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Zhou EH, Paolucci M, Dryja TP, Manley T, Xiang C, Rice DS, Prasanna G, Chen A. A Compact Whole-Eye Perfusion System to Evaluate Pharmacologic Responses of Outflow Facility. Invest Ophthalmol Vis Sci 2017;58(7):2991-3003.Abstract
Purpose: To discover novel therapies that lower IOP by increasing aqueous humor outflow facility, ex vivo ocular perfusion systems provide a valuable tool. However, currently available designs are limited by their throughput. Here we report the development of a compact, scalable perfusion system with improved throughput and its validation using bovine and porcine eyes. Methods: At a fixed IOP of 6 mm Hg, flow rate was measured by flow sensors. We validated the system by measuring the outflow responses to Y-39983 (a Rho kinase inhibitor), endothelin-1 (ET-1), ambrisentan (an antagonist for endothelin receptor A [ETA]), sphigosine-1-phosphate (S1P), JTE-013 (antagonist for S1P receptor 2 [S1P2]), S-nitroso-N-acetylpenicillamine (SNAP, a nitric oxide [NO] donor), and 3-Morpholino-sydnonimine (SIN-1, another NO donor). Results: The instrument design enabled simultaneous measurements of 20 eyes with a footprint of 1 m2. Relative to vehicle control, Y-39983 increased outflow by up to 31% in calf eyes. On the contrary, ET-1 decreased outflow by up to 79%, a response that could be blocked by pretreatment with ambrisentan, indicating a role for ETA receptors. Interestingly, the effect of ET-1 was also inhibited by up to 70% to 80% by pretreatment with NO donors, SNAP and SIN-1. In addition to testing in calf eyes, similar effects of ET-1 and ambrisentan were observed in adult bovine and porcine eyes. Conclusions: The compact eye perfusion platform provides an opportunity to efficiently identify compounds that influence outflow facility and may lead to the discovery of new glaucoma therapies.
Sriram S, Tran JA, Guo X, Hutcheon AEK, Kazlauskas A, Zieske JD. Development of wound healing models to study TGFβ3's effect on SMA. Exp Eye Res 2017;161:52-60.Abstract
The goal of this study was to test the efficacy of transforming growth factor beta 3 (TGFβ3) in reducing α-smooth muscle actin (SMA) expression in two models-an ex vivo organ culture and an in vitro 3D cell construct-both of which closely mimic an in vivo environment. For the ex vivo organ culture system, a central 6.0 mm corneal keratectomy was performed on freshly excised rabbit globes The corneas were then excised, segregated into groups treated with 1.0 ng/ml TGFβ1 or β3 (T1 or T3, respectively), and cultured for 2 weeks. The corneas were assessed for levels of haze and analyzed for SMA mRNA levels. For the 3D in vitro model, rabbit corneal fibroblasts (RbCFs) were cultured for 4 weeks on poly-transwell membranes in Eagle's minimum essential media (EMEM) + 10% FBS + 0.5 mM vitamin C ± 0.1 ng/ml T1 or T3. At the end of 4 weeks, the constructs were processed for analysis by indirect-immunofluorescence (IF) and RT-qPCR. The RT-qPCR data showed that SMA mRNA expression in T3 samples for both models was significantly lower (p < 0.05) than T1 treatment (around 3-fold in ex vivo and 2-fold in constructs). T3 also reduced the amount of scarring in ex vivo corneas as compared with the T1 samples. IF data from RbCF constructs confirmed that T3-treated samples had up to 4-fold (p < 0.05) lower levels of SMA protein expression than samples treated with T1. These results show that T3 when compared to T1 decreases the expression of SMA in both ex vivo organ culture and in vitro 3D cell construct models. Understanding the mechanism of T3's action in these systems and how they differ from simple cell culture models, may potentially help in developing T3 as an anti-scarring therapy.
