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Glaucoma and uveitis are non-vascular ocular diseases which are among the leading causes of blindness and visual loss. These conditions have distinct characteristics and mechanisms but share a multifactorial and complex nature, making their management challenging and burdensome for patients and clinicians. Furthermore, the lack of symptoms in the early stages of glaucoma and the diverse aetiology of uveitis hinder timely and accurate diagnoses, which are a cause of poor visual outcomes under both conditions. Although current treatment is effective in most cases, it is often associated with low patient adherence and adverse events, which directly impact the overall therapeutic success. Therefore, long-lasting alternatives with improved safety and efficacy are needed. Gene therapy, particularly utilising adeno-associated virus (AAV) vectors, has emerged as a promising approach to address unmet needs in these diseases. Engineered capsids with enhanced tropism and lower immunogenicity have been proposed, along with constructs designed for targeted and controlled expression. Additionally, several pathways implicated in the pathogenesis of these conditions have been targeted with single or multigene expression cassettes, gene editing and silencing approaches. This review discusses strategies employed in AAV-based gene therapies for glaucoma and non-infectious uveitis and provides an overview of current progress and future directions.

Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are vascular diseases with high prevalence, ranking among the leading causes of blindness and vision loss worldwide. Despite being effective, current treatments for AMD and DR are burdensome for patients and clinicians, resulting in suboptimal compliance and real risk of vision loss. Thus, there is an unmet need for long-lasting alternatives with improved safety and efficacy. Adeno-associated virus (AAV) is the leading vector for ocular gene delivery, given its ability to enable long-term expression while eliciting relatively mild immune responses. Progress has been made in AAV-based gene therapies for not only inherited retinal diseases but also acquired conditions with preclinical and clinical studies of AMD and DR showing promising results. These studies have explored several pathways involved in the disease pathogenesis, as well as different strategies to optimise gene delivery. These include engineered capsids with enhanced tropism to particular cell types, and expression cassettes incorporating elements for a targeted and controlled expression. Multiple-acting constructs have also been investigated, in addition to gene silencing and editing. Here, we provide an overview of strategies employing AAV-mediated gene delivery to treat AMD and DR. We discuss preclinical efficacy studies and present the latest data from clinical trials for both diseases., (© 2023. The Author(s).)

Introduction: The diagnosis of dry eye is challenging for eye health practitioners (EHP) and recently, a variety of new diagnostic tests have emerged. This study assesses the attitudes of EHP to dry eye and testing and compares these with attitudes in 2003., Methods: An electronic questionnaire was disseminated to EHP in Australasia between December 2020 to March 2021. Participants rated the likelihood that presenting symptoms/signs were associated with dry eye, the utility of diagnostic tests, the value of test characteristics, and their satisfaction with dry eye diagnostics. Qualitative responses were categorised into positive, negative, or neutral themes., Results: 144 responses were received, with 117 (81.3%) from Australia and 27 (18.7%) from New Zealand. Posterior blepharitis was significantly more likely to be associated with dry eye than other factors (p < 0.01). Clinical history, fluorescein staining and FBUT were judged significantly more useful in diagnosing dry eye compared to other tests (p < 0.01). Test validity was judged significantly more important in choosing a test than other qualities. Qualitative attitudes towards dry eye presentations and diagnostic tests were positive in 42.2% and 24.3%, negative in 32.4% and 41.9%, and neutral in 25.5% and 33.8% respectively., Conclusions: The opinions of EHP regarding dry eye tests were variable, but most favour history and corneal staining for diagnosis. Patterns of responses were similar to that reported by Turner et al 16-years ago, however, there is a higher satisfaction with available tests and therapeutic options. There is a need to develop a consensus amongst real-world clinicians regarding an optimum diagnostic pathway for dry eye, particularly in relation to newer diagnostic tests., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Uveal melanoma (UM) is the second-most-common melanoma in humans and has a high age-standardized incidence rate (ASR) in Australia. Regional patterns of UM ASRs in Australia are unknown. The aim of this study was to determine and compare UM ASRs in two geographically disparate eastern states, Queensland (QLD) and Victoria (VIC), by using cancer registry data that was obtained from 2001 to 2013. World-standardized UM ASRs and incidence-rate ratios (IRRs) were calculated. Higher UM ASR was also observed in anterior UM compared to posterior UM ASR. UM ASR remained unchanged from 2001 to 2013 in QLD but decreased in VIC. A south-to-north latitude trend in UM ASR along the east of Australia is weakly evident, and rural populations have higher UM ASRs than major city populations in both states. Differences in ultraviolent radiation (UVR) susceptibility, indigenous populations, social behaviours, chemical exposure, and socioeconomic status could all be contributing to differences in UM rates between QLD and VIC and between rural compared to major city areas. It is possible that a minority of cases in QLD and VIC might be prevented by sun-protective behaviours. This is important, because these findings suggest that QLD, which is already known to have one of the highest cutaneous melanoma (CM) ASRs in the world, also has one of the highest UM ASRs.

Neutralising antibodies (NAbs), caused by past adeno-associated virus (AAV) infection, represent a critical challenge for AAV-mediated gene therapy, with even low NAb titres capable of inhibiting gene transfer, however in protein-rich environments such as the vitreous it is expected that other constituents could also interact with the transduction process. Inhibition of AAV2/2, AAV2/5, AAV2/6 and AAV2/8 transduction by human vitreous humour (VH) obtained from 80 post-mortem eye cups was investigated in this report, with clinically relevant vitreous dilutions as low as 1:2. Unexpectedly, the highest prevalence of inhibition of transduction was observed against AAV2/6, with 66% of tested samples displaying neutralisation at a 1:2 VH dilution. Only two samples showed inhibition of AAV2/8, indicating this serotype is an attractive vector for use in non-vitrectomised eyes of unscreened individuals. Levels of anti-AAV NAbs observed in the VH were much lower than previously observed in serum of a similar Australian population. Among ten tested eye cup pairs, we observed only small variation in anti-AAV NAbs levels between the left and right eye cups. Interaction with 1:2 diluted VH had an augmentation effect on AAV2/8 transduction (p = 0.004), a phenomenon which was not due to albumin or transferrin and which, if developed, might benefit the use of AAV2/8 in clinical settings.

