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1.  Klebsormidium flaccidum genome reveals primary factors for plant terrestrial adaptation 
Nature Communications  2014;5:3978.
The colonization of land by plants was a key event in the evolution of life. Here we report the draft genome sequence of the filamentous terrestrial alga Klebsormidium flaccidum (Division Charophyta, Order Klebsormidiales) to elucidate the early transition step from aquatic algae to land plants. Comparison of the genome sequence with that of other algae and land plants demonstrate that K. flaccidum acquired many genes specific to land plants. We demonstrate that K. flaccidum indeed produces several plant hormones and homologues of some of the signalling intermediates required for hormone actions in higher plants. The K. flaccidum genome also encodes a primitive system to protect against the harmful effects of high-intensity light. The presence of these plant-related systems in K. flaccidum suggests that, during evolution, this alga acquired the fundamental machinery required for adaptation to terrestrial environments.
Plant colonization of land is an important evolutionary event. Here, the authors sequence the genome of a filamentous terrestrial alga and, through a comparative analysis with related algae and land plant species, provide insight into how aquatic algae adapted to terrestrial environments.
PMCID: PMC4052687  PMID: 24865297
3.  A magnetic anti-cancer compound for magnet-guided delivery and magnetic resonance imaging 
Scientific Reports  2015;5:9194.
Research on controlled drug delivery for cancer chemotherapy has focused mainly on ways to deliver existing anti-cancer drug compounds to specified targets, e.g., by conjugating them with magnetic particles or encapsulating them in micelles. Here, we show that an iron-salen, i.e., μ-oxo N,N'- bis(salicylidene)ethylenediamine iron (Fe(Salen)), but not other metal salen derivatives, intrinsically exhibits both magnetic character and anti-cancer activity. X-Ray crystallographic analysis and first principles calculations based on the measured structure support this. It promoted apoptosis of various cancer cell lines, likely, via production of reactive oxygen species. In mouse leg tumor and tail melanoma models, Fe(Salen) delivery with magnet caused a robust decrease in tumor size, and the accumulation of Fe(Salen) was visualized by magnetic resonance imaging. Fe(Salen) is an anti-cancer compound with magnetic property, which is suitable for drug delivery and imaging. We believe such magnetic anti-cancer drugs have the potential to greatly advance cancer chemotherapy for new theranostics and drug-delivery strategies.
PMCID: PMC4361848  PMID: 25779357
4.  Clinical impacts of additive use of olmesartan in hypertensive patients with chronic heart failure: the supplemental benefit of an angiotensin receptor blocker in hypertensive patients with stable heart failure using olmesartan (SUPPORT) trial 
Sakata, Yasuhiko | Shiba, Nobuyuki | Takahashi, Jun | Miyata, Satoshi | Nochioka, Kotaro | Miura, Masanobu | Takada, Tsuyoshi | Saga, Chiharu | Shinozaki, Tsuyoshi | Sugi, Masafumi | Nakagawa, Makoto | Sekiguchi, Nobuyo | Komaru, Tatsuya | Kato, Atsushi | Fukuchi, Mitsumasa | Nozaki, Eiji | Hiramoto, Tetsuya | Inoue, Kanichi | Goto, Toshikazu | Ohe, Masatoshi | Tamaki, Kenji | Ibayashi, Setsuro | Ishide, Nobumasa | Maruyama, Yukio | Tsuji, Ichiro | Shimokawa, Hiroaki | Shimokawa, H. | Fukuchi, M. | Goto, T. | Hiramoto, T. | Inoue, K. | Kato, A. | Komaru, T. | Ohe, M. | Sekiguchi, N. | Shiba, N. | Shinozaki, T. | Sugi, M. | Tamaki, K. | Hiramoto, T. | Inoue, K. | Kato, A. | Ogata, M. | Sato, S. | Sugi, M. | Ishide, N. | Ibayashi, S. | Maruyama, Y. | Ohno, I. | Tamaki, K. | Ogawa, H. | Kitakaze, M. | Tsuji, I. | Watanabe, T. | Sugiyama, K. | Oyama, S. | Nozaki, E. | Nakamura, A. | Takahashi, T. | Endo, H. | Fukui, S. | Nakajima, S. | Nakagawa, M. | Nozaki, T. | Yagi, T. | Horiguchi, S. | Fushimi, E. | Sugai, Y. | Takeda, S. | Fukahori, K. | Aizawa, K. | Ohe, M. | Tashima, T. | Sakurai, K. | Kobayashi, T. | Goto, T. | Matsui, M. | Tamada, Y. | Yahagi, T. | Fukui, A. | Takahashi, K. | Takahashi, K. | Kikuchi, Y. | Akai, K. | Kanno, H. | Kaneko, J. | Suzuki, S. | Takahashi, K. | Akai, K. | Katayose, D. | Onodera, S. | Hiramoto, T. | Komatsu, S. | Chida, M. | Iwabuchi, K. | Takeuchi, M. | Yahagi, H. | Takahashi, N. | Otsuka, K. | Koseki, Y. | Morita, M. | Shinozaki, T. | Ishizuka, T. | Onoue, N. | Yamaguchi, N. | Fujita, H. | Katoh, A. | Namiuchi, S. | Sugie, T. | Saji, K. | Takii, T. | Sugimura, A. | Ohashi, J. | Fukuchi, M. | Ogata, M. | Tanikawa, T. | Kitamukai, O. | Matsumoto, Y. | Inoue, K. | Koyama, J. | Tomioka, T. | Shioiri, H. | Ito, Y. | Kato, H. | Takahashi, C. | Kawana, A. | Sakata, Y. | Ito, K. | Nakayama, M. | Fukuda, K. | Takahashi, J. | Miyata, S. | Sugimura, K. | Sato, K. | Matsumoto, Y. | Nakano, M. | Shiroto, T. | Tsuburaya, R. | Nochioka, K. | Yamamoto, H. | Aoki, T. | Hao, K. | Miura, M. | Kondo, M. | Tatebe, S. | Yamamoto, S. | Suzuki, H. | Nishimiya, K. | Yaoita, N. | Sugi, M. | Yamamoto, Y. | Toda, S. | Minatoya, Y. | Takagi, Y. | Hasebe, Y. | Nihei, T. | Hanawa, K. | Fukuda, K. | Sakata, Y. | Takahashi, J. | Miyata, S. | Nochioka, K. | Miura, M. | Tadaki, S. | Ushigome, R. | Yamauchi, T. | Sato, K. | Tsuji, K. | Onose, T. | Abe, R. | Saga, C. | Suenaga, J. | Yamada, Y. | Kimura, J. | Ogino, H. | Oikawa, I. | Watanabe, S. | Saga, M. | Washio, M. | Nagasawa, K. | Nagasawa, S. | Kotaka, S. | Komatsu, W. | Hashimoto, R. | Ikeno, Y. | Suzuki, T. | Hamada, H.
European Heart Journal  2015;36(15):915-923.
We examined whether an additive treatment with an angiotensin receptor blocker, olmesartan, reduces the mortality and morbidity in hypertensive patients with chronic heart failure (CHF) treated with angiotensin-converting enzyme (ACE) inhibitors, β-blockers, or both. In this prospective, randomized, open-label, blinded endpoint study, a total of 1147 hypertensive patients with symptomatic CHF (mean age 66 years, 75% male) were randomized to the addition of olmesartan (n = 578) to baseline therapy vs. control (n = 569). The primary endpoint was a composite of all-cause death, non-fatal acute myocardial infarction, non-fatal stroke, and hospitalization for worsening heart failure. During a median follow-up of 4.4 years, the primary endpoint occurred in 192 patients (33.2%) in the olmesartan group and in 166 patients (29.2%) in the control group [hazard ratio (HR) 1.18; 95% confidence interval (CI), 0.96–1.46, P = 0.112], while renal dysfunction developed more frequently in the olmesartan group (16.8 vs. 10.7%, HR 1.64; 95% CI 1.19–2.26, P = 0.003). Subgroup analysis revealed that addition of olmesartan to combination of ACE inhibitors and β-blockers was associated with increased incidence of the primary endpoint (38.1 vs. 28.2%, HR 1.47; 95% CI 1.11–1.95, P = 0.006), all-cause death (19.4 vs. 13.5%, HR 1.50; 95% CI 1.01–2.23, P = 0.046), and renal dysfunction (21.1 vs. 12.5%, HR 1.85; 95% CI 1.24–2.76, P = 0.003). Additive use of olmesartan did not improve clinical outcomes but worsened renal function in hypertensive CHF patients treated with evidence-based medications. Particularly, the triple combination therapy with olmesartan, ACE inhibitors and β-blockers was associated with increased adverse cardiac events. This study is registered at
PMCID: PMC4466154  PMID: 25637937
Heart failure; Hypertension; Angiotensin II receptor blocker; Olmesartan
5.  Caenorhabditis elegans chaperonin CCT/TRiC is required for actin and tubulin biogenesis and microvillus formation in intestinal epithelial cells 
Molecular Biology of the Cell  2014;25(20):3095-3104.
Caenorhabditis elegans chaperonin CCT is required for the biogenesis of actin and tubulin and is thereby essential for microvillus formation in intestinal cells. Loss of CCT/TRiC had no strong effects on intermediate filament protein IFB-2 or cytoskeletal linker protein ERM-1, suggesting that CCT/TRiC exhibits clear substrate specificity in living animals.