Taniguchi EV, Paschalis EI, Crnej A, Ren A, Colby KA, Chodosh J, Pasquale LR, Shen LQ, Dohlman CH, Cruzat A. The Role of the Back Plate in Angle Anatomy with the Boston Type I Keratoprosthesis. Cornea 2017;36(9):1096-1101.Abstract
PURPOSE: To quantitatively evaluate the angle anatomy in eyes with the Boston type I keratoprosthesis (B-KPro) differing in the back plate (BP) material and size using anterior segment optical coherence tomography. METHODS: B-KPro eyes with poly(methyl methacrylate) (PMMA) (7.0 and 8.5 mm) and titanium (7.0, 8.5, and 9.5 mm) BPs were imaged with anterior segment optical coherence tomography. The angle opening distance at 500 μm from the scleral spur (AOD500), trabecular iris surface area at 500 μm from the scleral spur (TISA500), and trabecular iris angle at 500 μm from the scleral spur (TIA500) were measured. Among the visible quadrants, the average, the temporal, the widest, and the narrowest angle of each eye were included in the analysis. Average time between B-KPro implantation and imaging was 7.5 ± 1.4 years for a PMMA BP and 2.4 ± 2.3 years for a titanium BP (P < 0.0001). RESULTS: We analyzed 17 B-KPro eyes with PMMA BPs and 24 B-KPro eyes with titanium BPs. The average AOD500 (394.1 ± 226.9 vs. 454.5 ± 255.6 μm, P = 0.44), average TIA500 (26.2 ± 14.2 vs. 29.8 ± 13.9 degrees, P = 0.43), and average TISA500 (0.15 ± 0.08 vs. 0.17 ± 0.10 μm, P = 0.52) were not statistically different between eyes with PMMA and titanium BPs, nor were the temporal, the narrowest, and the widest angle measurements of each eye (all P > 0.05). Similarly, no significant differences were found between the angle measurements of B-KPro eyes with a titanium BP diameter of 8.5 or 9.5 mm (all P > 0.05). CONCLUSIONS: We successfully visualized the angle anatomy in 66.1% of the imaged eyes, including all BPs studied. Neither the material nor the size of the B-KPro BP had a significant impact on the angle anatomy.
Richarme G, Liu C, Mihoub M, Abdallah J, Leger T, Joly N, Liebart J-C, Jurkunas UV, Nadal M, Bouloc P, Dairou J, Lamouri A. Guanine glycation repair by DJ-1/Park7 and its bacterial homologs. Science 2017;357(6347):208-211.Abstract
DNA damage induced by reactive carbonyls (mainly methylglyoxal and glyoxal), called DNA glycation, is quantitatively as important as oxidative damage. DNA glycation is associated with increased mutation frequency, DNA strand breaks, and cytotoxicity. However, in contrast to guanine oxidation repair, how glycated DNA is repaired remains undetermined. Here, we found that the parkinsonism-associated protein DJ-1 and its bacterial homologs Hsp31, YhbO, and YajL could repair methylglyoxal- and glyoxal-glycated nucleotides and nucleic acids. DJ-1-depleted cells displayed increased levels of glycated DNA, DNA strand breaks, and phosphorylated p53. Deglycase-deficient bacterial mutants displayed increased levels of glycated DNA and RNA and exhibited strong mutator phenotypes. Thus, DJ-1 and its prokaryotic homologs constitute a major nucleotide repair system that we name guanine glycation repair.
Sun Y, Liu C-H, Wang Z, Meng SS, Burnim SB, SanGiovanni JP, Kamenecka TM, Solt LA, Chen J. RORα modulates semaphorin 3E transcription and neurovascular interaction in pathological retinal angiogenesis. FASEB J 2017;31(10):4492-4502.Abstract
Pathological proliferation of retinal blood vessels commonly causes vision impairment in proliferative retinopathies, including retinopathy of prematurity. Dysregulated crosstalk between the vasculature and retinal neurons is increasingly recognized as a major factor contributing to the pathogenesis of vascular diseases. Class 3 semaphorins (SEMA3s), a group of neuron-secreted axonal and vascular guidance factors, suppress pathological vascular growth in retinopathy. However, the upstream transcriptional regulators that mediate the function of SEMA3s in vascular growth are poorly understood. Here we showed that retinoic acid receptor-related orphan receptor α (RORα), a nuclear receptor and transcription factor, is a novel transcriptional regulator of SEMA3E-mediated neurovascular coupling in a mouse model of oxygen-induced proliferative retinopathy. We found that genetic deficiency of RORα substantially induced Sema3e expression in retinopathy. Both RORα and SEMA3E were expressed in retinal ganglion cells. RORα directly bound to a specific ROR response element on the promoter of Sema3e and negatively regulated Sema3e promoter-driven luciferase expression. Suppression of Sema3e using adeno-associated virus 2 carrying short hairpin RNA targeting Sema3e promoted disoriented pathological neovascularization and partially abolished the inhibitory vascular effects of RORα deficiency in retinopathy. Our findings suggest that RORα is a novel transcriptional regulator of SEMA3E-mediated neurovascular coupling in pathological retinal angiogenesis.-Sun, Y., Liu, C.-H., Wang, Z., Meng, S. S., Burnim, S. B., SanGiovanni, J. P., Kamenecka, T. M., Solt, L. A., Chen, J. RORα modulates semaphorin 3E transcription and neurovascular interaction in pathological retinal angiogenesis.
