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1.  Therapeutic targets in the ASK1-dependent stress signaling pathways 
Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that activates downstream MAP kinases (MAPKs), c-Jun N-terminal kinases (JNKs) and p38 MAPKs, in response to various stresses, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, lipopolysaccharide, and calcium overload. Activation of the JNK and p38 pathways induces stress responses such as cell death, differentiation, and the production of inflammatory cytokines. A series of studies using ASK1-deficient mice have indicated that ASK1 plays important roles in many stress-related diseases, including cardiovascular and neurodegenerative diseases, suggesting that small compounds that inhibit ASK1 activity could possibly be used for the amelioration of the development and/or progression of these diseases. In this review, we provide an overview of the pathophysiological roles of ASK1-dependent signaling pathways and discuss the mechanistic basis for how these could serve as potential therapeutic targets.
PMCID: PMC3491083  PMID: 23060232
ASK1; inhibitors; MAP kinase; signal transduction; stress
2.  Background electroencephalographic (EEG) activities of very preterm infants born at less than 27 weeks gestation: a study on the degree of continuity 
AIMS—To clarify the features of the background electroencephalographic (EEG) activities in clinically well preterm infants born at less than 27 weeks gestation and to outline their chronological changes with increasing postconceptional age (PCA).
METHODS—EEGs of clinically well premature infants born at less than 27 weeks gestation were recorded during the early postnatal period. The infants were separated into three groups according to their PCA at the time of EEG recording (21-22 weeks PCA, 23-24 weeks PCA, and 25-26 weeks PCA). The mean and maximum duration of interburst intervals (IBIs), the mean duration of bursts, and the percentage of continuous and discontinuous patterns in each PCA group were evaluated.
RESULTS—There were three infants at 21-22 weeks PCA, seven at 23-24 weeks PCA, and five at 25-26 weeks PCA. Eighteen EEG recordings were obtained. The mean and maximum IBI duration decreased with increasing PCA. The percentage of continuous patterns increased with increasing PCA. Conversely, the percentage of discontinuous patterns decreased with increasing PCA.
CONCLUSIONS—In premature infants born at less than 27 weeks gestation, the characteristics of the background EEG activities were similar to those of older premature infants. These changes reflect the development of the central nervous system in this period.

At less than 27 weeks gestational age, the characteristics of background EEG activities were found to be as follows:
PMCID: PMC1721237  PMID: 11320041
3.  Characterization and Quantification of Diacylglycerol Species in Biological Extracts after One-step Derivatization: A Shotgun Lipidomics Approach 
Analytical chemistry  2014;86(4):2146-2155.
Diacylglycerols (DAGs) are important intermediates of lipid metabolism and cellular signaling. It is well known that the mass levels of DAG are altered under disease states. Therefore, quantitative analysis of DAGs in biological samples can provide critical information to uncover underlying mechanisms of various cellular functional disorders. Although great efforts on the analysis of individual DAG species have recently been made by utilizing mass spectrometry with or without derivatization, cost effective and high throughput methodology for identification and quantification of all DAG species including regioisomers, particularly in an approach of shotgun lipidomics, are still missing. Herein, we described a novel method for directly identifying and quantifying DAG species including regioisomers present in lipid extracts of biological samples after facile one-step derivatization with dimethylglycine based on the principles of multi-dimensional mass spectrometry-based shotgun lipidomics. The established method provided substantial sensitivity (low limit of quantification at amol/µl), high specificity, and broad linear dynamics range (2,500 folds) without matrix effects. By exploiting this novel method, we revealed a 16-fold increase of total DAG mass in the livers of ob/ob mice compared to their wild type controls at 4 months of age (an insulin-resistant state) vs. a 5-fold difference between 3-month old mice (with normal insulin). These results demonstrated the importance and power of the method for studying biochemical mechanisms underpinning disease states.
PMCID: PMC3954745  PMID: 24432906
brain injury; diacylglycerol; dimethylglycine derivatization; lipid metabolism; mass spectrometry; obesity; shotgun lipidomics
4.  SIRT1 Suppresses the Senescence-Associated Secretory Phenotype through Epigenetic Gene Regulation 
PLoS ONE  2015;10(1):e0116480.
Senescent cells develop a pro-inflammatory response termed the senescence-associated secretory phenotype (SASP). As many SASP components affect surrounding cells and alter their microenvironment, SASP may be a key phenomenon in linking cellular senesence with individual aging and age-related diseases. We herein demonstrated that the expression of Sirtuin1 (SIRT1) was decreased and the expression of SASP components was reciprocally increased during cellular senescence. The mRNAs and proteins of SASP components, such as IL-6 and IL-8, quickly accumulated in SIRT1-depleted cells, and the levels of these factors were also higher than those in control cells, indicating that SIRT1 negatively regulated the expression of SASP factors at the transcriptional level. SIRT1 bound to the promoter regions of IL-8 and IL-6, but dissociated from them during cellular senescence. The acetylation of Histone H3 (K9) and H4 (K16) of the IL-8 and IL-6 promoter regions gradually increased during cellular senescence. In SIRT1-depleted cells, the acetylation levels of these regions were already higher than those in control cells in the pre-senescent stage. Moreover, these acetylation levels in SIRT1-depleted cells were significantly higher than those in control cells during cellular senescence. These results suggest that SIRT1 repressed the expression of SASP factors through the deacetylation of histones in their promoter regions.
