dataesr/scientific-paragraphs-categorization huggingface dataset:
magellanicus is sister to all other pectinids, its placement is never supported as such, rather the topology of the Pectinidae should be considered as a trichotomy represented by the subfamilies Palliolinae, Chlamydinae and a Pectininae+Aequipectini group. The B dataset resulted in the most well-supported tree (Fig. 4A ). All eight ML trees presented with proportional branch lengths are shown in Fig. 3. Every Bayesian phylogenetic reconstruction run on every dataset reached convergence (ESS>200). The results of these analyses slightly differ from the results of the ML method (Fig. 4B ). Support values are generally higher than in the ML approach. Pl. magellanicus is placed in the same clade with Chlamydinae in the trees B, E, F, G and H with moderate to high posterior probabilities (0. 81-0.
Annotation of pectinid genomes The fact that we were able the annotate both the atp8 gene and some new tRNA genes in most pectinid accessions downloaded for this study show that the lack of these genes were most likely artifacts emerging from the difficulty of annotating these features in bivalves. With the expansion of annotation databases and the advancement of annotation tools, these difficulties are getting easier and easier to overcome, leading to progressively more precise annotations. While variations in tRNA gene content presented by singular species (especially those with only one mitogenome annotated: Pe. albicans and Mim. varia) might well be resulting from sequencing errors, incomplete genomes, or simply misannotations. While the possibility of these cannot be completely excluded, even in the case of species that have multiple genomes published (Mim.
[40] , who concluded Aequipectini to be the basal clade in Pectinidae (similarly as shown in our rRNA datasets, G and H), we have found that Aequipectini always form a monophyletic group with Pectininae in both of our ML and Bayesian approaches, when we included PCGs in the analysis. It appears that while mitochondrial rRNA genes and the nuclear H3 gene are in accordance  [40] , mitochondrial PCGs paint a slightly different picture, as can also be seen in, for example, Marín et al. [22]  and Yao et al. [27]. However, this could at least be partly caused by the small number of taxa involved in our study, and the different choice of outgroups, as both taxon sampling and outgroup selection are known to influence topologies  [40]. Despite the Pectinidae being one of the largest families in Bivalvia, containing around 350 species  [72] , existing phylogenies usually contain only a handful of species, with the most making up only 31% of all species  [31].
Sequencing of the mitogenome of reference for the variegated scallop One individual of Mim. varia (adult male, shell length: 46 mm, shell height: 40mm) was collected in Angoulins, France, in order to sequence and annotate a reference transcriptome (nuclear and mitochondrial) for the species. RNA was purified from five tissues (digestive gland, mantle, gills, adductor muscle and gonads) using the Nucleospin RNA Set for Nucleozol kit (Macherey-Nagel). After quality control, extractions were pooled in equal amounts (4 μg of RNA per tissue type). The library was sequenced on Illumina HiSeq 2500 (2 × 300 bp). Full sample preparation and sequencing information is provided in Viricel et al. [50] ; raw reads can be accessed on the NBCI Sequence Read Archive (Acc. SRP127478), and transcripts can be accessed at the Transcriptome Shotgun Assembly database (GGGO01000000).
1; [49] ). When NOVOPlasty runs resulted in multiple contigs (mitogenome fragments), they were assembled in Geneious v2021. 0. 3 (https://www. geneious. com). Reads were mapped onto the assembly using bowtie2 v2. 3. 5. 1  [86]  and coverage statistics were generated with samtools v1. 10  [87]. Once the reference mitogenome was assembled, we used NOVOPlasty to detect intra-individual heteroplasmy  [88]  using a minimum Minor Allele Frequency (MAF) of 0. 1 and 0. 01 in two separate runs. Finally, the MITOS2 web server (http://mitos2. bioinf. uni-leipzig. de/index. py) [89]  was used to annotate the reference with default parameters, using the RefSeq 63 Metazoa database as reference and the protein-coding gene prediction method of Al Arab et al. [90]. The mitogenome was loaded into Geneious v2021.
Assembly and annotation of pectinid mitogenomes from GenBank. All currently available pectinid mitogenomes (as of January 2021) were downloaded from GenBank (Table  4 ) along with Ostrea lurida (Ostreidae) as an outgroup. We chose an oyster species as outgroup as there are no published mitogenomes of taxa more closely related to Pectinidae. Using only one outgroup made interpreting comparisons of mitogenome rearrangements substantially less difficult. The largest mitogenome from each species was reannotated using the MITOS2 web server with the same settings as for the Mim. varia mitogenome. New annotations of pectinid mitogenomes were reviewed manually, in the same way as it was described for Mim. varia. If there were multiple annotations for a gene, they were filtered manually based on length and sequence similarity to other species.
