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LINE1 and its host: one cell's junk is another cell's treasure. LINE1 及其宿主:一个细胞的垃圾是另一个细胞的宝藏。
IF 9.4 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-01 Epub Date: 2024-07-29 DOI: 10.1038/s44318-024-00175-5
John C Martinez, Andrei Seluanov, Vera Gorbunova
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引用次数: 0
Deubiquitinating enzyme mutagenesis screens identify a USP43-dependent HIF-1 transcriptional response. 去泛素化酶诱变筛选确定了一种依赖于 USP43 的 HIF-1 转录反应。
IF 9.4 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-01 Epub Date: 2024-07-15 DOI: 10.1038/s44318-024-00166-6
Tekle Pauzaite, Niek Wit, Rachel V Seear, James A Nathan

The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1α isoform, and while USP43 does not alter HIF-1α stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.

低氧诱导因子(HIFs)的泛素化和蛋白酶体介导的降解是后生动物氧传感的核心,但去泛素化酶(DUBs)参与 HIF 信号传导的情况还不太清楚。在这里,我们利用定制的 DUBs sgRNA 文库进行 CRISPR/Cas9 诱变筛选,以确定 DUBs 如何参与 HIF 信号传导。在确定参与 HIF 激活或抑制的 DUBs 的同时,我们还发现 USP43 是有效激活 HIF 响应所需的 DUB。USP43 受缺氧调节,并选择性地与 HIF-1α 异构体结合,虽然 USP43 不会改变 HIF-1α 的稳定性,但它能促进 HIF-1 的核积累并与其靶基因结合。从机理上讲,USP43 以缺氧和磷酸化依赖的方式与 14-3-3 蛋白结合,以增加 HIF-1 的核库。我们的研究结果凸显了 DUBs 的多功能性,说明它们在发挥催化作用的同时还能提供重要的信号功能。
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引用次数: 0
Recycled melanoma-secreted melanosomes regulate tumor-associated macrophage diversification. 黑色素瘤分泌的黑色素小体可调节肿瘤相关巨噬细胞的多样化。
IF 9.4 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-01 Epub Date: 2024-05-08 DOI: 10.1038/s44318-024-00103-7
Roma Parikh, Shivang Parikh, Daniella Berzin, Hananya Vaknine, Shai Ovadia, Daniela Likonen, Shoshana Greenberger, Alon Scope, Sharona Elgavish, Yuval Nevo, Inbar Plaschkes, Eran Nizri, Oren Kobiler, Avishai Maliah, Laureen Zaremba, Vishnu Mohan, Irit Sagi, Ruth Ashery-Padan, Yaron Carmi, Chen Luxenburg, Jörg D Hoheisel, Mehdi Khaled, Mitchell P Levesque, Carmit Levy

Extracellular vesicles (EVs) are important mediators of communication between cells. Here, we reveal a new mode of intercellular communication by melanosomes, large EVs secreted by melanocytes for melanin transport. Unlike small EVs, which are disintegrated within the receiver cell, melanosomes stay intact within them, gain a unique protein signature, and can then be further transferred to another cell as "second-hand" EVs. We show that melanoma-secreted melanosomes passaged through epidermal keratinocytes or dermal fibroblasts can be further engulfed by resident macrophages. This process leads to macrophage polarization into pro-tumor or pro-immune cell infiltration phenotypes. Melanosomes that are transferred through fibroblasts can carry AKT1, which induces VEGF secretion from macrophages in an mTOR-dependent manner, promoting angiogenesis and metastasis in vivo. In melanoma patients, macrophages that are co-localized with AKT1 are correlated with disease aggressiveness, and immunotherapy non-responders are enriched in macrophages containing melanosome markers. Our findings suggest that interactions mediated by second-hand extracellular vesicles contribute to the formation of the metastatic niche, and that blocking the melanosome cues of macrophage diversification could be helpful in halting melanoma progression.