PMCID: PMC4312089  PMID: 25635860
5.  Impact of Different Antimicrobial Therapies on Clinical and Fiscal Outcomes of Patients with Bacteremia Due to Vancomycin-Resistant Enterococci 
Vancomycin-resistant enterococci (VRE) are a growing health problem, and uncertainties exist regarding the optimal therapy for bloodstream infection due to VRE. We conducted systematic comparative evaluations of the impact of different antimicrobial therapies on the outcomes of patients with bloodstream infections due to VRE. A retrospective study from January 2008 to October 2010 was conducted at Detroit Medical Center. Unique patients with blood cultures due to VRE were included and reviewed. Three major therapeutic classes were analyzed: daptomycin, linezolid, and β-lactams. Three multivariate models were conducted for each outcome, matching for a propensity score predicting the likelihood of receipt of one of the therapeutic classes. A total of 225 cases of bacteremia due to VRE were included, including 86 (38.2%) cases of VR Enterococcus faecalis and 139 (61.8%) of VR Enterococcus faecium. Bacteremia due to VR E. faecalis was more frequent among subjects treated with β-lactams than among those treated with daptomycin or linezolid. The median dose of daptomycin was 6 mg/kg of body weight (range, 6 to 12 mg/kg). After controlling for propensity score and bacteremia due to VR E. faecalis, differences in mortality were nonsignificant among the treatment groups. Therapy with daptomycin was associated with higher median variable direct cost per day than that for linezolid. This large study revealed the three therapeutic classes (daptomycin, linezolid, and β-lactams) are similarly efficacious in the treatment of bacteremia due to susceptible strains of VRE.
PMCID: PMC4068570  PMID: 24798267
6.  Defensive function of persecutory delusion and discrepancy between explicit and implicit self-esteem in schizophrenia: study using the Brief Implicit Association Test 
If delusions serve as a defense mechanism in schizophrenia patients with paranoia, then they should show normal or high explicit self-esteem and low implicit self-esteem. However, the results of previous studies are inconsistent. One possible explanation for this inconsistency is that there are two types of paranoia, “bad me” (self-blaming) paranoia and “poor me” (non-self-blaming) paranoia. We thus examined implicit and explicit self-esteem and self-blaming tendency in patients with schizophrenia and schizoaffective disorder. We hypothesized that patients with paranoia would show lower implicit self-esteem and only those with non-self-blaming paranoia would experience a discrepancy between explicit and implicit self-esteem.
Participants consisted of patients with schizophrenia and schizoaffective disorder recruited from a day hospital (N=71). Participants were assessed for psychotic symptoms, using the Brief Psychiatric Rating Scale (BPRS), and self-blaming tendency, using the brief COPE. We also assessed explicit self-esteem, using the Rosenberg Self-Esteem Scale (RSES), implicit self-esteem, using Brief Implicit Association Test (BIAT), and discrepancy between explicit and implicit self-esteem.
Contrary to our hypothesis, implicit self-esteem in paranoia and nonparanoia showed no statistical difference. As expected, only patients with non-self-blaming paranoia experienced a discrepancy between explicit and implicit self-esteem; other groups showed no such discrepancy.
These results suggest that persecutory delusion plays a defensive role in non-self-blaming paranoia.
PMCID: PMC4278784  PMID: 25565849
coping style; poor me paranoia; remitted paranoid delusion; external attribution
7.  LOX-1 Plays an Important Role in Ischemia-Induced Angiogenesis of Limbs 
PLoS ONE  2014;9(12):e114542.
