Journal of Molecular Biology最新文献

英文中文

Expanding the Protein Universe 社论：拓展蛋白质宇宙。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-10-04 DOI: 10.1016/j.jmb.2024.168812

引用次数: 0

Recent Advances in Expression Screening and Sample Evaluation for Structural Studies of Membrane Proteins 用于膜蛋白结构研究的表达筛选和样本评估的最新进展。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-10-01 DOI: 10.1016/j.jmb.2024.168809

Membrane proteins are involved in numerous biological processes and represent more than half of all drug targets; thus, structural information on these proteins is invaluable. However, the low expression level of membrane proteins, as well as their poor stability in solution and tendency to precipitate and aggregate, are major bottlenecks in the preparation of purified membrane proteins for structural studies. Traditionally, the evaluation of membrane protein constructs for structural studies has been quite time consuming and expensive since it is necessary to express and purify the proteins on a large scale, particularly for X-ray crystallography. The emergence of fluorescence detection size exclusion chromatography (FSEC) has drastically changed this situation, as this method can be used to rapidly evaluate the expression and behavior of membrane proteins on a small scale without the need for purification. FSEC has become the most widely used method for the screening of expression conditions and sample evaluation for membrane proteins, leading to the successful determination of numerous structures. Even in the era of cryo-EM, FSEC and the new generation of FSEC derivative methods are being widely used in various manners to facilitate structural analysis. In addition, the application of FSEC is not limited to structural analysis; this method is also widely used for functional analysis of membrane proteins, including for analysis of oligomerization state, screening of antibodies and ligands, and affinity profiling. This review presents the latest advances and applications in membrane protein expression screening and sample evaluation, with a particular focus on FSEC methods.

膜蛋白参与许多生物过程，占所有药物靶点的一半以上；因此，这些蛋白的结构信息非常宝贵。然而，膜蛋白表达水平低，在溶液中稳定性差，容易沉淀和聚集，是制备纯化膜蛋白用于结构研究的主要瓶颈。传统上，用于结构研究的膜蛋白构建物的评估相当耗时和昂贵，因为必须大规模表达和纯化蛋白质，特别是用于 X 射线晶体学。荧光检测尺寸排阻色谱法（FSEC）的出现彻底改变了这一局面，因为这种方法可用于快速评估小规模膜蛋白的表达和行为，而无需纯化。FSEC 已成为膜蛋白表达条件筛选和样品评估最广泛使用的方法，并成功确定了许多结构。即使在冷冻电镜时代，FSEC 和新一代 FSEC 衍生方法也以各种方式被广泛应用于结构分析。此外，FSEC 的应用不仅限于结构分析，它还广泛用于膜蛋白的功能分析，包括寡聚状态分析、抗体和配体筛选以及亲和性分析。本综述介绍了膜蛋白表达筛选和样品评估的最新进展和应用，尤其侧重于 FSEC 方法。

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引用次数: 0

iACP-DFSRA: Identification of Anticancer Peptides Based on a Dual-channel Fusion Strategy of ResCNN and Attention iACP-DFSRA：基于 ResCNN 和 Attention 双通道融合策略的抗癌肽鉴定。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-10-01 DOI: 10.1016/j.jmb.2024.168810

Anticancer peptides (ACPs) have been widely applied in the treatment of cancer owing to good safety, rational side effects, and high selectivity. However, the number of ACPs that have been experimentally validated is limited as identification of ACPs is extremely expensive. Hence, accurate and cost-effective identification methods for ACPs are urgently needed. In this work, we proposed a deep learning-based model, named iACP-DFSRA, for ACPs identification. Specifically, we adopted two kinds of sequence embedding technologies, ProtBert_BFD pre-training language model and handcrafted features to encode protein sequences. Then, the LightGBM was used for feature selection, and the selected features were input into ResCNN and Attention mechanism, respectively, to extract local and global features. Finally, the concatenate features were deeply fused by using the Attention mechanism to allow key features to be paid more attention to by the model and make predictions by fully connected layer. The results of 10-fold cross-validation demonstrated that the iACP-DFSRA model delivered improved results in most metrics with Sp of 94.15%, Sn of 95.32%, Acc of 94.74% and MCC of 89.48% compared to the latest AACFlow model. Indeed, the iACP-DFSRA model is the only model with Acc > 90% and MCC > 80% on this independent test dataset. Furthermore, we have further demonstrated the superiority of our model on additional datasets. In addition, t-SNE and SHAP interpretation analysis demonstrated that it is crucial to use two channels for feature extraction and use the Attention mechanism for deep fusion, which helps the iACP-DFSRA to predict ACPs more effectively.

