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1.  Gene amplification of EGFR, HER2, FGFR2 and MET in esophageal squamous cell carcinoma 
International Journal of Oncology  2013;42(4):1151-1158.
Molecular targeted therapy is expected to be a promising therapeutic approach for the treatment of esophageal squamous cell carcinoma (ESCC); however, the gene amplification status of molecular targeted genes in ESCC remains largely unclear. The gene amplification of EGFR, HER2, FGFR2 and MET was examined using a real-time PCR-based copy number assay of 245 ESCC surgical specimens of formalin-fixed, paraffin-embedded samples. Fluorescence in situ hybridization (FISH) and comparative genomic hybridization analyses verified the results of the copy number assay. EGFR mutation was detected using the Scorpions-ARMS method. The EGFR status and drug sensitivity to an EGFR tyrosine kinase inhibitor was then evaluated in vitro. Gene amplification of EGFR and HER2 was observed in 7% (16/244) and 11% (27/245) of the ESCC specimens. A multivariate analysis revealed that HER2 amplification was a significant predictor of a poor prognosis in patients with stage III post-operative ESCC. The L861Q type of EGFR mutation with hypersensitivity to EGFR tyrosine kinase inhibitor was found in one of the eight ESCC cell lines and one del745 type of EGFR mutation was identified in 107 clinical samples. In addition, we demonstrated for the first time that FGFR2 amplification was observed in 4% (8/196) of the ESCC specimens. MET amplification was observed in 1% (2/196). In conclusion, the frequent gene amplification of EGFR, HER2 and FGFR2 and the presence of active EGFR mutations were observed in ESCC specimens. Our results strongly encourage the development of molecular targeted therapy for ESCC.
PMCID: PMC3622677  PMID: 23426935
EGFR; HER2; FGFR2; MET; esophageal squamous cell carcinoma
2.  Estimation of autistic children by metallomics analysis 
Scientific Reports  2013;3:1199.
Clarification of the pathogenesis and treatment of autism spectrum disorders is one of the challenges today. In this study, we examine scalp hair concentrations of 26 trace elements for 1,967 children with autistic disorders (1,553 males and 414 females). Five-hundred and eighty-four (29.7%), 347 (17.6%) and 114 (5.8%) subjects was found deficient in zinc, magnesium and calcium, respectively, and 2.0% or less in the other essential metals. The incidence rate of mineral deficiency was highly observed in infants aged 0-3 year-old. In contrast, 339 (17.2%), 168 (8.5%) and 94 (4.8%) individuals was found suffering from high burden of aluminium, cadmium and lead, and 2.8% or less from mercury and arsenic burden. These findings suggest that infantile zinc- and magnesium-deficiency and/or toxic metal burdens may epigenetically play principal roles as environmental factors in autistic disorders and that metallomics approach may lead to early screening and prevention of the neurodevelopment disorders.
PMCID: PMC3563033  PMID: 23383369
3.  Infantile zinc deficiency: Association with autism spectrum disorders 
Scientific Reports  2011;1:129.
Elucidation of the pathogenesis and effective treatment of autism spectrum disorders is one of the challenges today. In this study, we examine hair zinc concentrations for 1,967 children with autistic disorders (1,553 males and 414 females), and show considerable association with zinc deficiency. Histogram of hair zinc concentration was non-symmetric with tailing in lower range, and 584 subjects were found to have lower zinc concentrations than −2 standard deviation level of its reference range (86.3–193ppm). The incidence rate of zinc deficiency in infant group aged 0–3 year-old was estimated 43.5 % in male and 52.5 % in female. The lowest zinc concentration of 10.7 ppm was detected in a 2-year-old boy, corresponding to about 1/12 of the control mean level. These findings suggest that infantile zinc deficiency may epigenetically contribute to the pathogenesis of autism and nutritional approach may yield a novel hope for its treatment and prevention.
PMCID: PMC3216610  PMID: 22355646
4.  Metallomics study using hair mineral analysis and multiple logistic regression analysis: relationship between cancer and minerals 
The objective of this metallomics study is to investigate comprehensively some relationships between cancer risk and minerals, including essential and toxic metals.
Twenty-four minerals including essential and toxic metals in scalp hair samples from 124 solid-cancer patients and 86 control subjects were measured with inductively coupled plasma mass spectrometry (ICP-MS), and the association of cancer with minerals was statistically analyzed with multiple logistic regression analysis.
Multiple logistic regression analysis demonstrated that several minerals are significantly correlated to cancer, positively or inversely. The most cancer-correlated mineral was iodine (I) with the highest correlation coefficient of r = 0.301, followed by arsenic (As; r = 0.267), zinc (Zn; r = 0.261), and sodium (Na; r = 0.190), with p < 0.01 for each case. In contrast, selenium (Se) was inversely correlated to cancer (r = −0.161, p < 0.05), followed by vanadium (V) (r = −0.128). Multiple linear regression value was highly significantly correlated with probability of cancer (R2 = 0.437, p < 0.0001), and the area under the receiver-operating characteristic (ROC) curve was calculated to be 0.918. In addition, using contingency table analysis and the chi-square test, the precision of discrimination for cancer was estimated to be 0.871 (chi-square = 99.1, p < 0.0001).
