dataesr/scientific-paragraphs-categorization huggingface dataset:
Statistical analysis The statistical methods and analysed population were selected a posteriori. Baseline characteristics are shown as means ± standard deviation for continuous variables and numbers with percentages for categorical variables. Comparisons were done with a Kruskal-Wallis test for continuous variables and a Chi-square test for categorical variables. The estimate of the hazard ratio (HR) and its 95% confidential interval (CI) using an adjusted Cox proportional hazards model was based on the adjudication criteria and the trial was conducted as time to event under the intention-to-treat principle. All analyses were performed by the sponsor after agreeing on a statistical analysis plan using SAS version 9. 2 (SAS Institute Inc. , Cary, NC, USA). P-values reported are two-sided and p < 0.
3. Launching real-time acquisitions with the IncuCyteâ S3 live-cell analysis system and analysis of the scans. a. Place the IncuCyteâ ClearView 96-well chemotaxis cell migration plates on the vessel tray placed in the IncuCyteâ live-cell analysis instrument (which itself is placed in a Heracell 240i cell incubator) and allow the plate to warm to 37 C for about 10 min. b. If necessary, wipe away any condensation remaining on the outside of the cover plate. c. In the chemotaxis module of the IncuCyteâ software, schedule 24-h scans every 30 min for one plate for a total time of 3 days.
Scan analysis. Note: Once the scan parameters are set, they can be saved and reused, and the analysis can be performed automatically after each scan during the acquisition period. For this purpose, at the time of launch, load the parameters of the pre-saved analysis in the Analysis Setup window. a. Use the IncuCyteâ software (Chemotaxis Migration Top/Bot analysis) to perform the scan analysis. b. Use the following analysis settings as a basis for determining segmentation parameters: for the phase, seed threshold of 50, grow threshold of 100, no clean-up, and no area and eccentricity filters; for the red fluorescence, segmentation with a seed threshold (RCU) of 10 -5 , a grow threshold of 50, an edge sensitivity of 0, no clean-up filter, area filter of minimum 20 mm 2 and maximum 600 mm 2 , eccentricity filter of maximum 0.
Note: The IncuCyteâ software enables exporting images and movies from the same well. This can be done from unanalyzed and analyzed scans, which in the latter case allows displaying the masks of the objects and thus visualizing the segmented objects at each time point. a. After opening an experiment or an analyzed experiment, click on the Export Images and Movies icon. b. In the Export Type window, choose ''As Displayed. '' c. In the Wells Selection window, select one or more wells and choose the Image Channel(s) and the Channel Mask(s) to display. d. In the Sequence Type and Scans window, select ''Single Movie of Images as Displayed'' and select the scans to include in the movie. e. In the Export Design window, adjust cropping and scaling, customize the movie, and choose the number of frames per second.
EXPECTED OUTCOMES In our experiments, the number of THP-1 detected under the membrane increases continuously and linearly over time for the first 20-24 h, then the curve bends and this number reaches a plateau. A maximum number is reached in 24-48 h, is dependent on the concentration of the chemoattractant MCP1 in the reservoir wells, and reaches about 200-300 events in normal conditions for a concentration of 100 nM MCP1. This maximum number can double (about 600 events) when endothelial cells are previously exposed to ionizing radiation at a dose of 20 Gy. See Figure  3  for example data from experiments with increasing concentrations of chemoattractant and increasing doses of ionizing radiation. See Methods videos S1 and S2 for real-time visualization of THP1 passage through membrane pores and THP-1 detection by IncuCyteâ software in the absence or presence of MCP1 chemoattractant, and after exposure or no exposure to ionizing radiation.
Note: We usually choose a scan performed around 24 h after transmigration initialization. At this time, the plateau has not yet been reached and THP-1 cells have not yet begun to multiply significantly in the upper compartment, an effect that could render the assay definition parameters inaccurate due to excessive fluorescence. 3. Adjusted the settings n the ''Red bottom'' image channel chosen in the Layers menu. 4. Segmentation. Note: As described for the interface of the IncuCyteâ software, a Seed-and-Grow technique is used to mask the cells from the background (Figure  5A ). This technique uses two thresholds, the Seed Threshold and the Grow Threshold. a. Select the Seed Threshold first. b. Start with a Red Calibrated Unit (RCU) of the objects at 0 (default). c. Launch a preview. d. Use the mouse pointer to open the tooltip to evaluate the RCU of the non-segmented red objects or the wrongly segmented objects (Figure  5A , image on top right).
