Publications by authors named "Naoto Tsuchiya"

58 Publications

Ultrasound image-guided gene delivery using three-dimensional diagnostic ultrasound and lipid-based microbubbles.

J Drug Target 2021 Jul 22:1-8. Epub 2021 Jul 22.

Faculty of Pharma-Science, Laboratory of Drug and Gene Delivery Research, Teikyo University, Tokyo, Japan.

Gene therapy is a promising technology for genetic and intractable diseases. Drug delivery carriers or systems for genes and nucleic acids have been studied to improve transfection efficiency and achieve sufficient therapeutic effects. Ultrasound (US) and microbubbles have also been combined for use in gene delivery. To establish a clinically effective gene delivery system, exposing the target tissues to US is important. The three-dimensional (3D) diagnostic probe can three-dimensionally scan the tissue with mechanical regulation, and homogenous US exposure to the targeted tissue can be expected. However, the feasibility of therapeutically applying 3D probes has not been evaluated, especially gene delivery. In this study, we evaluated the characteristics of a 3D probe and lipid-based microbubbles (LB) for gene delivery and determined whether the 3D probe in the diagnostic US device could be used for efficient gene delivery to the targeted tissue using a mouse model. The 3D probe RSP6-16 with LB delivered plasmid DNA (pDNA) to the kidney after systemic injection with luciferase activity similar to that of probes used in previously studies. No toxicity was observed after treatment and, therefore, the combined 3D probe and LB would deliver genes to targeted tissue safely and efficiently.
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http://dx.doi.org/10.1080/1061186X.2021.1953510DOI Listing
July 2021

[Surgical Case of Accidental Infantile Acute Subdural Hematoma Caused by Household Minor Head Trauma:Hyperperfusion during Postoperative Hemispheric Hypodensity, Namely "Big Black Brain"].

No Shinkei Geka 2020 Sep;48(9):841-847

Department of Neurosurgery, Nagano Red Cross Hospital.

We experienced a case of an accidental infantile acute subdural hematoma caused by household minor head trauma(Nakamura type I intracranial hemorrhage)with postoperative hemispheric hypodensity lesion(Big Black Brain)whose pathophysiology was analyzed using perfusion MRI. A ten-month-old boy was admitted to our hospital in a comatose state. His mother revealed that the boy suffered a fall from a sofa bed. A CT scan indicated massive acute subdural hematoma in the left cerebral hemisphere. Emergency craniotomy and hematoma evacuation were performed. On postoperative day 3, CT revealed hemispheric hypodensity, and the boy suffered from status epilepticus. MRI on the following day showed widespread white matter hyperintensity in diffusion-weighted images, and MRA demonstrated dilation of the middle cerebral artery. Perfusion MRI using the dynamic susceptibility contrast method revealed a marked increase in cerebral blood flow in the left hemisphere. These abnormal MRI and MRA findings disappeared on postoperative day 13. Status epilepticus also improved upon administration of multi-antiepileptic drugs. Fundoscopy findings on postoperative day 3 showed small bilateral petechial or brush retinal hemorrhages. However, whole-body examination did not show any problems, and was consistent with the mother's account. Thus, we judged non-abusive head trauma. Although follow-up MRI showed diffuse atrophy of the left cerebral hemisphere, the boy aged well without obvious paresis or verbal developmental delay as judged by a follow-up more than a year later. Based on these results, we speculated that hyperperfusion caused by dilation of the cerebral artery was related to the postoperative hemispheric hypodensity, namely "Big Black Brain".
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http://dx.doi.org/10.11477/mf.1436204281DOI Listing
September 2020

A serum microRNA classifier for the diagnosis of sarcomas of various histological subtypes.

Nat Commun 2019 03 21;10(1):1299. Epub 2019 Mar 21.

Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan.

Due to their rarity and diversity, sarcomas are difficult to diagnose. Consequently, there is an urgent demand for a novel diagnostic test for these cancers. In this study, we investigated serum miRNA profiles from 1002 patients with bone and soft tissue tumors representing more than 43 histological subtypes, including sarcomas, intermediate tumors, and benign tumors, to determine whether serum miRNA profiles could be used to specifically detect sarcomas. Circulating serum miRNA profiles in sarcoma patients were clearly distinct from those in patients with other types of tumors. Using the serum levels of seven miRNAs, we developed a molecular detector, Index VI, that could distinguish sarcoma patients from benign and healthy controls with remarkably high sensitivity (90%) and specificity (95%), regardless of histological subtype. Index VI provides an approach to the early and precise detection of sarcomas, potentially leading to curative treatment and longer survival.
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http://dx.doi.org/10.1038/s41467-019-09143-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428849PMC
March 2019

A Nucleolar Stress-Specific p53-miR-101 Molecular Circuit Functions as an Intrinsic Tumor-Suppressor Network.

EBioMedicine 2018 Jul 7;33:33-48. Epub 2018 Jul 7.

Laboratory of Molecular Carcinogenesis, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. Electronic address:

Background: Activation of intrinsic p53 tumor-suppressor (TS) pathways is an important principle underlying cancer chemotherapy. It is necessary to elucidate the precise regulatory mechanisms of these networks to create new treatment strategies.

Methods: Comprehensive analyses were carried out by microarray. Expression of miR-101 was analyzed by clinical samples of lung adenocarcinomas.

