*Department of Neurology, University of Miami, School of Medicine;
Institut für Anthropologie und Humangenetik, Ludwig Maximilians Universität München, Munich, Germany;
Laboratory of Genomic Diversity, National Cancer Institute, Frederick, Maryland;
and
§Department of Cell Biology and Anatomy, University of Miami, School of Medicine
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Abstract |
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Introduction |
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Materials and Methods |
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Transfer of Chromosomes and mtDNA from Orangutan to Human ° Cells
Microcell Fusions
Microcell isolation and fusion with ° cells were performed as described (Barrientos and Moraes 1998
). After fusion, hybrid cells were allowed to grow overnight and then selected for the presence of oxidative phosphorylation (OXPHOS) function with a medium lacking uridine and supplemented with 10% dialyzed serum (only cells with at least partial OXPHOS function can grow in the absence of uridine). These hybrid clones, denoted M#, were maintained continuously in selective medium.
Whole-Cell Fusions
Orangutan fibroblasts were fused with the human 143B/206 ° cell line, following a procedure similar to that used for microcell fusion. The hybrid cells were also selected for the presence of orangutan mtDNA with a medium lacking uridine. Whole-cell hybrid clones, denoted H#, were maintained continuously in selective medium. To reduce the number of chromosomes to the minimum necessary to maintain a functional orangutan mtDNA in the hybrid cells, two of these hybrid clones (H1 and H2) were incubated with 50 ng/ml colcemide (Gibco-BRL) for 24 or 48 h. Colcemide arrests the cells in mitosis, which can result in chromosomal loss (Tsutsui et al. 1990
). Cells were allowed to recover in two different media selective for respiratory function: a medium rich in glucose but lacking uridine, and a more stringent one in which glucose was substituted with galactose as a carbon source. Those clones were denoted H#c.
Quantitation of Fusion Products
Human, gorilla, and orangutan fibroblasts, as well as a human ° cell line (used as a negative control), were fused with the human 143B/206
° cell line stably transfected with the plasmid pSV2-Neo (Southern and Berg 1982
) containing a gene for neomycin resistance. Four independent fusions were made for each species. One day after fusion, cells were split into twenty 60-mm2 dishes. Cells were selected in 450 µg/ml of G418 (Gibco-BRL) and, for the presence of functional mitochondria, with a medium lacking uridine. The number of independent colonies formed was determined in 50% of the dishes after 13 days by staining the dishes with toluidine blue. After 25 days, the second half was counted to estimate the number and stability of the respiratory competent hybrids formed.
Chromosomal Identification by Microsatellite Markers
Human microsatellite markers have been used for analysis of genetic variation in apes (Coote and Bruford 1996
). We selected 33 microsatellite primer pairs for potential use in distinguishing chromosomes from human 143B/206
° cells and orangutan fibroblasts. We used MAPPAIRS primers (Research Genetics, Huntsville, Ala.) references D1S551, D1S552, D2S2739, D2S427, D3S3038, D3S1535, D4S2639, D4S2431, D5S1470, D5S820, D6S1056, D7S820, D7S1820, D8S1113, D9S934, D10S677, D11S1985, D11S2369 D12S391, D13S788, D14S587, D15S642, D16S753, D16S2621, D17S1293, D17S1290, D18S851, D19S246, D20S481, D21S1437, D21S1270, D22S683, and DXS6797. MAPPAIRS primer references D7S820, D16S753, and D21S1437 were not informative and were not considered in the study. Basically, the forward primer of each pair was end-labeled with [
-32P]dATP using T4-polynucleotide kinase (New England Biolabs), the loci were PCR amplified, and 4 µl of the product was loaded onto a 6% denaturing polyacrylamide gel, followed by autoradiography.
