Background Sepsis has a high mortality rate, but no specific drug has been proven effective, prompting the development of new drugs. Immunologically, sepsis can involve hyperinflammation, immune paralysis, or both, which might pose challenges during drug development. Recently, mitochondrial transplantation has emerged as a treatment modality for various diseases involving mitochondrial dysfunction, but it has never been tested for sepsis. Methods We isolated mitochondria from L6 muscle cells and umbilical cord mesenchymal stem cells and tested the quality of the isolated mitochondria. We conducted both in vivo and in vitro sepsis studies. We investigated the effects of intravenous mitochondrial transplantation on cecal slurry model in rats in terms of survival rate, bacterial clearance rate, and the immune response. Furthermore, we observed the effects of mitochondrial transplantation on the immune reaction regarding both hyperinflammation and immune paralysis. To do this, we studied early- and late-phase cytokine production in spleens from cecal slurry model in rats. We also used a lipopolysaccharide (LPS)-stimulated human PBMC monocyte model to confirm the immunological effects of mitochondrial transplantation. Apoptosis and the intrinsic apoptotic pathway were investigated in septic spleens. Results Mitochondrial transplantation improved survival and bacterial clearance. It also mitigated mitochondrial dysfunction and apoptosis in septic spleens and attenuated both hyperinflammation and immune paralysis in the spleens of cecal slurry model in rats. This effect was confirmed with an LPS-stimulated human PBMC study. Conclusions In rat polymicrobial cecal slurry model, the outcome is improved by mitochondrial transplantation, which might have an immunomodulatory effect.
Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer. Besides genetic and environmental factors, epigenetic alterations contribute to the tumorigenesis of NSCLC. Epigenetic changes are considered key drivers of cancer initiation and progression, and altered expression and activity of epigenetic modifiers reshape the epigenetic landscape in cancer cells. Euchromatic histone-lysine N-methyltransferase 2 (EHMT2) is a histone methyltransferase and catalyzes monoand di-methylation at histone H3 lysine 9 (H3K9me1 and H3K9me2, respectively), leading to gene silencing. EHMT2 overexpression has been reported in various types of cancer, including ovarian cancer and neuroblastoma, in relation to cell proliferation and metastasis. However, its role in NSCLC is not fully understood. In this study, we showed that EHMT2 gene expression was higher in NSCLC than normal lung tissue based on publicly available data. Inhibition of EHMT2 by BIX01294 (BIX) reduced cell viability of NSCLC cell lines via induction of autophagy. Through RNA sequencing analysis, we found that EHMT2 inhibition significantly affected the cholesterol biosynthesis pathway. BIX treatment directly induced the expression of SREBF2, which is a master regulator of cholesterol biosynthesis, by lowering H3K9me1 and H3K9me2 at the promoter. Treatment of a cholesterol biosynthesis inhibitor, 25-hydroxycholesterol (25-HC), partially recovered BIX-induced cell death by attenuating autophagy. Our data demonstrated that EHMT2 inhibition effectively induced cell death in NSCLC cells through altering cholesterol metabolism-dependent autophagy.
ABSTRACT:We studied the preparation of antimicrobial silicone rubbers of improved interfacial strength, which could be formed with the ion beam assisted deposition (IBAD) technique for coating metallic or inorganic materials (silver (Ag), Copper (Cu), and Hydroxyapatite(HAp)/TiO 2 ) on the silicone surface. Those coating materials provide high product safety as well as outstanding antimicrobial activity. The deposition methodology is composed of pre-etching with oxygen gas, vaporizing the coating materials, and posttreatment with Ar ion. With the evaporation of the coating materials, the Ar beam was focused on the substrate to assist deposition. It was found out that the ion assisting depositions in the IBAD process give a prominent enhancement in adhesion between silicone rubbers and coatings of Ag and Cu. The HAp/TiO 2 coating layer was easily dissolved in aqueous saline solution. All deposited layers display high antimicrobial activities against Staphlococcus aureus (ATCC 6538) and Escherichia coil (ATCC 25,922), showing 99.9% reduction of bacteria, respectively. In a cytotoxicity test, the Ag and HAp/TiO 2 coated silicone shows a decrease of cytotoxicity, while the Cu coating leads to a slight increase of cytotoxicity. The result on the surface modifications of silicone rubber will be employed in further study for applications of medical or rehabilitation devices.
