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1.  Positive Association Between Serum Level of Glyceraldehyde-Derived Advanced Glycation End Products and Vascular Inflammation Evaluated by [18F]Fluorodeoxyglucose Positron Emission Tomography 
Diabetes Care  2012;35(12):2618-2625.
Advanced glycation end products (AGEs) evoke inflammatory reactions, contributing to the development and progression of atherosclerosis. We investigated the relationship between serum AGE level and vascular inflammation.
The study involved 275 outpatients at Kurume University, Japan (189 males and 86 females; mean age 61.2 ± 8.8 years) who underwent complete history and physical examinations and determinations of blood chemistry and anthropometric variables, including AGEs. Serum AGE level was examined by enzyme-linked immunosorbent assay. Vascular [18F]fluorodeoxyglucose (FDG) uptake, an index of vascular inflammation, was measured as blood-normalized standardized uptake value, known as the target-to-background ratio (TBR), by FDG–positron emission tomography (FDG-PET). Furthermore, we examined whether the changes in serum AGE level after treatment with oral hypoglycemia agents (OHAs) were correlated with those of TBR in another 18 subjects whose AGE value was >14.2 units/mL (mean ± 2 SD).
Mean serum AGE level and carotid TBR values were 9.15 ± 2.53 and 1.43 ± 0.22 units/mL, respectively. Multiple stepwise regression analysis revealed that TBR was independently correlated with AGEs (P < 0.001), carotid intima-media thickness (P < 0.01), and BMI (P < 0.02). When age- and sex-adjusted AGE values stratified by TBR tertiles were compared using ANCOVA, a significant trend was observed (P < 0.01). In addition, the changes in AGEs after OHA treatment were positively (r = 0.50, P < 0.05) correlated with those in TBR value.
The current study reveals that serum AGE level is independently associated with vascular inflammation evaluated by FDG-PET, suggesting that circulating AGE value may be a biomarker that could reflect vascular inflammation within an area of atherosclerosis.
PMCID: PMC3507595  PMID: 22912424
2.  Werner Syndrome-specific induced pluripotent stem cells: recovery of telomere function by reprogramming 
Werner syndrome (WS) is a rare human autosomal recessive premature aging disorder characterized by early onset of aging-associated diseases, chromosomal instability, and cancer predisposition. The function of the DNA helicase encoded by WRN, the gene responsible for WS, has been studied extensively. WRN helicase is involved in the maintenance of chromosome integrity through DNA replication, repair, and recombination by interacting with a variety of proteins associated with DNA repair and telomere maintenance. The accelerated aging associated with WS is reportedly caused by telomere dysfunction, and the underlying mechanism of the disease is yet to be elucidated. Although it was reported that the life expectancy for patients with WS has improved over the last two decades, definitive therapy for these patients has not seen much development. Severe symptoms of the disease, such as leg ulcers, cause a significant decline in the quality of life in patients with WS. Therefore, the establishment of new therapeutic strategies for the disease is of utmost importance. Induced pluripotent stem cells (iPSCs) can be established by the introduction of several pluripotency genes, including Oct3/4, Sox2, Klf4, and c-myc into differentiated cells. iPSCs have the potential to differentiate into a variety of cell types that constitute the human body, and possess infinite proliferative capacity. Recent studies have reported the generation of iPSCs from the cells of patients with WS, and they have concluded that reprogramming represses premature senescence phenotypes in these cells. In this review, we summarize the findings of WS patient-specific iPSCs (WS iPSCs) and focus on the roles of telomere and telomerase in the maintenance of these cells. Finally, we discuss the potential use of WS iPSCs for clinical applications.
PMCID: PMC4310323
Werner syndrome (WS); accelerated aging; chromosomal instability; telomere dysfunction; induced pluripotent stem cells (iPSCs); reprogramming; telomerase; premature senescence phenotypes
3.  Endoscopic Transsphenoidal Cisternostomy for Nonneoplastic Sellar Cysts 
BioMed Research International  2015;2015:389474.
Background and Importance. Sellar arachnoid cysts and Rathke's cleft cysts are benign lesions that produce similar symptoms, including optochiasmatic compression, pituitary dysfunction, and headache. Studies have reported the use of various surgical treatment methods for treating these symptoms, preventing recurrence, and minimizing operative complications. However, the postoperative cerebrospinal fluid (CSF) fistula and recurrence rate remain significant. Clinical Presentation. In this paper, we present 8 consecutive cases involving arachnoid cysts and Rathke's cleft cysts, which were managed by using drainage and cisternostomy, the intentional fenestration of the cyst into the subarachnoid space, and then meticulously closing sellar floor using dural sutures. The postoperative images, CSF fistula rate, and the recurrence rate were favorable. Conclusion. We report this technique and discuss the benefit of this minimally invasive approach.
