Kazuma Sekiba, MD, PhD

All Publications

• Satellite double-stranded RNA induces mesenchymal transition in pancreatic cancer by regulating alternative splicing. The Journal of biological chemistry Iwata, T., Kishikawa, T., Seimiya, T., Notoya, G., Suzuki, T., Shibata, C., Miyakawa, Y., Odawara, N., Funato, K., Tanaka, E., Yamagami, M., Sekiba, K., Otsuka, M., Koike, K., Fujishiro, M. 2024: 105742

  Abstract

  Human satellite II (HSATII), composed of tandem repeats in pericentromeric regions, is aberrantly transcribed in epithelial cancers, particularly pancreatic cancer. Dysregulation of repetitive elements in cancer tissues can facilitate incidental double-stranded RNA (dsRNA) formation; however, it remains controversial whether dsRNAs play tumor-promoting or tumor-suppressing roles during cancer progression. Therefore, we focused on the double-stranded formation of HSATII RNA and explored its molecular function. The overexpression of double-stranded HSATII (dsHSATII) RNA promoted mesenchymal-like morphological changes and enhanced the invasiveness of pancreatic cancer cells. We identified an RNA-binding protein, STRBP, which preferentially binds to dsHSATII RNA rather than single-stranded HSATII RNA. The mesenchymal transition of dsHSATII-expressing cells was rescued by STRBP overexpression. Mechanistically, STRBP is involved in the alternative splicing of genes associated with epithelial-mesenchymal transition (EMT). We also confirmed that isoform switching of CLSTN1, driven by dsHSATII overexpression or STRBP depletion, induced EMT-like morphological changes. These findings reveal a novel tumor-promoting function of dsHSATII RNA, inducing EMT-like changes and cell invasiveness, thus enhancing our understanding of the biological significance of aberrant expression of satellite arrays in malignant tumors.

  View details for DOI 10.1016/j.jbc.2024.105742

  View details for PubMedID 38346537

• A small molecule iCDM-34 identified by in silico screening suppresses HBV DNA through activation of aryl hydrocarbon receptor. Cell death discovery Furutani, Y., Hirano, Y., Toguchi, M., Higuchi, S., Qin, X., Yanaka, K., Sato-Shiozaki, Y., Takahashi, N., Sakai, M., Kongpracha, P., Suzuki, T., Dohmae, N., Kukimoto-Niino, M., Shirouzu, M., Nagamori, S., Suzuki, H., Kobayashi, K., Masaki, T., Koyama, H., Sekiba, K., Otsuka, M., Koike, K., Kohara, M., Kojima, S., Kakeya, H., Matsuura, T. 2023; 9 (1): 467

  Abstract

  IFN-alpha have been reported to suppress hepatitis B virus (HBV) cccDNA via APOBEC3 cytidine deaminase activity through interferon signaling. To develop a novel anti-HBV drug for a functional cure, we performed in silico screening of the binding compounds fitting the steric structure of the IFN-alpha-binding pocket in IFNAR2. We identified 37 compounds and named them in silico cccDNA modulator (iCDM)-1-37. We found that iCDM-34, a new small molecule with a pyrazole moiety, showed anti-HCV and anti-HBV activities. We measured the anti-HBV activity of iCDM-34 dependent on or independent of entecavir (ETV). iCDM-34 suppressed HBV DNA, pgRNA, HBsAg, and HBeAg, and also clearly exhibited additive inhibitory effects on the suppression of HBV DNA with ETV. We confirmed metabolic stability of iCDM-34 was stable in human liver microsomal fraction. Furthermore, anti-HBV activity in human hepatocyte-chimeric mice revealed that iCDM-34 was not effective as a single reagent, but when combined with ETV, it suppressed HBV DNA compared to ETV alone. Phosphoproteome and Western blotting analysis showed that iCDM-34 did not activate IFN-signaling. The transcriptome analysis of interferon-stimulated genes revealed no increase in expression, whereas downstream factors of aryl hydrocarbon receptor (AhR) showed increased levels of the expression. CDK1/2 and phospho-SAMHD1 levels decreased under iCDM-34 treatment. In addition, AhR knockdown inhibited anti-HCV activity of iCDM-34 in HCV replicon cells. These results suggest that iCDM-34 decreases the phosphorylation of SAMHD1 through CDK1/2, and suppresses HCV replicon RNA, HBV DNA, and pgRNA formation.

