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Stonewall: Biological Overview | References

The association of genomic loci to the nuclear periphery is proposed to facilitate cell type-specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. This study used an Oligopaint-based HiDRO screen targeting ~1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. The heterochromatin-associated protein Stonewall (Stwl) as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwl's known role in GSC maintenance and ovary homeostasis. Thus, this study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate (Chavan, 2024).

The distribution of the genome within the interphase nucleus can tune cell-specific gene expression. In both plant and animal cells, dense-staining heterochromatin and repressed tissue-specific genes are typically found near the inner nuclear membrane (INM). In metazoans, an INM-associated network involving the intermediate filament protein lamin and other associated proteins serves as a scaffold for the organization of peripheral chromatin. This chromatin, which is associated with the nuclear lamina, is referred to as lamina-associated domains (LADs) and is usually gene-poor, transcriptionally silent, and rich in repressive histone marks. Experiments using LAD-embedded transcriptional reporters have shown that perinuclear positioning is generally associated with reduced transcriptional output, although exceptions can occur. Functionally, perinuclear positioning of a locus has been speculated to preserve the inactive transcriptional state and stabilize cell-specific gene expression programs. Consistently, detachment of specific loci from the nuclear periphery in multiple cell types is associated with ectopic gene expression and alterations in cell fate decisions. While the nuclear lamina, nuclear pore complex (NPC) protein, and epigenetic modifications are known to influence chromatin association to the nuclear periphery, very few chromatin-binding perinuclear anchors have been identified thus far. As a result, the precise relationships between perinuclear positioning, gene expression, and cell fate remain enigmatic (Chavan, 2024).

This study leveraged recently developed HiDRO technology to conduct an RNAi screen in Drosophila cells aimed at identifying perinuclear anchors for heterochromatin. Approximately 1000 genes known to possess characteristic DNA-binding domains or nuclear localization sequences were individually depleted and then changes in the spatial positioning of genomic regions located both at the periphery and center of the nucleus were measured. Among the hits, a significant hit—the heterochromatin-associated MADF-BESS domain-containing protein Stonewall (Stwl)— was isolated as a factor important for the peripheral positioning of LAD-enriched chromatin. MADF-BESS proteins are transcriptional regulators that bind DNA through an N-terminal MADF (Myb-SANT-like in ADF) domain, whereas the C-terminal BESS motif mediates protein–protein interactions. Previous studies have demonstrated that stwl has a cell-autonomous function in female germline stem cell (GSC) maintenance as well as later stages of oogenesis, likely through gene repression. Notably, Stwl-depleted GSCs are reported to differentiate precociously (as determined by fusome-containing germline cysts) even in the absence of critical differentiation genes, suggesting that stwl plays an important role in the balance between GSC self-renewal and differentiation. However, the mechanism by which stwl fine-tunes this vital regulatory step in GSC fate has remained unclear. This study shows that stwl is crucial for perinuclear chromatin positioning in female GSCs. Using RNA sequencing, chromatin profiling, and single-molecule FISH (smFISH), stwl was shown to promote repression of canonical GSC differentiation genes such as benign gonial cell neoplasm (bgcn) by positioning these gene loci at the nuclear periphery. Overall, the HiDRO screen has identified multiple factors regulating nuclear architecture in Drosophila. Furthermore, this study has pinpointed stwl as an important factor that links perinuclear chromatin organization to female GSC fate (Chavan, 2024).

The regulation of gene expression is a primary mechanism that dictates cell fate. In addition to local factors influencing gene expression such as enhancer–promoter contacts and sequence-specific transcription factors, the position of a gene within the nucleus can also influence expression. In many organisms, the enrichment of dense and compact heterochromatin at the nuclear periphery gives rise to a gene-repressive nuclear subcompartment. Consistently, genes anchored to the nuclear periphery are generally transcriptionally inactive, while repositioning the same genes to the nuclear interior is associated with their expression In many species, INM-associated proteins and repressive chromatin modifications mediate large-scale chromatin tethering to the nuclear envelope. However, chromatin-associated proteins that position specific gene loci at the nuclear periphery are largely unidentified, even in powerful multicellular model organisms such as Drosophila (Chavan, 2024).

