dataesr/scientific-paragraphs-categorization huggingface dataset:
U N D I N G This work was supported by the French Government under the "Investissements d'avenir" programme managed by the National Agency for Research (ANR), Méditerranée-Infection 10-IAHU-03. In addition, collaborative work conducted by IHU Méditerranée Infection and the Hitachi High-Tech Corporation is funded by the Hitachi High Tech Corporation. O R C I D Djamal Brahim Belhaouari https://orcid. org/0000-0002-1112-9921 Jean-Pierre Baudoin https://orcid. org/0000-0001-7361-5653 Jean-Christophe Lagier https://orcid. org/0000-0003-0771-8245 Virginie Monnet-Corti https://orcid. org/0000-0002-3642-6981 Bernard La Scola https://orcid. org/0000-0001-8006-7704 Angéline Antezack https://orcid. org/0000-0002-4424-1573 R E F E R E N C E S.
F I G U R E 5 5 Anti-SARS-CoV-2 immunofluorescence staining on saliva sample from Participant 1 (A-C), Participant 2 (A1-C1) and Participant 3 (A2-C2). (A, A1, A2) DNA was stained using Hoechst 3342 (blue). (B, B1, B2) SARS-CoV-2 particles were stained using anti-SARS-CoV-2 antibody (orange-red), respectively. (C, C1, C2) Colocalization of Hoechst 3342 and anti-SARS-CoV-2 antibody (scale bar: 10 μm). A C K N O W L E D G E M E N T SWe thank Takashi Irie, Kyoko Imai, Shigeki Matsubara, Taku Sakazume, Toshihide Agemura, Yusuke Ominami, and the Hitachi team in Japan from Hitachi High-Tech Corporation and Hitachi Ltd. for the collaborative study conducted with IHU Méditerranée Infection, and for the installation of a SU5000 microscope at the IHU Méditerranée Infection facility. This work was supported by the French Government under the "Investissements d'avenir" programme managed by the National Agency for Research (ANR), Méditerranée-Infection 10-IAHU-03.
F U N D I N G This work was supported by the French Government under the "Investissements d'avenir" programme managed by the National Agency for Research (ANR), Méditerranée-Infection 10-IAHU-03. In addition, collaborative work conducted by IHU Méditerranée Infection and the Hitachi High-Tech Corporation is funded by the Hitachi High Tech Corporation. O R C I D Djamal Brahim Belhaouari https://orcid. org/0000-0002-1112-9921 Jean-Pierre Baudoin https://orcid. org/0000-0001-7361-5653 Jean-Christophe Lagier https://orcid. org/0000-0003-0771-8245 Virginie Monnet-Corti https://orcid. org/0000-0002-3642-6981 Bernard La Scola https://orcid. org/0000-0001-8006-7704 Angéline Antezack https://orcid. org/0000-0002-4424-1573 R E F E R E N C E S.
Patients Clinical data of all patients enrolled in this study are listed in Table  1. Patients were screened in participating centres. Patients were referred to our centres after consultation in primary or secondary care facilities. ASyS patients in this study were required to fulfil current ACR/EULAR criteria for IIM  [3]. Further, diagnosis was established by clinical and serological data as well as results from muscle biopsies as recommended in 224 th  [5]  and 239 th  [45]  European Neuromuscular Centre workshops  [72]. All patients were required to have received a muscle biopsy to verify diagnosis. A total of four centres participated in this study (Berlin (Germany), Duesseldorf (Germany), Lyon (France) and Paris (France). Patients were recruited from 2008 until 2019. No epidemiological or clinical differences were detected between centres (data not shown).
Clinical and demographic data is in line with previous reports on ASyS patients. In addition to the ASyS cohort, three control groups were included as follows: • Healthy controls (HC, n = 40): The HC group were healthy individuals without known diseases. HC were used as control group for analysis of the peripheral immune response in ASyS. HC were sex-and agematched to ASyS patients. • Diseased controls (DC, n = 40): The DC group consisted of patients diagnosed with IIM other than ASyS (IMNM = 9, DM = 21, IBM = 10). The diagnosis was made according to current criteria  [42]. DC patients were required to have no antibodies associated with ASyS.