These findings suggest that some minerals such as arsenic, selenium, and probably iodine, zinc, sodium, and vanadium contribute to regulation of cancer and also that metallomics study using multiple logistic regression analysis is a useful tool for estimating cancer risk.
PMCID: PMC2728251  PMID: 19568830
Cancer; Metallomics; Arsenic; Selenium; Multiple logistic regression analysis
Neuroscience  2008;156(1):71-80.
Cortical surface evoked potentials (SEPs) are larger during sleep and characterize a sleep-like state in cortical columns. Since tumor necrosis factor alpha (TNF) may be involved in sleep regulation and is produced as a consequence of waking activity, we tested the hypothesis that direct application of TNF to the cortex will induce a sleep-like state within cortical columns and enhance SEP amplitudes. We found that microinjection of TNF onto the surface of the somatosensory cortex enhanced whisker stimulation-induced SEP amplitude relative to a control heat-inactivated TNF microinjection. We also determined if whisker stimulation enhanced endogenous TNF expression. TNF immunoreactivity (IR) was visualized after 2 h of bilateral deflection of a single whisker bilaterally. The number of TNF-IR cells increased in layers II–IV of the activated somatosensory barrel column. In two separate studies, unilateral deflection of multiple whiskers for 2 h increased the number of TNF-IR cells in layers II–V in columns that also exhibited enhanced Fos-IR. TNF-IR also colocalized with NeuN-IR suggesting that TNF expression was in neurons. Collectively these data are consistent with the hypotheses that TNF is produced in response to neural activity and in turn enhances the probability of a local sleep-like state as determined by increases in SEP amplitudes.
PMCID: PMC2654198  PMID: 18694809
barrel field; evoked response potentials; surface evoked potentials; local sleep; cortical columns; cytokine
6.  Papillo-Choledochectomy in the Operative Management of Mucosal Neoplasms of the Periampullary Region 
HPB Surgery  1993;6(3):211-217.
Two patients with mucosal cancer of the periampullary region were treated with papillocholedochectomy, which entails removal of the papilla of Vater and the whole length of the common bile duct. The neoplasm is dissected out through the plane between the duodenal circular and longitudinal muscles, deep to the sphincter of Oddi and the fibromuscular layer of the bile duct. Pathological examination showed that cancer was confined to the mucosal layer without stromal invasion, and that the operation achieved radical cure. For mucosal cancer, papillo-choledochectomy is an alternative to pancreatoduodenectomy, provided that repeated frozen-section studies confirm the completeness of excision.
PMCID: PMC2443052  PMID: 8489969
7.  Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones 
Imanishi, Tadashi | Itoh, Takeshi | Suzuki, Yutaka | O'Donovan, Claire | Fukuchi, Satoshi | Koyanagi, Kanako O | Barrero, Roberto A | Tamura, Takuro | Yamaguchi-Kabata, Yumi | Tanino, Motohiko | Yura, Kei | Miyazaki, Satoru | Ikeo, Kazuho | Homma, Keiichi | Kasprzyk, Arek | Nishikawa, Tetsuo | Hirakawa, Mika | Thierry-Mieg, Jean | Thierry-Mieg, Danielle | Ashurst, Jennifer | Jia, Libin | Nakao, Mitsuteru | Thomas, Michael A | Mulder, Nicola | Karavidopoulou, Youla | Jin, Lihua | Kim, Sangsoo | Yasuda, Tomohiro | Lenhard, Boris | Eveno, Eric | Suzuki, Yoshiyuki | Yamasaki, Chisato | Takeda, Jun-ichi | Gough, Craig | Hilton, Phillip | Fujii, Yasuyuki | Sakai, Hiroaki | Tanaka, Susumu | Amid, Clara | Bellgard, Matthew | de Fatima Bonaldo, Maria | Bono, Hidemasa | Bromberg, Susan K | Brookes, Anthony J | Bruford, Elspeth | Carninci, Piero | Chelala, Claude | Couillault, Christine | de Souza, Sandro J. | Debily, Marie-Anne | Devignes, Marie-Dominique | Dubchak, Inna | Endo, Toshinori | Estreicher, Anne | Eyras, Eduardo | Fukami-Kobayashi, Kaoru | R. Gopinath, Gopal | Graudens, Esther | Hahn, Yoonsoo | Han, Michael | Han, Ze-Guang | Hanada, Kousuke | Hanaoka, Hideki | Harada, Erimi | Hashimoto, Katsuyuki | Hinz, Ursula | Hirai, Momoki | Hishiki, Teruyoshi | Hopkinson, Ian | Imbeaud, Sandrine | Inoko, Hidetoshi | Kanapin, Alexander | Kaneko, Yayoi | Kasukawa, Takeya | Kelso, Janet | Kersey, Paul | Kikuno, Reiko | Kimura, Kouichi | Korn, Bernhard | Kuryshev, Vladimir | Makalowska, Izabela | Makino, Takashi | Mano, Shuhei | Mariage-Samson, Regine | Mashima, Jun | Matsuda, Hideo | Mewes, Hans-Werner | Minoshima, Shinsei | Nagai, Keiichi | Nagasaki, Hideki | Nagata, Naoki | Nigam, Rajni | Ogasawara, Osamu | Ohara, Osamu | Ohtsubo, Masafumi | Okada, Norihiro | Okido, Toshihisa | Oota, Satoshi | Ota, Motonori | Ota, Toshio | Otsuki, Tetsuji | Piatier-Tonneau, Dominique | Poustka, Annemarie | Ren, Shuang-Xi | Saitou, Naruya | Sakai, Katsunaga | Sakamoto, Shigetaka | Sakate, Ryuichi | Schupp, Ingo | Servant, Florence | Sherry, Stephen | Shiba, Rie | Shimizu, Nobuyoshi | Shimoyama, Mary | Simpson, Andrew J | Soares, Bento | Steward, Charles | Suwa, Makiko | Suzuki, Mami | Takahashi, Aiko | Tamiya, Gen | Tanaka, Hiroshi | Taylor, Todd | Terwilliger, Joseph D | Unneberg, Per | Veeramachaneni, Vamsi | Watanabe, Shinya | Wilming, Laurens | Yasuda, Norikazu | Yoo, Hyang-Sook | Stodolsky, Marvin | Makalowski, Wojciech | Go, Mitiko | Nakai, Kenta | Takagi, Toshihisa | Kanehisa, Minoru | Sakaki, Yoshiyuki | Quackenbush, John | Okazaki, Yasushi | Hayashizaki, Yoshihide | Hide, Winston | Chakraborty, Ranajit | Nishikawa, Ken | Sugawara, Hideaki | Tateno, Yoshio | Chen, Zhu | Oishi, Michio | Tonellato, Peter | Apweiler, Rolf | Okubo, Kousaku | Wagner, Lukas | Wiemann, Stefan | Strausberg, Robert L | Isogai, Takao | Auffray, Charles | Nomura, Nobuo | Gojobori, Takashi | Sugano, Sumio
PLoS Biology  2004;2(6):e162.
The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.
An international team has systematically validated and annotated just over 21,000 human genes using full-length cDNA, thereby providing a valuable new resource for the human genetics community
PMCID: PMC393292  PMID: 15103394
8.  Neurocytotoxic effects of iron-ions on the developing brain measured in vivo using medaka (Oryzias latipes), a vertebrate model 
Purpose: Exposure to heavy-ion radiation is considered a critical health risk on long-term space missions. The developing central nervous system (CNS) is a highly radiosensitive tissue; however, the biological effects of heavy-ion radiation, which are greater than those of low-linear energy transfer (LET) radiation, are not well studied, especially in vivo in intact organisms. Here, we examined the effects of iron-ions on the developing CNS using vertebrate organism, fish embryos of medaka (Oryzias latipes).
Materials and methods: Medaka embryos at developmental stage 28 were irradiated with iron-ions at various doses of 0-1.5 Gy. At 24 h after irradiation, radiation-induced apoptosis was examined using an acridine orange (AO) assay and histo-logically. To estimate the relative biological effectiveness (RBE), we quantified only characteristic AO-stained rosette-shaped apoptosis in the developing optic tectum (OT). At the time of hatching, morphological abnormalities in the irradiated brain were examined histologically.
Results: The dose-response curve utilizing an apoptotic index for the iron-ion irradiated embryos was much steeper than that for X-ray irradiated embryos, with RBE values of 3.7-4.2. Histological examinations of irradiated medaka brain at 24 h after irradiation showed AO-positive rosette-shaped clusters as aggregates of condensed nuclei, exhibiting a circular hole, mainly in the marginal area of the OT and in the retina. However, all of the irradiated embryos hatched normally without apparent histological abnormalities in their brains.
Conclusion: Our present study indicates that the medaka embryo is a useful model for evaluating neurocytotoxic effects on the developing CNS induced by exposure to heavy iron-ions relevant to the aerospace radiation environment.
PMCID: PMC3169016  PMID: 21770703
High-LET radiation; apoptosis; embryonic brain; medaka; relative biological effectiveness (RBE)
9.  Evidence for Avian H9N2 Influenza Virus Infections among Rural Villagers in Cambodia 
Southeast Asia remains a critical region for the emergence of novel and/or zoonotic influenza, underscoring the importance of extensive sampling in rural areas where early transmission is most likely to occur.
