Heiner Kuhl
email: kuhl@igb-berlin.de

Yann Guiguen
email: yann.guiguen@inrae.fr

Christin Höhne
email: hoehne@igb-berlin.de

Eva Kreuz
email: kreuz@igb-berlin.de

Du Kang

Christophe C Klopp
email: christophe.klopp@inrae.fr

Celine Lopez-Roques
email: celine.lopez-roques@inrae.fr

Elena Santidrian
email: santidrian@futuregenomics.tech

Mitica Ciorpac
email: ciorpac.mitica@gmail.com

Jörn Gessner

Kang Du

Céline Lopez-Roques

Elena Santidrian Yebra-Pimentel

Daniela Holostenco
email: dana.holostenco@gmail.com

Wibke Kleiner
email: wibke.kleiner@igb-berlin.de

Klaus Kohlmann
email: kohlmann@igb-berlin.de

Dunja K Lamatsch
email: dunja.lamatsch@uibk.ac.at

Dmitry Prokopov
email: dprokopov@mcb.nsc.ru

Anastasia Bestin
email: anastasia.bestin@inrae.fr

Emmanuel Bonpunt
email: emmanuelb@caviar-perlita.com

Bastien Debeuf
email: b.debeuf@kaviar.com

Pierrick Haffray
email: pierrick.haffray@inrae.fr

Romain Morvezen
email: romain.morvezen@inrae.fr

Pierre Patrice
email: pierre.patrice@inrae.fr

Radu Suciu
email: radu.suciu34@gmail.com

Ron Dirks
email: dirks@futuregenomics.tech

Sven Wuertz
email: wuertz@igb-berlin.de

Werner Kloas
email: werner.kloas@igb-berlin.de

Manfred Schartl

Matthias Stöck
email: matthias.stoeck@igb-berlin.de

A 180 My-old female-specific genome region in sturgeon reveals the oldest known vertebrate sex determining system with undifferentiated sex chromosomes

Keywords: Acipenseridae, sturgeon, sex chromosomes, female-specific, polyploidy, evolution

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

Introduction

Sexual reproduction is an old feature of life. In vertebrates, sexual development is determined by environmental triggers (Environmental Sex determination, ESD) or genotypic sex determination (GSD) (e.g. [START_REF] Beukeboom | The evolution of sex determination[END_REF]) or a combination thereof. In an existing GSD-system, the paradigmatic evolutionary model of sex chromosome evolution suggests the rise of a new sex chromosome from an autosome by mutation or translocation of a gene, which becomes a trigger of the sex-determining regulatory networks. Heterozygotes encode one sex and homozygotes the other [START_REF] Ohno | Sex chromosomes and sex-linked genes[END_REF][START_REF] Rice | Evolution of the Y sex chromosome in animals[END_REF][START_REF] Charlesworth | The degeneration of Y chromosomes[END_REF]. According to this classical view, mutations and/or linkage to genes with sexually antagonistic effects may be favoured near this gene, i.e. mainly in unpaired parts of Y and W chromosomes, profiting from linkage disequilibrium. To preserve epistasis, recombination between such mutations and the sex-determining locus would then be suppressed in the heterogametic sex [START_REF] Bull | Evolution of sex determining mechanisms[END_REF][START_REF] Rice | The accumulation of sexually antagonistic genes as selective agent promoting the evolution of reduced recombination between primitive sex chromosomes[END_REF]. Such nonrecombining sex chromosomes are expected to degenerate through the progressive accumulation of deleterious mutations [START_REF] Charlesworth | The degeneration of Y chromosomes[END_REF][START_REF] Charlesworth | The timing of genetic degeneration of sex chromosomes[END_REF], except for small pseudo-autosomal regions. Recently, as an alternative or additional cause of degeneration, instability and divergence of cis-regulatory sequences in non-recombining genome regions, which become selectively haploidised to mask deleterious mutations on coding sequences, have been suggested [START_REF] Lenormand | Sex chromosome degeneration by regulatory evolution[END_REF]. In contrast to the conservation of differentiated sex chromosomes in most mammals and birds, in the majority of poikilothermic vertebrates differentiated sex chromosomes are rarely observed [START_REF] Kikuchi | Novel sex-determining genes in fish and sex chromosome evolution[END_REF][START_REF] Eggert | Sex determination: the amphibian model[END_REF][START_REF] Schartl | Dynamics of vertebrate sex chromosome evolution: from equal size to giants and dwarfs[END_REF] (but see [START_REF] Schartl | Sex chromosome evolution in non-mammalian vertebrates[END_REF]). Several hypotheses have been offered to explain the prevalence of undifferentiated sex chromosomes. The high-turnover model proposes that the emergence of new master sex-determining genes on new chromosomes would prevent sex chromosome degeneration [START_REF] Gamble | Restriction site-associated DNA sequencing. (RAD-seq) reveals an extraordinary number of transitions among gecko sex-determining systems[END_REF][START_REF] Bachtrog | Sex determination: why so many ways of doing it?[END_REF][START_REF] Sarre | Transitions between sex-determining systems in reptiles and amphibians[END_REF][START_REF] Jeffries | A rapid rate of sex-chromosome turnover and nonrandom transitions in true frogs[END_REF]. The "jumping sex locus"-hypothesis [START_REF] Woram | Comparative genome analysis of the primary sex-determining locus in salmonid fishes[END_REF] implies that the sex determination gene is maintained but changes its position to different chromosomes, preventing evolutionary decay of the original sex chromosome [START_REF] Yano | The sexually dimorphic on the Ychromosome gene. (sdY) is a conserved male-specific Y-chromosome sequence in many salmonids[END_REF]. The 'fountain of youth' hypothesis [START_REF] Perrin | Sex reversal: a fountain of youth for sex chromosomes[END_REF] suggests that sex chromosomes may escape from degeneration by occasional recombination Because sex-specific recombination depends on phenotypic rather than genotypic sex, homomorphic X and Y chromosomes might recombine in sex-reversed females. These rare events should generate bursts of new Y haplotypes, which will be quickly sorted out by natural or sexual selection [START_REF] Perrin | Sex reversal: a fountain of youth for sex chromosomes[END_REF]. Seemingly young sex chromosomes may thus carry old-established sexdetermining genes, challenging the opinion that sex chromosomes unavoidably decay [START_REF] Stöck | Ever-young sex-chromosomes in European tree frogs[END_REF][START_REF] Rodrigues | Sex-Chromosome recombination in common frogs brings water to the fountain-of-youth[END_REF]. Beyond mammals and birds, conserved sex chromosomes have recently been discovered in several amniote (specifically reptile) clades [START_REF] Rovatsos | Conservation of sex chromosomes in lacertid lizards[END_REF][START_REF] Iannucci | Conserved sex chromosomes and karyotype evolution in monitor lizards (Varanidae)[END_REF][START_REF] Rovatsos | Cretaceous park of sex determination: sex chromosomes are conserved across iguanas[END_REF][START_REF] Rovatsos | Evolutionary stability of sex chromosomes in snakes[END_REF], all of which feature differentiated sex chromosomes. Evolutionarily very old, conserved and homomorphic ZZ/ZW sex chromosomes are known in some ratite birds (Ratidae), dating back >130 My [START_REF] Vicoso | Sex-biased gene expression at homomorphic sex chromosomes in emus and its implication for sex chromosome evolution[END_REF][START_REF] Yazdi | Old but not (so) degenerated-slow evolution of largely homomorphic sex chromosomes in Ratites[END_REF]. Similarly, skinks (Scincidae) share homologous, mostly poorly differentiated XX/XY sex chromosomes across a wide phylogenetic spectrum for at least 85 million years [START_REF] Kostmann | Poorly differentiated XX/XY sex chromosomes are widely shared across skink radiation[END_REF]. Recent findings in the teleost family Esocidae report undifferentiated sex chromosomes of similar evolutionary age (65-90 My) in teleosts [START_REF] Pan | The rise and fall of the ancient northern pike master sex determining gene[END_REF]. The Salmonidae family, which experienced a whole genome duplication ca. 90 Mya [START_REF] Macqueen | A well-constrained estimate for the timing of the salmonid whole genome duplication reveals major decoupling from species diversification[END_REF], presumably harbours a conserved sex determination gene, perhaps as old as 50 My, on different chromosomes in different species [START_REF] Woram | Comparative genome analysis of the primary sex-determining locus in salmonid fishes[END_REF][START_REF] Yano | The sexually dimorphic on the Ychromosome gene. (sdY) is a conserved male-specific Y-chromosome sequence in many salmonids[END_REF], which encodes a homomorphic XX/XY sex chromosome system. Here we report the discovery of a sex-specific region in sturgeon (Acipenseridae), which is preserved in many extant species with a common ancestor about 180 Mya. Sturgeons and paddlefish comprise 27 living species [START_REF] Hochleithner | The sturgeon and paddlefishes of the world-Biology and Aquaculture[END_REF], which branched off 330 Mya [START_REF] Du | The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization[END_REF], from the root of the more than 31,000 living teleosts. The evolutionary history of vertebrates is characterized by several rounds of whole genome duplications (WGD). The 1R and 2R WGD happened early on, before the jawed vertebrates appeared, but the sturgeon branch diverged before the teleost 3R WGD [START_REF] Meyer | From 2R to 3R: evidence for a fish-specific genome duplication (FSGD)[END_REF][START_REF] Braasch | Polyploidy in fish and the teleost genome duplication[END_REF]. Later on, sturgeon experienced several clade-specific polyploidizations, resulting in chromosome numbers from ~120 up to ~380 [START_REF] Havelka | Extensive genome duplications in sturgeons: new evidence from microsatellite data[END_REF]. Based on a chromosome-scale genome assembly of Acipenser ruthenus, Du et al. [START_REF] Du | The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization[END_REF] have recently shown a WGS, early in the sturgeon radiation. Although subsequent rediploidization of the genome caused the loss of entire chromosomes or large fragments (i.e. segmental deduplication), structural and functional tetraploidy was maintained over 180 million years, and a decrease of redundancy in the tetraploid genome follows mostly random processes. Sturgeons possess undifferentiated microscopically indistinguishable sex chromosomes [START_REF] Du | The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization[END_REF][START_REF] Keyvanshokooh | A review of sex determination and searches for sex-specific markers in sturgeon[END_REF]. Although the sex ratio of offspring from experimental gynogenesis led to the

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assumption that all sturgeons possess female heterogametic (ZZ/ZW) sex chromosome systems [START_REF] Fopp-Bayat | Induction of meiotic gynogenesis in sterlet (Acipenser ruthenus) using UV-irradiated bester sperm[END_REF], a sex-specific marker, confirming female heterogamety and allowing genetic studies, has not been found [START_REF] Du | The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization[END_REF].