8-fold increase; however, all others were normal *** Symptoms were subjective; in muscle biopsies no irregularities were found and no immune reactions and no signs of inflammation. CK levels were normal and no MSA or MAA were detected 300 field-emission scanning electron microscope (Zeiss, Oberkochen, Germany), equipped with a scanning transmission electron microscopy detector (STEM) and Atlas 5 software (Fibics) with a scanning transmission electron microscopy detector (STEM)  [22].
After the last washing step, the instrument was allowed to equilibrate for 20 min. The acquisition of MS data was conducted in DIA (data independent acquisition) mode using a spectral library built in-house. An appropriate amount of iRT standard (Biognosys) was added to each sample analyzed. Full MS scans were acquired from 300 to 1100 m/z at a resolution of 60,000 (Orbitrap) using the polysiloxane ion at 445. 12002 m/z as lock mass. The automatic gain control (AGC) was set to 3E6 and the maximum injection time to 20 ms. Full MS scans were followed by 23 DIA windows, each covering a range of 28 m/z with 1 m/z overlap, starting at 400 m/z, acquired at a resolution 30,000 (Orbitrap) with an AGC set to 3E6 and nCE of 27 (CID).
Analysis of DIA data Samples acquired with nano-LC-MS/MS in DIA mode were analyzed by introducing the data to the Spectronaut software (Biognosys) and analyzed with a library-based search. As the library, spectral library built in-house was used. Search and extraction settings were kept as standards (BGS Factory settings). As proteome background the human proteome data were selected from UniProt (www. unipr ot. org) containing 20,374 entries. For reliable label-free quantification, only proteins identified with ≥ 2 unique peptides were considered for further analysis. Normalized relative intensities were obtained by Spectronaut and averages were calculated for each protein to determine the ratios between patient muscle samples and their respective controls. To investigate interactive networks, protein-protein interactions were visualized using STRING v11.
Clinical description of anti-Jo-1, -PL-7 and -PL-12 autoantibody positive ASyS patients 80 patients with ASyS were included in this study. In 52. 5% (n = 42) of cases, anti-Jo-1-autoantibodies were detected, followed by 22. 5% (n = 18) anti-PL-7-and 25% (n = 20) anti-PL-12-autoantibodies. To elucidate potential clinical differences between serological subgroups, we analyzed clinical data according to the associated autoantibody status. 62% (26 patients) of anti-Jo-1 + and 55% (11 patients) of anti-PL-12 + patients were female, while in anti-PL-7 + patients there was an equal proportion of each gender (50:50). The mean age at time of biopsy did not differ significantly between the subgroups (Jo-1 + 45y ± 14y, PL-7 + 55y ± 12y, PL-12 + 51y ± 18y). CK levels varied among patients, with higher levels in anti-Jo1 + patients when compared to -PL-7 + and -PL-12 + patients (40% > tenfold vs.
Blood analysis of cellular and soluble compartment reveals pronounced B cell related immune responses in ASyS To characterize the immunological phenotype of ASyS, different immune cell subsets of peripheral blood mononuclear cells and soluble inflammatory factors in the serum were investigated. For analysis of the peripheral immune response, we analyzed ASyS (n = 36), HC (n = 40) and DC (n = 40). We performed analysis of PBMCs to obtain flow cytometric data on immune cell composition. For soluble factors, we employed both bead arrays and multiplex analysis to investigate serum levels. At the cellular level, a subtle reduction in lymphocytes counts with similar granulocyte and monocyte levels were detected in ASyS and DC compared to HCs (Fig. 1a ). In the lymphocyte compartment, frequencies of CD4 + , CD8 + T, B and NK cells were comparable across groups (Fig.
