dataesr/scientific-paragraphs-categorization huggingface dataset:
Representative result from a biological triplicate; methylene blue staining serves as loading control. (D) Ten previously unknown positions (tRNAmodviz) on six E. coli tRNA species are found by Rho-seq to be dihydrouridinated. (E) Left panel: distribution of the 63 unique tRNA D-sites identified by Rho-seq. NA, not assigned due to sequence incompatibility (D22 on tRNA Ser GGA ). Right panel: distribution of the 74 unique tRNA D-sites as reported by the tRNAmodviz database. (Figure  5B ; Table  S2 ). These data indicate that the complete absence of D does not globally impact TEs, at least in the tested condition. Examination of individual genes revealed that nda2, cct3, cct7, and eng1 that were isolated in Rho-seq were translationally upregulated when D was absent (Figures  5B  and S8C ). Considering that the modified uridine represents only a subset of the population for a given codon, we did not expect strong effects on the relative codon occupancy of all codons in the D4 compared to the WT.
Another question arising from our work is the role of D within mRNAs. Our in vitro and in vivo data are compatible with D affecting translation kinetics, which in turn may affect co-translational folding. In this respect, it is striking that mRNA encoding cytoskeleton-related proteins that form highly structured complexes are found in the Rho-seq dataset  (Buhr et al. , 2016; Zhang et al. , 2009; Zhou et al. , 2013). Finally, the present work reveals the biological relevance of the D modification of one codon within the nda2 mRNA that encodes a-tubulin by showing that it is critical for meiotic progression, though it does not seem to affect mitotic progression. A similar meiotic-specific defect was also reported for the F200Y mutation in b-tubulin encoded by the nda3 gene. Intriguingly, Rho-seq also identified nda3 as a dihydrouridylated mRNA with the modified codon (U 586 CU encoding S196) being in the immediate proximity of F200.
Materials availability All unique/stable reagents generated in this study are available from the Lead Contact without restriction. Data and code availability d The Rho-seq, Ribo-seq and RNA-seq raw and processed data are available on GEO under the accession number GSE145686 and are publicly available as of the date of publication. Original blot images reported in this paper have been deposited at Mendeley and are publicly available as of the date of publication. The DOI is listed in the Key resources table. d This paper does not report original code. d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
Gene list and motif analyses The S. pombe lists of genes were analyzed by the fission yeast-specific platform called AnGeLi  (Bitton et al. , 2015). The sequence motif analyses were performed with the web-based application WebLogo  (Crooks et al. , 2004). Codon occurrence in the S. pombe transcriptome The probability to get a D-site on the coding S. pombe transcriptome was calculated based on the codon usage database as follows; there are 27 codons containing a single U, representing 1,099,338 occurrences, 9 codons containing two U residues, representing 612,597 occurrences and 1 codon containing three U residues, representing 92,872 occurrences. There are therefore 2,603,148 possibilities to get a D-site on a coding region  [(1 3 1, 099, 338) + (2 3 612, 597) + (3 3 92, 872) = 2, 603, 148].
Libraries were mapped to the S. pombe transcriptome. The sequencing data was analyzed with Xtail  (Xiao et al. , 2016) , subcodon analysis was performed as described in  (Nedialkova and Leidel, 2015; Zinshteyn and Gilbert, 2013). TMT-based proteomic analyses (Creative Proteomics) Tandem mass tag (TMT) relies on chemical label allowing sample multiplexing in mass spectrometry-based quantification and identification. Isobaric mass tags are molecules with the same mass yielding reporter ions of differing mass after fragmentation. The relative ratio of the measured reporter ions represents the relative abundance of the tagged molecule. TMT6plex Isobaric Label Reagent Set, Pierce Quantitative Colorimetric Peptide Assay, was purchased from Thermo Fisher Science. An Ultimate 3000 nano UHPLC system (Thermo Scientific, Waltham, MA) coupled online to a Q Exactive HF mass spectrometer (Thermo Scientific) equipped with a Nanospray Flex Ion Source (Thermo Scientific).
