dataesr/scientific-paragraphs-categorization huggingface dataset:
Gene Expression Analysis by qPCR Dissected hippocampi were snap frozen in liquid nitrogen and ground into powder for total mRNA extration using the RNAble kit (Eurobio laboratories, Les Ulis France). Quantitative real-time PCR (qPCR) analysis was carried out using the ABI Prism R 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA) as described previously  (Bieche et al. , 2004). As an endogenous RNA control, transcripts of the TATA-box binding protein gene (TBP) were measured. Primers for TBP and the other gene were chosen with the assistance of the Oligo 5. 0 computer program (National Biosciences, Plymouth, MN) (Supplementary Table  S2 ). Target transcript levels (N target ) were normalized according to TBP content and then to a basal mRNA level following the equation N target = 2.
, 2010). Data were collected separately both in positive (ESI + ) and negative (ESI -) ion mode. ESI source parameters were as follows: source temperature 120 • C, desolvation temperature 450 • C, cone gas flow 20 L/h, desolvation gas flow 800 L/h, capillary voltage 2,400 V (ESI -), 3,000 V (ESI + ), cone voltage 45 V (ESI -), and 30 V for (ESI + ). The mass spectrometer was operated in the MS E mode of acquisition for both polarities. Two independent acquisitions functions are automatically created in MS E mode. The first function set at 5 eV collects data on unfragmented ions while the second function collects fragmentation data by using a collision energy ramp from 20 to 50 eV. In addition MS 2 experiments were performed to confirm structural identification of ions of interest. Centroided accurate mass spectra were acquired over the m/z range 50-1,000 with a scan time of 0.
Data Processing Raw data files (. raw format) acquired on the UPLC-MS E platform were converted to NetCDF format (Waters Databridge software). Deconvolution of data was performed on XCMS online software with parameters settings suitable for high resolution UPLC-MS data acquired in centroid mode  (Smith et al. , 2006; Tautenhahn et al. , 2012b). Subsequent to data pre-processing, a table listing peak intensity associated to a unique retention time and m/z as identifiers (tR_m/z data pairs) vs. samples, blank and QC, was generated in both ionization modes. After normalization of the variables to the total intensity and protein concentration, the final dataset was analyzed by multivariate data analysis. The discriminating lipid species identified after supervised analyses were quantified by normalizing the intensities of their peaks to the intensity of the peaks of the corresponding internal standards spiked into the sample prior to lipid extraction and expressed as pmol/mg proteins.
Lipid Nomenclature The abbreviations used were according to LipidMaps recommendations  (Fahy et al. , 2005 (Fahy et al. , , 2009)). Glycerophospholipids and diacylglycerol were annotated as <lipid subclass>(<total fatty acyl chain length>: <total number of unsaturated bonds>). Sphingolipids were annotated as <lipid subclass>(<sphingoid base residue>/<fatty acyl residue>). Sulfoglycosphingolipids or sulfatides were annotated according to their common name described in Lipid Maps structure database. Lipid identification Lipid species annotation was performed through the online database LIPID MAPS (http://www. lipidmaps. org/) and METLIN Metabolite Database (metlin. scripps. edu) using 5 ppm of mass accuracy as a tolerance window  (Sud et al. , 2007; Tautenhahn et al. , 2012a). Lipid species structure was confirmed by MS E fragmentation spectra analysis and selected MS 2 experiments.
MS 2 data analysis highlights product ions, which are characteristic of lipid class and can serve to discriminate between database hits. The analytical reliability of each ion signal of interest was checked by calculating its relative standard deviation (% r. s. d. ) in QC samples  (Chan et al. , 2011). Electron Microscopy 4 weeks after injection, C57Bl/6 mice were lethally anesthetized and perfused with 4% paraformaldehyde and 2. 5% glutaraldehyde in phosphate buffer (PB) 0. 12 M pH 7. 4. Brains were post-fixed in 2. 5% glutaraldehyde in PB. The eGFP fluorescent region from the hippocampus of C57Bl/6 mice injected with AAV-scramble or AAV-shCYP46A1 was excised under a fluorescence dissecting binocular microscope (Leica Z16 APO). Sections of 50 µm were cut with a vibratome and post-fixed in 1% osmium tetroxide for 30 min, rinsed in PB, dehydrated in a graded series of ethanol solutions (75, 80, 90, and 100%), infiltrated with EponTM812, placed in molds and the resin was cured at 60 • C in a dry oven during 48 h.