LOX-1, lectin-like oxidized low-density lipoprotein (LDL) receptor-1, is a single transmembrane receptor mainly expressed on endothelial cells. LOX-1 mediates the uptake of oxidized LDL, an early step in atherosclerosis; however, little is known about whether LOX-1 is involved in angiogenesis during tissue ischemia. Therefore, we examined the role of LOX-1 in ischemia-induced angiogenesis in the hindlimbs of LOX-1 knockout (KO) mice. Angiogenesis was evaluated in a surgically induced hindlimb ischemia model using laser Doppler blood flowmetry (LDBF) and histological capillary density (CD) and arteriole density (AD). After right hindlimb ischemia, the ischemic/nonischemic hindlimb blood flow ratio was persistently lower in LOX-1 KO mice than in wild-type (WT) mice. CD and AD were significantly smaller in LOX-1 KO mice than in WT mice on postoperative day 14. Immunohistochemical analysis revealed that the number of macrophages infiltrating ischemic tissues was significantly smaller in LOX-1 KO mice than in WT mice. The number of infiltrated macrophages expressing VEGF was also significantly smaller in LOX-1 KO mice than in WT mice. Western blot analysis and ROS production assay revealed that LOX- KO mice show significant decrease in Nox2 expression, ROS production and HIF-1α expression, the phosphorylation of p38 MAPK and NF-κB p65 subunit as well as expression of redox-sensitive vascular cell adhesion molecule-1 (VCAM-1) and LOX-1 itself in ischemic muscles, which is supposed to be required for macrophage infiltration expressing angiogenic factor VEGF. Reduction of VEGF expression successively suppressed the phosphorylation of Akt and eNOS, which accelerated angiogenesis, in the ischemic leg of LOX-1 KO mice. Our findings indicate that LOX-1 plays an important role in ischemia-induced angiogenesis by 1) Nox2-ROS-NF-κB activation, 2) upregulated expression of adhesion molecules: VCAM-1 and LOX-1 and 3) promoting macrophage infiltration, which expresses angiogenic factor VEGF.
PMCID: PMC4267738  PMID: 25514797
8.  Natural Anti-Intestinal Goblet Cell Autoantibody Production from Marginal Zone B Cells 
Expression of a germline VH3609/D/JH2 IgH in mice results in the generation of B1 B cells with anti-thymocyte/Thy-1 glycoprotein autoreactivity by coexpression of Vk21-5/Jk2 L chain leading to production of serum IgM natural autoantibody. In these same mice, the marginal zone (MZ) B cell subset in spleen shows biased usage of a set of Ig L chains different from B1 B cells, with 30% having an identical Vk19-17/Jk1 L chain rearrangement. This VH3609/Vk19-17 IgM is reactive with intestinal goblet cell granules, binding to the intact large polymatrix form of mucin 2 glycoprotein secreted by goblet cells. Analysis of a μκ B cell AgR (BCR) transgenic (Tg) mouse with this anti–goblet cell/mucin2 autoreactive (AGcA) specificity demonstrates that immature B cells expressing the Tg BCR become MZ B cells in spleen by T cell–independent BCR signaling. These Tg B cells produce AGcA as the predominant serum IgM, but without enteropathy. Without the transgene, AGcA autoreactivity is low but detectable in the serum of BALB/c and C.B17 mice, and this autoantibody is specifically produced by the MZ B cell subset. Thus, our findings reveal that AGcA is a natural autoantibody associated with MZ B cells.
PMCID: PMC4282382  PMID: 25480561
9.  Does the presence of coexisting diseases modulate the effectiveness of a low-dose estrogen/progestin, ethinylestradiol/drospirenone combination tablet in dysmenorrhea? Reanalysis of two randomized studies in Japanese women 
The purpose of this study was to investigate the effectiveness of a combination of ethinylestradiol (EE) and 0.02 mg/drospirenone (DRSP) 3 mg in Japanese women with dysmenorrhea and in particular to determine whether or not the presence of specific coexisting organic diseases (eg, endometriosis, uterine fibroids, uterine adenomyosis) has an impact on treatment.
Methods and results
Four hundred and ten patients with dysmenorrhea aged 20 years or older (315 without coexisting organic disease, 28 with endometriosis, 37 with uterine fibroids, and 46 with uterine adenomyosis [some patients had multiple coexisting organic diseases]) were enrolled and treated with EE/DRSP in either a 16-week comparator study or a 52-week long-term safety study. Evaluations included changes in total dysmenorrhea score, visual analog scale for dysmenorrhea, severity of symptoms, hormone levels, endometrial thickness, and safety outcomes. In both studies, the total dysmenorrhea score was significantly (P<0.001) decreased from baseline during treatment with EE/DRSP. Time-dependent changes in visual analog score for dysmenorrhea and alleviation of symptoms, such as lower abdominal pain, low back pain (lumbago), headache, and nausea/vomiting, were similar in all patient groups with and without any specific coexisting organic diseases. These improvements with EE/DRSP were observed for both short-term (16 weeks) and long-term (52 weeks) use. These effects were associated with suppressed increases in serum estradiol and progesterone levels and decreased endometrial thickness. The safety profile of EE/DRSP was similar in all patients, irrespective of the presence of coexisting organic diseases.
EE/DRSP may be prescribed for patients with dysmenorrhea irrespective of the presence of any specific coexisting organic diseases.
