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1.  MCM Paradox: Abundance of Eukaryotic Replicative Helicases and Genomic Integrity 
As a crucial component of DNA replication licensing system, minichromosome maintenance (MCM) 2–7 complex acts as the eukaryotic DNA replicative helicase. The six related MCM proteins form a heterohexamer and bind with ORC, CDC6, and Cdt1 to form the prereplication complex. Although the MCMs are well known as replicative helicases, their overabundance and distribution patterns on chromatin present a paradox called the “MCM paradox.” Several approaches had been taken to solve the MCM paradox and describe the purpose of excess MCMs distributed beyond the replication origins. Alternative functions of these MCMs rather than a helicase had also been proposed. This review focuses on several models and concepts generated to solve the MCM paradox coinciding with their helicase function and provides insight into the concept that excess MCMs are meant for licensing dormant origins as a backup during replication stress. Finally, we extend our view towards the effect of alteration of MCM level. Though an excess MCM constituent is needed for normal cells to withstand stress, there must be a delineation of the threshold level in normal and malignant cells. This review also outlooks the future prospects to better understand the MCM biology.
PMCID: PMC4217321  PMID: 25386362
2.  Epigenetic Silencing of CXCR4 Promotes Loss of Cell Adhesion in Cervical Cancer 
BioMed Research International  2014;2014:581403.
In the network of chemokine signaling pathways, recent reports have described the SDF-1α/CXCR4 axis and its role in cancer progression and metastasis. Interestingly, we found downregulation of CXCR4 at both transcript and protein level in cervical cancer cell lines and primary tumors. We also found CXCR4 promoter hypermethylation in cervical cancer cell lines and primary biopsy samples. DNA hypomethylating drug 5-AZA-2′-deoxycytidine and histone deacetylase inhibitor Trichostatin A treatments in cell lines reactivate both CXCR4 transcription and protein expression. Cell adhesion assay demonstrated that autocrine SDF-1α promotes the loss of cell adhesion while paracrine SDF-1α predominantly protects the normal cervical cells from loss of cell adhesion. Cervical cancer cell line C-33A having increased expression of CXCR4 after TSA treatment showed increased cell adhesion by paracrine source of SDF-1α in comparison to untreated C-33A. These findings demonstrate the first evidence that epigenetic silencing of CXCR4 makes the cells inefficient to respond to the paracrine source of SDF-1α leading to loss of cell adhesion, one of the key events in metastases and progression of the disease. Our results provide novel insight of SDF-1α/CXCR4 signaling in tumor microenvironment which may be promising to further delineate molecular mechanism of cervical carcinogenesis.
PMCID: PMC4119908  PMID: 25114911
3.  Role of ROBO4 Signalling in Developmental and Pathological Angiogenesis 
BioMed Research International  2014;2014:683025.
Transmembrane roundabout receptor family members (ROBO1–ROBO4) principally orchestrate the neuronal guidance mechanism of the nervous system. Secreted glycoprotein SLITs are the most appreciated ligands for ROBOs. Recently identified ROBO4 is the key mediator of SLIT-ROBO mediated developmental and pathological angiogenesis. Although SLIT2 has been shown to interact with ROBO4 as ligand, it remains an open question whether this protein is the physiologic partner of ROBO4. The purpose of this review is to summarise how reliable SLIT2 as ligand for ROBO4 is, if not what the other possible mechanisms demonstrated till date for ROBO4 mediated developmental and pathological angiogenesis are. We conclude that ROBO4 is expressed specially in vascular endothelial cells and maintains the vascular integrity via either SLIT2 dependent or SLIT2 independent manner. On the contrary, it promotes the pathological angiogenesis by involving different signalling arm(s)/unknown ligand(s). This review explores the interactions SLIT2/ROBO1, SLIT2/ROBO1–ROBO4, ROBO1/ROBO4, and ROBO4/UNC5B which can be promising and potential therapeutic targets for developmental angiogenesis defects and pathological angiogenesis. Finally we have reviewed the ROBO4 signalling pathways and made an effort to elaborate the insight of this signalling as therapeutic target of pathological angiogenesis.
PMCID: PMC3933320  PMID: 24689049
4.  Vimentin and Notch as biomarkers for breast cancer progression 
Oncology Letters  2014;7(3):721-727.
