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King R, Struebing FL, Li Y, Wang J, Koch AA, Cooke Bailey JN, Gharahkhani P, Gharahkhani P, Gharahkhani P, Macgregor S, Allingham RR, Hauser MA, Wiggs JL, Geisert EE. Genomic locus modulating corneal thickness in the mouse identifies POU6F2 as a potential risk of developing glaucoma. PLoS Genet 2018;14(1):e1007145.Abstract
Central corneal thickness (CCT) is one of the most heritable ocular traits and it is also a phenotypic risk factor for primary open angle glaucoma (POAG). The present study uses the BXD Recombinant Inbred (RI) strains to identify novel quantitative trait loci (QTLs) modulating CCT in the mouse with the potential of identifying a molecular link between CCT and risk of developing POAG. The BXD RI strain set was used to define mammalian genomic loci modulating CCT, with a total of 818 corneas measured from 61 BXD RI strains (between 60-100 days of age). The mice were anesthetized and the eyes were positioned in front of the lens of the Phoenix Micron IV Image-Guided OCT system or the Bioptigen OCT system. CCT data for each strain was averaged and used to QTLs modulating this phenotype using the bioinformatics tools on GeneNetwork (www.genenetwork.org). The candidate genes and genomic loci identified in the mouse were then directly compared with the summary data from a human POAG genome wide association study (NEIGHBORHOOD) to determine if any genomic elements modulating mouse CCT are also risk factors for POAG.This analysis revealed one significant QTL on Chr 13 and a suggestive QTL on Chr 7. The significant locus on Chr 13 (13 to 19 Mb) was examined further to define candidate genes modulating this eye phenotype. For the Chr 13 QTL in the mouse, only one gene in the region (Pou6f2) contained nonsynonymous SNPs. Of these five nonsynonymous SNPs in Pou6f2, two resulted in changes in the amino acid proline which could result in altered secondary structure affecting protein function. The 7 Mb region under the mouse Chr 13 peak distributes over 2 chromosomes in the human: Chr 1 and Chr 7. These genomic loci were examined in the NEIGHBORHOOD database to determine if they are potential risk factors for human glaucoma identified using meta-data from human GWAS. The top 50 hits all resided within one gene (POU6F2), with the highest significance level of p = 10-6 for SNP rs76319873. POU6F2 is found in retinal ganglion cells and in corneal limbal stem cells. To test the effect of POU6F2 on CCT we examined the corneas of a Pou6f2-null mice and the corneas were thinner than those of wild-type littermates. In addition, these POU6F2 RGCs die early in the DBA/2J model of glaucoma than most RGCs. Using a mouse genetic reference panel, we identified a transcription factor, Pou6f2, that modulates CCT in the mouse. POU6F2 is also found in a subset of retinal ganglion cells and these RGCs are sensitive to injury.
Vazirani J, Nair D, Shanbhag S, Wurity S, Ranjan A, Sangwan V. Limbal Stem Cell Deficiency - Demography And Underlying Causes. Am J Ophthalmol 2018;Abstract
PURPOSE: To determine the demographic features of patients affected by limbal stem cell deficiency (LSCD), and to identify the underlying causes of LSCD DESIGN: Retrospective, multi-center case series SETTING: Two large tertiary care ophthalmology hospitals SUBJECTS: Patients with a diagnosis of LSCD presenting from January 1, 2005 to December 31, 2014 METHODS: Records of patients with a clinical diagnosis of LSCD were reviewed. Demographic details and clinical features at presentation, as well as the underlying cause of LSCD (if identified) were noted. Descriptive statistical analysis and chart preparation were done. MAIN OUTCOME MEASURES: Type of LSCD (unilateral or bilateral), age and sex of patients, extent of LSCD (clock hours of limbus involved) and underlying cause of LSCD RESULTS: We found 1331 patients with LSCD in the ten year period under study. Unilateral LSCD was more common (791 patients) than bilateral LSCD (540 patients). Out of 1331 patients, 875 (65.74%) were male. The median age of patients was 24 years. Extent of LSCD could be determined in 1849 eyes, of which 1239 eyes (67%) had total LSCD. The underlying cause of LSCD could be identified in 1512 eyes. In cases of unilateral LSCD, ocular surface burns was the commonest identifiable cause ( 83.73%). The leading identifiable causes of bilateral LSCD were ocular surface burns (29.95%), allergic conjunctivitis (29.48%), Stevens Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) (23.11%), aniridia (9.43%) and mucous membrane pemphigoid (3.54%). Lime ("chuna") injury was responsible for ocular surface burns in 352 (62.08%) out of 567 cases in which the agent was identified. CONCLUSIONS: In our study, unilateral LSCD was more common than bilateral LSCD. Young males were commonly affected, with a majority of eyes suffering from total LSCD. Overall, ocular surface burns are the leading cause of LSCD.Unilateral and bilateral LSCD had a markedly different distribution of causes, necessitating different approaches to management.
