scholarly journals Differential Transcription Profiling in Bone Marrow Mononuclear Cells Between Myasthenia Gravis Patients With or Without Thymoma

2021 ◽  
Author(s):  
Jingqun Tang ◽  
Ziming Ye ◽  
Yi Liu ◽  
Mengxiao Zhou ◽  
chao qin
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Myasthenia Gravis ◽  
Differentially Expressed Genes ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Differentially Expressed ◽  
Pcr Analysis ◽  
Bone Marrow Mononuclear Cells ◽  
Qrt Pcr

Abstract PurposeDefective stem cells have been recognized as being associated with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, autoimmune cytopenias and myasthenia gravis (MG). However, the differential gene expression profile of bone marrow mononuclear cells (BMMCs) and the molecular mechanisms underlying MG pathogenesis have not been fully elucidated. Therefore, we investigated the abnormal expression and potential roles and mechanisms of mRNAs in BMMCs among patients with MG with or without thymoma.MethodsTranscription profiling of BMMCs in patients with MG without thymoma (M2) and patients with thymoma-associated MG (M1) was undertaken by using high-throughput RNA sequencing (RNA-Seq), and disease-related differentially expressed genes were validated by quantitative real-time polymerase chain reaction (qRT-PCR).ResultsRNA-Seq demonstrated 60 significantly upregulated and 65 significantly downregulated genes in M2 compared with M1. Five disease-related differentially expressed genes were identified and validated by qRT-PCR analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed to predict the functions of aberrantly expressed genes. Recombination activating 1 (RAG1), RAG2, BCL2-like 11, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform and repressor element-1-silencing transcription factor might play roles in MG pathogenesis involving the primary immunodeficiency signaling pathway, signaling pathways regulating pluripotency of stem cells and forkhead box O signaling pathway.ConclusionThe aberrantly expressed genes of BMMCs in M1 or M2 patients demonstrate the underlying mechanisms governing the pathogenesis of MG.

Comparison of Different Strategies of MSC Isolation Revels Advantage To Expand MSC Directly from Purified CD105+ and CD271+ Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2566-2566 ◽  
Author(s):  
Danuta Jarocha ◽  
Ewa Lesko ◽  
Mariusz Z. Ratajczak ◽  
Marcin Majka
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Lipoprotein Lipase ◽  
Mononuclear Cells ◽  
Mrna Level ◽  
Surface Antigens ◽  
Pcr Analysis ◽  
Adherent Cell ◽  
Bone Marrow Mononuclear Cells