Materials and Methods

Animal sampling and establishment of genetic families

Acipenser ruthenus: For pool-sequencing (pool-seq) 61 (31 female, 30 male) adult sterlets from the broodstock at the Leibniz-IGB (Berlin, Germany), representing the Danube River population [START_REF] Du | The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization[END_REF], were sexed by gonadal biopsies, evidence from reproduction and a few autopsies. In addition, 52 sterlets (table 1) were sampled from three aquaculture populations. These came from two other A. ruthenus brood stocks, originating from the Danube (Wöllershof, Bavaria, Germany, 20-25 cm, ca. 1 year old; and Beucha, Saxony, Germany, 10-12 cm, 6 months old), as well as A. ruthenus interpopulation hybrids from the Volga and Danube Rivers (12-20 cm, 8-10 months). They were euthanized and gonadal sex determined histologically, and finclips used for genotyping. In spring 2020, a genetic family was derived from the IGB-broodstock; at the age of 4 months, histological sections of gonads from 14 offspring were prepared, and finclips from them and their parents also used for genetics. Huso huso: Using minimal invasive biopsy samples from the gonads of 28 subadult and adult beluga sturgeon from two fish farms (Horia and Peceneaga, Romania, fish between 10 and 13 years old) were inspected by the unaided eye and/or microscopically after standard hematoxylin/eosin staining (described below). Finclips were sampled for genetic analyses (table 1). To generate genetic families, on April 6, 2018, a beluga sturgeon male (PIT-tag 000004691858; 200 cm, ~100 kg) was captured in the Danube delta region at river-Km 126 (distance from the Black Sea) in Romania. On April 13, 2018, a mature H. huso female (PIT 642099000568452; TL 260 cm, ~150 kg, near Km 100), and another male on April, 14, 2018 (PIT 968000004692054; TL 143 cm, ~22 kg, Km 126) were caught. All three were transferred to an aquaculture company (Horia, Romania). After stimulation of all the three wild-caught animals and one additional captive male with gonadotropinreleasing hormone (GnRH), ovulation started on April 16. Eggs were fertilized with sperm of each male separately. Fin clips of the four adult fish were taken, and the offspring of the individual matings were reared separately until the age of one year. All offspring fish were individually tagged with PIT-tags and then reared in common tanks. For this study, fish of one genetic family were sampled at the age of 15-24 months for gonadal histology and finclips. Wild-caught adults and, after two years, hundreds of their tagged offspring were released into the Danube to minimize the impact on this highly threatened species. A. oxyrinchus: Tissue samples of 27 adult Baltic sturgeon (table 1) from the broodstock in Mecklenburg-Pomerania (Born, Germany), kept for re-introduction into Oder River and Baltic Sea, were taken from tagged males and females, sexed using gonadal biopsies and evidence from reproduction. In 2017, genetic families were derived from breeding pairs during the yearly reproduction. Finclips from the parental animals were kept in ethanol. A subset of juveniles was raised in separate tanks at the Leibniz-IGB (Berlin, Germany) for three years. A. sturio: Tissue samples of 36 adult European sturgeon (table 1) from a broodstock of immature fish at the IGB, raised for conservation aquaculture to re-introduce the species into the Elbe River were initially sexed using ultrasonic evidence. Gonadal biopsies were taken if genotyping did not match the ultrasonic evidence. Few specimens came from autopsy of deceased fish. A. baerii: Finclips of 56 adult Siberian sturgeon (table 1) used as breeders with known phenotypic sex were sampled in an aquaculture breeding company (l'Esturgeonnière, France) and kept in 100% ethanol until DNA extraction. A. gueldenstaedtii: Finclips of 27 adult Russian sturgeons (table 1) were sampled in an aquaculture breeding company (Ecloserie de Guyenne, SCEA Sturgeon, France) and kept in 100% ethanol until DNA extraction. All fish were sexed based on evidence from ultrasound, reproduction and autopsy.

DNA processing for genotyping DNA from finclips or other tissue samples was extracted using the DNeasy Tissue Kit (Qiagen) or the BioSprint robotic workstation with the 96 DNA Plant Kit (Qiagen, Germany), according to the manufacturer's protocols.

Histological sexing of juvenile and subadult fish as well as offspring of genetic families

Offspring from genetic families of H. huso, A. ruthenus, and A. oxyrinchus was sacrificed by an overdose of buffered tricaine methanesulfonate (MS222; 0.3 g/L, PHARMAQ), individual length and wet weight were recorded, finclips (stored in 100% ethanol) and gonadal samples were taken immediately. The right gonad strand was fixed in phosphate-buffered formaldehyde solution (ROTI®Histofix, 4%, Carl Roth) for at least 24 h at room temperature, then rinsed three times for 24 h with 70% ethanol, and stored at room temperature. Gonad strands were dehydrated in an increasing series of ethanol (75%, 90%, 95%, 100%), rinsed in xylol (Carl Roth), and transferred into Paraplast® (Leica), using the Shandon Excelsior ES Tissue Processor (Thermo Fisher Scientific). Gonads were embedded in a spiral-like arrangement, to ideally cut the whole organ into 5 µm sections across its entire length (microtomes: Leica 2065 or Microm HM 340E, Thermo Fisher Scientific). Sections were mounted on glass slides, and stained using standard hematoxylin/eosin (0.1%, Carl Roth). Histological evaluation was made at various magnifications using light microscope Nikon Eclipse Ni-U with Nikon DS-Fi3 camera, and the corresponding software Nikon DS-L4 to archive images.

F o r R e v i e w O n l y

Pool-sequencing

The genomic DNA of 31 female and 30 male A. ruthenus specimens was extracted from 90% ethanol-preserved fin clips using a classical phenol/chloroform protocol, quantified using Qubit fluorimetry and analysed using the Fragment Analyzer (Advanced Analytical Technologies, Inc., Iowa, USA). DNA shorts read sequencing was performed at the GeT-PlaGe core facility, INRAe Toulouse (http://www.get.genotoul.fr). Two DNA pool-seq libraries were prepared according to manufacturer's protocols using the Illumina TruSeq Nano DNA HT Library Prep Kit (Illumina, California, USA). Briefly, from 200 ng of each sample, DNA was fragmented (550 bp) by sonication on a M220 Focused-ultrasonicator (COVARIS). Size selection was performed using SPB beads (kit beads) and 3'-ends of the blunt fragments were mono-adenylated. Then, adaptors and indexes were ligated and the construction amplified with Illumina-specific primers (8 cycles). Library-quality was assessed using Fragment Analyzer and libraries were quantified by qPCR using the Kapa Library Quantification Kit (Roche). Sequencing was performed on a NovaSeq S4 lane (Illumina, California, USA), using a paired-end read length of 2x150 bp with the Illumina NovaSeq Reagent Kits.

Analysis of sex specific variants based on a male reference genome

Pool-seq data were mapped to the male A. ruthenus reference genome (Acipenser_ruthenus.ARUT1.2.dna.toplevel.fa, which is nearly identical to the current reference genome at NCBI GCF_010645085.1; electronic supplementary material, table S1) by Minimap2 (with parameters -t 12 -x sr -a; [START_REF] Li | Minimap2: pairwise alignment for nucleotide sequences[END_REF]) and converted to sorted bam-files by samtools [START_REF] Li | 1000 genome project data processing subgroup, the sequence alignment/map format and SAMtools[END_REF]. Variants (SNPs, MNPs, INDELs) were called from both bam files using the Platypus variant caller [START_REF] Rimmer | Integrating mapping-, assembly-and haplotype-based approaches for calling variants in clinical sequencing applications[END_REF] with integrated duplicate fragment reads removal (--filterDuplicates=1) and reassembly of reads (--assembleAll=1). The resulting vcf-file was screened for sex-specific variants using awk-scripting and different variant read coverage cutoffs (i.e. maximum reads in males (0, 1 …), minimum reads in females (10, 13 …) or vice versa). Variants were clustered according to their distance in the genome (maximal distance of variants in a cluster: 4000 bp), counted using bedtools (merge, annotate; [START_REF] Quinlan | BEDTools: a flexible suite of utilities for comparing genomic features[END_REF]) and sorted by variant count. Regions with highest variant densities and neighbouring regions were inspected for genes related to sex determination.

