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Brain Research Bulletin
Volume 56, Issues 3–4,
1 November 2001
, Pages 319-329
Author links open overlay panel, ,
We produced eight anti-huntingtin (Htt) monoclonal antibodies (mAbs), several of which have novel binding patterns. Peptide array epitope mapping shows that mAbs MW1–6 specifically bind the polyQ domain of Htt exon 1. On Western blots of extracts from mutant Htt knock-in mouse brain and Huntington’s disease lymphoblastoma cell lines, MW1-5 all strongly prefer to bind to the expanded polyQ repeat form of Htt, displaying no detectable binding to normal Htt. These results suggest that the polyQ domain can assume different conformations that are distinguishable by mAbs. This idea is supported by immunohistochemistry with wild type (WT) and mutant Htt transgenic mouse (R6) brains. Despite sharing the same epitope and binding preferences on Western blots, MW1–5 display distinct staining patterns. MW1 shows punctate cytoplasmic and neuropil staining, while MW2–5 strongly stain the neuronal Golgi complex. MW6, in contrast, stains neuronal somas and neuropil. In addition, despite their preference for mutant Htt on blots, none of these mAbs show enhanced staining of R6 brains over WT, and show no binding of the Htt-containing nuclear inclusions in R6 brains. This suggests that in its various subcellular locations, the polyQ domain of Htt either takes on different conformations and/or is differentially occluded by Htt binding proteins. In contrast to MW1–6, MW7, and 8 can differentiate transgenic from WT mice by staining nuclear inclusions in R6/2 brain; MW8 displays no detectable staining in WT brain and stains only inclusions in R6/2 brain. Epitope mapping reveals that MW7 and 8 specifically bind the polyP domains and amino acids 83-90, respectively. As with MW1-6, the epitopes for MW7 and 8 are differentially available in the various subcellular compartments where Htt is found.
Huntington’s disease (HD) is caused by the extension of a polyglutamine (polyQ) tract in the protein huntingtin (Htt) to a length >40 units . Immunohistochemistry and subcellular fractionation indicate that Htt is normally located in the cytoplasm while the mutant form of Htt is also found in aggregates (inclusions) in the nucleus . While this correlation with the disease suggests that translocation and/or aggregation in the nucleus is important for the neuronal cell death that occurs in HD, the importance of the inclusions themselves remains controversial . A related question concerns the binding partners for Htt in the nucleus and the cytoplasm. Studies in yeast, Drosophila and mammalian cells have shown that various chaperone proteins can alter nuclear inclusion formation and/or the toxicity associated with expanded polyQ repeat Htt (as well as ataxin-3, another protein that causes neurodegenerative disease when its polyQ tract is extended) . Other proteins also bind Htt and ataxin-3, which can lead to alterations in transcription 1, 12, 25. Regarding its normal function, Htt has been localized to various subcellular sites, including the neuronal cytoplasm, nucleus, presynaptic vesicles, varicosities, Golgi network, dendritic plasma membranes, and cytoskeleton . Although it is not yet clear how much of this diversity in reported localization is genuine, it would not be surprising if a protein of >3,000 amino acids had multiple functions and sites of action , as well as many different binding partners in various locations.
Critical tools in many of such studies are anti-Htt antibodies, which can be used for immunohistochemistry, immunoprecipitation, structural studies, drug screening, and functional perturbation. Seeking to broaden the range of available anti-Htt monoclonal antibodies (mAbs), and to clarify the subcellular localization of Htt, we have produced eight new mAbs. We used as antigens several expanded polyQ constructs, including soluble Htt exon 1, as well as aggregates of Htt. Some of these mAbs display distinct immunohistochemical staining patterns not seen in prior studies. Coupled with Western blotting results, these staining patterns suggest that the conformation and/or the binding partners of Htt are different in various subcellular compartments and when it forms nuclear inclusions.