Mito-phylogenomics Phylogenetic analysis was conducted on the 13 protein coding genes (PCGs) and two Ribosomal RNA (rRNA) genes of all available pectinid species in GenBank, Mim. varia and O. lurida as an outgroup. This approach provides the most comprehensive dataset without the ambiguities arising from tRNA annotations, non-coding regions and genome rearrangements. Every gene was extracted and aligned separately in Geneious v2021. 0. 3. In the case of the atp8 gene, where more copies were identified in some species, a Maximum Likelihood (ML) tree was built with the RAxML  [95]  plugin of Geneious using default parameters, and the least divergent copy was selected for further analysis. The atp8 gene of two accessions of Argopecten irradians (EU023915; NC_009687) were not included in the phylogenetic analyses given their unusual structure, and divergent sequences.
The 13 PCG sequences were aligned separately based on translated amino-acids with MAFFT  [96]  with the FFT-NS-2 algorithm as implemented in TranslatorX  [97]. As the initial alignments contained indels and poorly aligned regions, these regions were masked with Gblocks  [98]  as implemented in TranslatorX, with default settings (i. e. , allowing smaller final blocks, gap positions within the final blocks and less strict flanking positions). The two rRNA genes were aligned with MAFFT in Geneious using the FFT-NS-2 algorithm. Regions of uncertainty in the alignment were then masked with Gblocks using the same settings as for the PCGs. Individual gene alignments were then concatenated in Geneious resulting in eight datasets used for phylogenetic analyses: A) all PCG and rRNA genes treated with Gblocks; B) all PCG amino acid sequences and rRNA genes treated with Gblocks; C) all PCGs; D) all PCGs treated with Gblocks; E) all PCG amino acid sequences; F) all PCG amino acid sequences treated with Gblocks; G) rRNA genes; H) rRNA genes treated with Gblocks.
These datasets were prepared in order to investigate the effect of rRNA and protein-coding genes as well as the presence/absence of ambiguously aligned regions on the topology and branch support of the phylogeny. Maximum Likelihood phylogenetic reconstruction was achieved using IQtree v2. 0. 6  [99]  (except for the B dataset, where IQtree v1. 6. 12 was used), with automatic model selection and using the best partition scheme in ModelFinder (option MFP+MERGE)  [100] , 10,000 Ultrafast Bootstrap replicates and 10,000 replicates of the SH-like approximate likelihood ratio test (SH-aLRT)  [101]  on all eight datasets. Partitioning and substitution model information for every run can be found in Additional file 6. Bayesian analyses were done using MrBayes v3. 2. 7  [102]. For each dataset, two runs were performed over 1.
Common interval analysis Two joint softwaresboth relying on the same algorithm -CREx  [105]  and TreeREx  [106]  were implemented for common interval analyses. Both software consider the following types of rearrangement events: transpositions, inversions, inverse transpositions and tandem-duplication/random loss (TDRL) involving the duplication of a gene block followed by the random loss of one copy of a gene in either of the two blocks. Both tools are only able to handle identical gene sets among species. To conform to this condition, only major genes (PCGs and rRNA genes) and the MNR were included in this analysis, as tRNA gene content is variable among pectinid mitogenomes. The MrBayes tree based on the most supported dataset (B), with only 8 pectinids and one outgroup species representing unique gene orders, along with the gene order dataset described above, was used in the first step, using the TreeREx  [106]  software.
TreeREx uses the phylogenetic information in the tree to ascertain rearrangement events among nodes of the tree, and to infer putative gene orders of common ancestors at internal nodes. TreeREx was used with default settings: strong consistency method applied (-s); weak consistency method applied (-w); parsimonious weak consistency method applied (-W); and the maximum number of inversions (-m) set to 2. Finally, the input dataset in the previous analysis, complemented with the putative gene orders inferred at internal nodes was put into CREx again, to visualize each rearrangement event between each pair of neighboring nodes.
Acknowledgements We thank  Nathalie Imbert  and  Denis Fichet  for coordinating axe 2 of the  DYPOMAR  project and  Benoit Simon-Bouhet  for additional funding. RNA extractions were prepared at the  Molecular Core Facility  at the  University of La Rochelle. We thank the bioinformatics platform  Toulouse Midi-Pyrenees  ( Bioinfo GenoToul ).
