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1.
Trends Biochem Sci ; 49(8): 654-657, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38777701

RESUMO

Designers' work processes are shaped by a four-phase 'discover, define, develop, and deliver' model that alternates between divergent and convergent thinking. We suggest consideration of this conceptual scaffold in 'design sprint' workshops for graduate students in the life sciences and in design to promote creativity, interdisciplinary collaboration, and knowledge cocreation.


Assuntos
Disciplinas das Ciências Biológicas , Criatividade , Humanos
2.
J Proteome Res ; 22(8): 2743-2749, 2023 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-37417926

RESUMO

Data-independent acquisition (DIA) mass spectrometry methods provide systematic and comprehensive quantification of the proteome; yet, relatively few open-source tools are available to analyze DIA proteomics experiments. Fewer still are tools that can leverage gas phase fractionated (GPF) chromatogram libraries to enhance the detection and quantification of peptides in these experiments. Here, we present nf-encyclopedia, an open-source NextFlow pipeline that connects three open-source tools, MSConvert, EncyclopeDIA, and MSstats, to analyze DIA proteomics experiments with or without chromatogram libraries. We demonstrate that nf-encyclopedia is reproducible when run on either a cloud platform or a local workstation and provides robust peptide and protein quantification. Additionally, we found that MSstats enhances protein-level quantitative performance over EncyclopeDIA alone. Finally, we benchmarked the ability of nf-encyclopedia to scale to large experiments in the cloud by leveraging the parallelization of compute resources. The nf-encyclopedia pipeline is available under a permissive Apache 2.0 license; run it on your desktop, cluster, or in the cloud: https://github.com/TalusBio/nf-encyclopedia.


Assuntos
Proteômica , Software , Proteômica/métodos , Fluxo de Trabalho , Peptídeos/análise , Proteoma/análise
3.
Anal Chem ; 95(12): 5187-5195, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36916610

RESUMO

Post-transcriptional modifications of RNA strongly influence the RNA structure and function. Recent advances in RNA sequencing and mass spectrometry (MS) methods have identified over 140 of these modifications on a wide variety of RNA species. Most next-generation sequencing approaches can only map one RNA modification at a time, and while MS can assign multiple modifications simultaneously in an unbiased manner, MS cannot accurately catalog and assign RNA modifications in complex biological samples due to limitations in the fragment length and coverage depth. Thus, a facile method to identify novel RNA modifications while simultaneously locating them in the context of their RNA sequences is still lacking. We combined two orthogonal modes of RNA ion separation before MS identification: high-field asymmetric ion mobility separation (FAIMS) and electrochemically modulated liquid chromatography (EMLC). FAIMS RNA MS increases both coverage and throughput, while EMLC LC-MS orthogonally separates RNA molecules of different lengths and charges. The combination of the two methods offers a broadly applicable platform to improve the length and depth of MS-based RNA sequencing while providing contextual access to the analysis of RNA modifications.


Assuntos
Espectrometria de Mobilidade Iônica , RNA , Sequência de Bases , Espectrometria de Massas/métodos , Cromatografia Líquida , Espectrometria de Mobilidade Iônica/métodos
4.
Mol Cell Proteomics ; 19(7): 1088-1103, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32312845

RESUMO

Data independent acquisition (DIA) is an attractive alternative to standard shotgun proteomics methods for quantitative experiments. However, most DIA methods require collecting exhaustive, sample-specific spectrum libraries with data dependent acquisition (DDA) to detect and quantify peptides. In addition to working with non-human samples, studies of splice junctions, sequence variants, or simply working with small sample yields can make developing DDA-based spectrum libraries impractical. Here we illustrate how to acquire, queue, and validate DIA data without spectrum libraries, and provide a workflow to efficiently generate DIA-only chromatogram libraries using gas-phase fractionation (GPF). We present best-practice methods for collecting DIA data using Orbitrap-based instruments and develop an understanding for why DIA using an Orbitrap mass spectrometer should be approached differently than when using time-of-flight instruments. Finally, we discuss several methods for analyzing DIA data without libraries.