Abstract The potential for multilineage differentiation together with the ability to expand in cultures are the reasons why Mesenchymal Stem Cells (MSC) are considered to be the population of stem cells for potential treatment for variety of disorders (e. g. Osteogenesis Imperfecta, Myocardium Infarction, GvHD). MSC are isolated from the bone marrow mononuclear cells (MNC) based on their adhesive properties. There have been few attempts to isolate MSC directly based on the expression of selected surface antigens, but these isolation strategies were not compared with “the gold standard” procedure which is still selection by plastic adherence. Nevertheless, it is obvious that a presence of different populations of cells “contaminating” MSC in adherent cell cultures (e.g., endothelial cells, macrophages, dendritic cells) may affect expansion of MSC. In this study we proposed new isolation strategies of bone marrow MSC based on RosetteSep Isolation Kit (Stem Cells Technologies Inc., Vancouver, Canada) and immunomagnetic isolation of CD105+ or CD271+ cell populations (Miltenyi Biotec, Germany). Four fractions of bone marrow mononuclear cells i) non-purified MNC, ii) MNC enriched in MSC by RosetteSep Isolation Kit, iii) sorted CD105+ and iv) sorted CD271+ cells were cultured for three passages. Subsequently, we evaluated i) number of CFU-F colonies, ii) expression of selected surface antigens (CD105, CD166, CD44, CD73, CD45, CD34), iii) in vitro osteogenic differentiation of expanded cells and iv) changes in the expression of genes related to osteogenesis (RQ-PCR). We found that the mean number of CFU-F colonies counted on the 9th day of culture was 26 (range 14,5–41,4), 49 (range 21,2–97,1), 105 (range 36,5–221) and 148 (range 55,3–211) per 107 MNC for non-purified MNC, MNC enriched in MSC by RosetteSep Isolation Kit, purified CD105+cells and purified CD271+cells, respectively. After 3rd passage the phenotype of cells was similar as we observed a comparable percentage of cells positive (over 90%) for CD105, CD166, CD44, CD73 and negative (below 5%) for CD45, CD34 surface antigens in all fractions. The RQ - PCR analysis of mRNA level of osteogenic (osteocalcin, PTHR, α1collagen), adipogenic (lipoprotein lipase, leptin, PPARγ2) and chondrogenic (aggrecan1) genes in all four populations revealed that MSC isolated by means of expression of 105 and CD271 antigens had higher level of mRNA for all assessed genes except for lipoprotein lipase and α1collagen prior to differentiation. After 30 days of osteogenic differentiation RQ - PCR analysis was repeated and compared with that before differentiation. We noticed an increased level of mRNA for osteocalcin and PTHR (markers of osteogenic differentiation) in all four populations, with the highest expression in MSC derived from non-purified MNC. However, this fraction had also the highest mRNA level of PPARγ2, lipoprotein lipase, and aggrecan genes (adipogenic and chondrogenic lineage respectively). Since the highest number of CFU-F was derived from purified CD105+ and CD271+ cells as well as these two populations seem to be the most homogenous based on RQ-PCR data, these cell fractions should be employed to expand most efficiently MSC for potential therapeutic purposes. Our data suggest that, non-MSC cells present in MNC and RosetteSep cultures may negatively affect both the expansion efficiency and differentiation along desired MSC lineage.

scholarly journals Bone marrow mononuclear cells versus mesenchymal stem cells from adipose tissue on bone healing in an Old World primate: can this be extrapolated to humans?

2019 ◽  
Vol 71 (3) ◽  
pp. 917-928
Author(s):  
E. Branco ◽  
C.M.F.C. Miranda ◽  
A.R. Lima ◽  
K.S.M. Silva ◽  
R.M. Cabral ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Iliac Crest ◽  
Mononuclear Cells ◽  
Bone Marrow Mononuclear Cells ◽  
Old World ◽  
Bone Injury ◽  
The Right

ABSTRACT In veterinary medicine, the cell therapy is still unexplored and there are many unanswered questions that researchers tend to extrapolate to humans in an attempt to treat certain injuries. Investigating this subject in nonhuman primates turns out to be an unparalleled opportunity to better understand the dynamics of stem cells against some diseases. Thus, we aimed to compare the efficiency of bone marrow mononuclear cells (BMMCs) and mesenchymal stem cells (MSCs) from adipose tissue of Chlorocebus aethiops in induced bone injury. Ten animals were used, male adults subjected, to bone injury the iliac crests. The MSCs were isolated by and cultured. In an autologous manner, the BMMCs were infused in the right iliac crest, and MSCs from adipose tissue in the left iliac crest. After 4.8 months, the right iliac crests fully reconstructed, while left iliac crest continued to have obvious bone defects for up to 5.8 months after cell infusion. The best option for treatment of injuries with bone tissue loss in old world primates is to use autologous MSCs from adipose tissue, suggesting we can extrapolate the results to humans, since there is phylogenetic proximity between species.

scholarly journals Transcriptome profiling of peanut (Arachis hypogaea) gynophores in gravitropic response

2013 ◽  
Vol 40 (12) ◽  
pp. 1249 ◽  
Author(s):  
Hai-fen Li ◽  
Xiao-Ping Chen ◽  
Fang-he Zhu ◽  
Hai-Yan Liu ◽  
Yan-Bin Hong ◽  
...  
Keyword(s):  
Differentially Expressed Genes ◽  
Arachis Hypogaea ◽  
Molecular Mechanisms ◽  
Carbon Fixation ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Pentose Phosphate ◽  
Differentially Expressed ◽  
Pcr Analysis