Breast cancer, the most common spontaneous malignancy diagnosed in women, is a classical model of hormone dependency as it is associated with prolonged exposure to female hormones. Different cytoplasmic proteins are important in the transformation of a normal cell to an invasive tumor cell, and these include vimentin and Notch. To investigate the importance of these two genes and proteins in breast carcinogenesis, we used an in vitro breast cancer model system, in which an immortalized human breast epithelial cell line, MCF-10F, was malignantly transformed by exposure to low doses of high linear energy transfer α particle (150 keV/μm) radiation and subsequent growth in the presence or absence of 17β-estradiol. This model consisted of human breast epithelial cells in different stages of transformation: i) a parental cell line (MCF-10F), ii) an Estrogen cell line (MCF-l0F continuously grown with estradiol at 10−8), iii) a malignant and non-tumorigenic cell line (Alpha3), iv) a malignant and tumorigenic cell line (Alpha5) and v) a Tumor2 cell line derived from a xenograft of the Alpha5 cell line injected into nude mice. Vimentin and Notch showed greater expression in the Alpha5 and Tumor2 cell lines compared with that in the non-tumorigenic cell lines, MCF-10F, Estrogen and Alpha3. In the present study, positive staining for vimentin was found in 21% of cases. Vimentin and Notch protein expression was negative in noninvasive ductal carcinoma biopsies from breast cancer patients. However, positive cell expression was observed in invasive ductal carcinoma biopsies. These biomarkers can be considered important indicators of breast cancer progression and can be added to the diagnostic panel when overall survival is a primary end-point.
PMCID: PMC3919898  PMID: 24527079
biomarkers; estrogen; radiation; breast cells; vimentin; Notch
5.  Down Regulation of FOXO1 Promotes Cell Proliferation in Cervical Cancer 
Journal of Cancer  2014;5(8):655-662.
The Forkhead transcription factor FOXO1, an important downstream target of phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway, regulates cellular homeostasis by maintaining cell proliferation, apoptosis and viability in normal cells. Though, the function and regulation of FOXO1 is well documented in many cancers, the molecular mechanism of its regulation in cervical cancer is largely unknown. In the present study we have investigated the role of PI3K inhibition on FOXO1 regulation. Expression profiling of primary tumors and cell lines show over expression of PIK3CA and AKT1; and down regulation of FOXO1. Lack of FOXO1 promoter methylation and inability of hypomethylating drug 5-Aza-2'-deoxycytidine and HDAC inhibitor trichostatin A to reactivate FOXO1 expression suggest that loss of FOXO1 expression is due to mechanisms other than promoter methylation/acetylation. Inhibition of PI3K by LY294002 decreased the level of p-AKT1 and activated FOXO1 transcription factor. We demonstrate that activation of FOXO1 induces apoptosis, cell proliferation arrest, and decreased cell viability in cervical cancer cell lines. Our data suggest that frequent down regulation of FOXO1 and its functional inactivation may be due to post-translational modifications in cervical cancer. Together, these observations suggest that activation of FOXO1 and its nuclear sequestration is critical in the regulation of cell proliferation, cell viability and apoptosis in cervical cancer. Hence, PI3K/AKT pathway may be a potential molecular target for cervical cancer therapy.
PMCID: PMC4142327  PMID: 25157276
Cervical cancer; PI3K/AKT; FOXO1; LY294002; Apoptosis.
6.  Over Expression of Minichromosome Maintenance Genes is Clinically Correlated to Cervical Carcinogenesis 
PLoS ONE  2013;8(7):e69607.
Minichromosome Maintenance (MCM) proteins play important roles in cell cycle progression by mediating DNA replication initiation and elongation. Among 10 MCM homologues MCM 2–7 form a hexamer and assemble to the pre-replication complex acting as replication licensing factors. Binding and function of MCM2-7 to pre-replication complex is regulated by MCM10 mediated binding of RECQL4 with MCM2-7. The purpose of this study is to explore the role of MCMs in cervical cancer and their correlation with the clinical parameters of cervical cancer. We have investigated sixty primary cervical cancer tissue samples, eight cervical cancer cell lines and thirty hysterectomised normal cervical tissue. The expression profiling of MCMs was done using semi-quantitative RT-PCR, immunoblotting and immunohistochemistry. MCM2, 4, 5, 6, 7, 10 and RECQL4 are significantly over-expressed in cervical cancer. Among these, MCM4, 6 and 10 show increased frequency of over expression along with advancement of tumor stages. MCM4, 5 and 6 also show differential expression in different types of lesion, while MCM2 and MCM10 are over expressed in cervical cancer irrespective of clinico-pathological parameters. Our data indicates the role of MCM4, MCM5, MCM6, MCM10 and RECQL4 in the progression of cervical cancer.