细胞外囊泡(EVs)是细胞间通信的重要媒介。在这里,我们揭示了黑色素小体进行细胞间通信的一种新模式,黑色素小体是黑色素细胞为运输黑色素而分泌的大型EV。与在接收细胞内分解的小EV不同,黑色素体在接收细胞内保持完整,并获得独特的蛋白质特征,然后可以作为 "二手 "EV进一步转移到另一个细胞。我们的研究表明,通过表皮角质细胞或真皮成纤维细胞传递的黑色素瘤分泌的黑色素小体可被驻留的巨噬细胞进一步吞噬。这一过程会导致巨噬细胞极化为亲肿瘤或亲免疫细胞浸润表型。通过成纤维细胞转移的黑色素小体可携带 AKT1,AKT1 可通过 mTOR 依赖性方式诱导巨噬细胞分泌血管内皮生长因子,促进体内血管生成和转移。在黑色素瘤患者中,与AKT1共定位的巨噬细胞与疾病的侵袭性相关,免疫疗法无反应者的巨噬细胞中富含黑色素体标记。我们的研究结果表明,二手细胞外囊泡介导的相互作用促成了转移龛的形成,阻断巨噬细胞多样化的黑色素体线索有助于阻止黑色素瘤的发展。
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引用次数: 0
A common mechanism for recruiting the Rrm3 and RTEL1 accessory helicases to the eukaryotic replisome. 真核生物复制体招募 Rrm3 和 RTEL1 辅助螺旋酶的共同机制。
IF 9.4 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-01 Epub Date: 2024-07-22 DOI: 10.1038/s44318-024-00168-4
Ottavia Olson, Simone Pelliciari, Emma D Heron, Tom D Deegan

The eukaryotic replisome is assembled around the CMG (CDC45-MCM-GINS) replicative helicase, which encircles the leading-strand DNA template at replication forks. When CMG stalls during DNA replication termination, or at barriers such as DNA-protein crosslinks on the leading strand template, a second helicase is deployed on the lagging strand template to support replisome progression. How these 'accessory' helicases are targeted to the replisome to mediate barrier bypass and replication termination remains unknown. Here, by combining AlphaFold structural modelling with experimental validation, we show that the budding yeast Rrm3 accessory helicase contains two Short Linear Interaction Motifs (SLIMs) in its disordered N-terminus, which interact with CMG and the leading-strand DNA polymerase Polε on one side of the replisome. This flexible tether positions Rrm3 adjacent to the lagging strand template on which it translocates, and is critical for replication termination in vitro and Rrm3 function in vivo. The primary accessory helicase in metazoa, RTEL1, is evolutionarily unrelated to Rrm3, but binds to CMG and Polε in an analogous manner, revealing a conserved docking mechanism for accessory helicases in the eukaryotic replisome.

真核生物的复制体是围绕 CMG(CDC45-MCM-GINS)复制螺旋酶组装的,它在复制分叉处环绕着前导链 DNA 模板。当 CMG 在 DNA 复制终止过程中或在前导链模板上的 DNA 蛋白交联等障碍处停滞时,就会在滞后链模板上部署第二个螺旋酶,以支持复制体的进展。这些 "附属 "螺旋酶是如何锁定复制体以介导障碍绕过和复制终止的,目前仍是未知数。在这里,通过结合 AlphaFold 结构建模和实验验证,我们发现芽殖酵母 Rrm3 辅助螺旋酶在其无序的 N 端含有两个短线性相互作用分子(SLIM),它们与 CMG 和复制体一侧的前导链 DNA 聚合酶 Polε 相互作用。这种柔性系链将 Rrm3 定位在其转位的滞后链模板附近,对于体外复制终止和体内 Rrm3 的功能至关重要。元古动物中的主要辅助螺旋酶 RTEL1 在进化上与 Rrm3 无关,但以类似的方式与 CMG 和 Polε 结合,揭示了真核生物复制体中辅助螺旋酶的保守对接机制。
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引用次数: 0
An ancient role for CYP73 monooxygenases in phenylpropanoid biosynthesis and embryophyte development. CYP73 单氧化酶在苯丙类生物合成和胚胎植物发育中的古老作用。
IF 9.4 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-01 Epub Date: 2024-08-01 DOI: 10.1038/s44318-024-00181-7
Samuel Knosp, Lucie Kriegshauser, Kanade Tatsumi, Ludivine Malherbe, Mathieu Erhardt, Gertrud Wiedemann, Bénédicte Bakan, Takayuki Kohchi, Ralf Reski, Hugues Renault

The phenylpropanoid pathway is one of the plant metabolic pathways most prominently linked to the transition to terrestrial life, but its evolution and early functions remain elusive. Here, we show that activity of the t-cinnamic acid 4-hydroxylase (C4H), the first plant-specific step in the pathway, emerged concomitantly with the CYP73 gene family in a common ancestor of embryophytes. Through structural studies, we identify conserved CYP73 residues, including a crucial arginine, that have supported C4H activity since the early stages of its evolution. We further demonstrate that impairing C4H function via CYP73 gene inactivation or inhibitor treatment in three bryophyte species-the moss Physcomitrium patens, the liverwort Marchantia polymorpha and the hornwort Anthoceros agrestis-consistently resulted in a shortage of phenylpropanoids and abnormal plant development. The latter could be rescued in the moss by exogenous supply of p-coumaric acid, the product of C4H. Our findings establish the emergence of the CYP73 gene family as a foundational event in the development of the plant phenylpropanoid pathway, and underscore the deep-rooted function of the C4H enzyme in embryophyte biology.