PMCID: PMC4259553  PMID: 25506249
dysmenorrhea; organic disease; ethinylestradiol; drospirenone; oral contraceptive
10.  Molecular and Epidemiological Characterization of IMP-Type Metallo-β-Lactamase-Producing Enterobacter cloacae in a Large Tertiary Care Hospital in Japan 
IMP-type metallo-β-lactamase enzymes have been reported in different geographical areas and in various Gram-negative bacteria. However, the risk factors and epidemiology pertaining to IMP-type metallo-β-lactamase-producing Enterobacter cloacae (IMP-producing E. cloacae) have not been systematically evaluated. We conducted a retrospective, matched case-control study of patients from whom IMP-producing E. cloacae isolates were obtained, in addition to performing thorough molecular analyses of the clinically obtained IMP-producing E. cloacae isolates. Unique cases with IMP-producing E. cloacae isolation were included. Patients with IMP-producing E. cloacae were matched to uninfected controls at a ratio of 1 to 3. Fifteen IMP-producing E. cloacae cases were identified, with five of the isolates being obtained from blood, and they were matched to 45 uninfected controls. All (100%) patients from whom IMP-producing E. cloacae isolates were obtained had indwelling devices at the time of isolation, compared with one (2.2%) uninfected control. Independent predictors for isolation of IMP-producing E. cloacae were identified as cephalosporin exposure and invasive procedures within 3 months. Although in-hospital mortality rates were similar between cases and controls (14.3% versus 13.3%), the in-hospital mortality of patients with IMP-producing E. cloacae-caused bacteremia was significantly higher (40%) than the rate in controls. IMP-producing E. cloacae isolates were frequently positive for other resistance determinants. The MICs of meropenem and imipenem were not elevated; 10 (67%) and 12 (80%) of the 15 IMP-producing E. cloacae isolates had a MIC of ≤1 μg/ml. A phylogenetic tree showed a close relationship among the IMP-producing E. cloacae samples. Indwelling devices, exposure to cephalosporin, and a history of invasive procedures were associated with isolation of IMP-producing E. cloacae. Screening for carbapenemase production is important in order to apply appropriate clinical management and infection control measures.
PMCID: PMC4068452  PMID: 24709261
11.  Performance of Rapid Diagnostic Tests for Plasmodium ovale Malaria in Japanese Travellers 
Tropical Medicine and Health  2014;42(4):149-153.
Background: Rapid diagnostic tests (RDTs) are used widely in the diagnosis of malaria. Although the effectiveness of RDTs for malaria has been described in many previous studies, the low performance of RDT particularly for Plasmodium ovale malaria in traveller has rarely been reported.
Methods: This was a retrospective cohort study conducted on Japanese travellers diagnosed with malaria at the National Center for Global Health and Medicine between January 2004 and June 2013. The diagnosis of malaria was confirmed by microscopic examination, RDT, and polymerase chain reaction in all patients. The RDTs used in our study were Binax NOW Malaria (Binax Inc., Scarborough, Maine, USA) (BN) and SD Malaria Antigen Pf/Pan (Standard Diagnostics Inc., Korea) (SDMA). We compared the sensitivity of the RDTs to P. ovale malaria and Plasmodium vivax malaria.
Results: A total of 153 cases of malaria were observed, 113 of which were found among Japanese travellers. Nine patients with P. ovale malaria and 17 patients with P. vivax malaria undergoing RDTs were evaluated. The overall sensitivity of RDTs for P. ovale malaria and P. vivax malaria was 22.2% and 94.1%, respectively (P < 0.001). The sensitivity of SDMA for P. ovale malaria and P. vivax malaria was 50% and 100%, respectively. The sensitivity of BN for P. vivax malaria was 90.0%, but it was ineffective in detecting the cases of P. ovale malaria.
Conclusions: The sensitivity of RDTs was not high enough to diagnose P. ovale malaria in our study. In order not to overlook P. ovale malaria, therefore, microscopic examination is indispensable.
PMCID: PMC4229013  PMID: 25473374
Plasmodium ovale; malaria; rapid diagnostic test; Japanese; traveller
12.  Nanoclusters Synthesized by Synchrotron Radiolysis in Concert with Wet Chemistry 
Scientific Reports  2014;4:7199.
Wet chemical reduction of metal ions, a common strategy for synthesizing metal nanoparticles, strongly depends on the electric potential of the metal, and its applications to late transition metal clusters have been limited to special cases. Here, we describe copper nanoclusters grown by synchrotron radiolysis in concert with wet chemistry. The local structure of copper aggregates grown by reducing Cu(II) pentanedionate using synchrotron x-ray beam was studied in situ by x-ray absorption spectroscopy. A detailed analysis of the XANES and EXAFS spectra, compared with DFT calculations and full-potential non-muffin-tin multiple scattering calculations, identified the nanocluster as Cu13 with icosahedral symmetry. The novel “charged” nanoclusters tightly bound to electron-donating amido molecules, which formed as a result of photo-induced deprotonation of ligand amines, were stabilized by irradiation. Monodispersive deposition of nanoclusters was enabled by controlling the type and density of “monomers”, in remarkable contrast to the conventional growth of metallic nanoparticles.