Production of mAbs
Six-week-old Balb/c female mice were primed and boosted at 2 week intervals by intraperitoneal injection of antigen emulsified in adjuvant (RIBI Immunochem, Hamilton, MT, USA). Test bleeds were obtained 7 days after every other injection. A final series of boosts was performed without adjuvant. Spleen cells were isolated from the mouse 3 days after the final boost and fused with HL-1 murine myeloma cells (Ventrex, Portland, ME, USA) using polyethylene glycol (PEG 1500, Boehringer-Mannheim,
Generation of the mAbs
For the first round of immunizations, we injected proteins expressed from two constructs containing the polyQ domain (19 or 35 repeats) and 34 amino acids of the dentatorubralpalliodoluysian atrophy (DRPLA) gene fused to glutathione-S-transferase (GST) . Using enzyme linked substrate assay (ELISA) to screen against these antigens versus GST alone, we selected three hybridomas for cloning. These mAbs are termed MW (for Milton Wexler) 1, 2, and 5. As described below, while each of these mAbs
We thank James Burke, Marie-Francoise Chesselet, Vivian Hook, George Jackson, Parsa Kazemi-Esfarjani, Alex Kanzantsev, Ali Khoshnan, George Lawless, Marcy MacDonald, Hemachandro Reddy, Allan Sharp, Gabriele Shilling, Peter Snow, Leslie Thompson, Peter Thumfort, Jonathon Wood, and Scott Zeitlin for generously providing antibodies, cell lines, mouse tissues, and DNA constructs. Peter Snow expressed proteins and Peter Thumfort generously provided the peptide arrays and advice on epitope mapping.
- J.-H.J Cha
Transcriptional dysregulation in Huntington’s disease
- D.R Davies et al.
Twisting into shape
- S.W Davies et al.
Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation
- P Ferrigno et al.
Polyglutamine expansionsProteolysis, chaperones, and the dangers of promiscuity
- J.F Gusella et al.
HuntingtinA single bait hooks many species
Curr. Opin. Neurobiol.
- J.A Lebron et al.
Tolerization of adult mice to immunodominant proteins before monoclonal antibody production
J. Immunol. Methods
- X Lin et al.
Expanding our understanding of polyglutamine diseases through mouse models
- L Mangiarini et al.
Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice
- L Menalled et al.
Decrease in striatal enkephalin mRNA in mouse models of Huntington’s disease
- O Onodera et al.
Toxicity of expanded polyglutamine-domain proteins in Escherichia coli
Recent advances in understanding the pathogenesis of Huntington’s disease
Nuclear accumulation of truncated atrophin-1 fragments in a transgenic mouse model of DRPLA
Huntington’s disease intranuclear inclusions contain truncated, ubiquinated huntingtin protein
SH3GL3 associates with the Huntington exon 1 protein and promotes the formation of polygln-containing protein aggregates
Huntington’s diseaseThe challenge for cell biologists
Trends Cell. Biol.
Molecular basis of Q-length selectivity for the MW1 antibody–huntingtin interaction
2023, Journal of Biological Chemistry
Huntington’s disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein. Huntingtin exon 1 (Httex1), as well as other naturally occurring N-terminal huntingtin fragments with expanded polyQ are prone to aggregation, forming potentially cytotoxic oligomers and fibrils. Antibodies and other N-terminal huntingtin binders are widely explored as biomarkers and possible aggregation-inhibiting therapeutics. A monoclonal antibody, MW1, is known to preferentially bind to huntingtin fragments with expanded polyQ lengths, but the molecular basis of the polyQ length specificity remains poorly understood. Using solution NMR, electron paramagnetic resonance, and other biophysical methods, we investigated the structural features of the Httex1–MW1 interaction. Rather than recognizing residual α-helical structure, which is promoted by expanded Q-lengths, MW1 caused the formation of a new, non-native, conformation in which the entire polyQ is largely extended. This non-native polyQ structure allowed the formation of large mixed Httex1-MW1 multimers (600–2900 kD), when Httex1 with pathogenic Q-length (Q46) was used. We propose that these multivalent, entropically favored interactions, are available only to proteins with longer Q-lengths and represent a major factor governing the Q-length preference of MW1. The present study reveals that it is possible to target proteins with longer Q-lengths without having to stabilize a natively favored conformation. Such mechanisms could be exploited in the design of other Q-length specific binders.
