dataesr/scientific-paragraphs-categorization huggingface dataset:
5 Mya and 37. 7 Mya. This order of magnitude is similar to the basal split estimation for the mammal Plasmodium 34 (12. 8 Mya) or all Plasmodium  35  (21. 0-29. 3 Mya) but this dating remains significantly posterior to the emergence of Nephropidae, whose lobster is part of  36, 37  (~180 Mya). The 18S SSU rDNA, for which the largest taxonomic sampling for gregarines is available in databases, was used to investigate P. cf. gigantea position within apicomplexan and gregarines, especially crustacean gregarines. Using a combination of specific primers amplifications, initially based on Simdyanov et al (2015)  38  and Schrével et al (2016)  39  then in part redesigned (Figure  S2 , Table  S7 ), and in silico clusterings, we were able to fully reconstruct complete ribosomal loci covering: 18S-ITS1-5.
B). For all contigs, the putative protein coding genes were then predicted by using Augustus 115 (version 3. 3) and the only gene model implemented for an Apicomplexa: T. gondii. All the predicted proteins were thus compared with the NCBI nonredondant protein database by using BLAST  116. The analysis of the taxonomic groups associated to the corresponding best hits, enabled us to identify five clusters as putative bacterial contaminants whereas the sixth cluster which included 1745 contigs (18. 0Mb), was identified as organisms closely related to Apicomplexa, referred as "apicomplexa" cluster later (Figure  2. B).
Prediction and annotation All de novo assembled transcripts were aligned against the "apicomplexa" cluster contigs with GMAP  122  within the PASA program  123. Then, two ab initio gene prediction tools, SNAP  124 (version 2017-11-15)  and Augustus  115  were trained using a subset of the PASA transcriptome assemblies. A specific gene model was trained with Augustus, including meta-parameter optimization and construction of hints for introns (allowing small introns length >10bp) using our "apicomplexan cluster" repeat-masked genome assembly as reference (RepeatMasker 125 , version 4. 0. 8). Gene predictions were then performed allowing the prediction of alternative transcripts and noncanonical intron bounds. An alternative model was also trained with SNAP (default protocol) and used for gene predictions.
The Augustus and SNAP outputs having sometimes predicted genes slightly differently, the predictions were then parsed by a home-made script in order to keep, for each prediction made, as many alternative genes and transcripts as possible. The completeness of the gene prediction was assessed by using BUSCO  29  (version 4. 0. 6). The predicted proteins, have been automatically annotated by using i) the best hit of a BLASTP search against VEupathdb (version 2019-20-01), ii) the results of KoFamScam against the KEGG pathway database  126  (version 2019-05-11) and iii) the signature domains obtained with Interproscan 127 (version 5. 39-77. 0). The ortholog groups were identified by using orthoMCL  128  (default parameters, version 2. 0. 9) applied to the proteome of a selection of representative organisms available on VEuPathDB (Table  S1 ).
The divergence time of genome A and genome B was estimated from the divergence time of P. falciparum and P. reichenowi as estimated in TimeTree  30. Then, the coding sequences of the orthologous groups/quartets including one and only one gene for the genome A, the genome B, P. falciparum and P. reichenowi were aligned by using MacSE  129. For each alignment, the number of synonymous substitutions per site (dS) between genomes A/B and between P. falciparum/reichenowi were computed with the maximum likelihood method of Yang and Nielsen (2000)  130  implemented in PAML4  131. The Infernal software  132.
Removal of contaminant sequences Host. All the "apicomplexa" cluster contigs were screened by using the short reads available for the sequencing project of the genome of the closely related Homarus americanus species (PRJNA486050) in order to identify host contaminants. This dataset was supposed to be free of sequences from apicomplexan species, since it has been obtained from DNA extracted from the non-intestinal tissues (the tail, the leg or the pleiopod appendices). The mapping was carried out with Bowtie2 135 and the coverages were calculated by using the Samtools 118 library. The contigs thus identified, that were covered over more than 60% of their length by Homarus's short reads, were considered as host contaminants and were removed. Prokaryote/Fungi. In parallel, predicted genes on the "apicomplexa" cluster contigs were also deeply analyzed for contamination by bacteria and fungi sequences.
On scaffolds of this cluster containing at least one predicted protein, a BLASTP against NCBI NR database was launched. For contigs displaying a hit with a e-value lower than 1e-30 and more than 30% of length covered by Prokaryotes/Fungi hits on these contigs, an additional BLASTN against NCBI NR/NT was performed. For the remaining scaffolds without predicted protein, a BLASTN vs nr/nt was directly performed. At the end of this procedure, the contigs with Prokaryotes/Fungi hits covering more of 70% of length were labeled as contaminants and were removed from the genome assembly.
