dataesr/scientific-paragraphs-categorization huggingface dataset:
Gene expression analysis Differential gene expression between tumors with PLAGfamily gene amplification and a selection of other CNS tumor types was examined using the R2 Genomics Analysis and Visualization Platform (http:// r2. amc. nl). In concordance with the observed gene amplification, ET, PLAGL tumors showed overexpression of the respective amplified PLAG-family gene as assessed by RNA-seq (Fig. 5a, c ), while both PLAGL1 and PLAGL2 are downregulated postnatally in normal brain and cerebellar tissues (https:// apps. kaess mannl ab. org/ evode voapp/)  [13]  (Supplementary Fig. 5 ). Leveraging our expression data set of 11 PLAGL1and PLAGL2-amplified tumor samples and 279 samples from other CNS tumor and normal tissue types (HGGs with H3 G34R/V or K27M mutation and GBM_pedRTK1 or 2 (n = 76), PA with BRAF fusion (n = 25), PXA (n = 25), normal brain tissue (n = 36), embryonal tumors such as ATRT, ETMR, or medulloblastomas (n = 117)), we first compared gene expression of the PLAGL-amplified tumors to our subset of embryonal tumors.
Survival analysis Clinical outcome data were available for 21 patients with PLAGL1/2-amplified tumors. Five-year and 10-year OS for patients with PLAGL1-and PLAGL2-amplified tumors as well as for male and female patients was determined. Survival rates across the cohort remained constant after 5 years, hence both 5-and 10-year OS was 66% for patients with PLAGL1-amplified tumors, 25% for patients with PLAGL2amplified tumors, 18% for male patients, and 82% for female patients, respectively. Although a trend towards a worse prognosis for patients with PLAGL2-amplified tumors was noticeable-with 5 out of 12 patients with a PLAGL2-amplified tumor being deceased compared to 2 out of 9 patients with a PLAGL1-amplified tumor (Fig. 6b )-PFS and OS did not differ significantly between the two different groups (Fig.
Acknowledgements We thank the  German Cancer Research Centre (DKFZ) Genomics and Proteomics Core Facility  and  Laura Dörner ,  Katharina Müller ,  Lea Hofmann  ( Department of Neuropathology, Institute of Pathology at the University Hospital Heidelberg ). This work was supported by the  German Childhood Cancer Foundation  ("Neuropath 2. 0-Increasing diagnostic accuracy in pediatric neurooncology"; DKS.
2g, h ). To evaluate levels of DNA damage after WGD, we compared DNA damage between tetraploid and diploid cells with replication stress. Replication stress results from the slowing or stalling of replication forks, which can be induced by high doses of aphidicolin (APH; a DNA polymerase inhibitor) or hydroxyurea  12, 13  (a ribonucleotide reductase inhibitor). APH or hydroxyurea generated similar levels of DNA damage in diploid cells, when compared with untreated tetraploid cells (Extended Data Fig. 2i ). In addition to γH2AX, we also observed a significant increase in the number of foci containing the double strand break repair factors FANCD2 and 53PB1  14  in the first interphase following WGD (Fig. 1i-l ). Further, tetraploid cells showed an increased olive tail moment in alkaline comet assays, indicating single and double strand breaks (Extended Data Fig.
DNA replication-dependent DNA damage We determined the cell cycle stage when the DNA damage occurs using the fluorescence ubiquitination cell cycle indicator (FUCCI). During G1, the number of γH2AX foci was quite low and similar to that found in controls. As tetraploid cells entered S phase, we observed a slight increase in the number of foci, which increased substantially at the end of S phase (Fig. 2a , b, Extended Data Fig. 3e, f ). These results were further confirmed by time-lapse imaging using H2B-GFP to visualize DNA and 53BP1-RFP (Extended Data Fig. 3g , h, Supplementary Videos 1, 2). To confirm that DNA damage in tetraploid cells appeared during S phase, we blocked cells at the G1/S transition using high doses of inhibitors of CDK4/6 or CDK2 for 16 h (Extended Data Fig. 3i, j ). We chose a 16-h period because this corresponds to the end of S phase in the cycling population (Fig.
