Dysregulation of DNA methylation and mRNA alternative cleavage and polyadenylation (APA) are both prevalent in cancer and have been studied as independent processes. We discovered a DNA methylation-regulated APA mechanism when we compared genome-wide DNA methylation and polyadenylation site usage between DNA methylation-competent and DNA methylation-deficient cells. Here, we show that removal of DNA methylation enables CTCF binding and recruitment of the cohesin complex, which, in turn, form chromatin loops that promote proximal polyadenylation site usage. In this DNA demethylated context, either deletion of the CTCF binding site or depletion of RAD21 cohesin complex protein can recover distal polyadenylation site usage. Using data from The Cancer Genome Atlas, we authenticated the relationship between DNA methylation and mRNA polyadenylation isoform expression in vivo. This DNA methylation-regulated APA mechanism demonstrates how aberrant DNA methylation impacts transcriptome diversity and highlights the potential sequelae of global DNA methylation inhibition as a cancer treatment.

Objective The intravitreal injection (IVI) of pharmacological agents is the most commonly performed ocular procedure and is associated with a host of complications. The majority of IVI-related complications data is derived from randomized controlled clinical trials, which report a high adverse event rate. The nature of these protocol-driven trials limit their applicability to the diverse circumstances seen in routine clinical practice. The goal of this study is to determine the prevalence of patient reported IVI-related complications, their risk factors, and the manner in which patients presented in a tertiary eye care center. Design A retrospective, IRB-approved review. Subjects, Participants and/or Controls 44,734 injections in 5,318 unique patients at the Cleveland Clinic Cole Eye Institute from 2012-2016. Methods, Intervention, or Testing Intravitreal injection. Main Outcome Measures Complication occurrence within 15 days of injection. Results From 2012-2016, a total of 44,734 injections were performed in 5,318 unique patients. Overall, complication rates were low, representing 1.9% of all injections, with 1031 unique complications in 685 (12.9%) patients. The most common minor complications, or those not requiring intervention, were irritation (n=312) and subconjunctival hemorrhage (n=284). The most common serious complications, or those requiring intervention, were corneal abrasion (n=46) and iritis (n=31). The majority of complications (66%) were adequately managed by a telephone/Epic electronic message encounter only. Importantly, no injection protocol parameter, such as type of anesthesia, preparation, or post-injection medication, increased the risk of a complication. However, patient sex, age, number of previous injections, and provider strongly influenced the risk of patient-reported complications. Conclusions Overall, complication rates seen in routine clinical practice were low compared to clinical trial reporting. Providers should feel confident in the safety and administration of IVI during times when follow-up office visits and resources may be limited. When performing an IVI, factors such as a patient’s sex, age, number of previous injections, and provider must be taken into account to ensure the best possible outcomes.

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Purpose To evaluate depth-resolved changes of corneal biomechanical properties in eyes with corneal ectasia after corneal crosslinking (CXL) using optical coherence elastography. Methods In a prospective pilot series of eyes with corneal ectasia, a custom high-speed swept source optical coherence tomography system was used to image the cornea before and 3 months after CXL during a low-speed applanating deformation while monitoring applanation force. Cross-correlation was applied to track frame-by-frame two-dimensional optical coherence tomography speckle displacements, and the slope of force versus local axial displacement behavior during the deformation was used to produce a two-dimensional array of axial stiffness ( k ). These values were averaged for anterior ( k a ) and posterior ( k p ) stromal regions and expressed as a ratio ( k a /k p ) to assess depth-dependent differences in stiffness. CXL was performed according to the Dresden protocol with a system approved by the U.S. Food and Drug Administration. Results Four eyes from four patients with keratoconus ( n = 3) or post-LASIK ectasia ( n = 1) underwent optical coherence elastography before and 3 months after CXL. The mean k a /k p was 1.03 ± 0.07 before CXL compared with 1.34 ± 0.17 after the CXL procedure. All four eyes demonstrated at least a 20% increase in the k a /k p . Conclusions Preferential stiffening of the anterior stroma with the standard CXL protocol was demonstrated with optical coherence elastography in live human subjects. Translational Relevance Although ex vivo studies have demonstrated anterior stiffening effects after CXL using various destructive and nondestructive methods, this report presents the first evidence of such changes in serial live human measurements.

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