In 2008, 800 adult participants from eight sites were enrolled in a prospective population-based study of avian influenza (AI) virus transmission where highly pathogenic avian influenza (HPAI) H5N1 virus had been reported in humans and poultry from 2006 to 2008. From their enrollment sera and questionnaires, we report risk factor findings for serologic evidence of previous infection with 18 AI virus strains.
Serologic assays revealed no evidence of previous infection with 13 different low-pathogenic AI viruses or with HPAI avian-like A/Cambodia/R0404050/2007(H5N1). However, 21 participants had elevated antibodies against avian-like A/Hong Kong/1073/1999(H9N2), validated with a monoclonal antibody blocking ELISA assay specific for avian H9.
Although cross-reaction from antibodies against human influenza viruses cannot be completely excluded, the study data suggest that a number of participants were previously infected with the avian-like A/Hong Kong/1073/1999(H9N2) virus, likely due to as yet unidentified environmental exposures. Prospective data from this cohort will help us better understand the serology of zoonotic influenza infection in a rural cohort in SE Asia.
PMCID: PMC3612269  PMID: 23537819
influenza A virus, avian; zoonoses; occupational exposure; communicable diseases, emerging; cohort studies
10.  Use of Cell-Free Circulating Schistosome DNA in Serum, Urine, Semen, and Saliva To Monitor a Case of Refractory Imported Schistosomiasis Hematobia 
Journal of Clinical Microbiology  2013;51(10):3435-3438.
This case of imported refractory schistosomiasis has highlighted the usefulness of cell-free parasite DNA as a diagnostic marker to assess active schistosome infection. In contrast to the rapid disappearance of ova in urine, parasite DNA remained persistent in several other specimen types even after the fourth treatment with praziquantel. This result was consistent with the presence of morphologically intact ova in bladder biopsy samples and with the corresponding symptoms.
PMCID: PMC3811636  PMID: 23884992
11.  Survey of Culture, GoldenGate Assay, Universal Biosensor Assay, and 16S rRNA Gene Sequencing as Alternative Methods of Bacterial Pathogen Detection 
Journal of Clinical Microbiology  2013;51(10):3263-3269.
Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens.
PMCID: PMC3811648  PMID: 23884998
12.  Cyclin B1 mRNA translation is temporally controlled through formation and disassembly of RNA granules 
The Journal of Cell Biology  2013;202(7):1041-1055.
Temporally regulated formation of RNA granules containing cyclin B1 transcript is critical for the precise timing of translational activation.
Temporal control of messenger RNA (mRNA) translation is an important mechanism for regulating cellular, neuronal, and developmental processes. However, mechanisms that coordinate timing of translational activation remain largely unresolved. Full-grown oocytes arrest meiosis at prophase I and deposit dormant mRNAs. Of these, translational control of cyclin B1 mRNA in response to maturation-inducing hormone is important for normal progression of oocyte maturation, through which oocytes acquire fertility. In this study, we found that dormant cyclin B1 mRNA forms granules in the cytoplasm of zebrafish and mouse oocytes. Real-time imaging of translation revealed that the granules disassemble at the time of translational activation during maturation. Formation of cyclin B1 RNA granules requires binding of the mRNA to Pumilio1 protein and depends on actin filaments. Disruption of cyclin B1 RNA granules accelerated the timing of their translational activation after induction of maturation, whereas stabilization hindered translational activation. Thus, our results suggest that RNA granule formation is critical for the regulation of timing of translational activation.
PMCID: PMC3787373  PMID: 24062337
13.  RNA Sequence Reveals Mouse Retinal Transcriptome Changes Early after Axonal Injury 
PLoS ONE  2014;9(3):e93258.
Glaucoma is an ocular disease characterized by progressive retinal ganglion cell (RGC) death caused by axonal injury. However, the underlying mechanisms involved in RGC death remain unclear. In this study, we investigated changes in the transcriptome profile following axonal injury in mice (C57BL/6) with RNA sequencing (RNA-seq) technology. The experiment group underwent an optic nerve crush (ONC) procedure to induce axonal injury in the right eye, and the control group underwent a sham procedure. Two days later, we extracted the retinas and performed RNA-seq and a pathway analysis. We identified 177 differentially expressed genes with RNA-seq, notably the endoplasmic reticulum (ER) stress-related genes Atf3, Atf4, Atf5, Chac1, Chop, Egr1 and Trb3, which were significantly upregulated. The pathway analysis revealed that ATF4 was the most significant upstream regulator. The antioxidative response-related genes Hmox1 and Srxn1, as well as the immune response-related genes C1qa, C1qb and C1qc, were also significantly upregulated. To our knowledge, this is the first reported RNA-seq investigation of the retinal transcriptome and molecular pathways in the early stages after axonal injury. Our results indicated that ER stress plays a key role under these conditions. Furthermore, the antioxidative defense and immune responses occurred concurrently in the early stages after axonal injury. We believe that our study will lead to a better understanding of and insight into the molecular mechanisms underlying RGC death after axonal injury.
