TLR5 signaling stimulates the innate production of IL-17 and IL-22 by CD3(neg)CD127+ immune cells in spleen and mucosa.

In adaptive immunity, Th17 lymphocytes produce the IL-17 and IL-22 cytokines that stimulate mucosal antimicrobial defenses and tissue repair. In this study, we observed that the TLR5 agonist flagellin induced swift and transient transcription of genes encoding IL-17 and IL-22 in lymphoid, gut, and lung tissues. This innate response also temporarily enhanced the expression of genes associated with the antimicrobial Th17 signature. The source of the Th17-related cytokines was identified as novel populations of CD3(neg)CD127(+) immune cells among which CD4-expressing cells resembling lymphoid tissue inducer cells. We also demonstrated that dendritic cells are essential for expression of Th17-related cytokines and so for stimulation of innate cells. These data define that TLR-induced activation of CD3(neg)CD127(+) cells and production of Th17-related cytokines may be crucial for the early defenses against pathogen invasion of host tissues.

Advances in Sequence Analysis: Theory, Methods, Applications

This book gives a general view of sequence analysis, the statistical study of successions of states or events. It includes innovative contributions on life course studies, transitions into and out of employment, contemporaneous and historical careers, and political trajectories. The approach presented in this book is now central to the life-course perspective and the study of social processes more generally. This volume promotes the dialogue between approaches to sequence analysis that developed separately, within traditions contrasted in space and disciplines. It includes the latest developments in sequential concepts, coding, atypical datasets and time patterns, optimal matching and alternative algorithms, survey optimization, and visualization. Field studies include original sequential material related to parenting in 19th-century Belgium, higher education and work in Finland and Italy, family formation before and after German reunification, French Jews persecuted in occupied France, long-term trends in electoral participation, and regime democratization. Overall the book reassesses the classical uses of sequences and it promotes new ways of collecting, formatting, representing and processing them. The introduction provides basic sequential concepts and tools, as well as a history of the method. Chapters are presented in a way that is both accessible to the beginner and informative to the expert.

Quantitative sequence analysis of FBN1 premature termination codons provides evidence for incomplete NMD in leukocytes.

We improved, evaluated, and used Sanger sequencing for quantification of single nucleotide polymorphism (SNP) variants in transcripts and gDNA samples. This improved assay resulted in highly reproducible relative allele frequencies (e.g., for a heterozygous gDNA 50.0+/-1.4%, and for a missense mutation-bearing transcript 46.9+/-3.7%) with a lower detection limit of 3-9%. It provided excellent accuracy and linear correlation between expected and observed relative allele frequencies. This sequencing assay, which can also be used for the quantification of copy number variations (CNVs), methylations, mosaicisms, and DNA pools, enabled us to analyze transcripts of the FBN1 gene in fibroblasts and blood samples of patients with suspected Marfan syndrome not only qualitatively but also quantitatively. We report a total of 18 novel and 19 known FBN1 sequence variants leading to a premature termination codon (PTC), 26 of which we analyzed by quantitative sequencing both at gDNA and cDNA levels. The relative amounts of PTC-containing FBN1 transcripts in fresh and PAXgene-stabilized blood samples were significantly higher (33.0+/-3.9% to 80.0+/-7.2%) than those detected in affected fibroblasts with inhibition of nonsense-mediated mRNA decay (NMD) (11.0+/-2.1% to 25.0+/-1.8%), whereas in fibroblasts without NMD inhibition no mutant alleles could be detected. These results provide evidence for incomplete NMD in leukocytes and have particular importance for RNA-based analyses not only in FBN1 but also in other genes.

Sequence analysis and expression of the attachment and fusion proteins of canine distemper virus wild-type strain A75/17.

The biological properties of wild-type A75/17 and cell culture-adapted Onderstepoort canine distemper virus differ markedly. To learn more about the molecular basis for these differences, we have isolated and sequenced the protein-coding regions of the attachment and fusion proteins of wild-type canine distemper virus strain A75/17. In the attachment protein, a total of 57 amino acid differences were observed between the Onderstepoort strain and strain A75/17, and these were distributed evenly over the entire protein. Interestingly, the attachment protein of strain A75/17 contained an extension of three amino acids at the C terminus. Expression studies showed that the attachment protein of strain A75/17 had a higher apparent molecular mass than the attachment protein of the Onderstepoort strain, in both the presence and absence of tunicamycin. In the fusion protein, 60 amino acid differences were observed between the two strains, of which 44 were clustered in the much smaller F2 portion of the molecule. Significantly, the AUG that has been proposed as a translation initiation codon in the Onderstepoort strain is an AUA codon in strain A75/17. Detailed mutation analyses showed that both the first and second AUGs of strain A75/17 are the major translation initiation sites of the fusion protein. Similar analyses demonstrated that, also in the Onderstepoort strain, the first two AUGs are the translation initiation codons which contribute most to the generation of precursor molecules yielding the mature form of the fusion protein.

