Publications by authors named "Motoko Ohno"

34 Publications

Mutant KRAS drives metabolic reprogramming and autophagic flux in premalignant pancreatic cells.

Cancer Gene Ther 2021 Apr 8. Epub 2021 Apr 8.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-8655, Japan.

Mutational activation of the KRAS gene occurs in almost all pancreatic ductal adenocarcinoma (PDAC) and is the earliest molecular event in their carcinogenesis. Evidence has accumulated of the metabolic reprogramming in PDAC, such as amino acid homeostasis and autophagic flux. However, the biological effects of KRAS mutation on metabolic reprogramming at the earlier stages of PDAC carcinogenesis are unclear. Here we report dynamic metabolic reprogramming in immortalized human non-cancerous pancreatic ductal epithelial cells, in which a KRAS mutation was induced by gene-editing, which may mimic early pancreatic carcinogenesis. Similar to the cases of PDAC, KRAS gene mutation increased the dependency on glucose and glutamine for maintaining the intracellular redox balance. In addition, the intracellular levels of amino acids were significantly decreased because of active protein synthesis, and the cells required greater autophagic flux to maintain their viability. The lysosomal inhibitor chloroquine significantly inhibited cell proliferation. Therefore, metabolic reprogramming is an early event in carcinogenesis initiated by KRAS gene mutation, suggesting a rationale for the development of nutritional interventions that suppress or delay the development of PDAC.
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http://dx.doi.org/10.1038/s41417-021-00326-4DOI Listing
April 2021

Inhibition of HBV Transcription From cccDNA With Nitazoxanide by Targeting the HBx-DDB1 Interaction.

Cell Mol Gastroenterol Hepatol 2019 24;7(2):297-312. Epub 2018 Oct 24.

Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

Background & Aims: Hepatitis B virus (HBV) infection is a major health concern worldwide. Although currently used nucleos(t)ide analogs efficiently inhibit viral replication, viral proteins transcribed from the episomal viral covalently closed circular DNA (cccDNA) minichromosome continue to be expressed long-term. Because high viral RNA or antigen loads may play a biological role during this chronicity, the elimination of viral products is an ultimate goal of HBV treatment. HBV regulatory protein X (HBx) was recently found to promote transcription of cccDNA with degradation of Smc5/6 through the interaction of HBx with the host protein DDB1. Here, this protein-protein interaction was considered as a new molecular target of HBV treatment.

Methods: To identify candidate compounds that target the HBx-DDB1 interaction, a newly constructed split luciferase assay system was applied to comprehensive compound screening. The effects of the identified compounds on HBV transcription and cccDNA maintenance were determined using HBV minicircle DNA, which mimics HBV cccDNA, and the natural HBV infection model of human primary hepatocytes.

Results: We show that nitazoxanide (NTZ), a thiazolide anti-infective agent that has been approved by the FDA for protozoan enteritis, efficiently inhibits the HBx-DDB1 protein interaction. NTZ significantly restores Smc5 protein levels and suppresses viral transcription and viral protein production in the HBV minicircle system and in human primary hepatocytes naturally infected with HBV.

Conclusions: These results indicate that NTZ, which targets an HBV-related viral-host protein interaction, may be a promising new therapeutic agent and a step toward a functional HBV cure.
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http://dx.doi.org/10.1016/j.jcmgh.2018.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357790PMC
May 2019

Pevonedistat, a Neuronal Precursor Cell-Expressed Developmentally Down-Regulated Protein 8-Activating Enzyme Inhibitor, Is a Potent Inhibitor of Hepatitis B Virus.

Hepatology 2019 05 13;69(5):1903-1915. Epub 2019 Mar 13.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-8655, Japan.

Hepatitis B virus (HBV) infection is a major health concern worldwide. To prevent HBV-related mortality, elimination of viral proteins is considered the ultimate goal of HBV treatment; however, currently available nucleos(t)ide analogs rarely achieve this goal, as viral transcription from episomal viral covalently closed circular DNA (cccDNA) is not prevented. HBV regulatory protein X was recently found to target the protein structural maintenance of chromosomes 5/6 (Smc5/6) for ubiquitination and degradation by DDB1-CUL4-ROC1 E3 ligase, resulting in enhanced viral transcription from cccDNA. This ubiquitin-dependent proteasomal pathway requires an additional ubiquitin-like protein for activation, neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8). Here, we show that pevonedistat, a NEDD8-activating enzyme inhibitor, works efficiently as an antiviral agent. Pevonedistat significantly restored Smc5/6 protein levels and suppressed viral transcription and protein production in the HBV minicircle system in in vitro HBV replication models and in human primary hepatocytes infected naturally with HBV. Conclusion: These results indicate that pevonedistat is a promising compound to treat chronic HBV infection.
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http://dx.doi.org/10.1002/hep.30491DOI Listing
May 2019

ISGF3 with reduced phosphorylation is associated with constitutive expression of interferon-induced genes in aging cells.

NPJ Aging Mech Dis 2018 15;4:11. Epub 2018 Nov 15.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-8655 Japan.

