dataesr/scientific-paragraphs-categorization huggingface dataset:
RESEARCH We conducted an in-depth characterization of the Nipah virus (NiV) isolate previously obtained from a Pteropus lylei bat in Cambodia in 2003 (CSUR381). We performed full-genome sequencing and phylogenetic analyses and confirmed CSUR381 is part of the NiV-Malaysia genotype. In vitro studies revealed similar cell permissiveness and replication of CSUR381 (compared with 2 other NiV isolates) in both bat and human cell lines. Sequence alignments indicated conservation of the ephrin-B2 and ephrin-B3 receptor binding sites, the glycosylation site on the G attachment protein, as well as the editing site in phosphoprotein, suggesting production of nonstructural proteins V and W, known to counteract the host innate immunity. In the hamster animal model, CSUR381 induced lethal infections. Altogether, these data suggest that the Cambodia bat-derived NiV isolate has high pathogenic potential and, thus, provide insight for further studies and better risk assessment for future NiV outbreaks in Southeast Asia.
Viruses In this study, we used 3 different NiV isolates: the NiV isolate CSUR381 from Cambodia (GenBank accession no. MK801755), NiV-Malaysia isolate UMMC1 (GenBank accession no. AY029767), and NiV-Bangladesh isolate SPB200401066 (GenBank accession no. AY988601). CSUR381 was isolated from P. lylei bat urine at the Institut Pasteur in Battambang, Cambodia, in 2003  (21) , and the other 2 isolates were obtained from infected patients. We produced and titrated all viruses on Vero E6 cells.
illumina. com), which produced read lengths of 2 × 150 nt. We carried out genome assembly de novo using SPAdes (http://cab. spbu. ru/software/spades) and predicted open reading frames with Prodigal (https:// omictools. com/prodigal-tool). We reconstructed the missing 5′ and 3′ extremities using the 5′ RACE System for Rapid Amplification of cDNA Ends (Invitrogen, https://www. thermofisher. com) according to the manufacturer's recommendations. We performed reverse transcription with SuperScript II Reverse Transcriptase (Invitrogen) using the primer GSP1leader (5′-GACCATTGATCCAACATC-3′) to recover the viral leader sequence and GSP-trailer (5′-AAAGT-GATTGTCTACTCACT-3′) to recover the trailer sequence. After column purification, we tailed cDNA sequences with cytidine triphosphate and terminal deoxynucleotidyl transferase.
Last, we amplified dC-tailed cDNA using the Abridged Anchor Primer provided in the 5′ RACE System for Rapid Amplification of cDNA Ends Kit and primers nested-GSP2leader (5′-TACAGCTTCAATGTCTGGGTCATT-3′) to amplify the viral leader sequence, and nested-GSP2trailer (5′-CAAGTTCAAGGACACCAAAAGT-3′) to amplify the viral trailer sequence. We sequenced PCR products using Sanger technology and submitted the complete genome sequence of CSUR381 to GenBank (accession no. MK801755).
Full-Genome Characterization and Phylogenetic Analyses Analysis of the assembled viral sequence of CSUR381 showed a total genome length of 18,246 nt, similar to the lengths of NiV isolates reported in Malaysia. The nucleotide composition was 27. 8% T or U, 18. 4% C, 33. 6% A, and 20. 2% G; total GC content was 38. 6%. To investigate genetic relationships between CSUR381 and other henipaviruses, we constructed distance matrices for the complete genome and for each gene using the p-distance method. When we compared the sequence of CSUR381 with those of other NiVs available in GenBank, the most similar sequences (with 97. 7% nucleotide identity) were from NiV-Malaysia human isolates (GenBank accession nos. NC002728. 1 and AY029768. 1; Table  1 ). We also calculated nucleotide identity and amino acid homology for each of the 6 structural genes (Table  2 ).
At 7 days postinfection, 100% lethality was observed in the CSUR381 group. In the Malaysia group, 1 animal survived until 10 days postinfection (Figure  5 , panel C); however, the difference between the 2 groups was not significant. These results demonstrate similar lethality of the 2 analyzed NiV isolates, supporting our other data and suggesting CSUR381 has a high pathogenic potential.
