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1.  PI3K in the ventromedial hypothalamic nucleus mediates estrogenic actions on energy expenditure in female mice 
Scientific Reports  2016;6:23459.
Estrogens act in the ventromedial hypothalamic nucleus (VMH) to regulate body weight homeostasis. However, the molecular mechanisms underlying these estrogenic effects are unknown. We show that activation of estrogen receptor-α (ERα) stimulates neural firing of VMH neurons expressing ERα, and these effects are blocked with intracellular application of a pharmacological inhibitor of the phosphatidyl inositol 3-kinase (PI3K). Further, we demonstrated that mice with genetic inhibition of PI3K activity in VMH neurons showed a sexual dimorphic obese phenotype, with only female mutants being affected. In addition, inhibition of VMH PI3K activity blocked effects of 17β-estradiol to stimulate energy expenditure, but did not affect estrogen-induced anorexia. Collectively, our results indicate that PI3K activity in VMH neurons plays a physiologically relevant role in mediating estrogenic actions on energy expenditure in females.
PMCID: PMC4796901  PMID: 26988598
2.  Estrogen receptor–α in medial amygdala neurons regulates body weight 
The Journal of Clinical Investigation  2015;125(7):2861-2876.
Estrogen receptor–α (ERα) activity in the brain prevents obesity in both males and females. However, the ERα-expressing neural populations that regulate body weight remain to be fully elucidated. Here we showed that single-minded–1 (SIM1) neurons in the medial amygdala (MeA) express abundant levels of ERα. Specific deletion of the gene encoding ERα (Esr1) from SIM1 neurons, which are mostly within the MeA, caused hypoactivity and obesity in both male and female mice fed with regular chow, increased susceptibility to diet-induced obesity (DIO) in males but not in females, and blunted the body weight–lowering effects of a glucagon-like peptide-1–estrogen (GLP-1–estrogen) conjugate. Furthermore, selective adeno-associated virus-mediated deletion of Esr1 in the MeA of adult male mice produced a rapid body weight gain that was associated with remarkable reductions in physical activity but did not alter food intake. Conversely, overexpression of ERα in the MeA markedly reduced the severity of DIO in male mice. Finally, an ERα agonist depolarized MeA SIM1 neurons and increased their firing rate, and designer receptors exclusively activated by designer drug–mediated (DREADD-mediated) activation of these neurons increased physical activity in mice. Collectively, our results support a model where ERα signals activate MeA neurons to stimulate physical activity, which in turn prevents body weight gain.
PMCID: PMC4563687  PMID: 26098212
3.  Estrogens stimulate serotonin neurons to inhibit binge-like eating in mice 
The Journal of Clinical Investigation  2014;124(10):4351-4362.
Binge eating afflicts approximately 5% of US adults, though effective treatments are limited. Here, we showed that estrogen replacement substantially suppresses binge-like eating behavior in ovariectomized female mice. Estrogen-dependent inhibition of binge-like eating was blocked in female mice specifically lacking estrogen receptor-α (ERα) in serotonin (5-HT) neurons in the dorsal raphe nuclei (DRN). Administration of a recently developed glucagon-like peptide-1–estrogen (GLP-1–estrogen) conjugate designed to deliver estrogen to GLP1 receptor–enhanced regions effectively targeted bioactive estrogens to the DRN and substantially suppressed binge-like eating in ovariectomized female mice. Administration of GLP-1 alone reduced binge-like eating, but not to the same extent as the GLP-1–estrogen conjugate. Administration of ERα-selective agonist propylpyrazole triol (PPT) to murine DRN 5-HT neurons activated these neurons in an ERα-dependent manner. PPT also inhibited a small conductance Ca2+-activated K+ (SK) current; blockade of the SK current prevented PPT-induced activation of DRN 5-HT neurons. Furthermore, local inhibition of the SK current in the DRN markedly suppressed binge-like eating in female mice. Together, our data indicate that estrogens act upon ERα to inhibit the SK current in DRN 5-HT neurons, thereby activating these neurons to suppress binge-like eating behavior and suggest ERα and/or SK current in DRN 5-HT neurons as potential targets for anti-binge therapies.