Statistical Analysis Statistical analyses were performed with RStudio software (version 1. 4. 1103). We used non-parametric Wilcoxon tests to compare mean CMV viral loads in whole blood and serum, and to compare mean CMV viral loads in serum after maternal PI. We compared CMV PCR positivity percentages with Chi-squared tests according to the estimated date of onset of PI in pregnant women. Differences were considered statistically different if the p value was <0. 05.
biogen. com/news-releases/news-release-details/ biogen-announces-topline-results-tofersen-phase-3-studyand-its). In animal models of SCAs caused by CAG repeat expansions, such ASOs proved to be effective, and phase 1/2 trials in polyglutamine SCAs are imminent (ClinicalTrials. gov: NCT05160558). 7, 8 ASOs that modulate splicing promise even wider applications, including individualized treatments in single patients. 9 uch interventions are currently being developed for ataxia telangiectasia and optic atrophy 1, a disease often associated with ataxia. 9, 10 Another line of development is adeno-associated virus-based gene therapies to deliver small interfering RNAs or microRNAs targeting genes coding for toxic proteins. 11 fficient delivery still presents a major bottleneck for the clinical application of ASOs and viral vectors.
The Ataxia Advisory Committee Therapy allows academic groups and companies to have their preclinical and clinical development plans evaluated by an international board of preclinical, clinical, regulatory, and industrial experts, as well as patient representatives, with the aim to de-risk therapy development (Fig. 2 ). In addition, the AGI started a collaboration with the Critical Path Institute resulting in the launch of the Critical Path to Therapeutics for the Ataxias (CPTA), which has a specific focus on data sharing and driving toward regulatory acceptance of clinical and biomarker outcomes for clinical trials (https://c-path. org/programs/cpta/). AGI is sharing information and resources via the AGI website (https://ataxia-global-initiative. net), a monthly AGI newsletter (received by more than 700 people), and scientific conferences and symposia specifically focused on therapy development and trial readiness.
Availability and Aggregation of Natural History Data for Robust Trial Design Reliable natural history data are key for trial design. Although longitudinal cohort data are available for the main CAG repeat SCAs (SCA1, SCA2, SCA3, and SCA6) and the most frequent ARCAs (Friedreich ataxia, ARSACS, SPG7, and RFC1 disease),  [16] [17] [18] [19]  they are missing for the large majority of other SCAs and ARCAs. Extensive efforts are thus undertaken by AGI partners to map the natural history of other SCAs and ARCAs. The AGI is in an ideal position to serve as a platform for sharing and exchanging these data. To facilitate this, the AGI is developing procedures and preparing templates that help to overcome the related legal and administrative hurdles. One ongoing initiative to bring together existing natural history data of genetic ataxias is coordinated by the CPTA.
Clinical Outcome Parameters: Sensitivity to Change and Patient Meaningfulness Given the relatively slow progression of most SCAs and ARCAs with survival times exceeding 20 years after ataxia onset,  20  clinical outcome parameters for trials need to be sufficiently sensitive to change. Although SARA serves as the most widely applied primary clinical outcome for genetic ataxias, there is substantial intraindividual variability, reaching almost 20% of the entire scale range on repeated testing within 14 days. 21 hus, various sample size calculations come to the unanimous conclusion that, in a trial of at least 1 year in duration, several hundreds of participants would be needed to detect a disease-slowing effect of an investigational drug. 16, 22 There is an ongoing debate whether modified SARA versions, such as the Modified Functional SARA that is used in an ongoing drug trial (https://clinicaltrials.
gov/ct2/show/NCT03701399), have higher sensitivity. A thorough analysis of properties of SARA, potential modification of the scale, and careful analysis and validation of possible new versions are some of the main tasks of the AGI together with CPTA. Nevertheless, there is an obvious need to follow new strategies to improve clinical assessment that go beyond application of clinical scales in the hospital. 4] [25]  Further improvement can be achieved by shifting the assessment into real life, either by repeated video capture (SARA home ) or bodyworn sensors, which has shown to dramatically reduce calculated sample sizes. 21, 25, 26 tandard clinical ataxia scales such as the SARA need to show that they capture not just neurological proxies of ataxia functions, but indeed reflect meaningful benefit of patients' lives.
