bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2022–03–27
fourteen papers selected by
Giovanny Rodriguez Blanco, University of Edinburgh
  1. Artifactual fatty acid dimers mimic FAHFA signals in untargeted metabolomics pipelines.
  2. Lipid Metabolism Interplay in CRC-An Update.
  3. A rapid and robust method for amino acid quantification using a simple N-hydroxysuccinimide ester derivatization and liquid chromatography-ion mobility-mass spectrometry.
  4. Clinical metabolomics for inborn errors of metabolism.
  5. Serum untargeted lipidomics by UHPLC-ESI-HRMS aids the biomarker discovery of colorectal adenoma.
  6. CD8+ T cells PUF(A)ing the flames of cancer ferroptotic cell death.
  7. Formation, Signaling and Occurrence of Specialized Pro-Resolving Lipid Mediators-What is the Evidence so far?
  8. High-Throughput UHPLC-MS to Screen Metabolites in Feces for Gut Metabolic Health.
  9. The central moTOR of metabolism.
  10. Ether Lipids in Obesity: From Cells to Population Studies.
  11. Data-Independent Acquisition Enables Robust Quantification of 400 Proteins in Non-Depleted Canine Plasma.
  12. Lipidomic Profiling Identifies Serum Lipids Associated with Persistent Multisite Musculoskeletal Pain.
  13. The glucose transporter GLUT3 controls T helper 17 cell responses through glycolytic-epigenetic reprogramming.
  14. Lipid Extraction and Analysis.
  1. J Lipid Res. 2022 Mar 18. pii: S0022-2275(22)00034-7. [Epub ahead of print] 100201
    Artifactual fatty acid dimers mimic FAHFA signals in untargeted metabolomics pipelines.
    Alisa B Nelson, Lisa S Chow, Curtis C Hughey, Peter A Crawford, Patrycja Puchalska.
      Fatty acid esters of hydroxy fatty acids (FAHFAs) are lipokines with extensive structural and regional isomeric diversity that impact multiple physiological functions, including insulin sensitivity and glucose homeostasis. Due to their low molar abundance, FAHFAs are typically quantified using highly sensitive liquid chromatography and tandem mass spectrometry (LC-MS/MS) methods. Numerous relevant MS databases house in silico-spectra that allow identification and speciation of FAHFAs. These provisional chemical feature assignments provide a useful starting point, but could lead to misidentification. To address this possibility, we analyzed human serum with a commonly applied high-resolution LC-MS untargeted metabolomics platform. We found that many chemical features are putatively assigned to the FAHFA lipid class based on exact mass and fragmentation patterns matching spectral databases. Careful validation using authentic standards revealed that many investigated signals provisionally assigned as FAHFAs are in fact fatty acid dimers formed in the LC-MS pipeline. These isobaric fatty acid dimers differ structurally only by the presence of an olefinic bond. Furthermore, stable isotope-labeled oleic acid spiked into human serum at sub-physiological concentrations showed concentration-dependent formation of a diverse repertoire of fatty acid dimers that analytically mimicked FAHFAs. Conversely, validated FAHFA species did not form spontaneously in the LC-MS pipeline. Together, these findings underscore that FAHFAs are endogenous lipid species. However, non-biological fatty acid dimers forming in the setting of high concentrations of free fatty acids can be misidentified as FAHFAs. Based on these results, we assembled a fatty acid dimer database to identify non-biological fatty acid dimers in untargeted metabolomics datasets.
    Keywords:  Insulin resistance; LC-MS/MS; adipose tissue; isobaric fatty acid dimers; lipidomics; lipids; lipokines; obesity; olefinic bond; spectral database
    DOI:  https://doi.org/10.1016/j.jlr.2022.100201
  2. Metabolites. 2022 Feb 26. pii: 213. [Epub ahead of print]12(3):
    Lipid Metabolism Interplay in CRC-An Update.
    Dana Krauß, Ourania Fari, Maria Sibilia.