PMCID: PMC4317582
4.  Relationship between Expression of Onco-Related miRNAs and the Endoscopic Appearance of Colorectal Tumors 
Accumulating data indicates that certain microRNAs (miRNAs or miRs) are differently expressed in samples of tumors and paired non-tumorous samples taken from the same patients with colorectal tumors. We examined the expression of onco-related miRNAs in 131 sporadic exophytic adenomas or early cancers and in 52 sporadic flat elevated adenomas or early cancers to clarify the relationship between the expression of the miRNAs and the endoscopic morphological appearance of the colorectal tumors. The expression levels of miR-143, -145, and -34a were significantly reduced in most of the exophytic tumors compared with those in the flat elevated ones. In type 2 cancers, the miRNA expression profile was very similar to that of the exophytic tumors. The expression levels of miR-7 and -21 were significantly up-regulated in some flat elevated adenomas compared with those in exophytic adenomas. In contrast, in most of the miR-143 and -145 down-regulated cases of the adenoma-carcinoma sequence and in some of the de novo types of carcinoma, the up-regulation of oncogenic miR-7 and/or -21 contributed to the triggering mechanism leading to the carcinogenetic process. These findings indicated that the expression of onco-related miRNA was associated with the morphological appearance of colorectal tumors.
PMCID: PMC4307318  PMID: 25584614
microRNA; endoscopic appearance; colorectal tumor
5.  Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles 
Journal of Extracellular Vesicles  2014;3:10.3402/jev.v3.26913.
Secreted membrane-enclosed vesicles, collectively called extracellular vesicles (EVs), which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non-vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology. Here, the International Society for Extracellular Vesicles (ISEV) provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs.
PMCID: PMC4275645  PMID: 25536934
extracellular vesicles; microvesicles; microparticles; exosomes; ectosomes; extracellular RNA
6.  Contextual Effect of Repression of Bone Morphogenetic Protein Activity in Prostate Cancer 
Endocrine-related cancer  2013;20(6):861-874.
Several studies have focused on the impact of bone morphogenetic protein (BMP) on prostate cancer homing and growth at distant metastatic sites, but very little on impact at the primary site. Here we used two cell lines, one (E8) isolated from a primary tumor and the other (cE1) from a recurrent tumor arising at the primary site, both from the conditional Pten deletion mouse model of prostatic adenocarcinoma. Over-expression of the BMP antagonist Noggin inhibited proliferation of cE1 cells in vitro while enhancing their ability to migrate. On the other hand cE1/Noggin grafts grown in vivo showed a greater mass and a higher proliferation index than the cE1/Control grafts. For suppression of BMP activity in the context of cancer associated fibroblasts (CAFs), we used Noggin-transduced CAFs from the same mouse model to determine their effect on E8 or cE1 induced tumor growth. CAF/Noggin led to increased tumor mass and greater de-differentiation of the E8 cell as compared to tumors formed in the presence of CAF/Control cells. A trend in increase in the size of the tumor was also noted for cE1 cells when inoculated with CAF/Noggin. Together, the results may point to a potential inhibitory role of BMP in the growth or re-growth of prostate tumor at the primary site. Additionally, results for cE1/Noggin, and cE1 mixed with CAF/Noggin suggested that suppression of BMP activity in the cancer cells may have a stronger growth enhancing effect on the tumor than its suppression in the fibroblastic compartment of the tumor microenvironment.
PMCID: PMC3885249  PMID: 24042462
BMP; Noggin; prostate cancer mouse model; prostate cancer cell lines; cancer-associated fibroblasts
7.  Haplotype defined by the MLH1-93G/A polymorphism is associated with MLH1 promoter hypermethylation in sporadic colorectal cancers 
BMC Research Notes  2014;7(1):835.
Methylation of the MLH1 promoter region has been suggested to be a major mechanism of gene inactivation in sporadic microsatellite instability-positive (MSI-H) colorectal cancers (CRCs). Recently, single-nucleotide polymorphism (SNP) in the MLH1 promoter region (MLH1-93G/A; rs1800734) has been proposed to be associated with MLH1 promoter methylation, loss of MLH1 protein expression and MSI-H tumors. We examined the association of MLH1-93G/A and six other SNPs surrounding MLH1-93G/A with the methylation status in 210 consecutive sporadic CRCs in Japanese patients.
Methylation of the MLH1 promoter region was evaluated by Na-bisulfite polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) analysis. The genotype frequencies of SNPs located in the 54-kb region surrounding the MLH1-93G/A SNP were examined by SSCP analysis.
Methylation of the MLH1 promoter region was observed in 28.6% (60/210) of sporadic CRCs. The proportions of MLH1-93G/A genotypes A/A, A/G and G/G were 26% (n = 54), 51% (n = 108) and 23% (n = 48), respectively, and they were significantly associated with the methylation status (p = 0.01). There were no significant associations between genotype frequency of the six other SNPs and methylation status. The A-allele of MLH1-93G/A was more common in cases with methylation than the G-allele (p = 0.0094), especially in females (p = 0.0067). In logistic regression, the A/A genotype of the MLH1-93G/A SNP was shown to be the most significant risk factor for methylation of the MLH1 promoter region (odds ratio 2.82, p = 0.003). Furthermore, a haplotype of the A-allele of rs2276807 located -47 kb upstream from the MLH1-93G/A SNP and the A-allele of MLH1-93G/A SNP was significantly associated with MLH1 promoter methylation.