PMCID: PMC4191033  PMID: 25157819
4.  Unique and facile solvothermal synthesis of mesoporous WO3 using a solid precursor and a surfactant template as a photoanode for visible-light-driven water oxidation 
Nanoscale Research Letters  2014;9(1):542.
Mesoporous tungsten trioxide (WO3) was prepared from tungstic acid (H2WO4) as a tungsten precursor with dodecylamine (DDA) as a template to guide porosity of the nanostructure by a solvothermal technique. The WO3 sample (denoted as WO3-DDA) prepared with DDA was moulded on an electrode to yield efficient performance for visible-light-driven photoelectrochemical (PEC) water oxidation. Powder X-ray diffraction (XRD) data of the WO3-DDA sample calcined at 400°C indicate a crystalline framework of the mesoporous structure with disordered arrangement of pores. N2 physisorption studies show a Brunauer-Emmett-Teller (BET) surface area up to 57 m2 g-1 together with type IV isotherms and uniform distribution of a nanoscale pore size in the mesopore region. Scanning electron microscopy (SEM) images exhibit well-connected tiny spherical WO3 particles with a diameter of ca. 5 to 20 nm composing the mesoporous network. The WO3-DDA electrode generated photoanodic current density of 1.1 mA cm-2 at 1.0 V versus Ag/AgCl under visible light irradiation, which is about three times higher than that of the untemplated WO3. O2 (1.49 μmol; Faraday efficiency, 65.2%) was evolved during the 1-h photoelectrolysis for the WO3-DDA electrode under the conditions employed. The mesoporous electrode turned out to work more efficiently for visible-light-driven water oxidation relative to the untemplated WO3 electrode.
PMCID: PMC4194058  PMID: 25313301
Tungsten trioxide; Mesoporous structure; Photoelectrocatalysis; Water oxidation
5.  Coffee intake mitigated inflammation and obesity-induced insulin resistance in skeletal muscle of high-fat diet-induced obese mice 
Genes & Nutrition  2014;9(3):389.
Epidemiologic findings offer the promise that coffee or its many constituents may be useful as a dietary intervention in type 2 diabetes (T2D) prevention. We aimed to elucidate the molecular mechanisms involved in the ameliorative effects of caffeinated coffee (CC), decaffeinated coffee (DC) and unroasted caffeinated green coffee (GC) on skeletal muscle gene expression profiles and their relationships in an obesity animal model. Eight-week-old male C57BL6 mice were raised for 9 weeks ad libitum on a normal diet, a high-fat diet, or high-fat diet containing 2 % freeze-dried CC, or DC, or GC. Total RNA and protein were extracted from skeletal muscle and subjected to microarray (Mouse Genome 430 2.0, Affymetrix) and western blotting analyses, respectively. Coffee intake mitigated the insulin resistance by decreasing plasma glucose levels during an insulin tolerance test and by increasing tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), p85/IRS-1 complex and pAkt/PKB (protein kinase B). In addition, coffee intake down-regulated the anti-inflammatory genes activating transcription factor 3, FBJ osteosarcoma oncogene, heat shock protein 1A, heat shock protein 1B and synuclein, gamma and the inflammation-associated insulin signaling genes stearoyl-coenzyme A desaturase 1 and secreted phosphoprotein 1. These results provide scientific insight on the probable positive effects of coffee intake on impaired insulin signaling, inflammation and obesity, thereby providing a new perspective on the prevention of obesity and T2D.
Electronic supplementary material
The online version of this article (doi:10.1007/s12263-014-0389-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4026430  PMID: 24599575
Coffee intake; Insulin resistance; Inflammation and obesity; Skeletal muscle; Transcriptomic analysis; High-fat diet
6.  An Integrated Multi-Omics Study Revealed Metabolic Alterations Underlying the Effects of Coffee Consumption 
PLoS ONE  2014;9(3):e91134.
