A White Campion (Silene latifolia) floral expressed sequence tag (EST) library: annotation, EST-SSR characterization, transferability, and utility for comparative mapping

<p>Abstract</p> <p>Background</p> <p>Expressed sequence tag (EST) databases represent a valuable resource for the identification of genes in organisms with uncharacterized genomes and for development of molecular markers. One class of markers derived from EST sequences are simple sequence repeat (SSR) markers, also known as EST-SSRs. These are useful in plant genetic and evolutionary studies because they are located in transcribed genes and a putative function can often be inferred from homology searches. Another important feature of EST-SSR markers is their expected high level of transferability to related species that makes them very promising for comparative mapping. In the present study we constructed a normalized EST library from floral tissue of <it>Silene latifolia </it>with the aim to identify expressed genes and to develop polymorphic molecular markers.</p> <p>Results</p> <p>We obtained a total of 3662 high quality sequences from a normalized <it>Silene </it>cDNA library. These represent 3105 unigenes, with 73% of unigenes matching genes in other species. We found 255 sequences containing one or more SSR motifs. More than 60% of these SSRs were trinucleotides. A total of 30 microsatellite loci were identified from 106 ESTs having sufficient flanking sequences for primer design. The inheritance of these loci was tested via segregation analyses and their usefulness for linkage mapping was assessed in an interspecific cross. Tests for crossamplification of the EST-SSR loci in other <it>Silene </it>species established their applicability to related species.</p> <p>Conclusion</p> <p>The newly characterized genes and gene-derived markers from our <it>Silene </it>EST library represent a valuable genetic resource for future studies on <it>Silene latifolia </it>and related species. The polymorphism and transferability of EST-SSR markers facilitate comparative linkage mapping and analyses of genetic diversity in the genus <it>Silene</it>.</p

Method for sequencing nucleic acids and analogues.

A method is for detg. the sequence of a nucleic acid, analog or fragment thereof, based on the detn. of the Tm or of physico-chem. parameters assocd. to the Tm of the duplex generated by repeated annealings of probes to the nucleic acid

Novel Combination of Surface Markers for the Reliable and Comprehensive Identification of Human Thymic Epithelial Cells by Flow Cytometry: Quantitation and Transcriptional Characterization of Thymic Stroma in a Pediatric Cohort

Thymic epithelial cells (TECs) are essential in supporting the development of mature T cells from hematopoietic progenitor cells and facilitate their lineage-commitment, proliferation, T-cell receptor repertoire selection and maturation. While animal model systems have greatly aided in elucidating the contribution of stromal cells to these intricate processes, human tissue has been more difficult to study, partly due to a lack of suitable surface markers comprehensively defining human TECs. Here, we conducted a flow cytometry based surface marker screen to reliably identify and quantify human TECs and delineate medullary from cortical subsets. These findings were validated by transcriptomic and histologic means. The combination of EpCAM, podoplanin (pdpn), CD49f and CD200 comprehensively identified human TECs and not only allowed their reliable distinction in medullary and cortical subsets but also their detailed quantitation. Transcriptomic profiling of each subset in comparison to fibroblasts and endothelial cells confirmed the identity of the different stromal cell subsets sorted according to the proposed strategy. Our dataset not only demonstrated transcriptional similarities between TEC and cells of mesenchymal origin but furthermore revealed a subset-specific distribution of a specific set of extracellular matrix-related genes in TECs. This indicates that TECs significantly contribute to the distinct compartmentalization - and thus function - of the human thymus. We applied the strategy to quantify TEC subsets in 31 immunologically healthy children, which revealed sex-specific differences of TEC composition early in life. As the distribution of mature CD4- or CD8-single-positive thymocytes was correspondingly altered, the composition of the thymic epithelial compartment may directly impact on the CD4-CD8-lineage choice of thymocytes. We prove that the plain, reliable strategy proposed here to comprehensively identify human TEC subpopulations by flow cytometry based on surface marker expression is suitable to determine their frequency and phenotype in health and disease and allows sorting of live cells for downstream analysis. Its use reaches from a reliable diagnostic tool for thymic biopsies to improved phenotypic characterization of thymic grafts intended for therapeutic use

Thermodynamic Studies on PNA and PNA/DNA Dendrimer Formation

In this work we report a kinetic and thermodynamic study relative to the formation of gel systems based on PNA and PNA/DNA dendrimers, useful for drug delivery or diagnostic applications. We realized two kinds of systems: a PNA-based monomolecular system formed by an autoassembling PNA tridendron (A) and a PNA/DNA bimolecular system based on a PNA tridendron with a mixed sequence and a DNA crosslinker (B). Both systems have the ability to form a three-dimensional network by means of specific W-C base pairing

DNA-based strategies for blocking HMGB1 cytokine activity: design, synthesis and preliminary in vitro/in vivo assays of DNA and DNA-like duplexes

In this work we report the design and synthesis of kinked oligonucleotide duplexes as potential inhibitors of HMGB1, a cytokine which triggers a broad range of immunological effects. We found that the designed ligands can interact with HMGB1, as evidenced by circular dichroism spectroscopy, and are able to block some extracellular effects induced by the protein, such as cellular proliferation and migration, as we demonstrated by in vitro biological assays. After selecting the most stable and active kinked duplex, we synthesized the corresponding PNA/DNA chimeric duplex which resulted to be more resistant to enzymatic degradation, and showed a biological activity comparable to that of the natural duplex. Preliminary in vivo assays in a mouse inflammatory model, showed a significant decrease of the mortality after administration of the PNA/DNA kinked duplex to LPS-treated mice

dabPna: Design, Synthesis, And Dna Binding Studies

In continuing our research efforts for developing new oligodeoxynucleotide (ODN)-like drugs and diagnostics, we designed diaminobutyric peptide nucleic acids (dabPNAs), nucleopeptides characterized by a diaminobutyric-based building block that is an isomer of the aminoethylglycyl PNA (aegPNA) unit and the acyclic modification of the aminoprolyl PNA (ampPNA) monomer. In this work we present the solid phase synthesis of a dabPNA oligomer and of two aegPNAs containing a single dabPNA unit. A study relative to their binding ability towards DNA is also reported even in comparison with the well known aegPNAs

Mild Cognitive Impairment in newly diagnosed Parkinson's disease: A longitudinal prospective study

In PD, Mild Cognitive Impairment (PD-MCI) occurs since early stages of disease. The aims were to assess presence of PD-MCI in untreated, drug-naive PD patients, and to follow-up the sample over 4 years to ascertain evolution of neurocognitive profile