N-terminal mutant huntingtin deposition correlates with CAG repeat length and symptom onset, but not neuronal loss in Huntington's disease
2022, Neurobiology of Disease
Citation Excerpt :
MAB5492+ aggregate number did not correlate with CAG repeat length, symptom onset, SMI-32+ pyramidal cell loss or 1C2+ aggregates (Fig. 8 HK). MW8 was generated against human Htt exon 1 containing a 67-residue polyQ tract, resulting in its specificity for Htt N-terminal amino acids 83–90 (Fig. 9 A) (Ko et al., 2001). Qualitative examination of MW8 immunoreactivity demonstrated diffuse immunoreactivity localized to cell bodies in both HD and control tissue (Fig. 9 BC).
Huntington's disease (HD) is caused by a CAG repeat expansion mutation in the gene encoding the huntingtin (Htt) protein, with mutant Htt protein subsequently forming aggregates within the brain. Mutant Htt is a current target for novel therapeutic strategies for HD, however, the lack of translation from preclinical research to disease-modifying treatments highlights the need to improve our understanding of the role of Htt protein in the human brain. This study aims to undertake an immunohistochemical screen of 12 candidate antibodies against various sequences along the Htt protein to characterize Htt distribution and expression in post-mortem human brain tissue microarrays (TMAs).
Immunohistochemistry was performed on middle temporal gyrus TMAs comprising of up to 28 HD and 27 age-matched control cases, using 12 antibodies specific to various sequences along the Htt protein. From this study, six antibodies directed to the Htt N-terminus successfully immunolabeled human brain tissue. Htt aggregates and Htt protein expression levels for the six successful antibodies were subsequently quantified with a customized automated image analysis pipeline on the TMAs. A 2.5–12 fold increase in the number of Htt aggregates were detected in HD cases using antibodies MAB5374, MW1, and EPR5526, despite no change in overall Htt protein expression compared to control cases, suggesting a redistribution of Htt into aggregates in HD. MAB5374, MW1, and EPR5526 Htt aggregate numbers were positively correlated with CAG repeat length, and negatively correlated with the age of symptom onset in HD. However, the number of Htt aggregates did not correlate with the degree of striatal degeneration or the degree of cortical neuron loss. Together, these results suggest that longer CAG repeat lengths correlate with Htt aggregation in the HD human brain, and greater Htt cortical aggregate deposition is associated with an earlier age of symptom onset in HD. This study also reinforces that antibodies MAB5492, MW8, and 2B7 which have been utilized to characterize Htt in animal models of HD do not specifically immunolabel Htt aggregates in HD human brain tissue exclusively, thereby highlighting the need for validated means of Htt detection to support drug development for HD.
Time-resolved FRET screening identifies small molecular modifiers of mutant Huntingtin conformational inflexibility in patient-derived cells
2022, SLAS Discovery
Huntington's disease (HD) is the most common monogenic neurodegenerative disease and is fatal. CAG repeat expansions in mutant Huntingtin (mHTT) exon 1encode for polyglutamine (polyQ) stretches and influence age of onset and disease severity, depending on their length. mHTT is more structured compared to wild-type (wt) HTT, resulting in a decreased N-terminal conformational flexibility. mHTT inflexibility may contribute to both gain of function toxicity, due to increased mHTT aggregation propensity, but also to loss of function phenotypes, due to decreased interactions with binding partners. High-throughput-screening techniques to identify mHTT flexibility states and potential flexibility modifying small molecules are currently lacking. Here, we propose a novel approach for identifying small molecules that restore mHTT's conformational flexibility in human patient fibroblasts. We have applied a well-established antibody-based time-resolved Förster resonance energy transfer (TR-FRET) immunoassay, which measures endogenous HTT flexibility using two validated HTT-specific antibodies, to a high-throughput screening platform. By performing a small-scale compound screen, we identified several small molecules that can partially rescue mHTT inflexibility, presumably by altering HTT post-translational modifications. Thus, we demonstrated that the HTT TR-FRET immunoassay can be miniaturized and applied to a compound screening workflow in patient cells. This automated assay can now be used in large screening campaigns to identify previously unknown HD drugs and drug targets.
