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1.  Smoking Cessation Reverses DNA Double-Strand Breaks in Human Mononuclear Cells 
PLoS ONE  2014;9(8):e103993.
Cigarette smoking is a major risk factor for atherosclerotic cardiovascular disease, which is responsible for a significant proportion of smoking-related deaths. However, the precise mechanism whereby smoking induces this pathology has not been fully delineated. Based on observation of DNA double-strand breaks (DSBs), the most harmful type of DNA damage, in atherosclerotic lesions, we hypothesized that there is a direct association between smoking and DSBs. The goal of this study was to investigate whether smoking induces DSBs and smoking cessation reverses DSBs in vivo through examination of peripheral mononuclear cells (MNCs).
Approach and Results
Immunoreactivity of oxidative modification of DNA and DSBs were increased in human atherosclerotic lesions but not in the adjacent normal area. DSBs in human MNCs isolated from the blood of volunteers can be detected as cytologically visible “foci” using an antibody against the phosphorylated form of the histone H2AX (γ-H2AX). Young healthy active smokers (n = 15) showed increased γ-H2AX foci number when compared with non-smokers (n = 12) (foci number/cell: median, 0.37/cell; interquartile range [IQR], 0.31–0.58 vs. 4.36/cell; IQR, 3.09–7.39, p<0.0001). Smoking cessation for 1 month reduced the γ-H2AX foci number (median, 4.44/cell; IQR, 4.36–5.24 to 0.28/cell; IQR, 0.12–0.53, p<0.05). A positive correlation was noted between γ-H2AX foci number and exhaled carbon monoxide levels (r = 0.75, p<0.01).
Smoking induces DSBs in human MNCs in vivo, and importantly, smoking cessation for 1 month resulted in a decrease in DSBs to a level comparable to that seen in non-smokers. These data reinforce the notion that the cigarette smoking induces DSBs and highlight the importance of smoking cessation.
PMCID: PMC4122368  PMID: 25093845
2.  A Compensatory Role of NF-κB to p53 in Response to 5-FU–Based Chemotherapy for Gastric Cancer Cell Lines 
PLoS ONE  2014;9(2):e90155.
Despite of remarkable improvement of postoperative 5-FU–based adjuvant chemotherapy, the relapse rate of gastric cancer patients who undergo curative resection followed by the adjuvant chemotherapy remains substantial. Therefore, it is important to identify prediction markers for the chemotherapeutic efficacy of 5-FU. We recently identified NF-κB as a candidate relapse prediction biomarker in gastric cancer. To evaluate the biological significance of NF-κB in the context of 5-FU–based chemotherapy, we analyzed the NF-κB-dependent biological response upon 5-FU treatment in gastric cancer cell lines. Seven genes induced by 5-FU treatment in an NF-κB-dependent manner were identified, five of which are known p53 targets. Knockdown of RELA, which encodes the p65 subunit of NF-κB, decreased both p53 and p53 target protein levels. In contrast, NF-κB was not affected by TP53 knockdown. We also demonstrated that cell lines bearing Pro/Pro homozygosity in codon72 of p53 exon4, which is important for NF-κB binding to p53, are more resistant to 5-FU than those with Arg/Arg homozygosity. We conclude that NF-κB plays an important role in the response to 5-FU treatment in gastric cancer cell lines, with a possible compensatory function of p53. These results suggest that NF-κB is a potential 5-FU-chemosensitivity prediction marker that may reflect 5-FU-induced stress-response pathways, including p53.
PMCID: PMC3937424  PMID: 24587255
3.  Phase II study of concurrent chemoradiotherapy with carboplatin and vinorelbine for locally advanced non-small-cell lung cancer 
Molecular and Clinical Oncology  2014;2(3):405-410.