Karyotype Analysis by Fluorescent In Situ Hybridization (FISH)
By cross-species color segmenting (Müller et al. 1998
), also termed RxFISH, a chromosome bar code on human and great ape chromosomes was generated, which assisted in the identification of human and orangutan chromosomes. The respective FISH probe set was composed of gibbon chromosomespecific painting probes, which were labeled with three fluors in various combinations. Gibbon chromosomes differ from all other great ape homologs by multiple translocations and inversions. For this reason, the RxFISH probe set produces a multicolored banding pattern on both human and orangutan homologs. Since the majority of both species' homologs are evolutionary conserved and cannot be distinguished by chromosome morphology and banding pattern alone, the interspecies comparative genomic hybridization (iCGH) technique was employed simultaneously. Originally, CGH was developed in order to visualize genomic imbalances in differentially labeled tumor versus control genomic DNA samples, particularly deletions and amplifications (Kallioniemi et al. 1992
). Adaptation of the experimental setup allowed the differential display of human and orangutan chromosomes, taking advantage of the overall sequence divergence between both genomes: human genomic probe DNA preferentially hybridizes to human chromosomes and vice versa. Both the RxFISH and the iCGH probe sets were hybridized simultaneously using M-FISH technology and five fluors (Speicher, Gwyn Ballard, and Ward 1996
). Approximately 400 ng of each genomic orangutan DNA (biotin-dUTP labeled) and human genomic DNA (digoxigenin-dUTP labeled) were ethanol-precipitated and subsequently resuspended in 10 µl commercially available RxFISH probe mixture (labeled with FITC-dUTP, Cy3-dUTP, and Cy5-dUTP; Applied Imaging Corp.). Hybridization in situ to metaphase preparations of human x orangutan hybrid cell lines was performed for 72 h at 37°C. Post-hybridization washes included 2 x 5 min 50% formamide/1 x SSC, 45°C; 2 x 5 min 2 x SSC, 45°C; and 2 x 5 min 0,1 x SSC, 60°C. Biotinylated probe was detected by avidin-Cy3.5, and digoxigenin labeled probe by mouse-anti-digoxigenin-Cy5.5 and visualized by a microscopic setup described in Speicher, Gwyn Ballard, and Ward (1996).
Mitochondrial DNA Identification and Quantification
To insure that the only mtDNA present in the human ° x orangutan
+ (H
° x Or) hybrids were from orangutan, a Southern blot analysis was performed using total DNA digested with PvuII and a mixture of a 956 bp human mtDNA (nt 33054261) and its homologous orangutan mtDNA fragment [
-32P]dCTP-labeled probes. PvuII restriction patterns differ in human mtDNA (a fragment of 16.5 kb) and orangutan mtDNA (two fragments of 8 and 8.3 kb, only one of them detected with the partial mtDNA probe used). To determine the level of repopulation by orangutan mitochondria in H
° x Or hybrid clones, the mtDNA content relative to the nuclear DNA (nDNA) was quantified by a slot blot experiment using the same mixture of mtDNA probes plus a 5.8-kb 18S rDNA nuclear (Wilson et al. 1978
) [
-32P]dCTP-labeled probe. For each probe, we used three slots, containing 100, 200, and 300 ng of total DNA. After laser-scanning the autoradiograms, band signals were quantified using NIH Image 1.6 software. The mtDNA/nDNA ratio was considered the division of the arbitrary densitometrical values of the signals using each probe.
Mitochondrial Function Studies
Exponentially growing cells were collected by trypsinization, pelleted, and resuspended in cold phosphate-buffered saline (PBS) medium. The KCN-sensitive endogenous cell respiration in intact cells was measured polarographically as described (Barrientos, Kenyon, and Moraes 1998
). Mitochondria were isolated from cells and resuspended in a medium consisting of 20 mM Tris (pH 7.2), 0.25 M sucrose, 40 mM KCl, 2 mM EGTA, and 1 mg/ml BSA. The measurement of the specific activity of the individual complexes of the electron transport chain was performed spectrophotometrically as described (Barrientos, Kenyon, and Moraes 1998
). The electron transport chain enzyme activities were normalized both by milligrams of protein and by citrate synthase activity. The protein content in the cell and mitochondria samples was determined according to Bradford's (1976) method. Complex IV activity was also assayed cytochemically on cells grown on coverslips, as described (Seligman et al. 1968
). Complex IV inhibition by KCN was titrated by measuring the reduction in cytochrome c oxidation in the presence of increasing concentrations (up to 20 mM) of the specific inhibitor KCN. All experiments were performed at least in triplicate.