These results indicate that individuals, and particularly females, carrying the A-allele at the MLH1-93G/A SNP, especially in association with the A-allele of rs2276807, may harbor an increased risk of methylation of the MLH1 promoter region.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-0500-7-835) contains supplementary material, which is available to authorized users.
PMCID: PMC4253604  PMID: 25421847
MSI; SNP; MLH1; Methylation; Colorectal cancer; Haplotype
8.  Reprogramming Suppresses Premature Senescence Phenotypes of Werner Syndrome Cells and Maintains Chromosomal Stability over Long-Term Culture 
PLoS ONE  2014;9(11):e112900.
Werner syndrome (WS) is a premature aging disorder characterized by chromosomal instability and cancer predisposition. Mutations in WRN are responsible for the disease and cause telomere dysfunction, resulting in accelerated aging. Recent studies have revealed that cells from WS patients can be successfully reprogrammed into induced pluripotent stem cells (iPSCs). In the present study, we describe the effects of long-term culture on WS iPSCs, which acquired and maintained infinite proliferative potential for self-renewal over 2 years. After long-term cultures, WS iPSCs exhibited stable undifferentiated states and differentiation capacity, and premature upregulation of senescence-associated genes in WS cells was completely suppressed in WS iPSCs despite WRN deficiency. WS iPSCs also showed recapitulation of the phenotypes during differentiation. Furthermore, karyotype analysis indicated that WS iPSCs were stable, and half of the descendant clones had chromosomal profiles that were similar to those of parental cells. These unexpected properties might be achieved by induced expression of endogenous telomerase gene during reprogramming, which trigger telomerase reactivation leading to suppression of both replicative senescence and telomere dysfunction in WS cells. These findings demonstrated that reprogramming suppressed premature senescence phenotypes in WS cells and WS iPSCs could lead to chromosomal stability over the long term. WS iPSCs will provide opportunities to identify affected lineages in WS and to develop a new strategy for the treatment of WS.
PMCID: PMC4229309  PMID: 25390333
9.  The Host Protease TMPRSS2 Plays a Major Role in In Vivo Replication of Emerging H7N9 and Seasonal Influenza Viruses 
Journal of Virology  2014;88(10):5608-5616.
Proteolytic cleavage of the hemagglutinin (HA) protein is essential for influenza A virus (IAV) to acquire infectivity. This process is mediated by a host cell protease(s) in vivo. The type II transmembrane serine protease TMPRSS2 is expressed in the respiratory tract and is capable of activating a variety of respiratory viruses, including low-pathogenic (LP) IAVs possessing a single arginine residue at the cleavage site. Here we show that TMPRSS2 plays an essential role in the proteolytic activation of LP IAVs, including a recently emerged H7N9 subtype, in vivo. We generated TMPRSS2 knockout (KO) mice. The TMPRSS2 KO mice showed normal reproduction, development, and growth phenotypes. In TMPRSS2 KO mice infected with LP IAVs, cleavage of HA was severely impaired, and consequently, the majority of LP IAV progeny particles failed to gain infectivity, while the viruses were fully activated proteolytically in TMPRSS2+/+ wild-type (WT) mice. Accordingly, in contrast to WT mice, TMPRSS2 KO mice were highly tolerant of challenge infection by LP IAVs (H1N1, H3N2, and H7N9) with ≥1,000 50% lethal doses (LD50) for WT mice. On the other hand, a high-pathogenic H5N1 subtype IAV possessing a multibasic cleavage site was successfully activated in the lungs of TMPRSS2 KO mice and killed these mice, as observed for WT mice. Our results demonstrate that recently emerged H7N9 as well as seasonal IAVs mainly use the specific protease TMPRSS2 for HA cleavage in vivo and, thus, that TMPRSS2 expression is essential for IAV replication in vivo.
IMPORTANCE Influenza A virus (IAV) is a leading pathogen that infects and kills many humans every year. We clarified that the infectivity and pathogenicity of IAVs, including a recently emerged H7N9 subtype, are determined primarily by a host protease, TMPRSS2. Our data showed that TMPRSS2 is the key host protease that activates IAVs in vivo through proteolytic cleavage of their HA proteins. Hence, TMPRSS2 is a good target for the development of anti-IAV drugs. Such drugs could also be effective for many other respiratory viruses, including the recently emerged Middle East respiratory syndrome (MERS) coronavirus, because they are also activated by TMPRSS2 in vitro. Consequently, the present paper could have a large impact on the battle against respiratory virus infections and contribute greatly to human health.
PMCID: PMC4019123  PMID: 24600012
10.  Noninvasive Molecular Imaging of Cell Death in Myocardial Infarction using 111In-GSAO 
Scientific Reports  2014;4:6826.