Reconstruction of male and female specific genomic regions from unmapped pool-seq data

Unmapped reads potentially representing regions not present in the reference genome were extracted from bam-files and assembled using the IDBA assembler [START_REF] Peng | IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth[END_REF]. The resulting male/female specific contigs were added to the reference genome and pool-seq data was mapped again (as described above) to this extended reference genome. Finally, sequencing coverage by female and male pools was compared in bedgraph files derived from the bam-files (bedtools genomecov -bga -splitibam … ; bedtools unionbedgraph). These were screened for putative haploid coverage in one sex and no or low, spurious coverage in the other sex to identify female/male specific regions. Another high-quality assembly of a female A. ruthenus has recently been published at NCBI by the Vertebrate Genomes Project (GCA_902713425.1). We repeated our analyses from above using this reference with an emphasis on detection of coverage differences. Using bedgraph-files we compared female and male pool-sequencing coverage at the bp-level along the genome. We used different filtering approaches to call regions which had no or low coverage in one sex and approximately haploid coverage in the other sex. The most successful filtering applied awk-scripting to filter regions in male and female genomes where 0 coverage in male pool and a coverage of larger than 0.3 in the female pool was observed (here 1.0 was the normalized diploid coverage). These regions were combined in larger blocks, if they were as close as 30 bp to each other (bedtools merge). Finally, we filtered for blocks that were larger than 160 bp and derived at a clear signal present only in female data and female genome.

Primer design and PCR conditions

Using the male vs. female A. ruthenus genome sequence as well as preliminary Illumina-data of a female A. oxyrinchus genome, we designed a number of primers, which were primarily tested in gradient-PCRs on phenotypically sexed A. ruthenus. While several primers showed sex-specific amplification in this species, one (AllWSex2) could be optimized to work across multiple species (primer sequences and PCR-conditions: electronic supplementary material, texts S1-S3).

Phylogenomic reconstruction

To build a phylogenetic tree for sturgeons, we download transcriptome assemblies of Acipenser baerii, A. oxyrinchus, A. schrencki, A. sinensis and A. transmontanus from the Public Sturgeon Transcripts Database (http://publicsturgeon.sigenae.org/home.html) and of Acipenser gueldenstaedtii from NCBI (https://www.ncbi.nlm.nih.gov/Traces/wgs/?page=1&view=tsa&search=acipenser). The transcriptome of Huso huso was de novo assembled using Trinity [START_REF] Haas | De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis[END_REF] from mixed organs. Annotated genomes of A. ruthenus and Latimeria chalumnae were also downloaded from NCBI (electronic supplementary material, table S2). To identify orthologs between two species, we used the "Reciprocal best hit (RBH)" method, and A. ruthenus as the hub to retrieve orthologous genes across all species. Protein sequences of the transcriptome contigs were predicted using GeneWise [START_REF] Birney | GeneWise and genomewise[END_REF] by aligning each contig to its orthologous protein in A. ruthenus. Contigs with less than 60% of the protein aligned were discarded. In total, we collected 1017 genes with orthology across all species. For each orthologous gene, the protein sequences were aligned across all species using MAFFT [START_REF] Nakamura | Parallelization of MAFFT for large-scale multiple sequence alignments[END_REF], and trimmed them, using trimAl [START_REF] Capella-Gutiérrez | trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses[END_REF]. All 1017 alignments were then combined in a concatenated alignment, consisting of 383506 aligned amino acid sites. Finally, this alignment was transferred to RAxML [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF] to construct a Maximum Likelihood-phylogeny.

Results

Analyses of sterlet (A. ruthenus) female and male pool-sequencing

Pool-sequencing resulted in 553,117,774 reads (83.5 Gbp) and 486,762,084 reads (73.5 Gb) for the female and the male A. ruthenus pools, respectively. Pool-sequencing Illumina reads are available in the Sequence Read Archive (SRA: SRX9341540; SRX9341541), under BioProject reference PRJNA670610. After mapping to the reference genomes, the peak of the unimodal coverage distribution was 42x for the female pool and 37x for the male pool. There was no significant difference in the percentage of mapped reads for both pools (96.9% reads mapped). We initially used two bioinformatic strategies to uncover female-specific pool-seq data in A. ruthenus. We first analysed sex-specific variants based on a male reference genome. The number of female or male specific variants was low, but their ratio already hinted at a ZW sex determination system (details: electronic supplementary material, text S4). In the top candidate region, which harboured 27 female-specific variants at HiC_scaffold_4:45,149,338-45,165,057 (electronic supplementary material, figure S1, table S1, text S5), we found a female-specific 15 bp-indel, which we used to design PCR-primers. These amplified a product concordant with phenotypic sex of some A. ruthenus, but not all (data not shown). We also analysed male-or female-specific pool-seq reads, not matching the male reference genome, and independently assembled these unmapped reads from the male/female pools. This resulted in 1,067 additional female-specific contigs spanning 658,732 bp. However, BlastX-searches using these contigs did not detect any sequences related to known sex determination genes.

Identification of a female-specific locus in A. ruthenus by coverage analysis using pool-seq data and a female reference genome

Due to a possible high repeat content of a W-locus, short-read assemblies might not be able to resolve it in large contigs but in rather fragmented small contigs, hindering further analysis. Recently, a long-read female haplotype reference of A. ruthenus has become available (GCA_902713425.1), which allowed remapping our pool-seq data and focusing on regions with different coverage between males and females. With stringent filtering parameters (no coverage in males; a minimum of 30% diploid coverage in females and minimum size of regions of 160 bp), we found 11 neighbouring matches (summing up to 2705 bp) in the female genome, while we did only find a single spurious match (185 bp) in the male genome. Vice versa, filtering for zero coverage in females and 30% of diploid coverage in males, also resulted in no hits (female genome) or two spurious hits (175 and 181 bp; male genome) The filtered regions in the female genome fell into the region CACTIG010000179.1:61,229,779-61,285,849 (including 20 kb up-and down-stream). This region corresponds to the region HiC_scaffold_4:43,938,061-43,987,561 in the male assembly as shown by the alignments of the 20 Kbp up-and down-stream sequences. Figure 1 illustrates the female and the male pool-seq coverage of the identified locus. The female specific locus is located 1.162 Mb upstream of the identified cluster of 27 female-specific variants, mentioned above. Furthermore, a single contig (559 bp) of the 1,067 female-specific contigs assembled from the female pool-seq data, did match the identified locus. Thus, three independent analyses of the pool-seq data, specifically sequence variants, assembled female-specific reads and differential sequence coverage of the female genome assembly, support HiC_scaffold_4 as a sex chromosomal sequence, albeit with different specificity and resolution.

Establishment of a W-linked PCR across the sturgeon species tree

Our PCR-marker AllWSex2 revealed female-specific products for all unambiguously phenotypically sexed females under identical PCR-conditions in six sturgeon species (table 1), spanning the entire phylogenetic tree and the most divergent extant lineages (A. sturio, A. oxyrinchus vs. A. ruthenus, H. huso), all of which are tetraploid (~120 chromosomes). The marker showed also sex-linkage in two octoploid species (A. gueldenstaedtii and A. baerii; table 1, figure 2 and figure 3). Table 1. Numbers of individuals tested with the sex-linked PCR marker AllWSex2 (p-values for association with sex were calculated using a Pearson's Chi-squared test with Yates' continuity correction; gel pictures in electronic supplementary material, figure S3 to figure S10).

Heredity of the sex-linked marker in three sturgeon species

In three genetic families, consisting of mother, father and their histologically sexed offspring, of A. oxyrinchus, A. ruthenus and H. huso, representing 180 My of sturgeon evolution, the marker AllWSex2 was exclusively present in females, demonstrating mother to daughter transmission (gel pictures and histology in electronic supplementary material, figure S8 to figure S16).

Discussion

We have discovered a female-specific (W) region, consistent with a potential ZZ/ZW system in sturgeon. This finding is in accordance with previous assumptions or indirect evidence that sturgeons exhibit a female-heterogametic sex determination system [START_REF] Keyvanshokooh | A review of sex determination and searches for sex-specific markers in sturgeon[END_REF][START_REF] Havelka | Hybridization and polyploidization in sturgeon[END_REF][START_REF] Fajkowska | Review: Molecular mechanisms of sex differentiation in sturgeons[END_REF]. This genomic region is conserved in at least six sturgeon species, spanning the entire phylogenetic tree, represented by the most divergent species A. sturio, A. oxyrinchus A. ruthenus, and H. huso, all of which