  View details for DOI 10.1038/s41420-023-01755-w

  View details for PubMedID 38135680

• Cabozantinib inhibits HBV-RNA transcription by decreasing STAT3 binding to the enhancer region of cccDNA. Hepatology communications Funato, K., Miyake, N., Sekiba, K., Miyakawa, Y., Seimiya, T., Shibata, C., Kishikawa, T., Otsuka, M. 2023; 7 (11)

  Abstract

  Precision medicine and customized therapeutics based on the features of each patient are important for maximizing therapeutic effects. Because most cases of HCC occur in the damaged liver through various etiologies, such as hepatitis virus infection, steatohepatitis, and autoimmune hepatitis, there should be a rationale for the choice of therapeutic options based on these etiologies. Although cabozantinib, an oral multikinase inhibitor, has demonstrated clinical effectiveness in advanced HCC, subgroup analyses showed a lower HR for death in HBV-related HCC. This study aimed to determine the therapeutic effects of cabozantinib in HBV-related HCC.Using HBV infection models and gene knockout cells, we determined the crucial signaling axis responsible for the effects of cabozantinib on HBV. A chromatin immunoprecipitation assay was performed to determine the interaction between the signaling molecules and HBV DNA. Agonists and inhibitors were used for confirmation.Cabozantinib inhibited HBV replication through the HGF-mesenchymal-epithelial transition factor-signal transducer and activator of transcription 3 (MET-STAT3) signaling axis. The importance of STAT3 in viral replication has been confirmed using gene-edited STAT3 knockout cells. The chromatin immunoprecipitation assay revealed that the binding levels of phosphorylated STAT3 to enhancer region 1 of HBV covalently closed circular DNA were significantly increased by HGF stimulation.Cabozantinib has favorable therapeutic effects on HBV-related HCC because it inhibits HCC not only directly but also indirectly by means of inhibitory effects on HBV.

  View details for DOI 10.1097/HC9.0000000000000313

  View details for PubMedID 37938099

• A widely distributed gene cluster compensates for uricase loss in hominids. Cell Liu, Y., Jarman, J. B., Low, Y. S., Augustijn, H. E., Huang, S., Chen, H., DeFeo, M. E., Sekiba, K., Hou, B. H., Meng, X., Weakley, A. M., Cabrera, A. V., Zhou, Z., van Wezel, G., Medema, M. H., Ganesan, C., Pao, A. C., Gombar, S., Dodd, D. 2023; 186 (16): 3400-3413.e20

  Abstract

  Approximately 15% of US adults have circulating levels of uric acid above its solubility limit, which is causally linked to the disease gout. In most mammals, uric acid elimination is facilitated by the enzyme uricase. However, human uricase is a pseudogene, having been inactivated early in hominid evolution. Though it has long been known that uric acid is eliminated in the gut, the role of the gut microbiota in hyperuricemia has not been studied. Here, we identify a widely distributed bacterial gene cluster that encodes a pathway for uric acid degradation. Stable isotope tracing demonstrates that gut bacteria metabolize uric acid to xanthine or short chain fatty acids. Ablation of the microbiota in uricase-deficient mice causes severe hyperuricemia, and anaerobe-targeted antibiotics increase the risk of gout in humans. These data reveal a role for the gut microbiota in uric acid excretion and highlight the potential for microbiome-targeted therapeutics in hyperuricemia.

  View details for DOI 10.1016/j.cell.2023.06.010

  View details for PubMedID 37541197

• UNBIASED MAPPING OF GUT MICROBIAL PATHWAYS THAT CONTRIBUTE TO INTESTINAL HEALTH Sekiba, K., Hou, B., Chen, H., Liu, Y., Dodd, D. W B SAUNDERS CO-ELSEVIER INC. 2023: S88

  View details for Web of Science ID 001040954700223

• Combination of serum human satellite RNA and miR-21-5p levels as a biomarker for pancreatic cancer. iScience Seimiya, T., Suzuki, T., Iwata, T., Kishikawa, T., Sekiba, K., Shibata, C., Ishigaki, K., Fujiwara, H., Oyama, H., Kanai, S., Sato, T., Nakai, Y., Ishibashi, R., Moriyama, M., Nakagawa, R., Ijichi, H., Otsuka, M., Koike, K. 2023; 26 (2): 106021

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis due to the difficulty of its diagnosis. Because human satellite II (HSATII) RNA, a satellite repeat RNA, is highly and specifically expressed in human PDAC, the serum HSATII RNA level may be a biomarker of PDAC. To measure the serum HSATII RNA level with high sensitivity and reproducibility, we previously developed a convenient method, tandem repeat amplification by nuclease protection (TRAP) combined with droplet digital PCR (ddPCR). Here, we refined the original method by simultaneously measuring the serum miR-21-5p level to enhance the detection of PDAC. The resulting PDAC-Index, constructed using serum HSATII RNA and miR-21-5p levels, discriminated patients with PDAC with high accuracy. We verified the clinical usefulness of the PDAC-Index as a supportive test in difficult-to-diagnose cases. The PDAC-Index has satisfactory diagnostic performance and may routinely be applied for detecting PDAC.