Assuntos
Cromatografia Gasosa/métodos , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Biblioteca de Peptídeos , Peptídeos/química , Proteômica/métodos , Bases de Dados de Proteínas , Células HeLa , Humanos , Proteoma/análise , Software
5.
J Proteome Res ; 20(4): 1918-1927, 2021 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-33764077

RESUMO

Stable isotope labeling by amino acids in cell culture (SILAC) coupled to data-dependent acquisition (DDA) is a common approach to quantitative proteomics with the desirable benefit of reducing batch effects during sample processing and data acquisition. More recently, using data-independent acquisition (DIA/SWATH) to systematically measure peptides has gained popularity for its comprehensiveness, reproducibility, and accuracy of quantification. The complementary advantages of these two techniques logically suggests combining them. Here we develop a SILAC-DIA-MS workflow using free, open-source software. We empirically determine that using DIA achieves similar peptide detection numbers as DDA and that DIA improves the quantitative accuracy and precision of SILAC by an order of magnitude. Finally, we apply SILAC-DIA-MS to determine protein turnover rates of cells treated with bortezomib, an FDA-approved 26S proteasome inhibitor for multiple myeloma and mantle cell lymphoma. We observe that SILAC-DIA produces more sensitive protein turnover models. Of the proteins determined to be differentially degraded by both acquisition methods, we find known proteins that are degraded by the ubiquitin-proteasome pathway, such as HNRNPK, EIF3A, and IF4A1/EIF4A-1, and a slower turnover for CATD, a protein implicated in invasive breast cancer. With improved quantification from DIA, we anticipate that this workflow will make SILAC-based experiments like protein turnover more sensitive.


Assuntos
Proteoma , Espectrometria de Massas em Tandem , Bortezomib/farmacologia , Proteólise , Reprodutibilidade dos Testes
6.
Expert Rev Proteomics ; 18(9): 757-765, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34496693

RESUMO

BACKGROUND: Proteins are highly dynamic and their biological function is controlled by not only temporal abundance changes but also via regulated protein-protein interaction networks, which respond to internal and external perturbations. A wealth of novel analytical reagents and workflows allow studying spatiotemporal protein environments with great granularity while maintaining high throughput and ease of analysis. AREAS COVERED: We review technology advances for measuring protein-protein proximity interactions with an emphasis on proximity labeling, and briefly summarize other spatiotemporal approaches including protein localization, and their dynamic changes over time, specifically in human cells and mammalian tissues. We focus especially on novel technologies and workflows emerging within the past 5 years. This includes enrichment-based techniques (proximity labeling and crosslinking), separation-based techniques (organelle fractionation and size exclusion chromatography), and finally sorting-based techniques (laser capture microdissection and mass spectrometry imaging). EXPERT OPINION: Spatiotemporal proteomics is a key step in assessing biological complexity, understanding refined regulatory mechanisms, and forming protein complexes and networks. Studying protein dynamics across space and time holds promise for gaining deep insights into how protein networks may be perturbed during disease and aging processes, and offer potential avenues for therapeutic interventions, drug discovery, and biomarker development.


Assuntos
Proteínas , Proteômica , Humanos , Espectrometria de Massas , Organelas , Mapas de Interação de Proteínas
7.
Mass Spectrom Rev ; 39(3): 229-244, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-28691345

RESUMO

Skyline is a freely available, open-source Windows client application for accelerating targeted proteomics experimentation, with an emphasis on the proteomics and mass spectrometry community as users and as contributors. This review covers the informatics encompassed by the Skyline ecosystem, from computationally assisted targeted mass spectrometry method development, to raw acquisition file data processing, and quantitative analysis and results sharing.


Assuntos
Espectrometria de Massas/métodos , Proteínas/química , Proteômica/métodos , Animais , Humanos , Software
8.
J Proteome Res ; 19(3): 1147-1153, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32037841

RESUMO

Mass spectrometry is a powerful tool for quantifying protein abundance in complex samples. Advances in sample preparation and the development of data-independent acquisition (DIA) mass spectrometry approaches have increased the number of peptides and proteins measured per sample. Here, we present a series of experiments demonstrating how to assess whether a peptide measurement is quantitative by mass spectrometry. Our results demonstrate that increasing the number of detected peptides in a proteomics experiment does not necessarily result in increased numbers of peptides that can be measured quantitatively.


Assuntos
Peptídeos , Proteômica , Calibragem , Espectrometria de Massas , Proteínas
9.
Biochem Soc Trans ; 48(5): 1953-1966, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33079175

RESUMO

Research into the basic biology of human health and disease, as well as translational human research and clinical applications, all benefit from the growing accessibility and versatility of mass spectrometry (MS)-based proteomics. Although once limited in throughput and sensitivity, proteomic studies have quickly grown in scope and scale over the last decade due to significant advances in instrumentation, computational approaches, and bio-sample preparation. Here, we review these latest developments in MS and highlight how these techniques are used to study the mechanisms, diagnosis, and treatment of human diseases. We first describe recent groundbreaking technological advancements for MS-based proteomics, including novel data acquisition techniques and protein quantification approaches. Next, we describe innovations that enable the unprecedented depth of coverage in protein signaling and spatiotemporal protein distributions, including studies of post-translational modifications, protein turnover, and single-cell proteomics. Finally, we explore new workflows to investigate protein complexes and structures, and we present new approaches for protein-protein interaction studies and intact protein or top-down MS. While these approaches are only recently incipient, we anticipate that their use in biomedical MS proteomics research will offer actionable discoveries for the improvement of human health.