This study deployed HiDRO in tandem with a high-throughput RNAi screen for factors influencing nuclear architecture in Drosophila. Nine hits affecting chromatin positioning at the nuclear periphery were identified, including multiple heterochromatin-associated proteins such as Su(var)3-7, HP2, and Jarid2, as well as transcription factors such as Su(H), Sry-δ, and Fer2, with many of these hits known to have important roles in specific cell types. Among these hits, Stonewall (Stwl), a MADF-BESS transcriptional regulator previously implicated in female GSC maintenance, is a novel factor positioning chromatin at the nuclear periphery in Drosophila cultured cells and female GSCs. Using a multimodal approach, this study identified that stwl binds and represses many genes in female GSCs, including canonical differentiation genes such as bgcn as well as genes implicated in differentiation such as Smr. It is propose that Stwl-mediated repression of multiple such genes through perinuclear positioning preserves the balance between self-renewal and differentiation, thereby ensuring the long-term maintenance of the GSC reservoir and preserving tissue homeostasis (Chavan, 2024).

Cytologically, a fraction of stwl was observed to localize to the nuclear periphery in both cultured Drosophila cells and female GSCs. Moreover, interactions were identified between stwl and NPC proteins (Nup62, Nup88, Nup214, and Tpr/Megator) through quantitative proteomics in cultured cells. While it is possible that these interactions could facilitate nuclear import of Stwl, recent studies have also shown that the perinuclear localization of active and repressive chromatin can occur through interactions with NPC proteins. Interestingly, three other "peripheral" hits from the screen (Reptin, Pontin, and CG4557) copurified with Stwl, suggesting that a Stwl-containing multiprotein complex may be required to facilitate perinuclear positioning of bound loci. At the same time, the discovery of multiple potential perinuclear anchors suggests a high degree of redundancy in the system. One example of this potential redundancy may be the male germline, where stwl depletion has no effect on GSC maintenance. It is therefore speculate that other proteins function in parallel to stwl outside of the female germline, and these proteins may include other "peripheral" hits identified in the screen [e.g., Jarid2 and Su(var)3-7] or one of the 45 Drosophila melanogaster MADF-BESS family members (e.g., Brwl or Hng2), which are known to function redundantly in other tissues (Chavan, 2024).

In the absence of Stwl, it was observed that GSCs undergo substantial changes in chromatin organization at the nuclear envelope, including gaps in the nuclear lamina and decreased electron-dense perinuclear chromatin foci. These phenotypes appear to be linked, since GSCs containing lamin gaps exhibit a stronger reduction in peripherally associated chromatin, while the lamin gap regions also rarely contain NE-associated electron-dense chromatin foci. Interestingly, the lamin gaps are only observed in female GSCs, which are exquisitely sensitive to stwl loss, and not seen in other cell types lacking stwl (e.g., cultured cells). In addition, they do not appear to be caused by decreased lamin levels, since ectopic lamin expression in stwl mutant GSCs does not rescue the gap phenotype. Rather, reduced chromatin associations with the nuclear periphery in the absence of stwl in GSCs could lead to the dissociation and degradation of the lamina, similar to what has been suggested in senescent mammalian cells. One reason for the decreased perinuclear chromatin association in Stwl-depleted GSCs could be a lack of bridging interactions between chromatin and the nuclear envelope. Since loss of stwl has been linked to lowered levels of heterochromatin modifications in whole larvae. Another possibility is that peripheral detachment of specific chromatin loci may occur in concert with defects in global genome and/or heterochromatin organization in cells lacking Stwl. For example, a parallel study has identified that stwl is enriched at the boundaries between active and inactive genomic regions in young ovaries in a manner reminiscent of insulator proteins that demarcate topologically associated domains (TADs). In the absence of Stwl, the chromatin states of these active–inactive regions are indistinct, which is suggestive of compartment mixing and is associated with gene misexpression. Intriguingly, previous studies have noted that transcriptionally silent lamina-associated domains (LADs) are separated from neighboring active genomic compartments by a sharp border. In addition, induced expression of peripherally positioned genes and alteration of their chromatin state results in relocalization to the nuclear interior have further shown that mixing of active and inactive chromatin states at the Rps19b locus in the absence of stwl is associated with detachment from the nuclear periphery in nurse cells. Therefore, it is postulated that heterochromatin–euchromatin compartment mixing in the absence of stwl may destabilize heterochromatin domains, perinuclear chromatin anchoring, and the nuclear lamina. Moving forward, it will be of great interest to characterize the cis-regulatory sequences and protein–protein interactions that shape 3D genome organization at stwl target loci in GSCs (Chavan, 2024).