Development of Validated Biomarkers For early-phase clinical trials, there is an urgent need for biomarkers that are sensitive to detect treatment efficacy in small groups of patients before moving to large and registration trials with primary clinical outcomes. Currently, blood concentrations of neurofilament light chain, a marker of axonal injury,  30, 31  gait-related sensor data,  25  and a number of MR imaging measures, including regional brain volumes,  32, 33  diffusion tensor imagingderived measures,  34  and neurochemical abnormalities detected by MR spectroscopy,  35  are the most promising biomarker candidates. In the future, preventive trials in preataxic mutation carriers will also be a realistic option. In such trials, the number of patients converted to manifest disease could serve as a primary outcome, as in the recently started ATLAS (Adults With a Confirmed Superoxide Dismutase 1 Mutation) trial for ALS (https://clinicaltrials.
Acknowledgments: This work was supported by the  European Joint Programme on Rare Diseases  as part of the  PROSPAX consortium  under the  EJP RD COFUND-EJP 825575  (via the  Deutsche Forschungsgemeinschaft (German Research Foundation ; to M. S. and B. B. ) and by Grant  779257  " Solve-RD " from the  Horizon 2020 research and innovation program  (to M. S. and H. G. ). T. K. , A. D. , H. G. , and M. S. are members of the  European Reference Network for Rare Neurological Diseases (ERN-RD , project number  739510 ). Open access funding enabled and organized by Projekt DEAL. David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA ;  Ian Harding  ( Department of Neuroscience, Central Clinical School, Monash University, Melbourne, Australia; Monash Biomedical Imaging, Monash University, Melbourne, Australia); Pierre-Gilles Henry (Center for Magnetic Resonance Research, University of Minnesota, MN, USA );  Heike Jacobi  ( Department of Neurology, University Hospital Heidelberg, Heidelberg, Germany );  Francesca Maltecca  ( Neurogenomics Unit, Division of Neuroscience, IRCCS Ospedale San Raffaele, Milan, Italy );  David Mengel  ( Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany );  Andrea Nemeth  ( Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom; Oxford Centre for Genomic Medicine, Oxford University Hospitals NHS Trust, Oxford, United Kingdom );  Puneet Opal  ( Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA );  Gulin Oz  ( Center for Magnetic Resonance Research, University of Minnesota, MN, USA );  Hélène Puccio  ( Institut Neuromyogène, Inserm, Lyon, France );  Filippo Santorelli  ( Molecular Medicine, IRCCS Fondazione Stella Maris, Pisa, Italy );  Andreas Traschütz  ( Division Translational Genomics of Neurodegenerative Diseases, Center for Neurology and Hertie-Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany );  Adam Vogel  ( Centre for Neuroscience of Speech, The University of Melbourne, Victoria, Australia );  Stephan Zuchner  ( Dr.
Tissues were freeze-substituted with acetone containing 2% (w/v) osmium tetroxide at -90 • C for 48 h, -60 • C for 12 h, and -30 • C for 8 h using a Reichert CS freeze-substitution device. Samples were then infiltrated with Epon-Araldite resin and polymerized at 60 • C. Ultrathin sections were mounted on Formvar-coated slot grids, stained with uranyl acetate and lead citrate, and viewed with a Hitachi H7000 TEM. Biotrophic hyphae of C. higginsianum were isolated from infected A. thaliana leaves by fluorescence-activated cell sorting, cryo-fixed and prepared for TEM as described previously  (Takahara et al. , 2009). The mean diameter and size ranges of vesicles observed using TEM were measured using ImageJ software. For negative staining, vesicles resuspended in 20 mM Tris-HCl pH 7. 5 were applied to glow-discharged 3.
