dataesr/scientific-paragraphs-categorization huggingface dataset:
Why some invasion events in HeLa result in CaTCT, while others result in damage remains an open question. We find that host membrane breaching identified via Gal-3 recruitment during HeLa cell invasion, coincides (within the temporal resolution of our setup) or is followed by host cell death in the vast majority of invasion events studied. Thus, membrane Fig. 4  Serial block face-scanning electron microscopy (SBF-SEM) of Caco-2 invasion. A A representative SBF-SEM volume is presented together with dataset size and resolution in each axis (left panel). A C. albicans hypha in the process of invasion is visualized in three-dimensions (white, middle panel). Three invaded host cells are colour coded according to the sequence of invasion, cell 1 (magenta), cell 2 (green), cell 3 (yellow). The direction of filament extension is noted (arrow, right panel).
Insets show in detail inferred membrane organization in accordance with this hypothesis, with each layer derived from a plasma membrane deformed upon hypha entry or exit from each corresponding invaded cell. The presented dataset corresponds to invasion site 9 in Supplementary Table  2  and is presented in Supplementary Movie 14. Scale bar is 1 µm. question. While the current view holds that only two mechanisms (induced endocytosis and active penetration) drive C. albicans intracellular epithelial invasion, our work, performed at the single cell level, has revealed several invasive lifestyles (CaTCT, damaging invasion associated with TCTs, and damaging invasion that may not be associated with TCTs) that may be manifestations of a single invasion mechanism, or alternatively, may be mechanistically distinct from one another at the molecular level.
Cross-section views (derived from dotted lines) show the membrane layers surrounding the hypha cell wall (black), with the exclusion layer in dark grey, and each membrane layer coloured according to its cell of origin, i. e. magenta-cell 1, green-cell 2, yellow-cell 3. Additional actin layers derived from non-apical cortical actin may also be present within the TCT structure, but they were not illustrated for clarity. The model presented applies to non-damaging invasion, and does not account for scenarios where host membrane breaching occurs. bacteria like L. monocytogenes and S. flexneri that must induce host membrane breaching followed by 'escape' into the host cytosol in every invaded cell before transitioning to its neighbour  64. Remarkably, an exclusion layer in the first invaded Caco-2 cell was observed in all SBF-SEM datasets quantified.
For initial investigations into HeLa, a 40X air objective was used (Plan-Neofluar NA 0. 6) (see Fig. 1 ). For all other live-cell imaging experiments a 40X oil immersion objective (Plan-Apochromat NA 1. 4) was used. Data was acquired using ZEN 2. 6 pro software (Zeiss). Movies were recorded over 7 h (such that total time post infection at the end of the acquisition was 7 h for HeLa and 9 h for Caco-2). Every 10 min, a z-stack of 15 planes with 0. 94 µm z steps (40X air objective) or a z-stack of 30 planes with 0. 37 µm z steps (40X oil objective) was acquired sequentially in two fluorescent channels, with a single plane acquired in phase. Gal-3 was excited with a 475 nm LED and detected with a 525 nm filter. The CellMask Orange Plasma Membrane Stain was excited with a 555 nm LED and detected with a 605 nm filter and the CellMask Deep Red Plasma Membrane Stain was excited with a 630 nm LED and detected with a 690 nm filter.
For imaging, samples on aluminum stubs were trimmed using an ultramicrotome and inserted into a TeneoVS SEM (ThermoFisher). Acquisitions were performed with a beam energy of 3 kV, 200 pA current, in LowVac mode at 40 Pa, a dwell time of 1 µs per pixel at 10 nm pixel size. Sections of 100 nm were serially cut between images. Overall, 11 different datasets were acquired, containing 11 invasion events with hyphae entirely or almost entirely within the acquisition volume, and 30 invasion events containing hyphae partially within the acquisition volume. Western Blot and immunoprecipitation. HeLa and Caco-2 cells expressing or not eGFP-galectin-3 were lysed for 30 min in RIPA-buffer (30 mM Tris-HCl pH 7. 4, 150 mM NaCl, 1% Triton X-100, 0. 5% sodium deoxycolate, 0. 1% SDS, 1 mM EDTA), supplemented with a protease inhibitor cocktail (Sigma-Aldrich).
