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Two-photon excitation fluorescence (TPEF) is emerging as a powerful imaging technique with superior penetration power in scattering media, allowing for functional imaging of biological tissues at a subcellular level. TPEF is commonly used in cancer diagnostics, as it enables the direct observation of metabolism within living cells. The technique is now widely used in various medical fields, including ophthalmology. The eye is a complex and delicate organ with multiple layers of different cell types and tissues. Although this structure is ideal for visual perception, it generates aberrations in TPEF eye imaging. However, adaptive optics can now compensate for these aberrations, allowing for improved imaging of the eyes of animal models for human diseases. The eye is naturally built to filter out harmful wavelengths, but these wavelengths can be mimicked and thereby utilized in diagnostics via two-photon (2Ph) excitation. Recent advances in laser-source manufacturing have made it possible to minimize the exposure of in vivo measurements within safety, while achieving sufficient signals to detect for functional images, making TPEF a viable option for human application. This review explores recent advances in wavefront-distortion correction in animal models and the safety of use of TPEF on human subjects, both of which make TPEF a potentially powerful tool for ophthalmological diagnostics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kaushik, Dąbrowski, Gessa, Kumar and Fernandes.)

Diabetic retinopathy (DR) is a severe disease with a growing number of afflicted patients, which places a heavy burden on society, both socially and financially. While there are treatments available, they are not always effective and are usually administered when the disease is already at a developed stage with visible clinical manifestation. However, homeostasis at a molecular level is disrupted before visible signs of the disease are evident. Thus, there has been a constant search for effective biomarkers that could signal the onset of DR. There is evidence that early detection and prompt disease control are effective in preventing or slowing DR progression. Here, we review some of the molecular changes that occur before clinical manifestations are observable. As a possible new biomarker, we focus on retinol binding protein 3 (RBP3). We argue that it displays unique features that make it a very good biomarker for non-invasive, early-stage DR detection. Linking chemistry to biological function and focusing on new developments in eye imaging and two-photon technology, we describe a new potential diagnostic tool that would allow rapid and effective quantification of RBP3 in the retina. Moreover, this tool would also be useful in the future to monitor therapeutic effectiveness if levels of RBP3 are elevated by DR treatments.

Protein methylation occurs primarily on lysine and arginine, but also on some other residues, such as histidine. METTL18 is the last uncharacterized member of a group of human methyltransferases (MTases) that mainly exert lysine methylation, and here we set out to elucidate its function. We found METTL18 to be a nuclear protein that contains a functional nuclear localization signal and accumulates in nucleoli. Recombinant METTL18 methylated a single protein in nuclear extracts and in isolated ribosomes from METTL18 knockout (KO) cells, identified as 60S ribosomal protein L3 (RPL3). We also performed an RPL3 interactomics screen and identified METTL18 as the most significantly enriched MTase. We found that His-245 in RPL3 carries a 3-methylhistidine (3MH; τ-methylhistidine) modification, which was absent in METTL18 KO cells. In addition, both recombinant and endogenous METTL18 were found to be automethylated at His-154, thus further corroborating METTL18 as a histidine-specific MTase. Finally, METTL18 KO cells displayed altered pre-rRNA processing, decreased polysome formation and codon-specific changes in mRNA translation, indicating that METTL18-mediated methylation of RPL3 is important for optimal ribosome biogenesis and function. In conclusion, we have here established METTL18 as the second human histidine-specific protein MTase, and demonstrated its functional relevance., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)