Intestinal epithelial cells have unique apical membrane structures, known as microvilli, that contain bundles of actin microfilaments. In this study, we report that Caenorhabditis elegans cytosolic chaperonin containing TCP-1 (CCT) is essential for proper formation of microvilli in intestinal cells. In intestinal cells of cct-5(RNAi) animals, a substantial amount of actin is lost from the apical area, forming large aggregates in the cytoplasm, and the apical membrane is deformed into abnormal, bubble-like structures. The length of the intestinal microvilli is decreased in these animals. However, the overall actin protein levels remain relatively unchanged when CCT is depleted. We also found that CCT depletion causes a reduction in the tubulin levels and disorganization of the microtubule network. In contrast, the stability and localization of intermediate filament protein IFB-2, which forms a dense filamentous network underneath the apical surface, appears to be superficially normal in CCT-deficient cells, suggesting substrate specificity of CCT in the folding of filamentous cytoskeletons in vivo. Our findings demonstrate physiological functions of CCT in epithelial cell morphogenesis using whole animals.
PMCID: PMC4196862  PMID: 25143409
6.  Rer1p regulates the ER retention of immature rhodopsin and modulates its intracellular trafficking 
Scientific Reports  2014;4:5973.
Rhodopsin is a pigment in photoreceptor cells. Some rhodopsin mutations cause the protein to accumulate in the endoplasmic reticulum (ER), leading to photoreceptor degeneration. Although several mutations have been reported, how mutant rhodopsin is retained in the ER remains unclear. In this study, we identified Rer1p as a modulator of ER retention and rhodopsin trafficking. Loss of Rer1p increased the transport of wild-type rhodopsin to post-Golgi compartments. Overexpression of Rer1p caused immature wild-type rhodopsin to accumulate in the ER. Interestingly, the G51R rhodopsin mutant, which has a mutation in the first transmembrane domain and accumulates in the ER, was released to the plasma membrane or lysosomes in Rer1-knockdown cells. Consistent with these results, Rer1p interacted with both wild-type and mutant rhodopsin. These results suggest that Rer1p regulates the ER retention of immature or misfolded rhodopsin and modulates its intracellular trafficking through the early secretory pathway.
PMCID: PMC4122963  PMID: 25096327
7.  Effect of baseline HbA1c level on the development of diabetes by lifestyle intervention in primary healthcare settings: insights from subanalysis of the Japan Diabetes Prevention Program 
To determine the effects of a lifestyle intervention on the development of type 2 diabetes mellitus (T2DM) among participants with impaired glucose tolerance (IGT), in particular in the subgroup with baseline glycated hemoglobin (HbA1c) levels ≥5.7%, in primary healthcare settings.
Randomized controlled trial.
32 healthcare centers in Japan.
Participants with IGT, aged 30–60 years, were randomly assigned to either an intensive lifestyle intervention group (ILG) or a usual care group (UCG).
During the initial 6 months, participants in the ILG received four group sessions on healthy lifestyles by public health providers. An individual session was further conducted biannually during the 3 years. Participants in the UCG received usual care such as one group session on healthy lifestyles.
Outcome measures
The primary endpoint was the development of T2DM based on an oral glucose tolerance test.
The mean follow-up period was 2.3 years. The annual incidence of T2DM were 2.7 and 5.1/100 person-years of follow-up in the ILG (n=145) and UCG (n=149), respectively. The cumulative incidence of T2DM was significantly lower in the ILG than in the UCG among participants with HbA1c levels ≥5.7% (log-rank=3.52, p=0.06; Breslow=4.05, p=0.04; Tarone-Ware=3.79, p=0.05), while this was not found among participants with HbA1c levels <5.7%.
Intensive lifestyle intervention in primary healthcare setting is effective in preventing the development of T2DM in IGT participants with HbA1c levels ≥5.7%, relative to those with HbA1c levels <5.7%.
Trial registration number
PMCID: PMC4212559  PMID: 25452854
HbA1c; Type 2 Diabetes; Impaired Glucose Tolerance; Intervention Groups
8.  Inhibition of Elastase-Pulmonary Emphysema in Dominant-Negative MafB Transgenic Mice 
Alveolar macrophages (AMs) play important roles in the pathogenesis of chronic obstructive pulmonary disease (COPD). We previously demonstrated upregulation of the transcription factor MafB in AMs of mice exposed to cigarette smoke. The aim of this study was to elucidate the roles of MafB in the development of pulmonary emphysema. Porcine pancreatic elastase was administered to wild-type (WT) and dominant-negative (DN)-MafB transgenic (Tg) mice in which MafB activity was suppressed only in macrophages. We measured the mean linear intercept and conducted cell differential analysis of bronchoalveolar lavage (BAL) cells, surface marker analysis using flow cytometry, and immunohistochemical staining using antibodies to matrix metalloproteinase (MMP)-9 and MMP-12. Airspace enlargement of the lungs was suppressed significantly in elastase-treated DN-MafB Tg mice compared with treated WT mice. AMs with projected pseudopods were decreased in DN-MafB Tg mice. The number of cells intermediately positive for F4/80 and weakly or intermediately positive for CD11b, which are considered cell subsets of matured AMs, decreased in the BAL of DN-MafB Tg mice. Furthermore, MMP-9 and -12 were significantly downregulated in BAL cells of DN-MafB Tg mice. Because MMPs exacerbate emphysema, MafB may be involved in pulmonary emphysema development through altered maturation of macrophages and MMP expression.
