scholarly journals Hypermethylation at CpG islands of GSTP1 gene’s promoter is the typical property of breast cancer in Vietnamese population

2015 ◽  
Vol 18 (2) ◽  
pp. 105-112
Author(s):  
Thao Thi Phuong Doan ◽  
Phuong Kim Truong ◽  
Thuan Duc Lao ◽  
Thuy Huyen Ai Le
Keyword(s):  
Breast Cancer ◽  
Breast Tissue ◽  
Cpg Island ◽  
Medical Center ◽  
Methylation Status ◽  
Cpg Islands ◽  
Potential Candidate ◽  
Potential Candidate Gene ◽  
Vietnamese Population ◽  
Near Future

The demand for biomarker that applied in prognosis, early diagnosis, predicting and/or monitoring the therapeutic response and detection of recurrent cancer is the worldwide expection. One of the biomarker, which the worldwide researches focused on, is the hypermethylation at CpG island of promoter which undergoes the DNA methylation changes in carcinogenesis. In current study, the target gene is GSTP1 (Glutathion-Stransferase pi 1 gene), with one of the most function is particularly involved in detoxification. Because of this function, GSTP1 (obviously based on the frequencies of methylation) has been chosen as a potential candidate gene for several clinical trials in predicting of the response to the demethylation drugs in several cancers, including breast cancer. In this study, we foscused on the evaluation of the methylation status of 115 biopsy specimens collected from university medical center HCMC, including 95 breast cancer specimens and 20 noncancerous breast tissue. The results showed that 41 of 95 (43.2 %) breast cancer specimens are hypermethylated, no methylation was found on the noncancerous breast tissue (p<0.01). These results allowed us to predict the totally corresponding to the application of the treatment, which based on the demethylation drug, one of the epi-drug line, in Vietnamese patients in the near future.

Relevance of Aromatase Inhibitors in Breast Cancer Treatment

2021 ◽  
Vol 21 ◽  
Author(s):  
Ankita Sood ◽  
Damanpreet Kaur Lang ◽  
Rajwinder Kaur ◽  
Balraj Saini ◽  
Sandeep Arora
Keyword(s):  
Breast Cancer ◽  
Aromatase Inhibitors ◽  
Breast Tissue ◽  
Positive Impact ◽  
Treatment Options ◽  
Living Standards ◽  
Efficacious Treatment ◽  
Clinical Resistance ◽  
Steroidal Aromatase Inhibitors ◽  
Near Future

: Efficacious treatment for breast cancer is still a challenge despite the presence of various treatment options. Aromatase enzyme present in the breast tissue is responsible for estrogen formation from androgens. Aromatase inhibitors manifest remarkably ameliorated therapeutic efficacy as compared to the current therapeutic options available and exhibit a better safety profile as compared to the other drugs. Clinical resistance to aromatase inhibitors is perceived as a lack of growth inhibition by aromatase inhibitors treatment and cancer therapy becomes ineffective in causing a decrease in the size of the tumor. Naturally extracted aromatase inhibitors have a huge positive impact on vitality and living standards. This review article highlights the particulars about the currently approved steroidal and non-steroidal aromatase inhibitors for clinical use, adverse effects associated with their use and approach to tackling the problem, various strategies to overcome aromatase inhibitors resistance, information on the synthesis of various peculiar aromatase inhibitors which can prove as highly efficient and potent drugs in the near future and the drugs of natural and semi-synthetic origin which can demonstrate to be more efficient, potent and less-toxic than conventional therapy.

scholarly journals DNA Methylation of Endoglin Pathway Genes in Pregnant Women With and Without Preeclampsia

2020 ◽  
Vol 13 ◽  
pp. 251686572095968
Author(s):  
Allison H Rietze ◽  
Yvette P Conley ◽  
Dianxu Ren ◽  
Cindy M Anderson ◽  
James M Roberts ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Control Participant ◽  
Sample Collection ◽  
Promoter Regions ◽  
Participant Group