Findings: We discovered a functional link between p53 and miR-101, which form a molecular circuit in response to nucleolar stress. Inhibition of RNA polymerase I (Pol I) transcription resulted in the post-transcriptional activation of miR-101 in a p53-dependent manner. miR-101 induced G2 phase-specific feedback regulation of p53 through direct repression of its target, EG5, resulting in elevated phosphorylation of ATM. In lung cancer patients, low expression of miR-101 was associated with significantly poorer prognosis exclusively in p53 WT cases. miR-101 sensitized cancer cells to Pol I transcription inhibitors and strongly repressed xenograft growth in mice. Interestingly, the most downstream targets of this circuit included the inhibitor of apoptosis proteins (IAPs). Repression of cIAP1 by a selective inhibitor, birinapant, promoted activation of the apoptosis induced by Pol I transcription inhibitor in p53 WT cancer cells.

Interpretation: Our findings indicate that the p53-miR-101 circuit is a component of an intrinsic TS network formed by nucleolar stress, and that mimicking activation of this circuit represents a promising strategy for cancer therapy. FUND: National Institute of Biomedical Innovation, Ministry of Education, Culture, Sports & Technology of Japan, Japan Agency for Medical Research and Development.
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http://dx.doi.org/10.1016/j.ebiom.2018.06.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085539PMC
July 2018

[Bilateral Internal Carotid Artery Dissection Caused by Elongated Styloid Processes:A Case Report].

No Shinkei Geka 2018 Jan;46(1):53-59

Department of Neurosurgery, Nagano Red Cross Hospital.

We report a case of bilateral internal carotid artery(ICA)dissection associated with bilateral elongated styloid processes(ESPs). A 46-year-old man presented with transient aphasia and left visual disturbance at a business meeting. He complained of a foreign body sensation in his throat during swallowing for two years. Magnetic resonance imaging(MRI)demonstrated fresh small infarcts in the left corona radiata. Magnetic resonance angiography(MRA)revealed string signs bilaterally in the cervical ICAs. The patient was diagnosed with bilateral idiopathic ICA dissection and was treated with ozagrel and clopidogrel. Three-dimensional computed tomographic angiogram(3DCTA)indicated bilateral ESPs and bilateral ICA stenosis. 3DCTA with the patient's head tilting and neck extension revealed that each ICA was compressed by the ipsilateral ESP. A follow-up MRA showed complete normalization of bilateral ICAs after neck rest and anti-platelet therapy, following which, clopidogrel was stopped. The patient wore a soft cervical collar until the operation, to avoid contact between the ESPs and ICAs due to changes in head position. Bilateral ESP resection was performed to prevent recurrence of cerebral ischemic events caused by ICA dissection. The patient was discharged one week after the surgery without any neurological deficit. There was no recurrence of symptoms during the next eight months after the operation.
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http://dx.doi.org/10.11477/mf.1436203675DOI Listing
January 2018

An Integrated Prognostic Classifier for Stage I Lung Adenocarcinoma Based on mRNA, microRNA, and DNA Methylation Biomarkers.

J Thorac Oncol 2015 Jul;10(7):1037-48

*Laboratory of Human Carcinogenesis, NCI-CCR, National Institutes of Health, Bethesda, Maryland; †Division of Molecular Pathology, National Cancer Center Research Institute, Tokyo, Japan; ‡Genetics Branch, NCI-CCR, National Institutes of Health, Bethesda, Maryland; §Division of Genome Biology, National Cancer Center Research Institute, Tokyo, Japan; ‖Department of Chemical and Biological Working Environment, National Institute of Occupational Health, Oslo, Norway; and ¶Genomics and Epigenomics of Cancer Prediction Program, Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona (Barcelona), Spain.

Introduction: Up to 30% stage I lung cancer patients suffer recurrence within 5 years of curative surgery. We sought to improve existing protein-coding gene and microRNA expression prognostic classifiers by incorporating epigenetic biomarkers.

Methods: Genome-wide screening of DNA methylation and pyrosequencing analysis of HOXA9 promoter methylation were performed in two independently collected cohorts of stage I lung adenocarcinoma. The prognostic value of HOXA9 promoter methylation alone and in combination with mRNA and miRNA biomarkers was assessed by Cox regression and Kaplan-Meier survival analysis in both cohorts.

Results: Promoters of genes marked by polycomb in embryonic stem cells were methylated de novo in tumors and identified patients with poor prognosis. The HOXA9 locus was methylated de novo in stage I tumors (p < 0.0005). High HOXA9 promoter methylation was associated with worse cancer-specific survival (hazard ratio [HR], 2.6; p = 0.02) and recurrence-free survival (HR, 3.0; p = 0.01), and identified high-risk patients in stratified analysis of stages IA and IB. Four protein-coding gene (XPO1, BRCA1, HIF1α, and DLC1), miR-21 expression, and HOXA9 promoter methylation were each independently associated with outcome (HR, 2.8; p = 0.002; HR, 2.3; p = 0.01; and HR, 2.4; p = 0.005, respectively), and when combined, identified high-risk, therapy naive, stage I patients (HR, 10.2; p = 3 × 10). All associations were confirmed in two independently collected cohorts.

Conclusion: A prognostic classifier comprising three types of genomic and epigenomic data may help guide the postoperative management of stage I lung cancer patients at high risk of recurrence.
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http://dx.doi.org/10.1097/JTO.0000000000000560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4493931PMC
July 2015

Carbon-ion beam irradiation kills X-ray-resistant p53-null cancer cells by inducing mitotic catastrophe.

PLoS One 2014 22;9(12):e115121. Epub 2014 Dec 22.

Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.

Background And Purpose: To understand the mechanisms involved in the strong killing effect of carbon-ion beam irradiation on cancer cells with TP53 tumor suppressor gene deficiencies.

Materials And Methods: DNA damage responses after carbon-ion beam or X-ray irradiation in isogenic HCT116 colorectal cancer cell lines with and without TP53 (p53+/+ and p53-/-, respectively) were analyzed as follows: cell survival by clonogenic assay, cell death modes by morphologic observation of DAPI-stained nuclei, DNA double-strand breaks (DSBs) by immunostaining of phosphorylated H2AX (γH2AX), and cell cycle by flow cytometry and immunostaining of Ser10-phosphorylated histone H3.

