Validation of extracellular miRNA quantification in blood samples using RT-qPCR.

Extracellular microRNAs (miRs) have been proposed as important blood-based biomarkers for several diseases. Contrary to proteins and other RNA classes, miRs are stable and easily detectable in body fluids. In this respect, miRs represent a perfect candidate for minimal invasive biomarkers which can hopefully become a complement for invasive histological examinations of tumor tissue. Despite the high number of miR biomarker studies, the specificity and reproducibility of these studies is missing. Therefore, the standardization of pre-analytical and analytical methods is urgently needed. Here, we validated miR analysis for RNA isolation and miR quantification by quantitative polymerase chain reaction (RT-qPCR) based on good laboratory practice (GLP). Validation was carried out exemplarily on four miRs, which had already been described as potential biomarkers in previous studies. As basis for RNA analysis using RT-qPCR, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments were applied and adapted on the analysis of circulating miRs from human plasma. In our study, we identified and solved several pitfalls from handling to normalization strategy in the analysis of extracellular miRs that lead to inconsistent and non-repeatable data. Principles of GLP set a framework of experimental design, performance and monitoring to ensure high quality and reliable data. Within this study, we appointed first acceptance criteria for circulating miR quantification during validation which set standards for future miR quantification in blood samples.

Evaluation of the plasmatic and parenchymal elution kinetics of two different irinotecan-loaded drug-eluting embolics in a pig model.

PURPOSE: To evaluate and compare irinotecan elution kinetics of two drug-eluting embolic agents in a porcine model. MATERIALS AND METHODS: Embolization of the left liver lobe was performed in 16 domestic pigs, with groups of two receiving 1 mL of DC Bead M1 (70-150 µm) or Embozene TANDEM (75 µm) loaded with 50 mg irinotecan. Irinotecan plasma levels were measured at 0, 10, 20, 30, 60, 120, 180, and 240 minutes after completed embolization and at the time of euthanasia (24 h, 48 h, 72 h, or 7 d). Liver tissue samples were taken to measure irinotecan tissue concentrations. RESULTS: The highest irinotecan plasma concentrations of both embolic agents were measured 10 and 20 minutes after embolization, and concentrations were significantly higher for DC Bead M1 versus Embozene TANDEM (P = .0019 and P = .0379, respectively). At 48 hours and later follow-up, no irinotecan was measurable in the plasma. For both embolic agents, the highest irinotecan tissue concentration was found after 24 hours and decreased in a time-dependent manner at later follow-up intervals. Additionally, SN-38 tissue levels for both agents were therapeutic at 24 hours, with therapeutic levels of SN-38 at 48 hours in one liver embolized with TANDEM particles. Histopathologic analysis revealed ischemic, inflammatory, and fibrotic tissue reactions. CONCLUSIONS: Irinotecan is measurable in plasma and hepatic tissue after liver embolization with both types of irinotecan-eluting embolic agents. DC Bead M1 shows early burst elution kinetics, whereas Embozene TANDEM has a lower and slower release profile. The initial burst is significantly greater after embolization with DC Bead M1 than with Embozene TANDEM.

Mitogen-activated protein kinase-activated protein kinases 2 and 3 regulate SERCA2a expression and fiber type composition to modulate skeletal muscle and cardiomyocyte function.

The mitogen-activated protein kinase (MAPK)-activated protein kinases 2 and 3 (MK2/3) represent protein kinases downstream of the p38 MAPK. Using MK2/3 double-knockout (MK2/3(-/-)) mice, we analyzed the role of MK2/3 in cross-striated muscle by transcriptome and proteome analyses and by histology. We demonstrated enhanced expression of the slow oxidative skeletal muscle myofiber gene program, including the peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1α (PGC-1α). Using reporter gene and electrophoretic gel mobility shift assays, we demonstrated that MK2 catalytic activity directly regulated the promoters of the fast fiber-specific myosin heavy-chain IId/x and the slow fiber-specific sarco/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) gene. Elevated SERCA2a gene expression caused by a decreased ratio of transcription factor Egr-1 to Sp1 was associated with accelerated relaxation and enhanced contractility in MK2/3(-/-) cardiomyocytes, concomitant with improved force parameters in MK2/3(-/-) soleus muscle. These results link MK2/3 to the regulation of calcium dynamics and identify enzymatic activity of MK2/3 as a critical factor for modulating cross-striated muscle function by generating a unique muscle phenotype exhibiting both reduced fatigability and enhanced force in MK2/3(-/-) mice. Hence, the p38-MK2/3 axis may represent a novel target for the design of therapeutic strategies for diseases related to fiber type changes or impaired SERCA2 function.

Extracellular signal-regulated kinase 1/2-mediated phosphorylation of p300 enhances myosin heavy chain I/beta gene expression via acetylation of nuclear factor of activated T cells c1.

The nuclear factor of activated T-cells (NFAT) c1 has been shown to be essential for Ca(2+)-dependent upregulation of myosin heavy chain (MyHC) I/ß expression during skeletal muscle fiber type transformation. Here, we report activation of extracellular signal-regulated kinase (ERK) 1/2 in Ca(2+)-ionophore-treated C2C12 myotubes and electrostimulated soleus muscle. Activated ERK1/2 enhanced NFATc1-dependent upregulation of a -2.4 kb MyHCI/ß promoter construct without affecting subcellular localization of endogenous NFATc1. Instead, ERK1/2-augmented phosphorylation of transcriptional coactivator p300, promoted its recruitment to NFATc1 and increased NFATc1-DNA binding to a NFAT site of the MyHCI/ß promoter. In line, inhibition of ERK1/2 signaling abolished the effects of p300. Comparison between wild-type p300 and an acetyltransferase-deficient mutant (p300DY) indicated increased NFATc1-DNA binding as a consequence of p300-mediated acetylation of NFATc1. Activation of the MyHCI/ß promoter by p300 depends on two conserved acetylation sites in NFATc1, which affect DNA binding and transcriptional stimulation. NFATc1 acetylation occurred in Ca(2+)-ionophore treated C2C12 myotubes or electrostimulated soleus. Finally, endogenous MyHCI/ß gene expression in C2C12 myotubes was strongly inhibited by p300DY and a mutant deficient in ERK phosphorylation sites. In conclusion, ERK1/2-mediated phosphorylation of p300 is crucial for enhancing NFATc1 transactivation function by acetylation, which is essential for Ca(2+)-induced MyHCI/ß expression.

Neuronal NO synthase and its inhibitor PIN are present and influenced by glucose in the human beta-cell line CM and in rat INS-1 cells.

Nitric oxide (NO) is synthesised by different nitric oxide synthases (NOS) from L-arginine and acts as a signal transducer in a variety of cells. The neuronal isoenzyme of NOS (nNOS) was recently found in rodent beta-cells and beta-cell lines. We provide evidence that nNOS is also present in the human beta-cell line CM and that the specific inhibitor of nNOS PIN is expressed in CM and INS-1 cells. Furthermore, we investigated the influence of glucose on the activity of nNOS and the expression of PIN and are able to show that both are increased by glucose stimulation in the beta-cell lines but not in the mouse fibroblastic cell line LTK. This indicates that nNOS and PIN play a role in the specific function of beta-cells, not only in rodents but also in humans.