PMCID: PMC4244621  PMID: 25425181
13.  Stand‐up exercise training facilitates muscle recovery from disuse atrophy by stimulating myogenic satellite cell proliferation in mice 
Physiological Reports  2014;2(11):e12185.
Determining the cellular and molecular recovery processes in inactivity – or unloading –induced atrophied muscles should improve rehabilitation strategies. We assessed the effects of stand‐up exercise (SE) training on the recovery of atrophied skeletal muscles in male mice. Mice were trained to stand up and press an elevated lever in response to a light‐tone cue preceding an electric foot shock and then subjected to tail suspension (TS) for 2 weeks to induce disuse atrophy in hind limb muscles. After release from TS, mice were divided into SE‐trained (SE cues: 25 times per set, two sets per day) and non‐SE‐trained groups. Seven days after the training, average myofiber cross‐sectional area (CSA) of the soleus muscle was significantly greater in the SE‐trained group than in the non‐SE‐trained group (1843 ± 194 μm2 vs. 1315 ± 153 μm2). Mean soleus muscle CSA in the SE trained group was not different from that in the CON group subjected to neither TS nor SE training (2005 ± 196 μm2), indicating that SE training caused nearly complete recovery from muscle atrophy. The number of myonuclei per myofiber was increased by ~60% in the SE‐trained group compared with the non‐SE‐trained and CON groups (0.92 ± 0.03 vs. 0.57 ± 0.03 and 0.56 ± 0.11, respectively). The number of proliferating myonuclei, identified by 5‐ethynyl‐2′‐deoxyuridine staining, increased within the first few days of SE training. Thus, it is highly likely that myogenic satellite cells proliferated rapidly in atrophied muscles in response to SE training and fused with existing myofibers to reestablish muscle mass.
Stand‐up exercise (SE) training facilitates recovery of myofiber size within 7 days and increases the number of myonuclei during atrophied muscle recovery. This rapid increase in the number of myonuclei derived from myogenic satellite cells may account for the rapid histological changes in atrophied muscles induced by SE training.
PMCID: PMC4255801  PMID: 25367692
5‐ethynyl‐2’‐deoxyuridine; myogenic satellite cells; myonuclei; operant conditioning; stand‐up exercise
14.  Effects of Normobaric Oxygen on the Progression of Focal Cerebral Ischemia in Rats 
Experimental neurology  2013;249:33-38.
Normobaric oxygen (NBO) reduces infarction at 24–48 hrs in experimental models of focal cerebral ischemia. However, to be clinically relevant, longer term safety and efficacy must be explored. Here, we assessed the effects of NBO on glial activation, neurovascular recovery, and behavioral outcomes at 2 weeks after transient focal ischemia in rats. 100 min transient focal ischemia was induced by intraluminal occlusion of the middle cerebral artery in adult male Sprague-Dawley rats. Animals were randomized into sham, controls or 85′NBO started 15 minutes after ischemic onset. Infarct volumes and behavioral outcomes were blindly quantified. Immunohistochemistry was used to examine the effects of NBO on glial activation and neurovascular responses. After 2 weeks of reperfusion the infarct volume was marked reduced in animals subjected to NBO. They also had better outcomes in forelimb placement test and in body-swing test and weight loss reduction. After 14 days, NBO decreased expression of Iba1, a marker of activated microglia, and GFAP, a marker of activated astrocytes. NBO treatment had no detectable effect on angiogenesis. These results suggest that protective effects of NBO may persist for up to 2 weeks post-stroke.
PMCID: PMC3805377  PMID: 23958492
normobaric oxygen therapy; stroke; neuroprotection
15.  HbA1c and the Risks for All-Cause and Cardiovascular Mortality in the General Japanese Population 
Diabetes Care  2013;36(11):3759-3765.
Associations between HbA1c and cardiovascular diseases (CVD) have been reported mainly in Western countries. It is not clear whether HbA1c measurements are useful for assessing CVD mortality risk in East Asian populations.
The risk for cardiovascular death was evaluated in a large cohort of participants selected randomly from the overall Japanese population. A total of 7,120 participants (2,962 men and 4,158 women; mean age 52.3 years) free of previous CVD were followed for 15 years. Adjusted hazard ratios (HRs) and 95% CIs among categories of HbA1c (<5.0%, 5.0–5.4%, 5.5–5.9%, 6.0–6.4%, and ≥6.5%) for participants without treatment for diabetes and HRs for participants with diabetes were calculated using a Cox proportional hazards model.
