dataesr/scientific-paragraphs-categorization huggingface dataset:
Long-Term GW2580 Treatment after SCI Has No Effect on Tissue Reorganization We next used ex vivo diffusion-weighted magnetic resonance imaging (DW-MRI) to analyze the effect of long-term GW2580 treatment on the lesion volume and the spinal cord microstructure. Using DW-MRI, we could clearly discriminate the lesion epicenter (Figure  2A , underlined in red) as well as the spared white and grey matters. Six weeks after SCI, the percentage of damaged tissue at the lesion epicenter, the rostro-caudal lesion extension, and the lesion volume (area under the curve 121. 1+/-9. 35 and 117. 5+/-9. 45 in the untreated and GW2580 treated mice, respectively) were similar in both groups (Figure  2B ). Using luxol fast blue and toluidine blue staining, we further confirmed the absence of difference in lesion size between the two groups (data not shown).
These data demonstrate that an increased astrocytic reactivity is induced caudal to the lesion upon prolonged GW2580 treatment 6 weeks after SCI. Representative micrograph of GFAP immuno-staining at the lesion epicenter (A). Zoomed-in micrographs in the lesion site (B), the grey matter (D), the white matter (G), and the dorsal funiculus (J) at the lesion epicenter. Six weeks after SCI, quantifications of GFAP immunoreactivity were done in the lesion site (C), the grey matter (E,F), the white matter (H,I), and the dorsal funiculus (K,L). Optical densities were measured rostral (E,H,K) and caudal to the lesion site (F,I,L) ipsilateral and contralateral to the lesion site. Results are expressed as mean ± SEM in untreated (blue) and GW2580-treated groups (green). Each dot corresponds to a minimum of 9 sections (630 µm interval between each section).
Acknowledgments: CX3CR1 mice were obtained from  Dan Littman  ( Howard Hughes Medical Institute, Skirball Institute, NYU Medical Center, New York, NY, USA ). We thank  BioNanoMRI platform  for MRI acquisition,  Montpellier Ressources Imagerie  and the animal facility RAM-CECEMA. We thank  Vesna Zunic  for her technical help for behavior acquisitions. We thank  Patrick Villette  for his constant help related to the use of behavioral software.
Image analysis TH-positive SNpc cells were counted by stereology, using a Leica DM-6000B microscope coupled with the Mercator Pro software (Explora Nova). The SNpc was delineated for each section, and probes for stereological counting were applied to the map obtained (probe size was 50 Â 40 spaced by 150 Â 120μm). Only TH-positive cells with its nucleus included in the probe or crossing the green line of the probe were counted. The total number of TH-positive cells within the whole nigra was estimated using the optical fractionator method. The TH staining in the striatum was quantified by optical density (OD). Sections were scanned with an Epson expression 10000XL highresolution scanner. Images were first opened in ImageJ open-source software (ImageJ. nih. gov). Brightness and contrast were set to optimize the histogram of images.
The same parameters were applied for all images of the same staining in order to compare the mean grey value for each striatum without any bias. For the α-synuclein and phosphorylated α-synuclein staining, highresolution whole color slide images were first acquired with the 3DHistech Panoramic Scanner at 20 Â magnification, with 5 layers in extended mode. Each image was opened in the off-line Mercator Pro software (Explora Nova), and the mapping of all regions of interest was made. Then the threshold surface analysis was conducted to quantify antibodybased staining in the region of interest as previously described  (Arotcarena et al. , 2019). Briefly, brightness and contrast rules were applied to the RGB pictures to optimize details without any saturation of the image. The color thresholding tool was then used to select the threshold corresponding to the brown color revealed by the DAB staining.
R258Q mutation. All four variants were absent or rare in the Genome Aggregation Database, were predicted to be deleterious on in silico analysis and were found to be highly conserved between species. The patient with both the previously unknown p. D791fs and p. Y232H mutations presented with dystonia-parkinsonism accompanied by a frontal syndrome and oculomotor disturbances at the age of 39. In addition, two siblings from an Algerian consanguineous family carried the homozygous p. R258Q mutation and presented generalized tonic-clonic seizures during childhood, with severe intellectual disability, followed by progressive parkinsonism during their teens. By contrast, the isolated patient with the homozygous p. Y832C mutation, diagnosed at the age of 20, had typical parkinsonism, with no atypical symptoms and slow disease progression.