Results: The p53-/- cells were more resistant than the p53+/+ cells to X-ray irradiation, while the sensitivities of the p53+/+ and p53-/- cells to carbon-ion beam irradiation were comparable. X-ray and carbon-ion beam irradiations predominantly induced apoptosis of the p53+/+ cells but not the p53-/- cells. In the p53-/- cells, carbon-ion beam irradiation, but not X-ray irradiation, markedly induced mitotic catastrophe that was associated with premature mitotic entry with harboring long-retained DSBs at 24 h post-irradiation.

Conclusions: Efficient induction of mitotic catastrophe in apoptosis-resistant p53-deficient cells implies a strong cancer cell-killing effect of carbon-ion beam irradiation that is independent of the p53 status, suggesting its biological advantage over X-ray treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115121PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4274003PMC
August 2015

A three-microRNA signature predicts responses to platinum-based doublet chemotherapy in patients with lung adenocarcinoma.

Clin Cancer Res 2014 Sep 20;20(18):4784-93. Epub 2014 Aug 20.

Division of Genome Biology, National Cancer Center Research Institute, Tokyo, Japan.

Purpose: To examine the clinical utility of intratumor microRNAs (miRNA) as a biomarker for predicting responses to platinum-based doublet chemotherapy in patients with recurring lung adenocarcinoma (LADC).

Experimental Design: The expression of miRNAs was examined in LADC tissues surgically resected from patients treated with platinum-based doublet chemotherapy at the time of LADC recurrence. Microarray-based screening of 904 miRNAs followed by quantitative reverse transcription-PCR-based verification in 40 test cohort samples, including 16 (40.0%) responders, was performed to identify miRNAs that are differentially expressed in chemotherapy responders and nonresponders. Differential expression was confirmed in a validation cohort (n = 63 samples), including 18 (28.6%) responders. An miRNA signature that predicted responses to platinum-based doublet chemotherapy was identified and its accuracy was examined by principal component and support vector machine analyses. Genotype data for the TP53-Arg72Pro polymorphism, which is associated with responses to platinum-based doublet chemotherapy, were subsequently incorporated into the prediction analysis.

Results: A signature comprising three miRNAs (miR1290, miR196b, and miR135a*) enabled the prediction of a chemotherapeutic response (rather than progression-free and overall survival) with high accuracy in both the test and validation cohorts (82.5% and 77.8%). Examination of the latter was performed using miRNAs extracted from archived formalin-fixed paraffin-embedded tissues. Combining this miRNA signature with the TP53-Arg72Pro polymorphism genotype marginally improved the predictive power.

Conclusion: The three-miRNA signature in surgically resected primary LADC tissues may by clinically useful for predicting responsiveness to platinum-based doublet chemotherapy in patients with LADC recurrence.
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http://dx.doi.org/10.1158/1078-0432.CCR-14-1096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6329384PMC
September 2014

NEK9-dependent proliferation of cancer cells lacking functional p53.

Sci Rep 2014 Aug 18;4:6111. Epub 2014 Aug 18.

Division of Genome Biology, National Cancer Center Research Institute.

Dysfunction of the p53 network is a major cause of cancer development, and selective elimination of p53-inactivated cancer cells therefore represents an ideal therapeutic strategy. In this study, we performed a microRNA target screen that identified NEK9 (NIMA-related kinase 9) as a crucial regulator of cell-cycle progression in p53-inactivated cancer cells. NEK9 depletion selectively inhibited proliferation in p53-deficient cancer cells both in vitro and in vivo. The resultant cell-cycle arrest occurred predominantly in G1 phase, and exhibited senescence-like features. Furthermore, NEK9 repression affected expression of a broad range of genes encoding cell-cycle regulators and factors involved in mRNA processing, suggesting a novel role for NEK9 in p53-deficient cells. Lung adenocarcinoma patients with positive staining for NEK9 and mutant p53 proteins exhibited significantly poorer prognoses, suggesting that expression of both proteins promotes tumor growth. Our findings demonstrate that a novel NEK9 network regulates the growth of cancer cells lacking functional p53.
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http://dx.doi.org/10.1038/srep06111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135330PMC
August 2014

Circulating exosomal microRNAs as biomarkers of colon cancer.

PLoS One 2014 4;9(4):e92921. Epub 2014 Apr 4.

Division of Genome Biology, National Cancer Center Research Institute, Tokyo, Japan.

Purpose: Exosomal microRNAs (miRNAs) have been attracting major interest as potential diagnostic biomarkers of cancer. The aim of this study was to characterize the miRNA profiles of serum exosomes and to identify those that are altered in colorectal cancer (CRC). To evaluate their use as diagnostic biomarkers, the relationship between specific exosomal miRNA levels and pathological changes of patients, including disease stage and tumor resection, was examined.

Experimental Design: Microarray analyses of miRNAs in exosome-enriched fractions of serum samples from 88 primary CRC patients and 11 healthy controls were performed. The expression levels of miRNAs in the culture medium of five colon cancer cell lines were also compared with those in the culture medium of a normal colon-derived cell line. The expression profiles of miRNAs that were differentially expressed between CRC and control sample sets were verified using 29 paired samples from post-tumor resection patients. The sensitivities of selected miRNAs as biomarkers of CRC were evaluated and compared with those of known tumor markers (CA19-9 and CEA) using a receiver operating characteristic analysis. The expression levels of selected miRNAs were also validated by quantitative real-time RT-PCR analyses of an independent set of 13 CRC patients.

