Methods in molecular biology最新文献

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Efficient and Sensitive Sample Preparation, Separations, and Data Acquisition for Label-Free Single-Cell Proteomics. 用于无标记单细胞蛋白质组学的高效、灵敏的样品制备、分离和数据采集。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3934-4_7
Thy Truong, Ximena Sanchez-Avila, Kei G I Webber, S Madisyn Johnston, Ryan T Kelly
{"title":"Efficient and Sensitive Sample Preparation, Separations, and Data Acquisition for Label-Free Single-Cell Proteomics.","authors":"Thy Truong, Ximena Sanchez-Avila, Kei G I Webber, S Madisyn Johnston, Ryan T Kelly","doi":"10.1007/978-1-0716-3934-4_7","DOIUrl":"10.1007/978-1-0716-3934-4_7","url":null,"abstract":"<p><p>We describe a sensitive and efficient workflow for label-free single-cell proteomics that spans sample preparation, liquid chromatography separations, and mass spectrometry data acquisition. The Tecan Uno Single Cell Dispenser provides rapid cell isolation and nanoliter-volume reagent dispensing within 384-well PCR plates. A newly developed sample processing workflow achieves cell lysis, protein denaturation, and digestion in 1 h with a single reagent dispensing step. Low-flow liquid chromatography coupled with wide-window data-dependent acquisition results in the quantification of nearly 3000 proteins per cell using an Orbitrap Exploris 480 mass spectrometer. This approach greatly broadens accessibility to sensitive single-cell proteome profiling for nonspecialist laboratories.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2817 ","pages":"67-84"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141437070","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Profiling Mouse Brain Single-Cell-Type Proteomes Via Adeno-Associated Virus-Mediated Proximity Labeling and Mass Spectrometry. 通过腺相关病毒介导的近距离标记和质谱法分析小鼠大脑单细胞型蛋白质组
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3934-4_10
Him K Shrestha, Huan Sun, Ju Wang, Junmin Peng
{"title":"Profiling Mouse Brain Single-Cell-Type Proteomes Via Adeno-Associated Virus-Mediated Proximity Labeling and Mass Spectrometry.","authors":"Him K Shrestha, Huan Sun, Ju Wang, Junmin Peng","doi":"10.1007/978-1-0716-3934-4_10","DOIUrl":"10.1007/978-1-0716-3934-4_10","url":null,"abstract":"<p><p>Single-cell-type proteomics is an emerging field of research that combines cell-type specificity with the comprehensive proteome coverage offered by bulk proteomics. However, the extraction of single-cell-type proteomes remains a challenge, particularly for hard-to-isolate cells like neurons. In this chapter, we present an innovative technique for profiling single-cell-type proteomes using adeno-associated virus (AAV)-mediated proximity labeling (PL) and tandem-mass-tag (TMT) mass spectrometry. This technique eliminates the need for cell isolation and offers a streamlined workflow, including AAV delivery to express TurboID (an engineered biotin ligase) controlled by cell-type-specific promoters, biotinylated protein purification, on-bead digestion, TMT labeling, and liquid chromatography-mass spectrometry (LC-MS). We examined this method by analyzing distinct brain cell types in mice. Initially, recombinant AAVs were used to concurrently express TurboID and mCherry proteins driven by neuron- or astrocyte-specific promoters, which was validated through co-immunostaining with cellular markers. With biotin purification and TMT analysis, we successfully identified around 10,000 unique proteins from a few micrograms of protein samples with high reproducibility. Our statistical analyses revealed that these proteomes encompass cell-type-specific cellular pathways. By utilizing this technique, researchers can explore the proteomic landscape of specific cell types, paving the way for new insights into cellular processes, deciphering disease mechanisms, and identifying therapeutic targets in neuroscience and beyond.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2817 ","pages":"115-132"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141437150","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Imaging and Analysis of the Dynamics of Filamentous Actin Structures in Live Endothelial Cells. 活内皮细胞丝状肌动蛋白结构动力学的成像和分析。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3429-5_11
Jerome W Breslin, Zeinab Y Motawe
