dataesr/scientific-paragraphs-categorization huggingface dataset:
#3302-12), cDNA was biotin labeled before hybridization on Affymetrix Genechip Mouse Gene 2. 0 ST Array. Two independent WT and three independent Mdr2 -/-primary or immortalized MEF clones were profiled via microarrays according to the 'Target Preparation for Affymetrix GeneChip® Eukaryotic Array Analysis' protocol. Microarray data are available through the Gene Expression Omnibus (GEO) database under accession number GSE72615. CAGE libraries (for Cap Analysis of Gene Expression) with total RNA extracted from WT or Mdr2 -/-livers were constructed following the protocol reported previously  (13). About 5 μg of total RNA was used to synthesize cDNA with random primers. Full-length cDNAs were biotinylated and captured by streptavidin-coated magnetic beads. The cDNAs were released from cap-trapped RNAs, ligated to 5′ linkers including barcode sequences, and digested with EcoP15I.
e. communities) were detected via the Walktrap Algorithm by  Pons and Latapy (16). This algorithm uses a hierarchical agglomerative method and the distance between two nodes is defined in terms of random walks. The idea is that shorts random walks tend to stay in the same communities. We assessed that 40% of the identified communities are enriched for a specific biological function by Gene Ontology analysis performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Functional Annotation Clustering tool  (17). The list of Gene Ontology enrichment for each of the community is available upon request.
Measurement of ROS and malondialdehyde (MDA) Intracellular ROS contents were measured by flow cytometry analysis using 2,7-dichlorodihydrofluorescein diacetate dye (H 2 DCFDA, Life Technologies) as described previously  (19). Briefly, MEFs were treated for 1 h with 0. 1 mM H 2 O 2, then allowed to recover in fresh media and harvested after 1 and 6 h. Then cells were incubated in pre-warmed Hanks' balanced salt solution in the presence of 10 µM H 2 DCFDA and incubated 1 h at room temperature. Dye loading buffer was then removed and replaced by pre-warmed complete growth media. Cells were incubated for 30 min at 37°C to allow H 2 DCFDA oxidation generating 2,7-dichlorofluorescein (DCF), a fluorescent compound. ROS accumulation was analyzed using a MACSQuant® (Miltenyi Biotec) flow cytometer and FlowJo software.
Deletion of Mdr2 increases intestinal polyposis in a mouse model of colitis-associated cancer To determine the in vivo effects of Mdr2 deficiency on cell transformation in other tissues than the liver, we took advantage of the mouse model of AOM/DSS-induced intestinal tumorigenesis to investigate how Mdr2 affects the phenotype associated with AOM/DSS-induced cancer  (32). The protocol of AOM/DSS administration was applied to Mdr2 -/-mice and their Mdr2 +/+ littermate control mice. Expression of Mdr2 mRNA in WT intestine was detectable by qPCR (data not shown). Because of enterohepatic circulation of bile, we first examined if the lack of phospholipids in the bile had impact on the intestine in Mdr2 -/-mice. As shown in Figure  5A -D, deletion of Mdr2 did not alter crypt-villus architecture of the small intestine.
Acknowledgements We are grateful to  Agnese Collino  and  Paola Nicoli in Gioacchino Natoli 's laboratory at the  European Institute of Oncology (IEO), Milan , for sample preparation for CAGE experiments,  Estelle Mignot  and  Laura Garnier  for technical help, and  Justine Bertrand-Michel  at  MetaToul-Lipidomique Inserm U1048, Toulouse , for lipidomic analysis. We thank  Marie-Annick Buendia  at  Inserm U785, Villejuif, Anne-Marie Cassard-Doulcier at Inserm UMR_S996, Clamart, Edith Heard, Jean-Pierre Quivy and Geneviève Almouzni at Institut Curie, Paris, France , for insightful discussion.
