VAMP2 and synaptotagmin mobility in chromaffin granule membranes: implications for regulated exocytosis

Granule-plasma membrane docking and fusion can only occur when proteins that enable these reactions are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may influence docking, and membrane fusion. We measured the mobility of vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field produced by total internal reflection (TIR) was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, enabling the measurement of diffusion coefficients. We found that VAMP2-EGFP and Syt7-EGFP are mobile with a diffusion coefficient of approximately 3 × 10-10 cm2/s. Syt1-EGFP mobility was below the detection limit. Utilizing these diffusion parameters, we estimated the time required for these proteins to arrive at docking and nascent fusion sites to be many tens of milliseconds. Our analyses raise the possibility that the diffusion characteristics of VAMP2 and Syt proteins could be a factor that influences the rate of exocytosis.

Negative electroretinograms: genetic and acquired causes, diagnostic approaches and physiological insights

The dark-adapted human electroretinogram (ERG) response to a standard bright flash includes a negative-going a-wave followed by a positive-going b-wave that crosses the baseline. An electronegative waveform (or negative ERG) results when the b-wave is selectively reduced such that the ERG fails to cross the baseline following the a-wave. In the context of a normally sized a-wave, it indicates a site of retinal dysfunction occurring after phototransduction (commonly at the photoreceptor to bipolar cell synapse). This is an important finding. In genetic disease, the pattern of ERG abnormality can point to variants in a small group of genes (frequently those associated with congenital stationary night blindness and X-linked retinoschisis, but negative ERGs can also be seen in other conditions including syndromic disease). In acquired disease, there are numerous causes, but specific features may point to melanoma-associated retinopathy (MAR). In some cases, the visual symptoms precede the diagnosis of the melanoma and so the ERG findings can initiate investigations facilitating early detection and treatment. Negative ERGs can occur in other paraneoplastic conditions, and in a range of other diseases. This review will outline the physiological basis for the negative ERG, report prevalences in the literature from different cohorts, discuss the range of causes, displaying examples of a number of ERG phenotypes, highlight features of a clinical approach to patients, and briefly discuss further insights relating to current flows shaping the a-wave trough and from single-cell transcriptome analysis.

Electrophysiological markers of preclinical diagnosis of glaucomatous optic neuropathy

Purpose: to evaluate the changes in electrophysiological indicators reflecting various aspects of the function of retinal ganglion cells (RGC) and their axons in the early diagnosis of glaucomatous optic neuropathy (GON).Material and methods. Two clinical groups, (1) 35 patients (60 eyes) aged 49 to 70 with suspected glaucoma and (2) 16 patients (30 eyes) aged 43–68 with initial primary open-angle glaucoma (POAG), and a comparison group of 38 relatively healthy subjects (45 eyes) aged 42–70 were tested for pattern-reversed visual evoked potentials (PVEP), transient and stationary pattern-ERGs (PERG) according to ISCEV, and photopic negative response (PhNR).Results. The P100 amplitudes in both clinical groups differed significantly from the norm in PVEP on small and large patterns. The elongation of peak latency (T) of P100 compared with norm was significant for the stimulus 1° in group 2. In both groups of patients, increased variability of the temporal parameters of PERG and PVEP for small patterns was found. In groups 1 and 2, a decrease in the amplitude of P50 and N95 peaks of transient PERG for all stimuli was revealed, which was the most significant for the 0.3° pattern. In group 1, the N95 peak was significantly delayed in PERG for large patterns. A statistically significant reduction in the steady-state PERG's amplitude was found in the groups of suspected glaucoma and initial POAG. The sharpest changes were found for small (0.8° and 0.3°) patterns. The elongation of T compared to the norm was most pronounced for PERG at 0.3°, but due to the high variability of temporary indicators within the group, it had no statistical significance. The amplitude of PhNR was significantly different from the norm in the ERG for a flash of 3.0 cd·sec/m2.Conclusion. In patients with suspected glaucoma, a decrease in the P100 VEP amplitude with the simultaneous elongation of T may be considered as a criteria for the plastic stage at the level of lateral geniculate nucleus. Markers of functional changes in RGCs are the decrease in the amplitude of PhNR in response to bright flash, and P50 and N95 of PERG for pattern size 0.3°. The results indicate a greater vulnerability of the parvocellular system to early events in the development of GON.

Transient electroretinographic abnormalities that mimic those of KCNV2 retinopathy: a case report

Purpose The purpose of this report was to describe the case of a 68-year-old male patient with stage IV colon cancer who exhibited electroretinographic abnormalities that are similar to those of KCNV2 retinopathy. Methods The patient presenting with photophobia, reduced visual acuity, and poor general conditions, the onset of which occurred ten days before presentation, was examined using fundoscopy, full-field electroretinography, blood tests, and abdominal computed tomography. Results The patient’s decimal best-corrected visual acuity (BCVA) was 0.4 in each eye. Fundoscopy showed bull's eye-like maculopathy in both eyes. Electroretinographic findings were similar to the characteristic findings of KCNV2 retinopathy: Rod electroretinogram showed delayed and preserved b-wave amplitudes; bright-flash electroretinogram showed double troughs of a-waves; b/a ratios shown by bright-flash electroretinogram were higher than those shown by standard-flash electroretinogram; and both cone and 30-Hz flicker electroretinograms showed extinguished responses. His serum potassium level increased to 6.2 mmol/L (normal range 3.6–4.8 mmol/L) owing to hydronephrosis resulting from disseminated carcinoma. After performing an emergency surgery to treat this condition, the serum potassium level immediately decreased to a normal range. Eleven days after presentation, rod and standard/bright-flash electroretinography showed improvement in the implicit time of the rod b-waves and the a-waves. Unexpectedly, the responses recorded by cone and 30-Hz flicker electroretinography became normal. The symptoms and maculopathy disappeared, and his BCVA improved to 1.2. Conclusions The abnormal electroretinographic findings might be associated with the transient increase in serum potassium level.