How Much Does It Cost? Optimization of Costs in Sequence Analysis of Social Science Data

One major methodological problem in analysis of sequence data is the determination of costs from which distances between sequences are derived. Although this problem is currently not optimally dealt with in the social sciences, it has some similarity with problems that have been solved in bioinformatics for three decades. In this article, the authors propose an optimization of substitution and deletion/insertion costs based on computational methods. The authors provide an empirical way of determining costs for cases, frequent in the social sciences, in which theory does not clearly promote one cost scheme over another. Using three distinct data sets, the authors tested the distances and cluster solutions produced by the new cost scheme in comparison with solutions based on cost schemes associated with other research strategies. The proposed method performs well compared with other cost-setting strategies, while it alleviates the justification problem of cost schemes.

Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution.

We present here a draft genome sequence of the red jungle fowl, Gallus gallus. Because the chicken is a modern descendant of the dinosaurs and the first non-mammalian amniote to have its genome sequenced, the draft sequence of its genome--composed of approximately one billion base pairs of sequence and an estimated 20,000-23,000 genes--provides a new perspective on vertebrate genome evolution, while also improving the annotation of mammalian genomes. For example, the evolutionary distance between chicken and human provides high specificity in detecting functional elements, both non-coding and coding. Notably, many conserved non-coding sequences are far from genes and cannot be assigned to defined functional classes. In coding regions the evolutionary dynamics of protein domains and orthologous groups illustrate processes that distinguish the lineages leading to birds and mammals. The distinctive properties of avian microchromosomes, together with the inferred patterns of conserved synteny, provide additional insights into vertebrate chromosome architecture.

Genomic, Epigenomic and Transcriptomic Molecular Characterization of Splenic Marginal Zone Lymphoma Reveals Recurrent Abnormalities in miR-182

Splenic marginal zone lymphoma (SMZL) is a low grade B-cell non-Hodgkin's lymphoma. The molecular pathology of this entity remains poorly understood. To characterise this lymphoma at the molecular level, we performed an integrated analysis of 1) genome wide genetic copy number alterations 2) gene expression profiles and 3) epigenetic DNA methylation profiles.We have previously shown that SMZL is characterised by recurrent alterations of chromosomes 7q, 6q, 3q, 9q and 18; however, gene resolution oligonucleotide array comparative genomic hybridisation did not reveal evidence of cryptic amplification or deletion in these regions. The most frequently lost 7q32 region contains a cluster of miRNAs. qRT-PCR revealed that three of these (miR-182/96/183) show underexpression in SMZL, and miR-182 is somatically mutated in >20% of cases of SMZL, as well as in >20% of cases of follicular lymphoma, and between 5-15% of cases of chronic lymphocytic leukaemia, MALT-lymphoma and hairy cell leukaemia. We conclude that miR-182 is a strong candidate novel tumour suppressor miRNA in lymphoma.The overall gene expression signature of SMZL was found to be strongly distinct fromthose of other lymphomas. Functional analysis of gene expression data revealed SMZL to be characterised by abnormalities in B-cell receptor signalling (especially through the CD19/21-PI3K/AKT pathway) and apoptotic pathways. In addition, genes involved in the response to viral infection appeared upregulated. SMZL shows a unique epigenetic profile, but analysis of differentially methylated genes showed few with methylation related transcriptional deregulation, suggesting that DNA methylation abnormalities are not a critical component of the SMZL malignant phenotype.

Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita.

Plant-parasitic nematodes are major agricultural pests worldwide and novel approaches to control them are sorely needed. We report the draft genome sequence of the root-knot nematode Meloidogyne incognita, a biotrophic parasite of many crops, including tomato, cotton and coffee. Most of the assembled sequence of this asexually reproducing nematode, totaling 86 Mb, exists in pairs of homologous but divergent segments. This suggests that ancient allelic regions in M. incognita are evolving toward effective haploidy, permitting new mechanisms of adaptation. The number and diversity of plant cell wall-degrading enzymes in M. incognita is unprecedented in any animal for which a genome sequence is available, and may derive from multiple horizontal gene transfers from bacterial sources. Our results provide insights into the adaptations required by metazoans to successfully parasitize immunocompetent plants, and open the way for discovering new antiparasitic strategies.

