Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

Response to antiretroviral treatment in HIV-1-infected individuals with allelic variants of the multidrug resistance transporter 1: a pharmacogenetics study.

BACKGROUND:HIV-1-infected patients vary considerably by their response to antiretroviral treatment, drug concentrations in plasma, toxic events, and rate of immune recovery. This variability could have a genetic basis. We did a pharmacogenetics study to analyse the association between response to antiretroviral treatment and allelic variants of several genes. METHODS:In 123 patients, we did PCR analyses of the gene for the multidrug-resistance transporter (MDR1), which codes for P-glycoprotein, of genes coding for isoenzymes of cytochrome P450, CYP3A4, CYP3A5, CYP2D6, and CYP2C19, and of the gene for the chemokine receptor CCR5. We measured concentrations in plasma of the antiretroviral agents efavirenz and nelfinavir by high-performance liquid-chromatography, and measured levels of P-glycoprotein expression, CD4-cell count, and HIV-1 viraemia. FINDINGS: Median drug concentrations in patients with the MDR1 3435 TT, CT, and CC genotypes were at the 30th, 50th, and 75th percentiles, respectively (p=0.0001). In patients with CYP2D6 extensive-metaboliser or poor-metaboliser alleles, median drug concentrations were at percentiles 45 and 62.5, respectively (p=0.04). Patients with the MDR1 TT genotype 6 months after starting treatment had a greater rise in CD4-cell count (257 cells/microL) than patients with the CT (165 cells/microL) and CC (121 cells/microL) genotype (p=0.0048), and the best recovery of naïve CD4-cells. INTERPRETATION:The polymorphism MDR1 3435 C/T predicts immune recovery after initiation of antiretroviral treatment. This finding suggests that P-glycoprotein has an important role in admittance of antiretroviral drugs to restricted compartments in vivo.

Species delimitation: A case study in a problematic ant taxon

Species delimitation has been invigorated as a discipline in systematics by an influx of new character sets, analytical methods, and conceptual advances. We use genetic data from 68 markers, combined with distributional, bioclimatic, and coloration information, to hypothesize boundaries of evolutionarily independent lineages (species) within the widespread and highly variable nominal fire ant species Solenopsis saevissima, a member of a species group containing invasive pests as well as species that are models for ecological and evolutionary research. Our integrated approach uses diverse methods of analysis to sequentially test whether populations meet specific operational criteria (contingent properties) for candidacy as morphologically cryptic species, including genetic clustering, monophyly, reproductive isolation, and occupation of distinctive niche space. We hypothesize that nominal S. saevissima comprises at least 4-6 previously unrecognized species, including several pairs whose parapatric distributions implicate the development of intrinsic premating or postmating barriers to gene flow. Our genetic data further suggest that regional genetic differentiation in S. saevissima has been influenced by hybridization with other nominal species occurring in sympatry or parapatry, including the quite distantly related Solenopsis geminata. The results of this study illustrate the importance of employing different classes of genetic data (coding and noncoding regions and nuclear and mitochondrial DNA [mtDNA] markers), different methods of genetic data analysis (tree-based and non-tree based methods), and different sources of data (genetic, morphological, and ecological data) to explicitly test various operational criteria for species boundaries in clades of recently diverged lineages, while warning against over reliance on any single data type (e.g., mtDNA sequence variation) when drawing inferences.

Second-hand tobacco smoke in prison: tackling a public health matter through research.

OBJECTIVES: This action-research study conducted in a Swiss male post-trial detention centre (120 detainees and 120 staff) explored the attitudes of detainees and staff towards tobacco smoking. Tackling public health matters through research involving stakeholders in prisons implies benefits and risks that need exploration. STUDY DESIGN: The observational study involved multiple strands (quantitative and qualitative components, and air quality measurements). This article presents qualitative data on participants' attitudes and expectations about research in a prison setting. METHODS: Semi-structured interviews were used to explore the attitudes of detainees and staff towards smoking before and after a smoke-free regulation change in the prison in 2009. Specific coding and thematic content analysis for research were performed with the support of ATLAS.ti. RESULTS: In total, 77 interviews were conducted (38 before the regulation change and 39 after the regulation change) with 31 detainees (mean age 35 years, range 22-60 years) and 27 prison staff (mean age 46 years, range 29-65 years). Both detainees and staff expressed satisfaction regarding their involvement in the study, and wished to be informed about the results. They expected concrete changes in smoke-free regulation, and that the research would help to find ways to motivate detainees to quit smoking. CONCLUSION: Active involvement of stakeholders promotes public health. Interviewing detainees and prison staff as part of an action-research study aimed at tackling a public health matter is a way of raising awareness and facilitating change in prisons. Research needs to be conducted independently from the prison administrators in order to increase trust and to avoid misunderstandings.