In summary, this HiDRO-based nuclear architecture screen has identified multiple potential chromatin-associated perinuclear anchors in the Drosophila genome. This study focused on Stwl, which is identified as a factor required for positioning chromatin at the nuclear periphery in female GSCs. Strikingly, this property of stwl is critical to promote female GSC fate through the anchoring of canonical differentiation genes at the repressive perinuclear subcompartment. Thus, this study makes a significant step toward dissecting causal relationships between the position of a gene, the regulation of its expression, and the effect on cell fate decisions in multiple tissues (Chavan, 2024).

Genome organization regulates nuclear pore complex formation and promotes differentiation during Drosophila oogenesis

Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in Drosophila involves changes in genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ cell genes during differentiation, and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, this study found that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, stwl accumulates at boundaries between silenced and active gene compartments. stwl at these boundaries plays a pivotal role in transitioning germ cell genes into a silenced state and activating a group of oocyte genes and nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, cross-talk between genome architecture and NPCs is essential for successful cell fate transitions (Kolb, 2024).

Germ cell differentiation into an oocyte involves significant changes in gene expression. Germ cell-specific genes are silenced during this transition, while maternally deposited genes are activated. Active genes tend to be in the nuclear interior, whereas inactive genes are mainly found near the nuclear periphery, close to the lamina. The mechanisms that promote such genomic organization during the germ cell-to-maternal transition had not been deciphered (Kolb, 2024).

This studying found that stwl accumulates at TAD/LAD boundaries delineating active and repressed genomic compartments during germline differentiation to an oocyte. The presence of stwl at these boundaries facilitates the establishment of specific chromatin states of these genomic compartments and the maintenance of chromatin marks. Demarcating these compartments is required for both silencing germ cell genes and activating a cohort of maternal genes. In addition, the Stwl-dependent formation of genomic boundaries also promotes the expression of Nups, which aid in the formation of NPCs. These NPCs in turn tether TADs/LADs to the nuclear lamina, promoting gene regulation. Consistent with Stwl's role in binding NPCs and being present at TAD/LAD boundaries, stwl is enriched at the nuclear periphery and colocalizes with NPCs. Thus, stwl regulates local and global genome organization to regulate the cell fate transition during oogenesis (Kolb, 2024).

Stwl is required for GSC maintenance and the proper development of egg chambers. Previously, it had been proposed that stwl silences differentiation genes potentially via epigenetic control by modulating H3K9me3 and H3K27me3. Indeed, this study found that stwl is critical for silencing early oogenesis genes, some of which promote GSC differentiation into an oocyte. However, this function of stwl is thought not to be direct. Instead, stwl plays a crucial role in demarcating silenced and active compartments within the genome in part by recruiting BEAF. By regulating the distribution of these chromatin marks, stwl indirectly promotes the silencing of early oogenesis genes during oogenesis. This silencing is mediated via modulating the enhancer activity (Kolb, 2024).

The genomic compartments that stwl demarcates during oogenesis are annotated as TADs or LADs in Kc cells and salivary gland cells. The function of TADs has been shown to constrain promoter–enhancer interactions to prevent enhancer capture to regulate proper gene expression. It is possible that ectopic expression of repressed genes (such as early oogenesis genes) due to loss of stwl could be because of enhancer capture caused by loss of partition between active and repressed chromatin. However, it is not known whether the genomic compartments that were identified during oogenesis are bona fide TADs/LADs during oogenesis. In addition, another caveat is that many of the chromatin changes occurring upon loss of stwl could be indirect and simply follow changes in gene transcription rather than being instructive or directly caused by loss of stwl (Kolb, 2024).

