A tripartite complex containing MRCK modulates lamellar actomyosin retrograde flow.

Actomyosin retrograde flow underlies the contraction essential for cell motility. Retrograde flow in both lamellipodia and lamella is required for membrane protrusion and for force generation by coupling to cell adhesion. We report that the Rac/Cdc42-binding kinase MRCK and myosin II-related MYO18A linked by the adaptor protein LRAP35a form a functional tripartite complex, which is responsible for the assembly of lamellar actomyosin bundles and of a subnuclear actomyosin network. LRAP35a binds independently to MYO18A and MRCK. This binding leads to MRCK activation and its phosphorylation of MYO18A, independently of ROK and MLCK. The MRCK complex moves in concert with the retrograde flow of actomyosin bundles, with MRCK being able to influence other flow components such as MYO2A. The promotion of persistent protrusive activity and inhibition of cell motility by the respective expression of wild-type and dominant-negative mutant components of the MRCK complex show it to be crucial to cell protrusion and migration.

Sustainable practice of ophthalmology during COVID-19: challenges and solutions.

PURPOSE: The Coronavirus (COVID-19) outbreak is rapidly emerging as a global health threat. With no proven vaccination or treatment, infection control measures are paramount. In this article, we aim to describe the impact of COVID-19 on our practice and share our strategies and guidelines to maintain a sustainable ophthalmology practice. METHODS: Tan Tock Seng Hospital (TTSH) Eye Centre is the only ophthalmology department supporting the National Centre for Infectious Diseases (NCID), which is the national screening center and the main center for management of COVID-19 patients in Singapore. Our guidelines during this outbreak are discussed. RESULTS: Challenges in different care settings in our ophthalmology practice have been identified and analyzed with practical solutions and guidelines implemented in anticipation of these challenges. First, to minimize cross-infection of COVID-19, stringent infection control measures were set up. These include personal protective equipment (PPE) for healthcare workers and routine cleaning of "high-touch" surfaces. Second, for outpatient care, a stringent dual screening and triaging process were carried out to identify high-risk patients, with proper isolation for such patients. Administrative measures to lower patient attendance and reschedule appointments were carried out. Third, inpatient and outpatient care were separated to minimize interactions. Last but not least, logistics and manpower plans were drawn up in anticipation of resource demands and measures to improve the mental well-being of staff were implemented. CONCLUSION: We hope our measures during this COVID-19 pandemic can help ophthalmologists globally and serve to guide and maintain safe access in ophthalmology clinics when faced with similar disease outbreaks.

Predictors of persistent disease activity following anti-VEGF loading dose for nAMD patients in Singapore: the DIALS study.

BACKGROUND: To determine the frequency of persistent disease activity following 3 loading doses of anti- vascular endothelial growth factor (VEGF) agents, and the anatomic and demographic predictors of early persistent disease activity among patients with neovascular age-related macular degeneration (nAMD). METHODS: In a retrospective real-world cohort study, 281 consecutive patients with nAMD were reviewed at baseline and after 3 anti-VEGF injections for pre-defined indicators of disease activity. Optical coherence tomography (OCT) features such as subretinal fluid, intraretinal cysts and intraretinal fluid were assessed by reading-center certified graders. Multiple logistic regression was performed on demographic and anatomic factors. RESULTS: At month 3, 66.1% of patients had persistent disease activity. The best-corrected visual acuity (BCVA) improvement was 0.16 LogMAR for those with no disease activity compared to 0 for patients with persistent activity (p < 0.001). The significant risk factors for persistent activity at 3 months were male gender (odds ratio [OR] 0.54, 95% confidence interval [CI] 0.32-0.93, p = 0.025), intraretinal cysts at baseline (OR 2.95, 95% CI 1.67-5.20, p < 0.001) and subretinal fluid at baseline (OR 3.17, 95% CI 1.62-6.18, p = 0.002). At 3 months, 58% of patients had features of activity on OCT. Patients with intraretinal cysts and intraretinal fluid at baseline had worse BCVA at month 3 compared to patients without these OCT features (0.69 vs. 0.43, p < 0.001, and 0.62 vs. 0.43, p < 0.001, respectively). CONCLUSIONS: In a real-world study, 66.1% of nAMD patients have persistent disease activity after the initial loading dose, with poorer BCVA compared to those without. Baseline OCT features (intraretinal cysts and subretinal fluid) are useful predictors of persistent disease activity at month 3.