Uncropped and unprocessed scans of the blot are included in the Source Data file. Differential staining invasion assay. HeLa expressing Gal-3 or Caco-2 not expressing Gal-3 were fixed with 4% PFA for 10 min at room temperature 4 h or 6 h post infection, respectively. Infected Hela were washed three times with PBS and the fungal cells were first labelled with 10 µg/mL of concanavalin-A coupled with tetramethylrhodamine (Invitrogen) during 20 minutes at room temperature, hence labelling non-invasive fungal cells and the external parts of invasive fungal cells as previously described  30. For fixed Caco-2, a rabbit anti-C. albicans polyclonal antibody at 2,5 µg/mL (OriGene) counterstained with a secondary goat antirabbit IgG conjugated with Alexa Fluor 555 at 0. 4 µg/mL (Invitrogen) was used as a first label as previously described  14.
5 mg/mL for Caco-2 expressing Gal-3 in EM medium during 10 min at room temperature. After removing the propidium iodide solution, the host cells were infected with C. albicans BWP17 wild-type strain at MOI 0. 1 (7 × 10 3 cell per well) as described above (see Infection procedure). Imaging was started 15 min post-infection for Hela with images acquired every 10 min for 7 h. For Caco-2 late time points study, imaging was started at 19 h post-infection with images acquired every 10 min for 10 h. For HeLa cell invasion datasets, a total number of 358 invasion events were recorded in five independent replicates. The Chi-square (χ2) test was used to assess the homogeneity in the size of distribution of invasion scenarios among the five independent replicates, with a p value threshold of 0. 05 (p value = 0.
025). To quantify the host cell death timing, cell death was assigned to an invasion stage if it occurred within 30 min (three frames) of that stage's onset when PI was observed. We considered host cells alive at the end of invasion if no host cell death was observed at least 90 min after the SC stage. Host cells that were alive after SC but were not recorded for an additional 90 min due to the end of the live imaging session were discarded for the purpose of cell death quantification (98 invasion events discarded). In total, 260 invasion events were used for cell death quantification. Image processing and analysis. Data acquired by fluorescent microscopy were analysed using ZEN 2. 6 pro software (Zeiss, blue edition) and Fiji  85. Data acquired by SBF-SEM were processed using Fiji and Amira (ThermoFisher).
Data alignment and manual segmentation were performed using Amira. Figures were prepared using Inkscape, with auto-rendering of images in Fig. 5 , and pixelated rendering of images in all other figures. Quantifications, statistical analysis and reproducibility. For HeLa invasion studies, a total number of 629 invasion events were recorded out of six independent replicates. The Chi-square (χ2) test was used to assess the homogeneity in the size of distribution of invasion scenarios among the six independent replicates, with a p value threshold of 0. 05 (p value < 0. 0001). For Caco-2 invasion studies, a total number of 153 invasion events were recorded in three independent experiments. The cell traversal (CT) time was defined as the time needed for a hypha to extend through one host cell, which was calculated for every invasion event as follows: CT time = time of SCtime of FC.
Acknowledgements We thank  R. Arkowitz  and  M. Bassilana  ( iBV, Nice, France ) for providing us with the C. albicans strains used in this study. We thank  J. Enninga ,  P. L. Lambert  and  N. Mellouk  ( Institut Pasteur, Paris, France ) for providing us with stably expressing epithelial cell lines and other resources. We thank  C. D'Enfert  and  S. Bachellier-Bassi  ( Institut Pasteur, Paris, France ) for support with experimental tools. We thank  M. Larsen  and  E. Rubinstein  ( Cimi-Paris, Paris, France ) for help with statistical analysis and Western blot respectively. We acknowledge the  ImagoSeine core facility , member of the  France BioImaging infrastructure supported by  grant  ANR-10-INBS-04  from the  French National Research Agency. This work was supported by the  ATIP-Avenir program  to AW and by the  Fondation pour la Recherche Médicale (FRM)  fellowship ( FDM202006011423 ) to AP.
