| 1. |
- Buck, Kerstin, et al.
(author)
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Ser129 phosphorylation of endogenous α-synuclein induced by overexpression of polo-like kinases 2 and 3 in nigral dopamine neurons is not detrimental to their survival and function.
- 2015
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In: Neurobiology of Disease. - : Elsevier BV. - 0969-9961. ; 78:Mar 25, s. 100-114
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Journal article (peer-reviewed)abstract
- Phosphorylation of the α-synuclein (α-syn) protein at Ser129 [P(S129)-α-] was found to be the most abundant form in intracellular inclusions in brains from Parkinson's disease (PD) patients. This finding suggests that P(S129)-α-syn plays a central role in the pathogenesis of PD. However, it is at present unclear whether P(S129)-α-syn is pathogenic driving the neurodegenerative process. Rodent studies using neither the phosphomimics of human α-syn nor co-expression of human wild-type α-syn and kinases phosphorylating α-syn at Ser129 gave consistent results. One major concern in interpreting these findings is that human α-syn was expressed above physiological levels inducing neurodegeneration in rat nigral neurons. In order to exclude this confounding factor, we took a different approach and increased the phosphorylation level of endogenous α-syn. For this purpose, we took advantage of recombinant adeno-associated viral (rAAV) vectors to deliver polo-like kinase 2 (PLK2) or PLK3 in the substantia nigra and investigated whether increased levels of P(S129)-α-syn compromised the function and survival of nigral dopaminergic neurons. Interestingly, we observed that hyperphosphorylated α-syn did not induce nigral dopaminergic cell death, as assessed at 1 and 4months. Furthermore, histological analysis did not show any accumulation of α-syn protein or formation of inclusions. Using in vivo microdialysis, we found that the only measurable functional alteration was the depolarisation-induced release of dopamine, while the in vivo synthesis rate of DOPA and dopamine baseline release remained unaltered. Taken together, our results suggest that phosphorylation of α-syn at Ser129 does not confer a toxic gain of function per se.
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| 2. |
- Decressac, Mickael, et al.
(author)
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GDNF fails to exert neuroprotection in a rat {alpha}-synuclein model of Parkinson's disease.
- 2011
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In: Brain. - : Oxford University Press (OUP). - 1460-2156 .- 0006-8950. ; 134:8, s. 2302-2311
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Journal article (peer-reviewed)abstract
- The neuroprotective effect of the glial cell line-derived neurotrophic factor has been extensively studied in various toxic models of Parkinson's disease. However, it remains unclear whether this neurotrophic factor can protect against the toxicity induced by the aggregation-prone protein α-synuclein. Targeted overexpression of human wild-type α-synuclein in the nigrostriatal system, using adeno-associated viral vectors, causes a progressive degeneration of the nigral dopamine neurons and the development of axonal pathology in the striatum. In the present study, we investigated, using different paradigms of delivery, whether glial cell line-derived neurotrophic factor can protect against the neurodegenerative changes and the cellular stress induced by α-synuclein. We found that viral vector-mediated delivery of glial cell line-derived neurotrophic factor into substantia nigra and/or striatum, administered 2-3 weeks before α-synuclein, was inefficient in preventing the wild-type α-synuclein-induced loss of dopamine neurons and terminals. In addition, glial cell line-derived neurotrophic factor overexpression did not ameliorate the behavioural deficit in this rat model of Parkinson's disease. Quantification of striatal α-synuclein-positive aggregates revealed that glial cell line-derived neurotrophic factor had no effect on α-synuclein aggregation. These data provide the evidence for the lack of neuroprotective effect of glial cell line-derived neurotrophic factor against the toxicity of human wild-type α-synuclein in an in vivo model of Parkinson's disease. The difference in neuroprotective efficacy of glial cell line-derived neurotrophic factor seen in our model and the commonly used neurotoxin models of Parkinson's disease, raises important issues pertinent to the interpretation of the results obtained in preclinical models of Parkinson's disease, and their relevance for the therapeutic use glial cell line-derived neurotrophic factor in patients with Parkinson's disease.
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| 3. |
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| 4. |
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| 5. |
- Lastres-Becker, Isabel, et al.