PMCID: PMC4147222  PMID: 25170302
Pulmonary emphysema; MafB; alveolar macrophage; matrix metalloproteinase; gene targeted mouse.
9.  Structural basis of a nucleosome containing histone H2A.B/H2A.Bbd that transiently associates with reorganized chromatin 
Scientific Reports  2013;3:3510.
Human histone H2A.B (formerly H2A.Bbd), a non-allelic H2A variant, exchanges rapidly as compared to canonical H2A, and preferentially associates with actively transcribed genes. We found that H2A.B transiently accumulated at DNA replication and repair foci in living cells. To explore the biochemical function of H2A.B, we performed nucleosome reconstitution analyses using various lengths of DNA. Two types of H2A.B nucleosomes, octasome and hexasome, were formed with 116, 124, or 130 base pairs (bp) of DNA, and only the octasome was formed with 136 or 146 bp DNA. In contrast, only hexasome formation was observed by canonical H2A with 116 or 124 bp DNA. A small-angle X-ray scattering analysis revealed that the H2A.B octasome is more extended, due to the flexible detachment of the DNA regions at the entry/exit sites from the histone surface. These results suggested that H2A.B rapidly and transiently forms nucleosomes with short DNA segments during chromatin reorganization.
PMCID: PMC3863819  PMID: 24336483
10.  Caenorhabditis elegans SNAP-29 is required for organellar integrity of the endomembrane system and general exocytosis in intestinal epithelial cells 
Molecular Biology of the Cell  2011;22(14):2579-2587.
Caenorhabditis elegans SNAP-29 is required for the proper morphology and functions of the Golgi and endosomes and general exocytosis.
It is generally accepted that soluble N-ethylmaleimide–sensitive factor attachment protein receptors mediate the docking and fusion of transport intermediates with target membranes. Our research identifies Caenorhabditis elegans homologue of synaptosomal-associated protein 29 (SNAP-29) as an essential regulator of membrane trafficking in polarized intestinal cells of living animals. We show that a depletion of SNAP-29 blocks yolk secretion and targeting of apical and basolateral plasma membrane proteins in the intestinal cells and results in a strong accumulation of small cargo-containing vesicles. The loss of SNAP-29 also blocks the transport of yolk receptor RME-2 to the plasma membrane in nonpolarized oocytes, indicating that its function is required in various cell types. SNAP-29 is essential for embryogenesis, animal growth, and viability. Functional fluorescent protein–tagged SNAP-29 mainly localizes to the plasma membrane and the late Golgi, although it also partially colocalizes with endosomal proteins. The loss of SNAP-29 leads to the vesiculation/fragmentation of the Golgi and endosomes, suggesting that SNAP-29 is involved in multiple transport pathways between the exocytic and endocytic organelles. These observations also suggest that organelles comprising the endomembrane system are highly dynamic structures based on the balance between membrane budding and fusion and that SNAP-29–mediated fusion is required to maintain proper organellar morphology and functions.
PMCID: PMC3135482  PMID: 21613542
11.  Comparison between surgical outcomes of colorectal cancer in younger and elderly patients 
AIM: To compare the outcome of surgical treatment of colorectal adenocarcinoma in elderly and younger patients.
METHODS: The outcomes of 122 patients with colorectal adenocarcinoma who underwent surgical treatment between January 2004 and June 2009 were analyzed. The clinicopathological and blood biochemistry data of the younger group (< 75 years) and the elderly group (≥ 75 years) were compared.
RESULTS: There were no significant differences between the two groups in operation time, intraoperative blood loss, hospital stay, time to resumption of oral intake, or morbidity. The elderly group had a significantly higher rate of hypertension and cardiovascular disease. The perioperative serum total protein and albumin levels were significantly lower in the elderly than in the younger group. The serum carcinoembryonic antigen level was lower in the elderly than in the younger group, and there was a significant decreasing trend after the operation in the elderly group.
CONCLUSION: The short-term outcomes of surgical treatment in elderly patients with colorectal adenocarcinoma were acceptable. Surgical treatment in elderly patients was considered a selectively effective approach.
PMCID: PMC3070137  PMID: 21472132
Colorectal tumor; Elderly patient; Morbidity; Carcinoembryonic antigen; C-reactive protein
12.  Prevention of type 2 diabetes in a primary healthcare setting: Three-year results of lifestyle intervention in Japanese subjects with impaired glucose tolerance 
BMC Public Health  2011;11:40.
A randomized control trial was performed to test whether a lifestyle intervention program, carried out in a primary healthcare setting using existing resources, can reduce the incidence of type 2 diabetes in Japanese with impaired glucose tolerance (IGT). The results of 3 years' intervention are summarized.
Through health checkups in communities and workplaces, 304 middle-aged IGT subjects with a mean body mass index (BMI) of 24.5 kg/m2 were recruited and randomized to the intervention group or control group. The lifestyle intervention was carried out for 3 years by public health nurses using the curriculum and educational materials provided by the study group.
