dataesr/scientific-paragraphs-categorization huggingface dataset:
PacBio SMRT sequencing and methylome analysis DNA isolation, purification, SMRT bell library preparation were performed as previously described  [35, 36]. SMRT sequencing was performed on a PacBio RS instrument (Pacific Biosciences, Menlo Park CA, USA). Sequencing reads were processed and mapped to the sequence of B. animalis subsp. lactis CNCM I-2494 (Genbank accession number CP002915). Interpulse durations were measured as previously described  [35]. To identify methylated positions the Pacific Biosciences SMRTPortal analysis platform (version 1. 4) was adopted, this employs an in silico kinetic reference and a t-test based kinetic score detection of modified base positions.
5 kb and subsequently converted into a SMRTbell library  [52, 53]. Four SMRT cells were run using the C2 polymerase enzyme and two 45 minute movies were captured. For each library the methylome was determined using SMRT Analysis software version 1. 4. Genome wide motif analysis resulted in the identification of 59-RTC 6 m AGG-39 and 59-GGW 5 m CC-39 as the 6 mA and 5 mC recognition sequences, respectively (Fig. 1 ). The extent of methylation across the genome at 59-RTC 6 m AGG-39 was expected to be 1576 positions, all of which were indeed detected by the methylome analysis, while for 59-GGW 5 m CC-3, the AvaII recognition sequence, 4420 positions were present on the genome, of which 4124 (93. 3%) were detected as being methylated based on our methylome analysis.
EcoKDam and M. EcoKI, methylated the plasmid complement of pWSK29 (control) or pWSK29-M. BanLI at 59-G 6 m ATC-39 and 59-A 6 m ACNNNNNNGTGC-39, respectively (data not shown). An additional single stranded 6 methyl adenine sequence, 59-RTC 6 m AGG-39, was detected at nine positions across the sequence of pWSK29-M. BanLI, thereby attributing M. BanLI to this novel methylation activity encoded by B. animalis subsp. lactis CNCM I-2494. In order to verify the prediction that B. animalis subsp. lactis also encodes a distinct MTase that protects, based on sequence similarity to characterised R-Ms and on the obtained methylome data, DNA sequences restricted by AvaII, genomic DNA of B. animalis subsp. lactis CNCM I-2494 was restricted by AvaII and analysed by agarose gel electrophoresis. The results obtained indeed demonstrated that the B.
INTRODUCTION In the last decade, adeno-associated-virus (AAV)-mediated gene delivery has emerged as an effective and safe tool for both preclinical and clinical studies of neurological disorders  (Bartus et al. , 2013; Weinberg et al. , 2013; Ojala et al. , 2014). Currently, AAV is the most widely used vector for clinical trials for neurological disorders (gene therapy database can be found at: http://www. abedia. com/wiley/index. html). To date, no adverse effects linked to the use of this vector have ever been reported from clinical trials. Adeno-associated virus is a non-pathogenic dependovirus from the parvoviridae family requiring helper functions from other viruses, such as adenovirus or herpes simplex virus, to fulfill its life cycle  (Dayton et al. , 2012). The wild-type (WT) AAV is characterized by a single-stranded DNA (ssDNA) genome, with inverted terminal repeats (ITR) at both ends, of approximately 5 kb surrounded by a capsid.
ACKNOWLEDGMENTS The authors thank  Cynthia Lebeaupin  for valuable comments on the manuscript. The  Université of Bordeaux  and the  Centre National de la Recherche Scientifique  provided infrastructural support. This work was supported by  Marie Curie Reintegration  Grant  FP7-PEOPLE-2009-ERG256303  from the  European Commission  (to Benjamin Dehay); by a grant from the  Fondation pour la Recherche Médicale  (to Benjamin Dehay); by  Agence Nationale de la Recherche  Grants  ANR-08-MNP-018  and  ANR-07 - MNP-Trafinlid  (to  Erwan Bezard ) and  Mathieu Bourdenx  is a recipient of an  MESR  fellowship.