Mitochondrial Protein Synthesis
Mitochondrial protein synthesis was determined by pulse-labeling cell cultures in the presence of emetine as described (Chomyn 1996
). Four H
° x Or hybrids (M1, M2, H1, and H1c) were used for this experiment. Orangutan fibroblasts and human 143B cells were used as a control for the mtDNA-coded protein pattern in both species, and the 143B derivative 206
° was used as a negative control. Semiconfluent cells were labeled and processed as described (Chomyn 1996
). Approximately 45 µg of total protein was resolved by electrophoresis in a 15%20% exponential gradient polyacrylamide gel (Chomyn 1996
). Gels were fixed in a 30% methanol/10% acetic acid solution and treated with Fluoro-Enhance (Research Products International), dried, and exposed to an X-ray film at -80°C.
Detection of Mitochondrial Respiratory Chain Enzyme Subunits
Immunoblottings were performed using monoclonal antibodies against the human succinate dehydrogenase flavoprotein subunit (SDH(Fp)), core-1 of complex III, COX I, COX II, COX IV, COX Va, and subunit of ATPase (Marusich et al. 1997
) (a gift from Dr. R. Capaldi, University of Oregon) and a polyclonal antibody against the human ND1 (a gift from Dr. A. Lombes, Groupe Hospitalier Pitiè-Salpètrière, Paris). A mitochondria-enriched pellet was prepared as described (Barrientos, Kenyon, and Moraes 1998
) from orangutan fibroblasts, from 143B and 143B/206
° cells, and from several H
° x Or hybrid clones (M1M4, H1, H2, H1c, and H2c). Ten to twenty micrograms of mitochondrial proteins were separated onto 15% SDS-PAGE gels and transferred to PVDP membranes (Immobilon, Bio-Rad). Membranes were incubated for 1 h with 10% milk in PBS with 0.05% Tween 20 and with antibodies against different mtDNA- or nDNA-coded respiratory chain subunits for 14 h at 4°C. The chemiluminescent detection of the proteins was performed with the Phototope-HRP Western blot detection kit using an anti-rabbit or anti-mouse IgG secondary antibody, which was HRP-linked (New England Biolabs, Beverly, Mass.) following the manufacturer's recommendations.
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Results |
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The chromosome content analyzed in four hybrids produced by orangutan microcell fusions displayed many orangutan chromosomes, as well as several chromosomal abnormalities, including frequent human-orangutan translocations and dicentric (same or different species) chromosomes (fig. 2A,
summarized in table 1
). These made very difficult, if not impossible, any genotype-phenotype association. The total number of chromosomes in the hybrids able to grow in uridineless medium after fusions between human ° x orangutan microcells was close to 100, and approximately 30% of them were from orangutan (table 1
).
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The apparent OXPHOS deficiency, suggested by the growth behavior of H° x Or hybrids in the different selective media used, prompted us to perform polarographic analyses of the hybrids' respiratory capacity. Intact cell oxygen consumption in the hybrids was decreased by 68%80% as compared with the human parental cell line (143B), and by 62%75% as compared with the orangutan parental line (fibroblast) (P < 0.001 in both cases). Figure 3A
summarizes the oxygen consumption data on intact cells. Mitochondrial respiratory chain enzyme activities, measured in isolated mitochondria, were normalized to citrate synthase (CS) activity (fig. 3B
). Cell respiration in the hybrids was always less than 40% of that in controls regardless of the selection strategy used. Complex IV/CS ratios were decreased by 87%95 % as compared with the human cell line, and by 85%94% as compared with the orangutan cell line (P < 0.001 in both cases). Complex I, II+III, and III activities were not significantly altered in H
° x Or hybrids (fig. 3B
). These data are consistent with a specific complex IV or cytochrome c oxidase (COX) deficiency in these cell lines. To investigate whether the enzyme deficiency was associated with structural changes that would affect binding to inhibitors, we studied the kinetics of KCN inhibition on COX activity (fig. 3C
). In all cell lines studied, the kinetics of COX inhibition was similar. The Ki of KCN in each case was obtained by plotting the concentration of KCN versus the ratio [KCN]/% inhibition (fig. 3C
). The Ki values of KCN inhibition were similar between the parental cells (1.67 ± 0.16 for 143B, and 1.48 ± 0.25 for orangutan fibroblasts) and the H
° x Or hybrids (1.73 ± 0.39), indicating that there were no changes in KCN affinity for its binding site in complex IV, but, rather, a different amount of either enzyme or activity susceptible to inhibition by KCN. Therefore, the biochemical basis of the galactose growth failure in our human
° x orangutan
+ hybrids seemed to consist in a specific decrease in COX activity that is otherwise similar to the activity observed in orangutan cells.
