active bleeding in 95.4% of cases. Amongst the four patients with non-active bleeding, two had Several limitations of our study can be outlined. First, this study was retrospective. This was a multicenter study adding to the difficulty in collecting data retrospectively, specifically regarding follow-up. Second, no consensus regarding the criteria used for decision-making (procedure indication and per endoscopy treatment) was established prior to the study. However, this study reports real-life use of Hemospray ® during non-working hours, in a setting where physicians do not always have much experience, where potential assistance (i.e. other physicians) is non-existent and where fatigue of the on-call endoscopist could play a significant role in the decision making process.

In conclusion, Hemospray ® application by on-call endoscopists outside regular hours is technically feasible, primarily used for UGIB, mostly secondary to peptic ulcers and malignancy related bleeding, and mainly as a salvage therapy. Its use carries a very low risk of complication. Although recurrence and mortality rates appear to be higher as compared to previously published studies, 40.5% of our patients did not require a further intervention and were alive at day 30 of follow-up, despite being severely ill.

Other information

HIGHLIGHTS

 Real life use of the hemostatic powder Hemospray ® , in the setting of urgent endoscopic procedures performed outside working hours, by mobile on-call endoscopists, has yet to be described.

 The use of Hemospray ® by our group was stable after becoming available and did not increase with the endoscopists experience.

 Hemospray ® was mostly used for upper GIB (95.0%), nearly half of which were due to PUD (47.7%).

 Hemospray ® was used as salvage therapy in 60.8% of cases. During follow up, 39.9% had a recurrence. At 30 days of follow-up, the mortality rate was 28.4%.

 40.5% of our patients did not require a further intervention and were alive at day 30 of follow-up, despite being severely ill.

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Table 1 :

 12341 Figure 1: Endoscopic portable equipment

Table 2 : 15 *

 215 Endoscopic management PUD: peptic ulcer disease; GAVE: Gastric antral vascular ectasia; GEJ: gastro-esophageal junction * GAVE, angioectasia ** endoscopic resection, biopsy, sphincterotomy *** salvage therapy was defined as the application of Hemospray ® for persistent bleeding after failure of other hemostatic modalities either during the same endoscopic procedure or after failure of previous endoscopic attempts **** argon plasma coagulation, and bipolar electrocautery Table 3: Patients follow-up EGD: esogastroduodenoscopy, ERCP: endoscopic retrograde cholangiopancreatography * argon plasma coagulation, and bipolar electrocautery Results Participants 13* (a) Report numbers of individuals at each stage of study-eg numbers potentially eligible, examined for eligibility, confirmed eligible, included in the study, completing follow-up, and analysed (b) Give reasons for non-participation at each stage (c) Consider use of a flow diagram Descriptive data 14* (a) Give characteristics of study participants (eg demographic, clinical, social) and information on exposures and potential confounders (b) Indicate number of participants with missing data for each variable of interest (c) Cohort study-Summarise follow-up time (eg, average and total amount) Outcome data Cohort study-Report numbers of outcome events or summary measures over time Case-control study-Report numbers in each exposure category, or summary measures of exposure Cross-sectional study-Report numbers of outcome events or summary measures Main results 16 (a) Give unadjusted estimates and, if applicable, confounder-adjusted estimates and their precision (eg, 95% confidence interval). Make clear which confounders were adjusted for and why they were included (b) Report category boundaries when continuous variables were categorized (c) If relevant, consider translating estimates of relative risk into absolute risk for a meaningful time period Other analyses 17 Report other analyses done-eg analyses of subgroups and interactions, and sensitivity analyses Discussion Key results 18 Summarise key results with reference to study objectives Limitations 19 Discuss limitations of the study, taking into account sources of potential bias or imprecision. Discuss both direction and magnitude of any potential bias Interpretation 20 Give a cautious overall interpretation of results considering objectives, limitations, multiplicity of analyses, results from similar studies, and other relevant evidence Generalisability 21 Discuss the generalisability (external validity) of the study results

  Funding 22 Give the source of funding and the role of the funders for the present study and, if applicable, for the original study on which the present article is based *Give information separately for cases and controls in case-control studies and, if applicable, for exposed and unexposed groups in cohort and cross-sectional studies. An Explanation and Elaboration article discusses each checklist item and gives methodological background and published examples of transparent reporting. The STROBE checklist is best used in conjunction with this article (freely available on the Web sites of PLoS Medicine at http://www.plosmedicine.org/, Annals of Internal Medicine at http://www.annals.org/, and Epidemiology at http://www.epidem.com/). Information on the STROBE Initiative is available at www.strobe-statement.org.