{"title":"Imaging and Analysis of the Dynamics of Filamentous Actin Structures in Live Endothelial Cells.","authors":"Jerome W Breslin, Zeinab Y Motawe","doi":"10.1007/978-1-0716-3429-5_11","DOIUrl":"10.1007/978-1-0716-3429-5_11","url":null,"abstract":"<p><p>The ability to view and record the movements of subcellular structures is a powerful tool that has accelerated the discovery and understanding of signaling mechanisms that control microvascular functions such as the control of endothelial permeability. Advances in molecular biology over the past few decades have facilitated the generation of fusion proteins in which fluorescent reporters based upon the structure of green fluorescent protein can be linked to proteins found in human endothelial cells, such as VE-cadherin or β-actin. These fusion proteins have been found to incorporate into structures alongside their native protein counterparts, allowing the dynamic visualization of how these subcellular structures are modified when cells are challenged with stimuli such as inflammatory mediators. The result of such studies has been a much more advanced view of the complex mechanisms by which endothelial cells maintain barrier properties than previously obtained by only viewing fixed cells labeled by immunofluorescence. Here, we describe our protocols that we have used to view the dynamics of actin filaments using time-lapse microscopy to record endothelial cells expressing GFP-actin and the analysis tools available to quantify dynamics of subcellular structures.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2711 ","pages":"129-146"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11369499/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41132881","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Bioorthogonal Labeling and Click-Chemistry-Based Visualization of the Tannerella forsythia Cell Wall. 基于生物正交标记和点击化学的连翘单宁细胞壁可视化研究。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3491-2_1
Stephen N Hyland, Sreedevi Chinthamani, Sushanta Ratna, Kimberly A Wodzanowski, Liam-Michael D Sandles, Kiyonobu Honma, Catherine Leimkuhler-Grimes, Ashu Sharma
{"title":"Bioorthogonal Labeling and Click-Chemistry-Based Visualization of the Tannerella forsythia Cell Wall.","authors":"Stephen N Hyland, Sreedevi Chinthamani, Sushanta Ratna, Kimberly A Wodzanowski, Liam-Michael D Sandles, Kiyonobu Honma, Catherine Leimkuhler-Grimes, Ashu Sharma","doi":"10.1007/978-1-0716-3491-2_1","DOIUrl":"10.1007/978-1-0716-3491-2_1","url":null,"abstract":"<p><p>The objective of this chapter is to provide a detailed protocol for the peptidoglycan (cell wall) labeling of the periodontal pathogen Tannerella forsythia and the development of a laboratory-safe Escherichia coli strain utilizing the N-acetylmuramic acid recycling enzymes AmgK, N-acetylmuramate/N-acetylglucosamine kinase, and MurU, N-acetylmuramate alpha-1-phosphate uridylyltransferase, from T. forsythia. The procedure involves bioorthogonal labeling of bacterial cells with an azido-modified analog of the amino sugar, N-acetylmuramic acid, through \"click chemistry\" with a fluorescent dye. The protocol is suitable for the generation of fluorescently labeled peptidoglycan molecules for applications in the study of bacterial and peptidoglycan trafficking in the host cells and cell wall recycling in complex microbiomes.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2727 ","pages":"1-16"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11513787/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41204777","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Organic Supplementation of Vaccinium corymbosum Micropropagation Media. 蔓越橘微繁殖培养基的有机添加剂。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3782-1_11
Marcelina Krupa-Małkiewicz, Ireneusz Ochmian, Monika Figiel-Kroczyńska
{"title":"Organic Supplementation of Vaccinium corymbosum Micropropagation Media.","authors":"Marcelina Krupa-Małkiewicz, Ireneusz Ochmian, Monika Figiel-Kroczyńska","doi":"10.1007/978-1-0716-3782-1_11","DOIUrl":"10.1007/978-1-0716-3782-1_11","url":null,"abstract":"<p><p>The best Vaccinium corymbosum plant growth under in vitro conditions can be achieved by using the right composition and pH of the medium. For the initial phase of in vitro culture, a combination of cytokinins-mostly zeatin-can usually be used. Organic supplementation of the medium enables the use of a replacement for the expensive natural cytokinin used in micropropagation of highbush blueberry. This chapter describes the experiments with silicon Hydroplus™ Actisil (Si), coconut water (CW), and different pH (5.0; 5.5, and 6.0) as a stress factor. The addition of 200 mg dm<sup>-3</sup> silicon solution and 15% coconut water strongly stimulated highbush blueberry plant growth in vitro. Moreover, silicon solution benefits the negative effects of higher pH of the medium used for micropropagation of V. corymbosum. Maximum vegetative development of blueberry explants was obtained at pH 5.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2788 ","pages":"197-207"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140857104","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Precision Genome Editing with CRISPR-Cas9. 利用 CRISPR-Cas9 进行精准基因组编辑。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3782-1_21