Results: The serum exosomal levels of seven miRNAs (let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a) were significantly higher in primary CRC patients, even those with early stage disease, than in healthy controls, and were significantly down-regulated after surgical resection of tumors. These miRNAs were also secreted at significantly higher levels by colon cancer cell lines than by a normal colon-derived cell line. The high sensitivities of the seven selected exosomal miRNAs were confirmed by a receiver operating characteristic analysis.

Conclusion: Exosomal miRNA signatures appear to mirror pathological changes of CRC patients and several miRNAs are promising biomarkers for non-invasive diagnosis of the disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0092921PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3976275PMC
June 2015

Gene expression signature-based prognostic risk score in patients with glioblastoma.

Cancer Sci 2013 Sep 5;104(9):1205-10. Epub 2013 Jul 5.

Biostatistic Center, Kurume University School of Medicine, Kurume, Japan.

The present study aimed to identify genes associated with patient survival to improve our understanding of the underlying biology of gliomas. We investigated whether the expression of genes selected using random survival forests models could be used to define glioma subgroups more objectively than standard pathology. The RNA from 32 non-treated grade 4 gliomas were analyzed using the GeneChip Human Genome U133 Plus 2.0 Expression array (which contains approximately 47 000 genes). Twenty-five genes whose expressions were strongly and consistently related to patient survival were identified. The prognosis prediction score of these genes was most significant among several variables and survival analyses. The prognosis prediction score of three genes and age classifiers also revealed a strong prognostic value among grade 4 gliomas. These results were validated in an independent samples set (n = 488). Our method was effective for objectively classifying grade 4 gliomas and was a more accurate prognosis predictor than histological grading.
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http://dx.doi.org/10.1111/cas.12214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657127PMC
September 2013

Dopamine D1 receptors control exercise hyperpnoea in mice.

Exp Physiol 2013 Feb 28;98(2):491-500. Epub 2012 Sep 28.

Department of Physiology, Showa University School of Medicine, Tokyo 142-8555, Japan.

Previously, we undertook simultaneous recording of ventilation and pulmonary gas exchange in mice and revealed that dopamine D(2) receptors participate in exercise hyperpnoea via behavioural control of ventilation with unchanged pulmonary gas exchange. Here, we examined the hypothesis that D(1) receptors also contribute to exercise hyperpnoea using a D(1) receptor antagonist (SCH 23390; SCH) that crosses the blood-brain barrier, with the same recording technique and protocol as in the previous study. The respiratory responses of mice injected with saline or SCH (50 μg (kg body weight)(-1), i.p.) were compared during constant-load exercise at 6 m min(-1). Each mouse was set in an airtight treadmill chamber equipped with a differential pressure transducer and open-circuit system with a mass spectrometer. At rest, SCH-injected mice had significantly reduced respiratory frequency, minute ventilation and pulmonary gas exchange compared with saline-injected mice. Ventilation during hyperoxic gas inhalation and hypercapnic ventilatory responses between groups were similar. Abrupt increases and sequential declines to the steady-state level were produced by treadmill exercise in both groups of mice. Treatment with SCH lowered the increased levels of respiratory frequency, tidal volume and minute ventilation during the steady state, as well as reducing the O(2) uptake, CO(2) output and body temperature throughout treadmill exercise. These data suggest that D(1) receptors contribute to a resting ventilation level and exercise hyperpnoea during the steady state in parallel with metabolic changes. Notably, the metabolic control of D(1) receptors was important for maintenance of the steady state, and D(1) receptors in hypothalamic nuclei could be involved in this modulation.
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http://dx.doi.org/10.1113/expphysiol.2012.068312DOI Listing
February 2013

Hyperpnoeic response independent of limb movements at exercise onset in mice.

Respir Physiol Neurobiol 2013 Jan 27;185(2):319-31. Epub 2012 Sep 27.

Department of Physiology, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.

We have shown that constant-load treadmill exercise in mice produces an abrupt ventilatory increase to a maximal level at exercise onset. We examined what caused this abrupt response. We measured ventilation during 30-min constant-load exercise on a treadmill, below the lactate threshold, in conscious mice. Video analysis showed that hyperpnoea started before locomotion began. Incremental changes in speed did not further increase ventilation during the early phase of exercise. Next, we measured ventilatory responses to a sudden movement of the treadmill belt on which the mice were kept in a stationary position by a mesh cover. Hyperpnoea started concurrently with the sudden belt movement. In the absence of locomotion, ventilation increased to the extent reached during exercise hyperpnoea. Finally, the abrupt response showed plasticity but was attenuated by experience. The present study shows the importance of factors independent of limb movements in the hyperpnoeic response during the early phase of treadmill exercise in mice.
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http://dx.doi.org/10.1016/j.resp.2012.09.010DOI Listing
January 2013

Gene expression signature-based prognostic risk score in patients with primary central nervous system lymphoma.

Clin Cancer Res 2012 Oct 20;18(20):5672-81. Epub 2012 Aug 20.

Biostatistics Center, Kurume University, Kurume, Fukuoka, Japan.

Purpose: Better understanding of the underlying biology of primary central nervous system lymphomas (PCNSL) is critical for the development of early detection strategies, molecular markers, and new therapeutics. This study aimed to define genes associated with survival of patients with PCNSL.

Experimental Design: Expression profiling was conducted on 32 PCNSLs. A gene classifier was developed using the random survival forests model. On the basis of this, prognosis prediction score (PPS) using immunohistochemical analysis is also developed and validated in another data set with 43 PCNSLs.

