Anti-Inflammatory Potential of Newly Synthesized 4-[(Butylsulfinyl)methyl]-1,2-benzenediol in Lipopolysaccharide-Stimulated BV2 Microglia

In this study, we investigated the anti-inflammatory effects of newly synthesized 4-[(butylsulfinyl)methyl]-1,2-benzenediol (SMBD) in lipopolysaccharide (LPS)-stimulated BV2 microglia and the subsequent signaling events. Following stimulation with LPS, elevated production of nitric oxide (NO) and prostaglandin E2 (PGE2) was detected in BV2 cells; however, SMBD pretreatment inhibited the production of NO and PGE2 through suppressing gene expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively, at non-toxic concentrations. LPS-stimulated gene expression and production of interleukin (IL)-1β and tumor necrosis factor (TNF)-α were also significantly reduced by SMBD. The anti-inflammatory effects of SMBD were associated with suppression of LPS-induced nuclear translocation of nuclear factor-kappa B (NF-κB), and phosphorylation of mitogen-activated protein kinases (MAPKs) and Akt, a phosphatidylinositol 3-kinase (PI3K) downstream effector. Therefore, the present results demonstrate that SMBD down-regulates inflammatory gene expression by inhibiting the activation of NF-κB through interference with the activation of MAPKs and PI3K/Akt signaling. Taken together, our data suggest that SMBD may have potential to be developed into an effective anti-inflammatory agent

Identification of Coronavirus Isolated from a Patient in Korea with COVID-19

OBJECTIVES: Following reports of patients with unexplained pneumonia at the end of December 2019 in Wuhan, China, the causative agent was identified as coronavirus (SARS-CoV-2), and the 2019 novel coronavirus disease was named COVID-19 by the World Health Organization. Putative patients with COVID-19 have been identified in South Korea, and attempts have been made to isolate the pathogen from these patients. METHODS: Upper and lower respiratory tract secretion samples from putative patients with COVID-19 were inoculated onto cells to isolate the virus. Full genome sequencing and electron microscopy were used to identify the virus. RESULTS: The virus replicated in Vero cells and cytopathic effects were observed. Full genome sequencing showed that the virus genome exhibited sequence homology of more than 99.9% with SARS-CoV-2 which was isolated from patients from other countries, for instance China. Sequence homology of SARS-CoV-2 with SARS-CoV, and MERS-CoV was 77.5% and 50%, respectively. Coronavirus-specific morphology was observed by electron microscopy in virus-infected Vero cells. CONCLUSION: SARS-CoV-2 was isolated from putative patients with unexplained pneumonia and intermittent coughing and fever. The isolated virus was named BetaCoV/Korea/KCDC03/2020

Silencing of MicroRNA-21 Confers Radio-Sensitivity through Inhibition of the PI3K/AKT Pathway and Enhancing Autophagy in Malignant Glioma Cell Lines

<div><p>Radiation is a core part of therapy for malignant glioma and is often provided following debulking surgery. However, resistance to radiation occurs in most patients, and the underlying molecular mechanisms of radio-resistance are not fully understood. Here, we demonstrated that microRNA 21 (miR-21), a well-known onco-microRNA in malignant glioma, is one of the major players in radio-resistance. Radio-resistance in different malignant glioma cell lines measured by cytotoxic cell survival assay was closely associated with miR-21 expression level. Blocking miR-21 with anti-miR-21 resulted in radio-sensitization of U373 and U87 cells, whereas overexpression of miR-21 lead to a decrease in radio-sensitivity of LN18 and LN428 cells. Anti-miR-21 sustained γ-H2AX DNA foci formation, which is an indicator of double-strand DNA damage, up to 24 hours and suppressed phospho-Akt (ser473) expression after exposure to γ-irradiation. In a cell cycle analysis, a significant increase in the G₂/M phase transition by anti-miR-21 was observed at 48 hours after irradiation. Interestingly, our results showed that anti-miR-21 increased factors associated with autophagosome formation and autophagy activity, which was measured by acid vesicular organelles, LC3 protein expression, and the percentage of GFP-LC3 positive cells. Furthermore, augmented autophagy by anti-miR-21 resulted in an increase in the apoptotic population after irradiation. Our results show that miR-21 is a pivotal molecule for circumventing radiation-induced cell death in malignant glioma cells through the regulation of autophagy and provide a novel phenomenon for the acquisition of radio-resistance.</p> </div

The radio-sensitivity of glioma cells according to the modulation of microRNA 21 (miR-21).