Availability of data and materials Raw Mimachlamys varia RNAseq reads are deposited in the NBCI Sequence Read Archive (Acc. SRP127478), and transcripts can be accessed at the Transcriptome Shotgun Assembly database (GGGO01000000). The assembled mitochondrial genome is uploaded to GenBank (MZ520326), and the GenBank accession numbers of all mitogenomes used in this study can be found in Table  A. The sequence alignments used this article will be uploaded upon acceptance to a public repository.
Species GenBank accession no. Start Stop Length AA 5' 3' neighbor neighbor Placopecten DQ088274-atp8-1 23484 23660 177 58 trnM atp6 magellanicus DQ088274-atp8-2 64 231 168 55 trnG trnM NC_007234-atp8-1 23484 23660 177 58 trnM atp6 NC_007234-atp8-2 64 231 168 55 trnG trnM Chlamys EF473269 10041 10187 147 48 nad5 trnD farreri EU715252 10041 10187 147 48 nad5 trnD NC_012138 10041 10187 147 48 nad5 trnD Mizuhopecten AB271769-atp8-1 9797 9943 147 48 nad4l trnD yessoensis AB271769-atp8-2 9239 9385 147 48 nad5 nad4l FJ595959-atp8-1 9797 9943 147 48 nad4l trnD FJ595959-atp8-2 9239 9385 147 48 nad5 nad4l.
1d, e ). The upregulation of ISG was also detected in fluidized MCF10A monolayers (Supplementary Fig. 1a, b ) and in MCF10. DCIS. com cells grown as tumoroid (Extended Data Fig. 2a ). In all these conditions, we have previously shown that RAB5A expression is sufficient to promote a solid-to-liquid transition via flocking and persistent rotational collective motion  2, 6. RAB5A upregulated CytoDR genes only mildly in sparse cells (Extended Data Fig. 2b ), suggesting that this response is an emergent property of epithelial cell collectives and associated with tissue fluidification. To further explore this property, we correlated the expression of CytoDR genes and flocking motion (measured using the average migration speed v m of the entire cell collectives) as a function of cell density. We found that above a critical density, which corresponds to a condition where cells form a system-spanning inter-connected cluster, there is a sharp increase in v m (Extended Data Fig.
3NLS-EGFP displayed a nuclear restricted expression in control cells, but a cytoplasmic distribution in fluidized RAB5A-expressing monolayers, indicative of NE ruptures (Fig. 3d ,e and Supplementary Video 6). Finally, we performed correlative-light electron microscopy (CLEM) tomography and immune EM to directly visualize the presence of NE ruptures. In RAB5A, but not control cells, EGFP-cGAS accumulated at sites of condensed chromatin, immediately adjacent to the region where both the inner and outer NE membranes were ruptured (Fig. 3f  and Extended Data Fig. 4b ). Immunofluorescent staining of Lamin A/C also revealed that nuclei in RAB5A-fluidized monolayers undergo large deformation and possibly ruptures as indicated by the accumulation of cGAS around distorted nuclei and at the apex of nuclear invagination (Extended Data Fig.
Nuclear force indentation showed an approximately twofold increase in the stiffness in RAB5A-fluidized monolayers as compared with control (Extended Data Fig. 5a ). We also probed the rheological properties of the cytoplasm, which may contribute to the difference in Young's modulus obtained by AFM indentation. We found that the mean square displacement and coefficient of diffusion of genetically encoded, cytoplasmic fluorescence particles (GEM)  37  were not significantly different in control-arrested versus flocking-fluid RAB5A monolayers (Extended Data Fig. 5b, c ). Thus, the increased stiffness of RAB5-fluidized cells is likely the result of the increased nuclei rigidity. Next, we subjected control and RAB5A monolayers plated on a deformable polydimethylsiloxane (PDMS) membrane to a predefined 22% biaxial stretch and measured the ensuing cell and nuclear deformations.
The increased rigidity of RAB5A nuclei might impact the nuclear shape at a steady state. Thus, we measured nuclear shape variations in control and fluidized monolayers by determining the dimensionless parameter excess of the perimeter of the projected nuclear shape (EOP) through immunofluorescent analysis of SUN2, an inner nuclear membrane protein  38. EOP values of a highly folded, presumably floppy, and soft object are expected to be close to 1, whereas EOP of a rigid object tends to be close to 0. The EOP values of RAB5A fluidized monolayers were significantly closer to 0 with respect to control-jammed ones (Extended Data Fig. 5f, g ). We also estimated the nuclear envelope shape fluctuations assuming that rigid nuclei should display reduced fluctuation with respect to softer or floppy ones.