抗癌肽（ACPs）因其安全性好、副作用小、选择性高而被广泛应用于癌症治疗。然而，由于 ACPs 的鉴定费用极其昂贵，经过实验验证的 ACPs 数量有限。因此，亟需准确且经济有效的 ACPs 识别方法。在这项工作中，我们提出了一种基于深度学习的 ACPs 识别模型，命名为 iACP-DFSRA。具体来说，我们采用了两种序列嵌入技术：ProtBert_BFD 预训练语言模型和手工特征来编码蛋白质序列。然后，利用 LightGBM 进行特征选择，并将所选特征分别输入 ResCNN 和 Attention 机制，以提取局部特征和全局特征。最后，利用注意力机制对并集的特征进行深度融合，使模型更加关注关键特征，并通过全连接层进行预测。10 倍交叉验证结果表明，与最新的 AACFlow 模型相比，iACP-DFSRA 模型在大多数指标上都有所改进，Sp 为 94.15%，Sn 为 95.32%，Acc 为 94.74%，MCC 为 89.48%。事实上，iACP-DFSRA 模型是该独立测试数据集上唯一一个 Acc > 90% 和 MCC > 80% 的模型。此外，我们还在其他数据集上进一步证明了我们模型的优越性。此外，t-SNE 和 SHAP 解释分析表明，使用双通道进行特征提取和使用 Attention 机制进行深度融合至关重要，这有助于 iACP-DFSRA 更有效地预测 ACP。

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引用次数: 0

Potential vs Challenges of Expanding the Protein Universe With Genetic Code Expansion in Eukaryotic Cells 真核细胞中遗传密码扩展蛋白质宇宙的潜力与挑战。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-09-30 DOI: 10.1016/j.jmb.2024.168807

Following decades of innovation and perfecting, genetic code expansion has become a powerful tool for in vivo protein modification. Some of the major hurdles that had to be overcome include suboptimal performance of GCE-specific translational components in host systems, competing cellular processes, unspecific modification of the host proteome and limited availability of codons for reassignment. Although strategies have been developed to overcome these challenges, there is critical need for further advances. Here we discuss the current state-of-the-art in genetic code expansion technology and the issues that still need to be addressed to unleash the full potential of this method in eukaryotic cells.

经过数十年的创新和完善，遗传密码扩增已成为体内蛋白质修饰的有力工具。必须克服的一些主要障碍包括：GCE特异性翻译元件在宿主系统中的性能不理想、竞争性细胞过程、宿主蛋白质组的非特异性修饰以及可用于重新分配的密码子有限。虽然已经开发出了克服挑战的策略，但仍亟需进一步改进。在此，我们将讨论遗传密码扩增技术的最新进展，以及要充分释放这种方法在真核细胞中的潜力仍需解决的问题。

引用次数: 0

Sen1: The Varied Virtues of a Multifaceted Helicase. Sen1：多面螺旋酶的各种优点。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-09-30 DOI: 10.1016/j.jmb.2024.168808