      Colorectal cancer (CRC) to date still ranks as one of the deadliest cancer entities globally, and despite recent advances, the incidence in young adolescents is dramatically increasing. Lipid metabolism has recently received increased attention as a crucial element for multiple aspects of carcinogenesis and our knowledge of the underlying mechanisms is steadily growing. However, the mechanism how fatty acid metabolism contributes to CRC is still not understood in detail. In this review, we aim to summarize our vastly growing comprehension and the accompanied complexity of cellular fatty acid metabolism in CRC by describing inputs and outputs of intracellular free fatty acid pools and how these contribute to cancer initiation, disease progression and metastasis. We highlight how different lipid pathways can contribute to the aggressiveness of tumors and affect the prognosis of patients. Furthermore, we focus on the role of lipid metabolism in cell communication and interplay within the tumor microenvironment (TME) and beyond. Understanding these interactions in depth might lead to the discovery of novel markers and new therapeutic interventions for CRC. Finally, we discuss the crucial role of fatty acid metabolism as new targetable gatekeeper in colorectal cancer.
    Keywords:  colorectal cancer; immunometabolism; lipid metabolism; metabolites
    DOI:  https://doi.org/10.3390/metabo12030213
  3. Anal Bioanal Chem. 2022 Mar 26.
    A rapid and robust method for amino acid quantification using a simple N-hydroxysuccinimide ester derivatization and liquid chromatography-ion mobility-mass spectrometry.
    Taylor M Domenick, Austin L Jones, Robin H J Kemperman, Richard A Yost.
      The vast majority of mass spectrometry (MS)-based metabolomics studies employ reversed-phase liquid chromatography (RPLC) to separate analytes prior to MS detection. Highly polar metabolites, such as amino acids (AAs), are poorly retained by RPLC, making quantitation of these key species challenging across the broad concentration ranges typically observed in biological specimens, such as cell extracts. To improve the detection and quantitation of AAs in microglial cell extracts, the implementation of a 4-dimethylaminobenzoylamido acetic acid N-hydroxysuccinimide ester (DBAA-NHS) derivatization agent was explored for its ability to improve both analyte retention and detection limits in RPLC-MS. In addition to the introduction of the DBAA-NHS labeling reagent, a uniformly (U) 13C-labeled yeast extract was also introduced during the sample preparation workflow as an internal standard (IS) to eliminate artifacts and to enable targeted quantitation of AAs, as well as untargeted amine submetabolome profiling. To improve method sensitivity and selectivity, multiplexed drift-tube ion mobility (IM) was integrated into the LC-MS workflow, facilitating the separation of isomeric metabolites, and improving the structural identification of unknown metabolites. Implementation of the U-13C-labeled yeast extract during the multiplexed LC-IM-MS analysis enabled the quantitation of 19 of the 20 common AAs, supporting a linear dynamic range spanning up to three orders of magnitude in concentration for microglial cell extracts, in addition to reducing the required cell count for reliable quantitation from 10 to 5 million cells per sample.
    Keywords:  Amino acids; Derivatization; Ion mobility spectrometry; Mass spectrometry; Metabolomics; Quantitation
    DOI:  https://doi.org/10.1007/s00216-022-03993-w
  4. Adv Clin Chem. 2022 ;pii: S0065-2423(21)00075-5. [Epub ahead of print]107 79-138
    Clinical metabolomics for inborn errors of metabolism.
    Lisa Ford, Matthew Mitchell, Jacob Wulff, Annie Evans, Adam Kennedy, Sarah Elsea, Bryan Wittmann, Douglas Toal.
      Metabolism is a highly regulated process that provides nutrients to cells and essential building blocks for the synthesis of protein, DNA and other macromolecules. In healthy biological systems, metabolism maintains a steady state in which the concentrations of metabolites are relatively constant yet are subject to metabolic demands and environmental stimuli. Rare genetic disorders, such as inborn errors of metabolism (IEM), cause defects in regulatory enzymes or proteins leading to metabolic pathway disruption and metabolite accumulation or deficiency. Traditionally, the laboratory diagnosis of IEMs has been limited to analytical methods that target specific metabolites such as amino acids and acyl carnitines. This approach is effective as a screening method for the most common IEM disorders but lacks the comprehensive coverage of metabolites that is necessary to identify rare disorders that present with nonspecific clinical symptoms. Fortunately, advancements in technology and data analytics has introduced a new field of study called metabolomics which has allowed scientists to perform comprehensive metabolite profiling of biological systems to provide insight into mechanism of action and gene function. Since metabolomics seeks to measure all small molecule metabolites in a biological specimen, it provides an innovative approach to evaluating disease in patients with rare genetic disorders. In this review we provide insight into the appropriate application of metabolomics in clinical settings. We discuss the advantages and limitations of the method and provide details related to the technology, data analytics and statistical modeling required for metabolomic profiling of patients with IEMs.