Arc flash macular hole

Macular holes are common. Lightening, direct electric shock induced and laser beam induced macular holes are though rare. Reporting a case of spark flashlight (Arc Flash) induced macular hole in an electrician, which has never been reported. A 19 year old male electrician by profession presented to our clinic with a history of exposure to a bright flash light from spark of wires while at work that led to decrease of vision in his both eyes. Examination revealed a full thickness macular hole in his right eye and loss of foveal photoreceptors in the left eye. Arc flash light exposure in electricians can lead to macular holes too adding a new entity to the already existing types of macular holes.

Calcium flares and compartmentalization in rod photoreceptors

Rod photoreceptors are composed of a soma and an inner segment (IS) connected to an outer segment (OS) by a thin cilium. OSs are composed of a stack of ∼800 lipid discs surrounded by the plasma membrane where phototransduction takes place. Intracellular calcium plays a major role in phototransduction and is more concentrated in the discs, where it can be incorporated and released. To study calcium dynamics in rods, we used the fluorescent calcium dye CaSiR-1 AM working in the near-infrared (NIR) (excitation at 650 and emission at 664 nm), an advantage over previously used dyes. In this way, we investigated calcium dynamics with an unprecedented accuracy and most importantly in semidark-adapted conditions. We observed light-induced drops in [Ca2+]iwith kinetics similar to that of photoresponses recorded electrophysiologically. We show three properties of the rods. First, intracellular calcium and key proteins have concentrations that vary from the OS base to tip. At the OS base, [Ca2+]iis ∼80 nM and increases up to ∼200 nM at the OS tip. Second, there are spontaneous calcium flares in healthy and functional rod OSs; these flares are highly localized and are more pronounced at the OS tip. Third, a bright flash of light at 488 nm induces a drop in [Ca2+]iat the OS base but often a flare at the OS tip. Therefore, rod OSs are not homogenous structures but have a structural and functional gradient, which is a fundamental aspect of transduction in vertebrate photoreceptors.

Bekenstein’s Entropy Bound-Particle Horizon Approach to Avoid the Cosmological Singularity

The cosmological singularity of infinite density, temperature, and spacetime curvature is the classical limit of Friedmann’s general relativity solutions extrapolated to the origin of the standard model of cosmology. Jacob Bekenstein suggests that thermodynamics excludes the possibility of such a singularity in a 1989 paper. We propose a re-examination of his particle horizon approach in the early radiation-dominated universe and verify it as a feasible alternative to the classical inevitability of the singularity. We argue that this minimum-radius particle horizon determined from Bekenstein’s entropy bound, necessarily quantum in nature as a quantum particle horizon (QPH), precludes the singularity, just as quantum mechanics provided the solution for singularities in atomic transitions as radius r → 0 . An initial radius of zero can never be attained quantum mechanically. This avoids the spacetime singularity, supporting Bekenstein’s assertion that Friedmann models cannot be extrapolated to the very beginning of the universe but only to a boundary that is ‘something like a particle horizon’. The universe may have begun in a bright flash and quantum flux of radiation and particles at a minimum, irreducible quantum particle horizon rather than at the classical mathematical limit and unrealizable state of an infinite singularity.

Machine Learning Approach to Detect Red-Eye Using Pixel Detection Technique

Sometimes in Flash Photography red colored patches occurred in human eyes. It is actually a reflection of bright flash light reflected from blood vessels in the eyes, giving the eye an unnatural red hue. Red-eye is a big problem in professional photography. Most red-eye reduction systems in many editing software needed the user to identify the red-eye and make an outline through the red-eye. Here we propose an Automatic Red-Eye Detection System instead. The system contains a red-eye detector that finds bunch of red pixels those are clustered to gather, a state of face detector that used to eliminate most false positives (pixel clusters that look red eyes but are not); and a redeye outline detector. All three detectors are automatically learned from the taken datasets and with a proper classifiers using boosting. For creating a fully Automatic Red-Eye Corrector this system needed to be combined with a functional Red-Eye Reduction model.

A quantitative account of mammalian rod phototransduction with PDE6 dimeric activation: responses to bright flashes

We develop an improved quantitative model of mammalian rod phototransduction, and we apply it to the prediction of responses to bright flashes of light. We take account of the recently characterized dimeric nature of PDE6 activation, where the configuration of primary importance has two transducin molecules bound. We simulate the stochastic nature of the activation and shut-off reactions to generate the predicted kinetics of the active molecular species on the disc membrane surfaces, and then we integrate the differential equations for the downstream cytoplasmic reactions to obtain the predicted electrical responses. The simulated responses recover the qualitative form of bright-flash response families recorded from mammalian rod photoreceptors. Furthermore, they provide an accurate description of the relationship between the time spent in saturation and flash intensity, predicting the transition between first and second ‘dominant time constants’ to occur at an intensity around 5000 isomerizations per flash, when the rate of transducin activation is taken to be 1250 transducins s −1 per activated rhodopsin. This rate is consistent with estimates from light-scattering experiments, but is around fourfold higher than has typically been assumed in other studies. We conclude that our model and parameters provide a compelling description of rod photoreceptor bright-flash responses.