Understanding Patterns of Preserved Retinal Ganglion Cell Layer in Advanced Glaucoma as seen with OCT.

PRECIS: Using OCT, eyes with advanced glaucoma were found to have a wide range of patterns of damage that were consistent with the natural history of progression based upon a model of macular progression. PURPOSE: To understand the patterns of preserved retinal ganglion cells in eyes with advanced glaucoma using OCT and a model of progression of the central macula. METHODS: OCT GCL thickness was measured in 94 eyes with advanced glaucoma, defined as glaucomatous eyes with a 24-2 MD (mean deviation) worse than -12 dB. A commercial report supplied the GCL thickness in 6 sectors of the thick, donut-shaped GCL region around the fovea. For each eye, the 6 sectors were coded as green (within normal limits, WNL), yellow (≤5th, ≥1st percentile), or red (<1st percentile). RESULTS: In all 94 eyes, one or more of the 6 sectors of the donut were abnormal (red or yellow), while all 6 sectors were red in 52 (55%) of the eyes. On the other hand, 33 eyes had one or more sectors WNL (green). While the pattern of donut damage varied widely across these 33 eyes, 61 of the 66 hemiretinas were consistent with a temporal-to-nasal progression of damage within each hemiretina as predicted by our model. CONCLUSION: All eyes with advanced glaucoma had damage to the critically important central, donut-shaped GCL region. This region showed a wide range of patterns of damage, but these patterns were consistent with the natural history of progression based upon a model of macular progression. These results have implications for the clinical identification of macular progression, as well as for inclusion criteria for clinical trials seeking to preserve central macular function.

The OCT RNFL Probability Map and Artifacts Resembling Glaucomatous Damage.

Purpose: The purpose of this study was to improve the diagnostic ability of the optical coherence tomography (OCT) retinal nerve fiber layer (RNFL) probability (p-) map by understanding the frequency and pattern of artifacts seen on the p-maps of healthy control (HC) eyes resembling glaucomatous damage. Methods: RNFL p-maps were generated from wide-field OCT cube scans of 2 groups of HC eyes, 200 from a commercial normative group (HC-norm) and 54 from a prospective study group, as well as from 62 patient eyes, which included 32 with early glaucoma (EG). These 32 EG eyes had 24-2 mean deviation (MD) better than -6 dB and perimetric glaucoma as defined by 24-2 and 10-2 criteria. For the HC groups, "glaucoma-like" arcuates were defined as any red region near the temporal half of the disc. Results: Seven percent of the 200 HC-norm and 11% of the 54 HC RNFL p-maps satisfied the definition of "glaucoma-like," as did all the patients' p-maps. The HC p-maps showed two general patterns of abnormal regions, "arcuate" and "temporal quadrant," and these patterns resembled those seen on some of the RNFL p-maps of the EG eyes. A "vertical midline" rule, which required the abnormal region to cross the vertical midline through the fovea, had a specificity of >99%, and a sensitivity of 75% for EG and 93% for moderate to advanced eyes. Conclusions: Glaucoma-like artifacts on RNFL p-maps are relatively common and can masquerade as arcuate and/or widespread/temporal damage. Translational Relevance: A vertical midline rule had excellent specificity. However, other OCT information is necessary to obtain high sensitivity, especially in eyes with early glaucoma.

Reverse engineering the yeast RNR1 transcriptional control system.

Transcription is controlled by multi-protein complexes binding to short non-coding regions of genomic DNA. These complexes interact combinatorially. A major goal of modern biology is to provide simple models that predict this complex behavior. The yeast gene RNR1 is transcribed periodically during the cell cycle. Here, we present a pilot study to demonstrate a new method of deciphering the logic behind transcriptional regulation. We took regular samples from cell cycle synchronized cultures of Saccharomyces cerevisiae and extracted nuclear protein. We tested these samples to measure the amount of protein that bound to seven different 16 base pair sequences of DNA that have been previously identified as protein binding locations in the promoter of the RNR1 gene. These tests were performed using surface plasmon resonance. We found that the surface plasmon resonance signals showed significant variation throughout the cell cycle. We correlated the protein binding data with previously published mRNA expression data and interpreted this to show that transcription requires protein bound to a particular site and either five different sites or one additional sites. We conclude that this demonstrates the feasibility of this approach to decipher the combinatorial logic of transcription.

Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element.

Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014s(-1). We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase.