A quantitative PCR (QPCR) assay based on the use of SYBR Green I was developed to assess the abundance of specific groups of picoeukaryotes in marine waters. Six primer sets were designed targeting four different taxonomic levels: domain (Eukaryota), division (Chlorophyta), order (Mamiellales) and genus (Bathycoccus, Micromonas, and Ostreococcus). Reaction conditions were optimized for each primer set which was validated in silico, on agarose gels, and by QPCR against a variety of target and non-target cultures. The approach was tested by estimating gene copy numbers for Micromonas, Bathycoccus, and Ostreococcus in seawater samples to which cultured cells were added in various concentrations. QPCR was then used to determine that rRNA gene (rDNA) copy number varied from one to more than 12,000 in 18 strains of phytoplankton. Finally, QPCR was applied to environmental samples from a Mediterranean Sea coastal site and the results were compared to those obtained by Fluorescent in situ hybridization (FISH). The data obtained demonstrate that Chlorophyta and more specifically Mamiellales were important in these waters, especially during the winter picoplankton bloom. The timing of major abundance peaks of the targeted species was similar by QPCR and FISH. When used in conjunction with other techniques such as FISH or gene clone libraries, QPCR appears as very promising to quickly obtain data on the ecological distribution of important phytoplankton groups. Data interpretation must take into account primer specificity and the varying rRNA gene copy number among eukaryotes.

Selectin-mediated adhesion of tumor cells to platelets, leukocytes, and endothelial cells may regulate their hematogenous dissemination in the microvasculature. We recently identified CD44 variant isoforms (CD44v) as functional P-, but not E-or L-, selectin ligands on colon carcinoma cells. Moreover, an ϳ180-kDa sialofucosylated glycoprotein(s) mediated selectin binding in CD44-knockdown cells. Using immunoaffinity chromatography and tandem mass spectrometry, we identify this glycoprotein as the carcinoembryonic antigen (CEA). Blot rolling assays and flow-based adhesion assays using microbeads coated with CEA immunopurified from LS174T colon carcinoma cells and selectins as substrate reveal that CEA possesses E-and L-, but not P-, selectin ligand activity. CEA on CD44-knockdown LS174T cells exhibits higher HECA-452 immunoreactivity than CEA on wild-type cells, suggesting that CEA functions as an alternative acceptor for selectin-binding glycans. The enhanced expression of HECA-452 reactive epitopes on CEA from CD44-knockdown cells correlates with the increased CEA avidity for E-but not L-selectin. Through the generation of stable knockdown cell lines, we demonstrate that CEA serves as an auxiliary L-selectin ligand, which stabilizes L-selectin-dependent cell rolling against fluid shear. Moreover, CEA and CD44v cooperate to mediate colon carcinoma cell adhesion to E-and L-selectin at elevated shear stresses. The novel finding that CEA is an E-and L-selectin ligand may explain the enhanced metastatic potential associated with tumor cell CEA overexpression and the supportive role of selectins in metastasis.Several lines of evidence suggest that selectins facilitate cancer metastasis and tumor cell arrest in the microvasculature by mediating specific interactions between selectin-expressing hosts cells and ligands on tumor cells. A variety of tumor cells, including colon carcinoma, express sialylated, fucosylated molecules that could be recognized by selectins (1-6). Enhanced expression of sialylated fucosylated glycans such as sialyl Le x and sialyl Le a on the tumor cell surface correlates with poor prognosis because of tumor progression and metastatic spread (7-10). Earlier studies hypothesized a simple model whereby E-selectin expressed on the surface of activated endothelial cells mediates binding of malignant cells, thereby facilitating their extravasation from the vasculature and the seeding of metastatic foci. This model was corroborated by ample experimental evidence. For instance, the ability of human colon carcinoma cell lines to form lung metastases in nude mice correlates with their adhesion to E-selectin and is markedly diminished by a soluble E-selectin fusion protein (11). Along these lines, the metastatic potential of colon carcinoma cell lines is attenuated by preincubating carcinoma cells with antisialyl Le a antibodies (10). The overexpression of E-selectin in the liver of a transgenic mouse model redirected the metastasis to this organ (12).During their transit into the circulatory system, tumor...

Elevated levels of prostaglandin (PG)E(2) and interleukin (IL)-6 have been reported in the cartilage and synovial fluid from patients with arthritic disorders. PGE(2) regulates IL-6 production in numerous different cells including macrophages and synovial fibroblasts. Although PGE(2) stimulates IL-6 expression in human chondrocytes, the underlying signaling pathway of this process has yet to be delineated. Here, we investigate the mechanism of IL-6 induction in human T/C-28a2 chondrocytes treated with exogenously added PGE(2). PGE(2) induces IL-6 mRNA and protein expression via a cAMP-dependent pathway, reaching maximal levels after 60 min of stimulation before declining to baseline levels at 6 h. Forskolin, an adenylyl cyclase activator, also stimulates IL-6 expression in human chondrocytes in a dose- and time-dependent fashion. Inhibition of downstream effectors of cAMP activity such as protein kinase A (PKA) or phosphatidylinositol 3 kinase (PI3K) blocks PGE(2)- and forskolin-induced IL-6 upregulation. Simultaneous inhibition of PKA and PI3K reduces IL-6 expression in stimulated chondrocytes well below the basal levels of untreated cells. Gel shift, supershift, and chromatin immunoprecipitation assays reveal the activation and binding of the nuclear factor (NF)-kappaB p65 subunit to the IL-6 promoter, which is markedly suppressed by selective PI3K or PKA pharmacological inhibitors. p65 knockdown completely abrogates IL-6 mRNA synthesis in PGE(2)- and forskolin-primed chondrocytes. Cumulatively, our data show that PGE(2) and forskolin induce IL-6 expression in human chondrocytes via cAMP/PKA and PI3K-dependent pathways, which in turn regulate the activation and binding of p65 to the IL-6 promoter.

Hemoglobinopathies are among the most common autosomal-recessive disorders worldwide. A comprehensive next-generation sequencing (NGS) test would greatly facilitate screening and diagnosis of these disorders. An NGS panel targeting the coding regions of hemoglobin genes and four modifier genes was designed. We validated the assay by using 2522 subjects affected with hemoglobinopathies and applied it to carrier testing in a cohort of 10,111 couples who were also screened through traditional methods. In the clinical genotyping analysis of 1182 β-thalassemia subjects, we identified a group of additional variants that can be used for accurate diagnosis. In the molecular screening analysis of the 10,111 couples, we detected 4180 individuals in total who carried 4840 mutant alleles, and identified 186 couples at risk of having affected offspring. 12.1% of the pathogenic or likely pathogenic variants identified by our NGS assay, which were undetectable by traditional methods. Compared with the traditional methods, our assay identified an additional at-risk 35 couples. We describe a comprehensive NGS-based test that offers advantages over the traditional screening/molecular testing methods. To our knowledge, this is among the first large-scale population study to systematically evaluate the application of an NGS technique in carrier screening and molecular diagnosis of hemoglobinopathies.