After 1 year, the intervention had significantly improved body weight (-1.5 ± 0.7 vs. -0.7 ± 2.5 kg in the control; p = 0.023) and daily non-exercise leisure time energy expenditure (25 ± 113 vs. -3 ± 98 kcal; p = 0.045). Insulin sensitivity assessed by the Matsuda index was improved by the intervention during the 3 years. The 3-year cumulative incidence tended to be lower in the intervention group (14.8% vs.8.2%, log-rank test: p = 0.097). In a sub-analysis for the subjects with a BMI > 22.5 kg/m2, a significant reduction in the cumulative incidence was found (p = 0.027).
The present lifestyle intervention program using existing healthcare resources is beneficial in preventing diabetes in Japanese with IGT. This has important implications for primary healthcare-based diabetes prevention.
Trial registration number
PMCID: PMC3037863  PMID: 21235825
13.  Growth Inhibition of Re-Challenge B16 Melanoma Transplant by Conjugates of Melanogenesis Substrate and Magnetite Nanoparticles as the Basis for Developing Melanoma-Targeted Chemo-Thermo-Immunotherapy 
Melanogenesis substrate, N-propionyl-cysteaminylphenol (NPrCAP), is selectively incorporated into melanoma cells and inhibits their growth by producing cytotoxic free radicals. Magnetite nanoparticles also disintegrate cancer cells and generate heat shock protein (HSP) upon exposure to an alternating magnetic field (AMF). This study tested if a chemo-thermo-immunotherapy (CTI therapy) strategy can be developed for better management of melanoma by conjugating NPrCAP on the surface of magnetite nanoparticles (NPrCAP/M). We examined the feasibility of this approach in B16 mouse melanoma and evaluated the impact of exposure temperature, frequency, and interval on the inhibition of re-challenged melanoma growth. The therapeutic protocol against the primary transplanted tumor with or without AMF exposure once a day every other day for a total of three treatments not only inhibited the growth of the primary transplant but also prevented the growth of the secondary, re-challenge transplant. The heat-generated therapeutic effect was more significant at a temperature of 43°C than either 41°C or 46°C. NPrCAP/M with AMF exposure, instead of control magnetite alone or without AMF exposure, resulted in the most significant growth inhibition of the re-challenge tumor and increased the life span of the mice. HSP70 production was greatest at 43°C compared to that with 41°C or 46°C. CD8+T cells were infiltrated at the site of the re-challenge melanoma transplant.
PMCID: PMC2760320  PMID: 19830247
14.  A phase I trial of cytotoxic T-lymphocyte precursor-oriented peptide vaccines for colorectal carcinoma patients 
British Journal of Cancer  2004;90(7):1334-1342.
PMCID: PMC2409683  PMID: 15054451
peptide; cancer vaccine; immunotherapy; colon cancer; CTL
15.  A rapid biosensor chip assay for measuring of telomerase activity using surface plasmon resonance 
Nucleic Acids Research  2003;31(2):e4.
Considerable interest has been focused on telomerase because of its potential use in assays for cancer diagnosis, and for anti-telomerase drugs as a strategy for cancer chemotherapy. A number of assays based on the polymerase chain reaction (PCR) have been developed for evaluation of telomerase activity. To overcome the disadvantages of the conventional telomerase assay [telomeric repeat amplification protocol (TRAP)] related to PCR artifacts and troublesome post-PCR procedures, we have developed a telomeric repeat elongation (TRE) assay which directly measures telomerase activity as the telomeric elongation rate by biosensor technology using surface plasmon resonance (SPR). 5′-Biotinylated oligomers containing telomeric repeats were immobilized on streptavidin-pretreated dextran sensor surfaces in situ using the BIACORE apparatus. Subsequently, the oligomers associated with the telomerase extracts were elongated in the BIACORE apparatus. The rate of TRE was calculated by measuring the SPR signals. We examined elongation rates by the TRE assay in 18 cancer and three normal human fibroblast cell lines, and 12 human primary carcinomas and matching normal tissues. The elongation rates increased in a concentration- and time-dependent manner. Those of cancer cells were two to 10 times higher than fibroblast cell lines and normal tissues. Telomerase activities and its inhibitory effects of anti-telomerase agents as measured by both the TRE and TRAP assays showed a good correlation. Our assay allows precise quantitative comparison of a wide range of human cells from somatic cells to carcinoma cells. TRE assay is suitable for practical use in the assessment of telomerase activity in preclinical and clinical trials of telomerase-based therapies, because of its reproducibility, rapidity and simplicity.
PMCID: PMC140529  PMID: 12527793
16.  Endoscopic features of early-stage signet-ring-cell carcinoma of the stomach 
AIM: To identify the features of early signet ring cell gastric carcinoma using magnification endoscopy with narrow band imaging (NBI).