Structural Model of the Proline-Rich Domain of Huntingtin Exon-1 Fibrils
2020, Biophysical Journal
Huntington’s disease is a heritable neurodegenerative disease that is caused by a CAG expansion in the first exon of the huntingtin gene. This expansion results in an elongated polyglutamine domain that increases the propensity of huntingtin exon-1 to form cross-β fibrils. Although the polyglutamine domain is important for fibril formation, the dynamic, C-terminal proline-rich domain (PRD) of huntingtin exon-1 makes up a large fraction of the fibril surface. Because potential fibril toxicity has to be mediated by interactions of the fibril surface with its cellular environment, we wanted to model the conformational space adopted by the PRD. We ran 800-ns long molecular dynamics simulations of the PRD using an explicit water model optimized for intrinsically disordered proteins. These simulations accurately predicted our previous solid-state NMR data and newly acquired electron paramagnetic resonance double electron-electron resonance distances, lending confidence in their accuracy. The simulations show that the PRD generally forms an imperfect polyproline (polyP) II helical conformation. The two polyP regions within the PRD stay in a polyP II helix for most of the simulation, whereas occasional kinks in the proline-rich linker region cause an overall bend in the PRD structure. The dihedral angles of the glycine at the end of the second polyP region are very variable, effectively decoupling the highly dynamic 12 C-terminal residues from the rest of the PRD.
Optimizing intracellular antibodies (intrabodies/nanobodies) to treat neurodegenerative disorders
2020, Neurobiology of Disease
Intrabodies (both single-chain Fv and single-domain VH, VHH, and VL nanobodies) offer unique solutions to some of the challenges of delivery and target engagement posed by immunotherapeutics for the brain and other areas of the nervous system. The specificity, which includes the recognition of post-translational modifications, and capacity for engineering that characterize these antibody fragments can be especially well-focused when the genes encoding only the binding sites of the antibody are expressed intracellularly. Multifunctional constructs use fusions with peptides that can re-target antigen-antibody complexes to enhance both pharmacodynamic activity and intracellular solubility simultaneously. Fusions with proteolytic targeting signals, such as the PEST degron, greatly enhance potency in some cases. Stem cell transplants can be protected from exogenous misfolded proteins by stable transfection with intrabodies. Tandem expression to target two or more misfolding proteins in one treatment may be especially valuable for proteostatic disruptions due to genetic, aging, or toxic triggers. Advances in bioinformatics, screening protocols, and especially gene therapy are showing great promise for intrabody/ nanobody treatments of a full range of neurological disorders, including Alzheimer's disease and related tau dementias, Parkinson's disease and Lewy body diseases, Huntington's disease, amyotrophic lateral sclerosis, and prion diseases, among others.
Brain-penetrant PQR620 mTOR and PQR530 PI3K/mTOR inhibitor reduce huntingtin levels in cell models of HD
One of the pathological hallmarks of Huntington disease (HD) is accumulation of the disease-causing mutant huntingtin (mHTT), which leads to the disruption of a variety of cellular functions, ultimately resulting in cell death. Induction of autophagy, for example by the inhibition of mechanistic target of rapamycin (mTOR) signaling, has been shown to reduce HTT levels and aggregates. While rapalogs like rapamycin allosterically inhibit the mTOR complex 1 (TORC1), ATP-competitive mTOR inhibitors suppress activities of TORC1 and TORC2 and have been shown to be more efficient in inducing autophagy and reducing protein levels and aggregates than rapalogs. The ability to cross the blood-brain barrier of first generation catalytic mTOR inhibitors has so far been limited, and therefore sufficient target coverage in the brain could not be reached. Two novel, brain penetrant compounds – the mTORC1/2 inhibitor PQR620, and the dual pan-phosphoinositide 3-kinase (PI3K) and mTORC1/2 kinase inhibitor PQR530 - were evaluated by assessing their potential to induce autophagy and reducing mHTT levels. For this purpose, expression levels of autophagic markers and well-defined mTOR targets were analyzed in STHdh cells and HEK293T cells and in mouse brains. Both compounds potently inhibited mTOR signaling in cell models as well as in mouse brain. As proof of principle, reduction of aggregates and levels of soluble mHTT were demonstrated upon treatment with both compounds. Originally developed for cancer treatment, these second generation mTORC1/2 and PI3K/mTOR inhibitors show brain penetrance and efficacy in cell models of HD, making them candidate molecules for further investigations in HD.