  View details for DOI 10.1016/j.isci.2023.106021

  View details for PubMedID 36798431

  View details for PubMedCentralID PMC9926314

• The RNA-Binding Protein ELAVL1 Regulates Hepatitis B Virus Replication and Growth of Hepatocellular Carcinoma Cells. International journal of molecular sciences Kanzaki, H., Chiba, T., Kaneko, T., Ao, J., Kan, M., Muroyama, R., Nakamoto, S., Kanda, T., Maruyama, H., Kato, J., Zen, Y., Kotani, A., Sekiba, K., Otsuka, M., Ohtsuka, M., Kato, N. 2022; 23 (14)

  Abstract

  Previous RNA immunoprecipitation followed by proteomic approaches successfully demonstrated that Embryonic Lethal, Abnormal Vision, Drosophila-Like 1 (ELAVL1) interacts with hepatitis B virus (HBV)-derived RNAs. Although ELAVL family proteins stabilize AU-rich element (ARE)-containing mRNAs, their role in HBV transcription remains unclear. This study conducted loss-of-function assays of ELAVL1 for inducible HBV-replicating HepAD38 cells and HBx-overexpressed HepG2 cells. In addition, clinicopathological analyses in primary hepatocellular carcinoma (HCC) surgical samples were also conducted. Lentivirus-mediated short hairpin RNA knockdown of ELAVL1 resulted in a decrease in both viral RNA transcription and production of viral proteins, including HBs and HBx, probably due to RNA stabilization by ELAVL1. Cell growth of HepAD38 cells was more significantly impaired in ELAVL1-knockdown than those in the control group, with or without HBV replication, indicating that ELAVL1 is involved in proliferation by factors other than HBV-derived RNAs. Immunohistochemical analyses of 77 paired HCC surgical specimens demonstrated that diffuse ELAVL1 expression was detected more frequently in HCC tissues (61.0%) than in non-tumor tissues (27.3%). In addition, the abundant expression of ELAVL1 tended to affect postoperative recurrence in HBV-related HCC patients. In conclusion, ELAVL1 contributes not only to HBV replication but also to HCC cell growth. It may be a potent therapeutic target for HBV-related HCC treatment.

  View details for DOI 10.3390/ijms23147878

  View details for PubMedID 35887229

• Hepatitis B virus-associated hepatocellular carcinoma with Smc5/6 complex deficiency is susceptible to PARP inhibitors. Biochemical and biophysical research communications Funato, K., Otsuka, M., Sekiba, K., Miyakawa, Y., Seimiya, T., Shibata, C., Kishikawa, T., Fujishiro, M. 2022; 607: 89-95

  Abstract

  DNA repair processes represent attractive synthetic lethal targets because many cancers exhibit impaired DNA repair pathways, which leads to dependence on specific repair proteins. The finding that poly (ADP-ribose) polymerase (PARP)-1 inhibitors are highly effective against cancers with deficient homologous recombination highlights the potential of this approach. In hepatitis B viral (HBV) infection, degradation of the structural maintenance of the chromosome 5/6 (Smc5/6) complex, which plays a key role in repairing double-stranded DNA breaks by homologous recombination, is induced by HBV regulatory protein X (HBx). Here, we hypothesized that a deficiency in the Smc5/6 complex in HBV-associated hepatocellular carcinoma (HCC) increases susceptibility to PARP inhibitors via a deficiency in homologous recombination. We confirmed impaired double-stranded DNA break repair in HBx-expressing HCC cells using a sensitive reporter to monitor homologous recombination. Treatment with a PARP inhibitor was significantly more effective against HBx-expressing HCC cells, and overexpression of Smc5/6 prevented these effects. Overall, our results suggest that homologous recombination deficiency in HBV-associated HCC leads to increased susceptibility to PARP inhibitors.

  View details for DOI 10.1016/j.bbrc.2022.03.137

  View details for PubMedID 35367833

• HBx-induced degradation of Smc5/6 complex impairs homologous recombination-mediated repair of damaged DNA JOURNAL OF HEPATOLOGY Sekiba, K., Otsuka, M., Funato, K., Miyakawa, Y., Tanaka, E., Seimiya, T., Yamagami, M., Tsutsumi, T., Okushin, K., Miyakawa, K., Ryo, A., Koike, K. 2022; 76 (1): 53-62

  Abstract

  HBV causes hepatocellular carcinoma (HCC). While it was recently shown that the ability of HBV X protein (HBx) to impair the Smc5/6 (structural maintenance of chromosome 5/6) complex is important for viral transcription, HBx is also a potent driver of HCC. However, the mechanism by which HBx expression induces hepatocarcinogenesis is unclear.Degradation of the Smc5/6 complex and accumulation of DNA damage were observed in both in vivo and in vitro HBV infection models. Rescue experiments were performed using nitazoxanide (NTZ), which inhibits degradation of the Smc5/6 complex by HBx.HBx-triggered degradation of the Smc5/6 complex causes impaired homologous recombination (HR) repair of DNA double-strand breaks (DSBs), leading to cellular transformation. We found that DNA damage accumulated in the liver tissue of HBV-infected humanized chimeric mice, HBx-transgenic mice, and human tissues. HBx suppressed the HR repair of DSBs, including that induced by the CRISPR-Cas9 system, in an Smc5/6-dependent manner, which was rescued by restoring the Smc5/6 complex. NTZ restored HR repair in, and colony formation by, HBx-expressing cells.Degradation of the Smc5/6 complex by HBx increases viral transcription and promotes cellular transformation by impairing HR repair of DSBs.The hepatitis B virus expresses a regulatory protein called HBV X protein (or HBx). This protein degrades the Smc5/6 complex in human hepatocytes, which is essential for viral replication. We found that this process also plays a key role in the accumulation of DNA damage, which contributes to HBx-mediated tumorigenesis.