Assuntos
Espectrometria de Massas/métodos , Proteômica/métodos , Animais , Cromatografia , Humanos , Inflamação , Isótopos , Camundongos , Medicina de Precisão , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteoma/metabolismo , Transdução de Sinais , Pesquisa Translacional Biomédica , Fluxo de Trabalho
10.
Mol Cell Proteomics ; 17(5): 913-924, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29438992

RESUMO

The need for assay characterization is ubiquitous in quantitative mass spectrometry-based proteomics. Among many assay characteristics, the limit of blank (LOB) and limit of detection (LOD) are two particularly useful figures of merit. LOB and LOD are determined by repeatedly quantifying the observed intensities of peptides in samples with known peptide concentrations and deriving an intensity versus concentration response curve. Most commonly, a weighted linear or logistic curve is fit to the intensity-concentration response, and LOB and LOD are estimated from the fit. Here we argue that these methods inaccurately characterize assays where observed intensities level off at low concentrations, which is a common situation in multiplexed systems. This manuscript illustrates the deficiencies of these methods, and proposes an alternative approach based on nonlinear regression that overcomes these inaccuracies. We evaluated the performance of the proposed method using computer simulations and using eleven experimental data sets acquired in Data-Independent Acquisition (DIA), Parallel Reaction Monitoring (PRM), and Selected Reaction Monitoring (SRM) mode. When the intensity levels off at low concentrations, the nonlinear model changes the estimates of LOB/LOD upwards, in some data sets by 20-40%. In absence of a low concentration intensity leveling off, the estimates of LOB/LOD obtained with nonlinear statistical modeling were identical to those of weighted linear regression. We implemented the nonlinear regression approach in the open-source R-based software MSstats, and advocate its general use for characterization of mass spectrometry-based assays.


Assuntos
Espectrometria de Massas/métodos , Dinâmica não Linear , Sequência de Aminoácidos , Bioensaio , Calibragem , Humanos , Limite de Detecção , Modelos Teóricos , Peptídeos/química , Análise de Regressão
11.
J Proteome Res ; 18(11): 3936-3943, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31556620

RESUMO

For the 2018 YPIC Challenge, contestants were invited to try to decipher two unknown English questions encoded by a synthetic protein expressed in Escherichia coli. In addition to deciphering the sentence, contestants were asked to determine the three-dimensional structure and detect any post-translation modifications left by the host organism. We present our experimental and computational strategy to characterize this sample by identifying the unknown protein sequence and detecting the presence of post-translational modifications. The sample was acquired with dynamic exclusion disabled to increase the signal-to-noise ratio of the measured molecules, after which spectral clustering was used to generate high-quality consensus spectra. De novo spectrum identification was used to determine the synthetic protein sequence, and any post-translational modifications introduced by E. coli on the synthetic protein were analyzed via spectral networking. This workflow resulted in a de novo sequence coverage of 70%, on par with sequence database searching performance. Additionally, the spectral networking analysis indicated that no systematic modifications were introduced on the synthetic protein by E. coli. The strategy presented here can be directly used to analyze samples for which no protein sequence information is available or when the identity of the sample is unknown. All software and code to perform the bioinformatics analysis is available as open source, and self-contained Jupyter notebooks are provided to fully recreate the analysis.


Assuntos
Escherichia coli/metabolismo , Peptídeos/análise , Processamento de Proteína Pós-Traducional , Proteínas/análise , Proteômica/métodos , Sequência de Aminoácidos , Biologia Computacional/métodos , Escherichia coli/genética , Peptídeos/metabolismo , Biossíntese de Proteínas/genética , Proteínas/genética , Proteínas/metabolismo , Análise de Sequência de Proteína/métodos , Software , Espectrometria de Massas em Tandem/métodos
12.
Anal Chem ; 90(21): 13112-13117, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30350613

RESUMO

Mass spectrometry (MS) measurements are not inherently calibrated. Researchers use various calibration methods to assign meaning to arbitrary signal intensities and improve precision. Internal calibration (IC) methods use internal standards (IS) such as synthesized or recombinant proteins or peptides to calibrate MS measurements by comparing endogenous analyte signal to the signal from known IS concentrations spiked into the same sample. However, recent work suggests that using IS as IC introduces quantitative biases that affect comparison across studies because of the inability of IS to capture all sources of variation present throughout an MS workflow. Here, we describe a single-point external calibration strategy to calibrate signal intensity measurements to a common reference material, placing MS measurements on the same scale and harmonizing signal intensities between instruments, acquisition methods, and sites. We demonstrate data harmonization between laboratories and methodologies using this generalizable approach.