0001. IncuCyteâ software offers the ability to fill the holes that may have been left in the objects (Holl Fill (mm 2 )), as well as to adjust the size of the objects (Adjust Size (pixels)) to the actual fluorescence area visualized (Figure  5C ). At this stage of the analysis definition, these parameters do not change the number of detected objects but allow a better visualization and a more accurate quantification of the fluorescence intensity of the segmented objects. For the quantification of transmigration rates, based only on the number of objects, these parameters have no impact. 6. Filter. Once segmented, objects can be selected using 4 filters based on area, eccentricity (i. e. , shape), mean intensity and integrated intensity. For each of these filters, minimum, maximum, or both values can be defined (Figure  5D ).
The parameter values of each object can be visualized by means of the mouse pointer to open the tooltip on the setting image. The use of filters obviously changes the number of detected objects. 7. Select scan times and wells to consider, and save and apply analysis definition to create the analysis. Note: We found high reproducibility of object size, fluorescence, and signal-to-noise ratio across experiments, allowing the same analysis definition parameters to be used for the different experiments. As already mentioned, however, it is necessary to verify that the segmentation is satisfactory after the analysis is complete. 8. Statistical analysis. The IncuCyteâ software includes a graph builder and a graph viewer that allows visualizing the curves of the number of detected objects for the different conditions versus time.
This allows generating two types of graphs, one to visualize the values of each well as a function of time (Microplate Graph) and the other to visualize the means and standard deviations in each condition as a function of time (Graph) (Figure  6 ). The graphs of mean values versus time are useful to track the experiment in real time, provided that the analysis is performed at the end of each scan (reuse of a saved analysis definition). They are also useful to quickly visualize the results after all scans have been analyzed. The Microplate Graph is used to visualize the data from each well and to identify data from the wells (i. e. , technical replicates) that should be excluded from the analysis due to technical problems. Statistical tests and graphical representation of data for publications are done in statistical analysis software such as GraphPad Prism after exporting data from IncuCyteâ software.
c. Graphical representation of data and statistical analysis. Data exported in. csv format are opened in a Microsoft Excel spreadsheet to organize them, then pasted into statistical analysis software such as GraphPad Prism to build graphs and perform statistical tests. The most useful graphs present curves of the average number of THP-1 objects as a function of time (Figures  3A  and 3B ), as well as individual values of the number of THP-1 objects at given times after transendothelial migration initialization in scatter plots with bar (Figure  3C ). To find out whether the means of the conditions differ statistically, classical parametric (ANOVA) or non-parametric (Kruskal-Wallis test) statistical tests with correction for multiple comparisons are performed at different times, e. g. , at 24, 48, and 72 h.
The images from the experiment can be exported and edited into a movie using IncuCyteâ software and Image J (see Methods video S1). Data are represented as mean +/À SEM derived from n = 8-16 technical replicates. (B) Number of mKate2-N THP-1 cells detected under the membrane by the IncuCyteâ S3 module from 0 to 72 h after initiation of transendothelial migration and after irradiation of HUVECs at different doses, in the absence (-MCP1) or in the presence (+MCP1) of 100 nM MCP1 in the wells of the reservoir plates (from Ladaigue et al. 1  ). HUVECs were seeded on the wells of an IncuCyteâ ClearView 96-well chemotaxis cell migration plate. Twenty-four hours after the seeding of HUVECs, the plates were irradiated at 0, 2, 5, 10, or 20 Gy. Forty-eight hours after irradiation, mKate2-N THP-1 cells were added to the wells, and culture medium containing 100 nM MCP1 was added to the wells of the plate reservoir.
ACKNOWLEDGMENTS We are grateful to  Dr. Philippe Peron  ( Sanofi-Aventis, Chilly-Mazarin, France ) for his availability and his precious advice on the transendothelial migration assay. This work is supported by  Electricitede France (Groupe Gestion Projet Radioprotection)  and the  Institut de Radioprotection et de Surete Ńucleaire (ROSIRIS program ). The funding agencies were not involved in any research plan relative to the study design, data collection or analysis, decision to publish, or drafting of the manuscript.