Water expressed from each cut stem was collected using an Eppendorf tube containing dry cotton wool. The amount of sap was determined by weighing the tube before and after collection. The sap flow (Jv), expressed as the quantity of water exuded from the cut stems, was monitored every 5 min for at least 45 min after it had reached a constant rate (reached in less than 25 min). Aliquots of expressed sap were collected from each root system for later analysis of K + content. In preliminary experiments (pressure-flux curves), five pressures were applied in sequentially increasing order (0. 1, 0. 2, 0. 3, 0. 4 and 0. 5 MPa) to whole root systems. Flow values were logged for 25 min at each pressure, allowing a 5-min equilibration period between pressures. Because the regression of sap flow on applied pressure was linear for all genotypes (data not shown), sap flows in both control and HgCl 2-treated plants were determined at a constant 0.
Statistics Data were analyzed with the PASW statistics 18 (IBM, New York, USA) package. We used the Least Significant Difference (LSD) test (P <0. 05) to make post-hoc comparisons between all means. Percentages of inhibition by HgCl 2 were calculated for each individual root system, and mean values and standard deviations were calculated for the three experiments. In total, the response of nine HgCl 2 -treated plants was compared with those of untreated plants for each genotype.
Acknowledgments Afifuddin Latif Adiredjo was supported by a  French Government scholarship (Bourse du Gouvernement Français, BGF)  and a co-funding by  Directorate General of Higher Education, Ministry of Education and Culture, Republic of Indonesia (Beasiswa Luar Negeri, BLN). Philippe Grieu  and Thierry Lamaze are Ph. D. supervisors of the first author.
Background The most common spinocerebellar ataxias (SCA)-SCA1, SCA2, SCA3, and SCA6-are caused by (CAG)n repeat expansion. While the number of repeats of the coding (CAG)n expansions is correlated with the age at onset, there are no appropriate models that include both affected and preclinical carriers allowing for the prediction of age at onset. Methods We combined data from two major European cohorts of SCA1, SCA2, SCA3, and SCA6 mutation carriers: 1187 affected individuals from the EUROSCA registry and 123 preclinical individuals from the RISCA cohort. For each SCA genotype, a regression model was fitted using a log-normal distribution for age at onset with the repeat length of the alleles as covariates. From these models, we calculated expected age at onset from birth and conditionally that this age is greater than the current age.
Results For SCA2 and SCA3 genotypes, the expanded allele was a significant predictor of age at onset (-0. 105±0. 005 and -0. 056±0. 003) while for SCA1 and SCA6 genotypes both the size of the expanded and normal alleles were significant (expanded: -0. 049 ±0. 002 and -0. 090±0. 009, respectively; normal: +0. 013±0. 005 and -0. 029±0. 010, respectively). According to the model, we indicated the median values (90% critical region) and the expectancy (SD) of the predicted age at onset for each SCA genotype according to the CAG repeat size and current age. Conclusions These estimations can be valuable in clinical and research. However, results need to be confirmed in other independent cohorts and in future longitudinal studies. ClinicalTrials. gov, number NCT01037777 and NCT00136630 for the French patients.
It is crucial that studies dealing with prediction of disease onset include both affected individuals and preclinical individuals, which has not been the case in previous models. Ignoring individuals who are free of disease symptoms, are the same age, and have the same number of CAG repeats as affected individuals creates an artificial tendency towards earlier disease onset. For the purposes of this study, we pooled genetic and age at onset data of a large group of SCA1, SCA2, SCA3, and SCA6 patients from the European EUROSCA registry with data of clinically unaffected carriers of SCA1, SCA2, SCA3, and SCA6 mutations from the RISCA study. The EUROSCA registry was established in 2004 to collect core data of European SCA patients. RISCA is a prospective, multicentric, multinational, observational cohort of clinically unaffected at-risk individuals for SCA1, SCA2, SCA3, and SCA6 (ie, first degree relatives of patients with one of these diseases).
