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1.  Invalid retro-cues can eliminate the retro-cue benefit: Evidence for a hybridized account 
The contents of visual working memory (VWM) are capacity limited and require frequent updating. The retrospective cueing (retro-cueing) paradigm clarifies how directing internal attention among VWM items boosts VWM performance. In this paradigm a cue appears prior to retrieval, but after encoding and maintenance. The retro-cue effect (RCE) refers to superior VWM after valid versus neutral retro-cues. Here we investigated the effect of the invalid retro-cues inclusion on VWM performance. We conducted two pairs of experiments changing both probe type (recognition/recall) and in the presence/absence of invalid retro-cue trials. Furthermore, to fully characterize these effects over time we also used extended post-retro-cue delay durations. In the first set of experiments probing VWM using recognition indicated that the RCE remained consistent in magnitude with or without invalid retro-cue trials. In the second set of experiments VWM was probed with recall. Here, the RCE was eliminated when invalid retro-cues were included. This finer-grained measure of VWM fidelity showed that all items were subject to decay over time. We conclude that the invalid retro-cues impaired the protection of validly cues items, but they remain accessible, suggesting greater concordance with a prioritization account.
PMCID: PMC4172509  PMID: 25045904
visual working memory; short-term memory; retro-cue; retrospective cueing; attention
2.  DEC2–E4BP4 Heterodimer Represses the Transcriptional Enhancer Activity of the EE Element in the Per2 Promoter 
The circadian oscillation of clock gene expression in mammals is based on the interconnected transcriptional/translational feedback loops of Period (Per) and Bmal1. The Per feedback loop initiates transcription through direct binding of the BMAL1–CLOCK (NPAS2) heterodimer to the E-box of the Per2 promoter region. Negative feedback of PER protein on this promoter subsequently represses transcription. Other circadian transcription regulators, particularly E4BP4 and DEC2, regulate the amplitude and phase of Per2 expression rhythms. Moreover, a direct repeat of E-box-like (EE) elements in the Per2 promoter is required for its cell-autonomous circadian rhythm. However, the detailed mechanism for repression of the two core sequences of the EE element in the Per2 promoter region is unknown. Here, we show that E4BP4 binds to the Per2 EE element with DEC2 to repress transcription and identify the DEC2–E4BP4 heterodimer as a key repressor of the tightly interlocked Per2 feedback loop in the mammalian circadian oscillator. Our results suggest an additional modulatory mechanism for tuning of the phase of cell-autonomous Per2 gene expression cycling.
PMCID: PMC4512152  PMID: 26257703
PER2; DEC2; E4BP4; circadian rhythm; E-box
4.  Association between length of hospital stay and implementation of discharge planning in acute psychiatric inpatients in Japan 
Japan has introduced an acute psychiatric care unit to the public healthcare insurance program, but its requirement of a shorter length of stay could lead to discharges without proper discharge planning. The aim of this study was to examine the association between the implementation of discharge planning and the length of stay of acute psychiatric inpatients in Japan.
This retrospective cross-sectional study included 449 patients discharged from the ‘psychiatric emergency ward’ of 66 hospitals during a two-week period from March 7 to 20, 2011. The assigned nurse or nursing assistant for each patient provided information on the implementation of discharge planning in the hospital stay.
Approximately one quarter of the 449 patients (n = 122) received no support for coordination with post-discharge community care resources. The 122 patients who had received no support for community care coordination had a significantly lower mean age at admission, a shorter length of stay, and a higher rate of either no follow-up or unidentified post-discharge outpatient service than the other 327 patients. Multilevel linear regression analysis demonstrated a significantly greater length of stay among patients who were older, those who had a primary diagnosis of schizophrenia, those who were admitted compulsorily, those who received hospital outpatient services, and those who received community care coordination support from the assigned nurse or nursing assistant. The implementation of support for community care coordination did not indicate a significant association with these factors, which have been related to an increased risk of psychiatric readmission.
Patients to whom the assigned nurse or nursing assistant provided support on community care coordination experienced a significantly greater length of hospital stay. The implementation of support for community care coordination did not indicate a significant association with these factors, which have been related to an increased risk of psychiatric readmission. The mental health policy should increase focus on discharge planning in the acute psychiatric setting to enhance a link between psychiatric inpatient care and post-discharge community care resources.
