In Vitro Reconstitution of Exocytosis from Sea Urchin Egg Plasma Membrane and Isolated Cortical Vesicles

We have succeeded in reconstituting an exocytotically active egg cortex fraction by recombining purified cortical vesicles (CVs) with egg plasma membrane (PM). CVs were dislodged from a suspension of egg cortex by gentle homogenization in a dissociative buffer with a pH of 9.1, and purified by two rounds of differential centrifugation. Egg PM was prepared by shearing the cortical vesicles from a cortical lawn preparation with a jet of isotonic buffer. PM lawns produced by this procedure consist of an array of CV-free PM fragments attached via their extracellular surface to a polylysine coated glass slide. When a neutralized suspension of CVs was recombined with a PM lawn, CVs reassociated with the cytoplasmic face of the plasma membrane to form a reconstituted lawn (RL). RLs undergo a morphological change in response to Ca2+-containing buffers that is similar to the exocytotic release of CV contents from cortical lawns. In both reactions CV contents are vectorially transferred from the cytoplasmic to the extracytoplasmic face of the egg PM. A quantitative binding assay was developed and used to show that adherence of CVs to a heterologous PM lawn prepared from human red blood cells is minimal

Visual Field Progression in Glaucoma What Is the Specificity of the Guided Progression Analysis?

Purpose: To estimate the specificity of the Guided Progression Analysis (GPA) (Carl Zeiss Meditec, Dublin, CA) in individual patients with glaucoma. Design: Observational cohort study. Participants: Thirty patients with open-angle glaucoma. Methods: In 30 patients with open-angle glaucoma, 1 eye (median mean deviation [MD], −2.5 decibels [dB]; interquartile range, −4.4 to −1.3 dB) was tested 12 times over 3 months (Humphrey Field Analyzer, Carl Zeiss Meditec; SITA Standard, 24-2). “Possible progression” and “likely progression” were determined with the GPA. These analyses were repeated after the order of the tests had been randomly rearranged (1000 unique permutations). Main Outcome Measures: Rate of false-positive alerts of “possible progression” and “likely progression” with the GPA. Results: On average, the specificity of the GPA “likely progression” alert was high—for the entire sample, the mean rate of false-positive alerts after 10 follow-up tests was 2.6%. With “possible progression,” the specificity was considerably lower (false-positive rate, 18.5%). Most important, the cumulative rate of false-positive alerts varied substantially among patients, from 0.31, P≤0.10). Conclusions: On average, progression criteria currently used in the GPA have high specificity, but some patients are more likely to show false-positive alerts than others. This is a natural consequence of population-based change criteria and may not matter in clinical trials and studies in which large groups of patients are compared. However, it must be considered when the GPA is used in clinical practice where specificity needs to be controlled for individual patients

Ophthalmic statistics note 5: diagnostic tests-sensitivity and specificity

This is the fifth statistics note produced by the Ophthalmic Statistics Group (OSG) which is designed to be a simple guide to ophthalmic researchers on a statistical issue with an applied ophthalmic example. The OSG is a collaborative group of statisticians who have come together with a desire to raise the statistical standards of ophthalmic researcher by increasing statistical awareness of common issues

Combining optical coherence tomography with visual field data to rapidly detect disease progression in glaucoma: A diagnostic accuracy study

