To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem-loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins.

Precis: Intraocular pressure (IOP) measurements, when used during telemedicine eye screening alongside nonmydriatic fundus photography, were shown to improve the likelihood of accurate glaucoma and glaucoma-related diagnoses at the follow-up eye examination. Purpose: To determine if IOP measurements, used as an adjunct to nonmydriatic fundus photography, are useful in glaucoma telemedicine screening. Materials and Methods: A total of 902 high-risk individuals were screened for glaucoma at 7 primary care practices and 4 Federally Qualified Health Centers using telemedicine. Screening at visit 1 included fundus photography, assessing family history of glaucoma, and IOP measurements using a hand-held rebound tonometer. Participants with suspicious nerve findings for glaucoma, IOP>21 mm Hg or other ocular pathologies were invited for a follow-up appointment with an ophthalmologist (visit 2). Results: Of the 902 individuals screened at visit 1, 19.6% (n=177/902) had elevated IOP (>21 mm Hg). Fifteen participants were found to have an IOP>30 mm Hg at visit 1, including 2 with an IOP of >40 mm Hg. Among all who attended visit 2 (n=347), 10.9% had glaucoma and 7.2% had ocular hypertension. For participants having both suspicious nerve findings and IOP>21 mm Hg compared with those with neither, the odds ratio (OR) of being diagnosed with glaucoma was 4.48 (95% CI, 1.50-13.93; P=0.007), whereas for participants with suspicious discs and IOP≤21 mm Hg the OR was 2.04 (95% CI, 0.83-5.53; P=0.15). Conclusions: In this telemedicine vision screening setting, having a higher IOP at the screening visit increased the likelihood of receiving a final diagnosis of glaucoma. Therefore, this study supports incorporating IOP measurements, using a portable tonometer, into vision screening programs in high-risk populations.

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Purpose: We dosimetrically compared pencil beam scanning (PBS) proton therapy and intensity-modulated radiation therapy (IMRT) for pelvic and para-aortic lymph node disease in endometrial carcinoma and present acute toxicities associated with extendedfield PBS. Patients and Methods: Twenty-five patients with locally advanced endometrial malignancies were enrolled in an image-guided registry study. Seven of these patients were treated with PBS, and 18 patients were treated with IMRT. Organs at risk included pelvic bone marrow (PBM), small bowel (SB), large bowel (LB), rectum, bladder, and kidneys. The IMRT and PBS dosimetric parameters were compared using Wilcoxon rank-sum tests. Results: Compared with IMRT PBM dose-volume histograms, PBS resulted in significantly lower dose volumes from 0 to 26.0 Gy (P , .05) and higher dose volumes from 33.9 to 42.9 Gy (P , .05). Overall, PBS resulted in 22% lower median PBM volume irradiated to 10 Gy (RBE) (PBS 71.3% versus IMRT 93.4%, P , .001) and 14% lower median volume irradiated to 20 Gy (RBE) (PBS 65.1% versus IMRT 79.4%, P , .001). Compared with IMRT, PBS also significantly reduced SB dose volumes from 0 to 27.5 Gy, LB dose volumes from 0 to 31.6 Gy, bladder dose volumes from 0 to 27.3 Gy, and rectal dose volumes from 0 to 7.6 Gy (all P , .05). However, PBS resulted in higher rectal dose volumes compared with IMRT from 26.0 to 48.4 Gy. Grade 3þ hematologic toxicities were present in 2 (11%) IMRT-treated patients and no PBS-treated patients. No grade 3þ gastrointestinal or genitourinary toxicities were present in either treatment group. Conclusion: In endometrial carcinoma, extended-field PBS is clinically feasible, resulting in statistically significant dose reduction to PBM as well as SB, LB, and bladder in the lower dose regions.

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