If proven useful, passive antibody transfer could help shortening anti-TB treatment and reducing the rate of disease progression  [39]. Although we did not find any evidence on human studies, neither in PubMed nor in the clinicaltrials. gov database, multiple reports are available on the TB protection achieved by the passive transfer of antibodies against TB in animal models  [40]. The administration of human intravenous immunoglobulins (IVIg) to mice has been shown to protect against TB, mainly due to antibody responses against the BCG vaccine, LTBI or exposure to environmental mycobacteria (table  3 )  [41] [42] [43]. In pooled IVIg treatment of MTB-infected mice, a significant reduction in bacterial load in tissues was demonstrated  [43]. In another study, passive transfer of intact but not de-glycosylated IVIg reduced lung bacillary load and pneumonia in a murine model, suggesting the importance of Fc glycosylation in protection against MTB  [42].
Other non-financial interests reported as follows: C. Vilaplana's lab hosts secondments from the EMJMD LIVE MsC students (Erasmus+ Mundus Joint Master Degree Leading International Vaccinology Education), co-funded by the EACEA (Education, Audiovisual and Culture Executive Agency, award 2015-2323). Support statement: S. T. Melkie, registered in the EMJMD LIVE (Erasmus+ Mundus Joint Master Degree Leading International Vaccinology Education), co-funded by the EACEA (Education, Audiovisual and Culture Executive Agency, award 2015-2323) of the European commission, receives a scholarship from the EACEA. This work was also supported by the Spanish Government-FEDER Funds through CPII18/00031 and the CIBER Enfermedades Respiratorias (CB06/06/0031). This project has received funding from the European Union's Horizon 2020 research and Innovation programme under grant agreement No 847762.
Source data are provided as a Source data file. MORC2 mRNA expression levels are solely dependent on TASOR expression under these experimental conditions, whereas TNFα mRNAs remain very lowly expressed under all conditions (Fig. 3g, h ). Second, we tested the TASOR-CNOT1 synergy under conditions where infected cells represent a population with diverse integration sites as previously achieved  13. Jurkat T cells were infected with a genetically marked HIV-1 virus  13  (Fig. 4a ). This vector retains the complete LTRs, tat, and rev, but has a frameshift mutation in env, ngfr in place of nef and egfp in place of gag, pol, vif, and vpr. Finally, the splicing sites are conserved. We sorted infected cells by cytometry (EGFP-positive cells), kept the sorted cells in culture for another two weeks until EGFP expression was silenced, and finally sorted the population of  latently infected cells (EGFP-negative cells; Fig.
J-Lat A1 miR Luc and miR CNOT1 were treated or not for 72 h with doxycycline 1 µg/mL and VLPs containing HA-Vpx WT or R42A or mock were delivered for 24 h and TNF (1 ng/mL) 16 h prior to flow cytometric analysis and RT-qPCR analyses (from d to h, n = 3 for miR Luc; n = 6 for miR CNOT1; each independent replicate, mean and SEM are shown; two-sided unpaired t test was applied). Source data are provided as a Source data file. MATR3, YTHDF2, MTR4 (Fig. 6e ), or with EXOSC10 (Fig. S3b ). In humans, MTR4 is found in at least three different complexes that use the exosome complex to eventually degrade the targeted RNA: the nucleolar TRAMP complex, the nucleoplasmic TRAMP-like NEXT and PAXT complexes  47  (reviewed in ref. 48 ). Of note, in our Y2H screen, we also identified ZFC3H1, a member of the PAXT complex, as a partner of TASOR (Fig.