To assess the type of karyotype generated in a single S phase after WGD, we used single-cell DNA sequencing (Methods, Supplementary Information, Supplementary Methods). We identified over-duplicated chromosomes (more than 10) in addition to frequent over-and under-replicated regions (9n, 7n and 4n) in G2/M tetraploid cells (Fig. 2h , Extended Data Fig. 7a, b ). Both aneuploidy and heterogeneity scores and the proportion of the genome affected by aneuploidies were increased in G2/M tetraploid cells (Fig. 2h , Extended Data Fig. 7a-d , Methods). Our data establish that WGD generates abnormal karyotypes within a single S phase.
Non-optimal S phase in tetraploid cells Tetraploid cells would be expected to 'scale up' RNA and protein content by a factor of two. However, we found no evidence of such an increase First G2/M tetraploid (normalized to G1)   in total RNA and protein content in newly born tetraploid cells using pyronin Y staining and quantitative phase imaging (Fig. 3a, b , Extended Data Fig. 8a-c ). We next tested the levels of key DNA replication factors. We developed protocols to sort tetraploids from diploids on the basis of FUCCI and DNA content from a common cell population (Fig. 3c , Extended Data Fig. 8d , e, Methods). The same number of cells was loaded for diploid and tetraploid conditions and total protein extracts and chromatin-bound extracts were probed by western blot. The chromatin-associated H2B variant, the cytoskeleton component actin and the membrane component β-catenin showed increases consistent with tetraploidization.
RPE-1 GFP and RFP-positive cells were then collected using Sony SH800 FACS (BD FACSDiva Software Version 8. 0. 1). RPE-1 or RPE-1 CCNB1 AID clones expressing FUCCI were selected and the cell lines were established from one single clone. pBOB-EF1-FastFUCCI-Puro 37 was a gift from K. Brindle and D. Jodrell (Addgene 86849).
Generation of an RPE-1 GFP-53BP1 RFP-H2B stable cell line This cell line was obtained as described below. In brief, to produce lentiviral particles, HEK 293 cells were transfected with 4 μg pSMPUW-IRIS-Neo-H2BmRFP (Fachinetti laboratory) + 4 μg pMD2. G (Addgene 12259) + 4 μg psPAX2 (Addgene 12260). Then, RPE-1 cells were incubated with viral particles and RPE-1 RFP-positive cells were collected using Sony SH800 FACS (BD FACSDiva Software Version 8. 0. 1). RPE-1 clones expressing RFP-H2B were selected, and the cell line was established from one single clone. Then, new lentiviral particles were produced by transfecting HEK 293 cells with 4μg Apple-53BP1trunc (Addgene 69531) + 4 μg pMD2. G (Addgene 12259) + 4 μg psPAX2 (Addgene 12260). RPE-1 RFP-H2B cells were incubated with viral particles, and RPE-1 clones expressing both RFP-H2B and GFP-53BP1 were selected using flow cytometry (Sony SH800 FACS).
25% n-propyl gallate (Sigma P3130), 75% glycerol (bidistilled, 99. 5%, VWR 24388-295), 23. 75% H 2 O. Images were acquired on an upright widefield microscope (DM6B, Leica Systems, Germany) equipped with a motorized xy stage and a 40× objective (HCX PL APO 40×/1. 40-0. 70 Oil from Leica). Acquisitions were performed using Metamorph 7. 10. 1 software (Molecular Devices) and a sCMOS camera (Flash 4V2, Hamamatsu). Stacks of conventional fluorescence images were collected automatically at a z-distance of 0. 5 μm (Metamorph 7. 10. 1 software; Molecular Devices, SCR 002368). Images are presented as maximum intensity projections generated with ImageJ software (SCR 002285).