Objective: We compared blood-based DNA methylation levels of endoglin ( ENG) and transforming growth factor beta receptor 2 ( TGFβR2) gene promoter regions between women with clinically-overt preeclampsia and women with uncomplicated, normotensive pregnancies. Methods: We used EpiTect Methyl II PCR Assays to evaluate DNA methylation of CpG islands located in promoter regions of ENG (CpG Island 114642) and TGFβR2 (CpG Island 110111). Preeclampsia was diagnosed based on blood pressure, protein, and uric acid criteria. N = 21 nulliparous preeclampsia case participants were 1:1 frequency matched to N = 21 nulliparous normotensive control participants on gestational age at sample collection (±2 weeks), smoking status, and labor status at sample collection. Methylation values were compared between case and control participant groups [( ENG subset: n = 20 (9 cases, 11 controls); TGFβR2 subset: n = 28 (15 cases, 13 controls)]. Results: The majority of the preeclampsia cases delivered at ⩾34 weeks’ gestation (83%). Average methylation levels for ENG ([M ± (SD)]; Case Participant Group = 6.54% ± 4.57 versus Control Participant group = 4.81% ± 5.08; P = .102) and TGFβR2 (Case Participant Group = 1.50% ± 1.37 vs Control Participant Group = 1.70% ± 1.40; P = .695) promoter CpG islands did not differ significantly between the participant groups. Removal of 2 extreme outliers in the ENG analytic subset revealed a trend between levels of ENG methylation and pregnancy outcome (Case Participant Group = 5.17% ± 2.16 vs Control Participant Group = 3.36% ± 1.73; P = .062). Conclusion: Additional epigenetic studies that include larger sample sizes, investigate preeclampsia subtypes, and capture methylation status of CpG island shores and shelves are needed to further inform us of the potential role that ENG and TGFβR2 DNA methylation plays in preeclampsia pathophysiology.

scholarly journals Integrated Analysis of mRNA Expression, CpG Island Methylation, and Polymorphisms in the MITF Gene in Ducks (Anas platyrhynchos)

2019 ◽  
Vol 2019 ◽  
pp. 1-9
Author(s):  
Ruiyi Lin ◽  
Weimin Lin ◽  
Shiye Zhou ◽  
Qiaohui Chen ◽  
Jiahua Pan ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Methylation Level ◽  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Plumage Coloration ◽  
Integrated Analysis ◽  
Nucleotide Polymorphisms ◽  
White Feather ◽  
Black Feather

Microphthalmia-associated transcription factor (MITF) is a key regulator for the development and function of melanocytes in skin, eye, and plumage pigmentations. Thus, the MITF was selected as a candidate gene associated with plumage coloration in ducks. This study analyzed the mRNA expression, promoter methylation, and polymorphisms in the MITF gene in ducks with different plumage colors (Putian Black, Putian White, Liancheng White, and Longsheng Jade-green). No expression of the MITF melanin-specific isoform (MITF-M) was detected in white feather bulbs. By contrast, the mRNA expression levels of MITF-M were high in black feather bulbs. Bioinformatics analysis showed that two CpG islands were present in the promoter region of the MITF gene. The methylation level of the second CpG island was significantly lower in black feather bulbs than in white feather bulbs. However, the methylation level of the first CpG island was not different among the feather bulbs with various colors except Liancheng White feather bulbs. The methylation status of the whole CpG island significantly and negatively correlated with the mRNA expression of MITF-M (P<0.05). Furthermore, four novel SNPs (single nucleotide polymorphisms) were identified in the 5′UTR, exon 4, intron 7, and intron 8 of the MITF gene. Allele T in g.39807T>G and allele G in g.40862G>A were the predominant alleles only found in Putian White, whereas the variant A allele in g.32813G>A exhibited a high allele frequency in Liancheng White. Collectively, these results contributed to the understanding of the function of the MITF gene in duck plumage coloration.