Stahl A, Krohne TU, Eter N, Oberacher-Velten I, Guthoff R, Meltendorf S, Ehrt O, Aisenbrey S, Roider J, Gerding H, Jandeck C, Smith LEH, Walz JM, for and in of Group CARDSERP (CARE-ROP) S. Comparing Alternative Ranibizumab Dosages for Safety and Efficacy in Retinopathy of Prematurity: A Randomized Clinical Trial. JAMA Pediatr 2018;172(3):278-286.Abstract
Importance: Anti-vascular endothelial growth factor (VEGF) therapies are a novel treatment option in retinopathy of prematurity (ROP). Data on dosing, efficacy, and safety are insufficient. Objective: To investigate lower doses of anti-VEGF therapy with ranibizumab, a substance with a significantly shorter systemic half-life than the standard treatment, bevacizumab. Design, Setting, and Participants: This randomized, multicenter, double-blind, investigator-initiated trial at 9 academic medical centers in Germany compared ranibizumab doses of 0.12 mg vs 0.20 mg in infants with bilateral aggressive posterior ROP; ROP stage 1 with plus disease, 2 with plus disease, or 3 with or without plus disease in zone I; or ROP stage 3 with plus disease in posterior zone II. Patients were recruited between September 2014 and August 2016. Twenty infants were screened and 19 were randomized. Interventions: All infants received 1 baseline ranibizumab injection per eye. Reinjections were allowed in case of ROP recurrence after at least 28 days. Main Outcomes and Measures: The primary end point was the number of infants who did not require rescue therapy at 24 weeks. Key secondary end points included time-to-event analyses, progression of physiologic vascularization, and plasma VEGF levels. Stages of ROP were photodocumented and reviewed by an expert committee. Results: Nineteen infants with ROP were enrolled (9 [47.4%] female; median [range] postmenstrual age at first treatment, 36.4 [34.7-39.7] weeks), 3 of whom died during the study (1 in the 0.12-mg group and 2 in the 0.20-mg group). Of the surviving infants, 8 (88.9%) (17 eyes [94.4%]) in the 0.12-mg group and 6 (85.7%) (13 eyes [92.9%]) in the 0.20-mg group did not require rescue therapy. Both ranibizumab doses were equally successful in controlling acute ROP (Cochran-Mantel-Haenszel analysis; odds ratio, 1.88; 95% CI, 0.26-13.49; P = .53). Physiologic intraretinal vascularization was superior in the 0.12-mg group. The VEGF plasma levels were not systematically altered in either group. Conclusions and Relevance: This pilot study demonstrates that ranibizumab is effective in controlling acute ROP and that 24% of the standard adult dose (0.12 mg) appears equally effective as 40% (0.20 mg). Superior vascularization of the peripheral retina with 0.12 mg of ranibizumab indicates that the lower dose may be favorable. Unchanged plasma VEGF levels point toward a limited systemic drug exposure after ranibizumab. Trial Registration: clinicaltrials.gov Identifier: NCT02134457 and clinicaltrialsregister.eu Identifier: 2013-002539-13.
