Physical Mapping of Bread Wheat Chromosome 5A: An Integrated Approach.

The huge size, redundancy, and highly repetitive nature of the bread wheat [Triticum aestivum (L.)] genome, makes it among the most difficult species to be sequenced. To overcome these limitations, a strategy based on the separation of individual chromosomes or chromosome arms and the subsequent production of physical maps was established within the frame of the International Wheat Genome Sequence Consortium (IWGSC). A total of 95,812 bacterial artificial chromosome (BAC) clones of short-arm chromosome 5A (5AS) and long-arm chromosome 5A (5AL) arm-specific BAC libraries were fingerprinted and assembled into contigs by complementary analytical approaches based on the FingerPrinted Contig (FPC) and Linear Topological Contig (LTC) tools. Combined anchoring approaches based on polymerase chain reaction (PCR) marker screening, microarray, and sequence homology searches applied to several genomic tools (i.e., genetic maps, deletion bin map, neighbor maps, BAC end sequences (BESs), genome zipper, and chromosome survey sequences) allowed the development of a high-quality physical map with an anchored physical coverage of 75% for 5AS and 53% for 5AL with high portions (64 and 48%, respectively) of contigs ordered along the chromosome. In the genome of grasses, Brachypodium [Brachypodium distachyon (L.) Beauv.], rice (Oryza sativa L.), and sorghum [Sorghum bicolor (L.) Moench] homologs of genes on wheat chromosome 5A were separated into syntenic blocks on different chromosomes as a result of translocations and inversions during evolution. The physical map presented represents an essential resource for fine genetic mapping and map-based cloning of agronomically relevant traits and a reference for the 5A sequencing projects.

NG2/CSPG4-collagen type VI interplays putatively involved in the microenvironmental control of tumour engraftment and local expansion.

In soft-tissue sarcoma patients, enhanced expression of NG2/CSPG4 proteoglycan in pre-surgical primary tumours predicts post-surgical metastasis formation and thereby stratifies patients into disease-free survivors and patients destined to succumb to the disease. Both primary and secondary sarcoma lesions also up-regulate collagen type VI, a putative extracellular matrix ligand of NG2, and this matrix alteration potentiates the prognostic impact of NG2. Enhanced constitutive levels of the proteoglycan in isolated sarcoma cells closely correlate with a superior engraftment capability and local growth in xenogenic settings. This apparent NG2-associated malignancy was also corroborated by the diverse tumorigenic behaviour in vitro and in vivo of immunoselected NG2-expressing and NG2-deficient cell subsets, by RNAi-mediated knock down of endogenous NG2, and by ectopic transduction of full-length or deletion constructs of NG2. Cells with modified expression of NG2 diverged in their interaction with purified Col VI, matrices supplemented with Col VI, and cell-free matrices isolated from wild-type and Col VI null fibroblasts. The combined use of dominant-negative NG2 mutant cells and purified domain fragments of the collagen allowed us to pinpoint the reciprocal binding sites within the two molecules and to assert the importance of this molecular interaction in the control of sarcoma cell adhesion and motility. The NG2-mediated binding to Col VI triggered activation of convergent cell survival- and cell adhesion/migration-promoting signal transduction pathways, implicating PI-3K as a common denominator. Thus, the findings point to an NG2-Col VI interplay as putatively involved in the regulation of the cancer cell-host microenvironment interactions sustaining sarcoma progression.

Emerging knowledge from genome sequencing of crop species.

Extensive insights into the genome composition, organization, and evolution have been gained from the plant genome sequencing and annotation ongoing projects. The analysis of crop genomes provided surprising evidences with important implications in plant origin and evolution: genome duplication, ancestral re-arrangements and unexpected polyploidization events opened new doors to address fundamental questions related to species proliferation, adaptation, and functional modulations. Detailed paleogenomic analysis led to many speculation on how chromosomes have been shaped over time in terms of gene content and order. The completion of the genome sequences of several major crops, prompted to a detailed identification and annotation of transposable elements: new hypothesis related to their composition, chromosomal distribution, insertion models, amplification rate, and evolution patterns are coming up. Availability of full genome sequence of several crop species as well as from many accessions within species is providing new keys for biodiversity exploitation and interpretation. Re-sequencing is enabling high-throughput genotyping to identify a wealth of SNP and afterward to produce haplotype maps necessary to accurately associate molecular variation to phenotype. Conservation genomics is emerging as a powerful tool to explain adaptation, genetic drift, natural selection, hybridization and to estimate genetic variation, fitness and population's viability.

Fluorescence-based assays for in vitro analysis of cell adhesion and migration.

Cell adhesion and cell migration are two primary cellular phenomena for which in vitro approaches may be exploited to effectively dissect the individual events and underlying molecular mechanisms. The use of assays dedicated to the analysis of cell adhesion and migration in vitro also afford an efficient way of conducting larger basic and applied research screenings on the factors affecting these processes and are potentially exploitable in the context of routine diagnostic, prognostic, and predictive tests in the biological and medical fields. Therefore, there is a longstanding continuum in the interest in devising more rationale such assays and major contributions in this direction have been provided by the advent of procedures based on fluorescence cell tagging, the design of instruments capable of detecting fluorescent signals with high sensitivity, and informatic tools allowing sophisticated elaboration of data generated through these instruments. In this report, we describe three representative fluorescence-based model assays for the qualitative and quantitative assessment of cell adhesion and cell locomotion in static and dynamic conditions. The assays are easily performed, accurate and reproducible, and can be automated for high-to-medium throughput screenings of cell behavior in vitro. Performance of the assays involves the use of certain dedicated disposable accessories, which are commercially available, and a few instruments that, due to their versatility, can be regarded as constituents of a more generic laboratory setup.

Cyclin-dependent kinase inhibitor seliciclib shows in vitro activity in diffuse large B-cell lymphomas.

Despite recent improvements in treatment, a significant fraction of patients with diffuse large B-cell lymphoma (DLBCL) still fail therapy. Therefore, new therapeutic modalities are needed to advance the cure rate. Seliciclib (CYC202, R-roscovitine) is a purine analog developed as an inhibitor of CDK2/cyclin E CDK7/cyclin H and CDK9/cyclin T. Seliciclib has been shown to be active in B-cell neoplasms, such as mantle cell lymphoma, chronic lymphocytic leukemia and in multiple myeloma in vitro. The aim of this study was to assess the in vitro activity of seliciclib in DLBCL. The anti-proliferative activity of seliciclib was tested in nine human DLBCL cell lines and six DLBCL primary cell cultures. The effects of seliciclib on the cell cycle and on apoptosis, as well as on transcription-related proteins were assessed. The cell viability of all DLBCL cell lines and primary cells was reduced by seliciclib treatment. The IC50 for the cell lines ranged from 13 - 36 microm. The effect of seliciclib was independent of the genetic aberrations characterizing the cell lines. After seliciclib exposure cells accumulated in G2/M or in G1 phase, with most of the cells showing signs of apoptosis. Despite the clear cytotoxic effect and induction of apoptosis, this study could not identify a unique mechanism of action. The in vitro data suggest that seliciclib is an active agent in DLBCL. Its efficacy is apparently independent of the underlying chromosomal translocations characteristic of DLBCL. The drug might represent a new therapeutic agent in this lymphoma sub-type.