The Negative Effects of Travel on Student Athletes Through Sleep and Circadian Disruption.

Collegiate athletes must satisfy the academic obligations common to all undergraduates, but they have the additional structural and social stressors of extensive practice time, competition schedules, and frequent travel away from their home campus. Clearly such stressors can have negative impacts on both their academic and athletic performances as well as on their health. These concerns are made more acute by recent proposals and decisions to reorganize major collegiate athletic conferences. These rearrangements will require more multi-day travel that interferes with the academic work and personal schedules of athletes. Of particular concern is additional east-west travel that results in circadian rhythm disruptions commonly called jet lag that contribute to the loss of amount as well as quality of sleep. Circadian misalignment and sleep deprivation and/or sleep disturbances have profound effects on physical and mental health and performance. We, as concerned scientists and physicians with relevant expertise, developed this white paper to raise awareness of these challenges to the wellbeing of our student-athletes and their co-travelers. We also offer practical steps to mitigate the negative consequences of collegiate travel schedules. We discuss the importance of bedtime protocols, the availability of early afternoon naps, and adherence to scheduled lighting exposure protocols before, during, and after travel, with support from wearables and apps. We call upon departments of athletics to engage with sleep and circadian experts to advise and help design tailored implementation of these mitigating practices that could contribute to the current and long-term health and wellbeing of their students and their staff members.

Methodologic Considerations for Studying the Ocular Surface Microbiome.

The ocular surface microbiome, unlike that of the skin or gut, has not been well characterized. Culture experiments historically suggested a nearly sterile ocular surface, but initial application of molecular methods such as 16S ribosomal RNA and high-throughput sequencing demonstrated a surprisingly rich ocular surface microbiome. However, a major limitation in studying such a low-biomass niche is the potential for artifactual results when amplification-based techniques such as ribosomal polymerase chain reaction and shotgun sequencing are used. It will be essential to establish standards across the field for sample collection, positive and negative controls, and limitation of contamination in both the laboratory setting and computational analysis. New developments in ocular microbiome research, including the generation of reference reagents and fluoroscopic imaging techniques, provide improved means to validate sequencing results and to visualize complex interactions between host cells and bacteria. Through more thorough characterization of the ocular surface microbiome, the connections between a dysregulated surface and ophthalmic disease may be better understood. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Translational roadmap for regenerative therapies of eye disease.

The translation of regenerative therapies to neuronal eye diseases requires a roadmap specific to the nature of the target diseases, patient population, methodologies for assessing outcome, and other factors. This commentary focuses on critical issues for translating regenerative eye therapies relevant to retinal neurons to human clinical trials.

Targeted adaptive long-read sequencing for discovery of complex phased variants in inherited retinal disease patients.

Inherited retinal degenerations (IRDs) are a heterogeneous group of predominantly monogenic disorders with over 300 causative genes identified. Short-read exome sequencing is commonly used to genotypically diagnose patients with clinical features of IRDs, however, in up to 30% of patients with autosomal recessive IRDs, one or no disease-causing variants are identified. Furthermore, chromosomal maps cannot be reconstructed for allelic variant discovery with short-reads. Long-read genome sequencing can provide complete coverage of disease loci and a targeted approach can focus sequencing bandwidth to a genomic region of interest to provide increased depth and haplotype reconstruction to uncover cases of missing heritability. We demonstrate that targeted adaptive long-read sequencing on the Oxford Nanopore Technologies (ONT) platform of the USH2A gene from three probands in a family with the most common cause of the syndromic IRD, Usher Syndrome, resulted in greater than 12-fold target gene sequencing enrichment on average. This focused depth of sequencing allowed for haplotype reconstruction and phased variant identification. We further show that variants obtained from the haplotype-aware genotyping pipeline can be heuristically ranked to focus on potential pathogenic candidates without a priori knowledge of the disease-causing variants. Moreover, consideration of the variants unique to targeted long-read sequencing that are not covered by short-read technology demonstrated higher precision and F1 scores for variant discovery by long-read sequencing. This work establishes that targeted adaptive long-read sequencing can generate targeted, chromosome-phased data sets for identification of coding and non-coding disease-causing alleles in IRDs and can be applicable to other Mendelian diseases.

Assessment of Murine Retinal Acuity Ex Vivo Using Multielectrode Array Recordings.