PMCID: PMC4449576  PMID: 26029254
5.  The novel EuroSCORE II algorithm predicts the hospital mortality of thoracic aortic surgery in 461 consecutive Japanese patients better than both the original additive and logistic EuroSCORE algorithms 
The European System for Cardiac Operative Risk Evaluation (EuroSCORE) II was developed to improve the overestimation of surgical risk associated with the original (additive and logistic) EuroSCOREs. The purpose of this study was to evaluate the significance of the EuroSCORE II by comparing its performance with that of the original EuroSCOREs in Japanese patients undergoing surgery on the thoracic aorta.
We have calculated the predicted mortalities according to the additive EuroSCORE, logistic EuroSCORE and EuroSCORE II algorithms in 461 patients who underwent surgery on the thoracic aorta during a period of 20 years (1993–2013).
The actual in-hospital mortality rates in the low- (additive EuroSCORE of 3–6), moderate- (7–11) and high-risk (≥11) groups (followed by overall mortality) were 1.3, 6.2 and 14.4% (7.2% overall), respectively. Among the three different risk groups, the expected mortality rates were 5.5 ± 0.6, 9.1 ± 0.7 and 13.5 ± 0.2% (9.5 ± 0.1% overall) by the additive EuroSCORE algorithm, 5.3 ± 0.1, 16 ± 0.4 and 42.4 ± 1.3% (19.9 ± 0.7% overall) by the logistic EuroSCORE algorithm and 1.6 ± 0.1, 5.2 ± 0.2 and 18.5 ± 1.3% (7.4 ± 0.4% overall) by the EuroSCORE II algorithm, indicating poor prediction (P < 0.0001) of the mortality in the high-risk group, especially by the logistic EuroSCORE. The areas under the receiver operating characteristic curves of the additive EuroSCORE, logistic EuroSCORE and EuroSCORE II algorithms were 0.6937, 0.7169 and 0.7697, respectively. Thus, the mortality expected by the EuroSCORE II more closely matched the actual mortality in all three risk groups. In contrast, the mortality expected by the logistic EuroSCORE overestimated the risks in the moderate- (P = 0.0002) and high-risk (P < 0.0001) patient groups.
Although all of the original EuroSCOREs and EuroSCORE II appreciably predicted the surgical mortality for thoracic aortic surgery in Japanese patients, the EuroSCORE II best predicted the mortalities in all risk groups.
PMCID: PMC3957283  PMID: 24368550
Risk stratification; Hospital mortality; Aortic surgery
6.  DNA Methyltransferase Inhibitor Zebularine Induces Human Cholangiocarcinoma Cell Death through Alteration of DNA Methylation Status 
PLoS ONE  2015;10(3):e0120545.
Cholangiocarcinoma (CCA) is a cancer arising from the neoplastic transformation of cholangiocytes. During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. In this study, we explore the effect and possible mechanism of action of zebularine on CCA cells. We demonstrate that zebularine exerts an antitumor effect on CCA cells. Zebularine treatment decreased the concentrations of DNA methyltransferase (DNMT) proteins, and DNMT1 knockdown led to apoptotic cell death in the CCA cell lines TFK-1 and HuCCT1. DNA methylation analysis demonstrated that zebularine induced DNA demethylation, and the GO Biological Process terms “hemophilic cell adhesion”, “regulation of transcription, DNA-dependent” and “Wnt signaling pathway” were found to be significantly enriched in association with demethylated genes. Furthermore, we observed that zebularine treatment decreased β-catenin protein levels in TFK-1 and HuCCT1 cells. These results suggest that zebularine alters DNA methylation status, and that some aspect of DNA demethylation by zebularine induces suppression of the Wnt signaling pathway, which leads to apoptotic cell death in CCA. We previously reported a novel mechanism of zebularine-induced cell growth arrest and apoptosis in hepatocellular carcinoma via a DNA methylation-independent pathway. Together, our present and previous studies indicate that zebularine could function as both a DNMT inhibitor and a non-DNMT inhibitor reagent, and that, while the optimal usage of zebularine may depend on cancer type, zebularine may be useful for chemotherapy against cancer.
PMCID: PMC4370694  PMID: 25799509
7.  ANGPTL2 increases bone metastasis of breast cancer cells through enhancing CXCR4 signaling 
Scientific Reports  2015;5:9170.