Results: We identified 23 genes in which expressions were strongly and consistently related to patient survival. A PPS was developed for overall survival (OS) using a univariate Cox model. Survival analyses using the selected 23-gene classifiers revealed a prognostic value for high-dose methotrexate (HD-MTX) and HD-MTX-containing polychemotherapy regimen-treated patients. Patients predicted to have good outcomes by the PPS showed significantly longer survival than those with poor predicted outcomes (P < 0.0001). PPS using immunohistochemical analysis is also significant in test (P = 0.0004) and validation data set (P = 0.0281). The gene-based predictor was an independent prognostic factor in a multivariate model that included clinical risk stratification (P < 0.0001). Among the genes, BRCA1 protein expressions were most strongly associated with patient survival.

Conclusion: We have identified gene expression signatures that can accurately predict survival in patients with PCNSL. These predictive genes should be useful as molecular biomarkers and they could provide novel targets for therapeutic interventions.
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http://dx.doi.org/10.1158/1078-0432.CCR-12-0596DOI Listing
October 2012

miR-493 induction during carcinogenesis blocks metastatic settlement of colon cancer cells in liver.

EMBO J 2012 Apr 28;31(7):1752-63. Epub 2012 Feb 28.

Division of Cancer Differentiation, National Cancer Center Research Institute, Tokyo, Japan.

Liver metastasis is a major lethal complication associated with colon cancer, and post-intravasation steps of the metastasis are important for its clinical intervention. In order to identify inhibitory microRNAs (miRNAs) for these steps, we performed 'dropout' screens of a miRNA library in a mouse model of liver metastasis. Functional analyses showed that miR-493 and to a lesser extent miR-493(*) were capable of inhibiting liver metastasis. miR-493 inhibited retention of metastasized cells in liver parenchyma and induced their cell death. IGF1R was identified as a direct target of miR-493, and its inhibition partially phenocopied the anti-metastatic effects. High levels of miR-493 and miR-493(*), but not pri-miR-493, in primary colon cancer were inversely related to the presence of liver metastasis, and attributed to an increase of miR-493 expression during carcinogenesis. We propose that, in a subset of colon cancer, upregulation of miR-493 during carcinogenesis prevents liver metastasis via the induction of cell death of metastasized cells.
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http://dx.doi.org/10.1038/emboj.2012.25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3321205PMC
April 2012

Dopaminergic modulation of exercise hyperpnoea via D(2) receptors in mice.

Exp Physiol 2012 Feb 31;97(2):228-38. Epub 2011 Oct 31.

Department of Physiology, Showa University School of Medicine, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.

Dopamine is related to behaviour (including arousal, motivation and motor control of locomotion), and its turnover in the brain is increased during exercise. We examined the hypothesis that dopamine D(2) receptors contribute to exercise hyperpnoea via central neural pathways using the D(2)-like receptor antagonist, raclopride. We simultaneously measured ventilation and pulmonary gas exchange for the first time in mice. Mice injected with saline and raclopride (2 mg (kg body weight)(-1); i.p.) were compared for respiratory responses to constant-load exercise at 6 m min(-1). Each mouse was set in an airtight treadmill chamber. In the resting state, raclopride-treated mice had reduced respiratory frequency (f(R)) and minute ventilation (V) compared with saline-treated mice, but arterial P(CO(2)) and pulmonary gas exchange were not affected, showing that alveolar ventilation was maintained. Inhalation of hyperoxic gas maintained V in saline-treated mice, and hypercapnic ventilatory responses between the two groups were similar. Treadmill exercise produced an abrupt increase in V to a maximal level within 1 min and declined to a steady-state level in both groups. Raclopride-treated mice had reduced f(R) and V compared with saline-treated mice during steady states, but showed a similar increase in f(R) and V at exercise onset. Minute ventilation in the steady state was controlled, along with the increase in pulmonary O(2) uptake in both groups, but was lowered in raclopride-treated mice. Thus, D(2) receptors participate in resting breathing patterns to raise f(R) and exercise hyperpnoea in the steady state, probably through behavioural control and not central motor command, at exercise onset.
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http://dx.doi.org/10.1113/expphysiol.2011.062703DOI Listing
February 2012

Identification and validation of a gene expression signature that predicts outcome in malignant glioma patients.

Int J Oncol 2012 Mar 21;40(3):721-30. Epub 2011 Oct 21.

Department of Neurosurgery, Niigata University, Kyoto, Japan.

Better understanding of the underlying biology of malignant gliomas is critical for the development of early detection strategies and new therapeutics. This study aimed to define genes associated with survival. We investigated whether genes selected using random survival forests model could be used to define subgroups of gliomas objectively. RNAs from 50 non-treated gliomas were analyzed using the GeneChip Human Genome U133 Plus 2.0 Expression array. We identified 82 genes whose expression was strongly and consistently related to patient survival. For practical purposes, a 15-gene set was also selected. Both the complete 82 gene signature and the 15 gene set subgroup indicated their significant predictivity in the 3 out of 4 independent external dataset. Our method was effective for objectively classifying gliomas, and provided a more accurate predictor of prognosis. We assessed the relationship between gene expressions and survival time by using the random survival forests model and this performance was a better classifier compared to significance analysis of microarrays.
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http://dx.doi.org/10.3892/ijo.2011.1240DOI Listing
March 2012

Onset of quiescence following p53 mediated down-regulation of H2AX in normal cells.

PLoS One 2011 12;6(8):e23432. Epub 2011 Aug 12.

Division of Genome Stability Research, National Cancer Center Research Institute, Tokyo, Japan.