The fine-scale architecture of structural variants in 17 mouse genomes.

BACKGROUND: Accurate catalogs of structural variants (SVs) in mammalian genomes are necessary to elucidate the potential mechanisms that drive SV formation and to assess their functional impact. Next generation sequencing methods for SV detection are an advance on array-based methods, but are almost exclusively limited to four basic types: deletions, insertions, inversions and copy number gains. RESULTS: By visual inspection of 100 Mbp of genome to which next generation sequence data from 17 inbred mouse strains had been aligned, we identify and interpret 21 paired-end mapping patterns, which we validate by PCR. These paired-end mapping patterns reveal a greater diversity and complexity in SVs than previously recognized. In addition, Sanger-based sequence analysis of 4,176 breakpoints at 261 SV sites reveal additional complexity at approximately a quarter of structural variants analyzed. We find micro-deletions and micro-insertions at SV breakpoints, ranging from 1 to 107 bp, and SNPs that extend breakpoint micro-homology and may catalyze SV formation. CONCLUSIONS: An integrative approach using experimental analyses to train computational SV calling is essential for the accurate resolution of the architecture of SVs. We find considerable complexity in SV formation; about a quarter of SVs in the mouse are composed of a complex mixture of deletion, insertion, inversion and copy number gain. Computational methods can be adapted to identify most paired-end mapping patterns.

Evolution of vitellogenin genes: comparative analysis of the nucleotide sequences downstream of the transcription initiation site of four Xenopus laevis and one chicken gene.

Electron microscopic analysis of heteroduplexes between the most distantly related Xenopus vitellogenin genes (A genes X B genes) has revealed the distribution of homologous regions that have been preferentially conserved after the duplication events that gave rise to the multigene family in Xenopus laevis. DNA sequence analysis was limited to the region downstream of the transcription initiation site of the Xenopus genes A1, B1 and B2 and a comparison with the Xenopus A2 and the major chicken vitellogenin gene is presented. Within the coding regions of the first three exons, nucleotide substitutions resulting in amino acid changes accumulate at a rate similar to that observed in globin genes. This suggests that the duplication event which led to the formation of the A and B ancestral genes in Xenopus laevis occurred about 150 million years ago. Homologous exons of the A1-A2 and B1-B2 gene pairs, which formed about 30 million years ago, show a quite similar sequence divergence. In contrast, A1-A2 homologous introns seem to have evolved much faster than their B1-B2 counterparts.