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Mitochondrial Complex IV Deficiency in Human ° x Orangutan
+ Hybrids Is Not a Common Feature of Human
°Nonhuman Ape Interspecific Hybrids
To determine if the interference between nuclear-coded factors was also present in other ape hybrids, human, gorilla, and orangutan fibroblasts were fused with the neomycin-resistant human 143B/206NEO ° cell line. Cells were selected in G418 (a neomycin analog) and uridine-lacking medium. After 13 days of selection, the efficiency of the fusions was determined by the number of growing clones and expressed as a ratio (%) to the clones obtained in the human
° x human fibroblast hybrid fusion. The efficiency for human
° x gorilla fibroblast fusions was 38.5 ± 10.9, and that for human
° x orangutan was 13.7 ± 5.0. After 25 days of selection, the percentages of clones which survived with respect to the number of colonies obtained from human
° x human fibroblast fusions dropped (28.2 ± 7.3 for human
° x gorilla, and 6.9 ± 3.8 for human
° x orangutan). These results indicate that a significant proportion of hybrid clones were unstable and died under prolonged culture conditions (
27% for human
° x gorilla and
50% for human
° x orangutan, respectively). In contrast to H
° x Or hybrids, hybrids generated with human fibroblasts, and also with gorilla fibroblasts, exhibited endogenous cell respiration (fig. 3A
), steady-state levels of mtDNA coded complex IV subunits (fig. 4D
), and cytochrome oxidase activity (semiquantitatively determined by cytochemistry, not shown) which were comparable to those of the parental human cell line.
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Discussion |
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We tried to identify the factor(s) involved in orangutan mtDNAhuman nuclear DNA incompatibilities by functional complementation. Since OXPHOS cannot be restored in a cell system consisting of a pure human nuclear background coexisting with orangutan mtDNA, it was reasonable to think that the respiratory capacity in that system could be reestablished if the human nucleus was supplemented with one or a few orangutan chromosomes. The rationale was to provide the system with the orangutan homolog(s) of the human factor(s) unable to interact properly with orangutan mtDNA or its products. We took advantage of our capacity to transfer in a single step mtDNA and a limited number of chromosomes from orangutan to human cells to create hybrid cell lines which could be selected in a medium selective for respiratory function. In half of our attempts, no respiring clones were obtained. This unusually low yield, compared with our previous experience with this technique (Barrientos and Moraes 1998
), suggested that the orangutan chromosomal supplement necessary to maintain functional orangutan mtDNA in a human
° was more than one chromosome and that only in rare cases, when the infrequent microcells containing many chromosomes were fused to the human
° cells, were the right combination of chromosomes achieved. The FISH and microsatellite analyses of the hybrids obtained supported this assumption. In all clones with some respiratory function, the presence of a high number of orangutan chromosomes was observed. The generation of these hybrids, resembling complete hybrids constructed by fusing whole orangutan fibroblasts with human
° cells, would occur only when a large piece of nucleus containing several chromosomes or a whole nucleus escaped the several filtration steps. We speculate that when these large portions of nucleus surrounded by a thin ring of cytoplasm containing mitochondria were fused to a human
° cell, a hybrid with some respiratory capacity was created, probably explaining the low efficiency observed. In addition, the low level of respiration in the hybrids obtained suggests interference between nuclear-coded subunits of orangutan and human origins, creating new interactions that are less stable or efficient in the assembly and function of the multimeric respiratory enzymes. Our attempts to reduce the total number of chromosomes in the hybrids by treating the cells with the antimitotic agent colcemide failed to generate galactose-resistant clones with a significantly reduced number of orangutan chromosomes. Our results suggest that orangutan mtDNA requires interactions with several nuclear-coded factors that cannot be replaced by human genes. However, we cannot rule out the possibility that the right combination of chromosomes was not achieved in our experiments. In any case, our experiments showed that human chromosomes present in the hybrids have a dominant negative effect on the orangutan COX function.
