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1.  Huge epithelioid malignant peripheral nerve sheath tumor in the left axilla: a case report 
Surgical Case Reports  2015;1(1):64.
This report describes a patient with a rare huge epithelioid malignant peripheral nerve sheath tumor (MPNST) in the left axilla. A male in his 70s was admitted to our hospital for evaluation of a growing tumor in his left axilla. The tumor was solid and immovable. Examination of a biopsy specimen resulted in a diagnosis of epithelioid MPNST. Two weeks after the biopsy was performed, the tumor grew to 20 cm and became painful, and the patient was unable to feel pressure on his upper arm. Immediately before surgery to remove the tumor, computed tomography suggested the presence of lung metastases. The patient and his family were informed of his disease state, and they elected surgical treatment to ease the symptoms associated with tumor enlargement. Systemic metastases appeared soon after the surgery, and the patient died within 11 weeks. Comparative genomic hybridization (CGH) analysis showed that this tumor was chromosomally unstable, with impairments in gene expression.
PMCID: PMC4560135  PMID: 26366361
Epithelioid malignant peripheral nerve sheath tumor; Extended resection; Chromosomally unstable
2.  Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2 
The Journal of Experimental Medicine  2003;198(7):1035-1042.
Toll-like receptors (TLRs) are innate recognition molecules for microbial products, but their direct interactions with corresponding ligands remain unclarified. LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4. Although TLR4-MD-2 recognizes LPS, little is known about the physical interaction between LPS and TLR4-MD-2. Here, we demonstrate cell surface LPS–TLR4-MD-2 complexes. CD14 greatly enhances the formation of LPS–TLR4-MD-2 complexes, but is not coprecipitated with LPS–TLR4-MD-2 complexes, suggesting a role for CD14 in LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. A tentative dissociation constant (Kd) for LPS–TLR4-MD-2 complexes was ∼3 nM, which is ∼10–20 times lower than the reported Kd for LPS–MD-2 or LPS–CD14. The presence of detergent disrupts LPS interaction with CD14 but not with TLR4-MD-2. E5531, a lipid A antagonist developed for therapeutic intervention of endotoxin shock, blocks LPS interaction with TLR4-MD-2 at a concentration 100 times lower than that required for blocking LPS interaction with CD14. These results reveal direct LPS interaction with cell surface TLR4-MD-2 that is distinct from that with MD-2 or CD14.
PMCID: PMC2194215  PMID: 14517279
innate immunity; cell surface molecule; activation; macrophage
3.  Sequence dependence of the hyperthermic potentiation of carboplatin-induced cytotoxicity and intracellular platinum accumulation in HeLa cells. 
British Journal of Cancer  1993;68(2):259-263.
We have examined the enhancement of cytotoxic effects of cis-diammine-1,1-cyclobutane dicarboxylate platinum(II) (carboplatin) by hyperthermia in HeLa cells using different regimes of timing and sequence. The results were compared with those obtained with cis-diamminedichloroplatinum(II) (cisplatin). We found that cisplatin simultaneously combined with heat was the most cytotoxic toward HeLa cells of the various timing and sequencing conditions studied. On the other hand, for carboplatin, drug treatment immediately following or during heat exposure showed the greatest effect. Intracellular platinum concentration in HeLa cells treated with heat before carboplatin showed a 2.75-fold increase over that in cells treated with the drug alone. The ratios for carboplatin given before, or during heating, were 0.67 and 1.42 respectively. Simultaneous exposure of cells to cisplatin and heat led to a 1.64-fold enhancement in cisplatin accumulation, compared to 0.92- and 1.24-fold increase for cells treated with cisplatin before and after heat respectively. Although each drug exposure prior to heat was less cytotoxic toward HeLa cells than any other heat/drug combination sequences, the platinum concentration was less than seen with each drug alone. Even though heat exposure prior to and during carboplatin showed a similar toxicity, platinum concentration in cells treated with heat prior to carboplatin was higher than that in cells treated with heat and carboplatin simultaneously. Thus, increased cytotoxicity cannot always be explained on the basis of intracellular platinum concentration. It is clear however that, differing from cisplatin, exposure of cells to heat prior to or during carboplatin administration results in the greatest cell kill.
PMCID: PMC1968573  PMID: 8347479
4.  Pleuropneumonectomy for a large thymoma with multiple pleural dissemination using median sternotomy followed by posterolateral thoracotomy 
Surgical Case Reports  2015;1(1):75.