During cellular aging, many changes in cellular functions occur. A hallmark of aged cells is secretion of inflammatory mediators, which collectively is referred to as the senescence-associated secretory phenotype (SASP). However, the mechanisms underlying such changes are unclear. Canonically, the expression of interferon (IFN)-stimulated genes (ISGs) is induced by IFNs through the formation of the tripartite transcriptional factor ISGF3, which is composed of IRF9 and the phosphorylated forms of STAT1 and STAT2. However, in this study, the constitutive expression of ISGs in human-derived senescent fibroblasts and in fibroblasts from a patient with Werner syndrome, which leads to premature aging, was mediated mainly by the unphosphorylated forms of STATs in the absence of INF production. Under homeostatic conditions, STAT1, STAT2, and IRF9 were localized to the nucleus of aged cells. Although knockdown of JAK1, a key kinase of STAT1 and STAT2, did not affect ISG expression or IFN-stimulated response element (ISRE)-mediated promoter activities in these senescent cells, knockdown of STAT1 or STAT2 decreased ISG expression and ISRE activities. These results suggest that the ISGF3 complex without clear phosphorylation is required for IFN-independent constitutive ISG transcription in senescent cells.
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http://dx.doi.org/10.1038/s41514-018-0030-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237867PMC
November 2018

Hepatitis B virus pathogenesis: Fresh insights into hepatitis B virus RNA.

World J Gastroenterol 2018 Jun;24(21):2261-2268

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Hepatitis B virus (HBV) is still a worldwide health concern. While divergent factors are involved in its pathogenesis, it is now clear that HBV RNAs, principally templates for viral proteins and viral DNAs, have diverse biological functions involved in HBV pathogenesis. These functions include viral replication, hepatic fibrosis and hepatocarcinogenesis. Depending on the sequence similarities, HBV RNAs may act as sponges for host miRNAs and may deregulate miRNA functions, possibly leading to pathological consequences. Some parts of the HBV RNA molecule may function as viral-derived miRNA, which regulates viral replication. HBV DNA can integrate into the host genomic DNA and produce novel viral-host fusion RNA, which may have pathological functions. To date, elimination of HBV-derived covalently closed circular DNA has not been achieved. However, RNA transcription silencing may be an alternative practical approach to treat HBV-induced pathogenesis. A full understanding of HBV RNA transcription and the biological functions of HBV RNA may open a new avenue for the development of novel HBV therapeutics.
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http://dx.doi.org/10.3748/wjg.v24.i21.2261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5989240PMC
June 2018

DHX9 regulates production of hepatitis B virus-derived circular RNA and viral protein levels.

Oncotarget 2018 Apr 20;9(30):20953-20964. Epub 2018 Apr 20.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Hepatitis B virus (HBV) infection, which is a major health concern worldwide, can lead to liver cirrhosis and hepatocellular carcinoma. Although current nucleos(t)ide analogs efficiently inhibit viral reverse transcription and viral DNA load clinically, episomal viral covalently closed circular DNA (cccDNA) minichromosomes and transcripts from cccDNA continue to be expressed over the long term. We hypothesized that, under these conditions, viral transcripts may have biological functions involved in pathogenesis. Here, we show that the host protein DExH-box helicase 9 (DXH9) is associated with viral RNAs. We also show that viral-derived circular RNA is produced during HBV replication, and the amount is increased by knockdown of the DHX9 protein, which, in turn, results in decreased viral protein levels but does not affect the levels of HBV DNA. These phenomena were observed in the HBV-producing cell culture model and HBV mini-circle model mimicking HBV cccDNA, as well as in human primary hepatocytes infected with HBV. Based on these results, we conclude that, in HBV infection, the RNA binding factor DHX9 is a novel regulator of viral circular RNA and viral protein levels.
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http://dx.doi.org/10.18632/oncotarget.25104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940377PMC
April 2018

Satellite RNA Increases DNA Damage and Accelerates Tumor Formation in Mouse Models of Pancreatic Cancer.

Mol Cancer Res 2018 08 10;16(8):1255-1262. Epub 2018 May 10.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Highly repetitive tandem arrays such as satellite sequences in the centromeric and pericentromeric regions of chromosomes, which were previously considered to be silent, are actively transcribed in various biological processes, including cancers. In the pancreas, this aberrant expression occurs even in Kras-mutated pancreatic intraepithelial neoplasia (PanIN) tissues, which are precancerous lesions. To determine the biological role of satellite RNAs in carcinogenesis , we constructed mouse major satellite (MajSAT) RNA-expressing transgenic mice. However, these transgenic mice did not show spontaneous malignant tumor formation under normal breeding. Importantly, however, DNA damage was increased in pancreatic tissues induced by caerulein treatment or high-fat diet, which may be due to impaired nuclear localization of Y-Box Binding Protein 1 (YBX1), a component of the DNA damage repair machinery. In addition, when crossed with pancreas-specific Kras-mutant mice, MajSAT RNA expression resulted in an earlier increase in PanIN formation. These results suggest that aberrant MajSAT RNA expression accelerates oncogenesis by increasing the probability of a second driver mutation, thus accelerating cells to exit from the breakthrough phase to the expansion phase. Aberrant expression of satellite RNAs accelerates oncogenesis through a mechanism involving increased DNA damage. .
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0139DOI Listing
August 2018

RASAL1 is a potent regulator of hepatic stellate cell activity and liver fibrosis.