Why Is Complex IV (COX) Deficient in Cells Containing Orangutan mtDNA and Chromosomes from Both Human and Orangutan?
Why was COX activity specifically affected in our hybrids? MtDNA-coded COX subunits are among the most conserved polypeptides in mitochondria, and the sequence similarity between human and orangutan subunits is higher than 90%. The rate of mtDNA evolution varies for each mitochondrial gene (Holmes 1991
; Jukes 1994
): for example, COX II has evolved at a high rate in primates (Adkins, Honeycutt, and Disotell 1996
). Nevertheless, some COX nuclear-coded subunits, like subunit IV (Wu et al. 1997
) and subunit VII (Schmidt, Goodman, and Grossman 1999
), also had high rates of variation, illustrating the coevolutionary pressure associated with nuclear-mitochondrial interactions. Although at this point we cannot determine which specific factor(s) causes the COX assembly defect in the H
° x Or hybrid system, our results showed that this incompatibility can result from competition between nuclear-coded proteins.
Cytochrome c oxidase (a multimeric enzyme complex) resides in the mitochondrial inner membrane and catalyzes the final step of electron transfer through the respiratory chain. In eukaryotic organisms, COX is composed of up to 13 subunits encoded by both the mitochondrial (subunits I, II, and III, which form the catalytic core of the enzyme; Tsukihara et al. 1996
) and the nuclear genomes. The enzyme comprises four redox-active metal centers (two heme and two copper centers), and proteins implicated in heme biosynthesis (Poyton and McEwen 1996
) or copper homeostasis (Glerum, Shtanko, and Tzagoloff 1996
) are also nuclear-coded. In addition, some nuclear-coded proteins are implicated in the transport of nuclear-coded subunits across mitochondrial membranes, and others mediate the assembly or stability of the enzyme (reviewed by Poyton 1998
). COX assembly is not completely understood, and new proteins have recently been associated with this function, such as surf-1 (Mashkevich et al. 1997
), a protein implicated in a human mitochondrial disease (Tiranti et al. 1998
; Zhu et al. 1998
). The complex appears to be initiated by the assembly of subunits I and IV, which is followed by the addition of most other subunits (Nijtmans et al. 1998
).
In H° x Or hybrids, mtDNA-coded COX subunits (COX I and COX II) were efficiently translated, but their steady-state levels were greatly reduced. The import into the mitochondria and expression of nuclear-coded COX subunits were not affected. These characteristics, together with a loose association observed between COX nuclear-coded proteins and mitochondrial membranes, are the hallmarks of cytochrome oxidase assembly-defective mutants, as has been described for yeast (Glerum and Tzagoloff 1997
). In humans, a pathogenic missense mutation in COX II was shown to disturb the assembly of the holoenzyme (Rahman et al. 1999
). Nonsense mutations in the COX I gene also disrupt COX assembly (Bruno et al. 1999
; Comi et al. 1998
). Moreover, most patients with undefined COX deficiency also show a decrease in the steady-state levels of several subunits, suggesting an assembly defect (Taanman et al. 1996
).
