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A general definition and nomenclature for alternative splicing events

  • Guigó Serra, Roderic
  • Sammeth, Michael
  • Foissac, Sylvain
Understanding the molecular mechanisms responsible for the regulation of the transcriptome present in eukaryotic cells is/none of the most challenging tasks in the postgenomic era. In this regard, alternative splicing (AS) is a key phenomenon/ncontributing to the production of different mature transcripts from the same primary RNA sequence. As a plethora of/ndifferent transcript forms is available in databases, a first step to uncover the biology that drives AS is to identify the/ndifferent types of reflected splicing variation. In this work, we present a general definition of the AS event along with a/nnotation system that involves the relative positions of the splice sites. This nomenclature univocally and dynamically assigns/na specific ‘‘AS code’’ to every possible pattern of splicing variation. On the basis of this definition and the corresponding/ncodes, we have developed a computational tool (AStalavista) that automatically characterizes the complete landscape of AS/nevents in a given transcript annotation of a genome, thus providing a platform to investigate the transcriptome diversity/nacross genes, chromosomes, and species. Our analysis reveals that a substantial part—in human more than a quarter—of/nthe observed splicing variations are ignored in common classification pipelines. We have used AStalavista to investigate and/nto compare the AS landscape of different reference annotation sets in human and in other metazoan species and found that/nproportions of AS events change substantially depending on the annotation protocol, species-specific attributes, and/ncoding constraints acting on the transcripts. The AStalavista system therefore provides a general framework to conduct/nspecific studies investigating the occurrence, impact, and regulation of AS., This work has been funded by a DAAD (German Academic Exchange Service) postdoctoral fellowship to MS. Further support has been provided by grants from the NHGRI Encode project, the European Union ATD project, and the Spanish Plan Nacional de I+D.
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Disease-corrected haematopoietic progenitors from Fanconi anemia induced pluripotent stem cells

  • Rodríguez Pizà, Ignasi
  • Verma, Inder M.
  • Veiga, Anna
  • Aasen, Trond
  • Izpisúa Belmonte, J. C.
  • Bueren, Juan
  • Garreta, Elena
  • Tiscornia, Gustavo
  • Sleep Ronquillo, Eduard
  • Raya Chamorro, Ángel
  • Río, Paula
  • Consiglio, Antonella
  • Barrero Núñez, María José
  • Navarro, Susana
  • Vassena, Rita
  • Guenechea, Guillermo
The generation of induced pluripotent stem (iPS) cells has enabled the derivation of patient-specific pluripotent cells and/nprovided valuable experimental platforms to model human disease. Patient-specific iPS cells are also thought to hold great/ntherapeutic potential, although direct evidence for this is still lacking. Here we show that, on correction of the genetic defect,/nsomatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency to generate patient-specific iPS cells. These cell lines appear indistinguishable from human embryonic stem cells and iPS cells from healthy individuals. Most importantly, we show that corrected Fanconi-anaemia-specific iPS cells can give rise to haematopoietic progenitors of the myeloid and erythroid lineages that are phenotypically normal, that is, disease-free. These data offer proof-of-concept that iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications.
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Reprogramming of human fibroblasts to induced pluripotent stem cells under xeno-free conditions