Ma L, Jakobiec FA, Wolkow N, Dryja TP, Borodic GE. Multiple Eyelid Cysts (Apocrine and Eccrine Hidrocystomas, Trichilemmal Cyst, and Hybrid Cyst) in a Patient With a Prolactinoma. Ophthalmic Plast Reconstr Surg 2018;34(3):e83-e85.Abstract
A 53-year-old man presented with smooth-domed, variegated cysts (polycystic disease) of all 4 eyelids, worse on the left side. Some of the cysts were clear, while others were creamy-white colored. In addition, multiple, very fine vesicopapules were noted along the eyelid margins. Histopathologic examination revealed a trichilemmal cyst, several pure apocrine hidrocystomas displaying multiple chambers, a hybrid cyst, and many small eccrine cysts of the deep dermis. The apocrine lesions, including the small ones at the eyelid margins, predominated. Smooth muscle actin sometimes positively stained outer myoepithelial cells in some of the apocrine cysts, which helped to distinguish them from eccrine cysts. Most noteworthy was the fact that the patient had been diagnosed with a prolactinoma 20 years earlier. There is only 1 previous report of multiple apocrine cysts and an antecedent prolactinoma in the dermatologic literature. This syndrome should be separated from that of Schöpf-Schulz-Passarge, which manifests multiple small eyelid apocrine cysts and other ectodermal dysplasias without any association with neoplasia, and from that of focal dermal hypoplasia (Goltz-Gorlin) syndrome with apocrine cysts but again without neoplasia.
Kaochar S, Dong J, Torres M, Rajapakshe K, Nikolos F, Davis CM, Ehli EA, Coarfa C, Mitsiades N, Poulaki V. ICG-001 Exerts Potent Anticancer Activity Against Uveal Melanoma Cells. Invest Ophthalmol Vis Sci 2018;59(1):132-143.Abstract
Purpose: Uveal melanoma (UM) is uniformly refractory to all available systemic chemotherapies, thus creating an urgent need for novel therapeutics. In this study, we investigated the sensitivity of UM cells to ICG-001, a small molecule reported to suppress the Wnt/β-catenin-mediated transcriptional program. Methods: We used a panel of UM cell lines to examine the effects of ICG-001 on cellular proliferation, migration, and gene expression. In vivo efficacy of ICG-001 was evaluated in a UM xenograft model. Results: ICG-001 exerted strong antiproliferative activity against UM cells, leading to cell cycle arrest, apoptosis, and inhibition of migration. Global gene expression profiling revealed strong suppression of genes associated with cell cycle proliferation, DNA replication, and G1/S transition. Gene set enrichment analysis revealed that ICG-001 suppressed Wnt, mTOR, and MAPK signaling. Strikingly, ICG-001 suppressed the expression of genes associated with UM aggressiveness, including CDH1, CITED1, EMP1, EMP3, SDCBP, and SPARC. Notably, the transcriptomic footprint of ICG-001, when applied to a UM patient dataset, was associated with better clinical outcome. Lastly, ICG-001 exerted anticancer activity against a UM tumor xenograft in mice. Conclusions: Using in vitro and in vivo experiments, we demonstrate that ICG-001 has strong anticancer activity against UM cells and suppresses transcriptional programs critical for the cancer cell. Our results suggest that ICG-001 holds promise and should be examined further as a novel therapeutic agent for UM.
Eslani M, Putra I, Shen X, Hamouie J, Tadepalli A, Anwar KN, Kink JA, Ghassemi S, Agnihotri G, Reshetylo S, Mashaghi A, Dana R, Hematti P, Djalilian AR. Cornea-Derived Mesenchymal Stromal Cells Therapeutically Modulate Macrophage Immunophenotype and Angiogenic Function. Stem Cells 2018;36(5):775-784.Abstract
Macrophages are crucial drivers of inflammatory corneal neovascularization and thus are potential targets for immunomodulatory therapies. We hypothesized that therapeutic use of cornea-derived mesenchymal stromal cells (cMSCs) may alter the function of macrophages. We found that cMSCs can modulate the phenotype and angiogenic function of macrophages. In vitro, cMSCs induce apoptosis of macrophages while preferentially promoting a distinct CD14 CD16 CD163 CD206 immunophenotype that has significantly reduced angiogenic effects based on in vitro angiogenesis assays. In vivo, application of cMSCs to murine corneas after injury leads to reduced macrophage infiltration and higher expression of CD206 in macrophages. Macrophages cocultured ("educated") by cMSCs express significantly higher levels of anti-angiogenic and anti-inflammatory factors compared with control macrophages. In vivo, injured corneas treated with cMSC-educated macrophages demonstrate significantly less neovascularization compared with corneas treated with control macrophages. Knocking down the expression of pigment epithelial derived factor (PEDF) in cMSCs significantly abrogates its modulating effects on macrophages, as shown by the reduced rate of apoptosis, decreased expression of sFLT-1/PEDF, and increased expression of vascular endothelial growth factor-A in the cocultured macrophages. Similarly, cMSCs isolated from PEDF knockout mice are less effective compared with wild-type cMSCs at inhibiting macrophage infiltration when applied to wild-type corneas after injury. Overall, these results demonstrate that cMSCs therapeutically suppress the angiogenic capacity of macrophages and highlight the role of cMSC secreted PEDF in the modulation of macrophage phenotype and function. Stem Cells 2018;36:775-784.