In H° x Or hybrids, the mitochondrial membranes could have several versions of complex IV: (1) formed exclusively by the orangutan subunits, (2) constructed with human nuclear subunits and the orangutan mtDNA-coded subunits, and (3) built with nuclear-coded subunits of both orangutan and human origins. Probably, most of the complex IV present in the hybrids falls into the last category. It is possible that the residual COX activity measured in the H
° x Or hybrids is derived from the first class of enzyme (orangutan enzyme) or even some from the third class with mostly orangutan subunits. The nuclear subunits cross-interact at different regions of the complex (e.g., COX IV interacts with COX Va, which interacts with COX VIc), and if those interactions are defective, the assembly of the enzyme can be compromised, as has been shown for yeast (Glerum and Tzagoloff 1998
). We can speculate that most of the assembly-defective complexes are associated with defective interactions caused by the dominant negative effect of human nuclear-coded subunits. Mitochondrial DNA from humans, gorillas, and chimpanzees were able to produce HXC with normal COX activity. Amino acid changes in mtDNA-coded COX subunits in gorillas or chimpanzees have to be considered neutral polymorphisms, as they do not affect the activity of the enzyme. Therefore, changes in the orangutan COX subunits that can be considered potentially deleterious for COX assembly are those differing from the other three ape species (table 3 ). COX I is located mainly in the transmembrane domain of the enzyme complex, consisting of 12 transmembrane helices and without any large extramembrane portion (Tsukihara et al. 1996
). There are 17 amino acid changes between COX I from the orangutan and its human, chimpanzee, or gorilla homologs, 9 of which are conservative changes which do not affect hydrophobicity and most probably do not affect interactions between COX I and other subunits. There are no changes in the first transmembrane helix of COX I, which interacts with COX VIIc and COX VIII, and no significant changes in transmembrane helix IX, which interacts with COX V, or in helix XII, which interacts with COX IV. More stringent changes are those from Y to H (in amino acid 260; helix VI), from L to P (in amino acid 483), from E to Q (in amino acid 487), and from S to P (in amino acid 513) in the CH2 terminal extramembrane part after helix XII, which is located in the matrix side. COX II contains two transmembrane helices. Helix I interacts with COX VIc. The single change observed in this helix (from I to V in amino acid 21) is conservative. The large extramembrane domain of COX II is located above COX I in the cytosolic side, having a barrel structure that holds the CuA site. The A-to-T change in amino acid 164 probably will not alter the binding of the metal ion per se, but it could affect the interaction between COX II and other nuclear-coded proteins implicated in the delivery of Cu++ to the COX (e.g., Sco1). COX III contains seven transmembrane helices without extensive extramembrane domains. COX VIIa cross-interacts with its helices I and II. Two changes occurred in helix II (from M/T to L in amino acid 44 and from L to T in amino acid 45), which could well be the result of an inversion of those two amino acids. There is a single but less conservative change (from S to A in amino acid 135) in helix IV of COX III, which interacts with COX VIa. The NH2 terminal of COX VIa is in an extended conformation in the transmembrane region that makes contact with helices V and VII of COX I from the other monomer. These contacts seem likely to stabilize the dimeric structure (Tsukihara et al. 1996
). Changes in COX III (the one in helix IV) and several stringent changes in helix VI (table 3
) could affect the interaction with COX VIa and the stability of the dimeric structure of the enzyme.
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In conclusion, the cellular hybrid system presented here showed that COX assembly in mammals is very sensitive to small changes in amino acid sequences not involved in catalytic function and that even minimal variations in mtDNA-coded subunits must be compensated by a change in nuclear-coded subunits.
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Acknowledgements |
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Footnotes |
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1 Present address: Department of Biological Sciences, Columbia University.
2 Abbreviations: COX, cytochrome c oxidase; CS, citrate synthase; H° x Or hybrids, human
° x orangutan
+ hybrids; HXC, human xenomitochondrial cybrids; MRC, mitochondrial respiratory chain; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; OXPHOS, oxidative phosphorylation.
3 Keywords: mtDNA
mitochondria
COX
hybrids
evolution
4 Address for correspondence and reprints: Department of Neurology, University of Miami, 1501 NW 9th Avenue, Miami, Florida 33136. E-mail: cmoraes{at}med.miami.edu
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