METHODS: A retrospective review was conducted of 12 cases of early signet ring cell gastric carcinoma who underwent treatment in a single institution between January 2009 and April 2013. All patients had magnification endoscopy with NBI and indigo carmine contrast to closely examine the mucosal architecture, including the microvasculature and arrangement of gastric pits. Histologic examination of the final endoscopic submucosal dissection or gastrectomy specimen was performed and compared with the endoscopic findings to identify patterns specific to signet ring cell carcinoma.
RESULTS: Twelve patients with early signet ring cell gastric carcinoma were identified; 75% were male, and average age was 61 years. Most of the lesions were stage T1a (83%), while the remainder were T1b (17%). The mean lesion size was 1.4 cm2. On standard endoscopy, all 12 patients had a pale, flat lesion without any evidence of mucosal abnormality such as ulceration, elevation, or depression. On magnification endoscopy with NBI, all of the patients had irregularities in the glands and microvasculature consistent with early gastric cancer. In addition, all 12 patients exhibited the “stretch sign”, an elongation or expansion of the architectural structure. Histologic examination of the resected specimens demonstrated an expanded and edematous mucosal layer infiltrated with tumor cells.
CONCLUSION: The “stretch sign” appears to be specific for signet ring cell carcinoma and may aid in the early diagnosis and treatment of this aggressive pathology.
PMCID: PMC4482834  PMID: 26140102
Signet ring cells; Early gastric cancer; Magnification endoscopy; Narrow band imaging; Stretch sign; Endoscopic submucosal dissection
17.  Subunit architecture and functional modular rearrangements of the transcriptional Mediator complex 
Cell  2014;157(6):1430-1444.
The multisubunit Mediator comprising ~30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism.
PMCID: PMC4104964  PMID: 24882805
18.  A Single-Nucleotide Polymorphism in an Endo-1,4-β-Glucanase Gene Controls Seed Coat Permeability in Soybean 
PLoS ONE  2015;10(6):e0128527.
Physical dormancy, a structural feature of the seed coat known as hard seededness, is an important characteristic for adaptation of plants against unstable and unpredictable environments. To dissect the molecular basis of qHS1, a quantitative trait locus for hard seededness in soybean (Glycine max (L) Merr.), we developed a near-isogenic line (NIL) of a permeable (soft-seeded) cultivar, Tachinagaha, containing a hard-seed allele from wild soybean (G. soja) introduced by successive backcrossings. The hard-seed allele made the seed coat of Tachinagaha more rigid by increasing the amount of β-1,4-glucans in the outer layer of palisade cells of the seed coat on the dorsal side of seeds, known to be a point of entrance of water. Fine-mapping and subsequent expression and sequencing analyses revealed that qHS1 encodes an endo-1,4-β-glucanase. A single-nucleotide polymorphism (SNP) introduced an amino acid substitution in a substrate-binding cleft of the enzyme, possibly reducing or eliminating its affinity for substrates in permeable cultivars. Introduction of the genomic region of qHS1 from the impermeable (hard-seeded) NIL into the permeable cultivar Kariyutaka resulted in accumulation of β-1,4-glucan in the outer layer of palisade cells and production of hard seeds. The SNP allele found in the NIL was further associated with the occurrence of hard seeds in soybean cultivars of various origins. The findings of this and previous studies may indicate that qHS1 is involved in the accumulation of β-1,4-glucan derivatives such as xyloglucan and/or β-(1,3)(1,4)-glucan that reinforce the impermeability of seed coats in soybean.
PMCID: PMC4454576  PMID: 26039079
19.  Vaginal Memory T Cells Induced by Intranasal Vaccination Are Critical for Protective T Cell Recruitment and Prevention of Genital HSV-2 Disease 
Journal of Virology  2014;88(23):13699-13708.
Protective immunity against genital pathogens causing chronic infections, such as herpes simplex virus 2 (HSV-2) or human immunodeficiency virus, requires the induction of cell-mediated immune responses locally in the genital tract. Intranasal immunization with a thymidine kinase-deficient (TK−) mutant of HSV-2 effectively induces HSV-2-specific gamma interferon (IFN-γ)-secreting memory T cell production and protective immunity against intravaginal challenge with wild-type HSV-2. However, the precise mechanism by which intranasal immunization induces protective immunity in the distant genital mucosa more effectively than does systemic immunization is unknown. Here, we showed that intranasal immunization with live HSV-2 TK− induced the production of effector T cells and their migration to, and retention in, the vaginal mucosa, whereas systemic vaccination barely established a local effector T cell pool, even when it induced the production of circulating memory T cells in the systemic compartment. The long-lasting HSV-2-specific local effector T cells induced by intranasal vaccination provided superior protection against intravaginal wild-type HSV-2 challenge by starting viral clearance at the entry site earlier than with intraperitoneal immunization. Intranasal immunization is an effective strategy for eliciting high levels of cell-mediated protection of the genital tract by providing long-lasting antigen (Ag)-specific local effector T cells without introducing topical infection or inflammation.