Uveal melanoma (UM) is currently classified by the World Health Organisation as a melanoma caused by risk factors other than cumulative solar damage. However, factors relating to ultraviolet radiation (UVR) susceptibility such as light-coloured skin and eyes, propensity to burn, and proximity to the equator, frequently correlate with higher risk of UM. These risk factors echo those of the far more common cutaneous melanoma (CM), which is widely accepted to be caused by excessive UVR exposure, suggesting a role of UVR in the development and progression of a proportion of UM. Indeed, this could mean that countries, such as Australia, with high UVR exposure and the highest incidences of CM would represent a similarly high incidence of UM if UVR exposure is truly involved. Most cases of UM lack the typical genetic mutations that are related to UVR damage, although recent evidence in a small minority of cases has shown otherwise. This review therefore reassesses statistical, environmental, anatomical, and physiological evidence for and against the role of UVR in the aetiology of UM.

Importance: Understanding the outcomes of interventions over time is essential for clinical decision making in surgical specialties., Background: Analysis of survival time (or time to event) is complicated when loss to follow up occurs. This article explores transparent data analysis methods where missing ("censored") data are present., Design: Manual search of the top 20 Ophthalmology journals from a recent year of the established literature (2014)., Samples: A total of 4565 articles were identified, of which 218 reported outcomes of treatment over time in humans., Methods: Pertinent details to assist the use of Kaplan-Meier and life table actuarial statistics are explained, and criteria that define whether each has high, acceptable or poor quality are explored. The quality of reporting from the literature sample is analysed., Main Outcome Measures: Reporting quality of survival curves and life tables from each sampled article is assessed according to the established criteria., Results: In total, 31.2% of samples (n = 68) presented survival curves, 53.2% (n = 116) presented life tables, 22% (n = 48) presented both, whilst 46.8% (n = 102) presented neither; 2% of survival curves and 13% of life tables were high quality, with quality of life tables significantly better than survival curves (P = .0042). 90.36% (n = 197) of articles reported time to event data which was classified as poor: due to poor analysis of survival curves (n = 50, 43.10%) poor analysis of life tables (n = 45, 66.18%); and complete omission of survival graphics (n = 102, 46.97%)., Conclusions and Relevance: Ophthalmology research that follows patient outcomes over time can be analysed with "time-to-event" statistics, and reported with transparency. This analysis showed that important contextural information was omitted from 90% of ophthalmic studies, and this could impact patient decision making., (© 2020 Royal Australian and New Zealand College of Ophthalmologists.)

Ultraviolet (UV) radiation is an accepted etiological factor in cutaneous melanoma (CM), however its role in uveal melanoma (UM) is controversial. Partly as a consequence, CM and UM are often considered to be separate conditions, and advances in the treatment of CM have not led to joint clinical trials or parallel improvements in survival of UM. This study hypothesized that a subset of UM tumors displays evidence of genetic changes consistent with UV-related damage similar to that shown in CM. Analysis of the Broad Institute's Firebrowse depository of 80 UM samples and 343 CM samples, together with the Sanger Institute's Catalogue of Somatic Mutations in Cancer depository of 995 UM and 12,447 CM samples was undertaken to identify the most frequently mutated genes, mutation types, and specific nucleotide variants (SNVs) in each condition. Somatic mutation data were cross-correlated and shared mutations assessed against known effects of UV radiation. The proportion of samples with C>T substitutions (a classic genetic marker of UV-related damage) was higher in UM than CM on both DNA strands (17.0% vs 13.1%, p=0.038). The most frequently encountered cross-correlated mutated genes between UM and CM were, in order, BRAF, NRAS, TP53, CDKN2A, TERT, PTEN, ARID2, and KMT2C, with multiple common BRAF point mutations. Each cross-correlated mutation, and each common point mutation in BRAF, was associated with UV-related mechanistic changes. These findings support the hypothesis that the etiology of a substantial minority of UMs may be more UV dependent than previously recognized.

One of the major challenges in studying ocular toxicology is a lack of clinically-relevant retinal experimental models. In this study we describe the use of an in vitro human retinal explant strategy to generate a reproducible experimental model with utility in neuro-toxicity retinal studies. A retinal dissection strategy, referred to as the 8 fold quadrant dissection, was developed by dissecting human donor retinas into 4 fragments through the fovea in order to obtain 8 experimentally reproducible retinal explants from a single donor. This quadrant dissection gave rise to equivalent proportions of CD73+ photoreceptors and CD90+ ganglion cells in 8 fragments from a single donor and this remained stable for up to 3 days in culture. Major retinal cell types continued to be observed after 8 weeks in culture, despite breakdown of the retinal layers, suggesting the potential to use this model in long-term studies where observation of individual cell types is possible. The utility of this system was examined in a proof of principle neuro-toxicology study. We showed reproducible induction of toxicity in photoreceptors and retinal ganglion cells by glutamate, cobalt chloride and hydrogen peroxide insults, and observed the therapeutic positive effects of the administration of memantine, formononetin and trolox. The quadrant dissected human retinal explants have the potential to be used in toxicology studies in human ocular diseases., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA-RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2. Despite prior findings of linear sequence motifs, our measurements revealed widespread residue flipping and instances of positional coupling. Application of our thermodynamic model to published in vivo crosslinking data reveals quantitative agreement between predicted affinities and in vivo occupancies. Our analyses suggest a thermodynamically driven, continuous Pumilio-binding landscape that is negligibly affected by RNA structure or kinetic factors, such as displacement by ribosomes. This work provides a quantitative foundation for dissecting the cellular behavior of RBPs and cellular features that impact their occupancies., (Copyright © 2019. Published by Elsevier Inc.)

