SPIDR: a highly multiplexed method for mapping RNA-protein interactions uncovers a potential mechanism for selective translational suppression upon cellular stress.

RNA binding proteins (RBPs) play crucial roles in regulating every stage of the mRNA life cycle and mediating non-coding RNA functions. Despite their importance, the specific roles of most RBPs remain unexplored because we do not know what specific RNAs most RBPs bind. Current methods, such as crosslinking and immunoprecipitation followed by sequencing (CLIP-seq), have expanded our knowledge of RBP-RNA interactions but are generally limited by their ability to map only one RBP at a time. To address this limitation, we developed SPIDR (Split and Pool Identification of RBP targets), a massively multiplexed method to simultaneously profile global RNA binding sites of dozens to hundreds of RBPs in a single experiment. SPIDR employs split-pool barcoding coupled with antibody-bead barcoding to increase the throughput of current CLIP methods by two orders of magnitude. SPIDR reliably identifies precise, single-nucleotide RNA binding sites for diverse classes of RBPs simultaneously. Using SPIDR, we explored changes in RBP binding upon mTOR inhibition and identified that 4EBP1 acts as a dynamic RBP that selectively binds to 5'-untranslated regions of specific translationally repressed mRNAs only upon mTOR inhibition. This observation provides a potential mechanism to explain the specificity of translational regulation controlled by mTOR signaling. SPIDR has the potential to revolutionize our understanding of RNA biology and both transcriptional and post-transcriptional gene regulation by enabling rapid, de novo discovery of RNA-protein interactions at an unprecedented scale.

SEC-TMT facilitates quantitative differential analysis of protein interaction networks.

The majority of cellular proteins interact with at least one partner or assemble into molecular-complexes to exert their function. This network of protein-protein interactions (PPIs) and the composition of macromolecular machines differ between cell types and physiological conditions. Therefore, characterizing PPI networks and their dynamic changes is vital for discovering novel biological functions and underlying mechanisms of cellular processes. However, producing an in-depth, global snapshot of PPIs from a given specimen requires measuring tens to thousands of LC-MS/MS runs. Consequently, while recent works made seminal contributions by mapping PPIs at great depth, almost all focused on just 1-2 conditions, generating comprehensive but mostly static PPI networks. In this study we report the development of SEC-TMT, a method that enables identifying and measuring PPIs in a quantitative manner from only 4-8 LC-MS/MS runs per biological sample. This was accomplished by incorporating tandem mass tag (TMT) multiplexing with a size exclusion chromatography mass spectrometry (SEC-MS) work-flow. SEC-TMT reduces measurement time by an order of magnitude while maintaining resolution and coverage of thousands of cellular interactions, equivalent to the gold standard in the field. We show that SEC-TMT provides benefits for conducting differential analyses to measure changes in the PPI network between conditions. This development makes it feasible to study dynamic systems at scale and holds the potential to drive more rapid discoveries of PPI impact on cellular processes.

Pix2pix Conditional Generative Adversarial Networks for Scheimpflug Camera Color-Coded Corneal Tomography Image Generation.

Purpose: To assess the ability of pix2pix conditional generative adversarial network (pix2pix cGAN) to create plausible synthesized Scheimpflug camera color-coded corneal tomography images based upon a modest-sized original dataset to be used for image augmentation during training a deep convolutional neural network (DCNN) for classification of keratoconus and normal corneal images. Methods: Original images of 1778 eyes of 923 nonconsecutive patients with or without keratoconus were retrospectively analyzed. Images were labeled and preprocessed for use in training the proposed pix2pix cGAN. The best quality synthesized images were selected based on the Fréchet inception distance score, and their quality was studied by calculating the mean square error, structural similarity index, and the peak signal-to-noise ratio. We used original, traditionally augmented original and synthesized images to train a DCNN for image classification and compared classification performance metrics. Results: The pix2pix cGAN synthesized images showed plausible subjectively and objectively assessed quality. Training the DCNN with a combination of real and synthesized images allowed better classification performance compared with training using original images only or with traditional augmentation. Conclusions: Using the pix2pix cGAN to synthesize corneal tomography images can overcome issues related to small datasets and class imbalance when training computer-aided diagnostic models. Translational Relevance: Pix2pix cGAN can provide an unlimited supply of plausible synthetic Scheimpflug camera color-coded corneal tomography images at levels useful for experimental and clinical applications.

Topical Difluprednate for Early Corneal Graft Rejection After Penetrating Keratoplasty.