During the study, there were 1,104 deaths, including 304 from CVD, 61 from coronary heart disease, and 127 from stroke (78 from cerebral infarction, 25 from cerebral hemorrhage, and 24 from unclassified stroke). Relations to HbA1c with all-cause mortality and CVD death were graded and continuous, and multivariate-adjusted HRs for CVD death in participants with HbA1c 6.0–6.4% and ≥6.5% were 2.18 (95% CI 1.22–3.87) and 2.75 (1.43–5.28), respectively, compared with participants with HbA1c <5.0%. Similar associations were observed between HbA1c and death from coronary heart disease and death from cerebral infarction.
High HbA1c levels were associated with increased risk for all-cause mortality and death from CVD, coronary heart disease, and cerebral infarction in general East Asian populations, as in Western populations.
PMCID: PMC3816883  PMID: 23877989
16.  Major Variation in MICs of Tigecycline in Gram-Negative Bacilli as a Function of Testing Method 
Journal of Clinical Microbiology  2014;52(5):1617-1621.
Tigecycline is one of the few remaining therapeutic options for extensively drug-resistant (XDR) Gram-negative bacilli (GNB). MICs of tigecycline to Acinetobacter baumannii have been reported to be elevated when determined by the Etest compared to determinations by the broth microdilution (BMD) method. The study aim was to compare the susceptibility of GNB to tigecycline by four different testing methods. GNB were collected from six health care systems (25 hospitals) in southeast Michigan from January 2010 to September 2011. Tigecycline MICs among A. baumannii, carbapenem-resistant Enterobacteriaceae (CRE), extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, and susceptible Enterobacteriaceae isolates were determined by Etest, BMD, Vitek-2, and MicroScan. Nonsusceptibility was categorized as a tigecycline MIC of ≥4 μg/ml for both A. baumannii and Enterobacteriaceae. The study included 4,427 isolates: 2,065 ESBL-producing Enterobacteriaceae, 1,105 A. baumannii, 888 susceptible Enterobacteriaceae, and 369 CRE isolates. Tigecycline nonsusceptibility among A. baumannii isolates was significantly more common as determined by Etest compared to that determined by BMD (odds ratio [OR], 10.3; P < 0.001), MicroScan (OR, 12.4; P < 0.001), or Vitek-2 (OR, 9.4; P < 0.001). These differences were not evident with the other pathogens. Tigecycline MICs varied greatly according to the in vitro testing methods among A. baumannii isolates. Etest should probably not be used by laboratories for tigecycline MIC testing of A. baumannii isolates, since MICs are significantly elevated with Etest compared to those determined by the three other methods.
PMCID: PMC3993642  PMID: 24599978
17.  Deficient eNOS phosphorylation is a mechanism for diabetic vascular dysfunction contributing to increased stroke size 
Stroke; a journal of cerebral circulation  2013;44(11):10.1161/STROKEAHA.113.002073.
Background and Purpose
Phosphorylation of eNOS, an important post-translational modulator of its enzymatic activity, is reduced in diabetes. We hypothesized that modulation of eNOS phosphorylation could overcome diabetic vascular dysfunction and improves the outcome to stroke.
We used the db/db mouse model of type 2 diabetes. We mated db/db mice with eNOS knockin mice that carry single-amino acid mutations at the S1176 phosphorylation site; the phosphomimetic SD mutation shows increased eNOS enzymatic activity, while the unphosphorylatable SA mutation shows decreased eNOS activity. We characterized the vascular anatomy, baseline physiologic parameters and vascular reactivity. We used the middle cerebral artery occlusion model of stroke and measured infarct volume and neurological deficits.
db/db mice showed diminished eNOS phosphorylation at S1176. eNOS SD and SA mutations do not change the vascular anatomy at the Circle of Willis, brain capillary density, heart rate, or arterial blood gases of db/db mice. The eNOS SD mutation, but not the SA mutation, lowers blood pressure and improves vascular reactivity to acetylcholine in db/db mice. The eNOS SD mutation reduces stroke size and neurologic deficit following middle cerebral artery occlusion.
Diminished eNOS phosphorylation is a mechanism of vascular dysfunction in db/db mice. We show here that modulation of the eNOS S1176 phosphorylation site in db/db mice is associated with improved vascular reactivity and improved outcome to stroke following middle cerebral artery occlusion.
PMCID: PMC3864831  PMID: 23988642
nitric oxide; endothelial dysfunction; diabetes mellitus
18.  Preoperative parameters to predict incidental (T1a and T1b) prostate cancer 
Canadian Urological Association Journal  2014;8(11-12):E815-E820.
Prostate cancer has been found incidentally in transurethral resection of the prostate (TURP) specimens without prior diagnosis in 5% to 13% of the patients. We evaluated whether incidental prostate cancer (stages T1a and T1b) could be predicted preoperatively.
TURP was performed in 307 patients between 2006 and 2011. Patient age, prostate-specific antigen (PSA) level, total prostate volume, transitional zone volume, PSA density, history of needle biopsy, and pathological diagnosis on TURP specimen were assessed. We analyzed the association between these parameters and prostate cancer detection.