PATIENTS AND METHODS Patients Two groups of individuals were included: affected patients (EUROSCA registry) and preclinical mutation carriers (RISCA cohort). The EUROSCA registry includes individuals with any form of spinocerebellar ataxia (SCA) from 17 European centres. For the current study, we selected 1187 patients with a positive molecular genetic test for SCA1, SCA2, SCA3 or SCA6, genotyped at a central laboratory, and with information available on age at onset of the disease (317 SCA1, 308 SCA2, 399 SCA3, and 163 SCA6) and, when possible, a SARA (Scale for the Assessment and Rating of Ataxia, with a maximal score of 40 indicating a very severe cerebellar ataxia) score ≥3. 10  Patients were included in the database with age at onset as indicated by self-report during their examination by the neurologist, and as indicated in their medical records.
Disease onset was defined by the onset of gait difficulties, as this is the most frequent first symptom. Data were obtained from patients by personal interview. Information obtained by interview was then compared to that from medical records, if available. The RISCA cohort included individuals at-risk for SCA from 14 European centres. 11 These included adult individuals, children or siblings of an individual with SCA1, SCA2, SCA3 or SCA6. Absence of ataxia was defined as having a score on the SARA scale <3. All individuals were genotyped in the same central laboratory as the EUROSCA registry, and of the 264 individuals included with DNA available, 123 (47%) were carriers of a disease-causing expansion (50 SCA1, 31 SCA2, 26 SCA3, and 16 SCA6). For these preclinical mutation carriers, the age at examination was recorded.
Acknowledgements We sincerely thank all the patients and their families for their participation. Caterina Mariotti  thanks the  AISA-Association (Associazione Italiana Sindromi Atassiche, Sezione Lombardia). We are grateful to  Drs Elzbieta Zdzienicka  and  Rafal Rola  ( Institute of Psychiatry and Neurology, Warsaw, Poland ) for contribution of patients and help in patient's assessment. Many thanks to  Sarah Boster  for critical reading. Contributors STdM and TK conceived the study; STdM and  J-LG  established the prediction of age at onset formulae and calculations. STdM and ID-G performed the analysis. TK and AB contributed to the financing and administration of the registry and cohorts. SF, HJ and GS managed the clinical and genetic data. PB and AS performed the genetic testing. AD, MR, LN, PC, CaM, RR, LS, HJ, SF,  TSH, AF, DT, BPvdW, CeM , JSK, PG, AC, LB, MB, SB, SS, JB, JI, KB, MM, RDF, CD, SR, TK and AB enrolled the participants.
N401 097536  ;  0975/ B/PO1/2009/36-(2009-2012 )  Polish Ministry of Science and Higher Education. TSH receives/ has received funding from the  European Union  ( EUROSCA  project) and  Deutsche Forschungsgemeinschaft (DFG, Klinische Forschergruppe Tiefe Hirnstimulation   KFO 247 ). Funding This study was supported by grants  EUROSCA/LSHM-CT-2004-503304  from the  European Union , grant from the  European Community 's  Seventh Framework Programme  (FP7/2007-2013 n°2012-305121 NEUROMICS),  GeneMove/ 01 GM 0503  from the  German Ministry of Education and Research , within the framework of the  ERA-Net for Research Programmes on Rare Diseases , grant  3 PO5B 019 24  from the  Polish Ministry of Scientific Research and Information Technology , and grant No  674N-RISCA/2010-2014  from  Polish Ministry of Science and Higher Education.
The research leading to these results has received funding from the  programme 'Investissements d'avenir'   ANR-10-IAIHU-06. BPvdW is supported by research grants from the  Netherlands Brain Foundation , the  Royal Dutch Society for Physical Therapy, BBMRI-NL , and the  Radboud University Medical Centre. MB is supported by the grant  OTKA K 103983. AF was supported by a grant from  POR CREME 2007-20013. AB was supported by the grant  EUROSCA/ LSHM-CT-2004-503304  and by the grant NEUROMICS (7th framework programme) from the  European Union. AB, AD and GS received funding from the  VERUM Foundation. AD received support from  ANR (French Research Agency)  and  Eranet  for the  Risca  project, PG and AC work at  University College London Hospitals/University College London  which receives a proportion of its funding from the  Department of Health's National Institute for Health Research Biomedical Research Centres funding scheme.
