The present study was set out to address the therapeutic efficacy of human adipose tissue stem cells derived extracellular vesicles (hADSC-Evs) in a mouse model of dry eye disease and to investigate the underlying mechanisms involved. hADSC-Evs eye drops were topically administered to mice that subjected to desiccating stress (DS). Clinical parameters of ocular surface damage were assessed with fluorescein staining, tear production and PAS staining. For in vitro studies, cell viability assay and TUNEL staining were performed in human corneal epithelial cells (HCECs) treated with hADSC-Evs under hyperosmotic media. In addition, immunofluorescent staining, Real-time PCR (qRT-PCR) and Western blots were used to evaluated NLRP3, ASC, caspase-1, and IL-1β expression levels. Compared with vehicle control mice, topical hADSC-Evs treated mice showed decreased corneal epithelial defects, increased tear production, decreased goblet cell loss, as well as reduced inflammatory cytokines production. In vitro, hADSC-Evs could protect HCECs against hyperosmotic stress-induced cell apoptosis. Mechanistically, hADSC-Evs treatment suppressed the DS induced rises in NLRP3 inflammasome formation, caspase-1 activation and IL-1β maturation. In conclusion, hADSC-Evs eye drops effectively suppress NLRP3 inflammatory response and alleviate ocular surface damage in dry eye disease. Dry eye disease (DED) is a highly prevalent ocular surface disorder in the world 1. It is estimated that more than 16 million adults are diagnosed DED in US, and the prevalence in Asia is even higher than in western countries 2,3. According to the reports of Dry Eye Workshop (DEWS II), DED is a multifactorial disease that characterized by loss of homeostasis of the tear film 4. The tear film instability causes symptoms of discomfort, itching, eye irritation, glare and blurry vision, leading to a reduction in quality of life. Although the pathogenesis of DED is not yet fully understood, mounting evidence showed that the "vicious cycle of inflammation", including tear film instability, tear hyperosmolarity, apoptosis of cornea/conjunctiva and elevated levels of pro-inflammatory cytokines, play a core driver in its initiation and progression 5,6. Accordingly, most of the treatments to date are focused on reducing inflammation and restoring normal tear film 7. Mesenchymal stem cells (MSCs) are self-renewing multipotent stromal cells that can be isolated from mesenchymal tissues such as bone marrow, adipose, umbilical cord, as well as other tissues 8. Due to their immunomodulatory and trophic characteristics, MSC-based therapeutic intervention has been explored in a variety of immune-mediated disorders, including dry eye disease 9-12. Although most of the results were promising, safety issues regarding MSC-based therapy are still a matter of concern. Extracellular vesicles, with nanosized diameter of 30-150 nm, are known as intercellular communication mediators by transferring various bioactive molecules (proteins, lipids and RNAs) 13. Recent studies revealed that MS...

Expression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) can be induced by estrogens at the posttranscriptional level. However, the molecular mechanism of the process is unclear. In this study, we found that the C terminus (CT) of PTEN is indispensable for 17-β-estradiol (E2)-increased PTEN expression. Therefore, we screened for PTEN-CT-associated proteins using a glutathione-S-transferase pull-down approach in combination with mass spectrometry-based proteomic analyses. Our experiments led to the identification of Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) as a major PTEN-CT binding partner. The first postsynaptic density protein-95/Discslarge/zonula occludens-1 homology domain of NHERF1 and the last four amino acids of PTEN were found to be key determinants of this interaction. By associating with PTEN, NHERF1 could enhance PTEN protein expression by retention of PTEN turnover, as demonstrated by NHERF1 overexpression and small interfering RNA-mediated knockdown experiments, respectively. Furthermore, NHERF1 inhibited ubiquitination of the PTEN protein upon competition with binding of PTEN to neural precursor cell expressed, developmentally down-regulated 4, an ubiquitin E3 ligase. E2 strongly induced the expression of NHERF1 and PTEN only in estrogen receptor (ER)-positive cells but not in ER-negative cells. ICI182780, an ER-specific inhibitor, decreased the expression of both NHERF1 and PTEN, and ICI182780 pretreatment also retarded E2-increased PTEN expression in ER-MDA-MB-231 cells. In both ER-MDA-MB-231 and MCF-7 cells, E2 failed to increase PTEN expression when NHERF1 was knocked down. Taken together, these are the first results that present a possible mechanism for E2-increased PTEN expression. In this process, E2 first induces NHERF1 expression by activating the ER. Upon competition with neural precursor cell expressed, developmentally down-regulated 4, NHERF1 then interacts with PTEN to inhibit PTEN degradation, through an ubiquitination-dependent pathway. This in turn leads to the increase of PTEN expression at the protein level.