Recombinant Adeno-associated viruses (AAVs) are an attractive vector for gene therapy delivery which may be blocked by AAV neutralising antibodies (NAbs). As Type 1 Diabetes (T1DM) is an endocrine disease of immunological origin, it is likely that NAb profiles are altered in the disease. In this study NAb to AAV2, AAV5, AAV6, and AAV8 in 72 subjects with T1DM and 45 non-diabetic patients were measured over a 4-year follow-up period. AAV2 NAb titres were significantly lower in non-diabetic subjects (P = 0.036). The T1DM group had more AAV8 NAb activity at baseline (P = 0.019), whilst after 4 years follow-up the T1DM group displayed developed increased AAV 5 (P = 0.03), 6 (P = 0.03) and 8 (P = 0.002) activity relative to the control group, however, overall AAV5 and 8 NAb levels were very low in patients <40. AAV NAb titre activity and prevalence generally appears higher in T1DM, however, low levels of AAV 5 and 8, particular in younger adult age groups at which T1DM can be targeted, could make these attractive vectors to target the disease.

Voretigene neparvovec-rzyl was recently approved for the treatment of Leber congenital amaurosis, and the use of gene therapy for eye disease is attracting even greater interest. The eye has immune privileged status, is easily accessible, requires a reduced dosage of therapy due to its size and is highly compartmentalized, significantly reducing systemic spread. Adeno-associated virus (AAV), with its low pathogenicity, prolonged expression profile and ability to transduce multiple cell types, has become the leading gene therapy vector. Target diseases have moved beyond currently untreatable inherited dystrophies to common, partially treatable acquired conditions such as exudative age-related macular degeneration and glaucoma, but use of the technology in these conditions imposes added obligations for caution in vector design. This review discusses the current status of AAV gene therapy trials in genetic and acquired ocular diseases, and explores new scientific developments, which could help ensure effective and safe use of the therapy in the future., (© 2018 Royal Australian and New Zealand College of Ophthalmologists.)

High-throughput DNA sequencing techniques have enabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3 × 10 4 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a "superFLAG" epitope variant. We also measured the enzymatic activity of 1.56 × 10 5 molecular variants of full-length human O 6 -alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates revealed amino acid interaction networks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The retina is the tissue responsible for light detection, in which retinal neurons convert light energy into electrical signals to be transported towards the visual cortex. Damage of retinal neurons leads to neuronal cell death and retinal pathologies, compromising visual acuity and eventually leading to irreversible blindness. Models of retinal neurodegeneration include 2D systems like cell lines, disassociated cultures and co-cultures, and 3D models like organoids, organotypic retinal cultures and animal models. Of these, ex vivo human retinal cultures are arguably the most suitable models for translational research as they retain complex inter-cellular interactions of the retina and precisely mimic in-situ responses. In this review, we summarize the distinguishing features of the human retina which are important to preserve in experimental culture, the historical development of human retinal culture systems, the factors affecting ex vivo human retinal culture and the applications and challenges associated with current methods of human retinal explant culture., (© 2018 Royal Australian and New Zealand College of Ophthalmologists.)

Purpose: Diabetic retinal neuropathy refers to retinal neural tissue damage occurring before the structural retinal changes of diabetic retinopathy and fulfils many of the criteria for causality for the subsequent vasculopathy. Developing reliable means of measuring neuronal damage in diabetes may be important in efforts to prevent retinopathy of a clinically significant and irreversible stage. This study aimed at systematically assessing current clinical measurements of diabetic retinal neuropathy so that future studies may utilise a consensual battery of tests in studying this poorly understood disease state between a healthy retina and one that is retinopathic., Methods: A systematic search of the medical literature since 1984 was performed on PUBMED and EMBASE, and the evidence supporting each identified method as an indicator for clinically important diabetic retinal neuropathy was graded relatively as compelling, medium, or weak according to criteria assessing its relationship to subsequent diabetic retinopathy, quality of supporting studies, and published reproducibility., Results: The systematic search yielded 6432 results. Subsequent assessment by two independent investigators identified 601 multiple subject studies in humans assessing clinical aspects of the retinal structure, function, or psychophysics in the prediabetic retina. The 933 separate instances of clinical methods assessed as being supported by relatively "compelling" evidence included colour vision changes, flash ERG b-wave latency, flash multifocal b-wave latency, scotopic b-wave and oscillatory potentials in ERG, and contrast sensitivity., Conclusion: The results showed moderately poor quality of extant evidence and indicate the best clinical methods for assessing diabetic retinal neuropathy that remain to be confirmed. This is the first systematic assessment of the medical literature aiming at assessing the breadth and validity of these methods and represents an early step in identifying and developing clinical endpoints for use in trials designed to identify at-risk patients or prevent diabetic retinopathy.