Study design and participants Data were from participants recruited as part of the Incidence of pNeumonia and related ConseqUences in nursing home Residents (INCUR) study, a prospective observational cohort study of 800 NH residents. The INCUR rationale, study design, and methodology have been previously described  (11). The primary aim of INCUR was to estimate the incidence of pneumonia and the associated health-related expenditures in this population. The 6-month recruitment period started in February 2012. The INCUR project ended on June 2013 after all participants had been followed-up over 12 months. Main eligibility criteria of INCUR included: age of 60 years and older; a functional status ranging from 2 to 5 at the Autonomie Gérontologie -Groupes Iso-Ressources (AGGIR) scale (i. e. the nationally recognized functional scale on which the allocation of social support is decided by public health authorities in France; a score between 2 and 5 excluded totally disabled patients as well as subjects with no impairment in basic activities of daily living)  (12)  residents living in the NH for more than 30 days.
An in situ hybridization (ISH) study was performed on 2000 murine genes representing around 10% of the protein-coding genes present in the mouse genome using data generated by the EURExpress consortium. This study was carried out in 25 tissues of late gestation embryos (E14. 5), with a special emphasis on the developing ear and on five distinct developing sensory organs, including the cochlea, the vestibular receptors, the sensory retina, the olfactory organ, and the vibrissae follicles. The results obtained from an analysis of more than 11,000 micrographs have been integrated in a newly developed knowledgebase, called ImAnno. In addition to managing the multilevel micrograph annotations performed by human experts, ImAnno provides public access to various integrated databases and tools. Thus, it facilitates the analysis of complex ISH gene expression patterns, as well as functional annotation and interaction of gene sets.
Introduction One of the main challenges of developmental biology is to decipher the interplay between specific pathways and gene networks involved in the different phases of the ontogenesis of an organism. Such an ambitious goal can now be envisioned thanks to the recent biotechnological and bioinformatics developments that are providing massive amounts of gene expression data, as well as the powerful data mining tools available in several atlases dedicated to gene expression in whole organisms or in specific organs/tissues  [1, 2, 3, 4, 5, 6, 7, 8]. A recent review of webbased gene expression atlases for the mouse stressed the practical usefulness of these approaches  [7]. Among these, the GenePaint  [9]  and EURExpress consortia (http://www. genepaint. org; http://www. eurexpress. org) aim to develop a powerful mouse transcriptome digital atlas, by means of in situ hybridization (ISH) in the whole animal at a specific stage of development: embryonic day 14.
5. In this context, as members of the consortia, we were responsible for producing a subset of in situ hybridization slides, as well as the corresponding micro-photographs, via an automated microscopy image acquisition system. Expert anatomists from EURExpress manually annotated the expression profiles for over 18000 protein-coding genes and 1420 anatomic terms. This atlas now provides a semi-quantitative estimate of mRNA abundance, allowing the localization of transcript distributions at the cellular level within anatomical substructures  [4, 5]. In complement to the EURExpress projects, we have annotated various tissues, corresponding to the eye, teeth, ear and other sensory organs, with additional specific anatomic details. Finally, since the ear has not been studied in great detail in the available expression atlases due to its anatomical complexity, we decided to produce images for 2000 randomly chosen genes representing *10% of all murine protein-coding genes  [10, 11].
Using the existing ISH plates, this led to 11,000 additional ISH micrographs with higher magnification focused on detailed analysis of 25 ear and sensory system tissues or structures of late gestation embryos (E14. 5). To address the challenges involved in performing such a detailed integrated annotation process, we developed the ImAnno knowledgebase (imanno. lbgi. fr) with user-oriented and customizable services to annotate, manage, query and analyze the genes with their ISH expression images. ImAnno is designed as a gene centric database to allow the expert biologist to annotate, with standardized terms, gene expression patterns of detailed tissues using ISH experimental data. ImAnno also provides multi-filter search tools, allowing the user to perform complex combinatorial queries producing lists of genes, which can then be used as input for subsequent integrated analyses linked to external databases related to interactomics, gene ontology, transcriptomic expression, mutation or pathological information.
Microphotographs from ISH data Template design was performed by the European GenePaint and EURExpress consortia (http://www. genepaint. org, http://www. eurexpress. org) and template sequences are available at http://www. genepaint. org [9]. No animals were used thereafter for the analytical and bioinformatics work reported in the present manuscript. Methods for animal care, tissue preparation and sectioning of E14. 5 mouse fetuses (C57BL/6J strain) are described in detail in  [5, 12]. Briefly, from each fetus six parallel sets were produced each spanning the entire body from eye to eye consisting of 24 cryosections, 25 μm thick and spaced 150 μm apart. The automated device for performing non-radioactive ISH using digoxigenin-labelled riboprobes on such sets has been previously described  [9, 13, 14].
