Randomized Trial to Assess the Safety and Tolerability of Daily Intake of an Allulose Amino Acid-Based Hydration Beverage in Men and Women.

BACKGROUND: Maintaining adequate hydration is critical to optimal health, well-being, and performance. Those who are physically active in stressful environments, such as warm and/or humid scenarios, may be at particular risk for dehydration with ensuing loss of electrolytes, leading to sluggishness and impaired physical performance. METHODS: We evaluated an electrolyte and amino acid product containing L-alanine and L-glutamine, as well as select vitamins [B3 (niacin), B5 (pantothenic acid), B6 (pyridoxine), B12 (cobalamin), and vitamin C (ascorbic acid)]. Subjects (n = 40; four groups, n = 10) were randomized to consume either a placebo packet or one, two, or three packets daily of the test product for 4 weeks with site visits at 0, 2, and 4 weeks. We tested safety and tolerability by analyzing hematological parameters (complete blood counts), metabolic parameters (hepatic, renal, acid-base balance), urinalysis end products, thyroid status [T3 (triiodothyronine), T4 (thyroxine), TSH (thyroid-stimulating hormone)], tolerability (via questionnaire), vital signs, and dietary intake. RESULTS: Statistical analyses displayed ten significant main effects (p < 0.05) with white blood cells, lymphocytes, neutrophils, urinary pH, thyroxine, urination frequency, calcium, calories, fat, and cholesterol. Interactions for time and group (p < 0.05) were observed for MCV, eGFR, potassium, overall tolerability, bloating, and cramping-demonstrating mild GA disturbances. Little to no change of physiological relevance was noted for any outcome variable, regardless of dosing level. CONCLUSIONS: Our results indicate the product was well-tolerated at all dosing levels and no significant adverse changes occurred in any of the test parameters compared to the placebo group, indicating relative safety of ingestion over a 4-week treatment period, at the volumes used, and outside the context of physical stress.

Systematic review and appraisal of quality, definitions and treatment recommendations of clinical guidelines for glaucoma suspects.

BACKGROUND: To appraise the quality of clinical practice guidelines for glaucoma suspects, and to assess their consistency for how a 'glaucoma suspect' is defined and their recommendations for treatment initiation for such individuals. METHODS: This study included all documents that self-identified as a 'guideline' and provided recommendation(s) for the clinical care of glaucoma suspects. The quality of eligible guidelines was assessed using the Appraisal of Guidelines for Research and Evaluation (AGREE) II instrument. RESULTS: From 1196 records retrieved from comprehensive searches and two records manually included, 20 clinical practice guidelines were deemed eligible. Based on an appraisal using the AGREE II instrument, 16 (80%) guidelines had ≤2 domains with scores >66%. Overall, the lowest scoring domains were for applicability, editorial independence and stakeholder involvement. There was relatively poor agreement across the guidelines for what defines a 'glaucoma suspect' or 'primary open angle glaucoma [POAG] suspect', as well as the recommendations and criteria for treatment initiation in these populations. There was better agreement for the definition and recommendations for treatment initiation for 'primary angle closure suspects'. CONCLUSIONS: There is substantial room to improve the methodological quality of most current international clinical guidelines for glaucoma suspects. Clinicians should consider this finding when using such guidelines to inform their care of glaucoma suspects. Substantial variation in the definition of a POAG suspect and recommendations for treatment initiation underscores important gaps in the current evidence for the accurate prediction of glaucoma development and treatment effectiveness in these individuals.

Increased Glaucoma Case-finding Through Routine Optical Coherence Tomography in Optometry Practice.