(author)
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alpha-Synuclein expression and Nrf2 deficiency cooperate to aggravate protein aggregation, neuronal death and inflammation in early-stage Parkinson's disease
- 2012
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In: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 21:14, s. 3173-3192
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Journal article (peer-reviewed)abstract
- Although -synuclein (-SYN) aggregation is a hallmark of sporadic and familial Parkinsons disease (PD), it is not known how it contributes to early events of PD pathogenesis such as oxidative and inflammatory stress. Here, we addressed this question in a new animal model based on stereotaxic delivery of an adeno-associated viral vector (rAAV) for expression of human -SYN in the ventral midbrain of mice lacking the transcription factor Nrf2 (Nrf2(/)). Two months after surgery, Nrf2(/) mice exhibited exacerbated degeneration of nigral dopaminergic neurons and increased dystrophic dendrites, reminiscent of Lewy neurites, which correlated with impaired proteasome gene expression and activity. Dopaminergic neuron loss was associated with an increase in neuroinflammation and gliosis that were intensified in Nrf2(/) mice. In response to exogenously added -SYN, Nrf2(/) microglia failed to activate the expression of two anti-inflammatory genes, heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate quinone oxidorreductase-1 (NQO1). This impaired Nrf2 response correlated with a shift in the microglial activation profile, towards increased production of proinflammatory markers, IL-6, IL-1 and iNOS and reduced phagocytic capacity of fluorescent beads, and lower messenger RNA levels for TAM receptors Axl and Mer. Postmortem brain tissue samples from patients in early- to middle-stage progression of PD showed increased HO-1 expression in astrocytes and microglia, suggesting an attempt of the diseased brain to compensate these hallmarks of PD through activation of the Nrf2 pathway. This study demonstrates that -SYN and Nrf2 deficiency cooperate on protein aggregation, neuroinflammation and neuronal death and provides a bifactorial animal model to study early-stage PD.
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| 6. |
- Ulusoy, Ayse, et al.
(author)
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Can overexpression of parkin provide a novel strategy for neuroprotection in Parkinson's disease?
- 2008
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In: Experimental Neurology. - : Elsevier BV. - 0014-4886. ; May 3, s. 258-260
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Journal article (peer-reviewed)abstract
- Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by selective degeneration of the dopamine producing neurons in the substantia nigra. There is currently no clinically applicable therapy for treating or preventing Parkinsonian neurodegeneration. Great effort is put behind the development of novel therapeutic approaches that aim to alter the natural progression of the disease. For example, a disease-modifying strategy based on the use of glial cell line-derived neurotrophic factor family of ligands have yielded successful results in animal models and later in initial clinical trials. More recently, identification of the gene mutations underlying the familial forms of the disease opened new frontiers in tackling the underlying neuropathological changes seen in PD brains. Overexpression of parkin, in particular, emerged as a powerful approach with complementary effects to those described with use of neurotrophic factors. In light of the fact that the mechanism of disease in the affected patient population might be significantly variable, the ability to intervene the disease process at multiple levels should be seen as a key point in devising effective treatments.
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| 7. |
- Ulusoy, Ayse, et al.
(author)
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Co-expression of C-terminal truncated alpha-synuclein enhances full-length alpha-synuclein-induced pathology.
