The mechanisms of resistance to the antimetabolite gemcitabine in non-small cell lung cancer have not been extensively evaluated. In this study, we report the generation of two gemcitabine-selected non-small cell lung cancer cell lines, H358-G200 and H460-G400. Expression profiling results indicated that there was evidence for changes in the expression of 134 genes in H358-G200 cells compared with its parental line, whereas H460-G400 cells exhibited 233 genes that appeared to be under-or overexpressed compared with H460 cells. However, only the increased expression of ribonucleotide reductase subunit 1 (RRM1), which appeared in both resistant cell lines, met predefined analysis criteria for genes to investigate further. Quantitative PCR analysis demonstrated H358-G200 cells had a greater than 125-fold increase in RRM1 RNA expression. Western blot analysis confirmed high levels of RRM1 protein in this line compared with the gemcitabine-sensitive parent. No significant change in the expression of RRM2 was observed in either cell line, although both gemcitabine-resistant cell lines had an approximate 3-fold increase in p53R2 protein. A partial revertant of H358-G200 cells had reduced levels of RRM1 protein (compared with G200 cells), without observed changes in RRM2 or p53R2. In vitro analyses of ribonucleotide reductase activity demonstrated that despite high levels of RRM1 protein, ribonucleotide reductase activity was not increased in H358-G200 cells when compared with parental cells. The cDNA encoding RRM1 from H358-G200 cells was cloned and sequenced but did not reveal the presence of any mutations. The results from this study indicate that the level of RRM1 may affect gemcitabine response. Furthermore, RRM1 may serve as a biomarker for gemcitabine response.

Expansion of a polyglutamine tract within ataxin-1 causes spinocerebellar ataxia type 1 (SCA1). In this study, we used the yeast two-hybrid system to identify an ataxin-1-interacting protein, A1Up. A1Up localized to the nucleus and cytoplasm of transfected COS-1 cells. In the nucleus, A1Up co-localized with mutant ataxin-1, further demonstrating that A1Up interacts with ataxin-1. Expression analyses demonstrated that A1U mRNA is widely expressed as an approximately 4.0 kb transcript and is present in Purkinje cells, the primary site of SCA1 cerebellar pathology. Sequence comparisons revealed that A1Up contains an N-terminal ubiquitin-like (UbL) region, placing it within a large family of similar proteins. In addition, A1Up has substantial homology to human Chap1/Dsk2, a protein that binds the ATPase domain of the HSP70-like Stch protein. These results suggest that A1Up may link ataxin-1 with the chaperone and ubiquitin-proteasome pathways. In addition, these data support the concept that ataxin-1 may function in the formation and regulation of multimeric protein complexes within the nucleus.

PURPOSE To compare refractive data and testability of Spot (PediaVision) and Plusoptix A09 (Plusoptix, Inc) photoscreeners and to compare each device with traditional cycloplegic retinoscopy. DESIGN Prospective, interventional case series. METHODS After informed consent, patients underwent testing with the Spot and Plusoptix photoscreeners before their examination by a pediatric ophthalmologist masked to the results. Data including testability and estimated refractions were entered into a Research Electronic Data Capture database for statistical analysis. RESULTS A total of 265 children were enrolled (mean age, 6.0 ± 3.4 years). Both devices produced a computer printout result in 250 (94.3%) of the patients. The Spot photoscreener provided a refractive estimate in all computer printouts, whereas the Plusoptix, used binocularly, provided a refractive estimate in 75.2% (188/250) of the printouts. Compared with cycloplegic retinoscopy, both devices underestimated hyperopia or overestimated myopia (−1.35 diopters [D] and −0.64 D, Spot and Plusoptix, respectively) and overestimated astigmatism (0.36 D and 0.32 D, Spot and Plusoptix, respectively). The intraclass correlation coefficient for spherical equivalents indicated good agreement between cycloplegic retinoscopy and Spot (0.806) and excellent agreement between cycloplegic retinoscopy and Plusoptix (0.898). CONCLUSIONS The Spot photoscreener provided refractive data on a greater percentage of children. The photorefractors correlated with cycloplegic retinoscopy refractive findings for sphere and spherical equivalents, but underestimated hyperopia or overestimated myopia and overestimated astigmatism. The binocular refractions of Plusoptix agreed more closely with the refractions of our pediatric ophthalmologists.

Background The purpose of this study was to evaluate the updated Spot Vision Screener (PediaVision, Welch Allyn, Skaneateles Falls, NY) in detecting amblyopia risk factors using the 2013 guidelines of the American Association for Pediatric Ophthalmology and Strabismus (AAPOS). Methods In this prospective study, patients seen from June 2012 to November 2013 were tested with the Spot prior to examination by a pediatric ophthalmologist who was masked to test results. The following data were analyzed: age, subject testability, examination findings, and systemic and ocular pathology. Children were divided into three age groups to determine gold standard results according to the AAPOS guidelines. Results A total of 444 children (average age, 72 months) were included. Compared to the ophthalmologist's examination, the Spot sensitivity was 87.7% and the specificity was 75.9% in detecting amblyopia risk factors. There were no significant differences in sensitivity between the age groups, although the positive predictive value improved in the older age groups. Conclusions In our study cohort, the Spot provided good specificity and sensitivity in detecting amblyopia risk factors according 2013 AAPOS criteria, with minor improvements with updated versions.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract within the SCA1 gene product, ataxin-1. Expansion of this tract is believed to result in a gain of function by the mutant protein, perhaps through altered self-associations or interactions with other cellular proteins. We have used the yeast two hybrid system to determine if ataxin-1 is capable of multimerization. This analysis revealed that ataxin-1 does have the ability to self-associate, however, this association does not appear to be influenced by expansion of the polyglutamine tract. Consistent with this finding, deletion analysis excluded the involvement of the polyglutamine tract in ataxin-1 self-association, and instead localized the multimerization region to amino acids 495-605 of the wild type protein. These results, while identifying an ataxin-1 self-interaction region, fail to support a proposed model of polar-zipper mediated multimerization involving the ataxin-1 polyglutamine tract.