We present 2 cases of a large thymoma with invasion to the hilum of the lung and pleural dissemination. Case 1: a 47-year-old woman was diagnosed with a type B3 thymoma with abundant left pleural effusion and multiple pleural masses, Masaoka stage IVa. A radical resection was planned after chemical pleurodesis and systemic chemotherapy. The left main pulmonary artery and left upper and inferior veins were dissected and resected in the pericardium, while the left main bronchus was cut behind the pericardium through a median sternotomy. Next, the median incision was closed and a left posterolateral thoracotomy was made, thus allowing the pleuropneumonectomy to be safely performed. Case 2: a 47-year-old woman was diagnosed with a type B3 thymoma with lymph node swelling and multiple pleural masses, indicating Masaoka stage IVb. Following induction chemotherapy, a thymothymectomy combined with a right pleuropneumonectomy was performed under a median sternotomy followed by a right posterolateral thoracotomy. The left brachiocephalic vein (BCV) was reconstructed with a ringed polytetrafluoroethylene (PTFE) graft, followed by resection of the right BCV. Next, the right main pulmonary artery and right upper and inferior veins were resected in the pericardium, and the right main bronchus was cut behind the pericardium, followed by reconstruction of the right BCV. Finally, the median incision was closed and a right posterolateral thoracotomy was made, thus allowing performance of a safe pleuropneumonectomy. The median sternotomy allowed safe dissection of pulmonary vessels surrounding the hilum of the lung and, in combination with a posterolateral thoracotomy, was required for performing a pleuropneumonectomy in patients with a huge thymoma with pleural dissemination.
Electronic supplementary material
The online version of this article (doi:10.1186/s40792-015-0071-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4560151  PMID: 26366371
Thymoma; Pleuropneumonectomy; Pleural dissemination; Multimodal treatment
5.  Increased rate of death related to presence of viremia among hepatitis C virus antibody-positive subjects in a community-based cohort study 
Hepatology (Baltimore, Md.)  2009;50(2):393-399.
The overall mortality of hepatitis C virus (HCV)-infected patients has not been fully elucidated. The aim of this study was to analyze mortality in subjects positive for antibody to HCV (anti-HCV) in a community-based, prospective cohort study conducted in an HCV hyperendemic area of Japan. During a 10-year period beginning in 1995, 1,125 anti-HCV seropositive residents of Town C were enrolled into the study and followed for mortality through 2005. Cause of death was assessed by death certificates. Subjects with detectable HCV core antigen (HCVcAg) or HCV RNA were considered as having hepatitis C viremia and were classified as HCV carriers; subjects who were negative for both HCVcAg and HCV RNA (i.e., viremia-negative) were considered as having had a prior HCV infection and were classified as HCV noncarriers. Among the anti-HCV-positive subjects included in the analysis, 758 (67.4%) were HCV carriers, and 367 were noncarriers. A total of 231 deaths occurred in these subjects over a mean follow-up of 8.2 years: 176 deaths in the HCV carrier group and 55 in the noncarrier group. The overall mortality rate was higher in HCV carriers than in noncarriers, adjusted for age and gender (hazard ratio [HR], 1.53; 95% confidence interval [CI], 1.13–2.07). Although liver-related deaths occurred more frequently among the HCV carriers (HR, 5.94; 95% CI, 2.58–13.7), the rates of other causes of death did not differ between HCV carriers and noncarriers. Among HCV carriers, a higher level of HCVcAg (≥100 pg/ml) and persistently elevated alanine aminotransferase levels were important predictors of liver-related mortality.
The presence of viremia increases the rate of mortality, primarily due to liver-related death, among anti-HCV seropositive persons in Japan.
PMCID: PMC4551403  PMID: 19585614
HCV; cause of death; cohort study; hyperendemic area; hepatocellular carcinoma
6.  Characterization of Novel Transcripts of Human Papillomavirus Type 16 Using Cap Analysis Gene Expression Technology 
Journal of Virology  2014;89(4):2448-2452.
We have performed cap-analysis gene expression (CAGE) sequencing to identify the regulatory networks that orchestrate genome-wide transcription in human papillomavirus type 16 (HPV16)-positive cervical cell lines of different grades: W12E, SiHa, and CaSki. Additionally, a cervical intraepithelial neoplasia grade 1 (CIN1) lesion was assessed for identifying the transcriptome expression profile. Here we have precisely identified a novel antisense noncoding viral transcript in HPV16. In conclusion, CAGE sequencing should pave the way for understanding a diversity of viral transcript expression.