Umberto Aiello, Odil Porrua, Domenico Libri

Several machineries concurrently work on the DNA, but among them RNA Polymerases (RNAPs) are the most widespread and active users. The homeostasis of such a busy genomic environment relies on the existence of mechanisms that allow limiting transcription to a functional level, both in terms of extent and rate. Sen1 is a central player in this sense: using its translocase activity this protein has evolved the specific function of dislodging RNAPs from the DNA template, thus ending the transcription cycle. Over the years, studies have shown that Sen1 uses this same mechanism in a multitude of situations, allowing termination of all three eukaryotic RNAPs in different contexts. In virtue of its helicase activity, Sen1 has also been proposed to have a prominent function in the resolution of co-transcriptional genotoxic R-loops, which can cause the stalling of replication forks. In this review, we provide a synopsis of past and recent findings on the functions of Sen1 in yeast and of its human homologue Senataxin (SETX).

DNA 上同时存在着多种机制，但其中 RNA 聚合酶（RNAP）是最广泛和最活跃的使用者。如此繁忙的基因组环境的平衡有赖于各种机制的存在，这些机制可以将转录的范围和速度限制在功能水平上。在这个意义上，Sen1 是一个核心角色：利用其易位酶活性，这种蛋白质进化出了将 RNAP 从 DNA 模板上移开的特殊功能，从而结束了转录周期。多年来的研究表明，Sen1 在多种情况下都能利用这一机制，在不同情况下终止所有三种真核生物 RNAP。凭借其螺旋酶活性，Sen1 还被认为在解决共转录基因毒性 R 环（可导致复制叉停滞）方面具有突出功能。在这篇综述中，我们概述了过去和最近关于酵母中 Sen1 及其人类同源物 Senataxin（SETX）功能的研究结果。

引用次数: 0

Cryo-EM Reveals the Mechanism of DNA Compaction by Mycobacterium smegmatis Dps2 低温电子显微镜揭示了分枝杆菌 Dps2 的 DNA 压实机制。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-09-28 DOI: 10.1016/j.jmb.2024.168806

DNA binding protein from starved cells (Dps) is a miniature ferritin complex, which plays a vital role in protecting bacterial DNA during starvation to maintain the integrity of bacteria under hostile conditions. Several approaches, including cryo-electron tomography, have been previously implemented by other research groups to decipher the structure of the Dps protein bound to DNA. However, none of the structures of the Dps-DNA complex was resolved to high resolution to identify the DNA binding residues. Like other bacteria, Mycobacterium smegmatis also expresses Dps2 (called MsDps2), which binds DNA to protect it under oxidative stress conditions. In this study, we implemented various biochemical and biophysical studies to characterize the DNA protein interactions of Dps2 protein from Mycobacterium smegmatis. We employed single-particle cryo-EM-based structural analysis of MsDps2-DNA complexes and identified that the region close to the N-terminus confers the DNA binding property. Based on cryo-EM data, we also pinpointed several arginine residues, proximal to the DNA binding region, responsible for DNA binding. We also performed mutations of these residues, which dramatically reduced the MsDps2-DNA interaction. In addition, we proposed a model that elucidates the mechanism of DNA compaction, which adapts a lattice-like structure. We performed single-molecule imaging of MsDps2-DNA interactions that corroborate well with our structural studies. Taken together, our results delineate the specific MsDps2 residues that play an important role in DNA binding and compaction, providing new insights into Mycobacterial DNA compaction mechanisms under stress conditions.

