Using RNA-Seq to Explore the Repair Mechanism of the Three Methods and Three-Acupoint Technique on DRGs in Sciatic Nerve Injured Rats

Objective. To study the effects of the three methods and three-acupoint technique on DRG gene expression in SNI model rats and to elucidate the molecular mechanism of the three methods and three-acupoint technique on promoting recovery in peripheral nerve injury. Methods. 27 male SD rats were randomly divided into three groups: a Sham group, the SNI group, and the Tuina group. The Tuina group was treated with a tuina manipulation simulator to simulate massage on points, controlling for both quality and quantity. Point-pressing, plucking, and kneading methods were administered quantitatively at Yinmen (BL37), Chengshan (BL57), and Yanglingquan (GB34) points on the affected side once a day, beginning 7 days after modeling. Intervention was applied once a day for 10 days, then 1 day of rest, followed by 10 more days of intervention, totally equaling 20 times of intervention. The effect of the three methods and three-point technique on the recovery of injured rats was evaluated using behavior analysis. RNA sequencing (RNA-Seq) analysis of differentially expressed genes in DRGs of the three groups of rats was also performed. GO and KEGG enrichment was analyzed and verified using real-time PCR. Results. RNA-Seq combined with database information showed that the number of differentially expressed genes in DRG was the largest in the Tuina group compared with the SNI group, totaling 226. GO function is enriched in the positive regulation of cell processes, ion binding, protein binding, neuron, response to pressure, response to metal ions, neuron projection, and other biological processes. GO function is also enriched in the Wnt, IL-17, and MAPK signaling pathways in the KEGG database. PCR results were consistent with those of RNA sequencing, suggesting that the results of transcriptome sequencing were reliable. Conclusion. The three methods and three-acupoint technique can promote the recovery of SNI model rats by altering the gene sequence in DRGs.

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Identification of Differentially Expressed and Spliced Genes with RNA Sequencing Analysis of CLL Specimens

Blood ◽

10.1182/blood.v120.21.4582.4582 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4582-4582

Author(s):

Wei Liao ◽

Gwen Jordaan ◽

Artur Jaroszewicz ◽

Matteo Pellegrini ◽

Sanjai Sharma

Keyword(s):

B Cells ◽

Rna Sequencing ◽

Differentially Expressed Genes ◽

Fold Change ◽

Differentially Expressed ◽

Pcr Analysis ◽

Sequencing Analysis ◽

Rna Seq ◽

Mrna Isoforms ◽

Core Set

Abstract Abstract 4582 High throughput sequencing of cellular mRNA provides a comprehensive analysis of the transcriptome. Besides identifying differentially expressed genes in different cell types, it also provides information of mRNA isoforms and splicing alterations. We have analyzed two CLL specimens and a normal peripheral blood B cells mRNA by this approach and performed data analysis to identify differentially expressed and spliced genes. The result showed CLLs specimens express approximately 40% more transcripts compared to normal B cells. The FPKM data (fragment per kilobase of exon per million) revealed a higher transcript expression on chromosome 12 in CLL#1 indicating the presence of trisomy 12, which was confirmed by fluorescent in-situ hybridization assay. With a two-fold change in FPKM as a cutoff and a p value cutoff of 0.05 as compared to the normal B cell control, 415 genes and 174 genes in CLL#1 and 676 and 235 genes in CLL#2 were up and downregulated or differentially expressed. In these two CLL specimens, 45% to 75% of differentially expressed genes are common to both the CLL specimens indicating that genetically disparate CLL specimens have a high percentage of a core set of genes that are potentially important for CLL biology. Selected differentially expressed genes with increased expression (selectin P ligand, SELPLG, and adhesion molecule interacts with CXADR antigen 1, AMICA) and decreased (Fos, Jun, CD69 and Rhob) expression based on the FPKM from RNA-sequencing data were also analyzed in additional CLL specimens by real time PCR analysis. The expression data from RNA-seq closely matches the fold-change in expression as measured by RT-PCR analysis and confirms the validity of the RNA-seq analysis. Interestingly, Fos was identified as one of the most downregulated gene in CLL. Using the Cufflinks and Cuffdiff software, the splicing patterns of genes in CLL specimens and normal B cells were analyzed. Approximately, 1100 to 1250 genes in the two CLL specimens were significantly differentially spliced as compared to normal B cells. In this analysis as well, there is a core set of 800 common genes which are differentially spliced in the two CLL specimens. The RNA-sequencing analysis accurately identifies differentially expressed novel genes and splicing variations that will help us understand the biology of CLL. Disclosures: No relevant conflicts of interest to declare.