Oncotarget 2017 Sep 4;8(39):64840-64852. Epub 2017 May 4.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Liver fibrosis, leading to cirrhosis and liver failure, can occur after chronic liver injury. The transition of hepatic stellate cells (HSCs) from quiescent cells into proliferative and fibrogenic cells is a central event in liver fibrosis. Here, we show that RAS protein activator like-1 (RASAL1), a RAS-GTPase-activating protein, which switches off RAS activity, is significantly decreased during HSC activation, and that HSC activation can be antagonized by forced expression of the RASAL1 protein. We demonstrate that RASAL1 suppresses HSC proliferation by regulating the Ras-MAPK pathway, and that RASAL1 suppresses HSC fibrogenic activity by regulating the PKA-LKB1-AMPK-SRF pathway by interacting with angiotensin II receptor, type 1. We also show that RASAL1-deficient mice are more susceptible to liver fibrosis. These data demonstrate that deregulated RASAL1 expression levels and the affected downstream intracellular signaling are central mediators of perpetuated HSC activation and fibrogenesis in the liver.
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http://dx.doi.org/10.18632/oncotarget.17609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630295PMC
September 2017

Increased oxytocin-monomeric red fluorescent protein 1 fluorescent intensity with urocortin-like immunoreactivity in the hypothalamo-neurohypophysial system of aged transgenic rats.

Neurosci Res 2018 Mar 30;128:40-49. Epub 2017 Aug 30.

Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan. Electronic address:

To visualize oxytocin in the hypothalamo-neurohypophysial system, we generated a transgenic rat that expresses the oxytocin-monomeric red fluorescent protein 1 (mRFP1) fusion gene. In the present study, we examined the age-related changes of oxytocin-mRFP1 fluorescent intensity in the posterior pituitary (PP), the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) of transgenic rats. The mRFP1 fluorescent intensities were significantly increased in the PP, the SON and the PVN of 12-, 18- and 24-month-old transgenic rats in comparison with 3-month-old transgenic rats. Immunohistochemical staining for urocortin, which belongs to the family of corticotropin-releasing factor family, revealed that the numbers of urocortin-like immunoreactive (LI) cells in the SON and the PVN were significantly increased in 12-, 18- and 24-month-old transgenic rats in comparison with 3-month-old transgenic rats. Almost all of urocortin-LI cells co-exist mRFP1-expressing cells in the SON and the PVN of aged transgenic rats. These results suggest that oxytocin content of the hypothalamo-neurohypophysial system may be modulated by age-related regulation. The physiological role of the co-existence of oxytocin and urocortin in the SON and PVN of aged rats remains unclear.
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http://dx.doi.org/10.1016/j.neures.2017.08.001DOI Listing
March 2018

Transcriptional activation of the MICA gene with an engineered CRISPR-Cas9 system.

Biochem Biophys Res Commun 2017 04 18;486(2):521-525. Epub 2017 Mar 18.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Major histocompatibility complex class I polypeptide-related sequence A (MICA) is a prototypical NKG2D ligand. Because immune cells, such as natural killer (NK) cells, recognize virally infected or transformed cells and eliminate them through the interaction between NKG2D receptors on NK cells and NKG2D ligands on pathogenic cells, MICA expression levels are associated with NK cell-mediated immunity. Here, we report that an engineered clustered regularly interspaced short palindromic repeats-Cas9-related complex targeting MICA gene promoter sequences activates transcription of the MICA gene from its endogenous locus. Inhibiting microRNA function, which targets the 3' untranslated region of the MICA gene, enhances this activation. These results demonstrate that the combination of Cas9-based transcriptional activators and simultaneous modulation of microRNA function may be a powerful tool for enhancing MICA protein expression and efficient anti-pathogenic cell immunity.
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http://dx.doi.org/10.1016/j.bbrc.2017.03.076DOI Listing
April 2017

Repression of MicroRNA Function Mediates Inflammation-associated Colon Tumorigenesis.

Gastroenterology 2017 02 5;152(3):631-643. Epub 2016 Nov 5.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Background & Aims: Little is known about the mechanisms by which chronic inflammation contributes to carcinogenesis, such as the development of colon tumors in patients with inflammatory bowel diseases. Specific microRNA (miRNAs) can function as suppressors or oncogenes, and widespread alterations in miRNA expression have been associated with tumorigenesis. We studied whether alterations in miRNA function contribute to inflammation-associated colon carcinogenesis.

Methods: We studied the effects of inflammatory cytokines, such as tumor necrosis factor, interleukin-1α (IL1A), and IL1β (IL1B), on miRNA function, measured by activity of reporter constructs containing miRNA-binding sites in their 3' untranslated regions, in human 293T embryonic kidney, Caco-2, HT29, and HCT116 colon carcinoma cells, as well as dicer and dicer, and Apobec3 and Apobec3 mouse embryonic fibroblasts. Cells were analyzed by immunoblots, immunohistochemistry, and flow cytometry. We generated transgenic mice expressing reporter constructs regulated by LET7B, MIR122, and MIR29b response elements; some mice were given injections of miRNA inhibitors (anti-MIR122 or anti-LET7B), a negative control, or tumor necrosis factor. Liver tissues were collected and analyzed by immunoblotting. Reporter mice were given azoxymethane followed by dextran sulfate sodium to induce colitis and colon tumors; some mice were given the ROCK inhibitor fasudil along with these agents (ROCK inhibitors increase miRNA function). Colon tissues were collected and analyzed by immunohistochemistry, immunoblots, and fluorescence microscopy.