PMCID: PMC3968129  PMID: 24676137
14.  Acute hepatitis B of genotype H resulting in persistent infection 
A 47-year-old man presented with general fatigue and dark urine. The laboratory data showed increased levels of hepatic transaminases. The patient was positive for hepatitis B virus (HBV) markers and negative for anti-human immunodeficiency virus. The HBV-DNA titer was set to 7.7 log copies/mL. The patient was diagnosed with acute hepatitis B. The HBV infection route was obscure. The serum levels of hepatic transaminases decreased to normal ranges without any treatment, but the HBV-DNA status was maintained for at least 26 mo, indicating the presence of persistent infection. We isolated HBV from the acute-phase serum and determined the genome sequence. A phylogenetic analysis revealed that the isolated HBV was genotype H. In this patient, the elevated peak level of HBV-DNA and the risk alleles at human genome single nucleotide polymorphisms s3077 and rs9277535 in the human leukocyte antigen-DP locus were considered to be risk factors for chronic infection. This case suggests that there is a risk of persistent infection by HBV genotype H following acute hepatitis; further cases of HBV genotype H infection must be identified and characterized. Thus, the complete determination of the HBV genotype may be essential during routine clinical care of acute hepatitis B outpatients.
PMCID: PMC3961985  PMID: 24659896
Acute hepatitis; Chronic hepatitis; Genotyping; Hepatitis B virus; Single nucleotide polymorphisms
15.  Alpha-fetoprotein-producing ovarian clear cell adenocarcinoma simulating fetal gut in a postmenopausal woman 
•A 59-year-old postmenopausal woman had ovarian clear cell adenocarcinoma producing AFP.•The tumor lacked a yolk sac component and formed ducts similar to the fetal gut.
PMCID: PMC3973822
Ovary; Postmenopausal; AFP-producing; Clear cell adenocarcinoma; Fetal gut appearance
16.  The involvement of endoplasmic reticulum stress in bile acid-induced hepatocellular injury 
Secondary bile acids produced by enteric bacteria accumulate to high levels in the enterohepatic circulation and may contribute to the pathogenesis of hepatocellular injury. Relative hydrophobicity has been suggested to be an important determinant of the biological properties of these compounds, although the mechanism by which bile acids induce pathogenesis is not fully understood. On the other hand, endoplasmic reticulum stress has been shown to be involved in the induction and development of various pathogenic conditions. In this report, we demonstrated that the intensities of cytotoxicity and endoplasmic reticulum stress in HepG2 cells triggered by the bile acids tested were largely dependent on their hydrophobicity. The activation of caspase-3 and DNA fragmentation by treatment with chenodeoxycholic acid showed the contribution of apoptosis to cytotoxicity. Increases in intracellular calcium levels and the generation of reactive oxygen species stimulated by treatment with chenodeoxycholic acid contributed to endoplasmic reticulum stress. Bile acids also induced transforming growth factor-β, a potent profibrogenic factor, which is known to induce hepatocyte apoptosis and ultimately liver fibrosis. In conclusion, our study demonstrated that bile acids induced endoplasmic reticulum stress, which in turn stimulated apoptosis in HepG2 cells, in a hydrophobicity-dependent manner.
PMCID: PMC3947968
bile acid; endoplasmic reticulum stress; apoptosis; transforming growth factor-β; hydrophobicity
17.  Haematopoietic cells produce BDNF and regulate appetite upon migration to the hypothalamus 
Nature communications  2013;4:1526.
Brain-derived neurotrophic factor (BDNF) suppresses food intake by acting on neurons in the hypothalamus. Here we show that BDNF-producing haematopoietic cells control appetite and energy balance by migrating to the hypothalamic paraventricular nucleus. These haematopoietic-derived paraventricular nucleus cells produce microglial markers and make direct contacts with neurons in response to feeding status. Mice with congenital BDNF deficiency, specifically in haematopoietic cells, develop hyperphagia, obesity and insulin resistance. These abnormalities are ameliorated by bone marrow transplantation with wild-type bone marrow cells. Furthermore, when injected into the third ventricle, wild-type bone marrow mononuclear cells home to the paraventricular nucleus and reverse the hyperphagia of BDNF-deficient mice. Our results suggest a novel mechanism of feeding control based on the production of BDNF by haematopoietic cells and highlight a potential new therapeutic route for the treatment of obesity.