Multicolour imaging for the detection of polypoidal choroidal vasculopathy and age-related macular degeneration.

IMPORTANCE: Multicolour is a new imaging technology and its sensitivity for detecting polypoidal choroidal vasculopathy (PCV) and age-related macular degeneration (AMD) has not been well described. BACKGROUND: To evaluate the accuracy of multicolour imaging compared to colour fundus photography (CFP) in differentiating AMD and PCV from normal eyes, and in detecting PCV. DESIGN: Prospective cohort study at a tertiary referral centre. PARTICIPANTS: Fifty consecutive patients with PCV or AMD. METHODS: Standardized multimodal imaging, including CFP, multicolour imaging, and fluorescein and indocyanine green angiography, were graded by a Central Reading Center using standardized grading protocols. MAIN OUTCOMES AND MEASURES: Sensitivity, specificity, positive and negative predictive values (PPV and NPV). RESULTS: Of 100 eyes, 44 had PCV, 33 had AMD, and 23 were normal. Multicolour imaging had higher specificity (73.9% vs 52.2%) and NPV (94% vs 85.7%) compared to CFP for detecting all types of AMD. For the detection of PCV, multicolour had higher sensitivity (86.4% vs 59.1%) and NPV (89.3% vs 74.3%). Polypoidal lesions were detected in 39 of 44 eyes (88.6%) using multicolour imaging, while the branching vascular network (BVN) was detected in 16 of 44 eyes (36.4%). Using BVN as a parameter, infrared imaging specificity and PPV for detecting PCV were 96.6% and 88.9%, respectively. CONCLUSIONS AND RELEVANCE: Multicolour imaging is superior to standard CFP in differentiating AMD and PCV from normal eyes, and in detecting features of PCV. Specific features seen on multicolour imaging can alert ophthalmologists to the likely presence of these diseases so that additional definitive investigations can be performed.

EVEREST study report 4: Fluorescein angiography features predictive of polypoidal choroidal vasculopathy.

IMPORTANCE: It is important to identify features of polypoidal choroidal vasculopathy (PCV) that differentiate it from typical neovascular age-related macular degeneration (nAMD) on various imaging modalities, including fluorescein angiography (FA). BACKGROUND: PCV was thought to be indistinguishable from nAMD using FA alone. In real-world practice, indocyanine-green angiography may often be unavailable or contraindicated. DESIGN: Analysis of FA images from a prospective, multicentre study. PARTICIPANTS: Study images of both PCV and nAMD patients from the EVEREST study. METHODS: FA features at baseline were independently graded by masked graders (fellowship-trained ophthalmologists) using standardized diagnostic algorithms. MAIN OUTCOME MEASURES: Predictive indicators (sensitivity, specificity, positive and negative predictive values) for PCV. RESULTS: Of the 95 patients screened, 61 had PCV. Of the 34 screening failures, 15 were diagnosed as nAMD. Hyperfluorescent nodules on FA were observed in 80% of patients with PCV vs 20% with nAMD (P < 0.001). Blocked fluorescence on FA, which corresponded to the presence of subretinal haemorrhage, occurred more frequently among patients with PCV vs nAMD (61.7% vs 13.3%, P = 0.001). Similarly, the leakage characteristic of occult choroidal neovascularization occurred more frequently among patients with PCV vs nAMD (95.0% vs 73.3%, P = 0.026). The positive predictive value for PCV was 94.1% for hyperfluorescent nodules, 94.9% for blocked fluorescence, 83.8% for occult choroidal neovascularization and 82.0% for pigment epithelial detachment. CONCLUSIONS AND RELEVANCE: This study demonstrated that certain FA features can be predictive of PCV and may be considered as an indication for retina specialists to perform indocyanine green angiography as confirmatory test.