Acute insult to the myocardium is associated with substantial loss of cardiomyocytes during the process of myocardial infarction. In this setting, apoptosis (programmed cell death) and necrosis may operate on a continuum. Because the latter is characterized by the loss of sarcolemmal integrity, we propose that an appropriately labeled tracer directed at a ubiquitously present intracellular moiety would allow non-invasive definition of cardiomyocyte necrosis. A trivalent arsenic peptide, GSAO (4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid), is capable of binding to intracellular dithiol molecules such as HSP90 and filamin-A. Since GSAO is membrane impermeable and dithiol molecules abundantly present intracellularly, we propose that myocardial localization would represent sarcolemmal disruption or necrotic cell death. In rabbit and mouse models of myocardial infarction and post-infarct heart failure, we employed In-111-labelled GSAO for noninvasive radionuclide molecular imaging. 111In-GSAO uptake was observed within the regions of apoptosis seeking agent- 99mTc-Annexin A5 uptake, suggesting the colocalization of apoptotic and necrotic cell death processes.
PMCID: PMC4212241  PMID: 25351258
11.  Infection with Porphyromonas gingivalis Exacerbates Endothelial Injury in Obese Mice 
PLoS ONE  2014;9(10):e110519.
A number of studies have revealed a link between chronic periodontitis and cardiovascular disease in obese patients. However, there is little information about the influence of periodontitis-associated bacteria, Porphyromonas gingivalis (Pg), on pathogenesis of atherosclerosis in obesity.
In vivo experiment: C57BL/6J mice were fed with a high-fat diet (HFD) or normal chow diet (CD), as a control. Pg was infected from the pulp chamber. At 6 weeks post-infection, histological and immunohistochemical analysis of aortal tissues was performed. In vitro experiment: hTERT-immortalized human umbilical vein endothelial cells (HuhT1) were used to assess the effect of Pg/Pg-LPS on free fatty acid (FFA) induced endothelial cells apoptosis and regulation of cytokine gene expression.
Weaker staining of CD31 and increased numbers of TUNEL positive cells in aortal tissue of HFD mice indicated endothelial injury. Pg infection exacerbated the endothelial injury. Immunohistochemically, Pg was detected deep in the smooth muscle of the aorta, and the number of Pg cells in the aortal wall was higher in HFD mice than in CD mice. Moreover, in vitro, FFA treatment induced apoptosis in HuhT1 cells and exposure to Pg-LPS increased this effect. In addition, Pg and Pg-LPS both attenuated cytokine production in HuhT1 cells stimulated by palmitate.
Dental infection of Pg may contribute to pathogenesis of atherosclerosis by accelerating FFA-induced endothelial injury.
PMCID: PMC4204882  PMID: 25334003
12.  DNA Methylation Status of Epithelial-Mesenchymal Transition (EMT) - Related Genes Is Associated with Severe Clinical Phenotypes in Ulcerative Colitis (UC) 
PLoS ONE  2014;9(10):e107947.
Epithelial-to-mesenchymal transition (EMT) is a phenomenon that allows the conversion of adherent epithelial cells to a mesenchymal cell phenotype, which enhances migratory capacity and invasiveness. Recent studies have suggested that EMT contributes to the pathogenesis of ulcerative colitis (UC). We investigated the promoter DNA methylation status of EMT-related genes in the colonic mucosa in UC.
Colonic biopsies were obtained from the rectal inflammatory mucosa of 86 UC patients and the non-inflammatory proximal colonic mucosa of 10 paired patients. Bisulfite pyrosequencing was used to quantify the methylation of 5 candidate CpG island promoters (NEUROG1, CDX1, miR-1247, CDH1, and CDH13) and LINE1.
Using an unsupervised hierarchical clustering analysis, inflamed rectal mucosa was well separated from mucosa that appeared normal. The CDH1 and CDH13 promoters were significantly associated with patient age (p = 0.04, 0.03, respectively). A similar trend was found between those genes and the duration of disease (CDH1: p = 0.07, CDH13: p = 0.0002, mean of both: p<0.00001). Several positive associations were found between hypermethylation and severe clinical phenotypes (CDX1 and miR-1247 and a refractory phenotype: p = 0.04 and 0.006, respectively. miR-1247 and CDH1 hyper methylation and a more severe Mayo endoscopic subscore: miR-1247: p = 0.0008, CDH1: p = 0.03, mean of both: p = 0.003). When the severe clinical phenotype was defined as having any of five phenotypes (hospitalized more than twice, highest Mayo endoscopic subscore, steroid dependence, refractory, or a history of surgery) miR-1247 hypermethylation was associated with the same phenotype (p = 0.008).
Our data suggest that variability in the methylation status of EMT-related genes is associated with more severe clinical phenotypes in UC.
PMCID: PMC4193736  PMID: 25303049
14.  Risky locations for out-of-hospital cardiopulmonary arrest in a typical urban city 
The aim of this study is to clarify the circumstances including the locations where critical events resulting in out-of-hospital cardiopulmonary arrest (OHCPA) occur.