Taniguchi T, Woodward AM, Magnelli P, McColgan NM, Lehoux S, Jacobo SMP, Mauris J, Argüeso P. N-Glycosylation affects the stability and barrier function of the MUC16 mucin. J Biol Chem 2017;292(26):11079-11090.Abstract
Transmembrane mucins are highly O-glycosylated glycoproteins that coat the apical glycocalyx on mucosal surfaces and represent the first line of cellular defense against infection and injury. Relatively low levels of N-glycans are found on transmembrane mucins, and their structure and function remain poorly characterized. We previously reported that carbohydrate-dependent interactions of transmembrane mucins with galectin-3 contribute to maintenance of the epithelial barrier at the ocular surface. Now, using MALDI-TOF mass spectrometry, we report that transmembrane mucin N-glycans in differentiated human corneal epithelial cells contain primarily complex-type structures with N-acetyllactosamine, a preferred galectin ligand. In N-glycosylation inhibition experiments, we find that treatment with tunicamycin and siRNA-mediated knockdown of the Golgi N-acetylglucosaminyltransferase I gene (MGAT1) induce partial loss of both total and cell-surface levels of the largest mucin, MUC16, and a concomitant reduction in glycocalyx barrier function. Moreover, we identified a distinct role for N-glycans in promoting MUC16's binding affinity toward galectin-3 and in causing retention of the lectin on the epithelial cell surface. Taken together, these studies define a role for N-linked oligosaccharides in supporting the stability and function of transmembrane mucins on mucosal surfaces.
Li H, Reksten TR, Ice JA, Kelly JA, Adrianto I, Rasmussen A, Wang S, He B, Grundahl KM, Glenn SB, Miceli-Richard C, Bowman S, Lester S, Eriksson P, Eloranta M-L, Brun JG, Gøransson LG, Harboe E, Guthridge JM, Kaufman KM, Kvarnström M, Cunninghame Graham DS, Patel K, Adler AJ, Farris DA, Brennan MT, Chodosh J, Gopalakrishnan R, Weisman MH, Venuturupalli S, Wallace DJ, Hefner KS, Houston GD, Huang AJW, Hughes PJ, Lewis DM, Radfar L, Vista ES, Edgar CE, Rohrer MD, Stone DU, Vyse TJ, Harley JB, Gaffney PM, James JA, Turner S, Alevizos I, Anaya J-M, Rhodus NL, Segal BM, Montgomery CG, Scofield HR, Kovats S, Mariette X, Rönnblom L, Witte T, Rischmueller M, Wahren-Herlenius M, Omdal R, Jonsson R, Ng W-F, for Registry UKPS's S, Nordmark G, Lessard CJ, Sivils KL. Identification of a Sjögren's syndrome susceptibility locus at OAS1 that influences isoform switching, protein expression, and responsiveness to type I interferons. PLoS Genet 2017;13(6):e1006820.Abstract
Sjögren's syndrome (SS) is a common, autoimmune exocrinopathy distinguished by keratoconjunctivitis sicca and xerostomia. Patients frequently develop serious complications including lymphoma, pulmonary dysfunction, neuropathy, vasculitis, and debilitating fatigue. Dysregulation of type I interferon (IFN) pathway is a prominent feature of SS and is correlated with increased autoantibody titers and disease severity. To identify genetic determinants of IFN pathway dysregulation in SS, we performed cis-expression quantitative trait locus (eQTL) analyses focusing on differentially expressed type I IFN-inducible transcripts identified through a transcriptome profiling study. Multiple cis-eQTLs were associated with transcript levels of 2'-5'-oligoadenylate synthetase 1 (OAS1) peaking at rs10774671 (PeQTL = 6.05 × 10-14). Association of rs10774671 with SS susceptibility was identified and confirmed through meta-analysis of two independent cohorts (Pmeta = 2.59 × 10-9; odds ratio = 0.75; 95% confidence interval = 0.66-0.86). The risk allele of rs10774671 shifts splicing of OAS1 from production of the p46 isoform to multiple alternative transcripts, including p42, p48, and p44. We found that the isoforms were differentially expressed within each genotype in controls and patients with and without autoantibodies. Furthermore, our results showed that the three alternatively spliced isoforms lacked translational response to type I IFN stimulation. The p48 and p44 isoforms also had impaired protein expression governed by the 3' end of the transcripts. The SS risk allele of rs10774671 has been shown by others to be associated with reduced OAS1 enzymatic activity and ability to clear viral infections, as well as reduced responsiveness to IFN treatment. Our results establish OAS1 as a risk locus for SS and support a potential role for defective viral clearance due to altered IFN response as a genetic pathophysiological basis of this complex autoimmune disease.