苯丙类途径是植物代谢途径之一,与向陆地生命的过渡有着最显著的联系,但其进化和早期功能仍然难以捉摸。在这里,我们发现在胚状植物的共同祖先中,t-肉桂酸 4-羟化酶(C4H)的活性与 CYP73 基因家族同时出现,这是该途径中植物特有的第一步。通过结构研究,我们确定了 CYP73 的保守残基,包括一个关键的精氨酸,这些残基从 CYP73 进化的早期阶段就开始支持 C4H 的活性。我们进一步证明,通过 CYP73 基因失活或抑制剂处理损害 C4H 的功能,会一致导致三个叶绿体物种--苔藓 Physcomitrium patens、肝草 Marchantia polymorpha 和角草 Anthoceros agrestis--出现苯丙酸类物质短缺和植物发育异常。在苔藓中,通过外源供应 C4H 的产物对香豆酸,可以挽救后者。我们的研究结果证实,CYP73 基因家族的出现是植物苯丙氨酸途径发展过程中的一个奠基事件,并强调了 C4H 酶在胚状植物生物学中根深蒂固的功能。
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引用次数: 0
Author Correction: Microglia-synapse engulfment via PtdSer-TREM2 ameliorates neuronal hyperactivity in Alzheimer's disease models. 作者更正:小胶质细胞通过 PtdSer-TREM2 吞噬突触可改善阿尔茨海默病模型中神经元的过度活跃。
IF 9.4 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-01 DOI: 10.1038/s44318-024-00159-5
Javier Rueda-Carrasco, Dimitra Sokolova, Sang-Eun Lee, Thomas Childs, Natália Jurčáková, Gerard Crowley, Sebastiaan De Schepper, Judy Z Ge, Joanne I Lachica, Christina E Toomey, Oliver J Freeman, John Hardy, Samuel J Barnes, Tammaryn Lashley, Beth Stevens, Sunghoe Chang, Soyon Hong
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引用次数: 0
LINE-1 RNA triggers matrix formation in bone cells via a PKR-mediated inflammatory response. LINE-1 RNA 通过 PKR 介导的炎症反应引发骨细胞中基质的形成。
IF 9.4 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-01 Epub Date: 2024-07-01 DOI: 10.1038/s44318-024-00143-z
Arianna Mangiavacchi, Gabriele Morelli, Sjur Reppe, Alfonso Saera-Vila, Peng Liu, Benjamin Eggerschwiler, Huoming Zhang, Dalila Bensaddek, Elisa A Casanova, Carolina Medina Gomez, Vid Prijatelj, Francesco Della Valle, Nazerke Atinbayeva, Juan Carlos Izpisua Belmonte, Fernando Rivadeneira, Paolo Cinelli, Kaare Morten Gautvik, Valerio Orlando

Transposable elements (TEs) are mobile genetic modules of viral derivation that have been co-opted to become modulators of mammalian gene expression. TEs are a major source of endogenous dsRNAs, signaling molecules able to coordinate inflammatory responses in various physiological processes. Here, we provide evidence for a positive involvement of TEs in inflammation-driven bone repair and mineralization. In newly fractured mice bone, we observed an early transient upregulation of repeats occurring concurrently with the initiation of the inflammatory stage. In human bone biopsies, analysis revealed a significant correlation between repeats expression, mechanical stress and bone mineral density. We investigated a potential link between LINE-1 (L1) expression and bone mineralization by delivering a synthetic L1 RNA to osteoporotic patient-derived mesenchymal stem cells and observed a dsRNA-triggered protein kinase (PKR)-mediated stress response that led to strongly increased mineralization. This response was associated with a strong and transient inflammation, accompanied by a global translation attenuation induced by eIF2α phosphorylation. We demonstrated that L1 transfection reshaped the secretory profile of osteoblasts, triggering a paracrine activity that stimulated the mineralization of recipient cells.