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Abstract 128: RNA-seq Based Non-Invasive Endovascular Biopsy of Brain Arteriovenous Malformations

Stroke ◽

10.1161/str.51.suppl_1.128 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Ethan Winkler ◽

David McCoy ◽

Zhengda Sun ◽

Daniel Cooke

Keyword(s):

Differentially Expressed Genes ◽

Pearson Correlation ◽

Rna Extraction ◽

Correlation Coefficients ◽

Mapk Signaling ◽

Differentially Expressed ◽

Rna Seq ◽

Detachable Coil ◽

Illumina Hiseq ◽

Non Invasive

Introduction: To-date, there is no accurate means to identify which bAVMs will bleed and treatment remains controversial. Hypothesis: We developed an endovascular biopsy (EB) technique to isolate endothelial cells (ECs) from bAVMs in patients. We hypothesized this technique would allow RNA-seq analysis of relevant bAVM-related molecular pathways. Methods: EB was performed during angiography for bAVM patients undergoing resection. Cells were obtained from a bAVM juxta-nidal feeding artery and iliac artery (control) with a detachable coil and 0.035 inch wire. ECs were isolated with fluorescence assisted cell sorting (FACS). bAVM tissue was obtained from surgery, dissociated and underwent FACS sorting. Total RNA extraction and library preparation was performed, and samples sequenced on an Illumina HiSeq 4000 sequencer. Reads were aligned with Kallisto, and differentially expressed genes identified between bAVM and control with Sleuth using likelihood ratio tests. Correlations between EB and resected tissues were calculated with Pearson correlation coefficients. Principle Component Analysis (PCA) was used to assess for cell clustering. Results: EB was performed in 4 patients without complication or adverse event. PCA showed separation of bAVM ECs from controls. Analysis demonstrated 106 differentially expressed genes (FDR p ≤ 0.05). KEGG pathway analysis on these genes revealed enrichment in bAVM-related RAS/MAPK cell signaling functionally related to trophic factor, chemokine and gap junction signaling pathways. Detected genes were strongly correlated between EB and ECs isolated from resected tissues (R 2 = 0.77 for artery, nidus, and vein tissue). Results shown in Figure 1 . Conclusions: EB is a safe technique to permit non-invasive sequencing of bAVMs. These results implicate dysregulated KRAS/MAPK signaling in adult bAVMs. Whether this technique will allow for better natural history prediction or targeted medical therapies requires future study.

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Comprehensive MicroRNA Expression Profile of the Mammary Gland in Lactating Dairy Cows With Extremely Different Milk Protein and Fat Percentages

Frontiers in Genetics ◽

10.3389/fgene.2020.548268 ◽

2020 ◽

Vol 11 ◽

Author(s):

Xiaogang Cui ◽

Shengli Zhang ◽

Qin Zhang ◽

Xiangyu Guo ◽

Changxin Wu ◽

...

Keyword(s):

