dataesr/scientific-paragraphs-categorization huggingface dataset:
These results were considered, along with compatible clinical and laboratory presentations, in addition to the patient's outcome after antibiotic therapy. The data collected comprised demographic characteristics, infection site, microorganism type, identification method, initial clinical characteristics, risk factors including concomitant cancer, immunosuppressive treatment, ongoing corticoids therapy, diabetes, kidney insufficiency with renal clearance <30 mL/min or ongoing dialysis, drug addiction, severe liver disease, entry site (if identified), and laboratory parameters upon admission. CoNS infection was considered confirmed if the same CoNS was isolated from two different samples (blood cultures, disco-vertebral biopsy, or another internal site) taken at different times. CoNS infection was considered probable if only one sample was positive.
Statistical analyses Statistical analyses were performed using Stata software (version 13, StataCorp, College Station, TX. US). All statistical tests were two-tailed with a type I error at 5%. Continuous data were expressed as mean and 95% confidence interval (95% CI), depending on the statistical distribution, with an assumption of normality studied by the Shapiro-Wilk test. For quantitative data, the comparisons between groups (SA and CoNS, and confirmed and probable CoNS cases) were carried out using either Student's t-test or Mann-Whitney test if the t-test conditions were not met ([i] normality and [ii] homoscedasticity studied by the Fisher-Snedecor test). For categorical parameters, the between-group comparisons were performed using Chi-squared test or Fisher's exact test, as appropriate.
5 μg/ml fungizone, 40 μg/ml gentamycin and 2% heat inactivated MycoPlex FCS (PAA, Les Mureaux, France). LentiX 293T cells were purchased from Clontech Laboratories (Terra Bella Avenue, Mountain View, CA, USA) and cultured in αMEM supplemented with 10% FCS (Invitrogen), 2 mM L-glutamine, 100 U/ml streptomycin, and 0. 1 mg/ml penicillin (complete αMEM). Tumor samples and clinicopathological data of patients with invasive ductal carcinoma (IDC) and ductal carcinoma in situ (DCIS) were obtained from the Department of Tumor Pathology, Center of Oncology Maria Sklodowska-Curie Memorial Institute, Cracow. The material for IHC investigations involved 27 paraffin blocks of DCIS and 95 paraffin blocks of IDC. Patients were diagnosed from 2000 to 2007, and were aged between 27 and 82 years (mean age: 56.
From each experimental condition, three to five random areas were chosen and Z stacks were taken with a 0. 5 μm step using 40-63× oil objectives (0. 167 μm and 0. 106 μm per pixel, respectively). The same acquisition settings were applied to all conditions. For analysis, cells' equatorial optical section was chosen and pre-processed in the ImageJ software using Subtract Background function with rolling ball radius of 50 pixels. The WGA and CTV channels were thresholded and binarized in order to create mask channels showing plasma membrane and cytoplasm, respectively. Any potential overlap between plasma membrane and cytoplasm was removed with Image Calculator function-subtraction of overlapping structures. Analyze Particles function was applied to the WGA and CTV masks and then identified regions were transferred onto ezrin and Rho-GDI channels to calculate fluorescence mean intensities within membrane and cytoplasm followed by quantification of their ratio.
Wound-healing assay To measure the migratory properties of fibroblasts and breast cancer cells, the ibidi Culture-Insert system (ibidi GmbH Munich, Germany) with two chambers to separate different cell types was used. The Culture Inserts were placed in the compartments of a 24-well plate, 2. 8x10 4 cells were seeded into each chamber and grown to confluence in OptiMEM containing 2% FCS for 24 h. Then the silicon insert was removed with tweezers and a cell-free gap (500 μm ± 50 μm) was generated. Pictures (three/well) of wound closure were taken every hour for 48 hours, and cell migration was assessed using ImageJ software (National Institute of Health, available from http://rsb. info. nih. gov/ij; access date: 01 Oct 2015).