F o r R e v i e w O n l y
are formally ancient tetraploid (~120 chromosomes) [START_REF] Du | The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization[END_REF]. In the A. ruthenus genome, the sequence is residing on chromosome 4 [START_REF] Du | The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization[END_REF]. Of note, this genomic region is also sex-linked in two octoploid species (A. gueldenstaedtii with 258±4 chromosomes, and A. baerii with 249±5 chromosomes), demonstrating maintenance of this region and apparently a female heterogametic sex determination system through at least one round of polyploidization. In the future, conservation and evolution of the region in the remaining extant sturgeons should be examined to evaluate the degree to which this ancient female-specific genomic region is also conserved in other species. In particular, identification of the master sex determining gene(s) or other regulatory elements are required. At the current state of research, we also cannot rule out a situation like in Salmonidae, where a "jumping sex locus", perhaps as old as 90 My [START_REF] Macqueen | A well-constrained estimate for the timing of the salmonid whole genome duplication reveals major decoupling from species diversification[END_REF], resides on different chromosomes in different species, all of which share a male heterogametic (XX/XY) system [START_REF] Woram | Comparative genome analysis of the primary sex-determining locus in salmonid fishes[END_REF][START_REF] Yano | The sexually dimorphic on the Ychromosome gene. (sdY) is a conserved male-specific Y-chromosome sequence in many salmonids[END_REF]. Our data reveal the oldest known vertebrate sex determining system with undifferentiated sex chromosomes. Conservation of a female-specific sequence for 180 My of sturgeon evolution, and across at least one additional polyploidization event, raises many interesting biological and evolutionary questions. We hypothesize that this region might be part of the sex locus. Given the slow protein evolution of the sturgeon genome, evolving distinctly slower than in teleosts, including basal species such as arowana and arapaima, but also slower than gar, coelacanth or elephant shark [START_REF] Du | The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization[END_REF], we speculate that the relatively conserved karyotypes of the ancient tetraploid species examined here (A. sturio, A. oxyrinchus, A. ruthenus, H. huso) may have even maintained the same undifferentiated sex chromosome for 180 My. It will be fascinating to examine the mechanisms, which stabilized the sex determination system and whether or not this involves undifferentiated homologues sex chromosomes. Results from previous RADseq [START_REF] Du | The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization[END_REF] and pool-sequencing of the same A. ruthenus (sampled from aquaculture populations, and with few if any family-effects expected), revealing 99.98 % of the chromosome as largely undifferentiated, let us assume that the sturgeon sex chromosome contains a large pseudo-autosomal region (PAR; [START_REF] Otto | About PAR: The distinct evolutionary dynamics of the pseudoautosomal region[END_REF][START_REF] Bachtrog | Are all sex chromosomes created equal?[END_REF]; electronic supplementary material, figure S1). This and the large chromosome number (~120 to ~260 for the 6 species) make it challenging to apply SNPs or microsatellites to examine whether AllWSex2 resides on the same or different linkage groups and therefore is beyond the framework of this study, despite we have generated genetic families from three species. The disruption of sex determination in gonochoristic animals after genome duplication is considered the main cause for the relatively rare occurrence of polyploid animals compared to plants [START_REF] Muller | Why polyploidy is rarer in animals than in plants[END_REF][START_REF] Orr | Why polyploidy is rarer in animals than in plants' revisited[END_REF]. In vertebrates, in which autotetraploidy may be rarer than (hybrid-origin) allopolyploidy [START_REF]Why polyploidy is rarer in animals than in plants': myths and mechanisms[END_REF], polyploidy occurs especially frequently in amphibians [START_REF] Schmid | Polyploidy in Amphibia[END_REF], with few of which having their mostly undifferentiated sex chromosome characterized. In clawed frogs (Xenopus), alloploidy ranges from diploid to do-decaploid (12n) and subgenome evolution in allopolyploids has only recently been studied [START_REF] Session | Genome evolution in the allotetraploid frog Xenopus laevis[END_REF][START_REF] Furman | Divergent subgenome evolution after allopolyploidization in African clawed frogs (Xenopus)[END_REF]. In X. laevis, the female-determining gene DM-W is a paralog of DMRT1 [START_REF] Yoshimoto | A ZZ/ZW-type sex determination in Xenopus laevis[END_REF][START_REF] Ito | Sex determination and differentiation in frogs[END_REF], and arose after (and perhaps in response to) tetraploidization [START_REF] Bewick | Evolution of the closely related, sex-related genes DM-W and DMRT1 in African clawed frogs (Xenopus)[END_REF][START_REF] Furman | Sequential turnovers of sex chromosomes in African clawed frogs (Xenopus) suggest some genomic regions are good at sex determination[END_REF][START_REF] Mawaribuchi | Sex chromosome differentiation and the W-and Z-specific loci in Xenopus laevis[END_REF]. However, it is also found in some related Xenopus [START_REF] Bewick | Evolution of the closely related, sex-related genes DM-W and DMRT1 in African clawed frogs (Xenopus)[END_REF][START_REF] Furman | Sequential turnovers of sex chromosomes in African clawed frogs (Xenopus) suggest some genomic regions are good at sex determination[END_REF] but not in the entire radiation [START_REF] Furman | Divergent evolutionary trajectories of two young, homomorphic, and closely related sex chromosome systems[END_REF][START_REF] Song | in press) Sex chromosome degeneration, turnover, and sex-biased expression of sex-linked transcripts in African clawed frogs (Xenopus)[END_REF]. The clade-wide conservation of a male heterogametic sex determination gene and system (but not chromosome) in the 50 My-old, ancient tetraploid family Salmonidae (see above) curiously also evolved in a system with a common ancestral polyploidization event, as suggested here for Acipenseridae, with apparently conserved female heterogamety. Our finding is too recent to allow comprehensive interpretation. Whole genome approaches are underway to address several of the most pressing questions, such as conservation of the region or the entire sex chromosomes in sturgeons. Conservation of the latter would even stronger challenge the classical paradigm of sex chromosome evolution [START_REF] Muller | A gene for the fourth chromosome of Drosophila[END_REF][START_REF] Muller | The differentiation of the sex chromosomes of Drosophila into genetically active and inert regions[END_REF][START_REF] Wright | How to make a sex chromosome?[END_REF] than the 180 Mylong conservation of this small sex-linked sequence.

Reasons for the challenges to identify a female-specific genomic region in sturgeon

The search for a sex chromosome or sex-linked markers in sturgeons has not been successful for a long time [START_REF] Keyvanshokooh | A review of sex determination and searches for sex-specific markers in sturgeon[END_REF][START_REF] Havelka | Hybridization and polyploidization in sturgeon[END_REF][START_REF] Fajkowska | Review: Molecular mechanisms of sex differentiation in sturgeons[END_REF]. We discuss four major challenges, why this has been the case. First, in A. ruthenus, the identified W-specific sequence is very short and resides on a large undifferentiated chromosome. It comprises only ~16 kb, corresponding to ~0.001% of the genome or ~0.02% of the corresponding chromosome (~106 Mb). It is impossible to detect such a small structural difference by karyotype imaging methods unless the W sequence is known and FISH probes can be designed. Secondly, the high repeat content of the W-specific locus (~43%; www.repeatmasker.org) complicates its assembly by short-read data alone. Thus, when reconstructing female-specific genomic regions from unmapped pool-seq data, we filtered female-specific reads and independently assembled them. Unfortunately, the resulting small contigs were of limited utility for female-specific primer design. Looking for an explanation, we mapped these contigs back to the female genome assembly and found that only 0.09% of the sequence (a single contig) matched the W region, which we have identified by a sequencing coverage analysis. Thirdly, short-read-based methods that call and analyse variants between female and male sequencing data suffered from the low number of sex-specific variants in sturgeons (few hundreds to thousands depending on filter parameters), especially when reduced representation methods like RADseq are used [START_REF] Du | The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization[END_REF]. Even our whole genome pool-sequencing approach was not able to define the W-specific region by SNP markers. This approach only uncovered a nearby region (distance 1.16 Mb, ~1% of chromosome length); a result, which could point to reduced recombination around the W-specific sequence (Wlinked SNPs). Finally, analysis of sequencing coverage enabled identification of the W-specific locus in A. ruthenus, but detection of haploid/diploid coverage differences were still complicated due to high variability (electronic supplementary material, figure S2), causing noisy signals for haploid genomic regions. Completely deleted regions in one sex compared to the other provided significant signals, but rely on the availability of a high-quality genome assembly of the heterogametic sex (figure 1).

Potential application of the W-specific marker for conservation measures, aquaculture production and ethical issues

F o r R e v i e w O n l y
Beyond the basic biological interest in this ancient sex determination mechanism, molecular sexing of sturgeons is of great relevance in studies on wild sturgeon populations, their conservation and conservation aquaculture as well as caviarproducing aquaculture. Currently, ultrasonic diagnosis or biopsies are used for sexing. For ultrasonic sexing to become reliable, sturgeons have to reach progressed stages of maturation, six to ten years in some species, while biopsies are invasive and stress or even harm the fish [START_REF] Chebanov | Sturgeon hatchery practices and management for release -guidelines[END_REF]. Our molecular marker's reliability outperforms methods like early biopsies or ultrasound-sexing. Future application of cotton skin swabs will make molecular sexing much less stressful for the fish. It will also aid conservation by reducing time and efforts to rear fish intended as future broodstock in ex situ-programs, reducing costs and improving the selection of candidates for living gene banks, not only based on rare alleles but also on sex. In this case, fish not selected for broodstock development can be released in the frame of recovery programs. In addition, in the caviar aquaculture, females, which are commercially more attractive, can be selected early on, and differentiated rearing for caviar (females) and meat (males) production becomes feasible. Releases of commercially reared fish are no option in conservation programs since they show restricted fitness due to intensive aquaculture [START_REF] Gessner | Summary report on the major topics addressed at ISS 8 (8th International Symposium on Sturgeons)[END_REF]. In any case, we strongly emphasize that the early selection of females must not lead to littering male sturgeons, since their meat is a valuable source of protein [START_REF] Bronzi | Sturgeon meat and caviar production: Global update to 2017[END_REF]. Detection of a female-specific locus in Acipenser ruthenus, when mapping female/male pool-sequencing reads to a female (ZW) reference genome assembly. Genome-wide signal detection was highly specific, when screening for larger regions (here >= 160 bp) with zero and non-zero coverage between sexes. The red block and arrow show the position of the AllWSex2-PCR-marker (CACTIG010000179.1: 61246236 -61246344), which distinguishes sex in six sturgeon species. The green block and arrow mark the location of the only contig matching the region (CACTIG010000179.1: 61261166 -61261703), when using the female-specific pool-seq read assembly approach. For final submissions, figures should be uploaded as separate, high resolution, figure files. The approximate position of A. sturio, for which reliable transcriptomic data were unavailable, was added according to www.timetree.org. The root of Acipenseridae was dated at ~180 Mya, according to [START_REF] Du | The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization[END_REF]. Red arrows mark those species, for we have obtained female-specific PCR-products, using primers AllWSex2_F/_R.

Permits and Licences

Electronic Supplementary material

Submitted in separate file: texts S1-S5, tables S1-S2, and figures S1-S16.

FiguresFigure 1 .

 1 Figures

Figure 2 .