We monitored nuclei in live cells expressing a mini-EGFP-Nesprin1, which encompasses the Calponin actin-binding domain and the c-terminus of the NE protein Nesprin1 (ref. 39 ), at high frame rates. The amplitude of NE fluctuations was next calculated by measuring the standard deviation of the NE from its mean position. The NE fluctuations were significantly reduced in RAB5A fluidized cells (Extended Data Fig. 5h  and Supplementary Video 8). Finally, we employed Brillouin microscopy to probe nuclear mechanical properties. We detected a clear Brillouin frequency shift that corresponds to a significant increase in the longitudinal elastic modulus, M, indicative of increased nuclear stiffness, of RAB5A-expressing cell nuclei as compared with control (Extended Data Fig. 5i-k ). We also showed that nuclear-projected areas are nearly 25% larger in RAB5A-fluidized monolayers as compared with control ones (Extended Data Fig.
RAB5A induces cGAS activation in invasive carcinoma Next, we studied whether the nuclear mechano-perturbations leading to cGAS activation and DNA damage observed in vitro are also relevant in pathological tissues. Control and RAB5A-MCF10. DCIS. com cells were injected into the mammary fat pads of immunocompromised animals to model DCIS. In these tumours, RAB5A induction increased CytoDR, as expected (Extended Data Fig. 5i ), and was associated with an elevated number of γH2AX-positive cells (Fig. 6a, b ), and increased levels of cGAS, which display a perinuclear dotted or crescent-like appearance (Fig. 6c, d ). This pattern is similar to the one seen in human DCIS with local infiltrative areas (Extended Data Fig. 8c, d ), likely reflecting its activation by cytoplasmic DNA. In human tumours, RAB5A displayed a graded increase in expression right at the margin of locally invasive foci (Fig.
DCIS. com (Extended Data Fig. 9a ). Several canonical mesenchymal factors, including the master EMT regulators, SNAIL1 and 2 and TWIST, were unaffected, suggesting the acquisition of what has been defined as plastic EMT state (EMP)  47. This was corroborated by the morphological analysis, which indicated that RAB5A expression leads to the acquisition of an elongated mesenchymal morphology (Extended Data Fig. 9b ), and by the nuclear accumulation of ZEB1 in RAB5A-expressing MCF10. DCIS. com spheroids (Extended Data Fig. 9c ). The expression of this set of genes was dependent on YAP/TAZ activity (Extended Data Fig. 9d, e ), suggesting that the EMP phenotype switch is, at least in part, a mechanoresponsive process  48, 49. As EMP is associated with invasion and metastasis, next, we tested whether the increased migratory and invasive capacity in RAB5A-fluidized collectives is mediated by the activation of cGAS and STING.
Wound healing For wound healing experiments, confluent monolayers of MCF10. DCIS. com control and RAB5A-positive cells, were plated on 12-well plates, and were wounded by scraping with a pipette tip and then transferred immediately to the microscope stage-top incubator. For monolayer invading assay, wounds were coated with Matrigel before the time-lapse. Time lapses were performed with a Leica Thunder Imaging System based on a Leica DMi8 inverted microscope equipped with a Leica DFC9000 GT sCMOS camera. The images were acquired with an HC PL APO 10× objective using Leica LAS X software. Wound healing maximal velocity of closure is calculated from the area covered over time extracted using a custom Fiji  51  and Matlab code. The area covered over time is fitted with two straight lines (https://github.
Image acquisition Time-lapse imaging of the motility of 3D organoids was performed using a Leica TCS SP8 laser confocal scanner mounted on a Leica DMi8 microscope equipped with motorized stage; a HC PL FLUOTAR 20×/0. 5 NA dry objective was used. A white-light laser was used as the illumination source. Leica Application Suite X (LAS X, https://www. leicamicrosystems. com/products/microscopesoftware/details/product/ leica-las-x-ls/) software was used for all acquisitions. Image acquisition conditions were set to remove channel crosstalk, optimizing spectral detection bands and scanning modalities. ImageJ software was used for data analysis. Image acquisition was performed using Operetta CLS, highthroughput imaging confocal microscopy system (Perkin Elmer) with Harmony software 4. 9. Cells are imaged with 20× water immersion objective NA 1.