The slides were then mounted in Vectashield R mounting medium containing DAPI (Vectorlabs) and examined by fluorescence or confocal microscopy. For fluorescence microscopy, images were taken with a Nikon microscope (Eclipse 800) and a digital QIMAGING camera (CCD QICAM cooled plus RGB filter pixel 4. 65 × 4. 65 µm). Control and test slices were processed the same day and under the same condition. Images were acquired by confocal microscopy using Zeiss LSM 510 Meta confocal laser microscope with a Plan apochromat 63x/1. 4 numeric aperture oil immersion objective using the LSM 10v4. osp2. The number of LAMP-1 positive lysosomes in pyramidal neurons of CA3a region was measured in 60 cells per mouse (n = 5 mice per vector, six sections) using Image J software.
Statistical Analysis Experimental values are presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism software version 5. 0 (San Diego, CA). The significance of cholesterol and oxysterols concentration was determined using Mann-Whitney test with Bonferroni correction. Unpaired t-test was performed for gene expression analysis. In all cases, * p < 0. 05, * * p < 0. 01, and * * * p < 0. 001.
Multivariate Data Analyses To investigate the consequences of neuronal cholesterol accumulation on hippocampus lipidome, we performed an untargeted lipidomic analysis 4 weeks after AAV5-vector injection. The lipid contents of hippocampus tissue from control or AAV-shCYP46A1 mice, 4 weeks after injection, were analyzed by UPLC-ESI-MS E in negative and positive ion mode (Supplementary Figure  2 ). First, UPLC-MS data set obtained from ESI + and ESI -were processed by XCMS software. This processing step generates a list of variables annotated with tR_m/z ions pairs. After data set normalization, the data analysis is followed by multivariate analysis to identify the most discriminating lipids between the two groups. To easily visualize any clustering between the different sample groups between AAV-scramble (control) and AAV-shCYP46A1 samples, an unsupervised analysis was performed.
Lipid Annotation and Identification The variables selected, characterized by tR_m/z ions pairs, were annotated using LipidMaps and Metlin online databases, according to the exact mass. Among 709 and 234 variables detected, respectively in ESI -and ESI + , 22 lipid species in total were annotated (Tables  1, 2 ). These 22 lipids species in AAV-shCYP46A1 hippocampus were increased compared to AAVscramble. No decrease of any lipid species was observed. The untargeted analysis revealed an increase in glycerophospholipids (PE, PE-P, PC) and glycerolipids (DG) and sphingolipids (Cer, GlcCer, sulfatide) in AAV-shCYP46A1 injected hippocampus (Tables  1, 2 ). Confirmation of lipid identity was achieved by the analysis of the spectra acquired in MS E and MS 2 modes. The identities of the glycerophospholipids PC, PE, and PE-plasmalogens (PE-P) were confirmed by monitoring their respective characteristic polar head group ions: m/z 184.
ACKNOWLEDGMENTS The post-doctoral position of SAy was funded by  Agence Nationale de la Recherche  ( French-Canadian Cooperation-Project Cholesterol Alzheimer disease ,  2010-MALZ-10303 ). The PhD position of FD was funded by the  Ministère de l'Enseignement Supérieur et de la Recherche  and the  University Paris Descartes. The PhD position of MG was funded by  Technologie Servier (Orléans, France). OL is indebted to  Fondation pour la Recherche Médicale, Région-Île-de-France  and  Centre National de la Recherche Scientifique  for their financial support.