饥饿细胞的 DNA 结合蛋白（Dps）是一种微型铁蛋白复合物，在饥饿过程中保护细菌 DNA 方面发挥着重要作用，从而在恶劣条件下保持细菌的完整性。此前，其他研究小组采用了包括低温电子断层扫描在内的多种方法来破译 Dps 蛋白与 DNA 结合的结构。然而，没有一个 Dps-DNA 复合物的结构被解析到高分辨率，无法确定 DNA 结合残基。与其他细菌一样，分枝杆菌也表达 Dps2（称为 MsDps2），它与 DNA 结合，在氧化压力条件下保护 DNA。在本研究中，我们进行了各种生化和生物物理研究，以确定烟曲霉分枝杆菌 Dps2 蛋白的 DNA 蛋白相互作用的特征。我们采用单颗粒低温电子显微镜对 MsDps2-DNA 复合物进行了结构分析，发现靠近 N 端的区域具有 DNA 结合特性。根据低温电子显微镜数据，我们还确定了几个精氨酸残基，它们位于 DNA 结合区的近端，负责 DNA 结合。我们还对这些残基进行了突变，结果大大降低了 MsDps2 与 DNA 的相互作用。此外，我们还提出了一个模型，该模型阐明了 DNA 的压实机制，它适应了一种类似晶格的结构。我们对 MsDps2-DNA 的相互作用进行了单分子成像，结果与我们的结构研究完全吻合。综上所述，我们的研究结果确定了在 DNA 结合和压实过程中发挥重要作用的特定 MsDps2 残基，为我们深入了解分枝杆菌 DNA 在应激条件下的压实机制提供了新的视角。

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引用次数: 0

Differential Responses in the Core, Active Site and Peripheral Regions of Cytochrome c Peroxidase to Extreme Pressure and Temperature 细胞色素 c 过氧化物酶的核心、活性位点和外围区域对极端压力和温度的不同反应。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-09-26 DOI: 10.1016/j.jmb.2024.168799

In consideration of life in extreme environments, the effects of hydrostatic pressure on proteins at the atomic level have drawn substantial interest. Large deviations of temperature and pressure from ambient conditions can shift the free energy landscape of proteins to reveal otherwise lowly populated structural states and even promote unfolding. We report the crystal structure of the heme-containing peroxidase, cytochrome c peroxidase (CcP) at 1.5 and 3.0 kbar and make comparisons to structures determined at 1.0 bar and cryo-temperatures (100 K). Pressure produces anisotropic changes in CcP, but compressibility plateaus after 1.5 kbar. CcP responds to pressure with volume declines at the periphery of the protein where B-factors are relatively high but maintains nearly intransient core structure, hydrogen bonding interactions and active site channels. Changes in active-site solvation and heme ligation reveal pressure sensitivity to protein–ligand interactions and a potential docking site for the substrate peroxide. Compression at the surface affects neither alternate side-chain conformers nor B-factors. Thus, packing in the core, which resembles a crystalline solid, limits motion and protects the active site, whereas looser packing at the surface preserves side-chain dynamics. These data demonstrate that conformational dynamics and packing densities are not fully correlated in proteins and that encapsulation of cofactors by the polypeptide can provide a precisely structured environment resistant to change across a wide range of physical conditions.

考虑到极端环境中的生命，静水压力在原子水平上对蛋白质的影响引起了人们的极大兴趣。与环境条件相比，温度和压力的巨大偏差会改变蛋白质的自由能景观，从而揭示出原本低密度的结构状态，甚至促进蛋白质的解折。我们报告了含血红素过氧化物酶、细胞色素 c 过氧化物酶（CcP）在 1.5 和 3.0 千巴条件下的晶体结构，并将其与在 1.0 巴和低温（100 K）条件下测定的结构进行了比较。压力会使 CcP 发生各向异性的变化，但在 1.5 千巴之后可压缩性会趋于稳定。在 B 因子相对较高的蛋白质外围，CcP 对压力的反应是体积下降，但几乎保持不变的核心结构、氢键相互作用和活性位点通道。活性位点溶解和血红素连接的变化揭示了压力对蛋白质-配体相互作用和底物过氧化物潜在对接位点的敏感性。表面压缩既不会影响交替侧链构象，也不会影响 B 因子。因此，类似于结晶固体的核心填料限制了运动并保护了活性位点，而表面的松散填料则保持了侧链的动态变化。这些数据表明，蛋白质中的构象动力学和堆积密度并不完全相关，多肽对辅因子的包裹可以提供一个结构精确的环境，使其在各种物理条件下都不会发生变化。

