dataesr/scientific-paragraphs-categorization huggingface dataset:
PI/Annexin V Staining and Flow Cytometry Analysis to Study Apoptosis. HUVECs were either treated or not treated with native LDL or Mox-LDL at a final concentration of 100 g/mL for 24 hours. Apoptosis was detected using BD Pharmingen FITC Annexin V Detection Kit 1. The assay was performed according to manufacturer's instructions. In brief, HUVECs were washed twice with cold PBS and resuspended in Binding Buffer at a concentration of 10 6 cells/mL. FITC Annexin V and PI were added and cells were vortexed gently and incubated in the dark at room temperature for 15 min. Flow cytometry analysis was performed within 1 hour using BD FACSCanto II flow cytometry, and data were analyzed using FlowJO (Tree Star, inc. ©). The experiments were performed in duplicate and the percentages of annexin V positive and PI positive cells were averaged between the duplicates.
HUVECs MicroRNA Profiling and Data Analysis. HUVECs were either treated or not treated with native LDL or Mox-LDL at a final concentration of 100 g/mL for 24 hours. Total RNA was extracted from cells using TRIzol total RNA isolation reagent (Roche Applied Science). To profile microRNA expression in the treated and nontreated HUVECs, a two-step procedure was then performed. First, for cDNA synthesis from the miRNAs, 1000 ng of the extracted total RNA was subjected to reverse transcription (RT) using the TaqMan microRNA reverse transcription kit (Applied Biosystems) and Megaplex RT primers (Human Pool A, Applied Biosystems) following the manufacturer's instructions, allowing simultaneous reverse transcription of 380 mature human miRNAs. RT was performed on a Mastercycler Epgradient thermocycler (VWR).
After the RT step, cDNA was diluted in RNase-free water, combined with TaqMan gene expression master mix, and loaded into the TaqMan Human MicroRNA Array A (Applied Biosystems). This microRNA array is a 384-well real-time PCR-based microfluidic card with embedded TaqMan primers and probes in each well corresponding to 380 different mature human miRNAs. Quantitative RT-PCR was performed on an ABI PRISM 7900HT sequence detection system (Applied Biosystems) according to the manufacturer's instructions. The relative expression levels of miRNAs were calculated by the SDS 2. 3 software (Applied Biosystems) using the comparative ΔΔCt method and RNU48 as endogenous control. 2. 12. TaqMan miRNA Assay for Individual miRNAs. Cells were either treated or not with native LDL or Mox-LDL at a final concentration of 100 g/mL for 24 hours and total RNA was extracted from cells using TRIzol total RNA isolation reagent (Roche Applied Science).
QRT-PCR Based StellARray System to Study Angiogenesis. Cells were either treated or not with native LDL or Mox-LDL at a final concentration of 100 g/mL for 24 hours. Total RNA was extracted from cells using TRIzol total RNA isolation reagent (Roche Applied Science) and reverse-transcribed using Reverse Transcriptase Core Kit (Eurogentec) according to the manufacturer's instructions. For expression analysis of 94 preselected genes involved in angiogenesis, Human Angiogenesis 96-well StellARray qPCR array (Lonza Ltd. , Switzerland) was used according to manufacturer's instruction with ABsolute qPCR SYBR Green ROX Mix (Thermo Scientific) on an ABI Prism 7900HT sequence detection system (Applied Biosystems). Data were analyzed with Global Pattern Recognition Data Analysis Tool (Bar Harbor Biotechnology, Trenton, ME, USA) using the internal array control housekeeping gene expression for normalization.
Mox-LDL Has No Effect on Adhesion Foci. Our data pointed at an effect of Mox-LDL on HUVEC motility. Key players in this phenomenon are the structures mediating cells/ECM interaction called adhesion plaques or adhesion foci. We therefore analyzed the effect of our treatments on HUVEC were seeded, allowed to reach confluence, and incubated for 24 hours more. HUVECs were then either treated or not with native LDL or Mox-LDL for 24 h. Wounds were then made by scratching the confluent cell monolayers with a yellow pipet tip and washed twice and cells were incubated for additional 8 hours in the same conditions. Pictures were taken at time of wounding and eight hours later in all conditions. Black lines defining wound edges were superimposed to the pictures. The results shown are representative of three independent experiments performed in duplicate.