    Keywords:  Clinical metabolomics; Inborn errors of metabolism; Mass spectrometry; Rare disease; Small molecule biomarkers
    DOI:  https://doi.org/10.1016/bs.acc.2021.09.001
  5. BMC Cancer. 2022 Mar 24. 22(1): 314
    Serum untargeted lipidomics by UHPLC-ESI-HRMS aids the biomarker discovery of colorectal adenoma.
    Hailin Zhou, Yanying Nong, Yifan Zhu, Yunxiao Liang, Jiahao Zhang, Hongwei Chen, Pingchuan Zhu, Qisong Zhang.
       BACKGROUND: Colorectal adenoma (CA) is an important precancerous lesion and early screening target of colorectal cancer (CRC). Lipids with numerous physiological functions are proved to be involved in the development of CRC. However, there is no lipidomic study with large-scale serum samples on diagnostic biomarkers for CA.
    METHODS: The serum lipidomics of CA patients (n = 50) and normal control (NR) (n = 50) was performed by ultra high performance liquid chromatography-high resolution mass spectrometry with electrospray ionization (UHPLC-ESI-HRMS). Univariate and multivariate statistical analyses were utilized to screen the differential lipids between groups, and combining the constituent ratio analysis and diagnostic efficiency evaluation by receiver operating characteristic (ROC) curve disclosed the potential mechanism and biomarkers for CA.
    RESULTS: There were obvious differences in serum lipid profiles between CA and NR groups. Totally, 79 differential lipids were selected by criterion of P < 0.05 and fold change > 1.5 or < 0.67. Triacylglycerols (TAGs) and phosphatidylcholines (PCs) were the major differential lipids with ratio > 60%, indicating these two lipid metabolic pathways showed evident disequilibrium, which could contribute to CA formation. Of them, 12 differential lipids had good diagnostic ability as candidate biomarkers for CA (AUC ≥ 0.900) by ROC analysis.
    CONCLUSIONS: To our knowledge, this is the first attempt to profile serum lipidomics and explore lipid biomarkers of CA to help early screening of CRC. 12 differential lipids are obtained to act as potential diagnostic markers of CA. PCs and fatty acids were the main dysregulated biomarkers for CA in serum.
    Keywords:  Biomarkers; Colorectal adenoma; Colorectal cancer; Early screening; Serum lipidomics; UHPLC-ESI-HRMS
    DOI:  https://doi.org/10.1186/s12885-022-09427-1
  6. Cancer Cell. 2022 Mar 15. pii: S1535-6108(22)00118-0. [Epub ahead of print]
    CD8+ T cells PUF(A)ing the flames of cancer ferroptotic cell death.
    José Pedro Friedmann Angeli, Thamara Nishida Xavier da Silva, Bastian Schilling.
      In this issue of Cancer Cell, Liao et al. demonstrate that CD8+ T cell-secreted interferon-gamma (IFN-γ) rewires cancer cell lipid metabolism via the enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4). ACSL4 activates polyunsaturated fatty acids and sensitizes cancer cells to ferroptosis in immunotherapy-relevant settings. These findings provide insights into how the metabolic and immune milieu could be used to promote ferroptosis.
    DOI:  https://doi.org/10.1016/j.ccell.2022.03.003
  7. Front Pharmacol. 2022 ;13 838782
    Formation, Signaling and Occurrence of Specialized Pro-Resolving Lipid Mediators-What is the Evidence so far?
    Nils Helge Schebb, Hartmut Kühn, Astrid S Kahnt, Katharina M Rund, Valerie B O'Donnell, Nicolas Flamand, Marc Peters-Golden, Per-Johan Jakobsson, Karsten H Weylandt, Nadine Rohwer, Robert C Murphy, Gerd Geisslinger, Garret A FitzGerald, Julien Hanson, Claes Dahlgren, Mohamad Wessam Alnouri, Stefan Offermanns, Dieter Steinhilber.