The latently HIV-1-infected Jurkat miR Luc and miR CNOT1 cells were treated or not for 72 h with doxycycline 1 µg/mL and VLPs containing HA-Vpx WT or R42A or mock were delivered for 24 h and TNF (1 ng/mL) 16 h prior to flow cytometric analysis and RT-qPCR analyses (for d-g, n = 3 independent experiments, mean and SEM are represented; two-sided unpaired t test was applied: p values are indicated in the graph). Source data are provided as a Source data file. the role of TASOR at the epigenetic level  6  , we propose a feedback control mechanism in which TASOR and CNOT1 would follow RNAPII during HIV-1 provirus transcription by association with the nascent transcript and recruit RNA metabolism proteins, leading in turn to the degradation of the transcript and the deposition of H3K9me3 marks (Fig.
2630344; p adjusted values < 0. 05, Wald test with Benjamini-Hochberg multiple testing correction). ns not significant. f Number of TE families that are significantly upregulated (by at least 20%), upon TASOR + CNOT1 co-silencing as compared to control, TASOR, or CNOT1 individual silencing in HeLa cells (log2 fold change > 0. 2630344; p adjusted values < 0. 05 after Benjamini-Hochberg multiple testing correction of Wald test p value). More information is available in Source data file. a TASOR isoform of 1512 amino acids (NCBI Reference Sequence: NP_001106207. 1). TASOR-ΔPARP construct was obtained by deleting the DNA sequence corresponding to the 106-319aa using the CloneAmp™ HiFi PCR Premix (639298-Takarabio) and the following 5′-3′-oriented primers: F-CCAGGAAGT ATGCAGTTGTGTCTTTTACTTACA, R-CTGCATACTTCCTGGGGATCTGA AAACTCC.
The non-targeting control siRNAs (MISSION siRNA Universal Negative Control #1, SIC001) were purchased from Sigma Aldrich. Luciferase activity assay. Cells were washed twice with phosphate-buffered saline (PBS) then lysed directly in wells using 1× cell culture lysis reagent (Promega). Cell lysates were clarified by centrifugation, luciferase activity was measured using a luciferase assay system (Promega) and a TECAN multimode reader Infinite F200 Pro and data were normalized on protein concentration with the use of Pierce BCA Protein Assay Kit (23225-ThermoFisher). Flow cytometric analyses. TNFα-treated (1 ng/ml) or untreated cells were collected and resuspended in PBS-EDTA (0. 5 mM). Data were collected and analyzed with a BD Accuri C6 cytometer or with a BD LSRFortessa Cell Analyzer and software CFlow Plus or with FlowJo V10.
For endogenous Immunoprecipitation, the same procedures were followed but a minimum of 400 µg of lysate was used for overnight IP with 4 µg of the corresponding antibody and pre-washed Pierce™ Protein A/G Magnetic Beads (88802, ThermoFisher) were added for 1 h at room temperature. Following transfer onto PVDF membranes, proteins were revealed by immunoblot. Signals were acquired with Fusion FX (Vilber). The following antibodies, with their respective dilutions in 5% skimmed milk in PBS-Tween 0. 1%, were used: anti-Flag M2 (F1804-200UG, lot SLCD3990, Merck) 1/1000; anti-TASOR (HPA006735, lots A106822, C119001, Merck) 1/1000-for IF assays: 1/ 500; anti-TASOR (HPA017142, Merck) for Immunoprecipitation experiments, anti-MPP8 (HPA040035, lot R38302, Merck) 1/500; anti-CNOT1 (For WB: 66507-1-Ig, Proteintech, 1/1000; For IP: 14276-1-AP, Proteintech); anti-CNOT7 (14102- anti-rabbit (31460, lots VC297287, UK293475 ThermoFisher) were used at a 1/ 10,000 dilution before reaction with Immobilon Forte Western HRP substrate (WBLUF0100, Merck Millipore).