PMCID: PMC4338893  PMID: 25505068
7.  Virulence characterization of Campylobacter jejuni isolated from resident wild birds in Tokachi area, Japan 
The prevalence of Campylobacter jejuni in wild birds is a potential hazard for human and animal health. The aim of this study was to establish the prevalence of C. jejuni in wild birds in Tokachi area, Hokkaido, Japan and investigate their virulence in vitro. In total, 173 cloacal swabs from individual wild birds were collected for the detection of Campylobacter spp. Thirty four samples (19.7%) were positive for Campylobacter of which 94.1% (32/34 samples) were C. jejuni. Additionally, one C. coli and one C. fetus were isolated. Seven C. jejuni isolates (one from crows and the other from pigeons) had important virulence genes including all three CDT genes (cdtA, cdtB and cdtC) and flaA, flaB, ciaB and cadF, and the other isolates were lacking cdtA gene. Further studies on in vitro virulence-associated phenotypes, such as motility assay on soft agar and invasion assay in Caco-2 cells, were performed. The wild bird C. jejuni isolates adhered and invaded human cells. Although the numbers of viable intracellular bacteria of wild bird isolates were lower than a type strain NCTC11168, they persisted at 48-hr and underwent replication in host cells.
PMCID: PMC4565820  PMID: 25843040
Campylobacter; Japan; virulence; wild bird
8.  Utilization of Platelet-Rich Plasma for a Fistula With Subcutaneous Cavity Following Septic Bursitis: A Case Report 
Eplasty  2015;15:e31.
In platelet-rich plasma (PRP) therapy, various growth factors and cytokines released the α-granules contained in platelets after activation can potentially enhance wound healing by delivering. We report a patient in whom treatment with PRP, prepared using a syringe-centrifugation-system PRP kit (KYOCERA Medical PRP Kit), for a fistula following bursitis of the lateral malleolus, which could not be healed with conventional wound therapy, led to successful healing. A 58-year-old man was on dialysis for type II diabetes and chronic renal failure. In the left lateral malleolus, septic bursitis developed, leading to a refractory fistula with a subcutaneous cavity measuring 4 × 3 cm, which persisted for more than 2 months. Platelet-rich plasma was prepared using the KYOCERA Medical PRP Kit (KYOCERA Medical Corporation, Osaka, Japan) and infused into the cavity twice to close it. After this procedure, the cavity size reduced, but the orifice and subcutaneous cavity were not closed. Therefore, additional PRP therapy was conducted after 10 weeks of the first PRP session. Complete closure was achieved 13 weeks after the first PRP therapy. In the present case, PRP was prepared using the KYOCERA Medical PRP Kit, and wound healing of a fistula with subcutaneous cavity following bursitis of the lateral malleolus was successfully cured. The KYOCERA Medical PRP Kit was useful, because PRP could be prepared simply and inexpensively using the syringe-centrifugation system.
PMCID: PMC4516017  PMID: 26221200
platelet-rich plasma; wound healing; fistula; subcutaneous cavity; septic bursitis
9.  Transplantation of Horseshoe Kidney from Living, Genetically Unrelated Donor 
We report a case of renal transplantation using a horseshoe kidney from a living, genetically unrelated donor. The recipient was a 60-year-old man with diabetic nephropathy, and the donor was the 63-year-old wife of the recipient with a horseshoe kidney free of complications. Computed tomography showed two renal arteries and one renal vein on the left side, and the isthmus was perfused by several accessory arteries and veins. To demarcate the boundary of the isthmus, the left renal artery was ligated and cannulated for in situ perfusion. Furthermore, the isthmus was clamped, and the boundary of the isthmus was confirmed. The kidney was divided at the left margin of the perfused boundary. The cut ends of the isthmus were closed by sutures. The left kidney was transplanted into the right iliac fossa of the recipient. Asymptomatic fluid collection occurred on the cut surface at the isthmus of the donor, and this fluid decreased in due course. On the other hand, the recipient experienced no surgical complication or rejection, while maintaining serum creatinine levels of 2.00–2.20 mg/dL over a 22-month follow-up period. Horseshoe kidneys may be used for transplantation in selected cases after a detailed preoperative evaluation.
PMCID: PMC4512584  PMID: 26240774
10.  Exploratory clinical trial of combination wound therapy with a gelatin sheet and platelet-rich plasma in patients with chronic skin ulcers: study protocol 
BMJ Open  2015;5(5):e007733.
Chronic skin ulcers, such as diabetic ulcers, venous leg ulcers and pressure ulcers, are intractable and increasing in prevalence, representing a costly problem in healthcare. We developed a combination therapy with a gelatin sheet, capable of providing sustained release of platelet-rich plasma (PRP). The objective of this study is to investigate the safety and efficacy of autologous PRP covered with a hydrocolloid dressing and PRP covered with a gelatin sheet in the treatment of chronic skin ulcers.
