Non-invasive Detection of Unique Molecular Signatures in Laser-Induced Retinal Injuries.

Unintentional laser exposure is an increasing concern in many operational environments. Determining whether a laser exposure event caused a retinal injury currently requires medical expertise and specialized equipment that are not always readily available. The purpose of this study is to test the feasibility of using dynamic light scattering (DLS) to non-invasively detect laser retinal injuries through interrogation of the vitreous humor (VH). Three grades of retinal laser lesions were studied: mild (minimally visible lesions), moderate (Grade II), and severe (Grade III). A pre-post-treatment design was used to collect DLS measurements in vivo at various time points, using a customized instrument. VH samples were analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) and relative protein abundances were determined by spectral counting. DLS signal analysis revealed significant changes in particle diameter and intensity in laser-treated groups as compared with control. Differences in protein profile in the VH of the laser-treated eyes were noted when compared with control. These results suggest that laser injury to the retina induces upregulation of proteins that diffuse into the VH from the damaged tissue, which can be detected non-invasively using DLS.

MicroRNA profiling in intraocular medulloepitheliomas.

PURPOSE: To study the differential expression of microRNA (miRNA) profiles between intraocular medulloepithelioma (ME) and normal control tissue (CT). MATERIAL AND METHODS: Total RNA was extracted from formalin fixed paraffin embedded (FFPE) intraocular ME (n=7) and from age matched ciliary body controls (n=8). The clinical history and phenotype was recorded. MiRNA profiles were determined using the Affymetrix GeneChip miRNA Arrays analyzed using expression console 1.3 software. Validation of significantly dysregulated miRNA was confirmed by quantitative real-time PCR. The web-based DNA Intelligent Analysis (DIANA)-miRPath v2.0 was used to perform enrichment analysis of differentially expressed (DE) miRNA gene targets in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. RESULTS: The pathologic evaluation revealed one benign (benign non-teratoid, n=1) and six malignant tumors (malignant teratoid, n=2; malignant non-teratoid, n = 4). A total of 88 miRNAs were upregulated and 43 miRNAs were downregulated significantly (P<0.05) in the tumor specimens. Many of these significantly dysregulated miRNAs were known to play various roles in carcinogenesis and tumor behavior. RT-PCR validated three significantly upregulated miRNAs and three significantly downregulated miRNAs namely miR-217, miR-216a, miR-216b, miR-146a, miR-509-3p and miR-211. Many DE miRNAs that were significant in ME tumors showed dysregulation in retinoblastoma, glioblastoma, and precursor, normal and reactive human cartilage. Enriched pathway analysis suggested a significant association of upregulated miRNAs with 15 pathways involved in prion disease and several types of cancer. The pathways involving significantly downregulated miRNAs included the toll-like receptor (TLR) (p<4.36E-16) and Nuclear Factor kappa B (NF-κB) signaling pathways (p<9.00E-06). CONCLUSIONS: We report significantly dysregulated miRNAs in intraocular ME tumors, which exhibited abnormal profiles in other cancers as well such as retinoblastoma and glioblastoma. Pathway analysis of all dysregulated miRNAs shared commonalities with other cancer pathways.

RNA connectivity requirements between conserved elements in the core of the yeast telomerase RNP.

Telomerase is a specialized chromosome end-replicating enzyme required for genome duplication in many eukaryotes. An RNA and reverse transcriptase protein subunit comprise its enzymatic core. Telomerase is evolving rapidly, particularly its RNA component. Nevertheless, nearly all telomerase RNAs, including those of H. sapiens and S. cerevisiae, share four conserved structural elements: a core-enclosing helix (CEH), template-boundary element, template, and pseudoknot, in this order along the RNA. It is not clear how these elements coordinate telomerase activity. We find that although rearranging the order of the four conserved elements in the yeast telomerase RNA subunit, TLC1, disrupts activity, the RNA ends can be moved between the template and pseudoknot in vitro and in vivo. However, the ends disrupt activity when inserted between the other structured elements, defining an Area of Required Connectivity (ARC). Within the ARC, we find that only the junction nucleotides between the pseudoknot and CEH are essential. Integrating all of our findings provides a basic map of functional connections in the core of the yeast telomerase RNP and a framework to understand conserved element coordination in telomerase mechanism.