Genetic Findings We identified a familial case (FPD-1458-9) with the recurrent homozygous missense mutation, p. R258Q (c. 773G>A in exon 5) in SYNJ1 (GenBank accession number for the longer 1,612 amino acid isoform: NM_003895. 3) and two isolated cases-one consanguineous patient (SPD-174-10) carrying a homozygous missense mutation (c. 2495A>G in exon 19, p. Y832C) and another patient (SPD-68-1) carrying two heterozygous mutations (a missense variant, c. 694T>C in exon 5, p. Y232H and a truncating deletion, c. 2371delG in exon 18, p. D791Ifs * 4) (Figure  1A ). No additional family members were available for determining parental phase for these last two mutations. The index case, FPD-1458-9, came from a consanguineous family consisting of two healthy parents, two affected and five unaffected siblings.
All four mutations were verified by Sanger sequencing. Cosegregation analyses performed in the FPD-1458 family revealed that the proband's affected sister harbored the same homozygous p. R258Q mutation, whereas both the unaffected parents and one of the five unaffected siblings for whom DNA was available were heterozygous for this mutation (Figure  1A ). No other rare homozygous or biallelic deleterious variants of PD-associated genes were identified on gene panel or whole exome analyses, for any of the three index cases. Both the SYNJ1 p. R258Q, and p. Y832C mutations were rare or absent from public databases and highly conserved between species  (6, 7, 10). By contrast, neither of the new SYNJ1 mutations, p. Y232H or p. D791Ifs * 4, is present in any public database, including the Genome Aggregation Database (GnomAD).
The missense variant p. Y232H is predicted to be pathogenic [SIFT: deleterious; Polyphen-2; probably damaging; MutationTaster: disease-causing; Align GVGD: Class C0 (GV: 122. 78-GD: 0. 00); combined annotation-dependent (CADD)_phred: 28] and has been shown to be conserved in orthologous sequences from C. elegans onwards (Alamut R Visual v. 2. 11 software, Interactive Biosoftware, Rouen, France) (Figure  1B ). Both these mutations are located in functional domains: p. Y232H in the Sac1 domain and p. D791Ifs * 4, in the 5 ′ phosphatase domain (Figure  2 ).
FUNDING This work was supported by the  Fondation de France  (FDF; Grant no. 76353 ),  France-Parkinson Association , la  Fédération pour la Recherche sur le Cerveau (FRC ; Grant no. AFP-FRC-R16058DD ), and the  French Investissements d'avenir program  ( ANR-10-IAIHU-06 ). This work was also supported in part by the  Intramural Research Program  of the  National Institute on Aging, National Institutes of Health, Department of Health and Human Services ; project number  ZO1 AG000949.
v. injections of siRNA PMP22-SQ NPs at the dose of 0. 5 mg/kg twice per week (the treatment duration was 20 days, cumulative dose 2. 5 mg/kg). The dose of the siPMP22-SQ NPs was calculated based on in vitro studies and previous studies done in our lab on prostate and thyroid carcinoma. The schedule of treatment was also chosen based on previous data from our research on cancer models  22, 23, 30. Mice treated with siRNA PMP22-SQ NPs showed restoration and normalization of the motor activity and muscular strength (Fig. 2a , b and Supplementary Videos 3 and 4). Importantly, the siRNA Ct-SQ NPs had no effect on the same parameters. Moreover, treatment of JP18/JY13 mice displaying a very severe disease phenotype and paraplegia by siRNA PMP22-SQ NPs resulted in a significant improvement in motor activity (Supplementary Video 5).
d Semi-thin section of sciatic nerves from WT B6, JP18 B6, and JP18/JY13 B6 mice scanned at 40×. Scale bar 20 µm. Fiber count quantification was done using image J software at 70×. To determine the cutoff between small and large fibers a quadratic model analysis was performed. The cutoff is: 7. 4 with a 95% CI at [6. 95-7. 85] for WT (blue line), 7. 20 CI [6. 39-7. 99] for JP18 (dark dashed) and 6. 35 CI [5. 54-7. 10] for JP18/JY13 (pink line). e Transmission electron microscopy (TEM) images (5kx) of ultrathin sciatic nerve sections of the tested mice groups and the g-ratios analysis of myelinated fibers. Scale bar 2 µm. High magnification images (120kx) were taken to show the inter myelin layer distance which is represented by yellow lines for WT and red lines for JP18 and JP18/JY13 group followed by interperiodic analysis graph.