.  Database search The LC-MS/MS data was searched using Proteome Discoverer 2. 5 (ThermoFisher Scientific). MS spectra were searched against a Colletotrichum higginsianum database downloaded from Uniprot on 06/2018. The database search parameters were set as follows: two missed tryptic cleavage sites were allowed for trypsin digested with 10 ppm precursor mass tolerance and 0. 05 Da for fragment ion quantification tolerance. Oxidation of methionine was set as a variable modification. Carbamidomethylation (C; +57 Da) was set as a static modification. Results were filtered using the Percolator node with a FDR of 0. 01.
To do this, we subjected vesicle samples to a protease protection assay. Vesicle samples were treated with either buffer, trypsin or detergent followed by trypsin. Proteins protected inside of vesicles are expected to remain intact after trypsin treatment unless detergent has been added to solubilize lipid membranes. Again, our marker proteins were detected in the lysate of transgenic mycelia and in crude vesicle samples. Importantly, the proteins were still detected in vesicle samples after treatment with trypsin but could not be detected when the samples were pretreated with detergent followed by trypsin (Figure  8 ). These results indicate that the marker fusion proteins are packaged inside of fungal EVs, where they are protected from degradation. This helps validate our proteomic data and suggests that ChSnc1, ChSso2 and ChBmh1 can all function as markers for C.
Funding information Saclay Plant Sciences- SPS, Grant/Award Number :  ANR-17-EUR-0007 ;  Agence Nationale de la Recherche , Grant/Award Number:  ANR-17-CAPS -0004-01 ;  National Science Foundation , Grant/Award Numbers: IOS-1645745, IOS-1842685;  U. S. Department of Energy (DOE) Office of Biological and Environmental Research , Grant/Award Number: DE-SC0020348.
7  , and are accessible via GenBank accession numbers. The SARS-CoV-2 M protein was tagged at the C-terminus placing the fluorescent protein within the lumen of the SARS-CoV-2 VLPs. To generate a functional tag, the following protein linker was used: Flex (Flexible protein linker): GGGGSGGGGSGGGGSGGGGD to generate both M-Flex-mCherry and M-Flex-GFP. The plasmids were generated by the synthesis of Flex-mCherry and Flex-eGFP and inserted into humanized M plasmids described in  7  using EZcloning from Genscript. The SARS-CoV-2 N protein was tagged at the N-terminus. The GFP and mEOS2 tags were amplified by PCR from peGFP and pmEOS2 using Pfu Polymerase (M7741, Promega), dNTP mix (10297018, Thermo Scientific) and primers designed to carry NheI and KpnI restriction sites (Sigma Aldrich). PCR amplified inserts were added at the N-terminus of N protein in pcDNA3.
Immunoblotting. Proteins from cell lysates (20 µg of total proteins) or VLPs (volume to volume) were loaded and separated on 8% and 12% SDS-PAGE gel and transferred onto a polyvinylidene difluoride transfer membrane (Thermo Fisher). Immunoblotting was performed by using the corresponding antibodies. Horse Radish Peroxidase signals were revealed by using Amersham ECL Prime (Sigma Aldrich). Images were acquired using the Chemidoc Imaging system (Bio-Rad). Each band intensity on the immunoblot was quantified using the ImageJ software. Quantification was established by the following formula for each viral protein: VLP/(VLP + cell lysate) relative to GAPDH as a loading control.
4 NA, oil immersion) and the appropriate filter cube for GFP fluorescence (Ex 466/40, DM 495, BA 525/50). A software module (DirectOverlay, JPK Bio-AFM, Bruker Nano GmbH, Berlin, Germany) was used to calibrate the tip position with the optical image. AFM topographic images were obtained in TNE using the quantitative imaging (QI) mode with BL-AC40TS cantilevers (mean cantilever spring constant kcant = 0. 1 N/m, Olympus). Before each set of acquisitions, the sensitivity and spring constant of the cantilever were calibrated (thermal noise method). The applied force was kept at 200 pN with a constant approach/retract speed of 25 µm/s (z-range of 100 nm). Using the JPK SPM-data processing software, images were flattened with a polynomial/histogram line fit. Low-pass Gaussian and/or median filtering was applied to remove minor noise from the images.
