Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak

<p>Abstract</p> <p>Background</p> <p>The Fagaceae family comprises about 1,000 woody species worldwide. About half belong to the <it>Quercus </it>family. These oaks are often a source of raw material for biomass wood and fiber. Pedunculate and sessile oaks, are among the most important deciduous forest tree species in Europe. Despite their ecological and economical importance, very few genomic resources have yet been generated for these species. Here, we describe the development of an EST catalogue that will support ecosystem genomics studies, where geneticists, ecophysiologists, molecular biologists and ecologists join their efforts for understanding, monitoring and predicting functional genetic diversity.</p> <p>Results</p> <p>We generated 145,827 sequence reads from 20 cDNA libraries using the Sanger method. Unexploitable chromatograms and quality checking lead us to eliminate 19,941 sequences. Finally a total of 125,925 ESTs were retained from 111,361 cDNA clones. Pyrosequencing was also conducted for 14 libraries, generating 1,948,579 reads, from which 370,566 sequences (19.0%) were eliminated, resulting in 1,578,192 sequences. Following clustering and assembly using TGICL pipeline, 1,704,117 EST sequences collapsed into 69,154 tentative contigs and 153,517 singletons, providing 222,671 non-redundant sequences (including alternative transcripts). We also assembled the sequences using MIRA and PartiGene software and compared the three unigene sets. Gene ontology annotation was then assigned to 29,303 unigene elements. Blast search against the SWISS-PROT database revealed putative homologs for 32,810 (14.7%) unigene elements, but more extensive search with Pfam, Refseq_protein, Refseq_RNA and eight gene indices revealed homology for 67.4% of them. The EST catalogue was examined for putative homologs of candidate genes involved in bud phenology, cuticle formation, phenylpropanoids biosynthesis and cell wall formation. Our results suggest a good coverage of genes involved in these traits. Comparative orthologous sequences (COS) with other plant gene models were identified and allow to unravel the oak paleo-history. Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 52,834 SSRs and 36,411 SNPs. All of these are available through the Oak Contig Browser <url>http://genotoul-contigbrowser.toulouse.inra.fr:9092/Quercus_robur/index.html</url>.</p> <p>Conclusions</p> <p>This genomic resource provides a unique tool to discover genes of interest, study the oak transcriptome, and develop new markers to investigate functional diversity in natural populations.</p

Llama-Derived Single Domain Antibodies to Build Multivalent, Superpotent and Broadened Neutralizing Anti-Viral Molecules

For efficient prevention of viral infections and cross protection, simultaneous targeting of multiple viral epitopes is a powerful strategy. Llama heavy chain antibody fragments (VHH) against the trimeric envelope proteins of Respiratory Syncytial Virus (Fusion protein), Rabies virus (Glycoprotein) and H5N1 Influenza (Hemagglutinin 5) were selected from llama derived immune libraries by phage display. Neutralizing VHH recognizing different epitopes in the receptor binding sites on the spikes with affinities in the low nanomolar range were identified for all the three viruses by viral neutralization assays. By fusion of VHH with variable linker lengths, multimeric constructs were made that improved neutralization potencies up to 4,000-fold for RSV, 1,500-fold for Rabies virus and 75-fold for Influenza H5N1. The potencies of the VHH constructs were similar or better than best performing monoclonal antibodies. The cross protection capacity against different viral strains was also improved for all three viruses, both by multivalent (two or three identical VHH) and biparatopic (two different VHH) constructs. By combining a VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with high affinity for subtype B, a biparatopic construct was made with low nanomolar neutralizing potency against both subtypes. Trivalent anti-H5N1 VHH neutralized both Influenza H5N1 clade1 and 2 in a pseudotype assay and was very potent in neutralizing the NIBRG-14 Influenza H5N1 strain with IC50 of 9 picomolar. Bivalent and biparatopic constructs against Rabies virus cross neutralized both 10 different Genotype 1 strains and Genotype 5. The results show that multimerization of VHH fragments targeting multiple epitopes on a viral trimeric spike protein is a powerful tool for anti-viral therapy to achieve "best-in-class" and broader neutralization capacity