Mammary Gland ◽

Rna Sequencing ◽

Differentially Expressed Genes ◽

Small Rna ◽

Milk Protein ◽

Milk Composition ◽

Differentially Expressed ◽

Small Rna Sequencing ◽

Rna Seq

A total of 31 differentially expressed genes in the mammary glands were identified in our previous study using RNA sequencing (RNA-Seq), for lactating cows with extremely high and low milk protein and fat percentages. To determine the regulation of milk composition traits, we herein investigated the expression profiles of microRNA (miRNA) using small RNA sequencing based on the same samples as in the previous RNA-Seq experiment. A total of 497 known miRNAs (miRBase, release 22.1) and 49 novel miRNAs among the reads were identified. Among these miRNAs, 71 were found differentially expressed between the high and low groups (p < 0.05, q < 0.05). Furthermore, 21 of the differentially expressed genes reported in our previous RNA-Seq study were predicted as target genes for some of the 71 miRNAs. Gene ontology and KEGG pathway analyses showed that these targets were enriched for functions such as metabolism of protein and fat, and development of mammary gland, which indicating the critical role of these miRNAs in regulating the formation of milk protein and fat. With dual luciferase report assay, we further validated the regulatory role of 7 differentially expressed miRNAs through interaction with the specific sequences in 3′UTR of the targets. In conclusion, the current study investigated the complexity of the mammary gland transcriptome in dairy cattle using small RNA-seq. Comprehensive analysis of differential miRNAs expression and the data from previous study RNA-seq provided the opportunity to identify the key candidate genes for milk composition traits.

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Transcriptome Analysis of Orbital Adipose Tissue in Active Thyroid Eye Disease Using Next Generation RNA Sequencing Technology

The Open Ophthalmology Journal ◽

10.2174/1874364101812010041 ◽

2018 ◽

Vol 12 (1) ◽

pp. 41-52 ◽

Cited By ~ 2

Author(s):

Bradford W. Lee ◽

Virender B. Kumar ◽

Pooja Biswas ◽

Audrey C. Ko ◽

Ramzi M. Alameddine ◽

...

Keyword(s):

Adipose Tissue ◽

Differential Expression ◽

Rna Sequencing ◽

Differentially Expressed Genes ◽

Thyroid Eye Disease ◽

Differentially Expressed ◽

Eye Disease ◽

Healthy Controls ◽

Rna Seq ◽

Next Generation

Objective: This study utilized Next Generation Sequencing (NGS) to identify differentially expressed transcripts in orbital adipose tissue from patients with active Thyroid Eye Disease (TED) versus healthy controls. Method: This prospective, case-control study enrolled three patients with severe, active thyroid eye disease undergoing orbital decompression, and three healthy controls undergoing routine eyelid surgery with removal of orbital fat. RNA Sequencing (RNA-Seq) was performed on freshly obtained orbital adipose tissue from study patients to analyze the transcriptome. Bioinformatics analysis was performed to determine pathways and processes enriched for the differential expression profile. Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) was performed to validate the differential expression of selected genes identified by RNA-Seq. Results: RNA-Seq identified 328 differentially expressed genes associated with active thyroid eye disease, many of which were responsible for mediating inflammation, cytokine signaling, adipogenesis, IGF-1 signaling, and glycosaminoglycan binding. The IL-5 and chemokine signaling pathways were highly enriched, and very-low-density-lipoprotein receptor activity and statin medications were implicated as having a potential role in TED. Conclusion: This study is the first to use RNA-Seq technology to elucidate differential gene expression associated with active, severe TED. This study suggests a transcriptional basis for the role of statins in modulating differentially expressed genes that mediate the pathogenesis of thyroid eye disease. Furthermore, the identification of genes with altered levels of expression in active, severe TED may inform the molecular pathways central to this clinical phenotype and guide the development of novel therapeutic agents.

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PSII-11 RNA sequencing analysis reveals differentially expressed genes and novel upstream transcriptional regulators in porcine longissimus dorsi muscle affected by dietary lysine restriction

Journal of Animal Science ◽

10.1093/jas/skz258.474 ◽

2019 ◽

Vol 97 (Supplement_3) ◽

pp. 233-233

Author(s):

Ying Wang ◽

Huaijun Zhou ◽

Shengfa F Liao

Keyword(s):