Bone metastasis of breast cancer cells is a major concern, as it causes increased morbidity and mortality in patients. Bone tissue-derived CXCL12 preferentially recruits breast cancer cells expressing CXCR4 to bone metastatic sites. Thus, understanding how CXCR4 expression is regulated in breast cancer cells could suggest approaches to decrease bone metastasis of breast tumor cells. Here, we show that tumor cell-derived angiopoietin-like protein 2 (ANGPTL2) increases responsiveness of breast cancer cells to CXCL12 by promoting up-regulation of CXCR4 in those cells. In addition, we used a xenograft mouse model established by intracardiac injection of tumor cells to show that ANGPTL2 knockdown in breast cancer cells attenuates tumor cell responsiveness to CXCL12 by decreasing CXCR4 expression in those cells, thereby decreasing bone metastasis. Finally, we found that ANGPTL2 and CXCR4 expression levels within primary tumor tissues from breast cancer patients are positively correlated. We conclude that tumor cell-derived ANGPTL2 may increase bone metastasis by enhancing breast tumor cell responsiveness to CXCL12 signaling through up-regulation of tumor cell CXCR4 expression. These findings may suggest novel therapeutic approaches to treat metastatic breast cancer.
PMCID: PMC4360633  PMID: 25773070
8.  Effects of One-Hour Training Course and Spirometry on the Ability of Physicians to Diagnose and Treat Chronic Obstructive Pulmonary Disease 
PLoS ONE  2015;10(2):e0117348.
In China, the prevalence of chronic obstructive pulmonary disease (COPD) in persons 40 years of age or older is estimated at 8.2%, but this is likely a substantial underestimate.
Eight secondary hospitals which didn’t have spirometries were chosen randomly in Hunan province of central south China. Physician subjects at these hospitals underwent a one-hour training course on the Chinese COPD guidelines. Physicians answered questionnaires assessing their knowledge of the guidelines before and after the training session. The mean correct scores of questionnaires were compared before and after training. Four out of the eight hospitals were given access to spirometry. Eligible patient subjects underwent spirometry testing prior to the physician visit. After seeing the patient, physicians were asked to answer a questionnaire relating to the diagnosis and severity of COPD. Physicians were then given the results of the spirometry, and asked to answer the same questionnaire. Physicians’ responses before and after receiving the spirometry results were compared.
225 physicians participated in the training session. 207 questionnaires were completed. Mean scores (out of 100) before and after the training were 53.1 ± 21.7 and 93.3 ± 9.8, respectively. 18 physicians and 307 patient subjects participated in the spirometry intervention. Based on spirometric results, the prevalence of COPD was 38.8%. Physicians correctly identified the presence of COPD without spirometric data in 85 cases (76.6%); this increased to 117 cases (97.4%) once spirometric data were available. Without spirometric data, physicians incorrectly diagnosed COPD in 38 patients; this decreased to 6 patients once spirometric data were available. Spirometric data also improved the ability of physicians to correctly grade COPD severity.
Simple educational training can substantially improve physicians’ knowledge relating to COPD. Spirometry combined with education improves the ability of physicians to diagnose COPD and to assess its severity.
PMCID: PMC4338104  PMID: 25706774
9.  Angiopoietin-like protein 2 renders colorectal cancer cells resistant to chemotherapy by activating spleen tyrosine kinase–phosphoinositide 3-kinase-dependent anti-apoptotic signaling 
Cancer Science  2014;105(12):1550-1559.
Angiopoietin-like protein 2 (ANGPTL2) plays an important role in inflammatory carcinogenesis and tumor metastasis by activating tumor angiogenesis and tumor cell chemotaxis and invasiveness. However, it is unclear whether ANGPTL2 expression has an effect on tumor cell survival. Here, we explored that possibility by determining whether ANGPTL2 expression altered survival of human colorectal cancer cell lines treated with antineoplastic drugs. To do so, we generated SW480 cells expressing ANGPTL2 (SW480/ANGPTL2) and control (SW480/Ctrl) cells. Apoptosis induced by antineoplastic drug treatment was significantly decreased in SW480/ANGPTL2 compared to control cells. Expression of anti-apoptotic BCL-2 family genes was upregulated in SW480/ANGPTL2 compared to SW480/Ctrl cells. To assess signaling downstream of ANGPTL2 underlying this effect, we carried out RNA sequencing analysis of SW480/ANGPTL2 and SW480/Ctrl cells. That analysis, combined with in vitro experiments, indicated that Syk-PI3K signaling induced expression of BCL-2 family genes in SW480/ANGPTL2 cells. Furthermore, ANGPTL2 increased its own expression in a feedback loop by activating the spleen tyrosine kinase–nuclear factor of activated T cells (Syk–NFAT) pathway. Finally, we observed a correlation between higher ANGPTL2 expression in primary unresectable tumors from colorectal cancer patients who underwent chemotherapy with a lower objective response rate. These findings suggest that attenuating ANGPTL2 signaling in tumor cells may block tumor cell resistance to antineoplastic therapies.