Endothelial tube formation was analyzed by taking photographs every half hour with videomicroscope Zeiss Axiovert 200M and AxioVision Software (Zeiss, Germany). A toolset of Image J software was used for analysis of cellular networks  [32]. The number of nodes and number of meshes were quantified on fluorescence images of HSkMEC, MSU1. 1 NC and MSU1. 1 PDPN cells after 12 hours. A node was defined as pixels that have at least 3 neighbors, corresponding to a bifurcation. The meshes were defined as areas enclosed by the segments. Quantitative statistical colocalization on two-color images using a software tool to calculate the Pearson and Spearman correlation coefficients ('Pearson-Spearman correlation colocalization' ImageJ plug-in) across regions of interest within the image was used. A quantitative estimate of the amount of colocalization in the images was given as the result.
Immunofluorescence (IF) For IF reaction, paraffin blocks were cut in sections 4 μm thick. Deparaffinisation, hydration and heat induced epitope demasking were carried out in Dako PT Link apparatus (Dako) in Target Retrieval Solution (Dako) (pH 9, 97˚C, 20 min). For the evaluation of podoplanin and CD34 co-expression in IDC immunofluorescence reactions were prepared with the use of specific anti-human monoclonal rabbit anti-CD34 antibody (dilution 1:400; clone EP373Y; code no. ab81289, Abcam) and mouse anti-PDPN antibody (dilution 1:50; clone D2-40, code no. M3619, Dako). Overnight incubation with primary antibodies was made at 4˚C. Slides were rinsed with PBS and were incubated for 1 h in room temperature with donkey anti-mouse Alexa-Fluor 594 conjugated (code no. R37115, Invitrogen) and donkey anti-rabbit Alexa-Fluor 488 secondary antibody (code no.
Statistical analysis Prism 5. 0 software (GraphPad, La Jolla, CA, USA) was utilized to analyze the data. The Mann-Whitney test was used to compare the groups of data that did not meet the assumptions of the parametric test. Two-way ANOVA with the Bonferroni multiple comparison test was used to analyze the differences between the analyzed cell lines. In all analyzes, the results were considered statistically significant when p < 0. 05. Image J software (open source) was used to calculate the amount of co-localization in the selected images. The Pearson and Spearman correlation coefficient was measured and the amount of co-localization in the images was given as the result.
Generation of human fibroblastic cell lines overexpressing podoplanin To obtain cellular models representing gain-of-function phenotype, human fibroblastic cell lines (MSU1. 1 and Hs578Bst) were analyzed by Western blotting for the presence of podoplanin. Using polyclonal antibodies directed against podoplanin, this glycoprotein was not detected in lysates of either cell line. Therefore, the cells were transduced with pRRL-CMV-PDPN-IRES-PURO expression vector and the resulting cell populations resistant to puromycin were screened for the presence of this mucin-type protein on the mRNA and protein levels  [34]  (S1A and S1B Fig). The generated cells with high expression of podoplanin were named MSU1. 1 PDPN and Hs578 PDPN. In the lysates of transduced cells, rabbit polyclonal antibodies bound to three protein bands with apparent molecular masses corresponding to 36 kDa, 23 kDa and 17 kDa, which agrees with data obtained by others  [8].
Acknowledgments Grant support for this work was provided by the  National Science Center (Poland) : grant no. N N401 570340. Publication was supported by  Wrocław Centre of Biotechnology , programme the  Leading National Research Centre (KNOW)  for years 2014-2018, French National league against cancer (CK), CNRS PAN agreement IITD/CBM (CK). AT was a PhD student funded by the  Ministry of Polish Education and Research and the French Ministry of Foreign Affairs. GC was supported by the  National Science Centre (Poland)  grant no. 2014/15/B/NZ6/03502.
Funding: Grant support for this work was provided by the  National Science Center (Poland) : grant no. N N401 570340. Publication was supported by  Wrocław Centre of Biotechnology , programme the  Leading National Research Centre (KNOW)  for years 2014-2018, French National league against cancer (CK), CNRS PAN agreement IITD/CBM (CK). AT was a PhD student funded by the  Ministry of Polish Education and Research and the French Ministry of Foreign Affairs. GC was supported by the  National Science Centre (Poland)  grant no. 2014/15/B/NZ6/03502.