      Formation of specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins usually involves arachidonic acid 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- and 15-lipoxygenating paralogues (15-LO1, ALOX15; 15-LO2, ALOX15B; 12-LO, ALOX12). Typically, SPMs are thought to be formed via consecutive steps of oxidation of polyenoic fatty acids such as arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid. One hallmark of SPM formation is that reported levels of these lipid mediators are much lower than typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g., 5-HETE), leukotrienes or certain cyclooxygenase-derived prostaglandins. Thus, reliable detection and quantification of these metabolites is challenging. This paper is aimed at critically evaluating i) the proposed biosynthetic pathways of SPM formation, ii) the current knowledge on SPM receptors and their signaling cascades and iii) the analytical methods used to quantify these pro-resolving mediators in the context of their instability and their low concentrations. Based on current literature it can be concluded that i) there is at most, a low biosynthetic capacity for SPMs in human leukocytes. ii) The identity and the signaling of the proposed G-protein-coupled SPM receptors have not been supported by studies in knock-out mice and remain to be validated. iii) In humans, SPM levels were neither related to dietary supplementation with their ω-3 polyunsaturated fatty acid precursors nor were they formed during the resolution phase of an evoked inflammatory response. iv) The reported low SPM levels cannot be reliably quantified by means of the most commonly reported methodology. Overall, these questions regarding formation, signaling and occurrence of SPMs challenge their role as endogenous mediators of the resolution of inflammation.
    Keywords:  FPR; LC-MS-based lipid mediator analysis; SPM; leukotriene; lipoxin; lipoxygenase; resolution of inflammation; resolvin
    DOI:  https://doi.org/10.3389/fphar.2022.838782
  8. Metabolites. 2022 Feb 25. pii: 211. [Epub ahead of print]12(3):
    High-Throughput UHPLC-MS to Screen Metabolites in Feces for Gut Metabolic Health.
    Andressa de Zawadzki, Maja Thiele, Tommi Suvitaival, Asger Wretlind, Min Kim, Mina Ali, Annette F Bjerre, Karin Stahr, Ismo Mattila, Torben Hansen, Aleksander Krag, Cristina Legido-Quigley.
      Feces are the product of our diets and have been linked to diseases of the gut, including Chron's disease and metabolic diseases such as diabetes. For screening metabolites in heterogeneous samples such as feces, it is necessary to use fast and reproducible analytical methods that maximize metabolite detection. As sample preparation is crucial to obtain high quality data in MS-based clinical metabolomics, we developed a novel, efficient and robust method for preparing fecal samples for analysis with a focus in reducing aliquoting and detecting both polar and non-polar metabolites. Fecal samples (n = 475) from patients with alcohol-related liver disease and healthy controls were prepared according to the proposed method and analyzed in an UHPLC-QQQ targeted platform in order to obtain a quantitative profile of compounds that impact liver-gut axis metabolism. MS analyses of the prepared fecal samples have shown reproducibility and coverage of n = 28 metabolites, mostly comprising bile acids and amino acids. We report metabolite-wise relative standard deviation (RSD) in quality control samples, inter-day repeatability, LOD (limit of detection), LOQ (limit of quantification), range of linearity and method recovery. The average concentrations for 135 healthy participants are reported here for clinical applications. Our high-throughput method provides a novel tool for investigating gut-liver axis metabolism in liver-related diseases using a noninvasive collected sample.
    Keywords:  bile acids; fecal metabolomics; gut-liver axis; sample preparation; targeted metabolomics
    DOI:  https://doi.org/10.3390/metabo12030211
  9. Dev Cell. 2022 Mar 15. pii: S1534-5807(22)00126-5. [Epub ahead of print]
    The central moTOR of metabolism.
    Judith Simcox, Dudley W Lamming.
      The protein kinase mechanistic target of rapamycin (mTOR) functions as a central regulator of metabolism, integrating diverse nutritional and hormonal cues to control anabolic processes, organismal physiology, and even aging. This review discusses the current state of knowledge regarding the regulation of mTOR signaling and the metabolic regulation of the four macromolecular building blocks of the cell: carbohydrate, nucleic acid, lipid, and protein by mTOR. We review the role of mTOR in the control of organismal physiology and aging through its action in key tissues and discuss the potential for clinical translation of mTOR inhibition for the treatment and prevention of diseases of aging.