Incidental prostate cancer was found in 31 patients (10.1%), and 13 cases (4.2%) had cancer with T1b and/or Gleason ≥7. Multivariate analysis demonstrated that age ≥75 years (odds ratio [OR] 2.58, p = 0.022), prostate volume ≤50 cc (OR 4.11, p < 0.001), and the absence of preoperative needle biopsy despite PSA ≥4 ng/mL (OR 2.65, p = 0.046) were independent risk factors. In patients who had 2 or 3 of these risk factors, incidental prostate cancer and cancer with T1b and/or Gleason ≥7 were observed in 25% to 50% and 16% to 25% cases, respectively.
Older patient age, small prostate volume, and the absence of previous needle biopsy (despite a high PSA level) might be independent risk factors for detecting incidental prostate cancer, although external validation is warranted to confirm our results.
PMCID: PMC4250246  PMID: 25485009
19.  Expression of Adrenergic and Cholinergic Receptors in Murine Renal Intercalated Cells 
Neurons influence renal function and help to regulate fluid homeostasis, blood pressure and ion excretion. Intercalated cells (ICCs) are distributed throughout the renal collecting ducts and help regulate acid/base equilibration. Because ICCs are located among principal cells, it has been difficult to determine the effects that efferent nerve fibers have on this cell population. In this study, we examined the expression of neurotransmitter receptors on the murine renal epithelial M-1 cell line. We found that M-1 cells express a2 and b2 adrenergic receptor mRNA and the b2 receptor protein. Further, b2 receptor-positive cells in the murine cortical collecting ducts also express AQP6, indicating that these cells are ICCs. M-1 cells were found to express m1, m4 and m5 muscarinic receptor mRNAs and the m1 receptor protein. Cells in the collecting ducts also express the m1 receptor protein, and some m1-positive cells express AQP6. Acetylcholinesterase was detected in cortical collecting duct cells. Interestingly, acetylcholinesterase-positive cells neighbored AQP6-positive cells, suggesting that principal cells may regulate the availability of acetylcholine. In conclusion, our data suggest that ICCs in murine renal collecting ducts may be regulated by the adrenergic and cholinergic systems.
PMCID: PMC4272982  PMID: 25069412
autonomic nervous system; nephrology; parasympathetic; renal epithelium; sympathetic
20.  RhoA Mutations Identified in Diffuse Gastric Cancer 
Cancer cell  2014;26(1):9-11.
The diffuse-type histologic variant of gastric cancer is characterized by highly invasive growth patterns and lack of cellular cohesion. Two recent studies have identified highly recurrent mutations of the gene encoding the small GTPase RhoA and suggest that RhoA activity may have a tumor suppressive role in this disease.
PMCID: PMC4206186  PMID: 25026207
21.  c-di-GMP Enhances Protective Innate Immunity in a Murine Model of Pertussis 
PLoS ONE  2014;9(10):e109778.
Innate immunity represents the first line of defense against invading pathogens in the respiratory tract. Innate immune cells such as monocytes, macrophages, dendritic cells, NK cells, and granulocytes contain specific pathogen-recognition molecules which induce the production of cytokines and subsequently activate the adaptive immune response. c-di-GMP is a ubiquitous second messenger that stimulates innate immunity and regulates biofilm formation, motility and virulence in a diverse range of bacterial species with potent immunomodulatory properties. In the present study, c-di-GMP was used to enhance the innate immune response against pertussis, a respiratory infection mainly caused by Bordetella pertussis. Intranasal treatment with c-di-GMP resulted in the induction of robust innate immune responses to infection with B. pertussis characterized by enhanced recruitment of neutrophils, macrophages, natural killer cells and dendritic cells. The immune responses were associated with an earlier and more vigorous expression of Th1-type cytokines, as well as an increase in the induction of nitric oxide in the lungs of treated animals, resulting in significant reduction of bacterial numbers in the lungs of infected mice. These results demonstrate that c-di-GMP is a potent innate immune stimulatory molecule that can be used to enhance protection against bacterial respiratory infections. In addition, our data suggest that priming of the innate immune system by c-di-GMP could further skew the immune response towards a Th1 type phenotype during subsequent infection. Thus, our data suggest that c-di-GMP might be useful as an adjuvant for the next generation of acellular pertussis vaccine to mount a more protective Th1 phenotype immune response, and also in other systems where a Th1 type immune response is required.
PMCID: PMC4198122  PMID: 25333720
22.  Progastrin Stimulates Colonic Cell Proliferation via CCK2R- and β-Arrestin–Dependent Suppression of BMP2 
Gastroenterology  2013;145(4):10.1053/j.gastro.2013.07.034.
Background & Aims
Progastrin stimulates colonic mucosal proliferation and carcinogenesis through the cholecystokinin 2 receptor (CCK2R)—partly by increasing numbers of colonic progenitor cells. However, little is known about the mechanisms by which progastrin stimulates colonic cell proliferation. We investigated the role of bone morphogenetic proteins (BMPs) in progastrin induction of colonic cell proliferation via CCK2R.