A gene therapeutic platform needs to be both efficient and safe. The criterion of safety is particularly important for diseases like hepatocellular carcinoma (HCC), which develop in a background of an already compromised liver. Gene vectors can be constructed either by targeting HCC or by detargeting liver and/or other major organs. miRNA-based negative detargeting has gained considerable attention in recent times due to its effectiveness and the ease with which it can be adapted into current gene delivery vectors. In this study, we provide a proof-of-concept using miRNA199a as a negative targeting agent. We introduced vectors harboring reporters with miRNA199a binding sites in cells expressing high endogenous levels of miRNA199a and compared the reporter expression in HCC cells with low endogenous miRNA199a. We observed that the expression of reporters with miRNA199a binding sites is significantly inhibited in miRNA199a-positive cells, whereas minimal effect was observed in miRNA199a-negative HCC cells. In addition, we created a post-transcriptionally regulated suicide gene therapeutic system based on cytosine deaminase (CD)/5-fluorocytosine (5-FC) exploiting miRNA199a binding sites and observed significantly lower cell death for miRNA199a-positive cells. Furthermore, we observed a decrease in the levels of miRNA199 in 3D tumorspheres of miRNA199a-positive Hepa1-6 cells and a reduction in the inhibition of reporter expression after transfection in these 3D models when compared with 2D Hepa1-6 cells. In summary, we provide evidence of miRNA199a-based post-transcriptional detargeting with relevance to HCC gene therapy., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Intravitreal injection of a therapeutic substance is the most common procedure performed in ophthalmology. It has a low incidence of serious complications but is associated with a small chance of endophthalmitis. Although the rate of endophthalmitis is between 0.019% and 0.09%, the associated visual morbidity is often devastating. Procedural changes have evolved over the years to improve patient comfort and reduce injection-related injury and infection. Despite the availability of published evidence, there remains considerable variations and lack of consensus in practical clinical settings. In addition, emerging literature concerning the use of speculums, the use of prophylactic topical antibiotics, and the setting of injections continues to impact the ophthalmologist's injection practice. This article provides an up to date assessment of various aspects of the procedure such as the setting, ventilation, type of anaesthetic, and control of sterility during the procedure; including discussions on performing bilateral eye same-day injections and the use of antibiotics.

In this study, we report a miRNA122a based targeted gene therapy for hepatocellular cancer stem cells (CSCs). First, we assessed the levels of miRNA122a in normal human hepatocytes, a panel of hepatocellular carcinoma (HCC) cell lines and hepatocellular CSCs observing its significant downregulation in HCC and CSCs. The miRNA122a binding site was then incorporated at the 3'-UTR of reporter genes gaussia luciferase (GLuc) and eGFP which resulted in significant hepatocyte detargeting. Using this strategy for the delivery of gene directed enzyme prodrug therapy (GDEPT) utilizing the cytosine deaminase/5-fluorocytosine (CD/5-FC) system, we showed significant killing in cells with low or no miRNA122a while those cells, such as hepatocytes with high miRNA122a were largely spared. Next, we showed that CSC enriched tumorspheres exhibit a significant downregulation of miRNA122a expression providing a rational to exploit its binding site for targeted gene delivery. Using plasmids harboring reporters GLuc and eGFP with or without miR122a binding sites, we showed high reporter expression in the CSCs and little reported expression in the non-enriched cultures. Finally, we demonstrate the efficacy of miRNA122a based post-transcriptionally targeted GDEPT for hepatocellular CSCs., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest to declare.

Hepatocellular carcinoma (HCC) is a major health problem as evidenced by its increasing incidence and high morbidity and mortality rates. Most patients with HCC have underlying liver disease and dysfunction which limits the current therapeutic options. Treatments that spare the liver and destroy the HCC are needed. Targeting transcriptional differences between HCC and liver cells may provide this therapeutic window. In this study, we examine the potential of the Glypican 3 (GPC3) promoter as a targeting strategy. GPC3 is an oncofetal protein belonging to the proteoglycan family which is normally only expressed during fetal development. However, in HCC, the expression of this protein is reactivated. Here, we show that GPC3 is expressed primarily in HCC and not in normal liver lines. We show that the GPC3 promoter can be used to drive expression of significantly more luciferase and eYFP in HCC cell lines compared to normal liver cells. Further, we show that vectors containing cytosine deaminase (CD) under GPC3 promotor control induced significantly more killing of HCC cell lines after treatment with 5-FC compared to normal liver cell lines. These data suggest that transcriptionally targeted delivery of transgene in HCC cells can be achieved using the GPC3 promoter and this targeting strategy produces limited toxicity to normal liver cells.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer with high incidence globally. Increasing mortality and morbidity rates combined with limited treatment options available for advanced HCC press for novel and effective treatment modalities. Gene therapy represents one of the most promising therapeutic options. With the recent approval of herpes simplex virus for advanced melanoma, the field of gene therapy has received a major boost. Adeno-associated virus (AAV) is among the most widely used and effective viral vectors today with safety and efficacy demonstrated in a number of human clinical trials. This review identifies the obstacles for effective AAV based gene delivery to HCC which primarily include host immune responses and off-target effects. These drawbacks could be more pronounced for HCC because of the underlying liver dysfunction in most of the patients. We discuss approaches that could be adopted to tackle these shortcomings and manufacture HCC-targeted vectors. The combination of transductional targeting by modifying the vector capsid and transcriptional targeting using HCC-specific promoters has the potential to produce vectors which can specifically seek HCC and deliver therapeutic gene without significant side effects. Finally, the identification of novel HCC-specific ligands and promoters should facilitate and expedite this process.

RNA-binding proteins (RBPs) control the fate of nearly every transcript in a cell. However, no existing approach for studying these posttranscriptional gene regulators combines transcriptome-wide throughput and biophysical precision. Here, we describe an assay that accomplishes this. Using commonly available hardware, we built a customizable, open-source platform that leverages the inherent throughput of Illumina technology for direct biophysical measurements. We used the platform to quantitatively measure the binding affinity of the prototypical RBP Vts1 for every transcript in the Saccharomyces cerevisiae genome. The scale and precision of these measurements revealed many previously unknown features of this well-studied RBP. Our transcribed genome array (TGA) assayed both rare and abundant transcripts with equivalent proficiency, revealing hundreds of low-abundance targets missed by previous approaches. These targets regulated diverse biological processes including nutrient sensing and the DNA damage response, and implicated Vts1 in de novo gene "birth." TGA provided single-nucleotide resolution for each binding site and delineated a highly specific sequence and structure motif for Vts1 binding. Changes in transcript levels in vts1 Δ cells established the regulatory function of these binding sites. The impact of Vts1 on transcript abundance was largely independent of where it bound within an mRNA, challenging prevailing assumptions about how this RBP drives RNA degradation. TGA thus enables a quantitative description of the relationship between variant RNA structures, affinity, and in vivo phenotype on a transcriptome-wide scale. We anticipate that TGA will provide similarly comprehensive and quantitative insights into the function of virtually any RBP.