Methods Screening of siRNA against PMP22. To restore the basal levels of PMP22 gene expression, we first obtained the mRNA PMP22 sequence for both homo sapiens and mus musculus, then defined the homology part between these two sequences (Supplementary Table  1a ). Different siRNAs were designed and blasted to check their specificity. The siRNAs were checked for scoring by using three different methods (Tafer software, Thermofisher software and Reynold method). Reynold scores were calculated respecting different criteria  25. Eight siRNAs against PMP22 (Supplementary Table  1b ) were chosen to continue the study, in addition to a siRNA control (siRNA Ct, a scrambled sequence). siRNAs and chemical modifications. The designed sequences of sense and antisense siRNA strands were purchased from Eurogentec, France.
Purification of the bioconjugate from the excess of unconjugated SQ was performed by RP-HPLC on a polymeric column. Purified products were lyophilized, and then solubilized in RNAse-free water at the desired molar concentration. Purification of siRNA-SQ bioconjugates via HPLC. HPLC purification was performed on a Thermo scientific high-performance liquid chromatography system (Dionex UHPLC-3000) equipped with a photodiode array detector (DAD-3000) whose wavelength ranged between 190 and 800 nm, a pump and a manual injector. The stationary phase consisted of a nonporous, alkylated polystyrene divinylbenzene column (Hamilton PRP-3 10 μm, 4. 6 × 250 mm, PEEK, Ref: 79574) protected by a pre-column (Hamilton). Thermofisher Chromeleon 7 software was used for data acquisition. Flow rate was of 1.
Fig. 2  siRNA PMP22-SQ NPs treatment normalized the motor and sensory activity of both JP18 and JP18/JY13 CMT1A mice. Behavioral tests performed to study the motor activity of age-matched JP18 (a) and JP18/JY13 mice (b). CMT1A mice of both strains treated with siRNA PMP22-SQ NPs showed normalization of the time taken by the mice to walk across the beam or the locotronic ladder when compared to the WT and they were significantly faster and showed stronger grip strength than mice receiving 5% dextrose or siRNA Ct-SQ NPs. c Electrophysiological analysis showed that CMAP (left panel) and sensory NCV (right panel) were normalized for both JP18 and JP18/JY13 mice treated with siRNA PMP22-SQ NPs. Data represent mean ± s. e. m. Before: represent the analysis of the data before treatment. After: is the analysis of the test performed after the end of treatment.
MALDI-TOF mass spectrometry. A MALDI-TOF/TOF UltrafleXtreme mass spectrometer (Bruker Daltonics, Bremen) was used for all experiments to verify the identity of the obtained bioconjugates. Mass spectra were obtained in linear positive ion mode. The laser intensity was fixed just above the ion generation threshold to obtain peaks with the highest possible signal-to-noise (S/N) ratio without a significant broadening of the peaks. All data were analyzed using the Flex Analysis software package (Bruker Daltonics). Annealing of siRNA-SQ bioconjugates to antisense siRNA strands. Annealing of both strands of siRNA PMP22 and siRNA Ct was performed after the bioconjugation of the sense strand to SQ and is the same as mentioned before for the generation of siRNA from single RNA strands. Precisely, equimolar amounts of both sense-strand siRNA PMP22-SQ bioconjugate and antisense siRNA PMP22 were mixed with an annealing buffer [30 mM HEPES-KOH (pH 7.
Three measures of 5 min for each sample were performed and the average diameter ±S. D. of at least three independent samples was calculated. Cryogenic Transmission Electron Microscopy (cryo-TEM) was performed with the JEOL 2100 electron microscope at the Electronic Microscopy Platform (IBPS/ Institut de Biologie Paris-Seine, Université P. et M. Curie, Paris, FRANCE). 4 μL of siRNA PMP22-SQ NPs (concentration of 2. 2 mg/mL) was placed on a carboncoated copper grid. A filter paper was used to remove the excess solvents, and the samples were directly dipped in liquid ethane solution using a guillotine-like frame and transferred to a cryo-sample holder. The siRNA-SQ NPs were observed at an acceleration voltage of 200 kV under a low electron dose. Analysis was performed with Image J software. Cell line.
Membranes were blocked with 5% BSA solution for 1 h and then incubated overnight at 4 °C with the following primary antibodies: anti PMP22 (1:750, Sigma: SAB4502217), antimyelin protein zero (P0) (1/1000 abcam: ab31851), anti-MBP (1:1000, Abcam: ab7349), monoclonal GAPDH-HRP linked (1:1000, Cell Signaling technology, Saint Quentin in Yvelines, France. Ref: 3683), and monoclonal anti tubulin (1:10000, Cell signaling: mAb#3873). Blots were then washed and incubated with the corresponding secondary anti-rabbit and anti-mouse antibody conjugated to HRP (horseradish peroxidase, 1:3000, Cell Signaling technology) for PMP22, P0, MBP, and tubulin, respectively. Bands were visualized by enhanced chemiluminescence reagent (BioRAD, France) and quantified using image J software. Cell viability test. The efficacy of siRNA PMP22, siRNA Ct, siRNA PMP22-SQ NPs, and siRNA Ct-SQ NPs were tested for viability on MSC80 cells using MTT assay.