Multi-dimensional acquisitions were performed using Metamorph 7. 10. 1 software (Molecular Devices). Stacks of conventional fluorescence images were collected automatically at a z-distance of 1. 5 μm (Metamorph 7. 10. 1 software; Molecular Devices SCR 002368). Images are presented as maximum intensity projections generated with ImageJ software (SCR 002285). Primary and secondary antibodies were used at the following concentrations: guinea pig anti-CEP192 antibody  40.
However, it is important to mention that in a fraction of these cells, γH2Av dots (small and of low fluorescence intensity) can be occasionally seen. 2D human cell lines. For DNA damage quantification, the signals obtained in cultured cells were different from the signals found in Drosophila neuroblasts. To asses DNA damage in human cells, we used an ImageJ software-based plugin developed by QUANTACELL, where γH2AX signals were measured using z-projection stacks after thresholding. Nuclear size, DAPI intensity, the number of γH2AX foci, Article γH2AX fluorescence intensity and the percentage of nuclear coverage by γH2AX signal were obtained for each nucleus.
10. 1 software (Molecular Devices). Stacks of conventional fluorescence images were collected automatically at a z-distance of 0. 5 μm (Metamorph 7. 10. 1 software; Molecular Devices, RRID SCR 002368). Images are presented as maximum intensity projections generated with ImageJ software (RRID SCR 002285), from stacks deconvolved with an extension of Metamorph 7. 10. 1 software.
FACS of diploid and tetraploid cells A mix of diploid and tetraploid cells (see 'Induction of tetraploidy in human cell lines') were incubated with 2 μg ml -1 Hoescht 33342 (Sigma Aldrich 94403) for 1 h at 37 °C, 5% CO 2. Then, a single cell suspension was generated. Cells were washed using PBS, the supernatant was removed and cells were resuspended in a cold cell culture medium at 1 × 10 7 cell per ml and kept at 4 °C during all the experiments. Fluorescence-activated cell sorting (FACS) was performed using Sony SH800 FACS (BD FACSDiva Software Version 8. 0. 1). Compensation was performed using the appropriate negative control samples. Experimental samples were then recorded and sorted using gating tools to select the populations of interest. RFP + GFP -cells (G1 cells) were first selected.
Cell cycle analysis and measure of RNA levels by flow cytometry Cells were detached by treatment with Accutase (Sigma), immediately washed in PBS, fixed in 2 ml 70% ethanol and stored at -20 °C overnight. They were then washed in PBS and staining buffer (BD Pharmingen 554656). For cell cycle analysis, DNA content was visualized by incubating the cells with 2 μg ml -1 Hoescht 33342 (Sigma Aldrich 94403) in staining buffer for 15 min at room temperature. Alternatively, to measure RNA levels, cells were incubated with 2 μg ml -1 Hoescht 33342 + pyronin 4 μg ml -1 (Santa Cruz sc-203755A) in a staining buffer for 20 min at room temperature. Flow cytometry analysis was done using LSRII (BD Biosciences), by analysing 10,000 cells per condition. Data were then analysed with FlowJo 10. 6. 0 software (Tree Star).
3D reconstruction and analysis 3D videos (see 'Time-lapse microscopy') were imported into Imaris software v. 9. 6. 0 (Bitplane, RRID SCR 007370). For chosen cells, the module 'Spot tracking' of Imaris v. 9. 6. 0 was used to detect the foci, as spots of diameter 0. 5 μm in the xy-direction and 1 μm in z-direction (modelling PSF elongation). Because the volume of the foci changes in time, the option 'Enable growing regions' was used. In each video, the threshold was chosen on the brightest frame (to detect a maximum of the correct spots) and then applied to the whole video. For each cell, at each time point, the number of spots and volumes were recorded. To determine DNA replication timing, we quantified the signal of PCNA fluorescence intensity in the nucleus. This replication timing was characterized independently of any particular behaviour of PCNA.
As soon as PCNA fluorescence intensity was detected in the nucleus, t = 0 (beginning of S phase) was defined, and when PCNA fluorescence intensity was not detected anymore the last time point was defined (end of S phase). For each condition, at least ten cells (Supplementary Data 1) were studied and the statistics from Imaris v. 9. 6. 0 were averaged at each time point using a MATLAB script.