PMCID: PMC3640364  PMID: 23443554
18.  Regular heartbeat rhythm at the heartbeat initiation stage is essential for normal cardiogenesis at low temperature 
The development of blood flow in the heart is crucial for heart function and embryonic survival. Recent studies have revealed the importance of the extracellular matrix and the mechanical stress applied to the valve cushion that controls blood flow to the formation of the cardiac valve during embryogenesis. However, the events that trigger such valve formation and mechanical stress, and their temperature dependence have not been explained completely. Medaka (Oryzias latipes) inhabits a wide range of East Asia and adapts to a wide range of climates. We used medaka embryos from different genomic backgrounds and analyzed heartbeat characteristics including back-and-forth blood flow and bradyarrhythmia in embryos incubated at low temperature. We also used high-speed imaging analysis to examine the heartbeat of these animals after transient exposure to low temperature.
Embryos of the Hd-rR medaka strain exhibited back-and-forth blood flow in the heart (blood regurgitation) after incubation at 15°C. This regurgitation was induced by exposure to low temperature around the heartbeat initiation period and was related to abnormalities in the maintenance or pattern of contraction of the atrium or the atrioventricular canal. The Odate strain from the northern Japanese group exhibited normal blood flow after incubation at 15°C. High-speed time-lapse analysis of the heartbeat revealed that bradyarrhythmia occurred only in Hd-rR embryos incubated at 15°C. The coefficient of contraction, defined as the quotient of the length of the atrium at systole divided by its length at diastole, was not affected in either strain. The average heart rate after removing the effect of arrhythmia did not differ significantly between the two strains, suggesting that the mechanical stress of individual myocardial contractions and the total mechanical stress could be equivalent, regardless of the presence of arrhythmia or the heart rate. Test-cross experiments suggested that this circulation phenotype was caused by a single major genomic locus.
These results suggest that cardiogenesis at low temperature requires a constant heartbeat. Abnormal contraction rhythms at the stage of heartbeat initiation may cause regurgitation at later stages. From the evolutionary viewpoint, strains that exhibit normal cardiogenesis during development at low temperature inhabit northern environments.
PMCID: PMC3936829  PMID: 24564206
Heartbeat; Medaka; Blood regurgitation; Cold adaptation; Cardiogenesis
19.  Thresholds of various glycemic measures for diagnosing diabetes based on prevalence of retinopathy in community-dwelling Japanese subjects: the Hisayama Study 
There has been controversy over the diagnostic thresholds of hemoglobin A1c (HbA1c) for diabetes. In addition, no study has examined the thresholds of glycated albumin (GA) and 1,5-anhydroglucitol (1,5-AG) for diagnosing diabetes using the presence of diabetic retinopathy (DR). We examined the optimal thresholds of various glycemic measures for diagnosing diabetes based on the prevalence of DR in community-dwelling Japanese subjects.
A total of 2,681 subjects aged 40-79 years underwent a 75-g oral glucose tolerance test, measurement of HbA1c, GA, and 1,5-AG, and an ophthalmic examination in 2007-2008. The associations of glycemic measures with DR status were examined cross-sectionally. DR was assessed by an examination of the fundus photograph of each eye and graded according to the International Clinical Diabetic Retinopathy Disease Severity Scale. We divided the values of glycemic measures into ten groups on the basis of deciles. The receiver operating characteristic (ROC) curve analysis was performed to determine the optimal threshold of each glycemic measure for detecting the presence of DR.
Of the subjects, 52 had DR. The prevalence of DR increased steeply above the ninth decile for fasting plasma glucose (FPG) (6.2-6.8 mmol/l), for 2-hour postload glucose (PG) (9.2-12.4 mmol/l), for HbA1c (5.9-6.2% [41-44 mmol/mol]), and for GA (16.2-17.5%), and below the second decile for 1,5-AG (9.6-13.5 μg/mL). The ROC curve analysis showed that the optimal thresholds for DR were 6.5 mmol/l for FPG, 11.5 mmol/l for 2-hour PG, 6.1% (43 mmol/mol) for HbA1c, 17.0% for GA, and 12.1 μg/mL for 1,5-AG. The area under the ROC curve (AUC) for 2-hour PG (0.947) was significantly larger than that for FPG (0.908), GA (0.906), and 1,5-AG (0.881), and was marginally significantly higher than that for HbA1c (0.919). The AUCs for FPG, HbA1c, GA, and 1,5-AG were not significantly different.
Our findings suggest that the FPG and HbA1c thresholds for diagnosing diabetes in the Japanese population are lower than the current diagnostic criterion, while the 2-hour PG threshold is comparable with the diagnostic criterion. 2-hour PG had the highest discriminative ability, whereas FPG, HbA1c, GA, and 1,5-AG were similar in their ability.
PMCID: PMC3936993  PMID: 24533962
Diagnostic criteria; Hemoglobin A1c; Glycated albumin; 1,5-anhydroglucitol; Fasting plasma glucose; 2-hour postload glucose; Retinopathy
20.  Post-recurrence survival of patients with non-small-cell lung cancer after curative resection with or without induction/adjuvant chemotherapy 
Recently, the prognosis of patients with non-small-cell lung cancer (NSCLC) has improved, thanks to the standardization of adjuvant chemotherapy and the introduction of molecular-targeted drugs, notably epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and other new anti-cancer agents. However, the survival characteristics and prognosis of patients with recurrent NSCLC after curative resection are not well understood.