Pyrosequencing Analysis of the Methylation Status of the CpG Island in the Retinoic Acid Receptor-β2 (RAR-β2) Gene Promoter.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4297-4297
Author(s):  
Da-Cheng Zhou ◽  
David Reynolds ◽  
Robert E. Gallagher
Keyword(s):  
Retinoic Acid ◽  
Cell Lines ◽  
Cpg Island ◽  
Retinoic Acid Receptor ◽  
Methylation Status ◽  
Cpg Islands ◽  
Leukemia Cell ◽  
Gene Promoter ◽  
Cpg Dinucleotides ◽  
Retinoic Acid Receptor Β2

Abstract CpG islands are associated with the 5′-ends of most housekeeping genes and many regulated genes. We have hypothesized that the methylation status of CpG islands in the promoter region of all-trans retinoic acid (ATRA) target genes such as retinoic acid receptor-β2 (RAR-β2) may be related to ATRA resistance and relapse of acute promyelocytic leukemia (APL). In the present study, we developed a highly quantitative method to assess the degree of DNA methylation at specific sites using PyrosequencingTM technology (Biotage, Uppsala, Sweden). This method is more quantitative than methylation-specific PCR, and is as accurate as but simpler and more robust than combined bisulfite restriction analysis (COBRA) or direct sequencing of plasmid clones of PCR products. We used this method to study 14 CpG dinucleotides in the CpG island of the RAR-β2 promoter. In reconstruction experiments in which 100% methylated and 100% unmethylated DNAs were admixed in different proportions (100:0; 80:20, 60:40, etc), a straightline graph was obtained over the entire range from 0 – 100% for each of the 14 CpG dinucleotides (r2 &gt; 0.98). The results were highly reproducible and the variation between the results obtained from repetitive pyrosequencing of the same DNA was very low (S.D.&lt;2%). Also the standard deviation between measurements of different PCR-amplified, bisulfite-converted DNAs prepared in separate experiments was &lt;5%. We then used this method to measure the methylation level of the CpG island of the RAR-β2 promoter in several leukemia cell lines. Of 3 APL cell lines, the two with PML-RARα mutations, i.e., UF-1 and AP-1060, had higher overall methylation, compared to the NB4 cell line with non-mutant PML-RARα (mean ± SD = 52 ± 25% and 55 ± 21%, versus 43 ± 20%; p = 0.04 and 0.08, respectively; SD calculated from the variation across the 14 CpG dinucleotides for each source). Two myeloid leukemia cell lines with predominantly erythroid lineage characteristics, K562 and TF-1, had much lower levels of RAR-β2 methylation (2.6 ± 0.9% and 8.9 ± 3.2%, respectively). In the AP-1060 culture system, recently developed in our lab, there was little difference in methylation status between the patient bone marrow source and an intermediate, non-immortalized cell strain AP-1060S (27 ± 13% vs. 31 ± 25%). Further, there was no difference between lower and higher passage generations of AP-1060S (31 ± 25% vs. 30 ± 26%), which had markedly different replicative potential, indicating that replicative senescence at higher AP-1060 passages was not associated with altered methylation of the RAR-β2 gene promoter. However, the established, immortalized AP-1060 cell line had significantly greater methylation (52 ± 25%) than either the bone marrow source or AP-1060S (p &lt;0.0001 and p = 0.0002, respectively), consistent with published reports of increased promoter methylation of cell lines. In conclusion, pyrosequencing is a high throughput method with great quantitative strength, and can be used for accurate and consistent analysis of methylation status in large numbers of samples.

Epigenetic Dysregulation of Candidate Cis-Regulatory Sequences in Hematological Malignancies.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2229-2229
Author(s):  
Reid F. Thompson ◽  
Maria E. Figueroa ◽  
Ari M. Melnick ◽  
John M. Greally
Keyword(s):  
Cytosine Methylation ◽  
Hematological Malignancies ◽  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Regulatory Sequences ◽  
Cpg Island Methylator Phenotype ◽  
Global Methylation ◽  
Methylator Phenotype ◽  
Tumor Types