Methods and analysis
Thirty patients with chronic skin ulcers who have not healed with conventional therapy for at least 1 month are being recruited. The patients will receive PRP after debridement, and the wounds will be covered with a hydrocolloid dressing or gelatin sheet. The efficacy will be evaluated according to the time from the beginning of PRP application to secondary healing or the day on which wound closure is achieved with a relatively simple surgical procedure, such as skin grafting or suturing. All patients will be followed up until 6 weeks after application to observe adverse events related to the application of PRP and the dressings. This study was designed to address and compare the safety and efficacy of PRP covered with a hydrocolloid dressing versus a gelatin sheet. If successful, this combination therapy may be an alternative to bioengineered skin substitutes containing living cells and lead to substantial progress in the management of chronic skin ulcers.
Ethics and dissemination
The study protocol was approved by the Institutional Review Board of Kansai Medical University (KMU Number 0649-1, 4 August 2014: V.1.0). The findings of this trial will be disseminated through peer-reviewed journals, and national and international scientific meetings as well as to the patients.
Trial registration number
PMCID: PMC4431137  PMID: 25968005
11.  Association Between Visit-to-Visit Variability in Blood Pressure and Cardiovascular Events in Hypertensive Patients After Successful Percutaneous Coronary Intervention 
Visit-to-visit variability (VVV) in blood pressure (BP) in addition to high BP has been shown to be a strong predictor of coronary events and stroke. Therefore, we investigated the associations between VVV in BP or BP levels and cardiovascular events after successful percutaneous coronary intervention (PCI).
We enrolled 176 hypertensive patients who had undergone successful PCI and who had four clinic visits to measure BP until follow-up coronary angiography (CAG) at 6 - 9 months after PCI. The patients were divided into those with acute coronary syndrome (ACS group; n = 50) and those with stable angina pectoris (SAP group; n = 126). We determined VVV in BP expressed as the standard deviation (SD) of average BP, average, and the maximum and minimum BP during the follow-up period. Major adverse cardiovascular events (MACEs) (myocardial infarction (MI), target lesion revascularization (TLR) and all-cause death) were also analyzed.
There were no significant differences in VVV in BP, average BP or maximum or minimum BP between the patients with and without MACE in all patients, the ACS and SAP groups. Interestingly, in the ACS group, VVV in SBP and maximum SBP in patients with MI were significantly higher than those in patients without MI. The cut-off levels for VVV in BP and maximum SBP that gave the greatest sensitivity and specificity for MI in the ACS group were 15.1 and 138 mm Hg, respectively.
Higher VVV in SBP and maximum SBP in patients with ACS after successful PCI were associated with the onset of MI.
PMCID: PMC4432897  PMID: 26015820
Visit-to-visit variability in blood pressure; Percutaneous coronary intervention; Major adverse cardiovascular events; Myocardial infarction; Acute coronary syndrome
12.  Whole-Genome Sequences of Eight Campylobacter jejuni Isolates from Wild Birds 
Genome Announcements  2015;3(2):e00315-15.
We present here the draft genome sequences of 8 Campylobacter jejuni strains isolated from wild birds. The strains were initially isolated from swabs taken from resident wild birds in the Tokachi area of Japan. The genome sizes range from 1.65 to 1.77 Mbp.
PMCID: PMC4408334  PMID: 25908133
13.  Review of the diagnosis and management of gastrointestinal bezoars 
The formation of a bezoar is a relatively infrequent disorder that affects the gastrointestinal system. Bezoars are mainly classified into four types depending on the material constituting the indigestible mass of the bezoar: phytobezoars, trichobezoars, pharmacobezoars, and lactobezoars. Gastric bezoars often cause ulcerative lesions in the stomach and subsequent bleeding, whereas small intestinal bezoars present with small bowel obstruction and ileus. A number of articles have emphasized the usefulness of Coca-Cola® administration for the dissolution of phytobezoars. However, persimmon phytobezoars may be resistant to such dissolution treatment because of their harder consistency compared to other types of phytobezoars. Better understanding of the etiology and epidemiology of each type of bezoar will facilitate prompt diagnosis and management. Here we provide an overview of the prevalence, classification, predisposing factors, and manifestations of bezoars. Diagnosis and management strategies are also discussed, reviewing mainly our own case series. Recent progress in basic research regarding persimmon phytobezoars is also briefly reviewed.