A total of 2961 transcripts were analyzed by our lab this way. The resulting micrographs were deposited on GenePaint and contributed to the EURExpress project. We used these data as well as the GenePaintEURExpress resources to annotate, 1667 genes for the eye, 1012 for teeth and 2000 randomly chosen genes for the ear and sensory system (present study). For this present study, the full set of sections for a given probe was re-analyzed and observed under a light microscope (Leica DMLB). Imaging was performed with a CoolSNAP digital camera and corresponding software at different magnifications up to 0. 2 μm/pixel, leading to the production of 11,000 micrographs for the 2000 randomly chosen genes.
ImAnno computational infrastructure ImAnno is based on a relational database architecture (Fig. 2 ), and incorporates original annotation and filtering/analysis tools. The database infrastructure is designed to manage simultaneously multiple annotation projects performed by experts and the annotation, storage, access and analysis processes have been conceived for exploitation through a web user interface. New project and database features can be instanciated in the framework of collaboration. To initiate a project, the project manager defines: i) the central object under study (in our case this is a gene, but it could be a patient, an image or a protein 3D structure, etc. ), ii) the list of criteria associated with each object (in our case, the expression in 25 tissues, for a patient it could be a list of symptoms), iii) a scoring system with exclusive values for all defined criteria (e.
g. negative, weak, medium, strong, not analyzed), iv) the source of the data (e. g. internet localization or filename of the ISH images). Once these four main items have been defined, the appropriate database schema is generated automatically and the annotation process can begin. The input data can be either stored on the local system or accessible via internet, and an automated referencing in the database is performed once the data or its localization is integrated in the project. The list of criteria and scoring system can be modified throughout the duration of the project. Within a project, in addition to the display facilities, ImAnno offers various querying and integrated analysis tools to search for objects or criteria using their associated annotations (see below). Annotation of ISH images.
lbgi. fr). As several images from different sections with different magnifications are often necessary to precisely analyze a gene expression pattern, users can also upload their own images or provide URLs to visualize images available on any digital atlas site, or any gene-related web site (Fig. 1A ). Data management, querying and visualization tools. Information about the annotators, the history of the annotation process, the genes, their corresponding images, the tissue-specific expression data as well as the free text comments or links to external sites are also stored in the ImAnno knowledgebase and can be queried through the ImAnno web-based interface. The web site includes a user authentication system with appropriate read/write access rights (public, private and user sub-group) defined by the project manager to allow different levels of processing, querying and visualization of the database.
Functional genomics analysis. ImAnno offers additional tools to characterize gene lists related to: i) Interactomics data obtained from the STRING database  [15] , containing known or predicted physical and functional protein-protein interactions. Only interactions with high confidence levels (>0. 7) are used, as suggested by the authors. Interactomic networks can be visualized using CytoscapeWeb  [16]  or downloaded (networks and colors) for input to the Cytoscape software  [17]. Starting with a gene set provided by the user (the "query genes"), the StringInteractome module provides three different "zooming levels": level 1, the standard output displaying the query genes and all the genes exhibiting at least one interaction with the query genes; level 2, the intermediate level (obtained after filtering the level 1 list) displays the query genes and the genes interacting with at least two query genes; level 3, the "Query network", displaying only direct interactions between query genes.
ii) Relationships with human disorders with Mendelian inheritance, based on the morbidmap data from the OMIM database (http://www. omim. org). iii) Gene ontology (version 9. 05) (http://www. geneontology. org). For a gene list, attached GO terms can be displayed. For two gene lists A and B, GO terms can be ordered according to the fold enrichment determined as the percentage of genes from A verifying a given GO term over the percentage of genes from B verifying the same GO term. This fold enrichment can be used to evaluate an over-or under-representation of GO term. In the present study, we use molecular function as the default term. iv) Dendrogram of tissue correlation. A distance matrix based on the expression values of the 2000 annotated genes in the 25 tissues was computed using a Spearman's rank correlation coefficient with the following numeric values: 0: negative, 2: weak, 3: medium and 4: strong.