Note:

a post-sphincterotomy bleeding, difficult to treat given the lack of proper equipment available here.

In the two other cases (gastric ulcerations with minor stigmata of recent hemorrhage), using Hemospray ® was not appropriate. Studies have described the use of Hemospray ® in various causes of lower GIB, with high rates of immediate hemostasis [START_REF] Chen | Hemostatic Powders in Gastrointestinal Bleeding: A Systematic Review[END_REF][START_REF] Holster | Hemospray treatment is effective for lower gastrointestinal bleeding[END_REF][START_REF] Ng | Topical haemostatic powder as a novel endoscopic therapy for severe colonic diverticular bleeding[END_REF]. Interesting results have been published in severe diverticular bleeding with immediate hemostasis in a series of ten patients, with no recurrence [START_REF] Ng | Topical haemostatic powder as a novel endoscopic therapy for severe colonic diverticular bleeding[END_REF]. In our experience, Hemospray ® was used for lower GIB (malignancy, varices, ulcer, ischemia) in nine patients. However, there is insufficient data to suggest its routine use in this setting.

In previously published studies, Hemospray ® was mainly used in GIB secondary to peptic ulcers, malignancies and post-endoscopy [START_REF] Hagel | The Application of Hemospray in Gastrointestinal Bleeding during Emergency Endoscopy[END_REF][START_REF] Haddara | A novel hemostatic powder for upper gastrointestinal bleeding: a multicenter study (the "GRAPHE" registry)[END_REF]. Our study shows a different clinical practice. Although Hemospray ® was used first in PUD and second in malignancy cases, a significant portion of applications were for esophagitis and post-endoscopy bleeding. In addition, it was used in five patients for esophageal variceal bleeding. Several studies have reported on the use of Hemospray ® in variceal GIB. In a review dating from 2015, hemostasis was achieved in 100% of nine cases of variceal GIB [START_REF] Chen | Hemostatic Powders in Gastrointestinal Bleeding: A Systematic Review[END_REF]. A randomized controlled trial including 86 patients compared early hemostatic powder application followed by early elective endoscopy to early elective endoscopy only. The rate of rescue endoscopy was significantly lower in the hemostatic powder group (12% vs 30%, p=0.034) [START_REF] Ibrahim | Early application of haemostatic powder added to standard management for oesophagogastric variceal bleeding: a randomised trial[END_REF] and the survival was higher (30% vs 7% p=0.006). Finally, in our cohort, Hemospray ® was used once in a case of an eso-aortic fistula, which was not known at the time.

Complications of Hemospray ® are infrequent. We report one case of perforation. The likelihood of Hemospray ® application being the cause of this perforation is difficult to establish but probably low. This is consistent with previously published data and concurs with the safe profile of Hemospray ® . In the study by Alzoubaidi et al., there were no complications [START_REF] Alzoubaidi | Outcomes from an International Multicentre Registry of patients with acute gastrointestinal bleeding undergoing endoscopic treatment with Hemospray[END_REF]. In the review by Chen et al. including 243 cases, 5 complications were reported [START_REF] Chen | Hemostatic Powders in Gastrointestinal Bleeding: A Systematic Review[END_REF]: pain, splenic infarct (unclear if related), transient biliary obstruction (post sphincterotomy bleeding), and hemoperitoneum (unclear if related). In theory, there is a risk of vascular embolization, bowel perforation, and bowel obstruction. Embolization has however never been described. Caution is warranted in case of a thin wall at risk for perforation, and near the ampulla because of the risk of biliary obstruction.

Conflict of interest statement