PMCID: PMC4400622  PMID: 25901212
Bezoars; Gastrointestinal endoscopy; Persimmon phytobezoar; Trichobezoar; Endoscopic removal; Gastric ulcer; Ileus
14.  Determining the relevance of different aspects of formant contours to intelligibility☆ 
Speech communication  2014;59:1-9.
Previous studies have shown that “clear” speech, where the speaker intentionally tries to enunciate, has better intelligibility than “conversational” speech, which is produced in regular conversation. However, conversational and clear speech vary along a number of acoustic dimensions and it is unclear what aspects of clear speech lead to better intelligibility. Previously, Kain et al. [J. Acoust. Soc. Am. 124 (4), 2308–2319 (2008)] showed that a combination of short-term spectra and duration was responsible for the improved intelligibility of one speaker. This study investigates subsets of specific features of short-term spectra including temporal aspects. Similar to Kain’s study, hybrid stimuli were synthesized with a combination of features from clear speech and complementary features from conversational speech to determine which acoustic features cause the improved intelligibility of clear speech. Our results indicate that, although steady-state formant values of tense vowels contributed to the intelligibility of clear speech, neither the steady-state portion nor the formant transition was sufficient to yield comparable intelligibility to that of clear speech. In contrast, when the entire formant contour of conversational speech including the phoneme duration was replaced by that of clear speech, intelligibility was comparable to that of clear speech. It indicated that the combination of formant contour and duration information was relevant to the improved intelligibility of clear speech. The study provides a better understanding of the relevance of different aspects of formant contours to the improved intelligibility of clear speech.
PMCID: PMC4041876  PMID: 24910484
speech intelligibility; vowel perception; speech synthesis
15.  The Rapid Inactivation of Porcine Skin by Applying High Hydrostatic Pressure without Damaging the Extracellular Matrix 
BioMed Research International  2015;2015:587247.
We previously reported that high hydrostatic pressure (HHP) of 200 MPa for 10 minutes could induce cell killing. In this study, we explored whether HHP at 200 MPa or HHP at lower pressure, in combination with hyposmotic distilled water (DW), could inactivate the skin, as well as cultured cells. We investigated the inactivation of porcine skin samples 4 mm in diameter. They were immersed in either a normal saline solution (NSS) or DW, and then were pressurized at 100 and 200 MPa for 5, 10, 30, or 60 min. Next, we explored the inactivation of specimens punched out from the pressurized skin 10 × 2 cm in size. The viability was evaluated using a WST-8 assay and an outgrowth culture. The histology of specimens was analyzed histologically. The mitochondrial activity was inactivated after the pressurization at 200 MPa in both experiments, and no outgrowth was observed after the pressurization at 200 MPa. The arrangement and proportion of the dermal collagen fibers or the elastin fibers were not adversely affected after the pressurization at 200 MPa for up to 60 minutes. This study showed that a HHP at 200 MPa for 10 min could inactivate the skin without damaging the dermal matrix.
PMCID: PMC4387949  PMID: 25879028
16.  Impaired respiratory function in MELAS-induced pluripotent stem cells with high heteroplasmy levels 
FEBS Open Bio  2015;5:219-225.
•We modeled the mitochondrial disease MELAS by generating patient-specific iPS cells.•MELAS-iPS cells show a wide variety of heteroplasmy levels.•MELAS-iPS cells with high heteroplasmy levels showed impaired complex I activity.
Mitochondrial diseases are heterogeneous disorders, caused by mitochondrial dysfunction. Mitochondria are not regulated solely by nuclear genomic DNA but by mitochondrial DNA. It is difficult to develop effective therapies for mitochondrial disease because of the lack of mitochondrial disease models. Mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is one of the major mitochondrial diseases. The aim of this study was to generate MELAS-specific induced pluripotent stem cells (iPSCs) and to demonstrate that MELAS-iPSCs can be models for mitochondrial disease. We successfully established iPSCs from the primary MELAS-fibroblasts carrying 77.7% of m.3243A>G heteroplasmy. MELAS-iPSC lines ranged from 3.6% to 99.4% of m.3243A>G heteroplasmy levels. The enzymatic activities of mitochondrial respiratory complexes indicated that MELAS-iPSC-derived fibroblasts with high heteroplasmy levels showed a deficiency of complex I activity but MELAS-iPSC-derived fibroblasts with low heteroplasmy levels showed normal complex I activity. Our data indicate that MELAS-iPSCs can be models for MELAS but we should carefully select MELAS-iPSCs with appropriate heteroplasmy levels and respiratory functions for mitochondrial disease modeling.