Materials and Methods:
Subjects of this population-based observational case series study were the clinical records of patients with nontraumatic and nonneck-hanging OHCPA.
Of all 1546 cases, 10.3% occurred in a public place (shop, restaurant, workplace, stations, public house, sports venue, and bus), 8.3% on the street, 73.4% in a private location (victim's home, the homes of the victims’ relatives or friends or cheap bedrooms, where poor homeless people live), and 4.1% in residential institutions. In OHCPA occurring in private locations, the frequency of asystole was higher and the outcome was poorer than in other locations. A total of 181 OHCPA cases (11.7%) took place in the lavatory and 166 (10.7%) in the bathroom; of these, only 7 (3.9% of OHCPA in the lavatory) and none in the bath room achieved good outcomes. The frequencies of shockable initial rhythm occurring in the lavatory and in bath room were 3.7% and 1.1% (lower than in other locations, P = 0.011 and 0.002), and cardiac etiology in OHCPA occurring in these locations were 46.7% and 78.4% (the latter higher than in other locations, P < 0.001).
An unignorable population suffered from OHCPA in private locations, particularly in the lavatory and bathroom; their initial rhythm was usually asystole and their outcomes were poor, despite the high frequency of cardiac etiology in the bathroom. We should try to treat OHCPA victims and to prevent occurrence of OHCPA in these risky spaces by considering their specific conditions.
PMCID: PMC4231265  PMID: 25400390
Bathroom; lavatory; OHCPA; out-of-hospital cardiopulmonary arrest; the relation between outcome and location
15.  Unusual biochemical features and follicular dendritic cell expression of human Fcα/μ receptor 
European journal of immunology  2007;37(12):3540-3550.
The Fc receptor for IgA and IgM (Fcα/μR) is of particular interest because it can bind antibodies of both IgM and IgA isotypes and thus may play a pivotal role in systemic and mucosal immunity. Using IgM and IgA ligands and newly generated Fcα/μR specific monoclonal antibodies we have defined biochemical features and cellular distribution of the human Fcα/μR. Both recombinant and native forms of human Fcα/μR are expressed on the cell surface as remarkably stable homodimeric transmembrane glycoproteins that can bind specifically polymeric IgM or IgA. The only human B cells to express Fcα/μR, albeit at very low levels, are found in the pre-germinal center subpopulation defined by the IgD+/CD38+ phenotype. Hence the expression pattern differs from that of the mouse wherein Fcα/μR is expressed by both circulating and resident B cell populations. Significantly, the predominant cell type expressing the Fcα/μR in humans is the follicular dendritic cell (FDC) of germinal centers. The Fcα/μR may thus function in antigen presentation and B cell selection in the germinal center response.
PMCID: PMC4160165  PMID: 18000956
B lymphocytes; Fc receptors; follicular dendritic cells; germinal centers; IgM and IgA receptors
16.  Reduced Renal Clearance of Cefotaxime in Asians with a Low-Frequency Polymorphism of OAT3 (SLC22A8) 
Journal of pharmaceutical sciences  2013;102(9):3451-3457.
Organic anion transporter 3 (OAT3, SLC22A8), a transporter expressed on the basolateral membrane of the proximal tubule, plays a critical role in the renal excretion of organic anions including many therapeutic drugs. The goal of this study was to evaluate the in vivo effects of the OAT3-Ile305Phe variant (rs11568482), present at 3.5% allele frequency in Asians, on drug disposition with a focus on cefotaxime, a cephalosporin antibiotic. In HEK293- Flp-In cells, the OAT3-Ile305Phe variant had a lower maximum cefotaxime transport activity, Vmax, [159 ± 3 nmol*(mg protein)−1/min (mean ± SD)] compared with the reference OAT3 [305 ± 28 nmol*(mg protein)−1/min, (mean ± SD), p < 0.01], whereas the Michaelis-Menten constant values (Km) did not differ. In healthy volunteers, we found volunteers that were heterozygous for the Ile305Phe variant and had a significantly lower cefotaxime renal clearance (CLR; mean ± SD: 84.8 ± 32.1 mL/min, n = 5) compared with volunteers that were homozygous for the reference allele (158 ± 44.1 mL/min, n = 10; p = 0.006). Furthermore, the net secretory component of cefotaxime renal clearance (CLsec) was reduced in volunteers heterozygous for the variant allele [33.3 ± 31.8 mL/min (mean ± SD)] compared with volunteers homozygous for the OAT3 reference allele [97.0 ± 42.2 mL/min (mean ± SD), p = 0.01]. In summary, our study suggests that a low-frequency reduced-function polymorphism of OAT3 associates with reduced cefotaxime CLR and CLsec.
PMCID: PMC4151246  PMID: 23649425
Organic anion transporter; cefotaxime; tubular secretion; OAT3; polymorphisms; pharmacogenomics; pharmacokinetics; renal clearance
17.  Relationship between the Amount of Bitter Substances Adsorbed onto Lipid/Polymer Membrane and the Electric Response of Taste Sensors 
Sensors (Basel, Switzerland)  2014;14(9):16274-16286.