Acknowledgments We thank  Jean-Marc Reynes  and  Pasteur Institute  staff for providing us with the NiV Cambodia isolate and  Doris Preininger ,  Anton Weissenbacher , and  Tiergarten Schönbrunn  for P. giganteus bat sampling. We thank  Amelia Charlotte Coggon  for English proofreading of the manuscript, and we also thank  François Enchéry ,  Kévin Dhondt ,  Mathieu Iampietro ,  Sylvain Baize , and  Géraldine Gourru-Lesimple  for help initiating and finalizing this work. This work was supported by  LABEX ECOFECT  ( ANR-11-LABX-0048 ) of  Lyon University , within the  program Investissements d'Avenir  ( ANR-11-IDEX-0007 ) operated by the  French National Research Agency ,  ANR-18-CE11-0014-02 ,  Aviesan Sino-French Agreement on Nipah Virus Study , and the  International Division of the Institut Pasteur in Paris (Actions Concertées Inter-Pasteurienne ).
Implementation and visualization of the simulations Transport equations are numerically solved using an explicit Euler method. For signal D, which diffuses in the apoplast, additional discretization nodes are regularly inserted in growing cells so that the Courant-Friedrichs-Lewy stability condition is always satisfied. We have developed a dedicated graphical user interface. The source code, written in Python, is freely available online (https://forgemia. inra. fr/felix. hartmann/xydys). Simulation outputs are visualized using the graphical convention explained in Fig. 1.
Acknowledgements FPH thanks  Mélanie Decourteix  for helpful discussions. FPH benefited from a starting grant from the  AgroEcoSystem department of INRAE  (https://www. inrae. fr). The UMR 1434 Silva is supported by a grant overseen by the  French National Research Agency (ANR)  as part of the  'Investissements d'Avenir' program  ( ANR-11-LABX-0002-01 ,  Lab of Excellence ARBRE ).
Individual P1 carries biallelic RAD50 mutations To determine the molecular etiology of the disease, we performed whole exome sequencing analysis in P1 by considering coding sequences carrying rare biallelic variants (frequency less than 0. 1% in 1000 genomes, EVS, dbSNP, and our in-house database (8319 individuals)) predicted to be deleterious (Figure  1A ). This approach highlighted two mutations in RAD50 that were confirmed by Sanger sequencing and present in a compound heterozygous configuration (Figure  1B ). One consisted in a nucleotide insertion leading to a frameshift and a premature stop codon (c. 2165dup; p. Glu723Glyfs*5) (Figures  1B, 1C ) inherited from the asymptomatic mother and present in both healthy sister and brother (Figure  S2A ). The other mutation was an in-frame deletion of three nucleotides resulting to the loss of a single glutamic acid residue at position 1035 (c.
3109_3111del; p. Glu1035del) located in the coiled-coil domain of RAD50 (Figures 1B, 1C and Figure  S2A ). This deletion corresponded to a de novo mutation since it was absent in siblings and parents (Figure  S2A ) while microsatellite analysis ascertained for the genetic paternity of the P1's father (Figure  S2B ). Both RAD50 mutations were absent from gnomAD database (>120,000 individuals tested). Sequencing of RAD50 cDNA from P1's blood cells did not detect the c. 2165dup; p. Glu723Glyfs*5 mutation likely owed to nonsense mediated decay (NMD) (not shown). Hence, the only RAD50 transcripts present in P1's cells encoded a RAD50 protein lacking a unique glutamic acid residue in the coiled-coil domain of RAD50 (p. Glu1035del, hereafter dubbed RAD50 E1035Δ ) (Figure  1C ). RAD50 immunoblots with B lymphoblastoid cell line (B-LCL) lysates from P1 and his mother revealed approximately half of RAD50 amount as compared to B-LCL lysate from a healthy donor (Figure  1D ).