Diabetes causes a panretinal neurodegeneration herein termed diabetic retinal neuropathy, which manifests in the retina early and progresses throughout the disease. Clinical manifestations include changes in the ERG, perimetry, dark adaptation, contrast sensitivity and colour vision which correlate with laboratory findings of thinning of the retinal neuronal layers, increased apoptosis in neurons and activation of glial cells. Possible mechanisms include oxidative stress, neuronal AGE accumulation, altered balance of neurotrophic factors and loss of mitohormesis. Retinal neural damage precedes and is a biologically plausible cause of retinal vasculopathy later in diabetes, and this review suggests that strategies to target it directly could prevent diabetes induced blindness. The efficacy of fenofibrate in reducing retinopathy progression provides a possible proof of concept for this approach. Strategies which may target diabetic retinal neuropathy include reducing retinal metabolic demand, improving mitochondrial function with AMPK and Sirt1 activators or providing neurotrophic support with neurotrophic supplementation., (© 2016 Royal Australian and New Zealand College of Ophthalmologists.)

Uveal melanoma (UM) is the most common primary ocular tumour in adults. Despite good local control of the primary tumour with current methods, survival after the development of metastasis has remained poor over the last 30 years. After cutaneous melanoma, UM is the most common type of melanoma, and an ongoing debate exists regarding whether these conditions should be considered separate entities, particularly in the context of targeted therapy, where many of the initial trials for patients with metatatic cutaneous melanoma excluded metastatic UM. This paper will review the recent and ongoing investigations designed to validate systemic targeted therapy and immunotherapy in patients with metastatic UM and suggests ways in which these developments may affect management of UM by ophthalmologists in the near future., (© 2015 Royal Australian and New Zealand College of Ophthalmologists.)

Clinical prognostic markers in atrophic age-related macular degeneration include the extent of existing atrophy, fundus autofluorescence (FAF) patterns and optical coherence tomography changes in the outer retina/retinal pigment epithelium interface. The prognostic implications of these findings may be used to determine not just the rate of disease progression but also influence the likelihood, magnitude and clinical relevance of therapy responses. FAF phenotypes have been extensively investigated; however, the pathophysiological mechanisms behind their appearance have not been fully elucidated. Optical coherence tomography imaging is additive to FAF imaging in atrophic age-related macular degeneration, allowing the visualization of detail not available through FAF imaging whilst also displaying subtle changes correlating with the FAF phenotypes themselves, thereby giving clues to their histological determinates. The developing understanding of these imaging modalities and consequent development of prognostically useful classification systems have widespread implication in clinical care and clinical trial design., (© 2015 Royal Australian and New Zealand College of Ophthalmologists.)

Background: The effect of excess glucose on retinal cellular health remains controversial, and cellular reducing equivalents, as indicators of cellular energy production, are widely used as substitute indicators of retinal cellular health. These investigations hypothesised that excess energy substrate availability, as occurs in the diabetic retina, increases the susceptibility of retinal neurons to injury in the presence of increased cellular reducing equivalents., Methods: The response of 661W cells to phototoxicity, oxidative stress induced by H2O2 and apoptosis induction by staurosporine was characterised in the presence of 5mM glucose and B27 defined media without insulin. Cellular insult was produced by phototoxicity, H2O2 and the apoptosis induction agent staurosporine. The effect of physiologically relevant alterations in environmental glucose on cellular reducing equivalents was assessed by MTT dye reduction and NAD(P)H assays, and cell survival was assessed via caspase 3/7 activation and Annexin V/PI flow cytometry., Results: 661W photoreceptor-like cells underwent dose dependent cell death primarily by apoptosis in response to phototoxic insult, H2O2, and staurosporine by all measures of cellular viability. Exposure of cells to 25mM glucose (diabetic-type conditions) increased cell death in response to all insults as measured by caspase 3/7 activation and Annexin V/PI flow cytometry. Cellular reducing equivalents were nonetheless increased in all models of injury in the presence of excess glucose. The mechanism of this increase was partly due to increased NADPH but not NADH levels in the presence of 25mM glucose., Conclusions: Acute exposure to 25mM glucose decreased the resilience of 661W photoreceptor-like cells to a range of cellular stressors whilst maintaining or increasing cellular reducing equivalents, partly be increasing NADPH levels. This shows that in 661W cells, diabetic levels of glucose decrease cellular resilience to injury. The decoupling of cellular reducing equivalents levels from cell survival has important implications when investigating the mechanisms of neuronal damage in diabetic retinal neuropathy.

RNA-protein interactions drive fundamental biological processes and are targets for molecular engineering, yet quantitative and comprehensive understanding of the sequence determinants of affinity remains limited. Here we repurpose a high-throughput sequencing instrument to quantitatively measure binding and dissociation of a fluorescently labeled protein to >10(7) RNA targets generated on a flow cell surface by in situ transcription and intermolecular tethering of RNA to DNA. Studying the MS2 coat protein, we decompose the binding energy contributions from primary and secondary RNA structure, and observe that differences in affinity are often driven by sequence-specific changes in both association and dissociation rates. By analyzing the biophysical constraints and modeling mutational paths describing the molecular evolution of MS2 from low- to high-affinity hairpins, we quantify widespread molecular epistasis and a long-hypothesized, structure-dependent preference for G:U base pairs over C:A intermediates in evolutionary trajectories. Our results suggest that quantitative analysis of RNA on a massively parallel array (RNA-MaP) provides generalizable insight into the biophysical basis and evolutionary consequences of sequence-function relationships.