The bitterness of bitter substances can be measured by the change in the membrane electric potential caused by adsorption (CPA) using a taste sensor (electronic tongue). In this study, we examined the relationship between the CPA value due to an acidic bitter substance and the amount of the bitter substance adsorbed onto lipid/polymer membranes, which contain different lipid contents, used in the taste sensor. We used iso-α-acid which is an acidic bitter substance found in several foods and beverages. The amount of adsorbed iso-α-acid, which was determined by spectroscopy, showed a maximum at the lipid concentration 0.1 wt % of the membrane, and the same phenomenon was observed for the CPA value. At the higher lipid concentration, however, the amount adsorbed decreased and then remained constant, while the CPA value decreased monotonically to zero. This constant adsorption amount was observed when the membrane potential in the reference solution did not change with increasing lipid concentration. The decrease in CPA value in spite of the constant adsorption amount is caused by a decrease in the sensitivity of the membrane as the surface charge density increases. The reason why the peaks appeared in both the CPA value and adsorption amount is based on the contradictory adsorption properties of iso-α-acid. The increasing charged lipid concentration of the membrane causes an increasing electrostatic attractive interaction between iso-α-acid and the membrane, but simultaneously causes a decreasing hydrophobic interaction that results in decreasing adsorption of iso-α-acid, which also has hydrophobic properties, onto the membrane. Estimates of the amount of adsorption suggest that iso-α-acid molecules are adsorbed onto both the surface and interior of the membrane.
PMCID: PMC4208174  PMID: 25184491
taste sensor; electronic tongue; global selectivity; CPA measurement; adsorption amount
18.  Histone deacetylase inhibitory effect of Brazilian propolis and its association with the antitumor effect in Neuro2a cells 
Food Science & Nutrition  2014;2(5):565-570.
Propolis is a resinous product produced by honey bees and is known to have antitumor functions. On the other hand, histone deacetylase (Hdac) inhibitors have recently attracted attention for their antitumor effects. In this study, we examined whether Brazilian green propolis has an Hdac inhibitory activity and its contribution on antitumor effects. By in vitro Hdac activity assay, Brazilian propolis extract (BPE) significantly inhibited the enzyme activity. Actually, BPE treatment increased the intracellular histone acetylation in Neuro2a cells. Regarding antitumor effect in Neuro2a cells, BPE treatment significantly decreased cell viability. An Hdac activator theophylline significantly attenuated the effect. Then, we analyzed whether the decreasing effect on cell number was caused by cell death or growth retardation. By live/dead cell staining, BPE treatment significantly increased the dead cell number. By cell cycle analysis, BPE treatment retarded cell cycle at the M-phase. Both of these cellular effects were suppressed by addition of theophylline. These data indicate that BPE induced both cell death and growth retardation via Hdac inhibitory activity. We demonstrated that Brazilian propolis bears regulatory functions on histone acetylation via Hdac inhibition, and the effect contributes antitumor functions. Our data suggest that intake of Brazilian propolis shows preventing effects against cancer.
PMCID: PMC4237486  PMID: 25473514
Brazilian propolis; cell cycle arrest; cell death; Hdac; Neuro2a
19.  Pancreaticoduodenectomy with preservation of gastric tube blood flow after esophagectomy: Report of a case 
•We performed a pancreaticoduodenectomy in which the gastroduodenal artery was preserved.•The patient who had previously undergone esophagectomy, was diagnosed as middle bile duct cancer.•In order to prevent gastric tube ischemia, the gastroduodenal artery and gastroepiploic artery had to be preserved.
During pancreaticoduodenectomy (PD), the gastroduodenal artery (GDA) is commonly divided. In this study, we described the clinical features of PD in which the GDA was preserved in order to avoid gastric tube ischemia in a patient who had previously undergone esophagectomy.
A 70-year-old man had previously undergone esophagectomy. Esophagectomy and gastric tube reconstruction were performed 10 years earlier due to superior thoracic esophageal cancer. The patient was referred to our hospital for the treatment of obstructive jaundice and was diagnosed with middle bile duct cancer. We performed PD and preserved the GDA. The postoperative course was uneventful, and the gastric tube continued functioning well.
In a patient with a prior esophagectomy and gastric tube reconstruction, the blood flow to the gastric tube is supplied only by the GDA via the right gastroepiploic artery (RGEA). Therefore, we carefully chose a technique that would preserve the GDA and avoid gastric tube ischemia. Oncologically, this procedure may be debatable because the efficiency of lymph node dissection along the GDA and RGEA may be compromised. PD involving GDA preservation in common bile duct (CBD) cancer may be acceptable because the CBD is behind the pancreatic head, and the CBD lymph flows into the para-aorta lymph nodes behind the pancreas.
This procedure is suitable for patients who have previously undergone esophagectomy and this procedure prevents digestive function disorders. Using this method, preoperative angiographic assessment and meticulous surgical technique may lead to successful outcomes.