- 2010
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In: European Journal of Neuroscience. - : Wiley. - 1460-9568 .- 0953-816X. ; 32:3, s. 409-422
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Journal article (peer-reviewed)abstract
- Lewy bodies, which are a pathological hallmark of Parkinson's disease, contain insoluble polymers of alpha-synuclein (alphasyn). Among the different modifications that can promote the formation of toxic alphasyn species, C-terminal truncation is among the most abundant alterations in patients with Parkinson's disease. In vitro, C-terminal truncated alphasyn aggregates faster and sub-stoichiometric amounts of C-terminal truncated alphasyn promote aggregation of the full-length alphasyn (alphasynFL) and induce neuronal toxicity. To address in vivo the putative stimulation of alphasyn-induced pathology by the presence of truncated alphasyn, we used recombinant adeno-associated virus to express either alphasynFL or a C-terminal truncated alphasyn (1-110) in rats. We adjusted the recombinant adeno-associated virus vector concentrations so that either protein alone led to only mild to moderate axonal pathology in the terminals of nigrostriatal dopamine neurons without frank cell loss. When these two forms of alphasyn were co-expressed at these pre-determined levels, it resulted in a more aggressive pathology in fiber terminals as well as dopaminergic cell loss in the substantia nigra. Using an antibody that did not detect the C-terminal truncated alphasyn (1-110) but only alphasynFL, we demonstrated that the co-expressed truncated protein promoted the progressive accumulation of alphasynFL and formation of larger pathological accumulations. Moreover, in the co-expression group, three of the eight animals showed apomorphine-induced turning, suggesting prominent post-synaptic alterations due to impairments in the dopamine release, whereas the mild pathology induced by either form alone did not cause motor abnormalities. Taken together these data suggest that C-terminal truncated alphasyn can interact with and exacerbate the formation of pathological accumulations containing alphasynFL in vivo.
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| 8. |
- Ulusoy, Ayse, et al.
(author)
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Dose Optimization for Long-term rAAV-mediated RNA Interference in the Nigrostriatal Projection Neurons.
- 2009
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In: Molecular Therapy. - : Elsevier BV. - 1525-0024 .- 1525-0016. ; 17, s. 1574-1584
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Journal article (peer-reviewed)abstract
- Short-hairpin RNA (shRNA)-mediated gene knockdown is a powerful tool for targeted gene silencing and an emerging novel therapeutic strategy. Recent publications, however, reported unexpected toxicity after utilizing viral-mediated shRNA knockdown in vivo. Thus, it is currently unclear whether shRNA-mediated knockdown strategy can be used as a safe and efficient tool for gene silencing. In this study, we have generated rAAV vectors expressing shRNAs targeting the rat tyrosine hydroxylase (TH) mRNA (shTH) for testing the efficacy of in vivo TH knockdown in the nigral dopaminergic neurons. At high titers, not only the shTH vectors but also the scrambled and green fluorescence protein (GFP)-only controls caused cell death. In a dose-response study, we identified a dose window leading to >60% decrease in TH(+) neurons without any change in vesicular monoamine transporter-2 (VMAT2) expression. Moreover, using the safe and efficient dose, we showed that dopamine (DA) synthesis rate was significantly reduced and this lead to emergence of motor deficits in the shTH-expressing rats. Interestingly, these animals showed very robust and long-lasting recovery after a single systemic L-3,4-dihydroxyphenylalanine (L-DOPA) administration beyond what can be achieved in 6-hydroxydopamine (6-OHDA)-lesioned rats. Our results have implications for both mechanistic and therapeutic studies utilizing long-term shRNA-mediated gene silencing in the nigrostriatal projection system.Molecular Therapy (2009); doi:10.1038/mt.2009.142.
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| 9. |
- Ulusoy, Ayse, et al.
(author)
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Dysregulated dopamine storage increases the vulnerability to alpha-synuclein in nigral neurons
- 2012
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In: Neurobiology of Disease. - : Elsevier BV. - 0969-9961. ; 47:3, s. 367-377
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Journal article (peer-reviewed)abstract
- Impairments in the capacity of dopaminergic neurons to handle cytoplasmic dopamine may be a critical factor underlying the selective vulnerability of midbrain dopamine neurons in Parkinson's disease. Furthermore, toxicity of alpha-synuclein in dopaminergic neurons has been suggested to be mediated by direct interaction between dopamine and alpha-synuclein through formation of abnormal alpha-synuclein species, although direct in vivo evidence to support this hypothesis is lacking. Here, we investigated the role of dopamine availability on alpha-synuclein mediated neurodegeneration in vivo. We found that overexpression of alpha-synuclein in nigral dopamine neurons in mice with deficient vesicular storage of dopamine led to a significant increase in dopaminergic neurodegeneration. Importantly, silencing the tyrosine hydroxylase enzyme - thereby reducing dopamine content in the nigral neurons - reversed the increased vulnerability back to the baseline level observed in wild-type littermates, but failed to eliminate it completely. Importantly, TH knockdown was not effective in altering the toxicity in the wild-type animals. Taken together, our data suggest that under normal circumstances, in healthy dopamine neurons, cytoplasmic dopamine is tightly controlled such that it does not contribute significantly to alpha-synuclein mediated toxicity. Dysregulation of the dopamine machinery in the substantia nigra, on the other hand, could act as a trigger for induction of increased toxicity in these neurons and could explain how these neurons become more vulnerable and die in the disease process. (C) 2012 Elsevier Inc. All rights reserved.
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| 10. |
- Ulusoy, Ayse, et al.
(author)
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In vivo gene delivery for development of mammalian models for Parkinson's disease
- 2008
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In: Experimental Neurology. - : Elsevier BV. - 0014-4886. ; 209:1, s. 89-100
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Research review (peer-reviewed)abstract
- During the last decade, identification of the genes involved in familial forms of Parkinson's disease (PD) has advanced our understanding of the mechanisms underlying the development of different aspects of PD. However the available animal models still remain as the main limiting factor for the development of neuroprotective therapies that can halt the progression of the disease, through which we wish to provide a better quality of life for the PD patients. Here, we review the recently developed animal models based on overexpression of PD-associated genes using recombinant viral vectors. Recombinant adeno-associated viral vectors, in particular, have been very useful in targeting the nigral dopamine neurons both in the rodent and the primate brain. In order to provide insights into the establishment of these models in the laboratory, we will not only give an overview of the results from these studies but also cover practical issues related to the production and handling of the viral vectors, which are critical for the successful application of this approach.
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| 11. |
- Ulusoy, Ayse
(author)
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Modeling pathophysiological aspects of Parkinson’s disease: Manipulating DA handling and alpha-synuclein expression in the nigrostriatal pathway using viral vectors
- 2010
-
Doctoral thesis (other academic/artistic)abstract
- The pathological hallmark of Parkinson’s disease is dopaminergic neurodegeneration in the substantia nigra pars compacta neurons and accumulation of α-synuclein containing aggregates in the surviving neurons. The cause of cell death in Parkinson’s disease and the involvement of α-synuclein in the pathophysiology of the disease are unknown. Although other neuronal cell types exhibit α-synuclein positive aggregates, substantia nigra dopamine neurons display a selective vulnerability to α-synuclein mediated neurodegeneration. In this thesis work I have focused on the possible mechanisms underlying the vulnerability of dopamine producing neurons against α-synuclein induced neurotoxicity. To study the molecular interactions playing role in α-synuclein mediated dopaminergic neurodegeneration, we investigated putative mechanisms that has been implicated in α-synuclein toxicity. Interaction of the α-synuclein protein with other molecules has been suggested to enhance the aggregation. We studied the interaction between the full-length α-synculein protein and truncated α-synuclein in the rat substantia nigra. When the two forms are co-expressed the truncated form promotes full-length α-syn aggregation and enhance the pathology caused by the full-length protein. We next investigated the specific role of dopamine handling machinery in Parkinson’s disease pathophysiology and treatment related motor complications. To study the involvement of cytosolic DA and age related increase in the reactive DA species on α-syn toxicity, we utilized a transgenic mouse model carrying a hypomorphic VMAT2 mutation. The elevated cytosolic dopamine in these mice led to an increased vulnerability to α-synuclein overexpression. To show that this vulnerability was indeed dopamine dependent, we generated recombinant adeno-associated viral vectors to transfer short hairpin RNA sequences targeting the rate-limiting enzyme, tyrosine hydroxylase. Reducing dopamine production using the shRNA approach in these mice rescued the vulnerability against α-synuclein in the nigral dopamine neurons. Our results implicate the critical role of dopamine handling in Parkinson’s disease pathophysiology, thus suggest that regulating the specific pathways through which DA mediates its toxic effects can prevent the potential neurodegeneration.
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| 12. |
- Ulusoy, Ayse, et al.
(author)
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Presynaptic dopaminergic compartment determines the susceptibility to L-DOPA-induced dyskinesia in rats.