Widespread occurrence of non-canonical transcription termination by human RNA polymerase III.

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3'-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T(≥4) stretch within 50 bp of 3'-flanking region. In vitro analysis of tDNAs with a distanced T(≥4) revealed the existence of non-canonical terminators resembling degenerate T(≥5) elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3' trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3'-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3'-trailer sequences with the potential to contribute novel functional ncRNAs.

Clinical and genetic findings in a large cohort of patients with congenital myopathies due to mutations in the skeletal muscle ryanodine receptor (RYR1) gene

RYR1 mutations are the most common cause of structural congenital myopathies and may exhibit both dominant and recessive inheritance. Histopathological findings are variable and include central cores, multi-minicores, type 1 predominance/ uniformity, fibre type disproportion, increased internal nucleation and fatty and connective tissue. Until recently, diagnostic RYR1 sequencing was limited to mutational hotspots due to the large size of the gene. Since the introduction of full RYR1 sequencing in 2007 we have detected pathogenic mutations in 77 families: 39 had dominant inheritance and 38 recessive inheritance. In some cases with presumably recessive inheritance, only one heterozygous mutation inherited from an asymptomatic parent was identified. Of 28 dominant mutations, 6 were novel; 37 of the 59 recessive mutations were also novel. Dominant mutations were more frequently in recognized hotspot regions, while recessive mutations were distributed throughout the coding sequence. Dominant mutations were predominantly missense, whereas recessive mutations included many nonsense and splice mutations expected to result in reduced RyR1 protein. There was wide clinical variability in patients with both dominant and recessive inheritance. As a group, those with dominant mutations were generally more mildly affected than those with recessive inheritance, who had earlier onset and were weaker with more functional limitations. Extraocular muscle involvement was almost exclusively observed in the recessive group. Bulbar involvement was also more prominent in this group, resulting in a larger number requiring gastrostomy insertion. In conclusion, genomic sequencing of the entire RYR1 leads to the detection of many novel mutations, but may miss large genetic rearrangements in some cases. Assigning pathogenicity to novel mutations is often difficult and interpretation of genetic results in the context of clinical, histological and, increasingly, muscle MRI findings is essential.

Circulating microRNAs as long-term biomarkers for the detection of erythropoiesis-stimulating agent abuse.

MicroRNAs (miRNAs) are small, non-protein coding transcripts involved in many cellular and physiological mechanisms. Recently, a new class of miRNA called 'circulating miRNAs' was found in cell-free body fluids such as plasma and urine. Circulating miRNAs have been shown to be very stable, specific, and sensitive biomarkers. In this paper, we investigate whether circulating miRNAs can serve as biomarkers for erythropoiesis-stimulating agent abuse. To this end, we analyzed miRNA levels in plasma by miRNA microarrays and quantitative real-time polymerase chain reaction (PCR). Plasma samples are derived from a clinical study with healthy subjects injected with erythropoiesis-stimulating agent (C.E.R.A.). Based on microarray results, we observed a significant difference in the levels of miRNAs in plasma after C.E.R.A. injection. We demonstrated that a specific miRNA, miR-144, exhibit a high increase that lasts 27 days after C.E.R.A. stimulation. Considering the fact that miR-144 is an essential erythropoiesis agent in different organisms, these findings suggest the possibility of using miR-144 as a sensitive and informative biomarker to detect C.E.R.A. abuse. Copyright © 2011 John Wiley & Sons, Ltd.

Compositional Constraints in the Extremely GC-poor Genome of Plasmodium falciparum

We have analyzed the compositional properties of coding (protein encoding) and non-coding sequences of Plasmodium falciparum, a unicellular parasite characterized by an extremely AT-rich genome. GC% levels, base and dinucleotide frequencies were studied. We found that among the various factors that contribute to the properties of the sequences analyzed, the most relevant are the compositional constraints which operate on the whole genome