Assuntos
Espectrometria de Massas/normas , Proteoma/normas , Proteômica/normas , Calibragem , Padrões de Referência , Saccharomyces cerevisiae/química
13.
Mol Cell Proteomics ; 15(5): 1622-41, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26912667

RESUMO

Profiling post-translational modifications represents an alternative dimension to gene expression data in characterizing cellular processes. Many cellular responses to drugs are mediated by changes in cellular phosphosignaling. We sought to develop a common platform on which phosphosignaling responses could be profiled across thousands of samples, and created a targeted MS assay that profiles a reduced-representation set of phosphopeptides that we show to be strong indicators of responses to chemical perturbagens.To develop the assay, we investigated the coordinate regulation of phosphosites in samples derived from three cell lines treated with 26 different bioactive small molecules. Phosphopeptide analytes were selected from these discovery studies by clustering and picking 1 to 2 proxy members from each cluster. A quantitative, targeted parallel reaction monitoring assay was developed to directly measure 96 reduced-representation probes. Sample processing for proteolytic digestion, protein quantification, peptide desalting, and phosphopeptide enrichment have been fully automated, making possible the simultaneous processing of 96 samples in only 3 days, with a plate phosphopeptide enrichment variance of 12%. This highly reproducible process allowed ∼95% of the reduced-representation phosphopeptide probes to be detected in ∼200 samples.The performance of the assay was evaluated by measuring the probes in new samples generated under treatment conditions from discovery experiments, recapitulating the observations of deeper experiments using a fraction of the analytical effort. We measured these probes in new experiments varying the treatments, cell types, and timepoints to demonstrate generalizability. We demonstrated that the assay is sensitive to disruptions in common signaling pathways (e.g. MAPK, PI3K/mTOR, and CDK). The high-throughput, reduced-representation phosphoproteomics assay provides a platform for the comparison of perturbations across a range of biological conditions, suitable for profiling thousands of samples. We believe the assay will prove highly useful for classification of known and novel drug and genetic mechanisms through comparison of phosphoproteomic signatures.


Assuntos
Células-Tronco Embrionárias/metabolismo , Fosfoproteínas/análise , Proteômica/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Ensaios de Triagem em Larga Escala , Humanos , Células MCF-7 , Camundongos , Fosfoproteínas/efeitos dos fármacos , Transdução de Sinais
14.
Artigo em Inglês | MEDLINE | ID: mdl-39356573

RESUMO

Covalent labeling methods coupled to mass spectrometry have emerged in recent years for studying the higher order structure of proteins. Quantifying the extent of modification of proteins in multiple states (i.e., ligand free vs ligand-bound) can provide information on protein interaction sites and regions of conformational change. Though there are several software platforms that are used to quantify the extent of modification, the process can still be time-consuming, particularly for proteome-wide studies. Here, we present an open-source software for quantitation called Covalent labeling Automated Data Analysis Platform for high Throughput in R (coADAPTr). coADAPTr tackles the need for more efficient data analysis in covalent labeling mass spectrometry for techniques such as hydroxyl radical protein footprinting (HRPF). Traditional methods like Excel's Power Pivot (PP) are cumbersome and time-intensive, posing challenges for large-scale analyses. coADAPTr simplifies analysis by mimicking the functions used in the previous quantitation platform using PowerPivot in Microsoft Excel but with fewer steps, offering proteome-wide insights with enhanced graphical interpretations. Several features have been added to improve the fidelity and throughput compared to those of PowerPivot. These include filters to remove any duplicate data and the use of the arithmetic mean rather than the geometric mean for quantitation of the extent of modification. Validation studies confirm coADAPTr's accuracy and efficiency while processing data up to 200 times faster than conventional methods. Its open-source design and user-friendly interface make it accessible for researchers exploring intricate biological phenomena via HRPF and other covalent labeling MS methods. coADAPTr marks a significant leap in structural proteomics, providing a versatile and efficient platform for data interpretation. Its potential to transform the field lies in its seamless handling of proteome-wide data analyses, empowering researchers with a robust tool for deciphering complex structural biology data.