Abstract Epigenetic changes (in particular, altered cytosine methylation) have been described in a variety of tumors. The CpG Island Methylator Phenotype (CIMP) is a well-known instance of this phenomenon wherein cytosine methylation is markedly dysregulated (normally hypomethylated loci shift to a methylated state). CIMP has been demonstrated in a number of different cancer types including hematological malignancies like AML. While methylation status has been studied predominantly at CpG islands, we used a novel assay (HELP; Khulan et al., Genome Res. 2006) to look for changes in cytosine methylation in large contiguous regions of the genome. We assessed global patterns of cytosine methylation by HELP analysis in a variety of tumor samples including leukemias and lymphomas. We found significant changes in the global methylation patterns of malignant cells, confirming prior observations of epigenetic dysregulation in these tumor types. We also discovered that the majority of the changes in cytosine methylation are occurring not at CpG islands but at other loci in the genome, including constitutively hypomethylated loci that we are finding to be candidate cis-regulatory sequences. We conclude that cytosine methylation changes in cancer occur much more extensively than analysis of CpG islands alone would indicate, and that the epigenetic dysregulation in cancer may be predominantly targeted to cis-regulatory sequences rather than to promoters.

Methylation Status of the Collagen Prolyl-3 Hydroxylases (C-P3H) and C-P4H Genes in Multiple Myeloma: P3H2 Is Selectively Methylated

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4639-4639
Author(s):  
Eleftheria Hatzimichael ◽  
Aggeliki Dasoula ◽  
Konstantinos Lagos ◽  
Georgianna Kartalou ◽  
George Dranitsaris ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Clinical Parameter ◽  
Small Population ◽  
Cpg Methylation ◽  
Post Translational Modification

Abstract Abstract 4639 Background–Aim: Prolyl hydroxylation is an important and the most common post-translational modification that affects the structure and function of collagen. Enzymes that participate in this process are the collagen prolyl-3 hydroxylases (C-P3H) and the C-P4H. We have previously showed that P3H3 is methylated in multiple solid tumour types, whereas methylation in P3H2 appears to be specific for breast cancer and multiple myeloma (MM). In this study we assessed the CpG methylation status of the P3H genes (P3H1, P3H2, P3H3) in a larger cohort of MM patients and for the first time the CpG methylation status of the P4H genes (P4HA1, P4HA2, P4HA3) and investigated for clinical relevance. Patients and Methods: Bone marrow aspirate samples from 47 MM patients (28 males, median age 66 years) were obtained at diagnosis. Methylation-specific PCR (MSP) was employed to study the CpG methylation in P3H1, P3H2, P3H3, P4HA1, P4HA2 and P4HA3 CpG islands. Genomic DNA was isolated and bisulphite modified using commercially available kits (QIAmp DNA mini kit, Qiagen and EZ DNA methylation kit, Zymo Research respectively). Control methylated and unmethylated genomic DNAs were included in each experiment. For the study of the P3H2 CpG methylation status we used 2 sets of independent primers, which map to different areas of P3H2 CpG island. Ten bone marrow samples from patients with border line thrombocytopenia that were proved to have no neoplasia served as negative controls. Logistic regression analyses were used to measure the association between gene methylation and age, b2 microglobulin, serum albumin, hypercalcemia, ISS stage, extramedullary disease, bone disease, anemia and renal failure (eGFR< 50 ml/min). Results: None of the six genes under study was methylated in the control bone marrow samples. Methylation in the CpG island of P3H2 was detected in 44 % of patients (95% CI 27.8–62.8%) No methylation was detected in the CpG islands of P3H1, P3H3, P4HA1, P4HA2 and P4HA3. Trends noted were that patients with methylated P3H2 were more likely to have ISS stage 3 (OR 2.2, p=0.1). Overall no significant association was found with any clinical parameter although the study might have been underpowered due to the small population size. Conclusions: P3H2 is methylated at high frequency in MM, whereas P3H1, P3H3, P4HA1, P4HA2, P4HA3 were unmethylated, implying a striking specificity for methylation in P3H2 in MM. These findings warrant further evaluation in a larger sample of patients in order to better define the prognostic and clinical utility of P3H2 methylation in MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase.