This result was consistent with the presence of a null allele in both P1 and his mother (i. e. c. 2165dup; p. Glu723Glyfs*5). Moreover, this finding indicated that RAD50 E1035Δ was correctly expressed and as stable as its WT counterpart. Congruent with an interdependent stability of the human MRN components  (Stewart et al. , 1999; Waltes et al. , 2009) , we also noticed a reduced amount of NBS1 and MRE11 in cell lysates from P1 and his mother (Figure  1D ). The RAD50 E1035Δ mutation was located in the coiled-coil structure composed of heptad amino acid repeats commonly labeled abcdefg, where a and d represent hydrophobic positions  (Truebestein and Leonard, 2016)  (Figure  1E ). The program Marcoil that calculates coiled-coil probability  (Delorenzi and Speed, 2002)  indicated that the loss of the residue E1035 led to a break in the heptad repeats (Blue arrow in Figure  1F ) that was predicted to disrupt the RAD50 coiled-coil structure nearby the mutation, as inferred by the drop in coiled-coil probability in this region (Blue arrow in Figure  1G ).
EXPERIMENTAL MODEL AND SUBJECT DETAILS Human fibroblasts used in this study were obtained from skin biopsies from pediatric healthy donors (3 years of age) and patients. When indicated transformed (with large T antigen from SV40T) and immortalized (after transduction with a hTERT-expressing vector, Addgene #85140) fibroblasts were used. All cell lines were from male individuals and were checked for mycoplasma contamination. Informed and written consent was obtained from donors and patients. The study and protocols comply with the 1975 Declaration of Helsinki as well as with the local legislation and ethical guidelines from the Comité de Protection des Personnes de l'Ile de France II and the French advisory committee on data processing in medical research. Saccharomyces cerevisiae strains used in this paper are listed in Table  S2.
Homology directed repair assay Cells were plated in 6 well plates and transfected in triplicate with jetPRIME (Polyplus transfection) according to the manufacturer's instructions. 1 µg of mCherry vector (PGK-H2BmCherry, Addgene #21217), 4 µg of CRISPR/cas9 vector (pX330-LMNA-gRNA1, Addgene #122507) and 1 µg of donor vector (pCR2. 1 Clover-LMNA donor, Addgene #122508) were used. The percent of clover-positive cells among the transfected cells (inferred by mCherry staining) was analyzed by flow cytometry using a LSR-Fortessa FACS (BD Biosciences) and results were ploted using the FlowJo software. Statistical significance was determined using the student's t-test.
Variant calling was carried out with the Genome Analysis Toolkit (GATK), SAMtools, and Picard Tools. Single nucleotide variants were called with GATK Unified Genotyper, whereas indel calls were made with the GATK IndelGenotyper_v2. All variants with a read coverage ≤ 2× and a Phred-scaled quality of ≤ 20 were filtered out. All the variants were annotated and filtered using Polyweb, an in-house developed annotation software.
Slides were washed 10 minutes 3 times under agitation with PBS/Tween 0. 05%, stained for 5 minutes with 0. 1μg/mL 4′-6-diamidino-2-phenylindole dihydrochloride and mounted in FluorSave (Calbiochem). Slides were analyzed by using epifluorescence microscopy (Axioplan; Zeiss, Oberkochen, Germany). Images were processed for quantification with ImageJ software.
Acknowledgments General : We thank the patient and his family for their generous assistance with samples and information, which made this research possible. We acknowledge  M. Garfa Traore  and the imaging facility of  Imagine Institute  for help with microlaser irradiation, the  Bioinformatics Department of Imagine Institute  for help with Whole exome sequencing,  N. Lambert  ( CEDI, AP-HP, Necker Hospital, Paris, France ) for microsatellite analysis,  A. Fernandes  from the  Centre de ressource biologique, Imagine Institute , who generated B-LCL. We thank  Dr B. Lopez  for RAD50 construct used as matrix and technical advice for RAD51 and RPA immunofluorescence study. We thank  Dr G. Dellaire  for the HDR assay components (Pinder et al. , 2015). Dr P. Pasero  is acknowledged for his advice with replication combing assays.