Purpose. To determine factors associated with the presence of significant ocular injury in subjects with orbital fractures. Subjects. A consecutive prospective cohort of 161 patients presenting to a general tertiary referral hospital with orbital fractures and undergoing initial conservative treatment was identified. Subjects were assessed at time of injury for the need for emergency surgery, and those initially treated conservatively were subsequently followed up by the Ophthalmology Department to assess for ocular injury requiring ophthalmic management at 1-7 days after injury. Associations between ocular injury and age, sex, visual acuity, presence of blowout fracture, extent of orbital involvement, and presence of distant facial fractures were assessed. Results. 142 male (average age of 32 [95% CI 30-35]) and 19 female (average age of 49 [95% CI 39-59]) subjects were identified. 17 subjects were diagnosed with significant ocular injury. Ocular injury was significantly associated with LogMAR VA worse than 0.2 (OR 49 [95% CI 11-217, P < 0.0001]), but no relationship was noted for age, sex, presence of blowout fracture, extent of fractures, or presence of distal facial fractures. LogMAR visual acuity worse than or equal to 0.2 had a 98% negative predictive value for ocular injury in the setting of orbital fractures. Conclusions. Demographic and nonophthalmic fracture characteristics were not useful predictors of ocular injury in orbital fractures. LogMAR visual acuity worse than or equal to 0.2 is a highly sensitive and useful guide of the need for ophthalmic referral in subjects with orbital fractures.

We present a case of a 33-year-old female who was incidentally found to have cutaneous leiomyomata during a routine skin examination. Further history revealed that she also suffered from uterine fibroids and that her mother had died at an early age from renal cell carcinoma. This case serves as a reminder of the often-subtle cutaneous clues, as well as the importance of a multidisciplinary approach, for early diagnosis of potentially fatal conditions.

A 14-year-old girl presented with a 3-week history of mucosal erosions, injected conjunctiva, dehydration, and respiratory distress. She had been treated with intravenous acyclovir for herpes simplex infection with positive herpes simplex virus immunoglobulin M and immunoglobulin G. Physical examination and imaging revealed a large abdominal mass. Incisional biopsy was obtained, and pathology demonstrated angiofollicular hyperplasia with hyalinized germinal centers and Castleman's syndrome-like features. Based on the mucosal erosions, herpes simplex virus serology and positive herpes simplex virus-1 direct fluorescent antibody, Castleman's disease secondary to overwhelming herpes simplex virus infection was the initial impression. The poor response to antivirals and subsequent development of a bullous eruption on the hands resulted in dermatology consultation. Skin biopsy was obtained from a bullae and revealed suprabasilar acantholysis with necrosis as well as upper dermal, perivascular, and interface infiltrate of lymphocytes and eosinophils. No viropathic changes were present. Direct immunofluorescence was significant for immunoglobulin G deposition intercellularly and along the dermoepidermal junction and focal trace C3 deposition along the dermoepidermal junction consistent with paraneoplastic pemphigus, later confirmed by indirect immunofluorescence. We report this case of paraneoplastic pemphigus secondary to Castleman's syndrome confounded by herpes simplex virus-1 positive mucosal erosions., (© 2011 Wiley Periodicals, Inc.)

Assays that integrate detection of binding with cell-free protein expression directly from DNA can dramatically increase the pace at which protein-protein interactions (PPIs) can be analyzed by mutagenesis. In this study, we present a method that combines in vitro protein production with an enzyme-linked immunosorbent assay (ELISA) to measure PPIs. This method uses readily available commodity instrumentation and generic antibody-affinity tag interactions. It is straightforward and rapid to execute, enabling many interactions to be assessed in parallel. In traditional ELISAs, reporter complexes are assembled stepwise with one layer at a time. In the method presented here, all the members of the reporter complex are present and assembled together. The signal strength is dependent on all the intercomponent interaction affinities and concentrations. Although this assay is straightforward to execute, establishing proper conditions and analysis of the results require a thorough understanding of the processes that determine the signal strength. The formation of the fully assembled reporter sandwich can be modeled as a competition between Langmuir adsorption isotherms for the immobilized components and binding equilibria of the solution components. We have shown that modeling this process provides semiquantitative understanding of the effects of affinity and concentration and can guide strategies for the development of experimental protocols. We tested the method experimentally using the interaction between a synthetic ankyrin repeat protein (Off7) and maltose-binding protein. Measurements obtained for a collection of alanine mutations in the interface between these two proteins demonstrate that a range of affinities can be analyzed., (Copyright © 2011 The Protein Society.)

Thermal stability shift analysis is a powerful method for examining binding interactions in proteins. We demonstrate that under certain circumstances, protein-protein interactions can be quantitated by monitoring shifts in thermal stability using thermodynamic models and data analysis methods presented in this work. This method relies on the determination of protein stabilities from thermal unfolding experiments using fluorescent dyes such as SYPRO Orange that report on protein denaturation. Data collection is rapid and straightforward using readily available real-time polymerase chain reaction instrumentation. We present an approach for the analysis of the unfolding transitions corresponding to each partner to extract the affinity of the interaction between the proteins. This method does not require the construction of a titration series that brackets the dissociation constant. In thermal shift experiments, protein stability data are obtained at different temperatures according to the affinity- and concentration-dependent shifts in unfolding transition midpoints. Treatment of the temperature dependence of affinity is, therefore, intrinsic to this method and is developed in this study. We used the interaction between maltose-binding protein (MBP) and a thermostable synthetic ankyrin repeat protein (Off7) as an experimental test case because their unfolding transitions overlap minimally. We found that MBP is significantly stabilized by Off7. High experimental throughput is enabled by sample parallelization, and the ability to extract quantitative binding information at a single partner concentration. In a single experiment, we were able to quantify the affinities of a series of alanine mutants, covering a wide range of affinities (∼ 100 nM to ∼ 100 μM)., (Copyright © 2011 The Protein Society.)

