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DNA methylation analysis of malignant and normal uveal melanocytes reveals novel molecular features of uveal melanoma

ABSTRACT: In order to address epigenetic regulation, NM and UM models were analyzed through OxBS sequencing pipeline (Cambridge Epigenetics). Whole genome DNA methylation analysis was carried out with a Cambridge Epigenetics kit (TrueMethyl kit) that corresponds to an oxidative bisulfite reaction, to identify and analyze only 5-methylcytosine (5-mC). This modification of bisulfite sequencing was found to be more robust in our samples, particularly for the normal melanocytes, most probably due to the abundance of melanin

ORGANISM(S): Homo sapiens

PROVIDER: GSE200316 | GEO | 2023/08/21

REPOSITORIES: GEO

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Project description:A. flavus keratitis is one of the predominant fungal infections affecting the tropical parts of the world. Melanin is a virulence factor in numerous pathogenic fungi and the melanin layer maintains the cell wall structure and provides chemical and physical protection to the organism. However, the molecular and biological mechanisms modulating melanin-mediated host-pathogen interaction in A. flavus keratitis are not well understood. This work aimed to compare the morphology, surface proteome profile and virulence of melanized and non-melanized conidia of A. flavus. An environmental isolate and two clinical isolates were included in this study. L-DOPA melanin pathway-specific inhibitor kojic acid (KA) as used to prepare non-melanized conidia. Conidial surface proteins were extracted using 100% formic acid at 0 ºC and the extracted proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Formic acid extracts were analyzed in an Orbitrap Velos pro-mass spectrometer. Conidial surface morphology difference was examined using scanning electron microscopy (SEM). Furthermore, lactophenol and calcofluor staining were performed to check the morphology and staining efficiency of melanized conidia (MC) and non-melanized conidia (NMC). A Galleria mellonella insect model was used to examine the virulence efficiency of MC and NMC. Kojic acid treatment inhibited melanin synthesis in A. flavus and the conidial surface protein profile was significantly different in kojic acid-treated non-melanized conidia. Several cell wall-associated proteins and proteins responsible for oxidative stress, carbohydrate, and chitin metabolic pathway were found only in the formic acid extracts of NMC. SEM analysis showed the conidial surface morphology difference between the NMC and MC indicating the role of melanin in the structural integrity of the conidial cell wall. The levels of calcofluor staining efficiency were different, but there was no microscopic morphology difference in lactophenol cotton blue staining of MC and NMC. Evaluation of virulence of MC and NMC in G. mellonella model showed NMC was less virulent compared to MC . Our results showed that the integrity of the conidial surface is controlled by the melanin layer. The alteration in the surface protein profile indicated that many surface proteins are masked by the melanin layer and hence melanin can modulate the host response by preventing the exposure of fungal proteins to the host immune defense system. G. mellonella in vivo virulence assay also confirmed that the non-melanized conidia were susceptible to host defense as in other Aspergillus pathogens.

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DNA methylation analysis of malignant and normal uveal melanocytes reveals novel molecular features of uveal melanoma

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