Genes and Chromosomes of Leishmania infantum

During recent years, several Leishmania infantum genes have been cloned and characterized. Here, we have summarized the available information on the gene organization and expression in this protozoan parasite. From a comparative analysis, the following outstanding features were found to be common to most of the genes characterized: tandemly organized genes with conserved coding regions and divergent untranslated regions, polycistronic transcription and post-transcriptional regulation of gene expression. The analysis of chromosomes of L. infantum by pulsed-field electrophoresis showed the existence of both size and number polymorphisms such that each strain has a distinctive molecular karyotype. Despite this variability, highly conserved physical linkage groups exists among different strains of L. infantum and even among Old World Leishmania species. Gene mapping on the L. infantum molecular karyotype evidenced a bias in chromosomal distribution of, at least, the evolutionary conserved genes

Analysis and optimization of the satellite-to-plane link of an aeronautical global system

En aquest projecte s’ha analitzat i optimitzat l’enllaç satèl·lit amb avió per a un sistema aeronàutic global. Aquest nou sistema anomenat ANTARES està dissenyat per a comunicar avions amb estacions base mitjançant un satèl·lit. Aquesta és una iniciativa on hi participen institucions oficials en l’aviació com ara l’ECAC i que és desenvolupat en una col·laboració europea d’universitats i empreses. El treball dut a terme en el projecte compren bàsicament tres aspectes. El disseny i anàlisi de la gestió de recursos. La idoneïtat d’utilitzar correcció d’errors en la capa d’enllaç i en cas que sigui necessària dissenyar una opció de codificació preliminar. Finalment, estudiar i analitzar l’efecte de la interferència co-canal en sistemes multifeix. Tots aquests temes són considerats només per al “forward link”. L’estructura que segueix el projecte és primer presentar les característiques globals del sistema, després centrar-se i analitzar els temes mencionats per a poder donar resultats i extreure conclusions.

Molecular and Antigenic Characterization of the Leishmania (Viannia) panamensis Kinetoplastid Membrane Protein-11

The kinetoplastid membrane protein 11 (KMP-11) has been recently described in Leishmania (Leishmania) donovani as a major component of the promastigote membrane. Two oligonucleotide primers were synthesized to PCR-amplify the entire coding region of New World Leishmania species. The Leishmania (Viannia) panamensis amplification product was cloned, sequenced and the putative amino acid sequence determined. A remarkably high degree of sequence homology was observed with the corresponding molecule of L. (L) donovani and L. (L) infantum (97% and 96%, respectively). Southern blot analysis showed that the KMP-11 locus is conformed by three copies of the gene. The L. (V) panamensis ORF was subsequently cloned in a high expression vector and the recombinant protein was induced and purified from Escherichia coli cultures. Immunoblot analysis showed that 80%, 77% and 100% sera from cutaneous, mucocutaneous and visceral leishmaniasis patients, respectively, recognized the recombinant KMP-11 protein. In a similar assay, 86% of asymptomatic Leishmania-infected individuals showed IgG antibodies against the rKMP-11. We propose that KMP-11 could be used as a serologic marker for infection and disease caused by Leishmania in America.

Selection, Recombination and History in a Parasitic Flatworm (Echinococcus) Inferred from Nucleotide Sequences

Three species of flatworms from the genus Echinococcus (E. granulosus, E. multilocularis and E. vogeli) and four strains of E. granulosus (cattle, horse, pig and sheep strains) were analysed by the PCR-SSCP method followed by sequencing, using as targets two non-coding and two coding (one nuclear and one mitochondrial) genomic regions. The sequencing data was used to evaluate hypothesis about the parasite breeding system and the causes of genetic diversification. The calculated recombination parameters suggested that cross-fertilisation was rare in the history of the group. However, the relative rates of substitution in the coding sequences showed that positive selection (instead of purifying selection) drove the evolution of an elastase and neutrophil chemotaxis inhibitor gene (AgB/1). The phylogenetic analyses revealed several ambiguities, indicating that the taxonomic status of the E. granulosus horse strain should be revised

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Non-coding small RNAs (sRNAs) have important regulatory functions in bacteria. In Pseudomonas spp., about 40 sRNAs have been reported until the end of 2008, a number that almost certainly is an underestimate. We provide a summary of the coding regions for these sRNAs is Pseudomonas aeruginosa. The functions of some Pseudomonas sRNAs can be deduced from those of homologous well-characterized sRNAs of Escherichia coli, e.g. 6S RNA (a stationary phase regulator of RNA polymerase) and tmRNA (a component of a machinery serving to eliminate truncated polypeptides). Two sRNAs of P. aeruginosa, PrrF1 and PrrF2, whose expression is repressed by the Fur repressor in the presence of iron, inhibit translation initiation of mRNAs specifying superoxide dismutase (sodB), succinate dehydrogenase (sdhABCD) and anthranilate degradation (antABC), via a base-paring mechanism. As a consequence, these mRNAs are poorly expressed under conditions of iron limitation. Two further sRNAs of P. aeruginosa, RsmY and RsmZ, whose expression is positively controlled by the GacS/GacA two-component system in response to unknown signals, act as scavengers of the RNA-binding protein RsmA. In this way, translational repression exerted by RsmA on target mRNAs can be relieved. The Gac/Rsm signal transduction pathway globally regulates motility and the formation of extracellular products in pseudomonas spp.