  View details for DOI 10.1016/j.jhep.2021.08.010

  View details for Web of Science ID 000732465900008

  View details for PubMedID 34478763

• Humanized virus-suppressing factor inhibits hepatitis B virus infection by targeting viral cell entry HELIYON Miyakawa, Y., Otsuka, M., Sekiba, K., Funato, K., Koike, K. 2021; 7 (7): e07586

  Abstract

  Although nucleos(t)ide analogs and interferons suppress hepatitis B virus (HBV) replication, they must be taken continuously and have a low response rate. Therefore, therapeutics for HBV with novel modes of action are needed. Humanized virus-suppressing factor (hzVSF) is a monoclonal antibody against vimentin that exhibits broad-spectrum antiviral activity. Here, hzVSF significantly inhibited HBV infection. Although hzVSF inhibited HBV RNA production, it did not affect viral transcription from minicircle DNA mimicking covalently closed circular DNA. Additionally, hzVSF did not inhibit viral protein or DNA release from infected cells. Rather, hzVSF inhibited the cell entry of viral preS1 peptides, possibly by altering intracellular vimentin localization, which is important for HBV cell entry. These results suggest that hzVSF has therapeutic potential for HBV infection with a novel mode of action.

  View details for DOI 10.1016/j.heliyon.2021.e07586

  View details for Web of Science ID 000687266500024

  View details for PubMedID 34345745

  View details for PubMedCentralID PMC8319007

• Potential of HBx Gene for Hepatocarcinogenesis in Noncirrhotic Liver SEMINARS IN LIVER DISEASE Sekiba, K., Otsuka, M., Koike, K. 2021; 41 (02): 142-149

  Abstract

  Current treatments for hepatitis B virus (HBV) using nucleos(t)ide analogs cannot eliminate the risk of hepatocellular carcinoma (HCC) development. As HBV-associated HCC can develop even in the absence of liver cirrhosis, HBV is regarded to possess direct oncogenic potential. HBV regulatory protein X (HBx) has been identified as a primary mediator of HBV-mediated hepatocarcinogenesis. A fragment of the HBV genome that contains the coding region of HBx is commonly integrated into the host genome, resulting in the production of aberrant proteins and subsequent hepatocarcinogenesis. Besides, HBx interferes with the host DNA or deoxyribonucleic acid damage repair pathways, signal transduction, epigenetic regulation of gene expression, and cancer immunity, thereby promoting carcinogenesis in the noncirrhotic liver. However, numerous molecules and pathways have been implicated in the development of HBx-associated HCC, suggesting that the mechanisms underlying HBx-mediated hepatocarcinogenesis remain to be elucidated.

  View details for DOI 10.1055/s-0041-1723033

  View details for Web of Science ID 000649857300001

  View details for PubMedID 33984871

• Mutant KRAS drives metabolic reprogramming and autophagic flux in premalignant pancreatic cells CANCER GENE THERAPY Suzuki, T., Kishikawa, T., Sato, T., Takeda, N., Sugiura, Y., Seimiya, T., Sekiba, K., Ohno, M., Iwata, T., Ishibashi, R., Otsuka, M., Koike, K. 2021

  Abstract

  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.

  View details for DOI 10.1038/s41417-021-00326-4

  View details for Web of Science ID 000638033200002

  View details for PubMedID 33833413

• The simplified Kyoto classification score is consistent with the ABC method of classification as a grading system for endoscopic gastritis JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Nishizawa, T., Toyoshima, O., Kondo, R., Sekiba, K., Tsuji, Y., Ebinuma, H., Suzuki, H., Tanikawa, C., Matsuda, K., Koike, K. 2021; 68 (1): 101-104

  Abstract

  The ABC method combined with Helicobacter pylori antibody and serum pepsinogen is a useful predictive method for stomach cancer. Kyoto classification is a new grading system for endoscopic gastritis. However, the consistency of the Kyoto score with the ABC method remains unclear. The Kyoto classification score, which ranges from 0 to 8, is based on the following findings: atrophy, intestinal metaplasia, diffuse redness, nodularity, and enlarged folds. Furthermore, we defined a simplified Kyoto classification score as the sum of scores of just atrophy and intestinal metaplasia. The association between the Kyoto classification score and the ABC method was analyzed using the Kruskal-Wallis and Steel-Dwass tests. A total of 307 subjects were enrolled. Kyoto classification scores were similar in groups B, C, and D, while scores in group A were significantly lower than those of the other groups. The simplified Kyoto classification score showed the same stepwise increase as the classification of the ABC method. In conclusion, unlike the Kyoto classification score, the simplified Kyoto score showed the same significant stepwise increase as the classification of the ABC method.