Results: Incubation of cell lines with inflammatory cytokines reduced the ability of miRNAs to down-regulate expression from reporter constructs; dicer was required for this effect, so these cytokines relieve miRNA-dependent reductions in expression. The cytokines promoted degradation of APOBEC3G, which normally promotes miRNA loading into argonaute 2-related complexes. Mice with colitis had reduced miRNA function, based on increased expression of reporter genes. Administration of fasudil to mice did not reduce the severity of colitis that developed but greatly reduced the numbers of colon tumors formed (mean 2 tumors/colon in mice given fasudil vs 9 tumors/colon in mice given control agent). We made similar observations in IL10-deficient mice.

Conclusions: We found inflammatory cytokines to reduce the activities of miRNAs. In mice with colitis, activities of miRNAs are reduced; administration of an agent that increases miRNA function prevents colon tumor formation in these mice. This pathway might be targeted to prevent colon carcinogenesis in patients with inflammatory bowel diseases.
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http://dx.doi.org/10.1053/j.gastro.2016.10.043DOI Listing
February 2017

Quantitation of circulating satellite RNAs in pancreatic cancer patients.

JCI Insight 2016 06 2;1(8):e86646. Epub 2016 Jun 2.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Pancreatic ductal adenocarcinoma (Pdac) is a malignancy with a poor prognosis due to difficulties in early detection. Although promising biomarkers are increasingly reported, such methods are not yet easy to apply clinically, mainly due to their low reproducibility or technical difficulties. In this study, we developed a convenient and sensitive method for quantifying aberrantly expressed satellite repeat RNAs in sera, which can be used to efficiently detect patients with Pdac. Here, we introduce a Tandem Repeat Amplification by nuclease Protection (TRAP) method combined with droplet digital PCR (ddPCR) to detect human satellite II (HSATII) RNAs, which are specifically expressed in human Pdacs at greater levels than normal tissues but are difficult to measure due to their repetitive sequences and irregularities. HSATII RNA core sequence levels in sera were significantly higher in Pdac patients compared with noncancer patients (median copy number: 14.75 and 3.17 per μl in the training set and 17.35 and 2.9 in the validation set, respectively). In addition, patients with intraductal papillary mucinous neoplasm (IPMN), a precancerous lesion of Pdac, could also be efficiently detected. This method can be routinely applied to screen patients with Pdac and high-risk patients, facilitating the development of preventive medicine for this disease.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033942PMC
http://dx.doi.org/10.1172/jci.insight.86646DOI Listing
June 2016

Satellite RNAs promote pancreatic oncogenic processes via the dysfunction of YBX1.

Nat Commun 2016 Sep 26;7:13006. Epub 2016 Sep 26.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Highly repetitive tandem arrays at the centromeric and pericentromeric regions in chromosomes, previously considered silent, are actively transcribed, particularly in cancer. This aberrant expression occurs even in K-ras-mutated pancreatic intraepithelial neoplasia (PanIN) tissues, which are precancerous lesions. To examine the biological roles of the satellite RNAs in carcinogenesis, we construct mouse PanIN-derived cells expressing major satellite (MajSAT) RNA and show increased malignant properties. We find an increase in frequency of chromosomal instability and point mutations in both genomic and mitochondrial DNA. We identify Y-box binding protein 1 (YBX1) as a protein that binds to MajSAT RNA. MajSAT RNA inhibits the nuclear translocation of YBX1 under stress conditions, thus reducing its DNA-damage repair function. The forced expression of YBX1 significantly decreases the aberrant phenotypes. These findings indicate that during the early stage of cancer development, satellite transcripts may act as 'intrinsic mutagens' by inducing YBX1 dysfunction, which may be crucial in oncogenic processes.
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http://dx.doi.org/10.1038/ncomms13006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5052683PMC
September 2016

MicroRNAs and liver disease.

J Hum Genet 2017 Jan 26;62(1):75-80. Epub 2016 May 26.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

The biological roles of microRNAs (miRNAs) have been extensively studied. miRNA122 represents more than half of the miRNAs expressed in the liver and has various physiological and pathological functions, which include enhancing hepatitis virus replication, regulating lipid metabolism and suppressing hepatocellular carcinoma. miRNAs, whether globally or individually, have been linked with hepatocarcinogenesis. Furthermore, some miRNAs have been shown to be involved in the pathogenesis of nonalcoholic steatohepatitis. Using nucleotide-based strategies, these miRNAs may be developed as potential therapeutic targets. Because changes in miRNA expression can be measured in sera, they may be used as non-invasive biomarkers if they correctly reflect the pathological state of the liver. In this review, we show the biological roles of representative miRNAs in liver disease and discuss the current issues that remain to be clarified for future clinical applications.
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http://dx.doi.org/10.1038/jhg.2016.53DOI Listing
January 2017

Mutual antagonism between hepatitis B viral mRNA and host microRNA let-7.