Mass spectrometry data acquisition and processing All time points of the A. algerae infection kinetics (1 hpi, 6 hpi, 12 hpi, 24 hpi and 8 dpi) and the two of the hypoxia samples (24 h and 8 d) were resolved on 12%SDS PAGE using the protean II xi cell system (Bio-Rad laboratories, Marnes-La-Coquette, France). Gels were stained with PAGE-Blue protein staining solution (Fermentas Vilnius, Lithuania) and scanned using a computer-assisted densitometer (EPSON Perfection V750PRO). Gel lanes were cut in 3 regular pieces and destained with two washes in 50% acetonitrile/50 mM triethylammonium bicarbonate. After protein reduction (10 mM dithiothreitol at 50uC for 1 h), and alkylation (55 mM iodoacetamide at room temperature for 30 min), proteins were processed for in-gel digestion over night at 25uC using LysC (2.
LC-MS/MS analysis was then performed and mass spectra were analyzed with the MaxQuant software to achieve the relative protein quantification. doi:10. 1371/journal. pone. 0100791. g001 tion of samples were performed on-line on a Pepmap pre-column (0. 3 mm610 mm, Dionex). A gradient consisting of 0-40% B in A for 60 min, followed by 80% B/20% A for 15 min (A = 0. 1% formic acid, 2% acetonitrile in water; B = 0. 1% formic acid in acetonitrile) at 300 nL/min was used to elute peptides from the capillary reverse-phase column (0. 075 mm6150 mm, Pepmap, Dionex). Eluted peptides were electrosprayed online at a voltage of 2. 2 kV into an LTQ Orbitrap mass spectrometer. A cycle of one full-scan mass spectrum (400-2000 m/z) at a resolution of 60,000 (at 400 m/z) in the orbitrap, followed by 5 data-dependent MS/ MS spectra (LTQ) was repeated continuously throughout the nanoLC separation.
Raw data analysis was performed using the MaxQuant software (V. 1. 3. 0. 5)  [25]. Retention time-dependent mass recalibration was applied with the aid of a first search implemented in the Andromeda software  [26]  and peak lists were searched against the UniProt human database (release2013_04; http://www. uniprot. org) for the host response analysis and a local A. algerae database (supplementary data) for the parasite proteins detection, 255 frequently observed contaminants as well as reversed sequences of all entries. The following settings were applied: spectra were searched with a mass tolerance of 7 ppm (MS) and 0. 5Th (MS/MS). Enzyme specificity was set to LysC. Up to two missed cleavages were allowed and only peptides with at least six amino acids in length were considered. Oxidation on methionine was set as a variable modification.
Bioinformatics analysis A biological process Gene Ontology analysis was performed for each kinetic time points of the host. Differentially expressed proteins of the host at each time point were annotated with their Gene Ontology Biological Process terms  [28]  (Table  S1 ). The annotation of the parasites proteome was conducted using Blast2Go (V. 2. 7. 1)  [29]  with a BlastP algorithm using an e-value of 0. 01 followed by an InterproScan step against the entire databases. The GO analysis was then finalized by merging the biological processes terms obtained after these two steps. The prediction of signal peptide cleavage sites was performed using the SignalP program, version 4. 1  [30]  set with the default parameters for eukaryotes.
