Identification of a non-canonical nuclear localization signal (NLS) in BRCA1 that could mediate nuclear localization of splice variants lacking the classical NLS.

The breast cancer type 1 susceptibility gene (BRCA1) is a tumor suppressor gene, mutations or loss of which lead to genomic instability and breast cancer. BRCA1 protein is part of a large multi-protein complex involved in a variety of DNA repair and transcription regulatory functions. At least four splice variants have been described and these differ in their function and tissue and spatio-temporal expression patterns. Structural analysis has revealed the presence of two nuclear localization signals (NLS) located in exon 11 of BRCA1. Interestingly, a splice variant of the protein that lacks both of the known NLS still manages to gain entry to the nucleus. While there is experimental proof for the translocation of these proteins by binding to other established nuclear proteins, we examined the possibility of a hitherto unidentified NLS in this particular variant. In this paper, we present evidence for the existence of a previously unreported non-canonical NLS contained within the first 39 amino acids of exon 11. A fusion protein with this 39mer and a reporter green fluorescent protein translocated into the nucleus when it was expressed in breast epithelial cells. We demonstrate the presence of a hitherto unreported noncanonical NLS in exon 11a of BRCA1. This NLS might aid proteins that were encoded by splice variants and lack the canonical NLS to localize to the nucleus.

Repeatability of corneal first-surface wavefront aberrations measured with Pentacam corneal topography.

PURPOSE: To assess the repeatability of corneal wavefront aberrations derived from Pentacam (Oculus) corneal topography. SETTING: Flinders Eye Centre, Flinders Medical Centre, Bedford Park, South Australia, Australia. METHODS: Forty-five normal participants and 10 participants with keratoconus were tested. Intraobserver and interobserver repeatability was determined using 4 observers within and between sessions. Topographical maps were exported to external software, and corneal first-surface wavefront aberrations were calculated using a 10th-order Zernike expansion over a 6.0 mm optical zone. Repeatability was determined with Bland-Altman limits of agreement and expressed as the coefficient of repeatability (COR). RESULTS: Initial data showed high wavefront aberrations in normal participants and poor repeatability. Topographical maps showed extrapolated topography in zones without data acquisition; maps with less than 6.0 mm of complete data were excluded in the final analysis. The mean wavefront aberrations for normal participants remained high, but repeatability improved. The COR relative to the magnitude of wavefront aberrations was high (average 100%) across all modal pairs and orders, although best for total higher-order root mean square. Participants with keratoconus had higher magnitude wavefront aberrations and poorer repeatability but similar COR to average wavefront aberration ratios. Examination of raw elevation data showed poor repeatability. CONCLUSIONS: Wavefront aberrations calculated from Pentacam corneal topography were large in magnitude, and reliability was poor, largely due to variability in corneal elevation data. Intraobserver and interobserver reliability within and between sessions was comparable. The Pentacam was not reliable in measuring corneal wavefront aberrations.

Anterior segment biometry with the Pentacam: comprehensive assessment of repeatability of automated measurements.

PURPOSE: To comprehensively assess the reliability of automated Pentacam (Oculus, Inc.) measurements. SETTING: Flinders Eye Centre, Flinders Medical Centre, Bedford Park, South Australia, Australia. METHODS: Both eyes of 35 normal volunteers were tested twice on the same day by 2 different observers. All automated values were recorded, and manual analysis of topographic maps was performed only to overrule variance in corneal thickness due to pupil decentration altering the central reference point. Repeatability was determined with Bland-Altman limits of agreement and reported as the coefficient of repeatability (COR = +/-1.96 standard deviation of differences). Relative repeatability (RR) was calculated as a percentage of the ratio of COR to the mean. RESULTS: Overall, repeatability was good. Corneal curvature, reported in diopters, showed good repeatability anteriorly (simulated keratometry mean COR+/-0.28D; RR=0.64%) and posteriorly (COR+/-011D; RR=1.85%). Peripheral corneal curvature was more reliable when calculated by the sagittal (axial) method (RR=1.57%) than by the tangential (meridional) method (RR=2.38%). Keratometric power deviation was less reliable (RR=16.39%). Anterior chamber measurements showed good reliability (RR=3.07%-5.68%) except for anterior chamber angle (RR=14.41%). Pupil diameter showed poor reliability (RR=25.77%). Central corneal thickness was comparable at pupil center and corneal vertex, but peripheral repeatability was much better when centered on the corneal vertex (COR+/-16.00microm; RR=2.56%) than at pupil center (COR+/-26.28microm; RR=4.23%). CONCLUSIONS: Pentacam corneal curvature and anterior chamber parameters were highly repeatable, but pupil measurements had poor repeatability. Peripheral pachymetry readings were affected by pupil decentration and required manual analysis using the corneal vertex as the point of reference to achieve good repeatability.

Critical flicker fusion test of potential vision.

PURPOSE: To continue developing a potential vision test based on the critical flicker fusion (CFF) phenomenon by using a brighter stimulus and optimizing its size. SETTING: Flinders Eye Centre, Flinders Medical Centre, Flinders University, Bedford Park, South Australia, Australia. METHODS: In a prospective nonrandomized study, 225 participants were assigned to 1 of 4 groups: normal, media opacity only, retinal/neural disease only, and cataract plus retinal/neural disease. Participants were recruited if they were 20 years or older but were excluded if they had a neurological disorder or medication known to affect CFF. The CFF thresholds were measured for 3 stimulus sizes: 0.5 degree, 1.0 degree, and 1.5 degrees. Discrimination between groups was tested by analysis of variance and receiver operating characteristic analysis. The relationship between visual acuity and CFF in eyes without media opacity was determined by linear regression and used to predict visual outcomes in 23 eyes having cataract surgery. RESULTS: The mean age of the 225 participants was 71.4 years +/- 13.2 (SD); 134 (59.8%) were women. The normal group had 41 participants, and the other 3 groups had 61 participants each. Critical flicker fusion thresholds were reduced in retinal/neural disease but resistant to image degradation from media opacity. The 1.5-degree stimulus had 88% sensitivity and 90% specificity for discriminating groups. Visual acuity after cataract surgery was accurately predicted within +/-1 line in 43% of eyes, +/-2 lines in 83%, and +/-3 lines in 100%. All eyes with poor visual acuity (>0.50 logMAR) or dense cataract (>4.0 Lens Opacities Classification System III) were predicted within +/-2 lines. CONCLUSIONS: The CFF phenomenon effectively discriminated between subjects with and without retinal/neural disease and accurately predicted visual outcome after cataract surgery. The use of a brighter stimulus enhanced performance in cases of dense media opacity.