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引用次数: 0

Nuclear Receptor Interdomain Communication is Mediated by the Hinge with Ligand Specificity 核受体域间通讯由具有配体特异性的铰链介导。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-09-25 DOI: 10.1016/j.jmb.2024.168805

Nuclear receptors are ligand-induced transcription factors that bind directly to target genes and regulate their expression. Ligand binding initiates conformational changes that propagate to other domains, allosterically regulating their activity. The nature of this interdomain communication in nuclear receptors is poorly understood, largely owing to the difficulty of experimentally characterizing full-length structures. We have applied computational modeling approaches to describe and study the structure of the full-length farnesoid X receptor (FXR), approximated by the DNA binding domain (DBD) and ligand binding domain (LBD) connected by the flexible hinge region. Using extended molecular dynamics simulations (>10 microseconds) and enhanced sampling simulations, we provide evidence that ligands selectively induce domain rearrangement, leading to interdomain contact. We use protein–protein interaction assays to provide experimental evidence of these interactions, identifying a critical role of the hinge in mediating interdomain contact. Our results illuminate previously unknown aspects of interdomain communication in FXR and provide a framework to enable characterization of other full-length nuclear receptors.

核受体是配体诱导的转录因子，可直接与目标基因结合并调节其表达。配体结合会引发构象变化，并传播到其他结构域，从而对其活性进行异构调节。人们对核受体中这种结构域间通信的性质知之甚少，这主要是由于难以通过实验鉴定全长结构。我们采用计算建模方法描述并研究了全长法尼类固醇 X 受体（FXR）的结构，该结构近似于 DNA 结合结构域（DBD）和配体结合结构域（LBD），两者通过柔性铰链区相连。利用扩展分子动力学模拟（> 10 微秒）和增强采样模拟，我们提供了配体选择性诱导结构域重排从而导致结构域间接触的证据。我们利用蛋白质-蛋白质相互作用实验提供了这些相互作用的实验证据，确定了铰链在介导结构域间接触中的关键作用。我们的研究结果揭示了 FXR 域间通讯以前未知的方面，并为其他全长核受体的特征描述提供了一个框架。

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引用次数: 0

Rational Design of Untranslated Regions to Enhance Gene Expression 合理设计非翻译区，提高基因表达。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-09-24 DOI: 10.1016/j.jmb.2024.168804

How to improve gene expression by optimizing mRNA structures is a crucial question for various medical and biotechnological applications. Previous efforts focus largely on investigation of the 5′ UTR hairpin structures. In this study, we present a rational strategy that enhances mRNA stability and translation by engineering both the 5′ and 3′ UTR sequences. We have successfully demonstrated this strategy using green fluorescent protein (GFP) as a model in Escherichia coli and across different expression vectors. We further validated it with luciferase and Plasmodium falciparum lactate dehydrogenase (PfLDH). To elucidate the underlying mechanism, we have quantitatively analyzed both protein, mRNA levels and half-life time. We have identified several key aspects of UTRs that significantly influence mRNA stability and protein expression in our system: (1) The optimal length of the single-stranded spacer between the stabilizer hairpin and ribosome binding site (RBS) in the 5′ UTR is 25–30 nucleotide (nt) long. An optimal 32% GC content in the spacer yielded the highest levels of GFP protein production. (2) The insertion of a homodimerdizable, G-quadruplex structure containing RNA aptamer, “Corn”, in the 3′ UTR markedly increased the protein expression. Our findings indicated that the carefully engineered 5′ UTRs and 3′ UTRs significantly boosted gene expression. Specifically, the inclusion of 5 × Corn in the 3′ UTR appeared to facilitate the local aggregation of mRNA, leading to the formation of mRNA condensates. Aside from shedding light on the regulation of mRNA stability and expression, this study is expected to substantially increase biological protein production.