Differential Protein Analysis The raw MS files were analyzed and searched against fission yeast protein database based on the species of the samples using Maxquant (1. 6. 2. 6). The parameters were set as follows: the protein modifications were carbamidomethylation (C) (fixed), oxidation (M) (variable), TMT-6plex; the enzyme specificity was set to trypsin; the maximum missed cleavages were set to 2; the precursor ion mass tolerance was set to 10 ppm, and MS/MS tolerance was 0. 6 Da. In total, 2193 proteins were identified in meiotic growth (WT, dus3D, D4), and 1974 proteins were identified in vegetative growth (WT, dus3D, D4). Proteins of relative quantitation were divided into two categories. Fold-change (FC) > 1. 2 was considered up-regulation while FC < 0. 83 (1/1. 2) was considered as downregulation.
ACKNOWLEDGMENTS We are grateful to  Eric Phizicky  for reagents and  Marc Graille  for comments on the manuscript. Research in the lab of D. L. J. L. is supported by the  Fonds de la Recherche Nationale (F. R. S. /FNRS) , the  UniversiteLibre de Bruxelles (ULB) , the  European Joint Programme on Rare Diseases (EJP-RD; RiboEurope and DBAGeneCure) , the  Region Wallone (RiboCancer) , and the  International Brachet Stift€ ung (IBS). High-throughput sequencing was performed by the  NGS platform of Institut Curie , supported by grants from the  Agence Nationale de la Recherche  (investissements d'avenir;  ANR-10-EQPX-03  and  ANR10-INBS-09-08 ) and by the  Canceropole Ile-de-France. This work was also supported by  NIH  grants  ES031529  and  AG063341  (to P. D. ) and  PDR T. 0012. 14 , CDR J. 0066.
coli Lab stock JC8679 E. coli dusA::cmR dusB-fis::kanR dusC::specR Lab stock PS5107 Chemicals Dynabeads Oligo(dT) 25 Life Technologies 61002 sodium borohydride Sigma 452882 rhodamine 110 chloride Sigma 83695 [Ɣ-32P]-ATP Perkin Elmer NEG002A250UC 35 S labeled methionine Perkin Elmer NEG709A500UC Critical commercial assays wheat germ extracts Promega L4380 Ribodepletion kit Thermo A15020 -K155005 Deposited data Rho-seq, Ribo-seq and RNA-seq raw and This work GEO: GSE145686; https://doi. org/ processed data This work 10. 17632/m6v9rcprz6. 1 Raw data (Mendeley data) Oligonucleotides tRNAiMet primer extension This work 2540 ACCTACGGGTTATGAGCCCGTCGG tRNAAspGUC (SPCTRNAASP. 06) primer extension This work 2743 GGGCTGCAAGCGTGAC SPMITTRNAARG. 02 primer extension This work 2746 CTAACAATTTCGAAGATTGTTAC gBLOCK for synthetic RNA This work N/A TAATACGACTCACTATAGGGGAGGCGAGAACA CACCACAACGAAAACGAGCAAAACCCGGTACGCA ACACAAAAGC GAACAACGCGAAAAAGGACACC GAAGCGGAAGCAAAGACAACCAACAGAAAACAA CCGCAAACAAACGGGACCAGA CAACGCACCAGCAAAA (Continued on next page).
We previously identified matrix metalloproteinase 2 (MMP2) and MMP9 plasma levels as candidate biomarkers of bevacizumab activity in patients with recurrent glioblastoma. The aim of this study was to assess the predictive value of MMP2 and MMP9 in a randomized phase III trial in patients with newly diagnosed glioblastoma and to explore their tumor source. In this post hoc analysis of the AVAglio trial (AVAGlio/NCT00943826), plasma samples from 577 patients (bevacizumab, n = 283; placebo, n = 294) were analyzed for plasma MMP9 and MMP2 levels by enzymelinked immunosorbent assay. A prospective local cohort of 38 patients with newly diagnosed glioblastoma was developed for analysis of tumor characteristics by magnetic resonance imaging and measurement of plasma and tumor levels of MMP9 and MMP2.