A functional requirement for PAK1 binding to the KH(2) domain of the fragile X protein-related FXR1.

Loss of fragile X mental retardation protein FMR1 is the most common genetic cause of mental deficiency in man. We find that both FMR1 and the related FXR1 serve as direct binding partners for the Cdc42 effector PAK1. This involves an 11 residue segment in the PAK1 autoinhibitory domain that is exposed upon kinase activation and binds the FXR1 KH2 domain. Active PAK1 can phosphorylate FXR1 at Ser420; antibodies to this site show increased phosphorylation when fragile X proteins are recruited to stress granules. During zebrafish muscle development, FXR1 Ser420 phosphorylation is needed for protein function. The familial FMR1(I304N) mutation is biologically inactive, and FXR1(I304N) fails to bind PAK1. A different PAK1 binding-deficient mutant, FXR1(Q348K/E352A), fails to rescue loss of Zf-FXR1 unless combined with a gain-of-function S420D phosphomimetic. This is the first documented protein partner for the KH(2) domain of FMR1 or FXR1, and it has several implications for signaling by fragile X proteins.

Validation study of WatchPat 200 for diagnosis of OSA in an Asian cohort.

To validate a wrist-worn portable device (WatchPat 200) to diagnose OSA in an Asian cohort. To evaluate its cost-effectiveness. Twenty patients with suspected OSA were recruited and had simultaneous polysomnography (PSG) and WatchPAT assessments concurrently within the hospital's sleep laboratory. The study population consisted of 18 male and 2 female patients, whose mean age was 39 (±16) years, mean BMI was 27.2 (±5.5) kg/m2, mean Epworth Sleepiness score was 8.55 (±4.8). The correlation between the AHI was assessed using Spearman's correlation coefficient and agreement was tested via the Bland Altman plot. Sensitivities and specificities were then applied to the various AHI groups. Spearman's coefficient was 0.94, which suggested that a very strong correlation between the AHI recorded by the WatchPat and the PSG. Bland-Altman plot showed good agreement with the AHI mean difference of about 4.23 with a slight tendency to overscore the AHI at the mild range of OSA and underscore the range at the severe end of OSA. The WatchPat showed 100% sensitivity when compared to PSG for mild OSA and is thus a good screening test for the undiagnosed general population. Furthermore, it also showed 100% specificity when compared to PSG for severe OSA suggesting that it is a good diagnostic test for people with a high suspicion of OSA. This facilitates a timelier diagnosis and a more economical approach with potential cost savings of up to $900 per patient.

Contrasting phase effects on vestibular evoked myogenic potentials (VEMPs) produced by air- and bone-conducted stimuli.

We have studied the effects of stimulus phase on the latency and amplitude of cVEMPs and oVEMPs by reanalysing data from Lim et al. (Exp Brain Res 224:437-445, 2013) in which alternating phase was used. Responses for the different initial stimulus phase, either positive or negative, were separated and reaveraged. We found that the phase (compressive or rarefactive) of AC 500-Hz stimuli had no significant effect on either latency or amplitude of the responses. Conversely, phase (positive = motor towards subjects) did alter the effects of BC 500-Hz stimulation. For cVEMPs, phase consistently affected initial latency with earlier responses for positive stimuli, while, for stimulation at the mastoid, negative onset phase gave larger responses. For the oVEMP, effects were different for the two sites of BC stimulation. At the forehead, the response appeared to invert, whereas at the mastoid there appeared to be a delay of the initial response. Related to this, the effect of phase for the two sites was opposite: at the mastoid, positive responses were earlier but negative were larger (particularly for long stimuli). At the forehead, the effect was the opposite: negative onset stimuli evoked earlier responses, whereas positive onset evoked larger responses. These findings indicate a basic difference in the way that AC and BC stimuli activate vestibular receptors and also indicate that the effects of phase of BC stimulation depend on location. Stimulus alternation does little to affect the response to AC stimulation but obscures the effects of BC stimuli, particularly for the oVEMP.