Peanut (Arachis hypogaea L.) produces flowers aerially, but the fruit develops underground. This process is mediated by the gynophore, which always grows vertically downwards. The genetic basis underlying gravitropic bending of gynophores is not well understood. To identify genes related to gynophore gravitropism, gene expression profiles of gynophores cultured in vitro with tip pointing upward (gravitropic stimulation sample) and downward (control) at both 6 and 12 h were compared through a high-density peanut microarray. After gravitropic stimulation, there were 174 differentially expressed genes, including 91 upregulated and 83 downregulated genes at 6 h, and 491 differentially expressed genes including 129 upregulated and 362 downregulated genes at 12 h. The differentially expressed genes identified were assigned to 24 functional categories. Twenty pathways including carbon fixation, aminoacyl-tRNA biosynthesis, pentose phosphate pathway, starch and sucrose metabolism were identified. The quantitative real-time PCR analysis was performed for validation of microarray results. Our study paves the way to better understand the molecular mechanisms underlying the peanut gynophore gravitropism.

Differentially Expressed and Novel Transcripts in Highly Purified Chronic Phase CML Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 193-193
Author(s):  
Yun Zhao ◽  
Allen Delaney ◽  
Afshin Raouf ◽  
Kamini Raghuram ◽  
Haiyan I Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Blood Cells ◽  
Molecular Mechanisms ◽  
Chronic Phase ◽  
Differentially Expressed ◽  
Pcr Analysis ◽  
Direct Role ◽  
Hematopoietic Stem ◽  
Transcript Levels

Abstract The chronic phase of CML is sustained by rare BCR-ABL+ stem cells. These cells share many properties with normal pluripotent hematopoietic stem cells, but also differ in critical ways that alter their growth, drug responsiveness and genome stability. Understanding the molecular mechanisms underlying the biological differences between normal and CML stem cells is key to the development of more effective CML therapies. To obtain new insights into these mechanisms, we generated Long Serial Analysis of Gene Expression (SAGE) libraries from paired isolates of highly purified lin-CD34+CD45RA-CD36- CD71-CD7-CD38+ and lin-CD34+CD45RA-CD36-CD71-CD7-CD38- cells from 3 chronic phase CML patients (all with predominantly Ph+/BCR-ABL+ cells in both subsets) and from 3 control samples: a pool of 10 normal bone marrows (BMs), a single normal BM and a pool of G-CSF-mobilized blood cells from 9 donors. In vitro bioassays showed the CD34+CD38+ cells were enriched in CFCs (CML: 3–20% pure; normal: 4–19% pure) and the CD34+CD38- cells were enriched in LTC-ICs (CML: 0.2–26% pure; normal: 12–52% pure). Each of the 12 libraries was then sequenced to a depth of ~200,000 tags and tags from libraries prepared from like phenotypes were compared between genotypes using DiscoverySpace software and hierarchical clustering. 1687 (355 with clustering) and 1258 (316 with clustering) transcripts were thus identified as differentially expressed in the CML vs control CD34+CD38− and CD34+CD38+ subsets, respectively. 266 of these transcripts (11 with clustering) were differentially expressed in both subsets. The differential expression of 5 genes (GAS2, IGF2BP2, IL1R1, DUSP1 & SELL) was confirmed by real-time PCR analysis of lin-CD34+ cells isolated from an additional 5 normal BMs and 11 CMLs, and lin-CD34+CD38− cells from an additional 2 normal BMs and 2 CMLs (with dominant Ph+ cells). GAS2 and IL1R1 transcript levels were correlated with BCR-ABL transcript levels in both primitive subsets, and predicted differences in expression of IL1R1 and SELL were apparent within 3 days in CD34+ cord blood cells transduced with a lenti-BCR-ABL-IRES-GFP vs a control lenti-GFP vector (n=3). These findings support a direct role of BCR-ABL in perturbing the expression of these 3 genes. Further comparison of the meta CD34+CD38− and CD34+CD38+ CML cell libraries with most publicly accessible SAGE data revealed 69 novel tags in the CD34+ CML cells that correspond to unique but conserved genomic sequences. Nine of these were recovered by 5′- and 3′- RACE applied to cDNAs pooled from several human leukemic cell lines. These results illustrate the power of SAGE to reveal key components of the transcriptomes of rare human CML stem cell populations including transcripts of genes not previously known to exist. Continuing investigation of their biological roles in primary CML cells and primitive BCR-ABL-transduced human cells offer important strategies for delineating their potential as therapeutic targets.