Prospective peri-operative patient cohorts To understand the relationship between plasmatic MMP9 and glioblastoma, we established a local prospective cohort composed by 38 adult (≥ 18 years) IDHwt glioblastoma patients included at initial diagnosis between June 2016 and October 2017 at Timone Hospital (Marseille, France). For these patients, the plasma samples were collected before and 48 h after surgical resection, as well as paraffin and frozen tumor samples. A concomitant MRI was performed at the time of diagnosis including T1, T1 with gadolinium, T2, FLAIR and perfusion sequences. Tumor volumes were analyzed using the Horos software ®. Perfusion sequences were analyzed using the Olea Medical software ®. This cohort was completed by 7 patients hospitalized for the resection of brain aneurysm and 12 healthy controls.
The following data were recorded: age, gender, type of surgery, Karnofsky Performance Status (KPS), oncological treatment, clinical symptom, steroid dose, and MRI characteristics. All samples were stored in the APHM Biological Resource Center (BRC) (authorization number AC2018-31053; CRB BB-0033-00097). Tumor and plasma samples were obtained after written consent according to a protocol approved by the local institutional review board and ethics committee. The present studies were conducted in accordance with the declaration of Helsinki.
Immunofluorescence (IF) Five µm frozen section of tumor samples were permeabilized with ethanol-acetone (19v-1v) before saturation in PBS-BSA5%. Sequential staining with anti-CD31/anti-MMP9 antibodies or anti-CD45/anti-MMP9 antibodies were done (CD31, JC70A, Dako, 1/50-CD45, HI30, Invitrogen, 1/200-MMP9, ab38898 Abcam, 1/1000). Secondary antibodies were purchased from Molecular Probes ™ , goat anti mouse-AlexaFluor 488 (A11001) for antibodies targeting CD31 and CD45 and goat anti rabbit-AlexaFluor 568 (A11011) for anti-MMP9 antibody. Nuclei were stained with Hoechst 33,342 (B2261, Sigma, 1/1000). Fluorescent microcopy pictures were done with Zeiss ® Observer. Z1 and ZenPRO software and analysis were done with ImageJ software.
Mann-Whitney U-test or Kruskal-Wallis test were used to compare quantitative values; qualitative values were analyzed using Fisher exact test and Chi 2 test. Correlations were analyzed using the Spearman test. All reported p-values are two-sided, and p < 0. 05 was considered statistically significant. This study followed the REMARK criteria. All statistical analyses were performed using SPSS software v22 ®.
Then, because MMP9 was reported to be involved in invasive processes through extracellular matrix remodeling, we analyzed the infiltrative pattern of glioblastoma using the ratio enhancement volume/FLAIR volume. This ratio was not correlated with MMP9 plasma level (Fig. 3E ). Finally, no correlation was found between circulating levels of MMP9 and perfusion characteristics (Fig. 3F, G ). Regarding the MMP9 activity evaluated by zymogram, no correlation was observed between MMP9 activity and FLAIR volume (p = 0. 284), contrast enhancement volume (p = 0. 325) or infiltrative pattern, as previously defined (p = 0. 774). Of note, no correlation was observed between MMP2 plasma level and neuroimaging characteristics (data not shown). These results suggest that tumor growth, invasion, and vascularization seem to be independent of MMP9 level, suggesting that MMP9 could be related to another glioblastoma microenvironment process.
The status of the disease (recurrent versus newly diagnosed) and the patient's dataset issue from a large prospective randomized study may contribute to the differences between these two studies. We also showed that these biomarkers were correlated with disease control under treatment with plasma level modifications at the time of recurrence  [7]. Taken together, these results would allow the use of these biomarkers both at baseline, to select responders, and during treatment, to identify bevacizumab escape early. Advantages of these markers are that they are accessible (a simple blood test), inexpensive, fast, easy to use, and reproducible. Validation of the predictive value of MMP9 in a prospective trial is a major step toward its potential routine clinical use. These results raise new questions about the appropriate cutoff for clinical use and the potential use of MMP9 with other antiangiogenic agents and in other tumor types.