Patients with non-small cell lung cancer (NSCLC) have locally advanced disease with poor prognosis. Although concurrent chemoradiotherapy is the standard treatment, more effective regimens are required. The aim of this study was to assess the safety and efficacy of concurrent chemoradiotherapy with a divided schedule of carboplatin and vinorelbine in patients with locally advanced NSCLC. Patients with unresectable, stage IIIA or IIIB NSCLC were eligible for enrollment if they exhibited a performance status of 0–2 and were ≤75 years of age. Patients were treated with carboplatin at an area under the plasma concentration vs. time curve of 2.5 mg/ml/min and vinorelbine at 20 mg/m2 on days 1 and 8 every 3 weeks. Thoracic radiotherapy at a total dose of 60 Gy was concurrently administered (2 Gy per fraction). Twenty-eight patients (23 men and 5 women; median age, 67 years; range 47–75 years) were enrolled in the present study. The overall response rate was 85.7% [95% confidence interval (CI), 67.3–96.0%] and the disease control rate was 96.4% (95% CI, 81.7–99.9%). The median survival time (MST) was 23 months and the median progression-free survival (PFS) time was 8 months. Grade 3–4 toxicities included neutropenia, thrombocytopenia, anemia and infection in 100, 14, 46 and 36% of patients, respectively. One patient (4%) developed grade 3 radiation esophagitis that resolved completely without residual dilation. Grade 3 radiation pneumonitis occurred in 2 patients (7%); however, the symptoms and radiographic abnormalities subsided with corticosteroid therapy. In conclusion, concurrent chemoradiotherapy with a divided schedule of carboplatin and vinorelbine is well-tolerated and effective in patients with locally advanced NSCLC.
PMCID: PMC3999118  PMID: 24772308
carboplatin; chemoradiotherapy; non-small-cell lung cancer; vinorelbine
4.  Repeated short-term daily exercise ameliorates oxidative cerebral damage and the resultant motor dysfunction after transient ischemia in rats 
Long-term exercise prior to brain ischemia enhances the activities of antioxidant enzymes and leads to a significant reduction in brain damage and neurological deficits in rats subjected to transient middle cerebral artery occlusion. However, it has not been established whether relatively short-term exercise generates similar results following middle cerebral artery occlusion. We aimed to determine whether short-term exercise could reduce oxidative damage and prevent sensori-motor dysfunction. Male Wistar rats were subjected to perform daily exercise on a treadmill for 30 min at a speed of 15 m/min for 3 weeks, followed by a 90-min middle cerebral artery occlusion. Animals were assessed after middle cerebral artery occlusion for neurological deficits and sensori-motor function. Brain tissues were processed to evaluate infarct volume and oxidative damage. Oxidative stress was assessed using immunohistochemistry for 4-hydroxy-2-nonenal-modified proteins and 8-hydroxy-2'-deoxyguanosine. Antioxidant enzymes were evaluated using immunohistochemistry for thioredoxin and activity assay for superoxide dismutase. Exercise for 3 weeks decreased the severity of paralysis and impairment in forelimb motor coordination. Furthermore, exercise had effect on superoxide dismutase and reduced the infarct volume and the number of cells immunopositive for 4-hydroxy-2-nonenal-modified proteins and 8-hydroxy-2'-deoxyguanosine. Our results suggest that pre-conditioning treadmill exercise for 3 weeks is useful for ameliorating ischemia-induced brain injury.
PMCID: PMC3705152  PMID: 23874064
middle cerebral artery occlusion; pre-conditioning exercise; oxidative stress; sensori-motor function; rat
5.  Nitric Oxide Synthase Gene Transfer Overcomes the Inhibition of Wound Healing by Sulfur Mustard in a Human Keratinocyte In Vitro Model 
ISRN Toxicology  2012;2012:190429.