PMCID: PMC3714251  PMID: 23874974
7.  Protocadherin PCDH10, Involved in Tumor Progression, is a Frequent and Early Target of Promoter Hypermethylation in Cervical Cancer 
Genes, chromosomes & cancer  2009;48(11):983-992.
Cervical cancer (CC) is the second most common cancer in women. Currently no tractable molecular based therapeutic targets exist for patients with invasive CC and no predictive markers of risk assessment for progression of precancerous lesions are identified. New molecular insights into CC pathogenesis are urgently needed. Towards this goal, we first determined the copy number alterations of chromosome 4 and then examined the role of PCDH10 mapped to 4q28 as a candidate tumor suppressor gene. We identified monosomy 4 in 47% of 81 invasive CC studied by SNP array and found that 91% of 130 invasive CC harboring methylation in the promoter region of the PCDH10 gene. We then showed that aberrant promoter hypermethylation of PCDH10 is associated with down-regulation of gene expression and cell lines exposed to demethylating agent resulted in profound reactivated gene expression. We also showed that the promoter methylation in the PCDH10 gene occurs at an earliest identifiable stage of low-grade squamous intraepithelial lesion (LSIL). Our studies demonstrate that inactivation of PCDH10 may be a critical event in CC progression and form a potentially useful therapeutic target for CC.
PMCID: PMC3430375  PMID: 19681120
8.  Integrative genomic approaches in cervical cancer: implications for molecular pathogenesis 
Future oncology (London, England)  2010;6(10):1643-1652.
Cervical cancer (CC) as a single diagnostic entity exhibits differences in clinical behavior and poor outcomes in response to therapy in advanced tumors. Although infection of high-risk human papillomavirus is recognized as an important initiating event in cervical tumorigenesis, stratification of CC into subclasses for progression and response to treatment remains elusive. Existing knowledge of genetic, epigenetic and transcriptional alterations is inadequate in addressing the issues of diagnosis, progression and response to treatment. Recent technological advances in high-throughput genomics and the application of integrative approaches have greatly accelerated gene discovery, facilitating the identification of molecular targets. In this article, we discuss the results obtained by preliminary integrative analysis of DNA copy number increases and gene expression, utilizing the two most common copy number-gained regions of 5p and 20q in identifying gene targets in CC. These analyses provide insights into the roles of genes such as RNASEN, POLS and SKP2 on 5p, KIF3B, RALY and E2F1 at 20q11.2 and CSE1L, ZNF313 and B4GALT5 at 20q13.13. Future integrative applications using additional datasets, such as mutations, DNA methylation and clinical outcomes, will raise the promise of accomplishing the identification of biological pathways and molecular targets for therapies for patients with CC.
PMCID: PMC3037979  PMID: 21062161
amplification; cervical carcinoma; chromosome 5p; chromosome 20q; chromosome alteration; gene expression; integrative genomics; precancerous lesion; single nucleotide polymorphism array
9.  Burrow characteristics of the co-existing sibling species Mus booduga and Mus terricolor and the genetic basis of adaptation to hypoxic/hypercapnic stress 
BMC Ecology  2009;9:6.
The co-existing, sibling species Mus booduga and Mus terricolor show a difference in site-preference for burrows. The former build them in flat portion of the fields while the latter make burrows in earthen mounds raised for holding water in cultivated fields. In northern India which experiences great variation in climatic condition between summer and winter, M. booduga burrows have an average depth of 41 cm, as against 30 cm in southern India with less climatic fluctuation.
M. terricolor burrows are about 20 cm deep everywhere. The three chromosomal species M. terricolor I, II and III have identical burrows, including location of the nest which is situated at the highest position. In contrast, in M. booduga burrows, the nest is at the lowest position.