Funding: Work performed in the GDIS lab was supported by institutional grants from  INSERM, Ligue Nationale contre le Cancer (Equipe Labellisée  " LIGUE 2020 "),  INCa  ( PLBIO19-027 ;  INCA_13766 ),  GIS-Institut  des maladies rares, and state funding from the  Agence Nationale de la Recherche  under " Investissements d'avenir" program  ( ANR-10-IAHU-01 ). This work was supported by  GM56888  and  MSK Cancer Center Core  Grant  P30 CA008748  (J. H. J. P. ) and  Swiss National Science Foundation  ( 31003A_17544 ) and  European Research Council  grant ( 681-630 ) to P. C. M. M. is supported by the  French National League Against Cancer  ( EL2028. LNCC/MaM ), the  French National Cancer Institute  ( PLBIO2017-167 ). M. C. D-C. beneficiated from scholarships from  Association Nationale Recherche Technologie (ANRT)  and  La Ligue  contre le Cancer.
This is in stark contrast to polynucleotides and polypeptides, both of which are accessible via automated chemical synthesis processes and through in vivo biological synthesis. Unfortunately, unlike these biopolymers, most carbohydrates cannot be obtained using straightforward, generic technologies  4  amenable to automation. Enzyme-catalysed glycosylation offers an alternative to chemical methods. The natural choice for this are glycosyltransferases (GTs) that are well-represented in a variety of families in the CAZy database (http://www. cazy. org/)  5  , with each family potentially harbouring numerous specificities  6. Nevertheless, GTs have proven to be rather difficult to handle in vitro and often require expensive nucleotide-glycoside donors  7. Therefore, glycoside hydrolases (GHs) offer an alternative for glycosynthesis.
These enzymes are particularly abundant and their diversity in terms of bond breaking ability matches carbohydrate complexity itself. GHs catalyse hydrolysis using a variety of mechanisms, but the majority perform two subsequent displacements at the anomeric carbon, thus yielding a product whose anomeric configuration is identical to that of the substrate  8, 9. GHs operating by such a mechanism are termed "retaining" GHs and represent 67% of all GHs (755496 classified modules) in the CAZy database, grouped in 83 out of its current 161 GH families (as of February 24 th , 2020). Allowing for rare exceptions  10  , all other GHs are inverting. A result of the double displacement mechanism is that retaining GHs possess the intrinsic potential to catalyse transglycosylation, thus to synthesize glycosidic bonds.
Fractions (0. 5 µL) were combined with 0. 5 µL matrix solution (10 mg•mL -1 2,5-dihydroxybenzoic acid in 5 mM sodium acetate pH 5. 3) directly on a stainless steel target and dried under warm air. The fractions were analysed on a 4700 Proteomics Analyzer (Applied Biosystems, USA) and data were analysed using DataExplorer software (Applied Biosystems, USA). This revealed that peaks 2 and 3 (eluted between 3. 3 and 3. 6 mL; Fig. S1 ) contained compounds with m/z corresponding to p-nitrophenyl-β-D-mannobioside (pNP-Man2). Similarly, MS revealed that fractions collected between 3. 8 and 4 mL (i. e. peaks 4 and 5) contained p-nitrophenyl-β-D-mannotrioside (pNP-Man3).
The sample was twice lyophilized and dissolved in 0. 6 mL D2O (99. 9 atom % deuterium, Sigma-Aldrich). NMR spectra were acquired at 318 K using an Agilent Varian VNMRS 500 MHz spectrometer equipped with a 5 mm HCN probe. This temperature was chosen to prevent interference from the resonance of residual HDO. The NMR chemical shifts were referenced with respect to the resonances of the anomeric CH-group (H-1) of α-D-mannose at 5. 17 ( 1 H) and 96. 8 ppm ( 13 C), these values from the NMR chemical shift database BioMagResBank 1 (www. bmrb. wisc. edu, entry no. bmse000018) were measured using a solution of α-D-mannose (0. 5 mM) in 50 mM sodium phosphate buffer, equivalent to pH 7. 4, in D2O at 298 K, referenced to sodium trimethylsilylpropane-sulfonate (DSS). Standard 2D experiments (DQF-COSY, z-TOCSY with 120 ms mixing time, NOESY with 500 ms mixing time,  13  C-HSQC, and 13 C-HMBC, optimized for 8 Hz) were used for chemical shift assignments, using SPARKY 2 software.