15.
Cell Metab ; 35(1): 166-183.e11, 2023 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-36599300

RESUMO

Microproteins (MPs) are a potentially rich source of uncharacterized metabolic regulators. Here, we use ribosome profiling (Ribo-seq) to curate 3,877 unannotated MP-encoding small ORFs (smORFs) in primary brown, white, and beige mouse adipocytes. Of these, we validated 85 MPs by proteomics, including 33 circulating MPs in mouse plasma. Analyses of MP-encoding mRNAs under different physiological conditions (high-fat diet) revealed that numerous MPs are regulated in adipose tissue in vivo and are co-expressed with established metabolic genes. Furthermore, Ribo-seq provided evidence for the translation of Gm8773, which encodes a secreted MP that is homologous to human and chicken FAM237B. Gm8773 is highly expressed in the arcuate nucleus of the hypothalamus, and intracerebroventricular administration of recombinant mFAM237B showed orexigenic activity in obese mice. Together, these data highlight the value of this adipocyte MP database in identifying MPs with roles in fundamental metabolic and physiological processes such as feeding.


Assuntos
Adipócitos Brancos , Tecido Adiposo Marrom , Humanos , Animais , Camundongos , Adipócitos Brancos/metabolismo , Tecido Adiposo Marrom/metabolismo , Fases de Leitura Aberta/genética , Tecido Adiposo Branco/metabolismo , Adipócitos Marrons/metabolismo , Micropeptídeos
16.
Mol Omics ; 18(10): 894-895, 2022 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-36168986

RESUMO

In this themed issue of Molecular Omics, in partnership with the U.S. Human Proteome Organization, we are proud to present the latest research featured at the 17th Annual US HUPO conference: Proteomics from Single Cell to Systems Biology in Health and Disease. This issue is a testament to the continuing contributions of proteomic research, particularly the application of modern mass spectrometry-based proteomic workflows, to the advancement of our understanding of the underlying human biology and mechanisms of disease.


Assuntos
Proteômica , Biologia de Sistemas , Humanos , Proteoma , Espectrometria de Massas
17.
Genome Biol ; 22(1): 134, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947439

RESUMO

BACKGROUND: The evolution of multicellularity is a critical event that remains incompletely understood. We use the social amoeba, Dictyostelium discoideum, one of the rare organisms that readily transits back and forth between both unicellular and multicellular stages, to examine the role of epigenetics in regulating multicellularity. RESULTS: While transitioning to multicellular states, patterns of H3K4 methylation and H3K27 acetylation significantly change. By combining transcriptomics, epigenomics, chromatin accessibility, and orthologous gene analyses with other unicellular and multicellular organisms, we identify 52 conserved genes, which are specifically accessible and expressed during multicellular states. We validated that four of these genes, including the H3K27 deacetylase hdaD, are necessary and that an SMC-like gene, smcl1, is sufficient for multicellularity in Dictyostelium. CONCLUSIONS: These results highlight the importance of epigenetics in reorganizing chromatin architecture to facilitate multicellularity in Dictyostelium discoideum and raise exciting possibilities about the role of epigenetics in the evolution of multicellularity more broadly.


Assuntos
Dictyostelium/citologia , Dictyostelium/genética , Epigênese Genética , Acetilação , Animais , Caenorhabditis elegans/citologia , Cromatina/metabolismo , Perfilação da Expressão Gênica , Histonas/metabolismo , Metilação , Schizosaccharomyces/citologia , Fatores de Transcrição/metabolismo
18.
Cell Rep ; 30(8): 2463-2471.e5, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32101728

RESUMO

Transcription factors and other chromatin-associated proteins are difficult to quantify comprehensively. Here, we combine facile nuclear sub-fractionation with data-independent acquisition mass spectrometry to achieve rapid, sensitive, and highly parallel quantification of the nuclear proteome in human cells. We apply this approach to quantify the response to acute degradation of BET bromodomains, revealing unexpected chromatin regulatory dynamics. The method is simple and enables system-level study of previously inaccessible chromatin and genome regulators.


Assuntos
Compartimento Celular , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Cromatina/metabolismo , Humanos , Células K562 , Cinética , Proteólise
19.
Elife ; 92020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32648542

RESUMO

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.


Assuntos
Envelhecimento/efeitos dos fármacos , Cardiopatias/tratamento farmacológico , Mitocôndrias/fisiologia , Oligopeptídeos/administração & dosagem , Estresse Oxidativo , Animais , Metabolismo Energético , Feminino , Cardiopatias/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução
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