Stwl not only is required for silencing gene expression but is also critical for activating a cohort of maternally supplied genes such as polar granule component and some Nups. These maternally provided genes are thought not to be directly activated by Stwl. Instead, the maternal genes regulated by stwl are proximal to genomic boundaries that separate active and repressed regions. A cohort of maternally supplied genes are thought not to to be require the activity of stwl to provide a barrier from silenced regions present proximally. While the cohort of maternal genes regulated by stwl is small compared with the number of genes supplied maternally, it is functionally critical. For example, pgc is required to specify a germ cell fate, and Nups are required to complete oogenesis and launch the next generation.. Thus, stwl coordinates stable silencing of early oogenesis genes to activate maternally provided genes by providing a barrier function between chromatin compartments, preventing the mixing of chromatin states (Kolb, 2024).

Stwl coordinates global genome reorganization by both promoting NPC formation and interacting with NPCs NPCs are required for global genome organization, but whether global genome organization itself promotes NPC formation was not known;. This study found that by acting as a barrier between active and repressive compartments, stwl promotes the expression of a cohort of Nups and interacts with them. As all Nups are required for NPC formation, stwl promotes NPC formation by promoting the transcription of some Nups that comprise the NPC. Previous work showed that H3K9me3-mediated chromatin marks are also required for Nup transcription. This suggests that NPC formation is sensitive to both the levels of heterochromatin and how it is demarcated (Kolb, 2024).

NPCs are known to help in a genomic organization by helping both tether silenced genes to the lamina to maintain their silenced state and position active genes under NPCs to allow for RNA export. stwl colocalizes with NPCs, promoting the anchoring of silenced genes to the nuclear periphery. This study also found that NPC components and stwl are present at TAD boundaries. Taken together, these data suggest that Stwl, through BEAF recruitment, regulates NPC formation and then, by interaction with NPCs, anchors TADs to the nuclear periphery to maintain their silencing. Indeed, loss of the NPC components stwl and BEAF results in loss of Lamin association of an early oogenesis gene, rpS19b. Thus, stwl modulates global genome organization by regulating the formation of and binding to NPCs (Kolb, 2024).

Taken together, this study found that stwl is a critical regulator of local and global genome architecture during the germ cell-to-maternal transition. By establishing boundaries between silenced and active regions, stwl ensures the confinement of a particular chromatin state and the proper expression of germ cell differentiation genes and Nups to regulate NPC formation. The NPCs in turn promote the tethering of silenced regions to the lamina. This work provides an essential framework for understanding the interplay between genome organization, NPCs, and cell fate determination (Kolb, 2024).

Stonewall prevents expression of ectopic genes in the ovary and accumulates at insulator elements in D. melanogaster

Germline stem cells (GSCs) are the progenitor cells of the germline for the lifetime of an animal. In Drosophila, these cells reside in a cellular niche that is required for both their maintenance (self-renewal) and differentiation (asymmetric division resulting in a daughter cell that differs from the GSC). The stem cell-daughter cell transition is tightly regulated by a number of processes, including an array of proteins required for genome stability. The germline stem-cell maintenance factor Stonewall (Stwl) associates with heterochromatin, but its molecular function is poorly understood. This study performed RNA-Seq on stwl mutant ovaries and found significant derepression of many transposon families but not heterochromatic genes. This study also discovered inappropriate expression of multiple classes of genes. Most prominent are testis-enriched genes, including the male germline sex-determination switch Phf7, the differentiation factor bgcn, and a large testis-specific gene cluster on chromosome 2, all of which are upregulated or ectopically expressed in stwl mutant ovaries. Surprisingly, this study also found that RNAi knockdown of stwl in somatic S2 cells results in ectopic expression of these testis genes. Using parallel ChIP-Seq and RNA-Seq experiments in S2 cells, stwl was found to be localized upstream of transcription start sites and at heterochromatic sequences including repetitive sequences associated with telomeres. stwl is also enriched at bgcn, suggesting that it directly regulates this essential differentiation factor. Finally, stwl binding motifs were identified that are shared with known insulator binding proteins. It is proposed that stwl affects gene regulation, including repression of male transcripts in the female germline, by binding insulators and establishing chromatin boundaries (Zinshteyn, 2022).