PMCID: PMC4383791  PMID: 25853038
bFGF, basic fibroblast growth factor; EB, embryoid body; ES, embryonic stem; iPSCs, induced pluripotent stem cells; KSR, Knock-out Serum Replacement; MEF, mouse embryonic fibroblast; MELAS, mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes; mtDNA, mitochondrial DNA; OXPHOS, oxidative phosphorylation system; MELAS; iPS cell; Mitochondrial disease; Disease modeling
18.  A Case of Cardiac Metastasis from Uterine Cervical Carcinoma 
Cases of cardiac metastasis from uterine cervical carcinoma are rare. While they are occasionally found on autopsy, antemortem recognition is extremely rare. We confirmed a case of cardiac metastasis from cervical carcinoma antemortem, because we observed a decrease in platelet count during the course of treatment. The patient was a 27-year-old woman diagnosed with stage Ib1 uterine cervical carcinoma. Radical hysterectomy with pelvic lymphadenectomy was performed. Para-aortic lymph node metastasis was detected on positron emission tomography/computed tomography (PET-CT). Adjuvant chemotherapy was started, and most of the metastatic lesions disappeared. Pelvic lymph node recurrence was suspected on PET-CT during continued chemotherapy; therefore, treatment was shifted to radiation therapy. Tumor shrinkage was recognized, and the initial therapy was completed. A noticeable decrease in platelet count was recognized seven months after treatment. Multidetector CT was performed, and an intracardiac tumor was detected. The patient did not desire any further treatment. She died three weeks after the intracardiac tumor was confirmed. Few previous autopsy studies have reported cardiac metastasis from cervical carcinoma. Thus, it is necessary to consider the possibility of cardiac metastasis for patients diagnosed with terminal cervical carcinoma.
PMCID: PMC4355340  PMID: 25830052
19.  Extensive amplification of GI-VII-6, a multidrug resistance genomic island of Salmonella enterica serovar Typhimurium, increases resistance to extended-spectrum cephalosporins 
GI-VII-6 is a chromosomally integrated multidrug resistance genomic island harbored by a specific clone of Salmonella enterica serovar Typhimurium (S.Typhimurium). It contains a gene encoding CMY-2 β-lactamase (blaCMY−2), and therefore contributes to extended-spectrum cephalosporin resistance. To elucidate the significance of GI-VII-6 on adaptive evolution, spontaneous mutants of S. Typhimurium strain L-3553 were selected on plates containing cefotaxime (CTX). The concentrations of CTX were higher than its minimum inhibition concentration to the parent strain. The mutants appeared on the plates containing 12.5 and 25 mg/L CTX at a frequency of 10−6 and 10−8, respectively. No colonies were observed at higher CTX concentrations. The copy number of blaCMY−2 increased up to 85 per genome in the mutants, while the parent strain contains one copy of that in the chromosome. This elevation was accompanied by increased amount of transcription. The blaCMY−2 copy number in the mutants drastically decreased in the absence of antimicrobial selection pressure. Southern hybridization analysis and short-read mapping indicated that the entire 125 kb GI-VII-6 or parts of it were tandemly amplified. GI-VII-6 amplification occurred at its original position, although it also transposed to other locations in the genome in some mutants, including an endogenous plasmid in some of the mutants, leading to the amplification of GI-VII-6 at different loci. Insertion sequences were observed at the junction of the amplified regions in the mutants, suggesting their significant roles in the transposition and amplification. Plasmid copy number in the selected mutants was 1.4 to 4.4 times higher than that of the parent strain. These data suggest that transposition and amplification of the blaCMY−2-containing region, along with the copy number variation of the plasmid, contributed to the extensive amplification of blaCMY−2 and increased resistance to CTX.
PMCID: PMC4322709  PMID: 25713569
resistance; genomic island; insertion sequence; gene duplication and amplification; Salmonella enterica serovar Typhimurium
20.  Dietary small bowel obstruction 
BMJ Case Reports  2013;2013:bcr2012007950.