scholarly journals Combining Effects of the SMO Inhibitor and Jak1 Inhibitor in Terminal Differentiation of MDS-Derived Induced Potent Stem Cells (iPSC)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3150-3150
Author(s):  
Tetsuzo Tauchi ◽  
Seiichi Okabe ◽  
Seiichiro Katagiri ◽  
Yuko Tanaka ◽  
Kazuma Ohyashiki
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Nuclear Translocation ◽  
Terminal Differentiation ◽  
Scid Mice ◽  
Cytokine Signaling ◽  
Bone Marrow Mononuclear Cells

Abstract Background: Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by no efficient hematopoiesis and frequent progression to acute myeloid leukemia (AML). Even in low risk MDS, clonal hematopoiesis already dominates at diagnosis, and clones found in secondary AML originate from the MDS stage of disease, highlighting the need to specifically target the MDS-initiating clone. PF-0449913 is a potent and selective hedgehog pathway inhibitor that act by binding Smoothened (SMO) and blocking signal transduction. PF-04449913 demonstrated preliminary antitumor activity in a phase I trial, when given as monotherapy in patients with several hematopoietic malignancy. Jak1 tyrosine kinase plays an important role in cytokine signaling. Jak1 functions to phosphorylate STAT3 transcription factor, which triggers their dimerization and nuclear translocation. In the present study, we investigated the combining effects of PF-04449913 and Jak1 inhibitor, PF-6667291 in terminal differentiation of MDS-derived induced potent stem cells (iPSC). Methods: We generated iPSCs from bone marrow mononuclear cells of two MDS patients (RAEB1 and RAEB2 by WHO classification) with chromosome 5 deletion and complex karyotypic abnormalities, respectively. Karyotyping analysis revealed that MDS-derived iPSCs have identical abnormalities to primary MDS cells. We also generated iPSCs from bone marrow mononuclear cells of normal volunteer as control. To investigate the effects of PF-04449913 on self-renewal and the relevance as a therapeutic target in MDS initiating cells, we examined the activity of PF-04449913 against MDS-derived iPSCs transferred NOD/SCID mice in vivo. NOD/SCID mice were injected subcutaneously with MDS-derived iPSCs or normal iPSCs then treated with PF-04449913 (100 mg/kg; p.o.) from day 10 for 28 days. We also used MDS-L, a myelodysplastic cell line established from MDS patient with del (5q) and complex karyotypic abnormalities for in vitro studies. In vitro re-differentiation of MDS-iPSCs was performed with differentiation media (30 ng/ml VEGF, 30 ng/ml BMP-4, 40 ng/ml SCF, 50 ng/ml Activin) for 4 days. At day 14, a single cell suspension expressing CD34+CD38- was achieved with hematopoietic cytokines (300 ng/ml Flt-3 ligand, 10 ng/ml IL-3, 10 ng/ml IL-6, 50 ng/ml G-CSF, 25 ng/ml BMP-4). Results: Both MDS-derived iPSCs transferred NOD/SCID mice and normal iPSCs transferred NOD/SCID mice demonstrated the engraftment of CD34+CD38- positive cells by flow cytometry. However, the treatment with PF-04449913 reduced the population of CD34+CD38- positive cells in MDS-derived iPSCs transferred NOD/SCID mice. We isolated human CD45+ cells from the spleen of mice from each treatment group and injected equivalent numbers of CD45+ cells into secondary recipients. Following 50 days, all mice treated with vehicle engrafted with CD34+CD38- positive cells. In contrast, CD34+CD38- positive cells engraftment was not detected in recipient mice (n=3) from PF-04449913-treated donors. These results demonstrate the persistent effects of PF-0449913 on long term self-renewing MDS-initiating cells. Next we performed in vitro re-differentiation of MDS-iPSCs, which express CD34+CD38- population. CD34+CD38- cells from MDS-derived iPSCs were cultured with 2 μM of PF-04449913 and 1 μM of PF-6667291 in STEMdiff APEL medium for 14 days for CFC activities. Treatments with PF-04449913 and PF-6667291 significantly reduced the colony formations of mature erythroid, granulocyte-macrophage, and mixed of these hematopoietic cells. To identify the mechanisms that limit the terminal differentiation of MDS-derived iPSC by PF-04449913 and PF-6667291, MDS-L cells were cultured with PF-04449913 and PF-6667291 for 72 hrs. The treatments with PF-04449913 and PF-6667291 induced the expressions of p21Cip1, cleaved PARP and reduced the expression of BMI-1, c-Myc, Nanog, and phospho-Stat3. Conclusion: Our preclinical results indicate that the combination with PF-04449913 and PF-6667291 have potential as an important option for controlling the terminal differentiation of MDS-initiating cells. It is expected that the combination with PF-04449913 and PF-6667291 may become extremely useful therapeutic interventions in a number of hematological neoplasms, including MDS. Disclosures Tauchi: Pfizer Inc.: Research Funding. Ohyashiki:Bristol-Myers Squibb: Research Funding; Novartis International AG,: Honoraria, Research Funding.