PMCID: PMC4317964  PMID: 25287946
ANGPTL2; apoptosis; BCL-2; chemoresistance; Syk
10.  Distinctive Tooth-Extraction Socket Healing: Bisphosphonate Versus Parathyroid Hormone Therapy 
Journal of periodontology  2013;85(1):24-33.
Patients with osteoporosis who receive tooth extractions are typically on either oral bisphosphonate or parathyroid hormone (PTH) therapy. Currently, the consequence of these therapies on hard- and soft-tissue healing in the oral cavity is not clearly defined. The aim of this study is to determine the differences in the therapeutic effect on tooth-extraction wound healing between bisphosphonate and PTH therapies.
Maxillary second molars were extracted in Sprague Dawley rats (n = 30), and either bisphosphonate (zoledronate [Zol]), PTH, or saline (vehicle control [VC]) was administered for 10 days (n = 10 per group). Hard-tissue healing was evaluated by microcomputed tomography and histomorphometric analyses. Collagen, blood vessels, inflammatory cell infiltration, and cathepsin K expression were assessed in soft tissue using immunohistochemistry, quantitative polymerase chain reaction, and immunoblotting.
Both therapies significantly increased bone fill and suppressed vertical bone loss. However, considerably more devital bone was observed in the sockets of rats on Zol versus VC. Although Zol increased the numbers of blood vessels, the total blood vessel area in soft tissue was significantly smaller than in VC. PTH therapy increased osteoblastic bone formation and suppressed osteoclasts. PTH therapy promoted soft-tissue maturation by suppressing inflammation and stimulating collagen deposition.
Zoledronate therapy deters whereas PTH therapy promotes hard- and soft-tissue healing in the oral cavity, and both therapies prevent vertical bone loss.
PMCID: PMC4245052  PMID: 23688101
Parathyroid hormone; tooth extraction; wound healing; zoledronic acid
11.  Regulation of gustatory physiology and appetitive behavior by the circadian clock in Drosophila melanogaster 
Current biology : CB  2010;20(4):300-309.
Circadian regulation of chemosensory processes is common in animals, but little is known about how circadian clocks control chemosensory systems or the consequences of rhythms in chemosensory system function. Taste is a major chemosensory gate used to decide whether or not an animal will eat, and the main taste organ in Drosophila, the proboscis, harbors autonomous circadian oscillators. Here we examine gustatory physiology, tastant-evoked appetitive behavior, and food ingestion to understand clock-dependent regulation of the Drosophila gustatory system.
Here we report that single-unit responses from labellar gustatory receptor neurons (GRNs) to attractive and aversive tastants show diurnal and circadian rhythms in spike amplitude, frequency and duration across different classes of gustatory sensilla. Rhythms in electrophysiological responses parallel behavioral rhythms in proboscis extension reflex (PrER). Molecular oscillators in GRNs are necessary and sufficient for rhythms in gustatory responses, and drive rhythms in G protein coupled receptor kinase 2 (GPRK2) expression that mediate rhythms in taste-sensitivity. Eliminating clock function in certain GRNs increases feeding and locomotor activity, mimicking a starvation response.
Circadian clocks in GRNs control neuronal output and drive behavioral rhythms in taste responses that peak at a time of day when feeding is maximal in flies. Our results argue that oscillations in GPRK2 levels drive rhythms in gustatory physiology and behavior, and that GRN clocks repress feeding. The similarity in gustatory system organization and feeding behavior in flies and mammals, and diurnal changes in taste sensitivity in humans, suggest that our results are relevant to the situation in humans.
PMCID: PMC4234688  PMID: 20153192
13.  Binding of a Third Metal Ion by the Human Phosphatases PP2Cα and Wip1 is Required for Phosphatase Activity 
Biochemistry  2013;52(34):10.1021/bi4005649.