OBJECTIVE: Timely detection of glaucoma is key to preventing or delaying vision loss. This study aimed to assess whether the routine use of optical coherence tomography (OCT) by optometrists for detection of glaucomatous changes in the optic nerve and retina increased glaucoma referrals to ophthalmologists. DESIGN: This study was a retrospective review of routinely-collected electronic medical records of patients from a chain of 331 optometry practices in Australia. PARTICIPANTS: Electronic medical records were reviewed for every patient aged 18-99 years who attended an included practice between January 1 and July 31, 2019. METHODS: Odds of referral for glaucoma assessment were compared between practices performing OCT routinely on all patients (OCT practices, n=175) and without OCT (non-OCT practices, n=20). A subset of referrals were assessed by ophthalmologists to determine the false positive referral rate. MAIN OUTCOME MEASURES: The primary outcome measure of this study was referral to an ophthalmologist for glaucoma assessment. A secondary outcome was the rate of false positive referrals, analysed in a subset of patients referred for glaucoma assessment. RESULTS: Records from 994,461 patients (59% female) were included and 10,475 (1.1%) were referred for glaucoma assessment. Most referrals were associated with normal intraocular pressure (non-OCT practices: n=496, 66%; OCT practices: n=6,603, 68%). Referral for glaucoma was higher in OCT practices (n=9,719, 1.1%) compared to non-OCT practices (n=756, 0.8%, age-, gender- and location-adjusted odds ratio 1.39, 95% confidence interval 1.10-1.76). Of 318 referred patients (3%, all from OCT practices) for whom ophthalmologist feedback was available, 68 (21%) were considered not to have glaucoma. CONCLUSIONS: The routine use of OCT in optometric practice may lead to more timely glaucoma detection and prevention of avoidable vision loss.

Systematic Underestimation of Visual Sensitivity Loss on Microperimetry: Implications for Testing Protocols in Clinical Trials.

Purpose: To examine whether systematic changes in visual sensitivity measurements on microperimetry occur over tests within the same session and whether these changes vary according to the level of visual sensitivity loss. Methods: Eighty individuals with glaucoma or atrophic age-related macular degeneration underwent three microperimetry tests in one eye during one session using the 4-2 staircase strategy. Changes in mean sensitivity (MS) and pointwise sensitivity (PWS) between the first and second test pairs were examined, with PWS was examined separately based on its average value across the three tests in 6-dB bins. The coefficient of repeatability (CoR) for MS between each sequential test pair was also calculated. Results: There was a significant decline in MS from the first to second test (P = 0.001), but no significant difference in MS was seen between the second and third tests (P = 0.562). This significant decline in the first test pair was observed in locations with an average PWS of <6 dB or between 6 to 12 dB and between 12 to 18 dB (P < 0.001), but not for all other average PWS bins (P ≥ 0.337). The CoR of MS was significantly lower in the second compared to the first test pair (1.4 dB and 2.5 dB, respectively; P < 0.001). Conclusions: The 4-2 staircase strategy conventionally used on microperimetry testing systematically underestimates visual sensitivity loss on the first test. Translational Relevance: The consistency and accuracy of visual sensitivity measurements on microperimetry in clinical trials could be markedly improved by using estimates from an initial test to seed subsequent tests and excluding this first test from analyses.

Immunobiology of a rationally-designed AAV2 capsid following intravitreal delivery in mice.

Adeno-associated virus serotype 2 (AAV2) is a viral vector that can be used to deliver therapeutic genes to diseased cells in the retina. One strategy for altering AAV2 vectors involves the mutation of phosphodegron residues, which are thought to be phosphorylated/ubiquitinated in the cytosol, facilitating degradation of the vector and the inhibition of transduction. As such, mutation of phosphodegron residues have been correlated with increased transduction of target cells, however, an assessment of the immunobiology of wild-type and phosphodegron mutant AAV2 vectors following intravitreal (IVT) delivery to immunocompetent animals is lacking in the current literature. In this study, we show that IVT of a triple phosphodegron mutant AAV2 capsid is associated with higher levels of humoral immune activation, infiltration of CD4 and CD8 T-cells into the retina, generation of splenic germinal centre reactions, activation of conventional dendritic cell subsets, and elevated retinal gliosis compared to wild-type AAV2 capsids. However, we did not detect significant changes in electroretinography arising after vector administration. We also demonstrate that the triple AAV2 mutant capsid is less susceptible to neutralisation by soluble heparan sulphate and anti-AAV2 neutralising antibodies, highlighting a possible utility for the vector in terms of circumventing pre-existing humoral immunity. In summary, the present study highlights novel aspects of rationally-designed vector immunobiology, which may be relevant to their application in preclinical and clinical settings.