Many epidemiological studies have indicated that coffee consumption may reduce the risks of developing obesity and diabetes, but the underlying mechanisms of these effects are poorly understood. Our previous study revealed the changes on gene expression profiles in the livers of C57BL/6J mice fed a high-fat diet containing three types of coffee (caffeinated, decaffeinated and green unroasted coffee), using DNA microarrays. The results revealed remarkable alterations in lipid metabolism-related molecules which may be involved in the anti-obesity effects of coffee. We conducted the present study to further elucidate the metabolic alterations underlying the effects of coffee consumption through comprehensive proteomic and metabolomic analyses. Proteomics revealed an up-regulation of isocitrate dehydrogenase (a key enzyme in the TCA cycle) and its related proteins, suggesting increased energy generation. The metabolomics showed an up-regulation of metabolites involved in the urea cycle, with which the transcriptome data were highly consistent, indicating accelerated energy expenditure. The TCA cycle and the urea cycle are likely be accelerated in a concerted manner, since they are directly connected by mutually providing each other's intermediates. The up-regulation of these pathways might result in a metabolic shift causing increased ATP turnover, which is related to the alterations of lipid metabolism. This mechanism may play an important part in the suppressive effects of coffee consumption on obesity, inflammation, and hepatosteatosis. This study newly revealed global metabolic alterations induced by coffee intake, providing significant insights into the association between coffee intake and the prevention of type 2 diabetes, utilizing the benefits of multi-omics analyses.
PMCID: PMC3949743  PMID: 24618914
7.  A Case of In-Situ Reconstruction with a Rifampicin-Bonded Gelatin-Sealed Woven Dacron Graft for Prosthetic Graft Infection with Pseudoaneurysms after Ascending Aortic Replacement for Type A Dissection 
Annals of Vascular Diseases  2014;7(1):68-71.
A 74-year-old woman underwent replacement of the ascending aorta for acute type A aortic dissection. The patient suffered from bacteremia postoperatively and repeated computed tomography showed an increasing diameter of pseudoaneurysms at the site of the proximal anastomosis due to graft infection. Re-mechanical Bentall operation and arch replacement were therefore performed using a composite graft of a rifampicin-bonded gelatin-sealed 24-mm woven Dacron graft and a mechanical valve. The postoperative course was uneventful. We report the successful in situ reconstruction using the above-mentioned Dacron graft and describe the preparation of the rifampicin solution using a surfactant.
PMCID: PMC3968420  PMID: 24719667
prosthetic graft infection; rifampicin-bonded gelatin-sealed woven Dacron graft; rifampicin solution
8.  World's first telepathology experiments employing WINDS ultra-high-speed internet satellite, nicknamed “KIZUNA” 
Recent advances in information technology have allowed the development of a telepathology system involving high-speed transfer of high-volume histological figures via fiber optic landlines. However, at present there are geographical limits to landlines. The Japan Aerospace Exploration Agency (JAXA) has developed the “Kizuna” ultra-high speed internet satellite and has pursued its various applications. In this study we experimented with telepathology in collaboration with JAXA using Kizuna. To measure the functionality of the Wideband InterNet working engineering test and Demonstration Satellite (WINDS) ultra-high speed internet satellite in remote pathological diagnosis and consultation, we examined the adequate data transfer speed and stability to conduct telepathology (both diagnosis and conferencing) with functionality, and ease similar or equal to telepathology using fiber-optic landlines.
Materials and Methods:
We performed experiments for 2 years. In year 1, we tested the usability of the WINDS for telepathology with real-time video and virtual slide systems. These are state-of-the-art technologies requiring massive volumes of data transfer. In year 2, we tested the usability of the WINDS for three-way teleconferencing with virtual slides. Facilities in Iwate (northern Japan), Tokyo, and Okinawa were connected via the WINDS and voice conferenced while remotely examining and manipulating virtual slides.
Network function parameters measured using ping and Iperf were within acceptable limits. However; stage movement, zoom, and conversation suffered a lag of approximately 0.8 s when using real-time video, and a delay of 60-90 s was experienced when accessing the first virtual slide in a session. No significant lag or inconvenience was experienced during diagnosis and conferencing, and the results were satisfactory. Our hypothesis was confirmed for both remote diagnosis using real-time video and virtual slide systems, and also for teleconferencing using virtual slide systems with voice functionality.
Our results demonstrate the feasibility of ultra-high-speed internet satellite networks for use in telepathology. Because communications satellites have less geographical and infrastructural requirements than landlines, ultra-high-speed internet satellite telepathology represents a major step toward alleviating regional disparity in the quality of medical care.