The PPM phosphatases require millimolar concentrations of Mg2+ or Mn2+ to activate phosphatase activity in vitro. The human phosphatases PP2Cα (PPM1A) and Wip1 (PPM1D) differ in their physiological function, substrate specificity, and apparent metal affinity. A crystallographic structure of PP2Cα shows only two metal ions in the active site. However, recent structural studies of several bacterial PP2C phosphatases have indicated three metal ions in the active site. Two residues that coordinate the third metal ion are highly conserved, suggesting that human PP2C phosphatases may also bind a third ion. Here, isothermal titration calorimetry analysis of Mg2+ binding to PP2Cα distinguished binding of two ions to high affinity sites from the binding of a third ion with a millimolar affinity, similar to the apparent metal affinity required for catalytic activity. Mutational analysis indicated that Asp239 and either Asp146 or Asp243 was required for low-affinity binding of Mg2+, but that both Asp146 and Asp239 were required for catalysis. Phosphatase activity assays in the presence of MgCl2, MnCl2, or mixtures of the two, demonstrate high phosphatase activity toward a phosphopeptide substrate when Mg2+ was bound to the low-affinity site, whether Mg2+ or Mn2+ ions were bound to the high affinity sites. Mutation of the corresponding putative third metal ion-coordinating residues of Wip1 affected catalytic activity similarly both in vitro and in human cells. These results suggest that phosphatase activity toward phosphopeptide substrates by PP2Cα and Wip1 requires the binding of a Mg2+ ion to the low-affinity site.
PMCID: PMC3824971  PMID: 23906386
14.  Genetic variants and mutations of PPM1D control the response to DNA damage 
Cell Cycle  2013;12(16):2656-2664.
The Wip1 phosphatase is an oncogene that is overexpressed in a variety of primary human cancers. We were interested in identifying genetic variants that could change Wip1 activity. We identified 3 missense SNPs of the human Wip1 phosphatase, L120F, P322Q, and I496V confer a dominant-negative phenotype. On the other hand, in primary human cancers, PPM1D mutations commonly result in a gain-of-function phenotype, leading us to identify a hot-spot truncating mutation at position 525. Surprisingly, we also found a significant number of loss-of-function mutations of PPM1D in primary human cancers, both in the phosphatase domain and in the C terminus. Thus, PPM1D has evolved to generate genetic variants with lower activity, potentially providing a better fitness for the organism through suppression of multiple diseases. In cancer, however, the situation is more complex, and the presence of both activating and inhibiting mutations requires further investigation to understand their contribution to tumorigenesis.
PMCID: PMC3865055  PMID: 23907125
Wip1; PPM1D; genetic variants; mutations; human cancer; ATM; Chk2
15.  Rab35, acting through ACAP2 switching off Arf6, negatively regulates oligodendrocyte differentiation and myelination 
Molecular Biology of the Cell  2014;25(9):1532-1542.
Oligodendrocyte precursor cells differentiate into oligodendrocytes to form myelin sheaths. Rab35/ACAP2 and cytohesin-2 antagonistically control oligodendrocyte differentiation and myelination through Arf6 on/off regulation, presenting a unique way of regulating oligodendrocyte differentiation and myelination by a small GTPase network.
Oligodendrocyte precursor cells differentiate to produce myelin sheaths that insulate axons to ensure fast propagation of action potentials. Many aspects of differentiation are regulated by multiple extracellular signals. However, their intracellular signalings remain elusive. We show that Rab35 and its effector, ACAP2, a GTPase-activating protein that switches off Arf6 activity, negatively regulate oligodendrocyte morphological differentiation. Knockdown of Rab35 or ACAP2 with their respective small interfering RNAs promotes differentiation. As differentiation initiates, the activities of Rab35 and ACAP2 are down-regulated. The activity of Arf6, in contrast, is up-regulated. Arf6 knockdown inhibits differentiation, indicating that Rab35 and ACAP2 negatively regulate differentiation by down-regulating Arf6. Importantly, as differentiation proceeds, the activity of cytohesin-2, a guanine nucleotide exchange factor that switches on Arf6 activity, is up-regulated. Pharmacological inhibition of cytohesin-2 inhibits differentiation, suggesting that cytohesin-2 promotes differentiation by activating Arf6. Furthermore, using oligodendrocyte-neuronal cocultures, we find that knockdown of Rab35 or ACAP2 promotes myelination, whereas inhibition of cytohesin-2 or knockdown of Arf6 inhibits myelination. Thus Rab35/ACAP2 and cytohesin-2 antagonistically control oligodendrocyte differentiation and myelination through Arf6 regulation, presenting a unique small GTPase on/off switching mechanism.
PMCID: PMC4004601  PMID: 24600047
16.  Differential frontal involvement in shifts of internal and perceptual attention 
Brain stimulation  2012;6(4):675-682.
Perceptual attention enhances the processing of items in the environment, whereas internal attention enhances processing of items encoded in visual working memory. In perceptual and internal attention cueing paradigms, cues indicate the to-be-probed item before (pre-cueing) or after (retro-cueing) the memory display, respectively. Pre- and retro- cues confer similar behavioral accuracy benefits (pre-: 14–19%, retro-: 11–17%) and neuroimaging data show that they activate overlapping frontoparietal networks (1). Yet reports of behavioral and neuroimaging differences suggest that pre- and retro-cueing differentially recruit frontal and parietal cortices (1).