可转座元件(Transposable elements,TEs)是由病毒衍生而来的移动遗传模块,已成为哺乳动物基因表达的调节器。可转座元件是内源性 dsRNA 的主要来源,而 dsRNA 是一种信号分子,能够在各种生理过程中协调炎症反应。在这里,我们提供了 TEs 积极参与炎症驱动的骨修复和矿化的证据。在新骨折的小鼠骨骼中,我们观察到重复序列的早期短暂上调与炎症阶段的启动同时发生。在人体骨活检组织中,分析显示重复序列表达、机械应力和骨矿物质密度之间存在显著相关性。我们通过向骨质疏松症患者间充质干细胞递送合成的 L1 RNA,研究了 LINE-1(L1)表达与骨矿化之间的潜在联系。这种反应与强烈而短暂的炎症有关,并伴随着由eIF2α磷酸化诱导的全局翻译衰减。我们证明,L1转染重塑了成骨细胞的分泌谱,引发了一种旁分泌活动,刺激了受体细胞的矿化。
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引用次数: 0
TDP1 phosphorylation by CDK1 in mitosis promotes MUS81-dependent repair of trapped Top1-DNA covalent complexes. 有丝分裂过程中 CDK1 磷酸化 TDP1 可促进 MUS81 依赖性修复被困的 Top1-DNA 共价复合物。
IF 9.4 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-01 Epub Date: 2024-07-16 DOI: 10.1038/s44318-024-00169-3
Srijita Paul Chowdhuri, Benu Brata Das

Topoisomerase 1 (Top1) controls DNA topology, relieves DNA supercoiling during replication and transcription, and is critical for mitotic progression to the G1 phase. Tyrosyl-DNA phosphodiesterase 1 (TDP1) mediates the removal of trapped Top1-DNA covalent complexes (Top1cc). Here, we identify CDK1-dependent phosphorylation of TDP1 at residue S61 during mitosis. A TDP1 variant defective for S61 phosphorylation (TDP1-S61A) is trapped on the mitotic chromosomes, triggering DNA damage and mitotic defects. Moreover, we show that Top1cc repair in mitosis occurs via a MUS81-dependent DNA repair mechanism. Replication stress induced by camptothecin or aphidicolin leads to TDP1-S61A enrichment at common fragile sites, which over-stimulates MUS81-dependent chromatid breaks, anaphase bridges, and micronuclei, ultimately culminating in the formation of 53BP1 nuclear bodies during G1 phase. Our findings provide new insights into the cell cycle-dependent regulation of TDP1 dynamics for the repair of trapped Top1-DNA covalent complexes during mitosis that prevents genomic instability following replication stress.

拓扑异构酶 1(Top1)控制 DNA 的拓扑结构,在复制和转录过程中缓解 DNA 的超卷曲,对于有丝分裂进入 G1 阶段至关重要。酪氨酰-DNA 磷酸二酯酶 1(TDP1)介导清除被困的 Top1-DNA 共价复合物(Top1cc)。在这里,我们发现了有丝分裂过程中 CDK1 依赖性磷酸化 TDP1 的残基 S61。S61磷酸化缺陷的TDP1变体(TDP1-S61A)被困在有丝分裂染色体上,引发DNA损伤和有丝分裂缺陷。此外,我们还发现有丝分裂中的Top1cc修复是通过依赖于MUS81的DNA修复机制进行的。喜树碱或蚜虫霉素诱导的复制应激会导致TDP1-S61A在常见脆性位点富集,从而过度刺激依赖MUS81的染色体断裂、无丝期桥和微核,最终在G1期形成53BP1核体。我们的发现为细胞周期依赖性的 TDP1 动态调控提供了新的见解,TDP1 可在有丝分裂过程中修复被困的 Top1-DNA 共价复合物,从而防止复制应激后的基因组不稳定性。
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引用次数: 0
Cytokinetic abscission in Toxoplasma gondii is governed by protein phosphatase 2A and the daughter cell scaffold complex. 弓形虫的细胞分裂受蛋白磷酸酶 2A 和子细胞支架复合体的调控。
IF 9.4 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-01 Epub Date: 2024-07-15 DOI: 10.1038/s44318-024-00171-9
Jean-Baptiste Marq, Margaux Gosetto, Aline Altenried, Oscar Vadas, Bohumil Maco, Nicolas Dos Santos Pacheco, Nicolò Tosetti, Dominique Soldati-Favre, Gaëlle Lentini