Acknowledgements We thank the  ARTC-Sud  patients's association ( Association pour la Recherche sur les Tumeurs Cérébrales-Sud ), the  ARC Foundation  and the  Cancéropole PACA  that granted this work (to ET). We thank the  AP-HM Tumor Bank  (Authorization Number:  AC2018-31053 ;  CRB BB-0033-00097 ) for providing tissue samples. We thank  ANR  for the  ANR-20-CE-CE14-0028-01  grant (to RJA).
Data analysis Image analysis. Manual shape measurements. Fiji  [66]  is used to measure cell, embryo, pipette sizes, and wave velocity. The circle tool is used to fit a circle onto cells, embryos, and pipettes. The line tool is used to fit lines onto curvature kymographs. PIV analysis. To detect PeCoWaCo in phase contrast images of embryos, we use PIV analysis followed by a Fourier analysis. As previously  [22, 40] , PIVlab 2. 02 running on MATLAB  [67, 68]  is used to process approximately 10 minutes long time lapses with images taken every 5 seconds using 2 successive passes through interrogation windows of 20/10 μm resulting in approximately 180 vectors per embryo. The x-and y-velocities of individual vectors from PIV analysis are used for Fourier analysis. A Fourier transform of the vector velocities over time is performed using MATLAB's fast Fourier transform function.
Since PeCoWaCo halt during mitosis (S2 MovieAU : PleasenotethatMovie2hasbeenchangedtoS2Mov ), time lapses including dividing cells were excluded from the analysis. Local curvature analysis. To measure PeCoWaCo period, amplitude, and velocity, we analyze the associated changes in surface curvature and perform Fourier analysis. Importantly, since we can only extract these parameters from oscillating blastomeres and embryos, data shown in Fig  3   To obtain the local curvature of isolated blastomeres and embryos, we developed an approach similar to that of  [22, 36, 70]. First, a Gaussian blur is applied to images using Fiji  [66]. Then, using ilastik  [71] , pixels are associated with cell surface or background. Segmentations of cells are then used in a custom made Fiji plug-in (called WizardofOz, found under the Mtrack repository) for computing the local curvature information using the start, center and end point of a 10-μm strip on the cell surface to fit a circle.
The strip is then moved by 1 pixel along the segmented cell, and a new circle is fitted. This process is repeated till all the points of the cell are covered. The radius of curvature of the 10-μm strip boundaries are averaged. Kymograph of local curvature values around the perimeter over time is produced by plotting the perimeter of the strip over time. Curvature kymographs obtained from local curvature tracking are then exported into a custom made Python script for 2D Fast Fourier Transform analysis. Spectra of individual cells are checked for the presence of a distinct amplitude peak to extract the oscillation period. The peak value between 50 seconds and 200 seconds was taken as the amplitude, as this oscillation period range is detectable by our imaging method. To measure the wave velocity, a line is manually fitted on the curvature kymograph using Fiji.
Cortex thickness measurement. Super resolution images obtained using airyscan microscopy are used to measure cortex thickness. The full width at half maximum of cortical intensity profiles were used to assess cortical thickness by using CortexThicknessAnalysis tool  [43]  available at https://github. com/PaluchLabUCL/CortexThicknessAnalysis. Statistics. Data are plotted using Excel (MAU : PleaseprovidethemanufacturerlocationforMicrosoft icrosoft) and R-based SuperPlotsOfData tool  [72]. Mean, standard deviation, median, 1-tailed Student t test, and chi-squared p-values are calculated using Excel (Microsoft) or R (R Foundation for Statistical Computing). Statistical significance is considered when p < 10 -2. The sample size was not predetermined and simply results from the repetition of experiments.