PMCID: PMC3604329  PMID: 23355579
21.  Culture-independent bacterial community analysis of the salty-fermented fish paste products of Thailand and Laos 
A bacterial community analysis, using a culture-independent method (polymerase chain reaction-denaturing gradient gel electrophoresis), detected 17 species of bacteria including species of the genera Tetragenococcus, Lactobacillus, Pediococcus, Weissella Halanaerobium, Clostridium, and Sphingomonas in a traditional salty-fermented fish paste known as pla-ra or pa-daek in Thailand and Laos, which is used as a storage-stable multi-purpose seasoning. The representative genus of lactic acid bacteria seemed to vary in the 10 products collected from Thailand and Laos. Tetragenococci were common in products from central Thailand and Vientiane in Laos which had salinities of not less than 11% and pH values ranging from 5.6 to 6.1. However, lactobacilli were common in products from northern Thailand which had the lowest salinities (8.3–8.6%) and pH values (4.5–4.8) of all the samples examined. Two Lactobacillus and one Tetragenococcus species were detected in one product from northeastern Thailand containing 10% salt. These results suggest that salinity in pla-ra/pa-daek is an important determinant of the representative genus of lactic acid bacteria such as, Tetragenococcus or Lactobacillus. Additionally, differences in the acidity between these two groups seemed to be related to the production of d-/l-lactic acid in the lactic acid bacteria in each product. This is the first study to report a correlation between bacterial community structure and taste components in pla-ra/pa-daek products from various regions. This scientific work on a traditional fermented food will be useful in helping local producers meet differing consumer preferences in various regions.
PMCID: PMC4405397  PMID: 25918672
fermented fish; pla-ra; pa-daek; lactic acid bacteria; bacterial community; PCR-DGGE
22.  Endothelin‐1 Induces Myofibrillar Disarray and Contractile Vector Variability in Hypertrophic Cardiomyopathy–Induced Pluripotent Stem Cell–Derived Cardiomyocytes 
Despite the accumulating genetic and molecular investigations into hypertrophic cardiomyopathy (HCM), it remains unclear how this condition develops and worsens pathologically and clinically in terms of the genetic–environmental interactions. Establishing a human disease model for HCM would help to elucidate these disease mechanisms; however, cardiomyocytes from patients are not easily obtained for basic research. Patient‐specific induced pluripotent stem cells (iPSCs) potentially hold much promise for deciphering the pathogenesis of HCM. The purpose of this study is to elucidate the interactions between genetic backgrounds and environmental factors involved in the disease progression of HCM.
Methods and Results
We generated iPSCs from 3 patients with HCM and 3 healthy control subjects, and cardiomyocytes were differentiated. The HCM pathological phenotypes were characterized based on morphological properties and high‐speed video imaging. The differences between control and HCM iPSC‐derived cardiomyocytes were mild under baseline conditions in pathological features. To identify candidate disease‐promoting environmental factors, the cardiomyocytes were stimulated by several cardiomyocyte hypertrophy‐promoting factors. Interestingly, endothelin‐1 strongly induced pathological phenotypes such as cardiomyocyte hypertrophy and intracellular myofibrillar disarray in the HCM iPSC‐derived cardiomyocytes. We then reproduced these phenotypes in neonatal cardiomyocytes from the heterozygous Mybpc3‐targeted knock in mice. High‐speed video imaging with motion vector prediction depicted physiological contractile dynamics in the iPSC‐derived cardiomyocytes, which revealed that self‐beating HCM iPSC‐derived single cardiomyocytes stimulated by endothelin‐1 showed variable contractile directions.
Interactions between the patient's genetic backgrounds and the environmental factor endothelin‐1 promote the HCM pathological phenotype and contractile variability in the HCM iPSC‐derived cardiomyocytes.
PMCID: PMC4338713  PMID: 25389285
disease modeling; ET‐1; HCM; iPS cells; MYBPC3
23.  Final safety and efficacy of erlotinib in the phase 4 POLARSTAR surveillance study of 10 708 Japanese patients with non-small-cell lung cancer 
Cancer Science  2014;105(12):1584-1590.
Interstitial lung disease (ILD) occurrence and risk factors were investigated in the Japanese non-small-cell lung cancer, post-marketing, large-scale surveillance study, POLARSTAR. All patients with unresectable, recurrent/advanced non-small-cell lung cancer who were treated with erlotinib in Japan between December 2007 and October 2009 were enrolled. Primary endpoints were patterns of ILD and risk factors for onset of ILD and ILD-related death. Overall survival, progression-free survival, and occurrence of adverse drug reactions were secondary endpoints. Interstitial lung disease was confirmed in 429 (4.3%) patients. Concurrent/previous ILD (hazard ratio, 3.19), emphysema or chronic obstructive pulmonary disease (hazard ratio, 1.86), lung infection (hazard ratio, 1.55), smoking history (hazard ratio, 2.23), and period from initial cancer diagnosis to the start of treatment (<360 days; hazard ratio, 0.58) were identified as significant risk factors for developing ILD by Cox multivariate analysis. Logistic regression analysis identified Eastern Cooperative Oncology Group performance status 2–4 (odds ratio, 2.45 [95% confidence interval, 1.41–4.27]; P = 0.0016), ≤50% remaining normal lung area (odds ratio, 3.12 [1.48–6.58]; P = 0.0029), and concomitant honeycombing with interstitial pneumonia (odds ratio, 6.67 [1.35–32.94]; P = 0.02) as poor prognostic factors for ILD death. Median overall survival was 277 days; median progression-free survival was 67 days. These data confirm the well-characterized safety profile of erlotinib. Interstitial lung disease is still an adverse drug reaction of interest in this population, and these results, including ILD risk factors, give helpful information for treatment selection and monitoring. Erlotinib efficacy was additionally confirmed in this population. (POLARSTAR trial ML21590.)