Intraocular Pressure Spikes Following iStent Inject and the Relationship to Aqueous Outflow in Open Angle Glaucoma.

PRCIS: Adding trabecular bypass surgery (TBS) to phacoemulsification creates unpredictable short-term intraocular pressure (IOP) control that may be undesirable for patients with advanced glaucoma. Aqueous outflow (AO) responses after TBS are complex and probably multifactorial. PURPOSE: To assess IOP spikes in patients with open angle glaucoma up to 1 month after iStent inject and their relationship to AO patterns measured by hemoglobin video imaging (HVI). PARTICIPANTS AND METHODS: We studied IOP for 4 weeks after TBS with iStent inject in 105 consecutive eyes with open angle glaucoma (6 TBS only and 99 combined with phacoemulsification). The change in IOP after surgery at each time point was compared with baseline measurements and the prior postoperative visit. IOP-lowering medications were stopped on the day of surgery in all patients. A smaller pilot study of 20 eyes (TBS only = 6 and combined = 14) underwent concurrent HVI to observe and quantify perioperative AO. Aqueous column cross-sectional area (AqCA) of one nasal and one temporal aqueous vein was calculated at each time point, and qualitative observations were documented. An additional 5 eyes were studied after phacoemulsification only. RESULTS: Mean IOP for the entire cohort (preoperative 17.3 ± 5.6 mm Hg) was lowest the day after TBS (13.1 ± 5.0 mm Hg) and peaked at 1 week (17.2 ± 8.0 mm Hg), before stabilizing by 4 weeks (15.2 ± 5.2 mm Hg; P < 0.00001). The same IOP pattern was seen when separating the group into a larger cohort without HVI (respectively 15.9 ± 3.2 mm Hg, 12.8 ± 4.9 mm Hg, 16.4 ± 7.4 mm Hg, and 14.1 ± 4.1 mm Hg; N = 85, P < 0.00001) and the smaller HVI pilot study (respectively 21.4 ± 9.9 mm Hg, 14.2 ± 4.9 mm Hg, 20.2 ± 9.7 mm Hg, and 18.9 ± 7.6 mm Hg; N = 20, P < 0.001). More than 30% IOP elevation above baseline occurred in 13.3% of the entire cohort at 1 week after surgery. This increased to 46.7% when IOP was compared with 1 day after surgery. Inconsistent AqCA values and patterns of aqueous flow were demonstrated after TBS. AqCA after phacoemulsification alone was maintained or increased within 1 week in all 5 eyes. CONCLUSION: After iStent inject surgery in patients with open angle glaucoma, intraocular spikes were most commonly seen at 1 week. AO patterns were variable and additional studies are needed to understand the pathophysiology underlying IOP responses after this procedure.

Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters.

Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken ß-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken ß-actin/short ß-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

Fast Progressors in Glaucoma: Prevalence Based on Global and Central Visual Field Loss.