Normal cells, both in vivo and in vitro, become quiescent after serial cell proliferation. During this process, cells can develop immortality with genomic instability, although the mechanisms by which this is regulated are unclear. Here, we show that a growth-arrested cellular status is produced by the down-regulation of histone H2AX in normal cells. Normal mouse embryonic fibroblast cells preserve an H2AX diminished quiescent status through p53 regulation and stable-diploidy maintenance. However, such quiescence is abrogated under continuous growth stimulation, inducing DNA replication stress. Because DNA replication stress-associated lesions are cryptogenic and capable of mediating chromosome-bridge formation and cytokinesis failure, this results in tetraploidization. Arf/p53 module-mutation is induced during tetraploidization with the resulting H2AX recovery and immortality acquisition. Thus, although cellular homeostasis is preserved under quiescence with stable diploidy, tetraploidization induced under growth stimulation disrupts the homeostasis and triggers immortality acquisition.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023432PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3155552PMC
February 2012

Systematic exploration of cancer-associated microRNA through functional screening assays.

Cancer Sci 2011 Sep 12;102(9):1615-21. Epub 2011 Jul 12.

Division of Cancer Development System, National Cancer Center Research Institute, Tokyo, Japan.

MicroRNA (miRNA), non-coding RNA of approximately 22 nucleotides, post-transcriptionally represses expression of its target genes. miRNA regulates a variety of biological processes such as cell proliferation, cell death, development, stemness and genomic stability, not only in physiological conditions but also in various pathological conditions such as cancers. More than 1000 mature miRNA have been experimentally identified in humans and mice, yet the functions of a vast majority of miRNA remain to be elucidated. Identification of novel cancer-associated miRNA seems promising considering their possible application in the development of novel cancer therapies and biomarkers. Currently, there are two major approaches to identify miRNA that are associated with cancer: expression profiling study and functional screening assay. The former approach is widely used, and a large number of studies have shown aberrant miRNA expression profiles in cancer tissues compared with their non-cancer counterparts. Although aberrantly expressed miRNA are potentially good biomarkers, in most cases a majority of them do not play causal roles in cancers when functional assays are performed. In contrast, the latter approach allows screening of 'driver' miRNA with cancer-associated phenotypes, such as cell proliferation and cell invasion. Thus, this approach might be suitable in finding crucial targets of novel cancer therapy. The combination of both types of approaches will contribute to further elucidation of the cancer pathophysiology and to the development of a novel class of cancer therapies and biomarkers.
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http://dx.doi.org/10.1111/j.1349-7006.2011.02007.xDOI Listing
September 2011

Tumor suppressor miR-22 determines p53-dependent cellular fate through post-transcriptional regulation of p21.

Cancer Res 2011 Jul 12;71(13):4628-39. Epub 2011 May 12.

Division of Cancer Development System and Cancer Differentiation, National Cancer Center Research Institute, Tsukiji, Japan.

Selective activation of p53 target genes in response to various cellular stresses is a critical step in determining the ability to induce cell-cycle arrest or apoptosis. Here we report the identification of the microRNA miR-22 as a p53 target gene that selectively determines the induction of p53-dependent apoptosis by repressing p21. Combinatorial analyses of the AGO2 immunocomplex and gene expression profiles identified p21 as a direct target of miR-22. Induction of p21 was inhibited by miR-22 after exposure to the genotoxic agent Adriamycin (doxorubicin; Bedford Laboratories), sensitizing cells to p53-dependent apoptosis. Interestingly, the activation of miR-22 depended on the intensity of the stresses that induced cells to undergo apoptosis in the presence of p21 suppression. Our findings define an intrinsic molecular switch that determines p53-dependent cellular fate through post-transcriptional regulation of p21.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-2475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425979PMC
July 2011

MicroRNA, SND1, and alterations in translational regulation in colon carcinogenesis.

Mutat Res 2010 Nov 29;693(1-2):94-100. Epub 2010 Sep 29.

Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.

Post-transcriptional regulation of gene expression by microRNA (miRNA) has recently attracted major interest in relation to its involvement in cancer development. miRNA is a member of small non-coding RNA, consists of 22-24 nucleotides and regulates expression of target mRNA species in a post-transcriptional manner by being incorporated with RNA-induced silencing complex (RISC). Staphylococcal nuclease homology domain containing 1 (SND1), a component of RISC, is frequently up-regulated in human colon cancers and also chemically induced colon cancers in animals. We here showed that SDN1 is involved in miRNA-mediated gene suppression and overexpression of SND1 in colon cancer cells causes down-regulation of APC without altering APC mRNA levels. As for the miRNA expression profile in human colon cancer, miR-34a was among the list of down-regulated miRNA. Expression of miR-34a is tightly regulated by p53, and ectopic expression of miR-34a in colon cancer cells causes remarkable reduction of cell proliferation and induces senescence-like phenotypes. MiR-34a also participates in the positive feedback loop of the p53 tumor suppressor network. This circuitry mechanism for p53 activation is of interest in understanding the tumor suppressive function of miR-34a in colon carcinogenesis. miRNA should also be considered as novel anti-cancer agents in tumor suppressive therapeutic applications.
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http://dx.doi.org/10.1016/j.mrfmmm.2010.09.001DOI Listing
November 2010

NADPH oxidase overexpression in human colon cancers and rat colon tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

Int J Cancer 2011 Jun 8;128(11):2581-90. Epub 2010 Oct 8.

Linus Pauling Institute, Oregon State University, Corvallis, Oregon 97331, USA.

