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Weill Cornell Medical College

31 ARTICLES PUBLISHED IN JoVE

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Biology

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties
Helgi Ingolfson 1, Ruchi Kapoor 2, Shemille A. Collingwood 2, Olaf Sparre Andersen 2
1Department of Physiology and Biophysics, Weill Cornell Medical College, 2Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

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Biology

Preparation of Artificial Bilayers for Electrophysiology Experiments
Ruchi Kapoor 1, Jung H. Kim 1, Helgi Ingolfson 1, Olaf Sparre Andersen 1
1Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

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Biology

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties
Helgi I. Ingólfsson 1, R. Lea Sanford 1, Ruchi Kapoor 1, Olaf S. Andersen 1
1Department of Physiology and Biophysics, Weill Cornell Medical College

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

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Neuroscience

Neural Tube Closure in Mouse Whole Embryo Culture
Jason Gray 1, M. Elizabeth Ross 1
1Department of Neurology/Neuroscience, Weill Cornell Medical College

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

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Bioengineering

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery
Jia Fan 1,2, James W. Gallagher 1, Hung-Jen Wu 1, Matthew G. Landry 1, Jason Sakamoto 1, Mauro Ferrari 1, Ye Hu 1
1Department of Nanomedicine, The Methodist Hospital Research Institute, 2CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

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Chemistry

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications
Carlos Tassa 1, Monty Liong 1, Scott Hilderbrand 1, Jason E. Sandler 1, Thomas Reiner 1, Edmund J. Keliher 1, Ralph Weissleder 1, Stanley Y. Shaw 1
1Center for Systems Biology, Massachusetts General Hospital

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

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Medicine

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells
Gunjan Gakhar 1, Neil H. Bander 2, David M. Nanus 1
1Department of Medicine, Weill Cornell Medical College, 2Department of Urology, Weill Cornell Medical College

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

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Neuroscience

Subtype-selective Electroporation of Cortical Interneurons
Natalia V. De Marco Garcia 1,2, Gord Fishell 1
1NYU Neuroscience Institute, New York University School of Medicine, 2Brain and Mind Research Institute, Weill Cornell Medical College

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

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Bioengineering

Porous Silicon Microparticles for Delivery of siRNA Therapeutics
Jianliang Shen *1,2, Xiaoyan Wu *1,3, Yeonju Lee 1, Joy Wolfram 1,4, Zhizhou Yang 1, Zong-Wan Mao 2, Mauro Ferrari 1,5, Haifa Shen 1,6
1Department of Nanomedicine, Houston Methodist Research Institute, 2MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-sen University, 3Pediatrics Department of Union Hospital, Huazhong University of Science and Technology, 4CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience & Technology of China, 5Department of Medicine, Weill Cornell Medical College, 6Department of Cell and Developmental Biology, Weill Cornell Medical College

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

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Medicine

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics
Adriana D. Corben *1, Mohammad M. Uddin *2, Brooke Crawford 3, Mohammad Farooq 4, Shanu Modi 5, John Gerecitano 5, Gabriela Chiosis 2, Mary L. Alpaugh 6
1Department of Pathology, Memorial Sloan Kettering Cancer Center, 2Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, 3Department of Radiology, Weill Cornell Medical College, 4Department of Medicine, Memorial Sloan Kettering Cancer Center, 5Department of Oncology, Memorial Sloan Kettering Cancer Center, 6Department of Surgery, Memorial Sloan Kettering Cancer Center

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

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Biology

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
Francine E. Garrett-Bakelman *1, Caroline K. Sheridan *1, Thadeous J. Kacmarczyk 1, Jennifer Ishii 1, Doron Betel 1,2, Alicia Alonso 1, Christopher E. Mason 3, Maria E. Figueroa 4, Ari M. Melnick 1
1Department of Medicine, Weill Cornell Medical College, 2Institute for Computational Biomedicine, Weill Cornell Medical College, 3Department of Physiology and Biophysics, Weill Cornell Medical College, 4Department of Pathology, University of Michigan

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

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Bioengineering

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging
Thomas Reiner *1, Jason S. Lewis 1, Brian M. Zeglis *1
1Department of Radiology, Memorial Sloan Kettering Cancer Center

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

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Biology

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters
Daniel Basilio 1, Alessio Accardi 1,2,3
1Department of Anesthesiology, Weill Cornell Medical College, 2Department of Physiology and Biophysics, Weill Cornell Medical College, 3Department of Biochemistry, Weill Cornell Medical College

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

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JoVE Core

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies
Brian M. Zeglis 1, Jason S. Lewis 1
1Department of Radiology, Memorial Sloan Kettering Cancer Center

Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described.