 2 Figure 2. PCR products using the AllWSex2 marker in six species across the sturgeon phylogeny under identical PCRconditions (electronic supplementary material, texts S1 -S3) for two females and two males of each species. PCR products migrated ca. 45 min at 70 V on a 2% agarose gel in 1x TAE-buffer. Symbols and abbreviations: ♀: female; ♂: male; B: Blank, i.e. no DNA template in PCR; L: ladder, 100 bp-size-marker (Quantitas, Biozym).

Figure 3 .

 3 Figure 3. Phylogenomic tree of sturgeon species built by using RAxML with Maximum Likelihood (details: Materials and Methods). Numbers at nodes refer to bootstrap support values. The bar above indicates branch length of sturgeon lineages, while the bar below indicates branch length of the outgroup lineage coelacanth (Latimeria chalumnae). The approximate position of A. sturio, for which reliable transcriptomic data were unavailable, was added according to www.timetree.org. The root of Acipenseridae was dated at ~180 Mya, according to[START_REF] Du | The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization[END_REF]. Red arrows mark those species, for we have obtained female-specific PCR-products, using primers AllWSex2_F/_R.

F

  (on Z ~10 kb; on W ~16 kb) AllWSex2 http://mc.manuscriptcentral.com/issue-ptrsb Submitted to Phil. Trans. R. Soc. B -Issue F o r R e v i e w O n l y !"#$%&'( !"#$%&"'()('*+,-."-!""#$%&' /01*23,4* 5-(6(7*(7*+5%8* -"'"78*'5++57* ,7'"%85-

Figure S5 .

 S5 Figure S5. PCR-Result using primers AllWSex2F/R for 28 Huso huso (12 females, 16 males) with known phenotypic sex.

Figure S6 .

 S6 Figure S6. PCR-Result using primers AllWSex2F/R for 18 A. gueldenstaedtii (9 females, 9 males) with known phenotypic sex.

Figure S7 .

 S7 Figure S7. PCR-Result using primers AllWSex2F/R for 18 A. baerii (9 females, 9 males) with known phenotypic sex.

Figure S10 .

 S10 Figure S10. Inheritance of the PCR-marker AllWSex2 in a genetic family of A. ruthenus, with mother (F52), father (M33) and 7 daughters, followed by 7 sons.

Table 1 .

 1 Permits to catch and propagate wild H. huso were provided by the Romanian Ministry of Agriculture and Rural Development (No. 7/19.03.2018). The license to keep and propagate sturgeon at the IGB is no. ZH 114 (LaGeSo, 18/02/2019). Numbers of individuals tested with the sex-linked PCR marker AllWSex2 (p-values for association with sex were calculated using a Pearson's Chi-squared test with Yates' continuity correction; gel pictures in electronic supplementary

	F
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	y

material, figure

S3

-S10). To avoid circular evidence, neither the 61 (30 females, 31 males) A. ruthenus used for poolsequencing, nor the offspring of the three genetic families of A. ruthenus, A. oxyrhinchus and H. huso (supplementary materials, figures S8-S10) but only their parents are counted here.

Acknowledgments

This work was funded through COFASP/ERANET (STURGEoNOMICS) by the German Federal Ministry of Food and Agriculture through the Federal Office for Agriculture and Food (grant nos. 2816ERA04G, 2816ERA05G) to M. Stöck, S.W., J.G. and M. Schartl; by the French National Research Agency (grant nos. ANR-16-COFA-0002) to YG, CK and CR, and by the Romanian Executive Agency for Higher Education, Research Development and Innovation Funding (UEFISCDI, contract 3/2017) to RS, MC and DH. This research was also in part supported by the Russian Science Foundation (RSF) to DP (grant number 18-44-04-007), by the Deutsche Forschungsgemeinschaft to MSch (DFG SCHA 408/14-1), and by a European Maritime and Fisheries Fund to YG (EMFF/FEAMP project SIBERSEX). Research on A. gueldenstaedtii was supported by a European Maritime and Fisheries Fund to YG (EMFF/FEAMP project S'STURGEON). We thank Marilena Maereanu, Katarina Tosic, and Marian Paraschiv for help with H. huso catching, propagation and raising of animals; Asja Vogt, Marcus Ebert, Jan Hallermann, Kevin Bäumler, Udo Wolf for help with A. oxyrinchus or A. ruthenus propagation and raising of animals; Elodie Dupin-de-Beyssat, Camille Eché, Antje Tillack, Sophia Lambert, Alina Heide, and Petra Kersten for help in the lab, Lukáš Kratochvíl for references, Jörg Plötner and Robert Schreiber for access to the Qiagen robotic workstation, and two anonymous reviewers for their constructive comments on a previous version of this paper..

Additional Information

Ethics

The research using sturgeon in France, Romania and Germany were carried out in accordance with approved guidelines and obtained ethical approval by the institutional animal care and use committees; permits and licences as shown above.

Data Accessibility

The pool-sequencing Illumina reads are available in the Sequence Read Archive (SRA: SRX9341540 and SRX9341541), under BioProject reference PRJNA670610; all other datasets supporting this article have been uploaded as part of the Supplementary Material.

Authors' Contributions

Designed the study: MSchartl, MStöck, YG, SW, JG; wrote manuscript draft: HK, MS, MSch, YG, CH, JG, CK; analysed data and performed molecular analyses: HK, MStöck, CH, EK, CK, CLR, KD, ESYP, RD, JG, DP, WKleiner, KK, DKL; provided biological samples: RS, DH, MC, JG, HK, MStöck, WKloas, AB, EB, BD, PH, RM, PP. All authors read, contributed to and approved the manuscript.

Competing Interests

We have no competing interests.

Text S4. Bioinformatics to obtain sex-specific variants based on a male reference genome of A. ruthenus

Variant calling using all data resulted in a number of 15,682.485 genome-wide variant calls (avg. 8.5 per kbp). Stringent filtering for variants with supporting reads, only present in the female or male pools, resulted in 1,682 female-specific variants and 318 male specific variants (ratio female/male = 5.3). Using different cut-offs for variant filtering, we consistently found the female pool to harbour more sex-specific variants, which hints at a ZW sex determination system. However, the variants did not match known genes related to sex determination. We clustered sex-specific variants by genomic distance and ranked these clusters by variant count. The region with the most female specific variants was HiC_scaffold_4:45,149,338-45,165,057, which contained 27 variants. The second-best cluster consisted of just 6 variants, observed on HiC-scaffold_7:44,514,447-44,516,696 followed by several other hits on HiC_scaffold_4. Looking at male-specific variants, the best region only consisted of 3 variants (HiC_scaffold_4). In the top candidate region, we found a female-specific 15 bp-indel, which we used to design PCR primers, these primers could already sex some of our sexed A. ruthenus samples, but not all (details not shown).

Text S5. Corresponding regions in the current A. ruthenus NCBI reference genome

The current NCBI reference genome (GCF_010645085.1, as of Oct 2020) corresponds to the male genome assembly used for read mapping in our study with a few differences: A) NCBI assemblies have different sequence identifiers B) The NCBI assembly has a different gap-size, leading to a shift in coordinates The changes of sequence identifiers and coordinates are shown in table S1 for the regions mentioned in this study.

Supplementary Tables


Jérôme Darmont
email: jerome.darmont@univ-lyon2.fr

Cécile Favre
email: cecile.favre@univ-lyon2.fr

Sabine Loudcher
email: sabine.loudcher@univ-lyon2.fr

Camille Noûs
email: camille.nous@cogitamus.fr

Data Lakes for Digital Humanities

Keywords: Information systems → Data management systems, Database administration, Information integration, • Applied computing, • Digital Humanities, Data Lakes, Digital Humanities, Metadata

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

INTRODUCTION

Traditional data management has long been adopted by many researchers involved in Digital Humanities (DH). However, it requires a substantial investment in data modeling, including, at the physical level, technologies such as relational and semi-structured Database Management Systems (DBMSs), various data formats, e.g., XML and JSON for semi-structured data, RDF for linked data, and query languages such as SQL and XQuery. This investment in computer science and the fact that initial data are inevitably transformed are presumably impediments to the adoption of DBMSs and related digital tools for DH.

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Moreover, most source information exploited by humanities and social sciences comes in textual format. Again, such textual documents are difficult to manage without substantial transformations: digitization, encoding and tagging, e.g., via the Text Encoding Initiative (TEI), and even lowercasing, stemming, lemmatization, stopword removal or normalization when it comes to text mining and natural language processing.

Another important methodological issue is the black box effect that occurs when resorting to computer scientists only "as a service". How can DH researchers work without mastering the whole process? Furthermore, designing and managing such processes also lead to research issues for computer scientists.

To leverage the above-mentioned issues, we propose the use of data lakes, a concept introduced by Dixon in 2010 as a solution to data siloing and big data variety problems [START_REF] Dixon | Pentaho, Hadoop, and Data Lakes[END_REF]. Even if data exploited by DH are not always big data in terms of volume, they can bear considerable variety, i.e., including structured and semi-structured data, as well as unstructured data such as texts, various types of images, sounds and videos. Traditional data management tends to manage such heterogeneity with different systems, thus separating data into so-called silos.

A data lake is a scalable storage and analysis system for data of any type, retained in their native format and used mainly (but not only) by data specialists (statisticians, data scientists or analysts) for knowledge extraction [START_REF] Sawadogo | Metadata Systems for Data Lakes: Models and Features[END_REF].

One of the main advantages of data lakes is that data are stored in their initial form, and are thus recognizable by their producers, such as DH researchers. A data lake does not propose a new data model nor new data formats for data archiving. Moreover, when data are transformed for processing, the data lineage is stored as metadata, thus enforcing traceability.