Isolation of Human Bone Marrow Mesenchymal Stem Cells by a Novel Monoclonal Antibody, ZUC3.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2562-2562
Author(s):  
Xiaoyu Lai ◽  
He Huang ◽  
Li Huang ◽  
Fenfang Zeng
Keyword(s):  
Stem Cells ◽  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Sorting ◽  
Mononuclear Cells ◽  
Human Mscs ◽  
Bone Marrow Mononuclear Cells ◽  
Multiple Differentiation

Abstract Objective: Due to absence of a single definitive marker of mesenchymal stem cells (MSCs) and low incidence in human bone marrow, the primary culture of MSCs, conventionally isolated with its characteristic of adherent, were considered to be heterogeneous containing of several subpopulations, which had currently limited our understanding of their biology and therapeutic applications. In our previous study, a novel murine monoclonal antibody (McAb) ZUC3 was produced by hybridoma technology, which was specifically reactive with human MSCs, while showed negative cross-reactivity when screened against a variety of human tissues. Now, ZUC3 antigen positive MSCs population would be further identified by magnetic-activated cell sorting (MACS). Methods: Bone marrow were taken from the iliac crest of normal healthy adult volunteers, and mononuclear cells were separated by density gradient centrifugation, then separated into positively- and negatively-labelled fractions with McAb ZUC3 by immunomagnetic activated cell sorting. The purity of positive cells was analyzed by flow cytometry, then ZUC3 antigen positive and negative cells were plated respectively in human MSCs medium consisting of 10% FBS, LG-DMEM. Characteristics of ZUC3 antigen positive cells phenotype was analyzed by flow cytometry, and proliferation and multiple differentiation potential of the cells was observed in vitro. Results: Flow cytometric analysis showed that ZUC3 antigen expression by cultured MSCs and mononuclear cells derived from bone marrow were 91.31±2.92%, 0.96±0.28% respectively, and western blotting showed the molecular mass of antigen was about 33KD. The purity of the recovered fractions for ZUC3 by MACS was 76.82±6.32%. The positive cells have adhered to culture flask in vitro, and the quantity of adhered cells that had fibroblast-like morphology increased and proliferated during primary expansion period, while the negative cells were observed as round shape cells without any proliferation. It was demonstrated that ZUC3 antigen positive cells continued growth with spindle-shape, extending beyond 30 population doublings in long-term culture. Analyzed by flow cytometry, the culture-expanded positive cells were uniformly positive for CD29, CD44, CD105, CD106, and lack typical hematopoietic antigens such as CD14, CD34, CD45, HLA-DR, which demonstrated that ZUC3 postive cells sorted from bone marrow mononuclear cells by McAb were MSCs. With proper medium, the ZUC3 antigen positive cells could be successfully induced to differentiate into adipocytes, osteoblasts, and neuro-like cells which were positive of neuron markers such as nestin, NSE and NF-M. Conclusion: ZUC3 McAb was a specific surface marker against human MSCs for cell sorting. The ZUC3 antigen positive cells separated from bone marrow mononuclear cells had potential capacity of high proliferation and multiple differentiation.