Of the 430 consecutive patients with NSCLC who underwent complete surgical resection at our institution between January 2004 and July 2011, we included 76 patients with recurrence whose post-recurrence treatment and outcome could be confirmed. We then retrospectively evaluated the effect of prognostic factors on post-recurrence survival.
There were 50 men and 26 women, and the median age at recurrence was 74.5 years. The median time from surgical resection to recurrence was 12.7 months. Thirty-eight of the 76 (50%) patients underwent multimodality treatment with surgery and preoperative and/or postoperative chemotherapy as their initial treatment. For recurrence, systemic chemotherapy was administered to 64 (84%) patients, and the disease control rate for first-line chemotherapy was 55%. The 1- and 2-year post-recurrence survival rates were 68.3 and 45.8%, respectively, and the median post-recurrence survival time was 17.7 months. Six independent prognostic factors were identified: wild-type EGFR, no adjuvant chemotherapy for the primary lung cancer, age ≥80 years at recurrence, a poor Eastern Cooperative Oncology Group performance status at recurrence, symptomatic at recurrence and no systemic chemotherapy for recurrence, which significantly decreased the post-recurrence survival.
The prognosis of patients with NSCLC recurrence after surgery is currently improving. Our results suggested two new prognostic factors, adjuvant chemotherapy and EGFR mutations, neither of which have been previously reported. Treatment strategies for postoperative recurrence should be established based on a more detailed subdivision of factors, such as histology and molecular markers, in the future.
PMCID: PMC3548532  PMID: 23143203
Non-small-cell lung cancer; Post-recurrence survival; Adjuvant chemotherapy
21.  Anesthesia with Propofol Induces Insulin Resistance Systemically in Skeletal and Cardiac Muscles and Liver of Rats 
Hyperglycemia together with hepatic and muscle insulin resistance are common features in critically ill patients, and these changes are associated with enhanced inflammatory response, increased susceptibility to infection, muscle wasting, and worsened prognosis. Tight blood glucose control by intensive insulin treatment may reduce the morbidity and mortality in intensive care units. Although some anesthetics have been shown to cause insulin resistance, it remains unknown how and in which tissues insulin resistance is induced by anesthetics. Moreover, the effects of propofol, a clinically relevant intravenous anesthetic, also used in the intensive care unit for sedation, on insulin sensitivity have not yet been investigated. Euglycemic hyperinsulinemic clamp study was performed in rats anesthetized with propofol and conscious unrestrained rats. To evaluate glucose uptake in tissues and hepatic glucose output [3H]glucose and 2-deoxy[14C]glucose were infused during the clamp study. Anesthesia with propofol induced a marked whole-body insulin resistance compared with conscious rats, as reflected by significantly decreased glucose infusion rate to maintain euglycemia. Insulin-stimulated tissue glucose uptake was decreased in skeletal muscle and heart, and hepatic glucose output was increased in propofol anesthetized rats. Anesthesia with propofol induces systemic insulin resistance along with decreases in insulin-stimulated glucose uptake in skeletal and heart muscle and attenuation of the insulin-mediated suppression of hepatic glucose output in rats.
PMCID: PMC3566703  PMID: 23274498
Glucose output (hepatic); hepatic glucose uptake; hyperinsulinemic clamp; insulin resistance; muscle glucose uptake; propofol; sedation
22.  Effects of pioglitazone on nonalcoholic steatohepatitis in a patient with anorexia nervosa: A case report 
Diseases associated with metabolic syndromes are of major concern in developed countries. Nonalcoholic steatohepatitis (NASH) is one of the manifestations of metabolic syndrome in the liver. Previous studies have shown that NASH is also caused by malnutrition. In the present study, a case of malnutrition-associated NASH in a 66-year-old female with anorexia nervosa is reported. The patient had a body mass index (BMI) of only 11.1 kg/m2 and serum alanine aminotransferase levels of 1,495 IU/l. Steatohepatitis with fibrosis was confirmed by percutaneous liver needle biopsy. Total parenteral nutrition was conducted at first, followed by the administration of Stronger Neo-Minophagen C (a glycyrrhizin-containing preparation), ursodeoxycholic acid and prednisolone. The abnormal elevation of aminotransferase levels of the patient was prolonged and total bilirubin levels increased. Pioglitazone (15 mg/day), which has been identified to be effective for nonalcoholic steatohepatitis, was then administered. This resulted in marked reductions in aminotransferase and bilirubin levels within three months. Histological improvement of the liver was also confirmed by percutaneous liver needle biopsy after one year. The observations in the present case suggest that pioglitazone may be useful for the treatment of malnutrition-associated NASH.