However, a drawback is that unprepared data are difficult to process and require data specialists who can program. Yet, we strongly advocate, with other researchers, for the "industrialization" of data lakes, i.e., providing a software layer that allows non-data scientists such as DH researchers to transform and analyze their own data in autonomy, just as dynamic reports are prepared on top of data warehouses for the use of business (i.e, non technical) users.

The remainder of this paper is organized as follows. In Section 2, we describe data lake projects we currently run in close collaboration with researchers in social sciences and humanities. In Section 3, we conclude this paper by discussing the lessons learned running these projects.

EXAMPLE DH PROJECTS INVOLVING DATA LAKES 2.1 HyperThesau

The "Hyper thesaurus and data lakes: Mine the city and its archaeological archives" (HyperThesau) project involves a multidisciplinary team consisting of two research laboratories of archaeology and of computer science, a digital library, two archaeological museums and a private company. This project has two main objectives:

(1) the design and implementation of an integrated platform to host, search, share and analyze archaeological data; (2) the design of a domain-specific thesaurus taking the whole archaeological data lifecycle into account, from data creation to publication. Archaeological data may bear many different types, e.g., textual documents, images (photographs, drawings...), sensor data, etc. Moreover, similar documents, e.g., excavation reports, are often created by various software tools that are not compatible with each other. The description of an archaeological object also differs with respect to users, usages and time. Such a variety of archaeological data induces many scientific challenges related to storing heterogeneous data in a centralized repository, guaranteeing data quality, cleaning and transforming the data to make them interoperable, finding and accessing data efficiently and cross-analyzing the data with respect to their spatial and temporal dimensions.

To overcome all these challenges, we implement a data lake. Our approach aims to collect all types of archaeological data, save them inside the data lake and propose metadata for better organizing data and for allowing users to easily find data for analysis purposes. Our data lake prototype is architectured in nine layers (Figure 1) [START_REF] Liu | A data lake architecture and implementation for archaeological data management and analytics[END_REF][START_REF] Liu | Metadata model for an archeological data lake[END_REF].

Figure 1: HyperThesau data lake's layered architecture [START_REF] Liu | A data lake architecture and implementation for archaeological data management and analytics[END_REF] (1) The data source layer gathers the basic properties of data sources, e.g., volume, format, velocity, connectivity, etc. Based on these properties, data engineers can determine how to import data into the lake. (2) The data ingestion layer provides a set of tools for performing batch or real-time data integration. Data engineers can choose the right tools and plans to ingest data into the data lake with respect to data source properties and the lake's capacity. During ingestion, metadata provided by data sources, e.g., name of excavation sites or instruments, must be gathered as much as possible.

(3) The data storage layer is core to a data lake. It must have the capacity to store all data in any format. (4) The data distillation layer provides a set of tools for data cleaning (eliminating errors such as duplicates and type violations) and encoding formalization (converting various data and character encoding). ( 5) The data insights layer provides a set of tools for data transformation (e.g., into models) and exploratory data analysis (e.g., pattern discovery). Note that transformed data may also be stored into the lake for later reuse. [START_REF] Liu | Metadata model for an archeological data lake[END_REF] The data application layer provides applications that allow users extracting value from data, e.g., through an interactive query system, reports or dataviz. [START_REF] Pardoën | Last accessed[END_REF] The workflow manager layer provides tools to automate the flow of data processes. [START_REF] Pons | Computing Communities in Large Networks Using Random Walks[END_REF] The communication layer provides tools that allow the other layers to communicate with each other. It must provide synchronous and asynchronous communication capability. ( 9) The data governance layer provides a set of tools to establish and execute plans and programs for data quality control [START_REF] Khatri | Designing data governance[END_REF]. Each of the above layers is implemented with one or more frameworks of the Apache Hadoop ecosystem, e.g., Atlas1 , HDFS2 , HIVE 3 , OpenLdap4 , Spark5 , etc. This prototype is operational and currently hosts the data of two archaeological research facilities. The metadata management system instantiates the MEtadata model for Data Lakes (MEDAL), which adopts a graph model [START_REF] Sawadogo | Metadata Systems for Data Lakes: Models and Features[END_REF]. It is implemented with Apache Atlas, which can host not only descriptive metadata, but also several thesauruses. With the help of a search engine, i.e., Solr 6 , users can find data through descriptive metadata, a thesaurus or the data lineage.

Bretez/STRATEGE

Bretez [START_REF] Pardoën | Last accessed[END_REF] is a multidisciplinary project aiming at a visual and sonorous restitution of the XVIII th -century Paris. It is also an exploratory project constituted of successive, interlinked modules that are (and must be) interoperable and open. The historical urban restitution is achieved with video game engines that bear their own respective characteristics, of course related to gaming. Yet, here, they are used for specific management and traceability needs. Moreover, Bretez' documentation is a voluminous corpus of heterogeneous and multimedia data.

Within project Bretez, the "Traceability and information management system for multimedia data" (STRATEGE) aims at designing, storing, querying and analyzing all the project's data. To master data heterogeneity, manage data quality and volume, warrant data interoperability and an efficient access while keeping data in their Figure 2: CODAL example screenshot [START_REF] Sawadogo | Metadata Management for Textual Documents in Data Lakes[END_REF] original form so that they remain usable reference for the project's researchers, we resort to a data lake.

STRATEGE is in its first stages: we catalogued all existing data, which included a database, textual documents, sounds, images and a 3D Unity7 (a game engine) model, and more. The database is particularly interesting, for it contains both data and metadata. While retaining it, we also restructured it so as to allow its metadata to interoperate with specific data lake metadata. In short, there are "business" metadata and technical metadata.

The remaining tasks include fully designing and integrating the metadata system, on the basis of MEDAL [START_REF] Sawadogo | Metadata Systems for Data Lakes: Models and Features[END_REF]; make the data from the Unity model accessible into the lake; formalize analysis needs; and design tools that must jointly handle textual, visual and audio content, as well as the heterogeneity of data sources. Such software tools must be accessible to all researchers involved in the Bretez project.

COREL and AURA PMI

Both the projects "At the heart of customer relationship" (COREL) and "Digital transformation, servicization and mutations of industrial SME business models" (AURA PMI) relate to management sciences and are carried out in collaboration with the Coactis laboratory 8 . Although their respective focus and scope are different, they are quite similar in terms of data: a corpus of various textual documents (e.g., annual reports from companies and organizations, interviews of senior or top executives, press articles; all in French or English) and data from various sources, including the Web, curated and inferred by researchers in management sciences from company legal information and performance indicators such as workforce, annual revenue, stock-exchange price and perceived level of digitization and servicization.

With such data handy, the objective is to cross-analyze the terms and expressions found in textual resources with structured, qualitative and quantitative data, in order to discover new insights regarding how companies communicate vs. their actual customer relationship management strategy (for COREL) and how digitization and servicization impact economic performance (for AURA PMI). The challenges here are to:

(1) leverage metadata that allow querying the whole corpus;

(2) jointly analyze structured and unstructured data;

(3) allow management science researchers to perform analyses by themselves.

To complete these tasks for the COREL project, we designed a metadata system that prefigured MEDAL [START_REF] Sawadogo | Metadata Systems for Data Lakes: Models and Features[END_REF] and proposed the lightweight COREL Data Lake architecture (CODAL) [START_REF] Sawadogo | Metadata Management for Textual Documents in Data Lakes[END_REF], which is composed of:

(1) a storage layer that notably includes Elasticsearch 9 for indexing textual contents; (2) a metadata layer leveraging and extending the Metadata

Encoding & Transmission Standard (METS) [START_REF]METS: An Overview and Tutorial[END_REF], stored in the BaseX XML DBMS 10 ; (3) an analysis layer, i.e., an intuitive Web-based graphical interface that allows management science researchers to perform analyses in autonomy, thus enforcing the "industrialization" of CODAL. The analysis layer features three kinds of analyses:

(1) data exploration akin to On-Line Analytical Processing (OLAP) [START_REF] Codd | Providing OLAP (On-line Analytical Processing) to User-Analysts: An IT Mandate[END_REF];

(2) proximity analyses such as similarity (what documents are similar or different [START_REF] Pons | Computing Communities in Large Networks Using Random Walks[END_REF]) and centrality (to identify the documents bearing a specific or common vocabulary, hinting at its importance [START_REF] Farrugia | Towards Social Network Analytics for Understanding and Managing Enterprise Data Lakes[END_REF]) analyses; (3) custom highlights of the context of terms and, optionally, their synonyms, in textual documents. All three types of analyses come with various dataviz (Figure 2).

The AURA PMI Data Lake (AUDAL) is currently being developed, and builds upon CODAL. Its metadata system will notably be a substantial evolution of MEDAL supported by the Neo4J 11 graph DBMS. Moreover, the AUDAL analysis layer, which lays on an Application Programming Interface (API), will be much more elaborate and efficient than CODAL's.

CONCLUSION

In all four data lake projects summarized in Section 2, we use different versions of the MEDAL metadata system, which is designed to be generic. However, although MEDAL is quite flexible, we do not believe in a single model for data lakes. There are indeed significant differences in data in only four projects, in terms of volume, variety and velocity, which imply different architectures and technologies. Thus, we think that much needed methodological tools for data lakes should be instantiated for each project rather than applied "as is".

Furthermore, the software layer we add to "industrialize" our data lakes might become yet another black box, while there is a strong stake for researchers in humanities and social sciences involved in DH projects not to be dispossessed of data by an analysis layer that would adopt a "click and go" approach. Data are indeed often partly constructed by said researchers themselves as a product of scientific work that takes time, thus giving a significant value to datasets.