Sci Rep 2016 Mar 16;6:23237. Epub 2016 Mar 16.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

The interplay between viral and host factors plays a major role in viral pathogenesis. Hepatitis B virus (HBV) infection is a global health problem that leads to liver cirrhosis and hepatocellular carcinoma (HCC). Although HBV proteins have been studied extensively about their implication in hepatocarcinogenesis, the molecular mechanisms of oncogenesis are still largely unknown. A recent concept in gene regulation, in which competitive endogenous RNAs compete for common microRNAs (miRNAs), suggests that mRNA targets are key elements in the regulation of miRNA availability. Here, we show that HBV mRNA in the preS2 region can be targeted by host miRNA let-7 g. This leads to the sequestration of let-7 g and inhibition of let-7 g function. The expression of HBV transcripts, including the preS2 region, de-repressed let-7 g targets, which may contribute to long-term oncogenesis. HBV transcript-expressing transgenic mice, but not non-targeted transcript-expressing mice, were more prone to chemically induced hepatoocarcinogenesis. Let-7 target protein expression was upregulated in human HCC tissues derived from HBV-infected patients. On the other hand, let-7 g inhibited HBV preS2 protein expression and viral products. These results suggest that the interplay between viral intermediate transcripts during HBV replication and host miRNAs is crucial to the pathogenesis of chronic viral infection.
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http://dx.doi.org/10.1038/srep23237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4793232PMC
March 2016

Development of a screening method to identify regulators of MICA shedding.

Biochem Biophys Res Commun 2015 Oct 20;465(4):764-8. Epub 2015 Aug 20.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Immune cells, such as natural killer (NK) cells, recognize virally infected and transformed cells, and eliminate them through the interaction between NKG2D receptors on NK cells and NKG2D ligands on pathogenic cells. Shedding of NKG2D ligands is thought to be a type of counter-mechanism employed by pathogenic cells to evade from NKG2D-mediated immune surveillance. MHC class I polypeptide-related sequence A (MICA) is a prototypical NKG2D ligand. We previously reported that, in soluble form, MICA expression levels are significantly associated with hepatitis virus-induced hepatocellular carcinoma. Here, we report a MICA shedding assay that utilizes membrane-bound MICA tagged at its N-terminus with a nano-luciferase reporter to quantify MICA shedding into culture media. Using this method, we screened a compound library and identified putative regulators of MICA shedding that have the potential to enhance the immune reaction by simultaneously increasing cell surface MICA levels and decreasing soluble MICA levels. This shedding assay may be useful for screening regulators of cell surface molecule shedding.
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http://dx.doi.org/10.1016/j.bbrc.2015.08.081DOI Listing
October 2015

Circulating RNAs as new biomarkers for detecting pancreatic cancer.

World J Gastroenterol 2015 Jul;21(28):8527-40

Takahiro Kishikawa, Motoyuki Otsuka, Motoko Ohno, Takeshi Yoshikawa, Akemi Takata, Kazuhiko Koike, Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Pancreatic cancer remains difficult to treat and has a high mortality rate. It is difficult to diagnose early, mainly due to the lack of screening imaging modalities and specific biomarkers. Consequently, it is important to develop biomarkers that enable the detection of early stage tumors. Emerging evidence is accumulating that tumor cells release substantial amounts of RNA into the bloodstream that strongly resist RNases in the blood and are present at sufficient levels for quantitative analyses. These circulating RNAs are upregulated in the serum and plasma of cancer patients, including those with pancreatic cancer, compared with healthy controls. The majority of RNA biomarker studies have assessed circulating microRNAs (miRs), which are often tissue-specific. There are few reports of the tumor-specific upregulation of other types of small non-coding RNAs (ncRNAs), such as small nucleolar RNAs and Piwi-interacting RNAs. Long ncRNAs (lncRNAs), such as HOTAIR and MALAT1, in the serum/plasma of pancreatic cancer patients have also been reported as diagnostic and prognostic markers. Among tissue-derived RNAs, some miRs show increased expression even in pre-cancerous tissues, and their expression profiles may allow for the discrimination between a chronic inflammatory state and carcinoma. Additionally, some miRs and lncRNAs have been reported with significant alterations in expression according to disease progression, and they may thus represent potential candidate diagnostic or prognostic biomarkers that may be used to evaluate patients once detection methods in peripheral blood are well established. Furthermore, recent innovations in high-throughput sequencing techniques have enabled the discovery of unannotated tumor-associated ncRNAs and tumor-specific alternative splicing as novel and specific biomarkers of cancers. Although much work is required to clarify the release mechanism, origin of tumor-specific circulating RNAs, and selectivity of carrier complexes, and technical advances must also be achieved, such as creating a consensus normalization protocol for quantitative data analysis, circulating RNAs are largely unexplored and might represent novel clinical biomarkers.
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http://dx.doi.org/10.3748/wjg.v21.i28.8527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4515835PMC
July 2015

ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression.

Nucleic Acids Res 2015 Sep 17;43(15):7577-89. Epub 2015 Jul 17.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

The reduced expression levels and functional impairment of global miRNAs are related to various human diseases, including cancers. However, relatively little is known about how global miRNA function may be upregulated. Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors. The regulation of miRNA function by ROCK inhibitors is mediated, at least in part, by poly(A)-binding protein-interacting protein 2 (PAIP2), which enhances poly(A)-shortening of miRNA-targeted mRNAs and leads to global upregulation of miRNA function. In the presence of a ROCK inhibitor, PAIP2 expression is enhanced by the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) through increased ROCK1 nuclear localization and enhanced ROCK1 association with HNF4A. Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription. ROCK inhibitors may be useful for the various pathologies associated with the impairment of global miRNA function.
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http://dx.doi.org/10.1093/nar/gkv728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551943PMC
September 2015

Novel therapeutic approaches for hepatitis B virus covalently closed circular DNA.