PURPOSE: To evaluate the safety and efficacy of topical difluprednate ophthalmic emulsion use for prophylaxis of corneal graft rejection in patients undergoing penetrating keratoplasty. METHODS: This study reviewed the charts of patients undergoing penetrating keratoplasty who were treated with difluprednate (DP) ophthalmic emulsion postoperatively. At each follow-up visit, patients were followed for signs of graft rejection, cataract development, and intraocular pressure rise in addition to routine ocular examination. RESULTS: The charts of 36 patients (38 eyes) who underwent penetrating keratoplasty (PKP) (27 eyes) and PKP triple (11 eyes) were reviewed. All eyes were followed up for at least 8 months postoperatively. Five grafts developed rejection and three grafts subsequently failed. Six eyes had an increase of IOP that required use of antiglaucoma drops. Three eyes were switched from difluprednate to prednisolone acetate (PA) after persistent rise of IOP failed to respond to antiglaucoma drops. None of these cases needed glaucoma surgery. Two patients developed cataract during the follow-up period (out of 12 phakic eyes). CONCLUSION: Topical difluprednate is potentially effective and safe in preventing graft rejection after penetrating keratoplasty. Larger prospective clinical trials are warranted.

Cataract surgery on the previous corneal refractive surgery patient.

Cataract surgery in cases with previous corneal refractive surgery may be a major challenge for the ophthalmologist. The refractive outcome of the case deserves special attention in the preoperative planning process, which should be tailored for the type of prior refractive procedure: incisional, ablative under a flap, or on the corneal surface. Avoiding refractive surprise after cataract surgery in these cases is principally dependent on the accuracy of the intraocular lens calculation, together with the selection of the appropriate biometric formula for each case. Modern techniques for cataract surgery help surgeons to move toward the goal of cataract surgery as a refractive procedure free from refractive error. We give practical guidelines for the cataract surgeon in the management of these challenging cases.

Optical Profile Following High Hyperopia Correction With a 500-Hz Excimer Laser System.

PURPOSE: To evaluate corneal higher order aberrations after LASIK for the correction of high hyperopia using a 500-Hz excimer laser and optimized ablation profile. METHODS: Retrospective consecutive study including 51 eyes of 28 patients (age range: 21 to 54 years) with high hyperopia or hyperopic astigmatism (sphere ≥ 5.00 diopters). All cases underwent LASIK using the sixth generation Amaris excimer laser (SCHWIND eye-tech-solutions, Kleinostheim, Germany) and a femtosecond laser platform for flap creation. Postoperative changes in corneal higher order aberrations for the 4-, 5-, and 6-mm pupil diameters and corneal asphericity for 4.5 and 8 mm were represented at the end of the 6-month follow-up. RESULTS: A significant increase in corneal root mean square higher order, spherical, and coma aberrations was observed 6 months after surgery (P < .01). Corneal asphericity for the 4.5-mm (Q45) and 8-mm (Q8) corneal diameter also changed significantly during the postoperative period (P < .01). Strehl ratio change was not statistically significant (P = .77). CONCLUSIONS: Correction of high hyperopia with LASIK significantly induces corneal higher order aberrations regardless of the physiologic level of photopic and mesopic pupil conditions.

Refractive surgery following corneal graft.

PURPOSE OF REVIEW: To review the different surgical procedures for management of postkeratoplasty refractive errors after total suture removal. RECENT FINDINGS: There are different surgical options to address residual refractive errors that frequently occur after corneal transplantation. The correction can be done on the corneal surface or intraocular with intraocular lens (IOL) implantation which requires complete tectonic and refractive stability after suture removal. The most commonly used procedures are photorefractive keratectomy, laser in-situ keratomileusis and Phakic IOLs. Keratoplasty has been profited by recent advances in refractive surgery. Custom excimer laser ablation is an alternative way to treat irregular errors. New IOL modalities are good practical options for a wide range of errors. Femtosecond laser, as a new option in the toolbox, can modify corneal grafting refractive results and assist corrective refractive procedures. SUMMARY: Although being the most successful organ transplantation, keratoplasty is usually followed by significant ametropia. Different corrective modalities exist and the choice should fit ocular conditions, patient requirements, surgeon skills and the available technologies. Recent advances in ophthalmic surgery have improved the outcomes.

Femtosecond laser cataract surgery: updates on technologies and outcomes.

PURPOSE: To describe femtosecond laser cataract surgery and discuss the published peer-reviewed articles to have a fair evaluation of this new technology and its comparisons to conventional phacoemulsification surgery. METHODS: The technology information released in this article comes from the key individuals in each of the U.S. Food and Drug Administration-approved companies and from the available commercial information. Bibliographic research was performed in PubMed and MEDLINE for the published prospective or retrospective clinical studies. RESULTS: The femtosecond laser has been reported to be safe intraoperatively and postoperatively, with less cornea and macula effect than conventional phacoemulsification. The incision is integrated, stable, and aberration free. Many studies reported the high precision of the capsulotomy over manual continuous curvilinear capsulorhexis, which favors less intraocular lens tilt, higher optical quality, and more accurate premium intraocular lens centration. The lens fragmentation is effective with significant reduction of the phacoemulsification power and effective phacoemulsification time. The refractive difference between femtosecond laser and standard phacoemulsification is minimal, with no difference in corrected distance visual acuity, but the optical quality and the internal aberrations results are significantly better for femtosecond laser phacoemulsification. CONCLUSIONS: Femtosecond laser cataract surgery is a good addition to cataract surgery despite its few remarkable advantages among experienced phacoemulsification surgeons.