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We have previously reported that baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) late expression factor 11 (lef-11) is associated with viral DNA replication and have demonstrated that it potentially interacts with itself; however, whether LEF-11 forms oligomers and the impact of LEF-11 oligomerization on viral function have not been substantiated. In this study, we first demonstrated that LEF-11 is capable of forming oligomers. Additionally, a series of analyses using BmNPV LEF-11 truncation mutants indicated that two distinct domains control LEF-11 oligomerization (aa 42–61 and aa 72–101). LEF-11 truncation constructs were inserted into a lef-11-knockout BmNPV bacmid, which was used to demonstrate that truncated LEF-11 lacking either oligomerization domain abrogates viral DNA replication. Finally, site-directed mutagenesis was used to determine that the conserved hydrophobic residues Y58&I59 (representing Y58 and I59), I85 and L88&L89 (representing L88 and L89) are required for LEF-11 oligomerization and viral DNA replication. Collectively, these data indicate that BmNPV LEF-11 oligomerization influences viral DNA replication.

To study the effects of nicotine on retinal pigment epithelium (RPE) cells in vivo and in vitro, (Balb/cÂC57Bl/6) F1 mice were given water containing 100 mg/mL nicotine for six months. Cultured fetal RPE cells were treated with nicotine or lipopolysaccharide for seventy-two hours. Expression of matrix metalloproteinase protein (MMP)2, MMP9, and VEGF was measured using Western blot. Expression of IL6 and IL8 was measured using real-time polymerase chain reaction or enzyme-linked immunosorbent assay. Electron microscopy was performed to observe the effects of nicotine on morphological changes of mice retina or cultured RPE cells, and filamentous actin in RPE cells was stained with phalloidin. Electron microscopy revealed that nicotine-treated mice showed thinner outer nuclear layers, fewer pigment granules in RPE cells, and a damaged photoreceptor-RPE interface when compared with age-control mice. When added to cultured RPE cells, nicotine induced accumulation of osmiophilic lamellated intracellular inclusions in cytoplasm, mitochondrion hypertrophy and vacuolar degeneration, and redistribution of actin in cells without affecting cell proliferation. Expression of MMP2 and MMP9 in nicotine-treated RPE cells was decreased. Nicotine-induced changes in RPE morphology and function provide insight into pathogenesis of smoking-related retinal diseases.

-2,4-Dichlorophenol (2,4-DCP) is an environmental pollutant exhibiting a wide spectrum of toxic effects. We investigated the toxic effects and potential mechanisms underlying 2,4-DCP-induced hepatotoxicity. In vitro, 2,4-DCP caused hepatotoxicity manifested by a decrease in cell viability and inhibition of colony formation. Bip and CHOP expression was up-regulated at the mRNA and protein levels. Moreover, 2,4-DCP induced eIF2α phosphorylation and Xbp1 mRNA splicing, indicating that endoplasmic reticulum (ER) stress was activated after exposure of HL7702 cells to 2,4-DCP for 12 hr. Furthermore, the mitochondrial membrane potential collapsed and apoptosis was triggered after exposure to 2,4-DCP for 24 hr. In vivo, 2,4-DCP caused histological changes in the liver, and dramatically elevated the serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels of mice. ER stress was also triggered in the liver of mice on days 1 and 3. The ER stress inhibitor TUDCA could partly relieve the liver damage, as indicated by the restoration of serum ALT and AST levels. Taken together, our results demonstrated that ER stress may serve as an early warning mechanism against 2,4-DCP-induced hepatotoxicity, and severe ER stress may lead to apoptosis.

SynopsisHistone H3 lysine 9 dimethylation (H3K9me2) hypermethylation is thought to be a major influential factor in cellular reprogramming, such as somatic cell nuclear transfer (SCNT) and induction of pluripotent stem cells (iPSCs). The diazepin-quinazolin-amine derivative (BIX-01294) specifically inhibits the activity of histone methyltransferase EHMT2 (euchromatic histone-lysine N-methyltransferase 2) and reduces H3K9me2 levels in cells. The imprinted gene small nuclear ribonucleoprotein N (Snrpn) is of particular interest because of its important biological functions. The objective of the present study was to investigate the effect of BIX-01294 on H3K9me2 levels and changes in Snrpn DNA methylation and histone H3K9me2 in mouse embryonic fibroblasts (MEFs). Results showed that 1.3 μM BIX-01294 markedly reduced global levels of H3K9me2 with almost no cellular toxicity. There was a significant decrease in H3K9me2 in promoter regions of the Snrpn gene after BIX-01294 treatment. A significant increase in methylation of the Snrpn differentially methylated region 1 (DMR1) and slightly decreased transcript levels of Snrpn were found in BIX-01294-treated MEFs. These results suggest that BIX-01294 may reduce global levels of H3K9me2 and affect epigenetic modifications of Snrpn in MEFs.

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