b) NMR results The CfMan2A-N428T reaction mixture contained the substrate β-D-ManpOpNP, transglycosylation products (Fig. S1 ) and D-mannose that results from hydrolysis of the substrate. Identification of the transglycosylation products was performed using 1 H-and  13  C-NMR spectroscopy. The 1D 1 H spectrum of the sample revealed the presence of ten anomeric resonances, corresponding to ten D- Manp moieties (labelled A-I). Three of the D-Man units could be identified as α-D-Manp, β-D-Manp, and β-D-Manp B OpNP, i. e. D-Manp unit B, from comparisons to 1 H-and  13  C-NMR spectra of the substrate β-D-ManpOpNP (NMR data acquired in-house), or using the NMR chemical shift database BioMagResBank 1 , for α-, and β-D-Manp residues. Each of the spin systems with the remaining anomeric resonances were identified, and virtually complete 1 H and  13  C resonance assignments of the corresponding D-Manp residues were obtained (Table  S1 ).
Acknowledgements This project was enabled by a grant from the  Novo Nordisk Foundation Synthesis and Production program  to  D. Teze  ( NNF17OC0025660 ). The PhD fellowship of J. Z. was supported by the  China Scholarship Council (CSC). The GH29 NMR spectra were recorded at the  NMR Center DTU , supported by the  Villum Foundation. The GH51 NMR analyses were performed using facilities at  MetaToul (Metabolomics & Fluxomics Facilities, Toulouse, France , www. metatoul. fr),which is part of the national infrastructure MetaboHUB (The French National infrastructure for metabolomics and fluxomics, www. metabohub. fr) and is supported by grants from the  Région Midi-Pyrénées , the  European Regional Development Fund , SICOVAL,  IBiSa-France ,  CNRS , and  INRA. Guy Lippens (TBI)  is gratefully acknowledged for insightful discussions and technical development of the NMR pseudo-2D kinetics experiments.
M. E. R. thanks  DTU  for a PhD fellowship. H. S. thank the  Swedish Foundation for Strategic Research  (grant  RBP14-0046 ), the  Swedish Research Council  (grant  2019-05605 ) and FORMAS (grant  942-2016-117 ) for financial support. We thank  Julia Tanas Tanasi ,  Karina Jansen ,  Teresa Mazarella  and  Haleema Saadia  for technical support and protein production. 1  Department of Biotechnology and Biomedicine. Technical University of Denmark , Kongens Lyngby, Denmark. 2  TBI ,  Université de Toulouse ,  CNRS ,  INRAE ,  INSA, Toulouse, France. 3 Department of Biochemistry and Structural Biology. Lund University, Lund, Sweden. 4 Biotechnology ,  Department of Chemistry. Lund University, Lund, Sweden. 5 Centre for Analysis and Synthesis, Department of Chemistry. Lund university, Lund, Sweden 6 Department of Chemistry.
Methods Materials. Genes and genes variants, all codon-optimized for use in E. coli and inserted in pET24a, pET28b(+) or pET28a(+), were ordered from GenScript (Piscataway, USA) or Biomatik (Ontario, Canada), respectively. Gene sequences can be found in the Uniprot database  70  under the codes Q9XCV4 (CfMan2A), P96988 (Xyn10A), D4QAP4 (BbHI), A0A125UD88 (AlfB), A0A422MHI3 (AlfC) and O69262 (TxAbf). Substrates were ordered from either Sigma-Aldrich, Carbosynth or Megazyme. General procedures Bioinformatics. Protein BLAST searches were performed on the NCBI server (https://blast. ncbi. nlm. nih. gov/Blast. cgi), from the non-redundant protein database  70  , using default options, except in the case of the "Max target sequences" parameter, which was set at 20000. Queries were made between January 31 st , 2019 and May 3 rd , 2019.