Mombaerts I, Bilyk JR, Rose GE, McNab AA, Fay A, Dolman PJ, Allen RC, Devoto MH, Harris GJ, of the Society EPO. Consensus on Diagnostic Criteria of Idiopathic Orbital Inflammation Using a Modified Delphi Approach. JAMA Ophthalmol 2017;Abstract
Importance: Current practice to diagnose idiopathic orbital inflammation (IOI) is inconsistent, leading to frequent misdiagnosis of other orbital entities, including cancer. By specifying criteria, diagnosis of orbital inflammation will be improved. Objective: To define a set of criteria specific for the diagnosis of IOI. Design, Setting, and Participants: A 3-round modified Delphi process with an expert panel was conducted from June 8, 2015, to January 25, 2016. Fifty-three orbital scientist experts, identified through membership in the Orbital Society, were invited to participate in on online survey and they scored, using 5-point Likert scales, items that are eligible as diagnostic criteria from the literature and from personal experience. The items were clustered around the anatomic subtypes of IOI: idiopathic dacryoadenitis and idiopathic orbital fat inflammation (2 nonmyositic IOIs), and idiopathic orbital myositis (myositic IOI). Items with dissensus were rescored in the second round, and all items with consensus (median, ≥4; interquartile range, ≤1) were ranked by importance in the third round. Main Outcomes and Measures: Consensus on items to be included in the criteria. Results: Of the 53 experts invited to participate, a multinational panel of 35 (66%) individuals with a mean (SD) years of experience of 31 (11) years were included. Consensus was achieved on 7 of 14 clinical and radiologic items and 5 of 7 pathologic items related to diagnosis of nonmyositic IOI, and 11 of 14 clinical and radiologic items and 1 of 5 pathologic items for myositic IOI. There was agreement among panelists to focus on surgical tissue biopsy results in the diagnosis of nonmyositic IOI and on a trial with systemic corticosteroids in myositic IOI. Panelists agreed that a maximum number of 30 IgG4-positive plasma cells per high-power field in the orbital tissue is compatible with the diagnosis of IOI. Conclusions and Relevance: An international panel of experts endorsed consensus diagnostic criteria of IOI. These criteria define a level of exclusion suggested for diagnosis and include tissue biopsy for lesions not confined to the extraocular muscles. This consensus is a step toward developing guidelines for the management of IOI, which needs to be followed by validation studies of the criteria.