Nuclei tracking and segmentation Tracking and segmentation of single nuclei in sequencies of fluorescent microscopy images of confluent monolayers of mCherry-H2B cells is performed with a custom Matlab code implementing the following procedure. Images are first corrected for background intensity inhomogeneities by applying the background removing algorithm described previously in detail  56. Random noise in each corrected image is then reduced by applying a Wiener filter, an adaptive noise-removal filtering that preserves nuclei edges while smoothing the white noise (see equations 9. 44-9. 46 in ref. 57  ). Nuclear segmentation is obtained by applying a seeded watershed transform to the spatial gradient of each filtered image  58. The 'seeds' (that is, the pixels that are set to zero in the image before applying the watershed transform) are determined as follows.
Particle image velocimetry Particle image velocimetry (PIV) of fluorescent microscopy images of confluent monolayers of mCherry-H2B cells fluorescent images is performed by using the Matlab PIVLab software  61. We choose an interrogation area with size slightly larger than the typical inter-nuclear distance, typically corresponding to 14 μm. Outliers in the reconstructed velocity field, whose modulus exceeds a fixed threshold value, are identified, and replaced with the median value of the velocity over neighbouring grid points.
Nuclear deformation dynamics To characterize nuclear shape fluctuations, we evaluate the mean square nuclear strain MSS (τ) = ⟨⟨Δa 2 i (τ|t)⟩ t ⟩ i for different delay times τ. The nuclear strain Δa i (τ|t) is estimated as [A i (t + τ) -A i (t)] / ⟨A i (t)⟩ t , where A i (t) is the projected area of the i-th nucleus at time t. To extract the key parameters characterizing nuclear deformation, we fit the model function MSS (τ) = σ w + γ 0 τ c [1 -e -τ/τ c ] to the data. This model, which includes a term σ w accounting for the random noise in determination of the projected area, describes a diffusive-like growth of the area fluctuations with a characteristic strain rate γ 0 for short delay times MSS (τ) ∼ γ 0 τ, followed by a saturation to a plateau value γ 0 τ c for long times. In Fig. 2g, h , the data and the best fitting curves are reported upon the subtraction of the baseline value σ w obtained from the fitting procedure.
Image acquisition was performed using a Confocal Spinning Disk system (Olympus) mounted on an IX83 inverted microscope provided with a motorized stage and an IXON 897 Ultra camera (Andor, 16 bit, pixel size 16 μm), and driven by CellSens Dimension software. Fluorescence images were acquired before and after application of 22% biaxial strain (reached in six stretching steps) through a 20× objective (UPlansApo, NA 0. 75) using the EPI-fluorescence mode (excitation wavelength: 530-550 nm). For both control and RAB5A-expressing cells, 30 regions (field of views) were considered within the central region of the cell stretching dish. For each field of view, we evaluated the fractional change Δa = (A f -A i ) /A i in the average nuclear-projected area upon stretching. Exploiting the mechanical model in ref.
Magnified labels on the right indicate genes validated in Fig. 1d. Normalized expression has been calculated for each gene starting from DeSeq2 regularized log-transformed expression value and then normalized by row mean values. https://doi. org/10. 1038/s41563-022-01431-x Extended Data Fig. 2 | Tissues fluidification induces CytoDR gene expression in various normal and tumoral mammary epithelia. a. mRNA expression levels of IFI27, IFI44, IFI44L, IFI6, IFIT1, IFIT3, ISG15, OASL and RAB5A in RAB5A-MCF10. DCIS. com spheroids over the respective control. Data are the mean (n = 3 independent experiments). b. mRNA expression levels of RAB5A, IFI44, IFI44L, IFI27, IFI6, IFIT1, IFIT3, ISG15 and OASL in RAB5A-MCF10. DCIS. com monolayers or sparsely seeded cells (sparse) over the corresponding control cells.
Data are the mean ± s. d. (n = 3 independent experiments). In a. and b. , Values were normalized to the controls of each experiment. c. Average migration speed, v m , of control (CTR) or RAB5A-(RAB5A)-MCF10. DCIS. com cells seeded at various densities, and monitored by time lapse (Supplementary Video 1). The values were obtained by PIV analysis evaluated over the time window 8-40 h. Data are the mean ± s. d. (n = 3 independent experiments). d. Relative expression levels of the indicated ISG genes of control (CTR) or RAB5A-(RAB5A)-MCF10. DCIS. com cells seeded at various densities as described in c. Data are the mean (n = 3 independent experiments). Values were normalized to the controls at each dilution and expressed as a relative expression with respect to the maximal level for each ISG at the highest seeded density.