High-quality SNPs from genic regions highlight introgression patterns among European white oaks (Quercus petraea and Q. robur)

International audienceIn the post-genomics era, non-model species like most Fagaceae still lack operational diversity resources for population genomics studies. Sequence data were produced from over 800 gene fragments covering ~530 kb across the genic partition of European oaks, in a discovery panel of 25 individuals from western and central Europe (11 Quercus petraea, 13 Q. robur, one Q. ilex as an outgroup). Regions targeted represented broad functional categories potentially involved in species ecological preferences, and a random set of genes. Using a high-quality dedicated pipeline, we provide a detailed characterization of these genic regions, which included over 14500 polymorphisms, with ~12500 SNPs −218 being triallelic-, over 1500 insertion-deletions, and ~200 novel di- and tri-nucleotide SSR loci. This catalog also provides various summary statistics within and among species, gene ontology information, and standard formats to assist loci choice for genotyping projects. The distribution of nucleotide diversity (θπ) and differentiation (FST) across genic regions are also described for the first time in those species, with a mean n θπ close to ~0.0049 in Q. petraea and to ~0.0045 in Q. robur across random regions, and a mean FST ~0.13 across SNPs. The magnitude of diversity across genes is within the range estimated for long-term perennial outcrossers, and can be considered relatively high in the plant kingdom, with an estimate across the genome of 41 to 51 million SNPs expected in both species. Individuals with typical species morphology were more easily assigned to their corresponding genetic cluster for Q. robur than for Q. petraea, revealing higher or more recent introgression in Q. petraea and a stronger species integration in Q. robur in this particular discovery panel. We also observed robust patterns of a slightly but significantly higher diversity in Q. petraea, across a random gene set and in the abiotic stress functional category, and a heterogeneous landscape of both diversity and differentiation. To explain these patterns, we discuss an alternative and non-exclusive hypothesis of stronger selective constraints in Q. robur, the most pioneering species in oak forest stand dynamics, additionally to the recognized and documented introgression history in both species despite their strong reproductive barriers. The quality of the data provided here and their representativity in terms of species genomic diversity make them useful for possible applications in medium-scale landscape and molecular ecology projects. Moreover, they can serve as reference resources for validation purposes in larger-scale resequencing projects. This type of project is preferentially recommended in oaks in contrast to SNP array development, given the large nucleotide variation and the low levels of linkage disequilibrium revealed

HIV/SIV Infection Primes Monocytes and Dendritic Cells for Apoptosis

Subversion or exacerbation of antigen-presenting cells (APC) death modulates host/pathogen equilibrium. We demonstrated during in vitro differentiation of monocyte-derived macrophages and monocyte-derived dendritic cells (DCs) that HIV sensitizes the cells to undergo apoptosis in response to TRAIL and FasL, respectively. In addition, we found that HIV-1 increased the levels of pro-apoptotic Bax and Bak molecules and decreased the levels of anti-apoptotic Mcl-1 and FLIP proteins. To assess the relevance of these observations in the context of an experimental model of HIV infection, we investigated the death of APC during pathogenic SIV-infection in rhesus macaques (RMs). We demonstrated increased apoptosis, during the acute phase, of both peripheral blood DCs and monocytes (CD14+) from SIV+RMs, associated with a dysregulation in the balance of pro- and anti-apoptotic molecules. Caspase-inhibitor and death receptors antagonists prevented apoptosis of APCs from SIV+RMs. Furthermore, increased levels of FasL in the sera of pathogenic SIV+RMs were detected, compared to non-pathogenic SIV infection of African green monkey. We suggest that inappropriate apoptosis of antigen-presenting cells may contribute to dysregulation of cellular immunity early in the process of HIV/SIV infection

Hygroma colli au premier trimestre de la grossesse (intérêt de la CGH array devant un hygroma colli isolé chez les foetus à caryotype normal.)

STRASBOURG-Medecine (674822101) / SudocSudocFranceF

Historical and contemporary dynamics of adaptive differentiation in European oaks

International audienc