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RNA 5-methylcytosine (m5C) is a widespread post-transcriptional modification involved in diverse biological processes through controlling RNA metabolism. However, its roles in uveal melanoma (UM) remain unknown. Here, we describe the biological roles and regulatory mechanisms of RNA m5C in UM. Initially, we identified significantly elevated global RNA m5C levels in both UM cells and tissue specimens using ELISA assay and dot blot analysis. Meanwhile, NOP2/Sun RNA methyltransferase family member 2 (NSUN2) was upregulated in both types of these samples, whereas NSUN2 knockdown significantly decreased RNA m5C level. Such declines inhibited UM cell migration and suppressed cell proliferation through cell cycle G1 arrest. Furthermore, bioinformatic analyses, m5C-RIP-qPCR, and luciferase assay identified β-Catenin (CTNNB1) as a direct target of NSUN2-mediated m5C modification in UM cells. Additionally, overexpression of miR-124a in UM cells diminished NSUN2 expression levels indicating that it is an upstream regulator of this response. Our study suggests that NSUN2-mediated RNA m5C methylation provides a potential novel target to improve the therapeutic management of UM pathogenesis. [ABSTRACT FROM AUTHOR]

Background: To compare outcomes of femtosecond laser-assisted deep anterior lamellar keratoplasty (FSL-DALK) and penetrating keratoplasty (FSL-PK) for the treatment of keratoconus.Methods: Twenty eight eyes underwent FSL-DALK (consisted of 12 eyes in the FSL-DALKa subgroup without baring the Descemet's membrane and 16 eyes in the FSL-DALKb subgroup baring the Descemet's membrane using big-bubble technique) were compared with 12 eyes that underwent FSL-PK for keratoconus. These patients underwent an ophthalmic examination preoperatively and 3, 6, 9, and 12 months postoperatively.Results: The postoperative BCVA in the FSL-PK group, and the FSL-DALKb subgroup were significantly better than that in the FSL-DALKa subgroup (P < 0.05), whereas no differences were found between the FSL-DALKb subgroup and the FSL-PK group (P > 0.05). There were no significant differences in the mean spherical equivalent (SE) and astigmatism between the FSL-DALK and the FSL-PK groups, nor between the subgroups of FSL-DALK during the follow-up period (P > 0.05). At the last follow-up, the mean endothelial cell loss in the FSL-DALK group (9.12 %) was significantly less than that in the FSL-PK group (20.79 %) (P < 0.001), while there was no difference between the FSL-DALKa (9.15 %) and the FSL-DALKb (9.10 %) subgroups (P = 0.15). The FSL-DALK group seemed to have fewer graft rejections (1/28 cases) than the FSL-PK group (2/12 cases), although Kaplan-Meier curve showed no significant difference between the two groups (P = 0.144).Conclusions: In this retrospective study, the results suggested that FSL-DALKb gives better visual outcome, and FSL-DALKb is a better option for keratoconus whose endothelium is not compromised. However, larger and prospective studies are further required. [ABSTRACT FROM AUTHOR]

RNA 5-methylcytosine (m 5 C) is a widespread post-transcriptional modification involved in diverse biological processes through controlling RNA metabolism. However, its roles in uveal melanoma (UM) remain unknown. Here, we describe the biological roles and regulatory mechanisms of RNA m 5 C in UM. Initially, we identified significantly elevated global RNA m 5 C levels in both UM cells and tissue specimens using ELISA assay and dot blot analysis. Meanwhile, NOP2/Sun RNA methyltransferase family member 2 (NSUN2) was upregulated in both types of these samples, whereas NSUN2 knockdown significantly decreased RNA m 5 C level. Such declines inhibited UM cell migration and suppressed cell proliferation through cell cycle G1 arrest. Furthermore, bioinformatic analyses, m 5 C-RIP-qPCR, and luciferase assay identified β-Catenin (CTNNB1) as a direct target of NSUN2-mediated m 5 C modification in UM cells. Additionally, overexpression of miR-124a in UM cells diminished NSUN2 expression levels indicating that it is an upstream regulator of this response. Our study suggests that NSUN2-mediated RNA m 5 C methylation provides a potential novel target to improve the therapeutic management of UM pathogenesis.

Aim: To test our hypothesis that activation of protein kinase A (PKA) signal pathway by β-adrenergic agonist plays an important role in the protecting of cultured retinal pigment epithelial (RPE) cells against the hydroxychloroquine (HCQ) toxicity., Methods: Cultured human RPE cells were treated with 1) HCQ, 2) HCQ with salbutamol (a β2-adrenergic receptor agonist), and 3) HCQ with salbutamol and a PKA inhibitor, and compared these to 4) untreated cells (controls). After treated for 24h, cell vacuolation, cells viability, PKA and PKA kinase activity levels were determined by the measurement of the size of vacuoles using Image J software, the cell counting with a dye-exclusion testing, Western blot and PKA kinase detection, respectively., Results: Cell vacuolation and cell death of cultured RPE cells were significantly increased by the treatment of HCQ. Salbutamol significantly elevated PKA and PKA activity levels and this was associated with the inhibition of the vacuolation and cell death. The PKA inhibitor significantly decreased the PKA levels and eliminated the protective effects of salbutamol on HCQ-treated RPE cells., Conclusion: The PKA pathway plays an important role in the protective effects of β2-adrenergic agonist on the RPE cells against HCQ toxicity. These findings reveal a novel potential strategy against HCQ retinopathy by treatment with PKA activating medications., (International Journal of Ophthalmology Press.)