1996 ◽  
Vol 16 (8) ◽  
pp. 4555-4565 ◽  
Author(s):  
P M Vertino ◽  
R W Yen ◽  
J Gao ◽  
S B Baylin
Keyword(s):  
Enzyme Activity ◽  
De Novo ◽  
Cpg Island ◽  
Human Fibroblasts ◽  
Methylation Status ◽  
Cpg Islands ◽  
Parental Cell Line ◽  
Direct Role ◽  
Cpg Island Methylation ◽  
De Novo Methylation

Recent studies showing a correlation between the levels of DNA (cytosine-5-)-methyltransferase (DNA MTase) enzyme activity and tumorigenicity have implicated this enzyme in the carcinogenic process. Moreover, hypermethylation of CpG island-containing promoters is associated with the inactivation of genes important to tumor initiation and progression. One proposed role for DNA MTase in tumorigenesis is therefore a direct role in the de novo methylation of these otherwise unmethylated CpG islands. In this study, we sought to determine whether increased levels of DNA MTase could directly affect CpG island methylation. A full-length cDNA for human DNA MTase driven by the cytomegalovirus promoter was constitutively expressed in human fibroblasts. Individual clones derived from cells transfected with DNA MTase (HMT) expressed 1- to 50-fold the level of DNA MTase protein and enzyme activity of the parental cell line or clones transfected with the control vector alone (Neo). To determine the effects of DNA MTase overexpression on CpG island methylation, we examined 12 endogenous CpG island loci in the HMT clones. HMT clones expressing > or = 9-fold the parental levels of DNA MTase activity were significantly hypermethylated relative to at least 11 Neo clones at five CpG island loci. In the HMT clones, methylation reached nearly 100% at susceptible CpG island loci with time in culture. In contrast, there was little change in the methylation status in the Neo clones over the same time frame. Taken together, the data indicate that overexpression of DNA MTase can drive the de novo methylation of susceptible CpG island loci, thus providing support for the idea that DNA MTase can contribute to tumor progression through CpG island methylation-mediated gene inactivation.

scholarly journals p38β - MAPK11 and its role in female cancers

2020 ◽  
Author(s):  
Periklis Katopodis ◽  
Rachel Kerslake ◽  
Athanasios Zikopoulos ◽  
Nefeli Eirini Beri ◽  
Vladimir Anikin
Keyword(s):  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Her2 Status ◽  
Tissue Specific ◽  
Cancer Data ◽  
Signalling Molecules ◽  
Significant Difference

Abstract Background The p38MAPK family of Mitogen Activated Protein Kinases are a group of signalling molecules involved in cell growth, survival, proliferation and differentiation. The widely studied p38α isoform is ubiquitously expressed and is implicated in a number of cancer pathologies, as are p38γ and p38δ. However, the mechanistic role of the isoform, p38β, remains fairly elusive. Recent studies suggest a possible role of p38β in both breast and endometrial cancer with research suggesting involvement in bone metastasis and cancer cell survival. Female tissue specific cancers such as breast, endometrial, uterine and ovary account for over 3,000,000 cancer related incidents annually; advancements in therapeutics and treatment however require a deeper understanding of the molecular aetiology associated with these diseases. This study provides an overview of the MAPK signalling molecule p38β (MAPK11) in female cancers using an in-silico approach. Methods A detailed gene expression and methylation analysis was performed using datasets from cBioportal, CanSar and MEXPRESS. Breast, Uterine Endometrial, Cervical, Ovarian and Uterine Carcinosarcoma TCGA cancer datasets were used and analysed.Results Data using cBioportal and CanSAR suggest that expression of p38β is lower in cancers: BRCA, UCEC, UCS, CESC and OV compared to normal tissue. Methylation data from SMART and MEXPRESS indicate significant probe level variation of CpG island methylation status of the gene MAPK11. Analysis of the genes’ two CpG islands shows that the gene was hypermethylated in the CpG1 with increased methylation seen in BRCA, CESC and UCEC cancer data sets with a slight increase of expression recorded in cancer samples. CpG2 exhibited hypomethylation with no significant difference between samples and high levels of expression. Further analysis from MEXPRESS revealed no significance between probe methylation and altered levels of expression. In addition, no difference in the expression of BRCA oestrogen/progesterone/HER2 status was seen. Conclusion This data provides an overview of the expression of p38β in female tissue specific cancers, showing a decrease in expression of the gene in BRCA, UCEC, CESC, UCS and OV, increasing the understanding of p38β MAPK expression and offering insight for future in-vitro investigation and therapeutic application.