Diploid controls and the tetraploid-enriched population were then pulse-labelled with 0. 1 mM CIdU and 0. 1 mM IdU for 30 min and 100,000-300,000 cells per condition were collected for further analysis. The DNA was extracted from cells and prepped following the manufacturer's instructions using the FiberPrep DNA Extraction Kit (Genomic Vision). Subsequently, the prepped DNA was stretched onto coated glass coverslips (CombiCoverslips, Genomic Vision) by using the FiberComb Molecular Combing System (Genomic Vision). The labelling was performed with antibodies against ssDNA, IdU and CldU using the Replication Combing Assay (RCA) (Genomic Vision). The imaging of the prepared cover slips was carried out by Genomic Vision and analysed using the FiberStudio 2. 0. 1 Analysis Software by Genomic Vision.
43, 44. Images were acquired by a Phasics camera every 20 min for 35 h for the duration of the experiment. To obtain the reference image, 32 empty fields were acquired on the dish and a median image was calculated. This reference image was subtracted from the interferograms (images acquired by phasics) by custom written MATLAB scripts to measure the optical path difference. They were then processed to calculate the phase, intensity and phase cleaned images (the background set to 1,000 and the field cropped to remove edges). Background normalization was performed using a gridfit method, and a watershed algorithm was used to separate cells which came in contact with each other. Mass was calculated by integrating the intensity of the whole cell.
Sequencing and AneuFinder analysis A mixed population of diploid and tetraploid RPE-1 CCNB1 AID FUCCI cells were synchronized in G1 using 1 μM palbociclib (Selleckchem S1579) for 16 h or released in S phase for 20 h in the presence of 10 μM RO3306 (Calbiochem 217699) in order to block cells in the subsequent G2/M. G1 and G2/M diploid and tetraploid cells were then isolated using cell sorting (see 'FACS sorting of diploid and tetraploid cells') and collected in a 96-well plate. Sequencing was performed using a NextSeq 500 (Illumina; up to 77 cycles; single end). The generated data were subsequently demultiplexed using sample-specific barcodes and changed into fastq files using bcl2fastq (Illumina; version 1. 8. 4). Reads were afterwards aligned to the human reference genome (GRCh38/hg38) using Bow-tie2 (version 2.
2. 4; ref. 45. Duplicate reads were marked with BamUtil (version 1. 0. 3; ref. 46. The aligned read data (bam files) were analysed with the copy number calling algorithm AneuFinder  47  (https://github. com/ataudt/aneufinder). Following GC correction and blacklisting of artefact-prone regions (extreme low or high coverage in control samples), libraries were analysed using the dnacopy and edivisive copy number calling algorithms with variable width bins (average bin size = 1 Mb; step size = 500 kb). The G1 samples were analysed with an euploid reference  48. The G1 samples were used as a reference for the analysis of the G2/M samples (G1 diploid for G2/M diploid and G1 polyploid for G2/M polyploid). Aneuploid libraries were not used as a reference and blacklists were constructed using the example from Bioconductor as a guideline.
Quantifications Image analysis and quantifications were performed using Image J software V2. 1. 0/1. 53c, https://imagej. net/software/fiji/downloads. To quantify the colocalizations between two signals (Extended Data Figs. 3i, m, 4g, j ) we calculated the Manders coefficient using the JACOP plugin with Image J V2. 1. 0/1. 53c software. We determined that the colocalizations between γH2AX signal and EdU, FANCD2 or RAD51 signals are not random using an home-made based Costes randomization on nuclear area with Image J software. 1000 randomizations of the pixel positions were performed for each condition (Supplementary data 2). 3D videos (Extended Data Figs. 3c, 6c, 9c, d) were corrected using the 3D correct drift plugin with Image J V2. 1. 0/1. 53c software to keep the cell of interest at the centre of the region of interest.