Nascent RNA IP. HeLa LTR-ΔTAR-Luc were grown in 10 cm dishes. Forty-eight hours post-transfections, the cells were recovered and labeling of nascent RNAs was undertaken with the Nuclear Run On protocol  68   After quality control analysis, 1. 5 µg of RNA from each sample was sent for sequencing. Libraries were prepared using the NEB Ultra II protocol (stranded polyA+ RNA-seq) and sequenced with an Illumina NextSeq 500 as 2 × 75 bp paired-ends (PE) at the Genom'IC platform. Raw and processed RNA-seq data were submitted to GEO access number GSE184399. Trimmed PE reads were mapped against the human reference genome, hg38 (Gencode and gene annotation version 29) using STAR (version 2. 7. 5c)  70. The parameters used for the mapping were as follows:-outFilterMultimapNmax 1000 (1000 alignments allowed per read-pair),-alignSJoverhangMin 8 (minimum overhang for unannotated junctions).
Read counting was performed using TEtranscripts and TElocal (version 1. 1. 1) (https://github. com/mhammelllaboratory/TElocal) from the TEToolkit suite 71 with recommended parameters. DESeq2 (version 1. 30. 1)  72 was used for the differential expression analysis of genes or transposable elements. TASOR and CNOT1 interactions on gene expression were modeled with a contrast matrix in DESeq2 using the following design: ~"siTASOR+siCtrl" + "siCNOT1+siCtrl" + "siTASOR+siCtrl":"siCNOT1+siCtrl". Only genes upregulated upon double silencing of TASOR and CNOT1, and with expression levels >1 TPM, were considered in the analysis. The log2 fold change (FC) values obtained represents the difference: log2(FC(siCNOT1+siCtrl vs siCtrl)) -log2(FC(siTASOR+ siCNOT1 vs siTASOR+siCtrl)). Thus, genes with a log2(FC) > 0 and an adjusted p value < 0.
1% for 10 min at room temperature for TASOR immunofluorescence staining. After PBS-mediated washes, a 1/500 anti-TASOR (HPA006735-Merck) dilution in IF buffer (PBS-Tween 0. 05%-BSA 0. 2%) was incubated on the cells for 1 h at RT in a dark chamber. The cells were then washed three times with PBS and anti-rabbit Alexa 594 was added at a dilution of 1/1000 in IF buffer for 1 h at room temperature in a dark chamber. After three PBS-mediated washes, the coverslips were mounted on slides with the use of ProLong™ Diamond Antifade Mountant (P36970-ThermoFisher). Imaging was performed with a Spinning-Disk IXplore Olympus confocal microscope, using a ×100 objective, a Hamamatsu sCMOS Orca flash 4. 0 V3 camera, and the cellSens Dimension acquisition software. GFP-positive cells were randomly captured.
Analysis of images was performed with ImageJ and with the help of the Python core analysis package big-fish available at https://fish-quant. github. io/. Fig. 7  TASOR recruits RNA-degradation factors onto elongating RNAPII to silence gene expression. a TASOR colocalizes with active HIV-1 transcriptional centers in HIV-1-infected Jurkat cells. Latently HIV-1-EGFP-infected Jurkat cells were transduced with VLPs containing Vpx R42A or WT for 24 h and treated with TNF (1 ng/mL). LTR-driven unspliced (us) RNAs were marked with egfp probes and TASOR by immunofluorescence. The graph corresponds to the signal intensities across the white line. b Vpx WT triggers a significant loss of TASOR at the HIV-1 transcriptional centers. TASOR signal at HIV-1 transcription centers in the Vpx R42A delivery condition was set as 100%.
6810% of nuclei were put aside for purification of BrUTP incorporated Nascent RNAs (input). From the remaining nuclei, RNA-IP was then undertaken with the MagnaRIP Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit protocol (17-700, Merck) from step I. 3. TASOR, CNOT1 or anti-Rabbit IgG immunoprecipitations were performed with 4 µg of anti-TASOR (HPA017142-Merck), anti-CNOT1 (14276-1-AP-Proteintech), anti-Rabbit IgG(12-370)on Nuclei Lysates at 4 °C overnight. From step III. 6 of the MagnaRIP protocol, 10% of IPed lysate was recovered and washed three times with IP wash buffer (50 mM Tris-HCl pH 7. 5, 150 mM NaCl) and loaded on SDS-PAGE gel to check DDK pull-down efficiency. The remaining IPed Lysate was then washed from step III. 6 of the MagnaRIP protocol. Finally, purification of BrUTP incorporated IPed Nascent RNAs was undertaken from step 25 of the Nuclear Run On protocol68.