  • Rodríguez Pizà, Ignasi
  • Vassena, Rita
  • Richaud Patin, Yvonne
  • Izpisúa Belmonte, J. C.
  • Raya Chamorro, Ángel
  • Veiga, Anna
  • Barrero Núñez, María José
  • González, Federico
The availability of induced pluripotent stem cells (iPSCs)/nhas created extraordinary opportunities for modeling and/nperhaps treating human disease. However, all reprogramming/nprotocols used to date involve the use of products of animal origin. Here, we set out to develop a protocol to generate and maintain human iPSC that would be entirely/ndevoid of xenobiotics. We first developed a xeno-free cell/nculture media that supported the long-term propagation of human embryonic stem cells (hESCs) to a similar extent as conventional media containing animal origin products or commercially available xeno-free medium. We also derived/nprimary cultures of human dermal fibroblasts under strict/nxeno-free conditions (XF-HFF), and we show that they can be used as both the cell source for iPSC generation as well as autologous feeder cells to support their growth. We also replaced other reagents of animal origin trypsin, gelatin, matrigel) with their recombinant equivalents. Finally, we used vesicular stomatitis virus G-pseudotyped retroviral particles expressing a polycistronic construct encoding Oct4, Sox2, Klf4, and GFP to reprogram XF-HFF cells under xeno-free conditions. A total of 10 xeno-free human/niPSC lines were generated, which could be continuously passaged in xeno-free conditions and aintained characteristics indistinguishable from hESCs, including colony/nmorphology and growth behavior, expression of pluripotency-/nassociated markers, and pluripotent differentiation/nability in vitro and in teratoma assays. Overall, the results/npresented here demonstrate that human iPSCs can be generated/nand maintained under strict xeno-free conditions and provide a path to good manufacturing practice (GMP) applicability that should facilitate the clinical translation of iPSC-based therapies.
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Derivation of human embryonic stem cells at the center for regenerative medicine in Barcelona

  • Rodríguez Pizà, Ignasi
  • Raya Chamorro, Ángel
  • Aran, Begoña
  • Veiga, Anna
  • Izpisúa Belmonte, J. C.
  • Barri, Pere N.
  • Muñoz, Yolanda
  • Consiglio, Antonella
We report here the legislative issues related to/nembryo research and human embryonic stem cell (hESC)/nresearch in Spain and the derivation of nine hESC lines at/nthe Center of Regenerative Medicine in Barcelona. You can/nfind the information for obtaining our lines for research/npurposes at blc@cmrb.eu.
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A protocol describing the genetic correction of somatic human cells and subsequent generation of IPS cell

  • Rodríguez Pizà, Ignasi
  • Raya Chamorro, Ángel
  • Navarro, Susana
  • Izpisúa Belmonte, J. C.
  • Bueren, Juan
  • Consiglio, Antonella
  • Sánchez Danés, Adriana, 1984-
  • Guenechea, Guillermo
  • Richaud Patin, Yvonne
The generation of patient-specific induced pluripotent stem cells (iPSCPSCPSCs) offers unprecedented opportunities for modeling and treating human disease. In combination with gene therapy, the iPSCPSCPSC technology can be used to generate disease-free progenitor cells of potential interest for autologous cell therapy. We explain a protocol for the reproducible generation of genetically corrected iPSCPSCPSCs starting from the skin biopsies of Fanconi anemia patients using retroviral transduction with OCT4, SOX2 and KLF4. Before reprogramming, the fibroblasts and/or keratinocytes of the patients are genetically corrected with lentiviruses expressing FANCA. The same approach may be used for other diseases susceptible to gene therapy correction. Genetically corrected, characterized lines of patient-specific iPSCPSCPSCs can be obtained in 4–5 months.
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GENCODE: producing a reference annotation for ENCODE

  • Harrow, Jennifer
  • Denoeud, France
  • Frankish, Adam
  • Reymond, Alexandre
  • Chen, Chao-Kung
  • Chrast, Jacqueline
  • Lagarde, Julien
  • Gilbert, James
  • Storey, Roy
  • Swarbreck, David
  • Rossier, Colette
  • Ucla, Catherine
  • Hubbard, Tim J.
  • Antonarakis, Stylianos E.
  • Guigó Serra, Roderic
Background: The GENCODE consortium was formed to identify and map all protein-coding genes within the ENCODE regions. This was achieved by a combination of initial manual/nannotation by the HAVANA team, experimental validation by the GENCODE consortium and a refinement of the annotation based on these experimental results./nResults: The GENCODE gene features are divided into eight different categories of which only/nthe first two (known and novel coding sequence) are confidently predicted to be protein-coding/ngenes. 5’ rapid amplification of cDNA ends (RACE) and RT-PCR were used to experimentally/nverify the initial annotation. Of the 420 coding loci tested, 229 RACE products have been/nsequenced. They supported 5’ extensions of 30 loci and new splice variants in 50 loci. In addition,/n46 loci without evidence for a coding sequence were validated, consisting of 31 novel and 15/nputative transcripts. We assessed the comprehensiveness of the GENCODE annotation by/nattempting to validate all the predicted exon boundaries outside the GENCODE annotation. Out/nof 1,215 tested in a subset of the ENCODE regions, 14 novel exon pairs were validated, only two/nof them in intergenic regions./nConclusions: In total, 487 loci, of which 434 are coding, have been annotated as part of the/nGENCODE reference set available from the UCSC browser. Comparison of GENCODE/nannotation with RefSeq and ENSEMBL show only 40% of GENCODE exons are contained within/nthe two sets, which is a reflection of the high number of alternative splice forms with unique/nexons annotated. Over 50% of coding loci have been experimentally verified by 5’ RACE for/nEGASP and the GENCODE collaboration is continuing to refine its annotation of 1% human/ngenome with the aid of experimental validation.
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Multiple non-collinear TF-map alignments of promoter regions