We performed microarray analysis to compare changes in gene expression in the colonic mucosa of mice that express a human progastrin transgene (hGAS), gastrin knockout (GAS−/−) mice, and C57BL/6 mice (controls); the effects of progastrin were also determined on in vitro colonic crypt cultures from cholecystokinin 2 receptor knockout (CCK2R−/−) and wild-type mice. Human colorectal and gastric cancer cells that expressed CCK2R were incubated with progastrin or Bmp2 protein; levels of β-arrestin-1 and -2 (ARRB1 and ARRB2) were knocked down using small interfering RNAs. Cells were analyzed for progastrin binding, proliferation, changes in gene expression, and symmetric cell division.
The BMP pathway was downregulated in the colons of hGAS mice, compared with controls. Progastrin suppressed transcription of Bmp2 through a pathway that required CCK2R and was mediated by ARRB1 and ARRB2. In mouse colonic epithelial cells, downregulation of Bmp2 led to decreased phosphorylation of Smads1/5/8 and suppression of Id4. In human gastric and colorectal cancer cell lines, CCK2R was necessary and sufficient for progastrin binding and induction of proliferation; these effects were blocked when cells were incubated with recombinant Bmp2. Incubation with progastrin increased the number of CD44+, bromodeoxyuridine+, and NUMB+ cells, indicating an increase in symmetric divisions of putative cancer stem cells.
Progastrin stimulates proliferation in colons of mice and cultured human cells via CCK2R- and ARRB1- and 2-dependent suppression of Bmp2 signaling. This process promotes symmetric cell division.
PMCID: PMC3829714  PMID: 23891976
Progastrin; CCK2R; BMP
23.  DNA sensing via the Stimulator of Interferon Genes (STING) adaptor in myeloid dendritic cells induces potent tolerogenic responses1 
Cytosolic DNA sensing via the STING adaptor incites autoimmunity by inducing type I IFN (IFNαβ). Here we show that DNA is also sensed via STING to suppress immunity by inducing indoleamine 2,3 dioxygenase (IDO). STING gene ablation abolished IFNαβ and IDO induction by dendritic cells (DCs) after DNA nanoparticle (DNP) treatment. Marginal zone macrophages, some DCs and myeloid cells ingested DNPs but CD11b+ DCs were the only cells to express IFNβ, while CD11b+ non-DCs were major IL-1β producers. STING ablation also abolished DNP-induced regulatory responses by DCs and regulatory T cells (Tregs), and hallmark regulatory responses to apoptotic cells were also abrogated. Moreover, systemic cyclic diguanylate monophosphate (c-diGMP) treatment to activate STING induced selective IFNβ expression by CD11b+ DCs and suppressed Th1 responses to immunization. Thus, previously unrecognized functional diversity amongst physiologic innate immune cells regarding DNA sensing via STING is pivotal in driving immune responses to DNA.
PMCID: PMC3788571  PMID: 23986532
24.  An Acute-phase Protein as a Regulator of Sperm Survival in the Bovine Oviduct: Alpha 1-acid-glycoprotein Impairs Neutrophil Phagocytosis of Sperm In Vitro 
We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E2 (PGE2) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE2 at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct.
PMCID: PMC4219990  PMID: 24931131
Alpha 1-acid glycoprotein (AGP); Bovine; Oviduct; Phagocytosis; Sperm
25.  Markedly improved outcomes and acceptable toxicity in adolescents and young adults with acute lymphoblastic leukemia following treatment with a pediatric protocol: a phase II study by the Japan Adult Leukemia Study Group 
Blood Cancer Journal  2014;4(10):e252-.
The superiority of the pediatric protocol for adolescents with acute lymphoblastic leukemia (ALL) has already been demonstrated, however, its efficacy in young adults remains unclear. The ALL202-U protocol was conducted to examine the efficacy and feasibility of a pediatric protocol in adolescents and young adults (AYAs) with BCR–ABL-negative ALL. Patients aged 15–24 years (n=139) were treated with the same protocol used for pediatric B-ALL. The primary objective of this study was to assess the disease-free survival (DFS) rate and its secondary aims were to assess toxicity, the complete remission (CR) rate and the overall survival (OS) rate. The CR rate was 94%. The 5-year DFS and OS rates were 67% (95% confidence interval (CI) 58–75%) and 73% (95% CI 64–80%), respectively. Severe adverse events were observed at a frequency that was similar to or lower than that in children treated with the same protocol. Only insufficient maintenance therapy significantly worsened the DFS (hazard ratio 5.60, P<0.001). These results indicate that this protocol may be a feasible and highly effective treatment for AYA with BCR–ABL-negative ALL.
PMCID: PMC4220650  PMID: 25325302

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