EVEREST study report 3: diagnostic challenges of polypoidal choroidal vasculopathy. Lessons learnt from screening failures in the EVEREST study.

PURPOSE: To describe screening failures in the EVEREST study by examining the imaging characteristics that enabled differentiation of polypoidal choroidal vasculopathy (PCV) from cases that were subsequently diagnosed not to be PCV. METHODS: Post-hoc analysis of 34 patients with PCV reported as screening failures from EVEREST study. Standardised confocal scanning laser indocyanine green angiography (ICGA) images were graded by the Central Reading Centre to confirm PCV diagnosis based on the presence of early focal sub-retinal hyperfluorescence on ICGA and at least one of the following six diagnostic criteria: (1) nodular appearance of polyp(s) on stereoscopic examination, (2) hypofluorescent halo around nodule(s), (3) presence of a branching vascular network, (4) pulsation of polyp(s) on dynamic ICGA, (5) orange sub-retinal nodules on colour fundus photography, or (6) massive sub-macular haemorrhage (≥4 disc areas in size). Additional detailed image grading was performed with stereo-imaging and dynamic early-phase ICGA. RESULTS: Of the 95 screened PCV cases, 34 were excluded: (1) cases not suitable for recruitment as per the study protocol (n = 14), (2) equivocal lesions on ICGA characterised by small hyperfluorescent dots (n = 9), and (3) cases that were definitely not PCV (non-PCV, n = 11), identified by definitive diagnoses which included one case each of micro-aneurysm, retinal angiomatous proliferation, retino-choroidal anastomosis, small type-2 choroidal neovascularisation, retinal pigment epithelial (RPE) window defect and disciform scar; two cases of lesions where the choroidal vessel changed its course; and three cases of late-onset RPE staining. CONCLUSIONS: Standardised image grading techniques used in EVEREST study enabled effective differentiation of non-PCV from actual PCV.

Comparability of retinal thickness measurements using different scanning protocols on spectral-domain optical coherence tomography.

Retinal thickness measurements obtained using optical coherence tomography (OCT) play an essential role both in multi-center clinical trials and in normal clinical practice. Different scanning protocols are available on most OCT devices, and it is important to ascertain whether the retinal thickness measurements obtained from these are comparable. This study aimed to compare retinal thickness measurements between raster and radial scanning protocols using spectral-domain OCT (SD-OCT). In a prospective study, 32 healthy subjects were scanned sequentially using raster and radial protocols from a SD-OCT device. For both the raster and radial OCT scans, retinal thicknesses were measured manually subfoveally and at 12 other points at 0.5 mm intervals temporally and nasally on the horizontal OCT B-scan passing through the fovea. The retinal thickness measurements were compared using intraclass correlation (ICC) and Bland-Altman plots. Subfoveal retinal thickness was 227.0 µm when measured on the raster scan and 229.2 µm on the radial scan, with a mean difference of 2.2 µm (P = 0.141).The ICC for agreement was 0.889 (95 % confidence interval 0.818-0.933). Similar results were observed for retinal thickness measurements at all other points, with mean differences ranging from -3.37 to 2.59 µm, and ICC values ranging from 0.837 to 0.972. The retinal thickness measurements obtained by the raster and radial scans of the same SD-OCT device are comparable, with differences of less than 4 µm. This is of relevance when measurements made using different OCT scan protocols are compared.

Differential effects of duration for ocular and cervical vestibular evoked myogenic potentials evoked by air- and bone-conducted stimuli.

We investigated the changes in cervical (cVEMP) and ocular (oVEMP) vestibular evoked myogenic potentials in response to differing stimulus durations. cVEMPs (n = 12 subjects) and oVEMPs (n = 13 subjects) were recorded using air-conducted (AC: 500 Hz) and bone-conducted (BC: 500 Hz) tone burst stimuli with durations varying from 2 to 10 ms. BC stimulation was applied both frontally and to the mastoid. AC cVEMPs showed an increase in amplitude with stimuli up to 6-ms duration associated with a prolonged latency, as previously reported. In contrast, AC oVEMP amplitude decreased with increasing stimulus duration. BC stimuli showed no significant increase in amplitude with increasing stimulus duration for either reflex using either location of stimulation. BC cVEMPS following forehead stimulation showed a significant decrease as duration increased, and BC oVEMPs to mastoid stimulation were largest at 2 ms and decreased thereafter. We conclude that an increase in amplitude with increasing stimulus duration, using 500 Hz stimuli, only occurs for AC cVEMPs. There is no definite benefit in using longer stimuli than 2 ms for BC or oVEMP studies. Shorter stimuli also minimise subject exposure to sound and vibration.