Systems biology of plants : from intraspecific genome variation to computational morphodynamics

Recently, the introduction of second generation sequencing and further advance-ments in confocal microscopy have enabled system-level studies for the functional characterization of genes. The degree of complexity intrinsic to these approaches needs the development of bioinformatics methodologies and computational models for extracting meaningful biological knowledge from the enormous amount of experi¬mental data which is continuously generated. This PhD thesis presents several novel bioinformatics methods and computational models to address specific biological questions in Plant Biology by using the plant Arabidopsis thaliana as a model system. First, a spatio-temporal qualitative analysis of quantitative transcript and protein profiles is applied to show the role of the BREVIS RADIX (BRX) protein in the auxin- cytokinin crosstalk for root meristem growth. Core of this PhD work is the functional characterization of the interplay between the BRX protein and the plant hormone auxin in the root meristem by using a computational model based on experimental evidence. Hyphotesis generated by the modelled to the discovery of a differential endocytosis pattern in the root meristem that splits the auxin transcriptional response via the plasma membrane to nucleus partitioning of BRX. This positional information system creates an auxin transcriptional pattern that deviates from the canonical auxin response and is necessary to sustain the expression of a subset of BRX-dependent auxin-responsive genes to drive root meristem growth. In the second part of this PhD thesis, we characterized the genome-wide impact of large scale deletions on four divergent Arabidopsis natural strains, through the integration of Ultra-High Throughput Sequencing data with data from genomic hybridizations on tiling arrays. Analysis of the identified deletions revealed a considerable portion of protein coding genes affected and supported a history of genomic rearrangements shaped by evolution. In the last part of the thesis, we showed that VIP3 gene in Arabidopsis has an evo-lutionary conserved role in the 3' to 5' mRNA degradation machinery, by applying a novel approach for the analysis of mRNA-Seq data from random-primed mRNA. Altogether, this PhD research contains major advancements in the study of natural genomic variation in plants and in the application of computational morphodynamics models for the functional characterization of biological pathways essential for the plant. - Récemment, l'introduction du séquençage de seconde génération et les avancées dans la microscopie confocale ont permis des études à l'échelle des différents systèmes cellulaires pour la caractérisation fonctionnelle de gènes. Le degrés de complexité intrinsèque à ces approches ont requis le développement de méthodologies bioinformatiques et de modèles mathématiques afin d'extraire de la masse de données expérimentale générée, des information biologiques significatives. Ce doctorat présente à la fois des méthodes bioinformatiques originales et des modèles mathématiques pour répondre à certaines questions spécifiques de Biologie Végétale en utilisant la plante Arabidopsis thaliana comme modèle. Premièrement, une analyse qualitative spatio-temporelle de profiles quantitatifs de transcripts et de protéines est utilisée pour montrer le rôle de la protéine BREVIS RADIX (BRX) dans le dialogue entre l'auxine et les cytokinines, des phytohormones, dans la croissance du méristème racinaire. Le noyau de ce travail de thèse est la caractérisation fonctionnelle de l'interaction entre la protéine BRX et la phytohormone auxine dans le méristème de la racine en utilisant des modèles informatiques basés sur des preuves expérimentales. Les hypothèses produites par le modèle ont mené à la découverte d'un schéma différentiel d'endocytose dans le méristème racinaire qui divise la réponse transcriptionnelle à l'auxine par le partitionnement de BRX de la membrane plasmique au noyau de la cellule. Cette information positionnelle crée une réponse transcriptionnelle à l'auxine qui dévie de la réponse canonique à l'auxine et est nécessaire pour soutenir l'expression d'un sous ensemble de gènes répondant à l'auxine et dépendant de BRX pour conduire la croissance du méristème. Dans la seconde partie de cette thèse de doctorat, nous avons caractérisé l'impact sur l'ensemble du génome des délétions à grande échelle sur quatre souches divergentes naturelles d'Arabidopsis, à travers l'intégration du séquençage à ultra-haut-débit avec l'hybridation génomique sur puces ADN. L'analyse des délétions identifiées a révélé qu'une proportion considérable de gènes codant était affectée, supportant l'idée d'un historique de réarrangement génomique modelé durant l'évolution. Dans la dernière partie de cette thèse, nous avons montré que le gène VÏP3 dans Arabidopsis a conservé un rôle évolutif dans la machinerie de dégradation des ARNm dans le sens 3' à 5', en appliquant une nouvelle approche pour l'analyse des données de séquençage d'ARNm issue de transcripts amplifiés aléatoirement. Dans son ensemble, cette recherche de doctorat contient des avancées majeures dans l'étude des variations génomiques naturelles des plantes et dans l'application de modèles morphodynamiques informatiques pour la caractérisation de réseaux biologiques essentiels à la plante. - Le développement des plantes est écrit dans leurs codes génétiques. Pour comprendre comment les plantes sont capables de s'adapter aux changements environnementaux, il est essentiel d'étudier comment leurs gènes gouvernent leur formation. Plus nous essayons de comprendre le fonctionnement d'une plante, plus nous réalisons la complexité des mécanismes biologiques, à tel point que l'utilisation d'outils et de modèles mathématiques devient indispensable. Dans ce travail, avec l'utilisation de la plante modèle Arabidopsis thalicinci nous avons résolu des problèmes biologiques spécifiques à travers le développement et l'application de méthodes informatiques concrètes. Dans un premier temps, nous avons investigué comment le gène BREVIS RADIX (BRX) régule le développement de la racine en contrôlant la réponse à deux hormones : l'auxine et la cytokinine. Nous avons employé une analyse statistique sur des mesures quantitatives de transcripts et de produits de gènes afin de démontrer que BRX joue un rôle antagonisant dans le dialogue entre ces deux hormones. Lorsque ce-dialogue moléculaire est perturbé, la racine primaire voit sa longueur dramatiquement réduite. Pour comprendre comment BRX répond à l'auxine, nous avons développé un modèle informatique basé sur des résultats expérimentaux. Les simulations successives ont mené à la découverte d'un signal positionnel qui contrôle la réponse de la racine à l'auxine par la régulation du mouvement intracellulaire de BRX. Dans la seconde partie de cette thèse, nous avons analysé le génome entier de quatre souches naturelles d'Arabidopsis et nous avons trouvé qu'une grande partie de leurs gènes étaient manquant par rapport à la souche de référence. Ce résultat indique que l'historique des modifications génomiques conduites par l'évolution détermine une disponibilité différentielle des gènes fonctionnels dans ces plantes. Dans la dernière partie de ce travail, nous avons analysé les données du transcriptome de la plante où le gène VIP3 était non fonctionnel. Ceci nous a permis de découvrir le rôle double de VIP3 dans la régulation de l'initiation de la transcription et dans la dégradation des transcripts. Ce rôle double n'avait jusqu'alors été démontrée que chez l'homme. Ce travail de doctorat supporte le développement et l'application de méthodologies informatiques comme outils inestimables pour résoudre la complexité des problèmes biologiques dans la recherche végétale. L'intégration de la biologie végétale et l'informatique est devenue de plus en plus importante pour l'avancée de nos connaissances sur le fonctionnement et le développement des plantes.