Khajavi M, Zhou Y, Birsner AE, Bazinet L, Rosa Di Sant A, Schiffer AJ, Rogers MS, Krishnaji ST, Hu B, Nguyen V, Zon L, D'Amato RJ. Identification of Padi2 as a novel angiogenesis-regulating gene by genome association studies in mice. PLoS Genet 2017;13(6):e1006848.Abstract
Recent findings indicate that growth factor-driven angiogenesis is markedly influenced by genetic variation. This variation in angiogenic responsiveness may alter the susceptibility to a number of angiogenesis-dependent diseases. Here, we utilized the genetic diversity available in common inbred mouse strains to identify the loci and candidate genes responsible for differences in angiogenic response. The corneal micropocket neovascularization assay was performed on 42 different inbred mouse strains using basic fibroblast growth factor (bFGF) pellets. We performed a genome-wide association study utilizing efficient mixed-model association (EMMA) mapping using the induced vessel area from all strains. Our analysis yielded five loci with genome-wide significance on chromosomes 4, 8, 11, 15 and 16. We further refined the mapping on chromosome 4 within a haplotype block containing multiple candidate genes. These genes were evaluated by expression analysis in corneas of various inbred strains and in vitro functional assays in human microvascular endothelial cells (HMVECs). Of these, we found the expression of peptidyl arginine deiminase type II (Padi2), known to be involved in metabolic pathways, to have a strong correlation with a haplotype shared by multiple high angiogenic strains. In addition, inhibition of Padi2 demonstrated a dosage-dependent effect in HMVECs. To investigate its role in vivo, we knocked down Padi2 in transgenic kdrl:zsGreen zebrafish embryos using morpholinos. These embryos had disrupted vessel formation compared to control siblings. The impaired vascular pattern was partially rescued by human PADI2 mRNA, providing evidence for the specificity of the morphant phenotype. Taken together, our study is the first to indicate the potential role of Padi2 as an angiogenesis-regulating gene. The characterization of Padi2 and other genes in associated pathways may provide new understanding of angiogenesis regulation and novel targets for diagnosis and treatment of a wide variety of angiogenesis-dependent diseases.
Lee JK, Shin SR, Desalvo A, Lee G, Lee JY, Polini A, Chae S, Jeong H, Kim J, Choi H, Lee HY. Nonmediated, Label-Free Based Detection of Cardiovascular Biomarker in a Biological Sample. Adv Healthc Mater 2017;6(17)Abstract
Direct electrochemical (EC) monitoring in a cell culture medium without electron transporter as called mediator is attractive topic in vitro organoid based on chip with frequently and long-time monitoring since it can avoid to its disadvantage as stability, toxicity. Here, direct monitoring with nonmediator is demonstrated based on impedance spectroscopy under the culture medium in order to overcome the limitation of mediator. The applicability of EC monitoring is shown by detecting alpha-1-anti trypsin (A1AT) which is known as biomarkers for cardiac damage and is widely chosen in organoid cardiac cell-based chip. The validity of presented EC monitoring is proved by observing signal processing and transduction in medium, mediator, medium-mediator complex. After the observation of electron behavior, A1AT as target analyte is immobilized on the electrode and detected using antibody-antigen interaction. As a result, the result indicates limit of detection is 10 ng mL(-1) and linearity for the 10-1000 ng mL(-1) range, with a sensitivity of 3980 nF (log [g mL])(-1) retaining specificity. This EC monitoring is based on label-free and reagentless detection, will pave the way to use for continuous and simple monitoring of in vitro organoid platform.
Mahmoud MM, Serbanovic-Canic J, Feng S, Souilhol C, Xing R, Hsiao S, Mammoto A, Chen J, Ariaans M, Francis SE, Van der Heiden K, Ridger V, Evans PC. Shear stress induces endothelial-to-mesenchymal transition via the transcription factor Snail. Sci Rep 2017;7(1):3375.Abstract
Blood flow influences atherosclerosis by generating wall shear stress, which alters endothelial cell (EC) physiology. Low shear stress induces dedifferentiation of EC through a process termed endothelial-to-mesenchymal transition (EndMT). The mechanisms underlying shear stress-regulation of EndMT are uncertain. Here we investigated the role of the transcription factor Snail in low shear stress-induced EndMT. Studies of cultured EC exposed to flow revealed that low shear stress induced Snail expression. Using gene silencing it was demonstrated that Snail positively regulated the expression of EndMT markers (Slug, N-cadherin, α-SMA) in EC exposed to low shear stress. Gene silencing also revealed that Snail enhanced the permeability of endothelial monolayers to macromolecules by promoting EC proliferation and migration. En face staining of the murine aorta or carotid arteries modified with flow-altering cuffs demonstrated that Snail was expressed preferentially at low shear stress sites that are predisposed to atherosclerosis. Snail was also expressed in EC overlying atherosclerotic plaques in coronary arteries from patients with ischemic heart disease implying a role in human arterial disease. We conclude that Snail is an essential driver of EndMT under low shear stress conditions and may promote early atherogenesis by enhancing vascular permeability.
