Comparisons of ELISA and Western blot assays for detection of autophagy flux

We analyzed autophagy/mitophagy flux in vitro (C2C12 myotubes) and in vivo (mouse skeletal muscle) following the treatments of autophagy inducers (starvation, rapamycin) and a mitophagy inducer (carbonyl cyanide m-chlorophenylhydrazone, CCCP) using two immunodetection methods, ELISA and Western blotting, and compared their working range, accuracy, and reliability. The ELISAs showed a broader working range than that of the LC3 Western blots (Table 1). Table 2 showed that data value distribution was tighter and the average standard error from the ELISA was much smaller than those of the Western blot, directly relating to the accuracy of the assay. Test-retest reliability analysis showed good reliability for three individual ELISAs (interclass correlation, ≥ 0.7), but poor reliability for three individual Western blots (interclass correlation, ≤ 0.4) (Table 3). Keywords: Autophagy, Mitophagy, ELISA, Western blot, Skeletal muscl

Characterization of Mammalian Selenoprotein O: A Redox-Active Mitochondrial Protein

Selenoproteins exhibit diverse biological functions, most of which are associated with redox control. However, the functions of approximately half of mammalian selenoproteins are not known. One such protein is Selenoprotein O (SelO), the largest mammalian selenoprotein with orthologs found in a wide range of organisms, including bacteria and yeast. Here, we report characterization of mammalian SelO. Expression of this protein could be verified in HEK 293T cells by metabolic labeling of cells with 75Se, and it was abolished when selenocysteine was replaced with serine. A CxxU motif was identified in the C-terminal region of SelO. This protein was reversibly oxidized in a time- and concentration-dependent manner in HEK 293T cells when cells were treated with hydrogen peroxide. This treatment led to the formation of a transient 88 kDa SelO-containing complex. The formation of this complex was enhanced by replacing the CxxU motif with SxxC, but abolished when it was replaced with SxxS, suggesting a redox interaction of SelO with another protein through its Sec residue. SelO was localized to mitochondria and expressed across mouse tissues. Its expression was little affected by selenium deficiency, suggesting it has a high priority for selenium supply. Taken together, these results show that SelO is a redox-active mitochondrial selenoprotein

Ca2+ Signaling Triggered by Shear-Autocrine P2X Receptor Pathway in Rat Atrial Myocytes

Background/Aims: The atrium is exposed to high shear stress during heart failure and valvular diseases. We aimed to understand atrial shear-induced Ca2+ signaling and its underlying mechanisms. Methods: Pressurized micro-flow was applied to single rat atrial myocytes, and Ca2+ signal, membrane potential, and ATP release were assessed using confocal imaging, patch clamp technique, and luciferin-luciferase assay, respectively. Results: Shear stress (∼16 dyn/cm2) induced global Ca2+ waves (∼0.1 events/s) from the periphery to the center of cells in a transverse direction (“T-wave”; ∼145 μm/s). Pharmacological interventions and simultaneous recording of membrane potential and Ca2+ demonstrated that shear-induced T-waves resulted from action potential (AP)-triggered Ca2+ release from the sarcoplasmic reticulum. T-waves were not sensitive to inhibitors of known shear signaling mechanisms except connexin hemichannels and ATP release. Shear stress caused ATP release from these myocytes (∼1.1x10-17 moles/unit membrane, µm2); ATP release was increased by enhancement of connexin hemichannels and suppressed by inhibition of the hemichannels, but not affected by inhibitors of other ATP release pathways. Blockade of P2X receptor, but not pannexin or the Na+-Ca2+ exchanger, eliminated shear-induced T-wave initiation. Conclusion: Our data suggest that shear stress triggers APs and concomitant Ca2+ signaling via activation of P2X receptors by connexin hemichannel-mediated ATP release in atrial myocytes

Eosinophilic myositis in a slaughtered Korean native cattle

Histopathological findings of eosinophilic myositis in the carcass of a slaughtered Korean native cow are presented. Lesions contained massive fibrous septae with vacuolar changes in some lesions, and the hypercontraction and rupturing of muscle bundles, with replacement by eosinophils. Necrosis and severe eosinophil infiltration were observed. Sarcoplasmic fragmentation and atrophy developed. Typical of granuloma, calcified myofibers were focally surrounded by macrophages and numerous inflammatory cells, and multinucleated giant cell formation was evident

Empirical Validation of Heat Transfer Performance Simulation of Graphite/PCM Concrete Materials for Thermally Activated Building System

To increase the heat capacity in lightweight construction materials, a phase change material (PCM) can be introduced to building elements. A thermally activated building system (TABS) with graphite/PCM concrete hollow core slab is suggested as an energy-efficient technology to shift and reduce the peak thermal load in buildings. An evaluation of heat storage and dissipation characteristics of TABS in graphite/PCM concrete has been conducted using dynamic simulations, but empirical validation is necessary to acceptably predict the thermal behavior of graphite/PCM concrete. This study aimed to validate the thermal behavior of graphite/PCM concrete through a three-dimensional transient heat transfer simulation. The simulation results were compared to experimental results from previous studies of concrete and graphite/PCM concrete. The overall thermal behavior for both materials was found to be similar to experiment results. Limitations in the simulation modeling, which included determination of the indoor heat transfer coefficient, assumption of constant thermal conductivity with temperature, and assumption of specimen homogeneity, led to slight differences between the measured and simulated results