Statistics and reproducibility At least two (n) independent experiments were carried out to generate each dataset, and the statistical significance of differences was calculated using GraphPad Prism (RRID SCR 002798) version 7. 00 for Mac (GraphPad Software). The statistical test used for each experiment is indicated in the figure legends. Each representative image (Figs. 2a, c, 3a, e, g, k, n, 4a , Extended Data Figs. 2a, c, e, l, 3a, g, 4c, 5g, 6c, 9c, d, 10a ) originates from a dataset composed of at least two (n) independent experiments.
Acknowledgements We authors acknowledge the Cell and  Tissue Imaging  platform ( PICT-IBiSA ), member of the  French National Research Infrastructure France-BioImaging  ( ANR10-INBS-04 ) and the  Nikon Imaging center from Institut Curie  for microscopy. We thank  L. Guyonnet ,  A. Chipont ,  C. Guerrin  and  C. Roulin  from the  Cytometry and RADEXP platforms of Institut Curie ; and  V. Marthiens ,  S. Lambert ,  E. Schwob ,  W. Marshall ,  G. Almouzni ,  J. S. Hoffmann ,  D. Fachinetti ,  M. Budzyk ,  F. Edwards ,  G. Fantozzi ,  R. Salamé ,  A. Goupil ,  C. Chen ,  F. Perez  and  N. Ganem  for helpful discussions and/or comments on the manuscript. This work was supported by  FOR2800/STO918-7  to Z. S. ,  ERC CoG  (ChromoNumber-725907) to R. B. ,  Institut Curie  and the  CNRS. The  Basto laboratory  is a member of the Cell(n)Scale Labex.
| CT scanning The available collection of material from Chéry-Chartreuve referable to Chelydropsis aubasi sp. nov. consists of 57 elements (see Referred material below for a complete list). To fully document their morphology, especially that of the peripherals, all specimens were subjected to high-resolution X-ray micro-computed tomography (μCT) using a Bruker Skyscan 2,211 at the University of Fribourg, Switzerland. Three scanning sessions were performed to scan all the specimens, with an exposure time of 90 ms, a voltage of 180 kV, and a current of 35 mA, resulting in 3,056, 3,020, and 2,122 coronal slices and a voxel size of 45 μm. The resulting coronal sections were then segmented using the software program Thermo Fisher Scientific Amira (6. 1. 1. ). 3D models were exported in. ply format and uploaded to MorphoSource (https://www.
ACKNOWLEDGMENTS We would like to thank  Charles Aubas , even if only posthumously, for providing generous access to the sandpit near Chéry-Chartreuve that yielded the specimens presented herein. We also thank  Loïc Costeur  for helping integrate these specimens into the collection of the NMB. Jason Bourque ,  Tyler Lyson , and  Evangelos Vlachos  are thanked for numerous comments that helped improve the quality of the final manuscript. Open access funding provided by  Universite de Fribourg.
In the parametrization of the total cross-section, contributions from this type of interference between the H boson and continuum-ZZ amplitudes scale as √ μ off-shell F and √ μ off-shell V for the gg and EW modes, respectively. In this Article we study off-shell H-boson decays to ZZ → 2ℓ2ν, and on-shell as well as off-shell H-boson decays to ZZ → 4ℓ (ℓ = μ or e), using a sample of pp collisions at 13 TeV collected by the CMS experiment at the LHC. The selection and analysis of the off-shell ZZ → 2ℓ2ν data sample is described in detail in this Article, and it is based on data collected between 2016 and 2018, corresponding to an integrated luminosity of 138 fb -1. For the ZZ → 4ℓ mode, we use previously published CMS off-shell (2016 and 2017 datasets, 78 fb -1 ; ref. 15  ) and on-shell (2015  15, 22  and 2016-2018  23  datasets, 2.