For the long-lasting efficacy experiment, beam walking test was performed at different time points. The test was repeated three times per animal and was recorded by a camera. Data were presented as average time spent by the mice per group ± s. e. m. Locotronic test. The locotronic apparatus (Intellibio innovations A-1805-00049) was used to test the motor coordination when walking (Supplementary Fig. 10, step 3 ). The mice were allowed to cross a 75 × 5 × 20 cm horizontal ladder with bars (7 mm in diameter), which were set 2 cm apart. Infrared photocell sensors situated above and below the bars monitored paw errors. The locotronic apparatus was linked to a software that automatically recorded the time taken by the mouse to cross the path. The time was assessed in three trials, with 20 min rest between trials.
The statistical analysis of the data was performed by calculating the mean of three trials for each animal. Data are presented as the mean ± s. e. m. For the regular treatment, the test was performed at the end of the treatment, while for the longlasting experiment it was performed at different time points. Grip strength test. Neuromuscular strength was assessed by using the Grip strength test (BIOSEB Innovation Model: BIO-GS3) (Supplementary Fig. 10, step  3). This test was performed using an automated grip strength meter. The apparatus consisted of a T-shaped metal bar and a rectangular metal bar connected to a strength transducer. To measure strength in the forepaws of the mice, each mouse was held gently by the base of the tail, allowing the animal to grasp the T-shaped metal bar with its forepaws.
For the regular treatment protocol, the test was performed at the end of the treatment, while for the longlasting experiment it was performed at different time points as detailed above. Data represent average strength per group ± s. e. m. Electrophysiological test. The test was performed with a standard EMG apparatus (Natus UltraPro S100 EMG) in accordance with the guidelines of the American Association of Neuromuscular and Electrodiagnostic Medicine. Anesthesia was performed by isoflurane inhalation where mice were placed in an induction chamber containing 1. 5-2% isoflurane in pure oxygen. During the whole procedure, anesthesia was maintained on the same level through a face mask. Mice were placed on their frontal side on a heating pad to maintain their body temperature between 34 and 36 °C.
Images were analyzed using image J software. Exploration of fibers count and g-ratio. Sciatic nerves were incubated in 3. 6% glutaraldehyde solution (Sigma-Aldrich cat# G5882) for 4 h at 4 °C. This was followed by a PBS 1X wash for 5 min and a post fixation step in 2% osmium tetroxide (Sigma-Aldrich cat# 251755-5 ml) for 2 h. Dehydration step consisted in 5 min washes, using respectively 50%, 80%, 95% and 100% ethanol. The samples were further incubated in acetone solution for 15 min twice, then in an acetone-EPON solution (50:50) for 15 min, followed by embedding in EPON solution twice for 30 and 60 min, respectively. Finally, the samples were carefully positioned in molds filled with liquid epoxy resin solution consisting of 25 mL EPON (Fluka, cat# 45345), 11 mL DDSA (Fluka, cat#45346), 15 ml MNA (Fluka, cat#45347), 0.
Images were used to calculate the g-ratio and the interperiodic distance of myelin sheath. Statistics and reproducibility. Statistics were computed with GraphPad Prism 8. 3. 0 software. Outliers were identified using Grubbs' test. Differences in group means were calculated by one-way ANOVA followed by Dunn's multiple comparison test. For studies requiring grouped analyses, a one-way ANOVA followed by Tukey's multiple comparison test was performed. When we have two groups to compare, a Mann-Whitney analysis was performed to assess the statistical difference. A value of p < 0. 05 was considered significant. All measures were taken from distinct samples, and the sample sizes are presented in figure legends. All the in vitro experiments were performed at least three times independently. Data are presented as mean ± s.
Acknowledgements This work was supported by a public grant overseen by the  French National Research Agency (ANR)  as part of the  "Investissements d'Avenir" program  ( Labex NanoSaclay , reference:  ANR-10-LABX-0035 ). Authors thank the team of the animal core facility of BioMedTech Facilities INSERM  US36 | CNRS UMS2009  |  Université de Paris  and,  Ghislaine Frebourg  for cryo-TEM observations  Electronic Microscopy Platform, IBPS/ Institut de Biologie Paris-Seine, Université P. et  M. Curie , Paris,  FRANCE. We are grateful for  Dr. Simon BATE ,  GSK ,  Peterborough ,  United Kingdom , for his help in the statistical analysis.
