The HECT domain of the ubiquitin ligase Rsp5 contributes to substrate recognition.

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 ligase-substrate 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.

miR-184 regulates ezrin, LAMP-1 expression, affects phagocytosis in human retinal pigment epithelium and is downregulated in age-related macular degeneration.

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.

Centerline tracking for quantification of reverse structural remodeling of the pulmonary veins following cardiac ablation therapy.

RATIONALE AND OBJECTIVES: Patients with atrial fibrillation undergo structural remodeling resulting in increased pulmonary vein sizes. Studies have demonstrated that these changes are reversible following successful ablation therapy. To date, analyses of pulmonary vein structure have focused on measurements at the pulmonary vein ostia, and the full extent of reverse remodeling along the length of the pulmonary veins has not yet been fully characterized. MATERIALS AND METHODS: An automated, three-dimensional method is proposed that quantifies pulmonary vein geometry starting at the ostia and extending several centimeters into the veins. A centerline is tracked along the length of the pulmonary vein, and orthogonal planes are computed along the curve. The method was validated against manual measurements on each of the four pulmonary veins for 10 subjects. The proposed methodology was used to analyze the pulmonary veins in 21 patients undergoing cardiac ablation therapy with preoperative and postoperative computed tomographic scans. RESULTS: Validation results demonstrated that the automated measurements closely followed the manual measurements, with an overall mean difference of 11.50 mm(2). Significant differences in cross-sectional area at the two time points were observed at all pulmonary vein ostia and extending for 2.0 cm (excluding the 0.5-cm interval) into the left inferior pulmonary vein, 3.5 cm into the left superior pulmonary vein, and 2.0 cm into the right superior pulmonary vein. CONCLUSIONS: Quantitative analysis along the length of the pulmonary veins can be accomplished using centerline tracking and measurements from orthogonal planes along the curve. The patient study demonstrated that reverse structural remodeling following ablation therapy occurs not only at the ostia but for several centimeters extending into the pulmonary veins.

Calibration and Evaluation of a Magnetically Tracked ICE Probe for Guidance of Left Atrial Ablation Therapy.

The novel prototype system for advanced visualization for image-guided left atrial ablation therapy developed in our laboratory permits ready integration of multiple imaging modalities, surgical instrument tracking, interventional devices and electro-physiologic data. This technology allows subject-specific procedure planning and guidance using 3D dynamic, patient-specific models of the patient's heart, augmented with real-time intracardiac echocardiography (ICE). In order for the 2D ICE images to provide intuitive visualization for accurate catheter to surgical target navigation, the transducer must be tracked, so that the acquired images can be appropriately presented with respect to the patient-specific anatomy. Here we present the implementation of a previously developed ultrasound calibration technique for a magnetically tracked ICE transducer, along with a series of evaluation methods to ensure accurate imaging and faithful representation of the imaged structures. Using an engineering-designed phantom, target localization accuracy is assessed by comparing known target locations with their transformed locations inferred from the tracked US images. In addition, the 3D volume reconstruction accuracy is also estimated by comparing a truth volume to that reconstructed from sequential 2D US images. Clinically emulating validation studies are conducted using a patient-specific left atrial phantom. Target localization error of clinically-relevant surgical targets represented by nylon fiducials implanted within the endocardial wall of the phantom was assessed. Our studies have demonstrated 2.4 ± 0.8 mm target localization error in the engineering-designed evaluation phantoms, 94.8 ± 4.6 % volume reconstruction accuracy, and 3.1 ± 1.2 mm target localization error in the left atrial-mimicking phantom. These results are consistent with those disseminated in the literature and also with the accuracy constraints imposed by the employed technology and the clinical application.