World J Gastroenterol 2015 Jun;21(23):7084-8

Motoko Ohno, Motoyuki Otsuka, Takahiro Kishikawa, Takeshi Yoshikawa, Akemi Takata, Kazuhiko Koike, Department of Gastroenterology, Graduate School of Medicine, the University of Tokyo, Tokyo 113-8655, Japan.

Hepatitis B virus (HBV) infection is a major global health problem. Although current therapies, such as the use of nucleos(t)ide analogs, inhibit HBV replication efficiently, they do not eliminate covalently closed circular DNA (cccDNA), which persists in hepatocyte nuclei. As HBV cccDNA is a viral transcription template, novel therapeutic approaches to directly target HBV cccDNA are necessary to completely eradicate persistent HBV infections. HBV cccDNA levels in HBV-infected human liver cells are extremely low; thus, more reliable and simple measurement methods are needed to correctly monitor their levels during therapeutic treatment. Although reverse transcription-polymerase chain reaction or Southern blot procedures are currently used in research studies, these methods are not completely reliable and are also time-consuming and labor-intensive. Genome editing technologies, such as zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, which are designed to target specific DNA sequences, represent highly promising potential therapeutic tools. In particular, the CRISPR/Cas9 system is an easily customizable sequence-specific nuclease with high flexibility and may be the most feasible approach to target HBV cccDNA. Further research to develop easier, safer, and more effective protocols should be pursued.
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http://dx.doi.org/10.3748/wjg.v21.i23.7084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476870PMC
June 2015

Decreased miR122 in hepatocellular carcinoma leads to chemoresistance with increased arginine.

Oncotarget 2015 Apr;6(10):8339-52

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Reduced expression of microRNA122 (miR122), a liver-specific microRNA, is frequent in hepatocellular carcinoma (HCC). However, its biological significances remain poorly understood. Because deregulated amino acid levels in cancers can affect their biological behavior, we determined the amino acid levels in miR122-silenced mouse liver tissues, in which intracellular arginine levels were significantly increased. The increased intracellular arginine levels were through upregulation of the solute carrier family 7 (SLC7A1), a transporter of arginine and a direct target of miR122. Arginine is the substrate for nitric oxide (NO) synthetase, and intracellular NO levels were increased in miR122-silenced HCC cells, with increased resistance to sorafenib, a multikinase inhibitor. Conversely, maintenance of the miR122-silenced HCC cells in arginine-depleted culture media, as well as overexpression of miR122 in miR122-low-expressing HCC cells, reversed these effects and rendered the cells more sensitive to sorafenib. Using a reporter knock-in construct, chemical compounds were screened, and Wee1 kinase inhibitor was identified as upregulators of miR122 transcription, which increased the sensitivity of the cells to sorafenib. These results provide an insight into sorafenib resistance in miR122-low HCC, and suggest that arginine depletion or a combination of sorafenib with the identified compound may provide promising approaches to managing this HCC subset.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480756PMC
http://dx.doi.org/10.18632/oncotarget.3234DOI Listing
April 2015

Diagnostic and therapeutic application of noncoding RNAs for hepatocellular carcinoma.

World J Hepatol 2015 Jan;7(1):1-6

Chikako Shibata, Motoyuki Otsuka, Takahiro Kishikawa, Motoko Ohno, Takeshi Yoshikawa, Akemi Takata, Kazuhiko Koike, Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate gene expression posttranscriptionally, targeting thousands of messenger RNAs. Long noncoding RNAs (lncRNAs), another class of noncoding RNAs, have been determined to be also involved in transcription regulation and translation of target genes. Since deregulated expression levels or functions of miRNAs and lncRNAs in hepatocellular carcinoma (HCC) are frequently observed, clinical use of noncoding RNAs for novel diagnostic and therapeutic applications in the management of HCCs is highly and emergently expected. Here, we summarize recent findings regarding deregulated miRNAs and lncRNAs for their potential clinical use as diagnostic and prognostic biomarkers of HCC. Specifically, we emphasize the deregulated expression levels of such noncoding RNAs in patients' sera as noninvasive biomarkers, a field that requires urgent improvement in the clinical surveillance of HCC. Since nucleotide-based strategies are being applied to clinical therapeutics, we further summarize clinical and preclinical trials using oligonucleotides involving the use of miRNAs and small interfering RNAs against HCC as novel therapeutics. Finally, we discuss current open questions, which must be clarified in the near future for realistic clinical applications of these new strategies.
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http://dx.doi.org/10.4254/wjh.v7.i1.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4295186PMC
January 2015

The flavonoid apigenin inhibits hepatitis C virus replication by decreasing mature microRNA122 levels.

Virology 2014 Aug 14;462-463:42-8. Epub 2014 Jun 14.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 5-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.