TGFBI (BIGH3) gene mutations in German families: two novel mutations associated with unique clinical and histopathological findings.

BACKGROUND: To report the clinical, histopathological and immunohistochemical findings of two novel mutations within the TGFBI gene. METHODS: The genotype of 41 affected members of 16 families and nine sporadic cases was investigated by direct sequencing of the TGFBI gene. Clinical, histological and immunohistochemical characteristics of corneal opacification were reported and compared with the coding region changes in the TGFBI gene. RESULTS: A novel mutation Leu509Pro was detected in one family with a geographic pattern-like clinical phenotype. Histopathologically we found amyloid together with non-amyloid deposits and immunohistochemical staining of Keratoepithelin (KE) KE2 and KE15 antibodies. In two families and one sporadic case the novel mutation Gly623Arg with a late-onset, map-like corneal dystrophy was identified. Here amyloid and immunohistochemical staining of only KE2 antibodies occurred. Further, five already known mutations are reported: Arg124Cys Arg555Trp Arg124His His626Arg, Ala546Asp in 13 families and five sporadic cases of German origin. The underlying gene defect within the TBFBI gene was not identified in any of the four probands with Thiel-Behnke corneal dystrophy. CONCLUSIONS: The two novel mutations within the TGFBI gene add another two phenotypes with atypical immunohistochemical and histopathological features to those so far reported.