The nest chamber of M. booduga is located at greater depth than the nest chamber of M. terricolor. Also, in the burrows of M. booduga the exchange of air takes place only from one side (top surface) in contrast to the burrows of M. terricolor where air exchange is through three sides. Hence, M. booduga lives in relatively more hypoxic and hypercapnic conditions than M. terricolor.
We observed the fixation of alternative alleles in M. booduga and M. terricolor at Superoxide dismutase-1 (Sod-1), Transferrin (Trf) and Hemoglobin beta chain (Hbb) loci. All the three are directly or indirectly dependent on oxygen concentration for function. In addition to these, there are differences in burrow patterns and site-preference for burrows suggesting difference in probable adaptive strategy in these co-existing sibling species.
The burrow structure and depth of nest of the chromosomal species M. terricolor I, II and III are same everywhere probably due to the recency of their evolutionary divergence. Moreover, there is lack of competition for the well-adapted 'microhabitats' since they are non-overlapping in distribution. However, the co-existing sibling species M. booduga and M. terricolor exhibit mutual "exclusion" of the 'microhabitats' for burrow construction. Thus, location, structure and depth of the burrows might have been the contributory factors for selection of alternative alleles at three loci Sod-1, Trf and Hbb, which reflect difference in probable adaptive strategy in M. booduga and M. terricolor.
PMCID: PMC2678975  PMID: 19358716
10.  Integrative genomics analysis of chromosome 5p gain in cervical cancer reveals target over-expressed genes, including Drosha 
Molecular Cancer  2008;7:58.
Copy number gains and amplifications are characteristic feature of cervical cancer (CC) genomes for which the underlying mechanisms are unclear. These changes may possess oncogenic properties by deregulating tumor-related genes. Gain of short arm of chromosome 5 (5p) is the most frequent karyotypic change in CC.
To examine the role of 5p gain, we performed a combination of single nucleotide polymorphism (SNP) array, fluorescence in situ hybridization (FISH), and gene expression analyses on invasive cancer and in various stages of CC progression.
The SNP and FISH analyses revealed copy number increase (CNI) of 5p in 63% of invasive CC, which arises at later stages of precancerous lesions in CC development. We integrated chromosome 5 genomic copy number and gene expression data to identify key target over expressed genes as a consequence of 5p gain. One of the candidates identified was Drosha (RNASEN), a gene that is required in the first step of microRNA (miRNA) processing in the nucleus. Other 5p genes identified as targets of CNI play a role in DNA repair and cell cycle regulation (BASP1, TARS, PAIP1, BRD9, RAD1, SKP2, and POLS), signal transduction (OSMR), and mitochondrial oxidative phosphorylation (NNT, SDHA, and NDUFS6), suggesting that disruption of pathways involving these genes may contribute to CC progression.
Taken together, we demonstrate the power of integrating genomics data with expression data in deciphering tumor-related targets of CNI. Identification of 5p gene targets in CC denotes an important step towards biomarker development and forms a framework for testing as molecular therapeutic targets.
PMCID: PMC2440550  PMID: 18559093
11.  The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers 
The aryl hydrocarbon receptor repressor (AHRR) is a bHLH/Per-ARNT-Sim transcription factor located in a region of chromosome 5 (5p15.3) that has been proposed to contain one or more tumor suppressor genes. We report here consistent downregulation of AHRR mRNA in human malignant tissue from different anatomical origins, including colon, breast, lung, stomach, cervix, and ovary, and demonstrate DNA hypermethylation as the regulatory mechanism of AHRR gene silencing. Knockdown of AHRR gene expression in a human lung cancer cell line using siRNA significantly enhanced in vitro anchorage-dependent and -independent cell growth as well as cell growth after transplantation into immunocompromised mice. In addition, knockdown of AHRR in non-clonable normal human mammary epithelial cells enabled them to grow in an anchorage-independent manner. Further, downregulation of AHRR expression in the human lung cancer cell line conferred resistance to apoptotic signals and enhanced motility and invasion in vitro and angiogenic potential in vivo. Ectopic expression of AHRR in tumor cells resulted in diminished anchorage-dependent and -independent cell growth and reduced angiogenic potential. These results therefore demonstrate that AHRR is a putative new tumor suppressor gene in multiple types of human cancers.