Comparative outcomes of bimanual MICS and 2.2-mm coaxial phacoemulsification assisted by femtosecond technology.

PURPOSE: To compare the efficacy and safety outcomes of bimanual microincision cataract surgery (MICS) versus 2.2-mm coaxial phacoemulsification assisted by Femtosecond LenSx (Alcon-LenSx Inc., Aliso Viejo, CA). METHODS: This prospective, randomized, observational, comparative case series comprised 50 cataractous eyes of 50 patients receiving femtosecond laser refractive lens surgery followed by a bimanual MICS technique with two 1-mm incisions (25 patients) (FemtoMICS group) and a coaxial phacoemulsification technique with a 1-mm paracentesis and a 2.2-mm principal incision (25 patients) (FemtoCoaxial group). The main outcomes measures were: ultrasound power, effective phacoemulsification time, postoperative spherical equivalent, higher-order aberrations (corneal and internal), corneal thickness, endothelial cell count, macular thickness, and complications during and after surgery. Both groups were absolutely comparable for all variables preoperatively. RESULTS: Mean ultrasound power was 1.8% ± 0.9% for MICS and 14.7% ± 4.9% for 2.2-mm incisions (P < .001). Effective phacoemulsification time values for MICS and 2.2-mm incisions were 1.5 ± 0.9 and 4.5 ± 2.9 sec, respectively (P = .002). Mean postoperative spherical equivalent was −0.26 for FemtoMICS and −0.33 for FemtoCoaxial (P > .05). The efficacy index at 1 month postoperatively was 160.2% for FemtoMICS and 149% for FemtoCoaxial. No significant differences were found in corneal thickness, endothelial cell count, and macular thickness. Complications included posterior capsule rupture (4%) and anterior capsule rupture with no posterior capsule tear (4%) for FemtoMICS and bridges due to incomplete capsulorhexis (4%) for FemtoCoaxial. CONCLUSIONS: MICS and coaxial phacoemulsification techniques assisted by the Femtosecond LenSx achieved excellent safety and efficient outcomes. The FemtoMICS technique was surgically and statistically more efficient than the FemtoCoaxial technique.

Femtosecond laser assisted cataract surgery followed by coaxial phacoemulsification or microincisional cataract surgery: differences and advantages.

PURPOSE OF REVIEW: This review outlines the advantages and the differences of femtosecond laser-assisted cataract surgery (FLACS) following a coaxial or microincision cataract surgery phacoemulsification in the surgical outcome and greater control of cataract surgery. RECENT FINDINGS: FLACS offers minimal tissue damage and extreme precision during corneal incision creation, continuous circular capsulorhexis (CCC) and nuclear fragmentation. It also allows diminishing the mean average ultrasound power to emulsify the nucleus followed by a coaxial or a biaxial procedure. The impact of reduced phacoemulsification energy on the corneal endothelium is an interesting topic that is being investigated. Despite its benefits, this technology has relevant financial issues and a high learning curve. SUMMARY: FemtoMICS appears to be surgically and statistically more efficient than the FemtoCoaxial technique and Femtoincisions prove to be stable and do not change the corneal high order aberration significantly with favorable results of the triplanar configuration.

Femtosecond laser cataract incision morphology and corneal higher-order aberration analysis.

PURPOSE: Analysis of the femtosecond laser refractive lens surgery corneal incision configuration and corneal higher-order aberration (HOA) effect from the first postoperative day. METHODS: High-resolution anterior segment optical coherence tomography was used to assess 20 eyes undergoing femtosecond laser refractive lens surgery with 2.2-mm minimal incision. The primary incision (tri-planar) actual length, cord length, surface angle, surface irregularity, and regional pachymetry values and the secondary incision (uni-planar) length, angle, surface irregularity, and pachymetry values were analyzed. Hartmann-Shack aberrometer was used to assess corneal HOAs to correlate the effect. Assessment was done preoperatively and 1 month postoperatively. RESULTS: The actual length, cord length, and surface angle means for the primary incision in the first postoperative day and month were 1.50 ± 0.1 and 1.47 ± 0.2 mm (P = .5), 1.41 ± 0.1 and 1.42 ± 0.2 mm (P = .8), and 27° ± 4° and 23° ± 5° (P = .07), respectively. The length and surface angle for the secondary incision in the first postoperative day and month were 1.17 ± 0.01 and 1.04 ± 0.1 mm (P = .05) and 52° ± 3° and 42° ± 5° (P = .007). The regional pachymetry values for the primary and secondary incisions were significantly increased in the first postoperative day and then significantly decreased after 1 month. All irregularities occurred in the posterior surface (endothelium): 2 cases of posterior gap (first day) and 1 case of posterior retraction (first month). The HOAs had not significantly changed preoperatively and after 1 month. CONCLUSIONS: The femtosecond laser refractive lens surgery incision is stable and does not significantly change the HOA.