PURPOSE: To determine the prevalence of fast global and central visual field (VF) progression in individuals with glaucoma under routine care. DESIGN: Observational study. PARTICIPANTS: Six hundred ninety-three eyes of 461 individuals with glaucoma followed up over a median of 4.5 years. METHODS: This study included (1) patients at a private ophthalmology clinic in Melbourne, Australia, and (2) individuals in 2 prospective longitudinal observational studies across 3 sites in the United States. All individuals had a diagnosis of glaucoma and were under routine care, and had performed 5 or more reliable 24-2 VF tests over a 1- to 5-year period. Ordinary least squares regression analyses were used to calculate the rate of global mean deviation (MD) change over time and the rate of the mean total deviation values of the 12 test locations within the central 10° region (MTD10) for each eye. MAIN OUTCOME MEASURES: Prevalence of progression based on the rate of MD and the MTD10 change across various fixed cutoffs and cutoffs based on the estimated normal distribution (from the positive slopes). RESULTS: Based on the MD and the MTD10, 12.5% and 11.7% of the eyes, respectively, exhibited a rate of change that was less than -1.0 dB/year (being a rate that typically is defined as "fast progression" for MD values), and 29.0% of the eyes showed a change of less than -0.5 dB/year on MTD10. Furthermore, 12.7% and 9.1% of the eyes exhibited a rate of change that exceeded the 1% cutoff of the estimated normal distribution MD and the MTD10 values, respectively. CONCLUSIONS: This study found that approximately 1 in 8 eyes with glaucoma receiving routine care showed fast progression based on global MD values (< -1.0 dB/year) and that nearly 1 in 3 eyes showed a < -0.5 dB/year decline centrally. These findings highlight the clinical importance of assessing progressive central VF loss and reinforce the need for new therapies to prevent functional disability in a notable proportion of individuals who continue to exhibit fast progression. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

RNA-targeting strategies as a platform for ocular gene therapy.

Genetic medicine is offering hope as new therapies are emerging for many previously untreatable diseases. The eye is at the forefront of these advances, as exemplified by the approval of Luxturna® by the United States Food and Drug Administration (US FDA) in 2017 for the treatment of one form of Leber Congenital Amaurosis (LCA), an inherited blindness. Luxturna® was also the first in vivo human gene therapy to gain US FDA approval. Numerous gene therapy clinical trials are ongoing for other eye diseases, and novel delivery systems, discovery of new drug targets and emerging technologies are currently driving the field forward. Targeting RNA, in particular, is an attractive therapeutic strategy for genetic disease that may have safety advantages over alternative approaches by avoiding permanent changes in the genome. In this regard, antisense oligonucleotides (ASO) and RNA interference (RNAi) are the currently popular strategies for developing RNA-targeted therapeutics. Enthusiasm has been further fuelled by the emergence of clustered regularly interspersed short palindromic repeats (CRISPR)-CRISPR associated (Cas) systems that allow targeted manipulation of nucleic acids. RNA-targeting CRISPR-Cas systems now provide a novel way to develop RNA-targeted therapeutics and may provide superior efficiency and specificity to existing technologies. In addition, RNA base editing technologies using CRISPR-Cas and other modalities also enable precise alteration of single nucleotides. In this review, we showcase advances made by RNA-targeting systems for ocular disease, discuss applications of ASO and RNAi technologies, highlight emerging CRISPR-Cas systems and consider the implications of RNA-targeting therapeutics in the development of future drugs to treat eye disease.

Survey of perspectives of people with inherited retinal diseases on ocular gene therapy in Australia.

Many gene therapies are in development for treating people with inherited retinal diseases (IRD). We hypothesized that potential recipients of gene therapy would have knowledge gaps regarding treatment. We aimed to assess knowledge, attitudes, and perceptions of genetic therapies among potential recipients with IRD, using a novel instrument we designed (Attitudes to Gene Therapy-Eye (AGT-Eye)) and their associations with demographic data, self-reported visual status, and tools assessing quality of life and attitudes toward clinical trials using a community-based cross-sectional survey of Australian adults with IRD. AGT-Eye, overall quality of life EQ-5D-5L, National Eye Institute Visual Functioning Questionnaire (NEI-VFQ-25) and Patient Attitudes to Clinical Trials (PACT-22) instruments were administered. Six hundred and eighty-one people completed the study, 51.7% women of mean age 53.5 years (SD ± 15.8). Most participants (91.6%) indicated they would likely accept gene therapy if it was available to them or family members. However, only 28.3% agreed that they had good knowledge of gene therapy. Most obtained information about gene therapy from the internet (49.3%). Respondents with post-graduate degrees scored highest compared to other educational levels on methods (p < 0.001) and outcomes (p = 0.003) and were more likely to see economic value of treatment (p = 0.043). Knowledge gaps were present regarding methods and outcomes of gene therapy. This survey has shown high level of interest in the IRD community for gene therapies, and highlights areas for improved clinician and patient education.