PMCID: PMC2157559  PMID: 18172554
12.  Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression 
Molecular Cancer  2006;5:16.
Cervical Cancer (CC) exhibits highly complex genomic alterations. These include hemizygous deletions at 4p15.3, 10q24, 5q35, 3p12.3, and 11q24, the chromosomal sites of Slit-Robo pathway genes. However, no candidate tumor suppressor genes at these regions have been identified so far. Slit family of secreted proteins modulates chemokine-induced cell migration of distinct somatic cell types. Slit genes mediate their effect by binding to its receptor Roundabout (Robo). These genes have shown to be inactivated by promoter hypermethylation in a number of human cancers.
To test whether Slit-Robo pathway genes are targets of inactivation at these sites of deletion, we examined promoter hypermethylation of SLIT1, SLIT2, SLIT3, ROBO1, and ROBO3 genes in invasive CC and its precursor lesions. We identified a high frequency of promoter hypermethylation in all the Slit-Robo genes resulting in down regulated gene expression in invasive CC, but the inhibitors of DNA methylation and histone deacetylases (HDACs) in CC cell lines failed to effectively reactivate the down-regulated expression. These results suggest a complex mechanism of inactivation in the Slit-Robo pathway in CC. By analysis of cervical precancerous lesions, we further show that promoter hypermethylation of Slit-Robo pathway occurs early in tumor progression.
Taken together, these findings suggest that epigenetic alterations of Slit-Robo pathway genes (i) play a role in CC development, (ii) further delineation of molecular basis of promoter methylation-mediated gene regulation provides a potential basis for epigenetic-based therapy in advanced stage CC, and (iii) form epigenetic signatures to identify precancerous lesions at risk to progression.
PMCID: PMC1482714  PMID: 16700909
13.  Role of promoter hypermethylation in Cisplatin treatment response of male germ cell tumors 
Molecular Cancer  2004;3:16.
Male germ cell tumor (GCT) is a highly curable malignancy, which exhibits exquisite sensitivity to cisplatin treatment. The genetic pathway(s) that determine the chemotherapy sensitivity in GCT remain largely unknown.
We studied epigenetic changes in relation to cisplatin response by examining promoter hypermethylation in a cohort of resistant and sensitive GCTs. Here, we show that promoter hypermethylation of RASSF1A and HIC1 genes is associated with resistance. The promoter hypermethylation and/or the down-regulated expression of MGMT is seen in the majority of tumors. We hypothesize that these epigenetic alterations affecting MGMT play a major role in the exquisite sensitivity to cisplatin, characteristic of GCTs. We also demonstrate that cisplatin treatment induce de novo promoter hypermethylation in vivo. In addition, we show that the acquired cisplatin resistance in vitro alters the expression of specific genes and the highly resistant cells fail to reactivate gene expression after treatment to demethylating and histone deacetylase inhibiting agents.
Our findings suggest that promoter hypermethylation of RASSF1A and HIC1 genes play a role in resistance of GCT, while the transcriptional inactivation of MGMT by epigenetic alterations confer exquisite sensitivity to cisplatin. These results also implicate defects in epigenetic pathways that regulate gene transcription in cisplatin resistant GCT.
PMCID: PMC420487  PMID: 15149548
14.  Chromosomal amplifications, 3q gain and deletions of 2q33-q37 are the frequent genetic changes in cervical carcinoma 
BMC Cancer  2004;4:5.
Carcinoma of uterine cervix is the second most common cancers among women worldwide. Combined radiation and chemotherapy is the choice of treatment for advanced stages of the disease. The prognosis is poor, with a five-year survival rate ranging from about 20–65%, depending on stage of the disease. Therefore, genetic characterization is essential for understanding the biology and clinical heterogeneity in cervical cancer (CC).
We used a genome-wide screening method – comparative genomic hybridization (CGH) to identify DNA copy number changes in 77 patients with cervical cancer. We applied categorical and survival analyses to analyze whether chromosomal changes were related to clinico-pathologic characteristics and patients survival.