Results and Discussion The ImAnno infrastructure provides a centralized, structured knowledgebase infrastructure for storing very precise information about the expression of genes in anatomically detailed tissues corresponding to the eye, the teeth and the ear and sensory organs. The knowledgebase is accessible via a user-friendly web interface, allowing multiple expert annotators to share project data within a framework defined according to the anatomical properties of the studied organ. As illustrated below, besides the annotation process, the ImAnno interface was used to perform integrated analyses combining the experimental expression data with other knowledge.
ISH expression patterns in 25 tissues from the ear and sensory systems Among the 2000 mouse genes analyzed, 16. 5% (331) did not exhibit an ISH signal in any of the 25 tissues investigated. This value is comparable to the 18% of genes with no expression in the whole embryo, as reported in the EURExpress database  [5]. The majority of the remaining transcripts exhibited a weak signal in the 25 tissues (Table  1 ), which is not surprising since previous analyses showed that most transcripts (86%) were expressed by less than five copies per cell  [20]. More than half of the genes were expressed in the hindbrain and sensory organs (notably the sensory retina, olfactory epithelium and vibrissae follicles). For most of the inner ear tissues, less than 50% of the gene transcripts were "expressed".
Ndufs3, the second example illustrated in Fig. 6C , is involved in oxidative phosphorylation and directly connected with 8 KUROV genes, among which 3 genes (Ndufb11, Ndufa9 and 1110020P15Rik) share 53 genes with Ndufs3 via a level 2 type interaction. This gene set exhibits striking enrichment in Human diseases. To allow the user to associate the genes with potential human diseases, ImAnno offers the possibility to directly query the Morbid Map Database from OMIM implemented in our infrastructure. As an example, we focused on the 623 KUROV genes and found that 74 are known to be related to diseases (S3 Table) with 15 corresponding to specific syndromes related to impairment of some sensory functions, such as Aldh3a2 for Sjogren-Larsson syndrome, Hsp4 for Hermansky-Pudlak syndrome 4, Slc12a1 for Bartter syndrome (type 1) and Crebbp for Rubinstein-Taybi syndrome.
Conclusions Initially designed to facilitate the detailed and integrated analysis of complex organs, notably the ear, ImAnno is now being used for other large scale annotation projects for instance, in the developing mouse eye and teeth or the human eye fundus project (imanno. lbgi. fr). This demonstrates the usefulness and ergonomy of the knowledgebase infrastructure, which can combine object-centric, multi-level annotation (gene, patient, etc. ) and analysis of image data through various data mining tools of GO annotation, interactomics or human disease features. As a proof of concept, using the expert annotation of ISH gene expression of 2000 randomly chosen genes, we focused our ImAnno study on the E14. 5 developmental stage using 25 annotated embryonic tissues with special emphasis on 5 developing sensory tissues (KUROV).
The present work was funded by the grants from the  European Union  ( 6th Framework Program ,  EURExpress  integrated project,  LSHG-CT-2004-512003 ), the  Agence Nationale de la Recherche  ( ANR Neurosciences 2007 and Programme blanc 2011 ),  Fondation pour la Recherche Médicale  (Equipe FRM 2007) (to P. D. );  ANR BIPBIP :  ANR-10-BINF-03-05  (to O. P. ) and the  CNRS, INSERM ,  Université de Strasbourg, Fédération de Médecine Translationnelle de Strasbourg, Région Alsace. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Blinding and randomization Both follow-on milk formulas (with or without FOS) have been supplied by Laboratorios Ordesa in identical packages labelled with randomization codes. The study remained in blinded conditions for all participants, investigators, monitors, data managers and statisticians. To ensure equal product repartition throughout the study, a computed, blocks of four, randomization list stratified by centre was generated by an automated random number system by the coordination Contract Research Organization (not involved in recruiting participants and in assigning treatment). Each investigator had his own randomization list on site providing him randomization codes. The randomization codes were assigned to  the infants following an accurate consecutive order as they were included in the centre.