In any case, our data also suggest that branched dendritic networks alone without the action of formins or additional polymerases will not be efficient in counteracting the forces needed for effective protrusion, as for instance squeezing of lamellipodia-like structures through narrow pores or in between cells in 3D migration in vivo. Consistent with this view, overexpression of both FMNL2 and FMNL3 formins was previously found to correlate with invasion and metastasis of colorectal carcinoma  59, 60. Instead of promoting the collective elongation of all filaments within the network, we conclude FMNL2/3 to nucleate and elongate individual filaments, the presence of which is essential for rapid and smooth protrusion as well as efficient cell migration. Thus, our data also imply that in spite of homogenized rates of polymerization at the membrane, lamellipodial networks cannot be simply viewed as sole mass of homogenous filaments, but instead as collection of branched and unbranched filament populations of various kinds all harbouring specific features.
Truncated variants such as EGFP-FMNL3DDAD were generated by PCR with respective primers using EGFP-FMNL3 as template. For generation of FMNL2 cDNA and EGFP-FMNL2DDAD, see ref. 29. EGFP-FMNL2-A272E and EGFP-FMNL3-A275E were generated by site-directed mutagenesis using Qiagen site-directed mutagenesis kit employing forward primer 5 0 -GTCTTAGAACTGTTGGCAGAGGTTTGTCTTGTCAGAG GCG-3 0 (in case of FMNL2) and 5 0 -TGGAGCTGCTGGCAGAGGTGTGTTTGG TGCGG-3 0 (in case of FMNL3), and the respective complementary sequences as reverse primers. For generation of FMNL1 expression constructs, the cDNA of full-length FMNL1 (isoform 1, residues 1-1100), UniProt accession number O95466, was purchased as synthetic expression construct from GeneArt (Life Technologies). For EGFP-tagging, the full length sequence was amplified using forward primer 5 0 -GAGGAATTCATGGGCAATGCTGCCGG-3 0 and reverse primer 5 0 -GAGGGATCCCTAGTGGTGGTGATGATGG-3 0 harbouring a stop codon, and ligated into EGFP-C2 using EcoRI and BamHI restriction enzymes.
Acknowledgements This work was supported in part by the  Deutsche Forschungsgemeinschaft (DFG) , grants  RO2414/3-2  and  RO2414/5-1  (to K. R. ),  FA330/9-1  and  FA33010/1  (to J. F. ),  GE976/7-2  (to M. G. ),  SCHN430/6-2  (to H. -J. S. ) as well as the  Seventh Framework Programme  grant FORCEFULACTIN of the  European Research Council  (to M. -F. C. and K. R. ). M. G. and H. -J. S. are members of DFG Excellence Clusters ImmunoSensation and Cells in Motion, respectively. We would like to thank  Lae ¨titia Kurzawa  and  Laurent Blanchoin  for sharing unpublished data,  Vic Small  and  Martin Falcke  for advice,  Pontus Aspenstro  ¨m,  Laura Machesky  and  Roland Wedlich-So  ¨ldner for DNA constructs,  Markus Sommerfeld  for help with AFM measurements, and  Gerd Landsberg  and  Brigitte Denker  for excellent technical assistance.
In strain MK1 this mutation was also present. However, in the third position of the same codon, another point mutation occurred. An adenine to thymine transversion changed the stop codon into a cysteine codon, TGT (Fig. 2 ). With the restoration of the gene, strain MK1 recovered the ability to utilize erythritol. Thus, we decided to further investigate YALI0F01562g. Comparative analysis with yeast databases. Comparing YALI0F01562g translated into the protein sequence with the NCBI databases via the BLASTp algorithm showed that the protein contains sequences resembling two putative conserved domains: a GAL-4 like binuclear cluster DNA-binding domain at the beginning of the sequence (approximately codons 20-65) and a fungal specific transcription factor domain (codons 279-560)  13, 14. Search for a homology to whole known proteins showed some similarity to the C6 transcriptional factor from Rhodotorula toruloides, and to a pathway-specific nitrogen regulator from Moniliophthora roreri and Cryptococcus neoformans.