Parasite proteome Among the 148 parasite proteins detected during the HFF cells-A. algerae temporal cross-talk (Table  S3 ). Most of these proteins were only detected for the 8 dpi time point, except for three: the 26S proteasome regulatory subunit 10 (detected at 6 hpi, 12 hpi, 24 hpi and 8 dpi), the endoplasmic reticulum membrane protein (detected at 6 hpi) and the glutamine amidotransferase (detected at 1 hpi, 24 hpi and 8 dpi). From the 148 A. algerae proteins, 20 showed no significant similarity with proteins in the RefSeq nonredundant database (nr)  [31] , even with an e-value threshold of 0. 01. This suggests that these sequences are quite divergent from other known organism sequences. The SignalP program predicted the presence of a N-terminal signal peptide for 18 A. algerae proteins (Table  S3 ).
Discussion To date, the available proteomics data on host-parasite interactions are mainly about the host's response to the parasite  [4, 5, 32, 33]. Both the low parasite size of intracellular parasites and the low amount of parasite proteins compared to their host cells can explain the difficulty to extract enough parasite proteins from host cells suitable for detection during the kinetics of the hostparasite cross-talk. For the first time the host-parasite cross-talk was investigating in the context of an eukaryotic intracellular parasite using the highly sensitive and quantitative SILAC approach. We identified 148 parasite proteins most of them being detected at 8 dpi. The parasite proteins with known functions were mostly involved in fundamental biological processes and reflect the successfully multiplication of the parasite inside the host.
This work was funded by a grant, Projets Exploratoires Pluridisciplinaires (PEPS) from the  Centre National de la Recherche Scientifique (France)  ( CNRS SILACFOOD ). J. P. was supported by a scholarship from the  Ministere de l'Education Nationale de l'Enseignement Superieur et de la Recherche (France). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
EXPERIMENTAL PROCEDURES Recombinant Production of PaAbf62A and UmAbf62A-Recombinant PaAbf62A was produced as previously described  (18). The U. maydis strain used in this study was obtained from the FGSC (Fungal Genetics Stock Center, Kansas City, KS) fungal collection. The strain was verified by internal transcribed spacer sequencing and archived under the number BRFM 1093 at the "Centre International de Resources Microbiennes," which is dedicated to filamentous fungi of biotechnological interest (CIRMCF), at the National Institute of Agricultural Research (INRA), Marseille, France. The U. maydis BRFM 1093 strain was grown on MA2 (malt extract at 2% w/v) medium. Genomic DNA was purified from the mycelium using the NucleoSpin Plant II kit (Macherey-Nagel GmbH and Co. , Düren, Germany). The NW_101116 sequence encoding the UM04309 protein (GenBank TM EAK85571) was amplified by PCR from genomic DNA using Pwo Super yield DNA polymerase (GE Healthcare) and the following primers: EcoRI, forward 5Ј-GAATTCAACCCCGAAACTGAACG-3Ј and XbaI, reverse 5Ј-TCTAGACCCAGACCGTACAAGGTG-3Ј.
0) at 37 °C in a reaction volume of 100 l. The reaction was stopped by adding 900 l of 1 M sodium carbonate (pH 9. 0). The release of 4-nitrophenol was quantified at 410 nm using a molar extinction coefficient for 4-nitrophenol of 18,300 M Ϫ1 cm Ϫ1. One unit of enzyme activity was defined as the amount of protein that released 1 mol of arabinose per min. The apparent optimal pH was estimated using pNP-Ara in the pH range 2. 6 to 7. 5. The apparent optimal temperature was estimated in the 40 to 70 °C range under the conditions described above. For determination of Michaelis-Menten constants, the initial velocities of the enzymes were measured in 50 mM sodium phosphate buffer (pH 5. 0) at 37 °C, with pNP-Ara concentrations ranging from 0. 1 to 20 mM. The kinetic parameters were estimated by using weighted nonlinear least squares regression analysis with Grafit software (Erithacus Software, Horley, United Kingdom).