1d ) and second in a simultaneous accumulation of merozoite proteins at different expression levels, depending on the parasite line. Thus, the merozoite reporter gene GRA81-mCherry was induced upon depletion of each AP2 individually, whereas GRA11b, a hallmark of merozoites  14  , was expressed exclusively after loss of AP2XII-1, and GRA80 levels were less pronounced in AP2XI-2-depleted parasites (Fig. 1b  and Extended Data Fig. 1e ). Thus, the loss of AP2XI-2 does not fully mimic the depletion of AP2XII-1 in eliciting expression of the merozoite protein markers used here. To examine the genome-wide transcriptional outcome of depleting each AP2 separately, we monitored mRNA levels using both Nanopore DRS and Illumina sequencing. After addition of IAA, substantial fractions of mRNAs were up-and down-regulated in a strain-specific manner, preprint (which was not certified by peer review) is the author/funder.
AP2XI-2 and AP2XII-1 colocalize extensively in the genome where they recruit MORC and HDAC3 to reduce chromatin accessibility. To further explore how AP2XI-2 or AP2XII-1 cooperate in Toxoplasma to silence gene expression, we examined their genome-wide distribution using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq; GSE222819) in the context of their conditional single or double knockdown. In parallel, we generated high-resolution profiles of MORC and HDAC3 using in-house ChIP-grade antibodies. The enrichment at chromatin of AP2XII-1, which is the highest, was used as a proxy to analyze the distribution of loci to which the repressive complex binds. As a result, we identified two clusters of genes with significant enrichment in their environment: Cluster 1 groups genes with a scattered distribution of peaks that span large preprint (which was not certified by peer review) is the author/funder.
6a ; Extended Data Fig. 5h ). AP2XI-2 and AP2XII-1 are expected to mediate chromatin compaction and accessibility, a function attributed to their partners MORC and HDAC3. To investigate this assumption, we performed ATAC-seq (GSE222832), a robust and streamlined method for profiling chromatin accessibility  35. ATAC-seq peaks represent regions of chromatin accessible to transposases and are therefore proxies for transcription factor occupancy on chromatin. At the genome level, there is a slight decrease in average accessibility between untreated and treated conditions (Fig. 6c ) with a majority of the peaks located at TSS (Extended Data Fig. 6a, b ). However, when we plotted ATAC-seq data for the subset of down-and up-regulated genes (as defined in Fig. 1f ), the changes in occupancy were more pronounced and coherent with expected increase or decrease in accessibility of induced or repressed clusters, respectively (Fig.
Reagents Immunofluorescence microscopy. Toxoplasma-infected HFF cells grown on coverslips were fixed in 3% formaldehyde for 20 minutes at room temperature, permeabilized with 0. 1% (v/v) Triton X-100 for 15 minutes, and blocked in phosphate buffered saline (PBS) containing 3% (w/v) BSA. Cells were then incubated with primary antibodies for 1 hour, followed by the addition of secondary antibodies conjugated to Alexa Fluor 488 or 594 (Molecular Probes). Nuclei were stained with Hoechst 33258 (2 μg/ml in PBS) for 10 minutes at room temperature. After washing four times in PBS, coverslips were mounted on a glass slide with Mowiol mounting medium, and images were acquired with a fluorescence microscope ZEISS ApoTome. 2 and processed with ZEN software (Carl Zeiss, Inc. ). For IFA of in vivo cat stages, small intestines of infected kittens from a previous study  15  embedded in paraffin were sectioned to 3 μm and dried over night at 37 °C.
MS and MS/MS data were acquired using Xcalibur version 4. 0 (Thermo Scientific). Peptides and proteins were identified using Mascot (version 2. 8. 0) through concomitant searches against the Toxoplasma gondii database (ME49 taxonomy, version 58 downloaded from ToxoDB), the Uniprot database (Homo sapiens taxonomy for interactomes or Trichoplusia ni for SEC fractions, 20220527 download), and a homemade database containing the sequences of classical contaminant proteins found in proteomic analyses (human keratins, trypsin. ). Trypsin/P was chosen as the enzyme and two missed cleavages were allowed. Precursor and fragment mass error tolerances were set at respectively at 10 and 20 ppm. Peptide modifications allowed during the search were: Carbamidomethyl (C, fixed), Acetyl (Protein N-term, variable) and Oxidation (M, variable).