This study examined whether perceptual and internal attention are equally disrupted by neurostimulation to frontal and parietal cortices. We hypothesized that neurostimulation applied to frontal cortex would disrupt internal attention to a greater extent than perceptual attention.
Cathodal transcranial direct current stimulation (tDCS) was applied to frontal or parietal cortices. After stimulation, participants completed a change detection task coupled with either pre- or retro- cues.
Cathodal tDCS across site (frontal, parietal) hindered performance. However, frontal tDCS had a greater negative impact on the retro-cued trials demonstrating greater frontal involvement during shifts of internal attention.
These results complement the neuroimaging data and provide further evidence suggesting that perceptual and internal attention are not identical processes. We conclude that although internal and perceptual attention are mediated by similar frontoparietal networks, the weight of contribution of these structures differs, with internal attention relying more heavily on the frontal cortex.
PMCID: PMC3608701  PMID: 23266133
Internal attention; perceptual attention; working memory
18.  Anatomical variations in termination of the uncal vein and its clinical implications in cavernous sinus dural arteriovenous fistulas 
Neuroradiology  2014;56(8):661-668.
The aim of the study was to investigate the variations in the uncal vein (UV) termination and its clinical implication in cavernous sinus dural arteriovenous fistulas (CSDAVFs).
Biplane cerebral angiography in 80 patients (160 sides) with normal cerebral venous return (normal group) was reviewed with special interest in the termination of the UV. Frequency and types of uncal venous drainage from CSDAVFs in consecutive 26 patients were also analyzed.
In the normal group, the UV was identified in 118 sides (74 %). The UV terminated into cavernous sinus (CS) in 41 sides (34 %), the superficial middle cerebral vein (SMCV) in 58 sides (48 %), the laterocavernous sinus (LCS) in 15 sides (13 %), and the paracavernous sinus (PCS) in 4 sides (3 %). Cerebral venous blood via the UV draining into the CS directly (n = 41) or through the SMCV and/or the LCS (n = 45) was observed in 86 sides (54 %). Uncal venous drainage from CSDAVFs was found in 13 patients (50 %). The CSDAVFs drained directly into the UV in two patients, drained via LCS into the UV in two patients, and drained through the SMCV into the UV in the remaining nine patients. All cases were successfully treated by transvenous embolization with special attention given to uncal venous drainage.
There are several variations in UV termination according to the embryological development of the primitive tentorial sinus and the deep telencephalic vein. Careful attention should be paid to uncal venous drainage for the treatment of CSDAVFs.
PMCID: PMC4125747  PMID: 24878594
Cerebral vein; Dural arteriovenous fistula; Cerebral angiography
19.  A Novel Protein, CHRONO, Functions as a Core Component of the Mammalian Circadian Clock 
PLoS Biology  2014;12(4):e1001839.
Two independent studies, one of them using a computational approach, identified CHRONO, a gene shown to modulate the activity of circadian transcription factors and alter circadian behavior in mice.
Circadian rhythms are controlled by a system of negative and positive genetic feedback loops composed of clock genes. Although many genes have been implicated in these feedback loops, it is unclear whether our current list of clock genes is exhaustive. We have recently identified Chrono as a robustly cycling transcript through genome-wide profiling of BMAL1 binding on the E-box. Here, we explore the role of Chrono in cellular timekeeping. Remarkably, endogenous CHRONO occupancy around E-boxes shows a circadian oscillation antiphasic to BMAL1. Overexpression of Chrono leads to suppression of BMAL1–CLOCK activity in a histone deacetylase (HDAC) –dependent manner. In vivo loss-of-function studies of Chrono including Avp neuron-specific knockout (KO) mice display a longer circadian period of locomotor activity. Chrono KO also alters the expression of core clock genes and impairs the response of the circadian clock to stress. CHRONO forms a complex with the glucocorticoid receptor and mediates glucocorticoid response. Our comprehensive study spotlights a previously unrecognized clock component of an unsuspected negative circadian feedback loop that is independent of another negative regulator, Cry2, and that integrates behavioral stress and epigenetic control for efficient metabolic integration of the clock.