Protein Synthesis ◽

Rna Sequencing ◽

Differentially Expressed Genes ◽

Muscle Protein ◽

Transcriptional Regulators ◽

Growing Pigs ◽

Differentially Expressed ◽

Longissimus Dorsi ◽

Sequencing Analysis ◽

Rna Seq

Abstract The objective of this research was to investigate the effects of dietary lysine restriction on the global gene expression of skeletal muscle in growing pigs. Twelve crossbred (Yorkshire × Landrace) barrows (initial BW 22.6 ± 2.04 kg) were randomly assigned to two dietary treatments (Diet I: a lysine-deficient diet; Diet II: a lysine-adequate diet) according to a completely randomized experiment design (n = 6). After feeding for 8 weeks, muscle samples were collected from longissimus dorsi of individual pigs (approximately 2 g/each). The total RNA isolated was used to prepare cDNA library for RNA sequencing (RNA-Seq) analysis. The RNA-Seq data was then analyzed using the CLC Genomics Workbench to identify differentially expressed genes (DEGs). Sixty-nine genes were found differentially expressed (Benjamin-Hochberg corrected P < 0.05) in Diet I vs. Diet II pigs, of which 29 genes were down-regulated (Log₂ fold change (FC) < - 0.58) and 40 genes were up-regulated (Log₂ FC > 0.58). Gene ontology (GO) analysis of these DEGs for functional annotation using DAVID found a total of 36 GO terms. The significantly enriched terms (Benjamin-Hochberg corrected P < 0.05) are associated with biological processes that include acute-phase response, platelet activation, and protein polymerization, and Molecular Functions that include serine-type endopeptidase inhibitor activity, small molecule binding, heme binding, and oxidoreductase activity. In addition, Ingenuity Pathway Analysis predicted some upstream transcriptional regulators that regulate several sets of DEGs. For example, lysine restriction may lead inhibition of insulin, EIF2AK4 (an eIF2α activator), and MYC (a transcript elongation factor), which are associated with the regulation of protein synthesis. It may also lead activation of STAT3 and HNF1A, which regulate cell movement and fatty acid metabolism, respectively. In summary, these novel results showed that dietary lysine restriction may compromise pig muscle protein synthesis through the aforementioned transcriptional regulators and their affected genes.

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Identification of hypoxia-specific biomarkers in salmonids using RNA-sequencing and validation using high-throughput qPCR

10.1101/2020.05.14.086090 ◽

2020 ◽

Author(s):

Arash Akbarzadeh ◽

Aimee Lee S. Houde ◽

Ben J.G. Sutherland ◽

Oliver P. Günther ◽

Kristina M. Miller

Keyword(s):

Gene Expression ◽

Dissolved Oxygen ◽

Rna Sequencing ◽

Differentially Expressed Genes ◽

Chinook Salmon ◽

Differentially Expressed ◽

Early Gene ◽

Hypoxic Stress ◽

Rna Seq ◽

Anthropogenic Habitat

AbstractIdentifying early gene expression responses to hypoxia (i.e., low dissolved oxygen) as a tool to assess the degree of exposure to this stressor is crucial for salmonids, because they are increasingly exposed to hypoxic stress due to anthropogenic habitat change, e.g., global warming, excessive nutrient loading, and persistent algal blooms. Our goal was to discover and validate gill gene expression biomarkers specific to the hypoxia response in salmonids across multi-stressor conditions. Gill tissue was collected from 24 freshwater juvenile Chinook salmon (Oncorhynchus tshawytscha), held in normoxia [dissolved oxygen (DO) > 8 mg L−1] and hypoxia (DO = 4□5 mg L−1) in 10 and 18°C temperatures for up to six days. RNA-sequencing (RNA-seq) was then used to discover 240 differentially expressed genes between hypoxic and normoxic conditions, but not affected by temperature. The most significantly differentially expressed genes had functional roles in the cell cycle and suppression of cell proliferation associated with hypoxic conditions. The most significant genes (n = 30) were selected for real-time qPCR assay development. These assays demonstrated a strong correlation (r = 0.88; p < 0.001) between the expression values from RNA-seq and the fold changes from qPCR. Further, qPCR of the 30 candidate hypoxia biomarkers was applied to an additional 322 Chinook salmon exposed to hypoxic and normoxic conditions to reveal the top biomarkers to define hypoxic stress. Multivariate analyses revealed that smolt stage, water salinity, and morbidity status were relevant factors to consider with the expression of these genes in relation to hypoxic stress. These hypoxia candidate genes will be put into application screening Chinook salmon to determine the identity of stressors impacting the fish.