Experimental reconstruction of 18S/28S loci First, a partial SSU rDNA locus was amplified by using JS-470 gDNA (including only the genome A) as DNA template and WL1 and EukP3 primers (Table  S7 ) in a classical PCR reaction; the amplified bands were systematically cloned and sequenced as previously described  39. This partial SSU rDNA sequence was further extended experimentally in the 3' direction still using JS-470 gDNA as DNA template and novel primers designed or re-designed based on the molecular data published for Cephaloidophora cf. communis and Heliospora cf. longissima 38 (Figure  S2. A). The resulting sequence (>4 kb) was then used as anchor to reconstruct a complete ribosomal locus with the program iSeGWalker  136. By clustering reads from JS-470 on this anchor, a 7322-bp theoretical sequence that corresponded to [partial 28S -18S -ITS1 -5.
Maximum likelihood analyses were performed using RAxML 139 (version 8. 2. 12); bootstraps were estimated from 1,000 replicates. Bayesian phylogenetic tree was constructed with MrBayes 140 (version 3. 2. 3) using the following parameters: lset nst = 6 rates = invgamma; mcmc ngen=10000000 relburnin=yes burninfrac=0. 25 samplefreq=1000 printfreq=10000 nchains=4 nruns=2 savebrlens=yes; sump burnin=2500000; sumt burnin=2500000 contype = allcompat. Trees were visualized and edited using FigTree 141 (version 1. 4. 4) and Inkscape (www. inkscape. org). Environmental phylogeny focused on crustacean gregarines. The 189 sequences phylogeny was built from the 18S SSU rDNA sequences of the genomes A and B, which were aligned with 14 sequences from crustaceans' gregarines, as well as 154 environmental sequences from several projects described in Rueckert et al (2011)  40  or gathered from NCBI Genbank.
Expert annotation for glideosome proteins A reference apicomplexan glideosome proteins dataset was elaborated based on glideosome protein repertoires described in the literature mainly for T. gondii and P. falciparum  26, 66, 67. This reference dataset was used as a seed for parsing the orthogroups made for 25 reference proteomes (Table  S1 ) and the proteomes of the two P. cf. gigantea genomes. The selection of these reference proteomes was made by considering the most recent data and associated publications, in order to have the most complete panorama of apicomplexan proteins and key functions/structures currently documented. For each orthogroup containing at least one of the reference proteins, the list of proteins was extracted and the protein sequences were recovered, as well as their respective coding nucleic sequences for both P.
cf. gigantea genomes. A BLASTP was performed for extracted proteins against the proteomes of P. cf. gigantea, as well as a BLASTP of the candidate proteins for each P. cf. gigantea genome against the 25 species reference proteomes. A BLASTN was performed against NCBI NR for the coding sequences of the candidate proteins of both P. cf. gigantea genomes. The sequences thus collected for each described protein were aligned with mafft  142. Maximum likelihood molecular phylogeny was performed on each alignment using RAxML software  139. Analyses were performed using the LG model; bootstraps were estimated from 1,000 replicates. Annotations of the conserved molecular domains were searched for in the automatic annotation and structure analyzed with SMART  143. For each protein, all the performed analyses were then manually examined to validate the candidate proteins within the proteomes of the two P.
cf. gigantea genomes. Presence/absence table of glideosome proteins was visualized using R using the tidyverse package  144. The identification of TRAP-like proteins was done by searching for the TSP1 molecular domain (IPR000884) within the two P. cf. gigantea genomes. The structure of each candidate protein was then carefully studied. If necessary, partially predicted proteins were re-edited with Genewise  145. Schematic representation of TRAP-like proteins was done using BioRender (biorender. com).
ACKNOWLEDGEMENTS Roscoff Marine Station  and service mer for the hosts;  Pauline Konga  for the ribosomal amplifications;  Geraldine Toutirais ,  Cyril Willig ,  Marc Gèze  and the  MNHN Platform (Plateau technique de Microscopie Électronique, Muséum National d'Histoire Naturelle MNHN, Paris, France , http://ptme. mnhn. fr/)for the SEM imagin ; the computational cluster of the MNHN for assemblies/phylogenies. This work was supported by a grant from the  French Agence Nationale de la Recherche  [LabEx  ANR-10-LABX-0003-BCDiv ], in the program " Investissements d'avenir"  [ ANR-11-IDEX-0004-02 ], by several interdisciplinary Programs of the  MNHN (ATM-Microorganismes, ATM-Génomique et Collections, ATM-Emergence, AVIV department ), the  CNRS  ( Julie Boisard 's PhD fellowship,  2018-2021 ). This work also benefited from access to the  Station Biologique de Roscoff , an  EMBRC-France  and  EMBRC-ERIC Site.