In consequence, we take great care of accompanying DH users in their appropriation of our analysis tools, not only by training, but especially by interweaving research methodologies from computer science and other disciplines by design, in close collaboration with partner researchers.

Moreover, the possibility of having both access to the raw data and the entire possible processing chain is necessary, because black boxes are seldom compatible with a sound methodological approach aiming at producing scientific knowledge. Data lakes precisely allow this much needed transparency.

Darmont & al.

https://atlas.apache.org

hhttps://hadoop.apache.org/docs/r1.2.1/hdfs_user_guide.html

https://hive.apache.org

https://www.openldap.org

http://spark.apache.org

https://lucene.apache.org/solr/

https://unity.com

http://coactis.org

https://imu.universite-lyon.fr

ACKNOWLEDGEMENTS

Projects HyperThesau and Bretez/STRATEGE are funded by the Laboratory of Excellence "Intelligence of Urban Worlds" (IMU) 12 . Project COREL was funded by the University of Lyon 2. Project AURA PMI is funded by the Auvergne-Rhône-Alpes Region.


Albert Cao

Dr Benjamin Rohaut
email: benjamin.rohaut@sorbonne.universite.fr

Loïc Le Guennec

Samir Saheb

Clémence Marois

Victor Altmayer

Vincent T Carpentier

Safaa Nemlaghi

Marie Soulie

Quentin Morlon

Loic Le Guennec

Cle ´mence Marois

Bryan Berthet-Delteil

Alexandre Bleibtreu

Mathieu Raux

Nicolas Weiss

Sophie Demeret

PhD Ho ˆpital Pitie ´-Salpe

Stephanie Delattre

Sandrine Carvalho

Bruno Sagnes

Vincent Dubois

Celine Navarro

Tanya Louapre

Ahmed Stojkovic

Charlotte Idbaih

David Rosso

Ana Zenovia Grabli

Bruno Gales

Benjamin Millet

Eleonore Rohaut

Sophie Bayen

Gaelle Dupont

Stephane Bruneteau

Danielle Lehericy

Alexandra Seilhean

Aurelie Durr

Foudil Kas

Marion Lamari

Vanessa Houot

Catherine Batista Brochard

Pascale Lubetzki

Khe Pradat-Diehl

Bertrand Hoang-Xuan

Lionel Fontaine

Philippe Naccache

Isabelle Fossati

Alexandre Arnulf

Yves Carpentier

Anna Edel

Di Luisa

Gilberte Stefano

Philippe Robain

Bertrand Thoumie

Tarek Degos

Sonia Sharshar

Emmanuelle Alamowitch

Charles-Siegried Apartis-Bourdieu

Renata Peretti

Nathalie Ursu

Dzierzynski

Kinugawa Kiyoka

Joel Bourron

Bruno Belmin

Eric Oquendo

Marc Pautas

Yves Verny

Sara Samson

Anne Leder

Sandrine Leger

Flore Deltour

Stephanie Baronnet

Mehdi Bombois

Marc Touat

Caroline Sanson

Caroline Dehais

Florence Houillier

Dimitri Laigle-Donadey

Agusti Psimaras

Nadia Alenton

Nicolas Younan

Maria Del Mar Villain

Louise- Laure Amador

Nicolas Mariani

Graziella Mezouar

Aurelie Mangone

Andreas Meneret

Clement Hartmann

David Tarrano

Pierre-Franc¸ois Bendetowicz

Michel Pradat

Sara Baulac

Franc¸ois Sambin

Nadine Salachas

Phintip Le Forestier

Florence Pichit

Adele Chochon

Bastien Hesters

An Hung Herlin

Valerie Nguyen

Alexandre Procher

Elise Demoule

Julien Morawiec

Morgan Mayaux

Claire Faure

Giulia Ewenczyk

Anna Coarelli

Perrine Heinzmann

Marion Charles

Guillaume Masingue

Isabelle Bassez

Yulia An

Virginie Worbe

Rabab Lambrecq

Esteban Munoz Debs

Timothee Musat

Aurelie Lenglet

Lydia Hanin

Nathalia Chougar

Nadya Shor

Damien Pyatigorskaya

Delphine Galanaud

Sophie Leclercq

Albert Demeret

Clemence Cao

Nicolas Marois

Salimata Weiss

Loic Gassama

Vincent Le Guennec

Alice Degos

Thomas Jacquens

Capucine Similowski

Jean-Yves Morelot-Panzini

Bertrand Rotge

Victor Saudreau

Nassim Pitron

Nathalie Sarni

Redwan Girault

Smaranda Maatoug

Lionel Leu

Karima Thivard

Isabelle Mokhtari

Bruno Plu

Laure Gonc¸alves

Marion Bottin

Gaelle Yger

Rebecca Ouvrard

Flora Haddad

Carmelo Ketz

Christel Lafuente

Bruno Oasi

Dominique Megabarne

Haysam Herve

Armelle Salman

Alize Rametti-Lacroux

Anais Chalanc¸on

Hugo Herve

Florence Royer

Valentine Beauzor

Christelle Maheo

Camille Laganot

Aurelie Minelli

Abel Fekete

Marie Grine

Rania Biet

Aurore Hilab

Meriem Besnard

Gwen Bouguerra

Saida Goudard

Saba Houairi

Christine Al-Youssef

Anissa Pires

Katarzyna Oukhedouma

Tal Seidel Siuda-Krzywicka

Hanane Malkinson

Hassen Agguini

Safia Douzane

Said

Severe COVID-19-related encephalitis can respond to immunotherapy

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

We read with great interest the article of Ross W. Paterson and colleagues in Brain [START_REF] Paterson | The emerging spectrum of COVID-19 neurology: clinical, radiological and laboratory findings[END_REF], in which they describe the emerging spectrum of coronavirus disease-2019 (COVID-19) neurological syndromes. This article provides major categories of COVID-19-related neurological syndromes, including patients with encephalitis, and reports corticosteroids and intravenous immunoglobulin response in some patients. Indeed, various COVID-19-related neurological syndromes have been reported since December 2019 [START_REF] Filatov | Neurological complications of coronavirus disease (COVID-19): Encephalopathy[END_REF][START_REF] Helms | Neurologic features in Severe SARS-CoV-2 infection[END_REF][START_REF] Khoo | Postinfectious brainstem encephalitis associated with SARS-CoV-2[END_REF][START_REF] Mao | Neurologic manifestations of hospitalized patients with Coronavirus Disease 2019 in Wuhan, China[END_REF][START_REF] Moriguchi | A first case of meningitis/encephalitis associated with SARS-Coronavirus-2[END_REF][START_REF] Oxley | Large-vessel stroke as a presenting feature of Covid-19 in the young[END_REF][START_REF] Poyiadji | COVID-19-associated acute hemorrhagic necrotizing encephalopathy: CT and MRI features[END_REF]. However, encephalitis has seldom been reported and the potential benefit of immunotherapy remains unclear (one of two patients improved in [START_REF] Paterson | The emerging spectrum of COVID-19 neurology: clinical, radiological and laboratory findings[END_REF]. Herein, we report a case series of five patients (from an observational cohort: the CoCo Neurosciences Study) with severe COVID-19-related encephalitis (impaired consciousness/unresponsive and mechanically ventilated) treated by therapeutic plasma exchanges (TPE) and corticosteroids. The dramatic improvement in three of five patients reinforces the hypothesis of an immune-related mechanism, as evoked by Paterson and colleagues. Neurologists and intensivists should be aware that this life-threatening COVID-19 neurological syndrome has a potentially favourable outcome after immunotherapy, and should not motivate systematic limitation in active patient care.

Patients were aged between 37 and 77 years with COVID-19-related encephalitis presenting with altered consciousness, and were treated by TPE and corticosteroids. They all fulfilled diagnosis criteria for possible immune encephalitis according to [START_REF] Graus | A clinical approach to diagnosis of autoimmune encephalitis[END_REF]. The clinical presentation and the time-course of the disease are summarized in Table 1, and complementary explorations findings are summarized in Table 2 (a detailed history is available for each patient in the Supplementary material).

Patients had no prior history of neurological disease. They were intubated and mechanically ventilated for COVID-19related acute respiratory distress syndrome (ARDS). After sedation withdrawal (ranging from Day 12-30 from initiation), they presented severe and persistent consciousness disorder (comatose state or unresponsive wakefulness syndrome), three had oculomotor disturbances (Cases 1-3) and one had peripheral symptoms attributed to Guillain-Barre ´syndrome (Case 3). CSF examinations were unremarkable except in one patient with albuminocytologic dissociation (Case 3), and one with mild pleocytosis (Case 4). Reverse transcription polymerase chain reaction (RT-PCR) assays of the CSF were negative for severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), as common viruses for all patients (Supplementary material). Onconeural antibodies were negative in serum and CSF. None of the patients had signs of thrombotic microangiopathy (no haemolysis, normal levels of ADAMTS13 activity and antigen). When performed, somatosensory evoked potentials showed bilateral presence of N20 (Cases 2-4). EEGs showed unspecific slow-wave activity. Brain MRIs mostly showed bilateral hyperintense lesions in the deep and periventricular supratentorial white matter, either punctiform and slightly diffuse (Cases 1-3) or diffuse and confluent (Cases 4 and 5), associated with lesions in the pons for two patients (Cases 1 and 2) (Supplementary Fig. 1).

All patients received immunotherapy combining corticosteroids infusions (1 g/day intravenous methylprednisolone for 5-10 days) and TPE with albumin (5 to 10 sessions). It is worth noting that neurological impairment remained unchanged in all patients with severe consciousness disorder despite cessation of sedation for 9-33 days. Three patients (Cases 1-3) showed dramatic neurological improvement few days after immunotherapy initiation (6, 2, and 7 days, respectively), with consciousness improvement allowing functional communication. Two patients (Cases 4 and 5) showed no signs of consciousness improvement and died after discontinuation of life-sustaining therapies.