Acknowledgments  Luc Vanhamme ,  Frank Dequiedt , and  Maud Martin  are Director of Research, Research Associate, and  Postdoctoral Researcher , respectively at the  Belgian Fund for Scientific Research (FRS-FNRS ). The authors thank  Anouk Bleuart  and  Jonathan Bruyr  for valuable help. This research was funded by the  Belgian Fund for Scientific Research (FRS-FNRS) , the  Fonds Jean Brachet , the  Fonds David et Alice Van Buuren , the  Fonds pour la Recherche Médicale en Hainaut (FRMH) , the  Fondation pour la Chirurgie Cardiaque , and the  EU , 7th  FP  under Grant agreement no. 269966-Thrombus, and partly supported by the  Interuniversity Attraction Poles Program  initiated by the  Belgian Science Policy Office  ( IUAP-BELSPO PVI/28  and  PVII/13 ). Written consent was obtained from the donors.
In such centrifuged eggs, coelenterazine-dependent, detergent-induced bioluminescence was emitted from the mitochondria-rich zone (figure  6c ), indicating that the maternal clytin as well as GFP is targeted to mitochondria. A further strong indication that GFP2 and clytin2 are targeted to egg mitochondria came from sequence analysis (figure  7 ). The predicted amino acid sequence of clytin2 and of some CheGFP2 variants displayed N-terminal leaders of 30-50 amino acids, which were shown using MITOPROT (http://ihg. gsf. de/ihg/mitoprot. html) [31]  or IPSORT (http:// ipsort. hgc. jp/) [32]  protein localization prediction software to have high probability (more than 0. 8) to direct protein targeting and internalization within the mitochondrial lumen. For GFP2, pre-sequences with putative cleavage sites were present in sequences of two of the three variants from our EST collection (figure  7a ).
The three variants were otherwise quasi-identical at the nucleotide level, suggesting that the putative targeting sequence may derive from an alternative spliced N terminal exon. This was confirmed by the analysis of preliminary raw genomic sequence data from the ongoing C. hemisphaerica genome sequence project. Concerning clytin2 protein, sequence analysis predicted targeting to mitochondria with 0. 9 probability. In contrast to Clytia GFP2, no leaderless cDNA variants were identified in our transcriptomic data, while genome sequence analysis confirmed that the 5 0 leader sequence to this independent gene is not coded by a separate exon. Biochemical studies had previously provided evidence that bioluminescence in certain cnidarian and ctenophore species is generated in membrane-bound organelles termed lumisomes, around 0.
In a survey of 21 hydrozoan Figure  7. Diversification of GFP and clytin genes. (a) Amino acid alignments of the N terminus of deduced Clytia GFP and clytin protein sequences. GFP2a, GFP2c and clytin2 sequences show an N terminal presequence, predicted with high probability to mediate targeting to mitochondria (MITOPROT software). Predicted cleaved sequences are shown in brackets. Black shading: identical amino acids; grey shading, similar amino acids. (b) Phylogenetic relationships of representative cnidarian GFP and clytin genes, deduced by ML from the alignments provided in the electronic supplementary material, figure S1. Sequences of arthropod and cephalochordate GFPs were used as an out-group for the GFPs tree (a), and sequences of calmodulin protein were used as an out-group for the clytin tree (b).
Superscript M indicates the presence of a predicted mitochondrial targeting sequence. Bootstrap percentages (500 replicates) over 50% are shown. The scale bar indicates the number of amino acid substitutions per site. Acoe, Aequorea coerulescens; Amac, Aequorea macrodactyla; Avic, Aequorea Victoria; Amil, Acropora millepora; Ant, Anthomedusae species; Bf, Branchiostoma floridae; Cgra, Clytia gracilis; Cgre, Clytia gregarium; Che, Clytia hemisphaerica; Dis, Discosoma species; Laes, Labidocera aestiva; Mcav, Montastraea cavernosa; Mcel, Mitrocoma cellularia; Nv, Nematostella vectensis; Olon, Obelia longissima; Phy, Phialidium species; Pplu, Pontellina plumata; Rren, Renilla reniformis. Sequence accession numbers are given in the electronic supplementary material, table  S2. rsob. royalsocietypublishing.
Clytia hemisphaerica green fluorescent protein and clytin sequences Clytia hemisphaerica cDNA clones containing complete ORFs were retrieved from a mixed-stage cDNA library following identification from our EST collection. For phylogenetic analysis, selected sequences were aligned using CLUSTALW in BIOEDIT and corrected by eye. Maximum-likelihood (ML) analysis was performed using PHYML, as described previously  [55]. GFP sequences from Arthopoda and Cephalochordata were used as out-groups for the GFP tree, and calcium-binding domain of calmodulin sequences for the aequorin one.