Individual Gene Expression Profiling of Bone Marrow CD34 Cells in Acquired Severe Aplastic Anemia (aSAA) in Children.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 978-978 ◽  
Author(s):  
Bernd Hubner ◽  
Sylvia Merk ◽  
Sonja Rauhut ◽  
Martin Dugas ◽  
Torsten Haferlach ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Differentially Expressed Genes ◽  
Expression Profiling ◽  
Cell Metabolism ◽  
Cell Communication ◽  
Differentially Expressed ◽  
Cd34 Cells ◽  
The Individual

Abstract Acquired SAA in children is a rare, life-threatening disease characterized by pancytopenia and bone marrow hypocellularity. There is good clinical and laboratory evidence that a T-cell mediated immune attack against stem and progenitor cells plays an important role in the pathogenesis of SAA. However, due to the paucity of residual CD34 positive cells at diagnosis still only little is known about the stem cells and their response to the autoimmune attack in SAA in children. To further investigate the characteristics of CD34 cells in SAA we compared the individual transcriptomes of CD34 cells of 9 newly diagnosed, untreated pediatric SAA patients with 8 pediatric healthy controls. Hematopoietic stem cells were isolated with high efficiency from bone marrow by Ficoll density centrifugation and subsequent affinity purification using Dynabeads (Dynal, Invitrogen). Expression profiling experiments were performed using the two cycle amplification system and the HG-U133 plus 2.0 array (Affymetrix). Gene expression data were analyzed using R 2.3.0 and Bioconductor 1.8. packages (Affymetrix, multtest). Raw data were normalized using robust multiarray average (RMA) algorithm. Probe sets with “absent” calls in more than 50% of samples in the smaller group were identified and omitted from further analysis. To determine differentially expressed genes, t-test was applied. P value adjustments for multiple comparisons were done using the step-up false discovery rate (FDR) controlling method proposed by Benjamini and Hochberg. Overall 402 genes were differentially expressed in children with SAA compared to controls (p < 0.05), 288 genes were downregulated and 114 were upregulated. Gene ontology analyses (FatiGO) indicated that biological processes in CD34 cells are significantly affected in pediatric SAA by mainly downregulation of genes for cell metabolism (78 down, 30 up), cell communication/adhesion (48 down, 25 up), growth and differentiation (15 down, 4 up) and stress response (16 down, 3 up). Unexpectedly only very few genes involved in cell death/apoptosis (5 down, 4 up) were differentially expressed. Genes encoding for DNA/RNA binding proteins (28 down, 14 up) and ion binding proteins (47 down, 18 up) were also mainly downregulated. Despite the extremely low numbers of residual CD34 cells present in the bone marrow of children with untreated SAA we were able to analyze the individual transcriptome pattern of single patients. These patterns showed homogeneously and significantly different gene expressions in the group of affected children when compared to controls. Genes involved in apoptosis seem to be less altered in there expression than expected from adult data. These observation might be consistent with the major clinical finding in these children of almost empty bone marrows where most of the apoptotic cell death has already taken place. In the tiny population of “survivors” most of the differentially expressed genes are involved in cell metabolism and cell communication or adhesion. These unexpected results provide new hints for further investigations regarding the involvement of CD34 cells in the pathogenesis of childhood aSAA.

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