Author Summary
The circadian clock has a fundamental role in regulating biological temporal rhythms in organisms, and it is tightly controlled by a molecular circuit consisting of positive and negative regulatory feedback loops. Although many of the clock genes comprising this circuit have been identified, there are still some critical components missing. Here, we characterize a circadian gene renamed Chrono (Gm129) and show that it functions as a transcriptional repressor of the negative feedback loop in the mammalian clock. Chrono binds to the regulatory region of clock genes and its occupancy oscillates in a circadian manner. Chrono knockout and Avp-neuron-specific knockout mice display longer circadian periods and altered expression of core clock genes. We show that Chrono-mediated repression involves the suppression of BMAL1–CLOCK activity via an epigenetic mechanism and that it regulates metabolic pathways triggered by behavioral stress. Our study suggests that Chrono functions as a clock repressor and reveals the molecular mechanisms underlying its function.
PMCID: PMC3988004  PMID: 24736997
20.  Induction of Colonic Regulatory T Cells by Indigenous Clostridium Species 
Science (New York, N.Y.)  2010;331(6015):337-341.
CD4+ T regulatory cells (Tregs), which express the Foxp3 transcription factor, play a critical role in the maintenance of immune homeostasis. Here, we show that in mice, Tregs were most abundant in the colonic mucosa. The spore-forming component of indigenous intestinal microbiota, particularly clusters IV and XIVa of the genus Clostridium, promoted Treg cell accumulation. Colonization of mice by a defined mix of Clostridium strains provided an environment rich in transforming growth factor–β and affected Foxp3+ Treg number and function in the colon. Oral inoculation of Clostridium during the early life of conventionally reared mice resulted in resistance to colitis and systemic immunoglobulin E responses in adult mice, suggesting a new therapeutic approach to autoimmunity and allergy.
PMCID: PMC3969237  PMID: 21205640
21.  Survivin-responsive conditionally replicating adenovirus kills rhabdomyosarcoma stem cells more efficiently than their progeny 
Effective methods for eradicating cancer stem cells (CSCs), which are highly tumorigenic and resistant to conventional therapies, are urgently needed. Our previous studies demonstrated that survivin-responsive conditionally replicating adenoviruses regulated with multiple factors (Surv.m-CRAs), which selectively replicate in and kill a broad range of cancer-cell types, are promising anticancer agents. Here we examined the therapeutic potentials of a Surv.m-CRA against rhabdomyosarcoma stem cells (RSCs), in order to assess its clinical effectiveness and usefulness.
Our previous study demonstrated that fibroblast growth factor receptor 3 (FGFR3) is a marker of RSCs. We examined survivin mRNA levels, survivin promoter activities, relative cytotoxicities of Surv.m-CRA in RSC-enriched (serum-minus) vs. RSC-exiguous (serum-plus) and FGFR3-positive vs. FGFR3-negative sorted rhabdomyosarcoma cells, and the in vivo therapeutic effects of Surv.m-CRAs on subcutaneous tumors in mice.
Both survivin mRNA levels and survivin promoter activities were significantly elevated under RSC-enriched relative to RSC-exiguous culture conditions, and the elevation was more prominent in FGFR3-positive vs. FGFR3-negative sorted cells than in RSC-enriched vs. RSC-exiguous conditions. Although Surv.m-CRA efficiently replicated and potently induced cell death in all populations of rhabdomyosarcoma cells, the cytotoxic effects were more pronounced in RSC-enriched or RSC-purified cells than in RSC-exiguous or progeny-purified cells. Injections of Surv.m-CRAs into tumor nodules generated by transplanting RSC-enriched cells induced significant death of rhabdomyosarcoma cells and regression of tumor nodules.
The unique therapeutic features of Surv.m-CRA, i.e., not only its therapeutic effectiveness against all cell populations but also its increased effectiveness against CSCs, suggest that Surv.m-CRA is promising anticancer agent.
PMCID: PMC3925355  PMID: 24467821
Cancer stem cells; Conditionally replicating adenovirus; Fibroblast growth factor receptor 3; Gene therapy; Oncolytic adenovirus; Promoter; Rhabdomyosarcoma; Survivin; Tumor-initiating cell; Virotherapy
22.  Laterality of cervical disc herniation 
European Spine Journal  2012;22(1):178-182.
Cervical disc herniation (CDH) is found more frequently at the lower cervical spine than at the upper or middle level. However, there is scarcity of data about the laterality of CDH. The aim of this study is to detect the laterality of CDH.
We retrospectively evaluated preoperative computed tomography myelograms and magnetic resonance images of 75 cases of CDH who underwent single level anterior cervical discectomy and fusion at C4–5, C5–6, or C6–7 levels from 2008 to 2010 in our institute. Statistical analyses were performed using the Chi-square test.