3, 20, 21 Tissue was then incubated in biotinylated goat anti-rabbit secondary antibody (1:500; Vector) in blocking solution for 90 min, and labeling was revealed using the Vectastain Elite and DAB kits (Vector), dehydrated, then coverslipped using Permount (Fisher). No-primary control did not produce staining. Sections were visualized on an Olympus BX51 microscope. To confirm our results, human ErbB3 immunohistochemistry data were also obtained from the Human Protein Atlas for a rabbit anti-ErbB3 antibody (HPA045396) with a demonstrated high degree of specificity. 22, 23 In addition, ErbB3 in situ hybridization data (which had been verified with positive and negative controls) were obtained from the Allen Human Brain Atlas (http://human. brain-map. org/ish/search). 24, 25 ereological quantifications of hippocampal granule cells Fixed hippocampal samples from 14 suicides and 9 controls were immersed in 30% sucrose until sinking, flash-frozen in isopentane (~-45 °C), sliced coronally at 50 μm on a freezing sliding microtome in PBS, placed on a shaker for 1 h, then transferred to a cryopreservative solution (glycerol : ethylene glycol : PBS, 3 : 3 : 4) for storage at -20 °C.
Intron retention (IR) occurs when an intron is transcribed into pre-mRNA and remains in the final mRNA. We have developed a program and database called IRFinder to accurately detect IR from mRNA sequencing data. Analysis of 2573 samples showed that IR occurs in all tissues analyzed, affects over 80% of all coding genes and is associated with cell differentiation and the cell cycle. Frequently retained introns are enriched for specific RNA binding protein sites and are often retained in clusters in the same gene. IR is associated with lower protein levels and intronretaining transcripts that escape nonsense-mediated decay are not actively translated.
Since then, other publications have highlighted the importance of IR in gene regulation  [8] , differentiation  [9] , and cancer  [10]. Despite increasing evidence that IR can regulate hundreds of genes in numerous systems, current studies still fail to identify IR events in their transcriptomic data. Here we have developed a significantly enhanced program in terms of sensitivity and speed for detecting retained introns and filtering samples that are inappropriate for IR analysis in terms of library preparation and quality. This novel program was validated using quantitative RT-PCR (RT-qPCR) against retained introns and a NMD knockdown experiment. IRFinder correctly identified IR events and measured the ratio of retained introns to correctly spliced introns with great accuracy. Using IRFinder, we analyzed 3435 human samples, of which 2573 were suitable for analysis.
Implementation Fair measurement of intronic expression is challenged by numerous factors. Within introns, highly expressed features such as snoRNAs, microRNAs, or unannotated exons may erroneously inflate count-based measures of intronic expression. Conversely, low complexity regions, common in introns, prevent unique mapping of reads. Because retained introns are generally expressed at a fraction of their flanking exons, uncorrected biases can massively disrupt IR estimation. IRFinder implements an end-to-end analysis of retained introns from mRNA sequencing data in multiple species. It includes alignment via the STAR algorithm, quality controls on the sample analyzed, IR detection, and quantification and statistics for comparing IR levels between multiple samples. We provide standalone scripts for each of these steps so they can be used independently and provide command line tools to chain them together for complete analysis.
Our approach also uses a series of programmatically fast steps written in C++ that automatically detect and trim adapters from sequencing reads. Of the 3435 initial samples we analyzed, 3096 (91%) still had adapters in the sequencing reads despite having been already processed by an adaptor-trimming algorithm. IRFinder was capable of estimating IR events with low coverage or low mappability as confirmed by RT-qPCR (Additional file 1: Figure  S1a-e ). When compared with the currently available tools MISO and DEXseq, our IRFinder had higher accuracy and precision (Additional file 2: Text, Figure  S2 , Tables  S2  and S3 ). The IRFinder algorithm and instruction manual are available at GitHub (https://github. com/williamritchie/ IRFinder).
IRFinder detects IR in over 80% of coding genes and in all samples tested To demonstrate the functionality of our method, we downloaded 3774 human samples from the European Nucleotide Archive (http://www. ebi. ac. uk/ena). These were annotated as mRNA-Seq experiments in the archive with over 50 million reads each. The IRFinder quality control filter (see "Methods") labeled 1201 of these as unfit for further analysis because they were either not RNA-seq experiments or not enriched for poly(A) tailed mRNAs. We searched for retained introns in 2573 remaining samples. We focused on introns that were retained in more than 10% of transcripts (IR ratio >0. 1) with at least a coverage of three reads across the entire intron after excluding non-measurable intronic regions (see "Methods"). We found that 87.