The average and standard deviation was taken for each condition. The calibration curves in Figure  S5  in the Supporting Information were fitted by using the Langmuir adsorption isotherm, since the catalysis of H 2 O 2 on the Pt-NPs is a surface reaction  [40]  NR = K * C h 1 + K * C h (1)   where K is the rate constant and C h the concentration of H 2 O 2. Figure  S6  in the Supporting Information, the transconductance versus the frequency was fitted with sinusoidal model by using a custom MATLAB function.
Acknowledgements X. S. and M. J. D. contributed equally to this work. The authors would like to thank  Marc Ramuz ,  Jonathan Rivnay ,  Marie Lefevre , and  David Moreau  for fruitful discussions. This work was supported by a bourse Cifre from  ANR-T  to X. S. and a  European Research Council Stg  grant to R. M. O. ERC-2010-StG Proposal No. 258966  (IONOSENSE).
Fibroblast reprogramming and pluripotent stem cell culture The CT1 and CT3 control iPSC lines have been reprogrammed at I-Stem and already published  15; 16  (CT1 corresponding to line 1869 and CT3 to line PB12). CT2 iPSC line was reprogrammed from IMR-90 lung fibroblast cell (ATCC® CCL-186). WS1 and WS2 iPSC lines were kindly given by the New York Stem Cell Foundation and have already been published  17. WS5 iPSC line was derived from fibroblasts provided by Dr Cécile Delettre and the late Pr Christian Hamel, isolated from one individual affected by WS biopsy after approval of the local ethical committee (NCT03853252, Institute for Neurosciences in Montpellier, France). All the cell lines used for this study as well as the experimental processes have been performed according to the French legal policy.
The Ion Proton reads (FASTQ files) were imported into the RNA-seq pipeline of Partek Flow software (v6 Partek Inc) using hg19 as a reference genome. The number of reads per sample was ranging from 7. 1 million to 10 million reads. To determine genes that are differentially expressed between groups mapped reads were quantified using Partek E/M algorithm after normalization by the Total count/sample (the resulting counts represent the gene expression levels on reads/millions for over 20,800 different genes present in the AmpliSeq Human Gene Expression panel). Differentially expressed genes were identified using Partek Gene Specific Analysis (GSA) algorithm. Gene lists were filtered at pvalue ≤ 1%; |Fold Change| ≥ 1. 5 minReads100. The use of pvalue instead of adjusted pvalue is justified by biological meaning and experimental validations (Figure  S4C )  22.
Biological interpretations of the list of differentially expressed genes were performed using dedicated software (Partek Genomics Suite (v6. 6), and the web-based gene list enrichment analysis tool EnrichR  23; 24  ). EnrichR analysis were performed on KEGG2016 data base and GO biological process 2017b. Ampliseq data produced in this study have been deposited on NCBI GEO under the accession number GSE156911.
Time-lapse imaging of neuronal differentiation NSCs were seeded at 50,000 cells/cm² in PO/LAM-coated 384-well culture plates in N2B27 medium to induce neuronal differentiation. The plates were incubated in an IncuCyte Zoom Live Cell Imaging System (Essen Bioscience) and phase contrast images were automatically taken under 10X microscopic magnification every 4 hours for the first two weeks of differentiation and then every 12 hours for the next two weeks. Pictures were extracted and piled up using the IncuCyte Zoom Software to create the movies.
Immunocytochemistry Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 minutes at room temperature. Cells were then placed in a blocking buffer (phosphate-buffered saline, 2% bovine serum albumin and 0. 1% Triton X-100). Primary antibodies were diluted in blocking buffer and applied overnight at 4 °C. Secondary antibodies conjugated to Alexa fluorophores (ThermoFisher Scientific) were diluted at 1:1000 in blocking buffer and applied for 1 hour at room temperature in the dark. Hoechst 33258 (Sigma-Aldrich) diluted at 1:3000 was applied in the same time to visualize the cell bodies. The cells were visualized on a Zeiss inverted fluorescence microscope and image acquisition was performed using the Zen Blue software (Zeiss). When higher resolution was necessary, confocal imaging was performed using a Zeiss LSM880-Airyscan Confocal Microscope drived by the Zeiss Zen black software.