Acknowledgements. We thank our colleagues, especially  R. Dumollard ,  A. McDougall ,  S. Chevalier ,  P. Lapebie ,  T. Momose ,  C. Barreau ,  M. Manuel , as well as  M. Whittaker  ( Newcastle, UK ) for useful advice and comments, and  L. Lecle  `re and  P. Dru  for help with phylogenetic analyses. We also thank  C. Rouvie  `re for help with imaging and  A. Amiel  for figure 1a.
003). This allele was also expressed preferentially (p,10 26 ) with a two-fold average increase in regulated samples. Differential expression is supported by data from Genevar MuTHER (p 1 = 0. 004; p 2 = 0. 0177). Evidence for influence of rs6136 on transcription factor binding was also found in silico and in public datasets reporting in vitro data. In summary, we found SELP rs6136-T to be associated with RA and with increased expression of SELP mRNA. SELP is located on the surface of endothelial cells and crucial for recruitment, adhesion, and migration of inflammatory cells into the joint. Genetically determined increased SELP expression levels might thus be a novel additional risk factor for RA.
In RA, gene expression of integrins and their ligands were found to be upregulated  [7]  and studies even suggest correlations with prognosis and disease activity for adhesion gene products such as selectin P which is encoded by the gene SELP  [8, 9]. Therefore, genes involved in cellular adhesion processes are highly relevant candidate genes for studying genetic association with RA. To identify genes involved in cellular adhesion relevant in RA, we performed a thorough literature and database analysis. We favored genes that are located in genomic regions identified in genome-wide linkage scans in RA families  [10]  and genes with polymorphisms known to be associated with autoimmune disorders. Table  1  displays relevant information on all 14 genes selected. In brief, some properties and functions of these genes are as follows: Integrin: Integrin ITGA4 is the alpha-4 subunit of integrin VLA-4, ITGB1 is the beta-1 subunit of the same molecule.
Gene and marker selection We selected genes in cellular adhesion processes following a thorough analysis of literature and databases at the time of candidate selection. For this purpose we used MeSH (medical subject headings) search terms in the PubMed database, available information on biological pathways (e. g. KEGG pathways, http:// www. genome. jp/kegg/), gene ontology terms (http://www. geneontology. org/), and genetic databases such as OMIM (Online Mendelian Inheritance in Man, http://www. omim. org/), GDPinfo (genomics and disease prevention information system, currently accessible at http://hugenavigator. net/HuGENavigator/home. do), and GAD (genetic association database, http://geneticassociationdb. nih. gov/). Genes associated with autoimmunity and genes located in genomic regions linked to RA in genome-wide linkage analysis were preferred  [10].
We note that our selection of genes must be considered essentially subjective. It represents a subset of genes for each of which a plausible link to cellular adhesion processes and to immune diseases is given. Haplotype structure of each gene was analyzed by Haploview  [25]  and SNPs located in different LD (linkage disequilibrium) blocks were selected. SNPs with previously published disease associations or known functional consequences (e. g. non-synonymous coding SNPs) if existent were preferred. Information on SNPs was retrieved from databases EnsMart (http://www. ensembl. org/), PupaSNP  [26] , UCSC (http://genome. ucsc. edu), HapMap (www. hapmap. org), and dbSNP (http://www. ncbi. nlm. nih. gov/SNP/).
For a marker with 35% minor allele frequency, we had 80% power to identify a genotype relative risk of 1. 66, 1. 44, and 1. 29 within 100, 200, and 407 trio families in the TDT test, respectively. For a marker with 10% minor allele frequency, we had the same power to identify a genotype relative risk of 2. 06, 1. 7, and 1. 47 within 100, 200, and 407 trio families in the TDT test, respectively. Power calculation was done using the R add-on package ''powerpkg'' using formula as described  [30].
Genotyping Genotyping was carried out using the Genosnip technique by Bruker Daltonics  [31]. PCR primers were designed using MuPlex Vs 2. 2  [32]. Primer design for SBE (single base extension) was carried out using PrimExtend, an in-house software tool based on CalcDalton  [33]. Primer-sequences are shown in Table  S1. Samples of the third set were genotyped by TaqMan 59 allelic discrimination assays (Applied Biosystems, Foster City, CA, USA) following manufacturer's protocols. In quality control Mendel's 1 st law of inheritance had to be fulfilled in at least 95% of all children. Hardy-Weinberg Equilibrium (HWE) in non-transmitted controls had not to be violated in a chi-square test with 1 degree of freedom (p. 0. 01), all SNPs fulfilled these criteria. Non-transmitted controls refer to a genotype comprising of the two alleles that were not transmitted from the parents to the child.