Sulfur mustard (SM) is a chemical warfare agent that causes extensive skin injury. Previously we reported that SM exposure resulted in suppression of inducible nitric oxide synthase (iNOS) expression to inhibit the healing of scratch wounds in a cultured normal human epidermal keratinocyte (NHEK) model. Based on this finding, the present study was to use adenovirus-mediated gene transfer of iNOS to restore the nitric oxide (NO) supply depleted by exposure to SM and to evaluate the effect of NO on wound healing inhibited by SM in NHEKs. The effect of the iNOS gene transfer on iNOS protein expression and NO generation were monitored by Western blot and flow cytometry, respectively. Wound healing with or without the iNOS gene transfer after SM exposure was assessed by light and confocal microscopy. The iNOS gene transfer via adenovirus resulted in overexpression of the iNOS and an increase in NO production regardless of SM exposure in the NHEK model. The gene transfer was also effective in overcoming the inhibition of wound healing due to SM exposure leading to the promotion of wound closure. The findings in this study suggest that the iNOS gene transfer is a promising therapeutic strategy for SM-induced skin injury.
PMCID: PMC3664197  PMID: 23762631
6.  A Japanese Family Suffering from Familial Juvenile Hyperuricemic Nephropathy due to a Rare Mutation of the Uromodulin Gene 
We report the case of a Japanese family suffering from familial juvenile hyperuricemic nephropathy (FJHN) due to a rare missense mutation of the uromodulin (UMOD) gene. An 18-year-old male presented with gout, hyperuricemia, and stage 3 chronic kidney disease. Mostly, FJHN is caused by a mutation altering the cystine residue of UMOD/Tamm-Horsfall protein. However, in the present case, a T688C mutation was identified in exon 4, resulting in amino acid substitution with arginine replacing tryptophan at position 230 (Trp230Arg). This mutation was also found in his brother and father with the same phenotype, indicating autosomal dominant inheritance. The affected amino acid was conserved in 200 healthy Japanese controls. Therefore, mutation T688C most likely causes rare structural and/or functional abnormalities in UMOD/Tamm-Horsfall protein.
PMCID: PMC3482064  PMID: 23197950
Chronic kidney disease; Familial juvenile hyperuricemic nephropathy; Gene; mutation; Uromodulin
8.  Simultaneous Detection of Antibodies to Mouse Hepatitis Virus Recombinant Structural Proteins by a Microsphere-Based Multiplex Fluorescence Immunoassay▿ 
We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.
PMCID: PMC3122517  PMID: 21430123
9.  Plasticity and Injury in the Developing Brain 
Brain & development  2008;31(1):1-10.
The child’s brain is more malleable or plastic than that of adults and this accounts for the ability of children to learn new skills quickly or recovery from brain injuries. Several mechanisms contribute to this ability including overproduction and deletion of neurons and synapses, and activity-dependent stabilization of synapses. The molecular mechanisms for activity dependent synaptic plasticity are being discovered and this is leading to a better understanding of the pathogenesis of several disorders including neurofibromatosis, tuberous sclerosis, Fragile X syndrome and Rett syndrome. Many of the same pathways involved in synaptic plasticity, such as glutamate-mediated excitation, can also mediate brain injury when the brain is exposed to stress or energy failure such as hypoxia-ischemia. Recent evidence indicates that cell death pathways activated by injury differ between males and females. This new information about the molecular pathways involved in brain plasticity and injury are leading to insights that will provide better therapies for pediatric neurological disorders.
PMCID: PMC2660856  PMID: 18490122
Plasticity; Injury; Fragile X Syndrome; Rett Syndrome; Hypoxia-Ischemia; NMDA; AMPA; Periventricular Leukomalacia
10.  Rhizonin, the First Mycotoxin Isolated from the Zygomycota, Is Not a Fungal Metabolite but Is Produced by Bacterial Endosymbionts▿  
Rhizonin is a hepatotoxic cyclopeptide isolated from cultures of a fungal Rhizopus microsporus strain that grew on moldy ground nuts in Mozambique. Reinvestigation of this fungal strain by a series of experiments unequivocally revealed that this “first mycotoxin from lower fungi” is actually not produced by the fungus. PCR experiments and phylogenetic studies based on 16S rRNA gene sequences revealed that the fungus is associated with bacteria belonging to the genus Burkholderia. By transmission electron microscopy, the bacteria were localized within the fungal cytosol. Toxin production and the presence of the endosymbionts were correlated by curing the fungus with an antibiotic, yielding a nonproducing, symbiont-free phenotype. The final evidence for a bacterial biogenesis of the toxin was obtained by the successful fermentation of the endosymbiotic bacteria in pure culture and isolation of rhizonin A from the broth. This finding is of particular interest since Rhizopus microsporus and related Rhizopus species are frequently used in food preparations such as tempeh and sufu.