PMCID: PMC3961108  PMID: 24669237
steatohepatitis; malnutrition; pioglitazone
23.  Frequent Seizures Are Associated with a Network of Gray Matter Atrophy in Temporal Lobe Epilepsy with or without Hippocampal Sclerosis 
PLoS ONE  2014;9(1):e85843.
Patients with temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) have diffuse subtle gray matter (GM) atrophy detectable by MRI quantification analyses. However, it is not clear whether the etiology and seizure frequency are associated with this atrophy. We aimed to evaluate the occurrence of GM atrophy and the influence of seizure frequency in patients with TLE and either normal MRI (TLE-NL) or MRI signs of HS (TLE-HS).
We evaluated a group of 172 consecutive patients with unilateral TLE-HS or TLE-NL as defined by hippocampal volumetry and signal quantification (122 TLE-HS and 50 TLE-NL) plus a group of 82 healthy individuals. Voxel-based morphometry was performed with VBM8/SPM8 in 3T MRIs. Patients with up to three complex partial seizures and no generalized tonic-clonic seizures in the previous year were considered to have infrequent seizures. Those who did not fulfill these criteria were considered to have frequent seizures.
Patients with TLE-HS had more pronounced GM atrophy, including the ipsilateral mesial temporal structures, temporal lobe, bilateral thalami and pre/post-central gyri. Patients with TLE-NL had more subtle GM atrophy, including the ipsilateral orbitofrontal cortex, bilateral thalami and pre/post-central gyri. Both TLE-HS and TLE-NL showed increased GM volume in the contralateral pons. TLE-HS patients with frequent seizures had more pronounced GM atrophy in extra-temporal regions than TLE-HS with infrequent seizures. Patients with TLE-NL and infrequent seizures had no detectable GM atrophy. In both TLE-HS and TLE-NL, the duration of epilepsy correlated with GM atrophy in extra-hippocampal regions.
Although a diffuse network GM atrophy occurs in both TLE-HS and TLE-NL, this is strikingly more evident in TLE-HS and in patients with frequent seizures. These findings suggest that neocortical atrophy in TLE is related to the ongoing seizures and epilepsy duration, while thalamic atrophy is more probably related to the original epileptogenic process.
PMCID: PMC3903486  PMID: 24475055
24.  The effects of mating status and time since mating on female sex pheromone levels in the rice leaf bug, Trigonotylus caelestialium 
Die Naturwissenschaften  2014;101:153-156.
Although mating status affects future mating opportunities, the biochemical changes that occur in response to mating are not well understood. This study investigated the effects of mating status on the quantities of sex pheromone components found in whole-body extracts and volatile emissions of females of the rice leaf bug, Trigonotylus caelestialium. When sampled at one of four time points within a 4-day postmating period, females that had copulated with a male had greater whole-body quantities of sex pheromone components than those of virgin females sampled at the same times. The quantities of sex pheromone components emitted by virgin females over a 24-h period were initially high but then steadily decreased, whereas 24-h emissions were persistently low among mated females when measured at three time points within the 4 days after mating. As a result, soon after mating, the mated females emitted less sex pheromones than virgin females, but there were no significant differences between mated and virgin females at the end of the experiment. Thus, postmating reduction in the rate of emission of sex pheromones could explain previously observed changes in female attractiveness to male T. caelestialium.
Electronic supplementary material
The online version of this article (doi:10.1007/s00114-013-1141-3) contains supplementary material, which is available to authorized users.
PMCID: PMC3933747  PMID: 24419707
Sex pheromones; Postmating behavior; Sexual selection; Trigonotylus caelestialium
25.  Allocation of Attentional Resources toward a Secondary Cognitive Task Leads to Compromised Ankle Proprioceptive Performance in Healthy Young Adults 
The objective of the present study was to determine whether increased attentional demands influence the assessment of ankle joint proprioceptive ability in young adults. We used a dual-task condition, in which participants performed an ankle ipsilateral position-matching task with and without a secondary serial auditory subtraction task during target angle encoding. Two experiments were performed with two different cohorts: one in which the auditory subtraction task was easy (experiment 1a) and one in which it was difficult (experiment 1b). The results showed that, compared with the single-task condition, participants had higher absolute error under dual-task conditions in experiment 1b. The reduction in position-matching accuracy with an attentionally demanding cognitive task suggests that allocation of attentional resources toward a difficult second task can lead to compromised ankle proprioceptive performance. Therefore, these findings indicate that the difficulty level of the cognitive task might be the possible critical factor that decreased accuracy of position-matching task. We conclude that increased attentional demand with difficult cognitive task does influence the assessment of ankle joint proprioceptive ability in young adults when measured using an ankle ipsilateral position-matching task.
PMCID: PMC3910264  PMID: 24523966

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