Centre for Eye Research Australia (CERA) - Research Publications

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    Remote Control in Formation of 3D Multicellular Assemblies Using Magnetic Forces
    Jafari, J ; Han, X-L ; Palmer, J ; Tran, PA ; O'Connor, AJ (AMER CHEMICAL SOC, 2019-05)
    Cell constructs have been utilized as building blocks in tissue engineering to closely mimic the natural tissue and also overcome some of the limitations caused by two-dimensional cultures or using scaffolds. External forces can be used to enhance the cells' adhesion and interaction and thus provide better control over production of these structures compared to methods like cell seeding and migration. In this paper, we demonstrate an efficient method to generate uniform, three-dimensional cell constructs using magnetic forces. This method produced spheroids with higher densities and more symmetrical structures than the commonly used centrifugation method for production of cell spheroids. It was also shown that shape of the cell constructs could be changed readily by using different patterns of magnetic field. The application of magnetic fields to impart forces on the cells enhanced the fusion of these spheroids, which could be used to produce larger and more complicated structures for future tissue engineering applications.
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    Alterations in Retinal Microvascular Geometry in Young Type 1 Diabetes
    Sasongko, MB ; Wang, JJ ; Donaghue, KC ; Cheung, N ; Benitez-Aguirre, P ; Jenkins, A ; Hsu, W ; Lee, M-L ; Wong, TY (AMER DIABETES ASSOC, 2010-06)
    OBJECTIVE: To describe retinal microvascular geometric parameters in young patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: Patients with type 1 diabetes (aged 12-20 years) had clinical assessments and retinal photography following standardized protocol at a tertiary-care hospital in Sydney. Retinal microvascular geometry, including arteriolar and venular tortuosity, branching angles, optimality deviation, and length-to-diameter ratio (LDR), were measured from digitized photographs. Associations of these geometric characteristics with diabetes duration, A1C level, systolic blood pressure (SBP), and other risk factors were assessed. RESULTS: Of 1,159 patients enrolled, 944 (81.4%) had gradable photographs and 170 (14.7%) had retinopathy. Older age was associated with decreased arteriolar (P = 0.024) and venular (P = 0.002) tortuosity, and female subjects had larger arteriolar branching angle than male subjects (P = 0.03). After adjusting for age and sex, longer diabetes duration was associated with larger arteriolar branching angle (P 8.5 vs.
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    AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo
    Hung, SSC ; Chrysostomou, V ; Li, F ; Lim, JKH ; Wang, J-H ; Powell, JE ; Tu, L ; Daniszewski, M ; Lo, C ; Wong, RC ; Crowston, JG ; Pebay, A ; King, AE ; Bui, BV ; Liu, G-S ; Hewitt, AW (ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2016-06)
    PURPOSE: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. METHODS: Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. RESULTS: Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. CONCLUSIONS: Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.
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    Identifying circRNA-associated-ceRNA networks in retinal neovascularization in mice
    Cao, M ; Zhang, L ; Wang, J-H ; Zeng, H ; Peng, Y ; Zou, J ; Shi, J ; Zhang, L ; Li, Y ; Yoshida, S ; Tang, L ; Zhou, Y (IVYSPRING INT PUBL, 2019)
    Retinal neovascularization is a complication which caused human vision loss severely. It has been shown that circular RNAs (circRNAs) play essential roles in gene regulation. However, circRNA expression profile and the underlying mechanisms in retinal neovascular diseases remain unclear. In the present study, we identified altered circRNAs in the retinas of oxygen-induced retinopathy (OIR) mouse model by microarray profiling. Microarray analysis revealed that 539 circRNAs were significantly altered in OIR retinas compared with controls. Among them, 185 up-regulated and 354 down-regulated circRNAs were identified. The expression levels of 4 altered circRNAs including mmu_circRNA_002573, mmu_circRNA_011180, mmu_circRNA_016108 and mmu_circRNA_22546 were validated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Bioinformatic analysis with validated circRNAs such as competing endogenous RNA (ceRNA) regulatory networks with Gene Ontology (GO) enrichment analysis demonstrated that qRT-PCR validated circRNAs were associated with cellular process, cell part and phosphoric ester hydrolase activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that MAPK signaling pathway and renin-angiotensin system were related to validated circRNAs, suggesting these pathways may participate in pathological angiogenesis. The results together suggested that circRNAs were aberrantly expressed in OIR retinas and may play potential roles in retinal neovascular diseases.