Acknowledgments We thank the imaging platform of the  Genetics and Developmental Biology unit at the Institut Curie (PICT-IBiSA@BDD)  for their outstanding support and the animal facility of the  Institut Curie  for their invaluable help. We thank the  Matic Vignjevic, Plastino, Lennon-Dumenil , and Bardin teams for sharing reagents. We thank  Victoire Cachoux  and  Julie Firmin  for help with image and data analysis. We are grateful to  Ido Lavi ,  Sophie Louvet-Valle  ´e, and members of.
JLM was funded by the  Centre National de la Recherche Scientifique (CNRS) ;  Institut National de la Santeet de la Recherche Medicale (Inserm) ;  Fondation Schlumberger pour l'Education et la Recherche (FSER) ; EC | H2020 | H2020 Priority Excellent Science | H2020 European Research Council (ERC) ERC-2017-StG 757557 ;  European Molecular Biology Organization  - Young Investigator program ;  Universitede Recherche Paris Sciences et Lettres  (PSL)  17-CONV-0005 ;  Universitede Recherche Paris Sciences et Lettres (PSL)   ANR-11-LABX-0044 ;  Institut Curie. O ¨O ¨was funded by  EC | H2020 | H2020 Priority Excellent Science | H2020 Marie Skłodowska-Curie Actions  ( MSCA )  666003 ;  Fondation pour la Recherche Medicale (FRM)   FDT202001010796 ;  Institut Curie LP, VK, AFT  were funded by  Institut Curie.
Data can be downloaded and used under a CC BY-NC-SA license at https://ressources. curie. fr/pecowaco/. The code used to analyze the oscillation PLOS BIOLOGY frequencies from PIV and local curvature analyses can be found at https://github. com/MechaBlasto/ PeCoWaCo. git. The Fiji plugin for local curvature analysis WizardofOz can be found under the MTrack repository.
Clinical data We recruited 126 families with an undiagnosed GWMD. Based on cranial MRI findings, 86 cases (68%) were classified as non-hypomyelinating, whereas 40 of the cases showed a hypomyelinating picture. The index cases were 50 females and 76 males, with ages ranging from 1 month to 74 years (median 10. 3 years). The age at onset ranged from the first month of life to 72 years (median one year); age was lower than 18 years in 101 cases (80%) and higher in 25. The median evolution of disease before WES testing was 6. 3 years (1 month -34 years), and it was longer than 10 years in 37% of patients. Consanguinity was reported in 18 families (14%). Clinical characteristics, MRI patterns, studies performed and sWES-WGS results of every patient are summarized in Table  1  and eTable 1.
We also identified a uniparental disomy of maternal origin of chromosome 6 in LNF-68, harboring a loss-of-function homozygous variant in a novel candidate gene that was highly ranked by our prioritization method (in preparation). An added value of our study is that 73 of the 123 identified variants had not been previously reported in the literature, Human Gene Mutation Database (HGMD, public access), or ClinVar databases (eTable 7).
Results Detection of BrdU incorporated into replicating DNA. We previously developed the RepNano software to detect BrdU on sequencing reads produced by Oxford Nanopore Technologies (ONT) devices  13. Here we anchored our BrdU detection process in ONT's Megalodon program (https://github. com/nanoporetech/ megalodon) and estimated BrdU incorporation probability at each thymidine site rather than over 96 bp windows as in RepNano  13  (see the 'Methods' section). Megalodon combines Guppy (ONT) GPU-accelerated basecalling and read mapping to the reference genome into one single step, making it a fast, straightforward and easy-to-use pipeline. Its neural network was trained using nanopore reads of genomic DNA from thymidineauxotroph MCM869 yeast cells  14  grown with 11 different proportions of BrdU in the culture medium, from 0 to 100% with increments of 10% ('Methods').
Determining fork speed is mandatory in circumstances known or speculated to influence replication dynamics. 1, 25, 26, [47] [48] [49] [50]  ), mathematical modelling of DNA replication (e. g. , refs. 24, 44  ) and time-resolved (i) dense-isotope substitution experiments (for instance, see ref. 51  ), (ii) two-dimensional gels 27 , (iii) chromatin immunoprecipitation of replisome components  12, 23, 26, 27  or (iv) isolation of BrdU-or 5-ethynyl-2'-deoxyuridine-labelled DNA  52, 53. All these approaches are demanding and difficult to implement, and none of them allows the mapping of individual fork velocities on entire genomes. Not only does NFS have this ability, but it also combines a straightforward protocol with a ready-to-use analytical pipeline (available on GitHub at https:// github.