20) resulting in a 210 nm laser waist ( ω 0 ) in the object plane as calibrated using Rhodamine 6G in water at 25 °C (with D = 280 µm 2 /s). When considering a Gaussian profile for illumination ( ω z /ω 0 =5), an effective volume (Vo) of ~ 0. 3 fL is obtained, since the sample fluorescence is col- lected through a confocal pinhole (fixed at 1 Airy unit). Fluorescence intensity fluctuations traces (at least 120) were recorded for 10 s and correlated immediately with the built-in Zen software. Correlograms are then fitted with the PyCorrFit software  22  using a classical 3D free diffusion model (1) G(τ ) = 1 N 1 + τ τ D -1 1 + 25 τ τ D -1/2 in order to obtain the diffusion time τ D and the number of particle N (Eq. 1). All particles were assumed to have similar brightness. The VLPs concentration (in particle number/mL) is immediately obtained by Eq.
49 SR-100 × TIRF objective at room temperature. A 561 nm laser was used to excite the 405 nm photo-convertible mEOS-2 protein. The fluorescence is collected through a dedicated cube containing a dichroic mirror and an emission bandpass filter centered at 617 ± 73 nm. 20,000 images were captured by an iXon U897 camera from Andor at a rate of 50 Hz. PALM acquisitions were analyzed using the ThunderSTORM plugin in Fiji  23. The module DBSCAN of the super-resolution quantification software SR Tesseler  24  was used to quantify cluster diameters. Immuno-spotting assay on VLPs. FluoroDish cell culture Petri Dishes (WPI, USA) were coated overnight at room temperature with 0. 01% poly-l-lysine (Sigma) and rinsed three times with PBS. Purified fluorescent SARS-CoV-2 VLPs were diluted 10 times in TNE buffer and 100 µL were deposited 30 min at room temperature on the poly-l-lysine coated dish for adsorption.
PFA 4% was deposited 15 min at room temperature for fixation. NH 4 Cl was added for 5 min at room temperature. Dishes were rinsed once with BSA 3%PBS and were blocked with BSA 3%PBS for 15 min at room temperature. Immuno-spotting was performed by using corresponding antibodies. After incubation, dishes were rinsed three times with PBS and stored at 4 °C. Then, images of fluorescent VLP were acquired using the TIRF-Microscopy (Nikon) with the 100 × objective. 13 Images corresponding to 250-300 fluorescent VLPs were analyzed by ImageJ/Fiji software and ComDet Plugin was used for quantifying the number of fluorescent VLPs and the double GFP/mCherry colocalization. The image background was estimated at 10% of the fluorescent intensity signal of VLPs and was removed from images before colocalization quantification.
One point in the graph represents the % of GFP/mCherry colocalization in one frame. N = 2, 250 particles analyzed. N(GFP) was incorporated. The colocalization of the dual color GFP/mCherry VLPs was also quantified, which allowed us to monitor the relative incorporation of M(mCherry) and N(GFP) in a VLP (Fig. 3C ). We quantify that only 20 ± 3% and 11 ± 7% of the VLPs are dual color containing M(mCherry) and N(GFP) in the same MNE and MNES VLPs respectively (Fig. 3C ) when mixing label and unlabeled proteins. This indicates that VLPs could incorporate the two fluorescent labeled M and N proteins without impairing assembly. We also tried to produce M(mCherry)N(GFP)E VLP, i. e. in the presence of both tagged M and N, but this was totally preventing VLPs production (data not shown). Since the fluorescent protein tag is located at the C-ter of M and N-ter of N, it should be located inside the VLP.