Despite recent progress in the development of direct-acting antivirals against hepatitis C virus (HCV), chronic HCV infection remains an important health burden worldwide. MicroRNA122 (miR122), a liver-specific microRNA (miRNA), positively regulates HCV replication, and systemic application of antisense oligonucleotides against miR122 led to the long-lasting suppression of HCV viremia in human clinical trials. Here, we report that apigenin, a flavonoid and an inhibitor of maturation of a subset of miRNAs, inhibits HCV replication in vitro. Apigenin decreased the expression levels of mature miR122 without significantly affecting cell growth. Because supplementation of synthesized miR122 oligonucleotides or overexpression of constitutively active TRBP blocked these effects, the inhibitory effects of apigenin on HCV replication seemed to be dependent on the reduction of mature miR122 expression levels through inhibition of TRBP phosphorylation. Thus, apigenin intake, either through regular diet or supplements, may decrease HCV replication in chronically infected patients.
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http://dx.doi.org/10.1016/j.virol.2014.05.024DOI Listing
August 2014

Specific delivery of microRNA93 into HBV-replicating hepatocytes downregulates protein expression of liver cancer susceptible gene MICA.

Oncotarget 2014 Jul;5(14):5581-90

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC). To date, the lack of efficient in vitro systems supporting HBV infection and replication has been a major limitation of HBV research. Although primary human hepatocytes support the complete HBV life cycle, their limited availability and difficulties with gene transduction remain problematic. Here, we used human primary hepatocytes isolated from humanized chimeric uPA/SCID mice as efficient sources. These hepatocytes supported HBV replication in vitro. Based on analyses of mRNA and microRNA (miRNA) expression levels in HBV-infected hepatocytes, miRNA93 was significantly downregulated during HBV infection. MiRNA93 is critical for regulating the expression levels of MICA protein, which is a determinant for HBV-induced HCC susceptibility. Exogenous addition of miRNA93 in HBV-infected hepatocytes using bionanocapsules consisted of HBV envelope L proteins restored MICA protein expression levels in the supernatant. These results suggest that the rescued suppression of soluble MICA protein levels by miRNA93 targeted to HBV-infected hepatocytes using bionanocapsules may be useful for the prevention of HBV-induced HCC by altering deregulated miRNA93 expression.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4170619PMC
http://dx.doi.org/10.18632/oncotarget.2143DOI Listing
July 2014

The role of microRNAs in hepatocarcinogenesis: current knowledge and future prospects.

J Gastroenterol 2014 Feb 21;49(2):173-84. Epub 2013 Nov 21.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 5-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan,

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate gene expression post-transcriptionally through complementary base pairing with thousands of messenger RNAs. Although the precise biological functions of individual miRNAs are still unknown, miRNAs are speculated to play important roles in diverse biological processes through fine regulation of their target gene expression. A growing body of data indicates the deregulation of miRNAs during hepatocarcinogenesis. In this review, we summarize recent findings regarding deregulated miRNA expression and their possible target genes in hepatocarcinogenesis, with emphasis on inflammation-related hepatocarcinogenesis. Because miRNA-based strategies are being applied to clinical therapeutics, precise knowledge of miRNA functions is crucial both scientifically and clinically. We discuss the current open questions from these points of view, which must be clarified in the near future.
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http://dx.doi.org/10.1007/s00535-013-0909-8DOI Listing
February 2014

Effects of food deprivation on the hypothalamic feeding-regulating peptides gene expressions in serotonin depleted rats.

J Physiol Sci 2014 Mar 27;64(2):97-104. Epub 2013 Oct 27.

Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, 807-8555, Japan.

We examined the effects of serotonin (5-HT) depletion induced by peripheral injection of 5-HT synthesis inhibitor p-chlorophenylalanine (PCPA) on the expression of feeding-regulating peptides expressions by using in situ hybridization histochemistry in adult male Wistar rats. PCPA pretreatment had no significant effect on basal levels of oxytocin, corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH), pro-opiomelanocortin (POMC), cocaine and amphetamine-regulated transcript (CART), neuropeptide-Y (NPY), agouti-related protein (AgRP), melanin-concentrating hormone (MCH) or orexin in the hypothalamus. Food deprivation for 48 h caused a significant decrease in CRH, TRH, POMC, and CART, and a significant increase in NPY, AgRP and MCH. After PCPA treatment, POMC and CART did not decrease despite food deprivation. NPY was significantly increased by food deprivation with PCPA, but was attenuated compared to food deprivation without PCPA. These results suggest that the serotonergic system in the hypothalamus may be involved in the gene expression of POMC, CART, and NPY related to feeding behavior.
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http://dx.doi.org/10.1007/s12576-013-0296-1DOI Listing
March 2014

Regulation of the expression of the liver cancer susceptibility gene MICA by microRNAs.

Sci Rep 2013 ;3:2739

1] Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan [2].

Hepatocellular carcinoma (HCC) is a threat to public health worldwide. We previously identified the association of a single nucleotide polymorphism (SNP) at the promoter region of the MHC class I polypeptide-related sequence A (MICA) gene with the risk of hepatitis-virus-related HCC. Because this SNP affects MICA expression levels, regulating MICA expression levels may be important in the prevention of HCC. We herein show that the microRNA (miR) 25-93-106b cluster can modulate MICA levels in HCC cells. Overexpression of the miR 25-93-106b cluster significantly suppressed MICA expression. Conversely, silencing of this miR cluster enhanced MICA expression in cells that express substantial amounts of MICA. The changes in MICA expression levels by the miR25-93-106b cluster were biologically significant in an NKG2D-binding assay and an in vivo cell-killing model. These data suggest that the modulation of MICA expression levels by miRNAs may be a useful method to regulate HCCs during hepatitis viral infection.
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http://dx.doi.org/10.1038/srep02739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3781398PMC
July 2014

Unique haploinsufficient role of the microRNA-processing molecule Dicer1 in a murine colitis-associated tumorigenesis model.