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Bioengineering

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect
Daniel S. Oh 1, Alia Koch 1, Sidney Eisig 1, Sahng Gyoon Kim 2, Yoon Hyuk Kim 3, Do-Gyoon Kim 4, Jae Hyuck Shim 5
1Oral and Maxillofacial Surgery, Columbia University, 2Endodontics, Columbia University, 3Mechanical Engineering, Kyung Hee University, South Korea, 4Orthodontics, The Ohio State University, 5Pathology, Weill Cornell Medical College

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

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Medicine

VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma
Yanwen Jiang 1,2, Kui Nie 3, David Redmond 2, Ari M. Melnick 1, Wayne Tam 3, Olivier Elemento 2
1Department of Medicine, Weill Cornell Medical College, 2Institute for Computational Biomedicine, Weill Cornell Medical College, 3Department of Pathology and Laboratory Medicine, Weill Cornell Medical College

This protocol describes an approach to interrogate the recombined immunoglobulin heavy chain VDJ regions of lymphomas by deep-sequencing and retrieve VDJ rearrangement and somatic hypermutation status to delineate clonal architecture of individual tumor. Comparing clonal architecture between paired diagnosis and relapse samples reveals lymphoma relapse clonal evolution modes.

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Genetics

Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma
Jonathan B. Reichel 1, Jason McCormick 2, Jonathan R. Fromm 3, Olivier Elemento 4, Ethel Cesarman 5, Mikhail Roshal 6
1Innovation Laboratory, Center for Molecular Oncology, Memorial Sloan Kettering Cancer Center, 2Flow-Sorting Core Facility, Weill Cornell Medical College, 3Department of Laboratory Medicine, University of Washington, 4Department of Physiology and Biophysics, Institute for Computational Biomedicine, Weill Cornell Medical College, 5Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, 6Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center

Here, we describe a combined flow cytometric cell sorting and low-input, next-generation library construction protocol designed to produce high-quality, whole-exome data from the Hodgkin Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL).

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Biochemistry

A Fluorescence-based Assay of Phospholipid Scramblase Activity
Birgit Ploier 1, Anant K. Menon 1
1Department of Biochemistry, Weill Cornell Medical College

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

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Immunology and Infection

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria
Ben Gold 1, Julia Roberts *1, Yan Ling *1, Landys Lopez Quezada 1, Jou Glasheen 1, Elaine Ballinger 1, Selin Somersan-Karakaya 2, Thulasi Warrier 1, Carl Nathan 1
1Departments of Microbiology & Immunology, Weill Cornell Medical College, 2Medicine, Weill Cornell Medical College

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

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Bioengineering

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting
Gabriel E. Büchel 1,2, Brandon Carney 1,3, Jun Tang 1, Brian M. Zeglis 1,3, Jörg Eppinger 2, Thomas Reiner 1,4
1Department of Radiology, Memorial Sloan Kettering Cancer Center, 2KAUST Catalysis Center, King Abdullah University of Science and Technology, 3Department of Chemistry, Hunter College, and PhD Program in Chemistry, Graduate Center of City University of New York, 4Department of Radiology, Weill Cornell Medical College

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

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Cancer Research

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines
Jun Tang 1, Carlos Pérez-Medina 1,2, Yiming Zhao 2, Ahmad Sadique 1, Willem J. M. Mulder 2, Thomas Reiner 1
1Department of Radiology, Memorial Sloan Kettering Cancer Center, 2Translational and Molecular Imaging Institute, Icahn School of Medicine at Mount Sinai

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

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Biochemistry

A Tandem Liquid Chromatography–Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus
David J. Samuels 1, Zhe Wang 2, Kyu Y. Rhee 2,3, Shaun R. Brinsmade 1
1Department of Biology, Georgetown University, 2Division of Infectious Diseases, Weill Cornell Medical College, 3Department of Medicine, Weill Cornell Medical College

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

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Behavior

The Knob Supination Task: A Semi-automated Method for Assessing Forelimb Function in Rats
Samuel D. Butensky 1, Thelma Bethea 1, Joshua Santos 1, Anil Sindhurakar 1, Eric Meyers 2,3, Andrew M. Sloan 2,3, Robert L. Rennaker II 2,3, Jason B. Carmel 1,4,5
1Burke Medical Research Institute, 2Texas Biomedical Center, The University of Texas at Dallas, 3Erik Jonsson School of Engineering and Computer Science, The University of Texas at Dallas, 4Brain and Mind Research Institute, Weill Cornell Medical College, 5Departments of Neurology and Pediatrics, Weill Cornell Medical College

This manuscript describes a semi-automated task that quantifies supination in rats. Rats reach, grasp, and supinate a spherical manipulandum. The rat is rewarded with a pellet if the turn angle exceeds a criterion set by the user. This task increases throughput, sensitivity to injury, and objectivity compared to traditional tasks.