Immunostaining quantifications Three biologically independent differentiations for each conditions were investigated and 5 images at 20X magnification per each differentiation were quantified. Due to the high number of fibers on each image, the neurites thickness quantification was approached using a method of unbiased random sampling in ImageJ/FIJI. The method was based on the use of a grid (area per point: 8,000 µm²) apposed automatically on the image by the software. A random third of the squares composing the grid were quantified for each image. On each counted square of the grid, neurite thickness was measured manually using the selection tool of imageJ to delimitate the diameter of each fiber. ImageJ was then asked to measure the corresponding line length (i. e. the fiber thickness).
The results for each image were then compiled on a global table to be classified in 3 arbitrary thickness intervals (0 -3 µm; 3 -6 µm; > 6 µm). The same data were used to analyse the mean thickness of the neuronal network for each condition. To quantify the proportions/percentages of positive cells for each labelling, the CellInsight CX7 High-Content Screening (HCS) Platform and the HSC Studio software (Thermofisher Scientific) were used. Based on the CX7-Colocalization Bioapplication and the acquisition of images at 10X magnification, a homemade algorithm had allowed the individual detection and quantification of nuclei (Hoechst), HuC/D (green) and CUX2 (red), and finally the calculation of the proportion of each labelling to the total number of cells, as quantitative readouts.
mRNA extraction and Reverse-Transcription (RT) PCR Total RNA samples were isolated using the RNeasy Mini or Micro Plus extraction kit and QIAcube instrument (Qiagen) according to the manufacturer's protocol. RNA quantification and quality were assessed using the NanoDrop ND-1000 spectrophotometer (ThermoFisher Scientific) and the ND V33. 7. 1 software. The cDNA samples were synthetized from 500 ng of RNA with SuperScript III reverse transcriptase (ThermoFisher Scientific) using a mix of 500 ng/µL random primers (ThermoFisher Scientific) and 500 ng/µL Oligo(dT) (ThermoFisher Scientific).
The demonstrated loss of calcium cell homeostasis  40; 41  has thus led to a clinical trial based upon the ER calcium stabilizer dantrolene (NCT02829268). In parallel, the nonselective histone deacetylase inhibitor valproic acid (VPA) is currently tested in clinical trial (NCT03717909) after been shown to reverse the defective production of P21 cip in WFS1 knock-out cells  42. Other drugs under preclinical study include GLP1-agonists such as liraglutide that showed neuroprotective in Wolfram cell models in addition to its expected effect on insulin secretion  43; 44  , and the chemical chaperone with antioxidant activity 4-PBA  39. In our model, only VPA demonstrated a dose-dependent preventive action on the aberrant neurite outgrowth, and this was associated with the normalization of the expression of genes encoding axon guidance proteins.
Acknowledgements  I-Stem  is part of the  Biotherapies Institute for Rare Diseases (BIRD)  supported by the  Association Française contre les Myopathies (AFM-Téléthon ). This project was also supported by grants from  Association syndrome de Wolfram ,  Association Nationale de la Recherche et de la Technologie and Agence Nationale pour la Recherche : NeurATRIS   ANR-11-INBS-0011  and  Labex   REVIVE ANR-10-LABX-73. M. -G. B. -M. was supported by the programme "investissement d'avenir" INGESTEM. We thank  Yolande Masson  and  Lina El Kassar  for karyotyping the cell lines and  Alexandre Carteron  for the technical support with the ampliseq experiments. We thank  Dr Cécile Delettre  and the  late Pr Christian Hamel (Institut des neurosciences de Montpellier)  for the gift of the WS5 fibroblasts, and the  New York Stem Cell Foundation  for WS1 and WS2 iPSC lines.
1c ). As some non-myelinated cells (nmc) located in the nerve, such as non-myelinating Schwann cells and fibroblasts, are not easily seen in cross-sections, we teased sciatic nerve fibers on a glass slide and identified GFP-positive mSC, axons and nmc using their morphology and/ or specific markers (Supplementary Fig. S3 ). On 100 cells transduced with AAV2/9-CAG-GFP vector after intra-nerve injection in rat pup, 95 were mSC, 4 nmc and 1 was an axon (Table  1 ). The same specificity was observed after intra-nerve injection in adult and pup rat and mouse for AAV2/9-CAG-GFP and in adult mouse and rat pup for AAV2/rh10-CAG-GFP (Table  1 ). Taken together the data highlight the specificity of AAV2/9 and AAV2/ rh10 for mSC after intra-nerve injection. Next, as each large axon is surrounded by mSC in nerves, we used nerve cross-sections immunostained for MBP and Tuj1 to measure the number of transduced mSC (GFP and MBP positive) surrounding axons (Tuj1 positive), as a measure of the transduction rate of mSC.
