Characterization of Exosomes and Exosomal RNAs Isolated from Post-Mortem Body Fluids for Molecular Forensic Diagnosis.

Exosomes have been mainly studied for their potential applications in biomarker detection and drug delivery for diagnosis and treatment. However, in the field of forensic research, the potential value of exosomes derived from post-mortem body fluids has not been investigated to date. Here, we isolated the exosomes and exosomal RNAs from post-mortem body fluids, including cardiac blood, pericardial fluid, and urine. We also compared commercial exosome isolation kits to determine the optimal method for post-mortem exosome isolation. Transmission electron microscopy (TEM), the Agilent bioanalyzer system, and western blotting were used to evaluate the efficiencies of alternative isolation methods and the characteristics of isolated exosomes. There were no significant differences between exosomes obtained from post-mortem and ante-mortem body fluids in the expression of exosome surface markers or morphology. The exosomes were well-preserved even under simulated post-mortem conditions. Among the isolation procedures tested, the membrane affinity column-based method was the most suitable for post-mortem exosomal RNA isolation. These results suggest that exosomes are well-preserved in post-mortem body fluids and could be utilized for forensic diagnosis.

Virus-mimetic polymer nanoparticles displaying hemagglutinin as an adjuvant-free influenza vaccine.

The generation of virus-mimetic nanoparticles has received much attention in developing a new vaccine for overcoming the limitations of current vaccines. Thus, a method, encompassing most viral features for their size, hydrophobic domain and antigen display, would represent a meaningful direction for the vaccine development. In the present study, a polymer-templated protein nanoball with direction oriented hemagglutinin1 on its surface (H1-NB) was prepared as a new influenza vaccine, exhibiting most of the viral features. Moreover, the concentrations of antigen on the particle surface were controlled, and its effect on immunogenicity was estimated by in vivo studies. Finally, H1-NB efficiently promoted H1-specific immune activation and cross-protective activities, which consequently prevented H1N1 infections in mice.

From the Cover: Ethylmercury-Induced Oxidative and Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death: Involvement of Autophagosome-Lysosome Fusion Arrest.

Ethylmercury (EtHg) is derived from the degradation of thimerosal, the most widely used organomercury compound. In this study, EtHg-induced toxicity and autophagy in the mouse kidney was observed and then the mechanism of toxicity was explored in vitro in HK-2 cells. Low doses of EtHg induced autophagy without causing any histopathological changes in mouse kidneys. However, mice treated with high doses of EtHg exhibited severe focal tubular cell necrosis of the proximal tubules with autophagy. EtHg dose-dependently increased the production of reactive oxygen species, reduced the mitochondrial membrane potential, activated the unfolded protein response, and increased cytosolic Ca2+ levels in HK-2 cells. Cell death induced by EtHg exposure was caused by autophagy and necrosis. N-acetyl cysteine and 4-phenylbutyric acid attenuated EtHg-induced stress and ameliorated the autophagic response in HK-2 cells. Furthermore, EtHg blocked autophagosome fusion with lysosomes, which was demonstrated via treatment with wortmannin and chloroquine. Low doses of EtHg and rapamycin, which resulted in minimal cytotoxicity, increased the levels of the autophagic SNARE complex STX17 (syntaxin 17)-VAMP8-SNAP29 without altering mRNA levels, but high dose of EtHg was cytotoxic. Inhibition of autophagic flux by chloroquin increased autophagosome formation and necrotic cell death in HK-2 cells. Collectively, our results show that EtHg induces autophagy via oxidative and ER stress and blockade of autophagic flux. Autophagy might play a dual role in EtHg-induced renal toxicity, being both protective following treatment with low doses of EtHg and detrimental following treatment with high doses.

The Conserved Phenylalanine in the Transmembrane Domain Enhances Heteromeric Interactions of Syndecans.

The transmembrane domain (TMD) of the syndecans, a family of transmembrane heparin sulfate proteoglycans, is involved in forming homo- and heterodimers and oligomers that transmit signaling events. Recently, we reported that the unique phenylalanine in TMD positively regulates intramolecular interactions of syndecan-2. Besides the unique phenylalanine, syndecan-2 contains a conserved phenylalanine (SDC2-Phe-169) that is present in all syndecan TMDs, but its function has not been determined. We therefore investigated the structural role of SDC2-Phe-169 in syndecan TMDs. Replacement of SDC2-Phe-169 by tyrosine (S2F169Y) did not affect SDS-resistant homodimer formation but significantly reduced SDS-resistant heterodimer formation between syndecan-2 and -4, suggesting that SDC2-Phe-169 is involved in the heterodimerization/oligomerization of syndecans. Similarly, in an in vitro binding assay, a syndecan-2 mutant (S2(F169Y)) showed a significantly reduced interaction with syndecan-4. FRET assays showed that heteromolecular interactions between syndecan-2 and -4 were reduced in HEK293T cells transfected with S2(F169Y) compared with syndecan-2. Moreover, S2(F169Y) reduced downstream reactions mediated by the heterodimerization of syndecan-2 and -4, including Rac activity, cell migration, membrane localization of PKCα, and focal adhesion formation. The conserved phenylalanine in syndecan-1 and -3 also showed heterodimeric interaction with syndecan-2 and -4. Taken together, these findings suggest that the conserved phenylalanine in the TMD of syndecans is crucial in regulating heteromeric interactions of syndecans.