- 2010
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In: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 107:29, s. 13159-13164
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Journal article (peer-reviewed)abstract
- Drug-induced dyskinesias in dopamine-denervated animals are known to depend on both pre- and postsynaptic changes of the nigrostriatal circuitry. In lesion models used thus far, changes occur in both of these compartments and, therefore, it has not been possible to dissect the individual contribution of each compartment in the pathophysiology of dyskinesias. Here we silenced the nigrostriatal dopamine neurotransmission without affecting the anatomical integrity of the presynaptic terminals using a short-hairpin RNA-mediated knockdown of tyrosine hydroxylase enzyme (shTH). This treatment resulted in significant reduction (by about 70%) in extracellular dopamine concentration in the striatum as measured by on-line microdialysis. Under these conditions, the animals remained nondyskinetic after chronic L-DOPA treatment, whereas partial intrastriatal 6-hydoxydopamine lesioned rats with comparable reduction in extracellular dopamine levels developed dyskinesias. On the other hand, apomorphine caused moderate to severe dyskinesias in both groups. Importantly, single-dose L-DOPA challenge in apomorphine-primed shTH animals failed to activate the already established abnormal postsynaptic responses. Taken together, these data provide direct evidence that the status of the presynaptic, DA releasing compartment is a critical determinant of both the induction and maintenance of L-DOPA-induced dyskinesias.
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| 13. |
- Ulusoy, Ayse, et al.
(author)
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Viral vector-mediated overexpression of α-synuclein as a progressive model of Parkinson's disease
- 2010
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In: Progress in Brain Research. - 1875-7855. ; 184, s. 89-111
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Research review (peer-reviewed)abstract
- The discovery of the role of α-synuclein in the pathogenesis of Parkinson's disease (PD) has opened new possibilities for the development of more authentic models of Parkinson's disease. Recombinant adeno-associated virus (AAV) and lentivirus (LV) vectors are efficient tools for expression of genes locally in subsets of neurons in the brain and can be used to express human wild-type or mutated α-synuclein selectively in midbrain dopamine neurons. Using this approach, it is possible to trigger extensive PD-like cellular and axonal pathologies in the nigrostriatal projection, involving abnormal protein aggregation, neuronal dysfunction, and cell death that develop progressively over time. Targeted overexpression of human α-synuclein in midbrain dopamine neurons, using AAV vectors, reproduces many of the characteristic features of the human disease and provides, for the first time, a model of progressive PD that can be applied to both rodents and primates.
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| 14. |
- Xilouri, Maria, et al.
(author)
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Selective neuroprotective effects of the S18Y polymorphic variant of UCH-L1 in the dopaminergic system
- 2012
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In: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 21:4, s. 874-889
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Journal article (peer-reviewed)abstract
- Genetic studies have implicated the neuronal ubiquitin C-terminal hydrolase (UCH) protein UCH-L1 in Parkinson's disease (PD) pathogenesis. Moreover, the function of UCH-L1 may be lost in the brains of PD and Alzheimer's disease patients. We have previously reported that the UCH-L1 polymorphic variant S18Y, potentially protective against PD in population studies, demonstrates specific antioxidant functions in cell culture. Albeit genetic, biochemical and neuropathological data support an association between UCH-L1, PD, synaptic degeneration and oxidative stress, the relationship between the dopaminergic system and UCH-L1 status remains obscure. In the current study, we have examined the dopaminergic system of mice lacking endogenous UCH-L1 protein (gracile axonal dystrophy mice). Our findings show that the lack of wild-type (WT) UCH-L1 does not influence to any significant degree the dopaminergic system at baseline or following injections of the neurotoxin methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Furthermore, using a novel intrastriatal adenoviral injection protocol, we have found that mouse nigral neurons retrogradely transduced with S18Y UCH-L1, but not the WT protein, are significantly protected against MPTP toxicity. Overall, these data provide evidence for an antioxidant and neuroprotective effect of the S18Y variant of UCH-L1, but not of the WT protein, in the dopaminergic system, and may have implications for the pathogenesis of PD or related neurodegenerative conditions, in which oxidative stress might play a role.
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