Ϫ1/K d corresponds to the y value when x ϭ 0  (19). Inhibition Studies-Inhibition was assayed by measuring the arabinofuranosidase activity of the enzymes using pNP-Ara as substrate in the presence of cello-oligosaccharides. Cello-oligosaccharides were added to the reaction mixture at concentrations ranging from 0 to 300 mM (for cellobiose) and from 0 to 8 mM (for C3, C4, C5, and C6) at pH 5. 0 and 37 °C. The extent of inhibition was the relative enzyme activity (ratio of activity with inhibitor versus activity without inhibitor). Following the same procedure, the inhibition constant (K i ) was determined using C6 with 6 different concentrations ranging from 0. 5 to 8 mM. Data were fitted to the Michaelis-Menten equation to generate an estimate of K i , using the Grafit data analysis software.
The Mascot Daemon software (version 2. 2. 0, Matrix Science) was used to perform database searches against all entries in a homemade database containing the PaAbf62A protein sequence. Carbamidomethylation of cysteines, oxidation of methionines, acetylation on N-terminal protein, and HexNAc of asparagine were set as variable modifications for Mascot searches, without enzyme cleavage site. The mass tolerances in MS and MS/MS were set to 10 ppm and 0. 8 Da, respectively, and the instrument setting was specified as "ESI-Trap. " Size Exclusion Chromatography-200 l of 2 mg ml Ϫ1 of pro- teins in 50 mM sodium acetate buffer (pH 5. 0), 50 mM NaCl were loaded onto a Superdex 75 column (GE Healthcare, Life Sciences) and eluted with the same buffer at a flow rate of 0. 7 ml min Ϫ1. Calibration of the column was performed using the Gel Filtration LMW Calibration Kit (GE Healthcare, Life Sciences).
Conditions were further optimized manually using the hanging-drop vapor-diffusion method in 24-well plates. Best crystals of PaAbf62A were obtained with a 1:1 (v/v) ratio of protein (18 mg ml Ϫ1 in 50 mM sodium acetate (pH 5. 0) and 50 mM NaCl) to precipitant solution (25% (w/v) PEG 4000, 0. 2 M calcium chloride, and 0. 1 M Tris pH 8. 5). Crystals appeared within a week and grew to an approximated size of 0. 25 ϫ 0. 2 ϫ 0. 05 mm 3. Crystals of UmAbf62A were obtained by mixing a equal volume of protein (18 mg ml Ϫ1 in 50 mM sodium acetate (pH 5. 0) and 50 mM NaCl) and reservoir solution (20% (w/v) PEG 3350 and 0. 2 M sodium phosphate). Crystals of ϳ0. 30 ϫ 0. 30 ϫ 0. 20 mm 3 appeared within 6 days. Data Collection and Phasing-X-ray experiments were carried out at 100 K. Prior to flash cooling, optimized native crystals of UmAbf62A and PaAbf62A were soaked for a few seconds in the reservoir solution supplemented with 10% (v/v) glycerol to avoid ice formation.
Diffraction data were collected in-house (Xcalibur Nova, Agilent Technologies) for PaAbf62A in complex with cellotriose, on beamlines ID23-eh1 (PaAbf62a native structure) and ID14eh1 (UmAbf62a-L-arabinofuranose complex) at the European Synchrotron Radiation Facility (Grenoble, France) and on beamline PROXIMA 1 at.
Structural Characterization of GH62 ␣-L-Arabinofuranosidases FEBRUARY 21, 2014 • VOLUME 289 • NUMBER 8 SOLEIL (Gif sur Yvette, France) for the native UmAbf62A structure. The diffraction data were processed scaled and merged using the XDS package  (21). The native structure of UmAbf62A was solved by the sulfur-SAD phasing method using data collected in-house at 1. 7-Å resolution from a single crystal (Table  1 ). A highly redundant data set was collected at the Cu-K␣ wavelength covering 1425°o f total rotation with 1°oscillation width per frame. A native dataset was collected to 1. 0 Å on beamline PROXIMA 1 at SOLEIL. The anomalous signal of sulfur atoms from 6 methionine residues and 2 cysteine residues was evaluated and the eight sulfur atom positions and occupancies were determined and refined with SHELX C/D  (22, 23).