PMCID: PMC1800748  PMID: 17122400
11.  Agonist-stimulated cytoskeletal reorganization and signal transduction at focal adhesions in vascular smooth muscle cells require c-Src 
Journal of Clinical Investigation  1999;103(6):789-797.
Thrombin and angiotensin II (angII) have trophic properties as mediators of vascular remodeling. Focal adhesions and actin cytoskeleton are involved in cell growth, shape, and movement and may be important in vascular remodeling. To characterize mechanisms by which thrombin and angII modulate vessel structure, we studied the effects of these G protein–coupled receptor ligands on focal adhesions in vascular smooth muscle cells (VSMCs). Both thrombin and angII stimulated bundling of actin filaments to form stress fibers, assembly of focal adhesions, and protein tyrosine phosphorylation at focal adhesions, such as p130Cas, paxillin, and tensin. To test whether c-Src plays a critical role in focal adhesion rearrangement, we analyzed cells with altered c-Src activity by retroviral transduction of wild-type (WT) and kinase-inactive (KI) c-Src into rat VSMCs, and by use of VSMCs from WT (src+/+) and Src-deficient (src–/–) mice. Tyrosine phosphorylation of Cas, paxillin, and tensin were markedly decreased in VSMCs expressing KI-Src and in src–/– VSMCs. Expression of KI-Src did not inhibit stress fiber formation by thrombin. Surprisingly, actin bundling was markedly decreased in VSMCs from src–/– mice both basally and after thrombin stimulation, compared with src+/+ mice. We also studied the effect of KI-Src and WT-Src on VSMC spreading. Expression of KI-Src reduced the rate of VSMC spreading on collagen, whereas WT-Src enhanced cell spreading. In conclusion, c-Src plays a critical role in agonist-stimulated cytoskeletal reorganization and signal transduction at focal adhesions in VSMCs. c-Src kinase activity is required for the cytoskeletal turnover that occurs in cell spreading, whereas c-Src appears to regulate actin bundling via a kinase-independent mechanism.
PMCID: PMC408136  PMID: 10079099
12.  In Vitro and In Vivo Activities of Levofloxacin against Biofilm-Producing Pseudomonas aeruginosa 
Interactions between biofilm cells of Pseudomonas aeruginosa and levofloxacin were studied. P. aeruginosa incubated for 6 days with Teflon sheets formed a biofilm on its surface. Against the biofilm bacteria, levofloxacin at an MIC determined by the standard method for the strain was highly bactericidal whereas gentamicin, ceftazidime, and ciprofloxacin showed no significant killing activity. Levofloxacin, ciprofloxacin, and gentamicin, but not ceftazidime, exhibited killing activity against nongrowing cells of the strain incubated in phosphate buffer. In addition, levofloxacin, ciprofloxacin, and ceftazidime, but not gentamicin, showed the ability to penetrate an agar containing alginate. These findings may explain the efficacy of levofloxacin and the ineffectiveness of gentamicin and ceftazidime against biofilm bacteria; however, the cause of the ineffectiveness of ciprofloxacin still remains to be determined. In experimental pneumonia in guinea pigs, in which the biofilm mode of growth of the strain was observed in the lung, only levofloxacin exhibited substantial therapeutic efficacy. These findings suggest the significant role of levofloxacin in therapy of biofilm bacterium-associated infectious diseases.