Background: Progressive optic nerve damage in glaucoma results in vision loss, quantifiable with visual field (VF) testing. VF measurements are, however, highly variable, making identification of worsening vision (‘progression’) challenging. Glaucomatous optic nerve damage can also be measured with imaging techniques such as optical coherence tomography (OCT). Objective: To compare statistical methods that combine VF and OCT data with VF-only methods to establish whether or not these allow (1) more rapid identification of glaucoma progression and (2) shorter or smaller clinical trials. Design: Method ‘hit rate’ (related to sensitivity) was evaluated in subsets of the United Kingdom Glaucoma Treatment Study (UKGTS) and specificity was evaluated in 72 stable glaucoma patients who had 11 VF and OCT tests within 3 months (the RAPID data set). The reference progression detection method was based on Guided Progression Analysis™ (GPA) Software (Carl Zeiss Meditec Inc., Dublin, CA, USA). Index methods were based on previously described approaches [Analysis with Non-Stationary Weibull Error Regression and Spatial enhancement (ANSWERS), Permutation analyses Of Pointwise Linear Regression (PoPLR) and structure-guided ANSWERS (sANSWERS)] or newly developed methods based on Permutation Test (PERM), multivariate hierarchical models with multiple imputation for censored values (MaHMIC) and multivariate generalised estimating equations with multiple imputation for censored values (MaGIC). Setting: Ten university and general ophthalmology units (UKGTS) and a single university ophthalmology unit (RAPID). Participants: UKGTS participants were newly diagnosed glaucoma patients randomised to intraocular pressure-lowering drops or placebo. RAPID participants had glaucomatous VF loss, were on treatment and were clinically stable. Interventions: 24-2 VF tests with the Humphrey Field Analyzer and optic nerve imaging with time-domain (TD) Stratus OCT™ (Carl Zeiss Meditec Inc., Dublin, CA, USA). Main outcome measures: Criterion hit rate and specificity, time to progression, future VF prediction error, proportion progressing in UKGTS treatment groups, hazard ratios (HRs) and study sample size. Results: Criterion specificity was 95% for all tests; the hit rate was 22.2% for GPA, 41.6% for PoPLR, 53.8% for ANSWERS and 61.3% for sANSWERS (all comparisons p ≤ 0.042). Mean survival time (weeks) was 93.6 for GPA, 82.5 for PoPLR, 72.0 for ANSWERS and 69.1 for sANSWERS. The median prediction errors (decibels) when the initial trend was used to predict the final VF were 3.8 (5th to 95th percentile 1.7 to 7.6) for PoPLR, 3.0 (5th to 95th percentile 1.5 to 5.7) for ANSWERS and 2.3 (5th to 95th percentile 1.3 to 4.5) for sANSWERS. HRs were 0.57 [95% confidence interval (CI) 0.34 to 0.90; p = 0.016] for GPA, 0.59 (95% CI 0.42 to 0.83; p = 0.002) for PoPLR, 0.76 (95% CI 0.56 to 1.02; p = 0.065) for ANSWERS and 0.70 (95% CI 0.53 to 0.93; p = 0.012) for sANSWERS. Sample size estimates were not reduced using methods including OCT data. PERM hit rates were between 8.3% and 17.4%. Treatment effects were non-significant in MaHMIC and MaGIC analyses; statistical significance was altered little by incorporating imaging. Limitations: TD OCT is less precise than current imaging technology; current OCT technology would likely perform better. The size of the RAPID data set limited the precision of criterion specificity estimates. Conclusions: The sANSWERS method combining VF and OCT data had a higher hit rate and identified progression more quickly than the reference and other VF-only methods, and produced more accurate estimates of the progression rate, but did not increase treatment effect statistical significance. Similar studies with current OCT technology need to be undertaken and the statistical methods need refinement

A Measurement of the Electric Form Factor of the Neutron through d⃗(e⃗,e′n)p\vec{d}(\vec{e},e'n)p at Q2=0.5Q^2 = 0.5 (GeV/c)2^2

We report the first measurement of the neutron electric form factor $G_E^n$ via $\vec{d}(\vec{e},e'n)p$ using a solid polarized target. $G_E^n$ was determined from the beam-target asymmetry in the scattering of longitudinally polarized electrons from polarized deuterated ammonia, $^{15}$ND$_3$. The measurement was performed in Hall C at Thomas Jefferson National Accelerator Facility (TJNAF) in quasi free kinematics with the target polarization perpendicular to the momentum transfer. The electrons were detected in a magnetic spectrometer in coincidence with neutrons in a large solid angle segmented detector. We find $G_E^n = 0.04632\pm0.00616 (stat.) \pm0.00341 (syst.)$ at $Q^2 = 0.495$ (GeV/c)$^2$.Comment: Latex2e 5 pages, 3 figure

Effect of trabeculectomy on the rate of progression of visual field damage

Objectives This study quantifies the effect of trabeculectomy on the rate of progression (RoP) of visual field (VF) damage utilising pre- and post-operative visual function as the outcome instead of surrogate outcomes of success. Methods Clinical and VF data from 199 sequential patients who underwent trabeculectomy between 2015 and 2016 were extracted from the network of sites of Moorfields Eye Hospital NHS Foundation Trust. Of these, we analysed 80 eyes of 74 patients who met our inclusion criteria of at least three reliable VFs before and after surgery (false positive rate <15%). The change in mean RoP (dB/year) was tested using point-wise sensitivity values through a mixed effect model with random effects on both intercepts and slopes. A broken-stick regression of sensitivity over time, with a breakpoint at the day of surgery, modelled the individual change in RoP. Results We analysed 10 [9,12] VFs per subject (Median [Interquartile Range]). At surgery, the age was 67 [57, 72] years, mean deviation was −10.84 [−14.7, −5.6] dB and the IOP was 18 [15, 20] mmHg. One year after surgery, the IOP was 10 [8,13] mmHg (p = 0.002). Mean RoP before surgery was −0.94 [−1.20, −0.69] dB/year (Mean [95% credible intervals]) and it was slowed down by 0.62 [0.26, 0.97] dB/year (p < 0.001) after surgery. Conclusions Trabeculectomy leads to a significant reduction in the RoP of VF loss postoperatively