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Bioengineering

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery
Limor Man 1, Laura Park 1, Richard Bodine 1, Michael Ginsberg 2, Nikica Zaninovic 3, Glenn Schattman 1, Robert E. Schwartz 4, Zev Rosenwaks 1,3, Daylon James 1,3
1Center for Reproductive Medicine and Infertility, Weill Cornell Medical College, 2Angiocrine Biosciences, Inc., 3Tri-Institutional Stem Cell Derivation Laboratory, Weill Cornell Medical College, 4Joan and Sanford I. Weill Department of Medicine, Weill Cornell Medical College

For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules.

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Chemistry

Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction
Rosemery Membreno 1,2, Brendon E. Cook 1,2,3, Brian M. Zeglis 1,2,3,4
1Department of Chemistry, Hunter College of the City University of New York, 2Ph.D. Program in Chemistry, Graduate Center of the City University of New York, 3Department of Radiology, Memorial Sloan Kettering Cancer Center, 4Department of Radiology, Weill Cornell Medical College

This protocol describes the synthesis and characterization of a trans-cyclooctene (TCO)-modified antibody and a 177Lu-labeled tetrazine (Tz) radioligand for pretargeted radioimmunotherapy (PRIT). In addition, it details the use of these two constructs for in vivo biodistribution and longitudinal therapy studies in a murine model of colorectal cancer.

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Chemistry

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates
Maria Davydova 1, Guillaume Dewaele Le Roi 1,2, Pierre Adumeau 1, Brian M. Zeglis 1,2,3,4
1Department of Chemistry, Hunter College of the City University of New York, 2Ph.D. Program in Chemistry, Graduate Center of the City University of New York, 3Department of Radiology, Memorial Sloan Kettering Cancer Center, 4Department of Radiology, Weill Cornell Medical College

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

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Environment

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples
Zuha Anwar *1, Samantha B. Regan *1, Jennifer Linden 1
1Brain and Mind Institute, Weill Cornell Medical College

The objective of this protocol is to detect different Clostridium perfringens toxinotypes in locally purchased foods, particularly epsilon toxin producing strain types B and D, without the use of anaerobic chambers.

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Biochemistry

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation
Melanie Balbach 1, Jochen Buck 1, Lonny R. Levin 1
1Department of Pharmacology, Weill Cornell Medical College

We describe the application of an extracellular flux analyzer to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation.

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Neuroscience

A Psychophysics Paradigm for the Collection and Analysis of Similarity Judgments
Suniyya A. Waraich 1, Jonathan D. Victor 2
1Program in Neuroscience, Weill Cornell Graduate School of Medical Sciences, 2Feil Family Brain and Mind Research Institute, Weill Cornell Medical College

The protocol presents an experimental psychophysics paradigm to obtain large quantities of similarity judgments, and an accompanying analysis workflow. The paradigm probes context effects and enables modeling of similarity data in terms of Euclidean spaces of at least five dimensions.

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Biology

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles
Limor Man 1, Nicole Lustgarten Guahmich 1, Eleni Kallinos 1, Laury Arazi 1, Zev Rosenwaks 1,2, Daylon James 1,2
1Center for Reproductive Medicine and Infertility, Weill Cornell Medical College, 2Tri-Institutional Stem Cell Derivation Laboratory, Weill Cornell Medical College

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

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Cancer Research

Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids
Maud Charpentier *1, Norma Bloy *1, Silvia C. Formenti 1,2,3, Lorenzo Galluzzi 1,2,4, Sandra Demaria 1,5
1Department of Radiation Oncology, Weill Cornell Medical College, 2Sandra and Edward Meyer Cancer Center, 3Department of Medicine, Weill Cornell Medical College, 4Caryl and Israel Englander Institute for Precision Medicine, 5Department of Pathology and Laboratory Medicine, Weill Cornell Medical College

Here, we detail a straightforward live imaging approach for quantifying the sensitivity of patient-derived tumor organoids to ionizing radiation.

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