Daily Meditation Practice for Managing Glaucoma Patients' Attitudes and Acceptance.

PRCIS: Overall, 63% of glaucoma patients agreed to 45-60 minutes of daily meditation. Predictors of accepting meditation included previous meditation practice, a diagnosis of glaucoma <1 year, and having a marital status of "single". PURPOSE: To explore patients' acceptance and barriers towards 45-60 minutes daily meditation for glaucoma management and to identify glaucoma patients with higher perceived stress levels who may benefit more from meditation practice. METHODS: Glaucoma patients attending the Royal Victorian Eye and Ear Hospital, Melbourne, Australia outpatient department were invited to complete a patient survey. This survey explored if patients would agree to 45-60 minutes of daily meditation and included the Determinants of Meditation Practice Inventory and Perceived Stress Scale questionnaires. Questionnaire scores were compared across participants' clinical and demographic characteristics using Student t test, ANOVA, and multiple-linear-regression analysis. RESULTS: Of the 123 eligible patients screened, 100 completed the survey (81.3%). Sixty-three (63%) patients would agree to 45-60 minutes of daily meditation if advised by their doctor. Univariate analysis showed increased acceptance of meditation (lower Determinants of Meditation Practice Inventory scores) to be associated with agreeing to meditate 45-60 minutes daily ( P =0.002), currently or previously practicing meditation ( P =0.006 and P =0.0004 respectively), and having a marital status as "single" ( P =0.02). Multivariate regression analysis showed previous meditation practice and a glaucoma diagnosis of <1 year to be predictive of accepting meditation ( P =0.01 and P =0.03 respectively). There were no predictive factors of Perceived Stress Scale scores. CONCLUSION: Given the high acceptance rate of 45-60 minutes daily meditation (63% of glaucoma patients sampled), this may be recommended for the benefit of patients. Patients who have previously meditated, have a relatively new diagnosis of glaucoma, and are single (marital status) were more accepting of meditation practice.

Hemoglobin Video Imaging Detects Differences in Aqueous Outflow Between Eyes With and Without Glaucoma During the Water Drinking Test.

PRCIS: Hemoglobin video imaging (HVI) demonstrates increased aqueous outflow (AO) in response to the water drinking test (WDT) in patients with and without glaucoma. In glaucomatous eyes, increased AO was not sustained, and characteristic flow patterns were seen. PURPOSE: To observe how variations in intraocular pressure (IOP) correlate with the flow of aqueous in episcleral veins. DESIGN: Prospective observational cohort study. PARTICIPANTS: The WDT increased AO into the episcleral venous system in 30 eyes recruited from Sydney Eye Hospital. A comparison was made between glaucomatous (n=20) and nonglaucomatous eyes (n=10). METHODS: Each patient had baseline IOP and HVI before drinking 10 mL/kg body weight of water. IOP and HVI were then repeated every 15 minutes for 1 hour. Aqueous column cross-sectional area (AqCA) of the most prominent nasal and temporal aqueous veins was used to semi-quantify conventional AO. MAIN OUTCOME MEASURES: Change in IOP and AqCA from baseline during the WDT. Aqueous flow characteristics were also observed. RESULTS: Peak IOP elevation above baseline was significantly higher in the glaucoma group, with an average IOP rise of 39.7% on 1.6 1.1 medications, compared with 22.9% in the control group ( P =0.04). AqCA significantly increased for glaucomatous and nonglaucomatous eyes in response to water ingestion ( P <0.05). AqCA fell by 50% in glaucomatous eyes ( P =0.003) and 33% in nonglaucomatous eyes ( P =0.08) at study completion compared with the peak measurement. IOP remained >30% elevated in 8 glaucomatous eyes (40%) after 60 minutes and no control eyes. Variations in qualitative aqueous flow patterns were observed in glaucomatous eyes but not in controls. CONCLUSIONS: AO volume, estimated by AqCA, increases in response to IOP elevation induced by an ingested water bolus in patients with and without glaucoma. The increase in aqueous drainage was not sustained in glaucomatous eyes and may have led to incomplete recovery of IOP. Using HVI in combination with the WDT may assist with clinical decision-making and facilitate the monitoring of responses to treatment.