-Tolerance population: All randomized infants who had ingested least once the study product between V1 and V3. Data from all infants who met the inclusion and exclusion criteria were used for statistical analysis. Following the analysis of the results of the study, a posthoc analysis was performed in order to investigate if the effect of the scFOS could be related to the breastfeeding history. For each efficacy population (placebo and treatment) two subgroups were created (previously breastfed and never breastfed) and analysed. Both scFOS and control groups showed a similar distribution between previously breastfed and never breastfed infants. Statistical analyses were performed using the Statistical Analysis System (SAS) software, version 9. 2. The significance level for all statistic tests was considered 0.
05. Data from all clinical assessments, including baseline characteristics of the infants, were summarized by visit using descriptive techniques. The primary endpoint was the level of secretory poliovirus-specific sIgA in stools after 1 month and 2 months of supplementation. Normality was assessed using the Shapiro-Wilk test. sIgA concentrations were compared using a two-sample t-test or a Mann-Whitney test, when appropriate. All analyses were two-sided. For the secondary endpoints (tolerance and bifidobacteria) comparisons between groups were performed using a Chi square test or a Fisher's exact test as appropriate for categorical data (frequency of infectious diseases and digestive tolerance of the product), whereas continuous variables (bifidobacteria counts) were compared using a two-sample t-test or a Mann-Whitney test, depending on data distribution.
Study population A total of 75 infants were enroled in this study, of which 38 and 37 exclusively received a formula with scFOS or a control formula, respectively. The flowchart of enroled infants and the number of infant who provided individual data for the measurement of the different clinical outcomes is presented in Fig. 3  while the distribution of previously breastfed and never breastfed infants for each efficacy population is shown in Table  3. There were no differences between the two groups in the total numbers of withdrawals or in their reasons for withdrawing from the study.
Causal link between scFOS ingestion and gastro-enteritis, inguinal hernia repair and pyelonephritis was completely excluded. Other SAEs experienced in the scFOS but not in the control group (bronchitis, bronchiolitis or ear infections) were also analysed for their possible causality with the study product. Events of such type have been suspected to be related to regurgitation (referring to the passage of gastric contents into the oesophagus oropharynx) or GERD (Gastro-Esophageal Reflux Disease, i. e when regurgitation is the cause of troublesome symptoms and/or complications). However despite studies suggesting a possible association, current clinical evidence does not support this relationship  (Sherman et al. , 2009)  and our data as well as those from other trials in scFOS supplemented formulae  (Moro et al.
Acknowledgements Authors are grateful to the  Ordesa group  and to  Virginia Millan  and  Carmen Landa  from  Syral Iberia  for their technical support. We also thank the investigation centers ( ABS Gavá, ABS Viladecans, Clínica Quirón, CAP Les Hortes, Hospital Verge de la Cinta, Hospital Verge de la Cinta, Cap del Prat del Llobregat, Cap Sant Martí de Centelles, Cap de Centelles and Cap de Hostalets de Balenya ) and  RPS Research Iberica S. L. U  for their participation to the clinical study. CR, FG, AW and FR participated in the conception and design of the study. Data analysis and interpretation was performed by  CR, FR and FG. The manuscript was drafted by C. R. and critically revised by all authors for its final approval.
The incubation time with substrates was 5 min, that corresponds to the initial linear phase of substrate uptake, notably for OCT1  [28] , and is in the range of incubation periods already retained in previous transport studies  [32, 33]. Cells were next washed twice with ice-cold phosphate-buffered saline (PBS), and finally lysed in distilled water. DAPI content was next determined using a SpectraMax Gemini SX spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA); excitation and emission wavelengths were 355 and 460 nm, respectively. TEA and taurocholate accumulation were measured by scintillation counting. Data were usually expressed as % of substrate accumulation found in control cells; for OCT1-mediated transport inhibition by BIMs, data were also expressed as % of inhibition of OCT1 activity using the following equation: % inhibition of OCT1 activity ¼ 100 % À % TEA accumulation ðin the presence of BIMÞ Trans-stimulation assays [1-14 C] TEA trans-stimulation assays were performed in HEK-OCT1 cells as previously described  [34].