Tools and procedures for collection of the data The data for the first phase of the study were collected from patient files containing sociodemographic, clinical (questioning, neurological and physical examination), the data for the additional and the therapeutic examinations, and the follow-up data. The files were compiled according to the guidelines of the International Association for the Study of Pain (IASP) and the WHO. Files of patients suffering from NeuP were retained: persistent or recent pain lasting more than three (3) months  [25]. The files were categorized according to the 11th edition of the International Classification of Diseases (ICD-11)  [26, 27]. The first questionnaire for the collection of data in the consultation registries was in regard to the sociodemographic characteristics of the patients, the typologies, the clinical data, and the type of treatment received.
Statistical analysis of the data The data were collected and analyzed with Prism GraphPad version 8. 0 software. Frequency tables were generated, and calculation of the means were undertaken. The Chi 2 and Fisher's exact test were used to compare the proportions, with a threshold of significance set at p = 0. 05. Simple logistic regression was used for the measures of association, with presentation of the odds ratios and their 95% confidence intervals.
Ethical considerations The data in the patient files were collected in a strictly anonymous manner and with approval from the relevant authorities of the UHC. The Medical Work Committee of the UHC, which oversees the ethics committee and has an advisory role regarding the management of clinical research activities provided approval for this study to be undertaken. For active involvement of the comHC, the national health board of Mali was informed in writing. The AFAN administration was informed of the study by email. The practitioners did not receive any payment for participation in the study. The practitioners were informed of the objective and the benefits of the study, and their free and informed consent was obtained by email prior to their inclusion.
NeuP is a prominent issue in current practice This work, the first of its kind in Western Africa, was purposefully carried out in two phases (retrospective and prospective) to undertake a situational analysis of NeuP in our department and to carry out a KAP study of carers in terms of the treatment of NeuP, respectively. NeuP was, therefore, assessed from different perspectives in our context. Our results combined with the existing literature yielded preliminary data on the burden of NeuP in Mali and laid the foundation for context-tailored therapeutic strategies. The use of validated tools minimized the common selection bias seen in NeuP studies  [26]. Based on the ICD-11 guidelines, NeuP was categorized as peripheral or central. We included solely patient files with a valid neurological exam and a completed NeuP DN4 questionnaire, (if needed).
11. 2. ) with option -t exon -f bam -r pos -s reverse -m intersection-strict -nonunique none. Differential analyses were performed with SARTools (1. 4. 1) (65) using DESeq2 (1. 12. 3) (66) and default options. P-values were adjusted with Benjamini-Hochberg procedure  (35)  set for 5% of FDR. Heatmaps using a correlation matrix and boxplots were obtained with custom R script (3. 3. 0). The raw sequencing data used in this study are available in the National Center for Biotechnology Information's Gene Expression Omnibus (GEO) database and are accessible through GEO series accession number (pending).
Bioinformatics analyses: Gene ontology analyses on DEG in siCCM2 condition (fold change [FC] ≥ 2; P < 0. 05 with FDR corrected using Benjamini Horchberg method (35)) were made with PANTHER version 15,0 Released 2020-02-14 using slim cellular components and biological processes. Enriched pathways analyses were conducted with Reactome. DEGs (P < 0. 05, 5% FDR) rescued by additional silencing of ROCK1 or ROCK2 were recovered at the union of Venn diagrams of DEGs in siCCM2 vs siNT and DEGs in siCCM2 vs siCCM2+ROCK1 or ROCK2 respectively. Gene Set Enrichment Analyses were conducted using GSEA software from Broad Institute, UC San Diego  (67).
Quantitative RT-PCR: Purified RNA (1 μg) were reverse transcribed using the iScript Reverse Transcription Supermix (Biorad). Quantitative real-time PCR (Q-PCR) was performed with iTaq Universal SYBR Green Supermix (Biorad) in a 20 μl reaction on a thermal cycler (C-1000 Touch; Bio-Rad Laboratories). Product sizes were controlled by DNA gel electrophoresis and the melt curves were evaluated using CFX Manager (Bio-Rad Laboratories). A total of three housekeeping genes were selected for their stability in our HUVEC cell line under our experimental conditions, using the three analytical software programs, geNorm, Normfinder and Bestkeeper  (68, 69). We used the relative expression software tool CFX Manager for relative quantification, and normalization was achieved using a normalization factor from all reference genes  (68).