Browsing by Subject "Eye Proteins"
Now showing 1 - 3 of 3
- Results Per Page
- Sort Options
Item The Impact of Diurnal Changes and Inter-Visit Variability on the Concentration of Insulin-Like Growth Factor-1 in Human Tears(2015-01-26) Patel, Roshni; Zhu, Meifang; Robertson, Danielle M.INTRODUCTION: There is a growing body of research focused on the use of tear film-derived proteins as biomarkers of disease. Previous studies have reported quantitative changes in tear-derived growth factors and related proteins associated with various systemic and ocular diseases. Major challenges when working with human tears however, includes sample volume limitation and the high potential for reflex tearing. One method of tear collection that is increasingly being reported involves the use of microcapillary tubes to draw tears from the inferior tear reservoir. The purpose of this study was to investigate the impact of diurnal changes and inter-visit variability on the concentration of a known growth factor present in human tears, the insulin-like growth factor-1 (IGF-1). METHODS: Nine healthy volunteers without any reported symptoms of dry eye were recruited for this study. At visit 1 (baseline), all participants underwent a standard dry eye examination to assess tear volume, tear film break up time (TFBUT), and tear production. Subjects were asked to return to clinic for an additional 5 visits (morning and afternoon on a total of 3 days). Tears were collected at the start of each visit from the inferior temporal tear meniscus of both eyes using 1 - 10 μl glass microcapillary tubes and frozen at -80C until use. Total protein was measured for each patient using a bicinchoninic assay. IGF-1 levels wear assessed using ELISAs. RESULTS: 8.8 ± 2.1 μg/μl of total protein was obtained from each subject. Total protein was unchanged at each visit. There was no difference in IGF-1 between morning and afternoon. Tear levels of IGF-1 did vary with visit, with the final visit showing a 2 fold-increase over baseline (p<0.05). Tear levels of IGF-1 were correlated with TFBUT (R=0.856, p=0.007). DISCUSSION: While diurnal variation did not affect basal levels of IGF-1 in tears, there was a visit-dependent increase. This increase was likely due to a reduction in reflex tearing during tear collection as patients became more comfortable with the technique. Similarly, the decrease in IGF-1 that corresponded with increased tear evaporation was likely due to changes in reflex tearing. Together, these findings suggest that low abundant proteins, such as IGF-1, are highly susceptible to changes in reflex tearing. These findings also suggest that a participant training phase may be required.Item A Sensitive Assay for Monitoring Wild-Type and Mutant Myocilin Secretion(2016-01-19) Zadoo, Serena; Nguyen, Annie; Zode, Gulab; Hullerman, John D.Primary open angle glaucoma (POAG)-associated mutations in myocilin (MYOC) cause protein 'non-secretion', rendering secreted MYOC difficult to quantitatively detect using time-consuming conventional techniques. This study focused on developing an assay which could be used to quickly and easily detect mutant and wild-type (WT) MYOC secretion. We fused Gaussia luciferase (eGLuc2) to MYOC variants and expressed the constructs in HEK-293T cells. The secretion, intracellular soluble and insoluble portions of WT and Y437H MYOC eGLuc2 constructs were evaluated by western blotting and compared to FLAG-tagged constructs. Secreted and soluble MYOC eGLuc2 was measured by a GLuc assay. The secretion of nine additional MYOC mutants was assayed in conditioned media from HEK-293T and NTM-5 cells to test the general applicability of the assay. MYOC eGLuc2 behaved similarly to FLAG MYOC with respect to secretion, soluble intracellular levels, and in response to drug treatment. The GLuc assay could sensitively detect Y437H MYOC secretion 30 min after a media change. eGLuc2 fused variants followed predicted trends; non-pathogenic variants (D208E, G244V) were secreted at WT-like levels, whereas predicted disease-causing variants (C245Y, G246R, E300K, Y437H, I477N) demonstrated substantial secretion defects ranging from 0.20 - 4.0% of WT MYOC levels in HEK-293T cells. These variants were slightly more tolerated in NTM-5 cells, resulting in secretion levels ranging from 1.0 - 12% of WT MYOC levels. Secretion defects caused by the C245Y, G246R, and Y437H mutations were partially rescued by permissive growth temperatures. Interestingly, a combination of growth temperature reduction and cycloheximide treatment of transfected cells indicate that the pool of protein that is rescued at lower temperatures arises from intracellular stores, and is not from newly synthesized MYOC under permissive temperature. Fusion of eGLuc2 to MYOC does not significantly change the behavior of MYOC. The use of the eGLuc2-tagged version of MYOC can be utilized to develop a deeper understanding of MYOC folding and secretion. This newly developed MYOC reporter system has the potential to be used in small molecule and/or genetic high-throughput screens to identify modulators of MYOC secretion.Item Time Course of Disease Progression of PRPF31-Mediated Retinitis Pigmentosa(2019-04-04) Kiser, Kelly; Birch, David G.; Wang, Yi-Zhong; Ufret-Vincenty, RafaelPURPOSE: Variants in PRPF31, a splicing factor, are a common cause of autosomal dominant retinitis pigmentosa (RP). Deleterious variants are thought to cause disease by haploinsufficiency. In anticipation of upcoming replacement gene therapy trials, we present the phenotype and clinical progression of a large cohort of patients with PRPF31-mediated RP. DESIGN: Cross-sectional with retrospective review METHODS: A total of 26 patients with RP and 5 asymptomatic individuals, all with deleterious variants in PRPF31 (from 13 families), were selected from our database of patients followed longitudinally. Ages ranged from 9-77 years (mean 47 years old), with an average follow up time of 16 years. All patients underwent ophthalmic examination including psychophysical tests, electrophysiology, and imaging. All available records were reviewed retrospectively. Additionally, all patients were contacted, and all available patients (n=7) were examined in an additional prospective follow up visit. RESULTS: Age of onset ranged from 6 to 71 years of age, without apparent relationship to specific variant. Two adults (ages 42 and 77) and 3 teenaged children were found to harbor a mutation with no evidence of RP. In those with RP, visual field area (spot size III) declined exponentially at a rate of 8.1% per year of disease duration (p<0.001, 95% CI 5.6-10.6) , electroretinogram (ERG) cone amplitude declined exponentially at a rate of 7.3% per year of disease duration (p<0.001, 95% CI 5.4-9.1), and ellipsoid zone (EZ) area declined exponentially at a rate of 5.4% per year of disease duration (p<0.001, 95% CI 3.7-7.1). CONCLUSIONS: PRPF31-mediated retinitis pigmentosa is characterized by a variable age of onset. Once disease develops, it follows a predictable exponential time course.