Statistical test shows one-way ANOVA followed by Tukey's post hoc, two-sided. **p = 0. 0027 between WT ctr. sh and CMT1A ctr. sh, **p = 0. 0056 between CMT1A ctr. sh and CMT1A sh1, **p = 0. 008 between CMT1A ctr. sh and CMT1A sh2; ns, not significant; arb. units, arbitrary unit. All error bars show SEM. Source data are provided as a Source Data file. Finally, we investigated the myelin sheath structure using electron microscopy semi-thin nerve sections. Light microscopy imaging of these sections showed typical features of rat CMT1A disease: lower density of myelinated fibers, hypomyelination (Fig. 4d , green arrows) or demyelination of large axons (Fig. 4d , orange stars) and hypermyelination of small axons (Fig. 4d , blue stars). These features were still present in the nerve of treated animals but at lesser extent (Fig.
This limited distribution of the vector prompted us to measure the immune response against the vector capsid. A validated and standardized ELISA approach was used to measure AAV2/9 neutralizing factors in sera of ten injected animals (5 WT ctr. sh and 5 CMT1A ctr. sh) and four non-injected controls (2 WT and 2 CMT1A rats). Among these samples, only two showed neutralizing factors at low titer (1/500) (Table  4 ). Taken together these data show that intra-nerve injections of AAV2/9 vector results in a restricted transduction of the injected nerves with limited off-target tissues and a weak humoral immune response toward the vector.
Discussion CMT1A is the main hereditary peripheral neuropathy representing more than 50% of all these significantly disabling diseases  34. Since 2004, several therapeutic strategies have been proposed and tested preclinically and clinically but as of yet no treatment is available for this disease  4. At the present time, the most advanced strategy is a pharmacological treatment that reached the clinical phase III (NCT02579759). Another pharmacological treatment is reaching the clinical phase I (NCT03610334) and several others are in preclinical phases  [8] [9] [10] 27, [35] [36] [37]. As all t ese pharmacological treatments require a regular and permanent treatment with potential side effects on the long term, gene therapy represents an attractive alternative. Indeed, an indirect gene therapy approach involving the transduction of muscle cells to increase their production of neurotrophin-3 and promote axon survival is in clinical trial phase I/IIa (NCT03520751)  38, 39.
Results are expressed as the mean ± SD. NA, Not Applicable (< LOQ). Source data are provided as a Source Data file. The first column shows the number of positive sera over all tested samples. The second column shows the titer of positive sera nearly all mSC of the nerve are transduced with one vector copy. This was consistent with the high transduction rate of mSC that we observed using immunolabelling. This also indicated that the vector dose we used to transduce mSC of the rat pups is sufficient. Moreover, the high transduction rate that we obtained in the other species, and in particular in NHP, suggested that we also reached the sufficient dose in these conditions. Further optimizations of the treatment are therefore to be found in the extent of the vector diffusion along the nerve. We evaluated the diffusion of the vectors when injected in the nerve.
The percentage of transduced mSC (GFP and MBP positive cells surrounding Tuj1 positive axons) over all mSC (MBP positive but not GFP positive cells surrounding Tuj1 positive axons) in the full section was calculated using Zen software (Zeiss, France). For each species, this percentage was calculated at the injection site, proximally (toward the spinal cord) and distally (toward the paw) regarding the injection site (see Supplementary Fig. S5 ). Proximally distances of the injection site were 2 cm for mice, 3 cm for rats and 2 and 4 cm for NHP. Distally distances of the injection site were 0. 5 cm for mice, 1 cm for rats and 2 cm for NHP. Immunohistochemistry on teased fibers. After sciatic nerve fixation, fibers were gently teased, dried on glass slides, and stored at -20 °C. Teased fibers were permeabilized by immersion in -20 °C acetone for 10 min, blocked at room temperature for 1 h with NGS/Triton/PBS, then incubated overnight at 4 °C with primary antibodies diluted in NGS/Triton/PBS.