Association analysis Linkage and association analyses were performed using the Transmission Disequilibrium Test (TDT)  [34]. The TDT compares the transmission of SNP alleles from heterozygous parents to affected offspring with a transmission ratio of 50% as expected by Mendel's 1 st law. The allelic odds ratio (OR) compares differences in genotype distribution between RA cases and 'virtual controls' reconstructed from non-transmitted parental alleles. For gene-wide haplotype analysis the software Haploview 4. 1 was used  [25].
For each cell line, three replicates were analyzed. Each single replicate was quantified at least in triplicates applying Genosnip and averaged. As the Genosnip method results in quantitative signals for the genotyped alleles, data from genotyping heterozygous cDNA can be used to quantify gene expression in an allele specific manner. To identify DAE, the allelic ratio of genotyped cDNA is compared with the allelic ratio found in gDNA, which is expected to be around one. For robust quantification, signal to noise ratios were used and the natural logarithm of the ratios was calculated. We applied ANOVA to globally identify if allelic ratios differed in cDNA from cell lines and gDNA (implemented in the statistical software R (http://www. R-project. org) applying the R add-on package ''car'').
Then, we used Dunnett's test to identify ''regulated cell lines'', i. e. cell lines where DAE is present. This was defined to be the case when the allelic ratios of cDNA was statistically significant different from the allelic ratios of gDNA according to Dunnett's test (applying the R add-on package ''multcomp''  [35] ). In the context of this manuscript, ''cisregulation'' refers to DAE where regulated samples showed increased expression of always the same allele  [36]. This might result from some direct mechanism which is not influenced much by other aspects of the state of a cell. For verification of DAE by an independent method, purified PCR products (PCR-Purifying-Kit, Seqlab, Go ¨ttingen, Germany) originating from cDNA and genomic DNA of selected samples were analyzed by eurofins-MWG/operon (Ebersberg, Germany) with fluorescence-based sequencing (cycle sequencing technology with dideoxy chain termination using an Applied Biosystems ABI 3730XL-sequencer).
eQTLs and gene expression analysis Evidence for eQTL from public databases may be considered corroborative evidence for DAE. Therefore, eQTLs were assessed by comparing microarray expression data and genotyping data available via Genevar database (Version 3. 5. 0). Specifically, we used data from the pilot phase of The MuTHER Study  [37]  measured in lymphoid, skin, and adipose tissues derived from healthy female twin pairs. Supplemental microarray gene expression data was accessed through the Gene Expression Atlas 2. 0. 7. 5 05/11 (http://wwwtest. ebi. ac. uk/gxa/).
Evaluation of allele effects on transcription factor binding sites Genomatix SNPinspector software suite (v2. 1, matrix library 8. 3) was used to evaluate potential effects of SNP alleles on loss or gain of potential transcription factor binding sites (TFBS). Two scores, core similarity and matrix similarity, were analyzed. These scores are based on similarities between the highest conserved nucleotides of a predicted TFBS or a TFBS family matrix. Putative gains or losses of TFBS were deemed relevant for scores. 0. 8  [38]. Additionally, we reviewed in vitro data on TFBS at the genomic location of rs6136. Data are publicly available from the ENCODE project and accessible via the UCSC genome browser  [24].
002) when analyzed in joined data from both sets. In consequence, SNP rs6136 qualified for genotyping in the European RA family trio set for evaluation of broader relevance. Here again rs6136 showed over-transmission of the major T-allele, but significance was only reached if analyzed in combination with the other two sets (pTDT = 0. 003, Table  4 ). The power to detect the association of rs6136 with RA as reported in Table  4  for all sets combined was. 95%. In subgroup analysis, the observed association for rs6136 was stronger in females (OR 0. 58; 95%CI 0. 4-0. 8) than in males (OR 0. 9; 95%CI 0. 4-2. 2) and stronger in patients with radiographic erosion (OR 0. 71; 95%CI 0. 4-1. 4) than in those without erosion (OR 0. 9; 95%CI 0. 4-2. 2). However, differences in odds ratios between subgroups were not statistically significant (p.
Differential allelic expression As SELP rs6136 was associated with RA in our multi-step RA family trio study we analyzed SNP rs6136 for differential allelic expression. We observed a cis-effect on expression levels of SELP for rs6136 (ANOVA p,10 26 , figure  1 ). Dunnett's test revealed that three of the seven samples heterozygous for rs6136 individually showed significant DAE, always with the T-allele  being higher expressed than the C-allele. The average fold change in regulated samples was 2. Direction of allelic expression fold changes was verified by Sanger sequencing (Table  5 ). To further evaluate our finding of overexpression of the rs6136-T allele, we compared microarray expression data and genotype data available via the Genevar database. Within data from the pilot phase of The MuTHER Study  [37]  we observed a consistent cis-eQTL with overexpression of the rs6136-T allele (SELP probe: ILMN_1715417) in both (p 1 = 0.