PMCID: PMC105659  PMID: 9660997
13.  Recent Origin and Cultural Reversion of a Hunter–Gatherer Group 
PLoS Biology  2005;3(3):e71.
Contemporary hunter–gatherer groups are often thought to serve as models of an ancient lifestyle that was typical of human populations prior to the development of agriculture. Patterns of genetic variation in hunter–gatherer groups such as the !Kung and African Pygmies are consistent with this view, as they exhibit low genetic diversity coupled with high frequencies of divergent mtDNA types not found in surrounding agricultural groups, suggesting long-term isolation and small population sizes. We report here genetic evidence concerning the origins of the Mlabri, an enigmatic hunter–gatherer group from northern Thailand. The Mlabri have no mtDNA diversity, and the genetic diversity at Y-chromosome and autosomal loci are also extraordinarily reduced in the Mlabri. Genetic, linguistic, and cultural data all suggest that the Mlabri were recently founded, 500–800 y ago, from a very small number of individuals. Moreover, the Mlabri appear to have originated from an agricultural group and then adopted a hunting–gathering subsistence mode. This example of cultural reversion from agriculture to a hunting–gathering lifestyle indicates that contemporary hunter–gatherer groups do not necessarily reflect a pre-agricultural lifestyle.
Genes, language and culture reveal that the Mlabri reverted from an agricultural to a hunter-gatherer lifestyle, suggesting that hunter-gatherer groups might not always represent the pre- agricultural lifestyle of humans
PMCID: PMC1044832  PMID: 15736978
14.  Regulation of cargo-selective endocytosis by dynamin 2 GTPase-activating protein girdin 
The EMBO Journal  2014;33(18):2098-2112.
In clathrin-mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo-specific adaptors for distinct cellular functions. Here, we show that the actin-binding protein girdin is a regulator of cargo-selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase-activating protein. Interestingly, girdin depletion leads to the defect in clathrin-coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E-cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo-specific adaptor.
PMCID: PMC4195775  PMID: 25061227
clathrin-mediated endocytosis; dynamin; girdin; GTPase-activating protein; selective endocytosis
15.  Effects of oral tacrolimus as a rapid induction therapy in ulcerative colitis 
AIM: To determine the efficacy and safety of rapid induction therapy with oral tacrolimus without a meal in steroid-refractory ulcerative colitis (UC) patients.
METHODS: This was a prospective, multicenter, observational study. Between May 2010 and August 2012, 49 steroid-refractory UC patients (55 flare-ups) were consecutively enrolled. All patients were treated with oral tacrolimus without a meal at an initial dose of 0.1 mg/kg per day. The dose was adjusted to maintain trough whole-blood levels of 10-15 ng/mL for the first 2 wk. Induction of remission at 2 and 4 wk after tacrolimus treatment initiation was evaluated using Lichtiger’s clinical activity index (CAI).
RESULTS: The mean CAI was 12.6 ± 3.6 at onset. Within the first 7 d, 93.5% of patients maintained high trough levels (10-15 ng/mL). The CAI significantly decreased beginning 2 d after treatment initiation. At 2 wk, 73.1% of patients experienced clinical responses. After tacrolimus initiation, 31.4% and 75.6% of patients achieved clinical remission at 2 and 4 wk, respectively. Treatment was well tolerated.
CONCLUSION: Rapid induction therapy with oral tacrolimus shortened the time to achievement of appropriate trough levels and demonstrated a high remission rate 28 d after treatment initiation. Rapid induction therapy with oral tacrolimus appears to be a useful therapy for the treatment of refractory UC.
PMCID: PMC4323466
Ulcerative colitis; Tacrolimus; Rapid induction therapy; Steroid-refractory ulcerative colitis; Inflammatory bowel disease
16.  Real-Time Dynamic Adsorption Processes of Cytochrome c on an Electrode Observed through Electrochemical High-Speed Atomic Force Microscopy 
PLoS ONE  2015;10(2):e0116685.