Data were collected using CytExpert v2. 4. 0. 28 and analysed using FlowJo v10. 7. 1. Gating strategy is illustrated in Supplementary Fig. 13. Rad53 immunoblot analysis. Total protein extracts were obtained by trichloroacetic acid (TCA) precipitation, separated by SDS-PAGE on a 7. 5% gel and transferred to a nitrocellulose membrane. Rad53 immunoblot was performed with rabbit anti-Rad53 antibody at 1:10,000 (Abcam #104232, batch GR3353005-2), using HRP-conjugated anti-rabbit (Promega #W401B) at 1:5000 as secondary antibody. Detection was performed with SuperSignal West Pico (Thermo Scientific #34078) chemiluminescent reagents. ImageQuant LAS 4000 mini (GE Healthcare, software version 1. 3) was used for imaging. Samples used for neural network training. Neural network training was performed using nanopore-sequenced genomic DNA displaying variable BrdU substitution rates extracted from the thymidine-auxotroph MCM869 strain.
13. Samples were analysed in technical duplicates using the software TraceFinder (Thermo Scientific, version 5. 1). Pulse-chase labelling. In most experiments, BT1 cells and its derivatives as well as BT2 and BT3 strains were grown overnight in YPD, diluted in fresh medium at an OD 600 ≈ 0. 1 and pulsed after 3 doublings (OD 600 ≈ 0. 8) with 100 μM BrdU for 2 min followed by a 20 min incubation with 1 mM thymidine. For the experiment carried out with different BrdU concentrations, BT1 cells were pulsed in the same conditions as above with either 10, 25, 50, 100, 250, 500, 750 μM or 1 mM BrdU and chased with a ten-fold excess of thymidine. For experiments in the presence of HU, cells were grown for 2 doublings (OD 600 ≈ 0. 4) and treated for 1 h with HU prior to BrdU pulse-labelling. MCM869 cells were cultured overnight in YPD supplemented with 100 μM thymidine, diluted in fresh YPD with thymidine at an OD 600 ≈ 0.
1 and grown to OD 600 ≈ 0. 8 before being washed twice with YPD to remove thymidine, transferred to YPD for 30 min, pulsed with 100 μM BrdU for 2 min and chased with 1 mM thymidine for 45 min. Cells were pelleted after the thymidine chase, washed with water before DNA extraction as in ref. 57. Library preparation and data acquisition. All samples were sequenced using R9. 4. 1 chemistry flow cells from Oxford Nanopore Technology (ONT). MinION and GridION sequencing libraries were prepared using ligation sequencing kits SQK-LSK108 or SQK-LSK109 (ONT) in combination with ONT EXP-NBD103 Native Barcoding kit or ONT EXP-NBD104 Native Barcoding Expansion 1-12 pack in case of multiplexing according to ONT protocols with the modifications presented in ref. 57. PromethION sequencing libraries were prepared using the ultra-long DNA sequencing kit SQK-ULK001 (ONT) according to ONT protocols.
Data were acquired using MinKNOW (ONT, MinKNOW Core versions 1. 14. 1 to 4. 4. 13, Supplementary Data 1) with default parameters. Demultiplexing of barcoded nanopore reads was performed as described in ref. 57. Training of the BrdU basecalling model. Our model was built in three successive steps. First, we modified the architecture of RepNano convolutional neural network  13   RepNano v2 architecture. We used two convolutional layers with kernel size 7 and filter size 32 with no padding followed by a long short-term memory (LSTM) layer with 32 hidden units that output a vector of the same length as the input, then a convolutional layer with kernel size 1 and filter size 1 and finally an averaging layer outputting the BrdU percent of the segment. The initial length of the input vector was 112 so that after two convolutions without padding the final length was 100.