9% (16,307/ 18,560) of all multi-exonic protein coding genes with sufficient coverage had retained introns in at least three samples in our dataset. None of these 16,307 was retained in all of the samples and the majority (95%) of introns were retained in less than 7% of samples, suggesting that the IR events we detected were specific to tissue or cell types. All samples had over 100 retained introns with a median of 926 retained introns (Fig. 1a ) per sample. This means that IR is more widespread than previously expected with 87. 9% of protein coding genes and all tissue types showing high levels of IR. To determine how consistent IR levels were within the same tissue type we calculated the average IR ratio of retained introns in eight distinct tissue types for which we had over three replicates.
10-3; Additional file 3). Although in this study we were able to confirm that retained introns were generally surrounded by weaker splice sites  [8]  (Additional file 4: Text and Figure  S3 ), the above results on IR clusters indicate that IR is not solely regulated at each splicing junction but is also regulated by a more global means of regulation that encompasses multiple introns and even multiple genes. Multiple retained introns within the same gene could be regulated by transcription rate through the gene in agreement with the recent finding that individual retained introns are associated with an accumulation of the elongating form of RNA polymerase II  [8]. We created an online database of IR calculated in these samples, which is available at IRBase (http://mimirna. centenary. org. au/irfinder/database).
Although this dataset is restricted by the number of proteins measured, it demonstrated that IR could reduce protein output of hundreds of genes in multiple tissue types, thereby generalizing our previous findings. To determine whether retained introns were translated, we analyzed ribosome profiling data based on deep sequencing of ribosome-protected mRNA fragments using cycloheximide to stabilize all translating ribosomes (CHX Ribo-seq). These data were coupled with mRNA sequencing and quantitative translation initiation sequencing (QTI-seq), which uses lactimidomycin to preserve initiating ribosomes and puromycin to deplete elongating ribosomes  [12]. These data allowed us to simultaneously assess translation from all translating ribosomes (CHX Ribo-seq), or specifically from initiating ribosomes (QTI-seq), and IR in HEK293 cells (Fig.
2b ; Additional file 5). We found 429 IR events (IR ratio >10%) in this dataset. None of these displayed a signal from the CHX Ribo-seq or QTI-seq above background levels. This suggests that even though intron-retaining Fig. 1  Intron retention is widespread and its measurement is consistent within the same tissue type. a Number of retained introns in each of the 2573 downloaded samples that were retained in more than 10% (red), 20% (green), 30% (blue) of transcripts (IR ratio >0. 1, 0. 2, 0. 3). b Average IR ratio in eight different tissue types. hESC human embryonic stem cell. c Genes with IR events occur closer than expected. Genomic distance between genes with IR events (blue) compared to a control of the same number of randomly selected expressed genes. d Retained introns within the same gene are often adjacent to each other.
4a ) and U-rich motifs in their downstream exons. We found no significantly depleted motifs in the frequently retained set. To confirm that the RNA binding motifs that were enriched contributed to increased IR, we downloaded sequencing data from the ENCODE project (https://www. encodeproject. org/) consisting of a shRNAmediated knockdown (KD) of RNA binding proteins in HepG2 cells. We used IRFinder to calculate IR before and after KD of proteins for which the motifs were enriched in frequently retained introns (Fig. 4b ). We found that IR levels increased dramatically following KD for seven out of eight RNA binding proteins. Accordingly, their binding motifs were enriched in introns for which IR increased after KD (p values in Fig. 4b ). Interestingly, when we also searched for introns that were retained after KD but correctly spliced before, we found that there were less novel IR events than there were gains of IR levels (Fig.
Auto-detection of directionality Antisense RNA can confound the calculation of IR levels, so we developed a method to automatically detect if a library was prepared using a directional protocol such as ScriptSeq or using dUTPs. These protocols enable bioinformatics analyses to correctly attribute reads to a transcript or an overlapping antisense transcript. To detect directionality, IRFinder measures the coverage across splice junctions. For each splice junction crossed by more than eight reads, if one direction is more than fourfold the other, it is counted as evidence of directionality; if not, it is evidence against directionality. If the directional score is at least 90%, the directional analysis is output for this sample. In practice the directional score is well over 99% for directional data.