An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome c (cyt c) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry. This paper reports a real-time visualization of cyt c adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt c molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt c using EC-HS-AFM. The root mean square roughness (RRMS) from the AFM images and the number of the electrochemically active cyt c molecules adsorbed onto the electrode (Γ) simultaneously increased in positive cooperativity. Cyt c molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics.
PMCID: PMC4324961  PMID: 25671430
17.  The off-hour effect on trauma patients requiring subspecialty intervention at a community hospital in Japan: a retrospective cohort study 
Because most community hospitals in Japan do not maintain 24-h availability of in-house anesthesiologists, surgeons, and interventional radiologists, staffing dramatically declines during off hours. It is unclear whether, in such under-resourced hospitals, trauma patients presenting during off hours and requiring subspecialty intervention have worse outcomes than those who present during business hours.
This was a retrospective cohort study at a community hospital in Japan. Participants were all injured patients requiring emergency trauma surgery or transarterial embolization who presented from January 2002 to December 2013. We investigated whether outcomes of these patients differed between business hours (8:01 AM to 6:00 PM weekdays) and off hours (6:01 PM to 8:00 AM weekdays plus all weekend hours). The primary outcome measure was mortality rate, and the secondary outcome measures were duration of emergency room (ER) stay; unexpected death (death/probability of survival > 0.5); and adverse events occurring in the ER. We adjusted for potential confounders of age, sex, Injury Severity Score (ISS), Revised Trauma Score, presentation phase (2002–2005, 2006–2009, and 2010–2013), Charlson Comorbidity Index, and injury type (blunt or penetrating) using logistic regression models.
Of the 805 patients included, 379 (47.1%) presented during business hours and 426 (52.9%) during off hours. Off-hours presentation was associated with longer ER stays for patients with systolic blood pressure < 90 mmHg on admission (p = 0.021), ISS >15 (p = 0.047), and pelvic fracture requiring transarterial embolization (p < 0.001). Off-hours presentation was also associated with increased risk of adverse events in the ER (odds ratio [OR] 1.7, 95% confidence interval [CI] 1.1–2.7, p = 0.020). After adjustment for confounders, an increased risk of adverse events (OR 1.6, 95% CI 1.1–2.7, p = 0.049) persisted, but no differences were detected in mortality (p = 0.80) and unexpected death (p = 0.44) between off hours and business hours.
At a community hospital in Japan, presentation during off hours was associated with a longer ER stay for severely injured patients and increased risk of adverse events in the ER. However, these disadvantages did not impact mortality or unexpected outcome.
PMCID: PMC4329665
Complications; Emergency surgery; Night presentation; Preoperative period; Transarterial embolization; Unexpected trauma death; Weekend presentation
18.  Prognosis of patients with fulminant myocarditis managed by peripheral venoarterial extracorporeal membranous oxygenation support: a retrospective single-center study 
Peripheral venoarterial extracorporeal membranous oxygenation (ECMO) support is effective in patients with cardiogenic shock or fatal arrhythmia due to fulminant myocarditis. The clinical courses of fulminant myocarditis are still uncertain; therefore, it is difficult to determine the appropriate time for discontinuing ECMO or converting to a ventricular assist device. The purpose of this study was to investigate the prognosis of patients with fulminant myocarditis managed by ECMO.
Twenty-two consecutive patients with fulminant myocarditis managed by peripheral venoarterial ECMO between 1999 and 2013 were enrolled.
Survival to discharge was 59% (13 patients), and in-hospital mortality was 41% (9 patients). The age in the survivor group was significantly lower than that in the non-survivor group (survivor group vs. non-survivor group; 36.5 ± 4.1 vs. 60.2 ± 5.0 years, p = 0.001). Although the ECMO support duration was similar between the groups (181 ± 22 vs. 177 ± 31 h), the rate of complication related to ECMO was significantly lower in the survivor group (15.3% vs. 66.6%, p = 0.02). When comparing the laboratory data during ECMO management between the groups, the serum bilirubin level on day 7 was significantly lower in the survivor group (total: 4.6 ± 2.8 vs. 13.7 ± 10.8 mg/dL, p = 0.014; direct: 2.2 ± 0.5 vs. 9.8 ± 4.5 mg/dL, p = 0.009).