Heat profiling of phacoemulsification tip using a thermal scanning camera.

An experimental study to measure the heat profile of the phacoemulsification (phaco) tip using standard continuous phaco and hyperpulse phaco with and without waveform power modulation in the Millennium Microsurgical System with Custom Control Software (CCS). The phaco tip was imaged in air using a thermal camera. The highest temperature was measured 15 s after application of phaco power. Continuous, hyperpulse and waveform power modulations of the Millennium Microsurgical System were used with different power settings (20, 50 and 100 %) and duty cycles (40, 60 and 90 %), with the irrigation turned on and off. Using continuous phaco with the irrigation on, the phaco tip temperature remains <28.0 °C. With irrigation off, the temperature is higher compared to irrigation on but still remains <45.0 °C. Comparing the temperatures for all three power modulations when irrigation is on, at each phaco power and duty cycle setting, the temperature of the phaco tip is highest with continuous phaco, followed by hyperpulse with rise time 1, then hyperpulse with rise time 2. When irrigation is off, the highest temperatures are recorded using the hyperpulse with rise time 2, followed by continuous phaco, then hyperpulse with rise time 1. Hyperpulse and waveform modulations reduce heat generation compared to the continuous mode when irrigation is turned on. Lower duty cycles and lower ultrasound power produce less heat at the phaco tip.

Evolution of Polypoidal Lesions after Treatment of Polypoidal Choroidal Vasculopathy.

Purpose: To evaluate the status and evolution of polypoidal lesions during the course of treatment of patients with symptomatic macular polypoidal choroidal vasculopathy (PCV). Design: Comparative cohort study of randomly selected patients from a multicenter, randomized controlled clinical trial. Participants: Thirty randomly selected patients from the EVEREST II study who were treated with combination ranibizumab and verteporfin photodynamic therapy (n = 15) or ranibizumab monotherapy (n = 15). Methods: All patients were randomized at baseline and treated with a standardized treatment protocol. Indocyanine green angiography (ICGA) images were graded at the central reading center at baseline and months 3, 6, 12, and 24. Polypoidal lesions present at baseline were overlaid on ICGA images at subsequent visits to determine if these remained perfused or had regressed completely. New polypoidal lesions occurring at subsequent visits were similarly tracked to detail the evolution of each polypoidal lesion. Main Outcome Measures: Complete polypoidal lesion regression over time. Results: Complete polypoidal lesion regression was higher in the combination therapy group compared with the monotherapy group at all visits (month 12, 12 of 15 patients [80%] vs. 5 of 14 patients [35.7%]; P = 0.016). Persistence of baseline polypoidal lesions was lower in the combination therapy group: 1 of 15 patients (6.7%) versus 7 of 14 patients (50%) in the monotherapy group at month 12. Recurrences of polypoidal lesions that had regressed completely at an earlier time point were uncommon: 0% in the combination therapy group and 1 patient each at months 6 and 12 in the monotherapy group. Fewer new polypoidal lesions (arising after the baseline visit) were found in the combination therapy group at all visits (combination therapy: 2 of 15 [13.3%] vs. monotherapy: 4 of 14 eyes [28.6%] at month 12). Total polypoidal lesion area was significantly smaller in the combination therapy group compared with the monotherapy group throughout the study (0.013 mm2 vs. 0.110 mm2; P < 0.01 at month 12). Conclusions: Combination therapy was associated with higher rates of complete polypoidal lesion regression and fewer persistent polypoidal lesions compared with monotherapy. Closed polypoidal lesions rarely reopened, regardless of the treatment.