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    Rho/ROCK pathway is essential to the expansion, differentiation, and morphological rearrangements of human neural stem/progenitor cells induced by lysophosphatidic acid
    Frisca, F ; Crombie, DE ; Dottori, M ; Goldshmit, Y ; Pebay, A (ELSEVIER, 2013-05)
    We previously reported that lysophosphatidic acid (LPA) inhibits the neuronal differentiation of human embryonic stem cells (hESC). We extended these studies by analyzing LPA's effects on the expansion of neural stem/progenitor cells (NS/PC) derived from hESCs and human induced pluripotent stem cells (iPSC), and we assessed whether data obtained on the neural differentiation of hESCs were relevant to iPSCs. We showed that hESCs and iPSCs exhibited comparable mRNA expression profiles of LPA receptors and producing enzymes upon neural differentiation. We demonstrated that LPA inhibited the expansion of NS/PCs of both origins, mainly by increased apoptosis in a Rho/Rho-associated kinase (ROCK)-dependent mechanism. Furthermore, LPA inhibited the neuronal differentiation of iPSCs. Lastly, LPA induced neurite retraction of NS/PC-derived early neurons through Rho/ROCK, which was accompanied by myosin light chain (MLC) phosphorylation. Our data demonstrate the consistency of LPA effects across various sources of human NS/PCs, rendering hESCs and iPSCs valuable models for studying lysophospholipid signaling in human neural cells. Our data also highlight the importance of the Rho/ROCK pathway in human NS/PCs. As LPA levels are increased in the central nervous system (CNS) following injury, LPA-mediated effects on NS/PCs and early neurons could contribute to the poor neurogenesis observed in the CNS following injury.
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    Predictors of visual outcome and the role of early vitrectomy in streptococcal endophthalmitis
    Kurniawan, ED ; Rocke, JR ; Sandhu, SS ; Allen, PJ (WILEY, 2018-05)
    IMPORTANCE: Streptococcal endophthalmitis has devastating sequelae. This study aims to identify factors which may be targeted to optimize patient outcomes. BACKGROUND: This study investigated characteristics influencing visual outcomes and the role of early vitrectomy. DESIGN: Retrospective observational case series of consecutive patients was conducted. PARTICIPANTS: All patients with a culture-positive diagnosis of streptococcal endophthalmitis treated at a tertiary ophthalmology referral centre between July 1997 and February 2012 were included. METHODS: Patient records were reviewed and data collected on their presentation, examination, microbiology results, procedures and final outcome. MAIN OUTCOME MEASURES: Visual acuity (VA) and enucleation/evisceration were measured. RESULTS: Of the 101 patients, 35.6% presented with a VA of hand movements and 42.6% with light perception (LP). Final VA was poor (6/60 or worse) in 77.6% and 24.7% were enucleated/eviscerated. Presenting VA of LP or worse (P = 0.008), no view of fundus (P = 0.001), large number of organisms (P < 0.001), recognition of Streptococcus on Gram stain (P = 0.010), heavy growth on culture (P < 0.001) and more intravitreal injections (P = 0.038) were significantly associated with poor visual outcome (6/60 or worse). Presenting VA of LP or worse (P = 0.042) and non-viridans Streptococcus species (P = 0.002) were significantly associated with enucleation/evisceration. Fifteen patients (14.9%) had early vitrectomy within 48 h which was not associated with poor final VA or removal of the eye (P = 1.000). CONCLUSIONS AND RELEVANCE: Early vitrectomy did not influence visual outcome in this cohort. Microbiology results were useful in predicting poor outcomes, and may allow clinicians to make early treatment decisions and provide prognostic information for patients.
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    Clinical case of influenza A with outer retinal maculopathy
    Ong, D ; Harper, CA ; Fagan, X (WILEY, 2017-09)
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    Paracentral acute middle maculopathy as a finding in patients with severe vision loss following phacoemulsification cataract surgery
    Creese, K ; Ong, D ; Sandhu, SS ; Ware, D ; Harper, CA ; Al-Qureshi, SH ; Wickremasinghe, SS (WILEY, 2017-08)
    IMPORTANCE: Paracentral acute middle maculopathy (PAMM) diagnosed by spectral domain optical coherence tomography (SD-OCT) in patients with poor visual outcome post cataract surgery. BACKGROUND: Case series of severe vision loss due to PAMM after cataract surgery. DESIGN: Retrospective case series. PARTICIPANTS: Cases from five surgical centres in Victoria, Australia. METHODS: Retrospective analysis of cases with unexplained 'patch-off' vision loss post cataract surgery. All patients in our cohort had PAMM and presumed diagnosis of central or transient retinal artery occlusion. MAIN OUTCOME MEASURES: A review of the patient histories focusing on pre-operative ocular and systemic vascular risk factors, anaesthetic and operative factors. RESULTS: Ten cases were included. All patients had 6/72 Snellen visual acuity or worse noted on day one post surgery. Three patients had features of central retinal artery occlusion consisting of retinal pallor with a 'cherry red' macula but absent relative afferent pupillary defect. Seven had no features of retinal pallor or attenuation of retinal arterioles. On SD-OCT, all eyes had evident PAMM. Six patients had a history of cardiovascular disease or blood dyscrasia. CONCLUSIONS AND RELEVANCE: PAMM should be considered in patients with 'patch off' visual loss and absence of other fundal signs. We hypothesise that spasm or transient occlusion of central retinal artery leads to arterial hypoperfusion with subsequent ischaemia or infarction of the retina. Underlying arterial disease may have led to pre-existing hypoperfusion that may have been further compromised by raised intraocular pressure during the procedure itself or via raised orbital pressure from the anaesthesia.
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    Peeking into the molecular trove of discarded surgical specimens
    Hewitt, AW ; Cook, AL ; Pebay, A (WILEY-BLACKWELL, 2016-11)
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