Obtained sequences were clustered  24  to limit pairwise sequence identity at 80% by iterative cd-hit runs  25. Iterative multiple sequence alignments were performed using ClustalΩ 26 to progressively increase minimum pairwise sequence identity to a predefined threshold (10-20%) and reach convergence using make_msa. sh, a homemade bash script, available at https://gitlab. univnantes. fr/sanejouand-yh/Sequences/tree/master. The same script was used to analyse sequence conservation. Protein production and purification. Unless otherwise specified, pET24a, pET28a(+) or pET28b(+) plasmids bearing target genes were used to transform BL21(DE3) E. coli cells. Precultures of transformed cells were used to inoculate lysogeny broth media containing 30-50 mg•L⁻ 1 kanamycin. Cultures (0. 5-2 L) were incubated at 37℃ with shaking until OD600 reached ~0.
BL investigated the smoke bubble in the CALIOP data and diagnosed the vortex and its dynamics in the IFS analysis. SBu analysed the TROPOMI data. PS calculated the radiative forcing. LI analysed the forcing of the vortex by the IFS assimilation. FT, SBe and JJ have provided the estimates of injected mass of aerosols and gases using MLS and OMPS-LP data. AB, LR and DZ provided OMPS-LP data and a detailed insight into limb-scatter data quality aspects. SGB contributed to the results on aerosol perturbation in the stratosphere. SK, BL, SBu, PS, LI, SBe wrote the paper. All the authors contributed to the final version.
Mass estimation The stratospheric masses of CO, CH3CN, and H2O are derived from the MLS species mixing ratios vertical profiles combined with MLS vertical profiles of pressure and temperature on the standard 37 pressure levels. MLS data are filtered according to the recommendations from the MLS team (https://mls. jpl. nasa. gov/data/v4-2_data_quality_document. pdf). CH3CN measurements are not recommended below 46 hPa however have already been used successfully in a study on combustion products from Australian bush fires in the stratosphere  41. The mass calculation is performed as follows. First, we calculate for each profile the partial column of species (CO, CH3CN, H2O) and air for each measurement layer (vertical resolution of ∼3 km); the air partial column is derived from the difference in pressure between the top and bottom of the layer.
01 km -1. Profiles are therefore truncated below any altitude exceeding this value, which can lead to an underestimation of the early aerosol plume when it is at its thickest. This artifact, which explains the slower increase of aerosol mass than gases, persists until mid-February when the plume is sufficiently dispersed so that OMPS-LP extinction measurements no longer saturate. a) Selection of CALIOP attenuated scattering ratio profiles for clear intersections of the bubble by the orbit except the first panel of 1st of January that shows the dense and compact plume on its first day in the stratosphere. The attenuated scattering ratio is calculated by dividing the attenuated backscattering coefficient by the calculated molecular backscattering. The data are further filtered horizontally by an 81pixel moving median filter to remove the noise.
The centroid and the vertical boundaries of the aerosol bubble determined using CALIOP data are overplotted as circles and bars respectively. d) Composited temperature perturbation within the smoke bubble from Metop GNSS radio occultation (RO) temperature profiles collocated with the smoke bubble (see Methods). The black line shows the centroid of vortex detected from ECMWF data (see Supplementary notes 4. 1 and Supplementary Figure  7b ). the IFS. Supplementary Figure  4a  captures the vortex 18 days after the plume injection. Besides the clear aerosol confinement inside the vortex, the compact structure is very visible in the CO total column with values that are enhanced in the vortex with respect to the surrounding background ( 2 • 10 22 mol•m -2 with respect to a background lower than 1•10 22 mol•m -2 ).