    Keywords:  amino acids; lipids; mTOR; mTORC1; mTORC2; metabolism; protein; rapamycin
    DOI:  https://doi.org/10.1016/j.devcel.2022.02.024
  10. Front Physiol. 2022 ;13 841278
    Ether Lipids in Obesity: From Cells to Population Studies.
    Yvette L Schooneveldt, Sudip Paul, Anna C Calkin, Peter J Meikle.
      Ether lipids are a unique class of glycero- and glycerophospho-lipid that carry an ether or vinyl ether linked fatty alcohol at the sn-1 position of the glycerol backbone. These specialised lipids are important endogenous anti-oxidants with additional roles in regulating membrane fluidity and dynamics, intracellular signalling, immunomodulation and cholesterol metabolism. Lipidomic profiling of human population cohorts has identified new associations between reduced circulatory plasmalogen levels, an abundant and biologically active sub-class of ether lipids, with obesity and body-mass index. These findings align with the growing body of work exploring novel roles for ether lipids within adipose tissue. In this regard, ether lipids have now been linked to facilitating lipid droplet formation, regulating thermogenesis and mediating beiging of white adipose tissue in early life. This review will assess recent findings in both population studies and studies using cell and animal models to delineate the functional and protective roles of ether lipids in the setting of obesity. We will also discuss the therapeutic potential of ether lipid supplementation to attenuate diet-induced obesity.
    Keywords:  adipose tissue; alkylglycerols; ether lipids; obesity; plasmalogens
    DOI:  https://doi.org/10.3389/fphys.2022.841278
  11. Proteomes. 2022 Feb 28. pii: 9. [Epub ahead of print]10(1):
    Data-Independent Acquisition Enables Robust Quantification of 400 Proteins in Non-Depleted Canine Plasma.
    Halley Gora Ravuri, Zainab Noor, Paul C Mills, Nana Satake, Pawel Sadowski.
      Mass spectrometry-based plasma proteomics offers a major advance for biomarker discovery in the veterinary field, which has traditionally been limited to quantification of a small number of proteins using biochemical assays. The development of foundational data and tools related to sequential window acquisition of all theoretical mass spectra (SWATH)-mass spectrometry has allowed for quantitative profiling of a significant number of plasma proteins in humans and several animal species. Enabling SWATH in dogs enhances human biomedical research as a model species, and significantly improves diagnostic and disease monitoring capability. In this study, a comprehensive peptide spectral library specific to canine plasma proteome was developed and evaluated using SWATH for protein quantification in non-depleted dog plasma. Specifically, plasma samples were subjected to various orthogonal fractionation and digestion techniques, and peptide fragmentation data corresponding to over 420 proteins was collected. Subsequently, a SWATH-based assay was introduced that leveraged the developed resource and that enabled reproducible quantification of 400 proteins in non-depleted plasma samples corresponding to various disease conditions. The ability to profile the abundance of such a significant number of plasma proteins using a single method in dogs has the potential to accelerate biomarker discovery studies in this species.
    Keywords:  Data Independent Acquisition; FASP; SWATH; dog; plasma proteomics
    DOI:  https://doi.org/10.3390/proteomes10010009
  12. Metabolites. 2022 Feb 25. pii: 206. [Epub ahead of print]12(3):
    Lipidomic Profiling Identifies Serum Lipids Associated with Persistent Multisite Musculoskeletal Pain.
    Canchen Ma, Ming Liu, Jing Tian, Guangju Zhai, Flavia Cicuttini, Yvette L Schooneveldt, Peter J Meikle, Graeme Jones, Feng Pan.
