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Low-density Array (low-density + array)
Selected AbstractsMatriptase is a novel initiator of cartilage matrix degradation in osteoarthritisARTHRITIS & RHEUMATISM, Issue 7 2010Jennifer M. Milner Objective Increasing evidence implicates serine proteinases in pathologic tissue turnover. The aim of this study was to assess the role of the transmembrane serine proteinase matriptase in cartilage destruction in osteoarthritis (OA). Methods Serine proteinase gene expression in femoral head cartilage obtained from either patients with hip OA or patients with fracture to the neck of the femur (NOF) was assessed using a low-density array. The effect of matriptase on collagen breakdown was determined in cartilage degradation models, while the effect on matrix metalloproteinase (MMP) expression was analyzed by real-time polymerase chain reaction. ProMMP processing was determined using sodium dodecyl sulfate,polyacrylamide gel electrophoresis/N-terminal sequencing, while its ability to activate proteinase-activated receptor 2 (PAR-2) was determined using a synovial perfusion assay in mice. Results Matriptase gene expression was significantly elevated in OA cartilage compared with NOF cartilage, and matriptase was immunolocalized to OA chondrocytes. We showed that matriptase activated proMMP-1 and processed proMMP-3 to its fully active form. Exogenous matriptase significantly enhanced cytokine-stimulated cartilage collagenolysis, while matriptase alone caused significant collagenolysis from OA cartilage, which was metalloproteinase-dependent. Matriptase also induced MMP-1, MMP-3, and MMP-13 gene expression. Synovial perfusion data confirmed that matriptase activates PAR-2, and we demonstrated that matriptase-dependent enhancement of collagenolysis from OA cartilage is blocked by PAR-2 inhibition. Conclusion Elevated matriptase expression in OA and the ability of matriptase to activate selective proMMPs as well as induce collagenase expression make this serine proteinase a key initiator and inducer of cartilage destruction in OA. We propose that the indirect effects of matriptase are mediated by PAR-2, and a more detailed understanding of these mechanisms may highlight important new therapeutic targets for OA treatment. [source] In vivo Remote Delivery of DNA Encoding for Hypoxia-inducible Factor 1 Alpha Reduces Myocardial Infarct SizeCLINICAL AND TRANSLATIONAL SCIENCE, Issue 1 2009Gabor Czibik M.D. Abstract We tested if remote gene delivery of hypoxia-inducible factor 1 alpha (HIF-1,) protected hearts against induced ischemia, hypothesizing that gene delivery into skeletal muscle may lead to secretion of proteins with actions elsewhere. Murine quadriceps muscles were transfected with DNA encoding for human HIF-1,, which resulted in a local, but lasting expression (mRNA and protein, where the latter had nuclear localization). Subjection of isolated hearts to global ischemia and reperfusion 1, 4, and 8 weeks after gene delivery resulted in infarct size reduction (p < 0.05). Supporting that this was due to paracrine effects, HL-1 cells treated with conditioned media from cells transfected with HIF-1, or serum from HIF-1,-treated mice were protected against H2O2 -induced cell death (p < 0.05, respectively). The latter protection was reduced when a heme oxygenase activity blocker was used. Taqman low-density array of 47 HIF-1,-regulated genes at the treatment site showed nine specific upregulations (p < 0.05). Of the corresponding proteins, PDGF-B and adrenomedullin were upregulated in the heart. HIF-1, treatment induced an increased vascularization of the heart and skeletal muscle. In conclusion, remote delivery of DNAfor HIF-1, was cardioprotective, represented by consistent infarct size reduction, which may be due to release of paracrine factors from the transfected muscle. [source] Near real-time, autonomous detection of marine bacterioplankton on a coastal mooring in Monterey Bay, California, using rRNA-targeted DNA probesENVIRONMENTAL MICROBIOLOGY, Issue 5 2009Christina M. Preston Summary A sandwich hybridization assay (SHA) was developed to detect 16S rRNAs indicative of phylogenetically distinct groups of marine bacterioplankton in a 96-well plate format as well as low-density arrays printed on a membrane support. The arrays were used in a field-deployable instrument, the Environmental Sample Processor (ESP). The SHA employs a chaotropic buffer for both cell homogenization and hybridization, thus target sequences are captured directly from crude homogenates. Capture probes for seven of nine different bacterioplankton clades examined reacted specifically when challenged with target and non-target 16S rRNAs derived from in vitro transcribed 16S rRNA genes cloned from natural samples. Detection limits were between 0.10,1.98 and 4.43, 12.54 fmole ml,1 homogenate for the 96-well plate and array SHA respectively. Arrays printed with five of the bacterioplankton-specific capture probes were deployed on the ESP in Monterey Bay, CA, twice in 2006 for a total of 25 days and also utilized in a laboratory time series study. Groups detected included marine alphaproteobacteria, SAR11, marine cyanobacteria, marine group I crenarchaea, and marine group II euryarchaea. To our knowledge this represents the first report of remote in situ DNA probe-based detection of marine bacterioplankton. [source] Gene expression measurements in the context of epidemiological studiesALLERGY, Issue 12 2008C. Bieli Background:, Gene expression measurements became an attractive tool to assess biological responses in epidemiological studies. However, collection of blood samples poses various technical problems. We used gene expression data from two epidemiological studies to evaluate differences between sampling methods, comparability of two methods for measuring RNA levels and stability of RNA samples over time. Methods:, For the PARSIFAL study, PBLC of 1155 children were collected using EDTA tubes in two countries. In the PASTURE study, tubes containing RNA-stabilizing solutions (PAXgeneŽ Blood RNA Tubes; PreAnalytiX) were used to collect cord blood leucocytes of 982 children in five countries. Real-time PCR (conventional single tube assay and high-throughput low density arrays) was used to quantify expression of various innate immunity genes. In 77 PARSIFAL samples, gene expression was measured repeatedly during prolonged storage. Results:, In PARSIFAL (EDTA tubes) the median RNA yield after extraction significantly differed between the two centres (70 and 34 ng/,l). Collecting blood into an RNA-stabilizing solution markedly reduced differences in RNA yield in PASTURE (range of medians 91,107 ng/,l). The agreement [Spearman rank correlation (r)] between repeated measurements of gene expression decreased with increasing storage time [e.g., for CD14: r (first/second measurement) = 0.35; r (first/third measurement) = 0.03]. RNA levels measured with either the conventional method or low-density arrays were comparable (r > 0.9). Conclusion:, Collecting blood samples into tubes containing an RNA-stabilizing solution increases RNA yield and reduces its variability. Long-term storage of samples may lead to RNA degradation, requiring special attention in longitudinal studies. [source] |