Purpose: Visual acuity, measured by resolution of optotypes on a standard eye chart, is a critical clinical test for function of the visual system in humans. Behavioral tests in animals can be used to estimate visual acuity. However, such tests may be limited in the study of mutants or after synthetic vision restoration techniques. Because the total response of the retina to a visual scene is encoded in spiking patterns of retinal ganglion cells, it should be possible to estimate visual acuity in vitro from the retina by analyzing retinal ganglion cell output in response to test stimuli. Methods: We created a method, EyeCandy, that combines a visual stimulus-generating engine with analysis of multielectrode array retinal recordings via a machine learning approach to measure murine retinal acuity in vitro. Visual stimuli included static checkerboards, drifting gratings, and letter optotypes. Results: In retinas from wild-type C57Bl/6 mice, retinal acuity measurement for a drifting grating was 0.4 cycles per degree. In contrast, retinas from adult rd1 mice with outer retinal degeneration showed no detectable acuity. A comparison of acuities among different regions of the retina revealed substantial variation, with the inferior-nasal quadrant having highest RA. Letter classification accuracy of a projected Early Treatment Diabetic Retinopathy eye chart reached 99% accuracy for logMAR 3.0 letters. EyeCandy measured a restored RA of 0.05 and 0.08 cycles per degree for static and dynamic stimuli respectively from the retina of the rd1 mouse treated with the azobenzene photoswitch BENAQ. Conclusions: Machine learning may be used to estimate retinal acuity. Translational Relevance: The use of ex vivo retinal acuity measurement may allow determination of effects of mutations, drugs, injury, or other manipulations on retinal visual function.

Photochemical Restoration of Light Sensitivity in the Degenerated Canine Retina.

Photopharmacological compounds such as azobenzene-based photoswitches have been shown to control the conductivity of ionic channels in a light-dependent manner and are considered a potential strategy to restore vision in patients with end-stage photoreceptor degeneration. Here, we report the effects of DENAQ, a second-generation azobenzene-based photoswitch on retinal ganglion cells (RGC) in canine retinas using multi-electrode array (MEA) recordings (from nine degenerated and six WT retinas). DENAQ treatment conferred increased light sensitivity to RGCs in degenerated canine retinas. RGC light responses were observed in degenerated retinas following ex vivo application of 1 mM DENAQ (n = 6) or after in vivo DENAQ injection (n = 3, 150 µL, 3-10 mM) using 455 nm light at intensities as low as 0.2 mW/cm2. The number of light-sensitive cells and the per cell response amplitude increased with light intensity up to the maximum tested intensity of 85 mW/cm2. Application of DENAQ to degenerated retinas with partially preserved cone function caused appearance of DENAQ-driven responses both in cone-driven and previously non-responsive RGCs, and disappearance of cone-driven responses. Repeated stimulation slowed activation and accelerated recovery of the DENAQ-driven responses. The latter is likely responsible for the delayed appearance of a response to 4 Hz flicker stimulation. Limited aqueous solubility of DENAQ results in focal drug aggregates associated with ocular toxicity. While this limits the therapeutic potential of DENAQ, more potent third-generation photoswitches may be more promising, especially when delivered in a slow-release formulation that prevents drug aggregation.

Machine Learning Prediction of Adenovirus D8 Conjunctivitis Complications from Viral Whole-Genome Sequence.

Objective: To obtain complete DNA sequences of adenoviral (AdV) D8 genome from patients with conjunctivitis and determine the relation of sequence variation to clinical outcomes. Design: This study is a post hoc analysis of banked conjunctival swab samples from the BAYnovation Study, a previously conducted, randomized controlled clinical trial for AdV conjunctivitis. Participants: Ninety-six patients with AdV D8-positive conjunctivitis who received placebo treatment in the BAYnovation Study were included in the study. Methods: DNA from conjunctival swabs was purified and subjected to whole-genome viral DNA sequencing. Adenovirus D8 variants were identified and correlated with clinical outcomes, including 2 machine learning methods. Main Outcome Measures: Viral DNA sequence and development of subepithelial infiltrates (SEIs) were the main outcome measures. Results: From initial sequencing of 80 AdV D8-positive samples, full adenoviral genome reconstructions were obtained for 71. A total of 630 single-nucleotide variants were identified, including 156 missense mutations. Sequence clustering revealed 3 previously unappreciated viral clades within the AdV D8 type. The likelihood of SEI development differed significantly between clades, ranging from 83% for Clade 1 to 46% for Clade 3. Genome-wide analysis of viral single-nucleotide polymorphisms failed to identify single-gene determinants of outcome. Two machine learning models were independently trained to predict clinical outcome using polymorphic sequences. Both machine learning models correctly predicted development of SEI outcomes in a newly sequenced validation set of 16 cases (P = 1.5 × 10-5). Prediction was dependent on ensemble groups of polymorphisms across multiple genes. Conclusions: Adenovirus D8 has ≥ 3 prevalent molecular substrains, which differ in propensity to result in SEIs. Development of SEIs can be accurately predicted from knowledge of full viral sequence. These results suggest that development of SEIs in AdV D8 conjunctivitis is largely attributable to pathologic viral sequence variants within the D8 type and establishes machine learning paradigms as a powerful technique for understanding viral pathogenicity.