  View details for DOI 10.3164/jcbn.20-41

  View details for Web of Science ID 000687883500018

  View details for PubMedID 33536719

  View details for PubMedCentralID PMC7844658

• A single nucleotide polymorphism in Prostate Stem Cell Antigen is associated with endoscopic grading in Kyoto classification of gastritis JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Toyoshima, O., Nishizawa, T., Sekiba, K., Matsuno, T., Kondo, R., Watanabe, H., Suzuki, H., Tanikawa, C., Koike, K., Matsuda, K. 2021; 68 (1): 73-77

  Abstract

  The risk allele of a single nucleotide polymorphism (SNP) rs2294008 in the Prostate stem cell antigen (PSCA) gene is strongly associated with gastric cancer. Although the Kyoto classification score is believed to be an indicator of gastric cancer risk, it lacks supporting genetic evidence. We investigated the effect of this risk allele of PSCA SNP on the Kyoto score. Participants without a history of gastric cancer or Helicobacter pylori (H. pylori) eradication underwent esophagogastroduodenoscopy, H. pylori evaluation, and SNP genotyping. The Kyoto score is the sum of scores obtained from endoscopy-based atrophy, intestinal metaplasia, enlarged folds, nodularity, and diffuse redness. The Kyoto score is novel in the light of scoring for gastritis. A total of 323 patients were enrolled (number of individuals with genotype CC: 52; CT: 140; TT: 131, average age: 50.1 years, male: 50.8%). The patient baseline characteristics including age, sex, body mass index, smoking, drinking, family history of gastric cancer, and H. pylori status had no association with PSCA SNP. The Kyoto score was higher in T (CT or TT genotype; risk allele) carriers than in CC carriers. Atrophy, enlarged folds, and diffuse redness scores were higher in T allele carriers (risk allele) than in CC genotype individuals. In multivariate analysis, the Kyoto score was independently associated with PSCA SNP (OR: 1.30, p = 0.012). Thus, the Kyoto score was associated with a genetic predisposition.

  View details for DOI 10.3164/jcbn.20-67

  View details for Web of Science ID 000687883500014

  View details for PubMedID 33536715

  View details for PubMedCentralID PMC7844668

• The fatty-acid amide hydrolase inhibitor URB597 inhibits MICA/B shedding SCIENTIFIC REPORTS Sekiba, K., Otsuka, M., Seimiya, T., Tanaka, E., Funato, K., Miyakawa, Y., Koike, K. 2020; 10 (1): 15556

  Abstract

  MICA/B proteins are expressed on the surface of various types of stressed cells, including cancer cells. Cytotoxic lymphocytes expressing natural killer group 2D (NKG2D) receptor recognize MICA/B and eliminate the cells. However, cancer cells evade such immune recognition by inducing proteolytic shedding of MICA/B proteins. Therefore, preventing the shedding of MICA/B proteins could enhance antitumor immunity. Here, by screening a protease inhibitor library, we found that the fatty-acid amide hydrolase (FAAH) inhibitor, URB597, suppresses the shedding of MICA/B. URB597 significantly reduced the soluble MICA level in culture medium and increased the MICA level on the surface of cancer cells. The effect was indirect, being mediated by increased expression of tissue inhibitor of metalloproteinases 3 (TIMP3). Knockdown of TIMP3 expression reversed the effect of URB597, confirming that TIMP3 is required for the MICA shedding inhibition by URB597. In contrast, FAAH overexpression reduced TIMP3 expression and the cell-surface MICA level and increased the soluble MICA level. These results suggest that inhibition of FAAH could prevent human cancer cell evasion of immune-mediated clearance.

  View details for DOI 10.1038/s41598-020-72688-y

  View details for Web of Science ID 000573849300084

  View details for PubMedID 32968163

  View details for PubMedCentralID PMC7512021

• Aberrant expression of a novel circular RNA in pancreatic cancer JOURNAL OF HUMAN GENETICS Seimiya, T., Otsuka, M., Iwata, T., Tanaka, E., Sekiba, K., Shibata, C., Moriyama, M., Nakagawa, R., Maruyama, R., Koike, K. 2021; 66 (2): 181-191

  Abstract

  Circular RNAs (circRNAs) are single-stranded, covalently closed RNA molecules that are produced from pre-mRNAs through a process known as back-splicing. Although circRNAs are expressed under specific conditions, current understanding of their comprehensive expression status is still limited. Here, we performed a large-scale circRNA profiling analysis in human pancreatic ductal adenocarcinoma (PDAC) tissues, using circular RNA-specific RNA sequencing. We identified more than 40,000 previously unknown circRNAs, some of which were upregulated in PDAC tissues, compared with normal pancreatic tissues. We determined the full-length sequence of a circRNA upregulated in PDAC, which was derived from two noncoding RNA loci on chromosome 12. The novel circRNA, named circPDAC RNA, was not expressed in normal human cells, but was expressed in PDAC and other carcinoma cells. While postulated biological functions, such as peptide production from the circPDAC RNA, were not detected, its aberrant expression was confirmed in other PDAC tissues and in serum from a PDAC patient. These results demonstrate that comprehensive studies are necessary to reveal the expression status of circRNAs and that the circPDAC RNA identified here might serve as a novel biomarker for cancers, including PDAC.