Impact of AmaTea® Max on physiological measures and gaming performance in active gamers: A placebo-controlled, double-blind, randomized study.

Background and Aim: The activity of "gaming" has increased greatly in popularity in recent years, with many gamers using nutritional supplements to aid mood and gaming performance. We evaluated the impact of AmaTea® Max (referred to as AmaTea® throughout; a patented dietary supplement consisting of a blend of caffeine and polyphenol antioxidants), compared to both caffeine and a placebo, on gaming and cognitive performance in active gamers. Methods: Subjects reported to the lab on three occasions, separated by approximately 1 week. On each day, they had baseline measurements taken and then played the game Fortnite for four 1-h periods. Measures of cognitive performance, gaming performance, heart rate and blood pressure (BP), and blood cortisol were measured before and at selected times following gameplay. Results: Neither caffeine nor AmaTea® impacted gaming or cognitive performance in a statistically significant manner. However, a trend (P=0.075) was noted for the condition effect for kills/match, with values 21% higher for AmaTea® (1.84) compared to placebo (1.51), and 12% higher for AmaTea® compared to caffeine (1.63). Subjective mood was relatively unaffected, although a condition effect was noted for jittery (P=0.05), with values lower for placebo than for caffeine (P=0.02). BP was minimally elevated with both AmaTea® and caffeine, while cortisol followed the normal diurnal variation and was lower for placebo than AmaTea® and caffeine. Conclusion: AmaTea® modestly increased kills/match during gameplay. It is possible that a different gaming stimulus, varied time of gameplay, or different dosage of the supplement may have yielded different results. Relevance for Subjects: Active gamers who seek to use a dietary supplement for purposes of gaming performance may benefit slightly from ingestion of AmaTea® before gameplay while experiencing greater vigor and lower fatigue as compared to placebo.

Evaluating multidisciplinary glaucoma care: visual field progression and loss of sight year analysis in the community vs hospital setting.

BACKGROUND: A variety of shared care models have been developed, which aim to stratify glaucoma patients according to risk of disease progression. However, there is limited published data on the rate of glaucoma progression in the hospital vs community setting. Here we aimed to compare rates of glaucomatous visual field progression in the Cambridge Community Optometrist Glaucoma Scheme (COGS) and Addenbrooke's Hospital Glaucoma Clinic (AGC). METHODS: A retrospective comparative cohort review was performed. Patients with five or more visual field tests were included. Zeiss Forum software was used to calculate the MD progression rate (dB/year). Loss of sight years (LSY) were also calculated for both COGS and AGC. RESULTS: Overall, 8465 visual field tests from 854 patients were reviewed. In all, 362 eyes from the AGC group and 210 eyes from COGS were included. The MD deterioration rate was significantly lower in the COGS patients compared with the AGC group (-0.1 vs -0.3 dB/year; p < 0.0001). No patients in the COGS group were predicted to become blind within their lifetime by LSY analysis. Fifteen patients were at risk in the AGC group. CONCLUSION: This service evaluation shows that COGS is an effective scheme to stratify lower risk glaucoma patients, increasing the capacity within hospital eye services. COGS patients have a lower rate of visual field deterioration compared to AGC patients. Effective communication between community and tertiary schemes is essential to facilitate transfer of patients requiring further hospital management reliably and efficiently, with the potential for low-risk patients to be followed safely in the community.

Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes.

The COVID-19 pandemic caused by SARS-CoV-2 has infected millions worldwide, therefore there is an urgent need to increase our diagnostic capacity to identify infected cases. Although RT-qPCR remains the gold standard for SARS-CoV-2 detection, this method requires specialised equipment in a diagnostic laboratory and has a long turn-around time to process the samples. To address this, several groups have recently reported the development of loop-mediated isothermal amplification (LAMP) as a simple, low cost and rapid method for SARS-CoV-2 detection. Herein we present a comparative analysis of three LAMP-based assays that target different regions of the SARS-CoV-2: ORF1ab RdRP, ORF1ab nsp3 and Gene N. We perform a detailed assessment of their sensitivity, kinetics and false positive rates for SARS-CoV-2 diagnostics in LAMP or RT-LAMP reactions, using colorimetric or fluorescent detection. Our results independently validate that all three assays can detect SARS-CoV-2 in 30 min, with robust accuracy at detecting as little as 1000 RNA copies and the results can be visualised simply by color changes. Incorporation of RT-LAMP with fluorescent detection further increases the detection sensitivity to as little as 100 RNA copies. We also note the shortcomings of some LAMP-based assays, including variable results with shorter reaction time or lower load of SARS-CoV-2, and false positive results in some experimental conditions and clinical saliva samples. Overall for RT-LAMP detection, the ORF1ab RdRP and ORF1ab nsp3 assays have faster kinetics for detection but varying degrees of false positives detection, whereas the Gene N assay exhibits no false positives in 30 min reaction time, which highlights the importance of optimal primer design to minimise false-positives in RT-LAMP. This study provides validation of the performance of LAMP-based assays as a rapid, highly sensitive detection method for SARS-CoV-2, which have important implications in development of point-of-care diagnostics for SARS-CoV-2.

Receptor-ligand supplementation via a self-cleaving 2A peptide-based gene therapy promotes CNS axonal transport with functional recovery.

Gene replacement approaches are leading to a revolution in the treatment of previously debilitating monogenic neurological conditions. However, the application of gene therapy to complex polygenic conditions has been limited. Down-regulation or dysfunction of receptor expression in the disease state or in the presence of excess ligand has been shown to compromise therapeutic efficacy. Here, we offer evidence that combined overexpression of both brain-derived neurotrophic factor and its receptor, tropomyosin receptor kinase B, is more effective in stimulating axonal transport than either receptor administration or ligand administration alone. We also show efficacy in experimental glaucoma and humanized tauopathy models. Simultaneous administration of a ligand and its receptor by a single gene therapy vector overcomes several problems relating to ligand deficiency and receptor down-regulation that may be relevant to multiple neurodegenerative diseases. This approach shows promise as a strategy to target intrinsic mechanisms to improve neuronal function and facilitate repair.

Aqueous outflow imaging techniques and what they tell us about intraocular pressure regulation.

Recent advances in the medical and surgical management of open-angle glaucoma have increased the number of treatment options available. Several new intraocular pressure (IOP)-lowering treatments target the conventional aqueous outflow (AO) system. However, success rates are variable and outcomes in individual patients are often difficult to predict. Variable treatment responses remain unexplained and highlight deficiencies in our current understanding of AO regulation and IOP homeostasis. Imaging is often relied upon to confirm diagnoses and monitor treatment responses in other ocular and systemic pathologies. As yet no suitable AO imaging tool has been developed to fulfil this role in glaucoma. A variety of imaging techniques have been used to study the AO tracts of humans and animals in ex vivo and in vivo eyes. In this review, results from novel imaging techniques that assess aqueous drainage through the episcleral venous system are considered and we argue these provide new insights into AO regulation. We suggest that the ability to objectively measure AO responses to interventions would be a significant clinical advance, and we have demonstrated that this can be achieved with direct visualisation of aqueous drainage. We predict that the evolution of AO imaging technology will continue to reveal critical components of AO and IOP regulation, and that personalised IOP-lowering treatment in glaucoma care may well become a reality in the near future.