The effects of LSD1 inhibition on self-renewal and differentiation of human induced pluripotent stem cells
Experimental Cell Research, Volume 340, Issue 2, 2016, pp. 227-237
Human induced pluripotent stem cells (hiPSCs) are capable of unlimited self-renewal and can generate nearly all cells in the body. Changes induced by different LSD1 activities on the regulation of hiPSC self-renewal and differentiation and the mechanism underlying such changes were determined. We used two different LSD1 inhibitors (phenelzine sulfate and tranylcypromine) and RNAi technique to inhibit LSD1 activity, and we obtained hiPSCs showing 71.3%, 53.28%, and 31.33% of the LSD1 activity in normal hiPSCs. The cells still maintained satisfactory self-renewal capacity when LSD1 activity was at 71.3%. The growth rate of hiPSCs decreased and cells differentiated when LSD1 activity was at approximately 53.28%. The hiPSCs were mainly arrested in the G0/G1 phase and simultaneously differentiated into endodermal tissue when LSD1 activity was at 31.33%. Teratoma experiments showed that the downregulation of LSD1 resulted in low teratoma volume. When LSD1 activity was below 50%, pluripotency of hiPSCs was impaired, and the teratomas mainly comprised endodermal and mesodermal tissues. This phenomenon was achieved by regulating the critical balance between histone methylation and demethylation at regulatory regions of several key pluripotent and developmental genes.
Length of Uninterrupted CAG, Independent of Polyglutamine Size, Results in Increased Somatic Instability, Hastening Onset of Huntington Disease
The American Journal of Human Genetics, Volume 104, Issue 6, 2019, pp. 1116-1126
Huntington disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. Although the length of this repeat is inversely correlated with age of onset (AOO), it does not fully explain the variability in AOO. We assessed the sequence downstream of the CAG repeat in HTT [reference: (CAG)n-CAA-CAG], since variants within this region have been previously described, but no study of AOO has been performed. These analyses identified a variant that results in complete loss of interrupting (LOI) adenine nucleotides in this region [(CAG)n-CAG-CAG]. Analysis of multiple HD pedigrees showed that this LOI variant is associated with dramatically earlier AOO (average of 25 years) despite the same polyglutamine length as in individuals with the interrupting penultimate CAA codon. This LOI allele is particularly frequent in persons with reduced penetrance alleles who manifest with HD and increases the likelihood of presenting clinically with HD with a CAG of 36–39 repeats. Further, we show that the LOI variant is associated with increased somatic repeat instability, highlighting this as a significant driver of this effect. These findings indicate that the number of uninterrupted CAG repeats, which is lengthened by the LOI, is the most significant contributor to AOO of HD and is more significant than polyglutamine length, which is not altered in these individuals. In addition, we identified another variant in this region, where the CAA-CAG sequence is duplicated, which was associated with later AOO. Identification of these cis-acting modifiers have potentially important implications for genetic counselling in HD-affected families.
Structure of a Single-Chain Fv Bound to the 17 N-Terminal Residues of Huntingtin Provides Insights into Pathogenic Amyloid Formation and Suppression
Journal of Molecular Biology, Volume 427, Issue 12, 2015, pp. 2166-2178
Huntington's disease is triggered by misfolding of fragments of mutant forms of the huntingtin protein (mHTT) with aberrant polyglutamine expansions. The C4 single-chain Fv antibody (scFv) binds to the first 17 residues of huntingtin [HTT(1-17)] and generates substantial protection against multiple phenotypic pathologies in situ and in vivo. We show in this paper that C4 scFv inhibits amyloid formation by exon1 fragments of huntingtin in vitro and elucidate the structural basis for this inhibition and protection by determining the crystal structure of the complex of C4 scFv and HTT(1-17). The peptide binds with residues 3–11 forming an amphipathic helix that makes contact with the antibody fragment in such a way that the hydrophobic face of this helix is shielded from the solvent. Residues 12–17 of the peptide are in an extended conformation and interact with the same region of another C4 scFv:HTT(1-17) complex in the asymmetric unit, resulting in a β-sheet interface within a dimeric C4 scFv:HTT(1-17) complex. The nature of this scFv–peptide complex was further explored in solution by high-resolution NMR and physicochemical analysis of species in solution. The results provide insights into the manner in which C4 scFv inhibits the aggregation of HTT, and hence into its therapeutic potential, and suggests a structural basis for the initial interactions that underlie the formation of disease-associated amyloid fibrils by HTT.