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ViDGER: An R package for integrative interpretation of differential gene expression results of RNA-seq data

10.1101/268896 ◽

2018 ◽

Cited By ~ 4

Author(s):

Adam McDermaid ◽

Brandon Monier ◽

Jing Zhao ◽

Qin Ma

Keyword(s):

Gene Expression ◽

Rna Sequencing ◽

Differential Gene Expression ◽

Differentially Expressed Genes ◽

R Package ◽

Differentially Expressed ◽

Rna Seq ◽

Differential Gene

AbstractDifferential gene expression (DGE) is one of the most common applications of RNA-sequencing (RNA-seq) data. This process allows for the elucidation of differentially expressed genes (DEGs) across two or more conditions. Interpretation of the DGE results can be non-intuitive and time consuming due to the variety of formats based on the tool of choice and the numerous pieces of information provided in these results files. Here we present an R package, ViDGER (Visualization of Differential Gene Expression Results using R), which contains nine functions that generate information-rich visualizations for the interpretation of DGE results from three widely-used tools, Cuffdiff, DESeq2, and edgeR.

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Identification of Hypoxia-Specific Biomarkers in Salmonids Using RNA-Sequencing and Validation Using High-Throughput qPCR

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401487 ◽

2020 ◽

Vol 10 (9) ◽

pp. 3321-3336

Author(s):

Arash Akbarzadeh ◽

Aimee Lee S Houde ◽

Ben J G Sutherland ◽

Oliver P Günther ◽

Kristina M Miller

Keyword(s):

Gene Expression ◽

Dissolved Oxygen ◽

Rna Sequencing ◽

Differentially Expressed Genes ◽

Chinook Salmon ◽

Differentially Expressed ◽

Early Gene ◽

Hypoxic Stress ◽

Rna Seq ◽

Anthropogenic Habitat

Abstract Identifying early gene expression responses to hypoxia (i.e., low dissolved oxygen) as a tool to assess the degree of exposure to this stressor is crucial for salmonids, because they are increasingly exposed to hypoxic stress due to anthropogenic habitat change, e.g., global warming, excessive nutrient loading, and persistent algal blooms. Our goal was to discover and validate gill gene expression biomarkers specific to the hypoxia response in salmonids across multi-stressor conditions. Gill tissue was collected from 24 freshwater juvenile Chinook salmon (Oncorhynchus tshawytscha), held in normoxia [dissolved oxygen (DO) > 8 mg L-1] and hypoxia (DO = 4‒5 mg L-1) in 10 and 18° temperatures for up to six days. RNA-sequencing (RNA-seq) was then used to discover 240 differentially expressed genes between hypoxic and normoxic conditions, but not affected by temperature. The most significantly differentially expressed genes had functional roles in the cell cycle and suppression of cell proliferation associated with hypoxic conditions. The most significant genes (n = 30) were selected for real-time qPCR assay development. These assays demonstrated a strong correlation (r = 0.88; P < 0.001) between the expression values from RNA-seq and the fold changes from qPCR. Further, qPCR of the 30 candidate hypoxia biomarkers was applied to an additional 322 Chinook salmon exposed to hypoxic and normoxic conditions to reveal the top biomarkers to define hypoxic stress. Multivariate analyses revealed that smolt stage, water salinity, and morbidity status were relevant factors to consider with the expression of these genes in relation to hypoxic stress. These hypoxia candidate genes will be put into application screening Chinook salmon to determine the identity of stressors impacting the fish.

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Comparative analysis of gene expression profiles in differentiated subcutaneous adipocytes between Jiaxing Black and Large White pigs

BMC Genomics ◽

10.1186/s12864-020-07361-9 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Dawei Zhang ◽

Wenjing Wu ◽

Xin Huang ◽

Ke Xu ◽

Cheng Zheng ◽

...