Stonewall and Brickwall: Two partially redundant determinants required for the maintenance of female germline in Drosophila

Germline stem cells located at the anterior tip of the adult Drosophila melanogaster ovary are critical to the continuous production of mature eggs. Following germline stem cell division, one daughter cell remains a stem cell, while the other becomes a cystoblast committed to differentiation. In this study it was shown that mutations in the putative transcription factor stonewall (stwl) disrupted the maintenance of female germline stem cells. The stwl mutations resulted in a loss of germline stem cells, causing a rapid decrease in egg chamber production. The egg chambers developed only to a limited extent before degenerating. The four mitotic cystocyte divisions were frequently inhibited by stwl mutations. Furthermore, some stwl germaria from newly emerged females completely lacked both stem cells and developing cysts and had a strong reduction in size. The argument is presented that stwl is involved in the continuation of cell division during female germline development (Shukla, 2018).

Stonewall and Brickwall: Two partially redundant determinants required for the maintenance of female germline in Drosophila

Proper specification of germline stem cells (GSCs) in Drosophila ovaries depends on niche derived non-autonomous signaling and cell autonomous components of transcriptional machinery. Stonewall (Stwl), a MADF-BESS family protein, is one of the cell intrinsic transcriptional regulators involved in the establishment and/or maintenance of GSC fate in Drosophila ovaries. This stufy reports identification and functional characterization of another member of the same protein family, CG3838/Brickwall (Brwl) with analogous functions. Loss of function alleles of brwl exhibit age dependent progressive degeneration of the developing ovarioles and loss of GSCs. Supporting the conclusion that the structural deterioration of mutant egg chambers is a result of apoptotic cell death, activated caspase levels are considerably elevated in brwl(-) ovaries. Moreover, as in the case of stwl mutants, on several instances, loss of brwl activity results in fusion of egg chambers and misspecification of the oocyte. Importantly, brwl phenotypes can be partially rescued by germline specific over-expression of stwl arguing for overlapping yet distinct functional capabilities of the two proteins. Taken together with the phylogenetic analysis, these data suggest that brwl and stwl likely share a common MADF-BESS ancestor and they are expressed in overlapping spatiotemporal domains to ensure robust development of the female germline (Shukla, 2018).

Stonewalling Drosophila stem cell differentiation by epigenetic controls

During Drosophila oogenesis, germline stem cell (GSC) identity is maintained largely by preventing the expression of factors that promote differentiation. This is accomplished via the activity of several genes acting either in the GSC or in its niche. The translational repressors Nanos and Pumilio act in GSCs to prevent differentiation, probably by inhibiting the translation of early differentiation factors, whereas niche signals prevent differentiation by silencing transcription of the differentiation factor Bam. This study has found that the DNA-associated protein Stonewall (Stwl) is also required for GSC maintenance. stwl is required cell-autonomously; clones of stwl^- germ cells were lost by differentiation, and ectopic stwl caused an expansion of GSCs. stwl mutants acted as Suppressors of variegation, indicating that stwl normally acts in chromatin-dependent gene repression. In contrast to several previously described GSC maintenance factors, stwl probably functions epigenetically to prevent GSC differentiation. Stwl-dependent transcriptional repression does not target bam, but rather stwl represses the expression of many genes, including those that may be targeted by Nanos and Pumilio translational inhibition (Maines, 2007).