Human Pluripotent Stem Cell-Derived Striatal Interneurons: Differentiation and Maturation InVitro and in the Rat Brain
Stem Cell Reports, Volume 12, Issue 2, 2019, pp. 191-200
Striatal interneurons are born in the medial and caudal ganglionic eminences (MGE and CGE) and play an important role in human striatal function and dysfunction in Huntington's disease and dystonia. MGE/CGE-like neural progenitors have been generated from human pluripotent stem cells (hPSCs) for studying cortical interneuron development and cell therapy for epilepsy and other neurodevelopmental disorders. Here, we report the capacity of hPSC-derived MGE/CGE-like progenitors to differentiate into functional striatal interneurons. Invitro, these hPSC neuronal derivatives expressed cortical and striatal interneuron markers at the mRNA and protein level and displayed maturing electrophysiological properties. Following transplantation into neonatal rat striatum, progenitors differentiated into striatal interneuron subtypes and were consistently found in the nearby septum and hippocampus. These findings highlight the potential for hPSC-derived striatal interneurons as an invaluable tool in modeling striatal development and function invitro or as a source of cells for regenerative medicine.
The CAG–polyglutamine repeat diseases: a clinical, molecular, genetic, and pathophysiologic nosology
Handbook of Clinical Neurology, Volume 147, 2018, pp. 143-170
Throughout the genome, unstable tandem nucleotide repeats can expand to cause a variety of neurologic disorders. Expansion of a CAG triplet repeat within a coding exon gives rise to an elongated polyglutamine (polyQ) tract in the resultant protein product, and accounts for a unique category of neurodegenerative disorders, known as the CAG–polyglutamine repeat diseases. The nine members of the CAG–polyglutamine disease family include spinal and bulbar muscular atrophy (SBMA), Huntington disease, dentatorubral pallidoluysian atrophy, and six spinocerebellar ataxias (SCA 1, 2, 3, 6, 7, and 17). All CAG–polyglutamine diseases are dominantly inherited, with the exception of SBMA, which is X-linked, and many CAG–polyglutamine diseases display anticipation, which is defined as increasing disease severity in successive generations of an affected kindred. Despite widespread expression of the different polyQ-expanded disease proteins throughout the body, each CAG–polyglutamine disease strikes a particular subset of neurons, although the mechanism for this cell-type selectivity remains poorly understood. While the different genes implicated in these disorders display amino acid homology only in the repeat tract domain, certain pathologic molecular processes have been implicated in almost all of the CAG–polyglutamine repeat diseases, including protein aggregation, proteolytic cleavage, transcription dysregulation, autophagy impairment, and mitochondrial dysfunction. Here we highlight the clinical and molecular genetic features of each distinct disorder, and then discuss common themes in CAG–polyglutamine disease pathogenesis, closing with emerging advances in therapy development.
Reversal of Phenotypic Abnormalities by CRISPR/Cas9-Mediated Gene Correction in Huntington Disease Patient-Derived Induced Pluripotent StemCells
Stem Cell Reports, Volume 8, Issue 3, 2017, pp. 619-633
Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in HTT. Here we report correction of HD human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 and piggyBac transposon-based approach. We show that both HD and corrected isogenic hiPSCs can be differentiated into excitable, synaptically active forebrain neurons. We further demonstrate that phenotypic abnormalities in HD hiPSC-derived neural cells, including impaired neural rosette formation, increased susceptibility to growth factor withdrawal, and deficits in mitochondrial respiration, are rescued in isogenic controls. Importantly, using genome-wide expression analysis, we show that a number of apparent gene expression differences detected between HD and non-related healthy control lines are absent between HD and corrected lines, suggesting that these differences are likely related to genetic background rather than HD-specific effects. Our study demonstrates correction of HD hiPSCs and associated phenotypic abnormalities, and the importance of isogenic controls for disease modeling using hiPSCs.
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