Fulminant myocarditis is associated with high mortality rates despite ECMO. An older age and complications related to ECMO are associated with poor prognosis.
PMCID: PMC4336280
Fulminant myocarditis; Extracorporeal membranous oxygenation support; Prognosis
19.  Rab1A regulates anterograde melanosome transport by recruiting kinesin-1 to melanosomes through interaction with SKIP 
Scientific Reports  2015;5:8238.
Melanosomes are lysosome-related organelles in melanocytes that are transported from the perinucleus to the cell periphery by coordination between bidirectional (anterograde and retrograde) microtubule-dependent transport and unidirectional actin-dependent transport. Although the molecular machineries that mediate retrograde transport and actin-dependent transport have already been identified, little is known about the anterograde transport complex on microtubules in mammalian cells. Here we discovered that small GTPase Rab1A on melanosomes recruits SKIP/PLEKHM2 as a Rab1A-specific effector and that Rab1A, SKIP, and a kinesin-1/(Kif5b+KLC2) motor form a transport complex that mediates anterograde melanosome transport in melanocytes. Interestingly, Arl8, Arf-like small GTPase that also interacts with SKIP, is specifically localized at lysosomes and regulates their anterograde transport in melanocytes. Our findings suggest that the anterograde microtubule-dependent transport of melanosomes and lysosomes are differently regulated by independent cargo receptors, i.e., Rab1A and Arl8, respectively, but that a SKIP–kinesin-1 mechanism is responsible for the transport of both.
PMCID: PMC4316160  PMID: 25649263
22.  Emergent photovoltage on SmB6 surface upon bulk-gap evolution revealed by pump-and-probe photoemission spectroscopy 
Scientific Reports  2015;5:8160.
Recent studies suggest that an exemplary Kondo insulator SmB6 belongs to a new class of topological insulators (TIs), in which non-trivial spin-polarized metallic states emerge on surface upon the formation of Kondo hybridization gap in the bulk. Remarkably, the bulk resistivity reaches more than 20 Ω cm at 4 K, making SmB6 a candidate for a so-called bulk-insulating TI. We here investigate optical-pulse responses of SmB6 by pump-and-probe photoemission spectroscopy. Surface photovoltage effect is observed below ~90 K. This indicates that an optically-active band bending region develops beneath the novel metallic surface upon the bulk-gap evolution. The photovoltaic effect persists for >200 µs, which is long enough to be detected by electronics devices, and could be utilized for optical gating of the novel metallic surface.
PMCID: PMC4313084  PMID: 25640966
23.  Amoebal Endosymbiont Parachlamydia acanthamoebae Bn9 Can Grow in Immortal Human Epithelial HEp-2 Cells at Low Temperature; An In Vitro Model System to Study Chlamydial Evolution 
PLoS ONE  2015;10(2):e0116486.
Ancient chlamydiae diverged into pathogenic and environmental chlamydiae 0.7–1.4 billion years ago. However, how pathogenic chlamydiae adapted to mammalian cells that provide a stable niche at approximately 37°C, remains unknown, although environmental chlamydiae have evolved as endosymbionts of lower eukaryotes in harsh niches of relatively low temperatures. Hence, we assessed whether an environmental chlamydia, Parachlamydia Bn9, could grow in human HEp-2 cells at a low culture temperature of 30°C. The assessment of inclusion formation by quantitative RT-PCR revealed that the numbers of bacterial inclusion bodies and the transcription level of 16SrRNA significantly increased after culture at 30°C compared to at 37°C. Confocal microscopy showed that the bacteria were located close to HEp-2 nuclei and were actively replicative. Transmission electron microscopy also revealed replicating bacteria consisting of reticular bodies, but with a few elementary bodies. Cytochalasin D and rifampicin inhibited inclusion formation. Lactacystin slightly inhibited bacterial inclusion formation. KEGG analysis using a draft genome sequence of the bacteria revealed that it possesses metabolic pathways almost identical to those of pathogenic chlamydia. Interestingly, comparative genomic analysis with pathogenic chlamydia revealed that the Parachlamydia similarly possess the genes encoding Type III secretion system, but lacking genes encoding inclusion membrane proteins (IncA to G) required for inclusion maturation. Taken together, we conclude that ancient chlamydiae had the potential to grow in human cells, but overcoming the thermal gap was a critical event for chlamydial adaptation to human cells.