Cytokinetic abscission marks the final stage of cell division, during which the daughter cells physically separate through the generation of new barriers, such as the plasma membrane or cell wall. While the contractile ring plays a central role during cytokinesis in bacteria, fungi and animal cells, the process diverges in Apicomplexa. In Toxoplasma gondii, two daughter cells are formed within the mother cell by endodyogeny. The mechanism by which the progeny cells acquire their plasma membrane during the disassembly of the mother cell, allowing daughter cells to emerge, remains unknown. Here we identify and characterize five T. gondii proteins, including three protein phosphatase 2A subunits, which exhibit a distinct and dynamic localization pattern during parasite division. Individual downregulation of these proteins prevents the accumulation of plasma membrane at the division plane, preventing the completion of cellular abscission. Remarkably, the absence of cytokinetic abscission does not hinder the completion of subsequent division cycles. The resulting progeny are able to egress from the infected cells but fail to glide and invade, except in cases of conjoined twin parasites.

细胞脱落标志着细胞分裂的最后阶段,在这一阶段,子细胞通过产生新的屏障(如质膜或细胞壁)而物理分离。虽然收缩环在细菌、真菌和动物细胞的细胞分裂过程中发挥着核心作用,但这一过程在弓形虫中却出现了分化。在弓形虫中,两个子细胞通过内生作用在母细胞内形成。后代细胞在母细胞解体过程中获得质膜,从而使子细胞出现的机制仍不清楚。在这里,我们鉴定并描述了五种淋球菌蛋白,包括三种蛋白磷酸酶 2A 亚基,它们在寄生虫分裂过程中表现出独特的动态定位模式。单个下调这些蛋白可阻止质膜在分裂平面的聚集,从而阻止细胞脱落的完成。值得注意的是,细胞脱落的缺失并不妨碍后续分裂周期的完成。由此产生的后代能够从被感染的细胞中排出,但不能滑行和入侵,连体双胞胎寄生虫的情况除外。
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引用次数: 0
T4 DNA polymerase prevents deleterious on-target DNA damage and enhances precise CRISPR editing. T4 DNA 聚合酶可防止对目标 DNA 造成有害损伤,并提高 CRISPR 编辑的精确度。
IF 9.4 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-01 Epub Date: 2024-07-22 DOI: 10.1038/s44318-024-00158-6
Qiaoyan Yang, Jonathan S Abebe, Michelle Mai, Gabriella Rudy, Sang Y Kim, Orrin Devinsky, Chengzu Long

Unintended on-target chromosomal alterations induced by CRISPR/Cas9 in mammalian cells are common, particularly large deletions and chromosomal translocations, and present a safety challenge for genome editing. Thus, there is still an unmet need to develop safer and more efficient editing tools. We screened diverse DNA polymerases of distinct origins and identified a T4 DNA polymerase derived from phage T4 that strongly prevents undesired on-target damage while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing (termed CasPlus). CasPlus induced substantially fewer on-target large deletions while increasing the efficiency of correcting common frameshift mutations in DMD and restored higher level of dystrophin expression than Cas9-alone in human cardiomyocytes. Moreover, CasPlus greatly reduced the frequency of on-target large deletions during mouse germline editing. In multiplexed guide RNAs mediating gene editing, CasPlus repressed chromosomal translocations while maintaining gene disruption efficiency that was higher or comparable to Cas9 in primary human T cells. Therefore, CasPlus offers a safer and more efficient gene editing strategy to treat pathogenic variants or to introduce genetic modifications in human applications.

CRISPR/Cas9 在哺乳动物细胞中诱导的意外靶上染色体改变很常见,尤其是大缺失和染色体易位,给基因组编辑带来了安全挑战。因此,开发更安全、更高效的编辑工具的需求仍未得到满足。我们筛选了不同来源的DNA聚合酶,发现了一种来自噬菌体T4的T4 DNA聚合酶,它能有效防止不希望的靶上损伤,同时增加CRISPR/Cas9编辑过程中产生的1至2碱基对精确插入的比例(称为CasPlus)。与单用Cas9相比,CasPlus在人类心肌细胞中诱导的靶上大缺失大大减少,同时提高了DMD常见的换帧突变的纠正效率,并恢复了更高水平的肌营养不良蛋白表达。此外,在小鼠种系编辑过程中,CasPlus 大大降低了靶向大缺失的频率。在多导 RNA 介导的基因编辑中,CasPlus 可抑制染色体易位,同时在原代人类 T 细胞中保持高于或与 Cas9 相当的基因破坏效率。因此,CasPlus 是一种更安全、更高效的基因编辑策略,可用于治疗致病变异或在人类应用中引入基因修饰。
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引用次数: 0
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