Objectives G9A is a eukaryotic histone methyltransferase that contributes to transcriptional silencing of tumor suppressor genes by modulating histone 3 lysine 9 methylation (H3K9m). It has been recently suggested as a promising therapeutic target for breast cancer and neuroblastoma. This study was aimed to investigate the effect of G9A inhibition and its cellular metabolic mechanisms. Methods Using OncomineTM, G9A overexpression in lung cancer was assessed. Cell viability upon treatment of G9A inhibitor (BIX01294, BIX) and siG9A was measured by MTT and IncuCyteR assays. Additionally, apoptosis and autophagy were analyzed through western blots. In order to identify targets, transcriptomes using RNA sequencing was conducted upon BIX treatment. Further functional relevance of targets was validated using Chromatin IP and recovery tests. Results BIX-mediated inhibition of G9A reduced cell viability of lung cancer cells via induction of autophagy. Through RNA sequencing, we found that G9A inhibition mainly affected cholesterol biosynthesis pathway. BIX directly induced the expression of SREBF2 gene, by lowering H3K9me1 and H3K9me2 at the promoter. A cholesterol biosynthesis inhibitor, 25-HC, partially recovered BIX-induced cell death by attenuating autophagy. Our data suggests that cholesterol metabolism can be a potential therapeutic target by G9A inhibition and its induction of autophagic cell death. Conclusions Our data suggests that cholesterol metabolism can be a potential therapeutic target by G9A inhibition and its induction of autophagic cell death. Funding Sources NRF of Korea grant (2018R1D1A1B07051274); BK21 Plus Project (22A20130012143).
Objectives N6-Methyladenosine (m6A) is the most prevalent mRNA modification, which modulates mRNA export, splicing, and stability. Recent studies showed that m6A is involved in cell differentiation such as spermatogenesis, but the underlying mechanism is still unclear. This study was aimed to investigate whether alkylation repair homolog 5 (ALKBH5)-dependent m6A demethylation plays a role in metabolic-related cell differentiation. Methods Expression analysis upon differentiation was done using R studio on publicly available data from two independent cell differentiation models, adipogenic 3T3-L1 and myogenic C2C12. M6A levels in mRNA were measured using dot blot. Knockdown of ALKBH5 was conducted by small interference RNA at two days prior to differentiation induction. For phenotypic measures upon differentiation, staining analysis by Giemsa and Oil Red-O was conducted for 3T3-L1 and C2C12 cells, respectively, followed by histogram analysis using ImageJ. Total RNA was isolated from cells and was reverse-transcribed to cDNA, followed by qRT-PCR for gene expression analysis. Results Comparative analysis of adipogenesis and myogenesis models revealed common pathways, including oxidative phosphorylation. Among a-ketoglutarate-dependent demethylases, Alkbh5 expression was substantial and its substrate, m6A level was changed upon induction of differentiation. Staining analysis showed that Alkbh5 knockdown using siRNA significantly increased production of triglycerides and myotubes in 3T3-L1 and C2C12, respectively, indicating promotion of cell differentiation. To identify putative targets, expression data using microarray and qRT-PCR were analyzed. Expression of CEBPb and myogenin among adipogenic and myogenic markers was significantly altered upon Alkbh5 knockdown in 3T3-L1 and C2C12, respectively. Conclusions The results together showed that ALKBH repression promoted cell differentiation in both adipogenic and myogenic models, possibly regulating early differentiating markers such as CEBPb and myogenin, respectively. Funding Sources NRF of Korea (2018R1D1A1B07051274 & 22A20130012143).
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