如何通过优化 mRNA 结构来改善基因表达是各种医学和生物技术应用的关键问题。以往的研究主要集中在 5' UTR 发夹结构的研究上。在本研究中，我们提出了一种合理的策略，通过对 5' 和 3' UTR 序列进行工程设计来提高 mRNA 的稳定性和翻译能力。我们以绿色荧光蛋白（GFP）为模型，在大肠杆菌和不同的表达载体中成功验证了这一策略。我们还用荧光素酶和恶性疟原虫乳酸脱氢酶（PfLDH）进一步验证了这一策略。为了阐明其基本机制，我们对蛋白质、mRNA 水平和半衰期时间进行了定量分析。我们确定了 UTR 的几个关键方面，它们对我们系统中的 mRNA 稳定性和蛋白质表达有重大影响：（1）5' UTR 中稳定发夹和核糖体结合位点（RBS）之间的单链间隔的最佳长度为 25-30 个核苷酸（nt）。间隔中 32% 的最佳 GC 含量可产生最高水平的 GFP 蛋白。(2）在 3' UTR 中插入一个可同源二聚体化的、含有 G-四重结构的 RNA 类似物 "玉米"，可显著提高蛋白质的表达量。我们的研究结果表明，精心设计的 5' UTR 和 3' UTR 显著提高了基因的表达。具体来说，在 3' UTR 中加入 5×Corn 似乎能促进 mRNA 的局部聚集，从而形成 mRNA 凝聚体。除了揭示 mRNA 稳定性和表达的调控外，这项研究还有望大幅提高生物蛋白质的产量。

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引用次数: 0

π-π Interactions Drive the Homotypic Phase Separation of the Prion-like Diatom Pyrenoid Scaffold PYCO1 π-π相互作用驱动朊病毒样硅藻火绒素支架PYCO1的同型相分离。

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2024-09-24 DOI: 10.1016/j.jmb.2024.168800

CO₂ fixation in most unicellular algae relies on the pyrenoid, a biomolecular condensate, which sequesters the cell’s carboxylase Rubisco. In the marine diatom Phaeodactylum tricornutum, the pyrenoid tandem repeat protein Pyrenoid Component 1 (PYCO1) multivalently binds Rubisco to form a heterotypic Rubisco condensate. PYCO1 contains prion-like domains and can phase-separate homotypically in a salt-dependent manner. Here we dissect PYCO1 homotypic liquid–liquid phase separation (LLPS) by evaluating protein fragments and the effect of site-directed mutagenesis. Two of PYCO1′s six repeats are required for homotypic LLPS. Mutagenesis of a minimal phase-separating fragment reveals tremendous sensitivity to the substitution of aromatic residues. Removing positively charged lysines and arginines instead enhances the propensity of the fragment to condense. We conclude that PYCO1 homotypic LLPS is mostly driven by π-π interactions mediated by tyrosine and tryptophan stickers. In contrast π-cation interactions involving arginine or lysine are not significant drivers of LLPS in this system.

大多数单细胞藻类的二氧化碳固定都依赖于类肾上腺素（一种生物分子凝结物），它能封存细胞的羧化酶 Rubisco。在海洋硅藻 Phaeodactylum tricornutum 中，类肾上腺素串联重复蛋白类肾上腺素成分 1（PYCO1）可多价结合 Rubisco，形成异型 Rubisco 凝聚物。PYCO1含有朊病毒样结构域，能以盐依赖的方式进行同型相分离。在这里，我们通过评估蛋白质片段和定点突变的效果来剖析PYCO1同型液-液相分离（LLPS）。同型液-液相分离需要PYCO1六个重复序列中的两个。对最小相分离片段的突变显示了对芳香族残基替代的极大敏感性。去除带正电荷的赖氨酸和精氨酸反而会增强该片段的凝聚倾向。我们的结论是，PYCO1 同型 LLPS 主要是由酪氨酸和色氨酸粘合剂介导的 π-π 相互作用驱动的。相比之下，涉及精氨酸或赖氨酸的π-阳离子相互作用在该系统中并不是 LLPS 的重要驱动力。

引用次数: 0

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