Laíns I, Wang J, Providência J, Mach S, Gil P, Gil J, Marques M, Armstrong G, Garas S, Barreto P, Kim IK, Vavvas DG, Miller JW, Husain D, Silva R, Miller JB. Choroidal Changes Associated With Subretinal Drusenoid Deposits in Age-related Macular Degeneration Using Swept-source Optical Coherence Tomography. Am J Ophthalmol 2017;180:55-63.Abstract
PURPOSE: To compare choroidal vascular features of eyes with and without subretinal drusenoid deposits (SDD), using swept-source optical coherence tomography (SS OCT). DESIGN: Multicenter, cross-sectional study. METHODS: We prospectively recruited patients with intermediate age-related macular degeneration (AMD), without other vitreoretinal pathology. All participants underwent complete ophthalmic examination, color fundus photography (used for AMD staging), and spectral-domain OCT (to evaluate the presence of SDD). SS OCT was used to obtain automatic macular choroidal thickness (CT) maps, according to the Early Treatment Diabetic Retinopathy Study (ETDRS) sectors. For data analysis, we considered mean choroidal thickness as the arithmetic mean value of the 9 ETDRS sectors. SS OCT en face images of choroidal vasculature were also captured and converted to binary images. Choroidal vascular density (CVD) was calculated as a percent area occupied by choroidal vessels in a 6-mm-diameter submacular circular. Choroidal vessel volume was calculated by multiplying the average CVD by macular area and CT. Multilevel mixed linear models (to account for the inclusion of 2 eyes of same subject) were performed for analysis. RESULTS: We included 186 eyes (n = 118 subjects), 94 (50.5%) presenting SDD. Multiple regression analysis revealed that, controlling for age, eyes with SDD presented a statistically thinner mean CT (ß = -21.9, P = .006) and CT in all the individual ETDRS fields (ß ≤ -18.79, P ≤ .026). Mean choroidal vessel volume was also significantly reduced in eyes with SDD (ß = -0.003, P = .007). No significant associations were observed with mean CVD. CONCLUSION: In subjects with intermediate AMD, choroidal thickness and vessel volume are reduced in the presence of subretinal drusenoid deposits.
Jakobiec FA, Zakka FR, Lee NG. Exophytic Osteochondroma of the Brow. Ophthal Plast Reconstr Surg 2017;33(6):e166-e169.Abstract
Most bony and cartilaginous lesions of the orbit and periorbital compartments are benign, grow endophytically, and are composed of dense lamellar bone (eburnated or ivory osteomas). An 87-year-old woman had a well-circumscribed, firm, round, and exophytic lesion of the brow region for at least 15 years. Excisional surgery disclosed an osteocartilaginous lesion with an enveloping pseudocapsule (periosteum/perichondrium) and a narrow stalk connecting it to the frontal bone. The periphery of the lesion displayed lamellar bone which appeared to be replacing a central cartilaginous zone. The adjacent deep preaponeurotic fat displayed nodules of collagen with myxoid change and occasional CD34 spindle cells suggestive of a spindle cell lipoma. Because of the osteochondroma's deep location in the preaponeurotic fat, the lesion differs from an osteoma cutis found in the dermis which fails to exhibit a cartilaginous component or a periosteum. Other clinically simulating lesions are described.
da Silva S, Cepko CL. Fgf8 Expression and Degradation of Retinoic Acid Are Required for Patterning a High-Acuity Area in the Retina. Dev Cell 2017;42(1):68-81.e6.Abstract
Species that are highly reliant on their visual system have a specialized retinal area subserving high-acuity vision, e.g., the fovea in humans. Although of critical importance for our daily activities, little is known about the mechanisms driving the development of retinal high-acuity areas (HAAs). Using the chick as a model, we found a precise and dynamic expression pattern of fibroblast growth factor 8 (Fgf8) in the HAA anlage, which was regulated by enzymes that degrade retinoic acid (RA). Transient manipulation of RA signaling, or reduction of Fgf8 expression, disrupted several features of HAA patterning, including photoreceptor distribution, ganglion cell density, and organization of interneurons. Notably, patterned expression of RA signaling components was also found in humans, suggesting that RA also plays a role in setting up the human fovea.

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