Shahroz Rahman, Abdul Rehman Ikram, Farrukh Azeem, Muhammad Tahir Ul Qamar, Tayyaba Shaheen, Mehboob-Ur-Rahman
{"title":"Precision Genome Editing with CRISPR-Cas9.","authors":"Shahroz Rahman, Abdul Rehman Ikram, Farrukh Azeem, Muhammad Tahir Ul Qamar, Tayyaba Shaheen, Mehboob-Ur-Rahman","doi":"10.1007/978-1-0716-3782-1_21","DOIUrl":"10.1007/978-1-0716-3782-1_21","url":null,"abstract":"<p><p>The CRISPR/Cas9 system is a revolutionary technology for genome editing that allows for precise and efficient modifications of DNA sequences. The system is composed of two main components, the Cas9 enzyme and a guide RNA (gRNA). The gRNA is designed to specifically target a desired DNA sequence, while the Cas9 enzyme acts as molecular scissors to cut the DNA at that specific location. The cell then repairs the digested DNA, either through nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in either indels or precise modifications of DNA sequences with broad implications in biotechnology, agriculture, and medicine. This chapter provides a practical approach for utilizing CRISPR/Cas9 in precise genome editing, including identifying the target gene sequence, designing gRNA and protein (Cas9), and delivering the CRISPR components to target cells.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2788 ","pages":"355-372"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140870653","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Multicellular Tumoroids for Investigating Cancer Stem-Like Cells in the Heterogeneous Tumor Microenvironment. 用于研究异质肿瘤微环境中癌症干样细胞的多细胞肿瘤实体
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3730-2_8
Kathleen M Burkhard, Geeta Mehta
{"title":"Multicellular Tumoroids for Investigating Cancer Stem-Like Cells in the Heterogeneous Tumor Microenvironment.","authors":"Kathleen M Burkhard, Geeta Mehta","doi":"10.1007/978-1-0716-3730-2_8","DOIUrl":"10.1007/978-1-0716-3730-2_8","url":null,"abstract":"<p><p>Cancer stem-like cells (CSC) are a major contributing factor to chemoresistance, tumor recurrence, and poor survival outcomes in patients across cancer types. Signaling from non-tumor cells in the tumor microenvironment (TME) enriches for and supports CSC. This complex cell-cell signaling in the heterogeneous TME presents a challenge for patient survival; however, it also presents an opportunity to develop new targeted therapies that can inhibit survival of CSC. In this chapter, we report a multicellular tumoroid model which can be used to investigate the interactions between cancer cells and non-tumor cells in the TME to better understand the contribution of various cell types to cancer cell phenotypes, as well as the underlying mechanisms involved. The following methods allow for each cell type to be distinguished using FACS and studied individually. Gene expression can be analyzed for cancer cells, as well as the other non-tumor cells using qPCR following sorting. The response to chemotherapeutic agents and expression of stem markers can be determined for cancer cells using flow cytometry, excluding the other cell types to get an accurate view of the cancer cells. Furthermore, the viability of non-tumor cells can be analyzed as well to determine if there are cytotoxic effects of the drugs on non-tumor cells. Thus, the multicellular tumoroid model will reveal the interactions between the CSC and non-tumor cells in the heterogenous TME, resulting in discoveries in the fields of cancer biology, novel targeted therapies, and personalized drug screening for precision medicine.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2777 ","pages":"99-122"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140119948","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Prostate Cancer Organoids for Tumor Modeling and Drug Screening. 用于肿瘤建模和药物筛选的前列腺癌组织块。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3730-2_10
Amani Yehya, Fatima Ghamlouche, Sana Hachem, Wassim Abou-Kheir