  View details for DOI 10.1038/s10038-020-00826-5

  View details for Web of Science ID 000565493200001

  View details for PubMedID 32879441

• Expert endoscopists with high adenoma detection rates frequently detect diminutive adenomas in proximal colon ENDOSCOPY INTERNATIONAL OPEN Toyoshima, O., Nishizawa, T., Yoshida, S., Sekiba, K., Kataoka, Y., Hata, K., Watanabe, H., Tsuji, Y., Koike, K. 2020; 8 (6): E775-E782

  Abstract

  Background and study aims  Adenoma detection rate (ADR) is an important quality indicator in colonoscopy, and improved ADR decreases the incidence of colorectal cancer. We investigated differences in polyp detection according to the endoscopist's ADR. Patients and methods  We performed a propensity-score matching study using baseline patient characteristics of age, sex, body mass index, family history of colorectal cancer, smoking, drinking, indication for colonoscopy, bowel preparation, and colonoscope type. We compared polyp detection and colonoscopy procedures between patients who underwent colonoscopy by high-ADR endoscopists (high ADR group) and by low-ADR endoscopists (low ADR group). Results  We matched 334 patients in the high ADR group with 334 in the low ADR group. The ADR was 44.0 % and 26.9 % for the high-ADR and low-ADR endoscopists, respectively. Proximal, nonprotruding, and diminutive adenomas were more frequently detected by high-ADR endoscopists than by low-ADR endoscopists (all P  < 0.001); similarly, more high-risk adenomas were detected by high-ADR endoscopists ( P  = 0.028). Furthermore, more sessile serrated polyps detected by high-ADR endoscopists ( P  = 0.041). High-ADR endoscopists more frequently performed pancolonic chromoendoscopy ( P  < 0.001). Conclusions  Expert detectors often found nonprotruding and diminutive adenomas in the proximal colon along with increased detection rate of high-risk adenomas. Low-ADR endoscopists need to recognize the features of missed adenomas to improve their ADRs.

  View details for DOI 10.1055/a-1136-9971

  View details for Web of Science ID 000537109100012

  View details for PubMedID 32490163

  View details for PubMedCentralID PMC7247898

• Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Sekiba, K., Otsuka, M., Koike, K. 2019

  Abstract

  There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently inhibit viral replication, they have no direct effect on the expression of viral proteins transcribed from a viral covalently closed circular DNA (cccDNA). As high viral antigen load may play a role in this chronic and HBV-related carcinogenesis, the goal of HBV treatment is to eradicate viral proteins. HBV regulatory protein X (HBx) binds to the host DNA damage-binding protein 1 (DDB1) protein to degrade structural maintenance of chromosomes 5/6 (Smc5/6), resulting in activation of viral transcription from cccDNA. Here, using a split luciferase complementation assay system, we present a comprehensive compound screening system to identify inhibitors of the HBx-DDB1 interaction. Our protocol enables easy detection of interaction dynamics in real time within living cells. This technique may become a key assay to discover novel therapeutic agents for treatment of HBV infection.

  View details for DOI 10.3791/60652

  View details for Web of Science ID 000505787900052

  View details for PubMedID 31904023

• Detection of circulating colorectal cancer cells by a custom microfluid system before and after endoscopic metallic stent placement ONCOLOGY LETTERS Ishibashi, R., Yoshida, S., Odawara, N., Kishikawa, T., Kondo, R., Nakada, A., Hakuta, R., Takahara, N., Tanaka, E., Sekiba, K., Seimiya, T., Ohnaga, T., Otsuka, M., Koike, K. 2019; 18 (6): 6397-6404

  Abstract

  Although the detection of circulating tumor cells (CTCs) should be crucial for future personalized medicine, no efficient and flexible methods have been established. The current study established a polymeric custom-made chip for capturing CTCs with a high efficiency and flexibility. As an example of clinical application, the effects of self-expandable metallic stent (SEMS) placement on the release of cancer cells into the blood of patients with colorectal cancer and bowel obstruction were analyzed. This was assessed as the placement of SEMS may cause mechanical damage and physical force to malignant tissue, increasing the risk of cancer cell release into the bloodstream. The present study examined the number of CTCs using a custom-made chip, before, at 24 h after and at 4 days after SEMS placement in patients with colorectal cancer. The results revealed that, among the 13 patients examined, the number of CTCs was increased in three cases at 24 h after SEMS placement. However, this increase was temporary. The number of CTCs also decreased at 4 days after stent placement in most cases. The CTC chip of the current study detected the number of CD133-positive cancer stem-like cells, which did not change, even in the patient whose total number of CTCs temporarily increased. The results indicated that this custom-made microfluid system can efficiently and flexibly detect CTCs, demonstrating its potential for obtaining information during the management of patients with cancer.

  View details for DOI 10.3892/ol.2019.11047

  View details for Web of Science ID 000505576300083

  View details for PubMedID 31807163

  View details for PubMedCentralID PMC6876337

• Expression of circular RNA CDR1-AS in colon cancer cells increases cell surface PD-L1 protein levels ONCOLOGY REPORTS Tanaka, E., Miyakawa, Y., Kishikawa, T., Seimiya, T., Iwata, T., Funato, K., Odawara, N., Sekiba, K., Yamagami, M., Suzuki, T., Ishibashi, R., Otsuka, M., Koike, K. 2019; 42 (4): 1459-1466