PMCID: PMC4317960  PMID: 25287435
Erlotinib; interstitial lung disease; Japanese; non-small-cell lung cancer; surveillance
24.  Impact of antibody to hepatitis B core antigen on the clinical course of hepatitis C virus carriers in a hyperendemic area in Japan: a community-based cohort study 
Subjects positive for antibody to hepatitis B core antigen (HBcAb) and negative for hepatitis B surface antigen (HBsAg) are considered to have occult hepatitis B virus (HBV) infection. The aim of this study was to determine the impact of occult HBV infection on aggravation of the clinical course in hepatitis C virus (HCV) carriers.
A prospective cohort study was performed in 400 subjects who were positive for anti-HCV antibody and negative for HBsAg. Among these subjects, 263 were HCV core antigen-positive or HCV RNA-positive (HCV carriers). We examined whether the presence of HBcAb affected the clinical course in these HCV carriers from 1996 to 2005.
The HBcAb-positive rates were 53.6% and 52.6% in HCV carriers and HCV RNA-negative subjects, respectively. There were no differences in the incidence of hepatocellular carcinoma (HCC) and cumulative mortality associated with liver-related death between HCV carriers who were positive and negative for HBcAb. In multivariate analysis, age (≥65 years old) and alanin aminotransferase level (≥31 IU/L) emerged as independent risk factors for HCC development and liver-related death, but the HBcAb status was not a risk factor. In addition, increased serum hepatic fibrosis markers (measured from 2001 to 2004) were not associated with HBcAb status.
In our cohort study, the presence of HBcAb had no impact on HCC development, liver-related death and hepatic fibrosis markers in HCV carriers. Thus, our results indicate that occult HBV infection has no impact on the clinical course in HCV carriers.
PMCID: PMC3710530  PMID: 23413835
antibody to hepatitis B core antigen; occult hepatitis B virus infection; hepatitis C virus; hepatocellular carcinoma; mortality; hepatic fibrosis
25.  Influence of neglecting the curved path of the Achilles tendon on Achilles tendon length change at various ranges of motion 
Physiological Reports  2014;2(10):e12176.
Achilles tendon length has been measured using a straight‐line model. However, this model is associated with a greater measurement error compared with a curved‐line model. Therefore, we examined the influence of neglecting the curved path of the Achilles tendon on its length change at various ranges of motion. Ten male subjects participated in this study. First, the location of the Achilles tendon was confirmed by using ultrasonography, and markers were attached on the skin over the Achilles tendon path. Then, the three‐dimensional coordinates of each marker at dorsiflexion (DF) 15°, plantarflexion (PF) 0°, PF15°, and PF30° were obtained. Achilles tendon length in the curved‐line model was calculated as the sum of the distances among each marker. On the other hand, Achilles tendon length in the straight‐line model was calculated as the straight distance between the two most proximal and distal markers projected onto the sagittal plane. The difference of the Achilles tendon length change between curved‐line and straight‐line models was calculated by subtracting the Achilles tendon length change obtained in curved‐line model from that obtained in straight‐line model with three different ranges of motion (i.e., PF0°, PF15°, and PF30° from DF15°, respectively). As a result, the difference in Achilles tendon length change between the two models increased significantly as the range of motion increased. In conclusion, neglecting the curved path of the Achilles tendon induces substantial overestimation of its length change when the extent of ankle joint angle change is large.
This study examined the influence of neglecting the curved path of the Achilles tendon on its length change. As a result, neglecting the curved path of the Achilles tendon induces substantial overestimation of its length change when the extent of ankle joint angle change is large.
PMCID: PMC4254101  PMID: 25303951
Dorsiflexion; plantarflexion; three‐dimensional measurement; ultrasonography

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