PMCID: PMC4189085  PMID: 25240214
Bile duct cancer; Pancreaticoduodenectomy; Gastroduodenal artery; Esophagectomy; Gastric tube
20.  Change in DNA Methylation Patterns of SLC6A4 Gene in the Gastric Mucosa in Functional Dyspepsia 
PLoS ONE  2014;9(8):e105565.
The neurochemical serotonin (5-HT) is an important signaling molecule in the gastrointestinal motor and sensory functions. A key regulator of 5-HT levels is the transmembrane serotonin transporter (5-HTT; SLC6A4) that governs the reuptake of 5-HT. Recent studies have indicated 5-HTT expression may be regulated by epigenetic mechanisms. We investigated DNA methylation status of SLC6A4 gene in the gastric mucosa from functional dyspepsia (FD) because of their potential role in dyspeptic symptoms.
Endoscopic gastric biopsies were obtained from 78 subjects with no upper abdominal symptoms and 79 patients with FD. Bisulfite Pyrosequencing was carried out to determine the methylation status of promoter CpG islands (PCGIs), promoter non-CpG islands (PNCGIs) and gene body non-CpG islands (NPNCGIs) in the SLC6A4 gene. Gene expression was examined by real-time PCR.
In overall, methylation level of PCGIs was significantly lower in FD compared to control subjects (p = 0.04). On the other hand, methylation level of NPNCGIs was significantly higher in FD compared to control subjects (p = 0.03). Lower methylation level in PNCGIs was highlighted in the patients with PDS (p = 0.01), while higher methylation level in NPNCGIs was more prominent in the patients with EPS (p = 0.017). Methylation levels of PCGIs and PNCGIs were inversely correlated, while methylation levels of NPNCGIs was positively correlated with SLC6A4 mRNA levels in FD patients.
Our data suggest that change in DNA methylation pattern of SLC6A4 in the gastric mucosa may have a role for developing FD. A role of epigenetics for developing FD needs to be further evaluated.
PMCID: PMC4141787  PMID: 25148529
21.  Risk Factors and Surgical Refinements of Postresective Mandibular Reconstruction: A Retrospective Study 
Plastic Surgery International  2014;2014:893746.
Background. Postresective mandibular reconstruction is common in cases of oral and mandibular tumors. However, complications such as infection, plate exposure, or plate fracture can occur. We identified several significant risk factors of complications after reconstructive surgery and compared the effectiveness of different surgical techniques for reducing the incidence of complications. Methods. This study is a retrospective analysis of 28 oromandibular cancer cases that required reconstructive surgery between January 1999 and December 2011 at Kobe University Graduate School of Medicine in Japan. All cases were classified using Hashikawa's CAT and Eichner's classification methods. Then, we determined whether these classifications and different treatment or surgical methods were significantly related to complications. Results. Complications after mandibular reconstruction occurred in 10/28 patients (36%). Specifically, five patients had plate fractures, four had plate exposures, and one had an infection. Radiation therapy and closure without any flaps were significantly related to infection or plate exposure. The wrap-around technique of securing reconstruction plates was used in 14 cases, whereas the run-through technique was used in two cases. Conclusions. The success of mandibular reconstruction depends on both mechanical and biological factors, such as the location of defects, presence of occlusions, and the amount of vascularization of the flap.
PMCID: PMC4150385  PMID: 25228992
22.  Intravascular Optical Coherence Tomography Detection of Atherosclerosis and Inflammation in Murine Aorta 
The goal of this study was to evaluate the feasibility of imaging the aorta of apolipoprotein E–deficient (ApoE−/−) mice for the detection of atherosclerosis and macrophages using optical coherence tomography (OCT) compared with histology.
Methods and Results
Atherosclerosis was induced by high-fat diet in 7-week-old ApoE−/− mice for 10 (n=7) and 22 (n=7) weeks. Nine-week-old ApoE−/− mice (n=7) fed a standard chow diet were used as controls. OCT images of a 10-mm descending aorta in situ were performed in 4 mice for each, and plaque and macrophages were determined at 0.5-mm intervals. Automated detection and quantification of macrophages were performed independently using a customized algorithm. Coregistered histological cross-sections were stained with hematoxylin-eosin, Mac-3, and von Kossa. Three mice in each group had en face OCT imaging to detect macrophages, which were compared with lipid-positive area with Sudan IV. OCT images were successfully acquired in all mice. OCT and histology were able to discriminate macrophages and plaque among the 3 groups and showed excellent correlation for (1) visual detection of plaque (r=0.98) and macrophages (r=0.93), (2) automated detection and quantification of macrophages by OCT versus Mac-3-positive area (r=0.92), and (3) en face OCT detection of macrophages versus Sudan IV–positive area (r=0.92).
Murine intra-aortic OCT is feasible and shows excellent correlation with histology for detection of atherosclerotic plaque and macrophages.