  Abstract

  The expression of CDR1‑AS, a representative circular RNA, is closely linked with poor prognosis in gastrointestinal cancers, such as colon, liver, and pancreatic cancers. Although it is well known that CDR1‑AS antagonizes microRNA‑7 function through its sequence similarities in the brain, its biological function and link with the malignant potential of cancer cells remain unclear, partly due to the difficulties of ectopic expression of circular RNAs. In the present study, SW620, a colon cancer cell line that stably expresses CDR1‑AS RNA circularized, was established using the laccase 2 gene cassette, and its biological function associated with malignant behavior was determined. In contrast to previous studies, cell growth or invasion ability was not altered by CDR1‑AS expression. However, the expression levels of CMTM4 and CMTM6, which were recently recognized as critical regulators of PD‑L1 protein expression at the cell surface, were significantly increased. Accordingly, the cell surface PD‑L1 protein levels were increased in CDR1‑AS‑expressing cells. Notably, the effects were not canceled out by overexpressing microRNA‑7, indicating that the increase in cell surface PD‑L1 in CDR1‑AS‑expressing cells was not dependent on microRNA‑7 function. These results indicated that expression of this circular RNA in cancer cells may lead to poor prognosis by increasing cell surface PD‑L1 levels through microRNA‑7‑independent mechanisms.

  View details for DOI 10.3892/or.2019.7244

  View details for Web of Science ID 000487071800019

  View details for PubMedID 31322270

• Pevonedistat, a Neuronal Precursor Cell-Expressed Developmentally Down-Regulated Protein 8-Activating Enzyme Inhibitor, Is a Potent Inhibitor of Hepatitis B Virus HEPATOLOGY Sekiba, K., Otsuka, M., Ohno, M., Yamagami, M., Kishikawa, T., Seimiya, T., Suzuki, T., Tanaka, E., Ishibashi, R., Funato, K., Koike, K. 2019; 69 (5): 1903-1915

  Abstract

  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.

  View details for DOI 10.1002/hep.30491

  View details for Web of Science ID 000466449400007

  View details for PubMedID 30586159

• Inhibition of HBV Transcription From cccDNA With Nitazoxanide by Targeting the HBx-DDB1 Interaction CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY Sekiba, K., Otsuka, M., Ohno, M., Yamagami, M., Kishikawa, T., Suzuki, T., Ishibashi, R., Seimiya, T., Tanaka, E., Koike, K. 2019; 7 (2): 297-312

  Abstract

  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.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.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.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.

  View details for DOI 10.1016/j.jcmgh.2018.10.010

  View details for Web of Science ID 000459860200008

  View details for PubMedID 30704981

  View details for PubMedCentralID PMC6357790

• Inflammation and de-differentiation in pancreatic carcinogenesis WORLD JOURNAL OF CLINICAL CASES Seimiya, T., Otsuka, M., Iwata, T., Tanaka, E., Suzuki, T., Sekiba, K., Yamagami, M., Ishibashi, R., Koike, K. 2018; 6 (15): 882-891

  Abstract

  Pancreatic cancer is a malignancy with an extremely poor prognosis. Chronic pancreatitis is a well-known risk factor for pancreatic cancer. Inflammation is thought to influence carcinogenesis through DNA damage and activation of intracellular signaling pathways. Many transcription factors and signaling pathways co-operate to determine and maintain cell identity at each phase of pancreatic organogenesis and cell differentiation. Recent studies have shown that carcinogenesis is promoted through the suppression of transcription factors related to differentiation. Pancreatitis also demonstrates transcriptional changes, suggesting that multifactorial epigenetic changes lead to impaired differentiation. Taken together, these factors may constitute an important framework for pancreatic carcinogenesis. In this review, we discuss the role of inflammation and de-differentiation in the development of pancreatic cancer, as well as the future of novel therapeutic applications.

  View details for DOI 10.12998/wjcc.v6.i15.882

  View details for Web of Science ID 000452438600002

  View details for PubMedID 30568942

  View details for PubMedCentralID PMC6288496

• ISGF3 with reduced phosphorylation is associated with constitutive expression of interferon-induced genes in aging cells NPJ AGING AND MECHANISMS OF DISEASE Yamagami, M., Otsuka, M., Kishikawa, T., Sekiba, K., Seimiya, T., Tanaka, E., Suzuki, T., Ishibashi, R., Ohno, M., Koike, K. 2018; 4: 11

  Abstract

  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.

  View details for DOI 10.1038/s41514-018-0030-6

  View details for Web of Science ID 000461498400001

  View details for PubMedID 30455980

  View details for PubMedCentralID PMC6237867

• Circular RNA and exosomes in pancreatic cancer progression TRANSLATIONAL CANCER RESEARCH Seimiya, T., Otsuka, M., Liu, H., Suzuki, T., Sekiba, K., Yamagami, M., Tanaka, E., Ishibashi, R., Koike, K. 2018; 7: S745-S747

  View details for DOI 10.21037/tcr.2018.08.03

  View details for Web of Science ID 000442855200012

• Satellite RNA Increases DNA Damage and Accelerates Tumor Formation in Mouse Models of Pancreatic Cancer MOLECULAR CANCER RESEARCH Kishikawa, T., Otsuka, M., Suzuki, T., Seimiya, T., Sekiba, K., Ishibashi, R., Tanaka, E., Ohno, M., Yamagami, M., Koike, K. 2018; 16 (8): 1255-1262

  Abstract

  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 in vivo, 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.Implications: Aberrant expression of satellite RNAs accelerates oncogenesis through a mechanism involving increased DNA damage. Mol Cancer Res; 16(8); 1255-62. ©2018 AACR.