{"title":"Prostate Cancer Organoids for Tumor Modeling and Drug Screening.","authors":"Amani Yehya, Fatima Ghamlouche, Sana Hachem, Wassim Abou-Kheir","doi":"10.1007/978-1-0716-3730-2_10","DOIUrl":"10.1007/978-1-0716-3730-2_10","url":null,"abstract":"<p><p>Prostate cancer (PCa) is the second most common malignancy and the fifth leading cause of cancer death in men worldwide. Despite its prevalence, the highly heterogenic PCa has shown difficulty to establish representative cell lines that reflect the diverse phenotypes and different stages of the disease in vitro and hence hard to model in preclinical research. The patient-derived organoid (PDO) technique has emerged as a groundbreaking three-dimensional (3D) tumor modeling platform in cancer research. This versatile assay relies on the unique ability of cancer stem cells (CSCs) to self-organize and differentiate into organ-like mini structures. The PDO culture system allows for the long-term maintenance of cancer cells derived from patient tumor tissues. Moreover, it recapitulates the parental tumor features and serves as a superior preclinical model for in vitro tumor representation and personalized drug screening. Henceforth, PDOs hold great promise in precision medicine for cancer. Herein, we describe the detailed protocol to establish and propagate organoids derived from isolated cell suspensions of PCa patient tissues or cell lines using the 3D semisolid Matrigel™-based hanging-drop method. In addition, we highlight the relevance of PDOs as a tool for evaluating drug efficacy and predicting tumor response in PCa patients.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2777 ","pages":"135-144"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140119949","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Surface Markers for the Identification of Cancer Stem Cells. 用于识别癌症干细胞的表面标记。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3730-2_4
Tasfik Ul Haque Pronoy, Farhadul Islam, Vinod Gopalan, Alfred King-Yin Lam
{"title":"Surface Markers for the Identification of Cancer Stem Cells.","authors":"Tasfik Ul Haque Pronoy, Farhadul Islam, Vinod Gopalan, Alfred King-Yin Lam","doi":"10.1007/978-1-0716-3730-2_4","DOIUrl":"10.1007/978-1-0716-3730-2_4","url":null,"abstract":"<p><p>Cancer stem cells have genetic and functional characteristics which can turn them resistant to standard cancer therapeutic targets. Identification of these cells is challenging and is done mainly by detecting the expression of antigens specific to stem cells. Currently, there is a significant number of surface markers available which can detect cancer stem cells by directly targeting the specific antigens present in cells. These markers possess differential expression patterns and sub-localizations in cancer stem cells compared to nonneoplastic and somatic cells. In addition to these biomarkers, multiple analytical methods and techniques, including functional assays, cell sorting, filtration approaches, and xenotransplantation methods, are used to identify cancer stem cells. This chapter will overview the functional significance of cancer stem cells, their biological correlations, specific markers, and detection methods.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2777 ","pages":"51-69"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140119950","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Conformational Heterogeneity of β-Barrel Membrane Proteins Observed In Situ Using Orthogonal Spin Labels and Pulsed ESR Spectroscopy. 利用正交自旋标签和脉冲 ESR 光谱原位观测 β 管膜蛋白的构象异质性
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3734-0_15
Sophie Ketter, Aathira Gopinath, Benesh Joseph
{"title":"Conformational Heterogeneity of β-Barrel Membrane Proteins Observed In Situ Using Orthogonal Spin Labels and Pulsed ESR Spectroscopy.","authors":"Sophie Ketter, Aathira Gopinath, Benesh Joseph","doi":"10.1007/978-1-0716-3734-0_15","DOIUrl":"10.1007/978-1-0716-3734-0_15","url":null,"abstract":"<p><p>Outer membrane proteins (OMPs) of Gram-negative bacteria are involved in many essential functions of the cell. They are tightly packed in the outer membrane, which is an asymmetric lipid bilayer. Electron spin resonance (ESR) spectroscopic techniques combined with site-directed spin labeling (SDSL) enable observation of structure and conformational dynamics of these proteins directly in their native environments. Here we depict a protocol for site-directed spin labeling of β-barrel membrane proteins in isolated outer membranes and intact E. coli using nitroxide, triarylmethyl (trityl), and Gd<sup>3+</sup>-based spin tags. Furthermore, subsequent continuous wave (CW) and orthogonal pulsed electron-electron double resonance (PELDOR) measurements are described along with experimental setup at Q-band (34 GHz), the data analysis, and interpretation.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2778 ","pages":"237-257"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140119955","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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