The quantification of protein-ligand interactions is essential for systems biology, drug discovery, and bioengineering. Ligand-induced changes in protein thermal stability provide a general, quantifiable signature of binding and may be monitored with dyes such as Sypro Orange (SO), which increase their fluorescence emission intensities upon interaction with the unfolded protein. This method is an experimentally straightforward, economical, and high-throughput approach for observing thermal melts using commonly available real-time polymerase chain reaction instrumentation. However, quantitative analysis requires careful consideration of the dye-mediated reporting mechanism and the underlying thermodynamic model. We determine affinity constants by analysis of ligand-mediated shifts in melting-temperature midpoint values. Ligand affinity is determined in a ligand titration series from shifts in free energies of stability at a common reference temperature. Thermodynamic parameters are obtained by fitting the inverse first derivative of the experimental signal reporting on thermal denaturation with equations that incorporate linear or nonlinear baseline models. We apply these methods to fit protein melts monitored with SO that exhibit prominent nonlinear post-transition baselines. SO can perturb the equilibria on which it is reporting. We analyze cases in which the ligand binds to both the native and denatured state or to the native state only and cases in which protein:ligand stoichiometry needs to treated explicitly.

Objective: To determine factors associated with physician discovery of early melanoma in middle-aged and older men., Design: Survey., Setting: Three institutional melanoma clinics., Participants: A total of 227 male participants (aged > or =40 years) with invasive melanoma who completed surveys within 3 months of diagnosis. Intervention Survey., Main Outcome Measures: Factors associated with physician-detected thin melanoma., Results: Patients with physician-detected melanoma were older, 57% were 65 years or older compared with 34% for other-detected (odds ratio [OR], 2.57; 95% confidence interval [CI], 1.19-5.55) and 42% for patient-detected melanoma (P = .07). Physician-detected melanoma in the oldest patients (aged > or =65 years) had tumor thickness equal to that of self-detected melanoma or melanoma detected by other means in younger patients. Back lesions composed 46% of all physician-detected melanoma, 57% of those detected by other means, and 16% of self-detected lesions (physician- vs self-detected: OR, 4.25; 95% CI, 1.96-9.23). Ninety-two percent of all physician-detected back-of-the-body melanomas were smaller than 2 mm compared with 63% of self-detected lesions (P = .004) and 76% of lesions detected by other means (P = .07)., Conclusions: Skin screenings of at-risk middle-aged and older American men can be integrated into the routine physical examination, with particular emphasis on hard-to-see areas, such as the back of the body. "Watch your back" professional education campaigns should be promoted by skin cancer advocacy organizations.

Objectives: To identify factors related to the detection of melanoma and to determine those that differ between thinner vs thicker tumors in middle-aged and older men., Design: Survey., Setting: Three institutional melanoma clinics., Participants: Men 40 years or older who had newly diagnosed invasive melanoma., Main Outcome Measures: Differences in melanoma awareness, skin examination practices, discovery patterns, and social/medical care factors relative to tumor thickness., Results: Two hundred twenty-seven men completed surveys within 3 months of melanoma diagnosis; 57 (25.1%) had thicker tumors (>2.00 mm). Thicker tumors were associated with nodular histologic features (43.9%), a lack of atypical nevi, having less than a high school education, and patient vs physician (dermatologist or nondermatologist) detection. Knowledge of melanoma (P = .007), attention to skin cancer detection information (P = .02), an interest in health topics (P = .003), and knowing the importance of physician skin examination (P = .05) were more common in those with thin tumors. Tumor thickness did not correlate with age, anatomic location, marital/cohabitation status, prior skin cancer, or sun sensitivity. Overall patient awareness of melanoma warning signs, skin self-examination practices, and Internet use were poor (<20%, <50%, and <14%, respectively)., Conclusions: Physician discovery, the patient's higher level of education and detection-promoting awareness and attitudes, and the presence of clinically atypical nevi were related to thinner melanomas. Innovative outreach strategies and novel educational campaigns incorporating these factors, coupled with sharper messages regarding the importance of physician screening, are needed to improve early detection in middle-aged and older men.

Background: Diabetes inhibits dark adaptation and both processes alter the electroretinogram (ERG) in similar ways. This study aimed to investigate the relationship between oscillatory potentials (OPs) and the b-wave during dark adaptation and to determine if this relationship changes during the development of diabetes., Methods: Twenty-one rats were assigned to adaptation, control and diabetic groups. Rats were dark adapted for periods between 20 minutes and 4 hours, and ERGs recorded. Diabetes was induced with streptozotocin, and ERGs measured after 3, 6, 9 and 12 weeks after injection., Results: Increasing periods of dark adaptation led to a logarithmic increase in the amplitude of the b-wave and the OPs. This was accompanied by a decrease in the peak times of the OPs and b-wave. Total OP amplitude and b-wave amplitude were linearly related, allowing an empirical OP constant to be developed to describe the relationship between the two parameters. Diabetes led to a progressive decrease in the amplitude and increase in the peak time of all waves. The OP constant decreased in a linear fashion with increasing duration of diabetes., Conclusions: It is argued that OP masking of the b-wave could explain previous inconsistencies in reported ERG changes in diabetes and that a slowing of dark adaptation does not account for these ERG changes. The report concludes that the OPs and b-wave amplitudes and latencies are intimately related in the normal retina and that this correlation is lost predictably during the development of diabetes.

Digital dermoscopy systems employ computer-based algorithms to quantitate features of pigmented skin lesions (PSLs) and provide an assessment of malignancy risk. We evaluated interobserver concordance of PSL malignancy risk between a pigmented lesion specialist and an artificial neural network (ANN)-based automated digital dermoscopy system. While digital dermoscopy provides a reliable means of image capture, storage, and comparison of PSLs over time, the ANN algorithm requires further training and validation before the malignancy risk assessment feature can be widely used in clinical practice.