  • Blanco, Enrique
  • Guigó Serra, Roderic
  • Messeguer, Xavier
Background: The analysis of the promoter sequence of genes with similar expression patterns is/na basic tool to annotate common regulatory elements. Multiple sequence alignments are on the/nbasis of most comparative approaches. The characterization of regulatory regions from coexpressed/ngenes at the sequence level, however, does not yield satisfactory results in many/noccasions as promoter regions of genes sharing similar expression programs often do not show/nnucleotide sequence conservation./nResults: In a recent approach to circumvent this limitation, we proposed to align the maps of/npredicted transcription factors (referred as TF-maps) instead of the nucleotide sequence of two/nrelated promoters, taking into account the label of the corresponding factor and the position in the/nprimary sequence. We have now extended the basic algorithm to permit multiple promoter/ncomparisons using the progressive alignment paradigm. In addition, non-collinear conservation/nblocks might now be identified in the resulting alignments. We have optimized the parameters of/nthe algorithm in a small, but well-characterized collection of human-mouse-chicken-zebrafish/northologous gene promoters./nConclusion: Results in this dataset indicate that TF-map alignments are able to detect high-level/nregulatory conservation at the promoter and the 3'UTR gene regions, which cannot be detected/nby the typical sequence alignments. Three particular examples are introduced here to illustrate the/npower of the multiple TF-map alignments to characterize conserved regulatory elements in/nabsence of sequence similarity. We consider this kind of approach can be extremely useful in the/nfuture to annotate potential transcription factor binding sites on sets of co-regulated genes from/nhigh-throughput expression experiments.
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Heterogeneity and event dependence in the analysis of sickness absence

  • Torá Rocamora, Isabel, 1979-
  • Gimeno Ruiz de Porras, David
  • Manzanera, Rafael
  • Jardí, Josefina
  • Delclòs i Clanchet, Jordi, 1956-
  • Benavides, Fernando G. (Fernando García)
  • Alberti, Constança
  • Yasui, Yutaka
  • Martínez Martínez, José Miguel, 1974-
Sickness absence (SA) is an important social, economic and public health issue. Identifying and understanding the determinants, whether biological, regulatory or, health services-related, of variability in SA duration is essential for better management of SA. The conditional frailty model (CFM) is useful when repeated SA events occur within the same individual, as it allows simultaneous analysis of event dependence and heterogeneity due to unknown, unmeasured, or unmeasurable factors. However, its use may encounter computational limitations when applied to very large data sets, as may frequently occur in the analysis of SA duration. To overcome the computational issue, we propose a Poisson-based conditional frailty model (CFPM) for repeated SA events that accounts for both event dependence and heterogeneity. To demonstrate the usefulness of the model proposed in the SA duration context, we used data from all non-work-related SA episodes that occurred in Catalonia (Spain) in 2007, initiated by either a diagnosis of neoplasm or mental and behavioral disorders. As expected, the CFPM results were very similar to those of the CFM for both diagnosis groups. The CPU time for the CFPM was substantially shorter than the CFM. The CFPM is an suitable alternative to the CFM in survival analysis with recurrent events,/nespecially with large databases., This work was supported by grants from the Fondo de Investigación/nSanitaria [PI11/01470], Operating Grant of the Canadian Institute of Health Research entitled 'Statistical Methods for Epidemiologic Investigations' and the Institut Català d'Avaluacions Mèdiques.
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Gender inequalities in occupational health related to the unequal distribution of working and employment conditions: a systematic review