Relation between immunotherapy and clinical improvement

Although a neuro-invasive potential of SARS-CoV-2 is suspected-as for others coronaviruses-there are surprisingly few reports of COVID-19-associated encephalitis [START_REF] Huang | SARS-CoV-2 Detected in cerebrospinal fluid by PCR in a case of COVID-19 encephalitis[END_REF][START_REF] Guennec | Orbitofrontal involvement in a neuroCOVID-19 patient[END_REF][START_REF] Moriguchi | A first case of meningitis/encephalitis associated with SARS-Coronavirus-2[END_REF][START_REF] Paterson | The emerging spectrum of COVID-19 neurology: clinical, radiological and laboratory findings[END_REF]. An immune-mediated mechanism has been proposed to explain coronavirusesassociated encephalitis [START_REF] Weyhern | Early evidence of pronounced brain involvement in fatal COVID-19 outcomes[END_REF], and TPE has shown promising results in a recent case series of COVID-19 mild meningoencephalitis [START_REF] Dogan | Plasmapheresis treatment in COVID-19-related autoimmune meningoencephalitis: case series[END_REF].

Reports on patients with positive SARS-CoV-2 RT-PCR assay in the CSF are scarce [START_REF] Huang | SARS-CoV-2 Detected in cerebrospinal fluid by PCR in a case of COVID-19 encephalitis[END_REF][START_REF] Moriguchi | A first case of meningitis/encephalitis associated with SARS-Coronavirus-2[END_REF] and most patients had moderate acute cognitive impairment without pleocytosis [START_REF] Helms | Neurologic features in Severe SARS-CoV-2 infection[END_REF] or mildly elevated CSF cell counts [START_REF] Bernard-Valnet | Two patients with acute meningo-encephalitis concomitant to SARS-CoV-2 infection[END_REF]. Likewise, Guillain-Barre ´and Miller Fisher syndromes, acute necrotizing haemorrhagic encephalopathy, and acute disseminated encephalomyelitis have also been described in COVID-19 patients, suggesting a host-immune response mechanism rather than a direct neuro-invasion of the SARS-CoV-2 (Gutie ´rrez-Ortiz et al., 2020; [START_REF] Novi | Acute disseminated encephalomyelitis after SARS-CoV-2 infection[END_REF][START_REF] Toscano | Guillain-Barre ´Syndrome associated with SARS-CoV-2[END_REF]. In the Paterson cohort, 10 patients were treated with corticosteroids, and three of these patients also received intravenous immunoglobulin; one made a full recovery, 10 of 12 made a partial recovery, and one patient died [START_REF] Paterson | The emerging spectrum of COVID-19 neurology: clinical, radiological and laboratory findings[END_REF].

In our cases, the secondary neurological involvement (no prior neurological initial symptoms), associated with the MRI abnormalities and the absence of SARS-CoV-2 in the CSFs point towards a post-infectious antibody or cell-mediated immune mechanism rather than a direct viral neuro-invasion, as suggested by [START_REF] Weyhern | Early evidence of pronounced brain involvement in fatal COVID-19 outcomes[END_REF], although no oligoclonal bands and low interleukin-6 were found in the CSF.

The rapid clinical improvement (i.e. 6, 2, and 7 days for Cases 1, 2 and 3, respectively) after immunotherapy was in striking contrast with the protracted persistence of neurological impairment (24, 30, and 31 days, respectively, after sedation withdrawal) before treatment initiation. Such a feature supports an inflammatory or immune process. In the instance of critical illness, delayed awakening and cognitive impairment, such as delirium, may result from many factors, such as hypoxic encephalopathy, metabolic disturbances, or side effects of sedation in the case of ICU patients [START_REF] Mazeraud | Sepsis-associated encephalopathy is septic[END_REF]. However, ICU-related brain injuries had never been reported to be responsive to immunotherapy. Although we cannot rule out a spontaneous recovery [START_REF] Fischer | Intact brain network function in an unresponsive patient with COVID-19[END_REF], the rapid improvement observed after immunotherapy initiation in the present case series seems to point towards a therapeutic effect of immunotherapy.

Differences between responders and non-responders

TPE and corticosteroid responders (Cases 1-3) and non-responders (Cases 4 and 5) shared similar disease courses (severe COVID-19-related ARDS, mechanical ventilation and sedation for several weeks, severe consciousness impairment, which persisted several weeks after sedation withdrawal, unremarkable CSF findings). Differences in treatment response may be related to lesion intensity observed on MRI between the two groups. The responders mainly had small deep white matter lesions while non-responders had more diffuse confluent lesions of the deep white matter. Time of treatment from diagnosis does not seem to be a relevant factor since non-responders received immunotherapy earlier compared to responders (40 and 42 days after COVID-19 symptoms onset for the nonresponders, versus 48, 52 and 66 days for the responders). Another cause of treatment failure can also be related to the underlying mechanism: non-responders may have had irreversible necrotic lesions related to vasculopathy and coagulopathy as often seen after COVID-19 infection, especially in the lungs [START_REF] Helms | Neurologic features in Severe SARS-CoV-2 infection[END_REF].

Taken together, our findings support the hypothesis that immunotherapy combining TPE and corticosteroids can be effective in the treatment of severe COVID-19-related encephalitis. The exact pathophysiological mechanism underlying brain injury has yet to be clarified but a host-immune response to SARS-CoV-2 appears to be a plausible hypothesis.

Table

  = coronavirus disease 2019; ICU= intensive care unit; NP = not performed; SAPS II = simplified acute physiology score 2; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; TPE = therapeutic plasma exchange.Downloaded from https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awaa337/5926056 by guest on 03 December 2020

  = coronavirus disease 2019; FLAIR = fluid-attenuated inversion recovery; IL-6 = interleukin-6; NP = not performed; RT-PCR = reverse transcription polymerase chain reaction; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; SWAN = susceptibility weighted magnetic resonance sequences.

Table 1

 1 Clinical presentation and time course of the disease

	Patient 5	77 /F	Obesity	High blood pressure	Asthma	Fever, fatigue,	cough, shortness	of breath, head-	ache, anosmia	10			65		41	Yes	No	Yes		12	Unresponsive wake-	fulness syndrome				5 / 5		40 /50			18 /28
	Patient 4	37 /M	Obesity			Dry cough, odynophagia,	headache			10			60		50	Yes	Yes	Yes		23	Unresponsive wakefulness	syndrome				10 / 10		42 /45			9 /12
	Patient 3	61 /M	Pulmonary sarcoidosis	Heparin-induced	thrombocytopenia	Fever, fatigue, shortness of	breath, gait disturbances,	doubt about a paresis of	the right hand	6			83 (still ongoing at time of	writing)	65	No	Yes	Yes		17	Unresponsive wakefulness	syndrome	Brainstem impairment	Movement disorders	Dysautonomia	5 / 10		49 / 48			32 /31
	Patient 2	56 /M	High blood pressure			Fever, fatigue, short-	ness of breath			6			93		58	Yes	Yes	Yes		30	Coma	Brainstem impairment				5 / 5		66 / 69			30 /33
	Patient 1	Age (years) /sex 49 /M	Past medical history Kidney transplant (rheumatoid	purpura)	Clinical features at admission in ICU	COVID-19 symptoms Fever, cough, shortness of breath				Delay between COVID-19 10	onset and mechanical ventila-	tion (days)	Duration of mechanical ventila-59	tion (days)	SAPS II 38	Prone positioning sessions Yes	Renal replacement therapy Yes	Catecholamines a Yes	Clinical features at sedation withdrawal and treatments	18 Duration of sedation (days)	Unresponsive wakefulness Neurological symptoms after	syndrome sedation withdrawal	Brainstem impairment	Movement disorders		10 / 5 Corticosteroid injections (n) /	TPE sessions (n)	52 / 57 COVID-19 symptoms onset to	intravenous corticosteroids /	TPE (days)	24 /29 Sedation withdrawal to intra-	venous corticosteroids /TPE	(days)	First TPE to neurological im-	provement (verbal com-	mands following, days)

Letter to the Editor Downloaded from https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awaa337/5926056 by guest on 03 December 2020

Downloaded from https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awaa337/5926056 by guest on 03 December 2020

Acknowledgements

The authors thank the investigators of the Cohort COVID-19 Neurosciences (CoCo Neurosciences, see member list in Appendix 1 and full details in the Supplementary material) sponsored by Assistance Publique -Ho ˆpitaux de Paris (AP-HP) and, funded by the "Fe ´de ´ration Internationale de l'Automobile" (FIA) and the Paris Brain Institute -ICM. The authors also thank Prof. Didier Dreyfus (Department of Critical Care, Ho ˆpital Louis Mourier, AP-HP.Universite ´de Paris. Colombes, France) for his thorough review of the manuscript. We thank the tele-expertise plateform www.neu rocovid.fr for its assistance during the pandemic. Finally we thank all the caregivers of the Neuro-ICU and the residents of neurology who came to help the Neuro-ICU team during the epidemic outbreak (Jean-Baptiste Brunet de Courssou, Adam Celier, Gregoire Demoulin, Julia Devianne, Anceline Dong and Thomas Rambaud).

Funding

The research leading to these results received funding from the program "Investissements d'Avenir" ANR-10-IAIHU-06.

Data availability

Detailed data are available upon request to the corresponding author.

Competing interest

The authors report no competing interests.

Supplementary material

Supplementary material is available at Brain online.

Appendix 1

CoCo-Neurosciences Study Group

For full details, see the Supplementary material.

Cecile Delorme, Jean-Christophe Corvol, Jean-Yves