The Proline software (version 2. 2. 0) was used for the compilation, grouping, and filtering of the results (conservation of rank 1 peptides, peptide length ≥ 6 amino acids, false discovery rate of peptide-spectrum-match identifications < 1%, and minimum of one specific peptide per identified protein group). Intensity-based absolute quantification (iBAQ) values were calculated for each protein group in Proline using MS1 intensities of specific and razor peptides.
MS and MS/MS data were acquired using the Xcalibur software version 4. 0 (Thermo Scientific). Peptides and proteins were identified by Mascot (version 2. 8. 0, Matrix Science) through concomitant searches against the Toxoplasma gondii database (ME49 taxonomy, version 58 downloaded from ToxoDB), the Uniprot database (Homo sapiens taxonomy, 20220527 download), and a homemade database containing the sequences of classical contaminant proteins found in proteomic analyses (human keratins, trypsin. ). Trypsin/P was chosen as the enzyme and two missed cleavages were allowed. Precursor and fragment mass error tolerances were set at respectively at 10 and 20 ppm. Peptide modifications allowed during the search were: Carbamidomethyl (C, fixed), Acetyl (Protein N-term, variable) and Oxidation (M, variable).
The Proline software (version 2. 2. 0) was used for the compilation, grouping, and filtering of the results (conservation of rank 1 peptides, peptide length ≥ 6 amino acids, false discovery rate of peptide-spectrum-match identifications < 1%, and minimum of one specific peptide per identified protein group). Proline was then used to perform a MS1 label-free quantification of the identified protein groups based on razor and specific peptides. Statistical analyses were performed using ProStaR 49. Peptides and proteins identified in the reverse and contaminant databases or matched to human sequences were discarded. Only proteins identified by MS/MS in a minimum of two replicates of one condition and quantified in the three replicates of one condition were conserved. After log2 transformation, abundance values were normalized using the variance stabilizing normalization (vsn) method, before missing value imputation (SLSA algorithm for partially observed values in the condition and DetQuantile algorithm for totally absent values in the condition).
The enriched product of ~200-700 bp was excised from the gel and purified. Sequencing: the library was denatured with 0. 1 M NaOH to generate single-stranded DNA molecules and loaded into flow cell channels at a concentration of 8 pM and amplified in situ using TruSeq Rapid SR cluster kit (# GD -402-4001, Illumina). Sequencing was performed at 100 cycles on the Illumina HiSeq 4000 according to the manufacturer's instructions. Data Analysis. After the sequencing platform generated the sequencing images, the stages of image analysis and base calling were performed using Off-Line Basecaller software (OLB V1. 8). After passing Solexa CHASTITY quality filter, the clean reads were aligned to T. gondii reference genome (TGME49) using BOWTIE V2 then converted and sorted using Bamtools. Aligned reads were used for peak calling of the ChIP enriched peaks using MACS V2.
2 with a cutoff p-value of 10 -4. Data visualization: For IGB visualization and gene centered analysis using Deeptools, MACS2 generated bedgraph files were processed with the following command: "sort -k1,1 -k2,2n 5_treat_pileup. bdg > 5_treat_pileup-sorted. bdg" then converted using the BedGraphToBigWig program (ENCODE project). The Deeptools analysis were generated using "computeMatrix reference point" with the following parameters (--minThreshold 2, --binSize 10 and -averageTypeBins sum). Plotprofile or heatmap was then used with a k-mean clustering when applicable. Inter sample comparison were obtained using the nf-core chip-seq workflow with standard parameters  46. From this pipeline, HOMER (annotatePeaks) was used to analyze peak distribution relative to gene features. All these raw and processed files can be found at Series GSE222819.
After fragmentation, the first strand preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this this version posted January 17, 2023. ; https://doi. org/10. 1101/2023. 01. 16. 524187 doi: bioRxiv preprint cDNA was synthesized using random hexamer primers. Then the second strand cDNA was synthesized using dUTP, instead of dTTP. The directional library was ready after end repair, Atailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.