Systemic prime exacerbates the ocular immune response to heat-killed Mycobacterium tuberculosis.

Post-infectious uveitis describes the condition of chronic immune mediated ocular inflammation associated with pathogens such as Mycobacterium tuberculosis (Mtb). Mtb associated post-infectious uveitis can be modeled in mice by intravitreal injection of heat-killed Mtb (HKMtb). To better understand how prior systemic exposure to the pathogen alters the local immune response to Mtb, we used flow cytometry and multiplex ELISAs to compare ocular responses to intravitreal HKMtb in the presence or absence of a systemic "prime" of HKMtb. Priming resulted in exacerbation of local inflammation with significantly increased clinical and histologic inflammation scores and increased vitreous cytokines concentrations one day after intravitreal injection of HKMtb. Seven days after injection, uveitis in unprimed animals had largely resolved. In contrast in primed animals, clinical signs of chronic inflammation were associated with a significant increase in the number of ocular T cells, NK cells, and Ly6Chi macrophages and increasing vitreous concentrations of IL-17, VEGF, MIG(CXCL9), IP-10(CXCL10), IL-12p40 and MIP-1α(CCL3). In mice lacking mature T and B cells (RAG2 deficient), the impact of priming on the ocular immune response was ameliorated with significantly lower vitreous cytokine concentrations and spontaneous resolution of uveitis. Altogether these results suggest that the ocular response to Mtb is exacerbated by prior systemic Mtb infection and chronic post-infectious uveitis is mediated by local production of cytokines and chemokines that amplify Th17 and Th1 responses. This mouse model of chronic Mtb associated uveitis will help elucidate mechanisms of disease in patients with post-infectious uveitis.

Simultaneous presentation of idiopathic uveitis in monozygotic 4-year-old twin boys.

Purpose: To report monozygotic twin 4-year-old boys with chronic bilateral anterior uveitis with simultaneous onset. Observations: Here we report monozygotic twin 4-year-old boys with chronic bilateral anterior uveitis. The boys had simultaneous onset of uveitis and identical features. Evaluation, including whole exome sequencing (WES), failed to reveal a specific causative etiology. Each patient responded well to immune modulation and achieved uveitis remission on methotrexate monotherapy off topical glucocorticoids. Conclusions and Importance: From this case of monozygotic twin boys presenting with chronic uveitis, we conclude that monozygotic twins may warrant evaluation in the setting of idiopathic uveitis, especially in young patients unable to express an adequate history.

Deep Metagenomic Sequencing for Endophthalmitis Pathogen Detection Using a Nanopore Platform.

PURPOSE: To evaluate the utility of nanopore sequencing for identifying potential causative pathogens in endophthalmitis, comparing culture results against full-length 16S rRNA nanopore sequencing (16S Nanopore), whole genome nanopore sequencing (Nanopore WGS), and Illumina (Illumina WGS). DESIGN: Cross-sectional diagnostic comparison. METHODS: Patients with clinically suspected endophthalmitis underwent intraocular vitreous biopsy as per standard care. Clinical samples were cultured by conventional methods, together with full-length 16S rRNA and WGS using nanopore and Illumina sequencing platforms. RESULTS: Of 23 patients (median age 68.5 years [range 47-88]; 14 males [61%]), 18 cases were culture-positive. Nanopore sequencing identified the same cultured organism in all of the culture-positive cases and identified potential pathogens in two culture-negative cases (40%). Nanopore WGS was able to additionally detect the presence of bacteriophages in three samples. The agreements at genus level between culture and 16S Nanopore, Nanopore WGS, and Illumina WGS were 75%, 100%, and 78%, respectively. CONCLUSIONS: Whole genome sequencing has higher sensitivity and provides a viable alternative to culture and 16S sequencing for detecting potential pathogens in endophthalmitis. Moreover, WGS has the ability to detect other potential pathogens in culture-negative cases. Whilst Nanopore and Illumina WGS provide comparable data, nanopore sequencing provides potential for cost-effective point-of-care diagnostics.

Regenerative and restorative medicine for eye disease.