6%]), followed by generalized myoclonic seizures (16 subjects [40%]). Informed consent was obtained for all subjects in accordance with sitespecific institutional review boards. Our cohort represents a highly selected group of individuals without mutations in genes known to be associated with epilepsy (n ¼ 545), including the 51 established EEassociated genes. Variants within co-expressed candidate genes (n ¼ 20; Table  S1 ) were extracted from exome sequencing data. On average, 96% (83%-100%) of the coding region was sequenced >10-fold (Table  S1 ). Variant annotation was performed with ANNOVAR,  18  which integrated data from a variety of public databases. Highly penetrant de novo mutations are not expected in healthy individuals, and we assessed only extremely rare variants (%1 carrier in the Exome Aggregation Consortium [ExAC] Browser), including small indels and missense, nonsense, and splicing changes (55 bp).
Sanger sequencing in probands and both parents (when available) validated a total of seven non-synonymous variants in seven different genes (Table  1 ). Variants in four genes, GABBR2 (MIM: 607340), ANK3 (MIM: 600465), NBEA (MIM: 604889), and ACOT4 (MIM: 614314), were inherited from unaffected parents, so pathogenicity is doubtful. A variant of unknown significance was identified in PLXNA1 (MIM: 601055); whereas the mother is not a carrier, paternal DNA was not available. The remaining two mutations were de novo: (1) c. 244G>A (p. Gly82Arg) in SLC1A2 (solute carrier family 1 member 2 [MIM: 600300; GenBank: NM_004171. 3]) was identified in a 6-year-old boy (subject A) with seizure onset at 6 weeks and (2) c. 394C>T (p. Arg132Cys) in YWHAG (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma, also known as 14-3-3g [MIM: 605356; GenBank: NM_012479.
387C>G [p. Asp129Glu]),  4  subjects E and F (YWHAG c. 394C>T [p. Arg132Cys]), and DDD4K. 00159 with a neurodevelopmental disorder (YWHAG c. 398A>C [p. Tyr133Ser])  19  (Table  1 ). All mutations were de novo and resulted in substitutions that involve highly conserved amino acids and are predicted to be damaging by multiple in silico algorithms (Combined Annotation Dependent Depletion [CADD] and Rare Exome Variant Ensemble Learner [REVEL]). None have been reported in public databases (gnomAD Browser), and one mutation (YWHAG c. 394C>T) is recurrent in three subjects (B, E, and F). We used denovolyzeR  24  to evaluate the excess of de novo mutations in YWHAG. Assuming that 6,869 individuals were included, we observed a highly significant enrichment of heterozygotes (0. 2 expected versus 7 observed; p ¼ 5.
25 ] [28] [29]  EAAT2 p. Gly82Arg (subject A) substitutes a small hydrophobic and neutrally charged glycine with hydrophilic, positively charged arginine within the cytoplasmic loop joining the first pair of transmembrane domains (Figure  1 ). The same substitution is reported in the Epi4K-EPGP dataset in a subject with infantile spasms  4  and two additional individuals with a severe EE, and de novo variants (EAAT2 p. Gly82Arg and p. Leu85Pro) were reported  33  during the preparation of this manuscript. EAAT2 p. Pro289Arg (subject C) affects a highly conserved residue in all human EAATs (EAAT1-EAAT5) and their ancestral homolog. Pro289 leads to a hinge in the middle of the fifth transmembrane domain (Figure  1 ). Its substitution to positively charged arginine is predicted to cause a major structural change of the protein.