  • Campos-Serna, Javier, 1974-
  • Ronda-Pérez, Elena
  • Artazcoz Lazcano, Lucía, 1963-
  • Moen, Bente E
  • Benavides, Fernando G. (Fernando García)
Gender inequalities exist in work life, but little is known about their presence in relation to factors examined in occupation health settings. The aim of this study was to identify and summarize the working and employment conditions described as determinants of gender inequalities in occupational health in studies related to occupational health published between 1999 and 2010. A systematic literature review was undertaken of studies available in MEDLINE, EMBASE, Sociological Abstracts, LILACS, EconLit and CINAHL between 1999 and 2010. Epidemiologic studies were selected by applying a set of inclusion criteria to the title, abstract, and complete text. The quality of the studies was also assessed. Selected studies were qualitatively analysed, resulting in a compilation of all differences between women and men in the prevalence of exposure to working and employment conditions and work-related health problems as outcomes. Most of the 30 studies included were conducted in Europe (n=19) and had a cross-sectional design (n=24). The most common topic analysed was related to the exposure to work-related psychosocial hazards (n=8). Employed women had more job insecurity, lower control, worse contractual working conditions and poorer self-perceived physical and mental health than men did. Conversely, employed men had a higher degree of physically demanding work, lower support, higher levels of effort-reward imbalance, higher job status, were more exposed to noise and worked longer hours than women did. This systematic review has identified a set of working and employment conditions as determinants of gender inequalities in occupational health from the occupational health literature. These results may be useful to policy makers seeking to reduce gender inequalities in occupational health, and to researchers wishing to analyse these determinants in greater depth.
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Color differences among feral pigeons (Columba livia) are not attributable to sequence variation in the coding region of the melanocortin-1 receptor gene (MC1R)

  • Derelle, Romain
  • Kondrashov, Fyodor A., 1979-
  • Arkhipov, Vladimir Y.
  • Corbel, Hélène
  • Frantz, Adrien
  • Gasparini, Julien
  • Jacquin, Lisa
  • Jacob, Gwenaël
  • Thibault, Sophie
  • Baudry, Emmanuelle
Genetic variation at the melanocortin-1 receptor (MC1R) gene is correlated with melanin color variation in many birds. Feral pigeons (Columba livia) show two major melanin-based colorations: a red coloration due to pheomelanic pigment and a black coloration due to eumelanic pigment. Furthermore, within each color type, feral pigeons display continuous variation in the amount of melanin pigment present in the feathers, with individuals varying from pure white to a full dark melanic color. Coloration is highly heritable and it has been suggested that it is under natural or sexual selection, or both. Our objective was to investigate whether MC1R allelic variants are associated with plumage color in feral pigeons./n/nWe sequenced 888 bp of the coding sequence of MC1R among pigeons varying both in the type, eumelanin or pheomelanin, and the amount of melanin in their feathers. We detected 10 non-synonymous substitutions and 2 synonymous substitution but none of them were associated with a plumage type. It remains possible that non-synonymous substitutions that influence coloration are present in the short MC1R fragment that we did not sequence but this seems unlikely because we analyzed the entire functionally important region of the gene./nOur results show that color differences among feral pigeons are probably not attributable to amino acid variation at the MC1R locus. Therefore, variation in regulatory regions of MC1R or variation in other genes may be responsible for the color polymorphism of feral pigeons., Romain Derelle was supported by grant from Plan Nacional 004302 BFU2012-31329. Fyodor A Kondrashov was supported by grants HHMI (Howard Hughes Medical Institute) 003803 and EMBO 003691 EUI-EURYIP-2011-4320
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