Purpose: The diabetic retina exhibits decreases in endogenous nonangiogenic neurotrophins. This study hypothesized that deficiencies in systemic and retinal pigment epithelium-derived (RPE) neurotrophic factors also influence retinal changes in diabetes., Methods: Diabetes was established in Listar hooded rats with streptozotocin. Reverse transcriptase coupled polymerase chain reaction (RT-PCR) and immunoblotting were used to determine the expression of fibroblast growth factor-2 (FGF-2) in the retina and RPE, and glial fibrillary acid protein (GFAP) in the retina. In addition, primary human RPE cultures and a transformed Müller cell line were used to determine the effect of insulin, glucose, and insulin-like growth factor (IGF) on the expression of these substances., Results: FGF-2 and GFAP were increased in retina, but FGF-2 was decreased in the RPE of diabetic animals. Retinal GFAP correlated with RPE FGF-2 expression in these animals. Insulin produced a dose-dependent increase in FGF-2 in RPE cells and decrease in GFAP in Müller cells grown in 15 mM glucose. In 5 mM glucose, insulin had no effect on expression of either protein. Physiological levels of insulin inhibited changes induced by 15 mM glucose. The effect of 9 nM insulin on each culture was mimicked by 1 nM IGF, and blocked with an IGFR-1 inhibitor., Conclusions: It is suggested that decreased systemic insulin and high glucose levels contribute to decreased FGF-2 production in the RPE and increased glial cell activation in the diabetic retina. Addition of insulin and IGF act to reverse this effect through the IGFR-1. These mechanisms may contribute to the development of diabetic retinopathy.

Background: There is a wide variation in the use of diagnostic tests for dry eye disease. The purpose of this study was to survey the attitudes of eye practitioners towards both tests and therapies available for dry eye disease., Method: An anonymous, web-based questionnaire was used, containing both quantitative and qualitative sections., Results: Thirty-eight respondents, evenly distributed in experience, confirmed the wide variation of diagnostic tests in use. History and staining techniques were significantly preferred to tests of tear flow (P < 0.05). 'Evidence' was the most valued test characteristic, and was regarded as significantly more important than 'comfort' or 'cost' (P < 0.003). However, 'evidence' was not valued significantly higher than 'ease of use' or 'time taken to perform'. Overall, there was poor satisfaction with diagnostic and therapeutic options in dry eye, for both quantitative and qualitative measures., Conclusion: This is the first qualitative study of attitudes towards dry eye disease. Attitudes towards the adequacy of diagnostic tests vary considerably. As evidence supporting a diagnostic test's validity is the most highly valued test characteristic, a systematic review of the topic is justified.

Purpose: To determine the effect of diabetes on monocarboxylate transporter (MCT) expression in the rat retina., Methods: Diabetes was induced in Wistar rats by a single intraperitoneal injection of streptozotocin (62.5 mg/kg). Rats were killed after 10 weeks, and the retinal levels of PKCalpha, bFGF, glial fibrillary acidic protein (GFAP), caspase-3, and MCT1, -2, and -4 were assessed with immunoblot analysis and RT-PCR. Hippocampal samples were used as a comparison. In other animals, the retinas were processed histologically and sections stained for the breakdown of DNA (TUNEL procedure) and for the distribution of MCT1, -2, and -4., Results: Diabetic rats exhibited cataract formation, elevated blood glucose levels, and polydipsia. Retinal levels and distribution of MCT1, 2, and -4 were similar in the diabetic and age-matched control groups; however, the early retinopathy markers bFGF and PKCalpha were significantly elevated in the diabetic retinas but not in the hippocampal samples. ERG recordings showed decreased oscillatory potentials in the diabetic group, and TUNEL staining was most evident in the photoreceptor layer. Activated caspase-3 levels were elevated in the diabetic retina., Conclusions: The expression of retinal MCT1, -2, and -4 is unaffected after 10 weeks of diabetes in rats. It appears unlikely that diabetic retinal neuropathy is a result of a hyperglycemic induction of altered MCT expression.

The aim of this work was to investigate the interrelated effects of glucose, nitric oxide (NO) and erythropoietin on neuronal survival in retinal cultures, thereby exploring the mechanism of neuronal death in the diabetic retina. Rat retinal cells were cultured in low (5 mM) or high (15 mM) glucose concentrations. After 9 days, cell viability was assessed by (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and NO production was determined by the Griess reaction. Immunohistochemistry was used to quantify GABA-labelled neurones and cells staining for DNA breakdown. High or low glucose concentrations had no effect on basal NO production or the survival of neurones in culture, but treatment with N-nitro-L-arginine methyl ester reduced extracellular levels of NO and increased neuronal survival at both concentrations of glucose. Erythropoietin decreased cell death and NO levels, but only in cultures grown in low concentrations of glucose. It is concluded that erythropoietin's neurotrophic function in the retina is attenuated at glucose concentrations similar to those which occur in diabetes.

Background: Experimental studies have yielded a wealth of information related to the mechanism of ganglion cell death following injury either to the myelinated ganglion cell axon or to the ganglion cell body. However, no suitable animal models exist where injury can be directed to the optic nerve head region, particularly the unmyelinated ganglion cell axons. The process of relating the data from the various animal models to many different types of optic neuropathies in man must, therefore, be cautious., Results: Extensive studies on the isolated optic nerve have yielded valuable information on the way white matter is affected by ischaemia and how certain types of compounds can attenuate the process. Moreover, there are now persuasive data on how ganglion cell survival is affected when the ocular blood flow is reduced in various animal models. As a consequence, the molecular mechanisms involved in ganglion cell death are fairly well understood and various pharmacological agents have been shown to blunt the process when delivered before or shortly after the insult., Conclusions: A battery of agents now exist that can blunt animal ganglion cell death irrespective of whether the insult was to the ganglion cell body or the myelinated axon. Whether this information can be applied for use in patients remains a matter of debate, and major obstacles need to be overcome before the laboratory studies may be applied clinically. These include the delivery of the pharmacological agents to the site of ganglion cell injury and side effects to the patients. Moreover, it is necessary to establish whether effective neuroprotection is only possible when the drug is administered at a defined time after injury to the ganglion cells. This information is essential in order to pursue the idea that a neuroprotective strategy can be applied to a disease like glaucoma, where ganglion cell death appears to occur at different times during the lifetime of the patient.