Keyword(s):

Signaling Pathway ◽

Differentially Expressed Genes ◽

Expression Profiles ◽

Subcutaneous Fat ◽

Mapk Signaling ◽

Fat Deposition ◽

Differentially Expressed ◽

Rna Seq ◽

Large White ◽

Pig Breeds

Abstract Background Chinese domestic pig breeds are reputed for pork quality, but their low ratio of lean-to-fat carcass weight decreases production efficiency. A better understanding of the genetic regulation network of subcutaneous fat tissue is necessary for the rational selection of Chinese domestic pig breeds. In the present study, subcutaneous adipocytes were isolated from Jiaxing Black pigs a Chinese indigenous pig breed with redundant subcutaneous fat deposition and Large White pigs a lean-type pig breed with relatively low subcutaneous fat deposition. The expression profiles of mRNAs and lncRNAs were compared by RNA-seq analysis to identify biomarkers correlated with the differences of subcutaneous fat deposition between the two breeds. Results A total of 1058 differentially expressed genes and 221 differentially expressed lncRNAs were identified in subcutaneous adipocytes between Jiaxing Black and Large White pigs, which included 275 up-regulated mRNAs, 783 down-regulated mRNAs, 118 up-regulated lncRNAs and 103 down-regulated lncRNAs. Gene Ontology and KEGG pathway enrichment analyses revealed that the differentially expressed genes and differentially expressed lncRNAs were mainly involved in the immune response, cell fate determination, PI3K-Akt signaling pathway and MAPK signaling pathway, which are known to be related to adipogenesis and lipid metabolism. The expression levels of differentially expressed genes and differentially expressed lncRNAs according to the RNA-seq data were verified by quantitative PCR, which showed 81.8% consistency. The differences in MAPK pathway activity between Jiaxing Black and Large White pigs was confirmed by western blot analysis, which revealed elevated p38 phosphorylation in Jiaxing Black pigs. Conclusions This study offers a detailed characterization of mRNAs and lncRNAs in fat- and lean-type pig breeds. The activity of the MAPK signaling pathway was found to be associated with subcutaneous adipogenesis. These results provide new targets for further investigation of the molecular mechanisms regulating subcutaneous fat deposition in pigs.

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RNA-Seq based exploration of differentially expressed genes (DEGs) in cotton(Gossypium hirsutum. L) upon infection with Aspergillus tubingensis

10.21203/rs.3.rs-770815/v1 ◽

2021 ◽

Author(s):

Maria Khizar ◽

Jianxin Shi ◽

Urooj Haroon ◽

Musrat Ali ◽

Fiza Liaquat ◽

...

Keyword(s):

Gossypium Hirsutum ◽

Differentially Expressed Genes ◽

Defense Responses ◽

Mapk Signaling ◽

Differentially Expressed ◽

Ascorbate Oxidase ◽

Resistant Variety ◽

Potential Candidate ◽

Aspergillus Tubingensis ◽

Rna Seq

Abstract Differentially expressed genes help in exploring plant defense mechanism under variable stress conditions. In current investigation, RNA sequencing was executed to explore the differential gene expression in resistant and susceptible varieties of Cotton (Gossypium hirsutum), upon infection with Aspergillus tubingensis. Comparative RNA-Seq of control and infected plants was performed using Illumina HiSeq 2,500. Overall 79.84 G clean data was generated and 6,558 DEGs were identified in both varieties, in response to pathogen inoculation. Differentially expressed genes were found to be involved in defense, antifungal response, signaling pathways, oxidative burst and transcription. Genes involved in defense responses, MAPK signaling, cell wall fortification and signal transduction were highly induced in resistant variety. Real time PCR also revealed the up regulation of MAPKKK YODA like, L-ascorbate oxidase, late embryogenesis abundant protein (At1g64065) and flavonoid 3',5'-hydroxylase-like, in resistant variety. Elevated accumulation of such DEGs in resistant variety could function as the source for identifying biomarkers for breeding and these can be used as potential candidate genes for transgenic manipulation. Their study also helped in understanding complex plant-fungal interaction and advanced the understanding of plant-microbe interaction. Inclusively, our findings provide an indispensable foundation for advanced understanding of the plant resistance mechanisms of cotton.

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