  View details for DOI 10.1158/1541-7786.MCR-18-0139

  View details for Web of Science ID 000440602800005

  View details for PubMedID 29748382

• Hepatitis B virus pathogenesis: Fresh insights into hepatitis B virus RNA WORLD JOURNAL OF GASTROENTEROLOGY Sekiba, K., Otsuka, M., Ohno, M., Yamagami, M., Kishikawa, T., Suzuki, T., Ishibashi, R., Seimiya, T., Tanaka, E., Koike, K. 2018; 24 (21): 2261-2268

  Abstract

  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.

  View details for DOI 10.3748/wjg.v24.i21.2261

  View details for Web of Science ID 000434396400004

  View details for PubMedID 29881235

  View details for PubMedCentralID PMC5989240

• DHX9 regulates production of hepatitis B virus-derived circular RNA and viral protein levels. Oncotarget Sekiba, K., Otsuka, M., Ohno, M., Kishikawa, T., Yamagami, M., Suzuki, T., Ishibashi, R., Seimiya, T., Tanaka, E., Koike, K. 2018; 9 (30): 20953-20964

  Abstract

  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.

  View details for DOI 10.18632/oncotarget.25104

  View details for PubMedID 29765512

• A tiny but crucial player bridging microbes and colonic carcinogenesis TRANSLATIONAL CANCER RESEARCH Otsuka, M., Ishibashi, R., Tanaka, E., Seimiya, T., Suzuki, T., Sekiba, K., Yamagami, M., Ohno, M., Kishikawa, T., Koike, K. 2017; 6: S1467-S1470

  View details for DOI 10.21037/tcr.2017.11.13

  View details for Web of Science ID 000417970200021

• RASAL1 is a potent regulator of hepatic stellate cell activity and liver fibrosis ONCOTARGET Takata, A., Otsuka, M., Kishikawa, T., Yamagami, M., Ishibashi, R., Sekiba, K., Suzuki, T., Ohno, M., Yamashita, Y., Abe, T., Masuzaki, R., Ikenoue, T., Koike, K. 2017; 8 (39): 64840-64852

  Abstract

  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.

  View details for DOI 10.18632/oncotarget.17609

  View details for Web of Science ID 000410291200014

  View details for PubMedID 29029395

  View details for PubMedCentralID PMC5630295

• Transcriptional activation of the MICA gene with an engineered CRISPR-Cas9 system BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Sekiba, K., Yamagami, M., Otsuka, M., Suzuki, T., Kishikawa, T., Ishibashi, R., Ohno, M., Sato, M., Koike, K. 2017; 486 (2): 521-525

  Abstract

  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.

  View details for DOI 10.1016/j.bbrc.2017.03.076

  View details for Web of Science ID 000399261200046

  View details for PubMedID 28322797

• A new method for insertion of long intestinal tube for small bowel obstruction Nonendoscopic over-the-wire method via short nasogastric tube MEDICINE Sekiba, K., Ohmae, T., Odawara, N., Moriyama, M., Kanai, S., Tsuboi, M., Saito, T., Uchino, K., Akamatsu, M., Okamoto, M. 2016; 95 (47): e5449

  Abstract

  It is often difficult to insert a long intestinal tube (LT) in patients with small bowel obstruction (SBO). We developed a novel technique for inserting an LT without endoscopy called nonendoscopic over-the-wire method via short nasogastric tube (NEWSt). We evaluated the efficacy and safety of NEWSt.We performed a retrospective study of patients who underwent LT insertion for SBO without any indications of strangulation with either NEWSt (n = 16) or endoscopy (n = 17) between November 2011 and February 2015 at our hospital. Univariate analysis was used to assess the success rate of LT placement beyond the duodenojejunal flexure, time required for the procedure, clinical outcomes, and adverse events.The success rate was 100% in both groups. Procedure time was numerically, but not statistically, shorter in the NEWSt group compared with the endoscopy group (24 ± 13 vs 30 ± 13 min; P = 0.174). There were no statistically significant differences between the 2 groups in terms of surgery rate (31% vs 12%; P = 0.225), fasting period (11.3 ± 6.3 vs 9.9 ± 4.5 days; P = 0.482), hospital stay (26.4 ± 22.1 vs 18.7 ± 7.0 days; P = 0.194), and recurrence rate (19% vs 24%; P = 1.0). No serious adverse event was observed in the NEWSt group, whereas serious aspiration pneumonia was observed in 2 patients after LT insertion in the endoscopy group.Without endoscopy, NEWSt enabled the high success rate and the short procedure time for the LT insertion. Prospective, randomized controlled trials are needed.

  View details for DOI 10.1097/MD.0000000000005449

  View details for Web of Science ID 000389591000039

  View details for PubMedID 27893689

  View details for PubMedCentralID PMC5134882