IMPORTANCE Intranasal (i.n.) vaccines against sexually transmitted diseases that are caused by viruses such as herpes simplex virus 2 (HSV-2) have long been in development, but no vaccine candidate is currently available. Understanding the cellular mechanisms of immune responses in a distant vaginal mucosa induced by i.n. immunization with HSV-2 will contribute to designing such a vaccine. Our study demonstrated that i.n. immunization with an attenuated strain of HSV-2 generated long-lasting IFN-γ-secreting T cells in vaginal mucosa more effectively than systemic immunization. We found that these vaginal effector memory T cells are critical for the early stage of viral clearance at natural infection sites and prevent severe vaginal inflammation and herpes encephalitis.
PMCID: PMC4248987  PMID: 25231301
20.  Quantitative trait loci for whiteness of cooked rice detected in improved rice cultivars in Hokkaido 
Breeding Science  2015;65(3):201-207.
Improving the eating quality of cooked rice has been one of the most important objectives in rice breeding programs. Eating quality of cooked rice is a complex trait including several components, such as external appearance, taste, aroma, and texture. Therefore, dissection of these components followed by marker-assisted selection of detected QTL(s) may be a useful approach for achieving desirable eating quality in rice breeding. Whiteness of cooked rice (WCR) is an important factor related to the external appearance of cooked rice. WCR is known to be associated with the amylose and protein contents of the endosperm. However, the genetic basis of WCR remains unclear. In this study, we evaluated phenotypic variation in WCR among recently developed rice cultivars from Hokkaido, Japan. Then, we developed doubled haploid lines (DHLs) derived from a cross between two cultivars from Hokkaido, Joiku No. 462 (high WCR) and Jokei06214 (low WCR). Using the DHLs, we detected two QTLs for WCR, qWCR3 and qWCR11, on chromosomes 3 and 11, respectively. We also examined the dosage effect of the two QTLs based on both the categorized segregants in the DHLs and the relationship between the WCR phenotype and inheritance around the QTL regions in cultivars from Hokkaido.
PMCID: PMC4482169  PMID: 26175616
whiteness of cooked rice; Oryza sativa L.; eating quality; rice breeding programs
21.  Hearing loss caused by a P2RX2 mutation identified in a MELAS family with a coexisting mitochondrial 3243AG mutation 
We present a family with a mitochondrial DNA 3243A>G mutation resulting in MELAS, of which some members have hearing loss where a novel mutation in the P2RX2 gene was identified.
One hundred ninety-four (194) Japanese subjects from unrelated families were enrolled in the study. Targeted genomic enrichment and massively parallel sequencing of all known non-syndromic hearing loss genes were performed to identify the genetic causes of hearing loss.
A novel mutation in the P2RX2 gene, that corresponded to c.601G>A (p.Asp201Tyr) was identified. Two patients carried the mutation, and had severe SNHL, while other members with MELAS (who did not carry the P2RX2 mutation) had normal hearing.
This is the first case report of a diagnosis of hearing loss caused by P2RX2 mutation in patients with MELAS. A potential explanation is that decreasing ATP production due to MELAS with mitochondrial 3243A>G mutation might suppress activation of P2X2 receptors. We also suggest that hearing loss caused by the P2RX2 mutation might be influenced by the decrease in ATP production due to MELAS, and that nuclear genetic factors may play a modifying role in mitochondrial dysfunction.
PMCID: PMC4441871  PMID: 25788561
Hearing loss; genetics; P2X2; MELAS; massively parallel sequencing
22.  Neuregulin 1 Type II-ErbB Signaling Promotes Cell Divisions Generating Neurons from Neural Progenitor Cells in the Developing Zebrafish Brain 
PLoS ONE  2015;10(5):e0127360.
Post-mitotic neurons are generated from neural progenitor cells (NPCs) at the expense of their proliferation. Molecular and cellular mechanisms that regulate neuron production temporally and spatially should impact on the size and shape of the brain. While transcription factors such as neurogenin1 (neurog1) and neurod govern progression of neurogenesis as cell-intrinsic mechanisms, recent studies show regulatory roles of several cell-extrinsic or intercellular signaling molecules including Notch, FGF and Wnt in production of neurons/neural progenitor cells from neural stem cells/radial glial cells (NSCs/RGCs) in the ventricular zone (VZ). However, it remains elusive how production of post-mitotic neurons from neural progenitor cells is regulated in the sub-ventricular zone (SVZ). Here we show that newborn neurons accumulate in the basal-to-apical direction in the optic tectum (OT) of zebrafish embryos. While neural progenitor cells are amplified by mitoses in the apical ventricular zone, neurons are exclusively produced through mitoses of neural progenitor cells in the sub-basal zone, later in the sub-ventricular zone, and accumulate apically onto older neurons. This neurogenesis depends on Neuregulin 1 type II (NRG1-II)–ErbB signaling. Treatment with an ErbB inhibitor, AG1478 impairs mitoses in the sub-ventricular zone of the optic tectum. Removal of AG1478 resumes sub-ventricular mitoses without precedent mitoses in the apical ventricular zone prior to basal-to-apical accumulation of neurons, suggesting critical roles of ErbB signaling in mitoses for post-mitotic neuron production. Knockdown of NRG1-II impairs both mitoses in the sub-basal/sub-ventricular zone and the ventricular zone. Injection of soluble human NRG1 into the developing brain ameliorates neurogenesis of NRG1-II-knockdown embryos, suggesting a conserved role of NRG1 as a cell-extrinsic signal. From these results, we propose that NRG1-ErbB signaling stimulates cell divisions generating neurons from neural progenitor cells in the developing vertebrate brain.