PLoS One 2013 2;8(9):e71969. Epub 2013 Sep 2.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

A widespread downregulated expression of microRNAs (miRNAs) is commonly observed in human cancers. Similarly, deregulated expression of miRNA-processing pathway components, which results in the reduction of global miRNA expression, may also be associated with tumorigenesis. Here, we show that specific ablation of Dicer1 in intestinal epithelial cells accelerates intestinal inflammation-associated tumorigenesis. This effect was apparent only when a single copy of Dicer1 was deleted, but not with complete Dicer1 ablation. DICER expression and subsequent mature miRNA levels were inversely correlated with the number of intact Dicer1 alleles. Because the expression levels of DICER were retained in tumors and its surrounding tissues even after induction of colitis-associated tumors, the effects of Dicer1 deletion were cell-autonomous. Although the expression levels of representative oncogenes and tumor suppressor genes were in most cases inversely correlated with the expression levels of DICER, some genes were not affected by Dicer1 deletion. Thus, deregulating the delicate balance between the expression levels of tumor-promoting and -suppressive genes may be crucial for tumorigenesis in this unique haploinsufficient case.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071969PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3759383PMC
April 2014

The flavonoid apigenin improves glucose tolerance through inhibition of microRNA maturation in miRNA103 transgenic mice.

Sci Rep 2013 ;3:2553

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Polyphenols are representative bioactive substances with diverse biological effects. Here, we show that apigenin, a flavonoid, has suppressive effects on microRNA (miRNA) function. The effects were mediated by impaired maturation of a subset of miRNAs, probably through inhibition of the phosphorylation of TRBP, a component of miRNA-generating complexes via impaired mitogen-activated protein kinase (MAPK) Erk activation. While glucose intolerance was observed in miRNA103 (miR103)-overexpressing transgenic mice, administration of apigenin improved this pathogenic status likely through suppression of matured miR103 expression levels. These results suggest that apigenin may have favorable effects on the pathogenic status induced by overexpression of miRNA103, whose maturation is mediated by phosphorylated TRBP.
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http://dx.doi.org/10.1038/srep02553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3757358PMC
February 2014

Inhibition of microRNA122 decreases SREBP1 expression by modulating suppressor of cytokine signaling 3 expression.

Biochem Biophys Res Commun 2013 Aug 24;438(1):230-5. Epub 2013 Jul 24.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

While inhibition of microRNA122 (miR122) function in vivo results in reduced serum cholesterol and fatty acid levels, the molecular mechanisms underlying the link between miR122 function and lipid metabolism remains unclear. Because the expression of SREBP1, a central transcription factor involved in lipid metabolism, is known to be increased by suppressor of cytokine signaling 3 (SOCS3) expression, and because we previously found that SOCS3 expression is regulated by miR122, in this study, we examined the correlation between miR122 status and the expression levels of SOCS3 and SREBP1. SREBP1 expression decreased when SOCS3 expression was reduced by miR122 silencing in vitro. Conversely, SREBP1 expression in miR122-silenced cells was restored by enforced expression of SOCS3. Such correlations were observed in human liver tissues with different miR122 expression levels. These signaling links may explain one of the molecular mechanisms linking inhibition of miR122 function or decreased expression of miR122 to decreased fatty acid and cholesterol levels, in the inhibition of miR122 function, or in pathological status in chronic liver diseases.
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http://dx.doi.org/10.1016/j.bbrc.2013.07.064DOI Listing
August 2013

The gene expression of the hypothalamic feeding-regulating peptides in cisplatin-induced anorexic rats.

Peptides 2013 Aug 15;46:13-9. Epub 2013 May 15.

Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan.

Cisplatin has been widely used; however, various disadvantageous side effects afflict patients. Rikkunshito (RKT), a traditional Japanese herbal medicine, has been widely prescribed in Japan to improve anorexia; but the mechanisms are unknown. Here we studied whether RKT could improve anorexia induced by cisplatin and changes in feeding-regulating peptides in the hypothalamus in rats. Adult male rats were divided into 4 groups: water+saline (WS), water+cisplatin (WC), RKT+saline (RS), and RKT+cisplatin (RC) groups. Water or RKT (1g/kg) was intragastrically administered for 4 days, from day -1 to day 2, and saline or cisplatin (6mg/kg) was intraperitoneally (i.p.) administered at day 0. After i.p. administration, cumulative food intake, water intake, urine volume and body weight were measured. The rats were then decapitated, followed by removal of the brain, and feeding-regulating peptides in the hypothalamus were measured by in situ hybridization histochemistry. In the three-day measurements, there were no significant changes in cumulative water intake and urine volume. The body weight and cumulative food intake in WC significantly decreased compared to WS, whereas these were not observed in RC. Pro-opiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART) in the arcuate nucleus (ARC) in WC significantly increased, and neuropeptide Y (NPY) in the ARC decreased compared to WS, whereas those in RS and RC were comparable to WS. These results suggest that RKT may have therapeutic potential for anorexia induced by cisplatin.
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http://dx.doi.org/10.1016/j.peptides.2013.04.019DOI Listing
August 2013