The samples were sequenced on the Illumina NovaSeq platform (2 x 150 bp, strand-specific sequencing) and generated ~40 million paired-end reads for each sample. The quality of the raw sequencing reads was assessed using FastQC (www. bioinformatics. babraham. ac. uk/projects/fastqc/) and MultiQC. For the expression data quantification and normalization, the FASTQ reads were aligned to the ToxoDB-49 build of the T. gondii ME49 genome using Subread version 2. 0. 1 with the following options 'subread-align -d 50 -D 600 --sortReadsByCoordinates'. Read counts for each gene were calculated using featureCounts from the Subread package. Differential expression analysis was conducted using DESeq2 and default settings within the iDEP. 96 web interface  47. Transcripts were quantified and normalized using TPMCalculator.
The Illumina RNA-seq dataset generated during this study is available at the National Center for Biotechnology Information (NCBI): BioProject # PRJNA921935. Nanopore Direct RNA Sequencing (DRS). The mRNA library preparation followed the SQK-RNA002 kit (Oxford Nanopore)-recommended protocol, the only modification was the input mRNA quantity increased from 500 to 1000 ng, and all other consumables and parameters were standard. Final yields were evaluated using the Qubit HS dsDNA kit (Thermo Fisher Scientific, Q32851) with minimum RNA preps reaching at least 200 ng. For all conditions, sequencing was performed on FLO-MIN106 flow cells either using a MinION MK1C or MinION sequencer. All datasets were subsequently basecalled (high accuracy) with a Guppy version higher than 5. 0. 1 with a Q score cutoff of >7.
Long read alignment was performed by Minimap2 as previously described  48. Sam files were converted to bam and sorted using Samtools 1. 4. Alignments were converted and sorted using Samtools 1. 4. 1. For the three described samples, Toxoplasma aligned reads range between 600,000 and 800,000. The Nanopore DRS dataset is available at the National Center for Biotechnology Information (NCBI): BioProject # PRJNA921935. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this this version posted January 17, 2023. ; https://doi. org/10. 1101/2023. 01. 16. 524187 doi: bioRxiv preprint Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATACseq). Intracellular tachyzoites (non-treated or IAA treated for 24h) were prepared using HFF cells monolayers in a T175 format, which was freshly scrapped, gently homogenised by pipetting and centrifuged at 500*g.
Software and Statistical analyses. Volcano plots, scatter plots, and histograms were generated with Prism 7. Sample sizes were not predetermined and chosen according to previous literature. Experiments were performed in biological replicates and provided consistent statistically  Mean GRA80/Cas9-GFP(+) PV         mPM: mother plasma membrane, Go: Golgi apparatus, n: nucleus, DG: dense granule, m: mitochondrion, dc: daughter conoid. b, Tachyzoites (UT) and AP2XII-1/AP2XI-2-depleted meronts (24 hours post-IAA) were fixed and stained with H3K9me3 (red) and IMC7 (green) and Hoechst DNA-specific dye (white or blue). Yellow and pink arrows indicate mother and daughter conoids, respectively. c, Emphasis on daughter cell emergence. mi: microneme, m: mitochondrion, ER: endoplasmic reticulum, Go: Golgi apparatus, n: nucleus, rh: rhoptry, IMC: inner membrane complex.
Acknowledgments We are grateful to the developers of the ToxoDB. org Genome Resource. ToxoDB and EuPathDB are part of the  National Institutes of Health/National Institutes of Allergy and Infectious Diseases (NIH/NIAID) -funded  Bioinformatics Resource Center. We thank the excellent technical staff of the  Electron Microscopy Core Facility  at the  Johns Hopkins University School of Medicine Microscopy Facility. This work was supported by the  Laboratoire d'Excellence (LabEx) ParaFrap  [ ANR-11-LABX-0024 ], the  Agence Nationale pour la Recherche  [Project  HostQuest ,  ANR-18-CE15-0023 ], [Project  ApiNewDrug ,  ANR-21-CE35-0010-01 ], [Project  ApiMORCing ,  ANR-21-CE15-0002-01 ], and  Fondation pour la Recherche Médicale  [FRM Equipe,  EQU202103012571 ]. I. C. was supported by a  NIH  grant [ R01 AI060767 ].