<p>Anti-miR-21 (100 nM) or negative control (100 nM) was transiently transfected into U373 and U87 cells for loss-of-function. miR-21 (1 µg) or control plasmid (1 µg) was transiently transfected into LN18 and LN 428 cells for gain-of-function. The cells were plated onto 96 well microplates and incubated for a further 48 hours prior to γ-irradiation. After exposure to the indicated level of ionizing irradiation, the surviving cell fraction was measured using a sulforhodamin-B (SRB) assay at 5 days, and the effect of miR-21 modulation is illustrated for each cell line (A–F). Each error bar indicates the standard deviation of three independent experiments.</p

Cell cycle change after γ-irradiation in anti-microRNA 21 (miR-21)-treated U373 cells.

<p>(A) An illustrative case showing cell cycle changes 48 hours after exposure to 8 Gy radiation. (B) Statistical analysis of independent triplicate experiments showed significant differences between the negative-control and the antimiR-21 treated cells in terms of the G₂/M population (*p<0.05), but the subG₁ population representing apoptosis failed to reach statistical significance.</p

Suppression of γ-irradiation-induced phospho-Akt upregulation by antimicroRNA 21 (miR-21) in glioma cells.

<p>(A) Phospho-Akt (ser 473) expression was measured by Western blot at the indicated time intervals after γ-irradiation in U373 cells. Phospho-Akt (ser 473) expression at 4 hours after γ-irradiation showed a dose-dependent increase in the negative-control treated group and suppression in the anti-miR-21-treated U373 and U87 cells in both the Western blot (B) and the Image software analysis of their intensities (C and D, respectively). Each bar on a column represents the standard error of the mean for triplicate experiments.</p

Endogenous and radiation-induced microRNA 21 (miR-21) expression.

<p>(A) Northern blotting of endogenous miR-21 in U373, U87, and LN18 glioma cell lines without irradiation. (B) After exposure to the indicated dose (8 Gy) of γ-irradiation, radiation-induced increased miR-21 expression was observed in both phosphatase and tensin homolog (PTEN) wild type and deficit glioma cell lines. Real-time PCR data is plotted as relative expression compared to the endogenous level of each control. (C) Cell survival curve after irradiation shows that the observed radiosensitivity reversely correlated with miR-21 expression measured previously. (D) The relative expression of miR-21 after γ-irradiation in U373 and U87 cells is illustrated by real-time PCR data and shows a dose-dependent increase. Each error bar indicates the standard deviation of three independent experiments.</p

Measurement of autophagy and early apoptosis (Annexin-V) in anti-microRNA 21 (miR-21)-treated U373 cells.

<p>(A) A representative illustration showing the flow cytometry measurement of acidic vesicular organelles (AVOs) by acridine orange staining, representing autophagic activity 48 hours after exposure to 8 Gy of radiation. (B) Statistical analysis of independent triplicate values of the percentage of cells harboring AVO resulted in significant differences between the negative-control and anti-miR-21-treated cells (*P<0.05). (C) Western blot of the LC-3 protein, in which the appearance of the lower 1b band represents autophagosome-lysosome fusion. (D) Green fluorescent protein-labeled LC-3 protein (GFP-LC3) was stably over-expressed in U373 cells using a lentiviral vector, and GFP-LC3 granule-positive cells after irradiation (white arrow) were counted using a fluorescence microscope (10×20 high power fields; HPF). (E) The number of GFP-LC3 granule positive cells was counted as a percentage of total observed cells. Statistical analysis of the average percentage of ten HPFs showed significant differences (*P<0.05). (F) The flow cytometry measurements of Annexin-V staining representing apoptotic indices. The increased early apoptotic index 48 hours after 8 Gy of irradiation in anti-miR-21-treated U373 cells compared to that in negative control-treated cells (**p<0.01). Augmented apoptosis by transfection of anti-mir21 was neutralized by a 48 hour incubation with 10 mM 3-MA, an autophagy inhibitor. Each bar on a column represents the standard error of the mean for triplicate experiments.</p