Protrudin functions from the endoplasmic reticulum to support axon regeneration in the adult CNS.

Adult mammalian central nervous system axons have intrinsically poor regenerative capacity, so axonal injury has permanent consequences. One approach to enhancing regeneration is to increase the axonal supply of growth molecules and organelles. We achieved this by expressing the adaptor molecule Protrudin which is normally found at low levels in non-regenerative neurons. Elevated Protrudin expression enabled robust central nervous system regeneration both in vitro in primary cortical neurons and in vivo in the injured adult optic nerve. Protrudin overexpression facilitated the accumulation of endoplasmic reticulum, integrins and Rab11 endosomes in the distal axon, whilst removing Protrudin's endoplasmic reticulum localization, kinesin-binding or phosphoinositide-binding properties abrogated the regenerative effects. These results demonstrate that Protrudin promotes regeneration by functioning as a scaffold to link axonal organelles, motors and membranes, establishing important roles for these cellular components in mediating regeneration in the adult central nervous system.

The Effects of Trabecular Bypass Surgery on Conventional Aqueous Outflow, Visualized by Hemoglobin Video Imaging.

PRECIS: Hemoglobin Video Imaging (HVI) provides a noninvasive method to quantify aqueous outflow (AO) perioperatively. Trabecular bypass surgery (TBS) is able to improve, and in some cases re-establish, conventional AO. PURPOSE: The purpose of this study was to use HVI to illustrate and quantify effects of TBS on AO through the episcleral venous system. DESIGN: This is a prospective observational cohort study. PARTICIPANTS: Patients were recruited from Sydney Eye Hospital, Australia. The study included 29 eyes from 25 patients, 15 with glaucoma and 14 normal controls. TBS (iStent Inject) was performed on 14 glaucomatous eyes (9 combined phacoemulsification/TBS and 5 standalone TBS). Cataract surgery alone was performed on the remaining eye from the glaucoma group and 2 eyes from the control group. METHODS: We used HVI, a novel clinic-based tool, to visualize and quantify AO perioperatively during routine follow-up to 6 months. Angiographic blood flow patterns were observed within prominent aqueous veins on the nasal and temporal ocular surface. Aqueous column cross-section area (AqCA) was compared before and after surgery. MAIN OUTCOME MEASURES: AqCA, number of aqueous veins, intraocular pressure (IOP) before and after surgery, and number of IOP-lowering medications. RESULTS: Patients with glaucoma had reduced AqCA compared with normal controls (P=0.00001). TBS increased AqCA in 13 eyes at 1 month (n=14; P<0.002), suggesting improved AO. This effect was maintained at 6 months in 7 eyes (n=9, P≤0.05). All patients with unrecordable AO before surgery (n=3; 2 standalone TBS, 1 combined cataract/TBS) established measurable flow after TBS. IOP and/or medication burden became reduced in every patient undergoing TBS. Cataract surgery alone (n=3) increased AqCA in nasal and temporal vessels at 4 weeks after surgery. CONCLUSIONS: HVI provides a safe method for detecting and monitoring AO perioperatively in an outpatient setting. Improvement of AO into the episcleral venous system is expected after TBS and can be visualized with HVI. TBS is able to improve, and in some cases re-establish, conventional AO. Cataract surgery may augment this. Some aqueous veins were first seen after TBS and these patients had unstable postoperative IOP control, which possibly suggests reorganization of aqueous homeostatic mechanisms. HVI may confirm adequacy of surgery during short-term follow-up, but further work is required to assess the potential of HVI to predict surgical outcomes and assist with personalized treatment decisions.