Genome-Wide DNA Methylation Profile of CLL with 17p del Allowed Identification of Multiple Epigenetically Inactivated Molecular Pathways with Prognostic Value in Human Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 492-492
Author(s):  
Wei-Gang Tong ◽  
William G. Wierda ◽  
Neby Bekele ◽  
Shao-Qing Kuang ◽  
Michael J. Keating ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Cpg Island ◽  
Trichostatin A ◽  
Methylation Status ◽  
Cpg Islands ◽  
Human Leukemia ◽  
Dna Hypomethylation ◽  
Line P ◽  
Normal Controls

Abstract Aberrant DNA methylation of multiple promoter associated CpG islands is a very prevalent phenomenon in human leukemias. Data from our laboratory indicates that methylation profiling allows the identification of leukemia patients with different risk and prognosis. Despite the advances in the understanding of the molecular biology of CLL, few studies of DNA methylation have been performed in CLL. In the current study, we have developed a new assay combining MCA (Methylated CpG island Amplification) with the Agilent promoter CpG array to identify simultaneously hundreds of abnormally methylated CpG islands in CLL. To perform this, we compared DNA from two CLL patients with 17p del (tester) with that of CD19+ B cells from two age-matched controls (driver). We identified 280 promoter CpG islands differentially methylated in CLL compared to normal controls. Most of these genes are located on chromosomes 19 (16%), 16 (11%), 17 (10%) and 11 (9%). We also performed interaction pathway and functional analysis of these 280 genes using the online Ingenuity Pathway Analysis tools. The initial analysis divided these genes into 25 functional networks, with the majority of genes fall into top 10 networks. The major functions of genes in these interaction networks involve cancer, organ development, cell death, drug metabolism, DNA replication and repair. We validated 22 of these genes (ADCY5, R-spondin1, LHX1, GALGT2, TFAP2C, ING1, SOX11, SOX14, SALL1, LTBP2, APP, DXL1, DLX4, KLK10, BCL11B, NR2F2, FAM62T, HAND2, BNC1, SPOCK, Prima1 and MLL1) in samples from 78 CLL patients and 10 age-matched normal controls. The characteristics of the 78 patients are: median age 59 (range 39–79), male 70%, Rai stage 0–II/III–IV (83%/17%), IgVH unmutated 49%, ZAP-70 positive 33%. Our results indicate that most of the genes identified by the array are frequently hypermethylated in CLL patients compared with healthy controls. Methylation frequency ranged from 20%–100% in CLL patients. Expression analysis of four selected genes (LHX1, GALGT2, TFAP2C and Prima1) in human leukemia cell lines and CLL patient samples by real-time PCR further confirmed methylation associated gene silencing, and treatment of these cell lines with hypomethylating agent 5-aza-2′-deoxycitidine with or without the HDAC inhibitor Trichostatin A resulted in gene re-expression and induction of DNA hypomethylation. We also analyzed the association of methylation status of these genes with IgVH mutation status, ZAP70 expression and patient survival. Unmutated IgVH was associated with increased methylation levels of LINE (p<0.0001), which is a marker for global gene methylation and SALL1 (p=0.00008). Expression of ZAP-70 (>20%) was associated with increased methylation levels of LINE (p<0.00001), MLL (p=0.02) and SALL1 (p=0.048). Further analysis showed that methylation status of LINE (p=0.007), SALL1 (p=0.019), ADCY5 (p=0.021), R-spondin1 (p=0.002) and APP (p=0.002) correlated with survival. In conclusion, our studies indicate that MCA/promoter array technique allows the identification of large number of promoter CpG islands aberrantly methylated in CLL and also the identification of novel tumor suppressors and signaling pathways that could be important in the tumorigenesis of CLL and other hematological malignancies.

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