      Lipid mediators have been suggested to have a role in pain sensitivity and response; however, longitudinal data on lipid metabolites and persistent multisite musculoskeletal pain (MSMP) are lacking. This study was to identify lipid metabolic markers for persistent MSMP. Lipidomic profiling of 807 lipid species was performed on serum samples of 536 participants from a cohort study. MSMP was measured by a questionnaire and defined as painful sites ≥4. Persistent MSMP was defined as having MSMP at every visit. Logistic regression was used with adjustment for potential confounders. The Benjamini-Hochberg method was used to control for multiple testing. A total of 530 samples with 807 lipid metabolites passed quality control. Mean age at baseline was 61.54 ± 6.57 years and 50% were females. In total, 112 (21%) of the participants had persistent MSMP. Persistent MSMP was significantly associated with lower levels of monohexosylceramide (HexCer)(d18:1/22:0 and d18:1/24:0), acylcarnitine (AC)(26:0) and lysophosphatidylcholine (LPC)(18:1 [sn1], 18:2 [sn1], 18:2 [sn2], and 15-MHDA[sn1] [104_sn1]) after controlling for multiple testing. After adjustment for age, sex, body mass index, comorbidities, and physical activity, HexCer(d18:1/22:0 and d18:1/24:0) and LPC(15-MHDA [sn1] [104_sn1]) were significantly associated with persistent MSMP [Odds Ratio (OR) ranging from 0.25-0.36]. Two lipid classes-HexCer and LPC-were negatively associated with persistent MSMP after adjustment for covariates (OR = 0.22 and 0.27, respectively). This study identified three novel lipid signatures of persistent MSMP, suggesting that lipid metabolism is involved in the pathogenesis of persistent pain.
    Keywords:  biomarker; lipid classes; lipid species; lipidomics; persistent multisite musculoskeletal pain
    DOI:  https://doi.org/10.3390/metabo12030206
  13. Cell Metab. 2022 Mar 15. pii: S1550-4131(22)00087-0. [Epub ahead of print]
    The glucose transporter GLUT3 controls T helper 17 cell responses through glycolytic-epigenetic reprogramming.
    Sophia M Hochrein, Hao Wu, Miriam Eckstein, Laura Arrigoni, Josip S Herman, Fabian Schumacher, Christian Gerecke, Mathias Rosenfeldt, Dominic Grün, Burkhard Kleuser, Georg Gasteiger, Wolfgang Kastenmüller, Bart Ghesquière, Jan Van den Bossche, E Dale Abel, Martin Vaeth.
      Metabolic reprogramming is a hallmark of activated T cells. The switch from oxidative phosphorylation to aerobic glycolysis provides energy and intermediary metabolites for the biosynthesis of macromolecules to support clonal expansion and effector function. Here, we show that glycolytic reprogramming additionally controls inflammatory gene expression via epigenetic remodeling. We found that the glucose transporter GLUT3 is essential for the effector functions of Th17 cells in models of autoimmune colitis and encephalomyelitis. At the molecular level, we show that GLUT3-dependent glucose uptake controls a metabolic-transcriptional circuit that regulates the pathogenicity of Th17 cells. Metabolomic, epigenetic, and transcriptomic analyses linked GLUT3 to mitochondrial glucose oxidation and ACLY-dependent acetyl-CoA generation as a rate-limiting step in the epigenetic regulation of inflammatory gene expression. Our findings are also important from a translational perspective because inhibiting GLUT3-dependent acetyl-CoA generation is a promising metabolic checkpoint to mitigate Th17-cell-mediated inflammatory diseases.
    Keywords:  ACLY; ATP-citrate lyase; GLUT1; GLUT3; Th17 cells; acetyl-CoA; glucose metabolism; glycolysis; histone acetylation; immunometabolism
    DOI:  https://doi.org/10.1016/j.cmet.2022.02.015
  14. Methods Mol Biol. 2022 ;2468 271-281
    Lipid Extraction and Analysis.
    Henry H Harrison, Jennifer L Watts.
      Lipids are major components of cellular membranes and energy stores. Lipids contribute vital structural, energetic, and signaling functions. We have optimized methods to extract and analyze lipids from the nematode Caenorhabditis elegans based on standard methods. Here we describe a method to extract total lipids from C. elegans larvae, adults, or embryos. We describe a thin-layer chromatography method to separate major lipid classes and a gas chromatography method to analyze fatty acid composition from lipid extracts, lipid fractions, or directly from nematode larvae, adults, or embryos.
    Keywords:  Fatty acid methyl esters; Gas chromatography; Lipid extraction; Neutral lipids; Phospholipids; Thin-layer chromatography
    DOI:  https://doi.org/10.1007/978-1-0716-2181-3_14
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