PMCID: PMC4441363  PMID: 26001123
23.  Molecular typing of Salmonella enterica serovar 4,[5],12:i:- isolates from humans, animals and river water in Japan by multilocus variable-number tandem repeat analysis and pulsed-field gel electrophoresis 
Fifty-one Salmonella enterica serovar 4,[5],12:i:- (S. 4, [5],12:i:-) isolates (14 human strains, 34 animal strains and 3 river water strains) which are assumed to be monophasic variants of S. Typhimurium were analyzed using pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) in order to investigate their genetic diversities and relationships. PFGE, MLVA and combination of them identified 28, 27 and 34 profiles (Simpson’s diversity indices [DI]=0.94, 0.96 and 0.97), respectively. No correlations were detected between MLVA clustering and PFGE clustering or phage typing. These results suggested that S. 4,[5],12:i:- originated from multiple S. Typhimurium ancestors. Two cattle and one pig isolates showing identical phage types as well as PFGE and MLVA profiles to human isolates S. 4,[5],12:i:- suggested the existence of the links between human infections and animal reservoirs.
PMCID: PMC4478744  PMID: 25649169
MLVA; monophasic variant; PFGE; Salmonella 4,[5],12:i:-(S. 4, [5],12:i:-); S. Typhimurium
24.  Risk factors associated with late aneurysmal sac expansion after endovascular abdominal aortic aneurysm repair 
We aimed to identify the risk factors associated with late aneurysmal sac expansion after endovascular abdominal aortic aneurysm repair (EVAR).
We retrospectively reviewed contrast-enhanced computed tomography (CT) images of 143 patients who were followed for ≥6 months after EVAR. Sac expansion was defined as an increase in sac diameter of 5 mm relative to the preoperative diameter. Univariate and multivariate analyses were performed to identify associated risk factors for late sac expansion after EVAR from the following variables: age, gender, device, endoleak, antiplatelet therapy, internal iliac artery embolization, and preprocedural variables (aneurysm diameter, proximal neck diameter, proximal neck length, suprarenal neck angulation, and infrarenal neck angulation).
Univariate analysis revealed female gender, endoleak, aneurysm diameter ≥60 mm, suprarenal neck angulation >45°, and infrarenal neck angulation >60° as factors associated with sac expansion. Multivariate analysis revealed endoleak, aneurysm diameter ≥60 mm, and infrarenal neck angulation >60° as independent predictors of sac expansion (P < 0.05, for all).
Our results suggest that patients with small abdominal aortic aneurysms (<60 mm) and infrarenal neck angulation ≤60° are more favorable candidates for EVAR. Intraprocedural treatments, such as prophylactic embolization of aortic branches or intrasac embolization, may reduce the risk of sac expansion in patients with larger abdominal aortic aneurysms or greater infrarenal neck angulation.
PMCID: PMC4463258  PMID: 25858524
25.  In vivo gastric mucosal histopathology using endocytoscopy 
AIM: To study the ability of endocytoscopy to identify normal gastric mucosa and to exclude Helicobacter pylori (H. pylori) infection.
METHODS: Endocytoscopic examination of the gastric corpus and antrum was performed in 70 consecutive patients. Target biopsy specimens were also obtained from the assessed region and multiple H. pylori tests were performed. The normal endocytoscopy patterns of the corpus and antrum were divided into the normal pit-dominant type (n-Pit) or the normal papilla-dominant type (n-Pap), respectively characterized as either regular pits with capillary networks or round, smooth papillary structures with spiral capillaries. On the other hand, normal mucosa was defined as mucosa not demonstrating histological abnormalities, including inflammation and atrophy.
RESULTS: The sensitivity and specificity of n-Pit for normal mucosa in the gastric corpus were 94.4% and 97.1%, respectively, whereas those of n-Pap for normal mucosa in the antrum were 92.0% and 86.7%, respectively. The positive predictive values of n-Pit and n-Pap for H. pylori-negative tissue were 88.6% and 93.1%, respectively, and their negative predictive values for H. pylori-negative tissues were 42.9% and 41.5%, respectively. The inter-observer agreement for determining n-Pit and n-Pap for normal mucosa were 0.857 and 0.769, respectively, which is considered reliable.
CONCLUSION: N-Pit and n-Pap, seen using EC, are considered useful predictors of normal mucosa and the absence of H. pylori infection.
PMCID: PMC4408474  PMID: 25945015
Atrophy; Endocytoscopy; Gastric mucosa; Helicobacter pylori; In vivo histopathology

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