004; p 2 = 0. 0177; figure  2 ). The rs6136 variant was the strongest cis-eQTL hit within available Genevar expression data in a region of at least 200 kbp up-and downstream of SELP. For the closest neighbouring gene F5 (coagulating factor 5) no cis-eQTLs were detected. The Genomatix SNPinspector software suite  [38]  was applied to calculate possible gains or losses of transcription factor binding sites (TFBS) for rs6136 and a SNP, rs9332575. SNP rs9332575 is in perfect LD (D9 = 1, r 2 = 1) with exonic rs6136 within the Caucasian HapMap population, but located 40 kbp upstream of it (Table  6 ). The A-allele of rs9332575 corresponds to the T-allele of rs6136. Higher similarity values predict higher probability of transcription factor (TF) binding at this site. Table  6  shows several identified possible losses or gains of TFBS for rs6136 and rs9332575.
Discussion Adhesion molecules are potential candidate genes for involvement in the complex immune disease Rheumatoid Arthritis. We selected 14 genes involved in cellular adhesion processes and investigated possible association with RA. Two of these genes, CAST (encoding calpastatin) and SELP (encoding P selectin), were associated with RA in our discovery set of French RA family trios. Association of SNP rs6136 in SELP with RA could be successfully independently replicated in our replication set of French family trios and was still significant when including a larger European Caucasian family trio cohort (p Total = 0. 003). Within all three sets, the major T-allele was over-transmitted to RA patients in TDT, which is robust against ethnic heterogeneity  [39]. We compared our findings with data of a large meta-analysis of genome-wide association studies (GWAS) for RA  [40].
Within the Gene Expression Atlas (experiment E-GEOD-7307), SELP mRNA expression was found increased in two tissues most relevant for RA pathogenesis: joint synovium (p = 0. 007, N = 3) and synovial membrane (p = 4610 210 , N = 11) (figure  3 ). SELP expression was also increased in RA patients (p = 0. 033, N = 5) and in a rat model of pristane induced arthritis (experiment E-MEXP-782) (p = 0. 047). Functional studies focusing on cis-regulation of SELP expression would be required to further corroborate our findings and possibly reveal the mechanism and consequences of SELP DAE. The relevance of SELP in RA and other diseases is underlined by the finding of increased soluble SELP levels for many diseases, including atherosclerosis, in which soluble SELP is derived mostly from endothelial cells  [44] , and RA  [46].
Acknowledgments We like to thank the patients and their families for participating in our study. Material was collected by  ECRAF (European Consortium on Rheumatoid Arthitis Families :  Paola Migliorini ,  Alejandro Balsa ,  Rene Ẃesthovens ,  Pilar Barrera ,  Helena Alves ,  Carlos Vaz ,  Manuela Fernandes ,  Dora Pascuale-Salcedo ,  Stefano Bombardieri ,  Jan Dequeker ,  Timothy R Radstake ,  Piet Van Riel ,  Leo van de Putte, Antonio Lopes-Vaz, and Francois Cornelis ) with support of the  Association Francaise des Polyarthritiques, Association Rhumatisme et Travail ,  Societe Francaise de Rhumatologie ,  Association Polyarctique ,  Groupe Taitbout ,  Genopole  and  European Union AutoCure.
As previously described, the MWG array consists of 6,167 50-mer oligonucleotides covering the genomes of E. coli K-12 (MG1655) and E. coli O157:H7 strains Sakai (RIMD 0509952) and EDL933 (ATCC 700927)  [23]. Description of the array is available at the Gene Expression Omnibus (GEO) database (http://www. ncbi. nlm. nih. gov/projects/geo/) (platform accession number GPL6178). Two arrays were spotted per slide. Prior to hybridization, the slides were incubated for 50 min at 42uC in prehybridization buffer (56SSC, 0. 1% SDS, 0. 1% bovine serum albumin), washed twice at room temperature in distilled water for 10 min and dried by centrifugation for 5 min at 8006g. Cy3-and Cy5-labeled cDNA (200 pmol each) were then mixed, dried, resuspended in 50 mL hybridization buffer (56SSC, 0. 1% SDS, 50% deionized formamide, 0.