The arginine residue at position 132 is part of a highly conserved triad of two arginines and a tyrosine (Arg132-Arg57-Tyr133) (Figure  1 ) that normally form a positively charged pocket within a binding groove for interacting phosphopeptides. 57, 60 An individual (ND27637) with Lennox-Gastaut syndrome and a de novo p. Asp129Glu variant is present in the Epi4K-EPGP dataset. 4 The Asp129 residue is also located within the 14-3-3g binding groove (Figure  1 ) and plays an important role in determining the orientation of the phosphorylated peptides. 57 A de novo mutation affecting position p. Tyr133Ser has been reported in a Deciphering Developmental Disorders cohort individual (DDD4K. 00159) severely affected by a neurodevelopmental disorder,  19  but we were unable to contact the clinician who followed this subject.
GenBank accession numbers are as follows: Homo sapiens, NP_036611. 2; Macaca mulatta, NP_001181365. 1; Mus musculus, NP_061359. 2; Gallus gallus, NP_001026648. 1; Xenopus tropicalis, NP_001072309. 1; Dario rerio, NP_998187. 1; Caenorhabditis elegans, AAA61872. 1; human 14-3-3b, NP_003395. 1; human 14-3-3ε, NP_006752. 1; human 14-3-3 z, NP_001129174. 1; human 14-3-3 h, NP_003396. 1; human 14-3-3 q, NP_006817. 1; and human 14-3-3 NP_006133. 1. Sequences were aligned with CLUSTAL Omega. 32 Asterisks indicate positions with a single fully conserved residue, colons indicate conservation between groups with strongly similar properties, and periods indicate conservation between groups with weakly similar properties. fever. Subject D's epilepsy is characterized by episodes of status epilepticus with or without fever.
1; human EAAT1, NP_004163. 3; human EAAT3, NP_004161. 4; human EAAT4, NP_005062. 1; and human EAAT5, NP_006662. 3. (B) Top: crystal structure of 14-3-3g (PDB: 5D3E). Left: dimeric 14-3-3g is shown as green ribbons, and the phosphopeptide ligand is shown as an orange stick. Right: close-up view of the binding groove, side chains of the residues crucial for the phosphopeptide binding, (legend continued on next page).
Acknowledgments We thank the parents and children involved. We gratefully acknowledge  Dr. Suzanne Vercauteren, MD, PhD  (director),  Dr. William Gibson, MD, PhD  (chair and member of the  Biospecimen Advisory Committee ),  Tamsin Tarling  (BioBank administrative manager), and the technical expertise of  Katelin N. Townsend  of the  BC Children's Hospital BioBank , which is supported by  Mining for Miracles  through the  BC Children's Hospital Foundation. We also thank the  BC Children's Hospital Department of Pathology & Laboratory Medicine  and the  BC Children's Hospital Electroencephalogram Department. Clinical research was performed as part of the  Epilepsy Genomics (EPGEN) program  on the genetics of early-onset epilepsy at  BC Children's Hospital  and the  University of British Columbia.
a GenBank accession numbers: ACOT4, NM_152331. 3; ANK3, NM_001204403. 1; GABBR2, NM_005458; NBEA, NM_015678. 4; PLXNA1, NM_032242. 3; SLC1A2, NM_004171. 3; and YWHAG, NM_012479. 3.
Patient cohorts Protein tyrosine phosphatase receptor delta (PTPRD) expression was correlated with corresponding clinical data in three independent patient cohorts from Icahn School of Medicine at Mount Sinai, New York, USA (National Centre for Biotechnology Information Gene Expression Omnibus GSE10140) (cohort A),  17  from the Hiroshima University Hospital, Hiroshima, Japan (European Genome-phenome Archive, https://ega. crg. eu, accession number, EGAD00001001880) (cohort B)  18  and from the University Hospitals of Strasbourg and Reims, France (cohort C). Antibodies and western blotting P-STAT1 mAb (58D6) was obtained from Cell Signaling Technology, β-Actin mAb (AC-15) was obtained from Invitrogen and PTPRD pAb (C-18) from Santa Cruz Biotechnology. Western blots were performed using Hybond-P membranes (GE Healthcare), visualised using ECF substrate (GE Healthcare) and quantified with a fluorescence scanner (Typhoon Trio, GE Healthcare).
