4 research outputs found
Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast
Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry
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Soft Landing technique as a possible prevention strategy for proximal junctional failure following adult spinal deformity surgery
BackgroundThis cross sectional study describes a "Soft Landing" strategy utilizing hooks for minimizing proximal junctional kyphosis (PJK) and proximal junctional failure (PJF). The technique creates a gradual transition from a rigid segmental construct to unilateral hooks at the upper instrumented level and preservation of the soft tissue attachments on the contralateral side of the hooks. Authors devise a novel classification system for better grading of PJK severity.MethodsThirty-nine consecutive adult spinal deformity (ASD) patients at a single institution received the "Soft Landing" technique. The proximal junctional angle was measured preoperatively and at last follow-up using standing 36-inch spinal radiographs. Changes in proximal junctional angle and rates of PJK and PJF were measured and used to create a novel classification system for evaluating and categorizing ASD patients postoperatively.ResultsThe mean age of the cohort was 61.4 years, and 90% of patients were women. Average follow up was 2.2 years. The mean change in proximal junctional angle was 8° (SD 7.4°) with the majority of patients (53%) experiencing less than 10° and only 1 patients with proximal junctional angle over 20°. Four patients (10%) needed additional surgery for proximal extension of the uppermost instrumented vertebra (UIV) secondary to PJF.ConclusionsSoft Landing technique is a possibly effective treatment strategy to prevent PJK and PJF following ASD that requires further evaluation. The described classification system provides management framework for better grading of PJK. The "Soft Landing" technique warrants further comparison to other techniques currently used to prevent both PJK and failure