Causes of blindness differ across the globe; in higher-income countries, most blindness results from the degeneration of specific classes of cells in the retina, including retinal pigment epithelium (RPE), photoreceptors, and retinal ganglion cells. Advances over the past decade in retinal regenerative medicine have allowed each of these cell types to be produced ex vivo from progenitor stem cells. Here, we review progress in applying these technologies to cell replacement - with the goal of vision restoration in degenerative disease. We discuss the landscape of human clinical trials for RPE transplantation and advanced preclinical studies for other cell types. We also review progress toward in situ repair of retinal degeneration using endogenous progenitor cells. Finally, we provide a high-level overview of progress toward prosthetic ocular vision restoration, including advanced photovoltaic devices, opsin-based gene therapy, and small-molecule photoswitches. Progress in each of these domains is at or near the human clinical-trial stage, bringing the audacious goal of vision restoration within sight.

Molecular Diagnostics for Ocular Infectious Diseases: LXXVIII Edward Jackson Memorial Lecture.

PURPOSE: To review the use of molecular diagnostic techniques in the management of ocular infectious disease. DESIGN: Retrospective review. METHODS: A combination of literature review and personal recollections are used. RESULTS: Although the broad term molecular diagnostics may encompass techniques to identify pathogens via protein or metabolomic signatures, this review concentrates on detection of pathogen nucleic acid as an indicator of infection. The introduction of the polymerase chain reaction (PCR) in 1985 opened a new era in analysis of nucleic acids. This technique was soon applied to the detection of potential pathogen DNA and RNA, including viruses, bacteria, and parasites in infectious eye disease. Advances in PCR have allowed class-specific diagnostics (ie, pan-bacterial and pan-fungal), quantitation of pathogen DNA, and multiplexed testing. The Human Genome Project in the early 2000s greatly accelerated development of DNA sequencers, ushering in the era of "Next Generation Sequencing" and permitting pathogen-agnostic methods for the detection of potential infectious agents. Most recently, new technologies such as nanopore sequencing have reduced both cost and equipment requirements for whole-genome sequencing; when coupled with real-time sequence analysis methods, these methods offer the promise of true, real-time, point-of-service ocular infectious disease diagnostics. CONCLUSIONS: Molecular methods for pathogen detection have greatly advanced the diagnosis of ocular infectious disease. Further methodologic advances will have a direct impact on the management of these conditions.

Molecular and Clinical Characterization of Human Adenovirus E4-Associated Conjunctivitis.

PURPOSE: To determine the characteristics of conjunctivitis associated with human adenovirus E4 (AdV E4). METHODS: Samples and outcomes from 500 patients with conjunctivitis were obtained from the NVC-422 randomized controlled clinical trial comparing auriclosene to placebo. Molecular typing identified 36 cases associated with AdV E4. Signs and symptoms at presentation and at the day 18 endpoint were compared with the larger cohort of 262 subjects with conjunctivitis caused by due to AdV D8. Full viral genomes of 22 AdV E4 isolates were reconstructed. RESULTS: AdV E4 was the most frequently identified adenoviral type in conjunctivitis cases from the United States. Signs and symptoms at presentation were comparable to those associated with AdV D8. Viral load at presentation was comparable between groups but resolution was more rapid in the AdV E4 group. Clinical signs were fully resolved by day 18 in 26 of 36 (72%) patients with AdV E4. Subepithelial infiltrates developed in 12 of 36 (33%) patients with AdV E4 compared with 98 of 215 (45%) patients with AdV D8 (P = .0001). One hundred twenty-four polymorphisms were observed among 22 whole viral genome sequences, which clustered into 3 clades. Patients in each clade developed subepithelial infiltrates. Neither single nucleotide polymorphism analysis nor machine learning approaches identified specific sequence features predictive of presenting signs or outcome. CONCLUSIONS: AdV E4 conjunctivitis may be indistinguishable at presentation from AdV D8-associated disease. Resolution of viral load for AdV E4 appears more rapid than for AdV D8, and the risk for subepithelial infiltrates appears lower. Multiple substrains of AdV E4 are in circulation but all appeared equivalently pathogenic for conjunctivitis. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Anti-adalimumab Antibodies in Patients with Non-infectious Ocular Inflammatory Disease: A Case Series.

PURPOSE: To report the clinical course of patients with ocular inflammatory disease treated with adalimumab in whom anti-adalimumab antibodies (AAA) were detected. METHODS: Single center case series. RESULTS: Eight patients with initial response to adalimumab developed a disease flare associated with positive AAA testing after 5 to 76 months of therapy. Six patients were receiving no concurrent antimetabolite therapy at the time of AAA diagnosis and four had a temporary lapse in adalimumab therapy prior to AAA discovery. AAA resulted in undetectable drug levels in five of the seven patients for whom data were available, and adalimumab was discontinued in six of the eight patients. Of two patients continued on adalimumab, one maintained detectable serum adalimumab despite AAA and one had a low AAA titer. CONCLUSIONS: For patients receiving adalimumab for ocular inflammatory disease, a disease flare in the setting of previously well-controlled disease should prompt consideration of AAA testing.