PMCID: PMC3815045  PMID: 24244882
KIZUNA (絆); optical fiber; real-time video system; telepathology; ultra-high-speed internet satellite; virtual slide system
9.  Development of a teledermatopathology consultation system using virtual slides 
Diagnostic Pathology  2012;7:177.
An online consultation system using virtual slides (whole slide images; WSI) has been developed for pathological diagnosis, and could help compensate for the shortage of pathologists, especially in the field of dermatopathology and in other fields dealing with difficult cases. This study focused on the performance and future potential of the system.
In our system, histological specimens on slide glasses are digitalized by a virtual slide instrument, converted into web data, and up-loaded to an open server. Using our own purpose-built online system, we then input patient details such as age, gender, affected region, clinical data, past history and other related items. We next select up to ten consultants. Finally we send an e-mail to all consultants simultaneously through a single command. The consultant receives an e-mail containing an ID and password which is used to access the open server and inspect the images and other data associated with the case. The consultant makes a diagnosis, which is sent to us along with comments.
Because this was a pilot study, we also conducted several questionnaires with consultants concerning the quality of images, operability, usability, and other issues.
We solicited consultations for 36 cases, including cases of tumor, and involving one to eight consultants in the field of dermatopathology. No problems were noted concerning the images or the functioning of the system on the sender or receiver sides. The quickest diagnosis was received only 18 minutes after sending our data. This is much faster than in conventional consultation using glass slides. There were no major problems relating to the diagnosis, although there were some minor differences of opinion between consultants. The results of questionnaires answered by many consultants confirmed the usability of this system for pathological consultation. (16 out of 23 consultants.)
We have developed a novel teledermatopathological consultation system using virtual slides, and investigated the usefulness of the system. The results demonstrate that our system can be a useful tool for international medical work, and we anticipate its wider application in the future.
Virtual slides
The virtual slides for this article can be found here:
PMCID: PMC3557204  PMID: 23237667
Telepathology; Teledermatology; Consultation; Virtual slide; Whole slide image
10.  Omics and Integrated Omics for the Promotion of Food and Nutrition Science 
Transcriptomics, proteomics, and metabolomics are three major platforms of comprehensive omics analysis in the science of food and complementary medicine. Other omics disciplines, including those of epigenetics and microRNA, are matters of increasing concern. The increased use of the omics approach in food science owes much to the recent advancement of technology and bioinformatic methodologies. Moreover, many researchers now put the combination of multiple omics analysis (integrated omics) into practice to exhaustively understand the functionality of food components. However, data analysis of integrated omics requires huge amount of work and high skill of data handling. A database of nutritional omics data was constructed by the authors, which should help food scientists to analyze their own omics data more effectively. In addition, a novel tool for the easy visualization of omics data was developed by the authors’ group. The tool enables one to overview the changes of multiple omics in the KEGG pathway. Research in traditional and complementary medicine will be further facilitated by promoting the integrated omics research of food functionality. Such integrated research will only be possible with the effective collaboration of scientists with different backgrounds.
PMCID: PMC3942997  PMID: 24716102
Nutrigenomics; Transcriptomics; Proteomics; Metabolomics; Database
11.  Crystallization and preliminary X-ray diffraction study of an active-site mutant of pro-Tk-subtilisin from a hyperthermophilic archaeon 
Crystallization of and preliminary crystallographic studies on an active-site mutant of pro-Tk-subtilisin from the hyperthermophilic archaeon T. kodakaraensis were performed.
Crystallization of and preliminary crystallographic studies on an active-site mutant of pro-Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis were performed. The crystal was grown at 277 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 2.3 Å resolution using synchrotron radiation from station BL41XU at SPring-8. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 92.69, b = 121.78, c = 77.53 Å. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V M was calculated to be 2.6 Å3 Da−1 and the solvent content was 53.1%.
PMCID: PMC2242867  PMID: 16946475
pro-Tk-subtilisin; Thermococcus kodakaraensis
12.  Ca2+-Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis: the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding 
Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca2+ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca2+ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca2+ but was activated upon incubation with Ca2+ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ∼5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a Ki of 25 ± 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.
PMCID: PMC1489632  PMID: 16751527

Results 1-12 (12)