All qPCRs were performed following manufacturers' instructions. Specificities of phosphatase PCR products were validated by melting curve analysis and PCR product sequencing. Differential gene expression of patient biopsies was calculated after 2 -ΔCT transformation into individual data points. The transcriptome of Jc1E2 FLAG -infected Huh7. 5. 1 cells was measured by RNA-seq at the Broad Institute as previously described. 19 All microRNA (miRNA) indicated in this study are human (hsa-miR) if not indicated differently. miRNA expression was measured using miScript (Qiagen) with forward DNA primers derived from the miRNA sequence implemented in Sanger miRBase database (V. 21. 0) and referenced with a MIMAT accession number (see online supplementary table  S2 ). miRNA target sites were predicted using miRSystem.
Preamplification, amplification and detection were performed according to provider's protocol. Images were acquired with a laser scanning confocal microscope (LSM710, Carl Zeiss Microscopy) and Zen2 software, using the same settings for all the tissues analysed. Five random fields were acquired from each section. Image analysis was performed using ImageJ and CellProfiler software, with a customised pipeline. Total number of cells, frequency of HCV-positive and PTPRD-positive cells, and signal intensity were then evaluated and exported for statistical analysis (two-way analysis of variance (ANOVA), p≤0. 05).
Bioinformatics of gene expression database Paired gene expression of 62 formalin-fixed paraffin-embedded tumour and adjacent liver tissues was explored from cohort A  17  ). Downregulation of PTPRD expression in tumour tissue was analysed in silico for 46 patients with chronic HCV. Values of p were calculated with Wilcoxon signed-rank test (two-tailed). Probability of survival and tumour recurrence were evaluated by the log-rank test available on the GenePattern Survival Analysis module (http://www. broad. mit. edu/cancer/software/ genepattern). 22 Gene Set Enrichment Analysis (GSEA) was performed as described. 23 24 The normalised enrichment score (NES) is a measure expressing to what extent the members of a gene set shift towards the top or bottom in the gene list ranked on differences in expression between two phenotypes.
The analysed fields did not show an abnormal level of infiltrating non-parenchymal cells, and the level of PTPRD expression in those cells was marginal compared with hepatocytes (data not shown). This indicates that HCV impairs PTPRD expression directly in hepatocytes. To validate this observation, we measured PTPRD expression levels in isolated PHH infected in vitro with HCV. We observed that PHH robustly express PTPRD both at the mRNA and the protein level, providing a competent model system to study PTPRD. Two days after isolation, PHH were infected for 5 days with HCVcc strain JFH1, after which cells were lysed, and both mRNA and protein were isolated. With this established protocol, up to 10%-30% of hepatocytes can be infected by HCVcc (strain JFH1) as shown  29  (see online supplementary figure S2) using a reporter red fluorescence protein-nuclear localisation sequence-interferon-β promoter stimulator lentivirus (RFP-NLS-IPS).
However, PTPRD is not expressed in hepatoma cells and cannot be rescued in our hands (data not shown), precluding the in vitro study of PTPRD on the induction of the IL6-JAK-STAT3 signalling cascade. Therefore, we analysed a publicly accessible gene expression database of 82 adjacent non-tumour liver tissue specimens of patients with HCC (cohort A). 17  Those patients with the lowest 20 percentile of hepatic PTPRD expression in adjacent tissue showed a strong and marked enrichment (NES=1. 75, FDR=0. 009) of the STAT3 transcriptional programme compared with patients with the highest 20 percentile (see figure 4B, C and online supplementary figure  S3 ), which is testimony of a PTPRD-mediated STAT3 deactivation in vivo. For 62 of these 82 patients, HCV infection status was available. We could thus confirm PTPRD expression downregulation in 46 HCV(+) biopsies compared with 16 HCV(-) biopsies (figure  4D ) and show the enhanced STAT3 activity in HCV(+) through GSEA (NES=1.