The CGH analysis revealed a loss of 2q33-q37 (57.1%), gain of 3q (54.5%) and chromosomal amplifications (20.77%) as frequent genetic changes. A total of 15 amplified chromosomal sites were detected in 16 cases that include 1p31, 2q32, 7q22, 8q21.2-q24, 9p22, 10q21, 10q24, 11q13, 11q21, 12q15, 14q12, 17p11.2, 17q22, 18p11.2, and 19q13.1. Recurrent amplified sites were noted at 11q13, 11q21, and 19q13.1. The genomic alterations were further evaluated for prognostic significance in CC patients, and we did not find any correlation with a number of clinical or histological parameters. The tumors harboring HPV18 exhibited higher genomic instability compared to tumors with HPV 16.
This study demonstrated that 2q33-q37 deletions, 3q gains and chromosomal amplifications as characteristic changes in invasive CC. These genetic alterations will aid in the identification of novel tumor suppressor gene(s) at 2q33-q37 and oncogenes at amplified chromosomal sites. Molecular characterization of these chromosomal changes utilizing the current genomic technologies will provide new insights into the biology and clinical behavior of CC.
PMCID: PMC356916  PMID: 15018632
Comparative genomic hybridization (CGH); Chromosomal amplifications; DNA copy number changes; Clinical correlations; Cervical carcinoma
15.  Frequent Promoter Methylation of CDH1, DAPK, RARB, and HIC1 Genes in Carcinoma of Cervix Uteri: Its Relationship to Clinical Outcome 
Molecular Cancer  2003;2:24.
Cervical cancer (CC), a leading cause of cancer-related deaths in women worldwide, has been causally linked to genital human papillomavirus (HPV) infection. Although a host of genetic alterations have been identified, molecular basis of CC development is still poorly understood.
We examined the role of promoter hypermethylation, an epigenetic alteration that is associated with the silencing tumor suppressor genes in human cancer, by studying 16 gene promoters in 90 CC cases. We found a high frequency of promoter methylation in CDH1, DAPK, RARB, and HIC1 genes. Correlation of promoter methylation with clinical characteristics and other genetic changes revealed the following: a) overall promoter methylation was higher in more advanced stage of the disease, b) promoter methylation of RARB and BRCA1 predicted worse prognosis, and c) the HIC1 promoter methylation was frequently seen in association with microsatellite instability. Promoter methylation was associated with gene silencing in CC cell lines. Treatment with methylation or histone deacetylation-inhibiting agents resulted in profound reactivation of gene expression.
These results may have implications in understanding the underlying epigenetic mechanisms in CC development, provide prognostic indicators, and identify important gene targets for treatment.
PMCID: PMC156646  PMID: 12773202
cervical carcinoma; promoter hypermethylation; CDH1; DAPK; RARB; tumor suppressor gene; gene expression
16.  Mapping common deleted regions on 5p15 in cervical carcinoma and their occurrence in precancerous lesions 
Molecular Cancer  2002;1:3.
Previous studies have shown that the short arm of chromosome 5 (5p) exhibit frequent genetic changes in invasive cervical carcinoma (CC), and that these changes arise early during the carcinogenesis, in precancerous lesions. These data therefore suggest that loss of candidate tumor suppressor genes located on 5p is associated with the development of CC. However, the precise location of 5p deletions is not known.
We performed a detailed deletion mapping of 5p in 60 cases of invasive CC. We found that 60% of the tumors exhibit a 5p loss of heterozygosity (LOH). The patterns of LOH allowed us to identify two minimal regions of deletions, one at 5p15.3 spanning a 5.5 cM genetic distance and a second site of 7 cM at 5p15.2-15.3. In addition, we also identified 5p deletions in 16% lesions of high-grade cervical intraepithelial neoplasia (CIN). 5p LOH was found in 63% of HPV 16 positive tumors, while only 33% tumors with other HPV-types had 5p LOH. The differences in frequency of 5p LOH between tumors harboring HPV16 in combination with other HPV types and tumors harboring HPV16 DNA alone were significantly higher, suggesting a synergistic effect of high-risk types in causing genomic instability.
These findings implicate the presence of tumor suppressor gene(s) on 5p relevant to CC tumorigenesis.
PMCID: PMC140145  PMID: 12392596
Cervical carcinoma; cervical intraepithelial neoplasia; chromosome 5p; loss of heterozygosity; deletion mapping; human papilloma virus

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