Choi HJ, Wang R, Jakobs TC. Single-Cell Dissociation and Characterization in the Murine Retina and Optic Nerve. Methods Mol Biol 2018;1695:311-334.Abstract
Recent technological advances have extended the range of analytic tools to very small samples. It is now possible to assay the transcriptome, and in some cases even the proteome, of single cells reliably. This allows addressing novel questions, such as the genotype/phenotype relationships of single neurons, heterogeneity within individual cells of the same type, or the basis of differential vulnerability to injury. An important prerequisite for these kinds of studies is the ability to isolate well-defined individual cells without contamination by adjacent tissue. In the retina and optic nerve, cells of different types and functions are closely intermingled, limiting the use of standard methods such as laser capture microdissection. Here, we describe a simple method to isolate morphologically intact cells from the retina and the optic nerve and discuss considerations in recognizing and isolating different cell types after dissociation.
Wolkow N, Jakobiec FA, Hatton MP. A Common Procedure With an Uncommon Pathology: Triamcinolone Acetonide Eyelid Injection. Ophthalmic Plast Reconstr Surg 2018;34(3):e72-e73.Abstract
Local corticosteroid injections are frequently employed by ophthalmologists to treat a variety of ocular, periocular, and orbital inflammatory conditions. Triamcinolone acetonide is a slowly dissolving crystalline corticosteroid that is often used for this purpose because of its prolonged anti-inflammatory effect. On occasion, previously injected corticosteroid material persists in tissues longer than anticipated, creating nodules that may masquerade as other disease conditions, or appearing incidentally in excised lesions on histopathologic examination. The histopathologic features of corticosteroid residues are unfamiliar to most ophthalmic pathologists and general pathologists. These features are described herein. Triamcinolone acetonide deposits in the skin appear as pale eosinophilic lakes of acellular frothy material on hematoxylin-eosin staining and are occasionally surrounded by a mild inflammatory reaction.
Tao Y, Huang M, Shu Y, Ruprecht A, Wang H, Tang Y, Vandenberghe LH, Wang Q, Gao G, Kong W-J, Chen Z-Y. Delivery of Adeno-Associated Virus Vectors in Adult Mammalian Inner-Ear Cell Subtypes Without Auditory Dysfunction. Hum Gene Ther 2018;29(4):492-506.Abstract
Hearing loss, including genetic hearing loss, is one of the most common forms of sensory deficits in humans with limited options of treatment. Adeno-associated virus (AAV)-mediated gene transfer has been shown to recover auditory functions effectively in mouse models of genetic deafness when delivered at neonatal stages. However, the mouse cochlea is still developing at those time points, whereas in humans, the newborn inner ears are already fully mature. For effective gene therapy to treat genetic deafness, it is necessary to determine whether AAV-mediated therapy can be equally effective in the fully mature mouse inner ear without causing damage to the inner ear. This study tested several AAV serotypes by canalostomy in adult mice. It is shown that most AAVs transduce the sensory inner hair cells efficiently, but are less efficient at transducing outer hair cells. A subset of AAVs also transduces non-sensory cochlear cell types. Neither the surgical procedure of canalostomy nor the AAV serotypes damage hair cells or impair normal hearing. The studies indicate that canalostomy can be a viable route for safe and efficient gene delivery, and they expand the repertoire of AAVs to target diverse cell types in the adult inner ear.