PMCID: PMC4314085  PMID: 25643359
24.  Peanut oral immunotherapy results in increased antigen-induced Treg function and hypomethylation of FOXP3 
The mechanisms contributing to clinical immune tolerance remain incompletely understood. This study provides evidence for specific immune mechanisms that are associated with a model of operationally defined clinical tolerance.
Our overall objective was to study laboratory changes associated with clinical immune tolerance in antigen-induced T cells, basophils, and antibodies in subjects undergoing oral immunotherapy (OIT) for peanut allergy.
In a phase 1, single site study, we studied participants (n=23) undergoing peanut OIT and compared them to age-matched allergic controls (n=20) undergoing standard of care (abstaining from peanut) for 24 months. Participants were operationally defined as clinically immune tolerant (IT) if they had no detectable allergic reactions to a peanut oral food challenge after 3 months of therapy withdrawal (IT, n=7) while those that had an allergic reaction were categorized as non-tolerant (NT, n=13).
Antibody and basophil activation measurements did not statistically differentiate between NT vs. IT. However, T-cell function and demethylation of FOXP3 CpG sites in antigen-induced Treg were significantly different between IT vs. NT participants. When IT participants were withdrawn from peanut therapy for an additional 3 months (total of 6 months); only 3 participants remained "immune tolerant" and 4 participants regained sensitivity along with increased methylation of FOXP3 CpG sites in antigen-induced Treg.
In summary, modifications at the DNA level of antigen-induced T-cell subsets may be predictive of a state of operationally-defined clinical "immune tolerance" during peanut OIT.
PMCID: PMC4121175  PMID: 24636474
Food allergy; allergy; oral immunotherapy; peanut; T regulatory cells; desensitization; tolerance; epigenetics; Foxp3
25.  Efficacy of Repeated Intravenous Administration of Peramivir against Highly Pathogenic Avian Influenza A (H5N1) Virus in Cynomolgus Macaques 
Highly pathogenic avian influenza A (H5N1) viruses cause severe and often fatal disease in humans. We evaluated the efficacy of repeated intravenous dosing of the neuraminidase inhibitor peramivir against highly pathogenic avian influenza virus A/Vietnam/UT3040/2004 (H5N1) infection in cynomolgus macaques. Repeated dosing of peramivir (30 mg/kg/day once a day for 5 days) starting immediately after infection significantly reduced viral titers in the upper respiratory tract, body weight loss, and cytokine production and resulted in a significant body temperature reduction in infected macaques compared with that of macaques administered a vehicle (P < 0.05). Repeated administration of peramivir starting at 24 h after infection also resulted in a reduction in viral titers and a reduction in the period of virus detection in the upper respiratory tract, although the body temperature change was not statistically significant. The macaque model used in the present study demonstrated that inhibition of viral replication at an early time point after infection by repeated intravenous treatment with peramivir is critical for reduction of the production of cytokines, i.e., interleukin-6 (IL-6), tumor necrosis factor α, gamma interferon, monocyte chemotactic protein 1, and IL-12p40, resulting in amelioration of symptoms caused by highly pathogenic avian influenza virus infection.
PMCID: PMC4135987  PMID: 24913156

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