Eleven cases were found at C4–5 level, 42 cases at C5–6 level, and 22 cases at C6–7 level. At C4–5 level, CDH was recognized at the right side in 2 cases, at the left side in 2 cases, and at the center in 7 cases. At C5–6 level, CDH was found at the right side in 20 cases and at the left side in 22 cases. At C6–7 level, CDH was found at the right side in 3 cases and at the left side in 19 cases with significantly high frequency of left-sided CDH (p < 0.025).
In this study, it was revealed that the left-sided CDH was more frequent than the right-sided CDH at C6–7 level.
PMCID: PMC3540298  PMID: 23149494
Cervical disc herniation; C6–7 level; Handedness; Laterality
23.  Lung Cancer: Epidemiology, Etiology, and Prevention 
Clinics in chest medicine  2011;32(4):10.1016/j.ccm.2011.09.001.
PMCID: PMC3864624  PMID: 22054876
Lung cancer; Tobacco smoking; Epidemiology; Carcinogens
24.  Dietary fructose enhances the incidence of precancerous hepatocytes induced by administration of diethylnitrosamine in rat 
Nonalcoholic fatty liver disease (NAFLD) is a risk for hepatocellular carcinoma (HCC), but the association between a high-fructose diet and HCC is not fully understood. In this study, we investigated whether a high-fructose diet affects hepatocarcinogenesis induced by administration of diethylnitrosamine (DEN).
Seven-week-old male Sprague–Dawley rats were fed standard chow (controls), a high-fat diet (54% fat), or a high-fructose diet (66% fructose) for 8 weeks. All rats were given DEN at 50 μg/L in drinking water during the same period. Precancerous hepatocytes were detected by immunostaining of the placental form of glutathione-S-transferase (GST-P). The number of GST-P-positive hepatocytes was assessed in liver specimens.
Serum levels of total cholesterol were similar among the three groups, but serum triglyceride, fasting blood glucose, and insulin levels were higher in the high-fructose group compared to the high-fat group. In contrast, hepatic steatosis was more severe in the high-fat group compared with the high-fructose and control groups, but the incidence of GST-P-positive specimens was significantly higher in the high-fructose group compared to the other two groups. The average number of GST-P-positive hepatocytes in GST-P positive specimens in the high-fructose group was also higher than those in the other two groups. This high prevalence of GST-P-positive hepatocytes was accompanied by higher levels of 8-hydroxydeoxyguanosine in serum and liver tissue.
These results indicate that dietary fructose, rather than dietary fat, increases the incidence of precancerous hepatocytes induced by administration of DEN via insulin resistance and oxidative stress in rat. Thus, excessive fructose intake may be a potential risk factor for hepatocarcinogenesis.
PMCID: PMC4029300  PMID: 24321741
Diethylnitrosamine; Fructose; Hepatocarcinogenesis; Nonalcoholic steatohepatitis; Placental form of glutathione-S-transferase
25.  Induction of p53-Dependent p21 Limits Proliferative Activity of Rat Hepatocytes in the Presence of Hepatocyte Growth Factor 
PLoS ONE  2013;8(11):e78346.
Hepatocyte growth factor (HGF), a potent mitogen for hepatocytes, enhances hepatocyte function without stimulating proliferation, depending on the physiological conditions. p53, a transcription factor, suppresses the cell proliferation by expressing p21WAF1/CIP1 in various tissues.
To investigate the mechanism through which the hepatocytes maintain mitotically quiescent even in the presence of HGF.
We studied the relationship between p53 and p21 expression and the effect of p53-p21 axis on hepatocyte proliferation in primary cultured rat hepatocytes stimulated by HGF. Hepatic p21 levels are determined serially after partial hepatectomy or sham operation in rats.
DNA synthesis was markedly increased by HGF addition in rat hepatocytes cultured at low density but not at high density. Cellular p53 levels increased in the hepatocytes cultured at both the densities. p21 levels were increased and correlated with cellular p53 levels in hepatocytes cultured at high density but not at low density. When the activity of p53 was suppressed by a chemical inhibitor for p53, cellular p21 levels were reduced, and DNA synthesis was increased. Similarly, p21 antisense oligonucleotide increased the DNA synthesis. In rats after partial hepatectomy, transient elevation of hepatic p21 levels was observed. In contrast, in sham-operated rats, hepatic p21 levels were increased on sustained time scales.
p53-related induction of p21 may suppress hepatocyte proliferation in the presence of HGF in the setting that mitogenic activity of HGF is not elicitable.
PMCID: PMC3817248  PMID: 24223793

Results 1-25 (69)