15  ) in 4ℓ events are applied to enhance the H-boson signal contribution. The displayed values of integrated luminosity correspond to those included in the off-shell analyses of each final state. The bottom panels show the ratio of the data or dashed histograms to the SM prediction (stacked histogram). The black horizontal line in these panels marks unit ratio. , respectively. The dot-dashed and dashed contours enclose the 68% (-2Δ ln L = 2. 30) and 95% (-2Δ ln L = 5. 99) CL regions. The cross marks the minimum, and the blue diamond marks the SM expectation. The integrated luminosity reaches only up to 138 fb -1 as on-shell 4ℓ events are not included in performing this scan. Right: observed (solid) and expected (dashed) one-parameter likelihood scans over Γ H. Scans are shown for the combination of 4ℓ on-shell data with 4ℓ off-shell (magenta) or 2ℓ2ν off-shell data (green) alone, or with both datasets (black).
The selection requirements are tightened in the γ + jets CR, which leads to selection efficiencies in the range 50-75%, depending on p T and η. Event simulation. The signal Monte Carlo (MC) samples are generated for an undecayed H boson for gg, VBF, ZH and WH productions using the POWHEG 2 52-55 program at next-to-leading order (NLO) in QCD at various H-boson pole masses ranging from 125 GeV to 3 TeV. The generated H bosons are decayed to four-fermion final states through intermediate Z bosons using the JHUGen  26  program, with versions between 6. 9. 8 and 7. 4. 0. These samples are reweighted using the MELA matrix element package, which interfaces with the JHUGen and MCFM 13,56-58 matrix elements, following the same reweighting techniques used in ref. 15  to obtain the final ZZ event sample, including the H-boson contribution, the continuum and their interference.
The MelaAnalytics package developed for ref. 15  is used to automate matrix element computations and to account for the extra partons in the NLO simulation. The gg generation is rescaled with the next-to-NLO (NNLO) QCD K-factor, differential in m VV , and an additional uniform K-factor of 1. 10 for the next-to-NNLO cross-section computed at m H = 125 GeV (ref. 10  ). Furthermore, the pole mass values of the top quark (173 GeV) and the bottom quark (4. 8 GeV)  59  are used in the massive loop calculations for the generation of this process. The difference that would be introduced by using the MS renormalization scheme for these masses is found to be within systematic uncertainties after accounting for the effects on both the H-boson and continuum-ZZ amplitudes. The tree-level Feynman diagrams in Fig.
The parton shower and hadronization are modelled with Pythia (8. 205 or 8. 230) 63 , using tunes CUETP8M1 64 for the 2015 and 2016 datasets, and CP5 65 for the 2017 and 2018 periods. The PDFs are taken from NNPDF 3. 0 66 with QCD orders matching those of the cross-section calculations. Finally, the detector response is simulated with the GEANT4 67 package. Signal region selection requirements. Events in the 2ℓ2ν final state are required to have two opposite-sign, same-flavour leptons (μ + μ -or e + e -) satisfying tight isolation requirements with p T > 25 GeV, m ℓℓ within 15 GeV of m Z , and p ℓℓ T > 55 GeV. Additional requirements are imposed to reduce contributions from Z + jets and tt processes as follows. Events with b-tagged jets, additional loosely isolated leptons of p T > 5 GeV or additional loosely identified photons with p T > 20 GeV are vetoed.
acknowledgements We congratulate our colleagues in the CERN accelerator departments for the excellent performance of the LHC and thank the technical and administrative staffs at CERN and at other CMS institutes for their contributions to the success of the CMS effort. In addition, we gratefully acknowledge the computing centres and personnel of the Worldwide LHC Computing Grid and other centres for delivering so effectively the computing infrastructure essential to our analyses. Finally, we acknowledge the enduring support for the construction and operation of the → WZ (green) processes, and tZ+X production, where X refers to any other particle. Postfit refers to individual fits of the data (shown as black points with error bars as uncertainties at 68% CL) to the combined 2ℓ2ν + 4ℓ sample, including the WZ control region, and assuming either SM H boson parameters (stacked histogram with the hashed band as the total postfit uncertainty at 68% CL) or no off-shell H boson production (dot-dashed gold line).