NADPH oxidase/dual-oxidase (Nox/Duox) family members have been implicated in nuclear factor kappa-B (NFκB)-mediated inflammation and inflammation-associated pathologies. We sought to examine, for the first time, the role of Nox/Duox and NFκB in rats treated with the cooked meat heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). In the PhIP-induced colon tumors obtained after 1 year, Nox1, Nox4, NFκB-p50 and NFκB-p65 were all highly overexpressed compared with their levels in adjacent normal-looking colonic mucosa. Nox1 and Nox4 mRNA and protein levels also were markedly elevated in a panel of primary human colon cancers, compared with their matched controls. In HT29 human colon cancer cells, Nox1 knockdown induced G1 cell cycle arrest, whereas in Caco-2 cells there was a strong apoptotic response, with increased levels of cleaved caspase-3, -6, -7 and poly(ADP-ribose)polymerase. Nox1 knockdown blocked lipopolysaccharide-induced phosphorylation of IκB kinase, inhibited the nuclear translocation of NFκB (p50 and p65) proteins, and attenuated NFκB DNA binding activity. There was a corresponding reduction in the expression of downstream NFκB targets, such as MYC, CCND1 and IL1β. The results provide the first evidence for a role of Nox1, Nox4 and NFκB in PhIP-induced colon carcinogenesis, including during the early stages before tumor onset. Collectively, the findings from this investigation and others suggest that further work is warranted on the role of Nox/Duox family members and NFκB in colon cancer development.
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http://dx.doi.org/10.1002/ijc.25610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3262595PMC
June 2011

Functional screening using a microRNA virus library and microarrays: a new high-throughput assay to identify tumor-suppressive microRNAs.

Carcinogenesis 2010 Aug 4;31(8):1354-9. Epub 2010 Jun 4.

National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan.

MicroRNA (miRNA) is a class of non-coding RNAs that represses expression of target messenger RNAs posttranscriptionally. A growing body of evidence supports their roles in various normal cellular processes, as well as in pathological conditions, such as cancer. We established a functional screening assay that enables high-throughput identification of miRNAs that have a role in cancer phenotypes of interest, via the combination of pooled lentivirus vectors expressing several hundred miRNA precursors and a custom-made microarray. Self versus self-hybridization analysis using pooled polymerase chain reaction products generated highly linear and reproducible results. To test the feasibility of the assay, we focused on miRNAs that control proliferation of pancreatic cancer cells and successfully identified five miRNAs that negatively control cell proliferation, including miRNA-34a that was previously identified as a representative tumor-suppressive miRNA. The results were further validated using lentivirus vectors expressing each of the five miRNAs or synthetic miRNAs. The function-based nature of the assay enabled identification of miRNAs that were strongly linked to cell proliferation, but the relative ease and flexibility of the assay allow for future studies of cancer stem cells, metastasis and other cancer phenotypes of interest.
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http://dx.doi.org/10.1093/carcin/bgq112DOI Listing
August 2010

Inhibition of peroxisome proliferator-activated receptor gamma promotes tumorigenesis through activation of the beta-catenin / T cell factor (TCF) pathway in the mouse intestine.

J Pharmacol Sci 2008 Dec 10;108(4):535-44. Epub 2008 Dec 10.

Division of Gastroenterology, Yokohama City University School of Medicine, Japan.

Although peroxisome proliferator-activated receptor gamma (PPARgamma) is strongly expressed in the intestinal epithelium, the role of PPARgamma in intestinal tumorigenesis has not yet been elucidated. To address this issue, we investigated the effect of PPARgamma inhibition and its mechanism on intestinal tumorigenesis using a selective antagonist, T0070907. We treated Apc(Min/+) mice and carcinogen-induced colon cancer model C57BL/6 mice with T0070907 and counted the number of spontaneous polyps and aberrant crypt foci and observed cell proliferation and beta-catenin protein in the colon epithelium. To investigate its mechanism, the changes of beta-catenin/TCF (T cell factor) transcriptional activity and location of beta-catenin induced by T0070907 were investigated in the colon cancer cell lines. T0070907 promoted polyp formation in the small intestine of Apc(Min/+) mice and aberrant crypt foci in the colon of C57BL/6 mice. PPARgamma inhibition promoted cell proliferation and increased expressions of the c-myc and cyclin D1 genes and the beta-catenin protein in the colon epithelium. In vitro, cell proliferation was promoted, but it was inhibited by the transfection of dominant-negative Tcf4. T0070907 increased beta-catenin/TCF transcriptional activity and beta-catenin protein in the cytsol and nucleus, but relatively decreased it on the cell membrane. PPARgamma antagonist promotes tumorigenesis in the small intestine and colon through stimulation of epithelial cell proliferation. beta-Catenin contributes to the promotion of tumorigenesis by PPARgamma antagonist due to activation of TCF/LEF (lymphoid enhancer factor) transcriptional factor.
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http://dx.doi.org/10.1254/jphs.08193fpDOI Listing
December 2008

Results of treatment of 112 cases of primary CNS lymphoma.

Jpn J Clin Oncol 2008 May 15;38(5):373-80. Epub 2008 Apr 15.

Department of Neurosurgery, Brain Research Institute, Niigata University, Niigata, Japan.

Background: Chemotherapy with or without radiotherapy is the mainstay of treatment for primary central nervous system lymphoma (PCNSL). High-dose methotrexate (MTX) is the most effective drug available to treat these lesions, either as a single agent or in combination with other drugs. Due to the lack of well-conducted randomized trials, the optimal treatment remains controversial. Available retrospective studies are difficult to discuss, however, some common themes can be found.

Methods: One hundred and twelve patients with PCNSL were treated with four different regimens over a period of 24 years. Treatment regimens were: whole-brain irradiation (WBI) alone, MVP (MTX, vincristine, and predonisolone), ProMACE-MOPP hybrid (cyclophosphamide, pirarubicin, etoposide, vincristine, procarbazine, prednisone, and MTX) and R-MTX (rituximab, MTX, pirarubicin, procarbazine, and prednisone) combined-modality therapy.

