MicroRNA 184 (miR-184) is known to play a key role in neurological development and apoptosis and is highly expressed in mouse brain, mouse corneal epithelium, zebrafish lens and human retinal pigment epithelium (RPE). However, the role of miR-184 in RPE is largely unknown. We investigated the role of miR-184 in RPE and its possible implication in age-related macular degeneration (AMD). Proteomic analysis identified the ezrin (EZR) gene as a target of miR-184 in human RPE. EZR is a membrane cytoskeleton crosslinker that is also known to bind to lysosomal-associated membrane protein 1 (LAMP-1) during the formation of phagocytic vacuoles. In adult retinal pigment epithelium 19 (ARPE19) cells, inhibition of miR-184 resulted in upregulation of EZR mRNA and EZR protein, and induced downregulation of LAMP-1. The inhibition of miR-184 decreased EZR-bound LAMP-1 protein levels and affected phagocytic activity in ARPE19 cells. In primary culture of human RPE isolated from eyes of AMD donors (AMD RPE), miR-184 was significantly downregulated compared with control (normal) RPE. Downregulation of miR-184 was consistent with significantly lower levels of LAMP-1 protein in AMD RPE, and overexpression of MIR-184 in AMD RPE was able to rescue LAMP-1 protein expression to normal levels. Altogether, these observations suggest a novel role for miR-184 in RPE health and support a model proposing that downregulation of miR-184 expression during aging may result in dysregulation of RPE function, contributing to retinal degeneration.
Ubiquitin modification of endosomal membrane proteins is a signal for active inclusion into the Multivesicular Body (MVB) pathway, resulting in lysosomal degradation. However, the endosome represents a dynamic site of protein sorting with a majority of proteins destined for recycling, rather than MVB targeting. Substrate recognition by ubiquitin ligases is therefore highly regulated. We have investigated substrate recognition by the Nedd4 ortholog Rsp5 as a model for understanding ligasesubstrate interactions. Rsp5 interacts directly with its substrate Cps1 via a novel interaction mode. Perturbation of this mode of interaction revealed a compensatory role for the Rsp5 adaptor Bsd2. These results highlight the ability of Rsp5 to interact with substrates via multiple modalities, suggesting additional mechanisms of regulating this interaction and relevant outcomes.The post-translational addition of ubiquitin to protein substrates is a regulatory modification of remarkable scope in eukaryotic biology. Cellular processes as diverse as protein degradation, protein trafficking, DNA repair, and nuclear signaling are regulated by ubiquitination, and as a consequence, numerous pathologies and developmental defects have been linked to defects in the ubiquitin system (reviewed in Refs. 1-3). A cascade of reactions culminates in the formation of an isopeptide bond between the C-terminal glycine of ubiquitin and an acceptor amine within the substrate. Ubiquitin modification is tightly regulated, with the third enzyme in the ubiquitination cascade, the ubiquitin ligase, responsible for substrate selection (reviewed in Refs. 4, 5).Ubiquitin modification of endosomal transmembrane proteins has previously been demonstrated to play a major role in targeting proteins into multivesicular bodies (MVBs) 3 en route to lysosomal degradation (6 -9). Entry into intralumenal vesicles during MVB sorting is tightly regulated, and Carboxypeptidase S (Cps1) has served as a model MVB cargo in analyses demonstrating the role of ubiquitin modification as a positive cis-acting MVB sorting determinant (6 -9). Considerable evidence supports a model wherein the HECT ubiquitin ligase Rsp5 plays the major role in targeting a number of MVB cargoes, including Cps1, into this pathway in Saccharomyces cerevisiae (9 -20). Rsp5 is the yeast ortholog of Nedd4 family ligases, all of which contain WW protein interaction domains involved in substrate recognition (21). These WW domains participate in substrate recognition either directly through "PY" motifs within the substrates (9, 18 -20, 22-26) or indirectly via adaptors that contain PY motifs (27-29). Bsd2 is one such cofactor that has been implicated in Cps1 ubiquitination and subsequent MVB targeting (17, 30). However, we have previously observed a direct interaction between Rsp5 and Cps1 in vitro (9), suggesting that the interactions leading to Cps1 ubiquitination may be more complicated. Cps1 contains MVB targeting information within the amino acid sequence "PVEKAPR" (6), which does not possess a PY mot...
Despite increased use of early detection methods and more aggressive treatment strategies, the worldwide incidence of colorectal cancer is still on the rise. Consequently, it remains urgent to identify novel agents with enhanced efficacy in prevention and/or therapeutic protocols. Our studies focused on the use of Plumbagin, a natural phytochemical that showed promising results against other tumor types, to determine its effectiveness in blocking the proliferation and survival of colon cancer cells in experimental protocols mimicking the environment in primary tumors (attached culture conditions) and in circulating tumor cells (unattached conditions). Under both experimental settings, exposure of HCT116 cells to Plumbagin concentrations in the low micromolar range resulted in cell cycle arrest at the G1 phase, apoptosis via the mitochondrial cell death pathway, and increased production of reactive oxygen species. The cell cycle effects were more noticeable in attached cells, whereas the induction of cell death was more evident in unattached cells. These effects were consistent with the nature and the magnitude of the alterations induced by Plumbagin on the expression levels of a set of proteins known to play key roles in the regulation of cell cycle dynamics, apoptosis mechanisms and cell proliferation. In light of its previously reported lack of toxicity on normal colon cells and the striking anti-survival effect on colon cancer cells observed in our study, Plumbagin should be considered a promising drug for the treatment of colon cancer.
In this work, we describe a model for evaluating the impact of noise variability on the input parameters of a registration algorithm in the context of cardiac ablation therapy. The model can be used to predict both registration error as well as assess which inputs have the largest effect on registration accuracy.
Geometric analysis of the left atrium and pulmonary veins is important for studying reverse structural remodeling following cardiac ablation therapy. It has been shown that the left atrium decreases in volume and the pulmonary vein ostia decrease in diameter following ablation therapy. Most analysis techniques, however, require laborious manual tracing of image cross-sections. Pulmonary vein diameters are typically measured at the junction between the left atrium and pulmonary veins, called the pulmonary vein ostia, with manually drawn lines on volume renderings or on image cross-sections. In this work, we describe a technique for making semi-automatic measurements of the left atrium and pulmonary vein ostial diameters from high resolution CT scans and multi-phase datasets. The left atrium and pulmonary veins are segmented from a CT volume using a 3D volume approach and cut planes are interactively positioned to separate the pulmonary veins from the body of the left atrium. The cut plane is also used to compute the pulmonary vein ostial diameter. Validation experiments are presented which demonstrate the ability to repeatedly measure left atrial volume and pulmonary vein diameters from high resolution CT scans, as well as the feasibility of this approach for analyzing dynamic, multi-phase datasets. In the high resolution CT scans the left atrial volume measurements show high repeatability with approximately 4% intra-rater repeatability and 8% inter-rater repeatability. Intra-and inter-rater repeatability for pulmonary vein diameter measurements range from approximately 2 to 4 mm. For the multi-phase CT datasets, differences in left atrial volumes between a standard slice-by-slice approach and the proposed 3D volume approach are small, with percent differences on the order of 3% to 6%.
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