PMCID: PMC4091044  PMID: 22308042
atherosclerosis; macrophages; optical coherence tomography
23.  Comparison of the Invasiveness Between Reduced-Port Laparoscopy-Assisted Distal Gastrectomy and Conventional Laparoscopy-Assisted Distal Gastrectomy 
International Surgery  2013;98(3):247-253.
It is unknown whether reduced-port gastrectomy has a less invasive nature than conventional laparoscopy-assisted distal gastrectomy (C-LADG). So we compared 30 cases of dual-port laparoscopy-assisted distal gastrectomy (DP-LADG; using an umbilical port plus a right flank 5-mm port) as a reduced-port gastrectomy with 30 cases of C-LADG alternately performed by a single surgeon. No significant differences were observed in blood loss, intraoperative complications, the number of dissected lymph nodes, postoperative complications, the day of first defecation, analgesic agents required, changes in body temperature, heart rate, white blood cell count, serum albumin level, or lymphocyte count between the 2 groups. The amounts of oral intake in the DP-LADG group were significantly higher on postoperative days 9 and 10. We concluded that the amount of oral intake in the DP-LADG group was superior to that in the C-LADG group; however, no other evidence of DP-LADG being less invasive than C-LADG was obtained.
PMCID: PMC3756848  PMID: 23971779
Laparoscopy; Gastrectomy; Single incision; Single port; Reduced port
24.  Rationale and design of LUX-Head & Neck 1: a randomised, Phase III trial of afatinib versus methotrexate in patients with recurrent and/or metastatic head and neck squamous cell carcinoma who progressed after platinum-based therapy 
BMC Cancer  2014;14:473.
Patients with recurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) receiving platinum-based chemotherapy as their first-line treatment have a dismal prognosis, with a median overall survival (OS) of ~7 months. Methotrexate is sometimes used following platinum failure or in patients not fit enough for platinum therapy, but this agent has not demonstrated any OS improvement. Targeted therapies are a novel approach, with the EGFR-targeting monoclonal antibody cetuximab (plus platinum-based chemotherapy) approved in the US and Europe in the first-line R/M setting, and as monotherapy following platinum failure in the US. However, there is still a high unmet medical need for new treatments that improve outcomes in the second-line R/M setting following failure on first-line platinum-containing regimens. Afatinib, an irreversible ErbB family blocker, was recently approved for the first-line treatment of EGFR mutation-positive metastatic non-small cell lung cancer. Afatinib has also shown clinical activity similar to cetuximab in a Phase II proof-of-concept HNSCC trial. Based on these observations, the Phase III, LUX-Head & Neck 1 study is evaluating afatinib versus methotrexate in R/M HNSCC patients following progression on platinum-based chemotherapy in the R/M setting.
Patients with progressive disease after one first-line platinum-based chemotherapy are randomised 2:1 to oral afatinib (starting dose 40 mg once daily) or IV methotrexate (starting dose 40 mg/m2 once weekly) administered as monotherapy with best supportive care until progression or intolerable adverse events. Efficacy of afatinib versus methotrexate will be assessed in terms of progression-free survival (primary endpoint). Disease progression will be evaluated according to RECIST v1.1 by investigator and independent central review. Secondary endpoints include OS, tumour response and safety. Health-related quality of life and biomarker assessments will also be performed.
If the LUX-Head & Neck 1 trial meets its primary endpoint, it will demonstrate the ability of afatinib to elicit an improved treatment benefit versus a commonly used chemotherapy agent in the second-line treatment of R/M HNSCC patients who have failed on first-line platinum-based therapy, confirm the clinical efficacy of afatinib observed in the Phase II proof-of-concept study, and establish a new standard of care for this patient population.
PMCID: PMC4079914  PMID: 24973959
Afatinib; Methotrexate; Head and neck; Phase III; Recurrent; Metastatic
25.  PPARβ/δ activation of CD300a controls intestinal immunity 
Scientific Reports  2014;4:5412.
Macrophages are important for maintaining intestinal immune homeostasis. Here, we show that PPARβ/δ (peroxisome proliferator-activated receptor β/δ) directly regulates CD300a in macrophages that express the immunoreceptor tyrosine based-inhibitory motif (ITIM)-containing receptor. In mice lacking CD300a, high-fat diet (HFD) causes chronic intestinal inflammation with low numbers of intestinal lymph capillaries and dramatically expanded mesenteric lymph nodes. As a result, these mice exhibit triglyceride malabsorption and reduced body weight gain on HFD. Peritoneal macrophages from Cd300a−/− mice on HFD are classically M1 activated. Activation of toll-like receptor 4 (TLR4)/MyD88 signaling by lipopolysaccharide (LPS) results in prolonged IL-6 secretion in Cd300a−/− macrophages. Bone marrow transplantation confirmed that the phenotype originates from CD300a deficiency in leucocytes. These results identify CD300a-mediated inhibitory signaling in macrophages as a critical regulator of intestinal immune homeostasis.
PMCID: PMC4067692  PMID: 24958459

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