Results: The median failure-free survival was 16 months, and the median overall survival (OS) was 24 months. The 2- and 5-year actuarial probability of survival was 52.4 +/- 4.8% [95% confidence intervals (CI)] and 30.2 +/- 4.8% (95% CI), respectively. The ProMACE-MOPP protocol, Karnofsky performance status (KPS), MTX dose and WBI were associated with good OS by univariate models. By multivariate analysis, MTX dose, WBI dose, and its square dose were significantly associated with good OS. 20-30 Gy WB, and 500 mg/m(2) of MTX dose appeared important determinants of OS.

Conclusions: A modest dose of MTX (500 mg/m(2)) followed by reduced-dose WBI for patients who respond appears a feasible treatment approach that minimizes serious toxicity.
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http://dx.doi.org/10.1093/jjco/hyn027DOI Listing
May 2008

SND1, a component of RNA-induced silencing complex, is up-regulated in human colon cancers and implicated in early stage colon carcinogenesis.

Cancer Res 2007 Oct;67(19):9568-76

Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, Japan.

Colon cancers have been shown to develop after accumulation of multiple genetic and epigenetic alterations with changes in global gene expression profiles, contributing to the establishment of widely diverse phenotypes. Transcriptional and posttranscriptional regulation of gene expression by small RNA species, such as the small interfering RNA and microRNA and the RNA-induced silencing complex (RISC), is currently drawing major interest with regard to cancer development. SND1, also called Tudor-SN and p100 and recently reported to be a component of RISC, is among the list of highly expressed genes in human colon cancers. In the present study, we showed remarkable up-regulation of SND1 mRNA in human colon cancer tissues, even in early-stage lesions, and also in colon cancer cell lines. When mouse Snd1 was stably overexpressed in IEC6 rat intestinal epithelial cells, contact inhibition was lost and cell growth was promoted, even after the cells became confluent. Intriguingly, IEC6 cells with high levels of Snd1 also showed an altered distribution of E-cadherin from the cell membrane to the cytoplasm, suggesting loss of cellular polarity. Furthermore, the adenomatous polyposis coli (Apc) protein was coincidentally down-regulated, with no significant changes in the Apc mRNA level. Immunohistochemical analysis using chemically induced colonic lesions developed in rats revealed overexpression of Snd1 not only in colon cancers but also in aberrant crypt foci, putative precancerous lesions of the colon. Up-regulation of SND1 may thus occur at a very early stage in colon carcinogenesis and contribute to the posttranscriptional regulation of key players in colon cancer development, including APC and beta-catenin.
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http://dx.doi.org/10.1158/0008-5472.CAN-06-2707DOI Listing
October 2007

Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells.

Proc Natl Acad Sci U S A 2007 Sep 17;104(39):15472-7. Epub 2007 Sep 17.

Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.

Accumulating evidence suggests a role for microRNAs in human carcinogenesis as novel types of tumor suppressors or oncogenes. However, their precise biological role remains largely elusive. In the present study, we aimed to identify microRNA species involved in the regulation of cell proliferation. Using quantitative RT-PCR analysis, we demonstrated that miR-34a was highly up-regulated in a human colon cancer cell line, HCT 116, treated with a DNA-damaging agent, adriamycin. Transient introduction of miR-34a into two human colon cancer cell lines, HCT 116 and RKO, caused complete suppression of cell proliferation and induced senescence-like phenotypes. Moreover, miR-34a also suppressed in vivo growth of HCT 116 and RKO cells in tumors in mice when complexed and administered with atelocollagen for drug delivery. Gene-expression microarray and immunoblot analyses revealed down-regulation of the E2F pathway by miR-34a introduction. Up-regulation of the p53 pathway was also observed. Furthermore, 9 of 25 human colon cancers (36%) showed decreased expression of miR-34a compared with counterpart normal tissues. Our results provide evidence that miR-34a functions as a potent suppressor of cell proliferation through modulation of the E2F signaling pathway. Abrogation of miR-34a function could contribute to aberrant cell proliferation, leading to colon cancer development.
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http://dx.doi.org/10.1073/pnas.0707351104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2000550PMC
September 2007

Salvage therapy and late neurotoxicity in patients with recurrent primary CNS lymphoma treated with a modified ProMACE-MOPP hybrid regimen.

Leuk Lymphoma 2007 Jun;48(6):1119-26

Department of Neurosurgery, Brain Research Institute, Niigata University, Niigata.

We report the efficacy of salvage therapy with a modified ProMACE-MOPP combined with radiation in patients with primary central nervous system lymphoma (PCNSL). Thirty-two immunocompetent patients were treated with a regimen of pirarubicin, cyclophosphamide, etoposide, vincristin, and methotrexate (MTX: 500 mg/m(2)) administered in 21-day cycles. Patients received 20 Gy of whole-brain radiotherapy after three cycles of chemotherapy. A single cycle of chemotherapy was repeated every four months for two years. Nine patients with CNS relapse were retreated with additional cycles of the ProMACE-MOPP hybrid regimen with a 90% objective response rate. Median complete response (CR) duration was 13.2 months and median survival time (MST) for the nine patients treated after initial relapse was 30 months. One of 17 patients (5.8%) who had less than 20 Gy of whole brain irradiation developed dementia. In contrast, six of seven (85.7%) patients who had more than 30 Gy of whole brain radiotherapy became demented. Maintaining a moderate dose of MTX, while adding chemotherapeutic agents and 20 Gy of whole brain radiation therapy, improved disease control and overall survival and lowered the incidence of delayed neurologic toxicity in patients with PCNSL. Additional treatment with a ProMACE-MOPP hybrid regimen is still effective for relapsed disease.
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http://dx.doi.org/10.1080/10428190701299564DOI Listing
June 2007
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