BAC library resources for map-based cloning and physical map construction in barley (Hordeum vulgare L.)

Background: Although second generation sequencing (2GS) technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC) libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place. Result: Five new BAC libraries were constructed for barley (Hordeum vulgare L.) cultivar Morex. These libraries were constructed in different cloning sites (HindIII, EcoRI, MboI and BstXI) of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast), empty wells and off-scale clones (clones with 250 fragments). Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X. Conclusion: BAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing.Daniela Schulte, Ruvini Ariyadasa, Bujun Shi, Delphine Fleury, Chris Saski, Michael Atkins, Pieter deJong, Cheng-Cang Wu, Andreas Graner, Peter Langridge and Nils Stei

Tumor-Like Stem Cells Derived from Human Keloid Are Governed by the Inflammatory Niche Driven by IL-17/IL-6 Axis

Alterations in the stem cell niche are likely to contribute to tumorigenesis; however, the concept of niche promoted benign tumor growth remains to be explored. Here we use keloid, an exuberant fibroproliferative dermal growth unique to human skin, as a model to characterize benign tumor-like stem cells and delineate the role of their "pathological" niche in the development of the benign tumor.Subclonal assay, flow cytometric and multipotent differentiation analyses demonstrate that keloid contains a new population of stem cells, named keloid derived precursor cells (KPCs), which exhibit clonogenicity, self-renewal, distinct embryonic and mesenchymal stem cell surface markers, and multipotent differentiation. KPCs display elevated telomerase activity and an inherently upregulated proliferation capability as compared to their peripheral normal skin counterparts. A robust elevation of IL-6 and IL-17 expression in keloid is confirmed by cytokine array, western blot and ELISA analyses. The altered biological functions are tightly regulated by the inflammatory niche mediated by an autocrine/paracrine cytokine IL-17/IL-6 axis. Utilizing KPCs transplanted subcutaneously in immunocompromised mice we generate for the first time a human keloid-like tumor model that is driven by the in vivo inflammatory niche and allows testing of the anti-tumor therapeutic effect of antibodies targeting distinct niche components, specifically IL-6 and IL-17.These findings support our hypothesis that the altered niche in keloids, predominantly inflammatory, contributes to the acquirement of a benign tumor-like stem cell phenotype of KPCs characterized by the uncontrolled self-renewal and increased proliferation, supporting the rationale for in vivo modification of the "pathological" stem cell niche as a novel therapy for keloid and other mesenchymal benign tumors

Cross-oncopanel study reveals high sensitivity and accuracy with overall analytical performance depending on genomic regions

BackgroundTargeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing.ResultsAll panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5-20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden.ConclusionThis comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.Peer reviewe

Three-dimensional numerical model study of the residual current in the South China Sea

A three-dimensional baroclinic shelf sea model is employed to simulate the tidal and non-tidal residual current in the South China Sea. The four most significant constituents, M-2, S-2, K-1 and O-1, are included in the experiments with tidal effect. At most stations, the computed harmonic constants agree well with the observed ones. The circulations of the South China Sea in summer (August) and winter (December) are mainly discussed. It is shown that the barotropic tidal residual current is too weak to affect the South China Sea circulation, whilst the contribution of the baroclinic tidal residual current to the South China Sea circulation would be important in the continental shelf sea areas, especially in the Gulf of Thailand and Gulf of Tonkin. In the deep-sea areas, the upper barotropic or baroclinic tidal residual current is relatively very weak, however, the speed order of the deep baroclinic tidal residual current can be the same as that of the mean current without tidal effect. Moreover. the baroclinic tidal residual current seems to be related to the different seasonal stratification of ocean.Un modèle barocline tridimensionnel est utilisé pour simuler le courant résiduel (avec ou sans marée) dans la mer de Chine du Sud. Les quatre composantes principales, M2, S2, K1 et O1 sont prises en compte dans les expériences avec effet de marée. À la plupart des stations, les constantes harmoniques calculées sont cohérentes avec les mesures. Les circulations en mer de Chine du Sud en été (août) et en hiver (décembre) sont discutées. Le courant résiduel barotrope avec effet de marée est trop faible pour affecter la circulation alors que la contribution du courant résiduel barocline avec effet de marée est important en zone côtière, spécialement dans le golfe de Thaïlande et le golfe du Tonkin. Au large, le courant résiduel barocline ou barotrope supérieur est très faible mais la vitesse du courant résiduel barocline profond est équivalent au courant moyen sans effet de marée. De plus, le courant résiduel barocline avec effet de marée paraît relié à la différence saisonnière de stratification de l'océan

Aberrant Polo-like kinase 1-Cdc25A pathway in metastatic hepatocellular carcinoma

Purpose: Most studies on pathogenesis of tumor metastasis focus on cell adhesion and migration. Little is understood of how cell cycle pathways critically affect cell fate of metastatic cells and their sensitivity to anticancer drugs. In this study, we investigated cell cycle checkpoint progression and regulation in the presence of cisplatin in metastatic hepatocellular carcinoma (HCC) cells. Experimental Design: Cisplatin-mediated cell cycle progression and Polo-like kinase 1 (Plk1)-Cdc25A pathway were compared between metastatic and nonmetastatic HCC cells by flow cytometry,Western blots, and reverse transcription-PCR. Cdc25A expression in clinical HCC samples was detected using immunohistochemistry and its association with clinical HCC metastasis was analyzed. Results: Cisplatin induced degradation of Cdc25A in nonmetastatic HCC cells but not in metastatic HCC cells. Hence, metastatic HCC cells showed defective S-M cell cycle phase arrest and continued to enter mitosis. Tumor expression of Cdc25A was strongly associated with metastatic diseases in HCC patients, and elevated Cdc25A expression significantly correlated with HCC tumor-node-metastasis staging and venous invasion. Metastatic HCC cells did not show down-regulation of Plk1 that was normally induced by DNA damage. Blockage of Plk1 expression in metastatic HCC cells initiated Cdc25A degradation in response to DNA damage, suggesting that Plk1 could be an upstream regulator of Cdc25A. Deregulated Plk1-Cdc25A pathway in metastatic HCC cells and primary tumors did not result in drug-induced mitotic catastrophe but rather in accumulation of damaged DNA due to checkpoint adaptation. Conclusions: Metastatic HCC cells showed a defective S-M checkpoint following cisplatin treatment and potential aberrant checkpoint adaptation, which might result from deregulation of Plk1-Cdc25A pathway. ©2008 American Association for Cancer Research.link_to_subscribed_fulltex