Transcriptome mapping Total RNA-seq of liver tissue from wild-type (Upf2 fl/fl ) and Upf2 knockout (Upf2 fl/fl ;Mx1Cre) mouse (GSE26561) was downloaded from the Gene Expression Omnibus (GEO). The single-end reads from both samples were mapped to the transcriptome annotation version 73 of Ensembl mouse genome GRCm38 using STAR (version and options described in IRFinder methods).
Transmembrane NADPH oxidase (NOX) enzymes have been so far only characterized in eukaryotes. In most of these organisms, they reduce molecular oxygen to superoxide and, depending on the presence of additional domains, are called NOX or dual oxidases (DUOX). Reactive oxygen species (ROS), including superoxide, have been traditionally considered accidental toxic by-products of aerobic metabolism. However, during the last decade it has become evident that both O 2 •Ϫ and H 2 O 2 are key players in complex signaling networks and defense. A well-studied example is the production of O 2 •Ϫ during the bactericidal respiratory burst of phagocytes; this production is catalyzed by NOX2. Here, we devised and applied a novel algorithm to search for additional NOX genes in genomic databases. This procedure allowed us to discover approximately 23% new sequences from bacteria (in relation to the number of NOX-related sequences identified by the authors) that we have added to the existing eukaryotic NOX family and have used to build an expanded phylogenetic tree.
Starting from the 78,037,012 ORFs found in the UniprotKB/Swiss-Prot and UniProt/TrEMBL databases, our algorithm selected 4,404 putative NOX amino acid sequences from 1,492 different organisms, over a third of which were bacteria (Table  1 ). Conversely, only one archaeal sequence was found that fulfilled all of the above selection criteria (Fig. 1C ). Subsequently, we selected a smaller set of sequences from all the represented domains of life (see Table  S2  in the supplemental material) by removing (i) sequences from related organisms with Ͼ99% homology and (ii) a few sequences where the above motifs were not correctly aligned. Finally, 162 ORFs were selected to build an extended phylogenetic tree for the NOX/FRE The NOX Family of Proteins Is Also Present in Bacteria family (see Table  S3  in the supplemental material).
The calculated DPI 50% inhibitory concentration was around 5 M for 1 M FAD in our assay (Fig. 4B ). Cyanide does not inhibit SpNOX activity. Cyanide is known to be a global inhibitor of hemoproteins, thanks to its axial binding to the iron ion. In contrast to this general rule, NOX enzymes are insensitive to both azide and cyanide; this feature represents a hallmark of NOX proteins, where both hemes are expected to be hexacoordinated  (6). We have also assessed this property for SpNOX by using catalase as a  The NOX Family of Proteins Is Also Present in Bacteria positive control. While cyanide incubation induced a shift of the 417 nm Soret band of catalase (data not shown), no modification was observed in the SpNOX spectrum upon NaCN addition (Fig. 4C ). Furthermore, its NADPH oxidase activity was identical with or without added cyanide or azide (Fig.
In conclusion, the work reported here has allowed us to identify for the first time, both theoretically and functionally, a significant number of bacterial protein sequences that are found in current databases and are counterparts of eukaryotic NOX proteins. 1 ), defined using the 101 protein sequences identified by Kawahara et al. (50). The third filter selected sequences that contained the four histidine residues responsible for coordinating the two-heme moieties of the cytochrome b-like motif. The TMHMM sequence (51) was used to locate transmembrane ␣-helices. Sequences with more than seven predicted ␣-helices (the number found in DUOX1 and -2) were rejected, whereas sequences with at least 3 predicted ␣-helices were kept to account for errors in the prediction.
NOX Amino acid sequence alignments and phylogenetic tree construction. Multiple sequence alignment of NOX sequences was carried out with Clustal Omega  (52). The tree was built from 162 putative NOX sequences (Table  S3 ) by using PhyML 3. 0. The sequences used for software validation are provided in Table  S1. Cloning of the bacterial nox genes in a pQLink N vector. Genes were optimized for expression in E. coli and synthetized via GenScript. They were inserted in a pUC57 vector conferring ampicillin resistance and providing a His tag coding sequence in frame with the 5= end of the inserted gene. The pUC57 vectors containing the bacterial nox genes were treated with Not1 and BamHI restriction enzymes for 2 h at 37°C and ligated to a NotI-BamHI-digested pQLink N vector to yield pQLink N-bacterial NOX expression plasmids.