Aim: To study the effects of curcumin on the secretion of interleukin (IL)-6 and IL-8 by corneal limbus epithelial cells., Methods: Human corneal limbus epithelial cells were isolated and cultured from donor eyes and irradiated by UVB at different dosages with or without curcumin. MTT test was used for studying the effects of UVB and curcumin on the cell viability. The role of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) pathways on the UVB-induced secretion of IL-6 and IL-8 were tested by addition of their inhibitors to the culture with or without UVB-radiation. Levels of various signal pathways, IL-6 and IL-8 in the cells and in the conditioned culture medium were measured by ELISA analysis., Results: UVB at 20 mJ/cm 2 or less and curcumin at 20 µmol/L or less did not affect the cell viability of cultured limbus epithelial cells ( P >0.05). UVB irradiation at 10 and 20 mJ/cm 2 induced a significant increase of secretion of IL-6 and IL-8 and upregulated NF-κB and phosphorylated MAPK pathways of cultured limbus epithelial cells ( P <0.05). Various signal pathway inhibitors, including SP600125 (JNK inhibitor), SB203580 (p38 MAPK inhibitor) and BAY11-7082 (NF-κB inhibitor) significantly decreased the UVB-induced secretion of IL-6 and IL-8 secretion ( P <0.05). Curcumin at 5-20 µmol/L significantly inhibited UVB-induced secretion of IL-6 and IL-8 by limbus epithelial cells in a dose-dependent manner; while curcumin alone did not affect the secretion of IL-6 and IL-8. The upregulation of NF-κB and MAPK pathways induced by UVB treatment was significantly inhibited by curcumin, suggesting that NF-κB and MAPK pathways are involved in the inhibitory effect of curcumin on UVB-induced production of IL-6 and IL-8., Conclusion: Curcumin may be a promising agent to be explored for the prevention and treatment of pterygium.

Aim: To examine the genetic profile of primary uveal melanoma (UM) as compared to UM in immune escape., Methods: Dendritic cells (DC) loaded with lysates of UM cells of high metastatic potential were used to stimulate CTLs(CTLs). When CTLs co-cultured with the UM cells, most UM cells could be eliminated. Survival UM cells grew slowly and were considered to be survival variants and examined by a microarray analysis. These differential genes were analyzed further with Venn Diagrams and functions related to immune escape. We additionally examined transcriptional changes of manually selected survival variants of UM cells and of clinical UM samples by quantitative real-time polymerase chain reaction (qRT-PCR), and analyzed the correlation of these expressions and patients' survival., Results: Gene expression analyses revealed a marked up-regulation of SLAMF7 and CCL22 and a significant down-regulation of KRT10 , FXYD3 and ABCC2 . The expression of these genes in the relapsed UM was significantly greater than those in primary UM. UM patients with overexpression of these genes had a shorter survival period as compared with those of their underexpression., Conclusion: Gene expression, in particular of SLAMF7, CCL22, KRT10, FXYD3 and ABCC2 , differed between primary UM cells and survival variants of UM cells.

Purpose: Recently, we reported finding that circulating melatonin levels in age-related macular degeneration patients were significantly lower than those in age-matched controls. The purpose of this study was to investigate the hypothesis that melatonin deficiency may play a role in the oxidative damage of the retinal pigment epithelium (RPE) by testing the protective effect of melatonin and its receptor antagonist on RPE cells exposed to H(2)O(2) damage., Methods: Cultured human RPE cells were subjected to oxidative stress induced by 0.5 mM H(2)O(2). Cell viability was measured using the microculture tetrazoline test (MTT) assay. Cells were pretreated with or without melatonin for 24 h. Luzindole (50 μM), a melatonin membrane-receptor antagonist, was added to the culture 1 h before melatonin to distinguish direct antioxidant effects from indirect receptor-dependent effects. All tests were performed in triplicate., Results: H(2)O(2) at 0.5 mM decreased cell viability to 20% of control levels. Melatonin showed dose-dependent protective effects on RPE cells against H(2)O(2). Cell viability of RPE cells pretreated with 10(-10), 10(-8), 10(-6), and 10(-4) M melatonin for 24 h was 130%, 160%, 187%, and 230% of cells treated with H(2)O(2) alone (all p<0.05). Using cells cultured without H(2)O(2) as the control, cell viability of cells treated with H(2)O(2) after pretreatment with 10(-10)-10(-4) M melatonin was still significantly lower than that of the controls, suggesting that melatonin significantly decreased but did not completely abolish the in vitro cytotoxic effects of H(2)O(2). Luzindole completely blocked melatonin's protective effects at low concentrations of melatonin (10(-10)-10(-8) M) but not at high concentrations (10(-6)-10(-4) M)., Conclusions: Melatonin has a partial protective effect on RPE cells against H(2)O(2) damage across a wide range of concentrations (10(-10)-10(-4) M). This protective effect occurs through the activation of melatonin membrane receptors at low concentrations (10(-10)-10(-8) M) and through both the direct antioxidant and indirect receptor activation effects at high concentrations (10(-6)-10(-4) M).