The role of CTCFL/BORIS in Meiosis

The complexity of mammalian genome organization demands a complex interplay of DNA and proteins to orchestrate proper gene regulation. CTCF, a highly conserved, ubiquitously expressed protein has been postulated as a primary organizer of genome architecture because of its roles in transcriptional activation/repression, insulation and imprinting. Diverse regulatory functions are exerted through genome wide binding via a central eleven zinc finger DNA binding domain and an array of diverse protein-protein interactions through N- and C- terminal domains. CTCFL has been identified as a paralog of CTCF expressed only in spermatogenic cells of the testis. CTCF and CTCFL have a highly homologous DNA-binding domain, while the flanking amino acid sequences exhibit no significant similarity. Genome- wide mapping of CTCF binding sites has been carried out in many cell types, but no data exist for CTCFL apart from a few identified loci. The lack of high quality antibodies prompted us to generate an endogenously flag-tagged CTCFL mouse model using BAC recombination. IHC staining using anti-flag antibodies confirmed CTCFL localization to type Β spermatogonia and preleptotene spermatocytes and a mutually exclusive pattern of expression with CTCF. ChIP followed by high-throughput sequencing identified 10,382 binding sites showing 70% overlap but representing only 20% of CTCF sites. Consensus sequence analysis identified a significantly longer binding motif with prominently less ambiguity of base calling at every position. The significant difference between CTCF and CTCFL genomic binding patterns proposes that their binding to DNA is differentially regulated. Analysis of CTCFL binding to methylated regions on a genome wide scale identified approximately 1,000 loci. Methylation-independent binding of CTCFL might be at least one of the mechanisms that ensures distinct binding patterns of CTCF and CTCFL since CTCF binding is methylation- sensitive. Co-localization of CTCF with cohesin has been well established and analysis of CTCFL and SMC3 overlap identified around 3,300 binding sites from which two related but distinct consensus sequence motifs were derived. Because virtually all data for cohesin binding originate from mitotically proliferating cells, the anticipated overlap is expected to be considerably higher in meiotic cells. Meiosis-specific cohesin subunit Rec8 is specific for spermatocytes and 6 out of the 12 identified binding sites are also bound by CTCFL. In conclusion, this was the first genome-wide mapping of CTCFL binding sites in spermatocytes, the only cell type where CTCF is not expressed. CTCFL has a unique binding site repertoire distinct from CTCF, binds to methylated sequences and shows a significant overlap with cohesin binding sites. Future efforts will be oriented towards deciphering the role CTCFL plays in conversion of chromatin structure and function from mitotic to meiotic chromosomes. - La complexité de l'organisation du génome des mammifères exige une interaction particulière entre ADN et protéines pour orchestrer une régulation appropriée de l'expression des gènes. CTCFL, une protéine ubiquitaire très conservée, serait le principal organisateur de l'architecture du génome de par son rôle dans l'activation / la répression de la transcription, la protection et la localisation des gènes. Diverses régulations sont opérées, d'une part au travers d'interactions à différents endroits du génome par le biais d'un domaine protéique central de liaison à l'ADN à onze doigts de zinc, et d'autre part par des interactions protéine-protéine variées au niveau de leur domaine N- et C-terminal. CTCFL a été identifié comme un paralogue de CTCF exprimé uniquement dans les cellules spermatiques du testicule. CTCFL et CTCF ont un domaine de liaison à l'ADN très homologue, tandis que les séquences d'acides aminés situées de part et d'autre de ce domaine ne présentent aucune similitude. Une cartographie générale des sites de liaison au CTCF a été réalisée pour de nombreux types cellulaires, mais il n'existe aucune donnée pour CTCFL à l'exception de l'identification de quelques loci. L'absence d'anticorps de bonne qualité nous a conduit à générer un modèle murin portant un CTCFL endogène taggué grâce à un procédé de recombinaison BAC. Une coloration IHC à l'aide d'anticorps anti-FLAG a confirmé la présence de CTCFL au niveau des spermatogonies de type Β et des spermatocytes au stade préleptotène, et une distribution mutuellement exclusive avec CTCF. Une méthode de Chromatine Immunoprecipitation (ChIP) suivie d'un séquençage à haut débit a permis d'identifier 10.382 sites de liaison montrant 70% d'homologie mais ne représentant que 20% des sites CTCF. L'analyse de la séquence consensus révèle un motif de fixation à l'ADN nettement plus long et qui comporte bien moins de bases aléatoires à chaque position nucléotidique. La différence significative entre les séquences génomiques des sites de liaison au CTCF et CTCFL suggère que leur fixation à l'ADN est régulée différemment. Appliquée à l'échelle du génome, l'étude de l'interaction de CTCFL avec des régions méthylées de l'ADN a permis d'identifier environ 1.000 loci. Contrairement à CTCFL, la liaison de CTCF dépend de l'état de méthylation de l'ADN ; cette modification épigénétique constitue donc au moins un des mécanismes de régulation expliquant une localisation de CTCF et CTCFL à des sites distincts du génome. La co- localisation de CTCF avec la cohésine étant établie, l'analyse de la superposition des séquences de CTCFL avec la sous-unité SMC3 identifie environ 3.300 sites de liaison parmi lesquels deux mêmes motifs consensus distincts par leur séquence sont mis en évidence. La presque quasi-totalité des données sur la cohésine ayant été établie à partir de cellules en prolifération mitotique, il est probable que la similitude au sein des séquences consensus soit encore plus grande dans le cas des cellules en méiose. La sous-unité Rec8 de la cohésine propre à l'état de méiose est spécifiquement exprimée dans les spermatocytes. Or 6 des 12 sites de liaison identifiés sont également utilisés par CTCFL. Pour conclure, ce travail constitue la première cartographie à l'échelle du génome des sites de liaison de CTCFL dans les spermatocytes, seul type cellulaire où CTCFL n'est pas exprimé. CTCFL possède un répertoire unique de sites de fixation à l'ADN distinct de CTCF, se lie à des séquences méthylées et présente un nombre important de sites de liaison communs avec la cohésine. Les perspectives futures sont d'élucider le rôle de CTCFL dans le remodelage de la structure de la chromatine et de définir sa fonction dans le processus de méiose.