Acknowledgements We thank all the  Retrovirus, Infection and Latency group members at Institut Cochin  for fruitful discussions. We warmly thank  Nancy Standart  for providing the Flag-CNOT7 plasmid and  Alfonso Rodriguez-Gil  for sharing the pINDUCER10 miR Luc and miR CNOT1 vectors. We thank  Adham Safieddine  for sharing his smRNA FISH protocol. We acknowledge the  CYBIO, GENOM'IC, and IMAG'IC platforms of the Institut Cochin. This work was supported by grants from the  "Agence Nationale de la Recherche sur le SIDA et les hépatites virales" (ANRS)  and SIDACTION. R. M. was supported by  ANRS  and SIDACTION. B. M. is supported by  ENS Paris Saclay  and P. L. by  Université de Paris. Work in the laboratory of G. C. is supported by grants from  Fondation pour la Recherche Médicale  (FRM,  DEQ20180339170 ), the  Agence Nationale de la Recherche  ( LABEX SIGNALIFE ,  ANR-11-LABX-0028-01 ;  RetroMet ,  ANR-16-CE12-0020 ;  ImpacTE ,  ANR-19-CE12-0032 ), and  CNRS  ( GDR 3546 ).
Data availability The RNA sequencing data generated in this study have been deposited in the GEO database under the accession code GSE184399. The CUT&Tag and CUT&RUN data of TASOR GSM4710610 and H3K9me3 GSM4710590 and GSM4710594, respectively, were published by Douse et al. 12  and deposited at GEO under the accession number GSE155693. ChIPseq and RNAseq were performed by Liu et al. 55  and deposited at GEO under the accession number GSE95374. Source data and full western blots are provided with this article.
1f ) and each experiment yields up to 234 individual replicates, of which we typically obtain 50 time-lapse movies, due to the small image acquisition time-intervals (2min/ frame). For higher time-intervals, more data points can be collected from the same chip. The movies are then analyzed using our custom-made scripts (see Supplementary Software 1). Three stages in the cell-cell interactions can be identified: the CTL exploration of the gel, their accumulation on the spheroid, and the killing of B16 Ova-expressing cells (see Supplementary Movie 1 for representative cases). These stages are studied in detail below.
Faulty wells (with for example mis-detected spheroids) are then thrown away before data analysis. The full image-analysis algorithm is available on the GitHub repository associated with this publication. λ in and λ out estimation and simulation. The attachment and detachment rates are estimated using a bootstrapping procedure. From a subset of the experimental accumulation plots (Fig. 3d ) we count the attachment and detachment events and estimate the values of λ in and λ out. We repeat the procedure 50 times selecting for each iteration 70% of the total number of spheroids in each condition (84 B16-OVA and 81 B16-WT spheroids). This gives us a distribution of the estimated parameters λ in and λ out represented in Fig. 3. The code necessary for this estimation is available on the GitHub repository associated with this publication.
(  11 ), (12), and (  13 ), provides a formula for λ in (n): λ in ðnÞ ¼ À logð1 À p n;nþ1 Þ ðN À nÞΔt : ð14Þ This formula allows us to directly estimate the attachment rate from the experimentally measured attachment events. To estimate the uncertainty of our estimations we conduct a bootstrapping scheme, where we randomly select droplets, pool them together and calculate the hitting parameters for this experiment subset. We then repeat the procedure over multiple subsets (typically 50) to get a distribution of arrival rates λ in (n), which enables us to calculate the mean and the variance of λ in for each n. The code to conduct the bootstrapping procedure is freely available on the GitHub repository associated with this publication. The process is repeated while counting leaving events to estimate the leaving rate λ out (n) as a function of the number of CTLs on the spheroid, also using Eq.