Moein H-R, Kheirkhah A, Muller RT, Cruzat AC, Pavan-Langston D, Hamrah P. Corneal nerve regeneration after herpes simplex keratitis: A longitudinal in vivo confocal microscopy study. Ocul Surf 2018;16(2):218-225.Abstract
PURPOSE: To evaluate the long-term alterations of corneal nerves in patients with herpes simplex virus (HSV) keratitis using in vivo confocal microscopy (IVCM). DESIGN: Prospective, longitudinal, cross sectional. METHODS: This study included 16 patients with a history of HSV keratitis and 15 age-matched normal controls. Slit-scanning IVCM was performed in all subjects at baseline and then after a mean follow-up of 37.3 ± 1.7 months in the patient group. Corneal subbasal nerve density and corneal sensation were compared between groups at baseline and follow-up. RESULTS: At baseline, the mean subbasal nerve density was significantly lower in both affected eyes (1.4 ± 0.6 mm/mm) and contralateral unaffected eyes (6.4 ± 0.7 mm/mm) compared with the controls (14.1 ± 1.6 mm/mm; all P < .001). At the end of follow-up, the mean nerve density in affected eyes increased to 2.8 ± 0.7 mm/mm (P = .006), with no significant change in contralateral unaffected eyes (6.5 ± 1.0 mm/mm, P = .72). However, both eyes had lower nerve density than controls (all P < .001). Corneal sensation was significantly lower in affected eyes (2.6 ± 0.6 cm) than in the control group (6.0 ± 0.0, P < .001) and showed no significant change at the end of follow-up (2.5 ± 0.6 cm, P = .80). Corneal sensation in contralateral unaffected eyes was not different in comparison with controls at both baseline and follow up (all p > .05). CONCLUSIONS: Our results demonstrate that although corneal nerve regeneration occurs in patients with HSV keratitis, this change is not clinically significant and does not results in changes of corneal sensation. Therefore, these patients need to be followed closely for complications of neurotrophic keratopathy and might benefit from neuro-regenerative therapies.
Kobashi H, Kamiya K, Shimizu K. Impact of Forward and Backward Scattering and Corneal Higher-Order Aberrations on Visual Acuity after Penetrating Keratoplasty. Semin Ophthalmol 2018;:1-9.Abstract
PURPOSE: To assess the relationship of forward and backward scattering and corneal higher-order aberrations (HOAs) with corrected distance visual acuity (CDVA) after penetrating keratoplasty (PK). METHODS: This retrospective study comprised 25 eyes of 25 consecutive patients who underwent PK using the VisuMax femtosecond laser system and age-matched 25 eyes of 25 healthy subjects. We quantitatively assessed objective scattering index (OSI) using the double-pass instrument (OQAS II, Visiometrics), corneal densitometry (CD) and corneal HOAs with the Scheimpflug rotating camera (Pentacam HR, Oculus) 1 year postoperatively. RESULTS: The OSI, CD, and corneal HOAs were significantly larger in the PK group than those in the control group (p ≤ 0.011). We found significant correlations of logMAR CDVA with the OSI (r = 0.477, p = 0.016), and with the anterior, posterior, and total corneal HOAs of the central 4-mm zone (anterior: r = 0.573, p = 0.003, posterior: r = 0.596, p = 0.002, total: r = 0.472, p = 0.017), but no significant association with the CD of the 0-2 mm zone at any layers (anterior: r = 0.236, p = 0.257, center: r = 0.139, p = 0.506, posterior: r = 0.073, p = 0.728, total: r = 0.212, p = 0.308). Similar results were obtained when the analysis was repeated with corneal HOAs of the central 6-mm zone and CDs in 2-6 mm zone. CONCLUSIONS: Our pilot study demonstrated that the postoperative CDVA was significantly correlated with OSI and corneal HOAs, but not with backward scattering in post-PK eyes, suggesting that OSI as well as corneal HOAs plays an essential role in postoperative visual performance after PK.