The myb-related gene stonewall induces both hyperplasia and cell death in Drosophila: rescue of fly lethality by coexpression of apoptosis inducers

Gain-of-function mutagenesis screening was carried out, and a mutant was identified in which GAL4 induction led to both hyperplasia and apoptosis. The gene involved was identified as stonewall (stwl), a myb-related gene involved in germ cell proliferation and differentiation during oogenesis. As observed with dmyb, the ectopic expression of stwl^UY823 inhibited endoreplication in salivary glands. This study also found that stwl^UY823 overexpression, like overexpression of the wild-type gene, activated G1/S transition and apoptosis. The apoptosis triggered by stwl^UY823 expression is correlated to induction of the proapoptotic gene reaper. Finally, the death of flies induced by ectopic stwl^UY823 expression is efficiently prevented in vivo by triggering cell death in stwl^UY823 -expressing cells. These results suggest that stwl^UY823 kills flies by causing inappropriate cell cycle entry, and that triggering the death of these overproliferating cells or slowing their proliferation restores viability (Brun, 2006).

Mutations of stonewall disrupt the maintenance of female germline stem cells in Drosophila melanogasters

Germline stem cells located at the anterior tip of the adult Drosophila melanogaster ovary are critical to the continuous production of mature eggs. Following germline stem cell division, one daughter cell remains a stem cell, while the other becomes a cystoblast committed to differentiation. In this study it was shown that mutations in the putative transcription factor stonewall (stwl) disrupted the maintenance of female germline stem cells. The stwl mutations resulted in a loss of germline stem cells, causing a rapid decrease in egg chamber production. The egg chambers developed only to a limited extent before degenerating. The four mitotic cystocyte divisions were frequently inhibited by stwl mutations. Furthermore, some stwl germaria from newly emerged females completely lacked both stem cells and developing cysts and had a strong reduction in size. The argument is presented that stwl is involved in the continuation of cell division during female germline development (Akiyama, 2002).

Search PubMed for articles about Drosophila Stonewall

Akiyama, T. (2002). Mutations of stonewall disrupt the maintenance of female germline stem cells in Drosophila melanogaster. Dev Growth Differ, 44(2):97-102 PubMed ID: 11940096

Brun, S., Rincheval-Arnold, A., Colin, J., Risler, Y., Mignotte, B., Guenal, I. (2006). The myb-related gene stonewall induces both hyperplasia and cell death in Drosophila: rescue of fly lethality by coexpression of apoptosis inducers. Cell Death Differ, 13(10):1752-1762 PubMed ID: 16456582

Chavan, A., Isenhart, R., Nguyen, S. C., Kotb, N. M., Harke, J., Sintsova, A., Ulukaya, G., Uliana, F., Ashiono, C., Kutay, U., Pegoraro, G., Rangan, P., Joyce, E. F., Jagannathan, M. (2024). A nuclear architecture screen in Drosophila identifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate. Genes Dev, 38(9-10):415-435 PubMed ID: 38866555

Choi, J. Y. and Aquadro, C. F. (2014). The coevolutionary period of Wolbachia pipientis infecting Drosophila ananassae and its impact on the evolution of the host germline stem cell regulating genes. Mol Biol Evol 31(9):2457-71. PubMed ID: 24974378

Clark, K.A. and McKearin, D. M. (1996). The Drosophila stonewall gene encodes a putative transcription factor essential for germ cell development. Development 122: 937-950. PubMed ID: 8631271

Kotb, N. M., Ulukaya, G., Chavan, A., Nguyen, S. C., Proskauer, L., Joyce, E. F., Hasson, D., Jagannathan, M., Rangan, P. (2024). Genome organization regulates nuclear pore complex formation and promotes differentiation during Drosophila oogenesis. Genes Dev, 38(9-10):436-454 PubMed ID: 38866556

Maines, J. Z., Park, J. K., Williams, M., McKearin, D. M. (2007). Stonewalling Drosophila stem cell differentiation by epigenetic controls. Development, 134(8):1471-1479 PubMed ID: 17344229

Shukla, V., Dhiman, N., Nayak, P., Dahanukar, N., Deshpande, G. and Ratnaparkhi, G. (2018). Stonewall and Brickwall: Two partially redundant determinants required for the maintenance of female germline in Drosophila. G3 (Bethesda). PubMed ID: 29669801

date revised: 8 September 2025

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