Fernandez-Godino R, Bujakowska KM, Pierce EA. Changes in extracellular matrix cause RPE cells to make basal deposits and activate the alternative complement pathway. Hum Mol Genet 2018;27(1):147-159.Abstract
The design of efficient therapies for age-related macular degeneration (AMD) is limited by our understanding of the pathogenesis of basal deposits, which form between retinal pigment epithelium (RPE) and Bruch's membrane (BrM) early in disease, and involve activation of the complement system. To investigate the roles of BrM, RPE and complement in an AMD, we generated abnormal extracellular matrix (ECM) using CRISPR-edited ARPE-19 cells. We introduced to these cells the p.R345W mutation in EFEMP1, which causes early-onset macular degeneration. The abnormal ECM binds active complement C3 and causes the formation of basal deposits by normal human fetal (hf)RPE cells. Human fetal RPE (hfRPE) cells grown on abnormal ECM or BrM explants from AMD donors show chronic activation of the alternative complement pathway by excessive deposition of C3b. This process is exacerbated by impaired ECM turnover via increased matrix metalloproteinase-2 activity. The local cleavage of C3 via convertase-independent mechanisms can be a new therapeutic target for early AMD.
Cruzat A, Gonzalez-Andrades M, Mauris J, AbuSamra DB, Chidambaram P, Kenyon KR, Chodosh J, Dohlman CH, Argüeso P. Colocalization of Galectin-3 With CD147 Is Associated With Increased Gelatinolytic Activity in Ulcerating Human Corneas. Invest Ophthalmol Vis Sci 2018;59(1):223-230.Abstract
Purpose: Galectin-3 is a carbohydrate-binding protein known to promote expression of matrix metalloproteinases, a hallmark of ulceration, through interaction with the extracellular matrix metalloproteinase inducer CD147. The aim of this study was to investigate the distribution of galectin-3 in corneas of patients with ulcerative keratitis and to determine its relationship to CD147 and the presence of gelatinolytic activity. Methods: This was an observational case series involving donor tissue from 13 patients with active corneal ulceration and 6 control corneas. Fixed-frozen sections of the corneas were processed to localize galectin-3 and CD147 by immunofluorescence microscopy. Gelatinolytic activity was detected by in situ zymography. Results: Tissue from patients with active corneal ulceration showed a greater galectin-3 immunoreactivity in basal epithelia and stroma compared with controls. Immunofluorescence grading scores revealed increased colocalization of galectin-3 and CD147 in corneal ulcers at the epithelial-stromal junction and within fibroblasts. Quantitative analysis using the Manders' colocalization coefficient demonstrated significant overlap in corneas from patients with ulcerative keratitis (M1 = 0.29; M2 = 0.22) as opposed to control corneas (M1 = 0.01, P < 0.01; M2 = 0.02, P < 0.05). In these experiments, there was a significant positive correlation between the degree of galectin-3 and CD147 colocalization and the presence of gelatinolytic activity. Conclusions: Our results indicate that concomitant stimulation and colocalization of galectin-3 with CD147 are associated with increased gelatinolytic activity in the actively ulcerating human cornea and suggest a mechanism by which galectin-3 may contribute to the degradation of extracellular matrix proteins during ulceration.
Carvalho LS, Xiao R, Wassmer SJ, Langsdorf A, Zinn E, Pacouret S, Shah S, Comander JI, Kim LA, Lim L, Vandenberghe LH. Synthetic Adeno-Associated Viral Vector Efficiently Targets Mouse and Nonhuman Primate Retina In Vivo. Hum Gene Ther 2018;29(7):771-784.Abstract
Gene therapy is a promising approach in the treatment of inherited and common complex disorders of the retina. Preclinical and clinical studies have validated the use of adeno-associated viral vectors (AAV) as a safe and efficient delivery vehicle for gene transfer. Retinal pigment epithelium and rods-and to a lesser extent, cone photoreceptors-can be efficiently targeted with AAV. Other retinal cell types however are more challenging targets. The aim of this study was to characterize the transduction profile and efficiency of in silico designed, synthetic Anc80 AAVs for retinal gene transfer. Three Anc80 variants were evaluated for retinal targeting in mice and primates following subretinal delivery. In the murine retina Anc80L65 demonstrated high level of retinal pigment epithelium and photoreceptor targeting with comparable cone photoreceptor affinity compared to other AAVs. Remarkably, Anc80L65 enhanced transduction kinetics with visible expression as early as day 1 and steady state mRNA levels at day 3. Inner retinal tropism of Anc80 variants demonstrated distinct transduction patterns of Müller glia, retinal ganglion cells and inner nuclear layer neurons. Finally, murine findings with Anc80L65 qualitatively translated to the Rhesus macaque in terms of cell targets, levels and onset of expression. Our findings support the use of Anc80L65 for therapeutic subretinal gene delivery.

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